EA044641B1 - COMPOUND CONTAINING A THERAPEUTIC ENZYME AND A TRANSPORT ELEMENT CONNECTED TO EACH OTHER DIRECTLY OR BY USING A LINKER - Google Patents

Description

Field of technology

The invention relates to the field of biotechnology, specifically to the delivery (transportation) of therapeutic enzymes, which can be used in medicine. The present invention relates to a compound containing a therapeutic enzyme and a transport element connected to each other directly or via a linker, wherein the transport element is a Fab fragment of an IgG immunoglobulin specific for an epitope in the insulin receptor, to the use of the said compound for the preparation of a pharmaceutical composition for treatment diseases and the use of the said compound for the treatment and prevention of diseases, in particular lysosomal storage diseases, including for the treatment and prevention of deficiency of the corresponding enzyme characteristic of the corresponding lysosomal storage disease, such as mucopolysaccharidosis, in particular, mucopolysaccharidosis types I and II .

Prior Art

The use of enzymes in therapy has long been known in the art. Modern medicine is increasingly using therapeutic enzymes in a wide variety of branches of medicine due to their high activity and specificity. Currently, the following directions of enzyme therapy have emerged (Kazanskaya N.F. et al., 1984): 1) elimination of enzyme deficiency in order to compensate for congenital or acquired functional deficiency; 2) removal of non-viable, denatured structures, cellular and tissue fragments; 3) lysis of blood clots; 4) complex therapy of malignant neoplasms; 5) detoxification of the body.

The use of therapeutic enzymes to eliminate enzyme deficiency in order to compensate for congenital or acquired functional deficiency has been practiced for a long time. Treatment of congenital enzyme deficiencies, of which more than 150 have been described, is an important problem in replacement therapy. Hereditary diseases such as glycogenosis, lipidosis, mucopolysaccharidosis and other lysosomal storage diseases are predominantly treated by intravenous administration of recombinant analogues of the corresponding native enzymes.

Lysosomal storage diseases are a group of rare (orphan) hereditary metabolic diseases caused by the absence or deficiency of lysosomal enzymes involved in the breakdown of complex molecules.

Currently (Novikov P.V., 2014) the following groups of lysosomal storage diseases are distinguished: 1) mucopolysaccharidoses; 2) lipidoses (sphingolipidoses - GM1- and GM2-gangliosidoses, Gaucher disease, galactosialidosis, Farber granulomatosis, leukodystrophies, Niemann-Pick disease types A and B, etc.); 3) mucolipidoses, 4) glycoproteinoses (fucosidosis, sialidosis, mannosidosis, glycogen storage disease type II - Pompe disease, Danon disease, etc.); 5) neuronal ceroid lipofuscinoses; 6) other storage diseases (Niemann-Pick disease type C, Wolman disease, cholesterol storage disease, cystinosis, Sala disease, pycnodysostosis, etc.).

Mucopolysaccharidoses (MPS) are a group that unites nine (I - IX, with 14 intermediate forms) metabolic diseases caused by more than 40 genetic disorders leading to the absence or functional deficiency of a number of lysosomal enzymes involved in the hydrolytic breakdown of glycosaminoglycans (mucopolysaccharides). Glycosaminoglycans are oligosaccharides involved in the formation of bones, cartilage, ligaments, cornea, skin, connective tissue and synovial fluid (Burrow T. A. et al., 2013).

In patients suffering from mucopolysaccharidosis, there is a deficiency or absence of at least one of the 11 enzymes of glycosaminoglycan catabolism, which over time leads to the gradual accumulation of these carbohydrates in cells and body fluids, incl. and in connective tissue. As a result, the permanent accumulation of mucopolysaccharides leads to cell damage, dysfunction of tissues and organs, most often manifested in hypertension-hydrocephalic syndrome, hepatosplenomegaly, cardiovascular failure, osteoarticular complications, functional disorders of the central nervous system (CNS) up to severe cognitive disorders and dementia (Table 1) (Burrow T. A. et al., 2013).

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Table 1

Characteristics of various MPS syndromes

Syndrome Enzyme defect Defective chromosome, type of inheritance Clinic, laboratory data Neurological component Gurler (MPS-1-N) a-L-iduronidase Chromosome 4, autosomal recessive type Children die at the age of 6-10 years from cardiac and pulmonary decompensation. Urinary levels of heparan and dermatan sulfates are elevated (Connock et al. 2006). Eat Scheie (MPS-1-S) a-L-iduronidase (incomplete) Chromosome 4, autosomal recessive type Soft version, normal intelligence, normal stature. They live up to 30 years. Urinary levels of heparan and dermatan sulfates are elevated (Connock et al. 2006, Pastores et al. 2007). No Hunter (MPS II) Iduronate-2sulfatase X-linked, recessive type Boys are sick. Mental development is reduced. They live up to 3040 years. No corneal opacity. There is no dorsolubial hump. Characterized by progressive deafness and hirsutism. Urinary levels of heparan and dermatan sulfates are elevated (Wraith et al. 2008, da Silva et al. 2016, Burrow and Leslie 2008). Eat Sanfilippo (MPS-SH) Type A - sulfamidase. Type B - b-Nacetyllucosaminidase Type C - acetylKoAglucosamini-Nacetyltransferase Type D - a-Nacetylglucosamine-6phosphatase Chromosomes 12 and 17, autosomal recessive type Severe retardation of intelligence, macrocephaly, very aggressive behavior. Relatively weak skeletal changes. Visceromegaly appears at school age. Clinically the types cannot be Eat

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differentiated. In urine - heparan sulfate (Valstar et al. 2008). Morquio (MPS-IV) N-acetylgalactosamine-6sulfatase (type A) and b-galactosidase (type B) Chromosomes 3 and 16, autosomal recessive Normal intelligence. Severe skeletal deformation. Cloudiness of the cornea. Less degree of damage to the central nervous system. The first clinical signs appear in the 2-3rd year of life. They die at the age of 20-35 years. Keratan sulfate in urine (Northover, Cowie and Wraith 1996). No MarotoLami (MPS-VI) N-acetylgalactosamine-4sulfatase Chromosome 5, autosomal recessive Slight growth retardation, pronounced hump, normal intelligence, mild facial lesions. Dermatan sulfate in urine (Garrido et al. 2008). No Slya (MPS-VII) L-glucuronidase Chromosome 7, autosomal recessive Variable mental development, pronounced hump, hepatosplenomegaly, mild facial lesions. Dermatan sulfate in urine. Eat

Mucopolysaccharidosis type I is caused by a deficiency of the lysosomal enzyme, a-L-iduronidase. This deficiency leads to the accumulation of mucopolysaccharides, especially dermatan sulfate, in tissues and organs. The accumulation of excess dermatan sulfate leads to the gradual development of numerous morphological abnormalities in tissues and organs. Mucopolysaccharidosis type I is characterized by an autosomal recessive mode of inheritance.

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

HSCT is used to treat only severe forms of MPS type I - Hurler syndrome, which makes it possible to correct the deficiency of the enzyme a-L-iduronidase and, in turn, leads to a significant improvement in the patient’s condition, although some severe complications of the disease do not completely regress. HSCT should be performed as early as possible, before the onset of severe neurological disorders. Despite the improvement in the patient's condition, HSCT is accompanied by a high risk of serious post-transplant complications and is a complex, multi-stage, high-cost procedure.

ERT is safe, well tolerated by patients, does not cause severe adverse events and leads to the decomposition of the non-hydrolyzed substrate. Possible rare reactions to the administration of the drug are caused by the formation of antibodies against the injected protein, but they are not permanent and, as a rule, are quickly relieved by standard medications. The principle of ERT is based on restoring the level of enzymatic activity sufficient to hydrolyze accumulated substrates and prevent their further accumulation.

The drug Aldurazyme (MHH: laronidase) (Genzyme, USA) is intended for ERT in patients with three clinical variants of type I mucopolysaccharidosis (IH, IH/S and IS types or Hurler, Hurler-Scheie and Scheie syndromes), as well as children with Hurler syndrome up to carrying out HSCT (during the search for a related/unrelated donor) and for 3-6 months after HSCT until the child’s condition is stabilized; Along with this, Aldurazym is indicated for patients suffering from Hurler syndrome after HSCT in cases where the level of the donor enzyme a-L-iduronidase remains low.

Laronidase (Biomarin Pharmaceutical Inc., USA) is a recombinant form of human a-Liduronidase, produced using recombinant DNA technology using cellular

- 3 044641 Chinese hamster ovary culture. It has been established that after intravenous administration, laronidase quickly leaves the systemic circulation and is taken up by cells through mannose-6-phosphate receptors (M6PR), entering lysosomes. The drug is administered intravenously (at a dose of 100 U/kg body weight), for 3-4 hours, once a week.

Laronidase is a recombinant protein of 628 amino acid residues with a molecular weight of approximately 83 kDa. Its molecule contains 6 sections that bind through the amino group to oligosaccharides of different structures, including those containing mannose-6phosphate (M6P) residues (Rodney J.Y. Ho, Biotechnology and biopharmaceuticals: transforming proteins and genes into drugs. - Wiley-Blackwell, 2013, p . 380). It has been established that the laronidase molecule does not penetrate the blood-brain barrier (the physiological barrier between the peripheral circulation and the brain, which is formed by the tight junctions of the plasma membranes of the endothelial cells of the brain capillaries, creating a tight barrier that limits the transfer of molecules into the brain, even very small molecules; hereinafter also indicated as BBB) and, thus, does not slow down or prevent the development of disorders of the central nervous system (CNS) characteristic of mucopolysaccharidosis type I (Pastores G.M. et al., 2007).

Mucopolysaccharidosis type II (MPS-II, Hunter syndrome), unlike all other forms of mucopolysaccharidosis that have an autosomal recessive type of inheritance, is the only form of mucopolysaccharidosis with an X-linked recessive type of inheritance. MPS II is a heterogeneous clinical manifestations, progressive pathology of lysosomal accumulation, resulting from deficiency of the enzyme iduronate-2-sulfatase (idursulfase). This enzyme is normally involved in the degradation of mucopolysaccharides dermatan sulfate and heparan sulfate due to the hydrolytic removal of the O-linked sulfate group. Accordingly, in the case of MPS II, the main pathogenetic mechanism is associated with the progressive accumulation of dermatan and heparan sulfates in lysosomes.

The most common clinical symptoms of MPS-P are mental retardation, enlarged tongue, characteristic pathologies of the facial skeleton, alopecia, dental anomalies, restrictive lung disease, hepatosplenomegaly, heart valve pathology, osteoarticular pathologies, severe short stature. Also, progressive neurological disorders are often observed, accompanied by hydrocephalus and increased intracranial pressure. Death usually occurs during the second to third decade of life, most often due to respiratory and/or heart failure. It is important to emphasize that the disease occurs with the involvement of the nervous system in the pathological process, including a decrease in intelligence.

Today, there are two main methods of treating MPS II - ERT and symptomatic therapy (includes the use of hepatoprotectors, cardiovascular and anti-inflammatory drugs, vitamins and drugs that improve antioxidant protection). HSCT for the treatment of MPS II, unlike mucopolysaccharidosis type I, is not effective.

Elaprase (INN: idursulfase) (Shayer, USA) is a medical product representing recombinant human iduronate-2-sulfatase. This enzyme is responsible for the hydrolysis of C2-sulfate ester bonds of iduronic acid residues, which are part of the glycosaminoglycans (mucopolysaccharides) of dermatan sulfate and heparan sulfate (Burrow T. A. et al., 2013). The usual regimen of Elaprase administration is 0.5 mg/kg body weight, once a week; administration is carried out by intravenous infusion over 3 hours (infusion time can be gradually reduced to 1 hour).

Idursulfase contains two disulfide bonds and eight N-linked glycosylation sites occupied by complex high-mannose oligosaccharides. The presence of M6P residues in the oligosaccharide chains allows the recombinant enzyme to bind to M6P receptors on the surface of target cells, which leads to the internalization of this enzyme into cells and its internalization into lysosomes, where it ensures the degradation of lysosomal mucopolysaccharides (Muenzer J., et al., Genet Med 2006 Aug;8(8):465 - 473).

When performing ERT, the life expectancy of patients with Hunter syndrome increases several times. Despite this, Elaprase also does not penetrate the blood-brain barrier; as a result, when using ERT Elaprase, patients die at the age of 20 from neurodegenerative consequences, while in the second decade of life, patients receiving Elaprase, as a rule, experience great learning difficulties and need assistants even in everyday life (da Silva E. M. et al., 2016; Wraith J. E. et al., 2008).

Thus, a common disadvantage of all currently existing drugs used for ERT of mucopolysaccharidosis types I and II is their inability to penetrate 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 mucopolysaccharidosis, including mucopolysaccharidosis types I and II.

In many lysosomal storage diseases, there is a need for improved internalization of the enzyme into the cells of certain peripheral tissues (diaphragm muscles in Pompe disease, liver and spleen in Hunter disease, kidneys in Fabry disease, etc.) (Hawkes C. et al., 2004) .

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Thus, there is an urgent need to develop a therapeutic compound that could be used to treat a lysosomal storage disease, such as MPS type I or MPS type II, and which would retain the functional properties of the corresponding therapeutic enzyme, while demonstrating high activity and improved ability to be transported to lysosomes tissue cells of various organs, including lysosomes of nervous tissue cells, and would make it possible to achieve a significant improvement in the quality and life expectancy of patients with MPS types I and II.

Advantages of the invention

The above objects are successfully solved in the present invention, which relates to a compound intended for the treatment of lysosomal storage disease, containing a therapeutic enzyme and a transport element connected to each other directly or by means of a linker, wherein the specified transport element is a Fab fragment of an IgG immunoglobulin, specific to an epitope in the insulin receptor. The invention is based on the unexpected discovery that a transport element, which is a Fab fragment of an IgG immunoglobulin specific for an epitope in the insulin receptor, connected directly or via a linker to a therapeutic enzyme, unexpectedly leads to an improvement in the ability of the enzyme to be transported to the lysosomes of various organs and tissues, including to the lysosomes of nervous tissue cells, and at the same time the enzymatic activity of the compound remains at a high level. Thanks to this unexpected effect, progress is being made in the field of enzyme replacement therapy for lysosomal storage diseases such as mucopolysaccharidosis types I and II, and increasing the duration and improving the quality of life of subjects suffering from lysosomal storage diseases and requiring therapy. The compound of the present invention also provides an expansion of the arsenal of agents used for the treatment and prevention of diseases, in particular lysosomal storage diseases such as mucopolysaccharidosis, in particular mucopolysaccharidosis types I and II.

In addition, an unexpected increase in the enzymatic activity of a compound containing a transport element, which is a Fab fragment of an IgG immunoglobulin specific for an epitope in the insulin receptor, connected directly or via a linker to a therapeutic enzyme, was found, compared with a therapeutic enzyme without a transport element; and also that the administration of a compound containing a transport element, which is a Fab fragment of an IgG immunoglobulin specific to an epitope in the insulin receptor, connected directly or via a linker to a therapeutic enzyme, provides a higher degree of replacement of the activity of the therapeutic enzyme in the human brain in comparison with compounds containing Mab fragment.

Brief description of the invention

This Brief Description offers a brief description of the present invention in order to briefly outline the subject matter and spirit of the present invention. The Brief Description is provided with the understanding that it should not be used to interpret or limit the scope or content of the claims.

The first object of the present invention is a compound intended for the treatment of lysosomal storage disease, containing a therapeutic enzyme and a transport element connected to each other directly or by a linker, wherein the transport element is a Fab fragment of an IgG immunoglobulin specific for an epitope in the insulin receptor.

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

In a specific embodiment, the present invention provides a compound for the treatment of a lysosomal storage disease comprising a therapeutic enzyme and a transport element, wherein the transport element is a Fab fragment of an IgG immunoglobulin specific for an epitope in the insulin receptor and is capable of transporting said enzyme across the blood-brain barrier , wherein the therapeutic enzyme and the transport element are connected to each other using 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.

In a specific embodiment, the present invention provides a compound for the treatment of a lysosomal storage disease, comprising a therapeutic enzyme and a transport element connected to each other directly or by a linker, wherein the transport element is a Fab fragment of an IgG immunoglobulin, wherein the IgG immunoglobulin is IgG1 , IgG2 or IgG4.

In a specific embodiment, the present invention provides a compound for the treatment of a lysosomal storage disease comprising a therapeutic enzyme and a transport element linked to each other directly or by a linker, wherein the transport element is a Fab fragment of an IgG immunoglobulin, wherein the immunoglobulin IgG is IgG1.

In a specific embodiment, the present invention provides a compound for the treatment of a lysosomal storage disease, comprising a therapeutic enzyme and a transport element connected to each other directly or by a linker, wherein the transport element is an IgG1 immunoglobulin Fab fragment consisting of IgG1 immunoglobulin light chains to the insulin receptor (SEQ ID NO: 2, 8, 9, 10 and 11) and the heavy chain (SEQ ID NO: 3). In a particular embodiment, the object of the present invention is a compound where the transport element is a Fab fragment of an IgG1 immunoglobulin, consisting of a light chain of an IgG1 immunoglobulin to the insulin receptor SEQ ID NO: 2 and a heavy chain SEQ ID NO: 3.

In a specific embodiment, the present invention is a compound for the treatment of a lysosomal storage disease, comprising a therapeutic enzyme and a transport element connected to each other directly or by a linker, wherein the therapeutic enzyme is used for the treatment of a lysosomal storage disease, including the treatment of an enzymatic deficiency characteristic of this lysosomal storage disease.

In a specific embodiment, the present invention provides a compound for the treatment of a lysosomal storage disease, comprising a therapeutic enzyme and a transport element connected to each other directly or by a linker, wherein the therapeutic enzyme is used to treat a lysosomal storage disease with a neurological component, including, for the treatment of enzyme deficiency characteristic of this lysosomal storage disease.

In a specific embodiment, the present invention provides a compound for the treatment of a lysosomal storage disease, comprising a therapeutic enzyme and a transport element connected to each other directly or by a linker, wherein the therapeutic enzyme for a lysosomal storage disease with a neurological component is selected from the group consisting of iduronate -2-sulfatases and a-L-iduronidases.

In a specific embodiment, the present invention provides a compound for the treatment of a lysosomal storage disease, comprising a therapeutic enzyme and a transport element connected to each other directly or by a linker, wherein the therapeutic enzyme for a lysosomal storage disease with a neurological component is selected from the group consisting of iduronate -2-sulfatase, a fragment of iduronate 2-sulfatase having iduronate 2-sulfatase activity, or an iduronate 2-sulfatase analogue.

In a specific embodiment, the present invention provides a compound for the treatment of a lysosomal storage disease, comprising a therapeutic enzyme and a transport element connected to each other directly or by a linker, wherein the therapeutic enzyme for a lysosomal storage disease with a neurological component is selected from the group consisting of a-L -iduronidase, a fragment of a-L-iduronidase having a-L-iduronidase activity, or an a-L-iduronidase analogue.

In a specific embodiment, the present invention provides a compound for the treatment of a lysosomal storage disease comprising a therapeutic enzyme and a transport element linked to each other directly or by a linker, wherein said linker is a peptide linker containing one or more amino acids.

In a specific embodiment, the present invention provides a compound for the treatment of a lysosomal storage disease comprising a therapeutic enzyme and a transport element linked to each other directly or by a linker, wherein said linker is a peptide linker containing one or more amino acids selected from glycine, serine and leucine.

In a specific embodiment, the present invention provides a compound for the treatment of a lysosomal storage disease comprising a therapeutic enzyme and a transport element connected to each other directly or by a linker, in which said transport element is capable of transporting said enzyme to lysosomes of tissue cells.

In a specific embodiment, the present invention provides a compound for the treatment of a lysosomal storage disease comprising a therapeutic enzyme and a transport element connected to each other directly or by a linker, wherein said transport element is capable of transporting said enzyme to lysosomes of neural tissue cells.

Another aspect of the invention is a compound for the treatment or prevention of an enzyme deficiency (iduronate-2-sulfatase deficiency) in a subject having mucopolysaccharidosis type II, represented by the first amino acid sequence selected from SEQ ID NO: 2, 8, 9, 10 or 11, and a second amino acid sequence selected from SEQ ID NO: 4, 5, 12, 13, 14 or 15. In a particular embodiment, the present invention provides a compound for the treatment or prevention of an enzyme deficiency (iduronate-2-sulfatase deficiency) in a subject having mucopolysaccharidosis type II, represented by the amino acid sequences SEQ ID NO: 2 and

SEQ ID NO: 4.

Another object of the invention is a compound for the treatment or prevention of an enzyme deficiency (iduronate-2-sulfatase deficiency) in a subject having mucopolysaccharidosis type II with a neurological component, represented by the amino acid sequences SEQ ID NO: 2 and SEQ ID NO: 4.

Another object of the invention is a compound for the treatment or prevention of an enzyme deficiency (iduronate-2-sulfatase deficiency) in a subject having mucopolysaccharidosis type II with a neurological component, represented by the amino acid sequences SEQ ID NO: 2 and SEQ ID NO: 5.

Another object of the invention is a compound for the treatment or prevention of an enzyme deficiency (α-L-iduronidase deficiency) in a subject having mucopolysaccharidosis type I, represented by the amino acid sequences SEQ ID NO: 2 and SEQ ID NO: 6.

Another object of the invention is a compound represented by the amino acid sequences SEQ ID NO: 2 and SEQ ID NO: 6, for the treatment or prevention of enzyme deficiency (α-L-iduronidase deficiency) in a subject having mucopolysaccharidosis type I.

Another object of the invention is a compound represented by the amino acid sequences SEQ ID NO: 2 and SEQ ID NO: 6, for the treatment or prevention of enzyme deficiency (α-L-iduronidase deficiency) in a subject having mucopolysaccharidosis type I with a neurological component.

Another object of the invention is a compound represented by the amino acid sequences SEQ ID NO: 2 and SEQ ID NO: 7, for the treatment or prevention of enzyme deficiency (α-L-iduronidase deficiency) in a subject having mucopolysaccharidosis type I with a neurological component.

In addition, the present invention relates to the use of a compound containing a therapeutic enzyme and a transport element connected to each other directly or by a linker to prepare a pharmaceutical composition containing the said compound in an effective amount and a pharmaceutically acceptable carrier.

In addition, the present invention relates to the use of a compound containing a therapeutic enzyme and a transport element connected to each other directly or by a linker for the treatment or prevention of a subject having a lysosomal storage disease, comprising administering to said subject an effective amount of said compound.

In a specific embodiment, the invention relates to the use of a compound comprising a therapeutic enzyme and a transport element connected to each other directly or via a linker for the treatment or prevention of enzyme deficiency in a subject having a lysosomal storage disease, wherein the lysosomal storage disease is mucopolysaccharidosis, wherein the compound is administered to the subject in an effective amount, preferably in conjunction with a pharmaceutically acceptable carrier.

In a specific embodiment, the invention relates to the use of a compound comprising a therapeutic enzyme and a transport element connected to each other directly or by a linker, for the treatment or prevention of an enzyme deficiency (iduronate-2sulfatase deficiency) in a subject having a lysosomal storage disease, wherein the lysosomal storage disease is a mucopolysaccharidosis type II with a neurological component, wherein the compound is administered to the subject in an effective amount, preferably in conjunction with a pharmaceutically acceptable carrier.

In a specific embodiment, the invention relates to the use of a compound comprising a therapeutic enzyme and a transport element connected to each other directly or by a linker, for the treatment or prevention of an enzyme deficiency (a-Liduronidase deficiency) in a subject having a lysosomal storage disease, wherein the lysosomal storage disease is a mucopolysaccharidosis type I with a neurological component, wherein the compound is administered to the subject in an effective amount, preferably in conjunction with a pharmaceutically acceptable carrier.

In addition, the present invention relates to the use of a compound containing a therapeutic enzyme and a transport element connected to each other directly or via a linker for the treatment or prevention of enzyme deficiency (iduronate-2sulfatase deficiency) in a subject having a lysosomal storage disease, wherein the lysosomal storage disease accumulation is mucopolysaccharidosis type II, wherein the compound represented by the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 4, for the preparation of a pharmaceutical composition containing the specified compound in an effective amount and a pharmaceutically acceptable carrier.

In addition, the present invention relates to the use of a compound containing therapeutic

- 7 044641 lysosomal storage disease, wherein the lysosomal storage disease is mucopolysaccharidosis type II, with this compound represented by the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 4, for the treatment or prevention of enzyme deficiency in a subject.

In other embodiments, the approach of the present invention can be used to treat other lysosomal storage diseases, in particular, to treat other types of mucopolysaccharidosis, such as mucopolysaccharidosis type III (Sanfilippo syndrome), including mucopolysaccharidosis type IIIA, type IIIB, type IIIC and IIID type; mucopolysaccharidosis type IV (Morquio syndrome), including mucopolysaccharidosis type IVA and type IVB; mucopolysaccharidosis type VI (Maroteaux-Lamy syndrome), mucopolysaccharidosis type VII (Sly syndrome) and mucopolysaccharidosis type IX. In these cases, instead of a-L-iduronidase or iduronate-2-sulfatase, another therapeutic enzyme is used as the corresponding therapeutic enzyme, the deficiency of which is encountered by patients with the corresponding form of mucopolysaccharidosis: heparan sulfamidase - for mucopolysaccharidosis type IIIA, N-acetylglucosaminidase - for mucopolysaccharidosis type IIIB, heparan- aglucosaminide-N-acetyltransferase - for mucopolysaccharidosis type IIIC, N-acetylglucosamine-6sulfatase - for mucopolysaccharidosis type IIID, galactose-6-sulfate sulfatase - for mucopolysaccharidosis type IVA, β-galactosidase - for mucopolysaccharidosis type IVB, N-acetylgalactosamine-4-sulfatase - for mucopolysaccharidosis type VI, β-glucuronidase for mucopolysaccharidosis type VII and hyaluronidase for mucopolysaccharidosis type IX. The amino acid sequences of these therapeutic enzymes can be obtained using any of a number of computer programs known in the art, for example, BLAST or FASTA, etc. Both BLAST and FASTA offer offline and online searches (see Ausubel et al., 1999 ibid, pages 7-58 to 7-60, National Center for Biotechnology Information website at the National Institutes of Health website).

The approach proposed in the present invention, in alternative embodiments of the invention, can also be used for the treatment of lysosomal storage diseases not related to mucopolysaccharidoses, by using instead of aL-iduronidase or iduronate-2-sulfatase of another lysosomal enzyme, the deficiency of which is encountered by patients with the corresponding lysosomal storage disease. In certain non-limiting embodiments of the present invention, the proposed approach can be, in particular, applied to the treatment of disorders of amino acid metabolism, such as cystinosis; for the treatment of carbohydrate metabolism disorders, such as glycogen storage diseases, in particular Pompe disease; disorders of sphingolipid metabolism and other lipid storage diseases, in particular GM ₂ gangliosidosis, including Sandhoff disease and Tau-Sachs disease; other gangliosidoses, in particular GM1 gangliosidosis and mucolipidosis IV; other sphingolipidoses, in particular Fabry disease, Gaucher disease, Krabbe disease, Niemann-Pick disease, Farber syndrome, metachromic leukodystrophy and multiple sulfatase deficiency; lipofuscinosis of neurons, in particular, Batten, Bilypovsky-Jansky, Kufs, Spielmeyer-Vogt diseases; other lipid storage disorders, in particular Wolman's disease, as well as for the treatment of disorders of glycoprotein metabolism, including mucolipidosis II (I-cell disease), mucolipidosis III (Hurler pseudolipodystrophy), and for the treatment of defects in glycoprotein degradation, including aspartylglucosaminuria, fucosidosis, mannosidosis and sialidosis.

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 disease, is attached to the transport element directly or via a linker.

The most preferred embodiments of the present invention are described below. However, these preferred embodiments are provided only for purposes of illustration of the present invention and not to limit the scope of the claims.

Brief description of the figures

The invention is illustrated by the following figures.

In fig. 1 shows a representative autoradiogram of a male cynomolgus monkey recorded 2 hours after a single intravenous administration of [ ¹²⁵ I]-HIR-Fab-IDS at a nominal dose level of 0.0020 mg/kg body weight.

In fig. 2 shows a representative autoradiogram of a male cynomolgus monkey recorded 2 hours after a single intravenous administration of [ ¹²⁵ I]-HIR-Mab-IDS at a nominal dose level of 0.0015 mg/kg body weight.

In fig. 3 shows a representative autoradiogram of a male cynomolgus monkey recorded 2 hours after a single intravenous administration of [ ¹²⁵ I]-IDS (control) at a nominal dose level of 0.0010 mg/kg body weight.

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Detailed description

Unless otherwise noted, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., molecular genetics, nucleic acid chemistry, protein chemistry, biochemistry, organic chemistry, immunology , microbiology, genetics, etc.).

As used herein, the term compound is understood in its broadest sense as at least one molecule that is a conjugate, fusion protein, protein construct, protein complex, etc.

As used herein, the term "comprising" means a compound comprising the listed elements without excluding others.

As used herein, the terms therapeutic enzyme and enzyme are synonymous and refer to an enzyme for treating diseases resulting from absence, deficiency, dysfunction of an enzyme, and so on, wherein a subject suffering from diseases can be treated by ERT, administration of an enzyme, and so on. In particular, the enzyme may be an enzyme for the treatment of diseases that may occur due to, but not limited to, absence, deficiency and dysfunction of a lysosomal enzyme.

The therapeutic enzyme that is included in the compound herein includes any enzyme that has a therapeutic effect against a lysosomal storage disease, including, 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-acetylIII-glucosaminidase (NAGLU) , glucocerebrosidase, butyrylcholinesterase, chitinase, glutamate decarboxylase, lipase, uricase, platelet activating factor acetylhydrolase, neutral endopeptidase, myeloperoxidase, acetyl-CoA-glucosaminide-N-acetyltransferase, N-acetylglucosamine-b-sulfatase, galactosamine-6sulfatase ( GALN), hyaluronidase, α-fucosidase, β-mannosidase, α-neuraminidase (sialidase), N-acetyl-glucosamine-1-phosphotransferase, mucolipin-1, α-N-acetyl-galactosaminidase, N-aspartyl-βglucosaminidase, LAMP-2 (lysosome-associated membrane protein 2) , cystinosin, sialin, ceramidase, acid β-glucosidase, galactosylceramidase, NPC1 (Niemann-Pick disease protein type C1), cathepsin A, SUMF-1 (sulfatase-modifying factor-1), lysosomal lipase acid (LIPA) and tripeptidyl peptidase 1.

In particular, the therapeutic enzyme includes the enzymes agalsidase, imiglucerase, galsulfase, iduronate-2-sulfatase and a-L-iduronidase. Most preferably, the therapeutic enzyme is iduronate-2-sulfatase or a-L-iduronidase.

However, the present description may also cover any therapeutic enzyme, without limitation, regardless of the type or origin of the enzyme.

In the present description, the term iduronate-2-sulfatase, as well as idursulfase, IDS, IDS, I2S, means a recombinant analogue of the lysosomal enzyme iduronate-2-sulfatase, which normally hydrolyzes the O-linked sulfate group of mucopolysaccharides - dermatan and heparan sulfates. In the present invention, the term iduronate-2-sulfatase can be used interchangeably with the term idursulfase.

As used herein, 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 the recombinant iduronate-2-sulfatase fusion protein, covalently connected to the Fab fragment of a monoclonal antibody to the human insulin receptor. As used herein, HIR-Fab-IDS includes, but is not limited to, iduronate-2-sulfatase with a monoclonal antibody fragment to the human insulin receptor. In particular (non-limiting) embodiments of the present invention, HIR-Fab-IDS variants are represented by a first amino acid sequence selected from SEQ ID NO: 2, 8, 9, 10 or 11, and a second amino acid sequence selected from SEQ ID NO: 4. 5, 12, 13, 14 or 15. In a particular embodiment of the present invention, the HIR-Fab-IDS is represented by the first amino acid sequence of SEQ ID NO: 2 and the second amino acid sequence of SEQ ID NO: 4.

As used herein, HIR-Mab-IDS is a recombinant iduronate-2sulfatase fusion protein covalently linked to a full-length monoclonal antibody to the human insulin receptor. In particular (non-limiting) embodiments of the present invention, variants of HIRMab-IDS are variants according to US patent document US8834874 B2, 09/16/2014. In a specific embodiment of the present invention, the HIR-Mab-IDS is the amino acid sequence of the ArmaGen Technologies Inc. project AGT-182 product.

As used herein, the term a-L-iduronidase, as well as iduronidase, laronidase, IDUA, refers to an enzyme involved in the hydrolysis of glycosaminoglycans such as dermatan sulfate

- 9 044641 and heparan sulfate. As used herein, the term a-L-iduronidase may be used interchangeably with the term laronidase. Deficiency (genetically determined deficiency) of iduronidase, which occurs in mucopolysaccharidosis type I, leads to the gradual accumulation of glycosaminoglycans - heparan sulfate and dermatan sulfate - in the cells and tissues of the body.

HIR-Fab-IDUA, as well as rIDUA-FAB-HI, rHI-FAB-IDUA, rIDUA-FAB-HIR, rHIR-FAB-IDS hybrid recombinant protein a-L-iduronidase, covalently linked to the Fab fragment of a monoclonal antibody to the human insulin receptor . In particular (non-limiting) embodiments of the present invention, HIR-Fab-IDUA variants are represented by a first amino acid sequence selected from SEQ ID NO: 2, 8, 9, 10 or 11, and a second amino acid sequence selected from SEQ ID NO: 6. 7.

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

Therapeutic enzymes that may be included in the compounds of the present invention may be in their native form, as well as in the form of fragments consisting of parts of enzymes, or analogues of enzymes in which a variation has arisen selected from the group consisting of substitution, addition, deletions, modifications of certain amino acids and combinations thereof, without limitation, provided that they have the same enzymatic activity as the native forms of the corresponding therapeutic enzymes. In particular (non-limiting) embodiments of the invention, enzyme fragments that have the activity of native forms of the corresponding enzymes can be used. Enzyme analogues are, without limitation, those enzymes that have different glycosylation characteristics and degrees of glycosylation due to expression of the known enzyme in different hosts, as well as varying degrees of substitution of specific amino acid residues of the corresponding enzyme relative to the standard sequence, where the degree of substitution is not 100% substitution . In private (non-limiting) embodiments of the invention, analogues of a-L-iduronidase, known from patents US 5932211 A, 08/03/1999, US 6153188 A, 11/28/2006 and US6541254 B1, 04/01/2003, as well as iduronate-2sulfatase analogues, can be used. One skilled in the art will understand that other fragments and analogues of the enzymes a-L-iduronidase and iduronate-2-sulfatase may be used, having the activity of native forms of the corresponding enzymes that are currently known in the art or will subsequently become known.

Enzymes can be produced by methods conventional in the art, for example, through genetic recombination in animal cells, E. coli, yeast, insects, plants and living animals, etc., using various expression vectors well known to one skilled in the art. The production methods are not limited to these and also include other methods for producing enzymes known to a person skilled in the art. For non-limiting examples of such methods for producing enzymes in cells of various organisms, as well as non-limiting examples 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 preferred embodiments of the invention, the enzymes are produced in mammalian cells. In the most preferred embodiments of the invention, the enzymes are produced in Chinese hamster ovary cells.

In certain preferred embodiments of the invention, the enzymes may be commercially available enzymes. In addition, enzymes may include an amino acid sequence that has at least 80%, more particularly 90%, and even more particularly 91, 92, 93, 94, 95, 96, 97, 98, or 99% homology or greater with the above enzymes or analogues thereof, and the enzymes may be obtained from microorganisms using recombinant technology or may be purchased from commercial sources, without limitation.

As used herein, the term homology means the degree of similarity to an amino acid sequence of a wild-type protein or a nucleotide sequence encoding amino acids, and covers sequences that have the above degree of sequence similarity, expressed as a percentage, to the amino acid sequences or nucleotide sequences of the present invention. Homology can be determined by comparing two given sequences with the naked eye or can be determined using a bioinformatics algorithm that allows homology analysis by aligning the sequences in question for comparison. The homology between two given amino acid sequences can be indicated as a percentage. There are effective automated algorithms that can be used in the GAP, BESTFIT, FASTA and TFASTA software modules of the Wisconsin Genetics software package (Genetics Computer Group, Madison, WI, USA). The alignment algorithm automated in these modules includes the sequence alignment algorithms of Needleman and Wunsch, Pearson and Lipman, and Smith and Waterman. Other algorithms that can be used for sequence alignment and homology determination are automated in the FASTP, BLAST, BLAST2, PSIBLAST and CLUSTAL W programs. Amino acid sequences and nucleotide sequences encoding enzymes and their analogs can be obtained from a known database , such as but not limited to GenBank NCBI.

In some embodiments, the compound transport element comprises a Fab fragment of an IgG1 immunoglobulin consisting of a first amino acid sequence of 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% or more identical to SEQ ID NO: 2.

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

In some embodiments, a compound represented by a first amino acid sequence of 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% or more identical to SEQ ID NO: 2, and a second amino acid sequence at least identical to SEQ ID NO: 4, used for the treatment or prevention of lysosomal enzyme deficiency in a subject with mucopolysaccharidosis type II.

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

The term amino acid refers to a group of carboxy-a-amino acids either naturally occurring, i.e. which may be encoded directly or in the form of a precursor by a nucleic acid, or which do not occur in nature. Individual naturally occurring amino acids are encoded by nucleic acids consisting of three nucleotides—called codons or base triplets. Each amino acid is encoded by at least one codon.

The term amino acid as used herein refers to naturally occurring carboxy-a-amino acids including the following: alanine (three letter code: Ala, one 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 (Tip, W), tyrosine (Tyr, Y) and valine (Val, V). Examples of amino acids that do not occur in nature (non-proteinogenic amino acids) include, Aad (alpha-aminoadipic acid), Abu (aminobutyric acid), Ach (alpha-aminocyclohexane-carboxylic acid), Acp (alpha-aminocyclopentane-carboxylic acid), Acpc (1-aminocyclopropane-carboxylic acid). 1-carboxylic acid), Aib (alpha-aminoisobutyric acid), Aic (2-aminoindan-2-carboxylic acid; also called 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-cyclohexylalanine), Cit (citrulline), cyclohexylglycine (Chg), cyclopentylalanine, beta-cyclopropylalanine, Dab (1,4-diaminobutyric acid), Dap (1,3diaminopropionic acid), p-(3 ,3-diphenylalanine-carboxylic acid), 3,3-diphenylalanine, di-npropylglycine (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-Apc (1-amino-4-(4-methoxyphenyl)cyclohexane-1 -carboxylic acid), nor-leucine (Nle), Nva (norvaline), omatine, 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 (4oxo-6-(1-piperazinyl)-3(4H)-quinazoline-acetic acid (CAS 889958-08-1)), pyridylalanine, quinolylalanine, sarcosine (Sar), thiazolylalanine, thienylalanine, Tic (alpha-amino-1 ,2,3,4-tetrahydroisoquinoline3-carboxylic acid), Tic(OH), Tle (tert-butylglycine) and Tyr(Me), but are not limited to.

The term amino acid sequence refers to polypeptides having amino acid sequences that differ to some extent from the polypeptide with the native sequence of the corresponding therapeutic enzyme. Typically, the amino acid sequence variants will have at least about 70% sequence identity to a polypeptide with the native sequence of the corresponding therapeutic enzyme. In one embodiment, the variant has about 80% or more sequence identity to a polypeptide with the native sequence of the corresponding therapeutic enzyme. In one embodiment, the variant has about 90% or more sequence identity to a polypeptide with the native sequence of the corresponding therapeutic enzyme. In one embodiment, the variant has about 95% or more sequence identity to a polypeptide with the native sequence of the corresponding therapeutic enzyme. In one embodiment, an embodiment has

- 11 044641 approximately 98% or more sequence identity with the polypeptide with the native sequence of the corresponding therapeutic enzyme. Amino acid sequence variants have substitutions, deletions and/or insertions at specific positions within the amino acid sequence of the native amino acid sequence of the corresponding therapeutic enzyme. Amino acids are designated by traditional names, one-letter and three-letter codes.

The term first amino acid sequence as used in this invention refers to the amino acid sequence of an IgG immunoglobulin in general, and the light chain of an IgG immunoglobulin in particular. In particular, non-limiting embodiments, the first amino acid sequence is an IgG1 immunoglobulin amino acid sequence, an IgG1 immunoglobulin light chain amino acid sequence, a fragment of an IgG1 immunoglobulin light chain amino acid sequence selected from SEQ ID NO: 2, 8, 9, 10 or 11. In a particular embodiment the first amino acid sequence is the amino acid sequence of the immunoglobulin IgG light chain - SEQ ID NO: 2.

The term second amino acid sequence as used in this invention refers to the amino acid sequence of an IgG immunoglobulin in general; to an IgG immunoglobulin heavy chain, to an IgG immunoglobulin heavy chain fragment, to an IgG immunoglobulin heavy chain fragment connected to an enzyme sequence directly or by means of a linker, in particular. In specific (non-limiting) embodiments, the second amino acid sequence is an immunoglobulin IgG1 amino acid sequence, a heavy chain fragment amino acid sequence - SEQ ID NO: 3, is an IgG1 immunoglobulin heavy chain fragment fused to an iduronate-2-sulfatase sequence selected from SEQ ID NO : 4, 5, 12, 13, 14 or 15.

The term transport element as used in this invention refers to a substance that is capable of carrying, transporting, delivering a therapeutic enzyme to the lysosomes of cells of various tissues. In particular, but not limited to, the transport element will specifically interact with an epitope, antigen, receptor or target in such a way as to ensure effective delivery to the lysosomes of neural tissue cells. Such an epitope, antigen, receptor or target in the context of the present description may be the human insulin receptor or a part thereof, through which the delivery of a drug, peptide or protein is carried out, including through the BBB.

Immunoglobulin is a tetrameric molecule, in the context of the present description the term is a full-length antibody. In naturally occurring immunoglobulin, each tetramer consists of two identical pairs of polypeptide chains, with each pair having one light chain (approximately 25 kDa) and one heavy chain (approximately 50-70 kDa). The N-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids, primarily responsible for antigen recognition. The carboxyl portion of each chain defines a constant region primarily responsible for effector function.

As used herein, the term antibody is used in its broadest sense and includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments, provided that they have the required biological activity.

As used herein, antibody fragments comprise a portion of an intact antibody that retains the ability to bind an antigen. Examples of antibody fragments are Fab, Fab', F(ab')2 and Fv fragments; dimeric antibodies (diabody); linear antibodies; single chain antibody molecules, such as single chain Fab, scFv and multispecific antibodies formed from antibody fragments. As a result of the cleavage of antibodies by papain, two identical antigen-binding fragments are formed, called Fab fragments, each of which has one antigen-binding site, and a residual Fc fragment, the name of which reflects its ability to easily crystallize.

Fab fragment, Fab, FAB is a monovalent fragment that contains heavy and light chain variable domains and also contains a light chain constant domain and a first heavy chain constant domain. As used herein, the transport element comprises the amino acid sequence of a Fab fragment of an IgG immunoglobulin, where the IgG immunoglobulin is IgG1, IgG2 or IgG4. In particular (non-limiting) embodiments of the present invention, the IgG immunoglobulin is IgG1. In a specific embodiment, the transport element comprises an amino acid sequence of an IgG1 immunoglobulin Fab fragment consisting of a first amino acid sequence selected from SEQ ID NO: 2, 8, 9, 10 or 11 and a second amino acid sequence of SEQ ID NO: 3. In a more specific embodiment the transport element contains the amino acid sequence of the Fab fragment of the IgG1 immunoglobulin, consisting of SEQ ID NO: 2 and SEQ ID NO: 3.

As used herein, the term linker refers to a chemical linker or single-strand peptide linker that connects the therapeutic enzyme and the transport element of the compound of the present invention. The linker connects, for example, the monovalent linker contains the CH2-CH3 domain of Ig and sFab, the target of which is the insulin receptor,

those. a linker connects sFab to the C-terminus of the CH3-CH2 domain of Ig.

In some embodiments, the linker is a chemical linker. Single chain peptide linkers containing from one to twenty amino acids linked by peptide bonds can be used. In specific embodiments, the amino acids are selected from twenty 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 particular (non-limiting) embodiments of the present invention, one or more amino acids are selected from glycine, serine and leucine.

In specific embodiments of the invention, said linker is a single chain peptide whose amino acid sequence consists of at least 1 amino acid, preferably 1-2 amino acids. In particular (non-limiting) embodiments of the present invention, the amino acid sequence of the linker may consist of more than 1-2 amino acids, for example, 3 or 15 amino acids.

The coupling of the therapeutic enzyme and the transport element can be accomplished directly or using a variety of chemical linkers known in the art. In certain preferred (non-limiting) embodiments of the invention, coupling of the therapeutic enzyme and transport element can be accomplished using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2pyridyldithio)propionate (SPDP), succinimidyl-4-(N- maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional imidoesters (such as dimethyl adipimidate-HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bisazido compounds (such as bis (p-azidobenzoyl)hexanediamine), bisdiazonium derivatives (such as bis(p-diazoniumbenzoyl)ethylenediamine), diisocyanates (such as toluene-2,6-diisocyanate) and bis-active fluorine compounds (such as 1,5-difluoro-2 ,4-dinitrobenzene). One skilled in the art will also understand that other chemical and peptide linkers known in the art that are not expressly mentioned herein may be used for the purposes of the present invention.

In certain preferred embodiments of the invention, the linker may be a cleavable linker that facilitates the release of the therapeutic enzyme after transport of the compound into cells and tissues. For example, an acid-labile linker, a peptidase-sensitive linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker can 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). One skilled in the art will also appreciate that other cleavable linkers known in the art to facilitate the release of the therapeutic enzyme once the compound has entered the cells and tissues of the central nervous system may be used for purposes of the present invention.

The term epitope is the region of an antigen that is bound by an antigen-binding protein, including an antibody. Epitopes can be defined as structural or functional. Functional epitopes are generally a subset of structural epitopes and have those residues that directly contribute to the affinity of the interaction. Epitopes can also be conformational, which are composed of non-linear amino acids, in other words, conformational epitopes are composed of non-sequential amino acids. Epitopes may include determinants, which are chemically active surface moieties of molecules, such as amino acids, sugar side chains, phosphoryl groups or sulfonyl groups, and they may have specific three-dimensional structural characteristics and/or specific charge characteristics. As used herein, the epitope is an epitope in the insulin receptor represented by SEQ ID NO: 1 described in Zhang B, Roth RA. (1991) Proc Natl Acad Sci U S A.; 88(21):9858–9862 and S A Prigent, K K Stanley, and K Siddle. (1990) J. Biol. 1991.

The insulin receptor (HIR) is a transmembrane glycoprotein (with a molecular weight of approximately 320,000 Da), which consists of two α subunits and two β subunits linked by disulfide bridges (α subunits are located extracellularly and contain an insulin-binding domain) and is involved in the regulation of glucose absorption and distribution , as well as the synthesis and accumulation of fats, proteins and carbohydrates in the human body. Insulin receptors and their extracellular insulin-binding domain (ECD) are widely known in the art, both structurally and functionally. The localization of insulin receptors most often refers to the cell surfaces of insulin-sensitive tissues, such as connective tissue cells, skeletal muscles, adipose tissue cells, liver cells, etc. See, for example, Yip et al. (2003), J Biol. Chem. 278 (30): 27329-27332; And

- 13 044641

Whittaker et al. (2005), J Biol Chem, 280 (22): 20932-20936. In one embodiment, the HIR herein is a human insulin receptor comprising the amino acid sequence set forth in Kasuya et al. (Biochemistry 32 (1993) 13531-13536).

The insulin receptor is expressed almost everywhere, and the use of such a delivery route can significantly increase the bioavailability of the recombinant enzyme and increase the effectiveness of therapy. Delivery to brain tissue is carried out through transcytosis through the capillary endothelium of the central nervous system, through interaction with the human insulin receptor.

In peripheral and brain tissues (Hawkes C. et al., 2004) expressing M6PR, internalization of the chimeric molecule can occur in both ways: antibody-HIR interaction and enzyme-M6PR interaction. In this case, targeted delivery to lysosomes occurs through interaction with M6P receptors. Internalization via HIR (human insulin receptor) results in entry into early order endosomes and subsequent transcytosis. In addition to delivering a functional enzyme to the CNS, many lysosomal storage diseases require improved internalization by certain peripheral tissues (diaphragm muscles in Pompe disease, liver and spleen in Hunter disease, kidneys in Fabry disease, etc.).

The term specific means that the molecule related to the 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 of a molecule with the binding site of another molecule (affinity of complex formation) is determined by the indicators k _a (rate constant for the association of compounds to form a complex), k _D (dissociation constant, dissociation of the complex) and K _D (k _D /k _a ). Binding or specific binding means a binding affinity (K _D ) of about 10 ^-7 M or less, in one embodiment from about 10 ^-8 to about 10 ^-13 M, in one embodiment from about 10 ^-9 to about 10 ^-13 M .

The term lysosome denotes a cellular organelle, one of the types of vesicles that contains a number of enzymes (acid hydrolases) capable of breaking down macromolecules either from the cell itself (for example, when structural components of the cell are processed) or captured from the outside. Inherited defects or deficiencies of lysosomal enzymes (or other lysosomal components) can lead to the accumulation of undegraded metabolites. Glycosaminoglycans (formerly called mucopolysaccharides) are common polysaccharides of cell surfaces and extracellular matrix and structures. Enzyme deficiencies that interfere with the breakdown of glycosaminoglycan cause accumulation of glycosaminoglycan fragments in lysosomes and cause widespread changes in bone, soft tissue, and the central nervous system.

The activity of the enzyme(s) of the invention can be measured using any suitable test. Typically, pH determination and temperature determination can be tailored to the enzyme in question. Examples of tested pH values are pH 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. Examples of tested 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, such as pH values of 4, 5, 6, 7 or 8 and temperatures of 30, 35, 37 or 40°C. For example, protease activity can be measured using any test that uses a substrate that includes peptide bonds relevant to the specificity of the protease in question.

Examples of suitable enzyme tests are included in the experimental section, in particular see Example 2. As used herein, the term activity refers to enzyme activity, specific activity, enzyme specific activity; specific activity depending on the context of the description.

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

A pharmaceutical composition refers to a mixture of one or more of the compounds described herein, or physiologically/pharmaceutically acceptable salts or prodrugs thereof, with 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.

The term effective amount of a compound, for example, in a pharmaceutical composition, refers to an amount that is effective in doses and for periods of time necessary to achieve the desired therapeutic or prophylactic result (effect) in the subject.

- 14 044641 treated, with reasonable benefit/risk.

The term pharmaceutically acceptable carrier refers to an ingredient of a pharmaceutical composition, other than the active ingredient (compound, agent, etc.), that is non-toxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer or preservative. In particular (non-limiting) embodiments of the present invention, a pharmaceutically acceptable carrier is administered together with the compound.

Pharmaceutical compositions that contain the compounds used according to the present invention are prepared for storage by admixture with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences, 16th ed., ed. Osol A., 1980), preferably in the form lyophilized compositions or aqueous solutions. Acceptable carriers, excipients or stabilizers are non-toxic to subjects at the dosages and concentrations employed and include buffers such as phosphate, citrate and other organic acid buffers; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol; butyl or benzyl alcohol; alkylparabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol and metacresol); low molecular weight polypeptides (containing less than about 10 residues); proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as poly(vinylpyrrolidone); amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal-containing complexes (eg Zn-protein complexes); and/or nonionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

The pharmaceutical compositions used herein may also optionally contain more than one active substance (drug) active against lysosomal storage disease, optionally substances with additional activities that do not adversely affect each other. The type and effective amounts of such drugs depend, for example, on the amount of the therapeutic enzyme and transport element containing compound present in the composition and the clinical parameters of the subjects.

Administration of doses of the claimed therapeutic compound can be carried out by any suitable route well known to one skilled in the art, for example, by injection, such as intravenous, intramuscular or subcutaneous injection. The choice of the specific route of administration of a therapeutic compound is made by one skilled in the art and depends, in part, on whether the administration is short-term or long-term. One skilled in the art will appreciate that other routes of administration known in the art may be used to administer dosages of the claimed therapeutic compound, such as, without limitation, intrathecal administration, intra-arterial administration, intraperitoneal administration, transdermal administration, inhalation administration, buccal administration, intranasal administration, oral administration, sublingual administration or transnasal administration (Felice B.R., Wright T.L., Boyd R.B. Safety Evaluation of Chronic Intrathecal Administration of Idursulfase-IT in Cynomolgus Monkeys. - Toxicol Pathol. 2011 Aug;39(5):879-9, WO 2011/044542 A1).

As used herein, various dosing schedules may be contemplated, including, but not limited to, single or multiple administrations at different times, bolus administration, and pulsatile infusion.

The subject, individual, or patient is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and primates such as monkeys, particularly great apes), rabbits, and rodents (e.g., mice and rats). In some embodiments, the subject or patient is a human.

As used herein, the term treatment (and its grammatical variations such as treat or process of treatment) refers to a clinical intervention for the purpose of altering the natural history of disease in the individual being treated, and may be carried out either for prevention or during the development of clinical pathology. The desired treatment actions include, but are not limited to, prevention of onset or recurrence of the disease, relief of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastases, reduction in the rate of disease progression, alleviation or temporary amelioration of the disease state, and remission or improvement of prognosis. In some embodiments, the compounds of the invention are used to delay the development of a disease or slow the progression of a disease.

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The term CNS or central nervous system refers to the complex of nerve tissues that control body function and includes the brain and spinal cord.

As used herein, the term lysosomal storage disease (LSD; may also be referred to as lysosomal storage disease) refers to a rare genetic disease resulting in loss of lysosomal functions described above due to partial or complete loss of activity of one of the lysosomal enzymes . In this case, ERT is necessary to replenish an enzyme whose activity is completely or partially lost, or a missing enzyme. As used herein, the term lysosomal storage disorder is used interchangeably with the term lysosomal storage disorder (also may be referred to as lysosomal storage disorder). Lysosomal storage diseases can be classified depending on the defect or deficiency of the enzyme into: (i) sphingolipidosis, (ii) mucopolysaccharidosis, (iii) glycogen storage disease, (iv) mucolipidosis, (v) oligosaccharidosis, (vi) lipidosis, (vii) disorder lysosomal transport and so on.

Below, lysosomal storage diseases will be described in more detail according to their classification.

As used herein, the term sphingolipidosis refers to a genetically determined deficiency syndrome of the lysosomal enzyme that hydrolyzes the carbohydrate side chains or choline side chains of sphingolipids. Diseases are classified according to the distribution of each stored lipid, such as Krabbe disease caused by galactocerebrosidase deficiency, Fabry disease caused by α-galactosidase A deficiency, Niemann-Pick disease caused by sphingomyelinase deficiency, Gaucher disease caused by glucocerebrosidase deficiency, Tau disease. Sachs, caused by hexosaminidase A deficiency and so on, and they are diseases that are inherited in an autosomal recessive manner, with the exception of Fabry disease, which is an X-linked genetic disease.

As used herein, the term mucopolysaccharidosis (MPS) refers to a syndrome of genetic deficiency of mucopolysaccharide hydrolase caused by deficiency of the carbohydrate chain degrading enzyme sulfatase, acetyltransferase, and so on. The main symptom of mucopolysaccharidosis (MPS) is excessive secretion of mucopolysaccharides in the urine. Currently, MPS is classified into 6 types, among which type I disease includes Hurler syndrome and Scheie syndrome, type II includes Hunter syndrome, type III includes Sanfilippo syndrome types A, B, C and D; Type IV includes Morquio syndrome A and B; Type VI includes Maroteaux-Lamy syndrome and type VII includes Sly syndrome.

As used herein, the term glycogen storage disease (also known as glycogenosis) refers to an inborn error of carbohydrate metabolism caused by glycogen storage and is divided into subtypes I-VII. The subtypes of glycogen storage disease associated with lysosomal storage disease (LSD) are types II (Pompe disease) and IIIb (Danon disease).

A detailed description of the lysosomal storage diseases given in this description, as well as those disclosed in table. 1, cited 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) .

Mucopolysaccharidosis type I (MPS I) is an inherited metabolic disease caused by a defect in the enzyme a-L-iduronidase (IDUA), which functions to break down the acidic mucopolysaccharides heparan sulfate and dermatan sulfate. Insufficient levels of IDUA lead to pathological accumulation of these mucopolysaccharides in the tissues and organs of the patient, for example, in the heart, liver and central nervous system. Symptoms, including neurodegeneration and mental retardation, begin in childhood and, due to organ damage, early death can occur. A genetic deficiency in the carbohydrate breakdown enzyme αL-iduronidase causes a lysosomal storage disease known as mucopolysaccharidosis type I (MPS I). Severe MPS I is commonly known as Hurler syndrome, and is associated with a variety of problems such as mental retardation, corneal clouding, coarse facial features, heart disease, respiratory disease, enlarged liver and spleen, hernias, and stiffness. joints. Patients suffering from Hurler syndrome usually die before the age of 10 years. In the moderate form, known as Hurler-Scheie syndrome, mental function is usually not greatly affected, but physical problems can also lead to death in adolescence or between the ages of twenty and twenty-nine. Scheie's syndrome is a mild form of MPS I. It is compatible with a normal life expectancy, but joint stiffness, corneal clouding and heart valve disease cause serious problems.

Mucopolysaccharidosis type II (MPS II) or Hunter syndrome is an X-linked inherited metabolic disorder caused by a deficiency of the enzyme iduronate 2 sulfatase (I2S). I2S is localized in lysosomes and plays an important role in the catabolism of glycosaminoglycans (GAGs) of heparan and dermatan sulfate. In the absence of the enzyme, these substrates accumulate in cells,

- 16 044641 ultimately causing stagnation, and then cell death and tissue destruction. Due to the widespread expression of the enzyme, different types of cells, organs and systems are affected in patients with MPS II. A characteristic clinical feature of this disease is degeneration of the central nervous system (CNS), which leads to cognitive impairment (eg, decreased IQ). In addition, MRI scans of patients revealed white matter damage, expansion of perivascular spaces in the brain parenchyma, ganglia, corpus callosum and brain stem, atrophy and ventriculomegaly (Wang et al. Molecular Genetics and Metabolism, 2009). The disease usually manifests itself in the first years of life with organomegaly and skeletal abnormalities. Some patients experience progressive loss of cognitive function, with most patients dying from disease-related complications in the first or second decade of life (Raluy-Callado M et al. Orphanet J Rare Dis. 2013;8:101;).

As used herein, the term neurological entity refers to a disease or disorder that affects the central nervous system and/or the etiology of which is related to the central nervous system. Examples of CNS diseases or disorders include, but are not limited to, neuropathy, amyloidosis, cancer, ocular disease or disorder, viral or microbial infection, inflammation, ischemia, neurodegenerative disease, seizure disorder, behavioral disorders, and lysosomal storage disease. Specific examples of neurological disorders include, but are not limited to, neurodegenerative diseases (including, but not limited to, Lewy body disease, postmyelitis syndrome, 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-Strössler disease Schenker disease, chronic wasting disease, and fatal familial insomnia), palsy, motor neuron disease, and heterodegenerative disorders of the nervous system (including, but not limited to, Canavan disease, Huntington disease, neuronal ceroid lipofuscinosis, Alexander disease, Tourette syndrome, Menkes curly hair, Cockayne syndrome, Hallervorden-Spatz syndrome, Lafora disease, Rett syndrome, hepatolenticular degeneration, Lesch-Nyhan syndrome and Unferricht-Lundborg syndrome), dementia (including, but not limited to, Pick's disease and spinocerebellar ataxia), cancer (eg , cancer of the central nervous system and/or brain, including brain metastases resulting from cancer in any area of the body).

The compounds of the invention can be used in therapy either individually or in combination with other agents. For example, a compound of the invention comprising a therapeutic enzyme and a transport element connected by a linker may be co-administered with at least one additional therapeutic agent. In specific embodiments, the additional therapeutic agent is a therapeutic agent effective in treating the same neurological disorder for which the compound of the invention is used, or another neurological disorder. Examples of additional therapeutic agents include, but are not limited to, the various neurological drugs described above, including cholinesterase inhibitors (such as donepezil, galantamine, rovastigmine and tacrine), NMDA receptor antagonists (such as memantine), amyloid beta peptide aggregation inhibitors, antioxidants, γ-secretase modulators, nerve growth factor (NGF) mimics or NGF gene therapy agents, PPARy agonists, HMS-CoA reductase inhibitors (statins), ampakines, calcium channel blockers, GABA receptor antagonists, glycogen synthase kinase inhibitors, immunoglobulin, intended for intravenous administration, muscarinic receptor agonists, nicotinic receptor modulators, active or passive immunization agents against amyloid beta peptide, phosphodiesterase inhibitors, serotonin receptor antagonists and antibodies to amyloid beta peptide. Such combination therapies as defined above include coadministration (where two or more therapeutic agents are included in the same or different compositions) and separate administration, in which case administration of a compound containing a therapeutic enzyme and a transport element connected by a linker of the invention , can be performed before, simultaneously and/or after the administration of an additional therapeutic agent and/or adjuvant.

Some terms defined above may appear more than once, in which case each term should be defined independently of the other.

Hereinafter, exemplary embodiments of the present invention will be described in detail. Moreover, each of the explanations and implementations given as an example in this document may be valid for other explanations and implementations given as an example. Thus, all combinations of the various factors described herein are within the scope of the present invention. Moreover, the scope of the present invention is not limited to the specific description given below.

- 17 044641

Examples of implementation of the invention

The present invention will now be described in more detail with reference to the following Examples.

However, the Examples described below are for illustrative purposes only and are not intended to limit this invention.

Example 1. Receipt.

To obtain the compounds, animal cells into which an animal cell expression vector was introduced were cultured and purified.

Construction of expression vectors.

Amino acid sequences first LC-HIR-FAB-IDS (SEQ ID NO: 2, 8, 9, 10 and 11) and second HC-HIR-FAB-IDS (SEQ ID NO: 4, 5, 12, 13, 14, 15 )) to obtain HIR-FAB-IDS variants, including the signal peptide MDWTWRVFCLLAVAPGAHS, were converted into nucleotide sequences. For gene synthesis followed by cloning into the pCLN-1 vector, the following HindIII restriction site and Kozak sequence were added to the 5' end of the sequences. Two stop codons and an XbaI restriction site were added to the 3' end of the sequences.

Optimization of the nucleotide sequence by codon composition for Chinese hamster cells was carried out using the resources: http://gcua.schoedl.de and http://www.kazusa.or.jp. The synthesis of the light and heavy chain LC-HIR-FAB-IDS and HC-HIR-FAB-IDS was carried out at GenArt (USA) and transferred as part of the vectors pRA1675 and pRA1673 (containing the genes LC-HIR-FAB-IDS and HC-HIR- FAB-IDS, respectively).

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

The vectors of the present invention were constructed in accordance with molecular biology techniques well known in the art. See 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), Schleef M, Ed., Plasmid for Therapy and Vaccination (Wiley-VCH Verlag GmbH, Weinheim, DE , 2001).

Obtaining a monoclonal cell line.

Parental cell line CHO-S. Cells were cultured at 37°C, 5% CO2, 70% humidity, in BalanCD CHO Growth A medium (Invitrogen). Stable transfection was carried out on a NEON instrument (Invitrogen) according to the standard protocol for CHO cells using two linearized plasmids pCLN-1-HC-HIR-FAB-IDS and pCLN-1-LC-HIR-FAB-IDS. The DNA ratio for transfection was equimolar.

After 48 hours, the transfected pools were seeded into minipools in 96-well plates in BalanCD CHO Growth A medium containing 600 μg/ml of the selective antibiotic neomycin. Minipools were cultured under stationary conditions for 10 days at 37°C, 5% CO2, 70% humidity, after which a series of productivity screenings were carried out using ELISA. The leader minipool was cloned into semi-solid ClonaCell Flex medium (STEMCELL) for single colony growth using 6-well plates. The plates were incubated for 10 days at 37°C, 5% CO2, 70% humidity.

During the screening, the concentration of the target protein HIR-FAB-IDS secreted into the culture liquid was determined. The determination was carried out by the sandwich ELISA method, using instead of primary antibodies a fragment of the recombinant human insulin receptor (5 μg/ml in a carbonate-bicarbonate buffer pH 9.6); rat antibodies conjugated to horseradish peroxidase to iduronate-2-sulfatase were used as second antibodies ( diluted to 1:10000). Developed with a solution of tetramethylbenzidine, the reaction was stopped with 0.5 M sulfuric acid.

Based on the results of screening 6 moons. tablets, 20 leading minipools with productivity from 0.4 to 2.1 mg/l were selected, which were transferred to flasks for adaptation to suspension cultivation. To adapt to suspension cultivation, 3 passages were carried out, after which 20 minipools were frozen.

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

- 18 044641 cells.

Cultivation of producer cells.

Cultivation of producer cells expressing HIR-FAB-IDS was carried out in a fed-batch mode using the BalanCD CHO Growth A nutrient medium (Irvine Scientific) and the nutrient supplement BalanCD CHO Feed 2 (Irvine Scientific) for 12 days in bioreactor with overhead stirrer at a temperature of 37°C and pH 6.9. After the cultivation process, the culture liquid was clarified by depth filtration and transferred for isolation.

Isolation and purification.

Isolation and purification are carried out using standard technologies for protein isolation and purification. The compound can be isolated or purified by any method known in the art to isolate or purify protein, for example, by chromatography (e.g., ion exchange, high performance liquid chromatography, affinity assisted, protein A, and size-sorting column chromatography), centrifugation , differential solubility, or any other standard protein isolation or purification technique.

Example 2. Determination of the level of enzymatic activity of the Fab fragment of an antibody to the insulin receptor connected to the amino acid sequence of iduronate-2-sulfatase.

The specific activity of HIR-FAB-IDS and HIR-MAB-IDS is determined by a fluorimetric method using 4-mutilumbelliferyl-b-iduronide-2-sulfate (4-MUS) (Moscerdam Substrates, the Netherlands) (Voznyi YV et al., 2001; Tolun AA , et al., 2012; 9. Johnson BA et al., 2013; Azadeh M et al., 2017). During the assay, the substrate is hydrolyzed by iduronate-2-sulfatase to produce 4-mutylumbelliferyl-b-iduronide (MUBI), which is hydrolyzed by iduronidase (IDUA, Aldurazyme, Genzyme, USA) to 4-methylumbelliferyl (4-MU), which is detected fluorimetrically using the following fluorimeter settings: fluorescence excitation at a wavelength of 450 nm and fluorescence detection at a wavelength of 365 nm. The calibration curve is constructed using a 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 IDS is added in an amount of 12 μg and the mixture is further incubated at 37°C for 24 hours. The incubation is stopped by adding 0.2 ml 0.5 M sodium carbonate pH=10.3. HIR-FAB-IDS exhibits specific enzymatic (iduronate-2-sulfatase) activity towards the substrate 4-MU-aIdoA-2S (4-methylumbeliferyl a-b-idopyranosiduronate-2sulfate).

Instruments and equipment.

1. Thermostatic shaker PST-60 HL-4 (BioSan, Latvia or similar).

2. Multifunctional reader Spectra Max M3 (Molecular Devices, USA or similar).

3. Software for multifunctional reader Soft Max Pro (Molecular Devices, USA or similar).

4. Analytical balance ML 204 (Mettler Toledo, Switzerland or similar quality).

5. 96-well black plate with non-absorbent surface (Corning, USA, cat. No. 3916 or similar).

Reagents.

4-methylumbelliferone sodium salt (Sigma-Aldrich, cat. no. M1508 or equivalent quality).

2. Recombinant human a-L-iduronidase (R&D SYSTEM, cat. no. 4119-GH or equivalent quality).

3. 4-methylumbelliferyl a-L-idopyranosiduronic acid 2-sulfate sodium salt, 0.5 mg/vial (USBiological, cat. no. 017551 or equivalent).

4. Sodium acetate anhydrous (AppliChem Panreac, cat. no. 141633.1211 or equivalent quality).

5. Acetic acid (Fluka, cat. no. 49199 or similar quality).

6. Bovine serum albumin (Sigma, cat. no. A7030 or equivalent quality).

7. Sodium carbonate (Sigma-Aldrich, Cat. No. S7795 or equivalent quality).

8. Sodium bicarbonate (Sigma-Aldrich, Cat. No. S6297 or equivalent quality).

9. Lead diacetate trihydrate (Aldrich, cat. no. 467863 or equivalent quality).

10. Sodium phosphate dihydrate (Sigma-Aldrich, cat. no. 71643 or equivalent quality).

11. Anhydrous citric acid (AppliChem Panreac, cat. no. 141808 or equivalent quality).

Preparation of solutions.

Sample dilution solution, pH (5.5 ± 0.2). About 4.1 g of anhydrous sodium acetate and 0.5 g of bovine serum albumin are placed in a 1-liter beaker and dissolved in 700 ml of purified water. Adjust the pH of the solution to a value of (5.5 ± 0.2) with acetic acid. The resulting solution is transferred to a 1-liter volumetric flask, the volume of the solution is adjusted to the mark with purified water, mixed and filtered through a membrane filter with a pore diameter of 0.45 μm.

Shelf life - 3 months at temperatures from 2 to 8°C.

Substrate dilution solution, pH (5.00 ± 0.05). 19 044641 place 4.1 g of anhydrous sodium acetate, 1.9 g of lead diacetate trihydrate into a beaker with a capacity of 500 ml, add 450 ml of purified water, stir until dissolved. Adjust the pH of the solution to a value of (5.00 ± 0.05) with acetic acid (about 1.1 ml). The resulting solution is transferred to a 500 ml volumetric flask, the volume of the solution is adjusted to the mark with purified water, mixed and filtered through a membrane filter with a pore diameter of 0.45 μm.

Shelf life - 3 months at temperatures from 2 to 8°C.

Substrate solution (1.25 mmol/l). 0.83 ml of a solution for diluting the substrate is added to a bottle containing the substrate (4-methylumbelliferyl a-L-idopyranosiduronic acid 2-sulfate sodium salt), and gently mixed. The resulting solution is divided into 0.2 ml aliquots and frozen.

Shelf life - 3 months at a temperature not exceeding minus 70°C.

Solution of recombinant human a-L-iduronidase. 400 μl of purified water is added to a bottle containing recombinant human aL-iduronidase (10 μg) and mixed. The resulting solution is divided into 0.1 ml aliquots and frozen.

Shelf life - 3 months at a temperature not exceeding minus 70°C.

0.1 M solution of citric acid. About 19.2 g of anhydrous citric acid is placed in a 1-liter volumetric flask, dissolved in 900 ml of purified water, the volume of the solution is adjusted to the mark with purified water, and filtered through a 0.22-μm membrane filter.

Shelf life - 3 months at temperatures from 15 to 25°C.

0.2 M solution of sodium phosphate disubstituted. About 35.6 g of sodium phosphate dihydrate is placed in a 1-liter volumetric flask, dissolved in 900 ml of purified water, the volume of the solution is adjusted to the mark with purified water, and filtered through a 0.22-μm membrane filter.

Shelf life - 3 months at temperatures from 15 to 25°C.

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

Shelf life - 3 months at temperatures from 15 to 25°C.

0.5 M sodium bicarbonate solution. About 42.0 g of sodium bicarbonate is placed in a 1-liter volumetric flask, dissolved in 900 ml of purified water, the volume of the solution is adjusted to the mark with purified water and mixed and filtered through a membrane filter with a pore diameter of 0.22 μm.

Shelf life - 6 months at temperatures from 15 to 25°C.

0.5 M sodium carbonate solution. About 53.0 g of sodium carbonate is placed in a 1-liter volumetric flask, dissolved in 900 ml of purified water, the volume of the solution is adjusted to the mark with purified water and mixed and filtered through a membrane filter with a pore diameter of 0.22 μm.

Shelf life - 6 months at temperatures from 15 to 25°C.

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 into a 1-liter volumetric flask, mix and filter through a membrane filter with a pore diameter of 0.22 μm.

Shelf life - 6 months at temperatures from 15 to 25°C.

Stock solution of 4-methylumbelliferone sodium salt (100 µmol/ml). About 1.0 g (exactly weighed) of 4-methylumbelliferone sodium salt is placed in a 50 ml volumetric flask, 30 ml of purified water is added, mixed, and adjusted to the mark with purified water. The solution is divided into 2.0 μl aliquots.

Shelf life - 6 months at a temperature not exceeding minus 18°C.

Working standard solution of 4-methylumbelliferone sodium salt (50 nmol/ml).

An aliquot of the stock solution of 4-methylumbelliferone sodium salt is kept in a water bath for 5 minutes at a temperature of 37°C. Serial dilutions are prepared as follows. diagram:

No. solution Initial solution 4-MU ^a Solution No. 2 Stop solution Concentration, nmol/ml 1 100 - 9900 1000 2 - 500 9500 50

^and 4-MU - 4-methylumbelliferone sodium salt.

Dilutions of the working standard solution of 4-methylumbelliferone sodium salt. Prepare dilutions of a working standard solution of 4-methylumbeliferone sodium salt according to the following scheme:

- 20 044641

Breeding No. Volume of working standard solution (50 nmol/ml), ml Stop solution volume, ml Concentration, nmol/ml 1 0 5.00 0.0 2 0.05 4.95 0.5 3 0.10 4.90 1.0 4 0.15 4.85 1.5 5 0.20 4.80 2.0 6 0.25 4.75 2.5 7 0.30 4.70 3.0 8 0.35 4.65 3.5 9 0.40 4.60 4.0

Dilutions of the test sample. The test sample is diluted with sample dilution solution to a concentration of 1 mg/ml based on the actual HIR-FAB-IDS content stated on the certificate. Then successive dilutions are prepared according to the following scheme:________________

No. of tubes HIRFABIDS concentration, ng/ml Volume of solution, µl HIRFABIDS solution (1 mg/ml) Test tube no. Sample dilution solution 1 2 3 4 5 6 7 1 100000 100 - - - - - - - 900 2 10000 100 - - - - - - 900 3 1000 100 - - - - - 900 4 100 100 - - - - 900 5 10 100 - - - 900 6 5 100 - - 100 7 2.5 100 - 100

All dilutions are stored on ice until testing begins.

In further studies, solutions from test tubes No. 5-7 are used.

Conducting analysis.

1st enzymatic reaction.

Add 10 μl of each dilution of the test sample (in 2 replicates) to the wells of the plate; the sample dilution solution is used as a reference solution. Add 20 μl of substrate solution (4-methylumbelliferyl a-L-idopyranosiduronic acid 2-sulfate sodium salt). Cover the plate with film and incubate in a thermostatic shaker for 4 hours at a temperature of (37.0 ± 0.2) ° C and a stirring speed of 250 rpm. The plate should be protected from light during the entire incubation period.

2nd enzymatic reaction.

At the end of incubation, add 20 μl of phosphate-citrate buffer solution to the wells of the plate to stop the enzymatic reaction. Then add 10 μl of a solution of recombinant a-L-iduronidase to each well and mix gently. Cover the plate with film and incubate in a thermostatic shaker for 22-26 hours at a temperature of 37°C and a stirring speed of 250 rpm. The plate should be protected from light during the entire incubation period.

To stop the reaction, add 200 μl of stop solution to each well with the incubated mixture.

Add 260 μl of dilutions of the working standard solution of 4methylumbelliferone sodium salt into the empty wells of the plate.

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

Evaluation of results.

Based on the results of measuring the fluorescence signal in wells with dilutions of a working standard solution 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 the linear function equation, calculate the concentration of 4-methylumbelliferone produced during the reaction in wells with dilutions of test samples and a reference solution (nmol/ml).

Specific activity A, in U/µg, for each dilution of test samples was calculated

-21 044641 are calculated according to the formula:

A = 0.26 X (m - m ₀ ) X 1000 240 X C X 0.01 where: m is the concentration of 4-methylumbeliferone produced during the reaction in wells with a given dilution of the test sample (average value of 2 replicates) , nmol/ml;

mo is the concentration of 4-methylumbeliferone produced during the reaction in wells with a reference solution (average value of 2 replicates), nmol/ml;

0.26 is the final volume of dilutions of the working standard solution and the test sample added to the wells of the plate to measure the fluorescence signal, ml;

240 - time of the 1st enzymatic reaction, min;

C is the content of HIR-Fab-IDS in each of the prepared dilutions of the test sample, ng/ml; 0.01 - volume of dilutions of the test sample added to the 1st enzymatic reaction, ml; 1000 is the conversion factor from ng to mcg.

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

The results obtained for determining the enzymatic activity of the HIR-FAB-IDS and HIRMAB-IDS variants in comparison with the drug Elapraza are presented in Table. 2.

table 2

Evaluation of iduronate-2-sulfatase activity of HIR-FAB-IDS and HIR-MAB-IDS variants against the unmodified iduronate-2-sulfatase enzyme (Elaprase)

Name of samples Activity, units (nmol/min)/mcg HIR-FAB-IDS (SEQ ID NO:2 and SEQ ID NO:4) 27.0 HIR-FAB-IDS (SEQ ID NO:2 and SEQ ID NO:5) 20.0 HIR-FAB-IDS (SEQ ID NO:8 and SEQ ID NO:12) 25.2 HIR-FAB-IDS (SEQ ID NO:9 and SEQ ID NO:13) 30.2 HIR-FAB-IDS (SEQ ID NO: 10 and SEQ ID NO: 14) 27.5 HIR-FAB-IDS (SEQ ID NO: 11 and SEQ ID NO: 15) 23.4 HIR-MAB-IDS 11.0 HIR-MAB-IDS (AGT-182) 0.85 (51.7+-7 (value taken from patent for AGT-182 US8834874 B2, Fig.12 example 4, page 35) nmol/hour/mcg) US8834874B2 Elaprase (Shayer Human Genetic Therapies Inc., USA) 14.6

As can be seen from the results presented in table. 2, the HIR-FAB-IDS drug exhibits specific enzymatic (iduronate-2-sulfatase) activity towards the substrate 4-MU-aIdoA-2S (4-methylumbeliferyl α-L-idopyranosiduronate-2-sulfate) 27.0 U/μg. In this case, the free recombinant enzyme iduronate-2-sulfatase exhibits an activity of 14.6 U/µg. Full-length insulin receptor antibody linked to the amino acid sequence of iduronate-2-sulfatase (HIR-MAB-IDS) exhibits lower specific activity (11.0 U/μg) compared to HIR-FABIDS.

As a result of the studies, it was shown that iduronate-2-sulfatase in HIRFAB-IDS retains the main functional (enzymatic) properties at the level of the free recombinant enzyme. Moreover, the specific activity of HIR-FAB-IDS is higher than for the drug Elaprase and HIR-MAB-IDS (AGT-182).

Thus, an increase in the enzymatic activity of a compound containing a transport element, which is a Fab fragment of an IgG immunoglobulin, specific to an epitope in the insulin receptor, connected directly or via a linker to a therapeutic enzyme, was detected, in comparison with a therapeutic enzyme without a transport element, and with a therapeutic enzyme an enzyme with a transport element represented by MAB.

Example 3. Analysis of the interaction of modified Fab fragments of antibodies to the insulin receptor with the human and mouse insulin receptor.

To quantitatively assess the interaction with the insulin receptor of full-length recombinant monoclonal antibodies, variants of Fab fragments, as well as their modifications with IDS, an enzyme-linked immunosorbent assay (ELISA) method in a sandwich format is used (Kim C, Seo J, Chung Y et al., 2017 ). The method is based on the binding of proteins HIR-MAB (83-14 Mab), HIR-MAB-IDS and HIR-FAB-IDS with a protein fragment of the human insulin receptor (Human Insulin Receptor protein fragment (Abcam cat#ab200510)) or mouse insulin receptor ( mInsR, R&D Systems, Inc., Cat. No. 7544-MR).

The detection process is carried out after incubation of the complex of the human insulin receptor and the protein under study with secondary antibodies - horseradish peroxidase-conjugated Fab-specific goat antibodies to human immunoglobulin (Anti-Human IgG (Fab specific) - Peroxidase

- 22 044641 (Sigma-Aldrich, cat#A0293)).

Previously described mouse antibodies to the human insulin receptor (INSR/Insulin Receptor alpha Antibody 83-14 AHR0221 Life technologies, cat#AHR0221) and to the mouse insulin receptor (Mouse Anti-Human Insulin Receptor Clone 83-14 mAb Cell sciences) were used as a positive control , cat# MAP). Detection is carried out using horseradish peroxidase-conjugated goat polyclonal antibodies to mouse immunoglobulin (Goat pAb to Ms IgG (HRP) Abeam, cat# ab97023).

The results obtained for determining the interaction of the tested molecules with the human and mouse insulin receptor are presented in Table. 3 and 4.

Table 3

Binding of modified Fab fragments of antibodies to the insulin receptor with the human insulin receptor (Abeam cat#ab200510)

No. Sample name EC50, ng/ml Receptor binding from antibody 83-14 mAb, % 1 INSR/Insulin Receptor alpha Antibody 83-14 (Life technologies, cat#AHR0221) 32.15 102 2 Mouse Anti-Human Insulin Receptor Clone 83-14 mAb (Cell sciences, cat#MAIl) 33.10 100 3 HIR-FAB-IDS (SEQ ID NO:2 and SEQ ID NO:4) 24.31 136 4 HIR-FAB-IDS (SEQ ID NO:2 and SEQ ID NO:5) 50.25 66 5 HIR-FAB-IDS (SEQ ID NO:8 and SEQ ID NO:12) 120.55 27 6 HIR-FAB-IDS (SEQ ID NO:9 and SEQ ID NO:13) 60.35 55 7 HIR-FAB-IDS (SEQ ID NO: 10 and SEQ ID NO: 14) 35.10 94 8 HIR-FAB-IDS (SEQ ID NO: 11 and SEQ ID NO: 15) 155.67 21 9 HIR-MAB-IDS 26.19 126

Table 4

Binding of modified Fab fragments of insulin receptor antibodies to the mouse insulin receptor (R&D Systems, Inc., cat. no. 7544-MR)

No. Sample name EC50, ng/ml Receptor binding, % 1 INSR/Insulin Receptor alpha >800 0 Antibody 83-14 2 Mouse Anti-Human Insulin Receptor Clone 83-14 mAb >800 0 3 HIR-FAB-IDS (SEQ ID NO:2 and SEQ ID NO:4) >800 0 4 HIR-FAB-IDS (SEQ ID NO:2 and SEQ ID NO:5) >800 0 5 HIR-FAB-IDS (SEQ ID NO:8 and SEQ ID NO:12) >800 0 6 HIR-FAB-IDS (SEQ ID NO:9 and SEQ ID NO:13) >800 0 7 HIR-FAB-IDS (SEQ ID NO: 10 and SEQ ID NO: 14) >800 0 8 HIR-FAB-IDS (SEQ ID NO: 11 and SEQ ID NO: 15) >800 0 9 HIR-MAB-IDS >800 0

Thus, it was demonstrated that HIR-FAB-IDS specifically interacts with the extracellular domain of the alpha subunit of the human insulin receptor, but does not interact with the extracellular domain of the mouse insulin receptor. The binding of Fab fragments of antibodies to the insulin receptor to the human insulin receptor is comparable to the control antibody (83-14 mAb) and exceeds the binding to the similar full-length antibody to the insulin receptor (HIR-MAB-IDS).

The interaction of HIR-FAB-IDS with the human insulin receptor is fundamental to ensure active transcytosis of the molecule to cell lysosomes, in general, through the BBB, in particular.

Example 4. Analysis of the distribution in tissues of radioactively labeled Fab fragments of the antibody to the insulin 23 044641 receptor, connected to the amino acid sequence of iduronate-2-sulfatase

[ ¹²⁵ I]-HIR-Fab-IDS (SEQ ID NO: 2 and SEQ ID NO: 4), [ ¹²⁵ I]-HIR-Mab-IDS, [ ¹²⁵ I]-IDS (control) after intravenous administration in cynomolgus monkey .

The experimental animals were injected once into the left femoral vein with the corresponding molecule of the test substance at a target dose of 2 ml/kg body weight by intravenous bolus injection over 1-2 minutes. The target radioactive dose level was 1 MBq/kg animal body weight for all molecules studied.

The animal receiving the test substance was sedated by intramuscular injection of ketamine hydrochloride before intravenous administration of an excess dose of Doletil (sodium pentaborbitone). Animals were humanely euthanized 2 hours after administration of the test substance. During sedation, 2 ml of blood was collected from each animal from the cephalic vein and centrifuged to obtain plasma. The concentration of radioactivity was measured in the blood plasma of animals. Once death was confirmed, each animal was quickly frozen in a mixture of solid carbon dioxide in hexane. Once completely frozen, the bodies were placed in a mold containing 2% (w/v) aqueous carboxymethylcellulose paste. Several longitudinal sagittal sections (nominally 30 μm) were taken at at least 5 body levels and, if necessary, 3 head levels for each individual.

Sections mounted on Filmolux 610 tape (Neschan) were lyophilized in a GVD03 benchtop freeze dryer (Girovac Ltd) and mounted on FUJI X-ray plates (type BASMS, Raytek Scientific Ltd). Blood standards labeled with the isotope [ ¹²⁵ I] with the appropriate activity, also prepared in the form of sections with a nominal thickness of 30 μm, were applied to radiographic plates.

After developing in a copper-coated lead box for 7 days, the radiographic plates were processed using a FUJI FLA-5000 radiographic system (Raytek Scientific Ltd). Electronic images were analyzed using a PC-based image analysis system (Seescan 2 software, LabLogic Ltd). [ ^125I ] standards included in each autoradiogram were used to construct calibration curves over a range of radioactivity concentrations.

The concentration of radioactivity in plasma was determined by gammametry.

Tissue concentration data were recorded as ng equivalents of [ ^125I ]HIR-Fab-IDS [ ^125I ]HIR-Mab-IDS and [ ^125I ]-IDS (control).

Results were expressed to three significant numbers with no more than two decimal places. Data tables were computer generated and individual data were rounded appropriately.

[ ¹²⁵ I]HIR-Fab-IDS, [ ¹²⁵ I]HIR-Mab-IDS and [ ¹²⁵ I]-IDS (control) were administered to male cynomolgus monkeys as a single intravenous dose at a nominal dose level of 0.0020 mg/kg, 0.0015 mg /kg and 0.0010 mg/kg body weight. Euthanasia of the animal was performed 2 hours after the administration of each drug, after which the animals were frozen for studies using whole-body autoradiography.

After intravenous administration, the drugs were absorbed and widely distributed throughout the tissues of the body, and at the time of slaughter, all tissues studied were exposed to it. The lower limit of quantification was 0.2, 0.03, and 0.097 ng drug equivalent/g for all animal tissue concentration measurements, respectively.

Radioactivity concentrations in plasma and blood of the animal treated with [ ¹²⁵ 1] 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) /g), arborescent substance of the cerebellum (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.

Radioactivity concentrations in plasma and blood of the [ ¹²⁵ I]HIR-Mab-IDS-treated animal were 2.13 and 1.86 ng eq/g, respectively (blood:plasma ratio 0.87). Quantifiable levels of radioactivity were present in a number of areas of the animal's brain. These areas 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). d)), medulla (0.288 ng eq/g) and hypothalamus (0.279 ng eq/g), TP concentration ratios ranged from 0.13 to 0.38

Concentrations in plasma and blood of the [ ¹²⁵ I]IDS-treated animal 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 limit of quantification (0.097 ng eq/g) in all regions examined: medulla oblongata, cerebral cortex, cerebellum, hypothalamus, cerebellar arboresum, pons and medulla. Concentrations in CNS tissues were significantly lower than in plasma and blood.

These results indicate that substances associated with HIR-Fab-IDS as well as HIRMab-IDS, compared with substance associated with IDS, penetrated the blood-brain barrier.

- 24 044641 CNS tissues were exposed to the test substance or its labeled metabolites, and blood-borne radioactivity had little effect on the CNS tissue concentration determination (FIGS. 1 and 2). Moreover, HIR-Fab-IDS demonstrated a wider distribution in body tissue compared to the HIR-Mab-IDS molecule (Table 5, Fig. 3).

It should be noted that in example 10 of the invention US8834874 B2 (AGT-182 or HIR-MAB-IDS) the penetration efficiency into the human brain is estimated at 1% of the administered dose per 1000 grams of brain tissue based on experimental data on the penetration of AGT-182 (HIRMAB -IDS) into the brain in rhesus monkeys. It is stated that at this level of penetration, it is expected to achieve 20% of iduronate-2-sulfatase (IDS) activity in the human brain, which will eliminate the accumulation of glycosaminoglycans in the brain of a sick person.

Taking into account the data obtained on penetration into the brain of cynomolgus monkeys (Table 5), we can conclude that the present compound penetrates into the brain of cynomolgus monkeys almost 2 times better than AGT-182 (HIR-MAB-IDS), so - 0 .84 ng eq/g HIR-FAB-IDS versus 0.43 ng eq/g for HIR-MAB-IDS. This accordingly means that HIR-FAB-IDS penetrates the human brain with 2% efficiency. Taking into account the calculations specified in example 10 of the invention US 8834874 B2, it is expected to achieve 40% of iduronate2-sulfatase activity in the human brain, without taking into account the increased activity of HIR-FAB-IDS in comparison with HIR-MAB-IDS (AGT-182), cm table 2.

If we take into account the fact of an increase in the specific activity of HIR-FAB-IDS over HIR-MAB-IDS (AGT-182), shown in example 2, table. 2-27 U/µg for the present compound and 11 U/µg for the opposed one, as well as greater penetration into the brain: 27/11=in 2.4 specific iduronate-2-sulfatase activity is higher, 0.84/0.43=in 1.95 times greater penetration into the brain, then we get a 20% restoration of IDS activity in the patient’s brain during therapy with HIR-MAB-IDS (AGT-182) and 20% * 2.4 * 1.95 = 93.6% restoration of iduronate 2-sulfatase activity in the brain of a patient with MPS type II from the level of IDS activity in the brain of a healthy person. Thus, the present invention will almost completely restore the enzymatic function of IDS in the brain of a sick person, while the opposed AGT-182 (HIR-MAB-IDS) only restores by 20% (93% vs 20%).

Thus, it has been found that the administration of a compound containing a transport element, which is a Fab fragment of an IgG immunoglobulin specific for an epitope in the insulin receptor, connected directly or via a linker to a therapeutic enzyme, provides a higher degree of replacement of the activity of the therapeutic enzyme in the human brain in comparison with compounds containing Mab.

- 25 044641

Table 5

Radioactivity concentrations in cynomolgus monkey tissues following a single intravenous administration of [ ¹²⁵ I]-HIR-Fab-IDS at a nominal dose level of 0.0020 mg/kg body weight

Textile Radioactivity concentrations in tissues [ ¹²⁵ I] HIR-Fab-IDS (SEQ ID NO:2 and SEQ GO NO:4) [ ¹²⁵ I] HIR-Mab-IDS (AGT-182) [ ¹²⁵ I]-IDS Plasma 2.55 2.13 0.91 Blood 3.11 1.86 0.75 Adrenal cortex 18.3 5.29 4.47 Adrenal medulla 11.5 2.17 3.26 Aortic wall 3.29 1.96 BLQ Bone marrow 6.37 3.26 4.39 Perioste 3.32 BLQ 0.95 Brown fat 2.51 0.97 0.54 Bulbourethral gland 0.98 BLQ 0.60 Mucosa of the cecum 1.72 0.19 0.25 Epididymis 3.09 0.31 0.53 Kidney bark 10.1 4.25 4.33 Kidney medulla 6.37 2.39 1.70 Colon mucosa 1.42 0.41 0.24 Liver 21.4 7.63 18.0 Lung 4.15 1.10 2.35 Muscles 0.51 0.09 0.28 Myocardium 3.78 0.91 1.25 Nasal mucosa 1.18 0.61 0.72 Esophageal wall 1.53 0.51 0.48 Pancreas 2.68 0.70 0.69 Rectal mucosa 1.15 0.39 0.27 Salivary glands 1.01 0.23 0.82 Seminal glands 2.08 BLQ BLQ Leather 1.20 0.20 0.39 Mucosa of the small intestine 3.14 0.62 1.62 Spinal cord 0.57 0.41 0.17 Spleen 17.9 10.4 11.6 Testicles 1.34 0.18 0.41 Language 0.92 0.30 0.64 Trachea 0.65 0.32 0.45 Bladder wall 7.30 3.49 4.18 Brain (whole) 0.84 0.43 0.11 Cortex 1.03 0.45 BLQ Brain matter 0.56 0.29 BLQ Cerebellum 0.91 0.49 BLQ Hypothalamus 0.87 0.28 BLQ Medulla 1.09 0.80 BLQ Bridge 0.79 0.44 BLQ Substantia arborescens of the cerebellum 0.80 0.61 BLQ Pineal gland 1.03 0.39 BLQ Hypophysis 0.80 0.60 BLQ

NA - not applicable;

BLQ - concentration below the lower limit of quantitation.

The present description presents compounds containing therapeutic enzymes that exhibit their specific enzymatic activity in these compounds, and transport elements capable of interacting with the insulin receptor, while having the ability to transport therapeutic enzymes to tissue lysosomes, including through the BBB to lysosomes of nervous tissue. Thus, the compounds exhibit high activity and improved ability

- 26 044641 be transported to the lysosomes of tissue cells of various organs, including to the lysosomes of nervous tissue cells, which suggests the use of the present invention for enzyme replacement therapy for the treatment or prevention of a subject with a lysosomal storage disease.

LIST OF SEQUENCES <110> LIMITED LIABILITY COMPANY MBC GENERIUM <120> COMPOUND CONTAINING A THERAPEUTIC ENZYME AND A TRANSPORT ELEMENT CONNECTED TO EACH OTHER DIRECTLY OR BY USING A LINKER A <130> GNR-055 <160> 7 <170> PatentIn version 2.0 <210 > 1 <211> 124 <212> Protein <213> Artificial sequence <220>

<223> Epitope in the insulin receptor recognized by immunoglobulin IgG <400> 1

Met Glu Glu 1

Ala Leu Lys

Lys Phe Ser 35

Glu Pro Tyr

Phe Tyr Lys 65

Asp Ala Cys

Leu Arg Ser

Arg Gly Leu

115

Val Ser 5

Thr Asn

Tyr Ile

Trp Pro

Glu Ala

Gly Ser 85

Asn Asp

100

Lys Pro

Gly Thr Lys Gly Arg Gln Glu Arg Asn Asp Ile 1015

Gly Asp Gln Ala Ser Cys Glu Asn Glu Leu Leu 2530

Arg Thr Ser Phe Asp Lys Ile Leu Leu Arg Trp 4045

Pro Asp Phe Arg Asp Leu Leu Gly Phe Met Leu 5560

Pro Tyr Gln Asn Val Thr Glu Phe Asp Gly Gln 70 7580

Asn Ser Trp Thr Val Val Asp Ile Asp Pro Pro 9095

Pro Lys Ser Gln Asn His Pro Gly Trp Leu Met 105110

Trp Thr Gln Tyr Ala Ile Phe

120 <210> 2 <211> 214 <212> Protein <213> Artificial sequence <220>

<223> Immunoglobulin IgG light chain to insulin receptor <400> 2

Asp Ile 1

Glu Arg

Leu Tyr

Tyr Ala

Ser Arg 65

Glu Asp

Thr Phe

Pro Ser

Gln Met

Val Ser

Trp Leu 35

Thr Ser

Ser Gly

PheVal

Gly Gly

100

Val Phe

Thr Gln 5

Leu Thr

Gln Gln

Ser Leu

Ser Asp

Asp Tyr 85

Gly Thr

Ile Phe

Ser Pro

CysArg

Gly Pro

Asp Ser 55

Tyr Ser

Tyr Cys

Lys Met

Pro Pro

Ser Ser

Ala Ser 25

Asp Gly

Gly Val

Leu Thr

Leu Gln

Glu Ile

105

Ser Asp

Leu Ser

Gln Asp

Thr Ile

Pro Lys

Ile Ser 75

Tyr Ser

Lys Arg

Glu Gln

Ala Ser

Ile Gly

Lys Arg 45

Arg Phe

Ser Leu

Ser Ser

Thr Val

110

Leu Lys

Leu Gly 15

Gly Asn

Leu Ile

Ser Gly

Glu Ser

Pro Trp 95

Ala Ala

Ser Gly

- 27 044641

115 120125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn SerGln

145 150 155160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser LeuSer

165 170175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200205

Phe Asn Arg Gly Glu Cys

210 <210> 3 <211> 221 <212> Protein <213> Artificial sequence <220>

<223> Fragment VH-CH1 of the heavy chain of immunoglobulin IgG to the insulin receptor <400> 3

Gln Val Gln Leu Gln Gln Ser Gly Pro 15

Leu Val Lys Ile Ser Cys Lys Ala Ser 2025

Asp Ile His Trp Val Lys Gln Arg Pro 3540

Gly Trp Ile Tyr Pro Gly Asp Gly Ser 5055

Lys Gly Lys Ala Thr Leu Thr Ala Asp 6570

Met His Leu Ser Ser Leu Thr Ser Glu 85

Ala Arg Glu Trp Ala Tyr Trp Gly Gln 100105

Ala Ala Ser Thr Lys Gly Pro Ser Val 115120

Lys Ser Thr Ser Gly Gly Thr Ala Ala 130135

Tyr Phe Pro Glu Pro Val Thr Val Ser 145150

Ser Gly Val His Thr Phe Pro Ala Val 165

Ser Leu Ser Ser Val Val Thr Val Pro 180185

Thr Tyr Ile Cys Asn Val Asn His Lys 195200

Lys Lys Val Glu Pro Lys Ser Cys Asp 210215

Glu Leu Val Lys Pro Gly Ala 10 15

Gly Tyr Thr Phe Thr Asn Tyr 30

Gly Gln Gly Leu Glu Trp Ile 45

Thr Lys Tyr Asn Glu Lys Phe 60

Lys Ser Ser Ser Thr Ala Tyr 75 80

Lys Ser Ala Val Tyr Phe Cys 90 95

Gly Thr Leu Val Thr Val Ser 110

Phe Pro Leu Ala Pro Ser Ser 125

Leu Gly Cys Leu Val Lys Asp

140

Trp Asn Ser Gly Ala Leu Thr

155 160

Leu Gln Ser Ser Gly Leu Tyr

170 175

Ser Ser Ser Leu Gly Thr Gln 190

Pro Ser Asn Thr Lys Val Asp 205

Lys Thr His Leu

220 <210> 4 <211> 748 <212> Protein <213> Artificial sequence <220>

<223> VH-CH1 fragment of the immunoglobulin IgG heavy chain to the insulin receptor, connected to the sequence of iduronate-2-sulfatase <400> 4

- 28 044641

Gln Val Gln Leu Gln Gln Ser Gly 15

Leu Val Lys Ile Ser Cys Lys Ala 20

Asp Ile His Trp Val Lys Gln Arg 3540

Gly Trp Ile Tyr Pro Gly Asp Gly 5055

Lys Gly Lys Ala Thr Leu Thr Ala 6570

Met His Leu Ser Ser Leu Thr Ser 85

Ala Arg Glu Trp Ala Tyr Trp Gly 100

Ala Ala Ser Thr Lys Gly Pro Ser 115120

Lys Ser Thr Ser Gly Gly Thr Ala 130135

Tyr Phe Pro Glu Pro Val Thr Val

145150

Ser Gly Val His Thr Phe Pro Ala 165

Ser Leu Ser Ser Val Val Thr Val 180

Thr Tyr Ile Cys Asn Val Asn His 195200

Lys Lys Val Glu Pro Lys Ser Cys 210215

Glu Thr Gln Ala Asn Ser Thr Thr

225230

Ile Val Asp Asp Leu Arg Pro Ser 245

Val Arg Ser Pro Asn Ile Asp Gln 260

Gln Asn Ala Phe Ala Gln Gln Ala

275280

Phe Leu Thr Gly Arg Arg Pro Asp 290295

Ser Tyr Trp Arg Val His Ala Gly 305310

Phe Lys Glu Asn Gly Tyr Val Thr 325

Pro Gly Ile Ser Ser Asn His Thr 340

Phe Pro Pro Tyr His Pro Ser Ser 355360

Cys Arg Gly Pro Asp Gly Glu Leu 370375

Asp Val Leu Asp Val Pro Glu Gly 385390

Glu Gln Ala Ile Gln Leu Leu Glu 405

Phe Phe Leu Ala Val Gly Tyr His 420

Pro Lys Glu Phe Gln Lys Leu Tyr 435440

Pro Asp Pro Glu Val Pro Asp Gly 450455

Trp Met Asp Ile Arg Gln Arg Glu 465470

Val Pro Tyr Gly Pro Ile Pro Val 485

Ser Tyr Phe Ala Ser Val Ser Tyr 500

Leu Ser Ala Leu Asp Asp Leu Gln 515520

Phe Thr Ser Asp His Gly Trp Ala 530535

Lys Tyr Ser Asn Phe Asp Val Ala 545550

Pro Glu Leu Val Lys Pro Gly Ala 1015

Ser Gly Tyr Thr Phe Thr Asn Tyr 2530

Pro Gly Gln Gly Leu Glu Trp Ile 45

Ser Thr Lys Tyr Asn Glu Lys Phe 60

Asp Lys Ser Ser Ser Thr Ala Tyr 7580

Glu Lys Ser Ala Val Tyr Phe Cys 9095

Gln Gly Thr Leu Val Thr Val Ser

105110

Val Phe Pro Leu Ala Pro Ser Ser

125

Ala Leu Gly Cys Leu Val Lys Asp 140

Ser Trp Asn Ser Gly Ala Leu Thr 155160

Val Leu Gln Ser Ser Gly Leu Tyr 170175

Pro Ser Ser Ser Leu Gly Thr Gln

185190

Lys Pro Ser Asn Thr Lys Val Asp 205

Asp Lys Thr His Leu Ser Ser Ser 220

Asp Ala Leu Asn Val Leu Leu Ile 235240

Leu Gly Cys Tyr Gly Asp Lys Leu 250255

Leu Ala Ser His Ser Leu Leu Phe

265270

Val Cys Ala Pro Ser Arg Val Ser 285

Thr Thr Arg Leu Tyr Asp Phe Asn 300

Asn Phe Ser Thr Ile Pro Gln Tyr 315320

Met Ser Val Gly Lys Val Phe His 330335

Asp Asp Ser Pro Tyr Ser Trp Ser

345350

Glu Lys Tyr Glu Asn Thr Lys Thr 365

His Ala Asn Leu Leu Cys Pro Val 380

Thr Leu Pro Asp Lys Gln Ser Thr 395400

Lys Met Lys Thr Ser Ala Ser Pro 410415

Lys Pro His Ile Pro Phe Arg Tyr 425430

Pro Leu Glu Asn Ile Thr Leu Ala 445

Leu Pro Pro Val Ala Tyr Asn Pro 460

Asp Val Gln Ala Leu Asn Ile Ser 475480

Asp Phe Gln Arg Lys Ile Arg Gln 490495

Leu Asp Thr Gln Val Gly Arg Leu

505510

Leu Ala Asn Ser Thr Ile Ile Ala

525

Leu Gly Glu His Gly Glu Trp Ala 540

Thr His Val Pro Leu Ile Phe Tyr

555 560

- 29 044641

Val Pro

Pro Tyr

Arg Gln

Ala Gly

610 Phe His 625 Arg Phe

Glu Leu

Trp Asn

Ser Ile

690 Pro Asp 705 Tyr Phe

Ser Gln

Gly Arg

Leu Asp

580 Ser Met 595

Leu Ala

Val Glu

Arg Asp

Ile Ala

660 Ser Asp 675

Arg Thr

Glu Phe

Val Asp

Gly Gly 740

Thr Ala Ser 565

Pro Phe Asp

Asp Leu Val

Gly Leu Gln 615

Leu Cys Arg 630

Leu Glu Glu 645

Tyr Ser Gln

Lys Pro Ser

Ile Asp Tyr 695

Leu Ala Asn 710

Ser Asp Pro 725

Asp Leu Phe

Leu Pro Glu Ala Gly Glu Lys Leu Phe 570575

Ser Ala Ser Gln Leu Met Glu Pro Gly 585590

Glu Leu Val Ser Leu Phe Pro Thr Leu

600605

Val Pro Pro Arg Cys Pro Val Pro Ser 620

Glu Gly Lys Asn Leu Leu Lys His Phe 635640

Asp Pro Tyr Leu Pro Gly Asn Pro Arg 650655

Tyr Pro Arg Pro Ser Asp Ile Pro Gln 665670

Leu Lys Asp Ile Lys Ile Met Gly Tyr 680685

Arg Tyr Thr Val Trp Val Gly Phe Asn 700

Phe Ser Asp Ile His Ala Gly Glu Leu 715720

Leu Gln Asp His Asn Met Tyr Asn Asp 730735

Gln Leu Leu Met Pro

745 <210> 5 <211> 760 <212> Protein <213> Artificial sequence <220>

<223> Fragment VH-CH1 of the heavy chain of immunoglobulin IgG to the insulin receptor, connected to the sequence of iduronate-2-sulfatase by means of a glycine-serine linker by <400> 5

Gln Val Gln Leu Gln Gln Ser Gly Pro Glu 1 510

Leu Val Lys Ile Ser Cys Lys Ala Ser Gly 2025

Asp Ile His Trp Val Lys Gln Arg Pro Gly 3540

Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr 5055

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys 6570

Met His Leu Ser Ser Leu Thr Ser Glu Lys 8590

Ala Arg Glu Trp Ala Tyr Trp Gly Gln Gly 100105

Ala Ala Ser Thr Lys Gly Pro Ser Val Phe 115120

Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130135

Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145150

Ser Gly Val His Thr Phe Pro Ala Val Leu 165170

Ser Leu Ser Ser Val Val Thr Val Pro Ser 180185

Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195200

Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210215

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 225230

Leu Val Lys

Tyr Thr Phe

Gln Gly Leu 45

Lys Tyr Asn 60

Ser Ser Ser 75

Ser Ala Val

Thr Leu Val

Pro Leu Ala 125

Gly Cys Leu 140

Asn Ser Gly 155

Gln Ser Ser

Ser Ser Leu

Ser Asn Thr

205

Thr His Gly 220

Ser Ser Glu

235

Pro Gly Ala 15

Thr Asn Tyr 30

Glu Trp Ile

Glu Lys Phe

Thr Ala Tyr 80

Tyr Phe Cys 95

Thr Val Ser 110

Pro Ser Ser

Val Lys Asp

Ala Leu Thr

160

Gly Leu Tyr 175

Gly Thr Gln 190

Lys Val Asp

Gly Gly Gly

Thr Gln Ala

240

- 30 044641

Asn Ser

Leu Arg

Asn Ile

Ala Gln 290 Arg Arg 305

Val His

Gly Tyr

Ser Asn

His Pro 370 Asp Gly 385

Val Pro

Gln Leu

Val Gly

Gln Lys 450

Val Pro 465

Arg Gln

Pro Ile

Ser Val

Asp Asp 530

His Gly 545

Phe Asp

Thr Ala

Pro Phe

Asp Leu 610

Gly Leu 625

Leu Cys

Leu Glu

Tyr Ser

Lys Pro 690

Ile Asp 705

Leu Ala

Ser Asp

Asp Leu

Thr

Pro

Asp 275 Gln

Pro

Ala

Val

His 355 Ser

Glu

Glu

Leu

Tyr 435 Leu

Asp

Arg

Pro

Ser 515 Leu

Trp

Val

Ser

Asp 595 Val

Gln

Arg

Glu

Gln 675 Ser

Tyr

Asn

Pro

Phe 755

Thr

Ser 260 Gln

Ala

Asp

Gly

Thr 340 Thr

Ser

Leu

Gly

Glu 420 His

Tyr

Gly

Glu

Val 500 Tyr

Gln

Ala

Ala

Leu 580 Ser

Glu

Val

Glu

Asp 660 Tyr

Leu

Arg

Phe

Leu 740 Gln

Asp 245 Leu

Leu

Val

Thr

Asn 325 Met

Asp

Glu

His

Thr 405 Lys

Lys

Pro

Leu

Asp 485 Asp

Leu

Leu

Leu

Thr 565 Pro

Ala

Leu

Pro

Gly 645 Pro

Pro

Lys

Tyr

Ser 725 Gln

Leu

Ala

Gly

Ala

Cys

Thr 310 Ph

Ser

Asp

Lys

Ala 390 Leu

Met

Pro

Leu

Pro 470 Val

Phe

Asp

Ala

Gly 550 His

Glu

Ser

Val

Pro 630 Lys

Tyr

Arg

Asp

Thr 710 Asp

Asp

Leu

Leu Asn Val Leu Leu Ile Ile Val Asp Asp 250255

Cys Tyr Gly Asp Lys Leu Val Arg Ser Pro 265270

Ser His Ser Leu Leu Phe Gln Asn Ala Phe 280285

Ala Pro Ser Arg Val Ser Phe Leu Thr Gly 295300

Arg Leu Tyr Asp Phe Asn Ser Tyr Trp Arg 315320

Ser Thr Ile Pro Gln Tyr Phe Lys Glu Asn 330335

Val Gly Lys Val Phe His Pro Gly Ile Ser 345350

Ser Pro Tyr Ser Trp Ser Phe Pro Pro Tyr 360365

Tyr Glu Asn Thr Lys Thr Cys Arg Gly Pro 375380

Asn Leu Leu Cys Pro Val Asp Val Leu Asp 395400

Pro Asp Lys Gln Ser Thr Glu Gln Ala Ile 410415

Lys Thr Ser Ala Ser Pro Phe Phe Leu Ala 425430

His Ile Pro Phe Arg Tyr Pro Lys Glu Phe 440445

Glu Asn Ile Thr Leu Ala Pro Asp Pro Glu 455460

Pro Val Ala Tyr Asn Pro Trp Met Asp Ile 475480

Gln Ala Leu Asn Ile Ser Val Pro Tyr Gly 490495

Gln Arg Lys Ile Arg Gln Ser Tyr Phe Ala 505510

Thr Gln Val Gly Arg Leu Leu Ser Ala Leu 520525

Asn Ser Thr Ile Ile Ala Phe Thr Ser Asp 535540

Glu His Gly Glu Trp Ala Lys Tyr Ser Asn 555560

Val Pro Leu Ile Phe Tyr Val Pro Gly Arg 570575

Ala Gly Glu Lys Leu Phe Pro Tyr Leu Asp 585590

Gln Leu Met Glu Pro Gly Arg Gln Ser Met 600605

Ser Leu Phe Pro Thr Leu Ala Gly Leu Ala 615620

Arg Cys Pro Val Pro Ser Phe His Val Glu 635640

Asn Leu Leu Lys His Phe Arg Phe Arg Asp 650655

Leu Pro Gly Asn Pro Arg Glu Leu Ile Ala 665670

Pro Ser Asp Ile Pro Gln Trp Asn Ser Asp 680685

Ile Lys Ile Met Gly Tyr Ser Ile Arg Thr 695700

Val Trp Val Gly Phe Asn Pro Asp Glu Phe 715720

Ile His Ala Gly Glu Leu Tyr Phe Val Asp 730735

His Asn Met Tyr Asn Asp Ser Gln Gly Gly 745750

Met Pro

760 <210> 6

- 31 044641 <211> 850 <212> Protein <213> Artificial sequence <220>

<223> Fragment VH-CH1 of the heavy chain of an antibody to the human insulin receptor, combined with the sequence of β-b-iduronidase <400> 6

Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

5 1015

Leu Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr AsnTyr

2530

Asp Ile His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

4045

Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Lys Tyr Asn Glu Lys Phe

5560

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr AlaTyr

70 7580

Met His Leu Ser Ser Leu Thr Ser Glu Lys Ser Ala Val Tyr PheCys

9095

Ala Arg Glu Trp Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser

100 105110

Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser

115 120125

Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp

130 135140

Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala LeuThr

145 150 155160

Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly LeuTyr

165 170175

Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln

180 185190

Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp

195 200205

Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Leu Ser Ser Glu

210 215220

Ala Pro His Leu Val Gln Val Asp Ala Ala Arg Ala Leu Trp ProLeu

225 230 235240

Arg Arg Phe Trp Arg Ser Thr Gly Phe Cys Pro Pro Leu Pro HisSer

245 250255

Gln Ala Asp Gln Tyr Val Leu Ser Trp Asp Gln Gln Leu Asn Leu Ala

260 265270

Tyr Val Gly Ala Val Pro His Arg Gly Ile Lys Gln Val Arg Thr His

275 280285

Trp Leu Leu Glu Leu Val Thr Thr Arg Gly Ser Thr Gly Arg Gly Leu

290 295300

Ser Tyr Asn Phe Thr His Leu Asp Gly Tyr Leu Asp Leu Leu ArgGlu

305 310 315320

Asn Gln Leu Leu Pro Gly Phe Glu Leu Met Gly Ser Ala Ser GlyHis

325 330335

Phe Thr Asp Phe Glu Asp Lys Gln Gln Val Phe Glu Trp Lys Asp Leu

340 345350

Val Ser Ser Leu Ala Arg Arg Tyr Ile Gly Arg Tyr Gly Leu Ala His

355 360365

Val Ser Lys Trp Asn Phe Glu Thr Trp Asn Glu Pro Asp His His Asp

370 375380

Phe Asp Asn Val Ser Met Thr Met Gln Gly Phe Leu Asn Tyr TyrAsp

385 390 395400

Ala Cys Ser Glu Gly Leu Arg Ala Ala Ser Pro Ala Leu Arg LeuGly

405 410415

Gly Pro Gly Asp Ser Phe His Thr Pro Pro Arg Ser Pro Leu Ser Trp

420 425430

Gly Leu Leu Arg His Cys His Asp Gly Thr Asn Phe Phe Thr Gly Glu

435 440445

Ala Gly Val Arg Leu Asp Tyr Ile Ser Leu His Arg Lys Gly Ala Arg

450 455460

Ser Ser Ile Ser Ile Leu Glu Gln Glu Lys Val Val Ala Gln Gln Ile co- 32 044641

465

Arg Gln Leu

Ala Asp Pro

Val Thr Tyr 515

Leu Leu Leu 530

Asn Asp Asn 545

Thr Leu Thr

Gln Leu Leu

Leu Asp Glu 595

Leu Asp Ser 610

Gln Gly Pro 625

Asp Asp Thr

Leu Arg Gly

Leu Asp Asn 675

Arg Pro Val 690

Glu Asp Pro 705

Leu Thr Leu

Val Cys Ala

Ala Leu Pro 755

His Val Gly 770

Asp Gly Lys 785

Leu Phe Val

Val Arg Ala

Val Pro Tyr 835

Asn Pro

850

Phe Pro

485 Leu Val 500 Ala Ala

Ala Asn

Ala Phe

Ala Arg

565 Arg Lys 580

Glu Gln

Asn His

Ala Asp

Arg Ala

645

Val Pro 660

Gly Leu

Phe Pro

Val Ala

Arg Pro

725 Arg Pro 740

Leu Thr

Ser Lys

Ala Tyr

Phe Ser

805 Leu Asp 820

Leu Glu

470 LysPhe

Gly Trp

Met Val

Thr Thr

535 Leu Ser 550

Phe Gln

Pro Val

Leu Trp

Thr Val

615 Ala Trp 630 His Pro

Pro Gly

Cys Ser

Thr Ala

695 Ala Ala 710

Ala Leu

Glu Lys

Gln Gly

Cys Leu

775 Thr Pro 790 Pro Asp

Tyr Trp

Val Pro

475480

Ala Asp Thr Pro Ile Tyr Asn Asp Glu 490495

Ser Leu Pro Gln Pro Trp Arg Ala Asp 505510

Val Lys Val Ile Ala Gln His Gln Asn

520525

Ser Ala Phe Pro Tyr Ala Leu Leu Ser 540

Tyr His Pro His Pro Phe Ala Gln Arg 555560

Val Asn Asn Thr Arg Pro Pro His Val 570575

Leu Thr Ala Met Gly Leu Leu Ala Leu 585590

Ala Glu Val Ser Gln Ala Gly Thr Val 600605

Gly Val Leu Ala Ser Ala His Arg Pro 620

Arg Ala Ala Val Leu Ile Tyr Ala Ser 635640

Asn Arg Ser Val Ala Val Thr Leu Arg 650655

Pro Gly Leu Val Tyr Val Thr Arg Tyr 665670

Pro Asp Gly Glu Trp Arg Arg Leu Gly 680685

Glu Gln Phe Arg Arg Met Arg Ala Ala 700

Pro Arg Pro Leu Pro Ala Gly Gly Arg 715720

Arg Leu Pro Ser Leu Leu Leu Val His 730735

Pro Pro Gly Gln Val Thr Arg Leu Arg 745750

Gln Leu Val Leu Val Trp Ser Asp Glu

760765

Trp Thr Tyr Glu Ile Gln Phe Ser Gln 780

Val Ser Arg Lys Pro Ser Thr Phe Asn 795800

Thr Gly Ala Val Ser Gly Ser Tyr Arg 810815

Ala Arg Pro Gly Pro Phe Ser Asp Pro 825830

Val Pro Arg Gly Pro Pro Ser Pro Gly 840845 <210> 7 <211> 872 <212> Protein <213> Artificial sequence <220>

<223> VH-CH1 fragment of the heavy chain of an antibody to the human insulin receptor united with the sequence of β-L-iduronidase via a glycine-serine core <400> 7

Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

5 10 15

Leu Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

25 30

Asp Ile His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile saline 33 044641

3540

Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr 5055

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys 6570

Met His Leu Ser Ser Leu Thr Ser Glu Lys 8590

Ala Arg Glu Trp Ala Tyr Trp Gly Gln Gly 100105

Ala Ala Ser Thr Lys Gly Pro Ser Val Phe 115120

Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130135

Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145150

Ser Gly Val His Thr Phe Pro Ala Val Leu 165170

Ser Leu Ser Ser Val Val Thr Val Pro Ser 180185

Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195200

Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210215

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 225230

Val Gln Val Asp Ala Ala Arg Ala Leu Trp 245250

Arg Ser Thr Gly Phe Cys Pro Pro Leu Pro 260265

Tyr Val Leu Ser Trp Asp Gln Gln Leu Asn 275280

Val Pro His Arg Gly Ile Lys Gln Val Arg 290295

Leu Val Thr Thr Arg Gly Ser Thr Gly Arg 305310

Thr His Leu Asp Gly Tyr Leu Asp Leu Leu 325330

Pro Gly Phe Glu Leu Met Gly Ser Ala Ser 340345

Glu Asp Lys Gln Gln Val Phe Glu Trp Lys 355360

Ala Arg Arg Tyr Ile Gly Arg Tyr Gly Leu 370375

Asn Phe Glu Thr Trp Asn Glu Pro Asp His 385390

Ser Met Thr Met Gln Gly Phe Leu Asn Tyr 405410

Gly Leu Arg Ala Ala Ser Pro Ala Leu Arg 420425

Ser Phe His Thr Pro Pro Arg Ser Pro Leu 435440

His Cys His Asp Gly Thr Asn Phe Phe Thr 450455

Leu Asp Tyr Ile Ser Leu His Arg Lys Gly 465470

Ile Leu Glu Gln Glu Lys Val Val Ala Gln 485490

Pro Lys Phe Ala Asp Thr Pro Ile Tyr Asn 510515

Val Gly Trp Ser Leu Pro Gln Pro Trp Arg 525530

Ala Met Val Val Lys Val Ile Ala Gln His 540545

Asn Thr Thr Ser Ala Phe Pro Tyr Ala Leu 555560

Phe Leu Ser Tyr His Pro His Pro Phe Ala 575580

Arg Phe Gln Val Asn Asn Thr Arg Pro Pro 590595

Lys Pro Val Leu Thr Ala Met Gly Leu Leu

Tyr Asn Glu Lys Phe 60

Ser Ser Thr Ala Tyr 80

Ala Val Tyr Phe Cys 95

Leu Val Thr Val Ser 110

Leu Ala Pro Ser Ser 125

Cys Leu Val Lys Asp 140

Ser Gly Ala Leu Thr

160

Ser Ser Gly Leu Tyr 175

Ser Leu Gly Thr Gln 190

Asn Thr Lys Val Asp 205

His Gly Gly Gly Gly 220

Glu Ala Pro His Leu

240

Leu Arg Arg Phe Trp 255

Ser Gln Ala Asp Gln 270

Ala Tyr Val Gly Ala 285

His Trp Leu Leu Glu 300

Leu Ser Tyr Asn Phe

320

Glu Asn Gln Leu Leu 335

His Phe Thr Asp Phe 350

Leu Val Ser Ser Leu 365

His Val Ser Lys Trp 380

Asp Phe Asp Asn Val

400

Asp Ala Cys Ser Glu 415

Gly Gly Pro Gly Asp 430

Trp Gly Leu Leu Arg 445

Glu Ala Gly Val Arg 460

Arg Ser Ser Ile Ser

480

Ile Arg Gln Leu Phe 500

Glu Ala Asp Pro Leu 520

Asp Val Thr Tyr Ala 535

Asn Leu Leu Leu Ala 550

Ser Asn Asp Asn Ala

570

Arg Thr Leu Thr Ala 585

Val Gln Leu Leu Arg 600

Leu Leu Asp Glu Glu

- 34 044641

605 610615

Gln Leu Trp Ala Glu Val Ser Gln Ala Gly Thr Val Leu Asp Ser Asn 620 625630

His Thr Val Gly Val Leu Ala Ser Ala His Arg Pro Gln Gly ProAla

635 640 645650

Asp Ala Trp Arg Ala Ala Val Leu Ile Tyr Ala Ser Asp Asp ThrArg

655 660665

Ala His Pro Asn Arg Ser Val Ala Val Thr Leu Arg Leu Arg Gly Val 670 675680

Pro Pro Gly Pro Gly Leu Val Tyr Val Thr Arg Tyr Leu Asp Asn Gly 685 690695

Leu Cys Ser Pro Asp Gly Glu Trp Arg Arg Leu Gly Arg Pro Val Phe 700 705710

Pro Thr Ala Glu Gln Phe Arg Arg Met Arg Ala Ala Glu Asp ProVal

715 720 725730

Ala Ala Ala Pro Arg Pro Leu Pro Ala Gly Gly Arg Leu Thr LeuArg

735 740745

Pro Ala Leu Arg Leu Pro Ser Leu Leu Leu Val His Val Cys Ala Arg 750 755760

Pro Glu Lys Pro Pro Gly Gln Val Thr Arg Leu Arg Ala Leu Pro Leu 765 770775

Thr Gln Gly Gln Leu Val Leu Val Trp Ser Asp Glu His Val Gly Ser 780 785790

Lys Cys Leu Trp Thr Tyr Glu Ile Gln Phe Ser Gln Asp Gly LysAla

795 800 805810

Tyr Thr Pro Val Ser Arg Lys Pro Ser Thr Phe Asn Leu Phe ValPhe

815 820825

Ser Pro Asp Thr Gly Ala Val Ser Gly Ser Tyr Arg Val Arg Ala Leu 830 835840

Asp Tyr Trp Ala Arg Pro Gly Pro Phe Ser Asp Pro Val Pro Tyr Leu 845 850855

Glu Val Pro Val Pro Arg Gly Pro Pro Ser Pro Gly Asn Pro 860 865870 <210> 8 <211> 214 <212> Protein <213> Artificial sequence <220>

<223> Light chain immunoglobulin IgG to insulin receptor LFR1 <400> 8

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 1015

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Gly Gly Asn 20 2530

Leu Tyr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 4045

Tyr Ala Thr Ser Ser Leu Asp Ser Gly Val Pro Ser Arg Phe Ser Gly 50 5560

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu GlnPro

70 7580

Glu Asp Val Ala Thr Tyr Tyr Cys Leu Gln Tyr Ser Ser Ser ProTrp

9095

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn SerGln

145 150 155160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser LeuSer

165 170175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200205

Phe Asn Arg Gly Glu Cys

- 35 044641

210 < 210> 9 < 211> 214 < 212> Protein < 213> Artificial sequence <220>

<223> Light chain immunoglobulin IgG to insulin receptor LFR2 <400> 9

Glu Ile Val Leu Thr Gln Ser Pro Asp Phe Gln Ser Val Thr ProLys

5 1015

Glu Lys Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Gly GlyAsn

2530

Leu Tyr Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu LeuIle

Lys Ala Thr Ser Ser Leu Asp Ser Gly Val Pro Ser Arg Phe SerGly

5560

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu GluAla

70 7580

Glu Asp Ala Ala Ala Tyr Tyr Cys Leu Gln Tyr Ser Ser Ser ProTrp

9095

Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Arg Thr Val Ala Ala 100 105110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn SerGln

145 150 155160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser LeuSer

165 170175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200205

Phe Asn Arg Gly Glu Cys

210 <210> 10 <211> 214 <212> Protein <213> Artificial sequence <220>

<223> Light chain immunoglobulin IgG to insulin receptor LFR3 <400> 10

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Gly Gly Asn Leu Tyr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile Tyr Ala Thr Ser Ser Leu Asp Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Ser Ser Ser Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val a laAla Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val a spAsn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Leu Ser Ser Lys Ser Ala Pro Asp Val Tyr Thr Glu Lys Lys Ser His Phe Lys Asn Val Tyr Arg Gly Ala Glu Cys Cys Glu Val Thr His Gln Gly

<210> 11 <211> 214 <212> Protein <213> Artificial sequence <220>

<223> Insulin receptor IgG light chain LFR4

- 36 044641 <400> 11

Glu Ile Val Leu Leu Ser Cys Arg Gly Gln Ala Pro Arg Phe Ser Gly Glu Asp Phe Ala Gly Thr Arg Leu Ser Asp Glu Gln Pro Arg Glu Ala Glu Ser Val Thr Leu Ser Lys Ala Leu Ser Ser Pro

Thr Gln Ser Pro Ala Ser Gln Asp Arg Leu Leu Ile Ser Gly Ser Gly Val Tyr Tyr Cys Glu Ile Lys Arg Leu Lys Ser Gly Lys Val Gln Trp Glu Gln Asp Ser Asp Tyr Glu Lys Val Thr Lys Ser

Ala Thr Leu Ser Ile Gly Gly Asn Tyr Ala Thr Ser Thr Asp Phe Thr Leu Gln Tyr Ser Thr Val a laAla Thr Ala Ser Val Lys Val a spAsn Lys Asp Ser Thr His Lys Val Tyr Phe Asn Arg Gly

Leu Ser Pro Gly Leu Tyr Trp Tyr Ser Leu Asp Ser Leu Thr Ile Ser Ser Ser Pro Trp Pro Ser Val Phe Val Cys Leu Leu Ala Leu Gln Ser Tyr Ser Leu Ser Ala Cys Glu Val Glu Cys

Glu Arg Ala Thr Gln Gln Lys Pro Gly Ile Pro Ala Ser Leu Glu Pro Thr Phe Gly Gln Ile Phe Pro Pro Asn Asn Phe Tyr Gly Asn Ser Gln Ser Thr Leu Thr Thr His Gln Gly <210> 12 <211> 748 < 212 > Protein <213> Artificial sequence <220>

<223> VH-CH1 fragment of the heavy chain of an antibody to the human insulin receptor, connected to the sequence of the iduronate-2-sulfatase HFR1 <400> 12

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg Ser Leu Arg Leu

Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asn Tyr Asp Ile His Trp Val Arg Gln Ala

Pro Gly Lys Gly Leu Glu Trp Val Ser Trp Ile Tyr Pro Gly Asp Gly Ser Thr Lys Tyr

Asn Glu Lys Phe Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Glu Trp

Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu GlyCys

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala LeuThr

Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu SerSer

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val AsnHis

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys ThrHis

Leu Ser Ser Ser Glu Thr Gln Ala Asn Ser Thr Thr Asp Ala Leu Asn Val Leu LeuIle

Ile Val a spAsp Leu Arg Pro Ser Leu Gly Cys Tyr Gly Asp Lys Leu Val Arg Ser Pro

Asn Ile Asp Gln Leu Ala Ser His Ser Leu Leu Phe Gln Asn Ala Phe Ala Gln Gln Ala

Val Cys Ala Pro Ser Arg Val Ser Phe Leu Thr Gly Arg Arg Pro Asp Thr Thr Arg Leu

Tyr Asp Phe Asn Ser Tyr Trp Arg Val His Ala Gly Asn Phe Ser Thr Ile Pro Gln Tyr

Phe Lys Glu Asn Gly Tyr Val Thr Met Ser Val Gly Lys Val Phe His Pro Gly Ile Ser

Ser Asn His Thr Asp Asp Ser Pro Tyr Ser Trp Ser Phe Pro Pro Tyr His Pro Ser Ser

Glu Lys Tyr Glu Asn Thr Lys Thr Cys Arg Gly Pro Asp Gly Glu Leu His Ala Asn Leu

Leu Cys Pro Val Asp Val Leu Asp Val Pro Glu Gly Thr Leu Pro Asp Lys Gln Ser Thr

Glu Gln Ala Ile Gln Leu Leu Glu Lys Met Lys Thr Ser Ala Ser Pro Phe Phe Leu Ala

Val Gly Tyr His Lys Pro His Ile Pro Phe Arg Tyr Pro Lys Glu Phe Gln Lys Leu Tyr

Pro Leu Glu Asn Ile Thr Leu Ala Pro Asp Pro Glu Val Pro Asp Gly Leu Pro ProVal

Ala Tyr Asn Pro Trp Met Asp Ile Arg Gln Arg Glu Asp Val Gln Ala Leu Asn IleSer

Val Pro Tyr Gly Pro Ile Pro Val Asp Phe Gln Arg Lys Ile Arg Gln Ser Tyr PheAla

Ser Val Ser Tyr Leu Asp Thr Gln Val Gly Arg Leu Leu Ser Ala Leu Asp Asp LeuGln

Leu Ala Asn Ser Thr Ile Ile Ala Phe Thr Ser Asp His Gly Trp Ala Leu Gly GluHis

Gly Glu Trp Ala Lys Tyr Ser Asn Phe Asp Val Ala Thr His Val Pro Leu Ile PheTyr

Val Pro Gly Arg Thr Ala Ser Leu Pro Glu Ala Gly Glu Lys Leu Phe Pro Tyr LeuAsp

Pro Phe Asp Ser Ala Ser Gln Leu Met Glu Pro Gly Arg Gln Ser Met Asp Leu ValGlu

Leu Val Ser Leu Phe Pro Thr Leu Ala Gly Leu Ala Gly Leu Gln Val Pro Pro ArgCys

Pro Val Pro Ser Phe His Val Glu Leu Cys Arg Glu Gly Lys Asn Leu Leu Lys His Phe

Arg Phe Arg Asp Leu Glu Glu Asp Pro Tyr Leu Pro Gly Asn Pro Arg Glu Leu Ile Ala

Tyr Ser Gln Tyr Pro Arg Pro Ser Asp Ile Pro Gln Trp Asn Ser Asp Lys Pro Ser Leu

Lys Asp Ile Lys Ile Met Gly Tyr Ser Ile Arg Thr Ile Asp Tyr Arg Tyr Thr Val Trp

Val Gly Phe Asn Pro Asp Glu Phe Leu Ala Asn Phe Ser Asp Ile His Ala Gly Glu Leu

Tyr Phe Val Asp Ser Asp Pro Leu Gln Asp His Asn Met Tyr Asn Asp Ser Gln Gly Gly

Asp Leu Phe Gln Leu Leu Met Pro <210> 13 <211> 748 <212> Protein <213> Artificial sequence

- 37 044641 <220>

<223> VH-CH1 fragment of the heavy chain of an antibody to the human insulin receptor, connected to the sequence of iduronate-2-sulfatase HFR2 <400> 13

Gln Val Gln Leu Ser Cys Ala Ala Pro Gly Lys Gly Asn Glu Lys Phe Leu Gln Met Asn Ala Tyr Trp Gly Val Phe Pro Leu Leu Val Lys Asp Ser Gly Val His Val Val Thr Val Lys Pro Ser Asn Leu Ser Ser Ser Ile Val Asp Asp Asn Ile Asp Gln Val Cys Ala Pro Tyr Asp Phe Asn Phe Lys Glu Asn Ser Asn His Thr Glu Lys Tyr Glu Leu Cys Pro Val Glu Gln Ala Ile Val Gly Tyr His Pro Leu Glu Asn Ala Tyr Asn Pro Val Pro Tyr Gly Ser Val Ser Tyr Leu Ala Asn Ser Gly Glu Trp Ala Val Pro Gly Arg Pro Phe Asp Ser Leu Val Ser Leu Pro Val Pro Ser Arg Phe Arg Asp Tyr Ser Gln Tyr Lys Asp Ile Lys Val Gly Phe Asn Tyr Phe Val Asp Asp Leu Phe Gln

Val Glu Ser Gly Ser Gly Phe Thr Leu Glu Trp Val Lys Gly Arg Phe Gly Leu Arg Ala Gln Gly Thr Leu Ala Pro Ser Ser Tyr Phe Pro Glu Thr Phe Pro Ala Pro Ser Ser Ser Thr Lys Val a

Glu Thr Gln Ala Leu Arg Pro Ser Leu Ala Ser His Ser Arg Val Ser Ser Tyr Trp Arg Gly Tyr Val Thr Asp Asp Ser Pro Asn Thr Lys Thr Asp Val Leu Asp Gln Leu Leu Glu Lys Pro His Ile Ile Thr Leu Ala Trp Met Asp Ile Pro Ile Pro Val Leu Asp Thr Gln Thr Ile Ile Ala Lys Tyr Ser Asn Thr Ala Ser Leu Ala Ser Gln Leu Phe Pro Thr Leu Phe His Val Glu Leu Glu Glu Asp Pro Arg Pro Ser Ile Met Gly Tyr Pro Asp Glu Phe Ser Asp Pro Leu Leu Leu Met Pro

Gly Gly Val Val Phe Ser Asn Tyr a laTrp Ile Tyr Thr Ile Ser Arg Glu Asp Thr Ala Val Thr Val Ser Lys Ser Thr Ser Pro Val Thr Val Val Leu Gln Ser Leu Gly Thr Gln spLys Arg Val Glu Asn Ser Thr Thr Leu Gly Cys Tyr Ser Leu Leu Phe Phe Leu Thr Gly Val His Ala Gly Met Ser Val Gly Tyr Ser Trp Ser Cys Arg Gly Pro Val Pro Glu Gly Lys Met Lys Thr Pro Phe Arg Tyr Pro Asp Pro Glu Arg Gln Arg Glu a spPhe Gln Arg Val Gly Arg Leu Phe Thr Ser Asp Phe Asp Val a

Pro Glu Ala Gly Met Glu Pro Gly Ala Gly Leu Ala Leu Cys Arg Glu Pro Tyr Leu Pro Asp Ile Pro Gln Ser Ile Arg Thr Leu Ala Asn Phe Gln Asp His Asn

Gln Pro Gly Arg Asp Ile His Trp Pro Gly Asp Gly Asp Asn Ser Lys Val Tyr Tyr Cys Ser Ala Ser Thr Gly Gly Thr Ala Ser Trp Asn Ser Ser Gly Leu Tyr Thr Tyr Ile Cys Pro Lys Ser Cys Asp Ala Leu Asn Gly Asp Lys Leu Gln Asn Ala Phe Arg Arg Pro Asp Asn Phe Ser Thr Lys Val Phe His Phe Pro Pro Tyr Asp Gly Glu Leu Thr Leu Pro Asp Ser Ala Ser Pro Pro Lys Glu Phe Val Pro Asp Gly Asp Val Gln Ala Lys Ile Arg Gln Leu Ser Ala Leu His Gly Trp Ala laThr His Val Pro

Glu Lys Leu Phe Arg Gln Ser Met Gly Leu Gln Val Gly Lys Asn Leu Gly Asn Pro Arg Trp Asn Ser Asp Ile Asp Tyr Arg Ser Asp Ile His Met Tyr Asn Asp

Ser Leu Arg Leu Val Arg Gln Ala Ser Thr Lys Tyr Asn Thr Leu Tyr Ala Arg Glu Trp Lys Gly Pro Ser Ala Leu Gly Cys Gly Ala Leu Thr Ser Leu Ser Ser Asn Val Asn His Asp Lys Thr His Val Leu Leu Ile Val a rgSer Pro Ala Gln Gln Ala Thr Thr Arg Leu Ile Pro Gln Tyr Pro Gly Ile Ser His Pro Ser Ser His Ala Asn Leu Lys Gln Ser Thr Phe Phe Leu Ala Gln Lys Leu Tyr Leu Pro Pro Pro Val Leu Asn Ile Ser Ser Tyr Phe Ala Asp Asp Leu Gln Leu Gly Glu His Leu Ile Phe Tyr Pro Tyr Leu Asp Asp Leu Val Glu Pro Pro Arg Cys Leu Lys His Phe Glu Leu Ile Ala Lys Pro Ser Leu Tyr Thr Val Trp Ala Gly Glu Leu Ser Gln Gly Gly <210 > 14 <211> 748 <212> Protein <213> Artificial sequence <220>

<223> VH-CH1 fragment of the heavy chain of an antibody to the human insulin receptor, connected to the sequence of iduronate-2-sulfatase HFR3 <400> 14

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Asp Phe Thr Asn Tyr Asp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Gly Trp Ile Tyr Pro Gly Asp Gly Ser Thr Lys Tyr Asn Glu Lys Phe Lys Gly Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Glu Trp Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Leu Ser Ser Ser Glu Thr Gln Ala Asn Ser Thr Thr Asp Ala Leu Asn Val Leu Leu Ile Ile Val Asp Asp Leu Arg Pro Ser Leu Gly Cys Tyr Gly Asp Lys Leu Val Arg Ser Pro

- 38 044641

Asn Ile Asp Gln Val Cys Ala Pro Tyr Asp Phe Asn Phe Lys Glu Asn Ser Asn His Thr Glu Lys Tyr Glu Leu Cys Pro Val Glu Gln Ala Ile Val Gly Tyr His Pro Leu Glu Asn Ala Tyr Asn Pro Val Pro Tyr Gly Ser Val Ser Tyr Leu Ala Asn Ser Gly Glu Trp Ala Val Pro Gly Arg Pro Phe Asp Ser Leu Val Ser Leu Pro Val Pro Ser Arg Phe Arg Asp Tyr Ser Gln Tyr Lys Asp Ile Lys Val Gly Phe Asn Tyr Phe Val Asp Asp Leu Phe Gln

Leu Ala Ser His Ser Arg Val Ser Ser Tyr Trp Arg Gly Tyr Val Thr Asp Asp Ser Pro Asn Thr Lys Thr Asp Val Leu Asp Gln Leu Leu Glu Lys Pro His Ile Ile Thr Leu Ala Trp Met Asp Ile Pro Ile Pro Val Leu Asp Thr Gln Thr Ile Ile Ala Lys Tyr Ser Asn Thr Ala Ser Leu Ala Ser Gln Leu Phe Pro Thr Leu Phe His Val Glu Leu Glu Glu Asp Pro Arg Pro Ser Ile Met Gly Tyr Pro Asp Glu Phe Ser Asp Pro Leu Leu Leu Met Pro

Ser Leu Leu Phe Phe Leu Thr Gly Val His Ala Gly Met Ser Val Gly Tyr Ser Trp Ser Cys Arg Gly Pro Val Pro Glu Gly Lys Met Lys Thr Pro Phe Arg Tyr Pro Asp Pro Glu Arg Gln Arg Glu a spPhe Gln Arg Val Gly Arg Leu Phe Thr Ser Asp Phe Asp Val a

Pro Glu Ala Gly Met Glu Pro Gly Ala Gly Leu Ala Leu Cys Arg Glu Pro Tyr Leu Pro Asp Ile Pro Gln Ser Ile Arg Thr Leu Ala Asn Phe Gln Asp His Asn

Gln Asn Ala Phe Arg Arg Pro Asp Asn Phe Ser Thr Lys Val Phe His Phe Pro Pro Tyr Asp Gly Glu Leu Thr Leu Pro Asp Ser Ala Ser Pro Pro Lys Glu Phe Val Pro Asp Gly Asp Val Gln Ala Lys Ile Arg Gln Leu Ser Ala Leu His Gly Trp Ala laThr His Val Pro

Glu Lys Leu Phe Arg Gln Ser Met Gly Leu Gln Val Gly Lys Asn Leu Gly Asn Pro Arg Trp Asn Ser Asp Ile Asp Tyr Arg Ser Asp Ile His Met Tyr Asn Asp

Ala Gln Gln Ala Thr Thr Arg Leu Ile Pro Gln Tyr Pro Gly Ile Ser His Pro Ser Ser His Ala Asn Leu Lys Gln Ser Thr Phe Phe Leu Ala Gln Lys Leu Tyr Leu Pro Pro Val Leu Asn Ile Ser Ser Tyr Phe Ala Asp Asp Leu Gln Leu Gly Glu His Leu Ile Phe Tyr Pro Tyr Leu Asp Asp Leu Val Glu Pro Pro Arg Cys Leu Lys His Phe Glu Leu Ile Ala Lys Pro Ser Leu Tyr Thr Val Trp Ala Gly Glu Leu Ser Gln Gly Gly <210> 15 <211> 748 <212> Protein <213> Artificial sequence <220>

<223> VH-CH1 fragment of the heavy chain of an antibody to the human insulin receptor, connected to the sequence of iduronate-2-sulfatase HFR4 <400> 15

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg Ser Leu Arg Leu

Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asn Tyr Asp Ile His Trp Val Arg Gln Ala

Pro Gly Lys Gly Leu Glu Trp Val Ser Trp Ile Tyr Pro Gly Asp Gly Ser Thr Lys Tyr

Asn Glu Lys Phe Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Ser Leu Tyr

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys Ala Lys Glu Trp

Ala Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

Val Phe Pro Leu Ala Pro Gly Ser Ser Lys Ser Thr Ser Gly Thr Ala Ala Leu GlyCys

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala LeuThr

Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu SerSer

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val AsnHis

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys ThrHis

Leu Ser Ser Ser Glu Thr Gln Ala Asn Ser Thr Thr Asp Ala Leu Asn Val Leu LeuIle

Ile Val a spAsp Leu Arg Pro Ser Leu Gly Cys Tyr Gly Asp Lys Leu Val Arg Ser Pro

Asn Ile Asp Gln Leu Ala Ser His Ser Leu Leu Phe Gln Asn Ala Phe Ala Gln Gln Ala

Val Cys Ala Pro Ser Arg Val Ser Phe Leu Thr Gly Arg Arg Pro Asp Thr Thr Arg Leu

Tyr Asp Phe Asn Ser Tyr Trp Arg Val His Ala Gly Asn Phe Ser Thr Ile Pro Gln Tyr

Phe Lys Glu Asn Gly Tyr Val Thr Met Ser Val Gly Lys Val Phe His Pro Gly Ile Ser

Ser Asn His Thr Asp Asp Ser Pro Tyr Ser Trp Ser Phe Pro Pro Tyr His Pro Ser Ser

Glu Lys Tyr Glu Asn Thr Lys Thr Cys Arg Gly Pro Asp Gly Glu Leu His Ala Asn Leu

Leu Cys Pro Val Asp Val Leu Asp Val Pro Glu Gly Thr Leu Pro Asp Lys Gln Ser Thr

Glu Gln Ala Ile Gln Leu Leu Glu Lys Met Lys Thr Ser Ala Ser Pro Phe Phe Leu Ala

Val Gly Tyr His Lys Pro His Ile Pro Phe Arg Tyr Pro Lys Glu Phe Gln Lys Leu Tyr

Pro Leu Glu Asn Ile Thr Leu Ala Pro Asp Pro Glu Val Pro Asp Gly Leu Pro ProVal

Ala Tyr Asn Pro Trp Met Asp Ile Arg Gln Arg Glu Asp Val Gln Ala Leu Asn IleSer

Val Pro Tyr Gly Pro Ile Pro Val Asp Phe Gln Arg Lys Ile Arg Gln Ser Tyr PheAla

Ser Val Ser Tyr Leu Asp Thr Gln Val Gly Arg Leu Leu Ser Ala Leu Asp Asp LeuGln

Leu Ala Asn Ser Thr Ile Ile Ala Phe Thr Ser Asp His Gly Trp Ala Leu Gly GluHis

Gly Glu Trp Ala Lys Tyr Ser Asn Phe Asp Val Ala Thr His Val Pro Leu Ile PheTyr

Val Pro Gly Arg Thr Ala Ser Leu Pro Glu Ala Gly Glu Lys Leu Phe Pro Tyr LeuAsp

Pro Phe Asp Ser Ala Ser Gln Leu Met Glu Pro Gly Arg Gln Ser Met Asp Leu ValGlu

Leu Val Ser Leu Phe Pro Thr Leu Ala Gly Leu Ala Gly Leu Gln Val Pro Pro ArgCys

Pro Val Pro Ser Phe His Val Glu Leu Cys Arg Glu Gly Lys Asn Leu Leu Lys His Phe

Claims (7)

Arg Phe Arg Asp Leu Glu Glu Asp Pro Tyr Leu Pro Gly Asn Pro Arg Glu Leu Ile Ala Tyr Ser Gln Tyr Pro Arg Pro Ser Asp Ile Pro Gln Trp Asn Ser Asp Lys Pro Ser Leu Lys Asp Ile Lys Ile Met Gly Tyr Ser Ile Arg Thr Ile Asp Tyr Arg Tyr Thr Val Trp Val Gly Phe Asn Pro Asp Glu Phe Leu Ala Asn Phe Ser Asp Ile His Ala Gly Glu Leu Tyr Phe Val Asp Ser Asp Pro Leu Gln Asp His Asn Met Tyr Asn Asp Ser Gln Gly Gly Asp Leu Phe Gln Leu Leu Met Pro CLAIM 1. A HIR-Fab-IDS compound formed by a first amino acid sequence LC-HIRFab-IDS selected from SEQ ID NO: 2, 8, 9, 10 or 11 and a second amino acid sequence HC-HIR-Fab-IDS selected from SEQ ID NO: 4, 5, 12, 13, 14 or 15. 2. The compound according to claim 1, formed by the first amino acid sequence LC-HIR-Fab-IDS presented in SEQ ID NO: 2, and the second amino acid sequence HC-HIR-Fab-IDS presented in SEQ ID NO: 4. 3. The compound according to claim 1, formed by the first amino acid sequence LC-HIR-Fab-IDS presented in SEQ ID NO: 2, and the second amino acid sequence HC-HIR-Fab-IDS presented in SEQ ID NO: 5. 4. Use of a compound according to claims 1 to 3 for the preparation of a pharmaceutical composition containing said compound in an effective amount and a pharmaceutically acceptable carrier. 5. Use of a compound according to claims 1 to 3 for the treatment or prevention of lysosomal enzyme deficiency in a subject having a lysosomal storage disease, wherein said use comprises administering to said subject said compound in an effective amount. 6. Use according to claim 5, wherein the lysosomal storage disease is mucopolysaccharidosis. 7. Use according to claim 5, wherein the lysosomal storage disease is mucopolysaccharidosis type II with a neurological component. -