RU2814280C1 - Pharmaceutical composition and method of prevention and treatment of lysosomal enzyme deficiency in subject with type ii mucopolysaccharidosis - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the peptide, the HIR-Fab-IDS compound, as well as to a composition containing it.

EFFECT: invention is effective for the prevention or treatment of lysosomal enzyme deficiency in a subject having a lysosomal storage disease.

21 cl, 8 dwg, 7 tbl, 7 ex

Description

Field of technology

The invention relates to the field of biotechnology, specifically to a pharmaceutical composition and method for the prevention and treatment of lysosomal enzyme deficiency in a subject, which can be used in medicine. The present invention relates to a HIR-Fab-IDS compound used for the prevention or treatment of lysosomal enzyme deficiency in a subject having the lysosomal storage disease mucopolysaccharidosis type II, wherein the subject is administered at least one dose of the HIR-Fab-IDS compound from 1 to 12 mg/kg, to a pharmaceutical composition for use in the prevention or treatment of lysosomal enzyme deficiency in a subject having a lysosomal storage disease, which is mucopolysaccharidosis type II, where the composition contains the compound HIR-Fab-IDS, as well as to a method for preventing or treating lysosomal enzyme deficiency enzyme in a subject having lysosomal storage disease mucopolysaccharidosis type II, comprising administering to the patient at least one dose of a HIR-Fab-IDS compound.

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 intermediate forms - 14) 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 hypertensive-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).

Mucopolysaccharidosis type I is caused by a deficiency of the lysosomal enzyme, α-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 α-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.

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 hepa ran 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 II 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 for administering Elaprase® 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 MbP 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 with Elaprase®, patients die at the age of 20 years from neurodegenerative consequences, while in the second decade of life, patients receiving Elaprase® as a rule, they experience great difficulties in learning and need helpers 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 S. et al., 2004) .

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

Advantages of the invention

The above objectives are successfully achieved in the present invention, which relates to a compound, a pharmaceutical composition 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 said transport element is a Fab fragment IgG immunoglobulin specific for 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 type II, and increasing the duration and improving the quality of life of subjects suffering from lysosomal storage disease, mucopolysaccharidosis type II, and in need of therapy.

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 (human insulin receptor, or HIR), 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 introduction 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 a Mab fragment.

It was found that the HIR-Fab-IDS compound penetrates the patient's brain tissue 2 times better than the HIR-Mab-IDS compound.

The HIR-Fab-IDS compound was found to restore IDS enzymatic function in patient brain tissue by 98%.

The dose at which the compound is safe and effective for subjects suffering from lysosomal storage disease, mucopolysaccharidosis type II, and in need of therapy is indicated.

A more effective pharmaceutical composition and/or regimen has been developed for the prevention or treatment of lysosomal enzyme deficiency in a subject having mucopolysaccharidosis type II.

A more effective dosage regimen has been developed for the prevention or treatment of lysosomal enzyme deficiency in a subject with mucopolysaccharidosis type II with a neurological component in humans.

A more effective administration regimen and/or pharmaceutical composition, taking into account increased doses, has been developed for the prevention or treatment of lysosomal enzyme deficiency in a subject having mucopolysaccharidosis type II with a neurological component related to the neuropathic form in humans.

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.

A first object of the present invention is a HIR-Fab-IDS compound represented by the first amino acid sequence of SEQ ID NO: 2 and the second amino acid sequence of SEQ ID NO: 4, for the prevention or treatment of lysosomal enzyme deficiency in a subject having the lysosomal storage disease of mucopolysaccharidosis II type.

Also subject to the present invention is a HIR-Fab-IDS compound represented by the first amino acid sequence of SEQ ID NO: 2 and the second amino acid sequence of SEQ ID NO: 4, used for the prevention or treatment of lysosomal enzyme deficiency in a subject having a lysosomal storage disease of mucopolysaccharidosis Type II, wherein the subject is administered at least one dose of the HIR-Fab-IDS compound from 1 to 12 mg/kg.

Another aspect of the invention is the HIR-Fab-IDS compound represented by the first amino acid sequence of SEQ ID NO: 2 and the second amino acid sequence of SEQ ID NO: 4, used for the prevention or treatment of lysosomal enzyme deficiency in a subject having a lysosomal storage disease, wherein the subject is administered at least one dose of the HIR-Fab-IDS compound, wherein the dose is selected from the group consisting of 3 mg/kg, 6 mg/kg, 12 mg/kg of the HIR-Fab-IDS compound.

In a particular embodiment, the present invention is a HIR-Fab-IDS compound used for the prevention or treatment of lysosomal enzyme deficiency in a subject having a lysosomal storage disease, wherein the lysosomal storage disease is mucopolysaccharidosis type II or mucopolysaccharidosis type II with a neurological component related to neuropathic form.

In a particular embodiment, the present invention is a HIR-Fab-IDS compound used for the prevention or treatment of lysosomal enzyme deficiency in a subject having a lysosomal storage disease of mucopolysaccharidosis type II, where the subject is a human.

Another aspect of the invention is a pharmaceutical composition for use in the prevention or treatment of lysosomal enzyme deficiency in a subject having lysosomal storage disease mucopolysaccharidosis type II, wherein the composition contains the compound HIR-Fab-IDS represented by the first amino acid sequence of SEQ ID NO: 2 and the second amino acid sequence SEQ ID NO: 4, wherein the HIR-Fab-IDS compound is administered to the subject at least one dose selected from the group consisting of 3 mg/kg, 6 mg/kg, 12 mg/kg of the HIR-Fab- IDS.

In a specific embodiment, the invention relates to a pharmaceutical composition for use in the prevention or treatment of lysosomal enzyme deficiency in a subject having lysosomal storage disease mucopolysaccharidosis type II, wherein the composition comprises a HIR-Fab-IDS compound, wherein the pharmaceutical composition further comprises sodium chloride, sodium dihydrogen phosphate dihydrate, sodium hydroxide, polysorbate.

In a specific embodiment, the invention relates to a pharmaceutical composition for use in the prevention or treatment of lysosomal enzyme deficiency in a subject having the lysosomal storage disease mucopolysaccharidosis type II, wherein the composition comprises a HIR-Fab-IDS compound, wherein the pharmaceutical composition further comprises a HIR-Fab compound -IDS 5.3 mg, sodium chloride in the amount of 8.0 mg, sodium dihydrogen phosphate dihydrate in the amount of 3.12 mg, sodium hydroxide for pH correction to pH 6.0, polysorbate 20 in the amount of 0.2 mg, water for injection up to 1, 0 ml.

In a specific embodiment, the invention relates to a pharmaceutical composition for use in the prevention or treatment of lysosomal enzyme deficiency in a subject having a lysosomal storage disease of mucopolysaccharidosis type II, wherein the composition contains a HIR-Fab-IDS compound, wherein the lysosomal storage disease is mucopolysaccharidosis type II with a neurological component related to the neuropathic form.

In a specific embodiment, the invention relates to a pharmaceutical composition for use in the prevention or treatment of lysosomal enzyme deficiency in a subject having lysosomal storage disease mucopolysaccharidosis type II, wherein the composition contains a HIR-Fab-IDS compound, wherein the subject is a human.

A further aspect of the invention is a method for preventing or treating lysosomal enzyme deficiency in a subject having a lysosomal storage disease mucopolysaccharidosis type II, comprising administering to the patient at least one dose of a HIR-Fab-IDS compound represented by the first amino acid sequence of SEQ ID NO: 2 and the second amino acid sequence is SEQ ID NO: 4, wherein the dose is selected from the group consisting of 3 mg/kg, 6 mg/kg, 12 mg/kg of the HIR-Fab-IDS compound.

In a specific embodiment, the invention relates to a method for preventing or treating lysosomal enzyme deficiency in a subject having lysosomal storage disease mucopolysaccharidosis type II, comprising administering to the patient at least one dose of a HIR-Fab-IDS compound represented by the first amino acid sequence of SEQ ID NO: 2 and the second amino acid sequence SEQ ID NO: 4, where the HIR-Fab-IDS compound as part of a pharmaceutical composition is administered intravenously for 3 hours once a week.

In a specific embodiment, the invention relates to a method for preventing or treating lysosomal enzyme deficiency in a subject having lysosomal storage disease mucopolysaccharidosis type II, comprising administering to the patient at least one dose of a HIR-Fab-IDS compound represented by the first amino acid sequence of SEQ ID NO: 2 and the second amino acid sequence SEQ ID NO: 4, where lysosomal storage disease is mucopolysaccharidosis type II with a neurological component related to the neuropathic form.

In a specific embodiment, the invention relates to a method for preventing or treating lysosomal enzyme deficiency in a subject having lysosomal storage disease mucopolysaccharidosis type II, comprising administering to the patient at least one dose of a HIR-Fab-IDS compound represented by the first amino acid sequence of SEQ ID NO: 2 and the second amino acid sequence of SEQ ID NO: 4, where the subject is human.

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 α-L-iduronidase or iduronate-2-sulfatase, another therapeutic enzyme is used as the appropriate 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-α-glucosaminide-N-acetyltransferase - for mucopolysaccharidosis type IIIC, N-acetylglucosamine-6-sulfatase - for mucopolysaccharidosis type IIID, galactose-6-sulfate sulfatase - for mucopolysaccharidosis type IVA, (3-galactosidase - for mucopolysaccharidosis type IVB, N -acetylgalactosamine-4-sulfatase - for mucopolysaccharidosis type VI, (3-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 have offline and online search capabilities (see Ausubel et al., 1999 ibid, pages 7-58-7-60, National Center for Biotechnology Information website on 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 α-L-iduronidase or iduronate-2-sulfatase, another lysosomal enzyme, which is deficient in patients 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 GM ₁ 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, Bielschowsky-Janeki, 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 (l-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 iduronate-2-sulfatase, the amino acid sequence of the corresponding enzyme, which is deficient in patients with lysosomal storage disease, is attached directly or via a linker to the transport element.

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. Figure 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, with numbers indicating:

1. Cerebral cortex - cerebral cortex

2. Cerebral medulla - brain substance

3. Eye - eye

4. Hypothalamus - hypothalamus

5. Pons - bridge

6. Cerebellum - cerebellum

7. Medulla oblongata - medulla oblongata

8. Spinal cord - spinal cord

9. Myocardium - myocardium

10. Blood - blood

11. Lung - light

12. Liver - liver

13. Stomach - stomach

14. Kidney - kidney

15. Small intestine - small intestine

16. Large intestine - large intestine

17. Muscle - muscles

18. Testis - testicles

19. Bone marrow - bone marrow.

In fig. Figure 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, with numbers indicating:

1. Cerebral cortex - cerebral cortex

2. Cerebral medulla - brain substance

3. Eye - eye

4. Hypothalamus - hypothalamus

5. Pons - bridge

6. Cerebellum - cerebellum

7. Medulla oblongata - medulla oblongata

8. Spinal cord - spinal cord

9. Myocardium - myocardium

10. Blood - blood

11. Lung - light

12. Liver - liver

13. Stomach - stomach

20. Adrenal - adrenal gland

14. Kidney - kidney

15. Small intestine - small intestine

19. Bone marrow - bone marrow

21. Bladder - gallbladder.

In fig. Figure 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, with numbers indicating:

1. Cerebral cortex - cerebral cortex

2. Cerebral medulla - brain substance

3. Eye - eye

4. Hypothalamus - hypothalamus

5. Pons - bridge

6. Cerebellum - cerebellum

7. Medulla oblongata - medulla oblongata

8. Spinal cord - spinal cord

9. Myocardium - myocardium

10. Blood - blood

11. Lung - light

12. Liver - liver

13. Stomach - stomach

14. Kidney - kidney

15. Small intestine - small intestine

16. Large intestine - large intestine

17. Muscle - muscles

18. Testis - testicles

19. Bone marrow - bone marrow.

In fig. Figure 4 shows the level of GAGs in the urine of animals during the study. Individual values. In Latin letters in Fig. 4 are marked:

A. Wild phenotype mice

B. Knockout mice, physical. solution

C. Knockout mice, 0.3 mg/kg

D. Knockout mice, 1.0 mg/kg

E. Knockout mice, 3.0 mg/kg.

In fig. Figure 5 shows the liver weight in the animal groups of the study. I - wild type group; II - group - knockout animals of the control group; III - knockout animals, 0.3 mg/kg; IV - knockout animals, 1.0 mg/kg; V - knockout animals, 3.0 mg/kg. Individual values.

In fig. Figure 6 shows the level of iduronate-2-sulfatase activity in the plasma of experimental animals. I - wild type group; II - group - knockout animals of the control group; III - knockout animals, 0.3 mg/kg; IV - knockout animals, 1.0 mg/kg, V - knockout animals, 3.0 mg/kg. Individual values.

In fig. 7 shows drug concentrations of HIR-FAB-IDS compound in the serum of patient 1001 determined during the dose escalation phase. The LLOQ of the method is 50 ng/ml.

In fig. Figure 8 shows the dynamics of heparan sulfate concentration in the CSF when using different doses of the drug.

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, fusion antibody, fusion protein, fusion protein, protein construct, protein complex, etc.

A compound intended for the treatment of lysosomal storage disease, containing a therapeutic enzyme and a transport element connected to each other directly or via a linker, where the transport element is a Fab fragment of an IgG1 immunoglobulin consisting of light chains of an IgG1 immunoglobulin to the insulin receptor (SEQ ID NO : 2) and heavy chain with idursulfase (SEQ ID NO: 4).

As used herein, the term “comprising” refers to a compound comprising the listed elements without excluding others.

In the present specification, the terms "therapeutic enzyme" and "enzyme" are synonymous and refer to an enzyme for treating diseases resulting from the absence, deficiency, dysfunction of the enzyme and so on, wherein the subject suffering from the diseases can be treated by ERT, administration of the enzyme and etc. 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-acetyl- D-glucosaminidase (NAGLU), glucocerebrosidase, butyrylcholinesterase, chitinase, glutamate decarboxylase, lipase, uricase, platelet activating factor acetylhydrolase, neutral endopeptidase, myeloperoxidase, acetyl-CoA-glucosaminide-N-acetyltransferase, N-acetylglucosamine-6-sulfatase, galactosamine-6 -sulfatase (GALN), hyaluronidase, α-fucosidase, β-mannosidase, α-neuraminidase (sialidase), N-acetyl-glucosamine-1-phosphotransferase, mucolipin-1, α-N-acetyl-galactosaminidase, N-aspartyl-β -glucosaminidase, 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 acid lipase (LIPA) and tripeptidyl peptidase 1.

In particular, the therapeutic enzyme includes the enzymes agalsidase, imiglucerase, galsulfase, iduronate-2-sulfatase and α-L-iduronidase. Most preferably, the therapeutic enzyme is iduronate-2-sulfatase or α-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 "rlDS-FAB-HI", "rHI-FAB-IDS", "rlDS-FAB-HIR" and "rHIR-FAB-IDS" are synonymous and refer to a hybrid recombinant protein of iduronate-2-sulfatase, covalently linked to the Fab fragment of a monoclonal antibody to the human insulin receptor. As used herein, "HIR-Fab-IDS" means, 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-2-sulfatase 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 HIR-Mab-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 "α-L-iduronidase" as well as "iduronidase", "laronidase", "IDUA" refers to an enzyme involved in the hydrolysis of glycosaminoglycans such as dermatan sulfate and heparan sulfate. As used herein, the term "α-L -iduronidase" can 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 α-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 α-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 α-L-iduronidase, known from patents US 5932211 A, 08/03/1999, US 6153188 A, 11/28/2006 and US 6541254 B1, 04/01/2003, as well as analogs of iduronate- 2-sulfatases. One skilled in the art will understand that other fragments and analogues of the enzymes α-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 later become known.

Enzymes can be produced by methods conventional in the art, for example, through genetic recombination in cells of animals, 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, the enzymes are produced in mammalian cells.In most preferred embodiments, 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% homology or 99% or higher 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 programs FASTP, BLAST, BLAST2, PSIBLAST and CLUSTAL W. Amino acid sequences and nucleotide sequences encoding enzymes and their analogs can be obtained from a well-known database such as GenBank NCBI, not limited to it.

In some embodiments, the compound transport element comprises an IgG1 immunoglobulin Fab fragment 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, the compound transport element comprises an IgG1 immunoglobulin Fab fragment 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 - so-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 (Gin, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H) , isoleucine (lie, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), series (Ser, S), threonine (Thr, T), tryptophan (Trp, 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), Acrs ( 1-aminocyclopropane-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 (allyl Cly), alloisoleucine (allo-lle), Asu (alpha-aminosuberic acid, 2-aminooctanedioic acid), Bip (4-phenyl-phenylalanine-carboxylic acid acid), BnHP ((2S,4R)-4-hydroxyproline), Cha (beta-cyclohexylalanine), Cit (citrulline), cyclohexylglycine (Chg), cyclopentylalanine, beta-cyclopropylalanine, Dab (1,4-diaminobutyric acid), Dap (1,3-diaminopropionic acid), p-(3,3-diphenylalanine-carboxylic acid), 3,3-diphenylalanine, di-n-propylglycine (Dpg), 2-furylalanine, homocyclopohexylalanine (HoCha), homocitrulline (HoCit) , homocycloleucine, homoleucine (HoLeu), homoarginine (HoArg), homoserine (HoSer), hydroxyproline, Lys(Ac), (1) Nal (1-naphthylalanine), (2) Nal (2-naphthylalanine), 4-MeO-Ars (1-amino-4-(4-methoxyphenyl)-cyclohexane-1-carboxylic acid), 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 (4-oxo-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-tetrahydroisoquinoline-3-carboxylic acid), Tic(OH), TIe (tert-butylglycine) and Tyr(Me ), but are not limited to them.

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 approximately 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, the variant has approximately 98% or more sequence identity to a polypeptide with the native sequence of the corresponding therapeutic enzyme. Amino acid sequence variants have substitutions, deletions and/or insertions at certain 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 light chain IgG-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 part thereof, through which the delivery of a drug, peptide or protein is carried out, including through the BBB.

"Immunoglobulin" is a tetrameric molecule, as used herein the term "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” contain 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. The cleavage of antibodies by papain produces two identical antigen-binding fragments, called "Fab fragments", each of which has one antigen-binding site, and a residual "Fc fragment", the name of which reflects its ability to readily 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-chain 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 linking element contains the CH2-CH3 domain of lg and sFab, the target of which is the insulin receptor, i.e. a linker connects sFab to the C-terminus of the CH3-CH2 domain of lg.

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-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-( N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional imidoesters (such as dimethyladipimidate-HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bisazido compounds (such 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 understand that other cleavable linkers known in the art to facilitate the release of the therapeutic enzyme after 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) that consists of two α subunits and two β subunits linked by disulfide bridges (the α subunits are located extracellularly and contain an insulin-binding domain) and is involved in the regulation absorption and distribution of glucose, 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 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 tissues and brain tissues (Hawkes S. 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 M6R receptors. Internalization via HIR (human insulin receptor) results in entry into early order endosomes and subsequent transcytosis. In addition to delivering functional enzyme to the CNS, many lysosomal storage diseases require improved internalization by certain peripheral tissues (diaphragm muscle in Pompe disease, liver and spleen in Hunter disease, kidney 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 (the affinity of complex formation) is determined by the indicators k _a (rate constant for the association of compounds to form a complex), k (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 M to about 10 ^-13 M, in one embodiment from about 10 ^-9 M to about 10 ^{- 13} M.

The term “lysosome” refers to 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 in 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” means “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, which 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.

The compounds of the present invention may be formulated into a composition, for example, a pharmaceutical composition containing one compound or combination of compounds or portion(s) thereof and a pharmaceutically acceptable carrier.

"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 pharmaceutical composition of the present invention may include one or more pharmaceutically acceptable salts, an antioxidant, aqueous and non-aqueous carriers and/or adjuvants such as preservatives, wetting agents, emulsifiers and dispersing agents.

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 being treated, while the benefit is reasonable. risk.

The term "pharmaceutically acceptable carrier" refers to an ingredient of a pharmaceutical composition, other than the active substance (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 mixing with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences, 16th ed., ed. Osol A., 1980), preferably in the form of 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 meta-cresol); 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).

Preferably, the pharmaceutical composition further contains sodium chloride, sodium dihydrogen phosphate dihydrate, sodium hydroxide, polysorbate.

In some embodiments of the invention, the pharmaceutical composition additionally contains the compound HIR-Fab-IDS 5.3 mg, sodium chloride in an amount of 8.0 mg, sodium dihydrogen phosphate dihydrate in an amount of 3.12 mg, sodium hydroxide for pH correction to pH6.0, polysorbate 20 in quantity 0.2 mg, water for injection up to 1.0 ml.

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.

The pharmaceutical compositions provided herein can be administered to a patient by any suitable route, for example, intravenously by bolus injection or continuous infusion over a period of time, intramuscularly, intraperitoneally, intracerebrospinal, transdermal, subcutaneously, intra-articularly, sublingually, intrasynovially, orally, by inhalation , locally or externally. Intravenous administration is preferred.

As used herein, the term “dose” refers, for example, to the dose applied when the pharmaceutical composition provided herein is administered to a patient for the first time as an “initial dose”, or when the pharmaceutical composition provided herein is administered as a dose for continuous use “therapeutic dose”.

In general, single doses ranging from approximately 0.3 to 30 mg/kg of patient body weight are contemplated, more commonly ranging from 1 to 12 mg/kg.

Typically, treatment is started with low doses of the HIR-FAB-IDS compound. For example, an initial dose of antibodies is administered to a patient, for example, by injection or infusion. The initial dose should contain approximately 3 to 12 mg/kg.

The starting dose ranges from 3 to 12 mg/kg and is, for example, about 3 mg/kg, about 3.5 mg/kg, about 4 mg/kg, about 4.5 mg/kg, about 5 mg/kg , approximately 5.5 mg/kg, approximately 6 mg/kg, approximately 6.5 mg/kg, approximately 7 mg/kg, approximately 7.5 mg/kg, approximately 8 mg/kg, approximately 8.5 mg/kg , approximately 9 mg/kg, approximately 9.5 mg/kg, approximately 10 mg/kg, approximately 10.5 mg/kg, approximately 11 mg/kg, approximately 11.5 mg/kg, approximately 12 mg/kg.

Since the chronic dose (therapeutic dose) is approximately equal to or less than or greater than the initial dose, the dose can be varied within these limits and several doses during a period of continuous use do not necessarily represent the same dose. For example, the dose can be gradually reduced or different doses can be randomly reintroduced.

The therapeutic dose ranges from 1 to 12 mg/kg and is, for example, about 1 mg/kg, about 1.5 mg/kg, about 2 mg/kg, about 2.5 mg/kg, about 3 mg/kg , approximately 3.5 mg/kg, approximately 4 mg/kg, approximately 4.5 mg/kg, approximately 5 mg/kg, approximately 5.5 mg/kg, approximately 6 mg/kg, approximately 6.5 mg/kg , approximately 7 mg/kg, approximately 7.5 mg/kg, approximately 8 mg/kg, approximately 8.5 mg/kg, approximately 9 mg/kg, approximately 9.5 mg/kg, approximately 10 mg/kg, approximately 10.5 mg/kg, approximately 11 mg/kg, approximately 11.5 mg/kg, approximately 12 mg/kg.

In various embodiments, other treatment regimens may be required, for example, a therapeutic dose may be 3 mg/kg, a therapeutic dose may be 6 mg/kg, a therapeutic dose may be 12 mg/kg, etc.

More preferably, the therapeutic dose is selected from the group consisting of 3 mg/kg, 6 mg/kg, 12 mg/kg of the HIR-Fab-IDS compound.

In addition to specifying the dose value in mg/kg, the dose value can be expressed as a fixed dose (mg/body) and/or as a dose per unit body surface area (mg/m2), corresponding to the above dose per unit body weight.

There is no specific limitation on the number of administrations of a chronic dosage, the number being, for example, one time, two times, three times, four times, five times, six times, seven times, eight times, nine times, ten times, 15 times, 20 times, 25 times, 35 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 500 times, 1000 times and 10000 times.

The “dosing interval” (interval between individual doses) is the interval between the initial dose and the first chronic dose and the interval between the nth chronic dose (n is an integer of 1 or greater) and the administration of (n +1) dose for continuous use. The interval between administrations may be one or more days, for example, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks , 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months or 1 year. The interval between administrations can be expressed in other terms, such as once a day, once every 2 days, once every 3 days, once every 4 days, once every 5 days, once every 6 days, once every a week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, once every 8 weeks, once every 9 weeks, once every 10 weeks, once every 11 weeks, once every 12 weeks, once every 13 weeks, once every 14 weeks, once every 15 weeks, once every 16 weeks, once every 17 weeks, once every 18 weeks, once every 19 weeks, once every 20 weeks, once every 21 weeks, once every 22 weeks, once every 23 weeks, once every 24 weeks, once every 25 weeks, once a month , once every 2 months, once every 3 months, once every 4 months, once every 5 months, once every 6 months, once every 7 months, once every 8 months, once every 9 months, one once every 10 months, once every 11 months or once a year. In addition, the interval between administrations can be expressed in other terms, such as every day, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, every week, every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, every 10 weeks, every 11 weeks, every 12 weeks, every 13 weeks, every 14 weeks, every 15 weeks, every 16 weeks , every 17 weeks, every 18 weeks, every 19 weeks, every 20 weeks, every 21 weeks, every 22 weeks, every 23 weeks, every 24 weeks, every 25 weeks, every month, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months or every year.

The interval between the administration of the initial dose and the administration of the first chronic dose and the interval between the administration of the nth chronic dose (n is an integer of 1 or higher) and the administration of the (n+1)th chronic dose may be the same; however, the specified intervals do not have to be the same. For example, the interval between the administration of the initial dose and the administration of the first chronic dose may be longer than the interval between the administration of the nth chronic dose and the administration of the (n+1)th chronic dose; or the interval between the administration of the initial dose and the administration of the first chronic dose may be shorter than the interval between the administration of the nth chronic dose and the administration of the (n+1)th chronic dose. In addition, the interval between the administration of the nth chronic dose and the administration of the (n+1)th chronic dose and the interval between the administration of the mth chronic dose (m denotes an integer of 1 or higher) and the administration of ( m+1)-th doses for continuous use may be the same; however, these intervals need not be the same or different. For example, as the number of times a dose is administered for chronic dosing increases, the interval between administrations can be lengthened or shortened.

More preferably, the interval between administrations was one week (7 days).

The dosing interval is also understood as the interval between administrations of pharmaceutical compositions.

The schedule of administration is determined, for example, based on considerations of exposure and safety. In addition, the dosage schedule is determined based on patient convenience, within a range that does not compromise efficacy and safety.

In the context of the present invention, "substantially the same" means that the difference is about 20% or less, preferably the difference is 10% or less, or more preferably the difference is 5% or less, 4% or less, 3% or less, 2% or less or 1% or less.

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.

For treatment, the effective dose of the HIR-FAB-IDS compound ranges from 1 mg/kg body weight to 12 mg/kg body weight, preferably from about 3 mg/kg body weight to 12 mg/kg body weight, when administered weekly (one once a week), preferably by intravenous administration, preferably over 3 hours.

Each vial of the drug is intended for single use only and contains 15 mg of the HIR-FAB-IDS compound in 3 ml of solution. Before intravenous administration, the drug concentrate must be diluted with 0.9% sodium chloride solution.

The terms “about” and “approximately” denote all values that are expressed as numbers with one or two decimal places.

"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 “to treat” or “the process of treatment”) refers to a clinical intervention with the purpose of changing the natural history of a disease in the individual being treated, and may be carried out either for prevention or in the process of 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 relief 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.

As used herein, the term “prophylaxis” or “prevention” covers prophylactic administration and may reduce the incidence or likelihood of occurrence of a disease (eg, MPS type II with a neurological component).

Delivery or transport of the HIR-FAB-IDS compound to a subject (e.g., by direct administration) and the practice of other methods according to the present disclosure can be used to reduce, treat, prevent, or otherwise reduce the severity of any suitable aspect of the progression of a lysosomal storage disease (namely, the progression of MPS Type II with a neurological component). Methods that reduce, prevent or otherwise reduce the intensity of such aspects of the progression of MPS type II with a neurological component, individually or collectively, are advantageous features.

In another aspect, a method is provided for reducing the risk of progression of MPS type II with a neurological component, reducing the risk of further progression of MPS type II with a neurological component that has undergone initiation, and/or providing a treatment regimen for reducing the progression of MPS type II with a neurological component in a person, which includes administering to a patient one or more first-line therapies in an amount and at a schedule sufficient to achieve a response (partial or complete response), and then administering an amount of the HIR-FAB-IDS compound to the patient or involves administering to the patient as first-line therapy.

In an additional aspect, there is provided a method of inducing remission of MPS type II with a neurological component in a subject, such as a human, comprising administering a composition containing HIR-FAB-IDS compounds to the subject for the purpose of inducing remission of MPS type II with a neurological component in the subject.

In yet another additional aspect, a method is provided for reducing the risk of developing MPS type II with a neurological component, reducing the time to MPS type II with a neurological component diagnosed in the early stages, comprising administering to the patient a prophylactically effective amount of a HIR-FAB-IDS compound.

In a further aspect, a method is provided for increasing the likelihood of survival over an appropriate period in a person who has been diagnosed with MPS type II. In another aspect, there is provided a method of improving the quality of life of a Type II MPS subject, comprising administering to the patient a composition in an amount effective to improve the patient's quality of life.

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 disorder 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 described herein, as well as those disclosed in Table 1, is given 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 α-L-iduronidase (IDUA), whose function is 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 α-L-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 functions are generally 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, eventually 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, multi 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's disease, Strossler-Scheinker, 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 syndrome, Cockayne syndrome, Hallerwarden-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 from cancer in any area of the body).

In the context of this description, the term “neurological component” represents “neuronopathic (with neurocognitive impairment)”, “non-neuronopathic (without neurocognitive impairment)”, “neurocognitive effects”, “neuropathic diseases”, “neuronopathic form” and any other disorders, including including peripheral ones, which reflect the essence of therapy with the penetration of the drug into/through the central nervous system.

MPS disorders are associated with a wide range of neurocognitive effects, from mild problems with attention and executive function to progressive and degenerative neuropathic diseases.

The neuropathic form of MPS II is challenging due to the variability of the disease course with differences in the timing of slowdown and decline. MPS II has the greatest uncertainty regarding the neurocognitive progression of the disease. It is divided into two forms: a severe neuropathic form with a large impact on neurocognitive function and an attenuated form that is considered to have little or no neurocognitive consequences (Shapiro, Elsa G., and Julie B. Eisengart. "The natural history of neurocognition in MPS disorders: a review." Molecular Genetics and Metabolism 133.1 (2021): 8-34).

Central nervous system problems in MPS II, in addition to neurocognitive impairment, include behavioral disturbances, communicating hydrocephalus, seizures, sleep apnea, and spinal cord compression. The higher risk of hydrocephalus and seizures in later stages of the disease may exacerbate neurocognitive decline (S. Al Sawaf, E. Mayatepek, B. Hoffmann, Neurological findings in Hunter disease: pathology and possible therapeutic effects reviewed, J. Inherit. Metab. Dis 31 (4) (2008) 473-480; I. V. Schwartz, et al., A clinical study of 77 patients with mucopolysaccharidosis type II, Acta Paediatr. 96 (455) (2007) 63-70).

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 true 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.

It will be clear to one skilled in the art that, without the cost of inventive creativity, additions and changes can be developed with respect to what is disclosed in the examples below within the framework of the present invention.

Examples

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.

Methods for obtaining human antibodies are currently known. For example, a human antibody of interest can be produced by immunizing a transgenic animal carrying the full spectrum of human antibody genes with the antigen of interest (see international patent application publications WO 93/12227, WO 92/03918, WO 94/02602, WO 94/ 25585, WO 96/34096 and WO 96/33735).

Genetically modified antibodies can also be obtained using known methods. A specific example is a chimeric antibody, which is an antibody that contains the H chain and L chain variable regions of an immunized animal antibody and the H chain and L chain constant regions of a human antibody. Chimeric antibodies can be produced by linking DNA encoding the variable regions of an antibody originating from an immunized animal with DNA encoding the constant regions of a human antibody, inserting them into an expression vector and then introducing it into host cells to produce antibodies.

A humanized antibody is a modified antibody, often also called a reshaped human antibody. A humanized antibody is constructed by transferring the CDRs of an antibody derived from an immunized animal into the hypervariable regions of a human antibody. General methods of genetic recombination for the production of such antibodies are also known (see published European patent application EP 239400; published international patent application WO 96/02576; Sato K. et al., Cancer Research, 53, 1993, pp. 851-856 ; published international patent application WO 99/51743).

Construction of Expression Vectors

The amino acid sequences of the first LC-HIR-FAB-IDS (SEQ ID NO: 2) and the second HC-HIR-FAB-IDS (SEQ ID NO: 4)) to obtain the 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 Hindi 11 restriction site and Kozak sequence were added to the 5' end of the sequences. Two stop codons and an Xbal 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 the Hindill/Xbal sites to obtain vectors pGNR-055-007 (pCLN-1-HC-HIR-FAB- IDS) and pGNR-055-008 (pCLN-1-LC-HIR-FAB-IDS). The resulting vectors were linearized at the BspHI/Pvul 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 device (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. The minipools were cultivated 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 using 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); horseradish peroxidase-conjugated rat antibodies to iduronate-2- were used as second antibodies. sulfatase (diluted 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 the clones were assessed according to the following parameters: the dynamics of culture viability, the dynamics of viable cell density, the concentration of the target HIR-FAB-IDS, and the dependence of volumetric production on the cumulative cell density.

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 at bioreactor with an 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 fluorometric method using 4-mutilumbelliferyl-L-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-L-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 value 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-aldoA-2S (4-methylumbeliferyl α-L-idopyranosiduronate-2-sulfate).

Instruments and equipment

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

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

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

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

5. 96-well black plate with a non-absorbing surface (Corning, USA, cat. No. 3916).

Reagents

1. 4-methylumbelliferone sodium salt (Sigma-Aldrich, cat. No. M1508).

2. Recombinant human α-L-iduronidase (R&D SYSTEM, cat. No. 4119-GH).

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

4. Sodium acetate anhydrous (AppliChem Panreac, cat. No. 141633.1211).

5. Acetic acid (Fluka, cat. No. 49199).

6. Bovine serum albumin (Sigma, cat. No. A7030).

7. Sodium carbonate (Sigma-Aldrich, cat. no. S7795).

8. Sodium bicarbonate (Sigma-Aldrich, cat. no. S6297).

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

10. Sodium phosphate disubstituted dihydrate (Sigma-Aldrich, cat. No. 71643).

11. Anhydrous citric acid (AppliChem Panreac, cat. No. 141808).

Preparation of solutions

Solution for diluting samples, 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.

Solution for diluting the substrate, pH (5.00±0.05). Place 4.1 g of anhydrous sodium acetate, 1.9 g of lead diacetate trihydrate in 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 α-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 α-L-iduronidase. 400 μl of purified water is added to a bottle containing recombinant human α-L-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, 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 higher than minus 18°C. Working standard solution of 4-methylumbelliferone sodium salt (50 nmol/ml).

An apiquot of the original 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 according to the following scheme:

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:

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. Serial dilutions are then prepared according to the following scheme:

All dilutions are stored on ice until testing begins.

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

Carrying out 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 α-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, 20 μl of phosphate-citrate buffer solution is added to the wells of the plate to stop the first enzymatic reaction. Then add 10 μl of a solution of recombinant α-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 4-methylumbelliferone 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 the wells with dilutions of the test samples and the reference solution (nmol/ml).

Specific activity A, in units/µg, for each dilution of test samples is calculated using the formula:

where: m is the concentration of 4-metipumbeliferone produced during the reaction in wells with a given dilution of the test sample (average value of 2 replicates), nmol/ml;

m _o - concentration of 4-methylumbeliferone produced during the reaction in wells with a reference solution (average value for 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 value of specific activity 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 HIR-MAB-IDS variants in comparison with the drug - are presented in table 2.

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-aldoA-2S (4-methylumbeliferyl α-L-idopyranosiduronate-2-sulfate ) 27.0 units/mcg. In this case, the free recombinant enzyme iduronate-2-sulfatase exhibits an activity of 14.6 U/µg. Full-length insulin receptor antibody coupled to the amino acid sequence of iduronate-2-sulfatase (HIR-MAB-IDS) exhibits lower specific activity (11.0 U/μg) compared to HIR-FAB-IDS.

As a result of the studies, it was shown that iduronate-2-sulfatase in the composition of HIR-FAB-IDS retains the main functional (enzymatic) properties at the level of the free recombinant enzyme. At the same time, 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. Study of the effectiveness of the drug HIR-Fab-IDS in peripheral tissues of mice line 24744: B6N.Cg-ldstm1Muen/J IDS KO

Sixty (60) hemizygous male iduronate-2-sulfatase knockout mice, B6N.Cg-ldstm1Muen/J (JAX line #024744) and twelve (12) wild-type animals, were used in the HIR-Fab-IDS drug efficacy study. (C57BL/6NJ) as control. Animals, grouped according to age at first dosing (11 weeks), were treated weekly with HIR-Fab-IDS compound at doses of 0.3, 1.0 and 3.0 mg/kg and saline as a control for 16 weeks. Blood and urine samples were taken during dosing. Control knockout mice demonstrated a 4.7-fold increase in urinary GAG levels compared to normal animals. As shown in Figure 4, in the HIR-Fab-IDS dosing groups, urinary GAG levels decreased to those of normal animals after the first dosing and were maintained throughout the dosing period.

An hour after the last administration, the animals were euthanized, and terminal blood and tissue sampling was performed. Histological analysis of tissues was carried out, as well as measurements of the level of iduronate-2-sulfatase activity and accumulation of GAGs in the tissues of experimental animals. The liver weight in knockout animals in the control group was 1.5 times higher than in wild-type animals. The administration of the drug normalized the liver weight in knockout mice of the dosage groups (Figure 5).

The level of iduronate-2-sulfatase activity in the plasma of knockout animals differed significantly (-70%) from wild-type animals. 1 hour after administration, all dosing groups of the HIR-Fab-IDS compound demonstrated significantly higher values of this indicator than knockout animals of the control group (263-2408 times), as well as wild phenotype animals (80-730 times). Mice treated with 0.3 mg/kg HIR-Fab-IDS had an average of 263-fold higher iduronate-2-sulfatase activity compared to knockout animals treated with exercise. solution. The results of the analysis of iduronate-2-sulfatase activity in the plasma of experimental animals are presented in Figure 6.

The level of iduronate-2-sulfatase activity in the tissues of the main organs (spleen, liver, kidneys and heart) of knockout animals that did not receive drug therapy was significantly (on average 76%) reduced compared to normal animals. Knockout animals of all dosage groups one hour after drug administration demonstrated significantly higher (on average 17-56 times) values of iduronate-2-sulfatase enzymatic activity compared to the group of animals receiving physical therapy. solution, and were 2.6-10 times higher than those in wild-type animals. The HIR-Fab-IDS compound preparation did not have a significant effect on the level of enzyme activity in the brain tissue of knockout animals when administered at low and medium doses. In the high dose group, there was an increase in iduronate-2-sulfatase activity in the brain tissues of knockout animals by an average of 50% compared to the placebo group, but 82% lower compared to wild-type animals.

Measurements of the level of GAG in the tissues of the main organs of knockout animals that did not receive drug therapy were significantly (on average 12 times) higher compared to normal animals. Knockout animals of all dosage groups demonstrated significantly lower (on average 70%) GAG levels compared to the group of animals receiving physical therapy. solution (on average 70% in all tissues and dose groups) and slightly (1.0-1.5 times in all tissues and dose groups) exceeded the values of this indicator in normal animals. The levels of GAGs in brain tissue did not differ between knockout and normal animals, as well as between knockout animals receiving the drug and the control group (receiving saline).

Thus, 16-week administration of the drug to mice knockout for the iduronate-2-sulfatase gene at doses of 0.3-3 mg/kg corrected the increase in GAG levels in the urine and tissues of the main organs, and also normalized the increase in liver weight to the level of wild-type animals . One hour after dosing, animals in all three dosing groups demonstrated increased levels of iduronate-2-sulfatase activity in plasma and tissues of major organs (except brain) compared to the knockout animals in the control group (263-2408 fold increase). In the brain tissues of knockout animals from the dosage groups, such a correction did not reach the values obtained for wild-type animals. The effectiveness of HIR-Fab-IDS was demonstrated at all doses tested in a mouse model of MPS II (Hunter syndrome).

Example 4. Analysis of tissue distribution of radiolabeled Fab fragments of an antibody to the insulin 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 L]-HIR-Mab-IDS, [ ¹²⁵ I]-IDS (control) after intravenous administration in cynomolgus monkeys.

The experimental animals were injected once into the left femoral vein with the corresponding molecule of the test substance at a dose of 2 ml/kg body weight by intravenous bolus injection over 1-2 minutes (or 30 seconds). 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 BAS-MS, Raytek Scientific Ltd). Blood standards labeled with the isotope [ ¹²⁵ 1] 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. The doses were different because all solutions had different levels of radioactivity, thus ensuring the same level of 0.0015 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 I]HIR-Fab-IDS were 2.55 and 3.11 ng eq/g, respectively (blood:plasma ratio 1.22). Quantifiable levels of radioactivity were present in several brain regions, including the medulla oblongata (1.09 ng eq/g), cerebral cortex (1.03 ng eq/g), cerebellum (0.908 ng eq/g), hypothalamus (0.869 ng eq/g) /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 suggest that HIR-Fab-IDS-bound substances, as well as HIR-Mab-IDS-bound substances, compared with IDS-bound substances, penetrated the blood-brain barrier, CNS tissues were exposed to the test substance or its labeled metabolites , and radioactivity transmitted through the blood had little effect on the determination of concentration in CNS tissues (Figures 1 and 2). Moreover, HIR-Fab-IDS showed a wider distribution in body tissue compared to the HIR-Mab-IDS molecule (Table 5, Figure 3).

It should be noted that in example 10 of the invention U S8834874 B2 (AGT-182 or HIR-MAB-IDS) the efficiency of penetration 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 ( HIR-MAB-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 HIR-MAB-IDS, so - 0.84 ng eq/ g HIR-FAB-IDS vs. 0.43 ng eq/g for HIR-MAB-IDS. This accordingly means that HIR-FAB-IDS penetrates the human brain with 2% efficiency. Based on the calculations shown in Example 10 of US8834874B2, 40% of the iduronate-2-sulfatase activity in human brain is expected to be achieved, excluding the increased activity of HIR-FAB-IDS compared to 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, shown in example 2, table 2 - 27 U/μg for the present compound and 11 U/μg for the contrasted one, as well as greater penetration into the brain: 27/ 11 = 2.4 times higher specific iduronate-2-sulfatase activity, 0.84/0.43 = 1.95 times greater penetration into the brain, which results in a 20% restoration of IDS activity in the patient’s brain during HIR-MAB-IDS therapy 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 contrasted HIR-MAB-IDS will only restore 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.

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 to be transported to lysosomes of tissue cells of various organs, including 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.

Example 5. Composition of a pharmaceutical composition

The pharmaceutical compositions of the present invention, which are used for therapeutic or prophylactic purposes, can be prepared by admixing, if necessary, with suitable pharmaceutically acceptable carriers, excipients and the like. and prepare them as freeze-dried preparations or preparations in solution form. Examples of suitable pharmaceutically acceptable carriers and excipients include sterilized water, physiological saline, stabilizers, excipients, antioxidants (such as ascorbic acid), buffers (such as phosphate, citrate, histidine or other organic acid buffers), antiseptics, surface active agents (such as PEG and Tween), chelating agents (such as EDTA) and binders. They may also contain other low molecular weight polypeptides, proteins such as serum albumin, gelatin and immunoglobulins, amino acids such as glycine, glutamine, asparagine, glutamic acid, aspartic acid, methionine, arginine and lysine, sugars and carbohydrates such as polysaccharides and monosaccharides , and sugar alcohols such as mannitol and sorbitol. When preparing an aqueous solution for injection, you can use, for example, physiological saline and isotonic solutions containing glucose and other adjuvants such as D-sorbitol, D-mannose, D-mannitol and sodium chloride, and appropriate stabilizers such as alcohol (for example , ethanol), polyhydric alcohols (such as propylene glycol and PEG) and nonionic surfactants (such as polysorbate 80, polysorbate 20, poloxamer 188 and HCO-50) in combination with each other. When hyaluronidase is mixed with the drug, large volumes of liquid can be administered subcutaneously (Expert Opin Drug Deliv., 4(4), July 2007; pp. 427-440). In addition, the pharmaceutical compositions of the present invention can be preloaded into a syringe. For this purpose, the preparation can be prepared in the form of a solution according to the method described in WO 2011/090088.

If desired, the antigen-binding molecules of the present invention can be included in microcapsules (for example, consisting of hydroxymethylcellulose, gelatin and poly(methyl methacrylate)) or included in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) (See, for example, Remington's Pharmaceutical Science, 16th edition, Oslo, 1980). Methods for preparing pharmaceuticals as controlled release pharmaceuticals are also well known, and such methods can be used for the antigen binding molecules of the present invention (Langer et al., J. Biomed. Mater. Res., 15, 1981, pp. 267-277; Langer, Chemtech., 12, 1982, pp. 98-105; US 3773919; publication of the European patent application EP 58481; Sidman et al., Biopolymers, 22, 1983, pp. 547-556; EP 133988) .

Pharmaceutical composition of the concentrate for the preparation of solution for infusion:

Compound HIR-FAB-IDS 5.3 mg;

Sodium chloride 8.0 mg;

Sodium dihydrogen phosphate dihydrate 3.12 mg;

Sodium hydroxide for pH correction to pH 6.0;

Polysorbate 20-0.2 mg;

Water for infusion up to 1.0 ml.

Example 6. Calculation and justification of the starting dose for the first use in humans Calculation and justification of the starting dose for the first use in humans were carried out based on the NOAEL (No Observed Adverse Effect Level) and MABEL (Minimal Anticipated) approaches Biological Effect Level; the dose that produces the minimum expected biological effect), since the phase I study planned to administer the drug once to healthy volunteers within the expected therapeutic range without titration to the maximum tolerated dose. Information on the efficacy and safety in humans of drugs with similar mechanisms of action was also taken into account.

The main results of the evaluation of efficacy and safety in preclinical studies, which reflect the risk-benefit criterion, are presented in Table 6.

In studies of the safety of the drug based on the HIR-FAB-IDS compound, it was found that in monkeys, when administered weekly for 8 weeks at doses of 3, 10 and 30 mg/kg, none of the doses caused adverse effects. In rats, when administered weekly for 8 weeks at doses of 3, 10 and 30 mg/kg, none of the doses tested also caused adverse effects. Thus, the highest non-toxic dose confirmed with 8-9 times weekly use in rodents and non-rodents is a dose of 30 mg/kg. The therapeutic index in preclinical studies, defined as the ratio of the highest safe dose (30 mg/kg) to the effective dose during multiple course administration (0.3 mg/kg) for the HIR-FAB-IDS compound, is 30 mg/kg / 0.3 mg/kg=100.

Interspecies dose transfer coefficients based on body surface area were not used to extrapolate the doses reported in the table to humans because the molecular weight of the HIR-FAB-IDS compound is greater than 100 kDa. In this regard, the human equivalent maximum safe dose (HED NOAEL) is 30 mg/kg. From a risk minimization point of view, the starting dose in clinical trials should be 0.3 mg/kg, which, according to in vivo studies, causes minimal pharmacological effect and is safe.

To improve the safety of human starting dose prediction, a calculation based on the MABEL approach was also performed. As a result of our in vitro pharmacological and micromics studies, the lowest concentration of the HIR-FAB-IDS compound that has a biological effect is detected in a test for determining the activity of iduronate-2-sulfatase in fibroblasts of an MPS II patient after treatment with the HIR-FAB-IDS compound. In this test, a threshold increase in iduronate-2-sulfatase activity (~20%) is detected at a concentration of 5 * 10-9 M of the HIR-FAB-IDS compound. Considering the molecular weight of the HIR-FAB-IDS compound (105 kDa), this corresponds to a concentration of the HIR-FAB-IDS compound equal to 5.25 * 10-4 g/l. This concentration can be considered as a starting concentration for a person, to achieve which in the blood of an average person weighing 70 kg and a fluid volume in tissues of 56 liters (80%) it is necessary to enter 56 l * 5.25 * 10-4 g/l = 294 * 10 -4 g of the HIR-FAB-IDS compound preparation (which would correspond to 4.2 * 10-4 g/kg or 0.42 mg/kg). Therefore, based on in vitro data, the MABEL approach allowed us to determine a starting effective dose of the HIR-FAB-IDS compound in humans of at least 0.42 mg/kg.

At the same time, the results of in vivo pharmacodynamics (PD) studies obtained in B6N mice. Cg-ldstm1Muen/J (JAX line no. 024744) knockout of the iduronate-2-sulfatase gene, indicate that the smallest dose of the HIR-FAB-IDS compound that causes a pharmacodynamic effect (reduces the content of glycosaminoglycans in tissues) is 0.3 mg/kg.

Thus, based on in vitro data, the MABEL approach allowed us to determine the starting dose of the HIR-FAB-IDS compound in humans of at least 0.42 mg/kg, and based on in vivo data, the MABEL approach determined the starting dose of the HIR-FAB-IDS compound in humans not less than 0.3 mg/kg. In accordance with existing rules, in this case, the lower of the indicated values should be taken as the starting dose, that is, the starting effective dose of the HIR-FAB-IDS compound when used in humans should be at least 0.3 mg/kg.

Thus, the choice of dose for the first use of the drug in humans was based on the following provisions (Table 7):

During infusions at all dose levels, transient dose-dependent hypoglycemia was observed with a frequency of 6.4%, which did not limit therapy.

Based on the above, the therapeutic dose in humans is expected to be in the range of 1-3 mg/kg, the minimum effective dose extrapolated to humans is 0.3 mg/kg, drug penetration through the BBB has been confirmed for doses of 0.0015-0.0020 mg/kg kg. The therapeutic index is 100.

The safe dose ranges calculated on the basis of preclinical safety data, reaching a maximum of 30 mg/kg of the drug for adults and 10 mg/kg for children with repeated use, include the expected therapeutic dose range with a safety factor of at least 10 times. A pharmacologically active dose of 0.3 mg/kg was adopted as the starting dose for the first use in humans in the phase I clinical trial IDB-MPS-I.

The drug can only be administered intravenously as a 3-hour infusion.

Administration of the drug must be carried out under the supervision of a physician or other medical personnel who have experience in treating patients with MPS type II or other hereditary metabolic disorders. During the study, patients will be closely monitored, with patients required to remain in the study center for at least 6 hours after completion of the first infusion for each new dose level of the HIR-FAB-IDS compound, and patients will be monitored for infusion reactions.

Detailed information about the dose of the drug, duration of infusion, preparation of the infusion solution and method of its use, as well as disposal of unused drug is provided in the Clinical Study Protocol.

Rationale for dose selection for a phase II-III clinical trial

Based on preclinical and toxicological data, the results of the phase I clinical trial IDB-MPS-I, which showed good tolerability and a favorable safety profile of the investigational drug of the compound HIR-FAB-IDS with a single dose in the dose range from 0.3 mg/kg to 3 mg/kg (0.3 mg/kg, 0.5 mg/kg, 1 mg/kg, 2 mg/kg and 3 mg/kg), and also, given the short half-life of the drug, the starting dose of the HIR-FAB compound -IDS for first use in patients with MPS II can be defined as <3 mg/kg.

The choice of a starting dose of 1 mg/kg is also due to the expected somatic effect of the HIR-FAB-IDS compound, which is equivalent to the therapeutic effect of the drug Elaprase®. HIR-FAB-IDS compounds do not differ from idursulfase in their interaction with M6PR, penetration into M6PR-presenting cells and enzymatic activity in cell cultures of MPS II patients. Study results indicate that plasma clearance of the HIR-FAB-IDS compound occurs primarily through interaction with the MePR receptor, which would provide comparable exposure in M6PR cells in vivo.

The HIR-FAB-IDS compound reduces the level of GAGs in fibroblasts from MPS II patients to the level of healthy donors at a concentration of 120 nM. Taking into account the blood volume in children over 3 years of age is about 75 ml/kg and in adolescents about 70 ml/kg, an effective concentration of 120 nM can be achieved when the HIR-FAB-IDS compound is administered at a dose of 0.9-1.0 mg /kg.

In the 26-week toxicity study, NOAEL was defined as 10 mg/kg/week based on transient episodes of hypoglycemia, which are a predictable and expected class effect. Hypoglycemia has been described for other fusion proteins using the insulin receptor (HIR) to mediate drug trafficking across the BBB, including two anti-HIR MAbs coupled to iduronidase or idursulfase. A similar toxicity profile in preclinical studies (the development of transient hypoglycemia when using high doses up to 30 mg/kg) and isolated cases of dose-dependent hypoglycemia in patients with MPS II in clinical studies confirm the safety of the selected starting dose of 1 mg/kg in children.

A starting dose of 1 mg/kg is also justified from an ethical point of view, as it avoids the administration of subtherapeutic doses and a decrease in the already achieved effect of ERT.

Example 7. Proof of the principle of drug penetration of the HIR-Fab-IDS compound across the BBB

Proof of principle was obtained from patient 1001, a participant in the IDS-MPS-II/III clinical trial.

Patient 1001, 18-year-old man. The diagnosis of MPS type II (Hunter disease) with a neurological component/neuropathic form was confirmed in 2010 by a decrease in the level of iduronate-2-sulfatase in plasma to 3.18 nM/4 h/ml (normal 297 -705), and genotypically: in the exon 09 of the IDS gene (OMIM 300823), a nucleotide substitution c. 1327C>T in a hemizygous state, leading to termination of translation of p.R443. The nucleotide substitution has not been previously described, but according to computer analysis (Alamut Visual, version 2.10) it may be pathogenic. Since 2011, the patient received enzyme replacement therapy with Elapraza® (INN idursulfase).

Before starting therapy with the HIR-FAB-IDS compound, the patient's urinary excretion of glycosaminoglycans (GAGs) was approaching normal values due to receiving enzyme replacement therapy. During treatment, the rates of GAG excretion in urine did not change significantly, which confirms comparable enzymatic activity of the drug in the periphery. The concentration of GAGs in the cerebrospinal fluid (CSF) was initially high: 3294.5 ng/ml heparan sulfate (HS) and 3.5 ng/ml dermatan sulfate (DS). It has been experimentally shown that in MPS II, predominantly GS accumulates in the central nervous system (CNS), and the concentration of GS in the CSF correlates with its accumulation in brain tissue (Tanaka N, Kida S, Kinoshita M, Morimoto H, Shibasaki T, Tachibana K , Yamamoto R. Evaluation of cerebrospinal fluid heparan sulfate as a biomarker of neuropathology in a murine model of mucopolysaccharidosis type II using high-sensitivity LC/MS/MS. Mol Genet Metab. 2018 Sep;125(1-2):53-58 doi: 10.1016/j.ymgme.2018.07.013. Epub 2018 Jul 23. PMID: 30064964).

In accordance with the protocol, the patient received the HIR-FAB-IDS compound in the form of weekly intravenous infusions lasting 3 hours. After 3 administrations of the drug at each dose and assessment of safety and tolerability, the dose was gradually increased from 1 to 3 mg/kg. During each first administration of the drug at a new dose, blood serum samples were taken to study pharmacokinetics. Cerebrospinal fluid samples to determine the drug content and GAG concentration were taken during screening (baseline), as well as after 3 weeks of drug administration at doses of 2 and 3 mg/kg. CSF was collected to assess drug concentrations 2.5 hours after the end of the infusion.

The pharmacokinetics of the drug is nonlinear. HIR-FAB-IDS compound concentrations over time are presented in Table 8 and Figure 7.

After administration of the drug in doses of 2 and 3 mg/kg 2.5 hours after the end of the infusion, the drug was detected in the CSF in concentrations of 25.939 and 272.569 pg/ml, respectively, the LLOQ of the method was 100 pg/ml (Table 9).

There was also a significant decrease in the accumulation of heparan sulfate (HS) in the CSF (Figure 8), which confirms the presence of enzymatic activity of the drug compound HIR-FAB-IDS in the tissues of the central nervous system and, therefore, indicates the effectiveness of the drug in the treatment of a patient with a neuropathic form MPS type II.

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LIST OF SEQUENCES

<110> JOINT STOCK COMPANY "GENERIUM"

<120> PHARMACEUTICAL COMPOSITION AND METHOD OF PREVENTION AND TREATMENT

LYSOSOMAL ENZYME DEFICIENCY IN A SUBJECT WITH

MUCOPOLYSACCHARIDOSIS TYPE II

<130> GNR-055

<160> 7

<170> Patent In version 2.0

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<211> 124

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<220>

<223> Epitope on the insulin receptor recognized by IgG

immunoglobulin

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Met Glu Glu Val Ser Gly Thr Lys Gly Arg Gln Glu Arg Asn Asp Ile

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Ala Leu Lys Thr Asn Gly Asp Gln Ala Ser Cys Glu Asn Glu Leu Leu

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

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Glu Pro Tyr Trp Pro Pro Asp Phe Arg Asp Leu Leu Gly Phe Met Leu

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Phe Tyr Lys Glu Ala Pro Tyr Gln Asn Val Thr Glu Phe Asp Gly Gln

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Asp Ala Cys Gly Ser Asn Ser Trp Thr Val Val Asp Ile Asp Pro Pro

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Leu Arg Ser Asn Asp Pro Lys Ser Gln Asn His Pro Gly Trp Leu Met

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Arg Gly Leu Lys Pro Trp Thr Gln Tyr Ala Ile Phe

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<220>

<223> IgG immunoglobulin light chain to the insulin receptor

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Glu Arg Val Ser Leu Thr Cys Arg Ala Ser Gln Asp Ile Gly Gly Asn

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Leu Tyr Trp Leu Gln Gln Gly Pro Asp Gly Thr Ile Lys Arg Leu Ile

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

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Ser Arg Ser Gly Ser Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser

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Glu Asp Phe Val Asp Tyr Tyr Cys Leu Gln Tyr Ser Ser Ser Pro Trp

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Thr Phe Gly Gly Gly Thr Lys Met Glu Ile Lys Arg Thr Val Ala Ala

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

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

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

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

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

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

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Phe Asn Arg Gly Glu Cys

210

<210> 3

<211> 221

<212> Protein

<213> Artificial Sequence

<220>

<223> VH-CH1 fragment of IgG immunoglobulin heavy chain to the

insulin receptor

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

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

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

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

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Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

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Met His Leu Ser Ser Leu Thr Ser Glu Lys Ser Ala Val Tyr Phe Cys

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

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

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

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

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

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

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

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

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<220>

<223> VH-CH1 fragment of IgG immunoglobulin heavy chain to the

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

1 5 10 15

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

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

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

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Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

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Met His Leu Ser Ser Leu Thr Ser Glu Lys Ser Ala Val Tyr Phe Cys

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

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

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

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

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

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

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

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

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

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

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Val Arg Ser Pro Asn Ile Asp Gln Leu Ala Ser His Ser Leu Leu Phe

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Phe Leu Thr Gly Arg Arg Pro Asp Thr Thr Arg Leu Tyr Asp Phe Asn

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

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

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Pro Gly Ile Ser Ser Asn His Thr Asp Asp Ser Pro Tyr Ser Trp Ser

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Phe Pro Pro Tyr His Pro Ser Ser Glu Lys Tyr Glu Asn Thr Lys Thr

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

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

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Phe Phe Leu Ala Val Gly Tyr His Lys Pro His Ile Pro Phe Arg Tyr

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Pro Lys Glu Phe Gln Lys Leu Tyr Pro Leu Glu Asn Ile Thr Leu Ala

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Pro Asp Pro Glu Val Pro Asp Gly Leu Pro Pro Val Ala Tyr Asn Pro

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Trp Met Asp Ile Arg Gln Arg Glu Asp Val Gln Ala Leu Asn Ile Ser

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Phe Thr Ser Asp His Gly Trp Ala Leu Gly Glu His Gly Glu Trp Ala

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Lys Tyr Ser Asn Phe Asp Val Ala Thr His Val Pro Leu Ile Phe Tyr

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Pro Tyr Leu Asp Pro Phe Asp Ser Ala Ser Gln Leu Met Glu Pro Gly

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Arg Gln Ser Met Asp Leu Val Glu Leu Val Ser Leu Phe Pro Thr Leu

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Ala Gly Leu Ala Gly Leu Gln Val Pro Pro Arg Cys Pro Val Pro Ser

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

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

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Glu Leu Ile Ala Tyr Ser Gln Tyr Pro Arg Pro Ser Asp Ile Pro Gln

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Trp Asn Ser Asp Lys Pro Ser Leu Lys Asp Ile Lys Ile Met Gly Tyr

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

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

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Ser Gln Gly Gly Asp Leu Phe Gln Leu Leu Met Pro

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<213> Artificial Sequence

<220>

<223> VH-CH1 fragment of IgG immunoglobulin heavy chain to the

insulin receptor, connected to the sequence of iduronate-2-sulfatase

through glycine-serine linker

<400> 5

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

1 5 10 15

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

20 25 30

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

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

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

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Met His Leu Ser Ser Leu Thr Ser Glu Lys Ser Ala Val Tyr Phe Cys

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

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

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

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Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Glu Thr Gln Ala

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Asn Ser Thr Thr Asp Ala Leu Asn Val Leu Leu Ile Ile Val Asp Asp

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Leu Arg Pro Ser Leu Gly Cys Tyr Gly Asp Lys Leu Val Arg Ser Pro

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

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

290 295 300

Arg Arg Pro Asp Thr Thr Arg Leu Tyr Asp Phe Asn Ser Tyr Trp Arg

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

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

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

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His Pro Ser Ser Glu Lys Tyr Glu Asn Thr Lys Thr Cys Arg Gly Pro

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Asp Gly Glu Leu His Ala Asn Leu Leu Cys Pro Val Asp Val Leu Asp

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

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

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

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Gln Lys Leu Tyr Pro Leu Glu Asn Ile Thr Leu Ala Pro Asp Pro Glu

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Val Pro Asp Gly Leu Pro Pro Val Ala Tyr Asn Pro Trp Met Asp Ile

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Arg Gln Arg Glu Asp Val Gln Ala Leu Asn Ile Ser Val Pro Tyr Gly

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Pro Ile Pro Val Asp Phe Gln Arg Lys Ile Arg Gln Ser Tyr Phe Ala

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

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

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His Gly Trp Ala Leu Gly Glu His Gly Glu Trp Ala Lys Tyr Ser Asn

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Phe Asp Val Ala Thr His Val Pro Leu Ile Phe Tyr Val Pro Gly Arg

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Thr Ala Ser Leu Pro Glu Ala Gly Glu Lys Leu Phe Pro Tyr Leu Asp

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

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Asp Leu Val Glu Leu Val Ser Leu Phe Pro Thr Leu Ala Gly Leu Ala

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Gly Leu Gln Val Pro Pro Arg Cys Pro Val Pro Ser Phe His Val Glu

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

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

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

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Lys Pro Ser Leu Lys Asp Ile Lys Ile Met Gly Tyr Ser Ile Arg Thr

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Ile Asp Tyr Arg Tyr Thr Val Trp Val Gly Phe Asn Pro Asp Glu Phe

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

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

740 745 750

Asp Leu Phe Gln Leu Leu Met Pro

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<210> 6

<211> 850

<212> Protein

<213> Artificial Sequence

<220>

<223> VH-CH1 fragment of IgG immunoglobulin heavy chain to the

insulin receptor, connected to the sequence of ?-L-iduronidase

<400> 6

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met His Leu Ser Ser Leu Thr Ser Glu Lys Ser Ala Val Tyr Phe Cys

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

Ser Tyr Asn Phe Thr His Leu Asp Gly Tyr Leu Asp Leu Leu Arg Glu

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

Ser Ser Ile Ser Ile Leu Glu Gln Glu Lys Val Val Ala Gln Gln Ile

465 470 475 480

Arg Gln Leu Phe Pro Lys Phe Ala Asp Thr Pro Ile Tyr Asn Asp Glu

485 490 495

Ala Asp Pro Leu Val Gly Trp Ser Leu Pro Gln Pro Trp Arg Ala Asp

500 505 510

Val Thr Tyr Ala Ala Met Val Val Lys Val Ile Ala Gln His Gln Asn

515 520 525

Leu Leu Leu Ala Asn Thr Thr Ser Ala Phe Pro Tyr Ala Leu Leu Ser

530 535 540

Asn Asp Asn Ala Phe Leu Ser Tyr His Pro His Pro Phe Ala Gln Arg

545 550 555 560

Thr Leu Thr Ala Arg Phe Gln Val Asn Asn Thr Arg Pro Pro His Val

565 570 575

Gln Leu Leu Arg Lys Pro Val Leu Thr Ala Met Gly Leu Leu Ala Leu

580 585 590

Leu Asp Glu Glu Gln Leu Trp Ala Glu Val Ser Gln Ala Gly Thr Val

595 600 605

Leu Asp Ser Asn His Thr Val Gly Val Leu Ala Ser Ala His Arg Pro

610 615 620

Gln Gly Pro Ala Asp Ala Trp Arg Ala Ala Val Leu Ile Tyr Ala Ser

625 630 635 640

Asp Asp Thr Arg Ala His Pro Asn Arg Ser Val Ala Val Thr Leu Arg

645 650 655

Leu Arg Gly Val Pro Pro Gly Pro Gly Leu Val Tyr Val Thr Arg Tyr

660 665 670

Leu Asp Asn Gly Leu Cys Ser Pro Asp Gly Glu Trp Arg Arg Leu Gly

675 680 685

Arg Pro Val Phe Pro Thr Ala Glu Gln Phe Arg Arg Met Arg Ala Ala

690 695 700

Glu Asp Pro Val Ala Ala Ala Pro Arg Pro Leu Pro Ala Gly Gly Arg

705 710 715 720

Leu Thr Leu Arg Pro Ala Leu Arg Leu Pro Ser Leu Leu Leu Val His

725 730 735

Val Cys Ala Arg Pro Glu Lys Pro Pro Gly Gln Val Thr Arg Leu Arg

740 745 750

Ala Leu Pro Leu Thr Gln Gly Gln Leu Val Leu Val Trp Ser Asp Glu

755 760 765

His Val Gly Ser Lys Cys Leu Trp Thr Tyr Glu Ile Gln Phe Ser Gln

770 775 780

Asp Gly Lys Ala Tyr Thr Pro Val Ser Arg Lys Pro Ser Thr Phe Asn

785 790 795 800

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

805 810 815

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

820 825 830

Val Pro Tyr Leu Glu Val Pro Val Pro Arg Gly Pro Pro Ser Pro Gly

835 840 845

Asn Pro

850

<210> 7

<211> 872

<212> Protein

<213> Artificial Sequence

<220>

<223> VH-CH1 fragment of IgG immunoglobulin heavy chain to the

insulin receptor, connected to the sequence of ?-L-iduronidase

through glycine-serine linker

<400> 7

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met His Leu Ser Ser Leu Thr Ser Glu Lys Ser Ala Val Tyr Phe Cys

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ala Pro His Leu

225 230 235 240

Val Gln Val Asp Ala Ala Arg Ala Leu Trp Pro Leu Arg Arg Phe Trp

245 250 255

Arg Ser Thr Gly Phe Cys Pro Pro Leu Pro His Ser Gln Ala Asp Gln

260 265 270

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

275 280 285

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

290 295 300

Leu Val Thr Thr Arg Gly Ser Thr Gly Arg Gly Leu Ser Tyr Asn Phe

305 310 315 320

Thr His Leu Asp Gly Tyr Leu Asp Leu Leu Arg Glu Asn Gln Leu Leu

325 330 335

Pro Gly Phe Glu Leu Met Gly Ser Ala Ser Gly His Phe Thr Asp Phe

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

Ser Met Thr Met Gln Gly Phe Leu Asn Tyr Tyr Asp Ala Cys Ser Glu

405 410 415

Gly Leu Arg Ala Ala Ser Pro Ala Leu Arg Leu Gly Gly Pro Gly Asp

420 425 430

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

435 440 445

His Cys His Asp Gly Thr Asn Phe Phe Thr Gly Glu Ala Gly Val Arg

450 455 460

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

465 470 475 480

Ile Leu Glu Gln Glu Lys Val Val Ala Gln Gln Ile Arg Gln Leu Phe

485 490 500

Pro Lys Phe Ala Asp Thr Pro Ile Tyr Asn Asp Glu Ala Asp Pro Leu

510 515 520

Val Gly Trp Ser Leu Pro Gln Pro Trp Arg Ala Asp Val Thr Tyr Ala

525 530 535

Ala Met Val Val Lys Val Ile Ala Gln His Gln Asn Leu Leu Leu Ala

540 545 550

Asn Thr Thr Ser Ala Phe Pro Tyr Ala Leu Leu Ser Asn Asp Asn Ala

555 560 565 570

Phe Leu Ser Tyr His Pro His Pro Phe Ala Gln Arg Thr Leu Thr Ala

575 580 585

Arg Phe Gln Val Asn Asn Thr Arg Pro Pro His Val Gln Leu Leu Arg

590 595 600

Lys Pro Val Leu Thr Ala Met Gly Leu Leu Ala Leu Leu Asp Glu Glu

605 610 615

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

620 625 630

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

635 640 645 650

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

655 660 665

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

670 675 680

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

685 690 695

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

700 705 710

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

715 720 725 730

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

735 740 745

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

750 755 760

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

765 770 775

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

780 785 790

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

795 800 805 810

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

815 820 825

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

830 835 840

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

845 850 855

Glu Val Pro Val Pro Arg Gly Pro Pro Ser Pro Gly Asn Pro

860 865 870

<210> 8

<211> 214

<212> Protein

<213> Artificial Sequence

<220>

<223> IgG immunoglobulin light chain to the insulin receptor LFR1

<400> 8

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 9

<211> 214

<212> Protein

<213> Artificial Sequence

<220>

<223> IgG immunoglobulin light chain to the insulin receptor LFR2

<400> 9

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

1 5 10 15

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

20 25 30

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

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

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Glu Ala

65 70 75 80

Glu Asp Ala Ala Ala Tyr Tyr Cys Leu Gln Tyr Ser Ser Ser Pro Trp

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 10

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> IgG immunoglobulin light chain to the 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 Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr

His Gln Gly

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

<210> 11

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> IgG immunoglobulin light chain to the insulin receptor LFR4

<400> 11

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu

Arg Ala Thr

Leu Ser Cys Arg Ala Ser Gln Asp Ile Gly Gly Asn Leu Tyr Trp Tyr Gln

Gln Lys Pro

Gly Gln Ala Pro Arg Leu Leu Ile Tyr Ala Thr Ser Ser Leu Asp Ser Gly

Ile Pro Ala

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser

Leu Glu Pro

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

Phe Gly Gln

Gly Thr Arg Leu 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 Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr

His Gln Gly

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

<210> 12

<211> 748

<212> Protein

<213> Artificial sequence

<220>

<223> VH-CH1 fragment of IgG immunoglobulin heavy chain to the human

insulin receptor, connected to the sequence of 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 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 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 Pro Val

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

Asn Ile Ser

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

Tyr Phe Ala

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

Asp Leu Gln

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

Gly Glu His

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

Ile Phe Tyr

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

Tyr Leu Asp

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

Leu Val Glu

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

Pro Arg Cys

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

<220>

<223> VH-CH1 fragment of IgG immunoglobulin heavy chain to the human

insulin receptor, connected to the sequence of iduronate-2-sulfatase

HFR2

<400> 13

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

Leu Arg Leu

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

Arg Gln Ala

Pro Gly Lys Gly Leu Glu Trp Val a laTrp Ile Tyr Pro Gly Asp Gly Ser

Thr Lys Tyr

Asn Glu Lys Phe Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn

Thr Leu Tyr

Leu Gln Met Asn Gly Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

Arg 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 a spLys Arg 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 a

rgSer 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 Pro Val

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

Asn Ile Ser

Val Pro Tyr Gly Pro Ile Pro Val a spPhe Gln Arg Lys Ile Arg Gln Ser

Tyr Phe Ala

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

Asp Leu Gln

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

Gly Glu His

Gly Glu Trp Ala Lys Tyr Ser Asn Phe Asp Val a laThr His Val Pro Leu

Ile Phe Tyr

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

Tyr Leu Asp

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

Leu Val Glu

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

Pro Arg Cys

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> 14

<211> 748

<212> Protein

<213> Artificial Sequence

<220>

<223> VH-CH1 fragment of IgG immunoglobulin heavy chain 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

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 Pro Val

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

Asn Ile Ser

Val Pro Tyr Gly Pro Ile Pro Val a spPhe Gln Arg Lys Ile Arg Gln Ser

Tyr Phe Ala

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

Asp Leu Gln

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

Gly Glu His

Gly Glu Trp Ala Lys Tyr Ser Asn Phe Asp Val a laThr His Val Pro Leu

Ile Phe Tyr

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

Tyr Leu Asp

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

Leu Val Glu

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

Pro Arg Cys

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> 15

<211> 748

<212> Protein

<213> Artificial Sequence

<220>

<223> VH-CH1 fragment of IgG immunoglobulin heavy chain 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 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 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 Pro Val

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

Asn Ile Ser

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

Tyr Phe Ala

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

Asp Leu Gln

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

Gly Glu His

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

Ile Phe Tyr

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

Tyr Leu Asp

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

Leu Val Glu

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

Pro Arg Cys

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

<---

Claims (21)

1. A HIR-Fab-IDS compound for the prevention or treatment of lysosomal enzyme deficiency in a subject having a lysosomal storage disease, comprising a first amino acid sequence of SEQ ID NO: 2 and a second amino acid sequence of SEQ ID NO: 4. 2. The compound according to claim 1, where the lysosomal storage disease is mucopolysaccharidosis type II or mucopolysaccharidosis type II with a neurological component. 3. The compound according to claim 2, where the neurological component is represented by a neuropathic form. 4. Connection according to paragraphs. 1-3, where the subject is a person. 5. Use of a compound according to claim 1 for the prevention or treatment of lysosomal enzyme deficiency in a subject having a lysosomal storage disease, wherein the subject is administered at least one dose of the HIR-Fab-IDS compound of about 1 to 12 mg/kg. 6. Use according to claim 5, wherein the dose is selected from the group consisting of about 3 mg/kg, about 6 mg/kg, about 12 mg/kg of the HIR-Fab-IDS compound. 7. Application according to paragraphs. 5, 6, where lysosomal storage disease is mucopolysaccharidosis type II or mucopolysaccharidosis type II with a neurological component. 8. Application according to claim 7, where the neurological component is represented by a neuropathic form. 9. Application according to paragraphs. 5-8, where the subject is a person. 10. A pharmaceutical composition for the prevention or treatment of lysosomal enzyme deficiency in a subject having a lysosomal storage disease, wherein the composition contains a HIR-Fab-IDS compound represented by the first amino acid sequence of SEQ ID NO: 2 and the second amino acid sequence of SEQ ID NO: 4. 11. The pharmaceutical composition according to claim 10, where the pharmaceutical composition additionally contains sodium chloride, sodium dihydrogen phosphate dihydrate, sodium hydroxide, polysorbate. 12. The pharmaceutical composition according to claim 11, where the pharmaceutical composition additionally contains the compound HIR-Fab-IDS 5.3 mg, sodium chloride in the amount of 8.0 mg, sodium dihydrogen phosphate dihydrate in the amount of 3.12 mg, sodium hydroxide to adjust the pH to pH 6 ,0, polysorbate 20 in an amount of 0.2 mg, water for injection up to 1.0 ml. 13. Pharmaceutical composition according to claims. 10, 11, where lysosomal storage disease is mucopolysaccharidosis type II or mucopolysaccharidosis type II with a neurological component. 14. Pharmaceutical composition according to claim 13, where the neurological component is represented by a neuropathic form. 15. Pharmaceutical composition according to claims. 10-14, where the subject is a person. 16. Use of a pharmaceutical composition according to claim 10 for the prevention or treatment of lysosomal enzyme deficiency in a subject having a lysosomal storage disease, wherein the HIR-Fab-IDS compound is administered to the subject at least one dose selected from the group consisting of about 3 mg/ kg, approximately 6 mg/kg, approximately 12 mg/kg of HIR-Fab-IDS compound. 17. A method of preventing or treating lysosomal enzyme deficiency in a subject having a lysosomal storage disease, comprising administering to the patient at least one dose of a HIR-Fab-IDS compound represented by the first amino acid sequence of SEQ ID NO: 2 and the second amino acid sequence of SEQ ID NO: 4 wherein the dose is selected from the group consisting of about 3 mg/kg, about 6 mg/kg, about 12 mg/kg of the HIR-Fab-IDS compound. 18. The method according to claim 16, where the HIR-Fab-IDS compound as part of a pharmaceutical composition is administered intravenously for 3 hours once a week. 19. The method according to claim 16, where the lysosomal storage disease is mucopolysaccharidosis type II or mucopolysaccharidosis type II with a neurological component. 20. The method according to claim 18, where the neurological component is represented by a neuropathic form. 21. Method according to paragraphs. 16-19, where the subject is a person.