Fusion comprising anti-tau antibody and peptide and uses thereof

Abstract

The present invention relates to a fusion comprising an anti-tau antibody or an antigen-binding fragment thereof and a blood-brain barrier receptor binding peptide, and uses thereof. The fusion of the present invention specifically binds to tau protein by crossing the blood brain barrier, and thus can effectively prevent or treat neurodegenerative diseases mediated by tau protein.

Description

Technical Field

[0001] This application claims priority from 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

[0003] This application contains a sequence listing submitted electronically in XML format and is incorporated herein by reference in its entirety. A copy of the sequence listing was created on November 28, 2023, is named KC23138-SEQ.xml, and is 44.6KB in size.

[0004] The present disclosure relates to fusions comprising anti-tau antibodies and peptides and uses thereof. In particular, the present disclosure relates to fusions with improved blood-brain barrier permeability and uses thereof for preventing or treating neurodegenerative diseases. Background Art

[0005] Tau protein is a protein that stabilizes microtubules, which are proteins that transport cellular substances. Tau protein exists in six isoforms in the human body and is abundant in neurons of the central nervous system. It is known that when tau protein mutates, tau protein becomes hyperphosphorylated, which causes abnormal aggregation of neurofibrillary tangles (NFTs) in nerve cells, leading to neurodegenerative diseases such as dementia, Parkinson's disease and tauopathy. Therefore, the development of therapeutic agents targeting tau protein has been reported (Korean Patent Application No.: 10-2020-0086341).

[0006] Meanwhile, the brain permeability of neurological disorder drugs (e.g., large biotherapeutic drugs such as antibodies or small molecule drugs with low brain permeability) is strictly limited by the extensive and impermeable blood-brain barrier (BBB) ​​and other cellular components in the neurovascular unit. Previous studies have reported that only a very small percentage (about 0.1%) of IgG injected into the bloodstream is able to penetrate into the central nervous system compartment (Felgenhauer, Klin. Wschr. 52 1158-1164 (1974)).

[0007] Therefore, there is a need for tau-targeting drugs that can cross the BBB and be effectively delivered to the brain. Summary of the Invention Technical issues

[0008] The present disclosure aims to solve all the above problems.

[0009] Another object of the present disclosure is to provide a fusion comprising an anti-tau antibody or an antigen-binding fragment thereof that specifically binds to tau protein and a blood-brain barrier receptor-binding peptide.

[0010] Another object of the present disclosure is to provide polynucleotides, expression vectors and host cells encoding the fusions.

[0011] Another object of the present disclosure is to provide a method for preparing the fusion.

[0012] Another object of the present disclosure is to provide a pharmaceutical composition for preventing or treating neurodegenerative diseases, comprising the fusion.

[0013] Another object of the present disclosure is to provide a method for preventing or treating a neurodegenerative disease, comprising administering the fusion to a subject in need of prevention or treatment of a neurodegenerative disease.

[0014] The objects of the present disclosure are not limited to the above objects, but will become more apparent from the following description and can be achieved by the means and combinations thereof described in the claims. Technical Solution

[0015] To achieve the above objectives, the representative layout of the present disclosure is as follows.

[0016] According to one aspect of the present disclosure, a fusion is provided, 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.

[0017] In one embodiment, a fusion is provided, wherein the anti-tau antibody is directly conjugated to the blood-brain barrier receptor binding peptide without a linker.

[0018] In another embodiment, 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).

[0019] In another embodiment, the APEP may comprise, consist of, or consist essentially of the amino acid sequence of SEQ ID NO:20.

[0020] In another embodiment, RVG29 may comprise, consist of, or consist essentially of the amino acid sequence of SEQ ID NO:22.

[0021] In another embodiment, the TfR may comprise, consist of, or consist essentially of the amino acid sequence of SEQ ID NO:24.

[0022] In another embodiment, the anti-tau antibody or antigen-binding fragment thereof may comprise a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region comprises: 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 the light chain variable region comprises: 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.

[0023] 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, wherein the amino acid at position 280 is acetylated.

[0024] In one embodiment, the anti-tau antibody or antigen-binding fragment thereof may comprise a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 8 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 16.

[0025] In one embodiment, the anti-tau antibody or antigen-binding fragment thereof may 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.

[0026] In another embodiment, a fusion is provided, wherein 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.

[0027] In another embodiment, the fusion can be one that crosses the blood-brain barrier and specifically binds to tau protein.

[0028] In another embodiment, the anti-tau antibody or antigen-binding fragment thereof can be any one selected from the group consisting of full-length antibody, Fab, scFv, F(ab')2 and Fv.

[0029] In another embodiment, the anti-tau antibody may be an IgG antibody.

[0030] According to another aspect of the present disclosure, provided are polynucleotides encoding the fusions disclosed herein or the heavy and / or light chains thereof.

[0031] According to another aspect of the present disclosure, an expression vector comprising the polynucleotide is provided.

[0032] According to another aspect of the present disclosure, a host cell is provided, comprising a polynucleotide encoding the fusion disclosed herein or an expression vector comprising the polynucleotide.

[0033] According to another aspect of the present disclosure, there is provided a method of producing the fusion disclosed herein, the method comprising culturing a host cell comprising a polynucleotide encoding the fusion or the light and heavy chain polypeptides comprised in the fusion.

[0034] According to another aspect of the present disclosure, provided is a pharmaceutical composition for preventing or treating a neurodegenerative disease, the pharmaceutical composition comprising the fusion disclosed herein.

[0035] According to another aspect of the present disclosure, provided is a method for preventing or treating a neurodegenerative disease, comprising administering an effective amount of the fusion or the pharmaceutical composition disclosed herein to a subject in need of prevention or treatment of a neurodegenerative disease.

[0036] In one embodiment, the neurodegenerative disease can be selected from the group consisting of: 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, Hallervorden-Spatz disease and lipofuscinosis.

[0037] In another embodiment, the tauopathy can be selected from the group consisting of Alzheimer's disease, progressive supranuclear palsy, corticobasal degeneration, Pick's disease, a group of related disorders collectively known as chromosome 17-linked frontotemporal dementia with Parkinsonism (FTDP-17), amyotrophic lateral sclerosis, Creutzfeldt-Jakob disease, dementia pugilistica, Gerstmann-Straussler-Scheinker syndrome, Lewy body disease, chronic traumatic encephalopathy, and Huntington's disease.

[0038] In another embodiment, the fusion or the pharmaceutical composition disclosed herein can be administered by any one route of administration selected from the group consisting of intramuscular, intravenous, intraarterial, intraperitoneal, transdermal, subcutaneous, intradural, intracerebral, intracerebroventricular, intrapulmonary or intranasal administration. Beneficial effects

[0039] As disclosed herein, the fusions prepared by coupling anti-tau antibodies to blood-brain barrier receptor-binding peptides demonstrate excellent effectiveness in enabling the anti-tau antibodies to effectively cross the BBB. In particular, when directly linked to the blood-brain barrier receptor-binding peptide without a linker, the anti-tau antibodies exhibit superior BBB permeability. The fusion polypeptides disclosed herein can inhibit abnormal aggregation of tau protein or effectively prevent or treat neurodegenerative diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] FIG1 shows a schematic diagram of a fusion protein according to an embodiment of the present disclosure. Figure 1a A fusion protein is shown in which an anti-tau antibody is directly linked to a blood-brain barrier receptor binding peptide without a linker; Figure 1b A fusion protein is shown in which an anti-tau antibody is linked to a blood-brain barrier receptor-binding peptide via a linker.

[0041] Figure 2 The pcDNA3.1(+) vector used to clone three blood-brain barrier (BBB) ​​receptor binding peptides according to embodiments of the present disclosure is shown. In this specification, the term "blood-brain barrier receptor binding peptide" is used interchangeably with "brain penetrating peptide (BPP)" and is labeled as BPP in the accompanying drawings.

[0042] FIG3 shows a schematic diagram showing an expression vector (pcDNA3.1(+)) into which the heavy chain and light chain genes of the H1 antibody (human anti-tau antibody) are respectively inserted. Figure 3a Shown is a schematic diagram showing the following expression vector, wherein the heavy chain gene of the H1 antibody is inserted upstream of the brain-penetrating peptide (or linker-brain-penetrating peptide) using restriction enzymes BamHI and XhoI; Figure 3b Shown is a schematic diagram showing an expression vector into which the light chain gene of the H1 antibody is inserted using restriction enzymes HindIII and XbaI.

[0043] Figure 4 Shown is a schematic diagram demonstrating the protocol for differentiating induced pluripotent stem cells ("iPSCs") into human brain microvascular endothelial cells ("BMECs").

[0044] Figure 5Images obtained by fluorescence microscopy (Magnification: 400x) on days 6 to 10 after induction of differentiation into iBMECs are shown, which demonstrate the expression of BBB-related proteins (Scale bar: 100μm).

[0045] Figure 6 A graph showing the results obtained by measuring the TEER values of individual transwells administered with one antibody and six fusions according to embodiments of the present disclosure is shown. L4 represents the linker, and its value is expressed in units of Ω×cm ,

[0049] and is shown.

[0046] Figure 7 shows a graph demonstrating the total Papp results (including batches #1 to #3) of the H1 antibody and three antibody-brain penetrating peptide fusions (H1-APEP, H1-RVG29, and H1-TfR) according to embodiments of the present disclosure. Figure 7a A graph showing the total Papp results is shown, where the average value is shown. Figure 7b A graph showing the PAPP ratio (relative value) is shown, where each ratio is obtained by dividing the total Papp result by the Papp value of the H1 antibody, and the average value is shown. * indicates 0.01 < P < 0.05 relative to the H1 antibody, and ** indicates 0.001 < P < 0.01 relative to the H1 antibody.

[0047] Figure 8 A graph showing the total Papp results (including batches #1 to #4) of the H1 antibody and three antibody-linker-brain penetrating peptide fusions (H1-L4-APEP, H1-L4-RVG29, and H1-L4-TfR) according to embodiments of the present disclosure is shown. Detailed Description

[0048] The following detailed description of the present disclosure will be elaborated with reference to specific drawings, which relate to specific embodiments in which the present disclosure can be implemented; however, the content of the present disclosure is not limited thereto and is only limited by the appended claims and any equivalent scope described in the claims. It should be understood that the various embodiments / examples of the present disclosure are different from each other but not necessarily mutually exclusive. For example, certain shapes, structures, and features described herein can be changed in different embodiments / examples or can be implemented in combinations of embodiments / examples without departing from the spirit and scope of the present disclosure. Unless otherwise specified, the terms used to describe the present disclosure should be understood in their ordinary meaning and apply to the same terms used herein and the aspects or embodiments of the present disclosure that define the term. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular form also include the plural form, and vice versa.

[0049] definition

[0050] "Blood-brain barrier" or "BBB" refers to an important biological barrier that protects the brain, which is composed of cerebral vascular endothelial cells and the basement membrane, astrocytes and pericytes that surround and support these cells. The blood-brain barrier prevents foreign substances (molecules or small molecules such as pathogens, pigments, drugs and toxins) from entering the brain through the tight junctions of vascular endothelial cells. The blood-brain barrier in the brain, the blood-spinal cord barrier in the spinal cord, and the blood-retinal barrier in the retina are all continuous capillary barriers in the central nervous system and are collectively referred to as blood-brain barrier or BBB. The blood-spinal cord barrier (choroid plexus) is composed of ependymal cells rather than capillary endothelial cells.

[0051] "Antibody" is used broadly and includes 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 fusion of an antibody with a compound); and antibody fragments (including antigen-binding fragments). As used herein, when the prefix "anti" is used in conjunction with an antigen, it indicates that the given antibody is reactive with the given antigen. Antibodies that react with a specific antigen can be prepared by synthetic and / or recombinant methods, for example, but not limited to, screening recombinant antibody libraries in phage or similar vectors, or by immunizing an animal 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, which are linked by disulfide bonds. Each heavy chain and light chain comprises a constant region and a variable region. Each heavy chain variable region (HVR) and light chain variable region (LVR) comprises three segments, called "complementarity determining regions (CDRs)" or "hypervariable regions," which are primarily responsible for binding to antigenic epitopes. They are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially from the N-terminus. The more highly conserved portions of the variable regions outside of the CDRs are called "framework regions (FRs)." As used herein, the antibody may be, for example, an animal antibody, a chimeric antibody, a humanized antibody, or a human antibody.

[0052] The term "anti-tau antibody" refers to an antibody that is capable of binding tau with sufficient affinity such that the antibody is useful as a therapeutic agent targeting tau.

[0053] The term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, wherein the individual antibodies constituting the antibody population are identical, except for possible naturally occurring mutations and / or post-translational modifications (e.g., isomerization and amidation) that may be present in small amounts. Monoclonal antibodies are highly specific and are directed against a single antigenic site. The modifier "monoclonal" indicates the characteristic of an antibody obtained from a substantially homogeneous antibody population and should not be construed as requiring the antibody to be prepared by any particular method. For example, the monoclonal antibodies used in accordance with the present disclosure can be made by a variety of techniques, including hybridoma methods, recombinant DNA methods, phage display methods, methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, and the like.

[0054] The term "antigen-binding fragment" refers to a portion of an antibody that has specific binding ability to an antigen, or a polypeptide comprising the portion. Unless the term "antibody" is understood from the context to explicitly exclude "antigen-binding fragment", the terms "antibody" and "antigen-binding fragment" are used interchangeably, and "antibody" can be interpreted as including "antigen-binding fragment". 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 by antibody fragments and single-domain antibodies.

[0055] As used herein, the term "Fc region" refers to the C-terminal region of an immunoglobulin heavy chain, comprising at least a portion of the constant region (e.g., CH2, CH3, or CH2 and CH3). The term includes native sequence Fc regions and variant Fc regions.

[0056] The term "blood-brain barrier receptor binding peptide" refers to a peptide that can selectively cross the blood-brain barrier by binding to a receptor present in the plasma membrane of brain capillary endothelial cells, otherwise the blood-brain barrier will restrict the transport of substances. The blood-brain barrier receptor binding peptide may be included in a brain-penetrating peptide (BPP) or a cell-penetrating peptide (CPP) because it has the ability to pass through brain capillary endothelial cells. Therefore, in this specification, the terms "blood-brain barrier receptor binding peptide" and "brain-penetrating peptide", "cell-penetrating peptide", "BPP" or "CPP" can be used interchangeably. 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.

[0057] 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-penetrating peptide. In some embodiments, the fusion is a fusion polypeptide comprising an anti-tau antibody and a blood-brain barrier receptor binding peptide.

[0058] The term "linker" may refer to a peptide linker of 1 to 100 amino acids, specifically 2 to 50 amino acids, and more specifically 5 to 30 amino acids in length. The linker may include, but is not limited to, one or more amino acids selected from the group consisting of, for example, Gly, Asn, Ser, Thr, Ala, Asp, etc. For example, the linker may be represented by (GGGGS)n, where n is the number of repetitions of the unit (GGGGS), and considering the effectiveness of the antibody, it may be 1 to 10, and specifically 1 to 5. For another example, the linker may be a peptide fragment known to be able to connect antibody fragment domains. For example, the linker may have the amino acid sequence of [GGGGS]4. In one embodiment, preferably, the linker may be the amino acid sequence GGGGSGGGGSGGGGSGGGSG.

[0059] The terms "subject" and "patient" are used interchangeably and may be mammals in need of prevention or treatment of tau protein-mediated neurological diseases or neurodegenerative diseases, such as primates (e.g., humans), companion animals (e.g., dogs, cats, etc.), livestock (e.g., cattle, pigs, horses, sheep, goats, etc.), and experimental animals (e.g., rats, mice, guinea pigs, etc.). In some embodiments, the subject is a human.

[0060] The term "treatment" generally refers to obtaining a desired pharmacological effect and / or physiological effect. Such an effect has a therapeutic effect because it partially or completely cures the disease and / or the adverse reactions caused by the disease. The desired therapeutic effect includes, but is not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, alleviating any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the speed of disease progression, alleviating or alleviating the disease state, and slowing down or improving prognosis. Preferably, "treatment" can refer to a medical intervention for a disease or condition that has occurred.

[0061] The term "prevention" refers to prophylactic treatment, i.e., measures or procedures intended to prevent a disease rather than treat it. "Prevention" means obtaining a desired prophylactic pharmacological and / or physiological effect in order to partially or completely prevent a disease or its symptoms.

[0062] The term "administering" refers to providing a substance to a subject for prophylactic or therapeutic purposes.

[0063] The term "therapeutically effective amount" or "effective amount" refers to the amount of an active ingredient (substance) sufficient to achieve treatment of a disease when the active ingredient (substance) is administered to a mammal or other subject for the treatment of a disease. The "therapeutically effective amount" or "effective amount" can be determined based on factors including the subject's (preferably human) weight, sex, age, health status and severity of the disease, drug activity, sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, concurrent medications, and other factors known in the medical field.

[0064] Fusion comprising an anti-tau antibody or antigen-binding fragment thereof and a blood-brain barrier receptor binding peptide

[0065] According to one aspect of the present disclosure, a fusion is provided, comprising an anti-tau antibody or an antigen-binding fragment thereof and a blood-brain barrier receptor-binding peptide. The anti-tau antibody or antigen-binding fragment thereof can be directly coupled to the blood-brain barrier receptor-binding peptide without a linker, or can be coupled through a linker. Preferably, the anti-tau antibody or antigen-binding fragment thereof can be directly coupled to the blood-brain barrier receptor-binding peptide without a linker.

[0066] In some embodiments, the anti-tau antibody or antigen-binding fragment thereof can specifically bind to an acetylated epitope of a wild-type tau protein, such as an epitope of amino acids 275 to 286 of a wild-type tau protein comprising SEQ ID NO: 25, wherein the amino acid at position 280 is acetylated.

[0067] In some embodiments, the anti-tau antibody or antigen-binding fragment thereof may comprise a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region comprises: a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:2 or consisting of the amino acid sequence of SEQ ID NO:2, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:4 or consisting of the amino acid sequence of SEQ ID NO:4, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:6 or consisting of the amino acid sequence of SEQ ID NO:6; and the light chain variable region comprises: a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:10 or consisting of the amino acid sequence of SEQ ID NO:10, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:12 or consisting of the amino acid sequence of SEQ ID NO:12, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:14 or consisting of the amino acid sequence of SEQ ID NO:14.

[0068] In some embodiments, the anti-tau antibody or antigen-binding fragment thereof may comprise a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises 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 the light chain variable region comprises 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.

[0069] In some embodiments, the anti-tau antibody or antigen-binding fragment thereof can be any one selected from the group consisting of:

[0070] (1) an anti-tau antibody or an 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;

[0071] (2) an anti-tau antibody or an 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;

[0072] (3) an anti-tau antibody or an 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;

[0073] (4) an anti-tau antibody or an 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;

[0074] (5) an anti-tau antibody or an 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;

[0075] (6) an anti-tau antibody or an 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;

[0076] (7) an anti-tau antibody or an 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;

[0077] (8) an anti-tau antibody or an 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;

[0078] (9) an anti-tau antibody or an 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;

[0079] (10) an anti-tau antibody or an 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;

[0080] (11) an anti-tau antibody or an 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;

[0081] (12) an anti-tau antibody or an 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: 37; and

[0082] (13) An anti-tau antibody or an 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.

[0083] Preferably, the anti-tau antibody or antigen-binding fragment thereof may comprise: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 8 or consisting of 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 or consisting of the amino acid sequence of SEQ ID NO: 16.

[0084] In some embodiments, the anti-tau antibody can be a full-length antibody. In addition, the anti-tau antibody can be IgG1, IgG2, IgG3, IgG4, IgA or IgA2.

[0085] For the anti-tau antibody, reference can also be made to Korean Patent No. 10-2196840, the entire disclosure of which is incorporated herein in its entirety.

[0086] In some embodiments, the blood-brain barrier receptor binding peptide can be any one selected from the group consisting of Angiopep-2 (APEP), RVG29 and TfR binding peptide (TfR). The APEP peptide can comprise the amino acid sequence of SEQ ID NO: 20 or consist of the amino acid sequence of SEQ ID NO: 20. The RVG29 peptide can comprise the amino acid sequence of SEQ ID NO: 22 or consist of the amino acid sequence of SEQ ID NO: 22. The TfR binding peptide can comprise the amino acid sequence of SEQ ID NO: 24 or consist of the amino acid sequence of SEQ ID NO: 24. In embodiments of the present disclosure, it has been identified that the anti-tau antibody exhibits excellent blood-brain barrier (BBB) ​​permeability when coupled to APEP, RVG29 or TfR binding peptide.

[0087] In some embodiments, in the fusion, the anti-tau antibody can be coupled to a blood-brain barrier receptor binding peptide via a linker. When coupling is achieved via a linker, the linker can be a known peptide capable of connecting antibody fragment domains, and can be, for example, a peptide having the amino acid sequence [GGGGS] 4. In one embodiment, preferably, the linker can be the amino acid sequence GGGGSGGGGSGGGGSGGGSG. Specifically, the blood-brain barrier receptor binding peptide can be coupled to the Fc region (e.g., the C-terminus of the heavy chain) of the anti-tau antibody via a linker.

[0088] The present disclosure is based in part on the surprising discovery that the anti-tau antibodies are able to cross the blood-brain barrier (BBB) ​​more effectively when coupled to a blood-brain barrier receptor binding peptide without a linker. Thus, in another embodiment, the fusion can couple the anti-tau antibody directly to the blood-brain barrier receptor binding peptide without a linker. For example, the anti-tau antibody can comprise a light chain and a heavy chain, and the brain-penetrating peptide can be bound to the Fc region of the heavy chain (e.g., the C-terminus of the Fc region). In embodiments of the present disclosure, significantly improved BBB permeability was observed when the APEP, RVG29 or TfR peptide (particularly the APEP or RVG29 peptide) was directly coupled to the anti-tau antibody.

[0089] The nucleotide and amino acid sequences of the anti-tau antibodies, linkers, and brain-penetrating peptides included in exemplary fusions of the present disclosure are as follows.

[0090] [Table 1]

[0091] [Table 2]

[0092] The amino acid sequences of exemplary anti-tau antibodies that can be included in the fusions of the present disclosure are shown in Table 3.

[0093] [Table 3]

[0094] In some embodiments, the anti-tau antibody or antigen-binding fragment thereof and the blood-brain barrier receptor binding peptide may comprise a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, and most preferably at least 98% sequence identity with the sequences listed in Tables 1 and 2.

[0095] In another embodiment, the anti-tau antibody or antigen-binding fragment thereof may comprise a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, and most preferably at least 98% sequence identity to the sequences listed in Table 3.

[0096] In some embodiments, amino acid sequence variants of the anti-tau antibody or antigen-binding fragment thereof and the blood-brain barrier receptor binding peptide can be used. For example, it may be desirable for the variant to improve the binding affinity and / or other biological properties of the antibody or peptide. Amino acid sequence variants of the antibody or peptide can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the molecule or by peptide synthesis. Such modifications include, for example, deletion of residues, and / or insertion of residues, and / or substitution of residues from the corresponding amino acid sequence. Various changes (including deletions, insertions, and substitutions) can be combined in any manner to obtain the final construct, provided that the final construct has the desired properties, such as antigen binding properties. Sites of interest for substitution mutations 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 for amino acid side chain categories (1) to (6). Amino acid substitutions can be introduced into the molecule of interest and the product screened for the desired activity (e.g., retained / improved antigen receptor binding, reduced immunogenicity, or improved ADCC or CDC).

[0097] [Table 4]

[0098] Amino acids can be grouped according to common side chain properties as follows:

[0099] (1) Hydrophobicity: norleucine, Met, Ala, Val, Leu, Ile;

[0100] (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln;

[0101] (3) Acidic: Asp, Glu;

[0102] (4) Basic: His, Lys, Arg;

[0103] (5) Residues that affect chain orientation: Gly, Pro;

[0104] (6) Aromatic: Trp, Tyr, Phe.

[0105] Non-conservative substitutions involve exchanging a member of one of these classes for a member of another class.

[0106] Nucleic acid, vector, host cell and preparation method

[0107] According to another aspect of the present disclosure, a polynucleotide is provided, which encodes the fusion comprising the anti-tau antibody or antigen-binding fragment thereof as disclosed herein and a blood-brain barrier receptor-binding peptide, or encodes the heavy chain and / or light chain polypeptide contained in the fusion.

[0108] In one embodiment, the nucleotide sequence encoding the fusion encodes a light chain variable region and may comprise a nucleotide sequence encoding a CDR sequence or the entire variable region sequence as listed in Table 1, or a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, and most preferably at least 98% sequence identity to the nucleotide sequence.

[0109] In one embodiment, the nucleotide sequence encoding the fusion encodes a heavy chain variable region and may comprise a nucleotide sequence encoding a CDR sequence or the entire variable region sequence as listed in Table 1, or a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, and most preferably at least 98% sequence identity to the nucleotide sequence.

[0110] 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 a CDR sequence or the entire variable region sequence as listed in Table 1 and a nucleotide sequence encoding the blood-brain barrier receptor binding peptide, or may comprise a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, and most preferably at least 98% sequence identity to the nucleotide sequence.

[0111] In one embodiment, when the blood-brain barrier receptor binding peptide is directly or via a linker linked to the heavy chain of an antibody, a nucleic acid encoding the blood-brain barrier receptor binding peptide (linker optional) and a heavy chain polypeptide comprising the heavy chain variable region of an antibody can be provided.

[0112] According to another aspect of the present disclosure, a vector comprising the polynucleotide sequence disclosed herein is provided. Unless otherwise indicated, the “vector” refers to a substance capable of transporting genetic material into a cell. In addition, in the present disclosure, the “vector” may be an “expression vector”, which comprises the necessary regulatory elements operably linked to the inserted gene so that the gene is normally expressed. In the case where the fusion comprising the anti-tau antibody comprises two separate polypeptides, the nucleotide sequences encoding the 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 fragments can be inserted. Another type of vector is a viral vector, in which a DNA or RNA sequence of viral origin is present in the vector for packaging into a virus. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors and additional mammalian vectors having a bacterial replication origin). Other vectors (e.g., non-additional mammalian vectors) can be integrated into the host cell genome after being introduced into the host cell, thereby replicating along with the host genome. Common and practical expression vectors in recombinant DNA technology are usually in the form of plasmids. In this specification, "plasmid" and "vector" are used interchangeably, as plasmid is the most commonly used form of vector.

[0113] In one embodiment, the vector may be a viral vector. Specifically, the viral vector may be at least one 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 poxvirus vector, a herpes simplex viral vector, and a phagemid vector.

[0114] In another embodiment, the vector may be a non-viral vector. Specifically, the non-viral vector may be at least one selected from the group consisting of, but not limited to: a plasmid, naked DNA, a DNA complex, mRNA (transcript), and an amplicon. For example, the plasmid may be selected from the group consisting of: pcDNA series, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series, and pUC19.

[0115] According to another aspect of the present disclosure, a host cell comprising the polynucleotide disclosed herein or an expression vector is provided. In some embodiments, the host cell can produce fusions. Any host cell known in the art allowing stable and continuous cloning and expression of the polynucleotide or expression vector can be used. Suitable prokaryotic host cells include Escherichia coli, Bacillus species (such as Bacillus subtilis and Bacillus thuringiensis), enterobacteria and bacterial strains (such as Salmonella typhimurium), Serratia marcescens, and various Pseudomonas species. Suitable eukaryotic host cells for conversion 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). In addition, the term "host cell" refers to a transformed cell or a cell that has been transformed with a nucleotide sequence and is capable of expressing a selected gene of interest. The term includes progeny of the original parental cell, whether or not the progeny is identical to the parental cell in morphology or genetic makeup, as long as the selected gene is present.

[0116] In addition, when introducing an expression vector into a host cell, a CaCl2 precipitation method, a Hanahan method (with enhanced efficiency by using DMSO (dimethyl sulfoxide) as a reducing agent in the CaCl2 precipitation method), electroporation, a calcium phosphate precipitation method, a protoplast fusion method, a stirring method using silicon carbide fiber, an Agrobacterium-mediated transformation method, a transformation method using PEG, dextran sulfate, liposomes, a drying / inhibition-mediated transformation method, etc. can be used, but the above methods are not limited thereto.

[0117] According to another aspect of the present disclosure, a method for producing the fusion disclosed herein is provided, the method comprising culturing a host cell. In some embodiments, the method comprising culturing a host cell to produce the fusion may comprise: (i) culturing the host cell to obtain a culture (e.g., a culture comprising the fusion); and (ii) recovering the fusion from the culture.

[0118] The host cells can be cultured using suitable culture media and culture conditions known in the art. Specifically, the culture can be carried out in a batch process, or continuously in a fed-batch process or a repeated fed-batch process.

[0119] Recovering the fusion from the culture can be performed by methods known in the art. The recovery method may include, but is not limited to, centrifugation, filtration, extraction, spraying, drying, precipitation, crystallization, electrophoresis, fractional precipitation (e.g., ammonium sulfate precipitation), chromatography (e.g., ion exchange, affinity, hydrophobic, and size exclusion), and the like.

[0120] Furthermore, the fusions disclosed herein can be prepared by any known method, for example, conventional synthetic methods for protein synthesis, recombinant DNA methods, and the like.

[0121] Pharmaceutical composition

[0122] According to another aspect of the present disclosure, a pharmaceutical composition comprising the fusion is provided, wherein the fusion comprises 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 preventive or therapeutically effective amount. The pharmaceutical composition can be administered to a subject to inhibit abnormal aggregation of tau protein, or to prevent or treat neurodegenerative diseases.

[0123] In some embodiments, the neurodegenerative disease can be a degenerative brain disease. In addition, the neurodegenerative disease can be a tau protein-mediated nervous system disease. The neurodegenerative disease can be selected from the group consisting of, but not limited to: 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, Hallervorden-Spatz disease and lipofuscinosis.

[0124] The tauopathy may be selected from the group consisting of, but not limited to, Alzheimer's disease, progressive supranuclear palsy, corticobasal degeneration, Pick's disease, a group of related disorders collectively known as chromosome 17-linked frontotemporal dementia with Parkinsonism (FTDP-17), amyotrophic lateral sclerosis, Creutzfeldt-Jakob disease, dementia pugilistica, Gerstmann-Straussler-Scheinker syndrome, Lewy body disease, chronic traumatic encephalopathy, and Huntington's disease.

[0125] To prepare the pharmaceutical composition, the fusion can be mixed with a pharmaceutically acceptable carrier and / or excipient. The pharmaceutical composition can be prepared in the form of a lyophilized formulation or an aqueous solution. For example, see Remington's Pharmaceutical Sciences and US Pharmacopeia: National Formulary (Mack Publishing Company, Easton, PA (1984)). The acceptable carrier and / or excipient (including stabilizers) are non-toxic to the subject at the dosage and concentration used, and may include, but are not limited to: buffers (e.g., phosphates, citrates, or other organic acids); antioxidants (e.g., ascorbic acid or methionine); preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethylammonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl alcohol, or benzyl alcohol; alkyl parabens such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight proteins (e.g., serum albumin, gelatin, or immunoglobulins); hydrophilic polymers (e.g., polyvinyl pyrrolidone); amino acids (e.g., glycine, glutamine, asparagine, histidine, arginine, or lysine); monosaccharides, disaccharides, and other carbohydrates such as glucose, mannose, or dextrins; chelating agents (e.g., EDTA); sugars (e.g., sucrose, mannitol, trehalose, or sorbitol); salt-forming counterions (e.g., sodium); metal complexes (e.g., zinc-protein complexes); and / or nonionic surfactants (e.g., or polyethylene glycol (PEG)).

[0126] The pharmaceutical composition can be used orally or parenterally according to the method for expectation.If the expectation local treatment, use in the focus can be used. Parenteral use can include but not limited to intramuscular, intravenous, intraarterial, intraperitoneal, intrapulmonary, percutaneous, subcutaneous, intradural, rectal, intravesical, intravaginal, intraarticular, intranasal, intrathecal, intracerebral or intraventricular use. Administration can be carried out by any suitable approach, for example, by injection, as intravenous injection or subcutaneous injection, and this part depends on whether to use is short-term or long-term. Different dosage regimens can be considered, include but not limited to single or multiple use at different time points, bolus use and pulse infusion.

[0127] The pharmaceutical composition can be formulated into a suitable form known in the art according to its administration route.

[0128] Prevention or treatment methods

[0129] According to another aspect of the present disclosure, a method for preventing or treating a neurodegenerative disease is provided, the method comprising administering to a subject in need thereof the fusion disclosed herein, the fusion comprising an anti-tau antibody or antigen-binding fragment thereof and a blood-brain barrier receptor binding peptide. Regarding the neurodegenerative disease, see the definition described above.

[0130] According to another aspect of the present disclosure, provided is a method for inhibiting abnormal aggregation of tau protein in a subject, the method comprising administering the fusion disclosed herein to the subject.

[0131] In some embodiments, the method for inhibiting abnormal aggregation of tau protein can be provided by administering a pharmaceutical composition comprising a fusion, wherein the anti-tau antibody or antigen-binding fragment thereof is directly coupled to the blood-brain barrier receptor-binding peptide without a linker or is coupled to the blood-brain barrier receptor-binding peptide through a linker. Preferably, the fusion can be provided by administering a pharmaceutical composition comprising a fusion, wherein the anti-tau antibody or antigen-binding fragment thereof and the blood-brain barrier receptor-binding peptide are directly linked to each other without a linker.

[0132] In some embodiments, the fusions of the present disclosure can be effectively delivered across the blood-brain barrier using various suitable compositions and methods described herein or known in the art.

[0133] In some embodiments, the fusions disclosed herein can be co-administered with one or more other agents that are effective in preventing or treating neurodegenerative diseases. The other agents include, but are not limited to, any agents that can improve or alleviate neurodegenerative diseases, such as compounds, gene therapy agents, and proteins (including antibodies). Embodiments of the invention

[0134] The present disclosure will be described in more detail below through the following examples. The following examples are provided to help understand the present disclosure. These examples are not intended to limit the scope of the present disclosure in any way and should not be interpreted as limiting the present disclosure.

[0135] Example

[0136] Example 1. Production of a fusion comprising an anti-tau antibody and a blood-brain barrier receptor binding peptide

[0137] Example 1.1. Construction of expression vector

[0138] like Figure 1a and Figure 1bAs shown in the figure, the production method of the fusion of anti-tau antibody and blood-brain barrier receptor binding peptide is as follows. First, apply to GenScript, Inc. to clone the gene sequences of three corresponding blood-brain barrier receptor binding peptides (APEP, RVG29 and TfR). The vector used for cloning is pcDNA3.1(+). For the nucleotide and amino acid sequences of these three peptides, see Table 5. For the nucleotide and amino acid sequences of the anti-tau antibody, see Table 1 and Table 2. Hereinafter, the blood-brain barrier receptor binding peptide is represented by brain penetrating peptide (BPP).

[0139] [Table 5]

[0140] The nucleotide sequences of the three brain-penetrating peptides were inserted into the vector using restriction enzymes XhoI and XbaI, and the vector thus constructed was as follows: Figure 2 As shown. Next, primers for inserting the linker were prepared as shown in Table 6 and used to amplify the nucleotide sequence of each peptide and a portion of the vector by PCR. The nucleotide sequence was inserted into the XhoI-XbaI site of the pcDNA3.1(+) vector. Specifically, TfR with a linker sequence ([GGGGS]4) was synthesized and cloned to remove the linker sequence.

[0141] [Table 6]

[0142] For the fusion, a total of six vectors were constructed using the three brain-penetrating peptides, depending on the presence or absence of a linker. Then, the heavy chain gene of the anti-tau antibody (H1) was inserted upstream of the brain-penetrating peptide (or linker-brain-penetrating peptide) using restriction enzymes BamHI and XhoI to construct an expression vector ( Figure 3a In addition, the light chain gene was inserted into the pcDNA3.1(+) vector using restriction endonucleases HindIII and XbaI to construct an expression vector ( Figure 3b ).

[0143] At the same time, for the anti-tau antibody (H1), the heavy chain gene was inserted into the pcDNA3.1(+) vector using restriction endonucleases BamHI and XhoI, and the light chain gene was inserted into the pcDNA3.1(+) vector using restriction endonucleases HindIII and XbaI, thereby constructing an expression vector that does not contain a brain-penetrating peptide (or linker-brain-penetrating peptide) sequence.

[0144] Example 1.2. Production of recombinant fusions by culturing Expi293F cells

[0145] The recombinant vector of Example 1.1 was introduced into Expi HEK293F cells to produce a fusion product comprising an antibody and a peptide.

[0146] First, ExpiHEK293F cells were subcultured by incubating them in a shaking incubator (37°C, 8% CO2, 120 rpm) for 2 to 3 days until the cells reached 5 × 10 5 The cell count was calculated using FreeStyle 500 cells / mL supplemented with 1X antibiotic-antimycotic. TM 293 expression medium.

[0147] Next, to introduce the gene, 200 μg of plasmid vector was added to 10 mL of 150 mM NaCl and mixed thoroughly. Then, 400 μL of PEI (polyethyleneimine hydrochloride, 1 mg / mL) (based on 100 mL of culture medium) was added thereto. After 15 minutes at room temperature, the solution was added dropwise to the cell culture medium. The cells were then cultured continuously for 5 to 7 days. Based on the cell culture medium, the number of cells for gene introduction was adjusted to 2 × 10 6 cells / mL.

[0148] The cell culture fluid was centrifuged at 13,000 rpm for 15 minutes at 4 ° C. Only the supernatant (cells have been removed therefrom) was collected, filtered through a 0.45 μm filter, and then purified by flowing through a MabSelect SURE column (Cytiva, HiTrap Protein G) balanced with 1X PBS. In order to elute the antibody bound to the column, 0.1 M glycine-HCl (pH 2.7) buffer was passed through the column while collecting the eluted antibody. In order to neutralize the pH, 1 M Tris-HCl (pH 9.0) buffer was added to restore the pH to between 7 and 8, and the final buffer was then exchanged with 1X PBS.

[0149] Through the above process, the fusion compound comprising the antibody and the brain-penetrating peptide was obtained, and the obtained antibody and fusion compound are shown in Table 7.

[0150] [Table 7]

[0151] Example 2. Generation of a blood-brain barrier (BBB) ​​model using induced pluripotent stem cells (iPSCs)

[0152] In order to examine the BBB permeability of the fusion according to the embodiments of the present disclosure, referring to 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), a BBB model was established as follows using differentiation of human brain microvascular endothelial cells.

[0153] Example 2.1. Induction of differentiation into human brain microvascular endothelial cells (iBMECs)

[0154] Monoculture

[0155] To create a blood-brain barrier (BBB) ​​model, induced pluripotent stem cells (iPSCs) (WiCell Research Institute Inc, IMR90-4) were induced to differentiate into human brain microvascular endothelial cells (BMECs).

[0156] First, iPSCs were cultured at 3 × 10 5 Cells were seeded / well in a Matrigel-coated 6-well plate and subsequently cultured by adding mTeSR (containing 10 μM Y27, 2 mL / well). The entire medium was replaced with mTeSR1 (without Y27) every 24 hours after seeding. When the cells reached 2.5×10 5 to 4×10 5 cells / well (preferably 3×10 5 When the cell count reached 10 cells / well, the culture medium was replaced with 2 mL of UM (DMEM / F12 culture medium (containing 15 mM HEPES), 20% KOSR, 1% NEAA, 0.5% glutamax and β-ME) culture medium per well to induce differentiation into induced BMECs (iBMECs) (UM period, D0). On days 1 to 5 (D1 to D5) after the start of differentiation induction, the culture medium was replaced with UM (containing 1% B27) every day (2 to 3 mL / well); and on day 6, the culture medium was replaced with EC (containing hESFM culture medium, 2% B27 and 0.02% bFGF) containing 0.1% retinoic acid (RA).

[0157] On day 7 after differentiation induction, the transwells (consisting of a culture plate and insert) used for subculturing iBMECs were coated with an ECM solution (collagen:fibronectin:water = 4:1:5) as follows. Prior to subculturing, the membrane filters in the transwell inserts were coated with the ECM solution at 37°C for at least 4 hours or up to 24 hours. Culture plates were coated only when performing triple culture.

[0158] On day 8 after differentiation induction, the ECM solution was removed from the transwell inserts, and the inserts were then dried. The cultured iBMECs were collected using Accutase and hESFM medium, resuspended in an appropriate volume of EC medium (containing 0.1% RA), and plated at 1×10 6 cells / cm 2 Seed into transwell inserts. Place the inserts seeded with iBMECs on a culture plate and rock the plate back and forth or side to side to prevent the cells from clumping and distribute them evenly. Then incubate the plate in a 37°C incubator and replace the culture medium with EC (containing hESFM medium with 2% B27) every 24 hours.

[0159] The above differentiation process is shown in the schematic diagram Figure 4 shown.

[0160] Triple cultivation

[0161] Triple culture was performed in the same manner as iBMECs monoculture until day 6 after differentiation induction. On day 7, astrocytes (ScienceCell, product number #1800) and pericytes (ScienceCell, product number #1200) were seeded onto 12-transwell culture plates using the following two methods. Astrocytes (4.4×10 4 cells) and pericytes (4.4×10 4 cells) were cultured in pericyte culture medium at a ratio of 1:1 or astrocytes (1.5×10 4 cells) and pericytes (3×10 4 cells) were cultured in 500 μL pericyte culture medium at a ratio of 1:2.

[0162] On day 8 after differentiation induction, the culture medium in the culture plate was replaced with endothelial cell culture medium (containing 10 μM RA), and iBMECs were seeded into transwell inserts in the same manner as monoculture to prepare a BBB model containing three types of cells (endothelial cells, astrocytes, and pericytes).

[0163] For monoculture or triculture models seeded into transwells, medium changes were performed by removing medium from the plate and then from the insert. hESFM medium (containing 2% B27) was added by adding medium to the insert and then to the plate.

[0164] Example 2.2. Performance evaluation of the BBB model

[0165] To validate the performance of iBMECs differentiated in transwells as a BBB model, iBMECs were immunostained and the expression of tight junction markers was observed by fluorescence microscopy. Furthermore, tight junction formation was verified by measuring the transendothelial electrical resistance (TEER) of the transwells in which iBMECs differentiated and by assessing their permeability to low-molecular-weight substances.

[0166] Identification of cell tight junction markers and BBB-related protein expression

[0167] On days 6 to 10 after the start of differentiation into iBMECs, to identify the differentiation of iBMECs and the formation of the BBB, immunostaining of marker proteins constituting the BBB was performed and observed under a fluorescence microscope (Nikon, ECLIPSE Ti). Figure 5 As shown, differentiated iBMECs were identified to express occludin, claudin 5, and ZO-1, which are markers of tight junctions and adherens junctions, and glut1, TfR, and LRP1, which are transporters expressed in the BBB.

[0168] Measurement of TEER values ​​and identification of permeability to low molecular weight substances

[0169] TEER values ​​were measured 9 to 11 days after initiating differentiation into iBMECs. The measurement method was as follows: First, the transwell was removed from the incubator and allowed to stand at room temperature for 20 to 30 minutes to ensure that its resistance did not fluctuate. Next, a measuring rod was placed in the middle of the gap between the culture plate wall and the insert, and the TEER value was then measured.

[0170] In order to evaluate the permeability of substances to iBMECs, TEER values ​​were measured in transwells seeded with iBMECs to preliminarily identify the correct formation of tight junctions. 3kDa dextran (labeled with Cascade Blue) at a concentration of 50 μg / mL was added to a total of five transwell inserts with differentiated iBMECs and controls (empty transwells) and allowed to permeate for 24 hours. The amount of dextran in the culture plates and inserts was then measured. The results are shown in Table 8. For the control, dextran was detected in the culture plate from 1 hour after addition, and 50% or more of the total dextran was detected in the culture plate within 24 hours. On the other hand, for transwells (wherein iBMECs differentiated), it was identified that no dextran escaped into the culture plate, which proves that tight junctions have been correctly formed and are sufficient to prevent the passage of low molecular weight substances.

[0171] [Table 8]

[0172] Example 3. Identification of BBB permeability of fusions

[0173] On day 9 to day 10 after induction of differentiation into iBMECs, the TEER value of the transwell was measured according to the TEER value measurement method of Example 2.2. Then, one antibody and six fusions produced in Example 1 were applied to the transwell inserts, and the BBB permeability of the antibodies and fusions was measured by permeability coefficient (Papp) measurement and enzyme-linked immunosorbent assay (ELISA).

[0174] At the same time, after the administration of the antibody and fusion, the TEER value of each transwell was measured to check the average value. The results showed that after the administration of the antibody and fusion, the average value was about 4000Ω×cm 2 ( Figure 6 ), which was similar to the average value before administration. Therefore, it was determined that the administration of the antibodies and fusions did not affect the performance of the BBB model and did not cause any variation between samples.

[0175] Example 3.1. Measurement of permeability coefficient (Papp)

[0176] The permeability coefficient (Papp) is an absolute value that indicates the rate at which a substance can pass through the transwell per unit area and per unit time. Papp is a comparable value regardless of the transwell surface area and time used in the experiment and can be expressed by the following formula.

[0177] Papp = (dQ / dt) / (C0×A)

[0178] Here, dQ / dt represents the amount of material that escapes into the culture plate (substrate) area per unit time; A represents the area of ​​the transwell insert (cm 2 ); and C0 represents the initial concentration of the sample introduced into the insert (top) region of the transwell.

[0179] 0.5 mL of hESFM culture medium (containing 2% B27) was prepared in a 1.5 mL test tube, and 7 types of antibodies and fusions (25 μg / mL) were added thereto to prepare antibody-containing solutions. 0.4 mL of culture medium was removed from the insert area of ​​the transwell, and 0.4 mL of the 0.5 mL antibody-containing solution was subsequently added thereto. 24 hours (D11) after application of the antibody-containing solution, 1.5 mL of culture medium in the culture plate area and 0.5 mL of culture medium in the upper chamber were collected for ELISA analysis. dQ / dt values ​​were calculated based on the results obtained by ELISA analysis. The area of ​​the wells used (whether 12-well transwell (insert: 1.12 cm) was used as the test tube. 2 , culture plate: 3.5cm 2 ), or 24-well transwell (insert: 0.33 cm 2 , culture plate: 2cm 2 )) replaced variable A; and the remaining 0.1 mL of antibody-containing solution after adding the transwell insert was used to measure the C0 value.

[0180] Example 3.2. Enzyme-linked immunosorbent assay (ELISA)

[0181] The ELISA assay for antibodies and fusions that penetrate from the insert area to the plate area is as follows.

[0182] Pep1 was diluted 1:2000 (0.83 mg / mL) using capture buffer (50 mM NaHCO3 and distilled water, pH 9.6). 50 μL of the diluted solution was added to the culture plate and incubated (37°C, 600 rpm, 2 hours) for coating. Then, 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 (37°C, 600 rpm, 2 hours).

[0183] Next, using the standard dilution analysis (STD) technique, 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). 50 μL of the standard solution or sample was added to each well and incubated (37°C, 600 rpm, 1 hour).

[0184] 50 μL of the antibody-containing solution (peroxidase-labeled human IgG, 1:2000) (Sigma-Aldrich, AP309P) was added to each well and incubated (37°C, 600 rpm, 1 hour). Then, 50 μL of substrate reagent (R&D Systems, #DY999) was added to each well and incubated at room temperature for 5 minutes. Subsequently, 50 μL of stop solution (R&D Systems, #DY994) was added to terminate the reaction, and the absorbance was measured using a spectrophotometer (Infinite F50) (culture plate shaking for 10 seconds / measurement wavelength 450 nm / reference wavelength 620 nm).

[0185] Washing was performed four times in the following manner: each well was filled with a wash buffer (PBS containing 0.05% Tween 20), and then the buffer was discarded between the steps except for the step of adding a stop solution after the reaction with the substrate reagent.

[0186] Example 3.3. BBB permeability measurement results of fusions

[0187] The single culture BBB model is composed of only vascular endothelial cells; whereas the triple culture BBB model presents a form comprising not only vascular endothelial cells but also astrocytes and pericytes, which is more similar to the microenvironment of the human blood-brain barrier. For the antibody-brain penetrating peptide fusion (without a joint) and the antibody-linker-brain penetrating peptide fusion (with a joint), its permeability was verified in a single culture BBB model. Then, for the antibody-brain penetrating peptide fusion that showed statistically significant results, its permeability was also verified in a triple culture BBB model, and the final data were presented as a total result combining the results from the two models. Based on the production date, producer, concentration and assay method of the antibody and fusion, a total of four batches were measured; and each batch was measured separately or in the form of a mixed batch (Figures 7 and Figure 8 ).

[0188] Antibody-brain penetrating peptide fusion (no linker)

[0189] The overall average Papp values of the H1 antibody and three antibody-brain penetrating peptide fusions (H1-APEP, H1-RVG29, and H1-TfR) in the single culture BBB model were divided by the average Papp value of H1 to represent the ratio of each fusion. As a result, all three fusions showed higher permeability than the H1 antibody. In addition, the same method was used to represent their permeability in the triple culture BBB model. As a result, all three fusions showed higher permeability than the H1 antibody (Table 9). Regarding the overall results combining the permeability results of the single culture and triple culture, H1-APEP, H1-RVG29, and H1-TfR showed permeabilities 2.9-fold, 2.7-fold, and 2.3-fold that of the H1 antibody, respectively. The overall results of the H1 antibody and the three antibody-brain penetrating peptide fusions are as Figure 7a shown, with the vertical axis representing the Papp value (* indicates 0.01 < P < 0.05, and ** indicates 0.001 < P < 0.01).

[0190] [Table 9]

[0191] The results obtained by statistically processing the ratio values (calculated by dividing the Papp value of each of the three fusions by the Papp value of the H1 antibody) also showed that in the BBB model, all three fusions showed higher permeability than the H1 antibody. Specifically, H1-APEP, H1-RVG29, and H1-TfR showed permeabilities 3.54-fold, 3.04-fold, and 2.74-fold that of the H1 antibody, respectively ( Figure 7b ).

[0192] Antibody-Linker-Brain Penetrating Peptide Fusions (with Linker)

[0193] The overall average Papp values of the H1 antibody and three antibody-linker-brain penetrating peptide fusions (H1-L4-APEP, H1-L4-RVG29, and H1-L4-TfR) in the single culture BBB model were divided by the average Papp value of H1 to represent the ratio of each fusion. As a result, all three fusions showed slightly higher permeability than the H1 antibody (1.9-fold for H1-L4-APEP, 2.4-fold for H1-L4-RVG29, and 1.8-fold for H1-L4-TfR); however, their permeability was lower than that of the fusions without the linker. Here, L4 represents the linker.

[0194] The results obtained by dividing the average Papp value of the fusion by the average Papp value of H1 are shown in Table 10. For the antibody-head-brain penetrating peptide fusion, the results of the single culture represent the overall results. The overall results (for the single culture) of the H1 antibody and the three antibody-head-brain penetrating peptide fusions are shown in Table 10. Figure 8 As shown, the vertical axis represents the Papp value.

[0195] [Table 10] Average value / average value of H1 H1 H1-L4-APEP H1-L4-RVG29 H1-L4-TfR Overall results 1.0 1.9 2.4 1.8 Monoculture 1.0 1.9 2.4 1.8

[0196] Statistical analysis

[0197] Statistical analysis was performed using GraphPad Software (GraphPad Software, San Diego, CA, USA). Data were analyzed using one-way analysis of variance and Kruskal-Wallis test (nonparametric analysis). In addition, Dunn's multiple comparison was used to compare the significance of the differences between the H1 antibody group alone and the H1 antibody group fused with three brain-penetrating peptides (APEP, RVG29, and TfR) (*: 0.01). <P<0.05,**:0.001<P<0.01,***:P<0.001,ns:P> 0.05) for analysis. Sequence Listing Free Text

[0198] Sequence information for the skipped sequences (less than 10 unambiguous nucleotides: SEQ ID NO: 11; and less than 4 unambiguous amino acids: SEQ ID NO: 12) in the electronic sequence listing file attached to this specification is provided below.

[0199] SEQ ID NO:11

[0200] Sequence length: 9

[0201] Sequence type: DNA

[0202] Organism Qualifier: Synthetic Construct

[0203] Sequence name: Nucleotide sequence of CDR2 of anti-tau antibody (VL)

[0204] sequence:

[0205] ctggtgtcc

[0206] SEQ ID NO:12

[0207] Sequence length: 3

[0208] Sequence type: AA

[0209] Organism Qualifier: Synthetic Construct

[0210] Sequence name: Amino acid sequence of CDR2 of anti-tau antibody (VL)

[0211] sequence:

[0212] 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 according to claim 1, wherein The anti-tau antibody is directly coupled to the blood-brain barrier receptor binding peptide without a linker.

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

24.

5. The fusion according to claim 1 or 2, wherein The anti-tau antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), The heavy chain variable region comprises: 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; The light chain variable region comprises: 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 according to claim 1 or 2, wherein The anti-tau antibody or antigen-binding fragment thereof binds to an epitope of amino acids 275 to 286 of the wild-type tau protein comprising SEQ ID NO: 25, wherein the amino acid at position 280 is acetylated.

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

16.

8. The fusion according to claim 1 or 2, wherein The anti-tau antibody or antigen-binding fragment thereof comprises: (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.

9. The fusion according to 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 according to claim 1 or 2, wherein The fusion crosses the blood-brain barrier and binds specifically to tau protein.

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

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

13. A polynucleotide encoding the fusion of claim 1 or 2. An expression vector comprising the polynucleotide according to claim 13 . A host cell comprising the expression vector according to claim 14 .

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

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

18. The pharmaceutical composition according to claim 17, wherein The neurodegenerative disease is a nervous system disease mediated by tau protein.

19. The pharmaceutical composition according to claim 17, wherein The neurodegenerative disease is selected from the group consisting of 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, Hallervorden-Spatz disease and lipofuscinosis.

20. The pharmaceutical composition according to claim 19, wherein The tauopathy is selected from the group consisting of Alzheimer's disease, progressive supranuclear palsy, corticobasal degeneration, Pick's disease, a group of related disorders collectively known as chromosome 17-linked frontotemporal dementia with Parkinsonism (FTDP-17), amyotrophic lateral sclerosis, Creutzfeldt-Jakob disease, dementia pugilistica, Gerstmann-Straussler-Scheinker syndrome, Lewy body disease, chronic traumatic encephalopathy, and Huntington's disease.

21. The pharmaceutical composition according to claim 17, wherein The pharmaceutical composition is administered by any one administration route selected from the group consisting of intramuscular, intravenous, intraarterial, intraperitoneal, transdermal, subcutaneous, intradural, intracerebral, intracerebroventricular, intrapulmonary or intranasal administration.

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

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