A fusion protein containing GLP-1, immunoglobulin FC, and IGF-1, and its uses.

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 permeability barrier that separates the circulating blood from the brain extracellular fluid in the central nervous system (CNS), and 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.

[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 and 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 permeate the blood-brain barrier.

[0004] Thus, the blood-brain barrier plays a role in preventing bacteria, pathogens, and potentially hazardous substances in the blood from reaching the brain. However, this vascular barrier results in low efficiency of transcranial delivery for most central nervous system drugs, and to compensate for this, these drugs are administered at high doses, which can cause serious side effects in surrounding organs. Therefore, although research is ongoing (Patent Document 1), there is a need to find an efficient drug delivery system that can cross the blood-brain barrier in order to prevent negative systemic effects and ensure the therapeutic effect of chemodrugs. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] Korean Registered Patent Publication No. 10-2211721 [Overview of the project] [Problems that the invention aims to solve]

[0006] The inventors have developed a fusion protein that can cross the blood-brain barrier, exert a sustained effect upon drug administration, and exhibit preventive and progression-delaying effects on neurological diseases, thereby completing the present invention. [Means for solving the problem]

[0007] The present invention aims to provide a fusion protein comprising GLP-1, immunoglobulin Fc, and IGF-1.

[0008] Furthermore, the present invention aims to provide a pharmaceutical composition for the prevention or treatment of neurological diseases, comprising a fusion protein containing GLP-1, immunoglobulin Fc, and IGF-1 as an active ingredient.

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

[0010] Furthermore, the present invention aims to provide a fusion protein comprising GLP-1, immunoglobulin Fc, and IGF-1 for use in the prevention, improvement, or treatment of neurological diseases.

[0011] Furthermore, the present invention aims to provide a pharmaceutical composition containing the aforementioned fusion protein for use in the prevention or treatment of neurological diseases. [Effects of the Invention]

[0012] The fusion protein of the present invention provides a candidate substance for the treatment of neurological diseases with improved blood-brain barrier permeability in a novel form, and can therefore be widely used for the effective treatment of neurological diseases. [Brief explanation of the drawing]

[0013] [Figure 1] This is a schematic diagram showing the structure of the fusion protein PF1802. [Figure 2] This graph shows the results of the Saos-2 proliferation assay, which confirmed the IGF-1 activity of the PF1802 candidate substance. [Figure 3] This graph shows the IGF-1 activity of candidate PF1802 substances confirmed by the IGF-1R phosphorylation assay. [Figure 4] This graph shows the GLP-1 activity of the PF1802 candidate substance confirmed by cAMP assay. [Figure 5] This graph shows the mouse PK results for the PF1802 candidate substance. [Figure 6] This graph shows the ELISA results confirming improved binding affinity to human FcRn for PF1802_M020, M021, and M022, which were introduced with an extended half-life Fc molecule. [Figure 7] This graph shows the mouse BBB transmission of PF1802_M008 confirmed by IVIS imaging analysis. [Figure 8]This is a graph showing the NMJ improvement effect of PF1802_M008 in an amyotrophic lateral sclerosis animal model. [Figure 9] This is a graph showing the SDS-PAGE results confirming the structures of PF1802_M023, M024, and M025 selected by substance optimization. [Figure 10] This is a graph showing the SPR results confirming the binding affinity of PF1802_M024 and M025 selected by substance optimization. [Figure 11] This is a graph showing the NMJ improvement effect of PF1802_M024 in an amyotrophic lateral sclerosis animal model. [Figure 12] This is a graph showing the reduction of Iba1 by PF1802_M024 in an amyotrophic lateral sclerosis animal model. [Figure 13] This is a graph showing the mouse BBB permeability of PF1802_M024.

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 relevant technical field 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, it is intended that such equivalents are also 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] To achieve the above objective, we provide a drug carrier containing IGF-1 (Insulin-like growth factor 1) for blood-brain barrier (BBB) ​​penetration.

[0018] In this invention, the "blood-brain barrier (BBB)" refers to a cellular barrier composed of tight junctions with very high electrical resistance of 0.1 Ω or more between vascular endothelial cells in contact with related pericytes and astrocytes. This barrier has a highly selective permeability that separates circulating blood from brain extracellular fluid in the central nervous system (CNS), and plays a role as a barrier that protects the central nervous system by regulating the inflow and outflow of nutrients and other substances to the brain.

[0019] In the present invention, "drug carrier" means a carrier for efficiently delivering a drug that exhibits therapeutic activity to a target tissue or organ, and the drug carrier of the present invention is particularly characterized by its ability to deliver the drug to the brain with high efficiency by crossing the blood-brain barrier.

[0020] Specifically, the drug carrier of the present invention contains IGF-1, and in 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 to these.

[0021] The drug carrier of the present invention not only exhibits therapeutic efficacy itself, but also efficiently delivers the drug bound to the carrier by allowing it to cross the blood-brain barrier, thereby enabling it to exhibit therapeutic activity in the brain. Furthermore, it has the effect of extending the half-life of the drug, resulting in a longer-lasting effect and a reduction in the number of administrations.

[0022] In one embodiment of the present invention, pharmacokinetic evaluation of a fusion protein containing GLP-1, immunoglobulin Fc, and IGF-1 was performed, and it was confirmed that the half-life was extended (Figure 5). When the fluorescently labeled substance was administered intraperitoneally to mice and the brain was subsequently observed, it was confirmed that the fluorescence intensity increased over time, and that it also exhibited excellent blood-brain barrier (BBB) ​​permeability (Figure 7).

[0023] In this invention, "IGF-1 (Insulin-like growth factor 1)" refers to a cell growth factor structurally similar to insulin, which plays an important role in normal growth and maintaining health. It is derived from a peptide present in the human body. The IGF-1 of this invention includes not only the natural form but also all of its derivatives and variants. The derivatives and variants refer to those in which at least one amino acid has been substituted, deleted, or added to the natural sequence, while maintaining its unique activity. The amino acid sequence of IGF-1 is not particularly limited, but includes the amino acid sequence of SEQ ID NO: 9, or a sequence in which at least one amino acid corresponding to the 3rd, 49th, 67th, 70th positions and combinations thereof has been substituted, based on the amino acid sequence of SEQ ID NO: 9. Specifically, this includes sequences in which the amino acid corresponding to the 3rd position is substituted with alanine, the amino acid corresponding to the 49th position is substituted with alanine, the amino acid corresponding to the 67th position is substituted with threonine, and the amino acid corresponding to the 70th position is substituted with thymine. More specifically, this includes the amino acid sequences of SEQ ID NOs. 10, 11, or 12. The amino acid sequences of SEQ ID NOs. 9, 10, 11, or 12 include amino acid sequences that have 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 NOs. 9, 10, 11, or 12. Furthermore, it goes without saying that proteins having amino acid sequences with partial deletions, modifications, substitutions, conservative substitutions, or additions are also included in this application, as long as they possess such homology or identity and exhibit efficacy equivalent to the proteins of this application.

[0024] Examples include amino acid sequences having additions or deletions of sequences that do not alter the function of the protein of this application, spontaneous mutations, silent mutations, or conservative substitutions at the N-terminus, C-terminus, and / or within the amino acid sequence.

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

[0026] In this application, "homology" or "identity" refers to 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 conserved polynucleotides or proteins is determined by standard sequence algorithms, which may also be used in conjunction with a default gap penalty established by the program used. Substantially, homologous or identical sequences generally hybridize in whole or in part under moderate to high stringent conditions. Hybridization includes hybridization with polynucleotides that have common codons or codons considering codon degeneracy.

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

[0029] For the purposes of the present invention, the IGF-1 of the present invention may include modifications to the wild-type IGF-1 amino acid sequence of SEQ ID NO: 9 for blood-brain barrier crossing. The type of modification introduced for blood-brain barrier crossing is not limited, and any modification introduced to inhibit IGFBP binding may be used. Specifically, the IGF-1 of the present invention may be a mutant containing amino acid substitutions to inhibit IGFBP binding, and more specifically, a mutant containing the amino acids of SEQ ID NO: 10, 11, or 12.

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

[0031] In this invention, "IGFBP (Insulin-like growth factor-binding protein)" plays the role of a transport protein for IGF-1 (insulin-like growth factor 1). In the body, most IGF exists bound to IGFBP proteins, thus inhibiting its target delivery ability. Therefore, the drug carrier of this invention is designed so as not to bind to IGFBP, thereby improving BBB permeability efficiency.

[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] The "Fc-IGF1 drug carrier" in this invention is used in combination with "Fc-IGF1 Hybrid". The Fc-IGF1 drug carrier contains IGF-1 in which binding to IGFBP is inhibited and blood-brain barrier permeability is improved, thereby exhibiting excellent blood-brain barrier permeability, as well as having an extended drug half-life due to binding to immunoglobulin Fc.

[0034] In one embodiment of the present invention, to evaluate the IGF-1 activity of a fusion protein containing GLP-1, immunoglobulin FC, and IGF-1 of the present invention, a proliferation assay and the PathHunter HEK293 IGF-1R bioassay kit were used in the Saos-2 cell line. The results showed that the IGF-1 activity was superior to that of the control group (wild) (Figures 2 and 3).

[0035] In this invention, "immunoglobulin Fc" is a general term for proteins among serum components that play an important role in immunity and possess antibody activity. The basic structure consists of one pair of light chains (L chains) with a molecular weight of approximately 23,000 and one pair of heavy chains (H chains) with a molecular weight of 50,000 to 70,000, linked by disulfide bonds. Depending on the type of H chain, they are classified as IgG, IgA, IgM, IgD, and IgE. The molecular shape is Y-shaped, with the two upper parts being equivalent antibody-binding sites, while the lower part (Fc portion) is the site where antibodies that bind to antigens exhibit biological activity, such as binding to complement or cells. In this invention, "immunoglobulin Fc" is used interchangeably with "immunoglobulin Fc region".

[0036] In the present invention, the immunoglobulin Fc fragment may consist of one to four domains selected from the group consisting of CH1, CH2, CH3, and CH4 domains, and the immunoglobulin Fc fragment may further include a hinge region. The immunoglobulin Fc fragment may also be selected from the group consisting of IgG, IgA, IgD, IgE, IgM, combinations thereof, and hybrids thereof. A specific example of immunoglobulin Fc in the present invention is trastuzumab, but is not limited thereto. Furthermore, the drug carrier of the present invention containing Fc may be modified to have reduced effector function in the body, for example, by modifying N297A.

[0037] In this invention, "IgG (immunoglobulin G)" is a type of immunoglobulin that can cross the placenta. It is divided into four subclasses, IgG1, IgG2, IgG3, and IgG4, based on the level of the constant portion of the H chain in its basic structure.

[0038] In this invention, "human IgG1 Fc" is a type of antibody that accounts for 75% of all immunoglobulins present in human serum. In this invention, the Fc region of human-derived antibody (human IgG1) is used, and this domain is a functional unit of protein used in various pharmaceuticals for the purpose of improving the water solubility of substances and extending their half-life. The immunoglobulin Fc of this invention includes not only the natural form but also all derivatives and variants thereof. The derivatives and variants refer to those in which a mutation such as substitution, deletion, or addition has occurred in at least one amino acid, while maintaining their unique activity. The amino acid sequence of human IgG1 Fc is not particularly limited, but includes the amino acids of SEQ ID NO: 4, or a sequence in which a mutation such as substitution, deletion, or addition has occurred in the amino acids corresponding to the 297th, 309th, 311th, 428th positions and one or more combinations thereof, based on the amino acid sequence of SEQ ID NO: 4. Specifically, it may include a sequence in which the amino acid at position 297 is substituted with alanine, the amino acid at position 309 is substituted with tyrosine, the amino acid at position 311 is substituted with methionine, and the amino acid at position 428 is substituted with leucine. More specifically, it may include the amino acid sequence of SEQ ID NOs. 5, 6, 7, or 8. The amino acid sequence of SEQ ID NOs. 4, 5, 6, 7, or 8 contains an amino acid sequence that has 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 sequence represented by SEQ ID NOs.

[0039] In the present invention, the human IgG1 Fc enhances the preventive or therapeutic effect of neurological diseases by having the function of improving the water solubility of the carrier and extending the in vivo half-life.

[0040] In this invention, FcRn refers to a protein that binds to the Fc region of an IgG antibody. FcRn can be 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 immunoglobulins (antibodies) is mediated by the binding of Fc to FcRn, and the blood half-life and persistence of antibodies largely depend on the binding of the antibody's Fc site to FcRn (neonatal Fc receptor), one of the IgG-binding ligands. Furthermore, the binding of Fc to FcRn also plays an important role in antibody transport.

[0041] In one embodiment of the present invention, a fusion protein containing GLP-1, immunoglobulin Fc, and IGF-1 was found to have excellent binding affinity to FcRn (Figure 6).

[0042] Because the drug carrier of the present invention has high blood-brain barrier permeability, by binding a therapeutically active drug to the drug carrier having an Fc-IGF1 structure, not only is the efficacy of the drug appropriately exerted, but the efficacy is also prolonged due to the extended half-life, thus providing excellent therapeutic effects.

[0043] Specifically, substances for the prevention or treatment of neurological diseases that are expected to have excellent therapeutic effects by being delivered by crossing the blood-brain barrier can be linked to the drug carrier of the present invention. Any substance that can cross the blood-brain barrier and exert a therapeutic effect can be linked to the drug carrier of the present invention, and is not limited to therapeutic agents for specific diseases.

[0044] The substance may be a therapeutic agent for neurological disorders, and examples of such therapeutic agents for neurological disorders include small molecule drugs for the treatment of neurological disorders, peptides, enzymes, antibodies, and proteins. Examples of therapeutic agents for neurological disorders include celecoxib, masitinib, (1-3)IGF1, exenatide BDNF, GDNF, CNTF, iduronate 2-sulfatase (IDS), glucocerebrosidase (GBA), and α-synuclein specific antibody. Specifically, GLP-1 is an example, and the drug carrier Fc-IGF1 of the present invention is in the form of a fusion protein to which GLP-1 is linked, but is not limited to these.

[0045] In this invention, "GLP-1" is a type of gastrointestinal hormone derived from the transcript of the glucagon gene. The GLP-1 of this invention includes not only the natural form but also all of its derivatives and mutants. The derivatives and mutants refer to those which have undergone mutations such as substitution, deletion, or addition of at least one amino acid, while maintaining their unique 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 this invention may include the natural sequence (SEQ ID NO. 2), or it may include SEQ ID NO. 3, which corresponds to the active form with amino acids 7 to 36, or it may be a mutant of GLP-1 that further includes substitution, addition, or deletion of at least one amino acid in those sequences.

[0046] Alternatively, the natural sequence may be modified to have resistance to DPPIV and inhibit its degradation in the body. Examples of modifications include, but are not limited to, substituting the second amino acid, alanine, with another amino acid (e.g., glycine (G)) or a non-natural amino acid.

[0047] In one embodiment of the present invention, the GLP-1 activity of the fusion protein of the present invention was evaluated using the cAMP Hunter™ Liraglutide bioassay kit, and it was confirmed that it exhibited activity at a level equivalent to that of exendin-4, a GLP-1 receptor agonist (Figure 4). In the present invention, the GLP-1 has an anti-inflammatory function in the nerve cells of the carrier, thereby improving the preventive or therapeutic effect of neurological diseases.

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

[0049] Furthermore, the drug carrier of the present invention may be linked to a therapeutic agent for amyotrophic lateral sclerosis (ALS). Examples of therapeutic agents for amyotrophic lateral sclerosis include small molecule drugs for the treatment of neurological diseases, peptides, enzymes, antibodies, and proteins. Examples of therapeutic agents for amyotrophic lateral sclerosis include celecoxib, masitinib, (1-3)IGF1, exenatide BDNF, GDNF, CNTF, iduronate 2-sulfatase (IDS), glucocerebrosidase (GBA), and α-synuclein specific antibody, and specifically GLP-1, but are not limited to these.

[0050] In one embodiment of the present invention, administration of the PF1802_M008 candidate substance of the present invention to a gene-modified mouse model of amyotrophic lateral sclerosis (ALS) resulted in an increase in fully innervated NMJs and a decrease in denervated NMJs, confirming excellent preventive and progression-delaying effects against ALS (Figures 8 and 11).

[0051] Furthermore, the microglia marker Iba1 was significantly reduced compared to the untreated sample, exhibiting activity equivalent to that of riluzole, a known ALS treatment agent, confirming that the substance of the present invention has excellent blood-brain barrier (BBB) ​​permeability (Figure 12).

[0052] The drug carrier and drug of the present invention are linked by various methods known in the art. For example, this includes all forms in which the drug is linked to IGF-1 or Fc via a linker, or directly linked by covalent or non-covalent bonds, and any linking method or final linked form is acceptable as long as it allows for blood-brain barrier penetration and therapeutic effects.

[0053] The fusion protein of the present invention may be in a form in which GLP-1, a first linker, an Fc region, a 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 a fusion of IGF-1 and an immunoglobulin Fc region via a peptide linker. One end of the linker is linked to a single chain of the dimeric immunoglobulin Fc region, but is not limited to this.

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

[0056] Specifically, in the present invention, the linker is GSAPAP(G4S)(SEQ ID NO: 38)(G4S)4GAHS(SEQ ID NO: 39), ASGAGSTTLEVLFQGP(SEQ ID NO: 40), (G)8(SEQ ID NO: 41), or (PA)5(SEQ ID NO: 42), but is not limited to these.

[0057] In the present invention, a fusion protein means a structure comprising GLP-1, immunoglobulin Fc, and IGF-1, and includes, but is not limited to, any of the amino acid sequences represented by SEQ ID NOs. 25 to 37. The amino acid sequences of SEQ ID 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 SEQ ID NOs. The terms "homology" and "identity" are as described above.

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

[0059] A further aspect of the present invention provides a method for producing a fusion protein, comprising the step of linking GLP-1, immunoglobulin Fc, and IGF-1.

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

[0061] A further aspect of the present invention provides a pharmaceutical composition for the prevention or treatment of neurological diseases, comprising a fusion protein containing GLP-1, immunoglobulin Fc, and IGF-1 as an active ingredient.

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

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

[0064] Since the drug carrier or fusion protein of the present invention has excellent blood-brain barrier (BBB) ​​permeability, it is expected that the efficacy of neurological disease treatment agents delivered to the brain by the drug carrier or fusion protein will be improved.

[0065] In this invention, "neurological disease" refers to a disease in which a problem occurs in the nervous system, and includes, but is not limited to, neuromuscular diseases and, more specifically, neuromuscular junction diseases.

[0066] In this invention, "neurological brain diseases" include brain tumors (glioma, meningioma, schwannoma, neurofibroma, pituitary adenoma, craniopharyngioma, metastatic cancer), cerebral infarction, hypertensive cerebral hemorrhage, subarachnoid hemorrhage, subdural hemorrhage, cerebral contusion, arteriovenous malformation, brain abscess, encephalitis, meningitis, varicella, epilepsy, arachnoid cyst, concussion, cerebral palsy, hemifacial spasm, Parkinson's disease, moyamoya disease, migraine, dementia, and the like.

[0067] In this invention, "neuromuscular disease" refers to a disease encompassing direct functional abnormalities of muscles or indirect functional abnormalities of muscles due to abnormalities in nerves and neuromuscular connections, and is also called neuromuscular disorder. When the central nervous system is affected, muscle spasms and paralysis symptoms occur, and the symptoms vary depending on the part of the brain that is affected, but this includes stroke, multiple sclerosis, Parkinson's disease, peripheral nerve diseases, muscle diseases, myasthenia gravis, amyotrophic lateral sclerosis, and spinal muscular atrophy.

[0068] The pharmaceutical composition of the present invention provides, but is not limited to, improved neuromuscular junction function, neuronal protective effects, improved blood-brain barrier permeability, and extended half-life in the body, thereby having preventive or therapeutic effects on nervous system diseases. In the present invention, nervous system diseases are diseases related to dysfunction of the neuromuscular junction, and administration of the fusion protein according to the present invention provides preventive or therapeutic effects on nervous system diseases by improving and restoring the function of the neuromuscular junction.

[0069] In the present invention, "prevention" means any action that prevents or delays neurological diseases by administering the composition of the present invention, and "treatment" means any action that improves or favorably alters the symptoms of neurological diseases by administering the composition of the present invention.

[0070] The pharmaceutical compositions of the present invention may further contain pharmaceutically acceptable carriers, excipients, or diluents. Such pharmaceutically acceptable carriers, excipients, or diluents may be non-naturally occurring. Specifically, the compositions are used by conventional methods in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, as well as topical preparations, suppositories, and sterile injection solutions. Examples of carriers, excipients, and diluents included in the pharmaceutical compositions of the present invention include lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. When formulations are prepared, they are typically prepared using fillers, bulking agents, binders, wetting agents, disintegrants, surfactants, and other diluents or excipients. Examples of oral solid formulations include tablets, pills, powders, granules, and capsules. These solid formulations are prepared by mixing the extract and its fractions with at least one excipient, such as starch, calcium carbonate, sucrose, lactose, or gelatin. In addition to the usual excipients, lubricants such as magnesium stearate and talc are also used. Examples of oral liquid formulations include suspensions, oral solutions, emulsions, and syrups. In addition to water and liquid paraffin, which are commonly used diluents, various excipients such as wetting agents, sweeteners, fragrances, and preservatives are used. Examples of parenteral formulations include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, and suppositories. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include witepsol, macrogol, tween 61, cocoa butter, lauric acid butter, and glycerol gelatin.

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

[0072] In this invention, "individual" refers to any animal, including humans, that possesses or has developed a neurological disease as defined in this invention. By administering the pharmaceutical composition of this invention to an individual, preventive and therapeutic effects for neurological diseases can be obtained.

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

[0074] In the present invention, "administration" means introducing the pharmaceutical composition of the present invention to a subject by any appropriate method, and the administration route can be any common route that can deliver to the target tissue. This includes, but is not limited to, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, and intranasal administration.

[0075] The term "pharmaceutically effective amount" means an amount sufficient to prevent or treat a neurological disorder with a reasonable benefit / risk ratio applicable to medical use. The effective dose level is determined by factors including the individual's species and severity, age, sex, drug activity, sensitivity to the drug, administration time, route of administration and elimination rate, duration of treatment, other drugs used concurrently, 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 to 500 mg / kg per day, specifically 10 to 100 mg / kg, and the administration may be once a day or divided into several doses.

[0076] The composition of the present invention may be administered alone or in combination with other therapeutic agents, or sequentially or simultaneously with conventional therapeutic agents. It may also be administered alone or in multiple doses. It is important to administer the amount that provides the greatest effect with the minimum amount without side effects, taking all of the above factors into consideration, and this can be easily determined by those skilled in the art.

[0077] The compositions 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 neurological diseases.

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

[0079] In this invention, the term "neuromuscular junction (NMJ)" refers to a special structure where the terminal of a motor nerve connects to a muscle, transmitting nerve excitation to the membrane of the muscle fiber; it is a type of synapse. Neuromuscular junction diseases include myasthenia gravis (MG), Lambert-Eaton myasthenic syndrome, botulism, and congenital myasthenic syndrome.

[0080] A further aspect of the present invention provides a pharmaceutical composition for the prevention or treatment of amyotrophic lateral sclerosis (ALS), comprising the drug carrier or fusion protein.

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

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

[0083] A further aspect of the present invention provides a fusion protein comprising GLP-1, immunoglobulin Fc, and IGF-1 for use in the prevention, improvement, or treatment of neurological diseases.

[0084] A further aspect of the present invention provides a pharmaceutical composition comprising the fusion protein for use in the prevention or treatment of neurological diseases.

[0085] The terms "GLP-1," "immunoglobulin Fc," "IGF-1," "neurological diseases," and "pharmaceutical compositions" are as previously described. [Examples]

[0086] The present invention will be described in more detail below with reference to examples. These examples are provided to illustrate the present invention more concretely, and the present invention is not limited to these examples. [Examples]

[0087] Fabrication of fusion proteins To secure candidate substances, GLP1-Fc-IGF1 or GLP1-Fc-mut.IGF1-TfR were synthesized and then cloned into pcDNA3.1 vectors using the Nhe I / Hind III site. The cloned vectors were transfected into CHO-S cells, and the supernatant was collected on day 5. Cells and suspensions were then removed by centrifugation and a 0.22 μm filter. Subsequently, the mixture was purified with protein A resin, and various PF1802_M003~M032 candidate substances (SEQ ID NOs. 13~37) were secured, as shown in Table 1 (Figure 1, Table 1).

[0088] [Table 1-1]

[0089] [Table 1-2] [Examples]

[0090] Activity evaluation of PF1802 candidate substance Example 2-1. Evaluation of IGF-1 activity of PF1802 candidate substance using Saos-2 cell line To confirm the IGF-1 activity of the PF1802 candidate substance prepared by the method of Example 1, an IGF-1 proliferation assay was performed in the Saos-2 cell line. 1 × 10⁶ cells were used per well of the Saos-2 cell line. 4 Each well was dispensed into a 96-well plate and incubated at 37°C and 5% CO2 for 16 hours, followed by 4 hours of starvation. Then, each well was treated with a PF1802 candidate substance diluted 5-fold from the highest concentration of 100 nM to 0.00256 nM, and the cells were incubated at 37°C and 5% CO2 for 48 hours. The cultured cells were stained with SRB (sulforhodamine B), and the absorbance was measured at 540 nm using a microplate reader. The PLA statics function was applied, setting IGF-1 as the reference, and then the relative potency values ​​of the PF1802 candidate substances were obtained (Table 2, Figure 2).

[0091] [Table 2]

[0092] As a result, as shown in Table 2, the IGF-1 activity of the mutant IGF-1 substances M007 and M008 was confirmed compared to the substance using wt.IGF1.

[0093] Example 2-2. Evaluation of IGF-1 activity of PF1802 candidate substance 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, the procedure was carried out according to the technical manual of the assay kit, and the results were obtained by applying a 4-parameter fit (Table 4, Figure 3).

[0094] [Table 3]

[0095] [Table 4]

[0096] As a result, as shown in Table 4, it was confirmed that M005, M008, and M010, which are candidate substances for PF1802 in the present invention, possess IGF-1 activity when compared with M011 (negative control group), which is a substance lacking IGF-1.

[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 Hunter™ Liraglutide bioassay kit (Discover X, 95-0062Y2-00100). Specifically, as shown in Table 5, the procedure was carried out according to the assay kit's technical manual, and the results were obtained by applying a 4-parameter fit (Table 6, Figure 4).

[0098] [Table 5]

[0099] [Table 6]

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

[0101] Examples 2-4. Pharmacokinetic (PK) evaluation of PF1802 candidate substance. To confirm the pharmacokinetics of the PF1802 candidate substance in blood, mice were subcutaneously administered a dose of 5 mpk, and blood samples were collected up to 144 hours later. Subsequently, PK parameter values ​​were obtained using WinNolin software based on the ELISA results (Table 7, Figure 5).

[0102] [Table 7]

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

[0104] Therefore, when the drug is actually administered, the candidate substance of the present invention exhibits sustained efficacy, which suggests that the drug administration interval can be shortened.

[0105] Example 2-5. Evaluation of the binding affinity of PF1802 candidate substances introduced with Fc-derived substances that extend the blood half-life to Human FcRn. To confirm the binding affinity of PF1802_M020, M021, and M022, which are PF1802_M008 candidate substances into which three types of Fc with extended blood half-lives have been introduced, to human FcRn, ELISA was performed. To confirm the difference in binding affinity due to pH, two types of samples were prepared at pH 6.0 and pH 7.4, and the binding affinity of each was measured. Specifically, PF1802_M020, M021, and M022 were each coated at 4 ug / mL on a 96-well immunoplate, and reacted in 4% skim milk-containing PBS (pH 6.0, 7.4) at room temperature for 1 hour to inhibit nonspecific binding. Subsequently, the human FcRn-GST protein was diluted fourfold from a maximum concentration of 5 ug / mL to 0.0003 ug / mL in 1% skim milk-containing PBS (pH 6.0, 7.4), dispensed into each well, and reacted at room temperature for 1 hour. Subsequently, the samples were treated with anti-GST Ab-HRP (cytiva, 27457701), followed by treatment with TMB solution and H2SO4, and the absorbance was measured at 450 nm to obtain the results (Figure 6).

[0106] As a result, it was confirmed that the candidate substances PF1802_M020, M021, and M022, which incorporated a blood half-life extension Fc, showed a significantly increased binding affinity to Human FcRn compared to PF1802_M008. Therefore, superior 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 that underwent material optimization as described in Examples 4 and 5 below, candidate substances PF1802_M030, M031, and M032 were obtained by introducing the same three types of blood half-life prolonging Fc compounds as described above (SEQ ID NOs: 35, 36, 37). As a result, candidate substances PF1802_M030, M031, and M032 also showed increased binding affinity and excellent binding affinity to Human FcRn. [Examples]

[0108] In vivo efficacy evaluation of candidate substance PF1802_M008 Example 3-1. Evaluation of Mouse Brain Blood Barrier (BBB) ​​permeability of candidate substance PF1802_M008. To confirm blood-brain barrier (BBB) ​​transmittance, the IVISense 680 NHS Fluorescent labeling kit (PerkinElmer) was used, and M011 (GLP1-Fc) candidate substances lacking IGF-1 were used as a negative control group. Specifically, the fluorescently labeled substances were administered once intraperitoneally to mice (Balb / C male), followed by perfusion with physiological saline at 24 and 72 hours later. The mouse brains were then extracted and imaged using a 2D optical imaging system (IVIS spectrum) (Figure 7).

[0109] As a result, it was confirmed that the fluorescence intensity of brain tissue was in the order of M008 > M005 > M011. In M005, the observed 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, 1.7 times (24 hours) and 2.6 times (72 hours) higher than that of M011, and this was confirmed to be statistically significant.

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

[0111] Example 3-2. Efficacy testing of the 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, 9-week-old SOD-1(G39A) mice were subcutaneously administered the PF1802_M008 candidate substance twice a week for 10 weeks, as shown in Table 8. On day 109 postnatology, the mice were sacrificed for immunohistochemical analysis. To evaluate the neuromuscular junction (NMJ), gastronemius muscle samples were stained with anti-synatotagmin 2 Ab and alpha-bungarotoxin, and then imaging analysis was performed using a fluorescence microscope (Figure 8).

[0112] [Table 8]

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

[0114] Therefore, it was found that treatment with the candidate substance PF1802_M008 has a preventive effect and a delay in the progression of amyotrophic lateral sclerosis (ALS). [Examples]

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

[0116] Example 4-2. Evaluation of binding strength of candidate PF1802_M024 and M025 materials using SPR. To confirm the binding affinity of the optimized PF1802_M024 and M025 prepared in Example 4-1 to 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 flow cells were activated by injecting 100 mM N-hydroxysuccinimide (NHS) and 400 mM 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) in a 1:1 ratio. Subsequently, anti-His tag antibodies were immobilized at levels of 8000-10000 RU. The recombinant GLP-1R-His and IGF-1R-His proteins used as ligands were injected at a concentration of 2.5 ug / mL in each cycle, captured at a level of 280-290 RU, and then the SPR binding affinity was measured after injecting candidate substances M024 and M025 under the conditions shown in Table 9 (Figure 10).

[0117] [Table 9]

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

[0119] Therefore, it was found that both candidate substances M024 and M025 bind to GLP-1R and IGF-1R, and no significant difference in binding affinity was observed between the two substances. This suggests that candidate substances M024 and M025 exhibit superior GLP-1 and IGF-1 activity. [Examples]

[0120] Efficacy testing of candidate substance PF1802_M024 Example 5-1. Efficacy testing of candidate substance PF1802_M024 in ALS gene-modified mouse model To confirm the efficacy of the PF1802_M024 candidate substance, which was optimized in Example 4, 10-week-old SOD-1(G39A) mice were subcutaneously administered the PF1802_M024 candidate substance twice a week for 8 weeks, as shown in Table 10. For immunohistochemical analysis, the mice were sacrificed at 115 days postnatal. Subsequently, gastronemius muscle samples were stained with anti-synatotagmin 2 Ab and alpha-bungarotoxin to evaluate the neuromuscular junction (Figure 11), and spinal cord samples were stained with anti-Iba1 Ab to confirm neuroinflammation (Figure 12). Imaging analysis was then performed using a fluorescence microscope.

[0121] [Table 10]

[0122] As a result, in the G4 and G5 groups administered with the PF1802_M024 candidate substance, it was confirmed that the number of fully innervated neuromuscular junctions (NMJs) increased, while the number of denervated NMJs decreased. Furthermore, in the G4 and G5 groups, Iba1, an indicator of inflammation, decreased significantly compared to the untreated group, and it was confirmed that the activity was equivalent to that of the positive control group.

[0123] Therefore, since the PF1802_M024 candidate substance showed 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. Furthermore, the result of Iba1 reduction in spinal cord samples also proves that the PF1802_M024 candidate substance can cross the blood-brain barrier (BBB).

[0124] Example 5-2. Evaluation of Mouse BBB transmission rate of candidate substance PF1802_M024 To confirm blood-brain barrier (BBB) ​​permeability, fluorescently labeled substances were administered intravenously once to a cranial imaging window mouse model (C57BL / 6N) using the Alexa fluor 647 antibody labeling kit (PerkinElmer), and imaging was then performed at various time intervals. Brain vessel regions are shown in red (Figure 13a), and the test substances (PF1802 & Human IgG) are shown in yellow-green (Figure 13b). The cortex is shown in purple, which is confirmed by the merged image of brain vessels and the test substances (Figure 13c).

[0125] As a result, it was confirmed that the PF1802_M024 candidate substance exhibits superior blood-brain barrier (BBB) ​​permeability compared to human IgG.

[0126] From the above description, those skilled in the art in the field to which the present invention pertains will understand that the present invention can be implemented in other specific forms without altering its technical idea or essential features. It should be understood that the above embodiments are merely illustrative and not limiting. The present invention should be interpreted as encompassing all modified or altered forms derived from the meaning and scope of the claims and their equivalent concepts, rather than the specification.

Claims

1. It contains GLP-1, immunoglobulin Fc, and IGF-1. IGF-1 is an IGF-1 variant containing the amino acid sequence of SEQ ID NO: 10, 11, or 12. Fusion protein.

2. The immunoglobulin Fc is derived from an immunoglobulin selected from the group consisting of IgG, IgA, IgD, IgE, and IgM. The fusion protein according to claim 1.

3. The immunoglobulin Fc is derived from IgG1. The fusion protein according to claim 1.

4. It contains a fusion protein containing GLP-1, immunoglobulin Fc, and IGF-1 as an active ingredient. IGF-1 is an IGF-1 variant containing the amino acid sequence of SEQ ID NO: 10, 11, or 12. Pharmaceutical compositions for the prevention or treatment of neurological disorders.

5. The aforementioned neurological disorders are 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. The pharmaceutical composition according to claim 4.

6. The aforementioned composition has the effect of restoring or improving the function of the neuromuscular junction (NMJ). The pharmaceutical composition according to claim 4.

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