Fusions containing anti-tau antibodies and peptides and uses thereof

Abstract

The present invention relates to a fusion comprising an anti-tau antibody or its antigen-binding fragment and a blood-brain barrier receptor-binding peptide, and uses thereof. The fusion of the present invention can cross the blood-brain barrier and specifically bind to tau protein, thereby effectively preventing or treating tau protein-mediated neurodegenerative diseases.

Description

[Technical Field]

[0001] This application claims priority to Korean Patent Application No. 2022-0171003, filed on December 8, 2022, the disclosure of which is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing, which has been submitted electronically in XML format and is incorporated herein by reference in its entirety. A copy of said Sequence Listing, created on November 28, 2023, is named KC23138-SEQ.xml and is 44.6 kilobytes in size.

[0003] The present invention relates to a fusion comprising an anti-tau antibody and a peptide and uses thereof. In particular, the present invention relates to a fusion having improved permeability through the blood-brain barrier and uses thereof for the prevention or treatment of neurodegenerative diseases. [Background technology]

[0004] Tau protein stabilizes microtubules, proteins that transport cellular materials. It exists in six isoforms in the human body and is abundant in neurons of the central nervous system. When tau protein is mutated, it becomes hyperphosphorylated, leading to the abnormal accumulation of neurofibrillary tangles (NFTs) in neurons, causing neurodegenerative diseases such as dementia, Parkinson's disease, and tauopathy. Accordingly, the development of therapeutic drugs targeting tau protein has been reported (Korean Patent Application No. 10-2020-0086341).

[0005] On the other hand, the brain delivery of neurological therapeutics, including large-molecule biopharmaceuticals such as antibodies and small-molecule drugs with low brain delivery rates, is severely restricted by the extensive and impermeable blood-brain barrier (BBB), along with other cellular components of the neurovascular unit. Previous studies have shown that only a small fraction (approximately 0.1%) of IgG administered into the bloodstream can penetrate into the central nervous system compartment (Felgenhauer, Klin.Wschr. 52: 1158-1164 (1974)).

[0006] Therefore, there is a need for tau-targeting drugs that can be effectively transported across the BBB to the brain. Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to solve all of the above problems.

[0008] Another object of the present invention is to provide a fusion comprising an anti-tau antibody or antigen-binding fragment thereof that specifically binds to tau protein and a blood-brain barrier receptor-binding peptide.

[0009] Another object of the present invention is to provide polynucleotides encoding said fusions, expression vectors and host cells.

[0010] Another object of the present invention is to provide a method for producing said fusion construct.

[0011] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating a neurodegenerative disease, comprising the fusion protein.

[0012] Another object of the present invention is to provide a method for preventing or treating a neurodegenerative disease, comprising the step of administering the fusion to a subject in need thereof.

[0013] The objectives of the present invention are not limited to the above-mentioned objectives, but will become clearer from the following description and can be realized by the means and combinations thereof described in the claims. [Means for solving the problem]

[0014] A typical configuration of the present invention to achieve the above object is as follows.

[0015] According to one aspect of the present invention, there is provided a fusion comprising (i) an anti-tau antibody or an antigen-binding fragment thereof that specifically binds to tau protein, and (ii) a blood-brain barrier (BBB) ​​receptor-binding peptide.

[0016] In one embodiment, a fusion is provided in which an anti-tau antibody is directly coupled to a blood-brain barrier receptor-binding peptide without a linker.

[0017] In other embodiments, the blood-brain barrier receptor-binding peptide may comprise one or more selected from the group consisting of Angiopep-2 (APEP), RVG29, and TfR-binding peptide (TfR).

[0018] In other embodiments, the APEP may comprise, consist of, or essentially contain the amino acid sequence of SEQ ID NO:20.

[0019] In other embodiments, RVG29 can comprise, consist of, or essentially contain the amino acid sequence of SEQ ID NO:22.

[0020] In other embodiments, the TfR can comprise, consist of, or essentially contain the amino acid sequence of SEQ ID NO:24.

[0021] In other embodiments, the anti-tau antibody or antigen-binding fragment thereof may comprise a heavy chain variable region (VH) comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 2, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 4, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6; and a light chain variable region (VL) comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 10, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 12, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 14.

[0022] In one embodiment, the anti-tau antibody or antigen-binding fragment thereof can bind to an epitope comprising amino acids 275 to 286 of the wild-type tau protein of SEQ ID NO: 25, in which amino acid 280 is acetylated.

[0023] In one embodiment, the anti-tau antibody or antigen-binding fragment thereof can comprise a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:8 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:16.

[0024] In one embodiment, the anti-tau antibody or antigen-binding fragment thereof can comprise (i) a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, and SEQ ID NO:33, and (ii) a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:16, SEQ ID NO:35, SEQ ID NO:37, and SEQ ID NO:39.

[0025] In another embodiment, a fusion is provided in which the anti-tau antibody comprises a light chain and a heavy chain, and the blood-brain barrier receptor-binding peptide is linked to the Fc region of the heavy chain.

[0026] In another embodiment, the fusion may be a fusion that crosses the blood-brain barrier and specifically binds to tau protein.

[0027] In other embodiments, the anti-tau antibody or antigen-binding fragment thereof may be any one selected from the group consisting of a full-length antibody, Fab, scFv, F(ab')2, and Fv.

[0028] In other embodiments, the anti-tau antibody may be an IgG antibody.

[0029] According to another aspect of the present invention, there are provided polynucleotides encoding the fusions disclosed herein, or the heavy and / or light chains thereof.

[0030] According to another aspect of the present invention, there is provided an expression vector comprising the polynucleotide.

[0031] According to another aspect of the present invention, there is provided a host cell comprising a polynucleotide encoding a fusion as disclosed herein or an expression vector comprising said polynucleotide.

[0032] According to another aspect of the present invention, there is provided a method of producing a fusion comprising culturing a host cell comprising a polynucleotide encoding the fusion disclosed herein or the light chain polypeptide and the heavy chain polypeptide comprised in the fusion.

[0033] According to another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating a neurodegenerative disease, comprising the fusion disclosed herein.

[0034] According to another aspect of the present invention, there is provided a method for preventing or treating a neurodegenerative disease, comprising the step of administering an effective amount of the fusion or pharmaceutical composition disclosed herein to a subject in need thereof.

[0035] In one embodiment, the neurodegenerative disease is a tauopathy, primary age-related tauopathy, chronic traumatic encephalopathy, progressive supranuclear palsy, corticobasal degeneration, frontotemporal dementia, Lytico-Bodig disease, Parkinson's disease, subacute sclerosing meningitis, lead encephalopathy, tuberous sclerosis, ganglioglioma, gangliocytoma, meningioangiomatosis, subacute sclerosing panencephalitis, panencephalitis, pantothenate kinase-associated neurodegeneration (Hallervorden-Spatz disease), and lipofuscinosis.

[0036] In other embodiments, the tauopathy is Alzheimer's disease, progressive supranuclear palsy, corticobasal degeneration, Pick's disease, a group of related disorders collectively known as frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), amyotrophic lateral sclerosis, Creutzfeldt-Jakob disease, dementia pugilistica, Gerstmann-Straussler-Scheinker syndrome, Lewy body disease, chronic traumatic encephalopathy, and Huntington's disease. It may be selected from the group consisting of:

[0037] In other embodiments, the fusions or pharmaceutical compositions disclosed herein may be administered via any route of administration selected from the group consisting of intramuscular, intravenous, intraarterial, intraperitoneal, transdermal, subcutaneous, intrathecal, intracerebral, intraventricular, intrapulmonary, or intranasal administration. [Effects of the Invention]

[0038] As disclosed herein, fusions prepared by coupling an anti-tau antibody with a blood-brain barrier receptor-binding peptide exhibit excellent effects in allowing the anti-tau antibody to cross the BBB. In particular, when the anti-tau antibody is directly linked to the blood-brain barrier receptor-binding peptide without a linker, it exhibits superior BBB penetration. Therefore, the fusion polypeptides of the present invention can inhibit abnormal aggregation of tau protein and effectively prevent or treat neurodegenerative diseases. [Brief explanation of the drawings]

[0039] [Figure 1] 1A shows a schematic diagram of a fusion protein according to one embodiment of the present invention, in which an anti-tau antibody is directly bound to a blood-brain barrier receptor-binding peptide without a linker, and FIG. 1B shows a fusion protein in which an anti-tau antibody is bound to a blood-brain barrier receptor-binding peptide via a linker. [Figure 2] 1 shows the pcDNA3.1(+) vector for cloning three blood-brain barrier (BBB) ​​receptor-binding peptides according to one embodiment of the present invention. Herein, the term "blood-brain barrier receptor-binding peptide" is used interchangeably with "brain-localizing peptide (BPP)" and is referred to as BPP in the drawings. [Figure 3a] This diagram shows an expression vector (pcDNA3.1(+)) into which the heavy and light chain genes of the H1 antibody (human anti-tau antibody) have been inserted. Figure 3a shows an expression vector into which the heavy chain gene of the H1 antibody has been inserted before the brain-localizing peptide (or linker / brain-localizing peptide) using BamHI and XhoI restriction enzymes. [Figure 3b] FIG. 3b shows an expression vector (pcDNA3.1(+)) into which the heavy and light chain genes of the H1 antibody (human anti-tau antibody) have been inserted. FIG. 3b shows an expression vector into which the light chain gene of the H1 antibody has been inserted using HindIII and XbaI restriction enzymes. [Figure 4] FIG. 1 shows a protocol for differentiating induced pluripotent stem cells ("iPSCs") into brain microvascular endothelial cells ("BMECs"). [Figure 5] This figure shows the expression of BBB-related proteins confirmed using a fluorescence microscope (magnification: 400x) on days 6 to 10 after induction of differentiation into iBMECs (Scale bar: 100 μm). [Figure 6]

[0033] Figure 1 shows the results of measuring the TEER values ​​of transwells to which one antibody and six fusions according to an example of the present invention were administered, where L4 represents the linker and the unit is Ω×cm. [Figure 7a] FIG. showing the results of the total Papp of the H1 antibody and three antibody-brain-transporting peptide fusions (H1-APEP, H1-RVG29, and H1-TfR) according to embodiments of the present invention (including batches #1-3). FIG. 7a is a graph showing the results of the total Papp, showing the average value. * indicates 0.01 < P < 0.05 for the H1 antibody, and ** indicates 0.001 < P < 0.01 for the H1 antibody. [Figure 7b] FIG. showing the results of the total Papp of the H1 antibody and three antibody-brain-transporting peptide fusions (H1-APEP, H1-RVG29, and H1-TfR) according to embodiments of the present invention (including batches #1-3). FIG. 7b is a graph showing the PAPP ratio (relative value) obtained by dividing the results of the total Papp by the Papp value of each H1 antibody, showing the average value. * indicates 0.01 < P < 0.05 for the H1 antibody, and ** indicates 0.001 < P < 0.01 for the H1 antibody. [Figure 8] FIG. showing the results of the total Papp of the H1 antibody and three antibody-linker-brain-transporting peptide fusions (H1-L4-APEP, H1-L4-RVG29, and H1-L4-TfR) according to embodiments of the present invention (including batches #1-4).

BEST MODE FOR CARRYING OUT THE INVENTION

[0040] The following detailed description of the present invention will be described with reference to specific drawings for specific embodiments in which the present invention can be implemented. However, the present invention is not limited to these, and as long as it is appropriately described, it is limited only by the appended claims over the entire scope of the matters described in the claims and their equivalents.

[0041] It should be understood that various embodiments or examples of the present invention are different from each other but do not necessarily exclude each other.

[0042] For example, specific shapes, structures, and characteristics described herein may be changed from one embodiment or example to another, or may be implemented in combination with other embodiments or examples, without departing from the spirit and scope of the invention.

[0043] The terms used to describe the present invention are to be understood in their ordinary sense unless otherwise specified, and apply to all instances of the same term used in this specification, regardless of the form or embodiment of the invention for which the term is defined.

[0044] In interpreting this specification, the following definitions will apply, and terms used in the singular will also include the plural where appropriate and vice versa.

[0045] definition The "blood-brain barrier" (BBB) ​​refers to an important biological barrier that protects the brain and is composed of cerebral vascular endothelial cells and the basement membrane, astrocytes, and pericytes that surround and support them. The BBB prevents foreign substances (molecular or small molecules such as pathogens, pigments, pharmaceuticals, and toxins) from entering the brain through tight junctions in vascular endothelial cells. The BBB in the brain, as well as the BBB in the spinal cord and the BBB in the retina, are adjacent capillary barriers in the central nervous system and are collectively referred to herein as the BBB. The BBB (choroid plexus) is composed of ependymal cells rather than capillary endothelial cells.

[0046] The term "antibody" is used broadly to encompass monoclonal antibodies (including full-length antibodies) of any isotype, such as IgG, IgM, IgA, IgD, and IgE, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), antibody fusions (e.g., fusions of an antibody with a (poly)peptide or a compound), and antibody fragments (including antigen-binding fragments). As used herein, the prefix "anti-" when used in reference to an antigen means that the antibody is reactive with that antigen. Antibodies reactive with a particular antigen can be generated by, but are not limited to, synthetic and / or recombinant methods, such as screening of recombinant antibody libraries in phage or similar vectors, or by immunization of animals with the antigen or a nucleic acid encoding the antigen. A typical IgG antibody consists of two identical heavy chains and two identical light chains linked by disulfide bonds. Each heavy and light chain contains a constant region and a variable region. The heavy chain variable region (HVR) and light chain variable region (LVR) each contain three segments called "complementarity-determining regions" (CDRs) or "hypervariable regions," which are primarily involved in binding to antigen epitopes. These are numbered sequentially from the N-terminus and are usually called CDR1, CDR2, and CDR3. The more conserved regions in the variable regions other than the CDRs are called "framework regions" (FRs). As used herein, antibodies may be, for example, animal antibodies, chimeric antibodies, humanized antibodies, or human antibodies.

[0047] The term "anti-tau antibody" refers to an antibody that can bind to tau with such affinity that it may be useful as a therapeutic agent to target tau.

[0048] The term "monoclonal antibody" is used interchangeably with "monoclonal antibody" and refers to an antibody obtained from a substantially homogeneous population of antibodies, the individual antibodies of which are identical except for natural mutations and / or post-translational modifications (e.g., isomerization, amidation) that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Because they are obtained from a substantially homogeneous population, monoclonal antibodies exhibit the characteristics of the antibody and are not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention can be produced by a variety of techniques, including hybridoma methods, recombinant DNA techniques, phage display techniques, and methods utilizing transgenic animals containing all or part of the human immunoglobulin locus.

[0049] The term "antigen-binding fragment" refers to a portion of an antibody or a polypeptide comprising the same that has the ability to specifically bind to an antigen. Unless "antibody" is specifically understood to exclude "antigen-binding fragments" in the context, "antibody" and "antigen-binding fragment" can be used interchangeably, and "antibody" can be interpreted as including "antigen-binding fragments." Examples of antigen-binding fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, triabodies, tetrabodies, cross-Fab fragments, linear antibodies, single-chain antibody molecules (e.g., scFv), and multispecific antibodies formed from antibody fragments and single-domain antibodies.

[0050] The term "Fc region," as used herein, refers to the C-terminal region of an immunoglobulin heavy chain that comprises at least a portion of a constant region (e.g., CH2, CH3, or CH2 and CH3). The term includes native sequence Fc regions and variant Fc regions.

[0051] The term "blood-brain barrier receptor-binding peptide" refers to a peptide that can selectively cross the blood-brain barrier, which restricts substance transport, by binding to a receptor present in the endothelial plasma membrane of brain capillaries. Blood-brain barrier receptor-binding peptides may be included in brain-penetrating peptides (BPPs) or cell-penetrating peptides (CPPs) because they possess the ability to cross the endothelial cells of brain capillaries. Therefore, the terms "blood-brain barrier receptor-binding peptide," "brain-penetrating peptide," "cell-penetrating peptide," "BPP," or "CPP" can be used interchangeably herein. In some embodiments, the peptide may comprise or consist of 2 to 50, specifically 8 to 40, more specifically 10 to 30, and even more specifically 12 to 29 amino acid residues.

[0052] The term "fusion" can be used interchangeably with "fusion protein" or "fusion polypeptide" and refers to a fusion polypeptide molecule comprising an immunoglobulin molecule and a brain-localizing peptide. In some embodiments, the fusion is a fusion polypeptide comprising an anti-tau antibody and a blood-brain barrier receptor-binding peptide.

[0053] The term "linker" refers to a peptide linker having a length of 1 to 100 amino acids, specifically 2 to 50 amino acids, and more specifically 5 to 30 amino acids. The linker may contain, for example, one or more amino acids selected from the group consisting of Gly, Asn, Ser, Thr, Ala, and Asp, but is not limited to these. For example, the linker may be represented by (GGGGS)n, where n is the number of repeats of the (GGGGS) unit, and may be 1 to 10, specifically 1 to 5, taking into account antibody potency. For another example, the linker may be a peptide fragment known to be capable of linking antibody fragment domains, and may have, for example, the amino acid sequence of [GGGGS]4. In one embodiment, the linker may preferably have the amino acid sequence GGGGSGGGGSGGGGSGGGSG.

[0054] The term "subject" is used interchangeably with "patient" and may refer to a mammal, such as a primate (e.g., a human), a companion animal (e.g., a dog, a cat, etc.), a livestock animal (e.g., a cow, a pig, a horse, a sheep, a goat, etc.), and a laboratory animal (e.g., a rat, a mouse, a guinea pig, etc.), in need of prevention or treatment of a tau protein-mediated neurological or neurodegenerative disease. In some embodiments, the subject is a human.

[0055] The term "treatment" generally refers to obtaining a desired pharmacological and / or physiological effect. Such an effect has a therapeutic effect in that it partially or completely cures diseases and / or the harmful effects caused by these diseases. Preferred therapeutic effects include, but are not limited to, prevention of disease onset or recurrence, amelioration of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, slowing the rate of disease progression, improvement or palliation of the disease state, and remission or improved prognosis. Preferably, "treatment" refers to medical intervention for an already existing disease or disorder.

[0056] The term "prevention" relates to prophylactic treatment, i.e., measures or procedures aimed at preventing rather than treating a disease. "Prevention" means obtaining a prophylactic pharmacological and / or physiological effect aimed at partially or completely preventing a disease or its symptoms.

[0057] The term "administration" means providing a substance to a subject to achieve a prophylactic or therapeutic purpose.

[0058] The term "therapeutically effective amount" or "effective amount" refers to the amount of an active ingredient (substance) that, when administered to a mammal or other subject for treating a disease, is sufficient to effect treatment of the disease. A "therapeutically effective amount" or "effective amount" can be determined based on factors including the subject's (preferably human) weight, sex, age, health condition, severity, drug activity, drug sensitivity, administration time, administration route and excretion rate, treatment duration, concurrently used drugs, and other factors well known in the medical field.

[0059] Fusions containing anti-tau antibodies or antigen-binding fragments thereof and blood-brain barrier receptor-binding peptides

[0060] According to one aspect of the present invention, there is provided a fusion comprising an anti-tau antibody or its antigen-binding fragment and a blood-brain barrier receptor-binding peptide. The anti-tau antibody or its antigen-binding fragment can be bound to the blood-brain barrier receptor-binding peptide directly without a linker or via a linker. Preferably, the anti-tau antibody or its binding fragment can be bound to the blood-brain barrier receptor-binding peptide directly without a linker.

[0061] In some embodiments, the anti-tau antibody or antigen-binding fragment thereof comprises an acetylated epitope of wild-type tau protein, for example, amino acids 275 to 286 of the wild-type tau protein of SEQ ID NO: 25, and amino acid 280 can specifically bind to the acetylated epitope.

[0062] In some embodiments, the anti-tau antibody or antigen-binding fragment thereof may comprise a heavy chain variable region (VH) comprising a heavy chain CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 2, a heavy chain CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 4, and a heavy chain CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 6, and a light chain variable region (VL) comprising a light chain CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 10, a light chain CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 12, and a light chain CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 14.

[0063] In some embodiments, the anti-tau antibody or antigen-binding fragment thereof may comprise a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, and SEQ ID NO:33, and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:16, SEQ ID NO:35, SEQ ID NO:37, and SEQ ID NO:39.

[0064] In some embodiments, the anti-tau antibody or antigen-binding fragment thereof may be any one selected from the group consisting of:

[0065] (1) an anti-tau antibody or antigen-binding fragment thereof, comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 8 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 16; (2) an anti-tau antibody or antigen-binding fragment thereof, comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 27 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 35; (3) an anti-tau antibody or antigen-binding fragment thereof, comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 27 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 37; (4) An anti-tau antibody or antigen-binding fragment thereof, comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 27 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 39. (5) An anti-tau antibody or antigen-binding fragment thereof, comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 29 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 35. (6) An anti-tau antibody or antigen-binding fragment thereof, comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 29 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 37. (7) An anti-tau antibody or antigen-binding fragment thereof, comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 29 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 39. (8) An anti-tau antibody or antigen-binding fragment thereof, comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 31 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 35. (9) An anti-tau antibody or antigen-binding fragment thereof, comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 31 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 37. (10) An anti-tau antibody or antigen-binding fragment thereof, comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 31 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 39. (11) An anti-tau antibody or antigen-binding fragment thereof, comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 33 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 35. (12) An anti-tau antibody or antigen-binding fragment thereof, comprising a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 33 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 37, and (13) An anti-tau antibody or antigen-binding fragment thereof, comprising a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 33 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 39.

[0066] Preferably, the anti-tau antibody or antigen-binding fragment thereof may comprise a heavy chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO:8 and a light chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO:16.

[0067] In some embodiments, the anti-tau antibody may be a full-length antibody, and may be an IgG1, IgG2, IgG3, IgG4, IgA, or IgA2 antibody.

[0068] For anti-tau antibodies, reference may also be made to the contents of Korean Patent Registration No. 10-2196840, the entire disclosure of which is incorporated herein by reference.

[0069] In some embodiments, the blood-brain barrier receptor-binding peptide may be any one selected from the group consisting of Angiopep-2 (APEP), RVG29, and TfR-binding peptide (TfR). The APEP peptide may comprise or consist of the amino acid sequence of SEQ ID NO: 20. The RVG29 peptide may comprise or consist of the amino acid sequence of SEQ ID NO: 22. The TfR-binding peptide may comprise or consist of the amino acid sequence of SEQ ID NO: 24. In one embodiment of the present invention, it has been confirmed that when APEP, RVG29, or TfR-binding peptide is coupled to an anti-tau antibody, it exhibits excellent BBB penetration effects.

[0070] In some embodiments, in the fusion, the anti-tau antibody can be coupled to the blood-brain barrier receptor-binding peptide via a linker. When coupled with a linker, the linker may be a peptide fragment known to be capable of linking antibody fragment domains, and may have, for example, the amino acid sequence of [GGGGS]4. In one embodiment, the linker may preferably have the amino acid sequence GGGGSGGGGSGGGGSGGGSG. Specifically, the blood-brain barrier receptor can be coupled to the Fc region of the anti-tau antibody, for example, the C-terminus of the heavy chain, via a linker.

[0071] The present invention is based, in part, on the surprising discovery that the anti-tau antibody can cross the BBB more effectively when coupled to a blood-brain barrier receptor-binding peptide without a linker. Thus, in another embodiment, the fusion can be such that the blood-brain barrier receptor-binding peptide is directly coupled to the anti-tau antibody without a linker. For example, the anti-tau antibody comprises a light chain and a heavy chain, and the brain-localizing peptide can be attached to the Fc region of the heavy chain (e.g., the C-terminus of the Fc region). In one embodiment of the present invention, when APEP, RVG29, and TfR peptides, particularly APEP and RVG29 peptides, are directly coupled to the anti-tau antibody, BBB penetration is significantly improved.

[0072] The nucleic acid and amino acid sequences of the anti-tau antibody, linker, and brain-localizing peptide contained in exemplary fusions of the present invention are as follows:

[0073] [Table 1]

[0074] [Table 2]

[0075] The amino acid sequences of exemplary anti-tau antibodies that can be included in the fusions of the invention are shown in Table 3.

[0076] [Table 3-1] [Table 3-2] [Table 3-3]

[0077] In some embodiments, the anti-tau antibodies or antigen-binding fragments and blood-brain barrier receptor-binding peptides can comprise sequences having 80% or more, preferably 90% or more, more preferably 95% or more, and most preferably 98% or more sequence identity to the sequences shown in Tables 1 and 2.

[0078] In other embodiments, the anti-tau antibody or antigen-binding fragment can comprise a sequence having 80% or more, preferably 90% or more, more preferably 95% or more, and most preferably 98% or more sequence identity to a sequence shown in Table 3.

[0079] In some embodiments, amino acid sequence variants of anti-tau antibodies or antigen-binding fragments and blood-brain barrier receptor-binding peptides can be used. For example, variants may be desired to improve the binding affinity and / or other biological properties of the antibody or peptide. Amino acid sequence variants of antibodies or peptides can be produced by introducing appropriate mutations into the nucleotide sequence encoding the molecule or by peptide synthesis. Such mutations include, for example, deletion of residues from the amino acid sequence, insertion of residues into such amino acid sequences, and / or substitution of residues within such amino acid sequences. Any combination of various mutations, including deletion, insertion, and substitution, can be performed to arrive at the final construct, provided that the final construct possesses the desired properties, e.g., antigen-binding properties. Target sites for substitutional mutagenesis include the heavy chain variable region (HVR) and framework region (FR). Conservative substitutions are listed under the heading "Preferred Substitutions" in Table 4 and are further described below in relation to amino acid side chain classes (1) to (6). Amino acid substitutions can be introduced into the molecule of interest and the products screened for the desired activity, e.g., maintained / improved antigen / receptor binding, reduced immunogenicity, or improved ADCC or CDC.

[0080] [Table 4]

[0081] Amino acids can be grouped according to the general properties of their side chains: (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues that affect chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe

[0082] Non-conservative substitutions involve exchanging a member of one of these classes for another class.

[0083] Nucleic acids, vectors, host cells and methods for producing the same Another aspect of the present invention provides a fusion comprising an anti-tau antibody or antigen-binding fragment thereof and a blood-brain barrier receptor-binding peptide as described herein, or a polynucleotide encoding the heavy chain polypeptide and / or the light chain polypeptide comprised in the fusion.

[0084] In one embodiment, the nucleotide sequence encoding the fusion encodes a light chain variable region and can comprise a nucleotide sequence encoding the entire CDR or variable region sequence set forth in Table 1, or a sequence having at least 80% or more, preferably 90% or more, more preferably 95% or more, and most preferably 98% or more sequence identity thereto.

[0085] In one embodiment, the nucleotide sequence encoding the fusion encodes a heavy chain variable region and can comprise a nucleotide sequence encoding the entire CDR sequence or variable region sequence set forth in Table 1, or a sequence having at least 80% or more, preferably 90% or more, more preferably 95% or more, and most preferably 98% or more sequence identity thereto.

[0086] In one embodiment, the nucleotide sequence encoding the fusion encodes a heavy chain variable region and a blood-brain barrier receptor-binding peptide, and may comprise a nucleotide sequence encoding the CDR sequences or the entire heavy chain region sequence listed in Table 1 and a nucleotide sequence encoding the blood-brain barrier receptor-binding peptide, or a sequence having at least 80% or more, preferably 90% or more, more preferably 95% or more, and most preferably 98% or more sequence identity with the above sequences.

[0087] In one embodiment, when the blood-brain barrier receptor-binding peptide is attached to the heavy chain of an antibody directly or via a linker, a nucleic acid can be provided that encodes a heavy chain polypeptide comprising the blood-brain barrier receptor-binding peptide, optionally a linker, and an antibody heavy chain variable region.

[0088] Another aspect of the present invention provides a vector comprising a polynucleotide sequence disclosed herein. Unless otherwise specified, a "vector" refers to a substance capable of transporting genetic material into cells. In the present invention, a "vector" may also be an "expression vector" that contains the necessary regulatory elements operatively linked to ensure normal expression of the inserted gene. When an anti-tau antibody-containing fusion comprises two separate polypeptides, the nucleotide sequences encoding these two polypeptides can be cloned into the same vector or separate vectors. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be inserted. Another type of vector is a viral vector, in which viral-derived DNA or RNA sequences are present in the vector for packaging within the virus. Certain vectors can replicate autonomously in a host cell into which they are introduced (e.g., bacterial vectors with a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of the host cell upon introduction and replicate along with the host genome accordingly. Common expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector.

[0089] In one embodiment, the vector may be a viral vector, specifically, one or more types selected from the group consisting of a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, a vaccinia viral vector, a poxviral vector, a herpes simplex viral vector, and a phagemid vector.

[0090] In other embodiments, the vector may be a non-viral vector. Specifically, the non-viral vector may be one or more selected from the group consisting of a plasmid, naked DNA, a DNA complex, mRNA (transcript), and an amplicon, but is not limited to these. For example, the plasmid may be selected from the group consisting of the pcDNA series, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, the pGEX series, the pET series, and pUC19.

[0091] According to another aspect of the present invention, there are provided host cells comprising the polynucleotides or expression vectors disclosed herein. In some embodiments, the host cells are capable of producing fusions. Any host cell known in the art that allows for stable, continuous cloning and expression of polynucleotides or expression vectors can be used. Suitable prokaryotic host cells may include Escherichia coli, strains of Bacillus (e.g., Bacillus subtilis and Bacillus thuringiensis), Enterobacteriaceae and strains thereof (e.g., Salmonella typhimurium, Serratia marcescens), and various Pseudomonas species. Suitable eukaryotic host cells for transformation include yeast (e.g., Saccharomyces cerevisiae), insect cells, plant cells, and animal cells (e.g., Sp2 / 0, Chinese hamster ovary (CHO) K1, CHO DG44, PER.C6, W138, BHK, COS-7, 293, HepG2, Huh7, 3T3, RIN, and MDCK cell lines). The term "host cell" is also used to refer to a cell that has been transformed, or that has been transformed with a nucleic acid sequence and is capable of expressing a selected gene of interest. The term includes the progeny of the original parent cell, whether or not the progeny are morphologically or genetically identical to the original parent cell, so long as the selected gene is present.

[0092] In addition, when introducing an expression vector into a host cell, methods that may be used include, but are not limited to, the CaCl2 precipitation method, the Hanahan method in which the efficiency of the CaCl2 precipitation method is increased by using a reducing substance called DMSO (dimethyl sulfoxide), electroporation, calcium phosphate precipitation, protoplast fusion, agitation using silicon carbide fibers, Agrobacterium-mediated transformation, PEG-mediated transformation, dextran sulfate, lipofectamine, and desiccation / repression-mediated transformation.

[0093] According to another aspect of the present invention, there is provided a method for producing a fusion product disclosed herein, comprising culturing a host cell. In some embodiments, the method for producing a fusion product comprising culturing a host cell comprises (i) culturing the host cell to obtain a culture (e.g., a culture containing the fusion product); and (ii) recovering the fusion product from the culture.

[0094] The host cells can be cultured using appropriate media and culture conditions known in the art. Specifically, the culture can be performed in a batch process, or continuously in a fed batch or repeated fed batch process.

[0095] The fusion product can be recovered from the culture by methods known in the art, including, but not limited to, centrifugation, filtration, extraction, spraying, drying, precipitation, crystallization, electrophoresis, differential lysis (e.g., ammonium sulfate precipitation), and chromatography (e.g., ion exchange, affinity, hydrophobic, and size exclusion).

[0096] Alternatively, the fusion proteins disclosed herein can be produced by any known method, such as conventional synthetic methods for protein synthesis, recombinant DNA techniques, and the like.

[0097] Pharmaceutical Composition According to another aspect of the present invention, there is provided a pharmaceutical composition comprising a fusion comprising an anti-tau antibody or antigen-binding fragment thereof as disclosed herein and a blood-brain barrier receptor-binding peptide. The fusion can be included in the composition in a prophylactically or therapeutically effective amount. The pharmaceutical composition can be administered to a subject to inhibit abnormal tau protein aggregation or to prevent or treat a neurodegenerative disease.

[0098] In some embodiments, the neurodegenerative disease may be a degenerative brain disease. Further, the neurodegenerative disease may be a neurological disease mediated by the tau protein. Neurodegenerative diseases include tauopathy, primary age-related tauopathy, chronic traumatic encephalopathy, progressive supranuclear palsy, corticobasal degeneration, frontotemporal dementia, Lytico-Bodig disease, Parkinson's disease, subacute sclerosing meningitis, lead encephalopathy, tuberous sclerosis, ganglioglioma, gangliocytoma, meningioangiomatosis, and subacute sclerosing panencephalitis. The present invention may be selected from the group consisting of, but not limited to, panencephalitis, pantothenate kinase-associated neurodegeneration (Hallervorden-Spatz disease), and lipofuscinosis.

[0099] Tauopathies include Alzheimer's disease, progressive supranuclear palsy, corticobasal degeneration, Pick's disease, a group of related disorders collectively known as frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), amyotrophic lateral sclerosis, Creutzfeldt-Jakob disease, dementia pugilistica, Gerstmann-Straussler-Scheinker syndrome, Lewy body disease, chronic traumatic encephalopathy, and Huntington's disease. The disease may be selected from the group consisting of, but not limited to:

[0100] To prepare a pharmaceutical composition, the fusion may be mixed with a pharmaceutically acceptable carrier and / or excipient. The pharmaceutical composition may be prepared in the form of a lyophilized preparation or an aqueous solution. See, for example, "Millington's Pharmaceutical Sciences and US Pharmacopeia: National Formulary," Mack Publishing Company, Easton, PA (1984). Acceptable carriers and / or excipients (including stabilizers) are non-toxic to a subject at the dosages and concentrations employed, and include buffers (e.g., phosphate, citric acid, or other organic acids); antioxidants (e.g., ascorbic acid or methionine); preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens, e.g., methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (about 10 residues or less) polypeptides; proteins (e.g., serum albumin, gelatin or immunoglobulin); hydrophilic polymers (e.g., polyvinylpyrrolidone); amino acids (e.g., glycine, glutamine, asparagine, histidine, arginine, or lysine), monosaccharides, disaccharides, and other carbohydrates, such as glucose, mannose, or dextrin; chelating agents (e.g., EDTA); sugars (e.g., sucrose, mannitol, trehalose, or sorbitol); salt-forming counterions (e.g., sodium), metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants (e.g., TWEEN®, PLURONICS®, or polyethylene glycol (PEG)).

[0101] The pharmaceutical composition may be administered orally or parenterally depending on the intended method. If desired, it may be administered intralesionally for localized treatment. Parenteral administration may include, but is not limited to, intramuscular, intravenous, intraarterial, intraperitoneal, intrapulmonary, transdermal, subcutaneous, intradural, intrarectal, intravesical, intravaginal, intraarticular, intranasal, intraspinal, intracerebral, or intraventricular administration. Administration may be by any suitable route, for example, intravenous or subcutaneous injection, depending in part on whether administration is short-term or long-term. Various administration schedules may be considered, including, but not limited to, single or multiple administrations over multiple time points, bolus administration, and pulse administration.

[0102] Pharmaceutical compositions can be formulated in appropriate forms known in the art depending on the route of administration.

[0103] Preventive or therapeutic methods According to another aspect of the present invention, there is provided a method for preventing or treating a neurodegenerative disease, comprising the step of administering to a subject in need thereof a fusion comprising an anti-tau antibody or antigen-binding fragment thereof as disclosed herein and a blood-brain barrier receptor-binding peptide. See above for details of neurodegenerative diseases.

[0104] According to another aspect of the invention, there is provided a method of inhibiting abnormal aggregation of tau protein in a subject, comprising the step of administering to the subject a fusion disclosed herein.

[0105] In some embodiments, a method for inhibiting abnormal aggregation of tau protein can be provided by administering a pharmaceutical composition comprising a fusion between an anti-tau antibody or its antigen-binding fragment and a blood-brain barrier receptor-binding peptide, in which the anti-tau antibody or its antigen-binding fragment and the blood-brain barrier receptor-binding peptide are linked directly without a linker or via a linker. Preferably, the fusion can be provided by administering a pharmaceutical composition comprising a fusion between an anti-tau antibody or its antigen-binding fragment and a blood-brain barrier receptor-binding peptide, in which the anti-tau antibody or its antigen-binding fragment and the blood-brain barrier receptor-binding peptide are linked directly without a linker.

[0106] In some embodiments, the fusions of the present invention can be effectively delivered across the blood-brain barrier using a variety of suitable compositions and methods described herein or known in the art.

[0107] In some embodiments, the fusions of the present invention can be administered in combination with one or more other agents effective in preventing or treating neurodegenerative diseases, including, but not limited to, any agent that can ameliorate or alleviate neurodegenerative diseases, such as chemical compounds, gene therapy agents, proteins (including antibodies), etc.

[0108] The present invention will be described in more detail below with reference to the following examples. Note that the following examples are presented to aid in understanding the present invention, and are not intended to limit the scope of the present invention in any way, and should not be construed as limiting the scope of the present invention.

[0109] Example Example 1. Preparation of a fusion comprising an anti-tau antibody and a blood-brain barrier receptor-binding peptide Example 1.1. Production of Expression Vectors The method for producing fusions containing the anti-tau antibody and blood-brain barrier receptor-binding peptide shown in Figures 1a and 1b is as follows. First, we requested GenScript, Inc. to clone the gene sequences of the three blood-brain barrier receptor-binding peptides (APEP, RVG29, and TfR). The vector used for cloning was pcDNA3.1(+), and the nucleic acid and amino acid sequences of the three peptides are shown in Table 5 below. The nucleic acid and amino acid sequences of the anti-tau antibody are shown in Tables 1 and 2. Hereinafter, the blood-brain barrier receptor-binding peptides are referred to as brain-localizing peptides (BPPs).

[0110] [Table 5]

[0111] The nucleic acid sequences of the three brain-localizing peptides were inserted into vectors using XhoI and XbaI restriction enzymes, and the resulting vectors are shown in Figure 2. Next, primers for linker insertion were prepared as shown in Table 6 below, and each peptide nucleic acid sequence and a portion of the vector were amplified by PCR and inserted into the XhoI-XbaI site of the pcDNA3.1(+) vector. Exceptionally, TfR was synthesized with a linker sequence ([GGGGS]4), which was then removed by cloning.

[0112] [Table 6]

[0113] For fusions, a total of six vectors were constructed, each containing three brain-localizing peptides and a linker, and then the heavy chain gene of an anti-tau antibody (H1) was inserted before the brain-localizing peptide (or linker and brain-localizing peptide) using BamHI and XhoI restriction enzymes to create an expression vector for the fusion containing the antibody and brain-localizing peptide (Figure 3a).The light chain gene was inserted into the pcDNA3.1(+) vector using HindIII and XbaI restriction enzymes to create an expression vector (Figure 3b).

[0114] On the other hand, for the anti-tau antibody (H1), the heavy chain gene was inserted into the pcDNA3.1(+) vector using BamHI and XhoI restriction enzymes, and the light chain gene was inserted into the pcDNA3.1(+) vector using HindIII and XbaI restriction enzymes to produce expression vectors that did not contain the brain-localizing peptide (or linker-brain-localizing peptide) sequence.

[0115] Example 1.2. Production of recombinant fusions in Expi293F cell culture The recombinant vector of Example 1.1 was introduced into Expi HEK293F cells to produce a fusion comprising the antibody and the peptide.

[0116] First, Expi HEK293F cells were subcultured to a cell number of 5x10 5The cells were cultured in a shaking incubator (37°C, 8% CO2, 120 rpm) for 2-3 days until the cell density reached 100 cells / mL. The culture medium used was FreeStyle® 293 Expression Medium supplemented with 1X Antibiotic-Antimycotic.

[0117] Next, for gene transfer, 200 μg of the plasmid vector was added to 10 mL of 150 mM NaCl, mixed well, and then 400 μL of PEI (polyethyleneimine hydrochloride, 1 mg / mL) was added (based on a 100 mL culture). After 15 minutes at room temperature, the solution was added dropwise onto the cell culture medium, and the culture was continued for 5 to 7 days. The number of cells for gene transfer was 2 x 10 based on the cell culture medium. 6 The cells / mL was filled.

[0118] The cell culture medium was centrifuged at 13,000 rpm at 4°C for 15 minutes. The supernatant, from which the cells were removed, was filtered through a 0.45 μm filter and then purified by passing it through a MabSelect SURE column (Cytiva, HiTrap Protein G) equilibrated with 1X PBS. To elute the antibody bound to the column, 0.1 M glycine-HCl, pH 2.7 buffer was passed through the column and the eluted antibody was collected. To neutralize the pH, 1 M Tris-HCl, pH 9.0 buffer was added to restore the pH to 7-8, and the buffer was finally exchanged with 1X PBS.

[0119] Through the above process, fusion products containing antibodies and brain-localizing peptides were obtained, and the antibodies and fusion products prepared are listed in Table 7 below.

[0120] [Table 7]

[0121] Example 2. Creation of a blood-brain barrier (BBB) ​​model using induced pluripotent stem cells (iPSCs) To confirm the BBB permeability of the fusion complex according to the embodiment of the present invention, a BBB model was constructed using differentiation of human brain microvascular endothelial cells, with reference to the paper [Lippmann, E., Azarin, S., Kay, J. et al. Derivation of blood-brain barrier endothelial cells from human pluripotent stem cells. Nat Biotechnol 30, 783-791 (2012)].

[0122] Example 2.1. Differentiation into human brain microvascular endothelial cells (iBMECs) Monoculture To create a blood-brain barrier (BBB) ​​model, induced pluripotent stem cells (iPSCs) (WiCell Research Institute Inc, IMR90-4) were induced to differentiate into brain microvascular endothelial cells (BMECs).

[0123] First, iPSCs were plated in a Matrigel-coated 6-well plate at 3x10 5 After seeding at 2.5 x 10 cells / well, mTeSR (containing 10 μM Y27, a total of 2 mL / well) was added and cultured. Every 24 hours after seeding, the entire medium was replaced with mTeSR1 (without Y27). When the cell number reached 2.5 x 10 cells / well, 5 ~4x10 5 cells / well (preferably 3 x 10 5Once the cells reached a density of 1000 cells / well, the medium was replaced with 2 mL of UM (DMEM / F12 medium (containing 15 mM HEPES), 20% KOSR, 1% NEAA, 0.5% glutamax, and β-ME) per well to induce differentiation into iBMECs (induced BMECs) (UM phase, D0). From 1 to 5 days after the start of differentiation induction (D1 to D5), the medium was replaced daily with UM (containing 1% B27) (2–3 mL / well), and on day 6, it was replaced with EC (hESFM medium containing 2% B27 and 0.02% bFGF) supplemented with 0.1% retinoic acid (RA).

[0124] On day 7 after differentiation induction, transwells (consisting of plates and inserts) for subculture of iBMECs were coated with ECM solution (collagen:fibronectin:water = 4:1:5) as follows: The membrane filter of the transwell insert was coated with ECM solution at 37°C for a minimum of 4 hours or a maximum of 24 hours before subculture. At this time, plates were coated only when performing triple culture.

[0125] On day 8 after differentiation induction, the ECM solution was removed from the transwell insert plate and allowed to dry. The cultured iBMECs were then recovered using Accutase and hESFM medium, resuspended in an appropriate volume of EC medium (containing 0.1% RA), and placed in the transwell insert at 1x10 6 cells / cm 2 The inserts seeded with iBMECs were attached to the plate, and the plate was shaken back and forth or side to side to prevent clumping of the cells and ensure even cell distribution. The cells were then cultured in a 37°C incubator, with the medium replaced with ECs (hESFM medium containing 2% B27) every 24 hours.

[0126] A schematic diagram of the above-mentioned eruption process is shown in Figure 4.

[0127] triple culture The triple culture was performed in the same manner until day 6 after the differentiation of monocultured iBMECs. On day 7, astrocytes (ScienceCell, Catalog #1800) and pericytes (ScienceCell, Catalog #1200) were seeded onto 12-well transwell plates using the following two methods. Astrocytes (4.4 x 10 cells) were cultured in pericyte medium. 4 cells) and pericytes (4.4x10 4 cells) at a 1:1 ratio, or astrocytes (1.5x10) in 500 μL of pericyte medium. 4 cells) and pericytes (3x10 4 cells) were cultured at a ratio of 1:2.

[0128] On the 8th day after differentiation induction, the medium in the plates was replaced with endothelial cell medium (containing 10 μM RA), and iBMECs were seeded onto the transwell inserts in the same manner as for monoculture, to prepare a BBB model containing three types of cells (endothelial cells, astrocytes, and pericytes).

[0129] For the monoculture model or triple culture model seeded in a transwell, medium changes were performed by removing the medium from the plate and then removing the medium from the insert. When adding hESFM (containing 2% B27) medium, medium was added to the insert and then added to the plate.

[0130] Example 2.2. Performance evaluation of the BBB model To verify the performance of iBMECs differentiated in Transwells as a BBB model, we observed the expression of tight junction markers in iBMECs using a fluorescence microscope after immunostaining. We also measured the transendothelial electrical resistance (TEER) value of the Transwells in which iBMECs were differentiated and confirmed the permeability of small molecules to verify the formation of tight junctions.

[0131] Confirmation of cellular tight junction marker and BBB-related protein expression To confirm iBMEC differentiation and BBB formation, we immunostained for BBB marker proteins and observed them under a fluorescence microscope (Nikon, ECLIPSE Ti) from days 6 to 10, when differentiation into iBMECs had progressed. As shown in Figure 5, we confirmed the expression of tight junctions and tight junction markers occludin, claudin 5, and ZO-1 in differentiated iBMECs, as well as the expression of transporter proteins glut1, TfR, and LRP1, which are expressed in the BBB.

[0132] Measurement of TEER value and confirmation of permeation of small molecular substances TEER values ​​were measured between days 9 and 11 after the start of differentiation induction into iBMECs. The measurement method was as follows. First, the transwell was removed from the incubator and left at room temperature for 20–30 minutes to prevent fluctuations in resistance. Next, the measuring rod was positioned in the center of the gap between the plate wall and the insert, and the TEER value was measured.

[0133] To confirm the permeability of iBMECs to substances, we measured the TEER value in Transwells seeded with iBMECs to confirm the proper formation of tight junctions. Then, we added 50 μg / mL of 3 kDa dextran (Cascade Blue-labeled) to the Transwell inserts of five Transwells containing differentiated iBMECs and a control (empty Transwell) for 24 hours. The amount of dextran was then measured in the plate and insert. As shown in Table 8, dextran was detected in the control group within 1 hour after addition, and over 50% of the total dextran was detected within 24 hours. In contrast, no dextran leakage was observed in the Transwells containing differentiated iBMECs, confirming the tight junctions were well formed, preventing the passage of small molecules.

[0134] [Table 8]

[0135] Example 3. Confirmation of BBB penetration ability of fusion On the 9th to 10th day after differentiation induction into iBMECs, the TEER value of the transwell was measured according to the TEER measurement method described in Example 2.2. One antibody and six fusions prepared in Example 1 were then administered to the transwell insert, and the BBB penetration ability of the antibody and fusion was measured by permeability coefficient (Papp) measurement and enzyme-linked immunosorbent assay (ELISA).

[0136] On the other hand, after administration of the antibody and the fusion compound, the TEER value of each transwell was measured and the average value was confirmed. The average value after administration of the antibody and the fusion compound was approximately 4000 Ωxcm 2 It was confirmed that the antibody and fusion compound administration did not affect the performance of the BBB model and there were no differences between samples (Figure 6).

[0137] Example 3.1. Measurement of Permeability Coefficient (Papp) The permeability coefficient (Papp) is an absolute value that indicates the rate at which a substance escapes per unit area and unit time. It is a comparable value regardless of the area of ​​the transwell used in the experiment and the time, and can be expressed as follows:

[0138] Papp=(dQ / dt) / (C0ХA)

[0139] Here, dQ / dt is the amount of fluid that escapes to the basal area of ​​the plate per unit time, and A is the area of ​​the insert in the transwell (cm 2 ) and C0 indicate the initial concentration of the sample introduced into the insert (apical) region of the transwell.

[0140] 0.5 mL of hESFM (containing 2% B27) medium was prepared in a 1.5 mL tube, and seven types of antibodies and fusions (25 μm / mL) were added to prepare antibody-containing solutions. After removing 0.4 mL of medium from the insert region of the transwell, 0.4 mL of the 0.5 mL antibody-containing solution was added. 24 hours after administration of the antibody-containing solution (D11), 1.5 mL of medium from the plate region and 0.5 mL of medium from the upper chamber were removed and subjected to ELISA analysis. The dQ / dt value was calculated as the ELISA analysis value, and A was the ratio of the number of cells in the 12-well transwell (insert: 1.12 cm). 2 , Plate: 3.5cm 2 ) or 24-well transwell (insert: 0.33 cm 2 , Plate: 2cm 2 ) was substituted with the area of ​​the well used, and 0.1 mL of the solution containing the antibody remaining in the transwell insert was used to measure the C0 value.

[0141] Example 3.2. Enzyme-linked immunosorbent assay (ELISA) The ELISA analysis method for the antibodies and fusion products that permeated from the insert region to the plate region was as follows.

[0142] Pep1 (0.83 mg / mL) was diluted 1:2000 in capture buffer (50 mM NaHCO3 and distilled water, pH 9.6) and 50 μL of the diluted solution was added to the plate. The plate was then incubated at 37°C, 600 rpm, for 2 hours. Next, 100 μL of blocking buffer (100 mM Tris, 0.1% Tween 20, distilled water, and 2.5% casein, pH 7.6) was added to each well and incubated at 37°C, 600 rpm, for 2 hours.

[0143] Then, using the standard dilution analysis (STD) method, the standard solution or sample was diluted in the order of 20, 10, 5, 2.5, 1.25, 0.625, 0.3125, and 0 (ng / mL), and 50 μL of the standard solution or sample was loaded into each well and incubated (37°C, 600 rpm, 1 hour).

[0144] 50 μL of antibody solution (peroxidase-labeled human IgG, 1:2000) (Sigma-Aldrich, AP309P) was added to each well and incubated at 37°C, 600 rpm, for 1 hour. 50 μL of substrate reagent (R&D Systems, #DY999) was added to each well and incubated at room temperature for 5 minutes. 50 μL of stop solution (R&D Systems, #DY994) was added to stop the reaction. Absorbance was measured using an infinite F50 spectrophotometer (plate agitation: 10 seconds, measurement wavelength: 450 nm, reference wavelength: 620 nm).

[0145] After the reaction of the substrate reagent, except for the step of adding the stop solution, washing was performed four times between each step by repeatedly filling and draining each well with washing buffer (PBS containing 0.05% Tween 20).

[0146] Example 3.3. Results of BBB permeability measurement of fusions While the monoculture BBB model consists solely of vascular endothelial cells, the triple-culture BBB model contains not only vascular endothelial cells but also astrocytes and pericytes, providing a more similar microenvironment to the human blood-brain barrier. Antibody-brain-localizing peptide fusions (without linkers) and antibody-linker-brain-localizing peptide fusions (with linkers) were tested for permeability in the monoculture BBB model. For antibody-brain-localizing peptide fusions that showed statistically significant results, permeability was also tested in the triple-culture BBB model, and the combined results were reported as a combined total. A total of four batches were prepared, each separated by the manufacturing date, manufacturer, concentration, and assay method of the antibody and fusion. Each batch was prepared separately or in a mixed form (Figures 7 and 8).

[0147] Antibody-brain-localizing peptide fusion (linker not included) The overall average Papp values of the H1 antibody and three antibody-brain-transporting peptide fusions (H1-APEP, H1-RVG29, and H1-TfR) for the single-culture BBB model were divided by the value of H1 and represented as item-by-item ratios. As a result, permeation rates were calculated to be higher for all three fusions compared to the H1 antibody. The same method was used to represent the permeability coefficients for the three culture BBB models, and the permeation rates were also higher for all three fusions compared to the H1 antibody (Table 9). In the total result obtained by summing the permeabilities for single-culture and the three cultures, H1-APEP was measured to be 2.9-fold, H1-RVG29 was 2.7-fold, and H1-TfR was 2.3-fold higher compared to the H1 antibody. The total results for the H1 antibody and the three antibody-brain-transporting peptide fusions are shown in Fig. 7a, with Papp on the vertical axis (* indicates 0.01 < P < 0.05, and ** indicates 0.001 < P < 0.01).

[0148]

Table 9

[0149] Even when statistical analysis was performed using the values obtained by dividing the Papp results of the three fusions by the Papp value of the respective H1 antibody, the three fusions showed high permeability to the BBB model compared to the H1 antibody. Specifically, H1-APEP was measured to be 3.54-fold, H1-RVG29 was 3.04-fold, and H1-TfR was 2.74-fold higher compared to the H1 antibody (Fig. 7b).

[0150] Antibody-linker-brain-localizing peptide fusion (including linker) The overall average Papp values of the H1 antibody and three antibody-linker-brain-transporting peptide fusions (H1-L4-APEP, H1-L4-RVG29, and H1-L4-TfR) for the single-culture BBB model were divided by the value of H1 and represented as ratios. As a result, permeation rates were calculated to be slightly higher for all three fusions compared to the H1 antibody (H1-L4-APEP was 1.9-fold, H1-L4-RVG29 was 2.4-fold, and H1-L4-TfR was 1.8-fold), but the permeation rates were lower compared to the fusions without a linker. Here, L4 means a linker.

[0151] The average Papp values ​​were divided by H1 and the results are shown in Table 10. For antibody-linker-brain-localizing peptide fusions, the results of single cultures represent the total results. The total results (single cultures) for the H1 antibody and the three antibody-linker-brain-localizing peptide fusions are shown in Figure 8, with Papp plotted on the vertical axis.

[0152] [Table 10]

[0153] statistical analysis Statistical analysis was performed using GraphPad software (GraphPad Software, San Diego, CA, USA). Data were subjected to one-way analysis of variance and Kruskal-Wallis test (non-parametric test). The significance of differences between the H1 antibody alone and the antibody groups fused with three brain-localizing peptides (APEP, RVG29, and TfR) was analyzed using Dunn's multiple comparison test (*: 0.01 <P<0.05、**:0.001<P<0.01、***:P<0.001、ns:P> 0.05).

[0154] Sequence Listing Free Text Sequence information for sequences omitted (skipped) in the electronic sequence listing file attached to this specification, i.e., those with fewer than 10 defined nucleotides (SEQ ID NO: 11) and those with fewer than 4 defined amino acids (SEQ ID NO: 12), is provided below.

[0155] SEQ ID NO:11 Array length: 9 Sequence type: DNA Sequence origin: Synthetic construct Sequence name: Nucleotide sequence for CDR2 of anti-tau antibody (VL) array: ctggtgtcc SEQ ID NO:12 Array length: 3 Sequence type: AA Sequence origin: Synthetic construct Sequence name: Amino acid sequence for CDR2 of anti-tau antibody (VL) array: LVS

Claims

1. A fusion comprising (i) an anti-tau antibody or an antigen-binding fragment thereof that specifically binds to tau protein, and (ii) a blood-brain barrier receptor-binding peptide.

2. The fusion of claim 1, wherein the anti-tau antibody is directly coupled to the blood-brain barrier receptor-binding peptide without a linker.

3. 3. The fusion of claim 1 or 2, wherein the blood-brain barrier receptor-binding peptide is selected from the group consisting of Angiopep-2 (APEP), RVG29, and TfR-binding peptide (TfR).

4. 4. The fusion of claim 3, wherein the APEP comprises the amino acid sequence of SEQ ID NO: 20, the RVG29 comprises the amino acid sequence of SEQ ID NO: 22, and the TfR comprises the amino acid sequence of SEQ ID NO:

24.

5. The fusion of claim 1 or 2, wherein the anti-tau antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 2, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 4, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6, and a light chain variable region (VL) comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 10, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 12, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:

14.

6. The fusion of claim 1 or 2, wherein the anti-tau antibody or antigen-binding fragment thereof binds to an epitope comprising amino acids 275 to 286 of the wild-type tau protein of SEQ ID NO: 25, in which amino acid 280 is acetylated.

7. The fusion of claim 1 or 2, wherein the anti-tau antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 8 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:

16.

8. The anti-tau antibody or antigen-binding fragment thereof (i) a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, and SEQ ID NO:33; and (ii) the fusion of claim 1 or 2, comprising a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 35, SEQ ID NO: 37, and SEQ ID NO:

39.

9. The fusion of claim 1 or 2, wherein the anti-tau antibody comprises a light chain and a heavy chain, and the blood-brain barrier receptor-binding peptide binds to the Fc region of the heavy chain.

10. The fusion of claim 1 or 2, which crosses the blood-brain barrier and specifically binds to tau protein.

11. The fusion construct according to claim 1 or 2, wherein the anti-tau antibody or antigen-binding fragment thereof is any one selected from the group consisting of a full-length antibody, Fab, scFv, F(ab')2, and Fv.

12. The fusion of claim 1 or 2, wherein the anti-tau antibody is an IgG antibody.

13. A polynucleotide encoding the fusion of claim 1 or 2.

14. An expression vector comprising the polynucleotide of claim 13.

15. A host cell comprising the expression vector of claim 14.

16. A method for producing the fusion of claim 1 or claim 2, comprising the step of culturing a host cell containing a polynucleotide encoding the fusion of claim 1 or 2, or the light chain polypeptide or the heavy chain polypeptide comprised in the fusion of claim 1 or claim 2.

17. A pharmaceutical composition for preventing or treating a neurodegenerative disease, comprising the fusion protein of claim 1 or 2.

18. 18. The pharmaceutical composition according to claim 17, wherein the neurodegenerative disease is a tau protein-mediated neurological disease.

19. The neurodegenerative diseases include tauopathy, primary age-related tauopathy, chronic traumatic encephalopathy, progressive supranuclear palsy, corticobasal degeneration, frontotemporal dementia, Lytico-Bodig disease, Parkinson's disease, subacute sclerosing meningitis, lead encephalopathy, tuberous sclerosis, ganglioglioma, gangliocytoma, meningioangiomatosis, subacute sclerosing panencephalitis, and 18. The pharmaceutical composition according to claim 17, wherein the disease is selected from the group consisting of panencephalitis, pantothenate kinase-associated neurodegeneration (Hallervorden-Spatz disease), and lipofuscinosis.

20. The tauopathies include Alzheimer's disease, progressive supranuclear palsy, corticobasal degeneration, Pick's disease, a group of related disorders collectively known as frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), amyotrophic lateral sclerosis, Creutzfeldt-Jakob disease, dementia pugilistica, Gerstmann-Straussler-Scheinker syndrome, Lewy body disease, chronic traumatic encephalopathy, and Huntington's disease.

20. The pharmaceutical composition of claim 19, wherein the compound is selected from the group consisting of:

21. 18. The pharmaceutical composition of claim 17, wherein the pharmaceutical composition is administered by any one of administration routes selected from the group consisting of intramuscular, intravenous, intraarterial, intraperitoneal, transdermal, subcutaneous, intrathecal, intracerebral, intraventricular, intrapulmonary, or intranasal administration.

22. A method for preventing or treating a neurodegenerative disease, comprising administering an effective amount of the fusion of claim 1 or 2 to a subject in need of such prevention or treatment.

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