A fusion protein comprising GLP-1, immunoglobulin FC, and IGF-1 and uses thereof

Abstract

The present invention relates to a fusion protein comprising GLP-1, immunoglobulin FC, and IGF-1, and uses thereof. Since the fusion protein of the present invention has excellent blood-brain barrier permeation efficiency, it can be widely used for the effective treatment of nervous system diseases.

Description

Technical Field

[0001] The present invention relates to a fusion protein comprising GLP-1, immunoglobulin FC, and IGF-1, and uses thereof.

Background Art

[0002] The blood-brain barrier (BBB) is a cellular barrier composed of tight junctions with a very high electrical resistance of 0.1 Ω·m or more between vascular endothelial cells in contact with related pericytes and astrocytes. It is a highly selective permeable barrier that separates the blood circulating from the brain extracellular fluid in the central nervous system (CNS), and plays the role of a gateway to protect the central nervous system by regulating the entry and exit of nutrients and other substances into and out of the brain.

[0003] Normally, the blood-brain barrier not only selectively transports molecules such as glucose and amino acids essential for brain function, but also allows water, several gases, and lipophilic molecules to pass through by passive diffusion. On the other hand, the blood-brain barrier blocks the entry and exit of lipophilic, potential neurotoxins by an active transport mechanism mediated by P-glycoprotein. Therefore, neurodisease drugs such as drugs with a large molecular weight acting inside the brain and low-molecular drugs with low brain permeability cannot penetrate the blood-brain barrier.

[0004] Thus, the blood-brain barrier plays a role in preventing bacteria, pathogens, and potentially dangerous substances in the blood from being delivered to the brain via the blood. However, due to such a vascular barrier, most central nervous system drugs show low efficiency for transcranial delivery, and in order to compensate for this, these drugs are administered at high doses, which may cause serious side effects in peripheral organs. Therefore, although research is ongoing (Patent Document 1), there is a need to find an efficient drug delivery system that can penetrate the blood-brain barrier while preventing negative systemic effects and ensuring the therapeutic effect of chemotherapeutic drugs.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0006] The inventors of the present invention have developed a fusion protein that penetrates the blood-brain barrier, exhibits a continuous efficacy during drug administration, and exerts a preventive and progression-delaying effect on nervous system diseases, thus completing the present invention.

Means for Solving the Problems

[0007] An object of the present invention is to provide a fusion protein comprising GLP-1, immunoglobulin Fc, and IGF-1.

[0008] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating a nervous system disease, comprising a fusion protein comprising GLP-1, immunoglobulin Fc, and IGF-1 as an active ingredient.

[0009] Furthermore, an object of the present invention is to provide a method for preventing or treating a nervous system disease, comprising the step of administering the composition to an individual.

[0010] Furthermore, an object of the present invention is to provide a use of a fusion protein containing GLP-1, immunoglobulin Fc, and IGF-1 for preventing, improving, or treating a neurological disease.

[0011] Furthermore, an object of the present invention is to provide a use of a pharmaceutical composition containing the fusion protein for preventing or treating a neurological disease.

Advantages of the Invention

[0012] The fusion protein of the present invention provides a novel form of a therapeutic candidate substance for neurological diseases with improved blood-brain barrier permeation efficiency, and thus can be widely used for effective treatment of neurological diseases.

Brief Description of the Drawings

[0013]

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Mode for Carrying Out the Invention

[0014] Hereinafter, these will be specifically described. Note that each description and embodiment disclosed in the present invention is also applicable to other descriptions and embodiments. That is, any combination of various elements disclosed in the present invention is included in the present invention. Also, the present invention is not limited to the following specific description.

[0015] Also, those having ordinary knowledge in the art will be able to recognize and confirm many equivalents of the specific aspects of the present invention described in the present invention using only ordinary experiments. Furthermore, such equivalents are also intended to be included in the present invention.

[0016] One aspect of the present invention for achieving the object of the present invention provides a fusion protein comprising GLP-1, immunoglobulin Fc, and IGF-1.

[0017] In order to achieve the above object, a drug carrier for permeating the blood-brain barrier (BBB) containing IGF-1 (Insulin-like growth factor 1) is provided.

[0018] The "blood-brain barrier (BBB)" in the present invention is a cell barrier composed of tight junctions having a very high electrical resistance of 0.1 Ω or more between vascular endothelial cells in contact with related pericytes and astrocytes, and is a highly selective permeable barrier that separates the blood circulating from the brain extracellular fluid in the central nervous system (CNS). It plays the role of a gateway that protects the central nervous system by regulating the entry and exit of nutrients and other substances into and out of the brain.

[0019] The "drug carrier" in the present invention means a carrier for efficiently delivering a drug showing therapeutic activity to a target tissue or organ. The drug carrier of the present invention is characterized in that it can penetrate the blood-brain barrier and deliver the drug to the brain with high efficiency.

[0020] Specifically, the drug carrier of the present invention contains IGF-1. As a more specific embodiment, it is in the form of a protein in which IGF-1 and immunoglobulin Fc are linked, but is not limited thereto.

[0021] The drug carrier of the present invention can not only show a therapeutic effect by itself, but also allow the drug linked to the drug carrier to penetrate the blood-brain barrier and be efficiently delivered, showing a therapeutic activity in the brain. In addition, it has the effect of prolonging the half-life of the drug, enabling the drug efficacy to last for a long time and reducing the number of administrations.

[0022] In one embodiment of the present invention, as a result of pharmacokinetic evaluation of a fusion protein comprising GLP-1 of the present invention, immunoglobulin Fc, and IGF-1, it was confirmed that the half-life was extended (Figure 5). As a result of administering a fluorescently labeled substance into the abdominal cavity of a mouse and then observing the brain, it was confirmed that the fluorescence intensity increased over time, and it was also confirmed that the BBB permeability was excellent (Figure 7).

[0023] "IGF-1 (Insulin-like growth factor 1)" in the present invention means a cell growth factor whose structure is similar to insulin and plays an important role in normal growth and maintenance of health. This is derived from a peptide present in the human body. The IGF-1 of the present invention includes not only natural ones but also all its derivatives or variants. The derivatives and variants mean those in which at least one amino acid substitution, deletion, addition or other mutations have occurred in the natural sequence and which maintain their unique activity. The amino acid sequence of the IGF-1 is not particularly limited, but includes the amino acid sequence of SEQ ID NO: 9, or is based on the amino acid sequence of SEQ ID NO: 9, and substitution, deletion, addition or other mutations have occurred in the amino acids corresponding to at least one position among the 3rd, 49th, 67th, 70th positions and combinations thereof. Specifically, the amino acid corresponding to the 3rd position is alanine, the amino acid corresponding to the 49th position is alanine, the amino acid corresponding to the 67th position is threonine, and the amino acid corresponding to the 70th position is thymine. More specifically, the amino acid sequences of SEQ ID NO: 10, 11 or 12 are included. The amino acid sequences of SEQ ID NO: 9, 10, 11 or 12 include amino acid sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% or more homology or identity with the amino acid sequences represented by SEQ ID NO: 9, 10, 11 or 12. Needless to say, the present application also includes proteins having an amino acid sequence in which some sequences are deleted, modified, substituted, conservatively substituted or added, as long as the amino acid sequence has such homology or identity and exhibits the efficacy corresponding to the protein of the present application.

[0024] For example, those having an addition or deletion of a sequence that does not change the function of the protein of the present application, a naturally occurring mutation, a silent mutation, or a conservative substitution at the N-terminus, C-terminus, and / or inside of the amino acid sequence are included.

[0025] "Conservative substitution" in the present application means that an amino acid is substituted with another amino acid having similar structural and / or chemical properties. The protein has at least one conservative substitution, for example, while still having at least one biological activity. Such amino acid substitutions can generally occur based on the similarity in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues. For example, among the amino acids having an electrically charged side chain, the positively charged (basic) amino acids include arginine, lysine, and histidine, the negatively charged (acidic) amino acids include glutamic acid and aspartic acid, among the amino acids having an uncharged side chain, the nonpolar amino acids include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline, the polar or hydrophilic amino acids include serine, threonine, cysteine, tyrosine, asparagine, and glutamine, and among the above amino acids, the aromatic amino acids include phenylalanine, tryptophan, and tyrosine.

[0026] "Homology" or "identity" in the present application means the degree to which two given amino acid sequences or nucleic acid base sequences are similar, and is expressed as a percentage. Homology and identity are often used interchangeably.

[0027] The sequence homology or identity of a conserved polynucleotide or protein is determined by standard sequence algorithms and may use the default gap penalties established by the programs used. Substantially homologous or identical sequences generally hybridize to all or part of the sequence under moderately or highly stringent conditions. It goes without saying that hybridization also includes hybridization with a polynucleotide having codons that take into account common codons or codon degeneracy in polynucleotides.

[0028] In the present invention, the IGF-1 has a function of protecting neurons, improving neuronal protection against excitatory substances, and blood-brain barrier (BBB) permeability, and improving the preventive or therapeutic effect on nervous system diseases.

[0029] For the purpose of the present invention, the IGF-1 of the present invention may contain modifications in the wild-type IGF-1 amino acid sequence of SEQ ID NO: 9 for blood-brain barrier penetration. The types of modifications introduced for blood-brain barrier penetration are not limited, and any modification introduced for inhibiting the binding ability to IGFBP may be used. Specifically, the IGF-1 of the present invention may be a variant containing an amino acid substitution so that the binding ability to IGFBP is inhibited, and more specifically, a variant containing the amino acids of SEQ ID NO: 10, 11, or 12.

[0030] For example, at least one amino acid substitution in the sequence of IGF-1 can inhibit binding to the IGFBP protein. Examples of amino acids included in the IGF-1 of the present invention include, but are not limited to, E3A, F49A, A67T, A70T, etc. Here, these notations indicate the amino acid before substitution, the substitution position, and the amino acid after substitution in order. For example, E3A indicates that the glutamic acid (E), which is the amino acid corresponding to the third position based on the sequence of wild-type IGF-1, is substituted with alanine (A). Since it contains at least one amino acid substitution selected from the above-described amino acid substitutions, the drug carrier containing the IGF-1 of the present invention has excellent blood-brain barrier permeability.

[0031] "IGFBP (Insulin-like growth factor-binding protein)" in the present invention serves as a transport protein for IGF-1 (insulin-like growth factor 1). Since most of the IGF in the body exists in a state bound to the IGFBP protein group, its target delivery ability is inhibited. Therefore, since the drug carrier of the present invention is designed not to bind to IGFBP, the BBB permeation efficiency is enhanced.

[0032] A specific embodiment of the drug carrier of the present invention provides a drug carrier having a structure of Fc-IGF1 in which IGF-1 is linked to immunoglobulin Fc.

[0033] "Fc-IGF1 drug carrier" in the present invention is used interchangeably with "Fc-IGF1 hybrid". The Fc-IGF1 drug carrier not only exhibits excellent blood-brain barrier permeation efficiency by including IGF-1 whose binding to IGFBP is inhibited and whose blood-brain barrier permeability is improved, but also has an extended drug half-life due to binding to immunoglobulin Fc.

[0034] In one embodiment of the present invention, in order to evaluate the IGF-1 activity of a fusion protein comprising GLP-1, immunoglobulin Fc, and IGF-1 of the present invention, using a proliferation assay and a PathHunter HEK293 IGF-1R bioassay kit in the Saos-2 cell line, it was confirmed that the IGF-1 activity was excellent compared to the control group (wild) (FIG. 2, FIG. 3).

[0035] The "immunoglobulin Fc" in the present invention is a general term for proteins that play an important role in immunity and have antibody activity among serum components. The basic structure is composed of a pair of L chains (light chains) with a molecular weight of about 23,000 and a pair of H chains (heavy chains) with a molecular weight of 50,000 to 70,000, which are bound by S-S bonds, and are classified into IgG, IgA, IgM, IgD, and IgE according to the type of H chain. The shape of the molecule is Y-shaped, with the two upper parts being equivalent antibody-binding sites, while the lower part (Fc part) is a site that exhibits biological activities such as the binding of antibodies that bind to antigens to complement or cells. In the present invention, "immunoglobulin Fc" is used interchangeably with "immunoglobulin Fc region".

[0036] In the present invention, the immunoglobulin Fc fragment may consist of 1 to 4 domains selected from the group consisting of CH1, CH2, CH3, and CH4 domains, and the immunoglobulin Fc fragment may further include a hinge region. Also, the immunoglobulin Fc fragment may be selected from the group consisting of IgG, IgA, IgD, IgE, IgM, their combinations, and their hybrids. Specific examples of the immunoglobulin Fc of the present invention include trastuzumab, but are not limited thereto. Also, the drug carrier of the present invention containing Fc may include modifications so as to have reduced effector functions in the body, for example, may include modifications such as N297A.

[0037] "IgG (immunoglobulin G)" in the present invention is a type of immunoglobulin and has placental permeability. It is divided into four subclasses, IgG1, IgG2, IgG3, and IgG4, according to the upper part of the constant portion of the H chain in the basic structure.

[0038] "Human IgG1 Fc" in the present invention is a type of antibody and accounts for 75% of all immunoglobulins present in human serum. In the present invention, the Fc site of a human-derived antibody (human IgG1) is used, and this domain is a functional unit of a protein used in various pharmaceuticals for the purpose of improving the water solubility of substances and extending the half-life. The immunoglobulin Fc of the present invention includes not only natural ones but also derivatives or mutants thereof. The derivatives and mutants mean those in which mutations such as substitution, deletion, or addition have occurred in at least one amino acid and which maintain their inherent activity. The amino acid sequence of the human IgG1 Fc is not particularly limited, but it includes those containing the amino acids of SEQ ID NO: 4, or sequences in which mutations such as substitution, deletion, or addition have occurred in the amino acids corresponding to one or more positions of the 297th, 309th, 311th, 428th positions and combinations thereof based on the amino acid sequence of SEQ ID NO: 4. Specifically, it may include those in which the amino acid corresponding to the 297th position is substituted with alanine, the amino acid corresponding to the 309th position is substituted with tyrosine, the amino acid corresponding to the 311th position is substituted with methionine, and the amino acid corresponding to the 428th position is substituted with leucine. More specifically, it may include those containing the amino acid sequence of SEQ ID NO: 5, 6, 7, or 8. The amino acid sequences of SEQ ID NO: 4, 5, 6, 7, or 8 include amino acid sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% or more homology or identity with the amino acid sequences represented by SEQ ID NO: 4, 5, 6, 7, or 8. The "homology" and "identity" are as described above.

[0039] In the present invention, the human IgG1 Fc has the functions of improving the water solubility of the carrier and prolonging the in-vivo half-life, thereby improving the preventive or therapeutic effect on nervous system diseases.

[0040] FcRn in the present invention means a protein that binds to the Fc region of an IgG antibody. FcRn is derived from any organism including, but not limited to, humans, mice, rats, rabbits, and monkeys. It has been reported that the in-vivo half-life of an immunoglobulin (antibody) is mediated by the binding of Fc to FcRn. The blood half-life and persistence of an antibody greatly depend on the binding of the Fc site of the antibody to FcRn (neonatal Fc receptor), which is one of the IgG-binding ligands. Also, the binding of Fc to FcRn plays an important role in antibody transport.

[0041] In one embodiment of the present invention, it was confirmed that the fusion protein containing GLP-1, immunoglobulin Fc, and IGF-1 has excellent binding ability to FcRn (Figure 6).

[0042] Since the drug carrier of the present invention has high blood-brain barrier permeation efficiency, by binding a drug having therapeutic activity to the drug carrier having an Fc-IGF1 structure, not only can the efficacy of the drug be preferably exerted, but also the efficacy can be exerted for a long time due to the extended half-life, so that an excellent therapeutic effect can be obtained.

[0043] Specifically, a substance for the prevention or treatment of a nervous system disease, for which an excellent therapeutic effect is expected by delivering the drug through the blood-brain barrier, can be linked to the drug carrier of the present invention. As long as it is a substance that exerts a therapeutic effect by permeating the blood-brain barrier, it is not limited to a therapeutic agent for a specific disease, and any substance can be bound to the drug carrier of the present invention.

[0044] The substance may be a therapeutic agent for nervous system diseases. Examples of the therapeutic agent for nervous system diseases include low-molecular-weight pharmaceuticals for treating nervous system diseases, peptides, enzymes, antibodies, proteins, etc. Examples of the therapeutic agent for nervous system diseases include celecoxib, masitinib, (1-3)IGF1, Exenatide BDNF, GDNF, CNTF, Iduronate 2-sulfatase (IDS), Glucocerebrosidase (GBA), α-synuclein specific antibody, etc. Specifically, GLP-1 is included. The drug carrier Fc-IGF1 of the present invention is in the form of a fusion protein linked with GLP-1, but is not limited thereto.

[0045] "GLP-1" in the present invention is a kind of hormone derived from the digestive tract induced from the transcription product of the glucagon gene. The GLP-1 of the present invention includes not only natural ones but also all of its derivatives or mutants. The derivatives and mutants mean those in which mutations such as substitution, deletion, addition of at least one amino acid occur and which maintain their inherent activity. The amino acid sequence of the GLP-1 is not particularly limited, but includes any of the amino acid sequences of SEQ ID NOs: 1 to 3. The GLP-1 of the present invention may include a natural sequence (SEQ ID NO: 2), may include SEQ ID NO: 3 corresponding to the 7th to 36th amino acids which are its active form, or may be a mutant of GLP-1 further including substitution, addition, deletion of at least one amino acid in these sequences.

[0046] Alternatively, it may be modified to have resistance to DPPIV and inhibit in vivo degradation in the natural sequence. Examples of the modification include substituting alanine, which is the second amino acid, with another amino acid (for example, glycine (G)) or a non-natural amino acid, but are not limited thereto.

[0047] In one embodiment of the present invention, in order to evaluate the GLP-1 activity of the fusion protein of the present invention, using the cAMP HunterTM Liraglutide bioassay kit, it was confirmed that it showed an activity level equivalent to that of exendin-4, which is a GLP-1 receptor agonist (Figure 4). In the present invention, the GLP-1 has a function of anti-inflammatory action in the nerve cells of the carrier, thereby improving the preventive or therapeutic effect on nervous system diseases.

[0048] In the present invention, a fusion protein containing GLP-1, immunoglobulin Fc, and IGF-1 is named PF1802.

[0049] Also, the drug carrier of the present invention may be linked to a therapeutic agent for amyotrophic lateral sclerosis (ALS). Examples of the therapeutic agent for amyotrophic lateral sclerosis include small molecule pharmaceuticals for treating nervous system diseases, peptides, enzymes, antibodies, proteins, etc. Examples of the therapeutic agent for amyotrophic lateral sclerosis include celecoxib, masitinib, (1-3)IGF1, Exenatide BDNF, GDNF, CNTF, Iduronate 2-sulfatase (IDS), Glucocerebrosidase (GBA), α-synuclein specific antibody, etc., and specifically GLP-1 is included, but it is not limited thereto.

[0050] In one embodiment of the present invention, as a result of administering the PF1802_M008 candidate substance of the present invention to an amyotrophic lateral sclerosis gene-modified mouse model, it was confirmed that the fully innervated neuromuscular junction (Fully innervated NMJ) increased and the denervated neuromuscular junction (denervated NMJ) decreased, and it was confirmed that it was excellent in the preventive and progression delay effects on amyotrophic lateral sclerosis disease (Figure 8, Figure 11).

[0051] In addition, microglial marker Iba1 was significantly decreased compared to the untreated one, showed the same activity as riluzole known as an ALS therapeutic agent, and it was confirmed that the substance of the present invention has excellent BBB permeability (Figure 12).

[0052] The drug carrier and the drug of the present invention are linked by various methods known in the art. For example, forms in which the drug is linked to IGF-1 or Fc via a linker, and forms directly linked by covalent or non-covalent bonds are all included. As long as it exhibits blood-brain barrier permeability and therapeutic effects, the linking method and the ultimately linked form can be any.

[0053] The fusion protein of the present invention may be in a form in which GLP-1, the first linker, the Fc region, the second linker, and IGF-1 are linked from the N-terminus.

[0054] The linker is a peptide linker, and the drug carrier of the present invention may be one in which IGF-1 and the immunoglobulin Fc region are fused via a peptide linker. One end of the linker is linked to one strand of the dimeric immunoglobulin Fc region, but is not limited thereto.

[0055] The peptide linker contains one or more amino acids, for example, contains 1 to 1000 amino acids, and any peptide linker known in the art, such as [GS]x linker, [GGGS]x linker, [GGGGS]x linker, etc. can be mentioned. Here, x is a natural number of 1 or more (for example, 1, 2, 3, 4, 5 or more), but is not limited thereto.

[0056] Specifically, in the present invention, the linker is GSAPAP(G 4 S)(SEQ ID NO: 38)(G 4 S) 4 GAHS(SEQ ID NO: 39), ASGAGSTTLEVLFQGP(SEQ ID NO: 40), (G) 8 (SEQ ID NO: 41) or (PA) 5Although it is (Sequence No. 42), it is not limited thereto.

[0057] The fusion protein in the present invention means a structure containing GLP-1, immunoglobulin Fc, and IGF-1, and includes any amino acid sequence represented by Sequence Nos. 25 to 37, but is not limited thereto. The amino acid sequences of Sequence Nos. 25 to 37 include amino acid sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% or more homology or identity with the amino acid sequences represented by Sequence Nos. 25 to 37. The "homology" and "identity" are as described above.

[0058] Another aspect of the present invention provides a method for producing a drug carrier, which includes the step of linking IGF-1 and immunoglobulin Fc.

[0059] Still another aspect of the present invention provides a method for producing a fusion protein, which includes the step of linking GLP-1, immunoglobulin Fc, and IGF-1.

[0060] The "GLP-1", "IGF-1", "Fc", and "drug carrier" are as described above.

[0061] Still another aspect of the present invention provides a pharmaceutical composition for preventing or treating a nervous system disease, which includes a fusion protein containing GLP-1, immunoglobulin Fc, and IGF-1 as an active ingredient.

[0062] The "GLP-1", "immunoglobulin Fc", "IGF-1", and "fusion protein" are as described above.

[0063] The pharmaceutical composition may contain the fusion protein in a pharmaceutically effective amount.

[0064] Since the drug carrier or fusion protein of the present invention has excellent blood-brain barrier (BBB) permeation efficiency, an improvement in the efficacy of a therapeutic agent for nervous system diseases delivered to the brain by the drug carrier or fusion protein is expected.

[0065] The "nervous system disease" in the present invention means a disease in which a problem occurs in the nervous system, including nervous system brain diseases, neuromuscular diseases, etc., and specifically includes neuromuscular junction diseases, but is not limited thereto.

[0066] The "nervous system brain diseases" in the present invention include brain tumors (glioma, meningioma, schwannoma, neurofibroma, pituitary adenoma, craniopharyngioma, metastatic cancer), cerebral infarction, hypertensive cerebral hemorrhage, subarachnoid hemorrhage, subdural hemorrhage, brain contusion, cerebral arteriovenous malformation, brain abscess, encephalitis, meningitis, chickenpox, epilepsy, arachnoid cyst, concussion, cerebral palsy, hemifacial spasm, Parkinson's disease, moyamoya disease, migraine, dementia, etc.

[0067] The "neuromuscular disease" in the present invention means a disease that includes direct functional abnormalities of muscles or indirect functional abnormalities of muscles due to abnormalities of nerves or neuromuscular junctions, and is also called neuromuscular disorder. When the central nervous system is affected, muscle spasm and paralysis symptoms occur, and the symptoms vary depending on the affected part of the brain. Stroke, multiple sclerosis, Parkinson's disease, peripheral nerve diseases, muscle diseases, myasthenia gravis, amyotrophic lateral sclerosis, spinal muscular atrophy, etc. fall under this category.

[0068] The pharmaceutical composition of the present invention brings about improvement in neuromuscular junction function, neuroprotective effect, improvement in blood-brain barrier permeability, and extension of the in vivo half-life, and has a preventive or therapeutic effect on nervous system diseases, but is not limited thereto. In the present invention, the nervous system disease is a disease related to abnormal function of the neuromuscular junction. When the fusion protein according to the present invention is administered, a preventive or therapeutic effect on the nervous system disease can be obtained by improving and restoring the function of the neuromuscular junction.

[0069] "Prevention" in the present invention means any act of preventing or delaying a neurological disease by administration of the composition of the present invention, and "treatment" means any act of improving or favorably changing the symptoms of a neurological disease by administration of the composition of the present invention.

[0070] The pharmaceutical composition of the present invention may further contain a pharmaceutically acceptable carrier, excipient or diluent. Such pharmaceutically acceptable carriers, excipients or diluents may be non-natural-occurring. Specifically, the composition is formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories and sterile injection solutions by ordinary methods. In the present invention, examples of the carrier, excipient and diluent contained in the pharmaceutical composition include lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil. When formulating, it is usually prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants. Examples of oral solid preparations include tablets, pills, powders, granules, capsules, etc. These solid preparations are prepared by mixing at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. with the extract and its fractions. In addition to ordinary excipients, lubricants such as magnesium stearate and talc are also used. Examples of oral liquid preparations include suspensions, oral solutions, emulsions, syrups, etc. In addition to water and liquid paraffin which are ordinary diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives, etc. are used. Examples of parenteral preparations include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, suppositories. As non-aqueous solvents and suspensions, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc. are used. As the base of suppositories, witepsol, macrogol, tween 61, cocoa butter, laurin fat, glycerogelatin, etc. are used.

[0071] Still another aspect of the present invention provides a method for preventing or treating a neurological disorder, comprising the step of administering to an individual the drug carrier, fusion protein, or pharmaceutical composition containing the same.

[0072] The "individual" in the present invention means any animal including humans who has or has developed the neurological disorder in the present invention. By administering the pharmaceutical composition of the present invention to an individual, a preventive and therapeutic effect on the neurological disorder can be obtained.

[0073] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount.

[0074] "Administration" in the present invention means introducing the pharmaceutical composition of the present invention into a subject by any suitable method, and the administration route can be any general route as long as it can deliver to the target tissue. Examples include, but are not limited to, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, and intranasal administration.

[0075] The "pharmaceutically effective amount" means an amount sufficient to prevent or treat a neurological disorder with a reasonable benefit / risk ratio applicable to medical use, and the effective dosage level is determined by factors including the type and severity of the individual, age, gender, activity of the drug, sensitivity to the drug, administration time, administration route and excretion rate, treatment period, factors including drugs used simultaneously, and other factors known in the medical field. For example, the drug carrier, fusion protein, or pharmaceutical composition containing the same may be administered at a dose of 0.01 - 500 mg / kg per day, specifically 10 - 100 mg / kg, and the administration may be carried out once a day or divided into several times a day.

[0076] The composition of the present invention may be administered alone or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. Also, single or multiple administrations may be performed. It is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects considering all the above factors, and this can be easily determined by those skilled in the art.

[0077] The composition of the present invention may be used alone or in combination with surgery, hormone therapy, drug therapy, methods using biological response modifiers, etc. for the prevention or treatment of nervous system diseases.

[0078] The composition in the present invention may have an effect of restoring or improving the function of the neuromuscular junction (NMJ).

[0079] The "neuromuscular junction (NMJ)" in the present invention means a special structure where the end of a motor nerve connects to a muscle and transmits the excitation of the nerve to the membrane of muscle fibers, and it is a type of synapse. Neuromuscular junction diseases include myasthenia gravis (MG), Lambert-Eaton myasthenic syndrome, botulism, and congenital myasthenic syndrome.

[0080] Still another aspect of the present invention provides a pharmaceutical composition for the prevention or treatment of amyotrophic lateral sclerosis (ALS) containing the above drug carrier or fusion protein.

[0081] The "drug carrier", "fusion protein", and "pharmaceutical composition" are as described above.

[0082] In the present invention, the fusion protein may be one in which GLP-1, which is a therapeutic agent, is linked to the drug carrier of the present invention. Specifically, it may be one in which GLP-1, immunoglobulin Fc, and IGF-1 are linked. Since the fusion protein according to the present invention has high efficiency in reaching the brain and an extended half-life, an excellent therapeutic effect on amyotrophic lateral sclerosis is expected.

[0083] Still another aspect of the present invention provides a use of a fusion protein comprising GLP-1, an immunoglobulin Fc, and IGF-1 for preventing, ameliorating, or treating a neurological disease.

[0084] Still another aspect of the present invention provides a use of a pharmaceutical composition comprising the fusion protein for preventing or treating a neurological disease.

[0085] The "GLP-1", "immunoglobulin Fc", "IGF-1", "neurological disease", and "pharmaceutical composition" are as described above.

Example

[0086] Hereinafter, the present invention will be described in more detail with reference to examples. These examples are for more specifically explaining the present invention, and the present invention is not limited to these examples.

Example

[0087] Preparation of Fusion Protein To secure candidate substances, GLP1-Fc-IGF1 or GLP1-Fc-mut.IGF1-TfR was synthesized and then cloned into the pcDNA3.1 vector using Nhe I / Hind III sites. The cloned vector was transfected into CHO-S cells, and the supernatant was collected on the 5th day. Then, cells and suspensions were removed using centrifugation and a 0.22 μm filter. Subsequently, purification was performed using protein A resin, and various PF1802_M003 to M032 candidate substances (SEQ ID NOs: 13 to 37) were secured as shown in Table 1 (Figure 1, Table 1).

[0088]

Table 1-1

[0089]

Table 1-2

Example

[0090] Activity evaluation of PF1802 candidate substances Example 2-1. IGF-1 activity evaluation of PF1802 candidate substances using Saos-2 cell line To confirm the IGF-1 activity of the PF1802 candidate substance prepared by the method of Example 1, a proliferation assay with IGF-1 was performed in the Saos-2 cell line. The Saos-2 cell line was dispensed into a 96-well plate at 1×10 4 per well and cultured at 37°C, 5% CO 2 for 16 hours, and then starved for 4 hours. Then, the PF1802 candidate substance was diluted 5-fold from a maximum concentration of 100 nM to 0.00256 nM and each well was treated, and then cultured at 37°C, 5% CO 2 for 48 hours. The cultured cells were stained with SRB (sulforhodamine B), and then the absorbance was measured at 540 nm using a microplate reader. The PLA statics function was applied, and IGF-1 was set as the reference, and then the relative potency value of the PF1802 candidate substance was obtained (Table 2, Figure 2).

[0091]

Table 2

[0092] As a result, as shown in Table 2, the IGF-1 activities of M007 and M008, which are Mutant IGF-1 substances, were confirmed as compared with the substances using wt.IGF1.

[0093] Example 2-2. IGF-1 activity evaluation of PF1802 candidate substances To confirm the IGF-1 activity of the PF1802 candidate substance prepared by the method of Example 1, the IGF-1 activity (phosphorylation activity) of the produced PF1802 substance was measured using the PathHunter HEK293 IGF-1R bioassay kit (Discover X, 95-0505Y1-000070), a kit that uses a cell line overexpressing IGF1R. Specifically, as shown in Table 3, it was performed according to the technical manual of the assay kit, and a 4-parameter fit was applied to obtain the results (Table 4, Figure 3).

[0094]

Table 3

[0095]

Table 4

[0096] As a result, as shown in Table 4, when compared with M011 (negative control group), a substance without IGF1, it was confirmed that M005, M008, and M010, which are the PF1802 candidate substances of the present invention, have IGF-1 activity.

[0097] Examples 2-3. Evaluation of GLP-1 activity of PF1802 candidate substances To confirm the GLP-1 activity of the PF1802 candidate substance prepared by the method of Example 1, the GLP-1 activity of the produced substance was measured using the cAMP HunterTM Liraglutide bioassay kit (Discover X, 95-0062Y2-00100). Specifically, as shown in Table 5, it was performed according to the technical manual of the assay kit, and a 4-parameter fit was applied to obtain the results (Table 6, Figure 4).

[0098]

Table 5

[0099]

Table 6

[0100] As a result, as shown in Table 6, it was confirmed that the GLP-1 activities of M005, M008, M010, and M011 were at the same level as exendin-4, so it was found that there was no problem with the GLP-1 activity of the produced substances.

[0101] Example 2-4. Pharmacokinetics (PK) Evaluation of PF1802 Candidate Substances To confirm the pharmacokinetics of the PF1802 candidate substance in blood, mice were subcutaneously administered at a dose of 5 mpk, and blood samples were collected until 144 hours later. Then, based on the ELISA results, PK parameter values were obtained using WinNolin software (Table 7, Figure 5).

[0102]

Table 7

[0103] As a result, as shown in Table 7, the median Tmax of candidate substances M005 and M010 were 48 hours and 16 hours, respectively, and it was confirmed that they were gradually absorbed, and the half-lives were confirmed to be 61 hours and 29.4 hours, respectively.

[0104] Therefore, during actual drug administration, the candidate substances of the present invention exhibit a continuous efficacy, which suggests that the drug administration interval is reduced.

[0105] Example 2-5. Evaluation of the Binding Affinity of PF1802 Candidate Substances Introduced with a Substance for Extending the Blood Half-Life to Human FcRn To confirm the binding affinity of PF1802_M020, M021, and M022 substances, which introduced three types of blood half-life extended Fc into the PF1802_M008 candidate substance, to human FcRn, ELISA was performed. To confirm the difference in binding affinity due to pH, two types at pH 6.0 and pH 7.4 were prepared and their respective binding affinities were measured. Specifically, the PF1802_M020, M021, and M022 substances were each coated on a 96-well immunoplate at 4 μg / mL and reacted at room temperature for 1 hour with PBS (pH 6.0, 7.4) containing 4% skim milk to inhibit non-specific binding. Then, the Human FcRn-GST protein was diluted 4-fold from a maximum concentration of 5 μg / mL to 0.0003 μg / mL with PBS (pH 6.0, 7.4) containing 1% skim milk and dispensed into each well and reacted at room temperature for 1 hour. Then, it was treated with anti-GST Ab-HRP (cytiva, 27457701), and then treated with TMB solution and H 2 SO 4 and treated with, and the absorbance was measured at 450 nm to obtain the results (Figure 6).

[0106] As a result, it was confirmed that in the PF1802_M020, M021, and M022 candidate substances into which blood half-life extended Fc was introduced, the binding affinity to human FcRn increased significantly compared to PF1802_M008. Therefore, excellent binding affinity to human FcRn was confirmed at pH 6.0, which is important for extending the blood half-life.

[0107] Furthermore, in the PF1802_M024 candidate substance subjected to substance optimization in Examples 4 and 5 described below, when the PF1802_M030, M031, and M032 candidate substances into which the same three types of blood half-life extended Fc as described above were introduced were secured (SEQ ID NOs: 35, 36, 37), the binding affinity of the PF1802_M030, M031, and M032 candidate substances also increased, showing excellent binding affinity to human FcRn.

Example

[0108] In vivo efficacy evaluation of PF1802_M008 candidate substance Example 3-1. Evaluation of the Permeability of PF1802_M008 Candidate Substance through the Mouse Brain Blood Barrier (BBB) To confirm the BBB permeability, the IVISense 680 NHS Fluorescent labeling kit (PerkinElmer) was used, and the M011 (GLP1-Fc) candidate substance without IGF-1 was used as the negative control group. Specifically, the fluorescently labeled substance was administered once intraperitoneally to mice (Balb / C male), and then perfusion was performed with physiological saline 24 and 72 hours later. Next, the brains of the mice were removed and imaging analysis was performed using a 2D optical imaging system (IVIS spectrum) (Figure 7).

[0109] As a result, it was confirmed that the fluorescence intensity of the brain tissue was in the order of M008 > M005 > M011. In M005, the confirmed fluorescence intensity was 1.1 times (24 hours) and 1.2 times (72 hours) higher than that of M011, but there was no statistically significant difference. In contrast, in M008, the fluorescence intensity was the highest at 1.7 times (24 hours) and 2.6 times (72 hours) that of M011, and it was confirmed to be statistically significant.

[0110] Therefore, it was found that the PF1802_M008 candidate substance has excellent BBB permeability, and it was found that the BBB permeability of mut.IGF1 is significantly higher than that of wt.IGF1.

[0111] Example 3-2. Efficacy Assay of PF1802_M008 Candidate Substance in a Genetically Modified Mouse Model of Amyotrophic Lateral Sclerosis (ALS) To confirm the efficacy of the PF1802_M008 candidate substance, as shown in Table 8, 9-week-old SOD-1(G39A) mice were subcutaneously administered the PF1802_M008 candidate substance twice a week for 10 weeks. On the 109th day after birth, the mice were sacrificed for immunohistological analysis. For the evaluation of the neuromuscular junction (NMJ), gastrocnemius muscle samples were stained with anti-synatotagmin 2 Ab and alpha-bungarotoxin, and then imaging analysis was performed with a fluorescence microscope (Figure 8).

[0112]

Table 8

[0113] As a result, in the G4 and G5 groups administered with the PF1802_M008 candidate substance, it was confirmed that, in a dose-dependent manner, the fully innervated neuromuscular junctions (NMJ) increased and the denervated NMJ decreased.

[0114] Therefore, it was found that treatment with the PF1802_M008 candidate substance has a preventive and progression-delaying effect on amyotrophic lateral sclerosis disease.

Example

[0115] Optimization of the PF1802 candidate substance Example 4-1. Structure analysis of the PF1802_M008 candidate substance and preparation of the optimized substance For the optimization of the PF1802_M008 candidate substance whose efficacy was confirmed in Example 2 and Example 3, three different types of linkers were introduced to design M023, M024, and M025 candidate substances, and the substances were prepared in the same manner as in Example 1. Next, as a result of performing SDS-PAGE gels under non-reducing and reducing conditions on the prepared M023, M024, and M025 candidate substances, it was confirmed that the M024 and M025 substances were normal except for M023 which was confirmed to be in the cleavage form (Figure 9).

[0116] Example 4-2. Evaluation of the Binding Affinity of PF1802_M024 and M025 Candidate Substances Using SPR To confirm the binding affinities of the optimized PF1802_M024 and M025 prepared in Example 4-1 for GLP-1R and IGF-1R, signal changes due to binding were measured using SPR. Specifically, a CM5 sensor chip was mounted on a BIAcore T200 (GE Healthcare), and then 100 mM N-hydroxysuccinimide (NHS) and 400 mM 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) were injected at a 1:1 ratio to activate the flow cell. Subsequently, an anti-His tag antibody was immobilized at a level of 8000 - 10000 RU. The GLP-1R-His and IGF-1R-His recombinant proteins used as ligands were each injected at a concentration of 2.5 μg / mL for each cycle, captured at a level of 280 - 290 RU, and then the M024 and M025 candidate substances were injected under the conditions shown in Table 9 to measure the SPR binding affinity (Figure 10).

[0117]

Table 9

[0118] As a result, it was confirmed that the SPR binding affinities of the M024 candidate substance were 12.2 nM (GLP-1R) and 18.1 nM (IGF-1R), and those of the M025 candidate substance were 10.6 nM (GLP-1R) and 20.7 nM (IGF-1R).

[0119] Therefore, it was found that both the M024 and M025 candidate substances bind to GLP-1R and IGF-1R, and it was confirmed that there was no significant difference in the binding affinity between the two substances. This suggests that the M024 and M025 candidate substances are excellent in GLP-1 and IGF-1 activities.

Example

[0120] Efficacy Test of PF1802_M024 Candidate Substance Example 5-1. Efficacy Test of PF1802_M024 Candidate Substance in ALS Gene-Modified Mouse Model For the purpose of confirming the efficacy of the PF1802_M024 candidate substance that underwent substance optimization in Example 4, as shown in Table 10, the PF1802_M024 candidate substance was subcutaneously administered to 10-week-old SOD-1(G39A) mice twice a week for 8 weeks. For immunohistological analysis, the mice were sacrificed on the 115th day after birth. Subsequently, for the evaluation of the neuromuscular junction, the gastrocnemius muscle samples were stained with anti-synatotagmin 2 Ab and alpha-bungarotoxin (Figure 11), and for the confirmation of neuroinflammation, the spinal cord samples were stained with anti-Iba1 Ab (Figure 12), and then imaging analysis was performed with a fluorescence microscope.

[0121] [Table 10]

[0122] As a result, in groups G4 and G5 administered with the PF1802_M024 candidate substance, it was confirmed that the fully innervated neuromuscular junctions (NMJ) increased and the denervated NMJ decreased. Also, in groups G4 and G5, it was confirmed that Iba1 indicating the presence or absence of inflammation significantly decreased compared to the untreated ones and showed the same activity as the positive control group.

[0123] Therefore, since the PF1802_M024 candidate substance showed the results of NMJ improvement and Iba1 reduction, it was found that the PF1802 candidate substance has a preventive and delaying effect on amyotrophic lateral sclerosis disease. Also, the result of Iba1 reduction in the spinal cord samples is also a result proving that the PF1802_M024 candidate substance passes through the BBB.

[0124] Example 5-2. Evaluation of the Mouse BBB Permeability of PF1802_M024 Candidate Substances To confirm the BBB permeability, a fluorescently labeled substance was intravenously administered once to a Cranial imaging window mouse model (C57BL / 6N) using an Alexa fluor 647 antibody labeling kit (PerkinElmer), and then imaging was performed at each time point. The brain vessel sites were shown in red (a in Figure 13), and the test substances (PF1802 & Human IgG) were shown in yellow-green (b in Figure 13). The cortex was shown in purple. This was confirmed in the merged image of the brain vessels and the test substances (c in Figure 13).

[0125] As a result, it was confirmed that the PF1802_M024 candidate substance was superior in BBB permeability compared to Human IgG.

[0126] From the above description, those skilled in the technical field to which the present invention pertains will understand that the present invention can be implemented in other specific forms without changing its technical idea and essential features. It should be understood that the above examples are merely illustrative and not restrictive. The present invention should be construed as including all modifications or variations derived from the meaning and scope of the claims and their equivalent concepts, rather than the description.

Claims

Claim 1 A fusion protein comprising GLP-1, an immunoglobulin Fc, and IGF-1. Claim 2 The fusion protein according to claim 1, wherein the immunoglobulin Fc fragment is selected from the group consisting of IgG, IgA, IgD, IgE, IgM, combinations thereof, and hybrids thereof. Claim 3 The fusion protein according to claim 1, wherein the immunoglobulin Fc fragment is derived from IgG1. Claim 4 A pharmaceutical composition for preventing or treating a neurological disease, comprising, as an active ingredient, a fusion protein comprising GLP-1, an immunoglobulin Fc, and IGF-1. Claim 5 The pharmaceutical composition according to claim 4, wherein the neurological disease is any one selected from the group consisting of Parkinson's disease, Alzheimer's disease (senile dementia), stroke, Lou Gehrig's disease, Pick's disease, Creutzfeldt-Jakob disease, Huntington's disease, progressive supranuclear palsy, spinocerebellar degeneration, cerebellar atrophy, multiple sclerosis, amyotrophic lateral sclerosis, peripheral neuropathy, myasthenia gravis, and spinal muscular atrophy. Claim 6 The pharmaceutical composition according to claim 4, wherein the composition has an effect of restoring or improving the function of the neuromuscular junction (NMJ). Claim 7 A method for preventing or treating a neurological disease, comprising the step of administering the composition according to claim 4 to an individual. ​ ​ ​ ​ ​ ​

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