Antibodies to alpha-synuclein and uses thereof

Abstract

The present invention provides an anti-alpha-synuclein antibody that preferentially recognizes alpha-synuclein (α-Syn) aggregates, and uses of the antibody for the detection, diagnosis, and / or treatment or prevention of various diseases caused by the accumulation of alpha-synuclein aggregates or their related symptoms and disorders. [Solution] An antibody or antigen-binding fragment thereof that specifically binds to α-Syn aggregates, wherein the antibody or antigen-binding fragment comprises a heavy chain CDR1 having a specific amino acid sequence, a heavy chain CDR2 having a specific amino acid sequence and a heavy chain CDR3 having a specific amino acid sequence, a light chain CDR1 having a specific amino acid sequence, a light chain CDR2 having a specific amino acid sequence, and a light chain CDR3 having a specific amino acid sequence.

Description

[Technical field]

[0001] The present invention relates to a technology relating to an alpha-synuclein antibody and uses thereof. [Background technology]

[0002] Alpha-Synuclein (α-syn) is expressed primarily in the presynaptic terminals of neurons and exists as a monomer in an unfolded state under normal conditions. Alpha-Synuclein helps regulate the release of dopamine, a neurotransmitter important for controlling the initiation and termination of voluntary and involuntary movements. In particular, the function of alpha-synuclein is important for increasing synaptic activity and as we age, it is an important factor in neurodegeneration.

[0003] However, in pathological conditions, alpha-synuclein undergoes structural changes upon binding and interaction with droplets, phospholipid bilayers or lipid membranes, etc., to fold or form a folded α-helical secondary structure to form aggregates containing dimers, oligomers and / or fibrillar morphologies of the molecule.

[0004] Such alpha-synuclein aggregates are known to induce toxicity in cells and are the main component of Lewy bodies, abnormal protein aggregates found in neurons in Parkinson's disease (PD), Parkinson's disease dementia (PDD), multiple system atrophy (MSA), dementia with Lewy bodies (DLB), and various other diseases. In addition, post-translational modifications such as phosphorylation and ubiquitination of alpha-synuclein are also known to be related to alpha-synuclein aggregation and neurotoxicity. In animal and cell experiments, alpha-synuclein has been shown to kill dopamine neurons, induce inflammatory responses, and induce motor symptoms similar to those of Parkinson's disease in experimental animals. Alpha-synuclein aggregation is also known to be involved in the pathogenesis of a group of neurodegenerative diseases called α-synucleinopathies, which include Parkinson's disease, Parkinson's disease dementia, dementia with Lewy bodies, multiple system atrophy, and many other axonal diseases.

[0005] Furthermore, oligomeric and monomeric forms of alpha-synuclein have all been found in cerebrospinal fluid and plasma samples from Parkinson's disease patients, indicating that small molecular weight alpha-synuclein aggregates can penetrate cell membranes and access the extracellular space. It has also been revealed that folded alpha-synuclein can be released from cells by exocytosis and then transferred from one region of the brain to another by cell-to-cell transmission, similar to prion protein.

[0006] Therefore, alpha-synuclein has become a target for the development of therapeutic agents for synucleinopathies such as Parkinson's disease. The main development strategies include inhibition of aggregate formation, gene silencing, and aggregate removal. In the former case, epigallocatechin-3-gallate (EGCG), 3-(1,3-benzodioxol-5-yl)-5-(3-bromophenyl)-1H-pyrazole (anle138b, CLR0120, and the prolyl oligopeptidase inhibitor KYP-20479.

[0007] In the case of small molecule compounds, the target specific binding strength is reduced and high doses must be administered due to their low half-life. Although antibodies are target specific and exhibit a high half-life compared to small molecule compounds, in order to increase the possibility of disease treatment, antibodies that preferentially bind to aggregates with high binding strength are required. Therefore, for the treatment of diseases associated with abnormal accumulation of alpha-synuclein aggregates, it is necessary to develop antibodies that can preferentially bind to alpha-synuclein, particularly alpha-synuclein aggregates. Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention provides an antibody or an antigen-binding fragment thereof that can specifically bind to alpha-synuclein, particularly to alpha-synuclein aggregates. The antibody or an antigen-binding fragment thereof against alpha-synuclein can specifically recognize and bind to alpha-synuclein, and may particularly bind to the C-terminal site of alpha-synuclein.

[0009] One embodiment of the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to alpha-synuclein or alpha-synuclein aggregates and reduces the level of alpha-synuclein in the nervous system of a subject. More specifically, the antibody or antigen-binding fragment thereof of the present invention relates to an antibody or antigen-binding fragment thereof that has high binding affinity to alpha-synuclein aggregates, particularly amyloid fibrils, protofibrils and oligomers, particularly amyloid fibrils, and low binding affinity to alpha-synuclein monomers.

[0010] The antibody or antigen-binding fragment thereof according to the present invention may have one or more activities selected from the group consisting of (i) specific binding to alpha-synuclein or alpha-synuclein aggregates, (ii) reducing the level of alpha-synuclein or alpha-synuclein aggregates in the nervous system of a subject, (iii) reducing or inhibiting cell-to-cell transmission of alpha-synuclein or its aggregates between neurons, and (vi) enhancing phagocytic uptake of alpha-synuclein or its aggregates by neurons. Preferably, the antibody or antigen-binding fragment thereof according to the present invention may have all of the activities (i) to (iv).

[0011] One example of the present invention relates to a composition for preventing, alleviating, ameliorating or treating a synucleinopathic disease comprising an antibody or antigen-binding fragment thereof capable of specifically binding to alpha-synuclein, in particular alpha-synuclein aggregates.

[0012] One embodiment of the present invention relates to a method for preventing, alleviating, ameliorating or treating a synucleinopathic disease comprising administering to a subject in need thereof an antibody or antigen-binding fragment thereof capable of specifically binding to alpha-synuclein, particularly alpha-synuclein aggregates, The antibody or antigen-binding fragment thereof may be administered in an amount effective to reduce the level of alpha-synuclein, particularly alpha-synuclein aggregates, in the nervous system of the subject, more particularly in extraneuronal regions of the nervous system.

[0013] One example of the present invention relates to a method for reducing the levels of alpha-synuclein, particularly alpha-synuclein aggregates, in the nervous system of a subject, and more particularly in extraneuronal regions of the nervous system, comprising an antibody or antigen-binding fragment thereof capable of specifically binding to alpha-synuclein, particularly alpha-synuclein aggregates, or use of the antibody for reducing the levels of alpha-synuclein, particularly alpha-synuclein aggregates, in the extraneuronal regions of the nervous system.

[0014] One example of the present invention relates to a method for reducing or inhibiting interneuronal cell-to-cell transmission of alpha-synuclein or aggregates thereof in the nervous system of a subject, more particularly, or use of the antibody or antigen-binding fragment thereof capable of specifically binding to alpha-synuclein, in particular alpha-synuclein aggregates, for reducing or inhibiting interneuronal cell-to-cell transmission of alpha-synuclein or aggregates thereof in the nervous system.

[0015] One example of the present invention relates to a method for increasing phagocytic uptake of alpha-synuclein or aggregates thereof by microglia in the nervous system of a subject, comprising an antibody or antigen-binding fragment thereof capable of specifically binding to alpha-synuclein, in particular alpha-synuclein aggregates, or use of increasing phagocytic uptake of alpha-synuclein or aggregates thereof by microglia in the nervous system of a subject. [Means for solving the problem]

[0016] The present invention will now be described in further detail. The present invention provides an antibody or antigen-binding fragment thereof capable of specifically binding to alpha-synuclein, particularly alpha-synuclein aggregates. The antibody or antigen-binding fragment thereof against alpha-synuclein may be capable of specifically recognizing and binding to alpha-synuclein but not to beta (β)- or gamma (γ)-synuclein, and may be capable of specifically binding to alpha-synuclein aggregates but not to amyloid beta and tau aggregates.

[0017] In the present application, the term "antigen" or "immunogen" refers to a molecule or a portion of a molecule that can be bound by an antigen-binding protein (e.g., an antibody or an immunologically functional antigen-binding fragment thereof) and can be used to generate an antibody capable of binding to the antigen in an animal. An antigen can include one or more epitopes that can interact with different antibodies or fragments thereof. In one embodiment according to the present application, the antigen can be an alpha-synuclein protein or an alpha-synuclein aggregate. Specifically, the alpha-synuclein aggregate can be amyloid fibrils, protofibrils, and oligomers, in particular amyloid fibrils. The antibody or antigen-binding fragment thereof that specifically binds to alpha-synuclein or its aggregates according to the present invention can specifically bind to the C-terminal portion of alpha-synuclein. Alpha-synuclein antibodies that recognize the N-terminus cannot recognize aggregates in other diseases such as multiple system atrophy, which belongs to the synucleinopathies commonly known as alpha-synuclein-related diseases, whereas alpha-synuclein antibodies that recognize the C-terminus have the advantage of recognizing aggregates not only in Parkinson's disease but also in a variety of other synucleinopathies.

[0018] As used herein, the term "neutralizing" refers to inhibiting or reducing the biological activity of an antigen when the binding protein specifically binds to the antigen. In one embodiment, the neutralizing protein or antibody binds to an antigen (alpha-synuclein or alpha-synuclein aggregates) and reduces the level of the antigen in the extracellular space of a subject's nervous system by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%, or can prevent an increase in the level of the antigen in the extracellular space of a subject's nervous system.

[0019] In the present application, "alpha-synuclein" refers to native α-Syn (NCBI ID: NP_000336) composed of 140 amino acid residues encoded by the SNCA gene, including full-length unprocessed forms as well as various processed forms. It also includes naturally occurring variants of alpha-synuclein, such as mutations, splice variants, or allelic variants. In addition to the 140-residue protein, there are also 126- and 112-residue protein forms with deletions of exons 3 and 5 (CAG3339.1). It is also known that certain polymorphisms or missense mutations in the SNCA gene are associated with the occurrence of Parkinson's disease, and such mutants are also included. There are beta- and gamma-synucleins, which are homologs of alpha-synuclein. In one embodiment, the antibody of the present application specifically recognizes alpha-synuclein. The amino acid sequence of alpha-synuclein is represented by SEQ ID NO: 225.

[0020] As used herein, "alpha-synuclein aggregates" refers to structures or aggregates that result from changes in the conformation of alpha-synuclein and include oligomers, protofibrils, fibrils, and / or structures containing one or more of the foregoing structures.

[0021] The alpha-synuclein recognized as an antigen by the antibody provided herein may be selected from mammalian alpha-synuclein such as human alpha-synuclein, monkey alpha-synuclein (e.g., Rhesus alpha-synuclein), mouse alpha-synuclein, and rat alpha-synuclein, for example, human alpha-synuclein may be, but is not limited to, alpha-synuclein (NCBI ID: NP_000336). Unless otherwise stated herein, alpha-synuclein may refer to human alpha-synuclein, and the antibody or antigen-binding fragment thereof provided herein may have specific binding ability not only to human alpha-synuclein, but also to monkey (e.g., Rhesus), rat, and / or mouse alpha-synuclein.

[0022] The antibody or antigen-binding fragment thereof binds to the C-terminal site of alpha-synuclein, and specifically, human alpha-synuclein protein may be a C-terminal region of the amino acid sequence of SEQ ID NO: 126, for example, a C-terminal region containing a peptide consisting of at least 11 or 12 consecutive amino acids including residues 110 to 120 or residues 111 to 122. It has been confirmed that the antibody or antigen-binding fragment thereof according to the present invention recognizes the antigen recognition site and binds to alpha-synuclein aggregates with high affinity.

[0023] As used herein, "affinity" refers to the strength of interaction between an antibody or antigen-binding fragment thereof and an antigen, and can be determined by the CDR sequence of the antibody or antigen-binding fragment, and / or the physicochemical properties of the antibody or antigen-binding fragment (e.g., hydrophilicity / hydrophobicity, electrostatic properties, etc.), and the characteristics of the antigen, such as the size, shape, and / or charge of the antigen. Methods for determining such affinity are publicly known in the art, and can usually be expressed as a dissociation constant (KD), but are not limited thereto.

[0024] As used herein, the term "specifically binds to alpha-synuclein protein or alpha-synuclein aggregates" means that the affinity for alpha-synuclein protein or alpha-synuclein aggregates is relatively high compared to other antigens, and for example, the affinity for alpha-synuclein aggregates, specifically amyloid fibrils, protofibrils, and oligomers, particularly amyloid fibrils, is 0.1 x 10 or higher by Octet and SPR analysis, respectively. -10 M~2×10 -10 M, or 0.05 x 10 -10 M~0.3×10 -9 M, but is not limited to this.

[0025] Humanized alpha-synuclein antibodies comprising light and heavy chains according to an embodiment of the present invention, such as Hu11F11_(ver.1), Hu11F11(ver.2), and Hu11F11_ABL2-4, exhibit higher phagocytosis-promoting activity than chimeric alpha-synuclein antibodies. Hu11F11(ver.1), Hu11F11(ver.2), Hu11F11(ver.3), Hu11F11(ver.4), and ABL2-4 exhibit higher inhibitory ability against fibril binding to nerve cell membranes than chimeric alpha-synuclein antibodies, and Hu11F11(ver.2), Hu11F11(ver.4), and ABL2-4 exhibit higher inhibitory ability against the spread of alpha-synuclein secreted from alpha-synuclein overexpressing cells to other nerve cells than chimeric alpha-synuclein antibodies. The binding avidity to alpha-synuclein aggregates, for example as measured in a cell-based assay, is similar to or superior to that of chimeric alpha-synuclein antibodies.

[0026] The alpha-synuclein antibody according to the present invention inhibits cell-to-cell transmission of aggregates, which are secreted outside of neurons in the nervous system of a subject, from moving to normal cells in the extracellular space and infecting the corresponding neurons, and also has the ability to promote phagocytosis of microglia against alpha-synuclein aggregates located in the extracellular space. Like prions, alpha-synuclein aggregates spread from one cell to another, and alpha-synuclein, especially alpha-synuclein aggregates, spread throughout the brain in corresponding normal cells, causing synucleinopathies. Therefore, it is well known that alpha-synuclein aggregates are toxic to brain neurons, causing neurodegeneration and neuroinflammation. As alpha-synuclein aggregates spread to various parts of the brain, brain cell death and brain inflammatory responses increase, which leads to brain cell death and the resulting behavioral and cognitive impairments that are seen as synucleinopathies, such as Parkinson's disease, progress.

[0027] Therefore, the alpha-synuclein antibody of the present invention can inhibit the interneuronal movement of alpha-synuclein or alpha-synuclein aggregates, thereby preventing the spread of alpha-synuclein aggregates to various regions of the brain, and can also promote the phagocytosis of microglia, thereby reducing or removing alpha-synuclein aggregates themselves present outside neurons in the target nervous system, thereby reducing the level of alpha-synuclein aggregates, which are an important cause of synucleinopathies, thereby reducing brain neuronal death and brain inflammatory responses, and can be expected to have the effect of improving, alleviating or preventing the symptoms and progression of synucleinopathies, such as Parkinson's disease.

[0028] In addition, the alpha-synuclein antibody according to the present invention has excellent activity in that it can perform both of the following two functions: (i) inhibiting the interneuronal movement of alpha-synuclein or alpha-synuclein aggregates, and (ii) reducing the level of alpha-synuclein aggregates in the brain and nervous system by promoting the phagocytosis of microglia (see the results of the cell-based assay of the present application). In particular, the alpha-synuclein antibodies currently undergoing clinical trials or published in papers have one of the activities (i) and (ii), which indicates that the alpha-synuclein antibody of the present application has an advantage in preventing or treating synucleinopathies compared to known alpha-synuclein antibodies. Therefore, the alpha-synuclein antibody according to the present invention has a better effect in removing and reducing alpha-synuclein aggregates and inhibiting their pathological action, and is therefore more effective in treating synucleinopathies or symptomatic diseases related thereto (e.g., cognitive impairment, etc.).

[0029] The antibodies or antigen-binding fragments of the present application that have high affinity for alpha-synuclein aggregates can reduce alpha-synuclein aggregate formation and lower the concentration of aggregates in the brain. Also, the antibodies or antigen-binding fragments of the present application that have high affinity for alpha-synuclein aggregates can reduce alpha-synuclein aggregate formation outside the central nervous system, ultimately altering the equilibrium between alpha-synuclein forms across the blood-brain barrier, resulting in lower concentrations of aggregates in the central nervous system.

[0030] This has a great clinical advantage since sufficient efficacy can be obtained even if the antibody is administered by a more convenient method such as, but not limited to, subcutaneous injection. Furthermore, the antibody or antigen-binding fragment of the present application having high affinity for alpha-synuclein aggregates can inhibit and / or reduce alpha-synuclein aggregate formation and / or accumulation and / or intercellular transmission, thereby reducing the concentration of aggregates in the brain. Furthermore, the antibody or antigen-binding fragment of the present application having high affinity for alpha-synuclein aggregates can reduce alpha-synuclein aggregate formation outside the central nervous system, which can ultimately change the equilibrium between alpha-synuclein forms at the blood-brain barrier, thereby reducing the concentration of aggregates in the central nervous system. Furthermore, without being limited by this theory, the antibody or antigen-binding fragment of the present application can inhibit aggregate formation by removing monomers, or remove both monomers and aggregates.

[0031] The antibodies or antigen-binding fragments thereof that specifically bind to alpha-synuclein protein or alpha-synuclein aggregates provided herein may be non-naturally occurring (e.g., chemically synthesized or recombinantly produced). Such recombinant techniques are widely known in the art.

[0032] As used herein, "antibody" refers to an intact immunoglobulin of any isotype, or an antigen-binding fragment that can compete with the intact antibody for binding to a target antigen, including, for example, chimeric, humanized, fully human, or bispecific antibodies, or antigen-binding fragments thereof. An antibody is also a type of antigen-binding protein in its own right. An intact antibody generally contains at least two full-length heavy chains and two full-length light chains, although in some cases an antibody may contain only heavy chains.

[0033] Antibodies or antigen-binding fragments thereof may be derived from just a single source or may be chimeric; chimeric antibodies contain portions derived from two different antibodies, as described in more detail below. Antibodies or antigen-binding fragments thereof may be produced by hybridomas, recombinant DNA techniques, or enzymatic or chemical cleavage of intact antibodies. Unless otherwise stated, in this application the term antibody includes antibodies containing two full-length heavy chains and two full-length light chains, as well as derivatives, variants, fragments, and mutants thereof, examples of which are described below.

[0034] In one embodiment, the antibody includes various forms of antibodies disclosed herein, including, but not limited to, monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, antibody mimetics (or synthetic antibodies), chimeric antibodies, humanized antibodies, human antibodies, antibody fusions (or antibody conjugates), and fragments thereof. In one embodiment, the antibody fragment of the antibody disclosed herein may be a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an Fv fragment, a single-body fragment (scFv), a diabody, or a single-chain antibody molecule in which the heavy chain variable region and the light chain variable region are linked via a spacer.

[0035] As used herein, "light chain" includes full-length light chains and fragments thereof having sufficient variable region sequence to provide binding specificity to an antigen or epitope. A full-length light chain includes a variable region domain, VL, and a constant region domain, CL. The variable region domain of a light chain is located at the amino-terminus of the light chain polypeptide. Types of light chains include kappa and lambda chains.

[0036] As used herein, "heavy chain" includes full-length heavy chains and fragments thereof having sufficient variable region sequence to provide binding specificity for an antigen or epitope. A full-length heavy chain includes a variable region domain, VH, and three constant region domains, CH1, CH2, and CH3. The VH domain is at the amino-terminus of the heavy chain polypeptide, the CH domain is at the carboxy-terminus, and CH3 is located proximal to the carboxy-terminus. Heavy chains include isotypes of IgG (including IgG1, IgG2, IgG3, and IgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgM, and IgE.

[0037] As used herein, an "antigen-binding fragment" refers to a portion of an antibody having specific binding ability to an antigen or a polypeptide comprising the same. For example, an antigen-binding fragment may be a portion of an antibody comprising amino acid residues that interact with an antigen (e.g., an epitope) to confer specificity and / or affinity to the antibody for the antigen, or a polypeptide comprising the same. Such antigen-binding fragments typically comprise one or more "complementary determining regions" (or CDRs), and may additionally comprise one or more "framework" regions. CDRs are amino acid sequences that contribute to the antigen-binding specificity and affinity of an antibody, and framework regions are amino acid sequence regions that contribute to maintaining the proper conformation of these CDRs, and can promote binding between the antigen-binding region and the antigen. As used herein, an "antigen-binding fragment" of an antibody or immunoglobulin chain (heavy or light chain) includes a portion of an antibody that lacks certain amino acids compared to the full-length chain but is still capable of specifically binding to an antigen. Such fragments are biologically active in that they are capable of specifically binding to a target antigen or of competing with other antibodies or antigen-binding fragments for binding to a particular epitope. In one aspect, such fragments include at least one CDR present in a full-length light or heavy chain, and in some embodiments, include a single chain, heavy and / or light chain, or a portion thereof. Such biologically active fragments can be produced by recombinant DNA techniques or, for example, by enzymatic or chemical cleavage of an intact antibody. Immunologically functional immunoglobulin fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, domain antibodies, and single chain antibodies (e.g., scFv, scFv-Fc, etc.). They can be derived from any mammal, including, but not limited to, human, mouse, rat, camelid, or rabbit. A functional portion of an antibody, such as one or more of the CDRs disclosed herein, can be covalently linked to a second protein or small molecule compound and used as a targeted therapeutic against a specific target.

[0038] As used herein, a "Fab fragment" is composed of one light chain and one heavy chain that contains only the CH1 and variable regions. The heavy chain of a Fab molecule cannot form disulfide bonds with other heavy chain molecules.

[0039] As used herein, an "Fc" region comprises two antibody heavy chain fragments comprising the CH2 and CH3 domains, held together by two or more disulfide bonds and hydrophobic interactions of the CH3 domains.

[0040] As used herein, a "Fab' fragment" refers to a Fab fragment that additionally contains a region located between the CH1 and CH2 domains of the heavy chain, such that interchain disulfide bonds can form between the two heavy chains of two Fab' fragment molecules to form an F(ab')2 molecule.

[0041] In the present application, the term "F(ab')2 fragment" refers to a fragment that contains two light chains and two heavy chain molecules containing a variable region, a portion of the constant region between the CH1 and CH2 domains, as described above, and an interchain disulfide bond is formed between the two heavy chain molecules. Thus, the F(ab')2 fragment is composed of two Fab' fragments, and the two Fab' fragments are associated by disulfide bonds between them.

[0042] As used herein, an "Fv region" is an antibody that comprises the variable regions of the heavy and light chains, but does not include the constant regions.

[0043] In the present application, a "single chain antibody" refers to a single polypeptide chain of an antigen-binding region in which the heavy and light chain variable regions are linked by a flexible linker. For example, the single chain antibody may be one or more selected from the group consisting of scFv in which the heavy chain variable region and the light chain variable region are linked in a single chain form, and scFv-Fc in which the heavy chain variable region, the light chain variable region, and Fc are linked in a single chain form. For information on single chain antibodies, see, for example, U.S. Patent No. 5,260,203.

[0044] As used herein, a "bivalent antigen-binding protein" or a "bivalent antibody" comprises two antigen-binding sites. The two antigen-binding sites in such a bivalent antibody may have the same antigen specificity or may be bispecific antibodies that each bind to a different antigen. As used herein, a "multispecific antigen-binding protein" or a "multispecific antibody" targets more than one antigen or epitope.

[0045] As used herein, a "bispecific" or "dual-specific" antigen-binding protein or antibody is a hybrid antigen-binding protein or antibody that has two different antigen-binding sites. Such bispecific antibodies are a type of multispecific antigen-binding protein or antibody and can be produced by a variety of known methods, such as fusion of hybridomas or linking of Fab' fragments.

[0046] As used herein, a "bispecific" or "dual-specific" antigen-binding protein or antibody is a hybrid antigen-binding protein or antibody having two different antigen-binding sites. Such a bispecific antibody is a type of multispecific antigen-binding protein or antibody, and can be produced by a variety of known methods, such as fusion of hybridomas or linking of Fab' or scFv fragments. See, for example, Songsivilai and Lachmann, Clin. Exp. Immunol. 1990, 79:315-321; Kostelny et al. J. Immunol. 1992, 148:1547-1553. The two different epitopes bound by the two antigen-binding sites of a bispecific antigen-binding protein or antibody can be located on the same or different protein targets. In one embodiment according to the present application, the antibody according to the present application can be in the form of a bispecific antibody that additionally includes binding to a vehicle for delivery across the blood-brain barrier. One method for delivering drugs across the blood-brain barrier involves the use of delivery systems endogenous to cells, such as receptor-mediated transcytosis of the glucose and amino acid transporters, insulin or transferrin.

[0047] As used herein, a "conjugate" refers to a chimeric molecule of an antibody or antigen-binding fragment thereof disclosed herein and another molecule, particularly a blood-brain barrier mediator or a therapeutic agent, as described below. In a conjugate, an antibody or antigen-binding fragment thereof according to the present application is physically bound to another molecule, for example, by a covalent bond or physical forces through van der Waals or hydrophobic interactions, encapsulation, embedding, or a combination thereof. In a conjugate according to one embodiment, an antibody or antigen-binding fragment thereof according to the present application can be linked through a peptide linker.

[0048] The present application also includes one or more amino acid sequences that have substantial sequence identity with one or more amino acid sequences disclosed herein. By substantial identity, the present application means that sequence variations will maintain the effects disclosed herein. In one embodiment, about 90%, 95%, or 99% identity with a heavy chain variable region disclosed. In another embodiment, about 90%, 95%, or 99% identity with a light chain variable region disclosed. For example, in the case of variants that exhibit 90%, 95%, or 99% identity with the sequences of the antibodies or antigen-binding fragments disclosed herein, any variations will occur in the framework of the variable region rather than in the CDRs.

[0049] The antibody or antigen-binding fragment thereof that specifically binds to alpha-synuclein or an aggregate thereof according to the present invention can comprise a heavy chain variable region comprising the complementarity determining sites of CDRH1, CDRH2 and CDRH3; and a light chain variable region comprising the complementarity determining sites of CDRL1, CDRL2 and CDRL3.

[0050] In one embodiment, the anti-alpha-synuclein antibody or antigen-binding fragment thereof may comprise the following CDR sequences: A heavy chain CDR1 (H-CDR1) comprising one of the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 6; SEQ ID NOs: 2 to 4 and 139, and A heavy chain CDR2 (H-CDR2) comprising one of the amino acid sequences of SEQ ID NOs: 7 to 8; A heavy chain CDR3 (H-CDR3) comprising one of the amino acid sequences of SEQ ID NO:5 and SEQ ID NO:9; A light chain CDR1 (L-CDR1) comprising one of the amino acid sequences of SEQ ID NO: 10 and SEQ ID NO: 13; A light chain CDR2 (L-CDR2) comprising one of the amino acid sequences of SEQ ID NO: 11 and SEQ ID NO: 14, and A light chain CDR3 (L-CDR3) comprising one of the amino acid sequences of SEQ ID NO:12 and SEQ ID NO:15.

[0051] The amino acid sequences of the heavy chain CDR1 to CDR3 and the light chain CDR1 to CDR3 are summarized in Tables 1 and 2: TIFF0007610279000001.tif250170TIFF0007610279000002.tif174170

[0052] In one embodiment, the anti-alpha-synuclein antibody or antigen-binding fragment thereof has a heavy chain CDR1 (H-CDR1) comprising the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 (H-CDR3) comprising the amino acid sequence of SEQ ID NO: 2-4, and 139 and a light chain variable region comprising a light chain CDR1 (L-CDR1) comprising the amino acid sequence of SEQ ID NO: 10, a light chain CDR2 (L-CDR2) comprising the amino acid sequence of SEQ ID NO: 11, and a light chain CDR3 (L-CDR3) comprising the amino acid sequence of SEQ ID NO: 12.

[0053] In addition, the anti-alpha-synuclein antibody or antigen-binding fragment thereof may comprise a heavy chain variable region comprising a heavy chain CDR1 (H-CDR1) comprising the amino acid sequence of SEQ ID NO: 6, a heavy chain CDR2 (H-CDR2) comprising one of the amino acid sequences of SEQ ID NO: 7 to 8, and a heavy chain CDR3 (H-CDR3) comprising the amino acid sequence of SEQ ID NO: 9; and a light chain variable region comprising a light chain CDR1 (L-CDR1) comprising the amino acid sequence of SEQ ID NO: 13, a light chain CDR2 (L-CDR2) comprising the amino acid sequence of SEQ ID NO: 14, and a light chain CDR3 (L-CDR3) comprising the amino acid sequence of SEQ ID NO: 15.

[0054] In other embodiments, the anti-alpha-synuclein antibody or antigen-binding fragment thereof is The heavy chain variable region framework (H-FR1) is located on the N-terminal side of H-CDR1, and the heavy chain variable region framework (H-FR1) comprises a polypeptide fragment having one of the amino acid sequences of SEQ ID NOs: 16 to 17 and 35 to 40, The heavy chain variable region framework (H-FR2) is located between H-CDR1 and H-CDR2, and comprises a polypeptide fragment comprising one of the amino acid sequences of SEQ ID NOs: 18 to 19 and 41 to 44; The heavy chain variable region framework (H-FR3) is located between H-CDR2 and H-CDR3, and comprises a polypeptide fragment comprising one of the amino acid sequences of SEQ ID NOs: 20 to 31 and 45 to 50; The heavy chain variable region framework (H-FR4) located at the C-terminus of H-CDR3 comprises a polypeptide fragment comprising one of the amino acid sequences of SEQ ID NOs: 32 to 34 and 51 to 52, A polypeptide fragment comprising a light chain variable region framework (L-FR1) located on the N-terminal side of L-CDR1 and comprising one of the amino acid sequences of SEQ ID NOs: 53 to 58 and 71 to 77, A polypeptide fragment including a light chain variable region framework (L-FR2) located between L-CDR1 and L-CDR2 and including one of the amino acid sequences of SEQ ID NOs: 59 to 61 and 78 to 81, A light chain variable region framework (L-FR3) located between L-CDR2 and L-CDR3, comprising a polypeptide fragment comprising one of the amino acid sequences of SEQ ID NOs: 62 to 67 and 82 to 88, and / or It may be a light chain variable region framework (L-FR4) located at the C-terminus of L-CDR3, and may include a polypeptide fragment including one of the amino acid sequences of SEQ ID NOs: 68 to 70 and 89.

[0055] As examples of amino acid sequences that can be used for the framework herein, Tables 3 and 4 show heavy chain variable region framework sequences, and Tables 5 and 6 show light chain variable region framework sequences. TIFF0007610279000003.tif218170TIFF0007610279000004.tif217170TIFF0007610279000005.tif154170TIFF0007610279000006.tif154170

[0056] In other embodiments, the anti-alpha-synuclein antibody or antigen-binding fragment thereof has a heavy chain CDR1 (H-CDR1) comprising one of the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 6, a heavy chain CDR2 (H-CDR3) comprising one of the amino acid sequences of SEQ ID NO: 2-4, 139 and It may include a heavy chain variable region including a heavy chain CDR2 (H-CDR2) including one of the amino acid sequences of SEQ ID NOs: 7 to 8, and a heavy chain CDR3 (H-CDR3) including one of the amino acid sequences of SEQ ID NOs: 5 and 9, The heavy chain variable region can include a heavy chain variable region framework (H-FR1) comprising one of the amino acid sequences of SEQ ID NOs: 16 to 17 and 35 to 40, a heavy chain variable region framework (H-FR2) comprising one of the amino acid sequences of SEQ ID NOs: 20 to 31 and 45 to 50, a heavy chain variable region framework (H-FR3) comprising one of the amino acid sequences of SEQ ID NOs: 20 to 31 and 45 to 50, and a heavy chain variable region framework (H-FR4) comprising one of the amino acid sequences of SEQ ID NOs: 32 to 34 and 51 to 52. More specifically, the heavy chain variable region can include H-FR1 comprising the amino acid sequence of SEQ ID NO: 17, H-FR2 comprising the amino acid sequence of SEQ ID NO: 18, H-FR3 comprising one of the amino acid sequences of SEQ ID NOs: 29 to 31, and H-FR4 comprising the amino acid sequence of SEQ ID NO: 34.

[0057] The anti-alpha-synuclein antibody or antigen-binding fragment thereof may also comprise a light chain variable region comprising a light chain CDR1 (L-CDR1) comprising one of the amino acid sequences of SEQ ID NO: 10 and SEQ ID NO: 13, a light chain CDR2 (L-CDR2) comprising one of the amino acid sequences of SEQ ID NO: 11 and SEQ ID NO: 14, and a light chain CDR3 (L-CDR3) comprising one of the amino acid sequences of SEQ ID NO: 12 and SEQ ID NO: 15; The light chain variable region may additionally comprise a light chain variable region framework (L-FR1) comprising one of the amino acid sequences of SEQ ID NOs: 53 to 58 and 71 to 77, a light chain variable region framework (L-FR2) comprising one of the amino acid sequences of SEQ ID NOs: 59 to 61 and 78 to 81, a light chain variable region framework (L-FR3) comprising one of the amino acid sequences of SEQ ID NOs: 62 to 67 and 82 to 88, and a light chain variable region framework (L-FR4) comprising one of the amino acid sequences of SEQ ID NOs: 68 to 70 and 89. More specifically, the light chain variable region may comprise an L-FR1 comprising the amino acid sequence of SEQ ID NO: 58, an L-FR2 comprising the amino acid sequence of SEQ ID NO: 59, an L-FR3 comprising the amino acid sequence of SEQ ID NO: 67, and an L-FR4 comprising the amino acid sequence of SEQ ID NO: 69. 68 The L-FR4 may include an L-FR4 comprising the amino acid sequence:

[0058] However, the anti-alpha-synuclein antibody or antigen-binding fragment thereof according to the present invention does not include a mouse antibody or chimeric antibody or antigen-binding fragment thereof having a heavy chain CDR1-CDR3 consisting of the amino acid sequences of SEQ ID NOs: 1, 2 and 5 and a light chain CDR1-CDR3 consisting of the amino acid sequences of SEQ ID NOs: 10, 11, and 12, and a heavy chain FR1-FR4 consisting of the amino acid sequences of SEQ ID NOs: 16, 18, 20 and 32 and a light chain FR1-FR4 consisting of the amino acid sequences of SEQ ID NOs: 53, 59, 62 and 68. Furthermore, the anti-alpha-synuclein antibody or antigen-binding fragment thereof according to the present invention does not include a mouse antibody or chimeric antibody or antigen-binding fragment thereof having a heavy chain CDR1-CDR3 consisting of the amino acid sequences of SEQ ID NOs: 6, 7, and 9 and a light chain CDR1-CDR3 consisting of the amino acid sequences of SEQ ID NOs: 13, 14, and 15, and a heavy chain FR1-FR4 consisting of the amino acid sequences of SEQ ID NOs: 35, 41, 45, and 51 and a light chain FR1-FR4 consisting of the amino acid sequences of SEQ ID NOs: 71, 78, 82, and 89. As a specific example, an antibody comprising a heavy chain variable region sequence (ch11F11-VH) of SEQ ID NO: 90 and a light chain variable region sequence (ch11F11-VL) of SEQ ID NO: 109, or a Ch3A9 antibody comprising a heavy chain variable region sequence (ch11F11-VH) of SEQ ID NO: 102 and a light chain variable region sequence (ch11F11-VL) of SEQ ID NO: 116 is not included in the anti-alpha-synuclein antibody according to the present invention.

[0059] According to a specific embodiment of the present invention, the anti-alpha-synuclein antibody or antigen-binding fragment thereof comprises a heavy chain CDR1 (H-CDR1) having the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 2 to 4, and 139 and a heavy chain variable region comprising a heavy chain CDR2 (H-CDR2) comprising one of the amino acid sequences of SEQ ID NO: 1, and a heavy chain CDR3 (H-CDR3) comprising the amino acid sequence of SEQ ID NO: 5; and a light chain variable region comprising a light chain CDR1 (L-CDR1) comprising the amino acid sequence of SEQ ID NO: 10, a light chain CDR2 (L-CDR2) comprising the amino acid sequence of SEQ ID NO: 11, and a light chain CDR3 (L-CDR3) comprising the amino acid sequence of SEQ ID NO: 12, The heavy chain variable region is SEQ ID NO: 16 、 17 and 38A heavy chain variable region framework (H-FR1) comprising one of the amino acid sequences of SEQ ID NO: 18. 、 19 and 42 a heavy chain variable region framework (H-FR2) comprising one of the amino acid sequences set forth in SEQ ID NOs: 10 to 31; a heavy chain variable region framework (H-FR3) comprising one of the amino acid sequences set forth in SEQ ID NOs: 20 to 31; and a heavy chain variable region framework (H-FR4) comprising one of the amino acid sequences set forth in SEQ ID NOs: 32 to 34; The light chain variable region may comprise a light chain variable region framework (L-FR1) comprising one of the amino acid sequences set forth in SEQ ID NOs: 53 to 58, a light chain variable region framework (L-FR2) comprising one of the amino acid sequences set forth in SEQ ID NOs: 59 to 61, a light chain variable region framework (L-FR3) comprising one of the amino acid sequences set forth in SEQ ID NOs: 62 to 67, and a light chain variable region framework (L-FR4) comprising one of the amino acid sequences set forth in SEQ ID NOs: 68 to 70.

[0060] Preferably, the anti-alpha-synuclein antibody or antigen-binding fragment thereof according to a specific embodiment of the present invention comprises a heavy chain variable region comprising heavy chain CDR1 to CDR3 comprising the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 5, and a light chain variable region comprising light chain CDR1 to CDR3 comprising the amino acid sequences of SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, and the heavy chain variable region may comprise H-FR1 comprising the amino acid sequence of SEQ ID NO: 17, H-FR2 comprising the amino acid sequence of SEQ ID NO: 18, H-FR3 comprising one of the amino acid sequences of SEQ ID NO: 29 to 31, and H-FR4 comprising the amino acid sequence of SEQ ID NO: 34, and the light chain variable region may comprise L-FR1 comprising the amino acid sequence of SEQ ID NO: 58, L-FR2 comprising the amino acid sequence of SEQ ID NO: 59, L-FR3 comprising the amino acid sequence of SEQ ID NO: 67, and H-FR4 comprising the amino acid sequence of SEQ ID NO: 68. 68 The L-FR4 may include an L-FR4 having an amino acid sequence of:

[0061] According to a specific embodiment of the present invention, an anti-alpha-synuclein antibody or antigen-binding fragment thereof may include a heavy chain variable region including a heavy chain CDR1 (H-CDR1) having the amino acid sequence of SEQ ID NO: 6, a heavy chain CDR2 (H-CDR2) having one of the amino acid sequences of SEQ ID NO: 7 to 8, and a heavy chain CDR3 (H-CDR3) having the amino acid sequence of SEQ ID NO: 9; and a light chain variable region including a light chain CDR1 (L-CDR1) having the amino acid sequence of SEQ ID NO: 13, a light chain CDR2 (L-CDR2) having the amino acid sequence of SEQ ID NO: 14, and a light chain CDR3 (L-CDR3) having the amino acid sequence of SEQ ID NO: 15, The heavy chain variable region comprises a heavy chain variable region framework (H-FR1) comprising one of the amino acid sequences of SEQ ID NOs: 35 to 40, a heavy chain variable region framework (H-FR2) comprising one of the amino acid sequences of SEQ ID NOs: 41 to 44, a heavy chain variable region framework (H-FR3) comprising one of the amino acid sequences of SEQ ID NOs: 45 to 50, and a heavy chain variable region framework (H-FR4) comprising one of the amino acid sequences of SEQ ID NOs: 51. and 52 and a heavy chain variable region framework (H-FR4) comprising one of the amino acid sequences of The light chain variable region may comprise a light chain variable region framework (L-FR1) comprising one of the amino acid sequences set forth in SEQ ID NOs: 71 to 77, a light chain variable region framework (L-FR2) comprising one of the amino acid sequences set forth in SEQ ID NOs: 78 to 81, a light chain variable region framework (L-FR3) comprising one of the amino acid sequences set forth in SEQ ID NOs: 82 to 88, and a light chain variable region framework (L-FR4) comprising the amino acid sequence set forth in SEQ ID NO: 89.

[0062] In full-length light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids in length, and the heavy chain also includes a "D" region of about 10 or more amino acids. See, for example, Fundamental Immunology, 2nd ed., Ch. 7 (Paul, W., ed.) 1989, New York: Raven Press. Typically, the variable regions of an antibody light / heavy chain pair form the antigen-binding site.

[0063] The variable regions of immunoglobulin chains are generally identical in overall structure and contain relatively conserved framework regions (FR) connected by three hypervariable regions called "complementarity determining sites or regions or domains" or CDRs. The CDRs of the variable regions from each chain of a heavy / light chain pair are typically aligned by the framework regions to form a structure that specifically binds to a particular epitope of a target protein (alpha-synuclein). These elements of naturally occurring light and heavy chain variable regions are typically comprised in the following order from N-terminus to C-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The position of each of these corresponding amino acid sequences in the variable region follows the Kabat numbering system (Kabat Sequences of Proteins of Immunological Interest (1987 and 1991, NIH, Bethesda, MD) or as described in Chothia & Lesk, 1987, J. Mol. Biol. 196:901-917; Chothia et al., 1989, Nature 342:878-883).

[0064] Various heavy and light chain variable regions disclosed herein are disclosed in Tables 7 and 8. Each of these variable regions can be combined with the heavy and light chain constant regions to form the respective heavy and light chains of a complete antibody. The respective heavy and light chain sequences thus generated can also be combined to form a complete antibody structure. For example, an anti-alpha-synuclein antibody or antigen-binding fragment thereof according to the present invention can comprise a heavy chain variable region of one of the amino acid sequences of SEQ ID NOs: 90-101 or 102-108 and a light chain variable region of one of the amino acid sequences of SEQ ID NOs: 109-115 or 116-123, more specifically, a heavy chain variable region of one of the amino acid sequences of SEQ ID NOs: 90-101 and a light chain variable region of one of the amino acid sequences of SEQ ID NOs: 109-115, or a heavy chain variable region of one of the amino acid sequences of SEQ ID NOs: 102-108 and a light chain variable region of one of the amino acid sequences of SEQ ID NOs: 116-123.

[0065] In a specific example of the present invention, an anti-alpha-synuclein antibody or antigen-binding fragment thereof may comprise a heavy chain variable region of one of the amino acid sequences of SEQ ID NO: 98 to 101 and a light chain variable region of the amino acid sequence of SEQ ID NO: 115 (e.g., Hu11F11_(ver.1), Hu11F11_(ver.2), Hu11F11_(ver.3), Hu11F11_(ver.4), etc.), or may comprise a heavy chain variable region having the amino acid sequence of SEQ ID NO: 92 and a light chain variable region having the amino acid sequence of SEQ ID NO: 113 (e.g., Hu11F11_(ABL2-4) antibody).

[0066] However, the anti-alpha-synuclein antibody or antigen-binding fragment thereof excludes those consisting of a heavy chain variable region of SEQ ID NO: 90 and a light chain variable region of SEQ ID NO: 109, and those consisting of a heavy chain variable region of SEQ ID NO: 102 and a light chain variable region of SEQ ID NO: 116.

[0067] The amino acid sequences of the heavy and light chain variable regions of an antibody or antigen-binding fragment according to one embodiment are illustrated in Tables 7 and 8. TIFF0007610279000007.tif250170TIFF0007610279000008.tif144170TIFF0007610279000009.tif252170TIFF0007610279000010.tif47170

[0068] Also set forth in Table 9 are exemplary antibodies, including combinations of heavy and light chain variable regions and constant regions of an antibody or antigen-binding fragment according to one embodiment. TIFF0007610279000011.tif253170TIFF0007610279000012.tif255170TIFF0007610279000013.tif76170

[0069] The heavy and light chain variable regions disclosed herein can be combined with heavy and light chain constant regions, respectively, to form the heavy and light chains of a complete antibody. The respective heavy and light chains thus generated can be appropriately combined to form heavy-light chain combinations, which can form multimers (e.g., dimers in the case of IgG type antibodies) to form a complete antibody structure.

[0070] The constant region can be appropriately selected from immunoglobulin (e.g., human immunoglobulin) heavy chain constant region and light chain constant region. For example, the heavy chain constant region can be an IgG1 heavy chain constant region, an IgG3 heavy chain constant region, or an IgG4 heavy chain constant region, and the light chain constant region can be a kappa constant region or a lambda light chain constant region, but is not limited thereto. For antibody stability, manufacturability, antigen affinity, and / or other desired characteristics, other types of constant regions or modified constant regions can be appropriately selected and used, which is clearly known to those skilled in the art.

[0071] In other embodiments, the heavy chain variable regions and light chain variable regions disclosed in Tables 7 and 8 can be freely combined with each other to form various antibodies, or can be linked in a single chain form to form a single chain antibody such as scFv.

[0072] The antibodies disclosed herein share certain regions or sequences with other antibodies disclosed herein. In one embodiment, the constant regions of the antibodies or antigen-binding fragments may be shared. In another embodiment, the Fc region may be shared.

[0073] In one example, the anti-alpha-synuclein antibody provided herein may be a monoclonal or polyclonal antibody. In another example, the anti-alpha-synuclein antibody may be a human antibody, a humanized antibody, a chimeric antibody, or an animal-derived antibody. In another example, the anti-alpha-synuclein antibody may be recombinantly or chemically synthesized.

[0074] In one embodiment, the antigen-binding fragment or antibody fragment of the antibody disclosed herein may be one or more selected from the group consisting of a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an Fv fragment, a minibody, a diabody, and a single chain antibody molecule such as an scFv.

[0075] In other embodiments, the antibodies provided herein are human or humanized and may be of various isotypes (e.g., IgA, IgG1, IgG2, IgG3, IgG4, IgE, or IgD), in particular IgG1-, IgG2-IgG3-, or IgG4-types, e.g., IgG1- or IgG2-types.

[0076] In yet other embodiments, the anti-alpha-synuclein antibody may consist only of the light chain or heavy chain described above, hi other embodiments, the anti-alpha-synuclein antibody may consist only of the light chain variable region or the heavy chain variable region.

[0077] Those skilled in the art will understand that when an antibody contains one or more CDRs disclosed herein, each of the disclosed CDRs can be independently selected and combined. Thus, antibodies can be generated with 1, 2, 3, 4, 5, or 6 independently selected CDRs. Those skilled in the art will also understand that when CDRs are selected for combination, the same type of CDR will not be used repeatedly, e.g., antibodies will not generally be made containing two CDRH2 regions.

[0078] In one embodiment, the antibody may be a monoclonal antibody or a polyclonal antibody. The antibodies disclosed herein include monoclonal antibodies that bind to alpha-synuclein. Monoclonal antibodies can be produced using any technique known in the art. For example, they can be produced by immortalizing spleen cells harvested from immunized transgenic animals. Monoclonal antibodies secreted by hybridoma cell lines can be purified using techniques known in the art.

[0079] In other embodiments, the antibody may be an animal-derived antibody (such as a mouse antibody), a chimeric antibody (such as a mouse-human chimeric antibody), a humanized antibody, or a human antibody. Antibodies can also be modified in a variety of ways for a variety of purposes. Chimeric and humanized antibodies are further provided. Chimeric antibodies are antibodies in which polypeptide fragments from different antibodies are covalently linked to form an immunologically functional light chain, heavy chain, or fragments thereof.

[0080] In such monoclonal antibodies, certain amino acid residues that typically constitute the non-antigen recognizing portions of the antibody are modified to become homologous to the corresponding residues of the corresponding isotype of a human antibody. Humanization can be accomplished, for example, by replacing at least a portion of a rodent variable region with the corresponding region of a human antibody using a variety of known methods (U.S. Patent Nos. 5,585,089 and 5,693,762; Jones et al., 1986, Nature 321:522-525; Riechmann et al., 1988, Nature 332:323-27; Verhoeyen et al., 1988, Science 239:1534-1536).

[0081] Fully human antibodies are produced by immunizing transgenic animals (usually mice) that lack endogenous immunoglobulin production and are capable of producing human antibodies. Fully human antibodies can also be derived from phage-display libraries. The use of fully human antibodies can minimize immunogenic and allergic responses that can be elicited by administering mouse or mouse-derived mAbs to humans.

[0082] In one embodiment, the human alpha-synuclein antibody of the present application is selected by a phage display method. A recombinant method is used to convert the alpha-synuclein-specific monoclonal phage antibody selected by phage screening into a full IgG form. The sequence of each monoclonal phage antibody is obtained for recombinant production of an anti-alpha-synuclein antibody. In the obtained sequence, the heavy chain variable region sequence is grafted to the heavy chain constant region sequence, and the light chain variable region sequence is grafted to the light chain constant region sequence, and the amino acid sequence is changed to a nucleic acid sequence by a codon optimization method. The obtained nucleic acid sequence is cloned into a vector for animal cell culture, and then the vector is used to transform a host cell for protein production, such as a CHO cell, and the cell is cultured. In order to purify the antibody contained in the culture solution, the recombinant antibody is separated and purified using a purification technique such as affinity chromatography.

[0083] In other embodiments, the antibody may have the typical structure of an antibody found in nature or may have a modified structure.

[0084] An antibody having the above-mentioned typical structure may have a multimeric structure that includes a structural unit that includes two different polypeptide chains (i.e., a heavy chain and a light chain). The two different polypeptide chains may include a full-length light chain (about 25 kDa) and a full-length heavy chain (about 50-70 kDa). Each chain exhibits a characteristic folding pattern and is composed of several immunoglobulin domains, each of which consists of about 90-110 amino acids. Such domains are the basic units that make up an antibody polypeptide. The amino-terminal portion of each chain typically includes a portion called a variable region or V region, which is the portion that recognizes the antigen. The carboxy-terminal portion is more evolutionarily conserved than the amino-terminus and includes a portion called a constant region or C region. Human light chains are generally classified as kappa (κ) and lambda (λ) light chains, each of which includes one variable region and one constant region. Heavy chains are generally classified as mu (μ), delta (δ), gamma (γ), alpha (α) or epsilon (ε) chains, which are defined as IgM, IgD, IgG, IgA and IgE isotypes, respectively. IgG has multiple subtypes, including but not limited to IgG1, IgG2, IgG3 and IgG4. The heavy chain constant region typically contains one or more domains that exhibit effector function. The number of heavy chain constant region domains varies with isotype. An IgG heavy chain, for example, contains three C region domains known as CH1, CH2 and CH3, respectively. The antibodies disclosed herein may be of any one of such isotypes and subtypes. In one embodiment, the anti-alpha-synuclein antibody may be of an IgG isotype, for example, an IgG1, IgG2 or IgG4 subtype.

[0085] The antibodies disclosed herein are also variants of the antibodies disclosed herein. For example, the antigenic part comprises conservative amino acid substitutions at one or more residues of the disclosed heavy or light chain, variable region or CDR sequences. Those skilled in the art can determine suitable variants of the polypeptides disclosed herein using known techniques. Those skilled in the art can target variants to regions of the polypeptide that are not believed to be important for activity, to find sites that can alter the protein without destroying activity.

[0086] The present application also provides derivatives of the antibodies disclosed herein. A derivatized antibody can include any molecule or substance that provides an antibody or fragment thereof with a desired property, such as increased half-life for a particular application. A derivatized antibody can include, for example, a detectable (or labeling) moiety (e.g., a radioactive, colorimetric, antigenic or enzymatic molecule, a detectable bead (e.g., a magnetic or electron-dense (e.g., gold) bead), or a molecule that binds to another molecule (e.g., biotin or streptavidin), a therapeutic or diagnostic moiety (e.g., a radioactive, cytotoxic, or pharma- ceutical active moiety), or a molecule that increases the suitability of the antibody for a particular application (e.g., administration to a subject, e.g., a human subject, or other in vivo or in vitro use).

[0087] Another embodiment relates to a dimer comprising two fusion proteins in which an alpha-synuclein binding protein is fused to the Fc region of an antibody, which can be produced, for example, by inserting a gene fusion encoding the fusion protein into an appropriate expression vector, expressing the gene fusion in a host cell transformed with the recombinant expression vector, and allowing the expressed fusion protein to assemble similarly to an antibody molecule, where interchain disulfide bonds are formed between the Fc residues to form a dimer.

[0088] As used herein, the term "Fc polypeptide" refers to a polypeptide derived from the Fc region of an antibody, including wild-type or mutant forms. Also included are truncated forms of the polypeptide that contain the hinge region that promotes dimerization. Fusion proteins containing Fc residues and oligomers formed therefrom have the advantage that they can be readily separated by affinity chromatography using Protein A or Protein G columns.

[0089] In another aspect, the present application also provides an isolated polynucleotide encoding the antibody or antigen-binding fragment according to the present application. In another aspect, the present application also provides an expression vector comprising the polynucleotide according to the present application. In yet another aspect, the present application also provides a prokaryotic or eukaryotic cell or cell line transformed with the expression vector according to the present application.

[0090] In yet another aspect, the present application also provides a method for producing an isolated antibody or antigen-binding fragment thereof that specifically binds to alpha-synuclein, comprising culturing a cell according to the present application under conditions sufficient for expression of the antibody or antigen-binding fragment; and isolating the antibody or antigen-binding fragment thereof from the cell line.

[0091] The antibodies or antigen-binding fragments disclosed herein also have a variety of utilities, such as in specific binding assays, affinity-based purification of alpha-synuclein, or screening methods for identifying alpha-synuclein antagonists. In one embodiment, the antibodies of the present application may be useful for inhibiting the formation of alpha-synuclein aggregates, removing alpha-synuclein aggregates from the nervous system, inhibiting the intercellular movement of alpha-synuclein aggregates, and removing and reducing extracellular alpha-synuclein aggregates by phagocytosis of alpha-synuclein aggregates.

[0092] The antibodies of the present application can be used to detect and / or treat a variety of diseases associated with alpha-synuclein or alpha-synuclein aggregates. For example, the diseases are useful for detecting, treating, alleviating, or ameliorating diseases, conditions, or disorders associated with alpha-synuclein or alpha-synuclein aggregates. The antibodies of the present application can also be used to diagnose a variety of diseases or conditions associated with alpha-synuclein or alpha-synuclein aggregates or to detect the presence or absence of alpha-synuclein or alpha-synuclein aggregates.

[0093] The antibody or antigen-binding fragment thereof according to the present invention may have one or more activities selected from the group consisting of (i) specific binding to alpha-synuclein or alpha-synuclein aggregates, (ii) reducing the level of alpha-synuclein or alpha-synuclein aggregates in the nervous system of a subject, (iii) reducing or inhibiting cell-to-cell transmission of alpha-synuclein or its aggregates between neurons, and (vi) enhancing phagocytic uptake of alpha-synuclein or its aggregates by neurons. Preferably, the antibody or antigen-binding fragment thereof according to the present invention may have all of the activities (i) to (iv).

[0094] One example of the present invention relates to a composition for preventing, alleviating, ameliorating or treating a synucleinopathic disease comprising an antibody or antigen-binding fragment thereof capable of specifically binding to alpha-synuclein, in particular alpha-synuclein aggregates.

[0095] One embodiment of the present invention relates to a method for preventing, alleviating, ameliorating or treating a synucleinopathic disease comprising administering to a subject in need thereof an antibody or antigen-binding fragment thereof capable of specifically binding to alpha-synuclein, particularly alpha-synuclein aggregates, The antibody or antigen-binding fragment thereof may be administered in an amount effective to reduce the level of alpha-synuclein, particularly alpha-synuclein aggregates, in the nervous system of the subject, more particularly in extraneuronal regions of the nervous system.

[0096] One example of the present invention relates to a method for reducing the levels of alpha-synuclein, particularly alpha-synuclein aggregates, in the nervous system of a subject, and more particularly in extraneuronal regions of the nervous system, comprising an antibody or antigen-binding fragment thereof capable of specifically binding to alpha-synuclein, particularly alpha-synuclein aggregates, or use of the antibody for reducing the levels of alpha-synuclein, particularly alpha-synuclein aggregates, in the extraneuronal regions of the nervous system.

[0097] In yet another aspect, the present application also provides compositions or kits comprising any of the antibodies or antigen-binding fragments thereof according to the present application, which may be provided as pharmaceutical or diagnostic compositions, where the pharmaceutical compositions may include a pharma- ceutically acceptable carrier or excipient, and the diagnostic compositions may additionally include reagents necessary for diagnosis or detection.

[0098] In this application, the "blood-brain barrier" or BBB is a barrier formed by tight junctions in the endothelial cell membrane of brain capillaries that exists between the brain and spinal cord and the peripheral circulatory system. Such a barrier is very strong and restricts the passage of low molecular weight substances with a molecular weight of about 60 Da to the brain. 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 continuous capillary barriers in the central nervous system and are commonly referred to as the BBB.

[0099] As used herein, a "blood-brain barrier transduction agent" includes a molecule, such as a protein, including peptides and polypeptides, a nucleic acid, an antibody, or a small molecule compound, that can deliver an antibody or antigen-binding fragment of the present application across the blood-brain barrier.

[0100] As used herein, a "therapeutic agent" refers to a molecule administered to a subject for a desired therapeutic effect. The subject includes a non-human mammal, such as a primate or a human. Examples of therapeutic agents include proteins, including peptides and polypeptides, nucleic acids, antibodies, or small molecule compounds. In another aspect, a therapeutic agent can be conjugated to an antibody according to the present application in one embodiment and used as a therapeutic agent for diseases associated with alpha-synuclein aggregates.

[0101] Although the exact mechanisms by which alpha-synuclein accumulation induces the typical characteristics of neurodegeneration in synucleinopathies and the specific symptoms of synucleinopathies remain poorly understood, recent studies implicate the abnormal formation and accumulation of alpha-synuclein aggregates in the development and degenerative progression of potential synucleinopathies.

[0102] According to the present invention, the term "synucleinopathies" includes all neurodegenerative disorders characterized by pathological synuclein aggregates. Several neurodegenerative disorders are collectively grouped as synucleinopathies, including Parkinson's disease, Parkinson's disease dementia (PDD), dementia with Lewy bodies (DLB), Lewy body disease, dementia with Lewy bodies, Parkinsonism with dementia, multiple system atrophy (MSA), multiple neurological atrophy and neurodegeneration with cerebral iron deposition type I (NBIA Type I), etc. Alpha-synuclein aggregates are also found secondarily in Alzheimer's disease (Kim et al. Alzheimer's Research & Therapy 2014, 6:73).

[0103] Synucleinopathies are a diverse group of neurodegenerative disorders that share common pathological characteristics: in neuropathological experiments, characteristic lesions can be detected, including abnormal aggregation of alpha-synuclein protein in selected populations of neurons and oligodendrocytes. Alpha-synuclein (originally identified as PARK1 and PARK4) is a 140 amino acid protein that is widely expressed in the neocortex, hippocampus, dentate gyrus, olfactory bulb, striatum, thalamus, and cerebellum. Alpha-synuclein is also highly expressed in hematopoietic cells, including B-, T-, and NK cells, as well as in proteinocytes and platelets. Its exact role in these cells is unknown, but it has been associated with the differentiation of megakaryocytes (platelet precursors).

[0104] It is shown that the alpha-synuclein antibody of the present application specifically recognizes alpha-synuclein aggregates with high affinity and can be useful for the diagnosis or detection of such diseases. Furthermore, it is shown that the alpha-synuclein antibody of the present application that specifically recognizes alpha-synuclein aggregates can be effectively used for the treatment of synucleinopathies, particularly Parkinson's disease, by inhibiting the formation of alpha-synuclein aggregates or degrading the aggregates and inhibiting the transmission of intercellular aggregates.

[0105] In the present application, "diseases associated with alpha-synuclein aggregates" refer to a group of neurodegenerative diseases called synucleinopathies, in which alpha-synuclein aggregates are found in lesions involving neuronal and glial cell (glia) populations and are characterized by degeneration of the dopaminergic system, altered motor skills, cognitive impairment, and formation of Lewy bodies and / or Lewy neurites (Kim et al. Alzheimer's Research & Therapy 2014, 6:73; McKeith et al., Neurology (1996) 47:1113-24). Such diseases include, but are not limited to, Parkinson's disease, Parkinson's disease dementia, Lewy body dementia, Lewy body variant Alzheimer's disease, combined Alzheimer's and Parkinson's disease, multiple system atrophy, and many other neuroaxonal diseases. In one embodiment, the antibody of the present application is effectively used to treat Parkinson's disease.

[0106] In the present application, an "effective amount" generally refers to an amount sufficient to reduce the severity and / or frequency of symptoms of a disease, particularly an alpha-synuclein-related disease, eliminate the underlying cause of symptoms and / or disease onset of a disease, particularly an alpha-synuclein-related disease, or prevent the onset of symptoms and / or underlying cause of a disease, particularly an alpha-synuclein-related disease, and / or ameliorate or correct damage caused by a disease, particularly an alpha-synuclein-related disease. In some embodiments, an effective amount is a therapeutically effective amount or a prophylactically effective amount. A "therapeutically effective amount" is an amount sufficient to treat a disease, particularly an alpha-synuclein-related condition or symptom, or prevent, slow, or reverse the progression of a disease, particularly an alpha-synuclein-related condition or symptom. A "prophylactically effective amount" is an amount that, when administered to a subject, prevents or slows the onset or recurrence of a disease, particularly an alpha-synuclein-related disease, or reduces the likelihood of the onset or recurrence of a disease, particularly an alpha-synuclein-related disease, or a disease, particularly an alpha-synuclein-related disease or associated symptom, as intended. The complete therapeutic or prophylactic effect may occur through the administration of several doses rather than through a single administration of the dose, and thus a therapeutically or prophylactically effective amount may be delivered through one or more administrations.

[0107] In another aspect of the present invention, the present application provides a use of an antibody or antigen-binding fragment according to the present application, or a composition comprising the same, for treating a synucleinopathy in a subject having the disease, wherein the antibody or antigen-binding fragment or composition comprising the same inhibits intercellular transmission of alpha-synuclein aggregates, degrades alpha-synuclein aggregates, inhibits formation of alpha-synuclein aggregates, or increases microglial phagocytosis of alpha-synuclein aggregates in the subject or in the subject's brain.

[0108] As used herein, "treatment" can mean any action associated with the lessening or elimination of a disease or a symptom of a disease or a pathological condition, including reducing, alleviating, ameliorating, or eliminating the disease or disease symptom, making the disease symptom or pathological condition more tolerable, or slowing the rate at which the disease symptom or pathological condition worsens, etc. The term "subject" or "patient" includes a human or human patient.

[0109] Further provided is a pharmaceutical composition comprising a therapeutically effective amount of the antibody and a pharma- ceutical acceptable diluent, carrier, solubilizer, emulsifier, preservative and / or adjuvant. Also included is a method of treating a patient associated with alpha-synuclein, for example, by administering such a pharmaceutical composition. The pharmaceutical composition used for in vivo administration is typically provided as a sterile formulation. Once the pharmaceutical composition is formulated, it can be stored in a sterile vial as a solution, suspension, gel, emulsion, solid, crystal, or dehydrated or lyophilized powder. Such formulations can be stored in a ready-to-use form or in a form that is reconstituted (e.g., lyophilized) immediately prior to administration.

[0110] The pharmaceutical composition can be administered by known methods, such as orally; by injection via intravenous, intraperitoneal, intracerebral (intracemall), intraventricular, intramuscular, intraocular, intraarterial, intraportal, or intralesional routes; by sustained release system or implantation device. In certain embodiments, the composition is administered by bolus injection or continuously by infusion or implantation device.

[0111] The pharmaceutical composition or kit according to the present application is for treating a synucleinopathy, for example, the synucleinopathy includes Parkinson's disease, Parkinson's disease dementia, Lewy body dementia, Lewy body variant Alzheimer's disease, combined Alzheimer's and Parkinson's disease, or multiple system atrophy.

[0112] In yet another aspect, there is provided a method for treating a synucleinopathy in a subject or modulating the level of alpha-synuclein aggregates in said subject, comprising administering to said subject an antibody or antigen-binding fragment according to the present application or a composition comprising the same.

[0113] In one embodiment, the antibodies of the present application can be used in conjunction with a vehicle for crossing the BBB. Numerous methods have been described for delivering drugs through the BBB. For example, bradykinin or HIFU (High Density Intracranial Fibrillation) are used. focused There are several methods to disrupt the osmotic pressure of the BBB using methods such as ultrasound, as well as using transport systems such as endogenous glucose and amino acid transporters, insulin or transferrin receptor-mediated transcytosis, or blocking the active efflux transporters of glycoproteins.

[0114] In yet other embodiments, the antibodies of the present application can be linked and used in combination with other therapeutic agents to treat diseases associated with alpha-synuclein aggregates. Pharmaceutical compositions comprising a therapeutically effective amount of the antibody and a pharma- ceutical acceptable diluent, carrier, solubilizer, emulsifier, preservative and / or adjuvant are further provided.

[0115] Acceptable formulation materials are non-toxic to recipients at the dosages and concentrations used. In certain embodiments, a pharmaceutical composition is provided that includes a therapeutically effective amount of a human alpha-synuclein antibody. In certain embodiments, acceptable formulation materials are preferably non-toxic to patients at the dosages and concentrations used. In one embodiment, the pharmaceutical composition can include specific formulation materials for, for example, modifying, maintaining or preserving the pH, osmolality, viscosity, clarity, color, isotonicity, flavor, sterility, stability, dissolution or release rate, absorption or penetration of the composition.

[0116] In certain embodiments, optimal pharmaceutical compositions can be determined by one of skill in the art depending, for example, on the intended route of administration, the method of delivery, and the intended dose (see REMINGTON'S PHARMACEUTICAL SCIENCES, supra). In certain embodiments, these compositions can affect the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the antibodies disclosed herein. In certain embodiments, the primary vehicle or carrier in a pharmaceutical composition can be aqueous or non-aqueous. For example, a suitable vehicle or carrier can be water for injection, physiological saline solution, possibly supplemented with other substances common in compositions for parenteral administration. Additionally, in certain embodiments, the human alpha-synuclein antibody can be formulated as a lyophilizate using a suitable excipient, for example, sucrose.

[0117] Pharmaceutical compositions may be delivered parenterally, and therapeutic compositions may contain the desired human α-Syn binding protein in a pharma- ceutically acceptable vehicle and be provided in the form of a pyrogen-free, parenterally acceptable aqueous solution.

[0118] Once the pharmaceutical composition is formulated, it can be stored in a sterile vial as a solution, suspension, gel, emulsion, solid, crystal, or dehydrated or lyophilized powder. Such formulations can be stored in a ready-to-use form or in a form (e.g., lyophilized) that is reconstituted immediately prior to administration. Kits for producing single-dose administration units are also provided. The frequency of administration depends on the pharmacokinetic parameters of the human alpha-synuclein antibody in the formulation used.

[0119] It may also be preferable to use the human alpha-synuclein antibody pharmaceutical composition ex vivo. In such cases, cells, tissues or organs removed from a patient are exposed to the human alpha-synuclein antibody pharmaceutical composition, and the cells, tissues and / or organs are then subsequently transplanted into the patient. In particular, human alpha-synuclein antibodies can be delivered by transplanting specific cells that have been genetically engineered using methods as described herein to express and secrete the polypeptide.

[0120] In yet another aspect, a method for detecting alpha-synuclein aggregates in a biological sample is provided, comprising the steps of: providing an antibody or antigen-binding fragment according to the present application; and contacting the antibody or antigen-binding fragment with a biological sample in which alpha-synuclein aggregates need to be detected. Biological samples include a variety of samples in which alpha-synuclein aggregates need to be detected, including, for example, cerebrospinal fluid, blood, including plasma, or urine, and also cells, tissues, or organs. The method can be performed in vitro or in vivo. In vivo imaging can be performed, for example, using positron emission tomography (PET), single photon emission tomography (SPECT), near infrared (NIR) optical imaging, or magnetic resonance imaging (MRI).

[0121] In yet another aspect, the present application provides a method for diagnosing synucleinopathies in a subject in need of such diagnosis, the method comprising the steps of measuring / detecting the concentration or subcellular location of alpha-synuclein aggregates in the subject with any of the antibodies or antigen-binding fragments of the present application; and comparing the concentration or subcellular location of alpha-synuclein aggregates measured in the subject with a result from a control sample, where a similarity or difference from the control result indicates that the subject suffers from a synucleinopathies. In the method, the control may be a normal or a synucleinopathies sample. For detection or diagnostic applications, the antibody may be labeled, typically with a detectable label.

[0122] The antibodies disclosed herein can be used for diagnostic purposes to detect, diagnose, or monitor diseases and / or conditions associated with alpha-synuclein. In one embodiment, a method according to the present application is a method for diagnosing a synucleinopathy in a subject in need of such diagnosis, the method comprising the steps of measuring the concentration or subcellular location of alpha-synuclein aggregates in the subject with an antibody or antigen-binding fragment according to the present application; and comparing the concentration or subcellular location of alpha-synuclein aggregates measured in the subject with a result from a control sample, wherein a similarity or difference from the control result indicates that the subject suffers from a synucleinopathy.

[0123] In the method, the subject includes a patient who is asymptomatic or presymptomatic. In one embodiment, the control group may be a patient with a synucleinopathy, including Parkinson's disease, dementia with Lewy bodies, or multiple system atrophy, in which case a similarity to the control group in the comparison step diagnoses the subject as a patient with a synucleinopathy. In another embodiment, the control group is a sample from a normal individual, and a difference from the control group in the comparison step, for example an increase in aggregate concentration, diagnoses the subject as a synucleinopathy. In yet another embodiment, the subject and the control group may be age-matched. The method can be performed in vivo or on a biological sample isolated from a subject, for example a blood, cerebrospinal fluid (CSF) or urine sample.

[0124] For diagnostic applications, the antibody may be labeled, typically with a detectable labeling substance. Suitable labeling substances include radioisotopes or radionuclides (e.g., 3 H, 14 C. 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131I), fluorophores (e.g., FITC, rhodamine, lanthanum group fluorophores), enzymes (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent groups, biotinyl groups, or polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags).

[0125] In another embodiment, the antibodies of the present application can be used to identify tissues containing alpha-synuclein aggregates. In a particular embodiment, the antibody is labeled with a labeling agent and binding of the labeled antibody to alpha-synuclein aggregates is detected. In one embodiment, binding of the antibody to alpha-synuclein aggregates is detected in vivo.

[0126] In another aspect, the present application discloses detecting or screening test substances that compete with the antibodies disclosed herein for binding to alpha-synuclein aggregates, for example by detecting the amount of free antibody in a solution containing alpha-synuclein aggregates in the presence or absence of the test substance.

[0127] An increase in the concentration of free antibody, i.e., antibody not bound to alpha-synuclein aggregates, indicates that the test molecule can compete with the antibody for binding to alpha-synuclein aggregates. In one embodiment, the antibody is labeled with a labeling group. Alternatively, the test substance is labeled and the amount of free test substance is monitored in the presence and absence of the antibody.

[0128] Additionally, the antibodies or antigen-binding fragments disclosed herein have a variety of utilities, such as in specific binding assays, affinity-based purification of alpha-synuclein, or screening methods for identifying alpha-synuclein antagonists.

[0129] The antibodies disclosed herein are useful for detecting alpha-synuclein, particularly alpha-synuclein aggregates such as Lewy bodies, in biological samples and for identifying cells or tissues containing alpha-synuclein aggregates. For example, alpha-synuclein antibodies can be used in diagnostic, e.g., assays to detect and / or quantify alpha-synuclein aggregates expressed in biological samples, e.g., blood, including plasma, cerebrospinal fluid (CSF), or urine, tissues, or cells, and for diagnosing synucleinopathies based thereon. Effect of the Invention

[0130] The antibodies disclosed herein preferentially bind to alpha-synuclein, particularly alpha-synuclein aggregates, with high avidity. The antibodies or antigen-binding fragments of the present application having high affinity can reduce the formation of alpha-synuclein aggregates and can reduce the concentration of aggregates in the brain. In addition, the antibodies or antigen-binding fragments of the present application having high affinity for alpha-synuclein aggregates can reduce the formation of alpha-synuclein aggregates outside the central nervous system, which can ultimately change the equilibrium between alpha-synuclein forms at the blood-brain barrier and reduce the concentration of aggregates in the central nervous system. In addition, the high affinity has the advantage of allowing administration at a low dose. This is a great clinical advantage since sufficient efficacy can be obtained even when the antibodies are administered by a more convenient method such as, but not limited to, subcutaneous injection.

[0131] The antibodies disclosed in the present application can effectively remove or promote the degradation of alpha-synuclein aggregates, inhibit the intercellular transmission of alpha-synuclein, and can be useful in treating diseases associated with the accumulation of alpha-synuclein aggregates. [Brief description of the drawings]

[0132] [Figure 1]1 shows the results of dot blot assays to determine whether a monoclonal antibody prepared in one embodiment of the present invention specifically recognizes aggregated native alpha-synuclein. [Diagram 2] 1 shows the results of measuring the affinity of a monoclonal antibody produced in one example of the present invention by ELISA. [Figure 3a] 1 shows the results of BIAcore analysis of the preferential binding specificity and affinity of a monoclonal antibody produced in one embodiment of the present invention to alpha-synuclein aggregates. [Figure 3b] The results of Figure 3a are presented in a table. [Figure 4a] 1 shows the results of Octet analysis of the preferential binding specificity of a monoclonal antibody produced in one embodiment of the present invention to alpha-synuclein aggregates. [Figure 4b] The results of Figure 4a are presented in a table. [Diagram 5] 1 shows the principle (top) and experimental results of analyzing whether a monoclonal antibody produced in one embodiment of the present invention inhibits cell-to-cell transmission of alpha-synuclein. [Figure 6a] The present invention provides a method for determining whether a monoclonal antibody prepared according to the present invention can remove alpha-synuclein aggregates in a mouse animal model (TG) that overexpresses human alpha-synuclein. After administering the antibody to the mouse, mouse brain tissue was stained with p-129 α-Syn antibody and assayed. In the figure, HP stands for Hippocampus, p-129 α-syn is a marker for aggregates in which the 129th residue is phosphorylated, and the arrow indicates phosphorylated alpha-synuclein. [Figure 6b] Similar experiment to Fig. 6a, but stained using an antibody against total alpha-synuclein as a marker. The arrow indicates human alpha-synuclein. The 9B11, 11F4, and 11F11 antibodies effectively inhibited the accumulation of total alpha-synuclein. [Figure 7a]The results of measuring whether the monoclonal antibody produced in one embodiment of the present invention can reduce microgliosis in vivo were obtained by administering the antibody to mice and then staining mouse brain tissue with Iba-1 (microgliosis) antibody as a marker. The arrows indicate activated microglia. [Figure 7b] FIG. 1 shows the results of measuring whether the monoclonal antibody produced in one example of the present invention can reduce astrogliosis in vivo by administering the antibody to mice and then staining mouse brain tissue with a GFAP (astrogliosis) antibody as a marker. The arrows indicate activated astrocytes. [Figure 7c] In one example of the present invention, the ability of a monoclonal antibody to reduce inflammatory cytokines in vivo was measured by administering the antibody to mice and then staining mouse brain tissue with an IL-1 beta (IL-1β) antibody as a marker, with the arrow indicating cells expressing IL-1 beta. [Figure 7d] FIG. 1 shows the results of measuring whether a monoclonal antibody according to one embodiment of the present invention can reduce inflammatory cytokines in vivo by administering the antibody to mice and then staining mouse brain tissue with an IL-6 antibody as a marker. The arrow indicates cells expressing the inflammatory cytokine IL-6. [Figure 8a] 1 shows the results of measuring whether monoclonal antibodies 3A9 and 11F11 according to an embodiment of the present invention can specifically recognize Lewy bodies and Lewy neurites in human brain tissue, respectively. It shows that the antibodies according to the present application bind to Lewy bodies (arrows) and Lewy neurites (thread-like structures at the lower left). [Figure 8b] 1 shows the results of measuring whether monoclonal antibodies 3A9 and 11F11 according to an embodiment of the present invention can specifically recognize Lewy bodies and Lewy neurites in human brain tissue, respectively. It shows that the antibodies according to the present application bind to Lewy bodies (arrows) and Lewy neurites (thread-like structures at the lower left). [Figure 9] FIG. 1 is a schematic diagram showing the epitope mapping results of an antibody according to an embodiment of the present invention, which reveals that the antibody according to the present application mostly binds to the C-terminal site. [Figure 10a] FIG. 10 shows the results of an analysis of the ability of a chimeric antibody and a humanized antibody according to an embodiment of the present invention to promote phagocytosis of alpha-synuclein aggregates in microglia, where FIG. 10a is for the humanized 11F11 antibody and FIG. 10b is for the humanized 3A9 antibody. [Figure 10b] FIG. 10 shows the results of an analysis of the ability of a chimeric antibody and a humanized antibody according to an embodiment of the present invention to promote phagocytosis of alpha-synuclein aggregates in microglia, where FIG. 10a is for the humanized 11F11 antibody and FIG. 10b is for the humanized 3A9 antibody. [Figure 11] 1 shows the results of an analysis of the ability of chimeric and humanized antibodies according to an embodiment of the present invention to inhibit the binding of alpha-synuclein aggregates to the surface of nerve cells. [Figure 12] 13 shows the results of an analysis of the efficacy of a chimeric antibody and a humanized antibody according to an embodiment of the present invention in inhibiting the intercellular propagation of alpha-synuclein aggregates secreted from neurons overexpressing alpha-synuclein and transporting them to normal neurons. [Figure 13] 1 shows the results of measuring the affinity of a chimeric antibody produced in one example of the present invention and humanized 11F11 antibody by ELISA. [Figure 14] 1 shows the results of a BIAcore analysis of the preferential binding specificity and affinity of a chimeric antibody and a humanized 11F11 antibody produced in an example of the present invention to alpha-synuclein aggregates. [Figure 15a] The results of ELISA and dot blot analysis show that the chimeric antibody produced in one embodiment of the present invention specifically binds to beta-synuclein, gamma-synuclein, or alpha-synuclein that is not amyloid beta (amyloid beta 1-42 or tau-derived aggregates), particularly alpha-synuclein aggregates. [Figure 15b]The results of ELISA and dot blot analysis show that the chimeric antibody produced in one embodiment of the present invention specifically binds to beta-synuclein, gamma-synuclein, or alpha-synuclein that is not amyloid beta (amyloid beta 1-42 or tau-derived aggregates), particularly alpha-synuclein aggregates. [Figure 15c] The results of ELISA and dot blot analysis show that the chimeric antibody produced in one embodiment of the present invention specifically binds to beta-synuclein, gamma-synuclein, or alpha-synuclein that is not amyloid beta (amyloid beta 1-42 or tau-derived aggregates), particularly alpha-synuclein aggregates. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0133] The present invention will be described in more detail below with reference to examples, but these are merely illustrative and are not intended to limit the scope of the present invention. EXAMPLES

[0134] Example 1: Production of mouse alpha-synuclein antibodies immunization As antigens, full-length (140 residues) or alpha-synuclein monomers truncated at the C-terminus by 21 residues were placed in a 37°C thermomixer C and shaken at 1050 rpm for 14 days to aggregate and sonicate. The 140- and 119-residue alpha-synuclein fibrils prepared above at 1 mg / ml concentration were mixed with adjuvant at 1:1 (vol:vol) and thoroughly mixed. TIFF0007610279000014.tif42170

[0135] Next, 200μL of the mixture prepared above was subcutaneously injected into 5-7 week old BALB / c female mice, and two weeks later, 200μL of the mixture prepared in the same manner was further subcutaneously injected for boosting. One week after boosting, blood was collected and immunization titeration was performed using ELISA method with the administered antigen. Next, for the third boosting, only the antigen was subcutaneously injected.

[0136] Hybridoma Production Next, the spleens of the mice that had been immunized as described above were removed, and cells were obtained from them. Next, the spleen cells were suspended in Hybridoma-SFM medium (Thermo Fisher Scientific, USA) supplemented with 10% FBS. To prepare hybridomas, mouse myeloma cells SP2 / 0-Ag14 and spleen cells were mixed in serum-free Hybridoma-SFM medium, and then centrifuged to remove the medium. Next, PEG was added to the cell pellet, and the mixture was incubated at 37°C for 1 minute to induce cell fusion.

[0137] Single-cell cloning Two weeks after fusion, fusion with mouse B cells producing antibodies was confirmed using an ELISA method with antigen administered to the mouse using cell culture medium. Next, single cell cloning was performed using the hybridomas to select a total of 16 hybridomas producing monoclonal antibodies. The 9B11 clone (IgG1 kappa, respectively) was obtained using full-length (140 residues) alpha-synuclein aggregates as antigens, and the 3A9 and 11F11 clones (IgG2b kappa, IgG2b kappa, respectively) were obtained using alpha-synuclein aggregates truncated at the C-terminus 21 residues as antigens.

[0138] Antibody purification Each hybridoma was cultured in RPMI1640 medium containing 10% FBS, and the culture medium was replaced with serum-free SFM medium for antibody production, after which the cells were cultured for about 4 days. The cell culture supernatant was separated and centrifuged, and then filtered through a 0.22μm filter. The IgG1 type was purified using a protein G column, and the remaining antibodies were purified using a protein A column.

[0139] Variable region sequencing The variable region and CDR sequences were determined by referring to the paper by Ahn et al., Mol.Cells 2004, 18(2):237-241. After culturing the hybridoma, the cells were separated by centrifugation. The isolated hybridoma was treated with Trizol to separate RNA, which was used as a template to synthesize cDNA, and the variable region and CDR sequences were confirmed by base sequence analysis. Thus, the antibodies 3A9, 9B11, and 11F11 were obtained.

[0140] Example 2. Antigen-binding specificity and avidity analysis using alpha-synuclein antibodies Example 2-1: Dot blot analysis using anti-alpha-synuclein antibody A dot blot experiment was performed to analyze whether the antibody of the present invention binds to monomers or aggregates in the native state. For this purpose, 50ng or 100ng of alpha-synuclein monomer or fibril protein (prepared in the lab of Professor Lee Seung-jae, Seoul National University; Bae et al., J.Neurosci 32:13454, 2012) was spot-loaded onto a nitrocellulose membrane using a dot blot apparatus (BioRad). Monomer or fibril proteins diluted 2-fold were loaded sequentially from right to left on the membrane (12.5, 25, 50, 100ng). The membrane was blocked with 5% non-fat dry milk in TBST at room temperature for 1 hour, and the alpha-synuclein antibody prepared in Example 1 was added to TBST containing 1% bovine serum albumin at a concentration of 1mg / ml, and the membrane and the antibody were incubated at room temperature for 1 hour. After washing with TBST, the cells were incubated with a secondary antibody conjugated with HRP (horse radish peroxidase) and chemiluminescence as a substrate. e Signals were analyzed using NEN scence substrate according to the manufacturer's protocol. Results were imaged using a LAS-3000 Luminescent Image Analysis System (FUJIFILM Life Science). The results are shown in Figure 1.

[0141] As shown in Figure 1, the alpha-synuclein antibodies of the present application were found to preferentially bind only to aggregates compared to alpha-synuclein monomers. In particular, 9B11, 3A9 and 11F11 were found to bind only to aggregates. The 274 antibody (Bae et al., J Neurosci. 2012 Sep 26; 32(39): 13454-13469) is a comparative antibody that binds to both monomers and aggregates.

[0142] Example 2-2: ELISA analysis using anti-alpha-synuclein antibody ELISA was performed to quantitatively analyze the binding strength of the antibody of the present invention to the antigen. For this, the alpha-synuclein antibody of the present invention was coated on a 96-well plate at a concentration of 1 mg / ml, and then alpha-synuclein fibril aggregates at concentrations of 10, 100, 1000, and 10,000 ng / ml were treated on the plate, washed with PBS, and then treated with a secondary antibody conjugated with biotin and streptavidin conjugated with HRP, and reacted with TMB as a substrate, and the absorbance was measured, and the measurement results are shown in Figure 2.

[0143] As shown in FIG. 2, the antibody of the present invention was found to preferentially bind to aggregates with high avidity. Analysis of the antibody through ELISA results showed that the antibody preferentially binds to aggregates at 0.1×10 -9 M~2×10 -9 Antibodies that bind to both monomers and aggregates have an affinity of about M, up to 1×10 -10 The M value was shown. The antibody according to the present invention preferentially binds to alpha-synuclein aggregates with high affinity, and binds to single bodies with lower affinity than aggregates or does not bind at all, so affinity could not be derived. These results indicate that the causative agent of neurodegenerative diseases with alpha-synuclein pathology such as Parkinson's disease can be effectively removed or the activity can be suppressed.

[0144] Example 2-3. BIAcore analysis using anti-alpha-synuclein antibody Quantitative analysis of monomer and aggregate antigen binding of the alpha-synuclein antibodies prepared in Example 1 was performed using BIAcore.

[0145] The instrument used was T200 (GE Healthcare, S / N: 1565888). The chip used was Protein A (GE Healthcare, Cat. 29-1275-56), the regeneration buffer was 10 mM Glycine-HCl pH 1.5 (GE Healthcare, Cat. BR-1003-54), and the running buffer, analyte dilution buffer, and sample dilution buffer were HBS-EP. The α-syn antibodies (3A9, 9B11, 11F11) prepared in Example 1 were diluted with 1X HBS-EP (GE Healthcare, Cat. BR-1006-69), and the α-syn monomer (1 mg / ml) or fibril protein (3 mg / ml) (analyte) was diluted two-fold and analyzed at a total of six concentrations (0, 0.39, 1.56, 6.25, 25, 100 nM) including 0 nM. In the case of capture, the target RU for monomers was 800 (theoretical), and the target RU for fibrils was 100 (theoretical). The capture phase was performed with a contact time of 60 seconds, a flow rate of 30 μl / min, and a stabilization period of 180 seconds. In the association phase, the association time was 120 seconds and the flow rate was 30 μl / min. In the dissociation phase, the dissociation time was 360 seconds and the flow rate was 30 μl / min. In the regeneration phase, the flow rate was 30 μl / min, and the regeneration time was 240 seconds (primary) and 60 seconds (secondary), performed twice. Fitting was performed using a 1:1 binding model, and the evaluation software used was BIACore T200 Evaluation software (GE Healthcare). The results are shown in Figure 3a and Figure 3b.

[0146] Figure 3a shows the results of a BIAcore analysis of the preferential binding specificity and affinity of a monoclonal antibody prepared according to an embodiment of the present invention to alpha-synuclein aggregates. It shows that the antibody according to the present application binds to aggregates with high affinity. These results indicate that the causative agent of a neurodegenerative disease with alpha-synuclein pathology such as Parkinson's disease can be effectively removed or its activity can be inhibited. Figure 3b shows the results of Figure 3a in a table.

[0147] As shown in the results of Figures 3a and 3b, of the four alpha-synuclein antibodies analyzed, 3A9, 9B11, and 11F11, which preferentially bind to aggregates by other methods described above, were found to bind only to aggregates by BIAcore, with a binding affinity of approximately 1–3 × 10 -9 It was revealed that it has a high affinity for M.

[0148] Example 2-4. Octet analysis using anti-alpha-synuclein antibodies Quantitative analysis of monomer and aggregate antigen binding of the alpha-synuclein antibodies (3A9, 9B11, 11F11) prepared in Example 1 was performed using Octet.

[0149] Specifically, the running buffer was 1X KB buffer (Cat.18-1092) or 1X PBS buffer at 1000 rpm, and the immobilization buffer was sodium acetate, pH 5 (10 mM, Cat.18-1068). For α-syn monomer, α-syn antigen was immobilized, and for fibrils, test antibody was immobilized. The target concentration was 20 μg / ml for monomer and 0.4 μg / ml for fibrils. The kinetics concentration was 2-fold diluted from 50 nM for monomer and 100 nM for fibrils, for a total of seven points. The association / dissociation times were 5 min / 20 min for monomer and 5 min / 25 min for fibrils, respectively, and ARG2 was used as Biosensor and 1:1 fitting was used. The results are shown in Figure 4. FIG. 4 shows the results of Octet analysis of the preferential binding specificity of a monoclonal antibody produced in one embodiment of the present invention to α-Syn aggregates.

[0150] As shown in Figure 4a, 3A9, 9B11 and 11F11 hardly bind to monomers (red dotted box) but bind well to aggregates (the graph in the red dotted box rises). These results are similar or consistent with the results of Dot Blot, Octet and ELISA, and it was revealed that 3A9, 9B11 and 11F11, which are antibodies that preferentially bind to aggregates by the other methods among the four α-syn antibodies tested, also bind only to aggregates by Octet. Figure 4b shows the results of Figure 4a in a table. The results of Figures 4a and 4b show preferential binding to alpha-synuclein aggregates and are consistent with the results of Figure 1. It was revealed that the #274 antibody, used as a comparison group, binds well to both monomers and aggregates.

[0151] Example 3. Analysis of the inhibitory effect of anti-alpha-synuclein antibodies on the transmission of intercellular alpha-synuclein aggregates Since intercellular transmission of alpha-synuclein aggregates has been proposed as one of the causes of alpha-synuclein aggregate-associated diseases, antibodies that can inhibit this should be useful as therapeutic agents. To this end, we performed a bimolecular fluorescence complementation (BiFC) analysis as follows. The BiFC principle is shown diagrammatically in the upper part of Figure 5.

[0152] Figure 5 shows the principle (top) and experimental results of analyzing whether a monoclonal antibody produced according to an embodiment of the present invention inhibits cell-to-cell transmission of alpha-synuclein. Here, blue indicates the nucleus, and green indicates the signal when alpha-synuclein released from the cell is transmitted into another cell and comes into contact with other alpha-synuclein to form aggregates.

[0153] Cell culture for BiFC analysis SH-SY5Y human neuroblastoma cell lines expressing alpha-synuclein and half of the Venus fluorescent protein (Venus 1-αSyn (V1S) and αSyn-Venus 2 (SV2)), respectively, were cultured as previously described (Lee HJ et al. J. Neurosci. 2004;24:1888-1896). Prior to the experiment, 180,000 cells expressing V1S and SV2 were mixed and spread on a cover slip and cultured for 3 days. To confirm the continuous intercellular transfer of alpha-synuclein, the co-culture was subcultured every 48 hours. In a separate experiment, the degree of transfer was confirmed to be maximum at about 6 passages (data not shown), so in this example, the inhibitory effect of the antibody on intercellular transfer was analyzed using co-culture for 6 passages.

[0154] BiFC experiment / antibody treatment The day before imaging, 50 μg / ml of IgG or test antibodies (3A9, 9B11, 11F11) were added to the co-culture. Signals in the Venus channel that appeared in each co-culture indicated aggregates due to the aggregation of alpha-synuclein, which were considered to be aggregates generated by the transfer of alpha-synuclein from one cell to another. These signals were automatically analyzed using an IN Cell Analyzer (GE Lifescience) (instrument settings: puncta size: 0.1-0.4 μm, intensity: 4000-7000). The results are shown in Figure 5.

[0155] Specifically, blue indicates the nucleus, and green indicates that alpha-synuclein released from one cell contacts alpha-synuclein in another cell to form aggregates. The graph on the right shows the signal strength. In the 9B11, 11F11, and 3A9 treatment groups, the number of cells showing green signals was reduced compared to the negative control group IgG, indicating that the antibody of the present application can effectively inhibit intercellular transmission of aggregates. In the 9B11, 3A9, and 11F11 antibody treatment groups, the number of cells showing green signals was significantly reduced compared to the negative control group IgG, indicating that the antibody of the present application, which specifically binds to aggregates with high affinity, can effectively inhibit intercellular transmission of alpha-synuclein.

[0156] Example 4. Analysis of the effect of anti-alpha-synuclein antibodies on removing alpha-synuclein aggregates in vivo To analyze the in vivo effects of the alpha-synuclein antibody produced in this application, 10 mg / kg of the antibody or IgG produced in this application was intraperitoneally administered weekly for three months to transgenic mice overexpressing human alpha-synuclein (mThy-1 human α-synuclein, UC San Diego). Six mice were used per group, and non-transgenic littermates were used as a control group. Then, perfusion was performed as follows.

[0157] After the last administration, the animals were anesthetized with chloral hydrate according to humane regulations and cardiac perfused with 0.9% saline for intracerebral pathological analysis. One half of the perfused brain (saggital section) was then stored in 4% paraformaldehyde in phosphate buffer (pH 7.4, 4°C) until analysis, and the other half was immediately stored frozen (-70°C).

[0158] Pathological analysis was performed as follows. Paraformaldehyde-fixed half brains were cut into 40 μm-thick serial sections using a vibratome in a free-floating manner. To confirm the expression level of alpha-synuclein in the brain of each treatment group, sections including the cortex, striatum, and hippocampus were incubated overnight at 4°C with alpha-synuclein antibodies (p129α-syn antibody, an aggregate marker, Abcam, ab59264, or total alpha-synuclein antibody, Cell Signaling Technology, #2642). To confirm the activity level of astrocytes and microglia, the sections were treated with GFAP (glial fibrillary acidic protein) (AB5804, Millipore) or Iba1 antibodies (019-19741, Wako), respectively. To confirm the degree of neuroinflammation, the sections were treated with IL-6 (NB600-1131, Novus Biologicals) or IL-1β antibody (ab9722, Abcam). After incubation with the primary antibody, the sections were treated with biotin-conjugated goat anti-rabbit IgG (1:100, Vector Laboratories) and Avidin D-horseradish peroxidase (1:200, ABC Elite, Vector Laboratories) and detected with DAB (diaminobenzidine). The immunostained sections were observed under a bright-field microscope to measure the optical density. The results are shown in Figures 6a and 6b. Figure 8a shows the results of staining mouse brain tissue with p-129α-Syn antibody after administration of the monoclonal antibody produced in one embodiment of the present invention to remove alpha-synuclein aggregates in a mouse animal model (TG) overexpressing human α-Syn. In the figure, HP is Hippocampus, p-129α-syn is a marker for the aggregate form phosphorylated at residue 129, and the arrow indicates phosphorylated alpha-synuclein.

[0159] FIG. 6a shows the results of analysis using p-129α-Syn antibody (an antibody that recognizes alpha-synuclein phosphorylated at Ser129), a marker of alpha-synuclein aggregates, and it was revealed that the amount of aggregates in CA1 and CA3 of the cortex and hippocampus of TG (TransGenic) mice administered with the antibody of the present application was significantly reduced compared to the control mice administered with IgG only (the relevant sites are indicated by arrowheads in the IgG sample). This result indicates that the antibody of the present application can significantly remove alpha-synuclein. WT and IgG are negative control groups, and antibody 274 is a comparison group that binds to both monomers and aggregates. These results indicate that the antibody of the present application can effectively inhibit the accumulation of aggregated alpha-synuclein and can be effectively used for the prevention and / or treatment of diseases associated with alpha-synuclein pathology.

[0160] FIG. 6b shows the same experiment as FIG. 6a, but stained with total alpha-synuclein antibody as a marker, with the arrow indicating human alpha-synuclein. Human alpha-synuclein increased in TG mice was effectively removed by antibody administration, indicating that the antibodies of the present application can effectively reduce alpha-synuclein aggregates and can be effectively used to treat synucleinopathies such as Parkinson's disease. The 9B11, 11F4, and 11F11 antibodies effectively inhibited the accumulation of total alpha-synuclein. Detection of total alpha-synuclein indicates that the antibodies of the present application have the ability to clear alpha-synuclein itself and inhibit cell-to-cell transmission of the antibody. From another perspective, this can be interpreted as the inhibition of the formation of monomer aggregates or the removal of all monomers.

[0161] Example 5. Analysis of the effect of anti-alpha-synuclein antibodies in reducing microgliosis and astrogliosis and reducing the release of inflammatory cytokines Gliosis is a non-specific reaction that occurs in glial cells in response to damage to the central nervous system, triggered by substances such as BBB damage, TGF-beta, or interleukins. Representative examples include microgliosis and astrogliosis, with Iba-1 and GFAP proteins used as markers, respectively. Therefore, as described in Example 4, the antibody of the present application was administered to mice to analyze its effect on reducing microgliosis and astrogliosis and reducing the release of inflammatory cytokines that trigger them. The analysis results are shown in Figures 7a, 7b, 7c, and 7d.

[0162] Figure 7a shows the results of staining mouse brain tissue with Iba-1 (microgliosis) antibody as a marker to determine whether the monoclonal antibody prepared in one embodiment of the present invention can reduce microgliosis in vivo. The arrows indicate activated microglia, and it can be seen that microglial activation is significantly reduced in the brains of mice administered with 3A9, 9B11, and 11F11 antibodies.

[0163] Figure 7b shows the results of staining mouse brain tissue with GFAP (astrogliosis) antibody as a marker to determine whether the monoclonal antibody produced in one embodiment of the present invention can reduce astrogliosis in vivo. The arrows indicate activated astrocytes, and it was confirmed that the 3A9, 9B11, and 11F11 antibodies effectively suppressed astrogliosis to a significant level.

[0164] Figure 7c shows the results of staining mouse brain tissue with IL-1 beta antibody as a marker to determine whether a monoclonal antibody according to an embodiment of the present invention can reduce inflammatory cytokines in vivo. The arrow indicates cells expressing IL-1 beta. IL-1 beta induces inflammation, leading to the death of various nerve cells and inflammatory responses, and shows that IL-1 beta was significantly reduced in the brain tissue of mice administered with the antibody according to the present invention.

[0165] Fig. 7d shows the results of staining mouse brain tissue with an IL-6 antibody as a marker to determine whether a monoclonal antibody according to an embodiment of the present invention can reduce inflammatory cytokines in vivo. The arrow indicates cells expressing IL-6, an inflammatory cytokine. This shows that IL-6 was reduced in the brain tissue of mice administered with the antibody according to the present application.

[0166] As shown in the figures, it was revealed that the antibody of the present application reduced microgliosis and astrogliosis, and reduced the release of the inflammatory cytokines IL-1beta and IL-6 that trigger microgliosis and astrogliosis, compared to the control group.

[0167] Example 6. Assay of anti-alpha-synuclein antibodies to detect Lewy bodies in human brain tissue Brain tissue sections of 10 micrometer-thick paraffin-embedded patients who died of Parkinson's disease were stained for Lewy bodies and Lewy neurites in the tissue sections using the antibody of the present application used in Example 5 as follows. The tissue sections were treated with 90% formic acid for 3 minutes to carry out antigen retrieval, and then peroxidase activity of the tissue itself was suppressed using 1% H2O2 (50% ethanol base). To prevent non-specific binding of the tissue, 10% normal horse serum was added. Next, after washing with phosphate buffer, the adjacent sections were treated with 3A9, 11F11 and 11F11 antibodies of the present application overnight at 4°C. After washing with phosphate buffer, the sections were treated with anti-human IgG antibody conjugated with biotin at 37°C for 30 minutes, and then reacted with avidin-biotin complex at room temperature for 30 minutes (Vectastatin Elite kit; Vector Laboratories). Then, the sections were developed with DAB containing 0.005% H2O2, and counterstained with 0.5% cresyl violet to separate the individual cells. The results are shown in Figures 8a and 8b.

[0168] 8a and 8b show the results of measuring whether the monoclonal antibodies 3A9 and 11F11 according to an embodiment of the present invention can specifically recognize Lewy bodies and Lewy neurites in human brain tissue, respectively. The antibodies according to the present application bind to Lewy bodies (arrows) and Lewy neurites (thread-like structures at the lower left).

[0169] As shown in Figures 8a and 8b, it was revealed that the antibody of the present application effectively binds to Lewy bodies and Lewy neurites (indicated by arrows). Such results indicate that the antibody can effectively bind to alpha-synuclein aggregates, which are components of Lewy bodies in human brain tissue. This indicates that the antibody delivered to human brain can effectively and specifically bind to alpha-synuclein aggregates. This result indicates that the antibody of the present application can specifically bind to alpha-synuclein aggregates present in actual human brain tissue, and can be effectively used for the prevention and / or treatment of diseases associated with alpha-synuclein pathology.

[0170] Example 7. Epitope analysis of anti-alpha-synuclein antibodies Epitope mapping for the 3A9 and 11F11 antibodies of the present application was performed by peptide array analysis requested from PEPSCAN (The Netherlands). The results are disclosed in Figure 9. Figure 9 is a schematic diagram showing the epitope mapping results of an antibody according to an example of the present invention.

[0171] As shown in FIG. 9, it was revealed that the antibody of the present application recognizes the C-terminus. It was revealed that the antibody of the present application binds mostly to the C-terminus. Alpha-synuclein antibodies that recognize the N-terminus cannot recognize aggregates of other diseases such as multiple system atrophy, which belongs to synucleinopathies commonly known as alpha-synuclein-related diseases, whereas alpha-synuclein antibodies that recognize the C-terminus have the advantage of recognizing aggregates of not only Parkinson's disease but also various other synucleinopathies. In particular, as described above, when recognizing the range between 110 and 122, it showed preferential binding to aggregates.

[0172] Example 8. Preparation of anti-alpha-synuclein (chimeric) antibodies Cloning Using the antibody nucleotide sequences of the heavy and light chain variable regions obtained after humanization, gblocks (m.biotech), which are short nucleotide fragments, were synthesized and used to clone into an animal cell culture vector (pcDNA3.4). The gblocks were synthesized containing approximately 20bp of overlapping nucleotides before and after the variable regions, and the portion of the pcDNA3.4 vector excluding the variable regions was prepared by amplifying it by PCR and cloned using the Gibson assembly method.

[0173] Transfection and expression The prepared vector was maxi-prepped (Qiagen) to obtain a large amount of plasmid DNA, which was then introduced into cells as follows. TM (Gibco, Cat: A29127) cells were cultured in ExpiCHO TM After adjusting the concentration to 3×10E6~4×10E6 viable cells / mL in expression medium (Gibco, Cat: A29100-01), the cells were cultured for 1 day at 8% CO2, 37℃, and 120 rpm. On the day of DNA transfection, the cells, which had grown to 7×10E6~10×10E6 viable cells / mL and had a survival rate of 95% or more, were diluted with fresh medium to 6×106 viable cells / mL.

[0174] ExpiFectamine for transfection into prepared mother cells TM CHO transfection kit (Gibco, Cat: A29129) was used to transfect the cells with ExpiFectamine. TM CHO & plasmid DNA complex was prepared. TM SFM® (Gibco, Cat: 12309019) medium was dispensed and titrated concentrations of DNA and ExpiFectamine were added. TM After inoculating each CHO reagent, mix them together and leave them at room temperature for 5 minutes, then inoculate them onto the mother cells, inject the transfection, and start culturing. TMThe enhancer and feed included in the CHO transfection kit were inoculated into the transfected cells, and after 5 days, the feed was additionally inoculated. The cells were then cultured for 10 days under conditions of 8% CO2, 37°C, and 120 rpm to complete the production.

[0175] To obtain the culture medium after the production was completed, the culture medium was transferred to a centrifuge bottle and centrifuged at 4°C and 6500 rpm for 30 minutes, and then filtered through a 0.2 μm filter to remove the suspended matter and obtain the culture medium, which was then subjected to a purification process.

[0176] Antibody purification The culture fluid was purified using HiTrap MabSelectSure (GE Healthcare, 11-0034-94). After equilibration using equilibration buffer (50 mM Tris-HCl pH 7.2, 100 mM NaCl), the collected culture fluid was loaded onto the column. After completion of loading, intermediate washing with 50 mM sodium citrate pH 5.0 was performed, followed by elution with 50 mM sodium citrate pH 3.4. The eluate was neutralized to pH 6.0 by adding 1 M Tris-HCl pH 9.0. The eluate was then buffer exchanged with PBS (phosphate buffered saline, pH 7.4) and concentrated, and stored at 4°C until use. If additional purification was required, the primary purified material was passed through a HiLoad 26 / 600 superdex 200 column using 1X PBS as a buffer, and secondary purification was performed based on the size of the eluted sample. The amino acid sequence of the purified antibody was analyzed by mass spectrometry, and it was confirmed to match the variable region of a mouse-derived monoclonal antibody.

[0177] The variable regions of the 3A9, 9B11, and 11F11 antibodies confirmed by the above method were replaced with the backbone variable region of the human IgG1 isotype to prepare chimeric human IgG1 antibodies. Among the obtained chimeric antibodies, the Ch11F11 antibody is an IgG type antibody and comprises a combination of the heavy chain variable region sequence (ch11F11-VH) of SEQ ID NO: 90 and the light chain variable region sequence (ch11F11-VL) of SEQ ID NO: 109, and the Ch3A9 antibody is an IgG type antibody and comprises a combination of the heavy chain variable region sequence (ch11F11-VH) of SEQ ID NO: 102 and the light chain variable region sequence (ch11F11-VL) of SEQ ID NO: 116.

[0178] Example 9: Production of humanized antibodies Library phage preparation A mini library was constructed in which a human framework was linked to the CDR1, CDR2, and CDR3 residues of a chimeric antibody, and mouse or human-derived sequences were introduced into each CDR residue. Competent cells of the library were inoculated into 2X YT medium [tryptone (CONDA, 1612.00) 17 g, yeast extract (CONDA, 1702.00) 10 g, NaCl (Sigma, S7653) 5 g] containing chloramphenicol (Sigma, C0857) 34 μg / ml, 2% glucose (Sigma, G5400), and 5 mM MgCl2 (Sigma, M2393), and then cultured at 37°C for about 3 hours until the OD600 value reached 0.5 to 0.7. The cells were then infected with helper phage and cultured at 30°C for 16 hours in 2X YT medium supplemented with chloramphenicol 34 μg / ml, 5 mM MgCl2, kanamycin (Sigma, K1876) 70 μg / ml, and 1 mM IPTG (ELPISBIO, IPTG025) to induce phage packaging. Next, the culture solution was centrifuged at 4500 rpm for 15 minutes at 4°C, and 4% PEG6000 (Fluka, 81253) and 3% NaCl (Sigma, S7653) were added to the supernatant, which was thoroughly dissolved and then reacted on ice for 1 hour. This was centrifuged again at 8000 rpm for 20 minutes at 4°C, and the pellet was suspended in PBS and centrifuged again at 12000 rpm for 10 minutes at 4°C to obtain the supernatant containing the library phage, which was then placed in a new tube and stored at 4°C until use.

[0179] Phage display panning Specifically, recombinant alpha-synuclein aggregates were added to an immunotube (maxisorp 444202) at a concentration of 10 μg / ml in PBS and allowed to adsorb proteins onto the surface of the tube at 4°C overnight. A 3% solution of bovine serum albumin (BSA) was then added to the tube to protect the surface to which alpha-synuclein aggregates had not been adsorbed. After emptying the tube, 10 12CFU of the antibody phage library was placed in an immunoassay tube adsorbing alpha-synuclein monomer protein and reacted at room temperature for 1 hour (negative selection). Phages that did not bind to alpha-synuclein monomer were collected and bound to the immunoassay tube adsorbing alpha-synuclein aggregates at room temperature for 2 hours. Non-specifically bound phages were then washed off 5 to 30 times with PBS-T (0.05% Tween 20) solution, and the remaining antigen-specific phage antibodies were collected using 100 mM triethylamine solution. The collected phages were neutralized with 1M Tris buffer (pH 7.4) and then infected with ER2537 E. coli at 37°C for 1 hour, and the infected E. coli was smeared on 2X YT agar medium containing carbenicillin and cultured overnight at 37°C. The next day, the cultured E. coli was suspended in 4 ml of 2X YT carbenicillin culture medium, 15% glycerol was added, and a portion was stored at -80°C, while the remaining portion was used to produce phages for the next round of panning. This process was repeated for a total of three rounds to amplify antigen-specific antibodies. As the panning rounds progressed, the number of washings with PBS-T was increased to amplify and concentrate antigen-specific phages.

[0180] Single clone screening In order to select monoclonal antibodies that specifically bind to alpha-synuclein aggregates from the phage pool obtained by the panning, the following experiment was carried out.

[0181] To isolate a single clone from the enriched pool, the phage pool was plated on LB-tetracycline / carbenicillin agar medium and cultured to obtain a single colony. The single clone was then inoculated into a 96-deep well plate containing 400 μl of 2X YT-tetracycline / carbenicillin medium per well and grown overnight, after which 10 μl of the culture was placed into a new 96-deep well plate containing 390 μl of 2X YT-tetracycline / carbenicillin medium and cultured at 37°C for 4 hours. 1 mM IPTG was added to the culture and cultured at 30°C overnight. The overnight culture was centrifuged to remove the supernatant.

[0182] Then, a clone expressing a single clone soluble scFv that binds to alpha-synuclein aggregates was selected using the ELISA method as follows (Steinberger. Rader and Barbas III. 2000. Phage display vectors. In: Phage Display Laboratory Manual. 1st. Cold Spring Harbor Laboratory Press. NY. USA. pp. 11.9-11.12). Specifically, the 7B7 antibody selected in Example 1-1 was placed in a 96-well plate and coated overnight at 4°C. 200 μL of 3% BSA was placed in each well and blocked at 37°C for 2 hours. Next, alpha-synuclein aggregates and monomers were loaded at a concentration of 100 ng / well, respectively, and reacted at 37°C for 2 hours. Then, the plate was washed five times with 300 μL of PBS-T. The prepared single clone supernatant was mixed with 3% BSA at 1:1 (vol:vol), and 100 μL of this was loaded onto the plate bound to the aggregates and monomers, and reacted at 37°C for 2 hours. Then, the plate was washed five times with 300 μL of PBS-T, and anti-HA HRP-conjugated antibody was added, reacted at 37°C for 1 hour, and washed five times with PBS-T. After adding 100 μL of TMB (Tetramethylbenzidine, Sigma, T0440) to develop color, 50 μL of 1N H2SO4 was added to stop the reaction, and the absorbance was measured at 450 nm. Clones with an absorbance of 0.5 or more were considered to be positive due to binding, and clones that nonspecifically bound to BSA were excluded.

[0183] The CDR residues of the clones found in the library were analyzed in silico to select clones that had serious problems binding to the framework or did not contain T-cell epitopes, B cell epitopes, or MHCII epitopes in the framework portion excluding the CDRs.

[0184] Then, for the five antibodies formed by the following combinations of heavy and light chain variable regions, the variable regions of the 11F11 antibody were replaced with the backbone variable region portion of the human IgG1 isotype to produce five IgG1 backbone humanized antibodies. Specifically, Hu11F11_(ABL2-4) is an IgG-type antibody and includes a combination of a heavy chain variable region sequence (Hu11F11-VH2) of SEQ ID NO: 92 and a light chain variable region sequence (Hu11F11-VL4) of SEQ ID NO: 113; Hu11F11_(ver.1) is an IgG-type antibody and includes a combination of a heavy chain variable region sequence (Hu11F11-VH-v1) of SEQ ID NO: 98 and a light chain variable region sequence (Hu11F11-VLv3 4c) of SEQ ID NO: 115; and Hu11F11_(ver.2) is an IgG-type antibody and includes a combination of a heavy chain variable region sequence (Hu11F11-VH-v2) of SEQ ID NO: 99 and a light chain variable region sequence (Hu11F11-VLv3 Hu11F11_(ver.3) is an IgG-type antibody that includes a combination of a heavy chain variable region sequence of SEQ ID NO: 100 (Hu11F11-VH-v3) and a light chain variable region sequence of SEQ ID NO: 115 (Hu11F11-VLv3 4c), and Hu11F11_(ver.3) is an IgG-type antibody that includes a combination of a heavy chain variable region sequence of SEQ ID NO: 100 (Hu11F11-VH-v3) and a light chain variable region sequence of SEQ ID NO: 115 (Hu11F11-VLv3 4c).

[0185] Hu11F11 (ver. 4) is a combination of the heavy chain variable region sequence of SEQ ID NO: 101 (Hu11F11-VH-v4) and the light chain variable region sequence of SEQ ID NO: 115 (Hu11F11-VLv3 4c).

[0186] Example 10. Analysis of phagocytosis-promoting ability of humanized antibodies In order to evaluate the efficacy of the chimeric antibody obtained in Example 8 and the humanized antibodies obtained in Example 9, hu11F11 (ver. 1), hu11F11 (ver. 2), hu11F11 (ver. 3), hu11F11 (ver. 4) and ABL2-4, in promoting phagocytosis of extracellular alpha-synuclein aggregates by microglia, a phagocytosis-promoting assay was performed as follows.

[0187] Specifically, mouse microglia line BV-2 was cultured in RPMI1640 and 10% FBS to prepare a concentration of 2X106cells / ml, and then dispensed into U-bottom 96-well plates in 100ul portions. In a separate container, alpha-synuclein aggregates and chimera 11F11 or Hu11F11_11F11(ver.1) or Hu11F11_11F11(ver.2) or Hu11F11_11F11(ver.3) or Hu11F11_11F11(ver.4) or Hu11F11_ABL2-4 antibody prepared in Example 8 and Example 9 were reacted at room temperature for 20 minutes, and the mixture was added to BV-2 in the 96-well plate and reacted for 15 minutes. After centrifugation at 1,200 rpm for 5 minutes to remove unbound alpha-synuclein aggregates and antibodies, the cells were fixed with 4% formaldehyde, washed three times with PBS, and permeablized with 0.5% Triton X-100. Synuclein-recognizing antibodies (Santa Cruz) were incubated for 1 hour, washed three times with PBS containing 0.1% Tween 20, incubated with anti-rabbit-Alexa-488 antibodies for 1 hour, washed, and analyzed by FACS caliber. The experimental results are shown in Figures 12a and 12b, which show the results of analyzing the ability of chimeric and humanized antibodies according to an embodiment of the present invention to promote phagocytosis of microglial alpha-synuclein aggregates. Figure 10a is for humanized 11F11 antibody, and Figure 10b is for humanized 3A9.

[0188] As shown in Figures 10a and 10b, compared to the IgG control group, the chimeric antibodies described in the present application increased the uptake of alpha-synuclein aggregates by BV-2 by about 2-fold, and the humanized antibodies increased it by up to 4-fold. Among these, the humanized antibody variants of 11F11, namely, Hu11F11_ABL2-4 (combination of Hu11F11-VH2 and Hu11F11-VL4), Hu11F11_ver.1 (combination of Hu11F11-VH-v1 and Hu11F11-VLv3 4c), and Hu11F11_(ver.2) (combination of Hu11F11-VH-v2 and Hu11F11-VLv3 4c) showed superior ability to promote phagocytosis of BV-2 compared to the chimeric 11F11 antibody. Hu11F11_ver.3) (combination of Hu11F11-VH-v3 and Hu11F11-VLv3 4c) and hu11F11(ver.4) (combination of Hu11F11-VH-v4 and Hu11F11-VLv3 4c) showed phagocytosis-promoting ability similar to that of the chimeric 11F11 antibody.

[0189] Example 11: Fibril binding assay A fibril binding assay was performed to analyze the efficacy of antibodies that inhibit neuronal binding, a precursor step in the process of alpha-synuclein aggregates spreading to neighboring neurons through cell-to-cell transmission. Specifically, SH-SY5Y cells, a neuronal cell line, were treated with alpha-synuclein aggregates at 1uM, chimeric 11F11 antibody, humanized 11F11 variants, namely Hu11F11_ver1, Hu11F11_ver2, Hu11F11_ver3, Hu11F11_ver4, and Hu11F11_ABL2-4 at 20nM each for 10 minutes, and then fixed with 4% formaldehyde. After washing, the cells were treated with alpha-synuclein antibody (BD) that recognizes all forms of alpha-synuclein under non-permeable conditions at 1:250, washed, and imaged under a confocal microscope. Just before imaging, DAPI was added to show the cell nuclei. As a result, the antibodies of the present application effectively reduced the degree of cell membrane binding of alpha-synuclein aggregates compared to the IgG control group, and in particular, the humanized antibody 11F11 reduced the degree of binding similarly or more efficiently compared to the 11F11 chimeric antibody. Figure 11 shows the results of an analysis of the ability of a chimeric antibody and a humanized antibody according to an embodiment of the present invention to inhibit the binding of alpha-synuclein aggregates to the surface of nerve cells. As shown in Figure 11, the antibodies of the present application effectively reduced the extent of cell membrane binding of alpha-synuclein aggregates by up to 23-fold compared to the IgG control group, and in particular, the humanized antibody 11F11 reduced the extent of binding similarly or even more efficiently than the 11F11 chimeric antibody. hu11F11(ver.1) (combination of Hu11F11-VH-v1 and Hu11F11-VLv3 4c), hu11F11(ver.3) (combination of Hu11F11-VH-v3 and Hu11F11-VLv3 4c), and hu11F11(ver.4) (combination of Hu11F11-VH-v4 and Hu11F11-VLv3 4c) showed superior efficacy compared to the chimeric 11F11 antibody, while ABL2-4 (combination of Hu11F11-VH2 and Hu11F11-VL4) and hu11F11(ver.2) (combination of Hu11F11-VH-v2 and Hu11F11-VLv3 4c) showed a reduction ability similar to that of the chimeric 11F11 antibody.

[0190] Example 12: Dual chamber assay In order to more directly analyze the efficacy of the humanized antibody according to the present invention in inhibiting the intercellular spread of alpha-synuclein aggregates, a dual chamber assay was performed. alpha-synuclein-overexpressing SH-SY5Y cells (donor cells) were plated on a permeable membrane insert, and normal SH-SY5Y cells (recipient cells) were plated on a coverslip in a 12-well plate, and then the insert in which the alpha-synuclein-overexpressing cell line was cultured was placed on top and cultured overnight. At the same time, the recipient cells were treated with 20 nM of the antibody of the present application. The recipient cells were then fixed with 4% formaldehyde, washed, and treated with 0.5% TritonX-100 to form a permeable condition. After washing, the cells were treated with the same detection antibody and DAPI as in Example 14, and imaged with a confocal microscope. The analyzed images are shown in FIG.

[0191] As a result, the humanized antibodies according to the present invention effectively reduced the extent of spread of alpha-synuclein aggregates to recipient cells compared to the IgG control group, and in particular, the 11F11 humanized antibody reduced the spread of alpha-synuclein aggregates to recipient cells similarly or even more efficiently than the 11F11 chimeric antibody. FIG. 12 shows the efficacy analysis results of chimeric and humanized antibodies according to an embodiment of the present invention for suppressing the intercellular transmission of alpha-synuclein aggregates secreted from neurons overexpressing alpha-synuclein and transferred to normal neurons. The alpha-synuclein-overexpressing SH-SY5Y neuronal cell line plated in the top chamber secretes alpha-synuclein extracellularly, which is transferred to the normal SH-SY5Y cell line plated in the lower 12-well plate. The antibody of the present application significantly reduced the amount of alpha-synuclein flowing into the normal SH-SY5Y cell line in the lower plate compared to the IgG control group. In particular, it was confirmed that the humanized antibody of the 11F11 clone among the humanized antibodies of the present application further increased the degree of reduction in alpha-synuclein flowing into the recipient cells in the lower plate compared to the chimeric antibody. More specifically, hu11F11(ver.2) (combination of Hu11F11-VH-v2 and Hu11F11-VLv3 4c) and hu11F11(ver.4) (combination of Hu11F11-VH-v4 and Hu11F11-VLv3 4c) showed superior inhibitory ability compared to the chimeric antibody, while hu11F11(ver.1) (combination of Hu11F11-VH-v1 and Hu11F11-VLv3 4c) and hu11F11(ver.3) (combination of Hu11F11-VH-v3 and Hu11F11-VLv3 4c) showed inhibitory ability similar to that of the chimeric antibody.

[0192] Compared to the IgG control group, it effectively reduced the extent of spread of alpha-synuclein aggregates by approximately 22-fold, and in particular, the 11F11 humanized antibody reduced the spread of alpha-synuclein aggregates to recipient cells similarly or even more efficiently than the 11F11 chimeric antibody.

[0193] Example 13. ELISA analysis of humanized antibodies In order to quantitatively analyze the binding ability of the chimeric antibody obtained in Example 8 and the humanized antibody obtained in Example 9, sandwich ELISA was performed in a manner substantially similar to that in Example 2-2.

[0194] Specifically, each antibody was diluted 1 / 10 at concentrations of 0.04 to 400 nM and coated on a 96-well plate, and then 2000 ng / ml of aggregate was added to each well. After washing with 1X PBS, the plate was treated with a capture antibody bound to biotin and streptavidin bound to HRP, and reacted with TMB as a substrate, and the absorbance was measured. The results are shown in Figure 13.

[0195] As shown in FIG. 13, it was confirmed that the humanized antibody according to the present invention, particularly the humanized antibody derived from chimera 11F11 (humanized 11F11 antibody), exhibits binding activity equivalent to that of the chimera 11F11 clone. It was confirmed that the humanized antibodies, particularly the 11F11-derived variants, hu11F11 (ver. 1), i.e., the combination of Hu11F11-VH-v1 and Hu11F11-VLv3 4c, hu11F11 (ver. 2), i.e., the combination of Hu11F11-VH-v2 and Hu11F11-VLv3 4c, hu11F11 (ver. 3), i.e., the combination of Hu11F11-VH-v3 and Hu11F11-VLv3 4c, and hu11F11 (ver. 4), i.e., Hu11F11-VH-v4 and Hu11F11-VLv3 4c, exhibit binding strength similar to that of the chimeric 11F11 clone, and their EC 50 The EC of the chimeric 11F11 antibody was 11.5 to 15.1 nM. 50 The value was similar to 12.5 nM.

[0196] Example 14: BIAcore analysis using anti-alpha-synuclein antibodies The binding affinity of the chimeric antibody obtained in Example 8 and the humanized antibody obtained in Example 9 was quantitatively analyzed using BIAcore in a manner substantially similar to that in Examples 2-3. The analysis results are shown in FIG. 14 and the table below. TIFF0007610279000015.tif33170

[0197] As a result, it was confirmed that the humanized antibodies described in the present application, in particular the variants of 11F11, i.e., hu11F11(ver.2) (combination of Hu11F11-VH-v2 and Hu11F11-VLv3 4c), hu11F11(ver.3) (combination of Hu11F11-VH-v3 and Hu11F11-VLv3 4c), and hu11F11(ver.4) (combination of Hu11F11-VH-v4 and Hu11F11-VLv3 4c), exhibited KD values ​​similar to those of the chimeric 11F11 clone, and the degree of binding of the humanized clone was 0.02 to 0.06 × 10 -9 The KD of M, chimeric 11F11 clone was 0.02 × 10 -9 M exhibited a low KD value, i.e., a high binding affinity to aggregates.

[0198] Example 15. Evaluation of humanized antibody productivity The 11F11 chimeric antibody and four types of 11F11 humanized antibody variants (Hu11F11 (ver. 1), Hu11F11 (ver. 2), Hu11F11 (ver. 3), Hu11F11 (ver. 4)) were transfected and cultured as described in Example 8, and the primary and secondary purification steps were completed. The antibody quantity and purity of the final purified sample were then analyzed.

[0199] The amount of antibody was measured using Thermo Scientific TM NanoDrop TM The measurement was performed using a spectrophotometer. First, 1XPBS was loaded to determine the zero point, and then a certain amount of purified sample was loaded and the sample was measured at a UV wavelength of 280 nm, which is used for protein quantification. NanoDrop TM The concentration of the sample confirmed in step 1 was multiplied by the total volume of the sample to obtain the total yield, and the total yield was divided by the total volume to obtain the productivity (yield (g / L)). The purity of the final purified product was analyzed by HLC-001 (Agilent 1200 series) HPLC, and the ratio of the main peak was quantified using a mobile phase of 1X PBS and 200 mM Arginine-HCl (pH 6.8) on TSK gel G3000SWXL. The productivity analysis results are shown in the table below. TIFF0007610279000016.tif48170

[0200] As a result, the productivity of the chimeric 11F11 antibody and the humanized variant of 11F11, hu11F11(ver.1), i.e., hu11F11(ver.1) (combination of Hu11F11-VH-v1 and Hu11F11-VLv3 4c), hu11F11(ver.2) (combination of Hu11F11-VH-v2 and Hu11F11-VLv3 4c), hu11F11(ver.3) (combination of Hu11F11-VH-v3 and Hu11F11-VLv3 4c), and hu11F11(ver.4) (Hu11F11-VH-v4 and Hu11F11-VLv3 4c), was 0.068 to 0.423 g / L, which was superior to the usual antibody production yield produced by transient transfection. In particular, hu11F11 (ver. 2), ie, the combination of Hu11F11-VH-v2 and Hu11F11-VLv3 4c, exhibited a productivity of 0.423 g / L, which was higher than the productivity of the chimeric 11F11 antibody, 0.282 g / L.

[0201] Example 16. Evaluation of alpha-synuclein specific binding of chimeric antibodies Three chimeric antibodies, 3A9, 9B11, and 11F11, were produced according to Example 8, and the binding specificity of the antibodies to alpha-synuclein was compared with that of its homologues, beta- and gamma-synuclein, by ELISA.

[0202] For this purpose, human beta-synuclein (Uniprot:Q16143) protein (CUSABIO, Cat:CSB-EP624090HU) or human gamma-synuclein (Uniprot:O76070) protein (CUSABIO, Cat:CSB-EP021915HU) was coated on a 96-well plate at a concentration of 100ng / ml, washed, and then blocked with 5% BSA for 2 hours. At this time, a pan-Syn antibody (Santacruz, Cat:FL-140, sc-10717, rabbit) that binds to all of alpha, beta, and gamma-synuclein was used as a positive control. After washing, the sections were treated with chimeric antibodies (3A9, 9B11, 11F11) diluted in 1 / 10 increments from 400nM to 0.04nM for 2 hours, then washed with PBS. Next, the sections were treated with a detection antibody consisting of a goat anti-hFc antibody conjugated with HRP, reacted with TMB as a substrate, and the absorbance was measured at 450nm and 650nm. In the case of the positive control group, a goat anti-rabbit antibody conjugated with HRP was used as the detection antibody.

[0203] The experimental results are shown in Figures 15a and 15b, which show the very low binding affinity of chimeric antibodies (3A9, 9B11, 11F11) according to an embodiment of the present invention to human beta-synuclein and human gamma-synuclein analyzed by ELISA. In contrast, the positive control pan-Syn antibody showed high binding affinity to beta- and gamma-synuclein. Figure 15a is for human beta-synuclein and Figure 15b is for human gamma-synuclein.

[0204] In addition, the three antibodies were used to treat amyloid beta 1-42 The specific binding ability of the antibody to alpha-synuclein aggregates was analyzed by reacting it with aggregates formed from amyloid beta (Uniprot: P05067; CAS number: 107761-42-2) and tau (Uniport: P10636-8) proteins in a dot blot.1-42 Amyloid beta and tau form aggregates, which are believed to be an important cause of degenerative neurological diseases, particularly Alzheimer's disease, and the aggregates are known to have structures such as oligomers, protofibrils, and fibrils, similar to those of alpha-synuclein aggregates. Therefore, the dot blot was performed to confirm that the chimeric antibodies 3A9, 9B11, and 11F11 recognize the specific sequence of alpha-synuclein and do not recognize the common structure of aggregates that alpha-synuclein, amyloid beta, and tau-derived aggregates have as aggregates.

[0205] The dot blot method of this example was carried out in a manner substantially similar to that of Example 2-3. 1-42 Tau protein and its corresponding aggregates were produced in the laboratory of Professor Lee Seung-jae at Seoul National University. Syn-1, 6E10, and Tau5 are alpha-synuclein and amyloid beta, respectively. 1-42 , an antibody known to bind to tau protein. The analysis results are shown in Figure 15c.

[0206] The experimental results are shown in FIG. 15c, which shows that the chimeric antibodies 3A9, 9B11, and 11F11 of the present application specifically bind only to alpha-synuclein-derived aggregates and not to amyloid beta. 1-42 In contrast, the results showed that amyloid beta- 1-42 The 6E10 and Tau5 antibodies, known to bind to tau, respectively, were shown to bind to the aggregates on dot blots.

[0207] The above results suggest that the chimeric antibody does not bind to alpha-synuclein homologs and aggregates derived from different proteins, suggesting that the antibody binds efficiently only to the target protein and is more effective than antibodies or drugs that bind to homologs and aggregates derived from different proteins.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to alpha-synuclein (α-Syn) aggregates, The antibody or antigen-binding fragment comprises: Heavy chain complementarity determining region (CDR) 1 (H-CDR1) comprising SEQ ID NO:1; Heavy chain CDR2 (H-CDR2) comprising SEQ ID NO: 2 or 4; Heavy chain CDR3 (H-CDR3) comprising SEQ ID NO:5; Light chain CDR1 (L-CDR1) comprising SEQ ID NO: 10; A light chain CDR2 (L-CDR2) comprising SEQ ID NO:11, and a light chain CDR3 (L-CDR3) comprising SEQ ID NO: 12; When the H-CDR2 comprises SEQ ID NO:2, The antibody or antigen-binding fragment may have the following framework regions (FR): i) a heavy chain FR1 (H-FR1) comprising SEQ ID NO: 17 or 38; ii) a heavy chain FR2 (H-FR2) comprising SEQ ID NO: 19 or 42; iii) a heavy chain FR3 (H-FR3) comprising an amino acid sequence selected from SEQ ID NOs: 21-31; iv) heavy chain FR4 (H-FR4) comprising SEQ ID NO: 33 or 34; v) a light chain FR1 (L-FR1) comprising an amino acid sequence selected from SEQ ID NOs: 54-58; vi) light chain FR2 (L-FR2) comprising SEQ ID NO: 60 or 61; vii) a light chain FR3 (L-FR3) comprising an amino acid sequence selected from SEQ ID NOs: 63-67; and viii) a light chain FR4 (L-FR4) comprising SEQ ID NO: 69 or 70; An antibody or antigen-binding fragment thereof that specifically binds to alpha-synuclein (α-Syn) aggregates, further comprising one or more of:

2. 2. The antibody or antigen-binding fragment of claim 1, wherein the antibody comprises two, three, four, five, six, seven, or all eight of i) to viii).

3. The antibody is H-CDR1 comprising SEQ ID NO: 1, H-CDR2 comprising SEQ ID NO:2, H-CDR3 comprising SEQ ID NO:5, L-CDR1 comprising SEQ ID NO: 10, L-CDR2 comprising SEQ ID NO: 11, and L-CDR3 comprising SEQ ID NO: 12 The antibody or antigen-binding fragment of claim 1 , comprising:

4. a) a heavy chain variable region (VH) comprising an amino acid sequence selected from SEQ ID NOs: 91-101, or an amino acid sequence which is at least 90% or at least 95% identical thereto; and / or b) a light chain variable region (VL) comprising an amino acid sequence selected from SEQ ID NOs: 110-115, or an amino acid sequence which is at least 90% or at least 95% identical thereto; The antibody or antigen-binding fragment of claim 1 , comprising:

5. a) a VH comprising an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NO: 98, and a VL comprising an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NO: 115; b) a VH comprising an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NO: 99, and a VL comprising an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NO: 115; c) a VH comprising an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NO: 100, and a VL comprising an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NO: 115; d) a VH comprising an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NO: 101, and a VL comprising an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NO: 115; or e) a VH comprising an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NO: 92, and a VL comprising an amino acid sequence that is at least 90% or at least 95% identical to SEQ ID NO: 113; The antibody or antigen-binding fragment of claim 4, comprising:

6. each SEQ ID NOs: 98 and 115, SEQ ID NOs: 99 and 115, SEQ ID NOs: 100 and 115, SEQ ID NOs: 101 and 115, or SEQ ID NOs: 92 and 113, The antibody or antigen-binding fragment of claim 4, comprising a VH and a VL comprising:

7. 5. The antibody or antigen-binding fragment of claim 4, comprising a VH and a VL comprising SEQ ID NOs: 99 and 115, respectively.

8. An antibody comprising a heavy chain and a light chain comprising SEQ ID NO:129 and SEQ ID NO:130, respectively.

9. Fab fragment, Fab' fragment, F(ab') ２ The antigen-binding fragment of any one of claims 1 to 7, which is a fragment, diabody or scFV.

10. The antibody of any one of claims 1 to 8, which is of the IgG1, IgG2, IgG3 or IgG4 isotype subtype.

11. The antibody or antigen-binding fragment of any one of claims 1 to 10, which inhibits intercellular transmission of α-Syn aggregates, degrades α-Syn, and / or inhibits the formation of α-Syn aggregates.

12. One or more isolated polynucleotides encoding the heavy and light chain amino acid sequences of the antibody or antigen-binding fragment of any one of claims 1 to 11.

13. 13. One or more expression vectors comprising one or more polynucleotides of claim 12.

14. A cell transformed with one or more expression vectors according to claim 13.

15. Culturing the cell of claim 14 under conditions allowing expression of the antibody or antigen-binding fragment of any one of claims 1 to 11, and Isolating the antibody or antigen-binding fragment from the cell culture. a method for producing said antibody or antigen-binding fragment, comprising:

16. A pharmaceutical composition for treating α-synucleinopathy or regulating the level of α-Syn aggregates in a subject in need thereof, comprising an antibody or antigen-binding fragment of any one of claims 1 to 11 and a pharma- ceutically acceptable excipient.

17. A pharmaceutical composition for diagnosing α-synucleinopathy in a subject, comprising the antibody or antigen-binding fragment of any one of claims 1 to 11 and a pharma- ceutically acceptable excipient, The diagnosis is determining the concentration and / or subcellular location of α-Syn aggregates in a sample from the subject by using the composition; and comparing the concentration and / or subcellular location of the α-Syn aggregates measured in the subject sample with that of a control sample, wherein a similarity or difference compared to the control sample result indicates whether the subject has α-synucleinopathy. Pharmaceutical compositions.

18. The α-synucleopathy includes Parkinson's disease (PD), Parkinson's disease dementia (PDD), dementia with Lewy bodies (DLB), Lewy body variant of Alzheimer's disease (LBV), combined Alzheimer's and Parkinson's disease, and multiple system atrophy (MSA).

18. The pharmaceutical composition according to claim 16 or 17, wherein the compound is selected from the group consisting of cyclosporine, ...

19. A method for detecting α-Syn aggregates in a biological sample, comprising contacting the biological sample with an antibody or antigen-binding fragment according to any one of claims 1 to 11.

Images