Cerebrovascular 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 having blood-brain barrier permeability 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 medical 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 plays a role in isolating the central nervous system, including the brain, from potentially dangerous substances in the blood. The BBB is a central structure of the brain blood vessels, consisting of cells such as brain endothelial cells, astrocytes, and pericytes, and is distributed throughout the brain blood vessels in brain tissue. The BBB endothelial cells are tightly connected by tight junctions, and astrocytes and astrocyte end feet surround the periphery of blood vessels, forming a barrier that selectively prevents substances flowing along the blood inside the brain blood vessels from penetrating the blood vessel barrier and being absorbed / transmitted to 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 in the case of water and oxygen essential for life support, they can pass through the blood-brain barrier by diffusion, and in the case of amino acids and glucose used as energy sources, they can be transported from the blood to the brain tissue by active transport. However, not only are toxic substances and pathogenic bacteria that can potentially affect the brain inhibited from being transported by the blood-brain barrier, but even if they are transported, they will return to the blood due to the pumping action of cells, preventing their absorption into the brain tissue and protecting the brain tissue.

[0004] However, the blood-brain barrier having such a structure acts as a major obstacle in the pharmacological treatment of diseases related to 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 having a molecular weight of 400 to 500 Daltons or less can permeate the blood-brain barrier, and most therapeutic drugs cannot be delivered to brain tissue or are delivered in only a very small amount due to the blood-brain barrier, and cannot exert their efficacy in the target organ, brain tissue. To solve such problems, a technology that directly passes through the cell membrane of endothelial cells through lipophilization of drugs, a delivery technology through the innate nutrient delivery system, and an antibody-based delivery technology using receptor-mediated transcytosis have been proposed. However, such technologies act not only on the brain but also on other organs, so there are concerns about side effects in the body, and it is difficult to determine the appropriate concentration or content due to differences in efficacy of delivery from patient to patient, so there are limitations to their clinical use.

[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 reality is that there are still insufficient results. [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 an IgG antibody and a fusion protein in which a helical domain-binding moiety of a tetravalent transferrin receptor is linked to the end of the IgG antibody. The present inventors confirmed that the fusion protein not only enhances the permeability of the cerebrovascular barrier and improves the 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 is selectively distributed at a high level in brain tissue compared to other tissues, and based on this, the present invention was completed.

[0008] Thus, one embodiment provides a blood-brain barrier-permeable fusion protein 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 said fusion proteins, vectors comprising said polynucleotides, and transfected cell lines transfected with said vectors.

[0010] In another embodiment, there is provided a pharmaceutical composition for preventing or treating a disease associated with brain dysfunction, comprising 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, so 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 of the brain blood vessels that strictly controls the movement of substances such as ions, molecules, and pathogens present in the blood to brain tissue while allowing selective absorption of vital components such as amino acids and glucose. The brain-brain barrier is composed of cells such as brain-brain barrier endothelial cells, astrocytes, and perivascular cells. The cells are arranged in a form that shares a common basement membrane, and on one side of the brain-brain barrier, the brain-brain barrier endothelial cells are tightly connected, and on the other side, the astrocytes that surround the periphery of blood vessels are distributed. The brain-brain barrier endothelial cells form the capillary wall, are very strong, and are connected by complex tight junctions. Such a structure forms a physical barrier and plays a role in inhibiting the simple diffusion of most substances, including molecules that are larger than the average size, such as insulin. In addition, the astrocytes are a type of glial cell in the central nervous system, and affect the function of brain endothelial cells, blood flow, and ion balance through interaction with the brain blood vessels. The astrocytes use processes called end feet to surround blood vessels at one end and to make close contact with neurons at synapses at the other end. The blood-brain barrier, which has such a structure, protects brain tissue from potentially dangerous substances in the blood and also prevents substances effective in treating diseases from being delivered to brain tissue, acting as a major obstacle in pharmacological treatment. Therefore, a technology to improve the permeability of the blood-brain barrier can improve the applicability or efficacy of therapeutic agents in the medical field targeting brain tissue.

[0014] IgG antibody As used herein, the term "IgG antibody" refers to an immunoglobulin molecule that is immunologically reactive with a specific antigen and is a protein molecule that acts as an antigen receptor to specifically recognize the antigen. The antibody has a heavy chain and a light chain, and each of the heavy chain and light chain includes a constant region and a variable region. The light chain and the variable region of the heavy chain include three variable regions called complementarity-determining regions (CDRs) and four framework regions. The CDRs mainly play a role in binding to an epitope of an antigen. The CDRs of each chain are typically called CDR1, CDR2, and CDR3, starting from the N-terminus, and are identified by the chain in which the specific CDR is located. The antibody also includes polyclonal antibodies, monoclonal antibodies, full-length antibodies, and antibody fragments or antigen-binding fragments that include an antigen-binding domain. The full-length antibody is a structure having 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 its subtypes include 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 a significantly lower risk of immunogenicity in vivo than a genetically modified antibody. A recombinant antibody refers to an antibody that has been genetically modified, and has the characteristic that antigen binding ability or 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. In addition, the antigen-binding fragment may illustratively include, but is not limited to, the following, and may include, but is not limited to, structures of IgG-like bispecific antibodies known in the art: (1) Fab: the fragment which 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 portions of the intact light and heavy chains; 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 bound 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] The antibody or antibody fragment is an immunoglobulin molecule having an 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, disulfide-linked Fvs (sdFVs), scFv fragments, scFv-Fc fragments, Fv fragments, diabodies, triabodies, tetrabodies, and the like. In the antibody fragments, Fab has a structure having a light chain and a heavy chain variable region, a light chain constant region, and a heavy chain first constant region (CH1), and has one antigen-binding site. Fab' is different 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 by forming disulfide bonds between the cysteine ​​residues in the hinge region of Fab'. Fv is the smallest antibody fragment that contains 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 proteolytic enzymes (for example, whole antibodies can be digested with papain to obtain Fab fragments, and can be digested 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 helical domain-binding moiety of the transferrin receptor, 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 helical domain-binding moiety of the transferrin receptor. Such an IgG antibody has enhanced selective permeability through the blood-brain barrier and can 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 that can be applied to treat, alleviate, or detect a disease associated with brain dysfunction, e.g., a pharmacologic 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), primary CNS lymphoma (PCNSL), and other conditions. lymphoma, glioma, neuroblastoma, brain metastases, brain cancers such as meningioma;Pick's disease;anti-IgLON5-related taupathy;Guadeloupean Parkinsonism;nodding syndrome;pain;epilepsy;autism;stroke;Guillain-Barre syndrome (GBS);Creutzfeldt-Jakob disease (CJD);The antibody may be 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 disease (LSD), including, but not limited to, αPD-L1 IgG antibody, anti-PD1 IgG antibody, anti-tau IgG antibody, anti-HER2 IgG antibody, anti-Aβ IgG antibody, anti-TDP-43 IgG antibody, anti-α-synuclein IgG antibody, anti-SIGLEC3 IgG antibody, or anti-TREM2 IgG antibody;

[0020] Transferrin receptor (TfR) In this specification, the term "transferrin receptor (TfR)" refers to a membrane glycoprotein expressed on the surface of cells that mediates intracellular absorption of iron from transferrin, a plasma glycoprotein, and is reported to be widely distributed in normal cells of various tissues, as well as being expressed in large amounts in activated immune cells and tumor cells. Therefore, the transferrin receptor-mediated transfer technology requires not only effective transfer to brain tissue, but also low transfer to other organs other than brain tissue or normal cells. In fact, it has been reported that transferrs or therapeutic agents mediated by the transferrin receptor may cause red blood cell-related toxicity, including a decrease in reticulocyte count, severe coma, intermittent limb stiffness, generalized rigidity, hemolysis, and hemoglobinuria. In addition, 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 a publicly known database 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 in which multiple transferrin receptors are densely present in a local 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 in a local region in which a cluster is formed are distributed at a particularly high level, compared to other organ tissues or cells, and such brain tissue-specific clusters and the expression pattern of transferrin receptors in the clusters may show a close relationship with high permeability to the cerebrovascular 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 to 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 a functional / structural region that is exposed externally so that a fusion protein and a plurality of transferrin receptors densely distributed in the brain tissue blood vessel clusters form an effective tetravalent bond, which may contribute to high permeability to the blood-brain barrier and selective delivery to brain tissue. Therefore, a binding moiety capable of binding to the helical region of the transferrin receptor can be linked to an IgG antibody to not only significantly increase the blood-brain barrier permeability of the IgG antibody, but also to impart functionality that enables selective delivery / absorption to brain tissue.

[0024] The binding moiety is intended to interact 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, with the helical region of the transferrin receptor, and more specifically, with at least one amino acid selected from the region from amino acid 606 to amino acid 665 based on the transferrin receptor of SEQ ID NO:1.

[0025] In one embodiment, the transferrin receptor helical region binding moiety is linked to the C-terminus of the light chain and the C-terminal region of the heavy chain of the IgG antibody, such that the transferrin receptor helical region binding moiety is linked to the IgG antibody in a tetravalent form and can exert the desired functionality.

[0026] In one embodiment, the transferrin receptor helical region binding moiety has a length of 6 to 250 amino acids, e.g., 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 a transferrin receptor helical domain binding moiety may 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 amphiphilic sequence is repeated 2 to 5 times, 2 to 4 times, or 2 to 3 times. The amphiphilic sequences may be linked by fusion or by a linker peptide. The linker peptide may be 2 to 50 amino acids long, 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 long, but is not limited thereto. The linker peptide may 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, and 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 NO: 3 to SEQ ID NO: 43, but may be extended to any unit sequence having effective interaction (hydrogen bond, electrostatic attraction, van der Waals force), 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 are the same or different.

[0031] Brain-blood barrier permeable fusion protein As used herein, the term "fusion protein" refers to a protein formed through the binding of two or more originally separate proteins or parts thereof, and optionally includes a linker or space located between two or more proteins. As used herein, the term "brain-blood vessel barrier-permeable fusion protein" refers collectively to a protein formed through the binding of an IgG antibody and a helical domain-binding moiety of a transferrin receptor, and includes a functional structure that allows an effective amount of IgG antibody to be selectively delivered or absorbed into brain tissue by penetrating the brain-blood vessel barrier. The functional structure is modified to have effective binding affinity, reflecting the structure of transferrin receptor clusters of vascular endothelial cells specifically distributed in the brain-blood vessel barrier and their expression patterns, and includes a binding moiety capable of binding to the helical domain of the transferrin receptor, and a tetravalent bond (linkage) between the binding moiety and the C-terminal domains of the heavy and light chains constituting the IgG antibody.

[0032] In one embodiment, the blood-brain barrier-permeable fusion protein also comprises a functional structure in which a tetravalent helical domain-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, the details of which are as described above.

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

[0035] According to one embodiment, the functional structure was devised to reflect the effective binding affinity with the helical region of the transferrin receptor and the specific distribution / expression pattern of the transferrin receptor cluster distributed in the cerebrovascular region, and a fusion protein was prepared in which a plurality of transferrin receptor helical region-binding moieties were linked to the C-terminal region of the light chain and the C-terminal region of the heavy chain of an IgG antibody, i.e., a total of four terminal regions. Thereafter, when a formulation containing the fusion protein was intravenously administered, not only was the delivery of the fusion protein containing an IgG antibody significantly enhanced in brain tissue, but it was also confirmed that the IgG antibody was distributed at a high level in brain tissue compared to other organs, and in particular, the effect on normal cells including reticulocytes and other organ tissues was negligible. In addition, it was found that the fusion protein is a function caused by a tetravalent moiety having an effective binding affinity with the helical region of the transferrin receptor and a functional structure containing the same, and that an effect such as high permeability to the cerebrovascular barrier was exhibited regardless of the type of IgG antibody and the specific sequence of the binding moiety. Therefore, in one embodiment, a new functional structure was identified that can increase 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 performed by any of the methods for administering general fusion protein or antibody-based preparations, including, but not limited to, intravenous administration, intramuscular administration, subcutaneous administration, peritoneal 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 in brain tissue through binding to a part of the receptor present on the surface of cells that constitute the blood-brain barrier, i.e., a helical domain of the transferrin receptor, which can provide an appropriate level of interaction or binding affinity with the helical domain 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] Polynucleotides encoding fusion proteins 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 the non-coding (antisense) strand, and the coding sequence may be the result of degeneracy or redundancy in the genetic code and may 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. The allelic variants are alternate forms of a polybase 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 one or more nucleotide codons, and the polynucleotides can be easily modified to produce alternate polynucleotides that encode the same peptide.

[0042] Vectors Containing Polynucleotides As used herein, the term "vector" refers to a DNA vehicle capable of expressing a target protein in a suitable host cell, and refers to a genetic construct that includes a regulatory element operably linked to express a gene insert. According to one embodiment, the vector may include expression regulatory elements such as a promoter, an operator, an initiation codon, a termination codon, a polyadenylation signal, and / or an enhancer, and the promoter of the vector may be constitutive or inducible. The vector may also be an expression vector that can stably express the fusion protein in a host cell. The expression vector may be a vector that is commonly used in the art to express a foreign protein in plants, animals, or microorganisms. The recombinant vector may be constructed through various methods known in the art. For example, the vector may include a selectable marker for selecting a host cell containing the vector, and may also include a replication origin if the vector is a replicable vector. The vector may also be autonomously replicating or integrated into a 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. According to one embodiment, suitable promoters include promoters derived from mammalian viruses and promoters derived from the genome of mammalian cells, such as the cytomegalovirus (CMV) promoter, the T7 promoter, the U6 promoter, the H1 promoter, the murine leukemia virus (MLV) LTR (long terminal repeat) promoter, and the like. repeat) promoter, adenovirus early promoter, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, HSV tk promoter, RSV promoter, EF1-α promoter, metallothionine promoter, β-actin promoter, human IL-2 gene promoter, human IFN gene promoter, human IL-4 gene promoter, human lymphotoxin gene promoter, human GM-CSF gene promoter, human phosphoglycerate kinase (PGK) promoter, mouse phosphoglycerate kinase (PGK) promoter, survivin promoter and CHEF1-α (Chinese hamster elongation factor-1α) promoter.

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

[0045] Transduced cell lines As used herein, the term "transduction" refers to a molecular biology technique in which a DNA fragment or a plasmid carrying a foreign gene different from that possessed by the original cell is infiltrated between cells and combined with the DNA present in the original cell to change the genetic traits of the cell. The transduction refers to the insertion of a polynucleotide encoding the cerebrovascular barrier-permeable fusion protein or a vector containing the same into a host cell to produce the cerebrovascular 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 cells, 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 pharmaceutical composition for preventing or treating a disease associated with brain dysfunction, its medical use, or the method for treating a disease associated with brain dysfunction described below, 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 in which symptoms associated with diseases associated with brain dysfunction are improved or favorably altered by administration of the pharmaceutical composition.

[0051] The "diseases associated with brain dysfunction" that can be prevented or treated 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 pathology, aging-associated tau astrogliosis, chronic traumatic encephalopathy, primary central nervous system lymphoma, glio-tau astrogliosis, and the like. Brain cancers such as cerebrospinal fluid tumors, neuroblastomas, brain metastases, and meningiomas; Pick's disease; Anti-IgLON5 associated tauopathy; Guadeloupe Parkinsonism; Nodding syndrome; Pain syndrome; Electroencephalopathy; Autism; Stroke; Guillain-Barre syndrome; Creutzfeldt-Jakob disease; Huntington's disease; Progressive multifocal leukoencephalopathy; Depression; Post-traumatic stress disorder; and Lysosomal storage disorders.

[0052] The pharmaceutical composition, i.e., pharmaceutical preparations containing the fusion protein, are prepared by mixing the antibody having the desired degree of purity with any pharma- ceutical 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 phosphates, citrates, 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; alkyl parabens, such as methyl paraben, propyl paraben; 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 non-ionic surfactants, such as Twin™, Pluronics. TM Or polyethylene glycol (PEG).

[0053] The pharmaceutical composition may also include 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 clinical parameters of the subject. The active ingredient may be delivered in the form of encapsulation by colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or macroemulsions, for example, in microcapsules prepared by droplet formation techniques or interfacial polymerization, for example, hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively.

[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 antibodies 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 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 may use the pharmaceutical composition alone or in combination with other agents. For example, the pharmaceutical composition may 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] In addition, the appropriate dosage of the pharmaceutical composition (when 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 administered for prophylactic or therapeutic purposes, previous therapy, the patient's clinical strength and response to the antibody, and the judgment of the attending physician. The antibody is suitably administered to the patient at one time or over a series of treatments, and for purposes of the present invention, the antibody is also 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, about 1 μg / kg to 100 mg / kg (e.g., 0.1 mg / kg to 100 mg / kg) of the antibody or fusion protein is also an initial candidate dosage for administration to the patient, whether by one or more separate administrations or by continuous infusion, for example. Typical daily dosages may 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 the 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, or 10 mg / kg, 20 mg / kg (or any combination thereof) may be administered to the patient. The doses may be administered intermittently, for example, every week or every three weeks (e.g., such that the patient is administered a dose of about 2 mg / kg to about 20 mg / kg, or, for example, a dose of about 6 mg / kg of the antibody or fusion protein). 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 humans or non-human primates, mice, dogs, cats, horses, and cows. Effect of the Invention

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

[0059] Furthermore, according to one embodiment of the fusion protein capable of permeating the blood-brain barrier, 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 blood vessel 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 description of the drawings]

[0061] [Figure 1] The transferrin receptor was imaged via cryo-electron microscopy, and the helical domain within the transferrin receptor was confirmed. [Diagram 2] 1 shows the structural results of the binding between a helical domain-binding moiety and the helical domain of the transferrin receptor, according to one embodiment, determined by cryo-electron microscopy. [Diagram 3] 1 shows the results of confirming the thermodynamic structural stability of the binding between helical domain-binding moiety #01 and transferrin receptor according to one embodiment. [Figure 4] 1 shows the results of confirming the intracellular delivery level of helical domain-binding moiety #01 using a human brain endothelial cell line according to one embodiment. [Diagram 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 having a skull window. [Figure 8] FIG. 1 shows the results of monitoring in real time the levels of the fusion protein present in the intravascular (ROI1), vascular wall (ROI2), and extravascular (ROI3) regions after intravenous administration of the blood-brain barrier permeable fusion protein F3#01 according to one embodiment to an animal model having a skull window. [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] FIG. 1 shows the results of one embodiment of the fusion protein that permeates the blood-brain barrier (F1#01, F3#01, F5#01) administered intravenously to an animal model, and the binding of the fusion protein to the transferrin receptor was confirmed by immunostaining. (a) shows the results for the fusion protein F5#01, (b) shows the results for the fusion protein F1#01, and (c) shows the results for the fusion protein F3#01. [Figure 11] FIG. 1 shows the results of comparing the binding levels of the fusion protein and the transferrin receptor quantified 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-specific 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-specific 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] 1 shows the results of intravenously administering the blood-brain barrier permeable fusion protein F3#01 according to one embodiment to an animal model, obtaining blood, and then confirming the ratio (%) of reticulocytes to total red blood cells. [Figure 17] 1 shows the results of confirming the concentration of the fusion protein in plasma after intravenous administration of the blood-brain barrier-permeable fusion protein F3#01 according to one embodiment to an animal model. [Figure 18] 1 shows the results of calculating the blood-to-plasma ratio after treating a plasma sample and a blood sample with the blood-brain barrier permeable fusion protein F3#01 according to one embodiment. [Figure 19] 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 20] 1 shows the results of examining the organ-specific 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-specific 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 amount of transferrin receptor expressed in brain tissue, vascular cells, liver, lung, kidney and spleen was compared via Western blot. [Figure 23] The expression profile of transferrin receptor in brain tissue, liver, spleen, lung and reticulocytes was confirmed through confocal microscopy images and super-high resolution STED confocal microscopy fluorescence images. [Figure 24] This shows the results of comparing the quantitative distribution of transferrin receptor clusters formed in cerebral blood vessels, reticulocytes, lungs, liver, and spleen based on the size of the clusters. [Diagram 25] This is the result of comparing the average intensity of the entire transferrin receptor clusters in cerebral blood vessels and reticulocytes. [Figure 26] FIG. 2 is a diagram showing parameters for calculating the inter-transferrin receptor distance, and is a schematic diagram 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 domain-binding moiety #2, #3, #5, #6, #7 or #8 according to one embodiment and a transferrin receptor. [Diagram 30] 1 shows the results of confirming the thermodynamic structural stability of the binding between helical domain-binding moiety #9, #10, #11, #12, #13 or #14 according to one embodiment and a transferrin receptor. [Diagram 31]1 shows the results of confirming the thermodynamic structural stability of the binding between helical domain-binding moiety #15, #16, #17, #18, #19 or #20 according to one embodiment and the transferrin receptor. [Diagram 32] 1 shows the results of confirming the thermodynamic structural stability of the binding between helical domain-binding moiety #21, #22, #23 or #24 according to one embodiment and a transferrin receptor. [Diagram 33] 1 shows the results of confirming the thermodynamic structural stability of the binding between helical domain-binding moiety #25, #27, #30, #32, #33 or #34 according to one embodiment and the transferrin receptor. [Diagram 34] 1 shows the results of confirming the thermodynamic structural stability of the binding between helical domain-binding moiety #36, #38 or #39 according to one embodiment and the transferrin receptor. [Diagram 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. [Diagram 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. [Diagram 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. [Diagram 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 antibody in brain tissue using a human IgG1 ELISA kit. [Diagram 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. [Diagram 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. [Diagram 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. [Diagram 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. [Diagram 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 confirming 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 F1#05, F5#05, or F3#05 according to one embodiment to an animal model. [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. [Diagram 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 confirming the change in 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, and the results were expressed as a fold increase compared to the control group. [Figure 62]In one embodiment, the blood-brain barrier-permeable fusion protein F3#27-Tau (Tau) or F3#36-Tau (Tau) containing 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, and the results were expressed 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, and the results were expressed 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 confirmed 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 confirmed using a human IgG1 ELISA kit, and the results were expressed as a fold increase compared to the control group. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0062] In the following, preferred examples are presented to aid in understanding the present invention. However, the following examples are provided only to facilitate understanding of the present invention, and are not intended to limit the scope of the present invention. EXAMPLES

[0063] Examples 1 to 41: Preparation of cerebrovascular barrier permeable fusion protein In this embodiment, a fusion protein with enhanced blood-brain barrier permeability was produced. To this end, a fusion protein was produced in which a helical domain-binding moiety of a transferrin receptor was linked to the C-terminal domain of the light chain and the C-terminal domain of the heavy chain of an IgG1 antibody, i.e., a total of four terminal domains. In this embodiment, a total of 24 binding moieties were derived as the helical domain-binding moieties of the transferrin receptor, each of which has a binding property to the helical domain but has a different amino acid sequence (first binding moiety group). In addition, a total of 17 binding moieties were derived as the helical domain-binding moieties of the transferrin receptor, each of which maintains a certain level of sequence identity with the binding moiety of SEQ ID NO: 3, but has a part of the amino acid sequence substituted, inserted or deleted (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 the four terminal domains of the IgG1 antibody was produced as follows. Specifically, a 1 mL sample was taken from the flask containing the cells with a pipette, and the cell mass was measured. After that, the cells were cultured at a cell viability of 95% or more and a cell mass of 4 to 6 × 10 6 If the cell mass level is 9X10 cells / mL or more, add culture medium stored in a 37°C incubator and culture it until the cell mass level is 9X10 6 The cells were then transfected under the following experimental conditions.

[0065] [Table 1]

[0066] Then, 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 the cell viability became 70% or less or 8 days had passed since the day of transfection, the transfected cells were obtained. Then, the sample containing the transfected cells was centrifuged at 4,500 rpm and 25°C for 15 minutes, and the supernatant was collected. Then, the supernatant was filtered using a 0.22 μm filter. Then, the filtrate was purified using a purification technique such as affinity chromatography or size exclusion chromatography to obtain each fusion protein. 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 domain-binding moiety of the transferrin receptor, the IgG1 antibody, and the like are as follows. EXAMPLES

[0068] [Table 2] EXAMPLES

[0069] [Table 3] EXAMPLES

[0070] [Table 4] EXAMPLES

[0071] [Table 5] EXAMPLES

[0072] [Table 6] EXAMPLES

[0073] [Table 7] EXAMPLES

[0074] [Table 8] EXAMPLES

[0075] [Table 9] EXAMPLES

[0076] [Table 10] EXAMPLES

[0077] [Table 11] EXAMPLES

[0078] [Table 12] EXAMPLES

[0079] [Table 13] EXAMPLES

[0080] [Table 14] EXAMPLES

[0081] [Table 15] EXAMPLES

[0082] [Table 16] EXAMPLES

[0083] [Table 17] EXAMPLES

[0084] [Table 18] EXAMPLES

[0085] [Table 19] EXAMPLES

[0086] [Table 20] EXAMPLES

[0087] [Table 21] EXAMPLES

[0088] [Table 22] EXAMPLES

[0089] [Table 23] EXAMPLES

[0090] [Table 24] EXAMPLES

[0091] [Table 25]

[0092] 2. Preparation of Fusion Proteins Containing Tetravalent Second Binding Moiety Groups The fusion protein containing the second binding moiety group was prepared in the same manner as described above. In the fusion protein of this example, the transferrin receptor helical domain binding moiety and the IgG1 antibody are as follows: EXAMPLES

[0093] [Table 26] EXAMPLES

[0094] [Table 27] EXAMPLES

[0095] [Table 28] EXAMPLES

[0096] [Table 29] EXAMPLES

[0097] [Table 30] EXAMPLES

[0098] [Table 31] EXAMPLES

[0099] [Table 32] EXAMPLES

[0100] [Table 33] EXAMPLES

[0101] [Table 34] EXAMPLES

[0102] [Table 35] EXAMPLES

[0103] [Table 36] EXAMPLES

[0104] [Table 37] EXAMPLES

[0105] [Table 38] EXAMPLES

[0106] [Table 39] EXAMPLES

[0107] [Table 40] EXAMPLES

[0108] [Table 41] EXAMPLES

[0109] [Table 42] Comparative Example

[0110] Comparative Examples 1 to 22: Preparation of fusion proteins linked to helical domain-binding moieties of bivalent transferrin receptors Fusion proteins were prepared in which two transferrin receptor helical domain-binding moieties were linked to either the C-terminal domain of the heavy chain or the C-terminal domain of the light chain of an IgG1 antibody (F1, F5). Specifically, the transferrin receptor helical domain-binding moieties used included six representative binding moieties (#01, #03, #05, #06, #12, #16) in the first binding moiety group and five representative binding moieties (#25, #27, #31, #37, #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 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 above 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 the IgG1 antibody using binding moiety #01, #03, #05, #06, #12, or #16 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 7 to 12: F5#01, F5#03, F5#05, F5#06, F5#12, F5#16).

[0113] 2. Preparation of Fusion Proteins Containing Divalent Second Binding Moiety Groups 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 above 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 are linked to the C-terminal region of the light chain of the 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). Experimental Examples

[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 through the evaluation of its binding to the helical domain of the transferrin receptor and the level of signaling mediated by it within human brain endothelial cells.

[0116] (1) Confirmation of binding between the helical domain of the transferrin receptor and the binding moiety In the prepared fusion protein F3#01, the binding of the helical domain binding moiety 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 50 mM Tris, 150 mM NaCl, and a pH 7.6 buffer solution. Then, vitrification of the L7 TfR sample was carried out using a Quantifoil Cu 1.2 / 1.3 400mesh grid. At this time, a Glow discharger was used to induce negative charge discharge on the grid surface in order to enhance the interaction between the sample and the grid. Then, 3 μl of L7 TfR sample was injected onto the discharged grid, and vitrification was carried out under the following conditions: blotting time (7 seconds), blotting force (0), waiting time (0 seconds), 4°C, and 95% humidity. Cryo-EM analysis was performed using 200 kV Glycios, 300 kV Krios G4, Falcon 4, and K3 Detector. The lens was set to the following conditions: spherical aberration (2.7), 0 50 μM aperture, magnification (120K), and exposure time (6.55 seconds). In addition, the analysis was performed under the conditions of 50 fractions per sample, 225 frames, pixel value 0.894Å / pix, dose rate 6.1e / px / s, and total dose 49.93e / Å2. The defocus range was -1.25 to -2.75 with 0.25 intervals, and after picking more than 5 million particles in total, the initial 2D classification was performed using them. After that, about 2 million particles were selected, 3D refinement was performed, and C1 and C2 symmetry were applied, respectively, to derive a 3D electron density map with a resolution of ~3.4Å.A co-complex model (PDB: 3s9n) of TfR and transferrin was superimposed on this map, and the binding site and core residues were predicted through comparison with the existing binding method and the binding method of the helical region binding moiety.

[0117] In addition, the binding of the helical region binding moiety #01 to the transferrin receptor was confirmed through a docking simulation. Specifically, a docking simulation was performed to confirm whether the binding moiety #01 has the ability to bind to the transferrin receptor helical region. The structure of the binding moiety #01 was modeled using the RosettaRelax program, and the position expected to interact with the transferrin receptor helical region was determined through structural information and thermodynamic calculation. Thereafter, the position of the binding moiety was arbitrarily changed, and the interaction with the helical region was calculated, and a docking simulation was performed using the Rosettadocking program to find the most stable position. 20,000 simulations were performed for each sequence number, and the resulting data was analyzed based on the homology to the initial modeling structure and the thermodynamic structural stability.

[0118] As a result, as shown in Figures 1 and 2, it was confirmed that the helical region applicable to the interaction with the transferrin receptor was exposed to the outside, and when viewed together with a model that maintains the endogenous binding mode between the transferrin receptor and transferrin in a 3D electron density map with a resolution of 3.4 Å, it was confirmed that there was electron density corresponding to glycosylation, which is not shown in the existing transferrin receptor, in the predicted ideal range. In addition, the bond with the binding moiety was formed to conform to the shape of the α-helical structure, and the interaction between the helical region of the transferrin receptor and the binding moiety was confirmed through it. In addition, as shown in Figure 3, it was confirmed that the 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 carried out to confirm whether the helical domain-binding moiety #01, which was confirmed to bind to the transferrin receptor in the above-mentioned 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 above-mentioned interaction. Specifically, the hCMEC / D3 cells were cultured at 37°C under 5% CO2 conditions using EBM2 (Endothelial Cell Basal Medium 2) synthetic culture medium containing growth factors. Thereafter, when the cell saturation reached 80%, the cells were separated and 4X10 3Cells were added, dispensed into a 384-well plate, centrifuged for 10 seconds, and then cultured at 37°C and 5% CO2 for 18 hours or more to allow the cells to adhere to the plate. 1 mg of binding moiety peptide was mixed with 500 μL of 1xPBS, and the peptide was diluted to a final concentration of 200 μM and stored at 4°C. The 200 μM peptide was then diluted in EBM2 culture medium to a concentration 5 times that of the treatment concentration. 10 μL of the peptide diluted to a 5-fold concentration 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 1xPBS, and 75 μL of 4% PFA was dispensed into each well and stored at room temperature for 30 minutes to immobilize them. At this time, Hoechst solution was added to the 4% PFA to simultaneously carry out nuclear staining, 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 FIG. 4, the intracellular delivery level was evaluated using a human brain endothelial cell line (hCMEC / D3), and effective intracellular delivery of the binding moiety #01 corresponding to sequence number 3 was observed.

[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 A preparation containing 20 mg / kg of the fusion protein F3#01 was administered intravenously via the tail vein to C57BL / 6 mice, and a control group was administered only IgG1 antibody intravenously. 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 brains of the mice were then removed, and the removed brain tissue was flash frozen with liquid nitrogen and stored in a deep freezer until use. The removed brain tissue was homogenized using a protein extraction solution, and the homogenized brain tissue sample was dissolved at 4°C using a rotary mixer. The dissolved brain tissue sample was then centrifuged to obtain a supernatant, from which a brain tissue lysate was prepared. The level of IgG1 antibody in the brain tissue lysate was then measured using a human IgG1 ELISA kit. Specifically, the protein extract solution was used to prepare STD (standard) by diluting to each concentration, and the protein extract solution was used to prepare a sample (SPL) containing brain tissue lysate according to each dilution ratio, and then 50 μL of each was added to each well. Then, 50 μL of Ab Cocktail included in the ELISA kit was added to each well, and the reaction was carried out at 4°C. After that, the plate was washed, and 100 μL of TMB substrate was added to each well. Next, a microplate reader was used to measure the OD value of STD1 at a wavelength of 600 nm, and a stop solution was added to measure the value at a wavelength of 450 nm, and a standard curve was obtained using 4-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 level in the brain tissue of the group administered F3#01 according to one embodiment was observed to be significantly higher than that of the control group up to 14 days after administration. Specifically, the F3#01 administered group showed a high level of transmission, ranging from about 25 to about 320 times, compared to the control group.

[0124] (2) Assessment of IgG antibody levels in brain tissue using ISF samples After the fusion protein penetrated the blood-brain barrier, the amount of the fusion protein present in a form not bound to the brain parenchyma (the fusion protein present in the ISF sample) was detected, and then recovery was performed on the detected value to evaluate the amount of the fusion protein flowing from the blood vessels to the brain parenchyma, i.e., the amount of the fusion protein permeating the blood-brain barrier. Specifically, after anesthetizing C57BL / 6 mice, the skin of the mouse head was incised, and a perforation was formed in the skull adjacent to the hippocampus region using a drill. Then, a guide cannula was inserted into the perforation in the skull and fixed with resin. The incised skin was sutured, and the perforation area in the skull was not exposed to the outside, and the mouse was allowed to recover for 2 weeks. Then, a formulation containing 20 mg / kg of the fusion protein (F3#01) was intravenously administered to the recovered mouse via the tail vein.

[0125] After 4 hours, 1 day, or 4 days had elapsed since the intravenous administration of the fusion protein to the mouse, the mouse was anesthetized and an activated probe for detecting IgG1 antibodies was inserted into the mouse's probe guide cannula. Then, while flowing cerebrospinal fluid (CSF) containing BSA at a constant rate, an interstitial fluid (ISF) sample containing the fusion protein was collected and stored at -20°C. The level of IgG1 antibodies in the ISF sample was then measured using a human IgG1 ELISA kit. Specifically, STD (standard) and sample (SPL) were prepared by diluting them to their respective concentrations using NS sample buffer, and then 50 μL of each of them was added to each well. Then, 50 μL of Ab Cocktail included in the ELISA kit was added to each well and incubated at 4°C. After that, the plate was washed, and 100 μL of TMB substrate was added to each well. Next, using a microplate reader, when the OD value of STD1 reached 1.0 at a wavelength of 600 nm, a stop solution was added and the value at a wavelength of 450 nm was measured. As a control group, a group in which only IgG1 antibody was administered intravenously was used.

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

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

[0128] The real-time transcytosis change on the first day after injection of the fusion protein according to the embodiment was observed in real time for 20 minutes in three ROIs. As shown in FIG. 7 and FIG. 8, the concentration change of the fusion protein in the blood vessel (ROI1) was not observed, and the fusion protein in the blood vessel wall (ROI2) where the clusters existed formed clusters for 10 minutes, then moved to the outside of the blood vessel, and the concentration of the fusion protein was observed to be in an equilibrium state from the moment when the clusters were no longer observed (>10 minutes). In addition, the concentration change of the fusion protein in the extravascular region (ROI3) was observed to increase as the fusion protein clusters first observed in the blood vessel wall (ROI2) reached the extravascular region about 6 minutes later, and then reached a maximum concentration at about 12 minutes. From the above results, it was found that the transcytosis of the fusion protein occurs through clustering near the blood-brain barrier, and the fusion protein is released to the outside of the blood vessel within a few minutes.

[0129] 1-3. Functionality evaluation based on valence change of binding moiety The fusion proteins F3#01, F1#01, and F5#01 were formulated at 20 mg / kg each and then intravenously administered to C57BL / 6 mice via the tail vein, and the level of IgG1 antibody in the brain tissue was measured four days later in the same manner as in Experimental Example 1-2 (1). A standard curve was then obtained using four-parameter logistic regression, and the concentration of IgG1 antibody present in the sample (SPL) was calculated from the standard curve. In addition, in order to observe the antibody transmission state inside the cerebral blood vessels, the brain of the animal was removed one day after intravenous administration of the fusion protein. After anesthetizing the animal, the animal's thoracic 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 from the body. Then, 4% paraformaldehyde (PFA) was injected to fix the cells, and the skull was incised to extract the brain. The extracted brain was stored in 4% PFA for one day, further fixed, and then stored in 30% sucrose solution for three days to prevent cell destruction during brain section preparation. The mouse brain stored in 30% sucrose solution for three days was placed in a brain section preparation frame, injected with optimal cutting temperature compound solution, and stored at -60°C to cool the brain. For the cooled brain sample, a micro-cryostat was used to prepare 40 μm-thick brain sections, and each brain section was used for immunostaining. After that, transferrin receptor antibody and antibody against the fusion protein were used for immunostaining, and the sections were photographed and confirmed with a confocal microscope.

[0130] As a result, as shown in Figure 9, the IgG1 antibody level in the brain tissue 4 days after intravenous administration was quantified and compared with the control group. As a result, it was found that the IgG1 antibody level in the brain tissue was increased by about 80 times in the tetravalent fusion protein administration group in which a binding moiety was linked to each of the four regions of the heavy chain C-terminus and the light chain C-terminus, i.e., the F3#01 administration group, compared to the control group. However, the IgG1 antibody level in the bivalent fusion protein administration groups in which a binding moiety was linked to two regions of the heavy chain C-terminus or the light chain C-terminus, i.e., the F1#01 administration group and the F5#01 administration group, 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 and F5#01 administration groups, whereas binding of the fusion protein to the transferrin receptor was confirmed in the cerebral blood vessels in the F3#01 administration group. Quantification of these results showed a significant difference in the levels of the fusion protein present in the area where the transferrin receptor is located.

[0132] 1-4. Evaluation of interaction between fusion protein and transferrin receptor Under the premise that the high permeability to the blood-brain barrier confirmed in Experimental Example 1-2 is due to the interaction between the binding moiety and the transferrin receptor present in the brain tissue, a 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 to which the fusion protein and the transferrin receptor (TfR) were both added was prepared as an experimental group, and the control groups were (1) a group to which only native TfR was added, (2) a group to which only an IgG1 antibody not containing a binding moiety was added, and (3) a group to which an IgG1 antibody not containing a binding moiety and TfR were both added. All samples were quantified at 15μM using a buffer solution of 50mM Tris, 150mM NaCl, pH 7.4, and the group to which fusion protein / IgG1 antibody and TfR were added was mixed at a molar ratio (1:2) and reacted at 4℃ for 1 hour. Then, all samples were diluted to the appropriate concentration and TEM grid sampling was performed. For the grid for electron microscope specimens, a 400 mesh F / C Cu grid was used, and 10μl of the prepared sample was poured onto the grid and reacted for 1 minute, and then removed using filter paper. Then, 10μl of 2% uranyl acetate solution was poured onto the grid, and the remaining solution was removed using filter paper. The grid was also exposed to room temperature for about 12 hours to remove all 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, in the group to which both the IgG1 antibody not containing a binding moiety and TfR were added (FIG. 12(c)), each component was relatively homogeneously distributed, but no interaction between them was observed, as in the groups to which only one of the native TfR and the IgG1 antibody not containing a binding moiety was added (FIG. 12(a) and (b)). In addition, in the group to which the fusion protein according to one embodiment and the native TfR were added (FIG. 12(d)), a complex was formed by interaction with the transferrin receptor. In addition, when inferred from such experimental results, as shown in FIG. 13, it was found that each of the tetravalent binding moieties according to one embodiment interacts with the helical region of the transferrin receptor to form a complex. In particular, when considering the experimental results in Experimental Examples 1 to 3 (see FIG. 9), the experimental results in this embodiment indicate that the interaction within the complex can be a major factor in improving permeability to the blood-brain barrier.

[0134] 1-5. Evaluation of selective delivery to brain tissue In this experimental example, the organ-specific distribution level of IgG1 antibody 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, and a control group was used in which only the IgG1 antibody was administered intravenously. After one or four days had elapsed, the IgG1 antibody levels in a total of six organs (brain, lung, spleen, kidney, liver, muscle) were measured and compared using the same method as in (1) of the experimental example 1-2 above.

[0135] As a result, the distribution level of IgG1 antibody in each organ one day after intravenous administration of the cerebrovascular barrier-permeable fusion protein F3#01 is shown in FIG. 14, and the distribution level of IgG1 antibody in each organ four days after intravenous administration of the cerebrovascular barrier-permeable fusion protein F3#01 is shown in FIG. 15. That is, the IgG1 antibody administered intravenously as the control group was observed to be distributed most highly in the lungs among the organs, and its distribution in the brain tissue was relatively low, whereas the F3#01 administration group was confirmed to have a very high level of IgG1 antibody distributed in the brain tissue compared to other organs. From the above results, it was found that the fusion protein including the functional structure according to one embodiment can contribute to reducing the side effects of antibody-based drugs by selectively distributing to the brain tissue when administered intravenously, thereby reducing the distribution of IgG antibody in tissues other than the brain tissue.

[0136] 1-6. Evaluation of the effect of reducing side effects by selective delivery to brain tissue (1) Evaluation of reticulocyte levels The present invention is to confirm the effect of administration of the fusion protein F3#01 permeable to the blood-brain barrier according to one embodiment on a reticulocyte in a cell expressing a large amount of transferrin receptor. Specifically, a preparation containing 20 mg / kg or 50 mg / kg of the fusion protein F3#01 was intravenously administered to a C57BL / 6 mouse via the tail vein. After intravenous administration of the fusion protein to the mouse, 50 μl of blood sample was obtained from the blood vessel inside the eye of the mouse using a heparinized capillary tube. Then, 30 μl of the blood sample was mixed with 100 μl of new methylene blue solution, and the mixed solution was left at room temperature for a certain period of time. Then, the stained blood sample was smeared on a slide glass and observed under a microscope to calculate the ratio (%) of reticulocytes to the total red blood cells. As a control, a group to which IgG1 antibody was administered intravenously was used.

[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 ratio of reticulocytes to total red blood cells was at a similar level to that of the control group.

[0138] (2) Pharmacokinetic evaluation After administration of the cerebrovascular barrier permeable fusion protein F3#01 according to one embodiment to mice, pharmacokinetic evaluation in plasma was performed. Specifically, a preparation containing 20 mg / kg of the fusion protein F3#01 was administered intravenously to the mice via the tail vein. After intravenous administration of the fusion protein to the mice, 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. After that, the plasma samples were separated by centrifugation, and then pretreatment for liquid chromatography mass spectrometry (LC-MS) was performed, followed by LC-MS analysis of the samples. As a control group, a group to which an IgG1 antibody was administered intravenously was used.

[0139] As a result, as shown in FIG. 17, the plasma PK profile of the group administered 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 In order to verify whether the above experimental results were due to selective transfer to brain tissue, i.e., non-binding between reticulocytes containing transferrin receptors and other organ tissues, the blood-to-plasma ratio (peak area ratio of blood supernatant / peak area ratio of plasma) was calculated. Specifically, the cerebrovascular barrier permeable fusion protein F3#01 according to one embodiment was added to the plasma sample and the blood sample to a final concentration of 40 μg / mL, and the sample was left at room temperature for 30 minutes. After that, the blood sample was centrifuged to obtain a blood supernatant separately. The above plasma and blood supernatant samples were mixed with a PBS solution containing a surfactant and magnetic beads, and the mixture was cultured, and the culture was washed twice with PBS containing a surfactant. RapiGest surfactant and dithiothreitol were added thereto, and the mixture was cultured at 60° C. for 50 minutes, and then left at room temperature for 10 minutes. Then, 1) iodoacetic acid was added thereto, followed by incubation in the dark at room temperature for 30 minutes, 2) trypsin was added thereto, followed by incubation at 60°C for 24 hours, and 3) HCl was added thereto, followed by incubation at 37°C for 30 minutes, in that order. The culture was then centrifuged to obtain a supernatant, after which trypsin was added and 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 was used as a control group.

[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 at a similar level to that of the control group, but it was found that the above experimental result was due to the fact that the reticulocytes containing the transferrin receptor were not bound to the fusion protein. Also, 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, confirming selective delivery to brain tissue.

[0142] 1-7. Evaluation of the functionality of fusion proteins containing repeated binding moieties In this experimental example, the objective of the present invention is to confirm whether the functionality of the fusion protein described above can be maintained even when the helical domain-binding moiety of the transferrin receptor is modified so as to be repeated.To this end, a fusion protein having a structure in which a total of four binding moieties are 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 binding moiety in which the moiety of SEQ ID NO:3 was repeated twice as the helical domain-binding moiety of the transferrin receptor, 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 level of IgG1 antibody in the brain tissue of the F3'#01-administered group was observed to be higher than that of the control group up to 7 days after administration. Specifically, the F3'#01-administered group showed a high level of transmission, ranging from about 18 to about 160 times that of the control group.

[0145] (2) Evaluation of selective delivery to brain tissue The distribution level of IgG1 antibody in each organ 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 in the F3'#01 administration group, a significantly higher level of IgG1 antibodies was 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, in the same manner as before.

[0148] 1-8. Confirmation of specific expression of transferrin receptor in brain tissue It was experimentally confirmed that the 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 remarkably enhances the delivery of IgG1 antibody to brain tissue due to its high permeability to the blood-brain barrier as confirmed in the above 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, the present 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 saline was perfused to remove the blood. After perfusion was completed, brain tissue was removed from the mice, and the brain vessel, parenchyma, and choroidplexus were separated from the brain tissue, and total protein was extracted using RIPA buffer. For other organs such as the liver, lung, kidney, and spleen, a small amount of tissue was taken and total protein was extracted using RIPA buffer. The extracted protein was mixed with SDS-PAGE sample buffer, denatured, loaded on SDS-PAGE gel, and transferred to a PVDF membrane. Then, anti-TfR antibody and β-actin antibody were used as primary antibodies to confirm 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 abundantly expressed in cerebral blood vessels and blood cells, and a significant level of transferrin receptor was also expressed in the spleen. The above experimental results indicate that the selective delivery of the fusion protein according to one embodiment to brain tissue is not initiated simply by the quantitative difference in transferrin receptor expression in each organ, but is mainly due to the interaction between the transferrin receptor having a specific expression pattern in cerebral blood vessels and the binding moiety according to one embodiment, or a functional structure capable of forming such an interaction.

[0151] (2) Comparison of expression patterns of transferrin receptor clusters Blood was collected from the orbital vein or abdominal vein of C57BL / 6 mice, and then saline was perfused to remove the blood. The brain tissue was perfused with saline and 4% paraformaldehyde in sequence to prepare brain tissue samples for brain section staining using immunohistochemistry. Then, for brain section preparation, the brain tissue was placed on a frame, and tissue freezing medium was added thereto so that the brain tissue was fully immersed, and the frame was stored in a deep freezer at -70°C for one day. The frozen brain block was sectioned into 40 μm thick slices using a cryostat. The slices were stored in PBS solution containing 0.1% sodium azide until use.

[0152] Then, the tissue slices were added to a 24-well plate and reacted with 500 μl of PBS solution containing 0.5% Triton-X100 at room temperature for 20 minutes. Then, the solution was replaced with PBS solution and mixed using a plate stirrer. Then, the tissue slices were added to a PBS solution containing 5% BSA and 0.1% Triton-X100, reacted at room temperature for 2 hours, and washed three times with PBS solution. Transferrin receptor antibody and CD31 antibody were added thereto, and the reaction was induced overnight at 4°C, and then washed three times with PBS solution. Then, anti-rat Alexa488 and anti-goat Alexa568 were added thereto, reacted at room temperature for 2 hours, and then washed three times with PBS solution. Next, PBS solution containing DAPI was added and reacted at room temperature for 10 minutes. The tissue sections treated as described above were placed on a slide glass, and 100 μl of mounting solution was applied to the slide glass. Then, the tissue sections treated as described above were covered with a cover glass, which was then sealed.

[0153] Fluorescent images of the stained samples were obtained using a confocal microscope. Images were taken with LAS-X software, and the light source intensity was adjusted to 4% UV intensity and 70% white laser power (488:15%, 568:10%). Magnification was performed using a 40X objective lens with a pixel resolution of 284 nm, and images were obtained with a total thickness of 20 μm, with each tissue section being 1 μm thick. Ultra-high resolution fluorescent images of the stained samples were also obtained using a STED confocal microscope. Images were taken with LAS-X software, and the light source intensity was adjusted to 6% UV intensity and 70% white laser power (488:15%, 568:10%). Magnification was performed using a 100X objective lens with 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 of 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 fluorescence image analysis and STED confocal fluorescence image analysis, the sigma value was set to 1.0, Gaussian blur was performed, 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, background subtraction was performed using the rolling ball algorithm using a plug-in in Image J software. The green signal, which is the signal of the transferrin receptor, was normalized to match the intensity histogram, and a binary image was obtained using the local thresholding method. The masked multiple transferrin receptor clusters were analyzed using the analyze particle function, with the minimum pixel unit of 2,500 nm. 2 Only pixels with an area of ​​100 or more were selected to obtain a cluster size distribution map. The distribution map was prepared by sampling transferrin receptor distribution maps observed in a total of 40 cells per organ for 10 cells and tissues in each organ in a total of 4 C57BL / 6 mice. In addition, the distribution map results obtained with Image J were converted into a histogram using MATLAB, and a trend line of the histogram was obtained using a smooth function.

[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. In addition, as a result of comparing the quantitative distribution of the size of transferrin receptor clusters expressed in brain tissue blood vessels, reticulocytes, lung, liver, and spleen, it was confirmed that relatively small transferrin receptor clusters were densely packed in the blood vessel region of brain tissue, unlike other tissues, as shown in Figure 24.

[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, the transferrin receptor clusters were imaged using the same method as in Experimental Example 1-8(2), and the masked transferrin receptor clusters were then analyzed using the analyze particle function to obtain a minimum pixel unit of 2,500 nm. 2 The green signal was extracted from pixels having a width of 1000 pixels or more. The extracted image was then converted into a mask image, and the mask image obtained by Image J was multiplied by the image obtained by background subtraction using MATLAB to calculate the intensity of the green signal in the cluster mask.

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

[0158] B. Quantitative assessment of confluency via immunoblotting and image analysis 50,000 cells were collected from the cerebrovascular tissue and reticulocytes of C57BL / 6 mice by FACS, and total protein was extracted using RIPA buffer. The extracted protein was mixed with sample buffer, denatured, loaded on SDS-PAGE gel, and transferred to a PVDF membrane. Anti-TfR antibody was then used as the primary antibody to confirm each protein band on the membrane, and their intensity was measured using Image lab software (Biorad). The intensity of the transferrin receptor protein band measured in the two cells was quantified as the number of transferrin receptors per single cell using a standard curve of recombinant transferrin receptor bands. Next, in order to calculate the transferrin receptor density per single cell, the average diameter and number of transferrin receptor clusters measured by the STED confocal microscope described above were used to calculate the total area of ​​the transferrin receptor clusters in a single cell, and the quantified number of receptors was 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 divided by Avogadro's number to convert it to a molar concentration per area. At this time, since the transferrin receptor exists in a dimer, the calculated molar concentration was converted to half the value. The distance between transferrin receptors present inside the cluster was calculated as the distance from the center point of the dimer of the transferrin receptor to the center point of the adjacent receptor under the premise that the receptors are packed in a square lattice shape, and as shown in FIG. 26, the distance from the center of the transferrin receptor to the surface is determined in the case of the long axis and the short axis. That is, when multiple transferrin receptors were arranged / aligned in a lattice form, the surface distance between a receptor and an adjacent receptor around the long axis of the dimer was calculated as the minimum value (min) and the surface distance between a receptor and an adjacent receptor around the short axis of the dimer as the maximum value (max), and then the average value (average) of these was evaluated.

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

[0160] Taken together, the above experimental results indicate that the distribution pattern of specific transferrin receptor clusters in the blood vessel region of brain tissue indicates that the tetravalent binding moiety according to one embodiment can affect the interaction 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. It was also found that the specific expression pattern of transferrin receptor clusters inducing such specific interaction in brain tissue acts as a factor enabling high permeability through the blood-brain barrier and selective absorption of IgG1 antibodies in brain tissue.

[0161] Experimental Example 2. Evaluation of the functionality of the cerebrovascular barrier permeability fusion proteins F3#02 and F3#41 In this experimental example, in 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, whether or not the functionality of the above-mentioned fusion protein can be exhibited even when the helical region-binding moiety of the transferrin receptor is 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 binding to the helical domain of the transferrin receptor and the transduction level mediated by the binding to the helical domain in human brain endothelial cells.

[0163] (1) Confirmation of binding between the helical domain 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 protein prepared in the above example to the transferrin receptor (TfR) was confirmed through docking simulation. The structure of the binding moiety was modeled using the RosettaRelax program, and the position expected to interact with the transferrin receptor helical region was modeled through structural information and thermodynamic calculation. Then, the position of the binding moiety was arbitrarily changed, and the interaction with the helical region was calculated, and a docking simulation was performed using the RosettaDocking program to find the most stable position. 20,000 simulations were performed for each sequence number, and the resulting data was analyzed based on the homology to the initial modeling structure and the thermodynamic structural stability.

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

[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 in the same manner as in Experimental Example 1-1(2) to see whether they could be delivered into hCMEC / D3 cells, which are human brain endothelial cells that constitute the human blood-brain barrier, through interaction with the transferrin receptor. As a control group, a group to which only the IgG1 antibody was intravenously administered was used.

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

[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 the blood-brain barrier-permeable fusion proteins F3#02 to F3#41 according to one embodiment was evaluated 2 or 4 days after intravenous administration in the same manner as in Experimental Example 1-2(1). As a control group, a group to which only the IgG1 antibody was intravenously administered was used.

[0168] As a result, as shown in Figures 41 to 45, it was confirmed that the IgG1 antibody level in brain tissue was significantly increased in the groups administered with the fusion proteins F3#02 to F3#24 of Examples 2 to 24, which were prepared using the first binding moiety group having different amino acid sequences while maintaining the binding property to the helical region, compared to the control group. Also, as shown in Figures 46 to 49, it was confirmed that the IgG1 antibody level in brain tissue was significantly increased in the groups administered with the fusion proteins F3#025 to F3#41 of Examples 25 to 41, which were prepared using the second binding moiety group in which a part of the amino acid sequence was substituted, inserted or deleted while maintaining a certain level of sequence identity with the binding moiety of SEQ ID NO:3, compared to the control group.

[0169] Taking the above experimental results into consideration, it has been 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 having effective binding affinity to the helical domain of the transferrin receptor.

[0170] Experimental Example 3. Functional evaluation of blood-brain barrier permeability fusion protein 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 IgG1 antibody absorption into brain tissue following intravenous administration of the fusion proteins were evaluated 2 or 4 days after intravenous administration in the same manner as in Experimental Example 1-2(1), using 1) a fusion protein (F3) in which a transferrin receptor helical region-binding moiety is tetravalently linked to the C-terminal region of an IgG1 antibody light chain and the C-terminal region of the heavy chain, 2) a fusion protein (F1) in which a transferrin receptor helical region-binding moiety is bivalently linked to the C-terminal region of an IgG1 antibody heavy chain, and 3) a fusion protein (F5) in which a transferrin receptor helical region-binding moiety is bivalently linked to the C-terminal region of an IgG1 antibody light chain.

[0172] As a result, as shown in Figures 50 to 59, the IgG1 antibody levels in the brain tissue were quantified and compared. As a result, the groups administered with fusion proteins having binding moieties linked to each of the four regions at the C-terminus of the heavy chain and the C-terminus of 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 having binding moieties linked to two regions at the C-terminus of the heavy chain. It was found that the IgG1 antibody level in brain tissue was significantly enhanced compared to the groups administered 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 together, it was confirmed 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 exert its inherent functionality.

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

[0175] Specifically, 100μl of 0.25μg / ml human PD-L1 protein was added to a 96-well plate, and the protein was coated on the plate at 4℃ for 16 hours. The coated plate was then washed 4 times with PBS-T, and 200μl of blocking buffer was added to the same well, which was then incubated at room temperature for 2 hours to prevent non-specific binding of the antibody. The plate was then washed 4 times with PBS-T, and 4nM of fusion protein (F3#01, F3'#01) was added to the well, which was further incubated at room temperature for 2 hours. Then, 100μl of the detection antibody anti-human IgG FCγ HRP conjugated antibody was added to each sample well, which was then 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, which was then allowed to react at room temperature for 10 minutes. Then, 100 μl of stop solution was added to stop the reaction, and the absorbance of each well was measured at a wavelength of 450 nm using a microplate reader. In this experimental example, the IgG1 antibody was an anti-PD-L1 antibody and transferrin receptor helical domain-binding moiety #01, and the control group was a group to which only the IgG1 antibody was added.

[0176] As a result, as shown in Table 2 and Figure 60, it was found 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 at a similar level to that of the control group. The above results suggest that the blood-brain barrier permeation and selective delivery to brain tissue by the fusion protein according to one embodiment is achieved while maintaining the inherent biological activity of the IgG antibody.

[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 used to confirm whether the functionality of the fusion protein can be exhibited even when the IgG1 antibody is modified.

[0179] 5-1. Anti-Tau IgG1 antibody In the fusion protein (F3) in which the helical domain binding moiety of the transferrin receptor is tetravalently linked to the C-terminal domain of the light chain and the C-terminal domain of the heavy chain in an IgG1 antibody, the IgG1 antibody was an IgG1 antibody (anti-tau) that binds to tau (Tau), and the helical domain binding moiety of the transferrin receptor was binding moiety #25, #27, or #36, and a cerebrovascular barrier permeable fusion protein was prepared in the same manner as in Example 1 (F3#25-tau (Tau), F3#27-tau (Tau), F3#36-tau (Tau)). Then, the level of absorption of the IgG1 antibody into ISF by intravenous administration of the prepared fusion protein was evaluated in the same manner as in Experimental Example 1-2 (2). As a control group, a group to which only the IgG1 antibody was added was used.

[0180] As a result, as shown in Figures 61 and 62, it was found that, while maintaining the above-mentioned functional structure of the present application, 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 is the fusion partner of the helical domain-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 moiety of the transferrin receptor is tetravalently linked to the C-terminal domain of the light chain and the C-terminal domain of the heavy chain in an IgG1 antibody, an IgG1 antibody that binds to PD1 (anti-PD1) is used as the IgG1 antibody, and binding moiety #25 is used as the helical domain-binding moiety of the transferrin receptor, and a fusion protein permeable to the blood-brain barrier was prepared in the same manner as in Example 1 (F3#25-PD1). Then, the level of absorption of the IgG1 antibody into brain tissue by intravenous administration of the prepared fusion protein was evaluated in the same manner as in Experimental Example 1-2 (1). As a control group, a group to which only the IgG1 antibody was added was used.

[0182] As a result, as shown in Figure 63, it was found that when the type of IgG1 antibody, which 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 application, 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 moiety of the transferrin receptor is tetravalently linked to the C-terminal domain of the light chain and the C-terminal domain of the heavy chain in 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, and a fusion protein permeable to the blood-brain barrier was prepared in the same manner as in Example 1 (F3#25-HER2). Then, the level of absorption of the IgG1 antibody into brain tissue by intravenous administration of the prepared fusion protein was evaluated in the same manner as in Experimental Example 1-2 (1). As a control group, a group to which only the IgG1 antibody was added was used.

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

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

[0186] As a result, as shown in Figure 65, it was found that, while the functional structure of the present application was maintained as described above, the level of the anti-Aβ IgG1 antibody in brain tissue was increased as compared to the control group, even when the type of IgG1 antibody, which is 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 antibody 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 a person having ordinary skill in the art to which the present invention pertains will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential features of the present invention. Therefore, it should be understood that the above-described embodiments are illustrative in all respects and are not limiting.

Claims

1. An IgG antibody; A blood-brain barrier permeable fusion protein comprising a tetravalent transferrin receptor (TfR) helical domain-binding moiety linked to the C-terminal domain of the light chain and the C-terminal domain of the heavy chain of the IgG antibody.

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

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 moiety has binding affinity with at least one or more amino acids selected from the helical region of SEQ ID NO:2 in the transferrin receptor.

5. The fusion protein of claim 1 , wherein each of the multiple binding moieties is the same or different.

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

7. 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.

8. A polynucleotide encoding a fusion protein according to any one of claims 1 to 7.

9. A vector comprising the polynucleotide of claim 8.

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

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