Brain-blood barrier permeable fusion protein and its use

Abstract

The present application relates to a fusion protein capable of permeating the blood-brain barrier and uses thereof, and provides a fusion protein capable of permeating the blood-brain barrier, a polynucleotide encoding the fusion protein, a vector containing the polynucleotide, a cell line transfected with the vector, and a pharmaceutical composition for preventing or treating diseases associated with brain dysfunction, comprising the fusion protein as an active ingredient.

Description

[Technical Field]

[0001] The present invention relates to a fusion protein that permeates the blood-brain barrier and its use, and more particularly to a fusion protein comprising an IgG antibody and a helical domain-binding moiety of a transferrin receptor linked to the end of the IgG antibody, and its pharmaceutical use.

[0002] This patent application claims priority to Korean Patent Application No. 10-2021-0194319 filed with the Korean Intellectual Property Office on December 31, 2021, and Korean Patent Application No. 10-2022-0189752 filed with the Korean Intellectual Property Office on December 29, 2022, the disclosures of which are incorporated herein by reference. [Background technology]

[0003] The blood-brain barrier (BBB) ​​is a vascular barrier that separates the brain from the blood and serves to isolate the central nervous system, including the brain, from potentially dangerous substances in the blood. The BBB is a central structure of the brain's blood vessels, composed of cells such as brain endothelial cells, astrocytes, and pericytes, and is distributed throughout the brain's blood vessels within the brain tissue. The BBB endothelial cells are tightly connected by tight junctions, and astrocytes and astrocyte endfeet surround the periphery of blood vessels, forming a barrier that selectively prevents substances flowing along the blood in the brain vessels from penetrating the blood-brain barrier and being absorbed / transmitted into brain tissue. Such a barrier-like structure selectively allows or inhibits the permeation of substances depending on the type and size of the substance, but water and oxygen, which are essential for life, can pass through the blood-brain barrier by diffusion, while amino acids and glucose, which are used as energy sources, can be transported from the blood to brain tissue by active transport. However, toxic substances and pathogenic bacteria that can potentially affect the brain are not only inhibited from passing through by the blood-brain barrier, but even if they do pass through, they will return to the blood due to the pumping action of cells, preventing their absorption into brain tissue and protecting the brain tissue.

[0004] However, this structure of the blood-brain barrier poses a major obstacle to the pharmacological treatment of diseases associated with brain dysfunction, such as Alzheimer's disease, Parkinson's disease, and brain cancer. In fact, it has been reported that only a very small number of therapeutic drugs with molecular weights of 400-500 Daltons or less can penetrate the blood-brain barrier. Most therapeutic drugs are either not delivered to brain tissue or delivered only in very small amounts, preventing them from exerting their effects in the target organ, the brain tissue. To address this issue, several techniques have been proposed: direct drug passage through the endothelial cell membrane via lipophilicity of the drug; delivery via the innate nutrient delivery system; and antibody-based delivery using receptor-mediated transcytosis. However, these techniques affect not only the brain but also other organs, raising concerns about side effects in the body. Furthermore, differences in delivery efficacy among patients make it difficult to determine the appropriate concentration or dosage, limiting their clinical application.

[0005] Under such technical background, various studies have been conducted to increase the permeability of the blood-brain barrier and improve selective delivery to brain tissue (Patent Document 1), but the current situation is still insufficient. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Korean Patent Publication No. 10-2021-0005647 Summary of the Invention [Problem to be solved by the invention]

[0007] In order to overcome such limitations of the conventional technology, the present inventors devised a fusion protein in which an IgG antibody and a helical domain-binding moiety of a tetravalent transferrin receptor are linked to the end of the IgG antibody. They confirmed that the fusion protein not only increased the permeability of the cerebrovascular barrier and improved delivery efficiency to brain tissue due to the specific transferrin receptor cluster expression pattern in the cerebrovascular region and the resulting specific interaction with the clusters in the cerebrovascular region, but also that the IgG antibody was selectively distributed at a higher level in brain tissue compared to other tissues, and based on this, they completed the present invention.

[0008] Accordingly, one embodiment provides a fusion protein having brain-blood barrier permeability, comprising an IgG antibody and a helical domain-binding moiety of a tetravalent transferrin receptor (TfR) linked to the C-terminal region of the light chain and the C-terminal region of the heavy chain of the IgG antibody.

[0009] Other aspects provide polynucleotides encoding the fusion proteins, vectors containing the polynucleotides, and transfected cell lines transfected with the vectors.

[0010] Another aspect provides a pharmaceutical composition for preventing or treating a disease associated with brain dysfunction, which comprises the fusion protein as an active ingredient.

[0011] Other objects and advantages of the present application will become more apparent from the following detailed description taken in conjunction with the appended claims and drawings. Contents not described in this specification can be fully understood and inferred by those skilled in the technical field of the present application or a similar technical field, and therefore, the description thereof will be omitted. [Means for solving the problem]

[0012] One embodiment provides a blood-brain barrier-permeable fusion protein comprising an IgG antibody and a tetravalent transferrin receptor (TfR) helical domain-binding moiety linked to the C-terminal region of the light chain and the C-terminal region of the heavy chain of the IgG antibody.

[0013] Blood-brain barrier (BBB) As used herein, the term "brain-brain barrier (BBB)" refers to a central structure in the brain's blood vessels that strictly controls the movement of substances such as ions, molecules, and pathogens present in the blood into brain tissue while allowing the selective absorption of vital components such as amino acids and glucose. The BBB is composed of cells such as BBB endothelial cells, astrocytes, and perivascular cells. These cells are arranged in a pattern that shares a common basement membrane, with tightly connected BBB endothelial cells on one side and astrocytes surrounding the blood vessels on the other side. The BBB endothelial cells, which make up the capillary walls, are very strong and connected by complex tight junctions. Such a structure forms a physical barrier and serves to inhibit the simple diffusion of most substances, including molecules larger than average in size, such as insulin. Astrocytes, a type of glial cell in the central nervous system, affect the function of brain endothelial cells, blood flow, and ionic balance through their interaction with the brain's blood vessels. Astrocytes utilize processes called endfeet to surround blood vessels at one end and to form close contact with neurons at synapses at the other end. The blood-brain barrier, which has such a structure, protects brain tissue from potentially harmful substances in the blood and also prevents the delivery of substances effective in treating diseases to brain tissue, acting as a major obstacle in pharmacological treatment. Therefore, technologies to improve the permeability of the blood-brain barrier could improve the applicability or efficacy of therapeutic agents in the field of medicine targeting brain tissue.

[0014] IgG antibody As used herein, the term "IgG antibody" refers to an immunoglobulin molecule immunologically reactive with a specific antigen and a protein molecule that acts as an antigen receptor, specifically recognizing the antigen. The antibody has a heavy chain and a light chain, each of which contains a constant region and a variable region. The variable regions of the light chain and the heavy chain contain three variable regions called complementarity-determining regions (CDRs) and four framework regions. The CDRs primarily function to bind to the antigen epitope. The CDRs of each chain are typically referred to sequentially, starting from the N-terminus, as CDR1, CDR2, and CDR3, and are identified by the chain in which the particular CDR is located. The antibody also includes polyclonal antibodies, monoclonal antibodies, full-length antibodies, and antibody fragments or antigen-binding fragments containing the antigen-binding domain. The full-length antibody has two full-length light chains and two full-length heavy chains, each light chain linked to a heavy chain by a disulfide bond. The full-length antibody is preferably an IgG, and may be any of its subtypes, including IgG1, IgG2, IgG3, and IgG4.

[0015] The antibody may be a natural antibody or a recombinant antibody. A natural antibody refers to an antibody that has not been genetically modified, and may have significantly less immunogenicity in vivo than genetically modified antibodies. A recombinant antibody refers to an antibody that has been genetically modified, and has the advantage that antigen binding ability or other desired characteristics can be added through genetic modification.

[0016] As used herein, the term "antigen-binding fragment" or "antibody fragment" refers to a fragment that retains antigen-binding function, and includes functional antibody fragments such as Fab, F(ab'), F(ab')2, Fv, single-chain Fv (scFv), and scFv-Fc bivalent molecules, or combinations thereof. Examples of the antigen-binding fragment include, but are not limited to, the following, and may include, but are not limited to, structures of IgG-like bispecific antibodies known in the art: (1) Fab: The fragment that contains a monovalent antigen-binding fragment of an antibody molecule and can be produced by digesting a whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain; (2) Fab': A fragment of an antibody molecule that can be obtained by treating a whole antibody with pepsin followed by reduction to obtain the intact light and heavy chain portions; two Fab' fragments are obtained per antibody molecule; (3) (Fab')2: A fragment of an antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction, and a dimer of two Fab' fragments linked together by two disulfide bonds; (4) Fv: a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; (5) Single-chain antibodies (SCAs or scFvs), genetically fused single-chain molecules, genetically engineered molecules containing the variable region of a light chain and the variable region of a heavy chain linked by a suitable polypeptide linker; (6) scFv-Fc, a molecule produced by fusing a single-chain Fv (scFv) with a hinge region from an immunoglobulin (Ig), such as IgG, and the Fc region.

[0017] Furthermore, the antibody or antibody fragment is an immunoglobulin molecule that retains antigen-binding function, and includes monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, mouse antibodies, chimeric antibodies, scFVs, single-chain antibodies, Fab fragments, F(ab') fragments, F(ab')2 fragments, disulfide-linked Fvs (sdFVs), scFv fragments, scFv-Fc fragments, Fv fragments, diabodies, triabodies, tetrabodies, etc. Among antibody fragments, Fab has a structure comprising light and heavy chain variable regions, a light chain constant region, and a heavy chain first constant region (CH1) 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 disulfide bonds. Fv is the smallest antibody fragment, containing only the heavy-chain variable region and the light-chain variable region. In a two-chain Fv, the heavy-chain variable region and the light-chain variable region are linked by a non-covalent bond. Such antibody fragments can be obtained using protease hydrolases (for example, whole antibodies can be digested with papain to obtain Fab fragments, or with pepsin to obtain F(ab')2 fragments), or can be produced through genetic recombination techniques.

[0018] The IgG antibody herein is a fusion complex with a transferrin receptor helical domain-binding moiety, and the C-terminus of the light chain and the C-terminus of the heavy chain constituting the IgG antibody are each linked or joined to a transferrin receptor helical domain-binding moiety. Such an IgG antibody can have enhanced selective permeability across the blood-brain barrier and be distributed at a relatively high level in brain tissue compared to other organs and cells.

[0019] In one embodiment, the IgG antibody is an antibody or antigen-binding fragment capable of binding to a target antigen known in the art and applicable to treating, alleviating, or detecting a disease associated with brain dysfunction, e.g., a pharmaceutically active substance for treating or alleviating a disease associated with brain dysfunction. The IgG antibody is intended to be used in the treatment of Alzheimer's disease, dementia with Lewy bodies, frontotemporal dementia, tangle-only dementia, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis (ALS), traumatic brain injury, progressive supranuclear palsy, corticobasal degeneration, globular glial tauopathy, aging-related tau astrogliopathy, chronic traumatic encephalopathy (CTE), and primary CNS lymphoma (PCNSL). Brain cancers such as lymphoma, glioma, neuroblastoma, brain metastases, and meningioma; Pick's disease; anti-IgLON5-related taupathy; Guadeloupean parkinsonism; nodding syndrome; pain; epilepsy; autism; stroke; Guillain-Barré syndrome (GBS); Creutzfeldt-Jakob disease (CJD);Antibodies for treating or alleviating diseases associated with brain dysfunction, such as Huntington's disease, progressive multifocal leukoencephalopathy (PML), depression, post-traumatic stress disorder (PTSD), and lysosomal storage diseases (LSDs), include, but are not limited to, αPD-L1 IgG antibodies, anti-PD1 IgG antibodies, anti-tau IgG antibodies, anti-HER2 IgG antibodies, anti-Aβ IgG antibodies, anti-TDP-43 IgG antibodies, anti-α-synuclein IgG antibodies, anti-SIGLEC3 IgG antibodies, and anti-TREM2 IgG antibodies;

[0020] Transferrin receptor (TfR) As used herein, the term "transferrin receptor (TfR)" refers to a membrane glycoprotein expressed on the cell surface that mediates the intracellular absorption of iron from the plasma glycoprotein transferrin. It is reported to be widely distributed in normal cells of various tissues, as well as being abundantly expressed in activated immune cells and tumor cells. Therefore, transferrin receptor-mediated delivery techniques require not only effective delivery to brain tissue, but also low delivery to other organs other than brain tissue or to normal cells. In fact, it has been reported that transferrin receptor-mediated delivery agents or therapeutic agents can cause red blood cell-related toxicity, including a decrease in reticulocyte count, severe coma, intermittent limb stiffness, generalized rigidity, hemolysis, and hemoglobinuria. The ectodomain of the transferrin receptor is divided into an apical domain, a helical domain, and a protease-like domain, which are known to have different binding affinities with target substances during receptor-mediated transcytosis.

[0021] The genetic information of the transferrin receptor can be obtained from publicly known databases such as GenBank of the National Center for Biotechnology Information (NCBI). For example, the transferrin receptor may have the amino acid sequence of SEQ ID NO: 1, but is not limited thereto.

[0022] Transferrin receptor cluster As used herein, the term "transferrin receptor cluster" refers to a group of multiple transferrin receptors densely packed in a localized region, and the transferrin receptor cluster may be distributed in tissues or cells, such as blood vessels, in the form of a cluster of multiple transferrin receptors. In particular, unlike transferrin receptor clusters distributed in other organ tissues or cells, transferrin receptor clusters present in the cerebrovascular region have a specific expression pattern. Specifically, the specific expression pattern refers to a state in which transferrin receptors are distributed at an exceptionally dense level within the localized region where the cluster is formed, compared to other organ tissues or cells. Such brain tissue-specific clusters and the expression pattern of transferrin receptors within the clusters may be closely related to high permeability through the blood-brain barrier and selective delivery to brain tissue.

[0023] Transferrin receptor helical domain binding moiety As used herein, the term "transferrin receptor helical region-binding moiety" refers to a functional unit that interacts with a receptor present on the surface of cells that constitute the blood-brain barrier, and specifically refers to a moiety that has effective binding affinity for the helical region in the region that constitutes the transferrin receptor. The transferrin receptor helical region-binding moiety reflects the expression pattern of transferrin receptor clusters specific to brain tissue blood vessels and may be configured in a tetravalent form. Here, the transferrin receptor helical region is an externally exposed functional / structural region that allows effective tetravalent binding between the fusion protein and multiple transferrin receptors densely distributed within the brain tissue blood vessel clusters, which may contribute to high permeability through the blood-brain barrier and selective delivery to brain tissue. Therefore, when linked to an IgG antibody, a binding moiety capable of binding to the helical region of the transferrin receptor can not only significantly enhance the blood-brain barrier permeability of the IgG antibody, but also confer functionality that enables selective delivery / absorption to brain tissue.

[0024] The binding moiety interacts with transferrin receptor clusters distributed in the vascular region of brain tissue to form a complex / fusion, and has binding affinity with multiple transferrin receptors in the local region where the clusters are formed. Specifically, the binding moiety has binding affinity with the helical region of the transferrin receptor, more specifically, with at least one amino acid selected from the region from amino acid 606 to amino acid 665 of the transferrin receptor of SEQ ID NO: 1.

[0025] In one specific example, the transferrin receptor helical region-binding moiety is linked to the C-terminal region of the light chain and the C-terminal region of the heavy chain of the IgG antibody, thereby allowing the transferrin receptor helical region-binding moiety to be linked to the IgG antibody in a tetravalent form and to exert the desired functionality.

[0026] In one embodiment, the transferrin receptor helical region binding moiety has a length of 6 to 250 amino acids. For example, the length of the transferrin receptor helical region binding moiety is 6 to 240, 6 to 220, 6 to 200, 6 to 180, 6 to 160, 6 to 140, 6 to 120, 6 to 100, 6 to 80, 6 to 60, 6 to 40, 6 to 20, 6 to 10, 10 to 250, 10 to 240, 10 to 220, 10 to 200, 10 to 180, 10 to 160, 10 to 140, 10 to 120, 10 to 100, 10 to 80, 10 to 60, 10 to 40, 10 to 20, 20 to 250 , 20 to 240, 20 to 220, 20 to 200, 20 to 180, 20 to 160, 20 to 140, 20 to 120, 20 to 100, 20 to 80, 20 to 60, 20 to 40, 40 to 250, 40 to 240, 40 to 220, 40 to 200, 40 to 180, 40 to 160, 40 or less and may be, but is not limited to, 140, 40 to 120, 40 to 100, 40 to 80, 40 to 60, 60 to 250, 60 to 240, 60 to 220, 60 to 200, 60 to 180, 60 to 160, 60 to 140, 60 to 120, 60 to 100, or 60 to 80 amino acids in length.

[0027] The length of the transferrin receptor helical region binding moiety can also be, for example, but not limited to, 10 to 48, 10 to 44, 10 to 40, 10 to 36, 10 to 32, 10 to 28, 10 to 24, 10 to 20, 10 to 16, 10 to 12, 12 to 48, 12 to 44, 12 to 40, 12 to 36, 12 to 32, 12 to 28, 12 to 24, 12 to 20, 12 to 16, 14 to 48, 14 to 44, 14 to 40, 14 to 36, 14 to 32, 14 to 28, 14 to 24, 14 to 20, or 14 to 16 amino acids in length.

[0028] In one embodiment, the transferrin receptor helical domain binding moiety also includes an amino acid sequence in which the unit sequence is repeated 2 to 5 times, 2 to 4 times, or 2 to 3 times. The unit sequences can be linked by fusion or by a linker peptide. The linker peptide can be 2 to 50 amino acids in length, for example, 2 to 40, 2 to 30, 2 to 20, 2 to 15, 2 to 10, 2 to 5, 5 to 50, 5 to 40, 5 to 30, 5 to 20, 5 to 15, or 5 to 10 amino acids in length, but is not limited thereto. The linker peptide can also be, for example, (G l S m ) n (l is 2 to 8, m is 1 to 5, n is 1 to 5), (G d S e AS) f (d is 2 to 8, e is 1 to 5, f is 1 to 5), (G4S) a (EAAAK) b (G4S) a (a and b are integers from 1 to 4), [(G4S) p (EAAAK) q ] r (p, q, r are integers from 1 to 4), but are not limited thereto.

[0029] In one embodiment, the unit sequence constituting the helical domain-binding moiety of the transferrin receptor also includes a cell-penetrating peptide. The unit sequence may be any one of SEQ ID NOs: 3 to 43, but may be any unit sequence that has effective interaction (hydrogen bonding, electrostatic attraction, van der Waals forces), i.e., effective binding affinity, with the helical domain of the transferrin receptor.

[0030] In one embodiment, each of the helical region binding moieties of the plurality of transferrin receptors is the same or different.

[0031] Brain-blood barrier permeable fusion protein As used herein, the term "fusion protein" refers to a protein formed by the binding of two or more originally separate proteins or portions thereof, and optionally includes a linker or space between the two or more proteins. As used herein, the term "vascular-brain barrier-permeable fusion protein" collectively refers to a protein formed by the binding of an IgG antibody and a transferrin receptor helical domain-binding moiety, and includes a functional structure that allows the fusion protein to penetrate the vascular-brain barrier and selectively deliver or absorb an effective amount of IgG antibody into brain tissue. The functional structure reflects the structure and expression pattern of transferrin receptor clusters in vascular endothelial cells that are specifically distributed in the vascular-brain barrier, and is modified to have effective binding affinity. The functional structure also includes a binding moiety capable of binding to the transferrin receptor helical domain, and tetravalent bonds (linkages) between the binding moiety and the C-terminal domains of the heavy and light chains of an IgG antibody.

[0032] In one embodiment, the blood-brain barrier-permeable fusion protein also comprises a functional structure in which a tetravalent helical region-binding moiety is linked to the C-terminal region of the light chain and the C-terminal region of the heavy chain of the IgG antibody.

[0033] In one specific example, the C-terminal region of the light chain and the C-terminal region of the heavy chain of the IgG antibody are linked to the helical region-binding moiety by fusion to the terminal region of the IgG antibody or by a linker peptide, and the details regarding the linker peptide are as described above.

[0034] In one embodiment, the blood-brain barrier-permeable fusion protein binds to and forms a complex with transferrin receptors that form transferrin receptor clusters that are specifically distributed in the blood vessels of the blood-brain barrier, for example, the blood-brain barrier-permeable fusion protein binds to multiple transferrin receptors that are densely distributed in transferrin receptor clusters compared to other organ tissues or cells.

[0035] According to one embodiment, the functional structure was designed to reflect the effective binding affinity for the helical domain of the transferrin receptor and the specific distribution / expression pattern of transferrin receptor clusters distributed in the cerebrovascular region. A fusion protein was prepared in which multiple transferrin receptor helical domain-binding moieties were linked to the C-terminal regions of the light chain and the heavy chain of an IgG antibody, i.e., a total of four terminal domains. Subsequently, intravenous administration of a formulation containing the fusion protein not only significantly enhanced delivery of the IgG antibody-containing fusion protein in brain tissue, but also demonstrated a higher level of distribution of the IgG antibody in brain tissue compared to other organs, with minimal effects on normal cells, including reticulocytes, and other organ tissues. Furthermore, it was found that the fusion protein exhibited effects such as high permeability across the cerebrovascular barrier, a function resulting from the tetravalent moiety with effective binding affinity for the helical domain of the transferrin receptor and the functional structure containing the same, regardless of the type of IgG antibody or the specific sequence of the binding moieties. Therefore, in one embodiment, a functional structure was newly identified that can enhance the blood-brain barrier permeability of the IgG antibody linked to the binding moiety, induce selective delivery to brain tissue, and contribute to reducing the side effects of antibody-based drugs.

[0036] In one embodiment, the blood-brain barrier-permeable fusion protein also comprises a first binding moiety linked to the C-terminal region of the light chain of the IgG antibody and a second binding moiety linked to the C-terminal region of the heavy chain of the IgG antibody.

[0037] In one embodiment, the blood-brain barrier-permeable fusion protein may be formulated and administered, and the administration may be carried out by any of the common administration methods for fusion proteins or antibody-based formulations, including, but not limited to, intravenous administration, intramuscular administration, subcutaneous administration, intraperitoneal administration, ocular administration, intrathecal administration, intracerebroventricular administration, intranasal administration, etc. Under such administration conditions, the fusion protein can enable the fusion partner IgG antibody to be delivered / distributed at a high level within brain tissue through binding to a portion of the receptor present on the surface of cells that constitute the blood-brain barrier, i.e., the helical domain of the transferrin receptor, which can provide an appropriate level of interaction or binding affinity with the helical domain-binding moiety of the transferrin receptor.

[0038] Other aspects provide a polynucleotide encoding said fusion protein, a vector comprising said polynucleotide, or a transfected cell line transfected with said vector.

[0039] As mentioned above, the terms and elements referred to in the following polynucleotides, vectors, and transfected cell lines are the same as those referred to in the description of the blood-brain barrier-permeable fusion protein.

[0040] Polynucleotide encoding the fusion protein The polynucleotide may be in the form of RNA or DNA, including cDNA and synthetic DNA. The DNA may be single-stranded or double-stranded. If single-stranded, it may be the coding strand or non-coding (antisense) strand, and the coding sequence may be the result of degeneracy or redundancy in the genetic code and encode the same polypeptide.

[0041] The polynucleotides also include variants of the polynucleotides described herein, which may be naturally occurring allelic variants of the polynucleotide or non-naturally occurring variants of the polynucleotide. Allelic variants are alternate forms of a polynucleotide sequence that may have one or more nucleotide substitutions, deletions, or additions that do not substantially alter the function of the encoded polynucleotide. It is well known in the art that a single amino acid is encoded by more than one nucleotide codon, and that the polynucleotides can be readily modified to produce alternate polynucleotides that encode the same peptide.

[0042] Vector containing the polynucleotide As used herein, the term "vector" refers to a DNA vehicle capable of expressing a target protein in an appropriate host cell, and refers to a genetic construct containing operably linked regulatory elements for expression of a gene insert. According to one embodiment, the vector may contain expression regulatory elements such as a promoter, operator, initiation codon, termination codon, polyadenylation signal, and / or enhancer, and the promoter of the vector may be constitutive or inducible. The vector may also be an expression vector capable of stably expressing the fusion protein in host cells. The expression vector may be a vector commonly used in the art for expressing foreign proteins in plants, animals, or microorganisms. The recombinant vector may be constructed by various methods known in the art. For example, the vector may contain a selectable marker for selecting host cells containing the vector, and, if the vector is replicable, may also contain a replication origin. The vector may be self-replicating or integrated into the host DNA, and may be selected from the group consisting of a plasmid, a lentivirus, an adenovirus, an adeno-associated virus, a retrovirus, a herpes simplex virus, and a vaccinia virus.

[0043] In one embodiment, the vector comprises a promoter operable in an animal cell, preferably a mammalian cell. Suitable promoters include promoters derived from mammalian viruses and promoters derived from the genome of mammalian cells, such as the cytomegalovirus (CMV) promoter, T7 promoter, U6 promoter, H1 promoter, and murine leukemia virus (MLV) long terminal repeat (LTR). These include the α- and β-actin promoters, ...

[0044] In the vector, the polynucleotide sequence encoding the vascular-brain barrier-permeable fusion protein is also operably linked to a promoter. As used herein, the term "operably linked" refers to the functional connection between a nucleic acid expression regulatory sequence (e.g., a promoter, a signal sequence, or an array of transcriptional regulatory factor binding sites) and another nucleic acid sequence, whereby the regulatory sequence regulates the transcription and / or translation of the other nucleic acid sequence.

[0045] Transduced cell lines As used herein, the term "transfection" refers to a molecular biological technique in which a DNA fragment or a plasmid carrying a foreign gene different from that originally present in the original cell is introduced into the cell and binds to the DNA present in the original cell, thereby changing the genetic traits of the cell. Transfection refers to the insertion of a polynucleotide encoding the vascular-brain barrier-permeable fusion protein or a vector containing the same into a host cell, resulting in the production of the vascular-brain barrier-permeable fusion protein.

[0046] The transfected cell line or host cell is preferably any one selected from the group consisting of microorganisms including prokaryotes such as bacteria and eukaryotes such as yeast, insect-derived cells such as Sf9 cells, mouse-derived cells such as CHO cells, human-derived cells such as HEK293, and antibody-producing hybridomas, but is not limited thereto.

[0047] Other aspects provide a pharmaceutical composition for preventing or treating a disease associated with brain dysfunction, comprising the fusion protein as an active ingredient; a medical use of the fusion protein for preventing or treating a disease associated with brain dysfunction; or a method for treating a disease associated with brain dysfunction, comprising administering the pharmaceutical composition to an individual.

[0048] Among the terms or elements mentioned in the following pharmaceutical compositions for preventing or treating diseases associated with brain dysfunction, their pharmaceutical uses, or methods for treating diseases associated with brain dysfunction, those that are the same as those mentioned in the description of the blood-brain barrier-permeable fusion protein are as described above.

[0049] Pharmaceutical composition for preventing or treating diseases associated with brain dysfunction As used herein, the term "prevention" refers to any action of suppressing or delaying the onset of a disease associated with brain dysfunction by administering the pharmaceutical composition.

[0050] As used herein, the term "treatment" refers to any action that improves or favorably alters symptoms associated with diseases associated with brain dysfunction by administering the pharmaceutical composition.

[0051] The "diseases associated with brain dysfunction" that are the targets of prevention or treatment by the pharmaceutical composition include Alzheimer's dementia, Lewy dementia, anterior temporal lobe dementia, neurofibrillary tangle-dominant dementia, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis, traumatic brain injury, progressive supranuclear palsy, corticobasal degeneration, glio-tau pathologies, aging-associated tau-astrogliopathy, chronic traumatic encephalopathy, primary central nervous system lymphoma, glio-tau disease, and the like. Brain cancers such as cerebrospinal fluid tumors, neuroblastoma, brain metastases, and meningiomas; Pick's disease; anti-IgLON5-associated tauopathy; Guadeloupe Parkinsonism; nodding syndrome; pain syndrome; electroencephalopathy; autism; stroke; Guillain-Barré syndrome; Creutzfeldt-Jakob disease; Huntington's disease; progressive multifocal leukoencephalopathy; depression; post-traumatic stress disorder; and lysosomal storage diseases.

[0052] The pharmaceutical composition, i.e., pharmaceutical preparation containing the fusion protein, is prepared by mixing an antibody having a desired degree of purity with any pharmaceutically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)) in the form of a lyophilized preparation or an aqueous solution. Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations used, and include buffers such as phosphate, citrate, and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl alcohol, benzyl alcohol; alkylparabens such as methylparaben and propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol). low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; monosaccharides, disaccharides, and other carbohydrates, such as glucose, mannose, or dextrins; chelating agents, such as ethylenediaminetetraacetic acid (EDTA); sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants, such as Twin™, Pluronics. TM or polyethylene glycol (PEG).

[0053] The pharmaceutical composition may also contain additional active ingredients, optionally with complementary activities that do not adversely affect each other. The type and effective amount of the pharmaceutical composition may be determined, for example, by the amount of antibody present in the formulation and the subject's clinical parameters. The active ingredient may be delivered in the form of an encapsulated form in microcapsules prepared by droplet formation technology or interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or macroemulsions.

[0054] The pharmaceutical compositions may be formulated, dosed, and administered in a manner consistent with medical practice. Factors to be considered in this regard include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the administration schedule, and other factors known to physicians. The antibody may, but need not, be formulated with one or more agents currently used to prevent or treat disease. The effective amount of the other agent(s) will depend on the amount of antibody present in the formulation, the type of disorder or treatment, and other factors. They will generally be used in the same dosages and by any route of administration as described herein, or about 1 to 99% of the dosages described herein, or any dosage and route empirically / clinically determined to be appropriate.

[0055] Methods for treating disorders associated with brain dysfunction The method for treating a disease associated with brain dysfunction can use the pharmaceutical composition alone or in combination with other agents, for example, the pharmaceutical composition can be co-administered with at least one additional therapeutic agent. The additional therapeutic agent is a therapeutic agent effective in treating diseases associated with brain dysfunction, including, but not limited to, cholinesterase inhibitors (e.g., donepezil, galantamine, rivastigmine, and tacrine), NMDA receptor antagonists (e.g., memantine), amyloid beta peptide aggregation inhibitors, antioxidants, gamma secretase modulators, nerve growth factor (NGF) mimics or NGF gene therapy agents, PPAR gamma agonists, HMS-CoA reductase inhibitors (statins), ampakines, calcium channel blockers, GABA receptor antagonists, glycogen synthase kinase inhibitors, intravenous immunoglobulin, muscarinic receptor agonists, nicotinic receptor modulators, active or passive amyloid beta peptide immunization, phosphodiesterase inhibitors, serotonin receptor antagonists, and anti-amyloid beta peptide antibodies.

[0056] The appropriate dosage of the pharmaceutical composition (used alone or in combination with one or more additional therapeutic agents) for the prevention or treatment of a target disease will depend on the type of disease being treated, the severity and course of the disease, whether the antibody is being administered for prophylactic or therapeutic purposes, previous therapy, the patient's clinical status, and response to the antibody, as well as the judgment of the treating physician. The antibody may be administered to the patient at one time or over a series of treatments, and for purposes of the present invention, the antibody may also be provided in the form of a fusion protein linked to a tetravalent transferrin receptor helical domain-binding moiety. Depending on the type and severity of the disease, an initial candidate dosage of about 1 μg / kg to 100 mg / kg (e.g., 0.1 mg / kg to 100 mg / kg) of antibody or fusion protein may be administered to the patient, for example, in one or more separate administrations or by continuous infusion. Typical daily dosages range from about 1 μg / kg to 100 mg / kg or more, depending on the factors mentioned above. For repeated administration over several days or longer, depending on the condition, treatment is generally sustained until the desired suppression of disease symptoms occurs. An exemplary dosage of an antibody or fusion protein ranges from about 0.05 mg / kg to about 100 mg / kg. Thus, one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg, 10 mg / kg, or 20 mg / kg (or any combination thereof) may be administered to the patient. The doses may be administered intermittently, e.g., weekly or every three weeks (e.g., such that the patient receives an antibody or fusion protein dose of about 2 mg / kg to about 20 mg / kg, or, e.g., about 6 mg / kg). An initial, higher loading dose may be followed by one or more smaller doses.

[0057] As used herein, the term "individual" refers to a subject in need of treatment for a disease, and more specifically refers to mammals such as human or non-human primates, mice, dogs, cats, horses, and cattle. [Effects of the Invention]

[0058] According to one embodiment of the blood-brain barrier-permeable fusion protein, when a formulation containing the fusion protein is administered intravenously, it has been found that the delivery of IgG antibodies in brain tissue is significantly enhanced due to the effective interaction between the binding moiety and the transferrin receptor.

[0059] Furthermore, according to one embodiment of the fusion protein having blood-brain barrier permeability, when a formulation containing the fusion protein is administered intravenously, IgG antibodies can be selectively delivered at a high level in brain tissue compared to other organs and cells due to the specific distribution of transferrin receptor clusters in the vascular region of brain tissue and effective interaction with the clusters, while exhibiting excellent biological safety.

[0060] Therefore, the fusion protein according to one embodiment can be utilized in the medical field requiring selective drug delivery to brain tissue, for example, as an active ingredient of a pharmaceutical composition for the prevention or treatment of diseases associated with brain dysfunction. [Brief explanation of the drawings]

[0061] [Figure 1] The transferrin receptor was imaged using cryo-electron microscopy, and the helical domain within the transferrin receptor was identified. [Figure 2] 1 shows the structural results of the binding between a helical domain-binding moiety and the helical domain of the transferrin receptor, determined by cryo-electron microscopy, according to one embodiment. [Figure 3] 1 shows the results of confirming the thermodynamic structural stability of the binding between helical region-binding moiety #01 and transferrin receptor according to one example. [Figure 4] 1 shows the results of confirming the intracellular delivery level of helical region-binding moiety #01 using a human brain endothelial cell line, according to one embodiment. [Figure 5]1 shows the results of intravenous administration of the blood-brain barrier-permeable fusion protein F3#01 according to one embodiment to an animal model, and then confirming the level of IgG1 antibody in brain tissue using a human IgG1 ELISA kit. [Figure 6] 1 shows the results of confirming the level of IgG1 antibodies in ISF using a human IgG1 ELISA kit after intravenous administration of the blood-brain barrier-permeable fusion protein F3#01 according to one embodiment to an animal model. [Figure 7] 1 shows the results of real-time imaging of the transcytosis phenomenon of the fusion protein in the blood-brain barrier after intravenous administration of the blood-brain barrier-permeable fusion protein F3#01 according to one embodiment to an animal model with a cranial window. [Figure 8] Figure 1 shows the results of intravenous administration of the blood-brain barrier permeable fusion protein F3#01 according to one embodiment to an animal model with a cranial window, followed by real-time monitoring of the levels of the fusion protein present in the intravascular (ROI 1), vascular wall (ROI 2), and extravascular (ROI 3) regions. [Figure 9] FIG. 1 shows the results of comparing the IgG1 antibody levels in brain tissue after intravenous administration of blood-brain barrier-permeable fusion proteins (F1#01, F3#01, F5#01) according to one embodiment to an animal model, which were quantified as relative values ​​compared to a control group. [Figure 10] The blood-brain barrier-permeable fusion proteins (F1#01, F3#01, F5#01) according to one embodiment were intravenously administered to an animal model, and then the binding of the fusion proteins to the transferrin receptor was confirmed by immunostaining. (a) shows the results for fusion protein F5#01, (b) shows the results for fusion protein F1#01, and (c) shows the results for fusion protein F3#01. [Figure 11] 1 shows the results of comparing the binding levels of the fusion proteins to the transferrin receptor after intravenous administration of blood-brain barrier permeable fusion proteins (F1#01, F3#01, F5#01) according to one embodiment to an animal model. [Figure 12]1 shows the results of negative staining TEM analysis of the interaction between the blood-brain barrier permeable fusion protein F3#01 and the transferrin receptor, according to one embodiment. [Figure 13] 1 shows an image of the structure of a complex formed by binding between a blood-brain barrier-permeable fusion protein and a transferrin receptor according to one embodiment. [Figure 14] 1 shows the results of examining the organ distribution level of IgG1 antibody using a human IgG1 ELISA kit after intravenously administering the blood-brain barrier-permeable fusion protein F3#01 according to one embodiment to an animal model. [Figure 15] 1 shows the results of examining the organ distribution level of IgG1 antibody using a human IgG1 ELISA kit after intravenously administering the blood-brain barrier-permeable fusion protein F3#01 according to one embodiment to an animal model. [Figure 16] The blood-brain barrier permeable fusion protein F3#01 according to one embodiment was intravenously administered to an animal model, and the percentage (%) of reticulocytes in total red blood cells was then measured after blood was collected. [Figure 17] 1 shows the results of measuring the plasma concentration of the fusion protein F3#01 according to one embodiment of the present invention after intravenous administration to an animal model. [Figure 18] 1 shows the results of calculating the blood-to-plasma ratio after treating plasma samples and blood samples with the blood-brain barrier permeable fusion protein F3#01 according to one embodiment. [Figure 19] 1 shows the results of measuring the level of IgG1 antibody in brain tissue using a human IgG1 ELISA kit after intravenous administration of the blood-brain barrier-permeable fusion protein F3'#01 according to one embodiment to an animal model. [Figure 20] 1 shows the results of examining the organ distribution level of IgG1 antibody using a human IgG1 ELISA kit after intravenously administering the blood-brain barrier-permeable fusion protein F3'#01 according to one embodiment to an animal model. [Figure 21]1 shows the results of examining the organ distribution level of IgG1 antibody using a human IgG1 ELISA kit after intravenously administering the blood-brain barrier-permeable fusion protein F3'#01 according to one embodiment to an animal model. [Figure 22] The amounts of transferrin receptor expressed in brain tissue, vascular cells, liver, lung, kidney, and spleen were compared via Western blot. [Figure 23] The expression patterns of transferrin receptors in brain tissue, liver, spleen, lung, and reticulocytes were confirmed using confocal microscope images and super-high resolution STED confocal microscope fluorescence images. [Figure 24] The results show a comparison of the quantitative distribution of transferrin receptor clusters formed in cerebral blood vessels, reticulocytes, lungs, liver, and spleen based on cluster size. [Figure 25] This shows the results of comparing the average intensity of the entire transferrin receptor clusters between cerebral blood vessels and reticulocytes. [Figure 26] FIG. 1 is a diagram showing parameters for calculating the inter-transferrin receptor distance, and schematically showing the minimum (min) and maximum (max) values ​​of the inter-transferrin receptor distance. [Figure 27] This is the result of comparing the total transferrin receptor density between cerebral blood vessels and reticulocytes. [Figure 28] This is the result of comparing the distance between transferrin receptors on cerebral blood vessels and reticulocytes. [Figure 29] 1 shows the results of confirming the thermodynamic structural stability of the binding between helical region-binding moiety #2, #3, #5, #6, #7 or #8 according to one embodiment and a transferrin receptor. [Figure 30] 1 shows the results of confirming the thermodynamic structural stability of the binding between helical region-binding moiety #9, #10, #11, #12, #13 or #14 according to one embodiment and the transferrin receptor. [Figure 31]1 shows the results of confirming the thermodynamic structural stability of the binding between helical region-binding moiety #15, #16, #17, #18, #19 or #20 according to one embodiment and the transferrin receptor. [Figure 32] 1 shows the results of confirming the thermodynamic structural stability of the binding between helical region-binding moiety #21, #22, #23 or #24 according to one embodiment and the transferrin receptor. [Figure 33] 1 shows the results of confirming the thermodynamic structural stability of the binding between helical region-binding moiety #25, #27, #30, #32, #33 or #34 according to one embodiment and the transferrin receptor. [Figure 34] 1 shows the results of confirming the thermodynamic structural stability of the binding between helical region-binding moiety #36, #38, or #39 according to one embodiment and the transferrin receptor. [Figure 35] 1 shows the results of confirming the intracellular delivery level of helical domain-binding moiety #04 according to one embodiment using a human brain endothelial cell line. [Figure 36] 1 shows the results of confirming the intracellular delivery level of helical domain-binding moiety #16, #19, or #20 according to one embodiment using a human brain endothelial cell line. [Figure 37] 1 shows the results of confirming the intracellular delivery level of helical domain-binding moiety #25, #26, or #27 according to one embodiment using a human brain endothelial cell line. [Figure 38] 1 shows the results of confirming the intracellular delivery level of helical domain-binding moiety #28, #29, or #31 according to one embodiment using a human brain endothelial cell line. [Figure 39] 1 shows the results of confirming the intracellular delivery level of helical domain-binding moiety #34, #35, #36, or #37 according to one embodiment using a human brain endothelial cell line. [Figure 40] 1 shows the results of confirming the intracellular delivery level of helical domain-binding moiety #38, #40, or #41 according to one embodiment using a human brain endothelial cell line. [Figure 41]FIG. 1 shows the results of intravenous administration of the blood-brain barrier-permeable fusion protein F3#02, F3#03, F3#04, F3#05, or F3#06 according to one embodiment to an animal model, and then confirming the level of IgG1 antibodies in brain tissue using a human IgG1 ELISA kit. [Figure 42] FIG. 1 shows the results of intravenous administration of the blood-brain barrier-permeable fusion protein F3#07, F3#08, F3#09, F3#10, or F3#11 according to one embodiment to an animal model, and then confirming the level of IgG1 antibody in brain tissue using a human IgG1 ELISA kit. [Figure 43] FIG. 1 shows the results of intravenous administration of the blood-brain barrier-permeable fusion protein F3#12, F3#13, F3#14, or F3#15 according to one embodiment to an animal model, and then confirming the level of IgG1 antibody in brain tissue using a human IgG1 ELISA kit. [Figure 44] FIG. 1 shows the results of intravenous administration of the blood-brain barrier-permeable fusion protein F3#16, F3#17, F3#18, F3#19, or F3#20 according to one embodiment to an animal model, and then confirming the level of IgG1 antibody in brain tissue using a human IgG1 ELISA kit. [Figure 45] FIG. 1 shows the results of intravenous administration of the blood-brain barrier-permeable fusion protein F3#21, F3#22, F3#23, or F3#24 according to one embodiment to an animal model, and then confirming the level of IgG1 antibody in brain tissue using a human IgG1 ELISA kit. [Figure 46] 1 shows the results of intravenous administration of the blood-brain barrier-permeable fusion protein F3#25, F3#26, or F3#27 according to one embodiment to an animal model, and then confirming the level of IgG1 antibody in brain tissue using a human IgG1 ELISA kit. [Figure 47] FIG. 1 shows the results of intravenous administration of the blood-brain barrier-permeable fusion protein F3#28, F3#29, F3#30, F3#31, or F3#32 according to one embodiment to an animal model, and then confirming the level of IgG1 antibody in brain tissue using a human IgG1 ELISA kit. [Figure 48]FIG. 1 shows the results of intravenous administration of the blood-brain barrier-permeable fusion protein F3#33, F3#34, F3#35, F3#36, or F3#37 according to one embodiment to an animal model, and then confirming the level of IgG1 antibody in brain tissue using a human IgG1 ELISA kit. [Figure 49] FIG. 1 shows the results of intravenous administration of the blood-brain barrier-permeable fusion protein F3#38, F3#39, F3#40, ​​or F3#41 according to one embodiment to an animal model, and then confirming the level of IgG1 antibody in brain tissue using a human IgG1 ELISA kit. [Figure 50] 1 shows the results of intravenous administration of the blood-brain barrier-permeable fusion protein F1#03, F5#03, or F3#03 according to one embodiment to an animal model, and then confirming the level of IgG1 antibody in brain tissue using a human IgG1 ELISA kit. [Figure 51] 1 shows the results of intravenous administration of the blood-brain barrier-permeable fusion protein F1#05, F5#05, or F3#05 according to one embodiment to an animal model, and then confirming the level of IgG1 antibody in brain tissue using a human IgG1 ELISA kit. [Figure 52] 1 shows the results of intravenous administration of the blood-brain barrier-permeable fusion protein F1#06, F5#06, or F3#06 according to one embodiment to an animal model, and then confirming the level of IgG1 antibody in brain tissue using a human IgG1 ELISA kit. [Figure 53] 1 shows the results of intravenous administration of the blood-brain barrier-permeable fusion protein F1#12, F5#12, or F3#12 according to one embodiment to an animal model, and then confirming the level of IgG1 antibody in brain tissue using a human IgG1 ELISA kit. [Figure 54] 1 shows the results of intravenous administration of the blood-brain barrier-permeable fusion protein F1#16, F5#16, or F3#16 according to one embodiment to an animal model, and then confirming the level of IgG1 antibody in brain tissue using a human IgG1 ELISA kit. [Figure 55]1 shows the results of intravenous administration of the blood-brain barrier-permeable fusion protein F1#25, F5#25, or F3#25 according to one embodiment to an animal model, and then confirming the level of IgG1 antibody in brain tissue using a human IgG1 ELISA kit. [Figure 56] 1 shows the results of intravenous administration of the blood-brain barrier-permeable fusion protein F1#27, F5#27, or F3#27 according to one embodiment to an animal model, and then confirming the level of IgG1 antibody in brain tissue using a human IgG1 ELISA kit. [Figure 57] 1 shows the results of intravenous administration of the blood-brain barrier-permeable fusion protein F1#31, F5#31, or F3#31 according to one embodiment to an animal model, and then confirming the level of IgG1 antibody in brain tissue using a human IgG1 ELISA kit. [Figure 58] 1 shows the results of intravenous administration of the blood-brain barrier-permeable fusion protein F1#37, F5#37, or F3#37 according to one embodiment to an animal model, and then confirming the level of IgG1 antibody in brain tissue using a human IgG1 ELISA kit. [Figure 59] 1 shows the results of intravenous administration of the blood-brain barrier-permeable fusion protein F1#40, F5#40, or F3#40 according to one embodiment to an animal model, and then confirming the level of IgG1 antibody in brain tissue using a human IgG1 ELISA kit. [Figure 60] 1 shows the results of examining the change in the binding strength of an IgG1 antibody to PD-L1 depending on the presence or absence of binding moiety #01 in the blood-brain barrier-permeable fusion proteins F3#01 and F3'#01 according to one example. [Figure 61] In one embodiment, the blood-brain barrier-permeable fusion protein F3#25-Tau (Tau), which contains an IgG1 antibody that specifically binds to Tau (Tau), was intravenously administered to an animal model, and the level of IgG1 antibody in the ISF was confirmed using a human IgG1 ELISA kit. The results are shown as a fold increase compared to the control group. [Figure 62]In one embodiment, the blood-brain barrier-permeable fusion protein F3#27-Tau or F3#36-Tau, which contains an IgG1 antibody that specifically binds to Tau, was intravenously administered to an animal model, and the level of IgG1 antibody in the ISF was confirmed using a human IgG1 ELISA kit. The results are shown as a fold increase compared to the control group. [Figure 63] According to one embodiment, the blood-brain barrier-permeable fusion protein F3#25-PD1 containing an IgG1 antibody that specifically binds to PD1 was intravenously administered to an animal model, and the level of IgG1 antibody in the brain tissue was measured using a human IgG1 ELISA kit. The results are shown as a fold increase compared to the control group. [Figure 64] In one embodiment, the blood-brain barrier-permeable fusion protein F3#25-HER2, which contains an IgG1 antibody that specifically binds to HER2, was intravenously administered to an animal model, and the level of IgG1 antibody in the brain tissue was determined using a human IgG1 ELISA kit. The results are shown as a fold increase compared to the control group. [Figure 65] In one embodiment, the blood-brain barrier-permeable fusion protein F3#25-Aβ, which contains an IgG1 antibody that specifically binds to Aβ, was intravenously administered to an animal model, and the level of IgG1 antibody in the brain tissue was determined using a human IgG1 ELISA kit. The results are shown as a fold increase compared to the control group. DETAILED DESCRIPTION OF THE INVENTION

[0062] In order to aid in understanding the present invention, preferred examples are presented below. However, the following examples are provided only to facilitate understanding of the present invention, and the contents of the present invention are not limited to the following examples. [Example]

[0063] Examples 1 to 41: Preparation of fusion proteins that permeate the blood-brain barrier In this example, a fusion protein with enhanced blood-brain barrier permeability was prepared. To this end, a fusion protein was prepared in which a transferrin receptor helical region-binding moiety was linked to the C-terminal region of the light chain and the C-terminal region of the heavy chain of an IgG1 antibody, i.e., a total of four terminal regions. In this example, a total of 24 binding moieties were derived as the transferrin receptor helical region-binding moieties, each possessing binding properties to the helical region but with different amino acid sequences (first binding moiety group). Furthermore, a total of 17 binding moieties were derived as the transferrin receptor helical region-binding moieties, each maintaining a certain level of sequence identity with the binding moiety of SEQ ID NO: 3 but with partial substitutions, insertions, or deletions in the amino acid sequence (second binding moiety group).

[0064] 1. Preparation of a fusion protein containing a tetravalent first binding moiety group A fusion protein with brain-blood barrier permeability, in which the helical domain-binding moieties of the transferrin receptor were linked to all four terminal domains of the IgG1 antibody, was prepared as follows. Specifically, a 1 mL sample was taken with a pipette from a flask containing cells, and the cell mass was measured. After that, the cells were cultured to determine whether the cell viability was 95% or higher and the cell mass was 4-6 × 10. 6 If the cell mass level is 9x10 cells / mL or more, culture medium stored in a 37°C incubator is added and cultured until the cell mass level is 9x10 6 The cells were then transfected under the following experimental conditions.

[0065] [Table 1]

[0066] On the first day of culture, the cells were treated with the feed media and enhancer, and on the fifth day of culture, the cells were treated with the feed media and cultured. When cell viability fell below 70% or 8 days had passed since the transfection, the transfected cells were harvested. The transfected cell sample was centrifuged at 4,500 rpm and 25°C for 15 minutes, and the supernatant was collected. The supernatant was then filtered through a 0.22 μm filter. The filtrate was then purified using techniques such as affinity chromatography and size exclusion chromatography to obtain the respective fusion proteins. The purified antibodies were analyzed by SEC-HPLC (size exclusion high performance liquid chromatography), SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis), and mass spectrometry to confirm the purification results.

[0067] In the fusion protein of this example, the matters relating to the helical region-binding moiety of the transferrin receptor, the IgG1 antibody, etc. are as follows. [Example]

[0068] [Table 2] [Example]

[0069] [Table 3] [Example]

[0070] [Table 4] [Example]

[0071] [Table 5] [Example]

[0072] [Table 6] [Example]

[0073] [Table 7] [Example]

[0074] [Table 8] [Example]

[0075] [Table 9] [Example]

[0076] [Table 10] [Example]

[0077] [Table 11] [Example]

[0078] [Table 12] [Example]

[0079] [Table 13] [Example]

[0080] [Table 14] [Example]

[0081] [Table 15] [Example]

[0082] [Table 16] [Example]

[0083] [Table 17] [Example]

[0084] [Table 18] [Example]

[0085] [Table 19] [Example]

[0086] [Table 20] [Example]

[0087] [Table 21] [Example]

[0088] [Table 22] [Example]

[0089] [Table 23] [Example]

[0090] [Table 24] [Example]

[0091] [Table 25]

[0092] 2. Preparation of Fusion Proteins Containing Tetravalent Second Binding Moiety Groups Fusion proteins containing a second binding moiety group were prepared in the same manner as described above. The details of the transferrin receptor helical domain-binding moiety and IgG1 antibody binding moiety in the fusion proteins of this example are as follows: [Example]

[0093] [Table 26] [Example]

[0094] [Table 27] [Example]

[0095] [Table 28] [Example]

[0096] [Table 29] [Example]

[0097] [Table 30] [Example]

[0098] [Table 31] [Example]

[0099] [Table 32] [Example]

[0100] [Table 33] [Example]

[0101] [Table 34] [Example]

[0102] [Table 35] [Example]

[0103] [Table 36] [Example]

[0104] [Table 37] [Example]

[0105] [Table 38] [Example]

[0106] [Table 39] [Example]

[0107] [Table 40] [Example]

[0108] [Table 41] [Example]

[0109] [Table 42] Comparative Example

[0110] Comparative Examples 1 to 22: Preparation of fusion proteins containing bivalent transferrin receptor helical domain-binding moieties Fusion proteins were prepared in which two transferrin receptor helical region-binding moieties were linked to either the C-terminal region of the heavy chain or the C-terminal region of the light chain of an IgG1 antibody (F1, F5). Specifically, the transferrin receptor helical region-binding moieties used included six representative binding moieties (#01, #03, #05, #06, #12, and #16) in the first binding moiety group and five representative binding moieties (#25, #27, #31, #37, and #40) in the second binding moiety group.

[0111] 1. Preparation of a Fusion Protein Containing a Divalent First Binding Moiety Group Using binding moiety #01, #03, #05, #06, #12, or #16 as the transferrin receptor helical domain-binding moiety and an anti-PD-L1 IgG1 antibody as the IgG1 antibody, fusion proteins having a structure in which two binding moieties are linked to the C-terminal region of the heavy chain of the IgG1 antibody were prepared in the same manner as in the previous examples (Comparative Examples 1 to 6: F1#01, F1#03, F1#05, F1#06, F1#12, F1#16).

[0112] In addition, fusion proteins having a structure in which two binding moieties are linked to the C-terminal region of the light chain of an IgG1 antibody using binding moiety #01, #03, #05, #06, #12, or #16 as the transferrin receptor helical domain-binding moiety and an anti-PD-L1 IgG1 antibody as the IgG1 antibody were prepared in the same manner as in the previous examples (Comparative Examples 7 to 12: F5#01, F5#03, F5#05, F5#06, F5#12, F5#16).

[0113] 2. Preparation of Fusion Proteins Containing a Divalent Second Binding Moiety Group Using binding moiety #25, #27, #31, #37, or #40 as the transferrin receptor helical domain-binding moiety and an anti-PD-L1 IgG1 antibody as the IgG1 antibody, fusion proteins having a structure in which two binding moieties are linked to the C-terminal region of the heavy chain of the IgG1 antibody were prepared in the same manner as in the previous examples (Comparative Examples 13 to 17: F1#25, F1#27, F1#31, F1#37, F1#40).

[0114] In addition, fusion proteins having a structure in which two binding moieties were linked to the C-terminal region of the light chain of an IgG1 antibody using binding moiety #25, #27, #31, #37, or #40 as the transferrin receptor helical domain-binding moiety and anti-PD-L1 IgG1 antibody as the IgG1 antibody were prepared in the same manner as in the above Examples (Comparative Examples 18 to 22: F5#25, F5#27, F5#31, F5#37, F5#40).

[0115] Experimental Example 1: Evaluation of the functionality of the blood-brain barrier permeability fusion protein F3#01 1-1. Functional evaluation of binding moieties In this experimental example, the functionality of the binding moiety #01 of the blood-brain barrier-permeable fusion protein F3#01 according to one embodiment is confirmed by evaluating its binding to the helical domain of the transferrin receptor and the level of transduction mediated by this in human brain endothelial cells.

[0116] (1) Confirmation of binding between the helical region of the transferrin receptor and the binding moiety The binding of the helical domain-binding moiety of the fusion protein F3#01 to the transferrin receptor (TfR) was confirmed by cryo-electron microscopy (cryo-EM). Specifically, to prepare a vitrified frozen specimen, a 10 μM L7 TfR sample was prepared using a 50 mM Tris, 150 mM NaCl, pH 7.6 buffer solution. The L7 TfR sample was then vitrified using a Quantifoil Cu 1.2 / 1.3 400 mesh grid. To enhance the interaction between the sample and the grid, a negative charge was discharged from the grid surface using a glow discharger. Then, 3 μl of the L7 TfR sample was injected onto the discharged grid and vitrification was performed under the following conditions: blotting time (7 s), blotting force (0), waiting time (0 s), 4°C, 95% humidity. Cryo-EM analysis was performed using a 200 kV Glycios microscope, a 300 kV Krios G4 microscope, a Falcon 4 microscope, and a K3 detector. The lens settings were spherical aberration (2.7), 0.50 μM aperture, magnification (120 K), and exposure time (6.55 s). The analysis was performed under the following conditions: 50 fractions per sample, 225 frames, pixel value 0.894 Å / pixel, dose rate 6.1 e / px / s, total dose 49.93 e / Ų. The defocus range was -1.25 to -2.75 with 0.25 intervals. After selecting over 5 million particles, initial 2D classification was performed using these. Approximately 2 million particles were then selected and subjected to 3D refinement. C1 and C2 symmetries were applied, respectively, to derive a 3D electron density map with a resolution of ~3.4 Å.A co-complex model of TfR and transferrin (PDB: 3s9n) was overlaid on this map, and the binding site and core residues were predicted by comparing the existing binding method with the binding method of the helical region binding moiety.

[0117] The binding of the helical domain-binding moiety #01 to the transferrin receptor was also confirmed through docking simulations. Specifically, docking simulations were performed to confirm whether the binding moiety #01 has the ability to bind to the helical domain of the transferrin receptor. The structure of the binding moiety #01 was modeled using the RosettaRelax program, and the positions predicted to interact with the helical domain of the transferrin receptor were identified through structural information and thermodynamic calculations. The position of the binding moiety was then arbitrarily changed, and docking simulations were performed using the Rosettadocking program to calculate the interaction with the helical domain and find the most stable position. 20,000 simulations were performed for each sequence number, and the resulting data were analyzed based on homology to the initial modeled structure and thermodynamic structural stability.

[0118] As a result, as shown in Figures 1 and 2, it was confirmed that the helical region applicable to interaction with the transferrin receptor was exposed to the outside. Furthermore, when a 3D electron density map at 3.4 Å resolution was viewed in conjunction with a model that maintains the endogenous binding mode between the transferrin receptor and transferrin, it was confirmed that electron density corresponding to glycosylation, which is not shown in existing transferrin receptors, was present within the predicted ideal range. Furthermore, it was confirmed that the bond with the binding moiety was formed to conform to the shape of the α-helical structure, and that the interaction between the helical region of the transferrin receptor and the binding moiety was mediated through this. Additionally, as shown in Figure 3, it was confirmed that binding moiety #01 corresponding to SEQ ID NO: 3 stably binds to the helical region of the transferrin receptor.

[0119] (2) Confirmation of transduction within human brain endothelial cells This experiment was conducted to confirm whether the helical domain-binding moiety #01, which was confirmed to bind to the transferrin receptor in Experimental Example 1-1(1), can be delivered into hCMEC / D3 cells, which are human brain endothelial cells that constitute the human blood-brain barrier, through the aforementioned interaction. Specifically, the hCMEC / D3 cells were cultured in EBM2 (Endothelial Cell Basal Medium 2) synthetic culture medium containing growth factors at 37°C under 5% CO2 conditions. Thereafter, when the cell saturation reached 80%, the cells were detached and then 4x10 3Cells were added to the plate, dispensed into a 384-well plate, centrifuged for 10 seconds, and then cultured at 37°C and 5% CO2 for at least 18 hours to allow cells to adhere to the plate. 1 mg of binding moiety peptide was added to 500 μL of 1x PBS, mixed, and the concentration was measured using a UV-visible spectrometer. The peptide was then diluted to a final concentration of 200 μM and stored at 4°C. The 200 μM peptide was then diluted in EBM2 medium to a concentration 5x the treatment concentration. 10 μL of the 5x diluted peptide was dispensed into each well, centrifuged at 1,000 rpm for 10 seconds, and cultured at 37°C and 5% CO2 for 2 hours. The cultured cells were then washed with 1x PBS, and 75 μL of 4% PFA was dispensed into each well and stored at room temperature for 30 minutes to fix them. At this time, Hoechst solution was added to the 4% PFA to simultaneously stain the nuclei, and 30 minutes later, the cells were imaged via Cytation 5 (Biotek) to evaluate the intracellular delivery level.

[0120] As a result, as shown in Figure 4, the intracellular delivery level was evaluated using a human cerebral endothelial cell line (hCMEC / D3), and effective intracellular delivery of binding moiety #01 corresponding to sequence number 3 was confirmed.

[0121] 1-2. Evaluation of the level of permeation through the blood-brain barrier In this experimental example, the level of absorption of IgG1 antibody into brain tissue following intravenous administration of the blood-brain barrier-permeable fusion protein F3#01 according to one embodiment was evaluated.

[0122] (1) Evaluation of IgG1 antibody levels in brain tissue at different times C57BL / 6 mice were intravenously administered a formulation containing 20 mg / kg of the fusion protein F3#01 via the tail vein. A control group received intravenous administration of IgG1 antibody alone. After 1, 4, 7, or 14 days, the mice were anesthetized and blood was collected from the medial or abdominal vein. Then, saline was perfused to remove the blood. The mouse brains were then excised, and the excised brain tissue was flash-frozen in liquid nitrogen and stored in a deep freezer until use. The excised brain tissue was homogenized using a protein extraction solution and lysed at 4°C using a rotary mixer. The lysed brain tissue samples were then centrifuged, and the supernatant was collected to prepare brain tissue lysates. The IgG1 antibody levels in the brain tissue lysates were then measured using a human IgG1 ELISA kit. Specifically, the protein extraction solution was diluted to the appropriate concentration to prepare the standard (STD). The brain tissue lysate sample (SPL) was prepared using the protein extraction solution at the appropriate dilution ratio, and 50 μL of each was added to each well. The Ab Cocktail included in the ELISA kit was then added at 50 μL per well and incubated at 4°C. After washing the plate, 100 μL of TMB substrate was added to each well. Using a microplate reader, the stop solution was added when the OD of STD1 reached 1.0 at 600 nm. The value at 450 nm was measured. A standard curve was then generated using four-parameter logistic regression, from which the concentration of IgG1 antibody present in the sample (SPL) was calculated.

[0123] As a result, as shown in Figure 5, the IgG1 antibody levels in the brain tissue of the F3#01-administered group according to one embodiment were observed to be significantly higher than those of the control group up to 14 days after administration. Specifically, the F3#01-administered group showed a high level of transduction, ranging from approximately 25 to 320 times that of the control group.

[0124] (2) Evaluation of IgG antibody levels in brain tissue using ISF samples After the fusion protein penetrated the blood-brain barrier, the amount of fusion protein present in the unbound form in the brain parenchyma (the fusion protein present in the ISF sample) was measured, and then recovery was performed on the detected value to evaluate the amount of fusion protein that entered the brain parenchyma from the cerebral blood vessels, i.e., the amount of fusion protein that penetrated the blood-brain barrier. Specifically, C57BL / 6 mice were anesthetized, and the skin on the head of the mouse was incised. A perforation was made in the skull adjacent to the hippocampus using a drill. A guide cannula was then inserted into the perforation and fixed with resin. The skin incision was sutured, and the perforation area was closed to prevent external exposure. The mice were then allowed to recover for two weeks. A formulation containing 20 mg / kg of the fusion protein (F3#01) was then intravenously administered via the tail vein of the recovered mice.

[0125] The fusion protein was intravenously administered to the mice, and four hours, one day, or four days later, the mice were anesthetized and an activated probe for IgG1 antibody detection was inserted into the mouse's probe guide cannula. Then, cerebrospinal fluid (CSF) containing BSA was constantly flowed through the mice, and interstitial fluid (ISF) samples containing the fusion protein were collected and stored at -20°C. The IgG1 antibody levels in the ISF samples were measured using a human IgG1 ELISA kit. Specifically, the standard (STD) and sample (SPL) were diluted to their respective concentrations using NS sample buffer, and 50 μL of each was added to each well. Then, 50 μL of the Ab cocktail included in the ELISA kit was added to each well and incubated at 4°C. After washing the plate, 100 μL of TMB substrate was added to each well. Using a microplate reader, when the OD value of STD1 reached 1.0 at 600 nm, a stop solution was added and the value at 450 nm was measured. A control group received only IgG1 antibody intravenously.

[0126] As a result, as shown in FIG. 6, it was observed that the IgG1 antibody level in the ISF sample of the F3#01 administration group according to one embodiment was significantly increased compared to the control group.

[0127] (3) Evaluation of IgG antibody levels through cerebrovascular imaging in animal models Using a cranial window animal model and two-photon microscopy, we captured real-time images of the cerebrovascular region and confirmed the transcytosis of the fusion protein at the blood-brain barrier. Specifically, we used a cranial window-equipped Tie2-GFP Tg mouse and captured structural images using two-photon microscopy from the region where the pial vessels originate to a depth of approximately 300 μm, including cortical layers 2 and 3. To observe the transcytosis of the Alexa-568-conjugated fusion protein, 26 mg / kg of the Alexa-568-conjugated fusion protein was injected via the tail vein. After the fusion protein injection, GFP was measured in one channel, and the fluorescence intensity of Alexa-568 conjugated to the fusion protein was measured in the second channel in the same three-dimensional region as the image captured on day 1. The fluorescence intensity was measured for 20 minutes in a region containing at least three blood vessel segments, starting from the postcapillary venule, with a spatial resolution of 100 nm or less and a temporal resolution of 1 min or less. All time-series images acquired to observe transcytosis were aligned to the first time-series image and then divided into intravascular, vascular wall, and extravascular regions based on the GFP signal in the first channel. Based on the intravascular, vascular wall, and extravascular regions defined by the GFP signal in the first channel, regions clustered near the vascular wall in the Alexa-568 signal bound to the fusion protein observed in the second channel were redesignated as intravascular (ROI (region of interest) 1), vascular wall (ROI 2), and extravascular (ROI 3) regions. Changes in the concentration of Alexa-568 bound to the fusion protein in the three ROIs were observed over time.

[0128] In one embodiment, real-time transcytosis changes were observed in three ROIs for 20 minutes one day after injection of the fusion protein. As shown in Figures 7 and 8, no changes in fusion protein concentration were observed within the blood vessels (ROI 1). The fusion protein in the blood vessel wall (ROI 2), where clusters were present, formed clusters within 10 minutes and then migrated to the extravascular space, resulting in a decrease in fusion protein concentration. The concentration remained stable until no further clusters were observed (>10 minutes). Furthermore, changes in fusion protein concentration in the extravascular space (ROI 3) were observed. As the fusion protein clusters initially observed in the blood vessel wall (ROI 2) reached the extravascular space after approximately 6 minutes, the fusion protein concentration increased, reaching a maximum at approximately 12 minutes. These results suggest that transcytosis of the fusion protein occurs via clustering near the blood-brain barrier, followed by extravascularization within a few minutes.

[0129] 1-3. Functionality evaluation based on changes in the valence of binding moieties C57BL / 6 mice were administered 20 mg / kg of fusion proteins F3#01, F1#01, and F5#01 intravenously via the tail vein. Four days later, IgG1 antibody levels in brain tissue were measured using the same method as in Experimental Example 1-2 (1). A standard curve was then obtained using four-parameter logistic regression, and the IgG1 antibody concentration in the sample (SPL) was calculated from the standard curve. Additionally, to observe antibody transmission within the cerebral vasculature, the animal's brain was removed one day after intravenous administration of the fusion proteins. After anesthetization, the animal's chest cavity was opened, a butterfly needle was inserted into the left ventricle, saline was injected, and the right atrium was drained to remove the blood. Then, 4% paraformaldehyde (PFA) was injected to fix the cells, and the skull was opened to extract the brain. The extracted brains were stored in 4% PFA for one day for further cell fixation, followed by storage in 30% sucrose solution for three days to prevent cell destruction during brain sectioning. Mouse brains stored in 30% sucrose solution for three days were placed in a brain sectioning frame, injected with optimal cutting temperature compound solution, and stored at -60°C to cool the brain. The cooled brain samples were sliced ​​into 40 μm-thick brain sections using a micro-cryostat, and each brain section was used for immunostaining. Subsequently, the brain sections were immunostained using transferrin receptor antibodies and antibodies against the fusion protein, and then photographed using a confocal microscope.

[0130] As a result, as shown in Figure 9, the IgG1 antibody levels in brain tissue were quantified relative to the control group four days after intravenous administration. The results showed that the IgG1 antibody levels in brain tissue were elevated by approximately 80-fold in the tetravalent fusion protein group in which a binding moiety was linked to each of the four regions at the C-terminus of the heavy chain and the C-terminus of the light chain (i.e., the F3#01 group) compared to the control group. Furthermore, the IgG1 antibody levels in the bivalent fusion protein groups in which a binding moiety was linked to either the C-terminus of the heavy chain or two regions at the C-terminus of the light chain (i.e., the F1#01 and F5#01 groups) did not show a significant difference from the control group, despite the use of the same binding moiety.

[0131] Furthermore, as shown in Figures 10 and 11, no binding of the fusion protein to the transferrin receptor was observed in the cerebral blood vessels in the F1#01-administered group and the F5#01-administered group, whereas binding of the fusion protein to the transferrin receptor was confirmed in the cerebral blood vessels in the F3#01-administered group. Quantification of these results showed a significant difference in the level of the fusion protein present in the region where the transferrin receptor is located.

[0132] 1-4. Evaluation of the interaction between the fusion protein and the transferrin receptor Based on the premise that the high blood-brain barrier permeability observed in Experimental Examples 1-2 is due to the interaction between the binding moiety and the transferrin receptor present in brain tissue, negative staining transmission electron microscope (TEM) analysis was performed to confirm the interaction between the fusion protein, i.e., the tetravalent binding moiety, and the transferrin receptor. Specifically, a sample containing both the fusion protein and the transferrin receptor (TfR) was prepared as the experimental group, and the following control groups were set up: (1) a group containing only native TfR, (2) a group containing only an IgG1 antibody without a binding moiety, and (3) a group containing both an IgG1 antibody without a binding moiety and TfR. All samples were quantified at 15 μM using a buffer solution of 50 mM Tris, 150 mM NaCl, pH 7.4. For the group containing both fusion protein / IgG1 antibody and TfR, they were mixed at a molar ratio of 1:2 and incubated at 4°C for 1 hour. All samples were then diluted to the appropriate concentration and subjected to TEM grid sampling. For electron microscopy, 400-mesh F / C Cu grids were used. 10 μl of the prepared sample was injected onto the grid, incubated for 1 minute, and then removed using filter paper. Next, 10 μl of 2% uranyl acetate solution was injected onto the grid, and the remaining solution was removed using filter paper. The grid was then exposed to room temperature for approximately 12 hours to remove any remaining moisture. The grid specimens prepared as described above were observed using a 60 kV Jeol Gatan TEM, and negative staining sample analysis was performed at 60K to 200K magnification.

[0133] As a result, as shown in FIG. 12, the group to which the IgG1 antibody containing no binding moiety and TfR were added (FIG. 12(c)) showed a relatively homogeneous distribution of each component, but no interaction between them, as in the groups to which only one of native TfR and the IgG1 antibody containing no binding moiety was added (FIG. 12(a) and (b)). Furthermore, the group to which the fusion protein of one example and native TfR were added (FIG. 12(d)) formed a complex through interaction with the transferrin receptor. Furthermore, based on these experimental results, as shown in FIG. 13, it was found that each of the tetravalent binding moieties of one example interacted with the helical region of the transferrin receptor to form a complex. In particular, considering the experimental results of Experimental Examples 1 to 3 (see FIG. 9), the experimental results of this example indicate that the interaction within the complex may be a major factor in improving permeability across the blood-brain barrier.

[0134] 1-5. Evaluation of selective delivery to brain tissue In this experimental example, the organ-specific distribution of IgG1 antibodies following intravenous administration of the blood-brain barrier-permeable fusion protein F3#01 according to one embodiment was evaluated. Specifically, a formulation containing 20 mg / kg of the fusion protein F3#01 was administered intravenously via the tail vein to C57BL / 6 mice, with a control group receiving intravenous administration of IgG1 antibodies alone. One or four days later, the IgG1 antibody levels in a total of six organs (brain, lung, spleen, kidney, liver, and muscle) were measured and compared using the same method as in Experimental Example 1-2(1).

[0135] As a result, the organ-specific distribution levels of IgG1 antibodies one day after intravenous administration of the blood-brain barrier-permeable fusion protein F3#01 are shown in Figure 14, and the organ-specific distribution levels of IgG1 antibodies four days after intravenous administration of the blood-brain barrier-permeable fusion protein F3#01 are shown in Figure 15. Specifically, the IgG1 antibody administered intravenously to the control group was observed to be distributed most highly in the lungs and relatively less in brain tissue, whereas the F3#01-administered group showed significantly higher levels of IgG1 antibodies in brain tissue compared to other organs. These results suggest that a fusion protein comprising a functional structure according to one embodiment selectively distributes to brain tissue upon intravenous administration, thereby reducing the distribution of IgG antibodies in tissues other than the brain, and thereby potentially contributing to reducing the side effects of antibody-based drugs.

[0136] 1-6. Evaluation of the side effect reduction effect by selective delivery to brain tissue (1) Evaluation of reticulocyte levels In one embodiment, the blood-brain barrier-permeable fusion protein F3#01 was administered to examine its effect on reticulocytes, a type of cell expressing a large amount of transferrin receptor. Specifically, a formulation containing 20 mg / kg or 50 mg / kg of the fusion protein F3#01 was intravenously administered via the tail vein to C57BL / 6 mice. At 0, 1, 4, or 7 days after intravenous administration of the fusion protein, 50 μl of blood samples were obtained from the inside of the mouse eye using a heparinized capillary tube. Then, 30 μl of the blood sample was mixed with 100 μl of new methylene blue solution, and the mixture was allowed to stand at room temperature for a certain period of time. The stained blood sample was then smeared on a glass slide and observed under a microscope to calculate the percentage of reticulocytes among total red blood cells. The control group was a group to which IgG1 antibody was administered intravenously.

[0137] As a result, as shown in Figure 16, in the group administered with the blood-brain barrier-permeable fusion protein F3#01 according to one embodiment, the proportion of reticulocytes in total red blood cells was at a level similar to that of the control group.

[0138] (2) Pharmacokinetic evaluation The fusion protein F3#01, which has the ability to permeate the blood-brain barrier according to one embodiment, was administered to mice, and its pharmacokinetics in plasma was evaluated. Specifically, a formulation containing 20 mg / kg of the fusion protein F3#01 was administered intravenously via the tail vein to the mice. After the intravenous administration of the fusion protein, blood samples were obtained from the mice 30 minutes, 120 minutes, 360 minutes, 1 day, 2 days, 4 days, 7 days, or 14 days later. The plasma samples were then separated by centrifugation and pretreated for liquid chromatography-mass spectrometry (LC-MS). The samples were then analyzed by LC-MS. A group administered intravenously with an IgG1 antibody served as a control.

[0139] As a result, as shown in Figure 17, the plasma PK profile of the group administered with the blood-brain barrier-permeable fusion protein F3#01 according to one embodiment was similar to that of the control group, but the above experimental results indicate that the distribution of the fusion protein in various organs, including the transferrin receptor, was negligible.

[0140] (3) Blood-to-plasma ratio calculation To verify whether the above experimental results were due to selective transfer to brain tissue, i.e., non-binding of reticulocytes containing transferrin receptors to other organ tissues, the blood-to-plasma ratio (peak area ratio of blood supernatant / peak area ratio of plasma) was calculated. Specifically, the blood-brain barrier-permeable fusion protein F3#01 according to one embodiment was added to plasma samples and blood samples to a final concentration of 40 μg / mL and then left at room temperature for 30 minutes. The blood samples were then centrifuged to obtain separate blood supernatants. The plasma and blood supernatant samples were mixed with a surfactant-containing PBS solution and magnetic beads, and the mixture was incubated. The culture was then washed twice with surfactant-containing PBS. RapiGest surfactant and dithiothreitol were added to the mixture, and the mixture was incubated at 60°C for 50 minutes and then left at room temperature for 10 minutes. The following steps were then carried out: 1) adding iodoacetic acid and incubating in the dark at room temperature for 30 minutes; 2) adding trypsin and incubating at 60°C for 24 hours; and 3) adding HCl and incubating at 37°C for 30 minutes. The culture was then centrifuged to obtain the supernatant, to which trypsin was added. The blood-to-plasma ratio (peak area ratio of blood supernatant / peak area ratio of plasma) was calculated using LC-MS. An IgG1 antibody-treated group served as a control.

[0141] As a result, as shown in Figure 18, the blood-to-plasma ratio of the group administered with the blood-brain barrier-permeable fusion protein F3#01 according to one embodiment was similar to that of the control group, but it was found that this experimental result was due to the fact that the fusion protein did not bind to reticulocytes containing transferrin receptors. Furthermore, the blood-brain barrier-permeable fusion protein according to one embodiment did not show a high level of transferrin receptor-mediated delivery in organs other than brain tissue, thereby confirming selective delivery to brain tissue.

[0142] 1-7. Evaluation of the functionality of fusion proteins containing repeated binding moieties This experimental example was conducted to verify whether the functionality of the fusion protein could be maintained even when the helical domain-binding moiety of the transferrin receptor was modified so that it was repeated.To this end, a fusion protein having a structure in which a total of four binding moieties were linked to the C-terminus of each of the heavy and light chains of the IgG1 antibody was prepared in the same manner as in the previous example (F3'#01), using a transferrin receptor helical domain-binding moiety consisting of two repeats of the moiety of SEQ ID NO: 3 and an anti-PD-L1 IgG1 antibody as the IgG1 antibody.

[0143] (1) Evaluation of IgG1 antibody levels in brain tissue by time period The level of absorption of IgG1 antibody into brain tissue following intravenous administration of the blood-brain barrier-permeable fusion protein F3'#01 according to one embodiment was evaluated in the same manner as in Experimental Example 1-2 (1).

[0144] As a result, as shown in Figure 19, the IgG1 antibody levels in the brain tissue of the F3'#01-administered group were observed to be higher than those of the control group up to 7 days after administration. Specifically, the F3'#01-administered group showed a high level of transmission, ranging from approximately 18 to 160 times higher than that of the control group.

[0145] (2) Evaluation of selective delivery to brain tissue The organ distribution level of IgG1 antibody following intravenous administration of the blood-brain barrier-permeable fusion protein F3'#01 according to one embodiment was evaluated in the same manner as in Experimental Example 1-5.

[0146] As a result, the distribution levels of IgG1 antibodies in each organ one day after intravenous administration of the blood-brain barrier-permeable fusion protein F3'#01 are shown in Figure 20, and the distribution levels of IgG1 antibodies in each organ four days after intravenous administration of the blood-brain barrier-permeable fusion protein F3'#01 are shown in Figure 21. That is, it was found that the F3'#01-administered group had significantly higher levels of IgG1 antibodies distributed in brain tissue compared to other organs.

[0147] From the above results, it was found that even when the helical domain-binding moiety of the transferrin receptor is repeatedly modified in a fusion protein including a functional structure according to one embodiment, it can still exhibit its inherent functionality, such as increased absorption level in brain tissue and selective delivery to brain tissue, just like existing fusion proteins.

[0148] 1-8. Confirmation of specific expression of transferrin receptor in brain tissue It was experimentally confirmed that a fusion protein comprising a functional structure in which a tetravalent binding moiety according to one embodiment is linked to the C-terminus of the light chain and the C-terminus of the heavy chain of an antibody not only significantly enhances delivery of an IgG1 antibody to brain tissue due to its high permeability through the blood-brain barrier, as confirmed in the previous examples, but also exhibits high biological safety due to its selective delivery to brain tissue compared to other organs. On the premise that such efficacy is due to the interaction between the binding moiety according to one embodiment and the transferrin receptor present in brain tissue, this experimental example confirms the expression pattern and characteristics of the transferrin receptor distributed in brain tissue.

[0149] (1) Confirmation of the distribution of transferrin receptors by organ Blood was collected from the orbital or abdominal vein of C57BL / 6 mice and then removed by perfusion with saline. After perfusion, brain tissue was removed from the mice. Brain vessels, parenchyma, and choroid plexus were isolated and total protein was extracted using RIPA buffer. For other organs, such as the liver, lung, kidney, and spleen, small amounts of tissue were collected and total protein was extracted using RIPA buffer. The extracted proteins were mixed with SDS-PAGE sample buffer, denatured, loaded onto an SDS-PAGE gel, and transferred to a PVDF membrane. Anti-TfR and β-actin antibodies were used as primary antibodies to visualize the respective protein bands on the membrane, and their intensity was measured using Image J software.

[0150] As a result, as shown in Figure 22, the transferrin receptor expressed in each organ was quantified and confirmed to be highly expressed in cerebral blood vessels and blood cells, and a significant level of transferrin receptor was also found to be expressed in the spleen. The above experimental results indicate that the selective delivery of a fusion protein according to one embodiment to brain tissue is not simply due to quantitative differences in transferrin receptor expression in each organ, but is primarily due to the interaction between a transferrin receptor with a specific expression pattern in cerebral blood vessels and a binding moiety according to one embodiment, or a functional structure capable of forming such an interaction.

[0151] (2) Comparison of the expression patterns of transferrin receptor clusters Blood was collected from the orbital or abdominal vein of C57BL / 6 mice, and then removed by perfusion with saline. Brain tissue samples were then perfused sequentially with saline and 4% paraformaldehyde to prepare brain tissue samples for immunohistochemical staining. The brain tissue was then placed on a frame, and tissue freezing medium was added to cover the brain tissue. The frame was then stored in a deep freezer at -70°C for one day. The frozen brain block was then sectioned into 40 μm-thick sections using a cryostat. The sections were stored in PBS containing 0.1% sodium azide until use.

[0152] The tissue sections were then placed in a 24-well plate and incubated with 500 μL of PBS containing 0.5% Triton-X100 for 20 minutes at room temperature. The solution was then replaced with PBS and mixed using a plate shaker. The tissue sections were then placed in a PBS solution containing 5% BSA and 0.1% Triton-X100 and incubated for 2 hours at room temperature, followed by washing three times with PBS. Transferrin receptor antibody and CD31 antibody were then added and incubated overnight at 4°C, followed by washing three times with PBS. Anti-rat Alexa488 and anti-goat Alexa568 antibodies were then added and incubated for 2 hours at room temperature, followed by washing three times with PBS. PBS containing DAPI was then added and incubated for 10 minutes at room temperature. The tissue sections were placed on a slide, and 100 μl of mounting solution was applied to the slide. Then, the tissue sections were covered with a cover glass and sealed.

[0153] Fluorescent images of the stained samples were obtained using a confocal microscope. Images were taken using LAS-X software, with the light source intensity adjusted to 4% UV intensity and 70% white laser power (488:15%, 568:10%). A 40X objective lens was used for magnification, with a pixel resolution of 284 nm. Images were acquired for a total of 20 μm thick tissue sections, with a thickness of 1 μm per section. Ultra-high-resolution fluorescent images of the stained samples were also obtained using an STED confocal microscope. Images were taken using LAS-X software, with the light source intensity adjusted to 6% UV intensity and 70% white laser power (488:15%, 568:10%). A 100X objective lens was used for magnification, with the software zoom and STED functions. The magnification was set to a pixel resolution of 50 nm, and images were taken, with each tissue section having a thickness of 100 nm, to obtain images with a total thickness of 3 μm.

[0154] The acquired images were then analyzed using Image J software and MATLAB software. As a preprocessing step for confocal and STED confocal fluorescence image analysis, the sigma value was set to 1.0, Gaussian blur was applied, and each image was cut to the same size to create a representative image that matched the hue channel. To increase the signal-to-noise ratio, a plugin in Image J software was used to perform background subtraction using the rolling ball algorithm. The green signal, which represents the transferrin receptor signal, was normalized to match the intensity histogram, and a binary image was obtained using the local thresholding method. Masked multiple transferrin receptor clusters were analyzed using the analyze particle function, with the minimum pixel unit being 2,500 nm. 2 Only pixels with an area of ​​≥ 100 were selected to obtain a cluster size distribution map. The distribution map was created from transferrin receptor distribution observed in 10 cells and tissues per organ in a total of 40 cells per organ in four C57BL / 6 mice. Using MATLAB, the distribution map results obtained with Image J were converted to a histogram, and a smooth function was used to obtain trend lines for the histogram.

[0155] As a result, it was confirmed that the transferrin receptor expressed in brain tissue, liver, spleen, lung, and reticulocytes was distributed in the form of transferrin receptor clusters, as shown in Figure 23. Furthermore, as shown in Figure 24, a comparison of the quantitative distribution of the size of transferrin receptor clusters expressed in brain tissue blood vessels, reticulocytes, lung, liver, and spleen confirmed that relatively small transferrin receptor clusters were densely concentrated in the blood vessel region of brain tissue, unlike in other tissues.

[0156] (3) Characterization of organ-specific transferrin receptor clusters A. Assessment of relative confluency via fluorescence intensity assessment To evaluate the characteristics of transferrin receptor clusters by organ, the average intensity of the entire transferrin receptor clusters in the brain tissue blood vessels and reticulocytes was evaluated. Specifically, after imaging the transferrin receptor clusters using the same method as in Experimental Example 1-8 (2), the masked transferrin receptor clusters were analyzed using the analyze particle function at the minimum pixel unit of 2,500 nm. 2 The extracted image was then converted into a mask image, and the mask image obtained with Image J was multiplied by the image subjected to background subtraction using MATLAB to calculate the intensity of the green signal in the cluster mask.

[0157] As a result, as shown in Figure 25, a higher intensity of the green signal was observed in the blood vessels of brain tissue, which indicates that the transferrin receptor clusters in the blood vessels of brain tissue are densely packed with a larger number of transferrin receptors than in reticulocytes.

[0158] B. Quantitative Confluency Assessment via Immunoblot and Image Analysis C57BL / 6 mouse cerebrovascular tissue and reticulocytes were harvested via FACS, and total protein was extracted using RIPA buffer. The extracted proteins were mixed with sample buffer, denatured, loaded onto an SDS-PAGE gel, and transferred to a PVDF membrane. Anti-TfR antibodies were then used as the primary antibody to visualize each protein band on the membrane, and their intensity was measured using Image Lab software (Biorad). The intensity of the transferrin receptor protein bands measured in the two cells was quantified as the number of transferrin receptors per cell using a standard curve for recombinant transferrin receptor bands. Next, to calculate the transferrin receptor density per single cell, the average diameter and number of transferrin receptor clusters measured by the STED confocal microscope were used to calculate the total area of ​​the transferrin receptor clusters in a single cell. The quantified number of receptors was then divided by the calculated total area to calculate the number of transferrin receptors per cluster area in a single cell. The calculated number of transferrin receptors was then divided by Avogadro's number to convert it to a molar concentration per area. Since the transferrin receptor exists as a dimer, the calculated molar concentration was halved. The distance between transferrin receptors present within a cluster was calculated as the distance from the center of the dimer to the center of the adjacent receptor, assuming that the receptors are packed in a square lattice pattern. As shown in Figure 26, the distance from the center of the transferrin receptor to the surface was determined for the major and minor axes. That is, when multiple transferrin receptors were arranged / aligned in a lattice form, the surface distance between the receptor and the adjacent receptor around the long axis of the dimer was calculated as the minimum value (min), and the surface distance between the receptor and the adjacent receptor around the short axis of the dimer was calculated as the maximum value (max), and then the average value of these was used for evaluation.

[0159] The results of evaluating the overall transferrin receptor density and the distance between transferrin receptors on cerebral blood vessels and reticulocytes are shown in Figures 27 and 28. Specifically, as shown in Figure 27, the distribution of transferrin receptor density expressed in individual cerebral blood vessel cells was observed to be statistically significantly higher than that in reticulocytes. Furthermore, the distribution of surface distances between transferrin receptors expressed within clusters of single cells was calculated, and as shown in Figure 28, the surface distances between transferrin receptors on cerebral blood vessel cells were observed to be statistically significantly shorter than that in reticulocytes. In other words, the transferrin receptor expression pattern within transferrin receptor clusters on cerebral blood vessel cells was observed to be more densely packed than that in reticulocytes.

[0160] Taken together, the above experimental results indicate that the distribution of specific transferrin receptor clusters in the vascular region of brain tissue can affect the interaction of a tetravalent binding moiety according to one embodiment with a fusion protein containing a functional structure linked to the C-terminus of the light chain and the C-terminus of the heavy chain of an antibody. Furthermore, it was found that the specific expression pattern of transferrin receptor clusters that induces such specific interaction in brain tissue acts as a factor that enables high permeability across the blood-brain barrier and selective absorption of IgG1 antibodies in brain tissue.

[0161] Experimental Example 2: Evaluation of the functionality of the vascular-brain barrier permeability fusion proteins F3#02 and F3#41 In this experimental example, a fusion protein in which the helical region-binding moiety of the transferrin receptor is tetravalently linked to the C-terminal region of the light chain and the C-terminal region of the heavy chain of an IgG1 antibody was examined to determine whether the functionality of the fusion protein could be exhibited even when the helical region-binding moiety of the transferrin receptor was modified.

[0162] 2-1. Evaluation of the functionality of binding moieties The functionality of the binding moiety of the blood-brain barrier permeable fusion protein according to one embodiment is confirmed by evaluating the level of binding to the helical domain of the transferrin receptor and the transduction level mediated by this in human brain endothelial cells.

[0163] (1) Confirmation of binding between the helical region of the transferrin receptor and the binding moiety The binding of the helical region-binding moieties #02, #03, #05 to #25, #27, #30, #32, #33, #34, #36, #38, and #39 of the fusion proteins prepared in the above examples to the transferrin receptor (TfR) was confirmed through docking simulations. The structures of the binding moieties were modeled using the RosettaRelax program, and the positions predicted to interact with the helical region of the TfR were modeled using structural information and thermodynamic calculations. The positions of the binding moieties were then arbitrarily changed, and docking simulations were performed using the RosettaDocking program to calculate interactions with the helical region and find the most stable positions. 20,000 simulations were performed for each SEQ ID NO., and the resulting data were analyzed based on homology to the initial modeled structure and thermodynamic structural stability.

[0164] As a result, as shown in Figures 29 to 34, it was confirmed that the binding moiety according to one embodiment stably binds to the helical domain of the transferrin receptor.

[0165] (2) Confirmation of transduction within human brain endothelial cells The helical domain-binding moieties #04, #16, #19, #20, #25 to #29, #31, #34 to #38, #40, and #41 of the fusion proteins prepared in the above Examples were evaluated for their ability to be delivered into hCMEC / D3 cells, which are human brain endothelial cells that make up the human blood-brain barrier, through interaction with the transferrin receptor, in the same manner as in Experimental Example 1-1(2). A control group received intravenous administration of IgG1 antibody alone.

[0166] As a result, as shown in Figures 35 to 40, an evaluation of the intracellular delivery level using a human brain endothelial cell line (hCMEC / D3) confirmed effective intracellular delivery of the binding moiety according to one embodiment.

[0167] 2-2. Evaluation of the permeability of the fusion protein through the blood-brain barrier The level of IgG1 antibody absorption into brain tissue following intravenous administration of one example of the blood-brain barrier-permeable fusion proteins F3#02 to F3#41 was evaluated 2 or 4 days after intravenous administration in the same manner as in Experimental Example 1-2(1). A control group received intravenous administration of IgG1 antibody alone.

[0168] As a result, as shown in Figures 41 to 45, the IgG1 antibody levels in brain tissue were significantly elevated compared to the control group in the groups administered with fusion proteins F3#02 to F3#24 of Examples 2 to 24, which were prepared using first binding moiety groups with different amino acid sequences but retaining helical region-binding properties. Furthermore, as shown in Figures 46 to 49, the IgG1 antibody levels in brain tissue were also significantly elevated compared to the control group in the groups administered with fusion proteins F3#025 to F3#41 of Examples 25 to 41, which were prepared using second binding moiety groups with partial substitution, insertion, or deletion of the amino acid sequence while maintaining a certain level of sequence identity with the binding moiety of SEQ ID NO: 3.

[0169] Taking the above experimental results into consideration, it was found that the fusion protein according to one embodiment exhibits effects such as high permeability through the blood-brain barrier and high delivery of IgG1 antibody to brain tissue, which are functions provided by a moiety that has effective binding affinity to the helical domain of the transferrin receptor.

[0170] Experimental Example 3: Functional evaluation of fusion proteins that permeate the blood-brain barrier by changing the valence of the binding moiety In this experimental example, the effect on the functionality of a fusion protein to which a transferrin receptor helical domain-binding moiety is linked when the valency of the binding moiety linked to an IgG1 antibody is altered is confirmed.

[0171] Specifically, the levels of brain tissue absorption of IgG1 antibody following intravenous administration of the fusion proteins were evaluated 2 or 4 days after intravenous administration using the same method as in Experimental Example 1-2(1). These fusion proteins were: 1) a fusion protein (F3) in which a transferrin receptor helical region-binding moiety was tetravalently linked to the C-terminal region of the light chain and the C-terminal region of the heavy chain of an IgG1 antibody; 2) a fusion protein (F1) in which a transferrin receptor helical region-binding moiety was bivalently linked to the C-terminal region of the heavy chain of an IgG1 antibody; and 3) a fusion protein (F5) in which a transferrin receptor helical region-binding moiety was bivalently linked to the C-terminal region of the light chain of an IgG1 antibody.

[0172] As a result, as shown in Figures 50 to 59, the IgG1 antibody levels in the brain tissues were quantified and compared. The results showed that the groups administered with fusion proteins in which binding moieties were linked to each of the four C-terminal regions of the heavy chain and the light chain (F3#03, F3#05, F3#06, F3#12, F3#16, F3#25, F3#27, F3#31, F3#37, or F3#40) showed significantly higher levels than the groups administered with fusion proteins in which binding moieties were linked to two C-terminal regions of the heavy chain. It was observed that the IgG1 antibody level in brain tissue was significantly enhanced compared to the groups administered with fusion proteins (F1#03, F1#05, F1#06, F1#12, F1#16, F1#25, F1#27, F1#31, F1#37, or F1#40) or fusion proteins in which binding moieties were linked to the two C-terminal regions of the light chain (F5#03, F5#05, F5#06, F5#12, F5#16, F5#25, F5#27, F5#31, F5#37, or F5#40).

[0173] Taking the above experimental results into consideration, it was confirmed once again that the fusion protein according to one embodiment has a structure that reflects the distribution pattern of specific transferrin receptor clusters in the vascular region of brain tissue, and that when a moiety having effective binding affinity for the helical region of the transferrin receptor is linked in a tetravalent manner to the end of an IgG1 antibody, it can exhibit its inherent functionality.

[0174] Experimental Example 4. Evaluation of the reactivity of IgG1 antibodies by linking binding moieties In this experimental example, the effect of linking a binding moiety to a transferrin receptor helical domain-binding moiety in a tetravalent fusion protein was confirmed on the reactivity of an IgG1 antibody, i.e., its ability to bind to the target.

[0175] Specifically, 100μl of 0.25μg / ml human PD-L1 protein was added to a 96-well plate and allowed to coat the plate at 4℃ for 16 hours. The coated plate was then washed four times with PBS-T, and 200μl of blocking buffer was added to each well, followed by incubation at room temperature for 2 hours to prevent nonspecific antibody binding. After washing the plate four times with PBS-T, 4nM of fusion protein (F3#01, F3'#01) was added to each well and incubated at room temperature for another 2 hours. Next, 100μl of the detection antibody, anti-human IgG FCγ HRP-conjugated antibody, was added to each sample well and incubated at room temperature for 1 hour. The incubated plate was then washed four times with PBS-T, and 100 μl of TMB was added to each well, followed by incubation at room temperature for 10 minutes. 100 μl of stop solution was then added to terminate the reaction, and the absorbance of each well was measured at 450 nm using a microplate reader. In this example, the IgG1 antibody was an anti-PD-L1 antibody, transferrin receptor helical domain-binding moiety #01, and the control group was treated with only the IgG1 antibody.

[0176] As a result, as shown in Table 2 below and Figure 60, it was confirmed that the IgG1 antibody reactivity, i.e., the binding ability to PD-L1, of the fusion proteins F3#01 and F3'#01 according to one embodiment was similar to that of the control group. The above results suggest that the blood-brain barrier penetration and selective delivery to brain tissues of the fusion protein according to one embodiment are achieved while maintaining the inherent biological activity of IgG antibodies.

[0177] [Table 43]

[0178] Experimental Example 5. Evaluation of applicability to various IgG1 antibodies In this experimental example, a fusion protein in which a transferrin receptor helical domain-binding moiety is tetravalently linked to the C-terminal region of the light chain and the C-terminal region of the heavy chain of an IgG1 antibody was prepared, and it was confirmed whether the functionality of the fusion protein could be exhibited even when the IgG1 antibody was modified.

[0179] 5-1. Anti-tau (Tau) IgG1 antibody In a fusion protein (F3) in which the helical domain-binding moieties of the transferrin receptor were tetravalently linked to the C-terminal domain of the light chain and the C-terminal domain of the heavy chain of an IgG1 antibody, an IgG1 antibody that binds to tau (anti-tau) was used as the IgG1 antibody, and binding moieties #25, #27, or #36 were used as the helical domain-binding moieties of the transferrin receptor. Fusion proteins with blood-brain barrier permeability were prepared in the same manner as in Example 1 (F3#25-tau, F3#27-tau, F3#36-tau). The level of ISF absorption of the IgG1 antibody following intravenous administration of the prepared fusion proteins was evaluated in the same manner as in Experimental Example 1-2(2). A control group received only the IgG1 antibody.

[0180] As a result, as shown in Figures 61 and 62, it was found that, while the functional structure of the present invention was maintained, the level of the anti-tau IgG1 antibody in the ISF was increased compared to the control group, even when the type of IgG1 antibody that was the fusion partner of the helical region-binding moiety of the transferrin receptor was changed to an anti-tau IgG1 antibody.

[0181] 5-2. Anti-PD1 IgG1 antibody In a fusion protein (F3) in which the helical domain-binding moieties of the transferrin receptor were tetravalently linked to the C-terminal domain of the light chain and the C-terminal domain of the heavy chain of an IgG1 antibody, an IgG1 antibody that binds to PD1 (anti-PD1) was used as the IgG1 antibody, and binding moiety #25 was used as the helical domain-binding moiety of the transferrin receptor. A fusion protein with blood-brain barrier permeability (F3#25-PD1) was prepared in the same manner as in Example 1. The level of IgG1 antibody absorption into brain tissue following intravenous administration of the prepared fusion protein was then evaluated in the same manner as in Experimental Example 1-2(1). A group to which only the IgG1 antibody was added was used as a control.

[0182] As a result, as shown in Figure 63, it was found that when the type of IgG1 antibody that is the fusion partner of the helical domain-binding moiety of the transferrin receptor was changed to an anti-PD1 IgG1 antibody while maintaining the above-mentioned functional structure of the present invention, the level of the anti-PD1 IgG1 antibody in brain tissue was increased compared to the control group.

[0183] 5-3. Anti-HER2 IgG1 antibody In a fusion protein (F3) in which the helical domain-binding moieties of the transferrin receptor were tetravalently linked to the C-terminal domain of the light chain and the C-terminal domain of the heavy chain of an IgG1 antibody, an IgG1 antibody that binds to HER2 (anti-HER2) was used as the IgG1 antibody, and binding moiety #25 was used as the helical domain-binding moiety of the transferrin receptor. A fusion protein with blood-brain barrier permeability (F3#25-HER2) was prepared in the same manner as in Example 1. The level of IgG1 antibody absorption into brain tissue following intravenous administration of the prepared fusion protein was then evaluated in the same manner as in Experimental Example 1-2(1). A group to which only the IgG1 antibody was added was used as a control.

[0184] As a result, as shown in Figure 64, it was found that, while the functional structure of the present invention was maintained, the level of the anti-HER2 IgG1 antibody in brain tissue was increased compared to the control group, even when the type of IgG1 antibody that was the fusion partner of the helical domain-binding moiety of the transferrin receptor was changed to an anti-HER2 IgG1 antibody.

[0185] 5-4. Anti-Aβ IgG1 antibody In a fusion protein (F3) in which the helical domain-binding moieties of the transferrin receptor were tetravalently linked to the C-terminal domain of the light chain and the C-terminal domain of the heavy chain of an IgG1 antibody, an IgG1 antibody that binds to Aβ (anti-Aβ) was used as the IgG1 antibody, and binding moiety #25 was used as the helical domain-binding moiety of the transferrin receptor. A fusion protein with blood-brain barrier permeability (F3#25-Aβ) was prepared in the same manner as in Example 1. The level of absorption of the IgG1 antibody into brain tissue following intravenous administration of the prepared fusion protein was then evaluated in the same manner as in Experimental Example 1-2(1). A group to which only the IgG1 antibody was added was used as a control.

[0186] As a result, as shown in Figure 65, it was found that, while the functional structure of the present invention was maintained, the level of the anti-Aβ IgG1 antibody in brain tissue was increased compared to the control group, even when the type of IgG1 antibody that served as the fusion partner of the helical domain-binding moiety of the transferrin receptor was changed to an anti-Aβ IgG1 antibody.

[0187] From the above results, it was found that the fusion protein according to one embodiment exhibits effects such as high permeability through the blood-brain barrier and high delivery of IgG1 antibodies to brain tissue, regardless of the type of IgG1 antibody, as functions resulting from the moiety having effective binding affinity to the helical domain of the transferrin receptor and the functional structure containing the same.

[0188] The above description of the present invention is for illustrative purposes only, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. Therefore, it should be understood that the above-described embodiments are illustrative in all respects and not limiting.

Claims

1. an IgG antibody; four binding moieties to the helical region of the transferrin receptor (TfR) linked to the C-terminal region of the light chain and the C-terminal region of the heavy chain of the IgG antibody; wherein the binding moiety has binding affinity for the helical region of SEQ ID NO: 2 in the transferrin receptor.

2. The fusion protein of claim 1, wherein the blood-brain barrier-permeable fusion protein binds to and forms a complex with transferrin receptors that form transferrin receptor clusters that are specifically distributed in blood vessels of the blood-brain barrier.

3. The fusion protein according to claim 1, wherein the blood-brain barrier-permeable fusion protein is selectively delivered to brain tissue.

4. The fusion protein of claim 1 , wherein the binding moieties are identical to each other or different from each other.

5. The fusion protein of claim 1 , wherein the IgG antibody is IgG1, IgG2, IgG3, or IgG4.

6. The fusion protein of claim 1 , wherein the binding moiety is linked to the C-terminal region of the light chain and the C-terminal region of the heavy chain of the IgG antibody by a linker peptide.

7. A polynucleotide encoding the fusion protein according to any one of claims 1 to 6.

8. A vector comprising the polynucleotide of claim 7.

9. A transduced cell line transduced with the vector of claim 8.

10. A pharmaceutical composition for preventing or treating a disease associated with brain dysfunction, comprising the fusion protein according to any one of claims 1 to 6 as an active ingredient, wherein the brain dysfunction is Alzheimer's disease, dementia with Lewy bodies, frontotemporal dementia, senile dementia with neurofibrillary tangles, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis, traumatic brain injury, progressive supranuclear palsy, corticobasal degeneration, globular glial tauopathy, or the like. tauopathy), age-related tauastrogliopathy, chronic traumatic encephalopathy, brain tumor, Pick's disease, anti-IgLON5 antibody-associated tauopathy, Guadeloupe Parkinsonism, nodding syndrome, pain, epilepsy, autism, stroke, Creutzfeldt-Jakob disease, Huntington's disease, progressive multifocal leukoencephalopathy, depression, post-traumatic stress disorder, or lysosomal storage disease.

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