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, an immunoglobulin Fc region and IGF-1, and uses thereof. The fusion protein provided by the invention has excellent efficiency in the aspect of blood-brain barrier permeability, so that the fusion protein can be widely applied to effective treatment of nervous system diseases.

Description

[Technical field]

[0001] The present invention relates to a fusion protein containing GLP-1, immunoglobulin Fc region and IGF-1 and use thereof. [Background Technology]

[0002] The blood-brain barrier (BBB) ​​is a cellular barrier composed of tight junctions with a high electrical resistance of at least 0.1 Ω·m between vascular endothelial cells adjacent to associated pericytes and astrocytes. The BBB acts as a barrier with high selective permeability, separating circulating blood from brain extracellular fluid in the central nervous system (CNS), thereby playing a role in protecting the pathways of the CNS by regulating the passage of nutrients and other substances into and out of the brain.

[0003] Normally, the blood-brain barrier allows not only the selective transfer of molecules essential for brain function, such as glucose and amino acids, but also the passage of water, some types of gases, and fat-soluble molecules by passive diffusion. However, the blood-brain barrier prevents the entry and exit of lipophilic, potential neurotoxins by an active transport mechanism mediated by the permeable p-glycoprotein. As a result, drugs for neurological diseases such as drugs with large molecular weights that need to act in the brain or low molecular weight drugs with low brain permeability cannot penetrate the blood-brain barrier.

[0004] Therefore, the blood-brain barrier is used to block bacteria, pathogens and potentially harmful substances in the blood from being transported to the brain through the blood, but due to this vascular barrier, most central nervous system drugs show low efficiency of transcranial delivery, and to compensate for this, these drugs are administered in high doses, which may cause serious side effects in peripheral organs. Therefore, although research is still ongoing (KR 10-2211721), there is still a need to find an effective drug delivery system that can penetrate the blood-brain barrier to prevent negative systemic effects and ensure the therapeutic effect of chemotherapeutic drugs.

[0005] [Disclosure]

[0006] [Technical issues]

[0007] The present invention has developed a fusion protein that can penetrate the blood-brain barrier, exhibits sustained efficacy after administration, and exhibits effects of preventing and delaying the progression of nervous system diseases, and thus the present invention has been completed.

[0008] [Technical solution]

[0009] According to one aspect of the present invention, a fusion protein containing GLP-1, an immunoglobulin Fc region and IGF-1 is provided.

[0010] According to another aspect of the present invention, a pharmaceutical composition for preventing or treating a nervous system disease is provided, wherein the pharmaceutical composition contains a fusion protein as an active ingredient, wherein the fusion protein contains GLP-1, an immunoglobulin Fc region and IGF-1.

[0011] According to another aspect of the present invention, there is provided a method for preventing or treating a nervous system disease, the method comprising administering the composition to a subject.

[0012] According to another aspect of the present invention, there is provided use of a fusion protein containing GLP-1, an immunoglobulin Fc region and IGF-1 for preventing, alleviating or treating nervous system diseases.

[0013] According to another aspect of the present invention, there is provided use of the pharmaceutical composition for preventing or treating nervous system diseases.

[0014] [Beneficial Effects]

[0015] The fusion protein of the present invention provides a novel candidate substance for treating nervous system diseases, which has enhanced blood-brain barrier penetration efficiency and can therefore be widely used in the effective treatment of nervous system diseases. [Brief Description of the Figures]

[0017] Figure 1 Schematic diagram showing the structure of fusion protein PF1802.

[0018] Figure 2 Shown are graphs illustrating the results of a Saos-2 proliferation assay to investigate IGF-1 activity in the PF1802 candidate substance.

[0019] Figure 3 A diagram showing the confirmation of the IGF-1 activity of the PF1802 candidate substance by an IGF-1R phosphorylation assay.

[0020] Figure 4 A graph showing the GLP-1 activity of the PF1802 candidate substance confirmed by a cAMP assay.

[0021] Figure 5 A schematic diagram showing the mouse PK results of the PF1802 candidate substance.

[0022] Figure 6 Shown are graphs illustrating the results of ELISA to investigate the increased binding affinity of PF1802_M020, M021, and M022 to human FcRn with the addition of Fc induced increased blood half-life.

[0023] Figure 7 Shown are graphs demonstrating mouse BBB penetration by PF1802_M008 by IVIS imaging analysis.

[0024] Figure 8 Shown are graphs demonstrating the NMJ improving effect of PF1802_M008 in an amyotrophic lateral sclerosis (ALS) animal model.

[0025] Fig. 9 SDS-PAGE results are shown to investigate the structures of PF1802_M023, M024, and M025 selected by substance optimization.

[0026] Fig.10 Shown are graphs illustrating the SPR results to investigate the binding affinities of PF1802_M024 and M025 selected by substance optimization.

[0027] Fig.11 Shown are graphs demonstrating the NMJ improving effect of PF1802_M024 in an amyotrophic lateral sclerosis (ALS) animal model.

[0028] Fig.12 Shown are graphs demonstrating Iba1 reduction by PF1802_M024 in an amyotrophic lateral sclerosis (ALS) animal model.

[0029] Fig.13 Shown are graphs demonstrating mouse BBB penetration of PF1802_M024.

[0030] [Best Practice]

[0031] The present application will be described in detail below. Each description and exemplary embodiment disclosed herein can also be applied to other descriptions and exemplary embodiments. That is, all combinations of various elements disclosed herein fall within the scope of the present invention. In addition, the scope of the present invention is not limited by the following specific description.

[0032] Furthermore, those skilled in the art will recognize or be able to ascertain many equivalents to the specific embodiments of the invention described herein using only routine experimentation. Additionally, such equivalents are intended to be encompassed by the present invention.

[0033] According to one aspect of the present invention, a fusion protein containing GLP-1, an immunoglobulin Fc region and IGF-1 is provided.

[0034] To achieve this aspect, a drug delivery system for blood-brain barrier (BBB) ​​penetration is provided, which contains insulin-like growth factor 1 (IGF-1).

[0035] The term "blood-brain barrier" or "(BBB)" as used herein refers to a cellular barrier composed of tight junctions having a remarkably high electrical resistance of at least 0.1Ω between vascular endothelial cells adjacent to associated pericytes and astrocytes, and which acts as a barrier with high selective permeability to separate circulating blood from brain extracellular fluid in the central nervous system (CNS), thereby playing a role in protecting the passage of nutrients and other substances into and out of the brain.

[0036] As used herein, the term "drug delivery system" refers to a delivery system for effectively delivering a drug showing therapeutic activity to a target tissue or organ, and the drug delivery system of the present invention can particularly penetrate the blood-brain barrier to efficiently deliver the drug to the brain.

[0037] Specifically, the drug delivery system of the present invention includes IGF-1, and in a more specific aspect, the drug delivery system may be in the form of a protein in which IGF-1 and an immunoglobulin Fc region are linked, but is not limited thereto.

[0038] The drug delivery system of the present invention can show therapeutic efficacy as a drug delivery system itself, and effectively delivers the drug connected to the drug delivery system by penetrating the blood-brain barrier, thereby allowing the drug to show therapeutic activity in the brain. In addition, the drug delivery system increases the half-life of the drug, so that the drug efficacy lasts longer and reduces the number of administrations.

[0039] In one embodiment of the present invention, the results of the pharmacokinetic evaluation of a fusion protein containing GLP-1, an immunoglobulin Fc region and IGF-1 show that the half-life of the fusion protein is increased ( Figure 5 ), and the results of brain observation after intraperitoneal injection of fluorescent markers in mice showed that the fluorescence intensity increased over time, indicating that the BBB penetration was excellent ( Figure 7 ).

[0040] The term "insulin-like growth factor 1" or "IGF-1" as used herein refers to a cell growth factor having a similar structure to insulin, which plays an important role in normal growth and health maintenance. IGF-1 is derived from a peptide present in the human body. The IGF-1 of the present invention includes not only the natural type, but also its derivatives or mutants. A derivative or mutant refers to a derivative or mutant in which at least one amino acid in the natural sequence is mutated by substitution, deletion, addition, etc., and retains its original activity. The amino acid sequence of IGF-1 is not particularly limited, and may include the amino acid sequence of SEQ ID NO:9, or may include a sequence in which a mutation such as substitution, deletion or addition occurs at the amino acid corresponding to at least one position of position 3, 49, 67, 70 and a combination thereof in the amino acid sequence of SEQ ID NO:9. Specifically, the amino acid corresponding to position 3 may be substituted by alanine, the amino acid corresponding to position 49 may be substituted by alanine, the amino acid corresponding to position 67 may be substituted by threonine, and the amino acid corresponding to position 70 may be substituted by thymine. More specifically, the amino acid sequence of SEQ ID NO:10, 11 or 12 may be included. The amino acid sequence of SEQ ID NO: 9, 10, 11 or 12 may include an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% homology or identity with the amino acid sequence listed in SEQ ID NO: 9, 10, 11 or 12. Obviously, any protein having an amino acid sequence having a deletion, modification, substitution, conservative substitution or addition in a part thereof may also be included in the scope of the present application, as long as the amino acid sequence has such homology or identity and exhibits the efficacy corresponding to the protein of the present application.

[0041] For example, the amino acid sequence may include an amino acid sequence with sequence additions or deletions, naturally occurring mutations, silent mutations, or conservative substitutions that do not change the function of the protein of the present application at the N-terminus, C-terminus, and / or within the amino acid sequence.

[0042] As used herein, the term "conservative substitution" refers to the replacement of one amino acid with another amino acid having similar structure and / or chemical properties. The protein may have, for example, one or more conservative substitutions while still retaining one or more biological activities. Such amino acid substitutions can generally be made based on the similarity of the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic properties of the residues. For example, among the charged amino acids, positively charged (basic) amino acids include arginine, lysine, and histidine; negatively charged (acidic) amino acids include glutamic acid and aspartic acid. Among the uncharged amino acids, non-polar 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. Among the amino acids, aromatic amino acids include phenylalanine, tryptophan, and tyrosine.

[0043] As used herein, the term "homology" or "identity" refers to the degree of similarity between two given amino acid sequences or nucleic acid base sequences, and can be expressed as a percentage. The terms homology and identity are often used interchangeably.

[0044] The sequence homology or identity of conservative polynucleotides or proteins can be determined by standard comparison algorithms, and can be used together with the default gap penalty established by the program to be used. Substantially homologous or identical sequences can hybridize along their entire sequence or part thereof under moderate or high stringency conditions. Obviously, hybridization also includes hybridization with polynucleotides containing common codons or codons that take into account the degeneracy of codons in the polynucleotides.

[0045] In the present invention, IGF-1 has the effects of protecting neurons, protecting neurons against excitatory substances, and improving the permeability of the blood-brain barrier (BBB), thereby enhancing the preventive or therapeutic effects on nervous system diseases.

[0046] For the purpose 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 to increase blood-brain barrier permeability. There is no limitation on the type of modification introduced to the blood-brain barrier permeability, but any modification introduced to inhibit the ability to bind to IGFBP may be included in the present invention without limitation. Specifically, the IGF-1 of the present invention may be a mutant including an amino acid substitution that inhibits the ability to bind to IGFBP, more specifically, a mutant including the amino acid sequence of SEQ ID NO: 10, 11 or 12.

[0047] For example, at least one amino acid substitution in the IGF-1 sequence can lead to inhibition of binding to the IGFBP protein, and non-limiting examples of amino acids that can be included in the IGF-1 of the present invention can include the following: E3A, F49A, A67T, A70T, etc. The above symbols represent the amino acid before substitution, the position of substitution, and the amino acid after substitution, respectively. For example, E3A refers to glutamic acid (E), which corresponds to the amino acid at the third position based on the wild-type IGF-1 sequence, which is replaced by alanine (A). The drug delivery system containing IGF-1 of the present invention can maintain excellent blood-brain barrier permeability by including at least one amino acid substitution selected from the above amino acid substitutions.

[0048] As used herein, the term "insulin-like growth factor binding protein" or "IGFBP" is used as a transporter for "insulin-like growth factor 1" or "IGF-1". Most of the IGF in the body exists in a state of binding to the IGFBP protein group, inhibiting its targeted delivery ability. Therefore, the drug delivery system of the present invention can be designed not to bind to IGFBP, thereby increasing the BBB penetration efficiency.

[0049] In a specific aspect of the present invention, a drug delivery system having an Fc-IGF1 structure may be provided, wherein IGF-1 is linked to an immunoglobulin Fc region.

[0050] Herein, the term "Fc-IGF1 drug delivery system" may be used interchangeably with "Fc-IGF1 hybrid." The Fc-IGF1 drug delivery system not only exhibits excellent blood-brain barrier penetration efficiency by including IGF-1, which has inhibited binding to IGFBP and improved blood-brain barrier permeability, but also exhibits increased drug half-life by binding to the immunoglobulin Fc region.

[0051] In one embodiment of the present invention, the IGF-1 activity of a fusion protein containing GLP-1, an immunoglobulin Fc region and IGF-1 was evaluated using a penetration assay and a PathHunterHEK293 IGF-1R bioassay kit in a Saos-2 cell line, and the results showed that the fusion protein exhibited superior IGF-1 activity compared to the control (wild type) ( Figure 2 and Figure 3 ).

[0052] As used herein, the term "immunoglobulin Fc" refers to a general term for a protein that plays an important role in immunity and acts as an antibody in serum components. Its basic structure consists 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 50,000-70,000, connected by SS bridges. According to the type of H chain, these chains are classified as IgG, IgA, IgM, IgD or IgE. The immunoglobulin Fc region has a Y-shaped molecule, in which the two top ends correspond to equivalent antigen binding sites, and the bottom end (Fc fragment) is a site that shows biological activity, and antibody binding antigens show this biological activity, such as binding complement or cells. In this article, "immunoglobulin Fc" can be used interchangeably with "immunoglobulin Fc region".

[0053] In the present invention, the immunoglobulin Fc fragment can be composed of one to four domains selected from CH1, CH2, CH3 and CH4 domains, and the immunoglobulin Fc fragment can further include a hinge region. The immunoglobulin Fc fragment can be selected from IgG, IgA, IgD, IgE, IgM, a combination thereof and a hybrid thereof. A specific example of the immunoglobulin Fc region of the present invention can be trastuzumab, but is not limited thereto. The immunoglobulin Fc region can be mutated so that the drug delivery system containing Fc of the present invention has a reduced effector function in vivo, and for example, can include a N297A mutation.

[0054] As used herein, the term "immunoglobulin G" or "IgG" refers to a type of immunoglobulin with placental permeability. According to the upper part of the H chain constant region, IgG is divided into four subclasses: IgG1, IgG2, IgG3 and IgG4.

[0055] As used herein, the term "human IgG1 Fc" refers to one of the antibody types, accounting for 75% of the total immunoglobulin in human serum. In the present invention, the Fc region of a human antibody (human IgG1) is used, and the corresponding domain is a protein functional group, which is used in various drugs to improve the water solubility and half-life of a substance. The immunoglobulin Fc region of the present invention includes not only the natural form, but also its derivatives or mutants. A derivative or mutant refers to a derivative or mutant in which at least one amino acid is mutated by substitution, deletion, addition, etc., and retains its original activity. The amino acid sequence of IgG1 Fc is not particularly limited, and may include the amino acid sequence of SEQ ID NO: 4, or may include a sequence in which a mutation such as substitution, deletion or addition occurs at an amino acid corresponding to at least one position in the amino acid sequence of SEQ ID NO: 4, 309, 311, 428, and a combination thereof. Specifically, the amino acid corresponding to position 297 may be substituted with alanine, the amino acid corresponding to position 309 may be substituted with tyrosine, the amino acid corresponding to position 311 may be substituted with methionine, and the amino acid corresponding to position 428 may be substituted with leucine. More specifically, the amino acid sequence of SEQ ID NO: 5, 6, 7 or 8 may be included. The amino acid sequence of SEQ ID NO: 4, 5, 6, 7 or 8 may include an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% homology or identity with the amino acid sequence listed in SEQ ID NO: 4, 5, 6, 7 or 8. "Homology" and "identity" are as described above.

[0056] In the present invention, human IgG1 Fc has the function of increasing the water solubility and in vivo half-life of the carrier, thereby enhancing the preventive or therapeutic effect on nervous system diseases.

[0057] In the present invention, FcRn refers to a protein that is combined with 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 is reported that the in vivo half-life of immunoglobulins (antibodies) is mediated by the combination of Fc and FcRn, and the half-life and persistence of antibodies in the blood depend to a great extent on the combination between the Fc region of the antibody and the neonatal Fc receptor (FcRn), which is one of the IgG binding ligands. In addition, the combination of Fc and FcRn also plays an important role in antibody delivery.

[0058] In one embodiment of the present invention, a fusion protein containing GLP-1, an immunoglobulin Fc region and IGF-1 exhibits excellent binding affinity to FcRn ( Figure 6 ).

[0059] The drug delivery system of the present invention has high blood-brain barrier penetration efficiency, so the combination of a therapeutically active drug with a drug delivery system having an Fc-IGF1 structure can ensure appropriate efficacy of the drug and long-term efficacy due to an increased half-life, resulting in excellent therapeutic effects.

[0060] Specifically, substances for preventing or treating nervous system diseases can be connected to the drug delivery system of the present invention, and the substances can be expected to show excellent therapeutic effects by delivering drugs to penetrate the blood-brain barrier, but any substance that penetrates the blood-brain barrier to show therapeutic effects can be connected to the drug delivery system of the present invention, without being limited to therapeutic agents for specific diseases.

[0061] The substance may be a therapeutic agent for a nervous system disease, and the therapeutic agent for a nervous system disease may be, for example, a small molecule drug, a peptide, an enzyme, an antibody, etc., for treating a nervous system disease. Examples of therapeutic agents for nervous system diseases may include celecoxib, masitinib, (1-3) IGF1, exenatide BDNF, GDNF, CNTF, iduronate 2-sulfatase (IDS), glucocerebrosidase (GBA), α-synuclein specific antibodies, etc., and a specific example thereof may be GLP-1. The drug delivery system Fc-IGF1 of the present invention may be in the form of a fusion protein connected to GLP-1, but is not limited thereto.

[0062] As used herein, the term "GLP-1" refers to a class of gastrointestinal hormones derived from glucagon gene transcripts. The GLP-1 of the present invention includes not only the natural form, but also its derivatives or mutants. A derivative or mutant refers to a derivative or mutant in which at least one amino acid is mutated by substitution, deletion, addition, etc., and retains its original activity. The amino acid sequence of GLP-1 may include, but is not limited to, any one of SEQ ID NOs: 1 to 3. The GLP-1 of the present invention may include the natural sequence (SEQ ID NO: 2), and may also include SEQ ID NO: 3 corresponding to amino acids 7-36 in its active form, or may also include GLP-1 mutants in which at least one amino acid in these sequences is substituted, added, deleted, etc.

[0063] Alternatively, GLP-1 may be mutated to have resistance to DPPIV in the native sequence, thereby inhibiting its degradation in vivo. As an example of mutation, the second amino acid alanine may be substituted with another amino acid (eg, glycine (G)) or a non-natural amino acid, but is not limited thereto.

[0064] In one embodiment of the present invention, the GLP-1 activity of the fusion protein of the present invention was evaluated using a cAMP HunterTM liraglutide bioassay kit, and the results showed that the fusion protein exhibited a similar level of activity to the GLP-1 receptor agonist exendin-4 ( Figure 4 ). In the present invention, GLP-1 acts as a carrier in neurons to exert anti-inflammatory effects and enhance the prevention or treatment effects on nervous system diseases.

[0065] In the present invention, the fusion protein containing GLP-1, immunoglobulin Fc region and IGF-1 is named PF1802.

[0066] In addition, the drug delivery system of the present invention can be connected to an amyotrophic lateral sclerosis (ALS) therapeutic agent. The therapeutic agent of amyotrophic lateral sclerosis can be, for example, a small molecule drug, peptide, enzyme, antibody, protein, etc. for treating amyotrophic lateral sclerosis. Examples of therapeutic agents for amyotrophic lateral sclerosis can include celecoxib, masitinib, (1-3) IGF1, exenatide BDNF, GDNF, CNTF, iduronate 2-sulfatase (IDS), glucocerebrosidase (GBA), alpha-synuclein specific antibodies, etc., and a specific example thereof can be GLP-1, but is not limited thereto.

[0067] In one embodiment of the present invention, since the PF1802_M008 candidate substance of the present invention is administered to a transgenic mouse model of amyotrophic lateral sclerosis, the fully innervated neuromuscular junction (NMJ) increases, while the denervated neuromuscular junction (NMJ) decreases. Therefore, it is confirmed that the candidate substance of the present invention exhibits excellent effects of preventing and delaying the progression of amyotrophic lateral sclerosis ( Figure 8 and Fig.11 ).

[0068] In addition, the substance of the present invention showed a significant decrease in the microglial marker Iba1 compared to untreated and had activity similar to riluzole, which is known as an ALS therapeutic agent, confirming excellent BBB penetration ( Fig.12 ).

[0069] The drug delivery system of the present invention and the drug can be connected by various methods known in the art. Examples of connection can include connecting the drug to IGF-1 or Fc through a linker, or directly connecting through covalent or non-covalent connection, and the connection is not limited to a specific connection method or the form of the final connection, as long as the connection allows blood-brain barrier penetration and therapeutic effects.

[0070] The fusion protein of the present invention may have a form in which GLP-1, the first linker, the Fc region, the second linker, and IGF-1 are linked from the N-terminus.

[0071] Each of the linkers is a peptide linker, and the drug delivery system of the present invention may be a drug delivery system in which IGF-1 and the immunoglobulin Fc region are fused via a peptide linker. One end of the linker may be connected to one chain of the immunoglobulin Fc region of the dimer, but is not limited thereto.

[0072] The peptide linker may contain one or more amino acids, for example, 1 to 1000 amino acids. The peptide linker may be any peptide linker known in the art, for example, including [GS]x linker, [GGGS]x linker, and [GGGGS]x linker, etc., wherein x may be a natural number of 1 or more (e.g., 1, 2, 3, 4, 5 or more), but is not limited thereto.

[0073] Specifically, the linker of the present invention can be 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 thereto.

[0074] In the present invention, the fusion protein may refer to a structure including GLP-1, an immunoglobulin Fc region, and IGF-1, and may include any one of the amino acid sequences shown in SEQ ID NOs: 25 to 37, but is not limited thereto. The amino acid sequences of SEQ ID NOs: 25 to 37 may include amino acid sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% homology or identity with the amino acid sequences listed in SEQ ID NOs: 25 to 37. "Homology" and "identity" are as described above.

[0075] According to another aspect of the present invention, there is provided a method for producing a drug delivery system, the method comprising linking IGF-1 and an immunoglobulin Fc region.

[0076] According to another aspect of the present invention, there is provided a method for producing a fusion protein, the method comprising linking GLP-1, an immunoglobulin Fc region and IGF-1.

[0077] "GLP-1", "IGF-1", "Fc" and "drug delivery system" are as described above.

[0078] According to another aspect of the present invention, a pharmaceutical composition for preventing or treating a nervous system disease is provided, wherein the pharmaceutical composition contains a fusion protein as an active ingredient, wherein the fusion protein contains GLP-1, an immunoglobulin Fc region and IGF-1.

[0079] "GLP-1", "immunoglobulin Fc" region, "IGF-1" and "fusion protein" are as described above.

[0080] The pharmaceutical composition may comprise a pharmaceutically effective amount of the fusion protein.

[0081] The drug delivery system or fusion protein of the present invention has excellent blood-brain barrier (BBB) ​​penetration efficiency, and thus the efficacy of a therapeutic agent for a nervous system disease delivered to the brain via the drug delivery system or fusion protein can be expected.

[0082] As used herein, the term "neurological disease" refers to a disease causing problems in the nervous system, and may include neurological brain diseases, neurological muscle diseases, etc., and may specifically include neuromuscular junction diseases, but is not limited thereto.

[0083] As used herein, the term "neurological brain disease" corresponds to brain tumors (glioma, meningioma, schwannoma, neurofibroma, pituitary adenoma, craniopharyngioma, metastatic brain tumor), cerebral infarction, hypertensive cerebral hemorrhage, subarachnoid hemorrhage, subdural hemorrhage, cerebral contusion, brain arteriovenous malformation, brain abscess, encephalitis, meningitis, hydrocephalus, epilepsy, arachnoid cyst, concussion, cerebral palsy, hemifacial spasm, Parkinson's disease, moyamoya disease, migraine, dementia, etc.

[0084] As used herein, the term "neuromuscular disease" refers to a disease that covers indirect muscle dysfunction caused by direct muscle disease or abnormal nerves or neuromuscular junctions, and is also referred to as a neuromuscular disorder. If the central nervous system is affected, muscle spasms and paralysis occur, and different symptoms appear depending on the affected area of ​​the brain. Examples thereof may include stroke, multiple sclerosis, Parkinson's disease, peripheral neuropathy, myopathy, myasthenia gravis, amyotrophic lateral sclerosis, and spinal muscular atrophy.

[0085] The pharmaceutical composition of the present invention can have a preventive or therapeutic effect on neurobiological diseases by showing improvement in neuromuscular junction function, neuron protection, improvement in blood-brain barrier permeability and increase in in vivo half-life, but is not limited thereto. The nervous system diseases of the present invention are related to dysfunction of the neuromuscular junction, and the administration of the fusion protein according to the present invention can have a preventive or therapeutic effect on nervous system diseases by improving and restoring the function of the neuromuscular junction.

[0086] As used herein, the term "prevention" refers to any action to inhibit or delay a neurological disease by administering the composition of the present invention, and the term "treatment" refers to any action to alleviate or favorably change the symptoms of a neurological disease by administering the composition.

[0087] The pharmaceutical composition of the present invention may further contain a pharmaceutically acceptable carrier, excipient or diluent. A pharmaceutically acceptable carrier, excipient or diluent may be non-natural. Specifically, the composition may be prepared in the following forms according to a common method: oral preparations, such as powders, granules, tablets, capsules, suspensions, emulsions, syrups or aerosols; external preparations; suppositories; and sterile injections. Examples of carriers, excipients and diluents that may be contained in the pharmaceutical composition of the present invention may include lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, etc. The pharmaceutical composition may be prepared by using a commonly used diluent or excipient, such as a filler, a bulking agent, a binder, a wetting agent, a disintegrant or a surfactant. Exemplary solid preparations for oral administration may include tablets, pills, powders, granules, capsules, etc., and such solid preparations may be prepared by mixing an extract or its fraction with at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. Alternatively, in addition to simple excipients, lubricants such as magnesium stearate and talcum powder may be used. Liquid preparations for oral administration are equivalent to suspensions, internal solution preparations, emulsions, syrups, etc., and may contain not only commonly used simple diluents such as water and liquid paraffin, but may also contain several types of excipients such as wetting agents, sweeteners, flavoring agents, and preservatives. Examples of preparations for parenteral administration include sterile aqueous solutions, nonaqueous solvents, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of nonaqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc. Examples of suppository bases include Witepsol, polyethylene glycol, Tween 61, cocoa butter, laurin butter, glycerinated gelatin and the like.

[0088] According to another aspect of the present invention, there is provided a method for preventing or treating a nervous system disease, the method comprising administering a drug delivery system, a fusion protein or a pharmaceutical composition containing the same to a subject.

[0089] As used herein, the term "subject" may refer to any animal, including humans, that suffers from or develops the nervous system disease of the present invention. Administering the pharmaceutical composition of the present invention to a subject may result in preventive and therapeutic effects on nervous system diseases.

[0090] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount.

[0091] As used herein, the term "administer" refers to introducing the pharmaceutical composition of the present invention into a subject by any suitable method, and the pharmaceutical composition can be administered by any typical route as long as the pharmaceutical composition can reach the target tissue. The pharmaceutical composition can be administered intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, orally, topically or intranasally, but is not limited thereto.

[0092] The term "pharmaceutically effective amount" as used herein refers to an amount sufficient to prevent or treat a nervous system disease at a reasonable benefit / risk ratio suitable for medical use, and the level of an effective dose can be determined based on factors including the type of disease, severity of the disease, age and sex of the subject, drug activity, sensitivity to the drug, time of administration, route of administration, excretion rate, duration of treatment, and drugs used in combination with the composition, as well as other factors known in the medical field. For example, a drug delivery system, a fusion protein, or a pharmaceutical composition containing them can be administered at a dose of 0.01 to 500 mg / kg, specifically 10 to 100 mg / kg / day, and administration can be performed once a day or divided into several times.

[0093] The composition of the present invention can be used as a single therapeutic agent or in combination with other therapeutic agents, and can be used sequentially or simultaneously with conventional therapeutic agents. In addition, the composition can be used once or multiple times. Taking all factors into consideration, it is important to use the smallest possible dose without any adverse reactions, thereby achieving the maximum effect. The dosage can be determined by those skilled in the art.

[0094] The composition of the present invention can be used alone or in combination with surgery, hormone therapy, chemotherapy and methods using biological response modifiers to prevent or treat nervous system diseases.

[0095] In the present invention, the composition may have the effect of restoring or improving the function of neuromuscular junctions (NMJs).

[0096] As used herein, the term "neuromuscular junction" or "NMJ" refers to a type of synapse with a specific structure in which a motor neuron is attached to a muscle at its terminal end and the nerve impulse is transmitted to the membrane of the muscle fiber. Exemplary neuromuscular junction diseases include myasthenia gravis (MG), Lambert-Eaton myasthenic syndrome, botulism, and congenital myasthenic syndrome.

[0097] According to another aspect of the present invention, a pharmaceutical composition for preventing or treating amyotrophic lateral sclerosis (ALS) is provided, wherein the pharmaceutical composition contains the drug delivery system or the fusion protein.

[0098] "Drug delivery system", "fusion protein" and "pharmaceutical composition" are as described above.

[0099] In the present invention, the fusion protein may be a fusion protein in which GLP-1 as a therapeutic agent can be linked to the drug delivery system of the present invention, and specifically, GLP-1, immunoglobulin Fc region and IGF-1 can be linked to the drug delivery system of the present invention. The fusion protein of the present invention is expected to have an excellent therapeutic effect on amyotrophic lateral sclerosis due to its high efficiency in reaching the brain and long half-life.

[0100] According to another aspect of the present invention, there is provided use of a fusion protein containing GLP-1, an immunoglobulin Fc region and IGF-1 for preventing, alleviating or treating nervous system diseases.

[0101] According to another aspect of the present invention, provided is a pharmaceutical composition containing the fusion protein for use in preventing or treating nervous system diseases.

[0102] "GLP-1", "immunoglobulin Fc region", "IGF-1", "neurological disease" and "pharmaceutical composition" are as described above.

[0103] [Best Practice]

[0104] The present invention will be described in detail below by way of examples. These examples are given for the purpose of specifically illustrating the present invention, and the scope of the present invention is not limited thereto.

[0105] Example 1: Preparation of fusion protein

[0106] To ensure candidate substances, GLP1-Fc-IGF1 or GLP1-Fc-mut. IGF1-TfR was synthesized and then cloned into the pcDNA3.1 vector using the NheI / Hind III site. The cloned vector was transfected into CHO-S cells and the supernatant was collected on day 5. Then, the cells and floating materials were removed by centrifugation and a 0.22-μm filter. Subsequently, various candidate substances PF1802_M003 to M032 (SEQ ID NO.13 to 37) ( Figure 1 and Table 1).

[0107] Table 1

[0108]

[0109]

[0110]

[0111]

[0112]

[0113] *TfR: transferrin receptor binding peptide

[0114] Example 2: Evaluation of the activity of PF1802 candidate substances

[0115] Example 2-1. Evaluation of IGF-1 activity of PF1802 candidate substance using Saos-2 cell line

[0116] In order to study the IGF-1 activity of the PF1802 candidate substance prepared by the method of Example 1, a proliferation test was performed on the saos-2 cell line using IGF-1. The Saos-2 cell line was cultured at 1×10 4 / well was distributed into a 96-well plate, incubated at 37°C and 5% CO2 for 16 hours, and starved for 4 hours. Then, the PF1802 candidate substance was diluted 5 times from the maximum concentration of 100nM to 0.00256nM, added to each well, and then incubated at 37°C and 5% CO2 for 48 hours. The cultured cells were stained with sulforhodamine B (SRB), and the absorbance was measured at 540nm using a microplate reader. The PLA statistical function was applied, and the relative potency values ​​of the PF1802 candidate substances were calculated with IGF-1 as a reference (Table 2 and Figure 2 ).

[0117] Table 2

[0118]

[0119] The results are shown in Table 2 above, and the IGF-1 activity of the mutant IGF-1 substances M007 and M008 was confirmed by comparison with the wt. IGF1 substance.

[0120] Example 2-2. Evaluation of IGF-1 activity of PF1802 candidate substance

[0121] In order to study 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), which is a kit using an IGF1R-overexpressing cell line. Specifically, as shown in Table 3, the assay was performed according to the technical manual of the assay kit, and the results were obtained by applying a 4-parameter fitting (Tables 4 and 5). Figure 3 ).

[0122] Table 3

[0123]

[0124]

[0125] Table 4

[0126] sample <![CDATA[EC 50 (nM)]]> IGF-1 3.889 / 2.773 M005(GLP1-Fc-IGF1) 780.8 M008(GLP1-Fc-mut IGF1) 13.93 M010(GLP1-Fc-mut IGF1-TfR) 9.914 M011(Fc-GLP1) ＞2000

[0127] The results are shown in Table 4. The PF1802 candidate substances M005, M008 and M010 were confirmed to show IGF-1 activity compared with the substance M011 (negative control) that does not contain IGF1.

[0128] Example 2-3. Evaluation of GLP-1 activity of PF1802 candidate substance

[0129] In order to study the GLP-1 activity of the PF1802 candidate substance prepared by the method of Example 1, cAMPHunter TM The GLP-1 activity of the produced material was measured using a liraglutide bioassay kit (Discover X, 95-0062Y2-00100). Specifically, as shown in Table 5, the measurement was performed according to the technical manual of the assay kit, and the results were obtained by applying a 4-parameter fit (Tables 6 and Figure 4 ).

[0130] Table 5

[0131]

[0132] Table 6

[0133]

[0134]

[0135] As a result, as shown in Table 6, the GLP-1 activities of M005, M008, M010, and M011 had a level similar to that of exendin-4, indicating that there was no problem with the GLP-1 activity of the produced substances.

[0136] Example 2-4. Evaluation of the pharmacokinetics (PK) of PF1802 candidate substances

[0137] To study the pharmacokinetics of the PF1802 candidate substance in the blood, the candidate substance was subcutaneously administered to mice at a dose of 5 mpk, and blood was then collected for up to 144 hours. Then, WinNolin software was used to obtain PK parameter values ​​based on the ELISA results (Table 7 and Figure 5 ).

[0138] Table 7

[0139]

[0140]

[0141] The results are shown in Table 7. The median Tmax values ​​of candidate substances M005 and M010 are 48 hours and 16 hours, respectively, indicating that the candidate substances are slowly absorbed. The half-life values ​​are 61 hours and 29.4 hours, respectively.

[0142] These results suggest that the candidate substances of the present invention exhibit sustained drug effects when actually administered and can shorten the dosing interval.

[0143] Example 2-5. Evaluation of Binding Affinity of PF1802 Candidates Introducing Fc with Increased Half-Life to Human FcRn

[0144] In order to study the binding affinity of PF1802_M020, M021, and M022 substances obtained by introducing three Fcs that increase the blood half-life into the PF1802-M008 candidate substance to human FcRn, ELISA was performed. In order to investigate the difference in binding affinity with pH changes, two conditions of pH 6.0 and pH 7.4 were prepared. Specifically, each of PF1802_M020, M021, and M022 substances was coated on a 96-well immunoplate at 4 μg / mL and reacted with PBS (pH 6.0 and 7.4) containing 4% skim milk at room temperature for 1 hour to block nonspecific binding. Then, human FcRn-GST protein was diluted 4 times from the 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 incubated at room temperature for 1 hour. Subsequently, the wells were treated with anti-GST Ab-HRP (cytiva, 27457701), and then treated with TMB solution and H2SO4, and the absorbance was measured at 450nm to obtain the results ( Figure 6 ).

[0145] As a result, PF1802_M020, M021 and M022 candidate substances introduced with Fc that increases blood half-life showed a significant increase in binding affinity to human FcRn compared to PF1802_M008. These results indicate excellent binding affinity to human FcRn at pH 6.0, which is important for the increase in blood half-life.

[0146] In addition, by introducing the same three Fcs that increase blood half-life as mentioned above into the PF1802_M024 candidate substance whose substance optimization was completed by Examples 4 and 5 described later, the PF1802_M030, M031 and M032 candidate substances (SEQID NOs: 35, 36 and 37) were ensured, and the PF1802_M030, M031 and M032 candidate substances also showed an increase in binding affinity and excellent binding affinity to human FcRn.

[0147] Example 3: Evaluation of the in vivo efficacy of the PF1802_M008 candidate substance

[0148] Example 3-1. Evaluation of BBB permeability of PF1802_M008 candidate substance

[0149] To investigate BBB permeability, the IVISense 680NHS fluorescent labeling kit (PerkinElmer) was used, and the M011 (GLP1-Fc) candidate substance without IGF-1 was used as a negative control. Specifically, the fluorescent labeling substance was administered once into the peritoneum of mice (Balb / C males) and then perfused with saline at 24 and 72 hours. Then, the brains of the mice were extracted and imaged and analyzed using a secondary optical imaging device (IVIS Spectrum) ( Figure 7 ).

[0150] The results showed that the order of brain tissue fluorescence intensity was M008>M005>M011. The fluorescence intensity of M005 was 1.1 times (24 hours) and 1.2 times (72 hours) that of M011, but there was no statistical difference. The fluorescence intensity of M008 was 1.7 times (24 hours) and 2.6 times (72 hours) that of M011, with the highest fluorescence intensity, which was statistically significant.

[0151] These results indicate that the BBB permeability of the PF1802_M008 candidate substance is good, and the BBB permeability of mut.IGF1 is significantly higher than that of wt.IGF1.

[0152] Example 3-2. PF1802_M008 candidate substance in an amyotrophic lateral sclerosis (ALS) transgenic mouse model Efficacy assay

[0153] To investigate the efficacy of the PF1802_M008 candidate substance, the PF1802_M008 candidate substance was administered subcutaneously to 9-week-old SOD-1(G39A) mice twice a week for 10 weeks as shown in Table 8. The mice were sacrificed at 109 days of age for immunohistological analysis, and to assess the neuromuscular junction (NMJ), gastrocnemius muscle samples were stained with anti-synaptotagmin 2 antibody and α-bungarotoxin, and then imaged using a fluorescence microscope ( Figure 8 ).

[0154] Table 8

[0155]

[0156] The results showed that the G4 and G5 groups administered with the PF1802_M008 candidate substance showed a dose-dependent increase in fully innervated neuromuscular junctions (NMJs) and a decrease in denervated neuromuscular junctions (NMJs).

[0157] These results indicate that treatment with the PF1802_M008 candidate substance exhibits preventive and progression-delaying effects on amyotrophic lateral sclerosis (ALS) disease.

[0158] Example 4: Optimization of PF1802 candidate substances

[0159] Example 4-1. Structural analysis of PF1802_M008 candidate substance and preparation of optimized substance

[0160] In order to optimize the PF1802_M008 candidate substance whose efficacy has been confirmed by Examples 2 and 3, M023, M024 and M025 candidate substances were designed by introducing three different types of linkers respectively. These substances were prepared by the same method as in Example 1. Then, SDS-PAGE gel analysis was performed on the prepared M023, M024 and M025 candidate substances under non-reducing and reducing conditions. The results showed that, except for M023, which had been confirmed to be in a cleaved form, M024 and M025 substances had no problems. ( Fig. 9 ).

[0161] Example 4-2. Evaluation of the binding affinity of PF1802_M024 and M025 candidate substances using SPR

[0162] In order to study the binding affinity of the optimized PF1802_M024 and M025 prepared in Example 4-1 to GLP-1R and IGF-1R, the signal changes caused by binding were measured using SPR. Specifically, the CM5 sensor chip was mounted on a BIAcoreT200 (GE Healthcare), and then 100mM N-hydroxysuccinimide (NHS) and 400mM1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) were injected at a ratio of 1: 1 to activate the flow cell, followed by fixing the anti-His tag antibody at a level of 8000 to 10000RU. The GLP-1R-His and IGF-1R-His recombinant proteins as ligands were injected at a concentration of 2.5ug / mL in each cycle, and captured at a level of 280 to 290RU. Then, M024 and M025 candidate substances were injected under the conditions of Table 9, and SPR binding affinity was subsequently measured ( Fig.10 ).

[0163] Table 9

[0164]

[0165]

[0166] The results showed that the SPR binding affinities of the M024 candidate substance were 12.2 nM (GLP-1R) and 18.1 nM (IGF-1R), and the SPR binding affinities of the M025 candidate substance were 10.6 nM (GLP-1R) and 20.7 nM (IGF-1R).

[0167] These results show that both the M024 and M025 candidate substances bind to GLP-1R and IGF-1R, and there is no significant difference in the binding affinity between the two candidate substances. This indicates that the M024 and M025 candidate substances show excellent GLP-1 and IGF-1 activities.

[0168] Example 5: Determination of the efficacy of the PF1802_M024 candidate substance

[0169] Example 5-1. Determination of the efficacy of the PF1802_M024 candidate substance in an ALS transgenic mouse model

[0170] In order to investigate the efficacy of the PF1802_M024 candidate substance optimized by 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. The mice were sacrificed at 115 days of age for immunohistological analysis. In order to evaluate the neuromuscular junction, gastrocnemius muscle samples were stained with anti-synaptotagmin 2 antibody and α-bungarotoxin ( Fig.11 ), to examine neuroinflammation, spinal cord samples were stained with anti-Iba1 antibody ( Fig.12 ) and then imaged and analyzed using a fluorescence microscope.

[0171] Table 10

[0172]

[0173] As a result, the G4 and G5 groups administered with the PF1802_M024 candidate substance showed an increase in fully innervated neuromuscular junctions (NMJs) and a decrease in denervated neuromuscular junctions (NMJs). In addition, Iba1 was significantly reduced in the G4 and G5 groups compared to the untreated group, confirming the presence or absence of inflammation, and exhibited activity at a similar level to the positive control.

[0174] The results of NMJ improvement and Iba1 reduction indicated that the PF1802_M024 candidate substance had a preventive and delaying effect on ALS, and the results of Iba1 reduction in spinal cord samples verified that the PF1802_M024 candidate substance penetrated the BBB.

[0175] Example 5-2. Evaluation of the BBB permeability of the PF1802_M024 candidate substance in mice

[0176] To investigate BBB permeability, substances fluorescently labeled with the Alexa fluor 647 antibody labeling kit (PerkinElmer) were intravenously administered once to a cranial imaging window mouse model (C57BL / 6N), and then imaged over time. Brain vascular sites were labeled with red ( Fig.13 a), test substances (PF1802 & Human IgG) are marked in green ( Fig.13b). The cortex is marked in purple and can be seen in the merged image of the cerebral vasculature and the test substance ( Fig.13 c).

[0177] The results showed that the PF1802_M024 candidate exhibited excellent BBB permeability compared with human IgG.

[0178] Although the present invention has been described with reference to specific illustrative embodiments, it will be appreciated by those skilled in the art that the present invention may be implemented in other specific forms without departing from its technical spirit or essential features. Therefore, the above embodiments should be understood as examples rather than limitations of the present invention. The scope of the present invention should be understood as based on the meaning and scope of the appended claims rather than a detailed description, and all changes or variations derived from equivalent concepts belong to the scope of the present invention.

Claims

1. A fusion protein comprising GLP-1, an immunoglobulin Fc region and IGF-1.

2. The fusion protein of claim 1, wherein the immunoglobulin Fc fragment is selected from the group consisting of IgG, IgA, IgD, IgE, IgM, combinations thereof, and hybrids thereof. The fusion protein of claim 1 , wherein the immunoglobulin Fc fragment is derived from IgG1.

4. A pharmaceutical composition for preventing or treating a nervous system disease, the pharmaceutical composition comprising a fusion protein as an active ingredient, the fusion protein comprising GLP-1, an immunoglobulin Fc region and IGF-1.

5. The pharmaceutical composition of claim 4, wherein the nervous system disease is any one selected from 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.

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

7. A method for preventing or treating a nervous system disease, the method comprising administering the composition of claim 4 to a subject.

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