Anti-apolipoprotein e4 (APOE4) antibodies and uses thereof

Abstract

The present invention relates to an anti-apolipoprotein E4 (ApoE4) antibody and use thereof and, more specifically, to: an anti-ApoE4 antibody or an antigen-binding fragment thereof; a fusion in which the anti-ApoE4 antibody or an antigen-binding fragment thereof and one or more blood-brain barrier (BBB) receptor-binding peptides are bound; a method for preparing the fusion; and a composition for preventing or treating neurodegenerative diseases or metabolic diseases, comprising the anti-ApoE4 antibody or an antigen-binding fragment thereof, or the fusion.

Description

Anti-apolipoprotein E4 (APOE4) antibody and its use

[0001] The present invention relates to an anti-apolipoprotein E4 (ApoE4) antibody and its use, and more particularly, to an anti-ApoE4 antibody or an antigen-binding fragment thereof, a fusion product in which the anti-ApoE4 antibody or an antigen-binding fragment thereof is bound to one or more blood-brain barrier (BBB) ​​receptor-binding peptides, a nucleic acid encoding the antibody or an antigen-binding fragment thereof, or a fusion product in which the antibody or an antigen-binding fragment thereof is bound to a BBB receptor-binding peptide, a vector and a cell comprising the nucleic acid, a method for producing the anti-ApoE4 antibody or an antigen-binding fragment thereof, or a fusion product in which the antibody or an antigen-binding fragment thereof is bound to a BB receptor-binding peptide using the same, and a composition for preventing or treating a neurodegenerative disease or metabolic disease comprising the anti-ApoE4 antibody or an antigen-binding fragment thereof, or a fusion product in which the antibody or an antigen-binding fragment thereof is bound to a BB receptor-binding peptide.

[0002]

[0003] Dementia is considered a disease with a large social burden due to the rapidly increasing number of patients worldwide and increasing management costs. However, most of the known treatments to date are developed as symptom relief rather than fundamental treatments, so it is a disease with a very high unmet medical need due to the lack of effective treatments and diagnostic methods.

[0004] In June 2021, the FDA granted conditional approval for Aduhelm, an antibody treatment that targets amyloid beta, raising expectations for a new drug for dementia with a new mechanism of action. However, Aduhelm's phase 3 clinical trial showed that its therapeutic effect compared to the placebo group was unclear, so it is known that it will need to be re-evaluated after 10 years by proving clinical benefits through a phase 4 clinical trial. In addition, the incidence of ARIA side effects, such as cerebral edema and microhemorrhage, was reported to be as high as 40%, and due to concerns about the difficulty of confirming clinical improvement and side effects, the sales company Biogen announced that it would reduce the commercial infrastructure for Aduhelm and focus on the follow-up pipeline (Knopman, DS. et al., Alzheimer's & Dementia, Vol. 17, pp. 696-701, 2021). Another amyloid beta-targeting antibody treatment, Leqembi, jointly developed by Biogen and Eisai, received FDA approval in January 2023. However, concerns about its safety remain, as it was reported that a patient participating in a clinical trial of Leqembi died of a cerebral hemorrhage while receiving treatment for ARIA, a major side effect.

[0005] In addition, in November 2022, it was reported that gantenerumab, which Roche was developing with technology licensed from its subsidiary Genentech, failed to demonstrate statistical significance in the first efficacy evaluation in the interim analysis of phase 3 clinical trials. As dementia treatments under development continue to fail in the clinical stage, the difficulty of developing treatments is becoming more apparent, and the need for new treatment targets is emerging.

[0006] About 70% of the causes of dementia are genetic factors, such as a family history of dementia and the ApoE4 genotype, known as a vulnerable gene. Other known causes include advanced age, depression, and high blood pressure. ApoE exists in three alleles (ApoE 2, 3, and 4) in humans, and is classified according to the type of amino acid located at positions 112 and 158. 65% of normal people have ApoE3, and the ApoE3 genotype exists as 112 Cys and 158 Arg amino acids. The ApoE4 genotype, in which the 112th amino acid is mutated to Arg, is the second most common after ApoE3, with a distribution rate of about 24%. ApoE4 is well known as the strongest genetic risk factor for the development of dementia in the elderly (Fortea, J. et al., Nat. Med. Vol. 30, pp. 1284-1291, 2024).

[0007] ApoE is also known to be the cause of many diseases related to brain and peripheral lipoprotein metabolism, such as Parkinson's disease, multiple sclerosis, and cardiovascular disease, in addition to dementia. It has been reported that having only one ApoE4 gene increases the risk of developing dementia by 3 to 4 times, and having both ApoE4 genes increases the risk by 8 to 12 times. People with ApoE4 have a 50% to 90% increased risk of developing dementia in their old age, but those without ApoE4 have only a 9% incidence rate. It has been reported that about 55 to 75% of dementia patients have the ApoE4 gene (Blumenfeld J. et al., Nat Rev Neurosci. Vol.(2), pp. 91-110, 2024.).

[0008] ApoE is also known to be expressed in various cells of the central nervous system, and to be involved in synapse formation, lipid metabolism, and inflammation, and to inhibit the clearance function of amyloid beta in the brain, promote aggregation, and plaque deposition. It has been reported that brain atrophy and tau lesions are worsened in tau animal models according to ApoE4 expression, which is explained by the mechanism that ApoE4 induces hyperphosphorylation of tau, promotes NFT formation, and increases inflammatory responses (Safieh M. et al.,, BMC Med., Vol. 17(64), 2019.).

[0009] Recently, it was analyzed that patients with dementia have higher amyloid beta and tau accumulation in PET images when they have the ApoE4 genotype, and it was reported that people with ApoE4 have a shorter time to dementia (Health Info Stat 2022;47(4):307-311.).

[0010] In addition, ApoE4 is known to induce neuronal cell death by disrupting the homeostasis of brain lipids and lipid metabolism products between astrocytes and neurons (Qi, Cell Reports 34, 2021), forming excessive lipid droplets and interfering with neutral fat removal (Lindner K. et al., Cell Rep. Vol. 38(9), 2022.), and ApoE4 secreted from pericytes within the blood-brain barrier (BBB) ​​has also been reported to contribute to BBB damage (Montagne, Nature, 2020.).

[0011] Thus, ApoE4 in the central nervous system is highly likely to act as a pathogenic factor in various diseases, including dementia, through various mechanisms. It has also been revealed that peripheral ApoE4 acts as a risk factor, increasing vascular inflammation and leading to BBB damage and cognitive decline. Therefore, ApoE4 contributes to the development of brain diseases, including dementia, through a wide variety of mechanisms in the central and peripheral nervous systems. Therefore, it is reported as a therapeutic target that can address the complexity of dementia pathology and offer promising therapeutic outcomes (U.S. Patent No. 11,124,562).

[0012] Meanwhile, the low blood-brain barrier permeability (approximately 0.1%) of antibodies, which are high-molecular-weight substances, is considered one of the factors contributing to the low clinical success rate of brain disease treatments. In particular, because ApoE is a protein that also exists in the periphery, the low brain permeability of antibodies could pose a significant obstacle to targeting ApoE in the brain and achieving dementia therapeutic effects using antibody administration methods. Therefore, strategies to improve brain permeability are required for the successful commercialization of ApoE4-targeting therapeutic antibodies.

[0013] Under this technical background, the present inventors have made great efforts to develop antibodies and fusions that specifically bind to ApoE4 and have high brain penetration, and as a result, developed an antibody that specifically binds to ApoE4 using mouse hybridoma technology, developed a humanized antibody based on this, and developed a fusion combined with a BBB binding peptide, and confirmed that the binding affinity, dementia improvement effect, and blood-brain barrier penetration ability of the antibody are superior, and confirmed that the half-life extension effect of the Fc region-mutant antibody is excellent, and completed the present invention.

[0014] The above information described in this background section is solely intended to enhance understanding of the background of the present invention and may not include information that constitutes prior art already known to a person of ordinary skill in the art to which the present invention pertains.

[0015]

[0016] Summary of the invention

[0017] The purpose of the present invention is to provide an antibody or an antigen-binding fragment thereof that specifically binds to apolipoprotein E4 (ApoE4).

[0018] Another object of the present invention is to provide a fusion product comprising the antibody or an antigen-binding fragment thereof and one or more blood-brain barrier (BBB) ​​receptor binding peptides.

[0019] Another object of the present invention is to provide a nucleic acid encoding an antibody or an antigen-binding fragment thereof that specifically binds to the ApoE4, or a fusion product in which the antibody or an antigen-binding fragment thereof that specifically binds to the ApoE4 is linked to a blood-brain barrier (BBB) ​​receptor binding peptide.

[0020] Another object of the present invention is to provide a method for producing an antibody or antigen-binding fragment thereof that specifically binds to ApoE4, or a fusion product in which an antibody or antigen-binding fragment thereof that specifically binds to ApoE4 is combined with a blood-brain barrier (BBB) ​​receptor binding peptide, the method comprising a recombinant expression vector comprising the nucleic acid, a cell transformed with the recombinant expression vector, and a step of culturing the cell.

[0021] Another object of the present invention is to provide a composition for preventing or treating a neurodegenerative disease or metabolic disease, comprising an antibody or antigen-binding fragment thereof that specifically binds to ApoE4, or a fusion product in which an antibody or antigen-binding fragment thereof that specifically binds to ApoE4 is combined with a blood-brain barrier (BBB) ​​receptor-binding peptide.

[0022] Another object of the present invention is to provide a diagnostic kit for a neurodegenerative disease or metabolic disease comprising an antibody or an antigen-binding fragment thereof that specifically binds to the ApoE4.

[0023] Another object of the present invention is to provide a method for detecting ApoE4, which comprises a step of contacting a sample isolated from a patient with an antibody that specifically binds to ApoE4, in order to provide information necessary for the diagnosis of a neurodegenerative disease or metabolic disease.

[0024] To achieve the above object, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to apolipoprotein E4 (ApoE4), comprising a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1; a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 2; and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 3; and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4; a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6.

[0025] The present invention also provides a fusion product comprising an antibody or an antigen-binding fragment thereof that specifically binds to the ApoE4 and one or more blood-brain barrier (BBB) ​​receptor binding peptides.

[0026] The present invention also provides a nucleic acid encoding a fusion protein comprising an antibody or an antigen-binding fragment thereof that specifically binds to the ApoE4 and one or more blood-brain barrier (BBB) ​​receptor binding peptides.

[0027] The present invention also provides a recombinant expression vector comprising the nucleic acid.

[0028] The present invention also provides a cell transformed with the recombinant expression vector.

[0029] The present invention also provides a method for producing an antibody or antigen-binding fragment thereof that specifically binds to ApoE4, or a fusion product comprising an antibody or antigen-binding fragment thereof that specifically binds to ApoE4 and a blood-brain barrier (BBB) ​​receptor binding peptide, comprising a step of culturing the cells.

[0030] The present invention also provides a composition for preventing or treating a neurodegenerative disease or metabolic disease, comprising an antibody or antigen-binding fragment thereof that specifically binds to the ApoE4, or a fusion product in which the antibody or antigen-binding fragment thereof that specifically binds to the ApoE4 and a blood-brain barrier (BBB) ​​receptor binding peptide are combined.

[0031] The present invention also provides a method for preventing or treating a neurodegenerative disease or metabolic disease, comprising administering an antibody or antigen-binding fragment thereof that specifically binds to ApoE4, or a fusion product in which an antibody or antigen-binding fragment thereof that specifically binds to ApoE4 and a blood-brain barrier (BBB) ​​receptor-binding peptide are combined.

[0032] The present invention also provides the use of an antibody or antigen-binding fragment thereof that specifically binds to ApoE4, or a fusion comprising an antibody or antigen-binding fragment thereof that specifically binds to ApoE4 and a blood-brain barrier (BBB) ​​receptor binding peptide for the manufacture of a medicament for preventing or treating a neurodegenerative disease or metabolic disease.

[0033] The present invention also provides the use of an antibody or antigen-binding fragment thereof that specifically binds to the ApoE4, or a fusion comprising an antibody or antigen-binding fragment thereof that specifically binds to the ApoE4 and a blood-brain barrier (BBB) ​​receptor binding peptide.

[0034] The present invention also provides the use of an antibody or antigen-binding fragment thereof that specifically binds to the ApoE4, or a fusion comprising an antibody or antigen-binding fragment thereof that specifically binds to the ApoE4 and a blood-brain barrier (BBB) ​​receptor binding peptide.

[0035] The present invention also provides a combined therapeutic use of an antibody or an antigen-binding fragment thereof that specifically binds to the ApoE4, or a fusion comprising an antibody or an antigen-binding fragment thereof that specifically binds to the ApoE4 and a blood-brain barrier (BBB) ​​receptor binding peptide.

[0036] The present invention also provides a diagnostic kit for a neurodegenerative disease or metabolic disease comprising an antibody or an antigen-binding fragment thereof that specifically binds to the ApoE4.

[0037] The present invention also provides a method for detecting ApoE4, comprising a step of contacting a sample isolated from a patient with an antibody that specifically binds to ApoE4, to provide information necessary for the diagnosis of a degenerative neurological disease or metabolic disease.

[0038]

[0039] Figure 1a shows the results of confirming the characteristics of an ApoE4 antibody manufactured according to one embodiment of the present invention, where (A) shows the results of measuring the sensitivity to each ApoE protein, and (B) to (D) show the results of measuring the affinity to each ApoE protein. Figure 1b shows the results of measuring the reactivity of an ApoE4 antibody manufactured according to one embodiment of the present invention to aggregated ApoE4 protein.

[0040] Figure 2 shows the results of confirming the phagocytic activity of an ApoE4 antibody manufactured according to one embodiment of the present invention against ApoE4. **** indicates P<0.0001.

[0041] Figure 3A is a photograph (A) of a stained brain section of a mouse injected with an ApoE4 antibody prepared according to one embodiment of the present invention, and a graph (B) quantifying the photograph. Figure 3B is a result comparing the expression levels of ApoE4 protein in the brain of a mouse injected with an ApoE4 antibody prepared according to one embodiment of the present invention, where (A) is a Western blot result for ApoE4 protein in the brain, and (B) is a graph quantifying the results. * means P<0.05, ** means P<0.01, and *** means P<0.001.

[0042] Figure 4a shows the results of a water maze test performed on dementia-induced mice injected with an ApoE4 antibody manufactured according to one embodiment of the present invention. Figure 4b shows the results of measuring the amount of tau protein in the sarkosyl-insoluble tau fraction of the brain tissue (cerebral cortex) of dementia-induced mice injected with an ApoE4 antibody manufactured according to one embodiment of the present invention. (A) is a Western blot result using AT8 antibody, (B) is a graph quantifying the result, (C) is a Western blot result using AT180 antibody, (D) is a graph quantifying the result. * means P<0.05, and ** means P<0.01.

[0043] (A) of Fig. 5a shows the result of staining the CA1 pyramidal cell layer of the mouse hippocampus injected with the ApoE4 antibody manufactured according to one embodiment of the present invention with NeuN protein, and (B) is a graph quantifying the result. (A) of Fig. 5b shows the result of confirming the amount of PSD95 protein in the mouse brain tissue (hippocampus) injected with the ApoE4 antibody manufactured according to one embodiment of the present invention by Western blot, and (B) is a graph quantifying the result. (A) of Fig. 5c shows the result of confirming the amount of Synapsin1 protein in the mouse brain tissue (hippocampus) injected with the ApoE4 antibody manufactured according to one embodiment of the present invention by Western blot, and (B) is a graph quantifying the result. Fig. 5d shows the result of staining for the sypnapsin1 protein in the mouse brain tissue (hippocampus) injected with the ApoE4 antibody manufactured according to one embodiment of the present invention. * indicates P<0.05, and *** indicates P<0.001.

[0044] (A) of Fig. 6a shows the results of staining with Iba1 protein to confirm the inflammatory status of mouse brain tissue (hippocampus) injected with ApoE4 antibody manufactured according to one embodiment of the present invention, and (B) is a graph quantifying the results. (A) of Fig. 6b shows the results of staining with GFAP protein to confirm the inflammatory status of mouse brain tissue (hippocampus) injected with ApoE4 antibody manufactured according to one embodiment of the present invention, and (B) is a graph quantifying the results. ** means P<0.01, and *** means P<0.001.

[0045] Figure 7a is a schematic diagram of the structure of an ApoE4 antibody and a BBB receptor-binding peptide fusion prepared according to one embodiment of the present invention. Figure 7b is a schematic diagram of a vector map (A) of a heavy chain portion and a vector map (B) of a light chain portion for preparing an ApoE4 antibody and a BBB receptor-binding peptide fusion prepared according to one embodiment of the present invention.

[0046] Figure 8 (A) shows the results of measuring the permeability coefficient of an ApoE4 antibody and a BBB receptor-binding peptide fusion prepared according to one embodiment of the present invention in a human brain microvascular endothelial cell-based blood-brain barrier model, and (B) shows the results normalized to the ApoE4 antibody prepared according to one embodiment of the present invention. ** means P<0.01, *** means P<0.001, and **** means P<0.0001.

[0047] Figure 9 shows the results of confirming the characteristics of a humanized ApoE4 antibody manufactured according to one embodiment of the present invention. (A) shows the results of measuring the sensitivity for each ApoE protein, and (B) to (D) show the results of measuring the affinity for each ApoE protein.

[0048] Figure 10 shows the results of confirming the phagocytic activity of a humanized ApoE4 antibody manufactured according to one embodiment of the present invention toward ApoE4. **** indicates P<0.0001.

[0049] (A) of Fig. 11 shows the results of confirming the sensitivity to ApoE4 of a fusion product in which a BBB receptor binding peptide is conjugated to a humanized ApoE4 antibody manufactured according to one embodiment of the present invention, (B) shows the results of measuring the permeability coefficient of the fusion product in a blood-brain barrier model based on human brain microvascular endothelial cells, and (C) is a graph showing the ratio for Y04h. **** indicates P<0.0001.

[0050] Figure 12 is a graph showing the BBB permeability calculated by measuring the concentration of the fusion in the plasma and cerebrospinal fluid using ELISA after injecting a humanized ApoE4 antibody and BBB receptor binding peptide fusion manufactured according to one embodiment of the present invention into a mouse.

[0051]

[0052] Detailed description of the invention and preferred embodiments

[0053] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Generally, the nomenclature used herein and the experimental methods described below are well known and commonly used in the art.

[0054] In the present invention, an antibody or a binding fragment thereof that specifically binds to apolipoprotein E4 (ApoE4) was discovered, and a fusion product was produced by combining the antibody or a binding fragment thereof with a blood-brain barrier (BBB) ​​receptor binding peptide. This fusion product exhibited not only high binding affinity for ApoE4 and BBB penetration ability, but also high therapeutic effects on degenerative and metabolic diseases.

[0055] That is, in one embodiment of the present invention, when an antibody or antigen fragment that specifically binds to ApoE4 is used, it was confirmed that the binding affinity for ApoE4 was excellent (Figs. 1 to 3), cognitive improvement and reduction of tau protein in dementia mice were shown (Fig. 4), and not only did it have neuroprotective and inflammation-reducing effects in the body (Figs. 5 to 6), but also the BBB penetration ability of the fusion combined with the BBB receptor binding peptide was significantly superior (Fig. 8).

[0056] Therefore, the present invention, from a consistent perspective,

[0057] It relates to an antibody or antigen-binding fragment thereof that specifically binds to apolipoprotein E4 (ApoE4) comprising:

[0058] A heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1; a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 2; and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 3; and

[0059] A light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4; a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6.

[0060]

[0061] In the present invention, the term "ApoE4", "apolipoprotein E4" or "Apolipoprotein E4" and similar terms refer to a variant of wild-type Apolipoprotein in which the 112th and 115th amino acids are mutated to arginine, and unless otherwise stated, refers to a polypeptide ("polypeptide" and "protein" are used interchangeably herein) from a vertebrate source, including mammals such as primates (e.g., humans, cynomolgus monkeys (cyno)), dogs and rodents (e.g., mice and rats), or any native ApoE4, and in certain embodiments, includes related ApoE4 polypeptides, including SNP variants thereof.

[0062] In the present invention, the antibody or antigen-binding fragment thereof that specifically binds to apolipoprotein E4 (ApoE4) may be characterized by including a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, or SEQ ID NO: 36; and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 8, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, or SEQ ID NO: 40, but is not limited thereto.

[0063] In the present invention, the antibody that specifically binds to the apolipoprotein E4 (ApoE4) is

[0064] (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 9; and a light chain comprising the amino acid sequence of SEQ ID NO: 10;

[0065] (ii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 41; and a light chain comprising the amino acid sequence of SEQ ID NO: 42;

[0066] (iii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 45; and a light chain comprising the amino acid sequence of SEQ ID NO: 42; and

[0067] (vi) a heavy chain comprising the amino acid sequence of SEQ ID NO: 46; and a light chain comprising the amino acid sequence of SEQ ID NO: 42;

[0068] It may be characterized by including any one selected from the group consisting of, but is not limited thereto.

[0069] The amino acid sequences of the CDR, variable region, heavy chain, and light chain of the antibody or antigen-binding fragment thereof that specifically binds to apolipoprotein E4 (ApoE4) according to the present invention are as described in Table 1 below.

[0070]

[0071]

[0072]

[0073] The base sequence of the nucleic acid encoding the antibody that specifically binds to apolipoprotein E4 (ApoE4) according to the present invention is as described in Table 2 below.

[0074]

[0075]

[0076]

[0077]

[0078]

[0079] The antibody or antibody fragment of the present invention may include not only the sequence of the anti-ApoE4 antibody of the present invention described herein, but also biological equivalents thereof, as long as it can specifically recognize ApoE4. For example, additional changes may be made to the amino acid sequence of the antibody to further improve the binding affinity and / or other biological properties of the antibody. Such modifications include, for example, deletions, insertions, and / or substitutions of amino acid sequence residues of the antibody. Such amino acid mutations are made based on the relative similarity of the amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, etc. Analysis of the size, shape, and type of amino acid side chain substituents reveals that arginine, lysine, and histidine are all positively charged residues; alanine, glycine, and serine have similar sizes; and phenylalanine, tryptophan, and tyrosine have similar shapes. Therefore, based on these considerations, arginine, lysine, and histidine; Alanine, glycine and serine; and phenylalanine, tryptophan and tyrosine are biologically functional equivalents.

[0080] Considering the mutations having the above-described biological equivalent activity, the antibody of the present invention or the nucleic acid molecule encoding the same is interpreted to also include a sequence showing substantial identity with the sequence described in the sequence number. The substantial identity means a sequence showing at least 90% homology, most preferably at least 95% homology, 96% or more, 97% or more, 98% or more, or 99% or more homology when the sequence of the present invention is aligned with any other sequence to the greatest extent possible and the aligned sequence is analyzed using an algorithm commonly used in the art. Alignment methods for sequence comparison are known in the art. NCBI Basic Local Alignment Search Tool (BLAST) is accessible from NBCI, etc., and can be used in conjunction with sequence analysis programs such as blastp, blasm, blastx, tblastn, and tblastx on the Internet.

[0081] The anti-ApoE4 antibody or antigen-binding fragment thereof according to the present invention also includes an antibody or antigen-binding fragment thereof in which a part of the amino acid sequence is substituted through conservative substitution in the anti-ApoE4 antibody or antigen-binding fragment thereof according to the present invention.

[0082] As used herein, the term "conservative substitution" refers to a modification of a polypeptide that involves replacing one or more amino acids with amino acids having similar biochemical properties, without causing a loss of biological or biochemical function of the polypeptide. A "conservative amino acid substitution" is a substitution that replaces an amino acid residue with an amino acid residue having a similar side chain. Classes of amino acid residues having similar side chains are well known and defined in the art. These classes include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with beta-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). It is anticipated that antibodies of the present invention may have conservative amino acid substitutions and still retain activity.

[0083]

[0084] In the present invention, “antibody” refers to a general term for a substance produced in the immune system by antigen stimulation, and its type is not particularly limited. The antibody is an immunoglobulin molecule that is immunologically reactive with a specific antigen, and refers to a protein molecule that acts as a receptor that specifically recognizes the antigen, and may include polyclonal antibodies, monoclonal antibodies, whole antibodies, and antibody fragments. The antibody may be non-naturally produced, for example, recombinantly or synthetically produced. The antibody may be an animal antibody (e.g., mouse antibody, etc.), chimeric antibody, humanized antibody, or human antibody. The antibody may be a monoclonal antibody. In addition, unless otherwise specified, the antibody may be understood to also include an antigen-binding fragment of an antibody that possesses antigen-binding ability.

[0085] The "variable region" of an antibody used in the present invention refers to the light chain and heavy chain portions of an antibody molecule, including the amino acid sequences of the complementarity determining regions (CDRs; i.e., CDR1, CDR2, and CDR3) and the framework regions (FR). VH refers to the variable domain of the heavy chain, and VL refers to the variable domain of the light chain.

[0086] In the present invention, the term “heavy chain” refers to both a full-length heavy chain and fragments thereof, which comprises a variable domain VH comprising an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen, and three constant region domains CH1, CH2, and CH3. In addition, the term “light chain” refers to both a full-length light chain and fragments thereof, which comprises a variable domain VL comprising an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen, and a constant region domain CL.

[0087] In the present invention, the term "complementarity determining region (CDR)" refers to the amino acid sequence of the hypervariable region of the immunoglobulin heavy and light chains. The heavy chains (CDRH1, CDRH2, and CDRH3) and light chains (CDRL1, CDRL2, and CDRL3) each contain three CDRs. The CDRs provide key contact residues for antibody binding to an antigen or epitope.

[0088] In the present invention, the term “framework region (FR)” refers to variable domain residues other than CDR residues. Each variable domain typically has four FRs: FR1, FR2, FR3, and FR4.

[0089] In one embodiment of the present invention, the anti-ApoE4 antibody may be, but is not limited to, a mouse antibody, a chimeric antibody, or a humanized antibody.

[0090] As used herein, “humanized antibody”, also referred to as a reshaped human antibody, refers to an antibody in which the complementarity determining region (CDR) of a non-human mammalian antibody, such as a mouse antibody, is grafted onto the complementarity determining region (CDR) of a human antibody. Humanized antibodies can be produced using known general genetic recombination methods (see, for example, European Patent Publication No. EP 0125023 A and WO 96 / 02576).

[0091] Specifically, a DNA sequence designed to link the CDRs of a mouse antibody and the framework regions (FRs) of a human antibody is synthesized. The framework regions of the human antibody linked to the CDRs are selected so that the CDRs form a favorable antigen-binding site. If necessary, amino acids in the framework regions of the variable region of the antibody can be substituted so that the complementarity-determining regions of the humanized antibody form a suitable antigen-binding site (see K Sato, et al., Cancer Res. 1993 Feb 15; 53(4):851-6).

[0092] The framework region of a humanized antibody uses the corresponding region of a human antibody with the highest similarity, based on information from an electroporated mouse antibody. In the present invention, sequence-based and structure-based similarity were utilized to investigate the sequence of human antibodies.

[0093] While humanizing antibodies while maintaining the efficacy or binding affinity of the original antibody is typically challenging, the present invention successfully yielded a humanized antibody with activity equivalent to that of the original mouse antibody by identifying key interaction sites through structural elucidation of the antigen-antibody complex and selecting amino acid substitutions based on this information. Because humanized antibodies exhibit reduced antigenicity in the human body, they are useful for human administration, particularly for therapeutic purposes.

[0094] Preferred examples of the humanized anti-ApoE4 antibody of the present invention include, but are not limited to, an antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 33 and a light chain variable region (SEQ ID NO: 37) comprising the amino acid sequence of SEQ ID NO: 34.

[0095] Another preferred example of a humanized anti-ApoE4 antibody of the present invention includes, but is not limited to, an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 35 and a light chain comprising the amino acid sequence of SEQ ID NO: 36.

[0096] In the present invention, the constant region of the chimeric antibody and humanized antibody may be that of a human antibody, and the human antibody constant region may be modified to improve the stability of the antibody or its production. The human antibody used in the humanized antibody may include any isotype of human antibody, such as IgG, IgM, IgA, IgE, and IgD, and in the present invention, IgG1 is preferred, but is not limited thereto. In addition, after the humanized antibody is produced, amino acids within the constant region may be substituted with other amino acids for the purpose of enhancing antibody stability.

[0097] Preferred examples of anti-ApoE4 antibodies with substituted constant regions in the present invention include, but are not limited to, antibodies comprising a heavy chain comprising an amino acid sequence of SEQ ID NO: 39 or antibodies comprising a heavy chain comprising an amino acid sequence of SEQ ID NO: 40.

[0098] In the present invention, the term “anti-ApoE4 antibody” refers to an antibody that binds to ApoE4 and causes inhibition of the biological activity of ApoE4, and is used interchangeably with “ApoE4-specific antibody” and “antibody that specifically binds to ApoE4.” In the present invention, the anti-ApoE4 antibody or antigen-binding fragment thereof may be characterized by having specific binding ability to human or mouse ApoE4, but is not limited thereto.

[0099] In the present invention, “anti-ApoE4 antibody” is a concept that includes both polyclonal antibodies and monoclonal antibodies (monoclonal antibodies), and is preferably a monoclonal antibody, and may have a complete whole antibody form. The whole antibody has a structure that includes two full-length light chains and two full-length heavy chains, and a structure that includes a constant region, and each light chain is connected to a heavy chain by a disulfide bond.

[0100] The entire antibody of the anti-ApoE4 antibody according to the present invention is a concept including IgA, IgD, IgE, IgM and IgG forms, and IgG is a subtype, including IgG1, IgG2, IgG3 and IgG4.

[0101] A full-length antibody consists of two full-length light chains and two full-length heavy chains, each light chain linked to a heavy chain by a disulfide bond. The heavy-chain constant region is of the gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types, and has subclasses of gamma1 (γ1), gamma2 (γ2), gamma3 (γ3), gamma4 (γ4), alpha1 (α1), and alpha2 (α2). The light-chain constant region is of the kappa (κ) and lambda (λ) types.

[0102] The “antigen-binding fragment” of the anti-ApoE4 antibody according to the present invention refers to a fragment that has the function of binding to the antigen of the anti-ApoE4 antibody, i.e., ApoE4, and is a concept that includes Fab, Fab', F(ab')2, scFv, (scFv)2, scFv-Fc, and Fv, etc., and is used interchangeably with the same meaning as “antibody fragment” in the present invention.

[0103] The Fab has a structure with variable regions of the light and heavy chains, a constant region of the light chain, and the first constant region (CH1 domain) of the heavy chain, and has one antigen-binding site. Fab' differs from Fab in that it has a hinge region containing one or more cysteine ​​residues at the C-terminus of the heavy chain CH1 domain. F(ab')2 antibodies are produced when the cysteine ​​residues in the hinge region of Fab' form a disulfide bond.

[0104] Fv (variable fragment) refers to the smallest antibody fragment containing only the heavy chain variable region and the light chain variable region. In double-chain Fv (dsFv), the heavy chain variable region and the light chain variable region are linked by a disulfide bond, and in single-chain Fv (scFv), the heavy chain variable region and the light chain variable region are covalently linked, usually through a peptide linker. These antibody fragments can be obtained using a proteolytic enzyme (for example, Fab can be obtained by restriction digestion of the whole antibody with papain, and F(ab')2 fragment can be obtained by digestion with pepsin), and can be produced through genetic recombination technology (for example, a DNA encoding the heavy chain or its variable region and a DNA encoding the light chain or its variable region are used as templates, and a primer pair is used to amplify by the PCR (Polymerase Chain Reaction) method, and a DNA encoding a peptide linker is combined with a primer pair so that both ends are linked to the heavy chain or its variable region and the light chain or its variable region, respectively, and amplified).

[0105] Antibodies of the present invention include, but are not limited to, monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, single-chain Fvs (scFV), single-chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFV) and anti-idiotypic (anti-Id) antibodies, or epitope-binding fragments of the above antibodies.

[0106] The above monoclonal antibody refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., identical except for possible naturally occurring mutations that may exist in trace amounts in individual antibodies comprising the population. Monoclonal antibodies are highly specific, being directed against a single antigenic site. In contrast to conventional (polyclonal) antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.

[0107] For example, monoclonal antibodies useful in the present invention can be produced by hybridoma methods, or can be produced using recombinant DNA methods in bacterial, eukaryotic, or plant cells (see U.S. Patent No. 4,816,567). Monoclonal antibodies can also be isolated from phage antibody libraries.

[0108] An "epitope" is a protein determinant to which an antibody can specifically bind. Epitopes typically consist of chemically active surface molecules, such as amino acids or sugar side chains, and typically have specific three-dimensional structural features as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished by the fact that binding to the former is lost in the presence of denaturing solvents, while binding to the latter is not.

[0109]

[0110] From another aspect, the present invention relates to a fusion comprising an antibody or an antigen-binding fragment thereof that specifically binds to the above ApoE4 and one or more blood-brain barrier (BBB) ​​receptor binding peptides.

[0111] In the present invention, the term “blood-brain barrier”, “blood-brain barrier” or “BBB” refers to an important biological barrier that protects the brain, which is composed of cerebral vascular endothelial cells and the basement membrane that surrounds and supports them, astrocytes and pericytes. The blood-brain barrier prevents foreign substances (molecules such as pathogens, pigments, drugs, and toxins or small molecules) from invading the brain by the tight junction of vascular endothelial cells. The blood-brain barrier in the brain, as well as the blood-spinal cord barrier in the spinal cord and the blood-retinal barrier in the retina, are adjacent capillary barriers in the central nervous system, and are collectively referred to herein as the blood-brain barrier or BBB. The blood-spinal cord barrier (choroid plexus) is composed of ependymal cells, not capillary endothelial cells.

[0112] As used herein, the term "fusion" may be used interchangeably with "fusion protein" or "fusion polypeptide" and refers to a fusion polypeptide molecule comprising an immunoglobulin molecule and a brain-penetrating peptide. In some embodiments, the fusion is a fusion polypeptide comprising an anti-ApoE4 antibody and a blood-brain barrier receptor-binding peptide.

[0113]

[0114] In the present invention, the blood-brain barrier receptor binding peptide may be characterized by being at least one selected from the group consisting of APEP2, RVG29, THR, APEP7, L57, AEP, L-CDX, HAI, CRT, Leptin30, G23, and IGF_C12, but is not limited thereto.

[0115] In the present invention, the blood-barrier receptor binding peptide is

[0116] (1) APEP2 comprising the amino acid sequence of sequence number 11;

[0117] (2) RVG29 comprising the amino acid sequence of sequence number 12;

[0118] (3) THR comprising the amino acid sequence of sequence number 13;

[0119] (4) APEP7 comprising the amino acid sequence of sequence number 14;

[0120] (5) L57 comprising the amino acid sequence of sequence number 15;

[0121] (6) AEP comprising the amino acid sequence of sequence number 16;

[0122] (7) L-CDX comprising the amino acid sequence of sequence number 17;

[0123] (8) HAI comprising the amino acid sequence of sequence number 18;

[0124] (9) CRT comprising the amino acid sequence of sequence number 19;

[0125] (10) Leptin30 comprising the amino acid sequence of sequence number 20;

[0126] (11) G23 comprising the amino acid sequence of sequence number 21; and

[0127] (12) IGF_C12 comprising the amino acid sequence of sequence number 22;

[0128] It may be characterized by being selected from the group consisting of, but is not limited to.

[0129] The amino acid sequence of the blood-brain barrier receptor binding peptide according to the present invention is as described in Table 3 below.

[0130]

[0131] In the present invention, the antibody specifically binding to ApoE4, or an antigen-binding fragment thereof, and the blood-brain barrier (BBB) ​​receptor-binding peptide may be characterized by direct binding, linker-mediated binding, direct fusion (genetic fusion), or linker-mediated fusion.

[0132] In the present invention, the linker may be a peptide linker and may have a length of about 1-40 aa. For example, it may include hydrophilic amino acids such as glycine and / or serine, but is not limited thereto.

[0133] Specifically, the linker is, for example, GGG, (GS), which provides structural flexibility without being cleaved by proteolytic enzymes. n , (GGS) n , (GGGGS) n (SSSSG) n or (G n S) m (n, m are 1 to 10 respectively) and more specifically, the linker may include, for example, GGG, (GGGGS) n or (SSSG) n (n and m can be 1 to 10, respectively).

[0134] In the present invention, the linker may be characterized as being an amino acid linker.

[0135] In the present invention, the antibody specifically binding to ApoE4, or an antigen-binding fragment thereof, and the blood-brain barrier (BBB) ​​receptor binding peptide may be characterized as being a fusion protein fused via an amino acid linker.

[0136] In the present invention, it may be characterized in that the antibody specifically binding to ApoE4, or an antigen-binding fragment thereof, is in the form of a fusion protein in which a blood-brain barrier (BBB) ​​receptor binding peptide is fused to at least one position of the C-terminus via an amino acid linker.

[0137] In the present invention, it may be characterized in that the antibody specifically binding to ApoE4, or an antigen-binding fragment thereof, is in the form of a fusion protein in which a blood-brain barrier (BBB) ​​receptor binding peptide is fused via an amino acid linker at one or more positions of the heavy chain C-terminus and / or the light chain C-terminus. For example, the BBB receptor binding peptide may bind to one or both positions of the heavy chain C-terminus and not to the light chain C-terminus, may bind to one or both positions of the light chain C-terminus and not to the heavy chain C-terminus, may bind to one position of the heavy chain C-terminus and one position of the light chain C-terminus, may bind to one position of the heavy chain C-terminus and one position of the light chain C-terminus, may bind to one position of the heavy chain C-terminus and two positions of the light chain C-terminus, may bind to two positions of the heavy chain C-terminus and one position of the light chain C-terminus, or may bind to both two positions of the heavy chain C-terminus and two positions of the light chain C-terminus.

[0138] In the present invention, it may be characterized in that the antibody specifically binding to ApoE4, or an antigen-binding fragment thereof, is in the form of a fusion protein in which a blood-brain barrier (BBB) ​​receptor binding peptide is fused to both C-terminal positions of the heavy chain via an amino acid linker.

[0139] In the present invention, it may be characterized in that the antibody specifically binding to ApoE4, or an antigen-binding fragment thereof, is in the form of a fusion protein in which a blood-brain barrier (BBB) ​​receptor binding peptide is fused via an amino acid linker at both two heavy chain C-terminal positions and both two light chain C-terminal positions.

[0140] In the present invention, the fusion body

[0141] (a) an amino acid sequence of SEQ ID NO: 23; and an amino acid sequence of SEQ ID NO: 24;

[0142] (b) the amino acid sequence of SEQ ID NO: 25; and the amino acid sequence of SEQ ID NO: 26;

[0143] (c) the amino acid sequence of SEQ ID NO: 27; and the amino acid sequence of SEQ ID NO: 28;

[0144] (d) the amino acid sequence of SEQ ID NO: 29; and the amino acid sequence of SEQ ID NO: 30;

[0145] (e) the amino acid sequence of SEQ ID NO: 31; and the amino acid sequence of SEQ ID NO: 32; and

[0146] (f) the amino acid sequence of SEQ ID NO: 43; and the amino acid sequence of SEQ ID NO: 44;

[0147] It may be characterized by being selected from the group consisting of, but is not limited to.

[0148] The amino acid sequence of the fusion product in which the antibody or antigen-binding fragment thereof that specifically binds to ApoE4 according to the present invention is combined with one or more blood-brain barrier (BBB) ​​receptor binding peptides is as described in Table 4 below.

[0149]

[0150]

[0151]

[0152] The nucleic acid sequence encoding the fusion product in which the antibody or antigen-binding fragment thereof that specifically binds to the ApoE4 according to the present invention is combined with one or more blood-brain barrier (BBB) ​​receptor binding peptides is as described in Table 5 below.

[0153]

[0154]

[0155]

[0156]

[0157]

[0158]

[0159]

[0160]

[0161]

[0162]

[0163] In another aspect, the present invention relates to a nucleic acid encoding an antibody or an antigen-binding fragment thereof that specifically binds to ApoE4, or a fusion product comprising an antibody or an antigen-binding fragment thereof that specifically binds to ApoE4 and a blood-brain barrier (BBB) ​​receptor binding peptide.

[0164] The nucleic acids used in the present invention may be present in cells, cell lysates, or in a partially purified or substantially pure form. A nucleic acid is “isolated” or “rendered substantially pure” if it has been purified from other cellular components or other contaminants, such as nucleic acids or proteins from other cells, by standard techniques, including alkaline / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and others well known in the art. The nucleic acids of the present invention may be, for example, DNA or RNA, and may or may not contain intronic sequences.

[0165] In the present invention, the nucleic acid encoding the antibody or antigen-binding fragment thereof that specifically binds to ApoE4 may be characterized as being any one selected from the following group:

[0166] A nucleic acid comprising a base sequence represented by SEQ ID NO: 41 and / or a base sequence represented by SEQ ID NO: 42;

[0167] A nucleic acid comprising a base sequence represented by SEQ ID NO: 53 and / or a base sequence represented by SEQ ID NO: 54;

[0168] A nucleic acid comprising a base sequence represented by SEQ ID NO: 57 and / or a base sequence represented by SEQ ID NO: 54; and

[0169] A nucleic acid comprising a base sequence represented by SEQ ID NO: 58 and / or a base sequence represented by SEQ ID NO: 54;

[0170] In the present invention, the nucleic acid encoding the antibody specifically binding to ApoE4 or the fusion product in which the antigen-binding fragment and the blood-brain barrier (BBB) ​​receptor binding peptide are combined may be characterized by being any one selected from the following group:

[0171] A nucleic acid comprising a base sequence represented by SEQ ID NO: 43 and / or a base sequence represented by SEQ ID NO: 44;

[0172] A nucleic acid comprising a base sequence represented by SEQ ID NO: 45 and / or a base sequence represented by SEQ ID NO: 46;

[0173] A nucleic acid comprising a base sequence represented by SEQ ID NO: 47 and / or a base sequence represented by SEQ ID NO: 48;

[0174] A nucleic acid comprising a base sequence represented by SEQ ID NO: 49 and / or a base sequence represented by SEQ ID NO: 50;

[0175] A nucleic acid comprising a base sequence represented by SEQ ID NO: 51 and / or a base sequence represented by SEQ ID NO: 52; and

[0176] A nucleic acid comprising a base sequence represented by SEQ ID NO: 55 and / or a base sequence represented by SEQ ID NO: 56;

[0177] The nucleic acid can be isolated to recombinantly produce an antibody or an antigen-binding fragment thereof or a fusion product comprising the antigen-binding fragment and a blood-brain barrier (BBB) ​​receptor-binding peptide.

[0178] In the present invention, the term "nucleic acid" has a comprehensive meaning including DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are the basic structural units of nucleic acids, include not only natural nucleotides but also analogs in which sugar or base moieties are modified. The sequence of a nucleic acid encoding an antibody of the present invention, or an antigen-binding fragment thereof, or a fusion product in which the antigen-binding fragment and a blood-brain barrier (BBB) ​​receptor-binding peptide are linked may be modified. The modifications include additions, deletions, non-conservative substitutions, or conservative substitutions of nucleotides.

[0179] DNA encoding the antibody or antigen-binding fragment thereof or a fusion product of the antigen-binding fragment and a blood-brain barrier (BBB) ​​receptor-binding peptide can be readily isolated or synthesized using conventional molecular biological techniques (for example, by using an oligonucleotide probe that can specifically bind to DNA encoding the antibody and its heavy and light chains), and the nucleic acid is isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or further expression.

[0180]

[0181] In another aspect, the present invention relates to a recombinant expression vector comprising the nucleic acid.

[0182] The term "vector" used in the present invention refers to a means for expressing a target gene in a host cell, and includes viral vectors such as plasmid vectors, cosmid vectors, bacteriophage vectors, adenovirus vectors, retrovirus vectors, and adeno-associated virus vectors.

[0183] For expression of the anti-ApoE4 antibody or antigen-binding fragment thereof according to the present invention or a fusion of the antigen-binding fragment and a blood-brain barrier (BBB) ​​receptor binding peptide, DNA encoding partial or full-length light and heavy chains can be obtained by standard molecular biology techniques (e.g., PCR amplification or cDNA cloning using a hybridoma expressing the antibody of interest) and inserted into an expression vector “operably linked” to transcriptional and translational control sequences.

[0184] The term “operatively linked” as used herein may mean that a gene encoding an antibody or fusion is ligated into a vector such that the transcriptional and translational control sequences within the vector perform their intended function of regulating the transcription and translation of the antibody or fusion gene. The expression vector and expression control sequences are selected to be compatible with the expression host cell used. The antibody light chain gene, the antibody heavy chain gene, and the fusion may be inserted into separate vectors, or all genes may be inserted into the same expression vector. The antibody or fusion may be inserted into the expression vector by standard methods (e.g., ligation of complementary restriction enzyme sites on the antibody gene or fusion fragment and the vector, or blunt-end ligation if no restriction enzyme sites are present at all).

[0185] In some cases, the recombinant expression vector may encode a signal peptide that facilitates secretion of the antibody chain or fusion from the host cell. The antibody chain or fusion gene may be cloned into the vector such that the signal peptide is linked in frame to the amino terminus of the antibody chain or fusion gene. The signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide derived from a protein other than immunoglobulin). The recombinant expression vector also carries regulatory sequences that control the expression of the antibody chain gene or fusion in the host cell. The “regulatory sequences” may include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain gene or fusion. Those skilled in the art will recognize that the design of the expression vector may vary by selecting different regulatory sequences depending on factors such as the choice of host cell to be transformed, the level of protein expression, etc.

[0186]

[0187] In another aspect, the present invention relates to a cell (or host cell) transformed with the recombinant expression vector.

[0188] The cells according to the present invention may be characterized by being selected from the group consisting of animal cells, plant cells, yeast, Escherichia coli, and insect cells, but are not limited thereto. The cells used to produce the antibodies or fusion constructs of the present invention may be prokaryotic, yeast, or higher eukaryotic cells, but are not limited thereto.

[0189] Specifically, the host cell according to the present invention may be a prokaryotic cell such as Escherichia coli, Bacillus subtilis, Streptomyces sp., Pseudomonas sp., Proteus mirabilis or Staphylococcus sp. In addition, it may be a eukaryotic cell such as a fungus such as Aspergillus sp., a yeast such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomycess sp. and Neurospora crassa, other lower eukaryotic cells and cells of higher eukaryotes such as cells from insects.

[0190] It may also be derived from plants or mammals. Preferably, monkey kidney cells 7 (COS7; monkey kidney cells), NSO cells, SP2 / 0 cells, Chinese hamster ovary (CHO; Chinese hamster ovary) cells, W138, baby hamster kidney (BHK; baby hamster kidney) cells, MDCK, myeloma cell lines, HuT 78 cells, and HEK293 cells are available, but are not limited thereto. However, animal cells are of greatest interest, and examples of useful host cell lines include, but are not limited to, COS-7, BHK, CHO, CHOK1, DXB-11, DG-44, CHO / -DHFR, CV1, COS-7, HEK293, BHK, TM4, VERO, HELA, MDCK, BRL 3A, W138, Hep G2, SK-Hep, MMT, TRI, MRC 5, FS4, 3T3, RIN, A549, PC12, K562, PER.C6, SP2 / 0, NS-0, U20S, or HT1080. CHO cells are particularly preferred.

[0191] The nucleic acid or the vector is transfected or transformed into a host cell. Any of a variety of techniques commonly used to introduce exogenous nucleic acids (DNA or RNA) into prokaryotic or eukaryotic host cells for “transfection” or “transfection” may be used, such as electrophoresis, calcium phosphate precipitation, DEAE-dextran transfection, or lipofection. Various expression host / vector combinations may be used to express the anti-ApoE4 antibody or fusion construct according to the present invention. Suitable expression vectors for eukaryotic hosts include, but are not limited to, expression control sequences derived from SV40, bovine papillomavirus, adenovirus, adeno-associated virus, cytomegalovirus, and retrovirus. Expression vectors that can be used in bacterial hosts include bacterial plasmids obtained from Escherichia coli, such as pET, pRSET, pBluescript, pGEX2T, pUC vectors, col E1, pCR1, pBR322, pMB9 and their derivatives; plasmids with a wider host range, such as RP4; phage DNA, exemplified by the numerous phage lambda derivatives, such as λgt10 and λgt11, NM989; and other DNA phages, such as M13 and filamentous single-stranded DNA phages. Useful expression vectors for yeast cells are the 2°C plasmids and their derivatives. A useful vector for insect cells is pVL941.

[0192]

[0193] In another aspect, the present invention relates to a method for producing an anti-ApoE4 antibody or an antigen-binding fragment thereof, comprising a step of culturing the cells. Preferably, the method may include a step of culturing the cells to express the anti-ApoE4 antibody or an antigen-binding fragment thereof, or a fusion product comprising the anti-ApoE4 antibody or an antigen-binding fragment thereof and a blood-brain barrier (BBB) ​​receptor-binding peptide.

[0194] When a recombinant expression vector capable of expressing the anti-ApoE4 antibody or antigen-binding fragment thereof or a fusion of the anti-ApoE4 antibody or antigen-binding fragment thereof and a blood-brain barrier (BBB) ​​receptor binding peptide is introduced into a mammalian host cell, the antibody or fusion can be produced by culturing the host cell for a period of time sufficient to cause the antibody to be expressed in the host cell, or more preferably, for a period of time sufficient to cause the antibody or fusion to be secreted into the culture medium in which the host cell is cultured.

[0195] The above cells can be cultured in various media. Any commercially available medium can be used as a culture medium. Any other essential supplements known to those skilled in the art may also be included at appropriate concentrations. Culture conditions, such as temperature and pH, are already used with the host cells selected for expression and will be readily apparent to those skilled in the art.

[0196] In some cases, the expressed antibody or fusion can be isolated from the host cell and purified to a homogeneous state. The isolation or purification of the antibody or fusion can be performed by a separation or purification method commonly used for proteins, such as chromatography. The chromatography can include, for example, affinity chromatography including a protein A column or a protein G column, ion exchange chromatography, or hydrophobic chromatography. In addition to the chromatography, the antibody can be isolated and purified by a combination of filtration, ultrafiltration, salting out, dialysis, etc.

[0197]

[0198] In another aspect, the present invention relates to a composition for preventing or treating a neurodegenerative disease or metabolic disease, comprising an antibody or an antigen-binding fragment thereof that specifically binds to ApoE4, or a fusion product in which an antibody or an antigen-binding fragment thereof that specifically binds to ApoE4 is linked to a blood-brain barrier (BBB) ​​receptor-binding peptide.

[0199] In the present invention, the degenerative neurological disease may be characterized by being selected from the group consisting of vascular dementia, multiple sclerosis, ischemic stroke, and Parkinson's disease, but is not limited thereto.

[0200] In the present invention, the metabolic disease may be characterized by being selected from the group consisting of cardiovascular disease, type 2 diabetes mellitus, hyperlipoproteinemia, and age-related hearing loss, but is not limited thereto.

[0201] The term “prevention” as used in the present invention means any act of inhibiting or delaying the onset of a degenerative neurological disease or metabolic disease by administering the pharmaceutical composition of the present invention.

[0202] The term "treatment" as used in the present invention means any act in which the symptoms of a degenerative neurological disease or metabolic disease are improved or beneficially changed by administration of the pharmaceutical composition of the present invention.

[0203] In the present invention, the composition may include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier included in the composition is one commonly used in formulations, and includes, but is not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above components, the pharmaceutical composition may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like.

[0204] The term "administration" of the present invention means introducing the pharmaceutical composition of the present invention to a patient by any appropriate method, and the pharmaceutical composition of the present invention can be administered orally or parenterally, and can be administered by, for example, infusion, intravenous injection, intramuscular injection, subcutaneous injection, intraperitoneal injection, intrarectal administration, topical administration, intranasal injection, etc., but is not limited thereto.

[0205] The appropriate dosage of the pharmaceutical composition for preventing or treating a degenerative neurological disease or metabolic disease of the present invention may be prescribed in various ways depending on factors such as the formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity. The preferred dosage of the composition is within the range of 0.001-100 mg / kg for adults. The term "pharmaceutically effective amount" means an amount sufficient to prevent or treat cancer, or to prevent or treat a disease caused by angiogenesis.

[0206] The composition may be prepared in a unit dose form or may be placed in a multi-dose container by formulating the composition using a pharmaceutically acceptable carrier and / or excipient according to a method that can be easily performed by a person skilled in the art. In this case, the formulation may be in the form of a solution, suspension, syrup or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, granule, tablet or capsule, and may additionally include a dispersing agent or a stabilizer. In addition, the composition may be administered as an individual therapeutic agent or in combination with another therapeutic agent, and may be administered sequentially or simultaneously with conventional therapeutic agents. Meanwhile, since the composition includes an antibody or an antigen-binding fragment, it may be formulated as an immunoliposome. Liposomes containing antibodies may be prepared according to methods widely known in the art. The immunoliposome may be a lipid composition containing phosphatidylcholine, cholesterol and polyethylene glycol-derivatized phosphatidylethanolamine, and may be prepared by a reverse phase evaporation method. For example, the Fab' fragment of an antibody can be conjugated to liposomes via a disulfide-exchange reaction. A chemotherapeutic agent, such as doxorubicin, can be additionally incorporated into the liposomes.

[0207]

[0208] In another aspect, the present invention relates to a method for preventing or treating a neurodegenerative disease or metabolic disease, comprising administering an antibody or an antigen-binding fragment thereof that specifically binds to ApoE4, or a fusion product comprising an antibody or an antigen-binding fragment thereof that specifically binds to ApoE4 and a blood-brain barrier (BBB) ​​receptor-binding peptide.

[0209] In another aspect, the present invention relates to the use of an antibody or antigen-binding fragment thereof that specifically binds to ApoE4, or a fusion comprising an antibody or antigen-binding fragment thereof that specifically binds to ApoE4 and a blood-brain barrier (BBB) ​​receptor binding peptide for the manufacture of a medicament for the prevention or treatment of a neurodegenerative disease or a metabolic disease.

[0210] In another aspect, the present invention relates to the use of an antibody or an antigen-binding fragment thereof that specifically binds to ApoE4, or a fusion comprising an antibody or an antigen-binding fragment thereof that specifically binds to ApoE4 and a blood-brain barrier (BBB) ​​receptor binding peptide.

[0211] In another aspect, the present invention relates to the use of an antibody or an antigen-binding fragment thereof that specifically binds to ApoE4, or a fusion comprising an antibody or an antigen-binding fragment thereof that specifically binds to ApoE4 and a blood-brain barrier (BBB) ​​receptor binding peptide.

[0212] In another aspect, the present invention relates to a combined therapeutic use of an antibody or an antigen-binding fragment thereof that specifically binds to ApoE4, or a fusion comprising an antibody or an antigen-binding fragment thereof that specifically binds to ApoE4 and a blood-brain barrier (BBB) ​​receptor-binding peptide.

[0213]

[0214] In another aspect, the present invention relates to a diagnostic kit for a neurodegenerative disease or metabolic disease comprising an antibody or an antigen-binding fragment thereof that specifically binds to ApoE4.

[0215] In the present invention, the kit can be used to diagnose a neurodegenerative disease or a metabolic disease by detecting ApoE4 through an antigen-antibody binding reaction, and can be in any form as long as it can be used in an analysis method for detecting the presence of ApoE4. The kit is well known to those skilled in the art, and can be easily reconstituted by those skilled in the art if a sample containing the antigen (e.g., a cell overexpressing ApoE4) is provided. The kit may include, but is not limited to, kits using immunohistochemistry, immunoblotting, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), and radioimmunoassay, for example.

[0216] The above kit can be manufactured by a conventional manufacturing method known to those skilled in the art, and generally includes an antibody in lyophilized form, a buffer, a stabilizer, and an inactive protein. Meanwhile, a description of the method for detecting the ApoE4 protein below can be provided as a protocol for the kit.

[0217]

[0218] In another aspect, the present invention relates to a method for detecting ApoE4, comprising the step of contacting a sample isolated from a patient with an antibody that specifically binds to ApoE4, in order to provide information necessary for the diagnosis of a neurodegenerative disease or metabolic disease.

[0219] In the present invention, the sample isolated from the patient may be a cell or tissue isolated from a human body to determine whether the patient has a neurodegenerative disease or metabolic disease caused by overexpression of ApoE4, or whether there is a risk of developing the neurodegenerative disease or metabolic disease, and may be a sample isolated from a patient with a neurodegenerative disease or metabolic disease or a normal person.

[0220] The step of contacting the antibody can be performed by reacting an antigen present on a sample isolated from the patient, i.e., ApoE4, with an antibody that specifically binds to ApoE4.

[0221] The above antigen-antibody reaction can be performed according to various immunoassay or immunostaining methods developed in the past. The immunoassay or immunostaining methods include, but are not limited to, radioimmunoassay, radioimmunoprecipitation, immunoprecipitation, ELISA, capture-ELISA, inhibition or competition assay, sandwich assay, flow cytometry, immunofluorescence staining, and immunoaffinity purification. The immunoassay or immunostaining methods are widely known in the art. For example, when the immunoassay method is performed according to the radioimmunoassay method, a radioisotope (e.g., C 14 , I 125 , P 32 and S 35 ) can be used to detect ApoE4.

[0222] When the above method is performed in the ELISA manner, it may include (i) a step of coating an unknown cell sample lysate to be analyzed on the surface of a solid substrate; (ii) a step of reacting the cell lysate with an antibody that specifically binds to ApoE4 as a primary antibody; (iii) a step of reacting the resultant with a secondary antibody conjugated to an enzyme; and (iv) a step of measuring the activity of the enzyme.

[0223] Suitable solid substrates include hydrocarbon polymers such as polystyrene and polypropylene, glass, metal or gels, and may be, for example, microtiter plates.

[0224] The enzyme bound to the secondary antibody includes, but is not limited to, enzymes that catalyze a color reaction, a fluorescent reaction, a luminescent reaction, or an infrared reaction, and examples thereof include alkaline phosphatase, β-galactosidase, horseradish peroxidase, luciferase, and cytochrome P. 450 In the case where alkaline phosphatase is used as an enzyme binding to the secondary antibody, a color reaction substrate such as bromochloroindolyl phosphate (BCIP), nitro blue tetrazolium (NBT), naphthol-AS-B1-phosphate and enhanced chemifluorescence (ECF) is used as a substrate, and in the case where horseradish peroxidase is used, chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (bis-N-methylacridinium nitrate), resorufin benzyl ether, luminol, Amplex Red reagent (10-acetyl-3,7-dihydroxyphenoxazine), HYR (hypersensitive reaction solution: p-phenylenediamine-HCl and pyrocatechol), TMB (tetramethylbenzidine), Substrates such as ABTS (2,2'-Azine-di[3-ethylbenzthiazoline sulfonate]), o-phenylenediamine (OPD), and naphthol / pyronine, glucose oxidase, and t-NBT (nitroblue tetrazolium) and m-PMS (phenzaine methosulfate) can be used.

[0225] The above detection antibody may have a label that generates a detectable signal. The label may be a chemical such as biotin, alkaline phosphatase, β-galactosidase, horseradish peroxidase, and cytochrome P. 450 Enzymes such as C 14 , I 125 , P 32 and S 35 These include, but are not limited to, radioactive substances such as fluorescein, fluorescent substances such as fluorescein, luminescent substances, chemiluminescent substances, and FRET (fluorescence resonance energy transfer).

[0226] In the above ELISA method, the final enzyme activity measurement or signal measurement can be performed using various methods known in the art. Detection of this signal enables qualitative or quantitative analysis of the c-Met protein. If biotin is used as a label, the signal can be easily detected with streptavidin, and if luciferase is used, the signal can be easily detected with luciferin.

[0227] When the above method is performed by immunohistochemistry, it includes the steps of (i) fixing an unknown cell or tissue sample to be analyzed and creating a section; (ii) reacting the section with an antibody that specifically binds to ApoE4 protein as a primary antibody; (iii) reacting the resultant with a secondary antibody conjugated to an enzyme; and (iv) measuring the activity of the enzyme.

[0228] Methods for fixing the sample and preparing sections are widely known in the art. For example, the sample can be fixed using a chemical substance such as formalin. Furthermore, the sections can be prepared by embedding the sample in a material such as paraffin. If paraffin is used in the preparation of the sections, a deparaffinization process can be performed to facilitate the reaction between the primary antibody and antigens within cells or tissues.

[0229] Since the contents of the above steps (iii) and (iv) have been described in the method performed using the above ELISA method, their description is omitted to avoid excessive complexity of the specification.

[0230] By analyzing the final signal intensity from the above-described immunoassay process, neurodegenerative or metabolic diseases can be diagnosed. Specifically, if the ApoE4 signal is stronger than that of a normal sample, a diagnosis of neurodegenerative or metabolic disease can be made.

[0231]

[0232] Example

[0233] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0234]

[0235] Example 1. Preparation of a fusion comprising an anti-ApoE4 antibody and a blood-brain barrier receptor binding peptide

[0236] 1-1. Anti-ApoE4 antibody screening

[0237] To develop antibodies that specifically bind to the ApoE4 protein, we commissioned GenScript, a company specializing in antibody development, to produce the antibodies. After four booster injections of ApoE4 fragments and the entire protein into mice, the production of ApoE4 antibodies was confirmed in three out of ten mice. Cell lines producing antibodies that specifically bind to the ApoE4 protein were then screened using enzyme-linked immunosorbent assay.

[0238] Cell fusion was performed from mice producing ApoE4 antibodies, and antibodies secreted from the resulting parental clones were rescreened to obtain clones that showed reactivity to the ApoE4 antigen. Among the obtained clones, a clone with high reactivity to the ApoE4 antigen was selected through enzyme-linked immunosorbent assay, and this was subcloned to establish a stable antibody-producing cell line. Finally, among the produced antibodies, an antibody that showed low reactivity to ApoE2 and ApoE3 and high specificity only for ApoE4 was selected, and this antibody was designated Y04m.

[0239] 1-2. Production and screening of humanized antibodies

[0240] We commissioned Absolute Antibody (UK), an overseas CRO, to humanize ApoE4 mouse antibodies. After obtaining the sequence of the mouse antibody produced from the #15E5 hybridoma cell line, we defined the CDR (Complementarity Determining Region) using the International ImMunoGeneTics Information System (IMGT). By inserting the defined CDR into the framework region of the human germline, we designed four heavy chain and four light chain humanized antibody genes. By combining these, we produced a total of 16 humanized antibodies.

[0241] The produced humanized antibodies were analyzed for expression patterns and purity, and their binding affinity to ApoE proteins (ApoE2, ApoE3, and ApoE4) was evaluated. As a result, a highly specific humanized antibody was selected that had an affinity for ApoE4 equivalent to or higher than that of a mouse antibody, while binding to ApoE2 and ApoE3 with negligible binding.

[0242] 1-3. Production of Fc domain-mutant antibodies

[0243] To extend the blood half-life of antibodies, we applied IgG variants with enhanced binding affinity to FcRn. The variants used were the YTE variant (M252Y / S254T / T256E) and the LS variant (M428L / N434S), selected based on relevant literature. These variants have been applied to commercialized antibody therapeutics, and it has been reported that increased binding affinity to FcRn extends the blood half-life of antibodies in mouse animal models (Ko S. et al., BioDrugs. Vol. 35(2), pp. 147-175, 2021).

[0244] 1-4. Production of anti-ApoE4 antibodies and fusions

[0245] The heavy and light chain genes (Tables 2 and 4) of antibodies (or peptide fusion antibodies) were each injected into the pcDNA 3.4 vector to prepare an expression vector with a structure as shown in Figure 7b. Antibodies and fusion constructs were produced by co-transfecting CHO cells (Gibco™, A29127) with the vectors for expressing the heavy and light chains of antibodies. Gibco's ExpiCHO cells and ExpiCHO transfection kit were used. ExpiCHO cells were adjusted to a certain concentration, mixed with lipofectamine and DNA, and transfected, then cultured in a shaking incubator for 8–10 days. After harvest, the cell culture medium was filtered, and the antibodies and peptide fusion constructs were purified using Protein A resin with FPLC (Fast Protein Liquid Chromatography) equipment. The purified samples were then dialyzed against 1X PBS and stored.

[0246]

[0247] Example 2. Characterization of a fusion comprising an anti-ApoE4 antibody and a blood-brain barrier receptor binding peptide.

[0248] 2-1. Confirming specificity (affinity)

[0249] The binding affinity of Y04 antibody (Y04m, Y04h) manufactured in Example 1 with ApoE2, ApoE3, and ApoE4 (manufactured from E. coli, ADEL, Inc.) was measured using an Octet device (Pall ForteBio, Octet K2). That is, ApoE protein was immobilized on a BLI AR2G biosensor (Satorius, 18-5092), and the KD value was measured using Y04 antibody as the analyte. After activating the AR2G biosensor with an EDC / NHS solution, the ApoE protein was immobilized on the biosensor using the amine group, and the association rate and dissociation rate for the analyte (antibody) were measured to calculate the KD value.

[0250] As a result, in the case of the Y04m antibody, it was confirmed that it specifically binds only to the ApoE4 protein, as described in B to D of Fig. 1a and Table 6 below.

[0251]

[0252] Additionally, for the Y04h antibody, it was confirmed that it specifically binds only to the ApoE4 protein, as described in Figures B to D of Figure 9 and Table 7 below.

[0253]

[0254] 2-2. Check sensitivity

[0255] To measure the sensitivity of the Y04m, Y04h, and Y04h-APEP(4) fusion antibodies manufactured in Example 1, the ApoE protein was diluted in coating buffer (sodium bicarbonate buffer, pH 9.6), dispensed into a 96-well plate at 50 μl per well, and coated at 4°C for one day. Then, the coating buffer was removed, and the plate was washed four times by adding and removing 160 μl of washing buffer (1xPBS with 0.05% Tween-20) per well. After that, 150 μl of blocking buffer (Thermo, 37532) was dispensed into each well, and blocking was performed at 37°C for 2 hours while shaking at 700 rpm, and then the blocking buffer was removed and washed four times in the same manner. Next, 50 ㎕ of the detection antibody solution of the desired concentration (diluted in blocking buffer, 1,000, 333.3, 111.1, 37.4, 12.35, 4.12 ng / mL) was dispensed into each well, and the reaction was performed while shaking at 700 rpm for 1 hour at 37℃. The detection antibody solution was removed and washed 4 times in the same manner. After this, the secondary antibody (Vector, PI-2000-1) was diluted in blocking buffer at a ratio of 1:10,000 and dispensed into each well 50 ㎕, and the reaction was performed while shaking at 700 rpm for 1 hour at 37℃, and the washing was performed 4 times in the same manner. Next, 50 μl of TMB substrate solution (TMB substrate solution, R&D systems, DY999) was added to each well, and the degree of color development was checked while reacting at room temperature. When appropriate color development was confirmed, 25 μl of stop solution (Stop solution, R&D systems, DY994) was added to each well to stop the reaction, and the absorbance was measured at 450 nm and 620 nm within 30 minutes.

[0256] As a result, as described in A of Fig. 1a, A of Fig. 9, and A of Fig. 11, it was confirmed that all of the Y04m antibody, Y04h antibody, and Y04h-APEP(4) fusion antibody could detect ApoE4 even at concentrations of less than 10 ng / ml.

[0257] 2-3. Check reactivity

[0258] The reactivity of the Y04m antibody prepared in Example 1 to aggregated ApoE4 was confirmed by the method of Example 2-2. As a result, it was confirmed that it reacted even to aggregated ApoE4 of less than 10 ng / ml, as described in Fig. 1b.

[0259]

[0260] Example 3. Confirmation of phagocytic activity of anti-ApoE4 antibodies against ApoE4.

[0261] Phagocytosis of target proteins by macrophages is a key functional assay in evaluating the activity of monoclonal antibodies. Monoclonal antibodies specifically recognize and bind to target proteins. The antibodies interact with immune cells via the Fc region, and macrophages possessing Fc receptors can remove the antibody-bound protein through phagocytosis. The purpose of this study was to determine whether the antibody prepared in Example 1 promotes phagocytosis of the target protein, ApoE4, in microglia, a type of brain macrophage.

[0262] 3-1. BV2 cell preparation

[0263] Before cell seeding, the plate was coated with PDL solution for 1 hour at 37°C, washed once with distilled water (DW), dried, and then BV2 cells (Accegen, ABC-TC212S) were added to the culture medium (DMEM, 5% FBS, 1% antibiotics) at a density of 4×10⁴ cells / well. 100 μl was dispensed into each well of a 96-well plate, and the plate was placed in a 37°C, 5% CO₂ incubator and cultured overnight (O / N).

[0264] 3-2. ApoE4 Biotinylation

[0265] The amounts of ApoE4 and Biotin used were calculated with reference to the information below.

[0266]

[0267] Afterwards, 170 ㎕ of distilled water was added to 2 mg of NHS-PEG4-Biotin (ThermoFisher, A39259) to prepare a stock solution with a concentration of 20 mM, and then the prepared NHS-PEG4-Biotin solution was added to the ApoE4 solution and reacted at room temperature for 30 minutes. NHS-PEG4-Biotin not bound to the ApoE4 protein was removed by dialysis against 1X PBS three times for 1 hour each and once overnight.

[0268] 3-3. Phagocytosis Analysis

[0269] ApoE4 protein and each antibody (IgG, Y04m, Y04h) were mixed at a molar ratio of 1:1 and reacted at room temperature for 1 hour to allow binding, then diluted in BV2 culture medium, and the culture medium was removed from the BV2 cell culture plate, and 100 ㎕ of the mixed culture medium was dispensed into each well. After that, the plate was placed in a 37℃, 5% CO₂ incubator and incubated for 10 minutes, the medium was removed, and the plate was washed once with 1X HBSS. Then, to remove the ApoE4 protein that was not incorporated into the cell but was attached to the surface, a 0.05% Trypsin-EDTA solution was treated for 3 minutes, the Trypsin-EDTA was neutralized with the culture medium, and then the plate was washed once with 1X HBSS.

[0270] 3-4. ApoE4 staining (Cy3 staining)

[0271] 4% paraformaldehyde was added to each well at room temperature and the cells were fixed by shaking for 30 minutes, washed three times with 1X PBS, treated with blocking buffer (2% BSA, 2% NGS, 0.1% Triton X-100 in 1X PBS) for 30 minutes at room temperature, and washed three times with 1X PBS. To observe intracellular ApoE4, streptavidin-Cy3, which binds to biotin, was diluted 1:200 in blocking buffer (excluding 0.1% Triton X-100) and treated to the cells, incubated for 1 hour at room temperature, and then the streptavidin-Cy3 solution was removed, washed three times with 1X PBS, and the nuclei were stained with DAPI for 5 minutes. The intracellular Cy3 signal was confirmed under a microscope, and the Cy3 signal was measured using CLARIOstar equipment under the following conditions.

[0272] Read condition: - Scan mode: spiral scan - Excitation, Emission: 530-20, 580-30 - Focal height [mm]: 4.1.

[0273] As a result, as described in Fig. 2, it was confirmed that the Y04m antibody had superior phagocytosis of ApoE4 compared to the control group, and as described in Fig. 10, it was confirmed that the Y04h antibody had high phagocytosis of ApoE4 similar to the Y04m antibody.

[0274]

[0275] Example 4. Experimental method for confirming in vivo efficacy of anti-ApoE4 antibody

[0276] In order to confirm the in vivo efficacy of the Y04m antibody manufactured in Example 1, TE4 (PS19 x ApoE4 KI) mice, a dementia mouse model (manufactured and produced in-house with reference to Yang Shi, et al., Nature. Vol. 20(549), pp. 523-527, 2017) and wild-type mice (same), were intraperitoneally administered 50 mg / kg of mouse IgG control and Y04m every week for 20 weeks, and the following analysis was performed to confirm the efficacy. That is, the mice used in the experiment were G1: 8 wild-type-mIgG control mice, G2: 10 TE4-mIgG control mice, and G3: 10 TE4-Y04m mice.

[0277] 4-1. Water maze experiment

[0278] The water hole was divided into four sections: northeast (NE), southeast (SE), southwest (SW), and northwest (NW). A platform was installed in the northwest, allowing the experimental rats to escape from the water hole. Specifically, all experimental rats were trained to find the visible platform for four days. Training was performed three times a day, with the starting position being the intersection of the northeast, southeast, and southwest. On the fifth day, the platform was removed, and movements, such as the time it took to reach the target point (northwest), were analyzed.

[0279] 4-2. Immunohistochemical staining

[0280] After brain extraction from the above mouse, the brain samples were perfused with cold phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde (PFA), washed with PBS, permeabilized with 30% sucrose for 48 hours, embedded in OCT complex buffer (Sakura Finetek USA, Inc.), and then cut into 30 μm-thick coronal sections using a Leica CM1860. For immunohistochemical staining, brain sections were washed several times with 1x PBS and peroxidase blocking buffer (PBS containing 20% ​​MeOH, 2% Triton X-100, and 5% H2O2), and then the brain sections washed with 1x PBS were placed in NGS-BSA blocking buffer (PBS containing 5% NGS, 0.2% Triton X-100, and 1% BSA) for 1 hour. The sections were then incubated with primary antibodies in PBS containing 5% NGS, 0.2% Triton X-100, and 1% BSA overnight at 4°C. The next day, after several washes, the sections were stored for 1 hour with fluorescent secondary antibodies, and after mounting, the brain samples were observed and cell counted using a Nikon ECLIPSE Ti microscope, and analyzed using ImageJ software (NIH, Bethesda, MD).

[0281] The antibodies used are as shown in Table 8 below.

[0282]

[0283] 4-3. Obtaining Sarkosyl Fraction

[0284] After brain extraction from the above mouse, insoluble tau fraction was isolated from the mouse brain tissue through sarkosyl fractionation. The brain tissue was homogenized by dissolving it in cold sample buffer (10 mM Tris-HCl pH 7.4, 0.8 M NaCl, 1 mM EDTA, 2 μM DTT, 0.1% Sarkosyl, 10% sucrose + protease / phosphatase inhibitors) equivalent to 5 times the tissue weight. The sample was then centrifuged at 10,000 g for 10 minutes at 4°C, and the supernatant was transferred to a new tube, followed by the addition of 1% sarkosyl (N-lauroylsarcosine sodium salt; Sigma-Aldrich). After that, the fraction was left at room temperature (RT) for 1 hour, then centrifuged at 300,000 g at 4°C for 1 hour, the supernatant was transferred to a new tube, and the remaining pellet (sarkosyl-insoluble tau fraction) was resuspended in cold PBS, sonicated, and stored at -80°C.

[0285] 4-4. Western Blot

[0286] All samples were lysed in lysis buffer (RIPA Lysis and Extraction Buffer, Thermo Fisher, USA) containing protease / phosphatase inhibitors (Sigma-Aldrich, St. Louis, MO, USA), and protein concentrations were measured using the Bradford assay. For denaturing conditions, samples were mixed with 4× sample buffer (60 mM Tris-HCl [pH 6.8], 2% [w / v] sodium dodecyl sulfate [SDS], 25% [v / v] glycerol, 14.4 mM [v / v] β-mercaptoethanol, bromophenol blue) and heat-denatured at 95°C for 5 min. The insoluble tau fraction obtained by the method of Example 4-3 was measured under semi-denaturing conditions. For semi-denaturing conditions, samples were mixed with 4× Laemmli sample buffer (62.5 mM Tris-HCl [pH 6.8], 1% [w / v] lithium dodecyl sulfate [LDS], 10% [v / v] glycerol, bromophenol blue) without β-mercaptoethanol. Proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA, USA). The membranes were blocked in 5% nonfat dry milk containing phosphate-buffered saline (PBS) and 0.1% Tween-20, and incubated with primary antibodies overnight at 4°C. The membranes were then incubated with HRP-conjugated secondary antibodies (Vector Laboratories, Burlingame, CA, USA) for 1 h at room temperature, and protein bands were detected with enhanced chemiluminescence reagent (Thermo Fisher Scientific, Rockford, IL, USA). The band intensities of each sample were measured by densitometry and analyzed using ImageJ software (NIH, Bethesda, MD, USA).

[0287] The antibodies used are as shown in Table 9 below.

[0288]

[0289] Example 5. Confirmation of in vivo efficacy of anti-ApoE4 antibodies

[0290] 5-1. Confirming ApoE4 concentration in brain tissue

[0291] As a result of confirming the concentration of ApoE4 in the brain tissue sample of the mouse using the method of Example 4-2, as described in Figures 3a and 3b, it was confirmed that in the case of the TE4 mouse administered with the Y04m antibody, ApoE4 in the brain tissue was reduced similarly to that of the wild type.

[0292] 5-2. Confirmation of cognitive improvement and Tau reduction

[0293] As a result of performing a water maze experiment on mice using the method of Example 4-1, as described in Fig. 4a, it was confirmed that in the case of TE4 mice administered with the Y04m antibody, the time taken to find the exit was reduced, similar to that of normal mice.

[0294] In addition, the amount of tau protein of each mouse was confirmed by the method of Example 3-4 in the insoluble tau fraction obtained by the method of Example 4-3, and as a result, it was confirmed that the amount of tau protein was drastically reduced in the TE4 mouse administered with the Y04m antibody, as described in Fig. 4b.

[0295] 5-3. Confirmation of neuroprotective effect

[0296] As a result of confirming the thickness of pyramidal neurons in the CA1 region of the hippocampus using the method of Example 4-2, it was confirmed that the thickness was restored in TE4 mice administered the Y04m antibody, as described in Fig. 5a.

[0297] In addition, the amount of PSD95, a key protein of excitatory synapses, was confirmed using the method of Example 4-4, and as described in Fig. 5b, it was confirmed that the amount increased in TE4 mice administered with the Y04m antibody.

[0298] In addition, the amount of synapsin1 protein, which plays an important role in regulating the storage and release of synaptic vesicles, was confirmed by the method of Example 3-4, and as described in Fig. 5c, it was confirmed that the amount increased in TE4 mice administered with the Y04m antibody, and as described in Fig. 5d, the amount was also confirmed to have increased by immunohistochemical staining.

[0299] 5-4. Confirmation of anti-inflammatory effect

[0300] As a result of confirming the Iba1 protein and GFAP protein related to the inflammatory response using the method of Example 4-2, it was confirmed that the amount was reduced in TE4 mice administered with the Y04m antibody, as described in Figures 6a and 6b.

[0301] That is, when the Y04m antibody manufactured in Example 1 was administered to the TE4 mouse, a dementia mouse model, it was confirmed that cognitive improvement, Tau reduction, neuroprotection, and inflammation reduction effects were observed.

[0302]

[0303] Example 6. Experimental method for confirming BBB penetration performance of a fusion comprising an anti-ApoE4 antibody and a blood-brain barrier receptor binding peptide.

[0304] In order to confirm the BBB permeability of the fusion prepared in Example 1, the BBB permeability performance was confirmed using the method described in Korean Patent No. 2023-0174973.

[0305] 6-1. Creation of a blood-brain barrier (BBB) ​​model using induced pluripotent stem cells (iPSCs)

[0306] 6-1-1. Induction of differentiation into human brain microvascular endothelial cells (iBMECs)

[0307] monoculture

[0308] To create a blood-brain barrier (BBB) ​​model, induced pluripotent stem cells (iPSCs) (WiCell Research Institute Inc, IMR90-4) were differentiated into human brain microvascular endothelial cells (BMECs). First, iPSCs were seeded at 3x10 in a Matrigel-coated 6-well plate. 5 After seeding to a cell density of 2.5 × 10 cells / well, mTeSR (containing 10 μM Y27, total 2 mL / well) was added and cultured. Every 24 hours after seeding, the entire medium was replaced with mTeSR1 (excluding Y27). When the cell count was 2.5 × 10 5 4x10 5 cells / well (preferably 3x10 5 When the number of cells / well reached 10, the medium was replaced with 2 mL of UM (DMEM / F12 medium (containing 15 mM HEPES), 20% KOSR, 1% NEAA, 0.5% glutamax, and β-ME) per well to induce differentiation into iBMECs (induced BMECs) (UM phase, D0). The medium was replaced (2–3 mL / well) with UM (containing 1% B27) every day for 1–5 days (D1–D5) after the start of differentiation induction, and on day 6, the medium was replaced with EC (hESFM medium, containing 2% B27 and 0.02% bFGF) containing 0.1% retinoic acid (RA).

[0309] On the 7th day after differentiation induction, the transwell (consisting of a plate and an insert) for subculturing iBMECs was coated with an ECM solution (collagen:fibronectin:water = 4:1:5) as follows. The membrane filter of the transwell insert was coated with the ECM solution at 37°C for a minimum of 4 hours or a maximum of 24 hours before subculturing. At this time, the plate was coated only when performing triplicate cultures.

[0310] On the 8th day after differentiation induction, the ECM solution was removed from the insert plate of the transwell, dried, and the cultured iBMECs were harvested using Accutase and hESFM medium, resuspended in an appropriate volume of EC medium (containing 0.1% RA), and seeded at 1x10 in the insert plate of the transwell. 6 cells / cm 2 were seeded. The insert seeded with iBMECs was placed on the plate, and the plate was shaken back and forth or left and right to evenly spread the cells so that they did not clump together. Then, the plate was cultured in a 37°C incubator, and the medium was replaced with EC (hESFM medium, containing 2% B27) every 24 hours.

[0311] Triple culture

[0312] The three-type culture was performed in the same manner until day 6 after inducing differentiation of iBMECs in a single culture, and on day 7, astrocytes (ScienceCell, Catalog #1800) and pericytes (ScienceCell, Catalog #1200) were seeded in 12 transwell plates using the following two methods. In pericyte medium, astrocytes (4.4x10 4 cells) and pericytes (4.4x10 4 cells) were cultured at a 1:1 ratio, or 1.5x10 astroglial cells (1.5x10 ) were cultured in 500 μL of pericyte medium. 4 cells) and pericyte cells (3x10 4 cells) were cultured at a ratio of 1:2.

[0313] On the 8th day after differentiation induction, the medium in the plate was replaced with endothelial cell medium (containing 10 μM RA), and iBMECs were seeded in the inserts of the transwell in the same manner as in single culture to prepare a BBB model containing three types of cells (endothelial cells, astrocytes, and pericytes).

[0314] Medium replacement for single culture models or triple culture models seeded on transwells was performed by removing the medium from the plate and then removing the medium from the insert. When adding hESFM (containing 2% B27) medium, the medium was added to the insert and then added to the plate.

[0315] 6-2. Measurement of permeability coefficient (Papp)

[0316] The permeability coefficient (Papp) is an absolute value that indicates the extent to which a substance escapes per unit area and per unit time. It is a comparable value regardless of the area and time of the transwell used in the experiment, and can be expressed as the following equation.

[0317] Papp = (dQ / dt) / (C0 Х A)

[0318] Here, dQ / dt means the amount that escapes into the plate (basal) region per unit time, and A is the insert area of ​​the transwell (cm 2 ) and C0 represent the initial concentration of the sample introduced into the insert (apical) region of the transwell.

[0319] 0.5 mL of hESFM (containing 2% B27) medium was prepared in a 1.5 mL tube, and seven types of antibodies and fusion constructs (25 μg / mL) were added to prepare a solution containing antibodies. After removing 0.4 mL of medium from the insert area of ​​the transwell, 0.4 mL of the 0.5 mL solution containing antibodies was added. 24 hours after administration of the solution containing antibodies (D11), 1.5 mL of the medium in the plate area and 0.5 mL of the medium in the upper chamber were obtained and subjected to ELISA analysis. The dQ / dt value was obtained from the ELISA analysis value, and A is 12 transwells (insert: 1.12 cm). 2 , Plate: 3.5 cm 2 ) or 24 Transwells (insert: 0.33 cm 2 , plate: 2 cm2 ) was used as the area of ​​the well, and 0.1 mL of the solution containing the remaining antibody placed in the transwell insert was used to measure the C0 value.

[0320] 6-3. Enzyme-linked immunosorbent assay (ELISA)

[0321] The ELISA analysis method for antibodies and fusions that penetrated from the insert area to the plate area is as follows. ApoE4 protein was diluted to 1 concentration using capture buffer (50 mM NaHCO3 and distilled water, pH 9.6) and 50 μL was added to the plate and incubated (37°C, 700 rpm, for 1 day) to coat it. Then, 130 μL of blocking buffer (100 mM Tris, 0.1% Tween20, distilled water and 2.5% Casein, pH 7.6) was added to each well and incubated (37°C, 700 rpm, 1 hour). Then, 50 μL of standard solution or sample was loaded into each well and diluted in the order of 20, 10, 5, 2.5, 1.25, 0.625, 0.3125, and 0 (ng / mL) using the standard dilution analysis (STD) technique. After incubation (2 hours), 50 μL of a solution containing antibodies (peroxidase-labeled mouse or human IgG, 1:10,000) (Sigma-Aldrich, AP309P) was added to each well and incubated (37°C, 600 rpm, 1 hour). Then, 50 μL of substrate reagent (R&D systems, #DY999) was added to each well and incubated at room temperature for 5 minutes. Afterwards, 50 μL of stop solution (R&D systems, #DY994) was added to terminate the reaction, and the absorbance was measured using a spectrometer (infinite F50) (plate shaking 10 seconds / measurement wavelength 450 nm / reference wavelength 620 nm).

[0322] Except for the step of adding stop solution after the substrate reagent reaction, each well was filled with wash buffer (PBS containing 0.05% Tween 20) and then discarded, and washed four times.

[0323]

[0324] Example 7. Confirmation of BBB penetration performance of a fusion comprising an anti-ApoE4 antibody and a blood-brain barrier receptor binding peptide.

[0325] 7-1. Y04m antibody-based fusion

[0326] As a result of confirming the BBB permeability of the Y04m antibody prepared in Example 1 by the method of Example 6 and the fusion product in which the antibody and BPP are fused (Y04m-APEP2(2): SEQ ID NO: 23+SEQ ID NO: 24; Y04m-THR(2): SEQ ID NO: 25+SEQ ID NO: 26; Y04m-RVG29(2): SEQ ID NO: 27+SEQ ID NO: 28; Y04m-APEP2(4): SEQ ID NO: 29+SEQ ID NO: 30; Y04m-THR(4): SEQ ID NO: 31+SEQ ID NO: 32), it was confirmed that the permeability was high in all the fusion products compared to the Y04m antibody, and in particular, it was confirmed that the permeability of the Y04m-APEP2(4) fusion product was the highest.

[0327] 7-2. Y04h antibody-based fusion

[0328] As a result of confirming the BBB permeability of the Y04h antibody and Y04h-APEP2(4) fusion antibody (SEQ ID NO: 37 + SEQ ID NO: 38) prepared in Example 1 by the method of Example 6, it was confirmed that the permeability of the Y04h-APEP2(4) fusion was higher than that of the Y04h antibody, as described in (B) and (C) of Fig. 11.

[0329]

[0330] Example 8. In vivo BBB penetration performance of a fusion comprising a humanized anti-ApoE4 antibody and a blood-brain barrier receptor binding peptide

[0331] 8-1. Sampling

[0332] After a single intravenous administration of 50 mg / kg of human IgG control, Y04h, and Y04h-APEP2(4), blood samples were collected via the tail vein and heart, and cerebrospinal fluid samples were collected via the cisterna magna. Approximately 0.2–0.9 mL of blood was collected using a K2EDTA tube, centrifuged at 10,000 rpm for 1 min, and plasma was collected and stored at -80°C until analysis. Approximately 0.05–0.1 mL of cerebrospinal fluid was collected using a glass tube and stored at -80°C until analysis.

[0333] 8-2. Sample Analysis

[0334] Human IgG control, Y04h, and Y04h-APEP2(4) concentrations in plasma and cerebrospinal fluid (CSF) were analyzed using an hIgG ELISA kit. 100 μL of the standard and sample were added to each well. The plates were covered and incubated at room temperature for 2.5 hours. The solution was discarded, and the plates were washed four times with 1X wash solution (300 μL). 100 μL of 1X biotinylated IgG detection antibody was added to each well and incubated at room temperature for 1 hour. The plates were washed, and 100 μL of 1X HRP-streptavidin was added and incubated at room temperature for 45 minutes. After washing, 100 μL of TMB One-Step Substrate Reagent was added and incubated at room temperature for 30 minutes with gentle shaking in the dark. 50 μL of Stop Solution was added to each well. Absorbance was measured immediately at 450 nm. The BBB penetration rate (%) was calculated as (cerebrospinal fluid concentration 24 hours after administration) / (plasma concentration 24 hours after administration * 1000) * 100, and the unit was set to ng / ml by multiplying the plasma concentration by 1000.

[0335] 8-3. Confirmation of BBB penetration performance

[0336] As a result of confirming the BBB permeability measured by the above method, as described in Fig. 12, it was confirmed that the permeability of the Y04h-APEP2(4) fusion was the highest compared to the control group and Y04h antibody.

[0337]

[0338] While specific aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

[0339]

[0340] The antibody specifically binding to ApoeE4 according to the present invention has a very high and specific binding affinity for ApoE4, and is superior in behavioral improvement effect, reduction of tau pathology in the brain, neuroprotection and inflammation reduction effect in a mouse model of dementia, and in the case of an Fc region-variant antibody, has an effect of extending the duration in the body, and a fusion product in which the antibody and a blood-brain barrier receptor binding peptide are combined exhibit excellent blood-brain barrier penetration ability and can be usefully used for the prevention or treatment of degenerative neurodegenerative diseases or metabolic diseases caused by ApoE4.

[0341]

[0342] Electronic file attached.

Claims

An antibody or antigen-binding fragment thereof that specifically binds to apolipoprotein E4 (ApoE4), comprising: A heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1; a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 2; and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 3; and A light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4; a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:

6. An antibody or antigen-binding fragment thereof that specifically binds to ApoE4 protein, characterized in that it comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, or SEQ ID NO: 36; and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 8, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, or SEQ ID NO:

40. In the first paragraph, the antibody (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 9; and a light chain comprising the amino acid sequence of SEQ ID NO: 10; (ii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 41; and a light chain comprising the amino acid sequence of SEQ ID NO: 42; (iii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 45; and a light chain comprising the amino acid sequence of SEQ ID NO: 42; and (vi) a heavy chain comprising the amino acid sequence of SEQ ID NO: 46; and a light chain comprising the amino acid sequence of SEQ ID NO: 42; An antibody that specifically binds to the ApoE4 protein, characterized in that it comprises any one selected from the group consisting of: A fusion product comprising an antibody or an antigen-binding fragment thereof that specifically binds to ApoE4 according to any one of claims 1 to 3 and one or more blood-brain barrier (BBB) ​​receptor binding peptides. In claim 4, a fusion protein characterized in that the blood-brain barrier receptor binding peptide is at least one selected from the group consisting of APEP2, RVG29, THR, APEP7, L57, AEP, L-CDX, HAI, CRT, Leptin30, G23, and IGF_C12. In the fifth paragraph, the blood brain barrier receptor binding peptide (1) APEP2 comprising the amino acid sequence of sequence number 11; (2) RVG29 comprising the amino acid sequence of sequence number 12; (3) THR comprising the amino acid sequence of sequence number 13; (4) APEP7 comprising the amino acid sequence of sequence number 14; (5) L57 comprising the amino acid sequence of sequence number 15; (6) AEP comprising the amino acid sequence of sequence number 16; (7) L-CDX comprising the amino acid sequence of sequence number 17; (8) HAI comprising the amino acid sequence of sequence number 18; (9) CRT comprising the amino acid sequence of sequence number 19; (10) Leptin30 comprising the amino acid sequence of sequence number 20; (11) G23 comprising the amino acid sequence of sequence number 21; and (12) IGF_C12 comprising the amino acid sequence of sequence number 22; A fusion characterized by being selected from the group consisting of: A fusion according to any one of claims 4 to 6, wherein the antibody specifically binding to ApoE4, or an antigen-binding fragment thereof, and the blood-brain barrier (BBB) ​​receptor binding peptide are characterized by direct binding, linker-mediated binding, direct fusion (genetic fusion), or linker-mediated fusion. A fusion product according to claim 7, characterized in that the linker is an amino acid linker. In claim 8, a fusion protein characterized in that the antibody specifically binding to ApoE4, or an antigen-binding fragment thereof, and the blood-brain barrier (BBB) ​​receptor binding peptide are fused via an amino acid linker. A fusion protein according to any one of claims 7 to 9, characterized in that the fusion protein is in the form of a blood-brain barrier (BBB) ​​receptor binding peptide fused to at least one position of the C-terminus of an antibody or antigen-binding fragment thereof that specifically binds to ApoE4 via an amino acid linker. A fusion protein characterized in that the fusion protein is in the form of a fusion protein in which a blood-brain barrier (BBB) ​​receptor binding peptide is fused via an amino acid linker at one or more positions of the heavy chain C-terminus and / or the light chain C-terminus of an antibody or antigen-binding fragment thereof that specifically binds to ApoE4. A fusion protein characterized in that the fusion protein is in the form of a blood-brain barrier (BBB) ​​receptor binding peptide fused via an amino acid linker at both C-terminal positions of the heavy chain of an antibody or antigen-binding fragment thereof that specifically binds to ApoE4 in claim 11. A fusion protein characterized in that the fusion protein is in the form of a fusion protein in which a blood-brain barrier (BBB) ​​receptor binding peptide is fused via an amino acid linker at both two C-terminal heavy chain positions and both two C-terminal light chain positions of an antibody or antigen-binding fragment thereof that specifically binds to ApoE4. In any one of claims 4 to 13, the fusion body, (a) an amino acid sequence of SEQ ID NO: 23; and an amino acid sequence of SEQ ID NO: 24; (b) the amino acid sequence of SEQ ID NO: 25; and the amino acid sequence of SEQ ID NO: 26; (c) the amino acid sequence of SEQ ID NO: 27; and the amino acid sequence of SEQ ID NO: 28; (d) the amino acid sequence of SEQ ID NO: 29; and the amino acid sequence of SEQ ID NO: 30; (e) the amino acid sequence of SEQ ID NO: 31; and the amino acid sequence of SEQ ID NO: 32; and (f) the amino acid sequence of SEQ ID NO: 43; and the amino acid sequence of SEQ ID NO: 44; A fusion characterized by being selected from the group consisting of: A nucleic acid encoding an antibody or an antigen-binding fragment thereof that specifically binds to ApoE4 according to any one of claims 1 to 14, or a fusion product comprising an antibody or an antigen-binding fragment thereof that specifically binds to ApoE4 and a blood-brain barrier (BBB) ​​receptor binding peptide. A recombinant expression vector comprising the nucleic acid of claim 15. A cell transformed with the recombinant expression vector of Article 16. A cell according to claim 17, characterized in that the transformed cell is selected from the group consisting of animal cells, plant cells, yeast, Escherichia coli, and insect cells. A method for producing an antibody or antigen-binding fragment thereof that specifically binds to ApoE4, or a fusion product comprising an antibody or antigen-binding fragment thereof that specifically binds to ApoE4 and a blood-brain barrier (BBB) ​​receptor-binding peptide, comprising a step of culturing the cell of claim 17. A composition for preventing or treating a neurodegenerative disease or metabolic disease, comprising an antibody or antigen-binding fragment thereof that specifically binds to ApoE4 according to any one of claims 1 to 14, or a fusion product comprising the antibody or antigen-binding fragment thereof that specifically binds to ApoE4 and a blood-brain barrier (BBB) ​​receptor-binding peptide. A composition characterized in that the composition is at least one selected from the group consisting of vascular dementia, multiple sclerosis, ischemic stroke, and Parkinson's disease, according to claim 20. A composition according to claim 20, wherein the metabolic disease is at least one selected from the group consisting of cardiovascular disease, type 2 diabetes mellitus, hyperlipoproteinemia, and age-related hearing loss.

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