Bispecific antibodies against alpha-syn / IGF1r and uses thereof

Abstract

Provided are bispecific antibodies against alpha-synuclein and IGF1R, and pharmaceutical compositions for preventing and / or treating synucleinopathies, comprising the bispecific antibodies.SOLUTION: The bispecific antibodies against α - synuclein and IGF1R are used for preventing, treating and / or diagnosing synucleinopathies, which are diseases associated with α - synuclein or aggregates thereof, so that the α - syn antibodies or antigen-binding fragments thereof can pass through the blood brain barrier to exert their action in the brain, and the half-lives are extended to maintain the drug efficacy for a long period of time.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a bispecific antibody against alpha-synuclein and IGF1R, a pharmaceutical composition for the prevention and / or treatment of synucleinopathies (α-synucleinopathies) comprising said bispecific antibody, and a method for detecting alpha-synuclein aggregates or providing information for the diagnosis of synucleinopathies comprising said bispecific antibody. [Background technology]

[0002] Alpha-synuclein (α-syn) is primarily expressed in the presynaptic terminals of neurons and exists normally as a monomer in an unfolded state. Alpha-synuclein helps regulate the release of dopamine, a key neurotransmitter that controls the initiation and termination of voluntary and involuntary movements. Alpha-synuclein function is particularly important for increased synaptic activity and as we age, it is an important factor in neurodegeneration.

[0003] However, in pathological conditions, alpha-synuclein undergoes structural changes through binding and interaction with droplets, phospholipid bilayers, or lipid membranes, forming folded or folded alpha-helical secondary structures and forming aggregates containing dimers, oligomers, and / or fibrillar molecules.

[0004] These alpha-synuclein aggregates are known to induce cell toxicity and are the main component of Lewy bodies, abnormal protein aggregates found in neurons in various diseases, including Parkinson's disease (PD), Parkinson's disease dementia (PDD), multiple system atrophy (MSA), dementia with Lewy bodies (DLB), and others. Post-translational modifications of alpha-synuclein, such as phosphorylation and ubiquitination, are also known to be associated with 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, Parkinsonism-related dementia, dementia with Lewy bodies, multiple system atrophy, and many other axonal disorders.

[0005] Antibodies to alpha-synuclein or fragments of alpha-synuclein to induce such antibodies have been proposed as immunotherapeutic methods for synucleinopathies, but brain penetration of antibodies can be limited by the blood-brain barrier (BBB). Furthermore, the lack of highly specific BBB transporters has slowed the development of new therapeutic and diagnostic agents for diseases that originate in the brain, including brain tumors and neurodegenerative diseases. There is clearly a need for methods for delivering therapeutic and diagnostic molecules to the brain in pharmaceutically effective doses without disrupting the physiology and homeostasis of the BBB. Summary of the Invention [Problem to be solved by the invention]

[0006] One example of the present invention provides an antibody comprising an antigen-binding site for alpha-synuclein (α-syn) and an antigen-binding site for IGF1R, or a method for producing said antibody. Other examples provide polynucleotides encoding the antibodies, recombinant vectors containing the same, and recombinant cells containing the same.

[0007] Yet another example provides a pharmaceutical composition for preventing and / or treating alpha-synucleinopathies, comprising the bispecific antibody against alpha-syn and IGF1R and a pharmaceutically acceptable excipient.

[0008] The present invention provides a method for delivering drugs to the brain for use in the diagnosis, treatment, or prevention of alpha-synucleinopathies using the antibody or antigen-binding fragment thereof.

[0009] One example of the present invention provides an anti-IGF1R antibody or antigen-binding fragment thereof that specifically recognizes IGF1R (Insulin-like Growth Factor 1 Receptor) without affecting the binding of IGF1R ligands, does not inhibit signal transduction through the IGF1R receptor, and is capable of transcytosis.

[0010] In another aspect, there is provided an isolated polynucleotide encoding an anti-IGF1R antibody or antigen-binding fragment according to the invention.

[0011] In another aspect, there is provided a composition for delivering a physiologically active substance across the blood-brain barrier, comprising the anti-IGF1R antibody or antigen-binding fragment thereof according to the present invention.

[0012] Another aspect of the present invention provides a blood-brain barrier transporter in which the anti-IGF1R antibody or antigen-binding fragment can transport a physiologically active substance, for example, a physiologically active substance that acts in the brain, to the brain through the IGF1R receptor and across the blood-brain barrier (BBB).

[0013] In yet another embodiment, the present invention provides a protein complex in which the anti-IGF1R antibody or antigen-binding fragment is conjugated to a biologically active molecule, for example, a physiologically active substance that acts in the brain, and which can be delivered to the brain through the blood-brain barrier (BBB).

[0014] In another aspect, there is provided a method for detecting IGF1R from a biological sample, comprising the steps of providing an anti-IGF1R antibody or antigen-binding fragment according to the present invention and contacting the anti-IGF1R antibody or antigen-binding fragment with a biological sample in which IGF1R expression needs to be detected.

[0015] The present invention provides a method for delivering a physiologically active substance used in the diagnosis, treatment, or prevention of brain diseases through the blood-brain barrier into the brain using an anti-IGF1R antibody or antigen-binding fragment thereof. [Means for solving the problem]

[0016] The present invention is described in more detail below. As used herein, the term "antibody" refers to an intact immunoglobulin of any isotype, an antigen-binding fragment capable of competing with the intact antibody for binding to a target antigen, or a combination thereof. This term includes, for example, chimeric, humanized, and fully human antibodies, as well as antigen-binding fragments and combinations thereof. An antibody is also a type of antigen-binding protein in itself. An antibody generally comprises at least two full-length heavy chains and two full-length light chains, although in some cases an antibody may comprise only a heavy chain. The antibody includes monospecific antibodies that specifically bind to one target, and multispecific antibodies (e.g., bispecific and trispecific antibodies) that specifically bind to multiple targets.

[0017] The antibody may be a monoclonal antibody or a polyclonal antibody, and the monoclonal antibody may be a human, humanized, or chimeric antibody that specifically binds to IGF1R. The monoclonal antibody may be an IgG1, IgG2, IgG3, or IgG4 type antibody that specifically binds to IGF1R.

[0018] As used herein, the term "light chain" includes full-length light chains and fragments thereof that contain sufficient variable region sequence to provide binding specificity for an antigen or epitope. A full-length light chain contains 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.

[0019] As used herein, the term "complementarity-determining regions (CDRs)" refers to the sites in the variable region of an antibody that confer antigen-binding specificity.

[0020] 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 closest 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.

[0021] The antibody may be selected from any subtype of immunoglobulin (e.g., IgA, IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4), IgM, etc.). The IgG antibody may be of the IgG1, IgG2, IgG3, or IgG4 subtype, e.g., IgG1 or IgG2 subtype. The IgG antibody comprises two heavy chains and two light chains, each of which is linked via a disulfide bond to form two heavy-light chain dimers, and the two heavy-light chains are linked via a disulfide bond in the Fc region of the heavy chain. The IgG antibody may be a monospecific antibody that targets one antigen, containing antigen-binding sites for the same antigen in both heavy-light chain structures, or a bispecific antibody that targets two antigens, containing antigen-binding sites for different antigens in both heavy-light chain structures.

[0022] Antibodies according to the present invention include, but are not limited to, bispecific antibodies, whole antibodies, minibodies, domain antibodies, antibody mimetics (or synthetic antibodies), antibody fusions (or antibody conjugates), fragments thereof, and combinations thereof. Various antibody structures are additionally disclosed in the present invention below.

[0023] As used herein, a "variant" of a polypeptide, such as an antigen-binding fragment, protein, or antibody, is a polypeptide that contains insertions, deletions, additions, and / or substitutions of one or more amino acid residues relative to another polypeptide sequence, including fusion polypeptides. For example, an antibody portion may contain conservative amino acid substitutions at one or more residues in the heavy or light chain, variable region, or CDR sequences.

[0024] A "derivative" of a polypeptide according to the present invention refers to a polypeptide that has been chemically altered through conjugation with other chemical moieties, as distinct from insertion, deletion, addition or substitution mutants.

[0025] Antibodies according to the present invention can be produced and selected using hybridoma technology, whereby antigen-specific human mAbs with the desired specificity are generated and selected from transgenic mice, such as those described above. Such antibodies can be cloned and expressed using appropriate vectors and host cells, or harvested from cultured hybridoma cells. Alternatively, the antibodies can be derived from a phage-display library. Phage display technology is a method mimicking immune selection, in which an antibody repertoire is displayed on the surface of filamentous bacteriophage, and phages that bind to the desired antigen are selected from the displayed antibody repertoire. For details of this technology, see the Examples of the present invention or PCT Publication WO 99 / 10494. In one embodiment, the humanized antibodies of the present invention are selected using a phage display method.

[0026] Antibodies or antigen-binding fragments thereof may be derived from a single source or may be chimeric. Chimeric antibodies contain portions derived from two different antibodies and are 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 specified, the term antibody herein includes antibodies containing two full-length heavy chains and two full-length light chains, as well as derivatives, variants, immunologically functional immunoglobulin fragments, mutants, and combinations thereof, including, for example, antibodies containing one or two scFvs in addition to two full-length heavy chains and two full-length light chains, examples of which are described below.

[0027] As used herein, an "antigen-binding fragment" refers to a portion of an antibody or a polypeptide comprising the same that retains the ability to specifically bind to an antigen. For example, an antigen-binding fragment can be a portion of an antibody or a polypeptide comprising the same that contains amino acid residues that interact with an antigen (e.g., an epitope) to confer specificity and / or affinity to the antibody for the antigen. Such fragments contain at least one CDR present in a full-length light or heavy chain, and in some embodiments, a single heavy and / or light chain, or portion thereof. Such biologically active fragments can be produced by recombinant DNA techniques or, for example, by enzymatic or chemical cleavage of intact antibodies.

[0028] 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.), and may be derived from any mammal, including, but not limited to, human, mouse, rat, camelid, or rabbit. Functional portions of the antibodies disclosed herein, such as one or more CDRs, can be covalently linked to a second protein or small molecule to be used as targeted therapeutics against specific targets.

[0029] As used herein, a "single-chain antibody" refers to a single polypeptide chain of antigen-binding domains in which heavy and light chain variable regions are linked by a flexible linker. For example, the single-chain antibody may be one or more types selected from the group consisting of scFv, in which the heavy chain variable region and light chain variable region are linked in a single chain, and scFv-Fc, in which the heavy chain variable region, light chain variable region, and Fc are linked in a single chain. For information on single-chain antibodies, see, for example, U.S. Patent No. 5,260,203.

[0030] As used herein, "affinity" refers to the strength of the interaction between an antibody or its antigen-binding fragment 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), and characteristics of the antigen, such as the size, shape, and / or charge of the antigen. Methods for determining affinity are known in the art and can usually be expressed as, but are not limited to, the dissociation constant (KD).

[0031] 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, with the heavy chain also including a "D" region of about 10 more amino acids.

[0032] 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.

[0033] One example of the present invention relates to an antibody that contains an antigen-binding site for alpha-synuclein (α-syn) and an antigen-binding site for IGF1R, specifically a bispecific antibody for alpha-synuclein (α-syn) and IGF1R (hereinafter referred to as an anti-α-syn / anti-IGF1R bispecific antibody). Thus, the bispecific antibody of the present invention can recognize and bind to both alpha-synuclein and IGF1R as antigens.

[0034] The anti-α-syn / anti-IGF1R bispecific antibody according to the present invention comprises the anti-α-syn antibody or an antigen-binding fragment thereof, and can specifically recognize and bind to alpha-synuclein, particularly the C-terminal region of alpha-synuclein, and can be used for the prevention, treatment and / or diagnosis of synucleinopathies, which are diseases associated with alpha-synuclein or its aggregates.

[0035] As used herein, a "bivalent antigen-binding protein" or "bivalent antibody" comprises two antigen-binding sites. The two antigen-binding sites in such a bivalent antibody may have the same antigen specificity, or they may be bispecific antibodies that bind to different antigens. As used herein, a "multispecific antigen-binding protein" or "multispecific antibody" targets more than one antigen or epitope.

[0036] As used herein, a "bispecific" or "dual-specific" antigen-binding protein or antibody refers to a hybrid antigen-binding protein or antibody having two different antigen-binding sites. Such bispecific antibodies are a type of multispecific antigen-binding protein or multispecific antibody and can be produced by various known methods, such as hybridoma fusion 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 distinct 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, the antibody of the present application can take 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 such as receptor-mediated transcytosis of the glucose and amino acid transporters, insulin or transferrin, contained within cells.

[0037] 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), and neurodegeneration with multiple neuronal atrophy and cerebral iron deposition type I (NBIA Type I). Alpha-synuclein aggregates are also found secondary to Alzheimer's disease (Kim et al., Alzheimer's Research & Therapy 2014, 6:73).

[0038] Synucleinopathies are a diverse group of neurodegenerative disorders that share common pathological characteristics: in neuropathological experiments, distinctive lesions can be detected, including abnormal aggregation of alpha-synuclein protein within select 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 proteocytes and platelets. The precise role of alpha-synuclein within these cells is unknown, but it is thought to be associated with the differentiation of megakaryocytes (platelet precursors).

[0039] As used herein, "alpha-synuclein aggregate-associated diseases" refer to a group of neurodegenerative diseases known as synucleinopathies, characterized by the discovery of alpha-synuclein aggregates in lesions involving neuronal and glial cell (glia) populations. Such diseases include, but are not limited to, Parkinson's disease, Parkinsonism-associated dementia, Lewy body dementia, Alzheimer-Lewy body disease, combined Alzheimer's and Parkinson's disease, multiple system atrophy, and numerous other neuroaxonal diseases. In one embodiment, the antibodies of the present application are effectively used to treat Parkinson's disease.

[0040] In addition, the anti-α-syn / anti-IGF1R bispecific antibody of the present invention comprises an anti-IGF1R antibody or its antigen-binding fragment, which enables the anti-α-syn antibody or its antigen-binding fragment to pass through the blood-brain barrier and exert its effect in the brain, and extends its half-life, thereby maintaining its efficacy for a long period of time.

[0041] Furthermore, the anti-α-syn / anti-IGF1R bispecific antibody of the present invention binds to IGF1R on the cell surface without affecting ligand binding or signaling pathways via IGF1R, and therefore can be used as a shuttle agent to cross the blood-brain barrier because it does not inhibit IGF1R-ligand binding or signaling via IGF1R.

[0042] In particular, the anti-IGF1R antibody or antigen-binding fragment according to the present invention specifically recognizes IGF1R (Insulin-like Growth Factor 1 Receptor), recognizes and binds to IGF1R, particularly human IGF1R, mouse IGF1R, rat IGF1R, and monkey IGF1R, does not interfere with the binding of IGF1R ligands IGF-1, IGF-2, and / or insulin to IGF1R, does not inhibit signal transduction through IGF1R, is capable of transcytosis and crosses the blood-brain barrier, does not exhibit ADCC (Antibody-dependent cell-mediated cytotoxicity), does not reduce brain IGF1R levels even when administered repeatedly to animals, and is non-toxic.

[0043] In particular, the anti-IGF1R antibody according to the present invention binds to IGF1R present on the surface of brain endothelial cells that constitute the BBB, and is internalized into the cells.

[0044] For example, the anti-IGF1R antibodies of the present invention may have an scFv structure and can be conjugated to therapeutic antibodies in various ways. For example, the scFv of an anti-IGF1R antibody can be conjugated to the C-terminus of a therapeutic antibody, such as an α-syn antibody, to form a bispecific antibody (i.e., a bivalent bispecific antibody), or to a single bispecific antibody (i.e., a monovalent bispecific antibody), and all of the bispecific antibodies are internalized into cells expressing IGF1R. The high binding affinity of the IGF1R antibody to cell surface antigens enhances internalization, leading to BBB crossing. If an antibody has BBB crossing ability but interferes with IGF1R signaling, it may cause side effects. Therefore, the anti-IGF1R antibodies of the present invention have the binding affinity to function as a BBB shuttle while also being non-blocking against IGF1R signaling.

[0045] The anti-IGF1R antibody or antigen-binding fragment has excellent ease of development. In this regard, we attempted to remove post-translational modifications, such as deamidation, that occur in the CDR regions of the anti-IGF1R antibody and reduce the stability and efficacy of the antibody.

[0046] Alternatively, at least one of the amino acids located on either side of the deamidation site of the antibody can be substituted, preferably the amino acid immediately C-terminal to the deamidation site of the antibody. For example, by substituting G located next to Asn at the deamidation site of the antibody with A or S located next to Asn with V, a deamidated antibody can be prepared that has similar binding strength to the parental anti-IGF1R antibody, as well as excellent stability and BBB crossing ability.

[0047] Additionally, when the anti-IGF1R antibody according to the present invention is linked to a physiologically active substance that acts in the brain, it can induce improved BBB crossing ability and efficacy compared to the physiologically active substance alone.

[0048] An anti-IGF1R antibody according to one embodiment of the present invention can be used as a bispecific antibody containing various therapeutic second antibodies. In a human iPSC-derived in vitro BBB penetration experiment, the bispecific antibody exhibited approximately 15-fold higher BBB penetration ability than a single antibody composed of only a therapeutic antibody. The anti-IGF1R antibody conjugated to the second antibody in the bispecific antibody may be monovalent or bivalent. For example, when a single dose of a bispecific antibody containing monovalent or bivalent anti-IGF1R antibodies was administered to normal rats and the antibody levels in the blood and CSF were analyzed, the bispecific antibody containing monovalent or bivalent anti-IGF1R antibodies exhibited up to 5-fold increased blood antibody levels and up to 5-fold increased CSF antibody levels compared to the parental anti-IGF1R antibody (1564 clone). The antibody levels in the brain were approximately 3-fold increased CSF and 4.5-fold increased compared to the parental anti-IGF1R antibody (1564 clone). Therefore, bispecific anti-IGF1R antibodies improved by the above method are expected to exhibit up to approximately 15-fold greater CSF and approximately 23-fold greater brain penetration ability than monoclonal antibodies consisting of only a therapeutic second antibody.

[0049] The anti-IGF1R antibody according to the present invention has been confirmed to bind to IGF1R, particularly to IGF1R of mammals including humans, monkeys, rats, and mice, and can be useful in screening and clinical trials for drug development.

[0050] The anti-IGF1R antibody or antigen-binding fragment according to the present invention is an antibody or antigen-binding fragment thereof that specifically recognizes IGF1R (Insulin-like Growth Factor 1 Receptor).

[0051] The anti-IGF1R antibodies or antigen-binding fragments of the present invention have a dissociation constant (K D ) is ≦1×10 -6 An antibody is said to "specifically bind" to its target, such as an antigen, when it binds with an affinity of M. D is ≦1×10 -8M, or an EC50 (effective concentration 50) of 2 nM or less. In one embodiment, an antibody or antigen-binding fragment thereof specifically binds to a target with high affinity. D ≦1×10 -8 It can bind to IGF1R or human IGF1R.

[0052] As used herein, the term "epitope" refers to an antigenic determinant, a portion of an antigen recognized by an antibody. According to one embodiment, the binding site of an anti-IGF1R antibody according to the present invention may be the extracellular domain of an IGF1R protein, e.g., human IGF1R protein. More specifically, the binding site of an anti-IGF1R antibody according to the present invention, e.g., clone 1564 antibody, to human IGF1R protein comprises Y775, P776, F778, R650, S791, L798, and Glu779; binding site 2 comprises L641, H808, E809, and L813; and binding site 3 comprises V397, D435, W434, Y460, and C488. Therefore, the epitope of the IGF1R antibody of the present invention may be a conformational epitope, which may include all or part of the above three binding sites.

[0053] The anti-IGF1R antibodies or antigen-binding fragments according to the present invention do not interfere with the binding of IGF1R ligands IGF-1, IGF-2, and / or insulin to IGF1R. Specifically, the anti-IGF1R antibodies or antigen-binding fragments do not interfere with the binding of IGF1R ligands to IGF1R located on the cell membrane in IGF1R-expressing cells, do not inhibit signal transduction through IGF1R, and do not affect IGF1R expression on the cell surface. Therefore, the anti-IGF1R antibodies or antigen-binding fragments according to the present invention can be effectively used to cross the blood-brain barrier through transcytosis. Compared to other transcytosis targets known to be expressed in brain endothelial cells, which have been primarily used to improve BBB crossing ability, IGF1R expression has been shown to be relatively high in the brain. In one embodiment, IGF1R has been found to be expressed at relatively low levels in peripheral tissues, such as the liver, lungs, and colon, compared to other targets currently being developed for improving the BBB crossing ability of therapeutic antibodies, such as transferrin receptor and insulin receptor.

[0054] IGF1R is a target of receptor-mediated transcytosis (RMT), which can deliver useful substances to the brain through the blood-brain barrier (BBB). However, to be used as a drug delivery target for crossing the BBB, it is preferable that the antibody binds to IGF1R on the cell surface without affecting ligand binding or the signal transduction pathway through IGF1R. Therefore, the anti-IGF1R antibodies and antigen-binding fragments thereof according to the present invention can be used as a shuttle for crossing the BBB because they do not inhibit IGF1R-ligand binding or IGF1R-mediated signal transduction.

[0055] The anti-IGF1R antibodies or antigen-binding fragments thereof according to the present invention are capable of transcytosis and can pass through brain endothelial cells. Furthermore, when intravascularly injected into mice, the antibodies according to the present invention are localized to the same location as the mouse's cerebral blood vessels. These results demonstrate that the antibodies or antigen-binding fragments according to the present invention can be effectively used as drug delivery vehicles that cross the blood-brain barrier.

[0056] Therefore, the anti-IGF1R antibody or antigen-binding fragment thereof according to the present invention allows physiologically active substances acting in the brain to pass through the blood-brain barrier. In the present invention, a biological barrier refers to a cell, tissue, membrane, or structure that prevents the effective passage, diffusion, or transmission of a biological molecule. Such biological barriers include nerve cells / tissues, connective tissue, muscle, membrane, or epithelial (e.g., mucosal or vascular) cells. A representative example is the blood-brain barrier.

[0057] In the present invention, the "blood-brain barrier" or BBB is a barrier formed by tight junctions in the endothelial cell membranes of brain capillaries that exist between the brain and spinal cord and the surrounding circulatory system. This barrier is so strong that it even restricts the passage of low-molecular-weight substances with a molecular weight of approximately 60 Da into 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 within the central nervous system, and are commonly referred to as the BBB.

[0058] In the present invention, a "blood-brain barrier transmitter" is capable of passing through the blood-brain barrier to deliver the brain-acting factor substance, and the brain-acting factor substance includes, for example, compounds, proteins including peptides and polypeptides, nucleic acids, antibodies, or small molecular weight compounds.

[0059] The present invention relates to an isolated antibody or antigen-binding fragment thereof that specifically binds to IGF1R, wherein the antibody or antigen-binding fragment may be a polypeptide, protein, or antibody or antigen-binding fragment thereof that specifically binds to IGF1R and comprises a heavy chain complementarity-determining region and a light chain complementarity-determining region.

[0060] In one specific example, the anti-IGF1R antibodies and antigen-binding fragments thereof are (i) one or more heavy chain complementarity-determining regions selected from the group consisting of H-CDR1, H-CDR2, and H-CDR3 listed in Table 1, or a heavy chain variable region containing said one or more heavy chain complementarity-determining regions; (ii) one or more light chain complementarity-determining regions selected from the group consisting of L-CDR1, L-CDR2, and L-CDR3 listed in Table 1, or a light chain variable region containing said one or more light chain complementarity-determining regions; a combination of said one or more heavy chain complementarity determining regions and said one or more light chain complementarity determining regions; or The combination of the heavy chain variable region and the light chain variable region It may also include.

[0061] Additionally, in the heavy chain variable region, the light chain variable region, or the combination of the heavy chain variable region and the light chain variable region, the heavy chain variable region may comprise one or more heavy chain frameworks selected from the group consisting of H-FR1, H-FR2, H-FR3, and H-FR4, and the light chain variable region may comprise one or more light chain frameworks selected from the group consisting of L-FR1, L-FR2, L-FR3, and L-FR4.

[0062] In one embodiment of the present invention, removal of deamidated amino acids in the anti-IGF1R antibody reduces the risk of antibody degradation, which can be detrimental to process development, storage, etc., without affecting the binding affinity to the ECD of the IGF1R antigen. The amino acid positions at which deamidation is removed in the anti-IGF1R antibody may be, for example, N51D, N51Q, or S52V in the light chain LCDR2 of 1564 (IgG), 1564 (scFv), or 1564-3; N95aK, N95aH, N95aR, N95aD, or G95bA in the LCDR3; or N54D, N54Q, or G55A in the heavy chain HCDR2. Removal of deamidation is not limited to the clones mentioned above, but can also be performed on other clones using the method described in Table 14.

[0063] [Table 1]

[0064] [Table 2]

[0065] the anti-IGF1R antibody comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises a heavy chain CDR1 (H-CDR1) comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 10, a heavy chain CDR2 (H-CDR2) comprising one selected from the amino acid sequences of SEQ ID NOs: 2 to 7 and SEQ ID NOs: 11 to 18, and a heavy chain CDR3 (H-CDR3) comprising one selected from the amino acid sequences of SEQ ID NOs: 8 to 9 and SEQ ID NO: 19; The light chain variable region may comprise a light chain CDR1 (L-CDR1) comprising the amino acid sequence of SEQ ID NO: 20, a light chain CDR2 (L-CDR2) comprising one of the amino acid sequences selected from SEQ ID NOs: 21 to 23, and a light chain CDR3 (L-CDR3) comprising one of the amino acid sequences selected from SEQ ID NOs: 24 to 28 and SEQ ID NOs: 29 to 31.

[0066] In one embodiment of the present invention, the anti-IGF1R antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises a heavy chain CDR1 (H-CDR1) comprising the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 (H-CDR2) comprising one selected from the amino acid sequences of SEQ ID NO: 3 and SEQ ID NOs: 5 to 7, and a heavy chain CDR3 (H-CDR3) comprising one selected from the amino acid sequences of SEQ ID NOs: 8 to 9; The light chain variable region may comprise a light chain CDR1 (L-CDR1) comprising the amino acid sequence of SEQ ID NO: 20, a light chain CDR2 (L-CDR2) comprising one of the amino acid sequences selected from SEQ ID NOs: 22 and 23, and a light chain CDR3 (L-CDR3) comprising one of the amino acid sequences selected from SEQ ID NOs: 26 to 28.

[0067] The heavy chain variable region of the anti-IGF1R antibody according to the present invention may comprise H-CDR1, H-CDR2, and H-CDR3 described in Table 1 above, or may additionally comprise H-FR1 comprising the amino acid sequence of SEQ ID NO: 32, H-FR2 comprising the amino acid sequence of SEQ ID NO: 33 or SEQ ID NO: 34, H-FR3 comprising the amino acid sequence of SEQ ID NO: 35, and H-FR4 comprising the amino acid sequence of SEQ ID NO: 36.

[0068] The light chain variable region of the anti-IGF1R antibody according to the present invention may comprise L-CDR1, L-CDR2, and L-CDR3 shown in Table 1, or may additionally comprise L-FR1 comprising the amino acid sequence of SEQ ID NO: 37, L-FR2 comprising the amino acid sequence of SEQ ID NO: 38, L-FR3 comprising the amino acid sequence of SEQ ID NO: 39 or SEQ ID NO: 40, and L-FR4 comprising the amino acid sequence of SEQ ID NO: 41 or SEQ ID NO: 42. Specific examples include the light chain frameworks of L-FR1, L-FR2, L-FR3, and L-FR4 shown in Table 3.

[0069] Of the heavy chain or light chain frameworks 1 to 4, framework 1 (FR1) is located on the N-terminal side of CDR1, framework 2 (FR2) is located between CDR1 and CDR2, framework 3 (FR3) is located between CDR2 and CDR3, and framework 4 (FR4) is located at the C-terminus of CDR3.

[0070] Specifically, the framework sequence of the heavy chain variable region of the 1564 (IgG) clone comprises the amino acid sequences of SEQ ID NOs: 32, 33, 35, and 36, and the remaining clones listed in Table 1, except for the 1564 (IgG) clone, comprise the amino acid sequences of SEQ ID NOs: 32, 34, 35, and 36.

[0071] Specifically, the framework sequences of the light chain variable region of the anti-IGF1R antibody according to the present invention are as shown in Table 3 below. [Table 3]

[0072] The anti-IGF1R antibody according to the present invention may be an antibody comprising a heavy chain variable region and a light chain variable region, and various heavy and light chain variable regions disclosed herein are exemplarily shown in Tables 4 and 5. The heavy chain variable regions and light chain variable regions shown in Tables 4 and 5 below can be freely combined to produce various forms of antibodies. Each of these variable regions can be combined with the heavy and light chain constant regions to form the heavy and light chains of a complete antibody.

[0073] [Table 4]

[0074] [Table 5]

[0075] More specifically, the heavy chain variable region of the anti-IGF1R antibody or antigen-binding fragment thereof according to the present invention can comprise one selected from the group consisting of the amino acid sequences of SEQ ID NO: 43 to SEQ ID NO: 87. The light chain variable region of the anti-IGF1R antibody or antigen-binding fragment thereof according to the present invention can comprise one selected from the group consisting of the amino acid sequences of SEQ ID NO: 88 to SEQ ID NO: 132. Examples of the heavy chain variable region and light chain variable region are listed in Tables 4 and 5 above.

[0076] The anti-IGF1R antibody or antigen-binding fragment thereof may be an anti-IGF1R antibody or antigen-binding fragment thereof that specifically recognizes and binds to at least one amino acid selected from the group consisting of Y775, P776, F778, R650, S791, L798, Glu779, L641, H808, E809, L813, V397, D435, W434, Y460, and C488 in a human IGF1R protein having the amino acid sequence of SEQ ID NO: 174. Specifically, the anti-IGF1R antibody or antigen-binding fragment thereof according to the present invention binds to at least one or more binding sites selected from the group consisting of binding site 1 to binding site 3 in a protein comprising the amino acid sequence of SEQ ID NO: 174 for human IGF1R, wherein binding site 1 may comprise one or more amino acids selected from the group consisting of Y775, P776, F778, R650, S791, L798, and Glu779; binding site 2 may comprise one or more amino acids selected from the group consisting of L641, H808, E809, and L813; and binding site 3 may comprise one or more amino acids selected from the group consisting of V397, D435, W434, Y460, and C488.

[0077] The heavy chain variable regions and light chain variable regions listed in Tables 4 and 5 can be used as separate domain antibodies or 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.

[0078] As used herein, a "domain antibody" is an immunologically functional immunoglobulin fragment containing only the variable region of a heavy chain and / or the variable region of a light chain. In one embodiment, two or more VH regions are covalently linked by a peptide linker to form a bivalent domain antibody. The two VH regions of such a bivalent domain antibody can target the same or different antigens.

[0079] The antigen-binding fragment of the anti-IGF1R antibody according to the present invention can be selected from the group consisting of antibody fragments containing one or more complementarity-determining regions, such as scFv, (scFv)2, scFv-Fc, Fab, Fab', F(ab')2, minibody, and diabody.

[0080] Among the antigen-binding fragments, Fab has a structure comprising a light chain variable region, a heavy chain variable region, a light chain constant region, and the first constant region of the heavy chain (CH1), and has one antigen-binding site. Fab' has a hinge region containing one or more cysteine ​​residues at the C-terminus of the heavy chain CH1 domain of Fab. F(ab')2 antibodies are produced by forming disulfide bonds between two Fab's via the cysteine ​​residues in the Fab' hinge regions.

[0081] Fv is the minimum antibody fragment containing only the heavy and light chain variable regions, and includes single-chain Fv (scFv) and two-chain Fv (two-chain variable fragment). In a two-chain Fv, the heavy chain variable region and the light chain variable region can be linked non-covalently. In a single-chain Fv, the heavy chain variable region and the light chain variable region can be linked covalently directly or via a peptide linker, or can be linked directly at the C-terminus, forming a structure similar to an scFv dimer (di-scFv) like a two-chain Fv. In the present invention, a single-chain Fv is a single polypeptide chain of an antigen-binding region in which the heavy and light chain variable regions are linked directly or via a linker, and may be one or more types 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, a structure similar to an scFv dimer (di-scFv), and scFv-Fc in which the heavy chain variable region, light chain variable region, and Fc are linked in a single chain form.

[0082] The peptide linker may be as described above, for example, 1 to 100, e.g., 2 to 50, or 5 to 25 amino acids long. The length of the peptide linker can be determined in various ways as long as it does not affect the function of the antibody. The amino acids contained in the peptide linker can be, for example, one or more amino acids selected from the group consisting of Gly, Ser, and Leu. Specific examples include Gly and Ser residues, or leucine (Leu) and serine (Ser). As a specific example, the peptide linker may be (G4S)n, where n is the number of (G4S) repeats and may be expressed as an integer of 1 to 10, e.g., 2 to 5, particularly 3 or 4. An example of the peptide linker may be a peptide consisting of the amino acids of SEQ ID NO: 133 or 134. SEQ ID NO: 133: GGGGSGGGGSGGGGS SEQ ID NO: 134: GGGGSGGGGSGGGGSGGGGS

[0083] Such single-chain Fvs (scFvs) can be produced by fusing DNA encoding a peptide linker between DNA encoding two variable domain polypeptides (VL and VH). The produced polypeptides can fold to form antigen-binding monomers, or they can form multimers (e.g., dimers, trimers, or tetramers) depending on the length of the flexible linker between the two variable domains. By combining polypeptides containing different VL and VH, multimeric scFvs that bind to different epitopes can be formed.

[0084] The antigen-binding fragment can be obtained using protease (for example, whole antibodies can be digested with papain to obtain Fab fragments, or with pepsin to obtain F(ab')2 fragments), or can be produced through genetic recombination techniques. The single chain antibodies disclosed in the present invention include, but are not limited to, scFvs comprising a combination of heavy and light chain variable domains, or a combination of light and heavy chain variable domains comprising CDRs.

[0085] The antigen-binding fragments of the anti-IGF1R antibodies can be linked with or without a linker, e.g., a peptide linker. Furthermore, the heavy and light chain portions of the antigen-binding fragments, e.g., the heavy and light chain variable regions of the scFv fragments, can also be linked with or without a peptide linker. The peptide linker may be as described above.

[0086] In the bispecific antibody, the anti-IGF1R antibody and its antigen-binding fragment can transport a second antibody or its antigen-binding fragment targeting a different antigen or epitope across the blood-brain barrier to the brain. The second antibody may be an antibody that exerts its effect in the brain, including, but not limited to, the anti-alpha-synuclein antibody or its binding fragment according to the present invention.

[0087] The anti-IGF1R antibodies or antigen-binding fragments thereof of the present invention can share specific regions or sequences with a different second antibody, for example, the anti-IGF1R antibodies can share constant regions or Fc regions of the antibody or antigen-binding fragment of the second antibody.

[0088] Furthermore, the structure of the bispecific antibody in the present invention includes both bivalent bispecific antibodies in which an anti-IGF1R antibody scFv is linked to each Fc region of two heavy chains of an intact immunoglobulin, for example, to the end of each heavy chain, either directly or via a linker, and monovalent bispecific antibodies in which an anti-IGF1R antibody scFv is linked to only the end of one of the two heavy chains of an intact immunoglobulin, either directly or via a linker, although monovalent bispecific antibodies are preferred.

[0089] Specifically, in one example of the present invention, a monovalent clone may have a longer half-life than a bivalent clone. In this case, the structure of the monovalent clone is a complete immunoglobulin in which a domain antibody (scFv) that binds to IGF1R is linked via a linker to only the end of one heavy chain. For example, the complete immunoglobulin may be in the form of a heterodimer formed by applying the knob-in-hole technique, in which one heavy chain is linked to a domain antibody that binds to the IGF1R antigen and contains a linker at its C-terminus, and the remaining heavy chain contains two different heavy chains with no linkage after the C-terminus of the constant region.

[0090] In the bispecific antibody, the second antibody that binds to the anti-IGF1R antibody or its antigen-binding fragment may be a human antibody, a humanized antibody, or a chimeric antibody. The second antibody includes, but is not limited to, a complete antibody, a bispecific antibody, a minibody, a domain antibody, an antibody mimic (or synthetic antibody), an antibody fusion (or antibody conjugate), and fragments thereof. In the bispecific antibody, an example of the second antibody that binds to the anti-IGF1R antibody or its antigen-binding fragment may be an anti-syn antibody and its antigen-binding fragment according to the present invention.

[0091] The following relates to anti-syn antibodies and antigen-binding fragments thereof according to the present invention.

[0092] The alpha-synuclein recognized as an antigen by the antibodies 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 specified herein, alpha-synuclein may refer to human alpha-synuclein, and the antibodies or antigen-binding fragments thereof provided herein may have the ability to specifically bind not only to human alpha-synuclein, but also to monkey (e.g., Rhesus), rat, and / or mouse alpha-synuclein.

[0093] The antibody or antigen-binding fragment thereof binds to the C-terminal site of alpha-synuclein, and specifically, the human alpha-synuclein protein may be the C-terminal region of the amino acid sequence of SEQ ID NO: 173, 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.

[0094] As used herein, "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 such that the dissociation constants (K D )0.1×10 -10 M~2×10 -10 M, or 0.05 x 10 -10 M~0.3×10 -9 M, but is not limited to this.

[0095] Humanized alpha-synuclein antibodies comprising light and heavy chains according to one embodiment of the present invention, such as Hu11F11 (ver. 2), 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 activity against fibril binding to neuronal membranes than chimeric alpha-synuclein antibodies, and Hu11F11 (ver. 2), Hu11F11 (ver. 4), and ABL2-4 exhibit higher inhibitory activity against radioactivity of alpha-synuclein secreted from alpha-synuclein-overexpressing cells to other neurons than chimeric alpha-synuclein antibodies. The binding avidity to alpha-synuclein aggregates, for example, the binding avidity measured in a cell-based assay, is similar to or superior to that of chimeric alpha-synuclein antibodies.

[0096] The alpha-synuclein antibody according to the present invention inhibits cell-to-cell transmission of alpha-synuclein aggregates secreted outside neurons in the nervous system of a subject, preventing them from migrating to other normal cells in the extracellular space and infecting those neurons. It also promotes the phagocytosis of microglia against alpha-synuclein aggregates located in the extracellular space. Like prions, alpha-synuclein aggregates spread from one cell to another, even within normal cells. As a result, alpha-synuclein, particularly alpha-synuclein aggregates, spread throughout the brain, causing synucleinopathies. Therefore, alpha-synuclein aggregates are known to be toxic to brain neurons, causing neurodegeneration and inflammatory responses in the brain. Therefore, as alpha-synuclein aggregates spread to various parts of the brain, brain cell death and brain inflammatory responses increase, resulting in the brain cell death and resulting behavioral and cognitive impairments that are observed as synucleinopathies, such as Parkinson's disease, progress.

[0097] Therefore, the alpha-synuclein antibodies 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. They also promote the phagocytosis of microglia, thereby reducing or eliminating alpha-synuclein aggregates present outside of neurons in the target nervous system, thereby reducing the level of alpha-synuclein aggregates, which are an important cause of synucleinopathies, and thereby reducing brain neuron death and brain inflammatory responses, and are therefore expected to have the effect of improving, alleviating, or preventing the symptoms and progression of synucleinopathies, such as Parkinson's disease.

[0098] Furthermore, the alpha-synuclein antibodies of the present invention have excellent activity in that they can perform both of 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 central nervous system by promoting the phagocytosis of microglia. In particular, alpha-synuclein antibodies currently undergoing clinical trials or published in academic papers have at least one of the activities (i) and (ii), indicating that the alpha-synuclein antibodies of the present invention have superior advantages in preventing or treating synucleinopathies compared to known alpha-synuclein antibodies. Therefore, the alpha-synuclein antibodies of the present invention have superior efficacy in removing and reducing alpha-synuclein aggregates and inhibiting their pathogenic effects, and are therefore more effective in treating synucleinopathies or related symptomatic diseases (e.g., cognitive impairment, etc.).

[0099] 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. Furthermore, 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 and reducing the concentration of aggregates in the central nervous system. This has significant clinical advantages, as sufficient efficacy can be achieved even when the antibody is administered via a more convenient method such as, but not limited to, subcutaneous injection.

[0100] The antibodies or antigen-binding fragments of the present application can inhibit aggregate formation through removal of monomers, or can remove both monomers and aggregates.

[0101] 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), and such recombinant techniques are widely known in the art.

[0102] The alpha-synuclein antibody or bispecific antibody comprising the same according to the present invention can be used as a pharmaceutical composition for preventing or treating alpha-synucleinopathy, which may include, but is not limited to, 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, or multiple system atrophy (MSA).

[0103] The antibody or antigen-binding fragment thereof that specifically binds to alpha-synuclein or an aggregate thereof according to the present invention may 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.

[0104] In one embodiment, the anti-alpha-synuclein antibody or antigen-binding fragment thereof comprises the following CDR sequences: a heavy chain CDR1 (H-CDR1) comprising the amino acid sequence of SEQ ID NO: 135; a heavy chain CDR2 (H-CDR2) comprising the amino acid sequence of SEQ ID NO: 136 or 137; a heavy chain CDR3 (H-CDR3) comprising the amino acid sequence of SEQ ID NO: 138; a light chain CDR1 (L-CDR1) comprising the amino acid sequence of SEQ ID NO: 139; a light chain CDR2 (L-CDR2) comprising the amino acid sequence of SEQ ID NO: 140, and A light chain CDR3 (L-CDR3) comprising the amino acid sequence of SEQ ID NO: 141.

[0105] The amino acid sequences of the heavy chain CDR1 to CDR3 and the light chain CDR1 to CDR3 are summarized in Tables 6 and 7. The light chains of Hu11F11-VLv3 4c and Hu11F11-VL4 shown in Table 7 have the same amino acid sequences of CDR1 to CDR3 but different framework sequences.

[0106] [Table 6]

[0107] [Table 7]

[0108] The various heavy and light chain variable regions disclosed herein can be combined with the heavy and light chain constant regions to form the respective heavy and light chains of a complete antibody, and the respective heavy and light chain sequences so generated can also be combined to form a complete antibody structure.

[0109] For example, an anti-alpha-synuclein antibody or antigen-binding fragment thereof according to the present invention comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 142 to 146, and a light chain variable region comprising an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 147 to 148, and exemplary sequences of the heavy chain variable region and light chain variable region are shown in Table 8 below.

[0110] [Table 8]

[0111] Also listed in Table 9 are exemplary antibodies through combinations of heavy chain variable regions and light chain variable regions of antibodies or antigen-binding fragments according to one embodiment. [Table 9]

[0112] In other embodiments, the anti-alpha-synuclein antibody may consist only of the light chain or heavy chain described above, or may consist only of the light chain variable region or the heavy chain variable region.

[0113] In other embodiments, the heavy and light chain variable regions disclosed in Table 8 can be combined to form various antibodies, or can be linked in a single chain form to form a single-chain antibody such as an scFv.

[0114] 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.

[0115] It can include a heavy chain comprising the heavy chain variable region and a light chain comprising the light chain variable region. Specifically, the heavy chain variable region and light chain variable region can be combined with a heavy chain constant region and a light chain constant region, and the heavy and light chain sequences can also be combined to form a complete antibody structure.

[0116] Such constant region sequences to be combined with the variable regions of the present invention are exemplary, and the constant regions can be appropriately selected from heavy and light chain constant regions of immunoglobulins (e.g., human immunoglobulins). 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, but is not limited to, a kappa constant region or a lambda light chain constant region.

[0117] An exemplary antibody comprising the variable and constant regions of an anti-alpha-syn antibody or antigen-binding fragment according to one embodiment, the anti-alpha-synuclein antibody hu11F11(ver.2) clone, may be an antibody having a heavy chain comprising the amino acid sequence of SEQ ID NO: 149 and a light chain comprising the amino acid sequence of SEQ ID NO: 150.

[0118] The anti-alpha-synuclein antibody of the present application may be used alone as a therapeutic antibody, or may be used as a bispecific antibody in combination with another antibody capable of crossing the blood-brain barrier and delivering the antibody to the brain. An example of an antibody capable of crossing the blood-brain barrier and delivering the antibody to the brain may be an anti-IGF1R antibody and its antigen-binding fragment. The anti-IGF1R antibody and its antigen-binding fragment, which can be used to prepare the bispecific antibody, may include any of the above-mentioned anti-IGF1R antibodies and their antigen-binding fragments, and may be, for example, a complete antibody. The antigen-binding fragment may be selected from the group consisting of domain antibodies, scFv, (scFv)2, scFvFc, Fab, Fab', and F(ab')2.

[0119] The antigen-binding fragments of the anti-IGF1R antibodies can be linked with or without a linker, e.g., a peptide linker. Furthermore, the heavy and light chain portions of the antigen-binding fragments, e.g., the heavy and light chain variable regions of the scFv fragments, can also be linked with or without a peptide linker. The peptide linker may be as described above.

[0120] The anti-alpha-synuclein antibody or antigen-binding fragment thereof according to the present invention can be used to produce a bispecific antibody. Examples of heavy chains of anti-alpha-synuclein antibodies used to produce heavy chain combinations for producing bispecific antibodies relate to the anti-alpha-synuclein antibody hu11F11 (ver. 2), and include hu11F11 (ver. 2) (IGG) having the amino acid sequence of SEQ ID NO: 149 and hu11F11 (ver. 2) (IGG) WITH HOLE MUTATION AT FC having the amino acid sequence of SEQ ID NO: 151.

[0121] Examples of anti-alpha-synuclein antibody heavy chains used in the preparation of heavy chain combinations for producing bispecific antibodies, including the aforementioned anti-alpha-synuclein antibody hu11F11 (ver. 2), are listed in Table 10 below, and these heavy chain sequences are represented by SEQ ID NOs: 151 to 172, but are not intended to be limiting. Specific components of bispecific antibody clones that combine heavy chain combinations using the heavy chains of the anti-alpha-synuclein antibodies with light chains are also listed in Table 10 below. The bispecific antibodies listed below are listed by way of example, and even if they are not listed by SEQ ID NOs, their configurations are clear from the description of the bispecific antibody heavy chain combinations. Exemplary bispecific antibodies are specifically listed in Table 10 below. [Table 10] TIFF2026004290000012.tif253170TIFF2026004290000013.tif253170TIFF2026004290000014.tif209170

[0122] A pharmaceutical composition for preventing or treating an alpha-synucleinopathy comprising an alpha-synuclein antibody or an antigen-binding fragment thereof, or a bispecific antibody comprising the same according to the present invention may contain a pharmaceutically effective amount of the alpha-synuclein antibody or bispecific antibody.

[0123] As used herein, "treatment" can mean any action associated with the alleviation 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.

[0124] Pharmaceutical compositions comprising a therapeutically effective amount of an antibody and a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative, and / or adjuvant are also provided. Also included are methods for treating patients associated with alpha-synuclein, for example, by administering such pharmaceutical compositions. Pharmaceutical compositions used for in vivo administration are typically provided as sterile formulations. Once formulated, the pharmaceutical composition 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.

[0125] The pharmaceutical compositions can be administered by known methods, such as orally; by injection via intravenous, intraperitoneal, intracerebral (intraparenchymal), intraventricular, intramuscular, intraocular, intraarterial, intraportal, or intralesional routes; by sustained release system or implantation device. In certain embodiments, the compositions can be administered by bolus injection or continuously by infusion or implantation device.

[0126] The alpha-synuclein antibodies or antigen-binding fragments thereof, or bispecific antibodies comprising the same, according to the invention, described herein, can be used for diagnostic purposes to detect, diagnose, or monitor diseases and / or conditions associated with alpha-synuclein. For diagnostic uses, the antibodies can typically be labeled with a detectable label. [Effects of the Invention]

[0127] The antibodies prepared in accordance with one embodiment of the present invention specifically bind to IGF1R with an affinity optimized for brain endothelial transcytosis, making them useful for delivering therapeutic antibodies for degenerative brain diseases and brain cancers, whose therapeutic efficacy has been limited due to their low blood-brain barrier penetration ability. In particular, the antibodies disclosed herein exhibit benefits related to blood-brain barrier penetration, such as not affecting the binding of IGF1R ligands IGF-1, IGF-2, and their homolog insulin, and not inhibiting signaling through the IGF1R receptor. The antibodies disclosed herein can effectively remove or promote the degradation of alpha-synuclein aggregates and inhibit the intercellular transmission of alpha-synuclein, making them useful for treating diseases associated with the accumulation of alpha-synuclein aggregates. The alpha-synuclein antibody or bispecific antibody containing the same according to the present invention can be used as a pharmaceutical composition for preventing or treating alpha-synucleinopathy. [Brief explanation of the drawings]

[0128] [Figure 1] 1 shows the results of ELISA measurement of the affinity of an anti-α-syn chimeric antibody produced in one example of the present invention to the humanized 11F11 antibody. [Figure 2a] 1 shows the results of BIAcore analysis of the preferential binding specificity and affinity of an anti-α-syn chimeric antibody and a humanized 11F11 antibody produced in one example of the present invention for α-synuclein aggregates. [Figure 2b] 1 shows the results of BIAcore analysis of the preferential binding specificity and affinity of an anti-α-syn chimeric antibody and a humanized 11F11 antibody produced in one example of the present invention for α-synuclein aggregates. [Figure 2c] 1 shows the results of BIAcore analysis of the preferential binding specificity and affinity of an anti-α-syn chimeric antibody and a humanized 11F11 antibody produced in one example of the present invention for α-synuclein aggregates. [Figure 3a]1 shows binding data for IGF1R protein of an anti-IGF1R antibody produced in one example of the present application. [Figure 3b] 1 shows binding data for IGF1R protein of an anti-IGF1R antibody produced in one example of the present application. [Figure 4a] 1 shows binding data for an anti-IGF1R antibody produced in one example of the present application to an IGF1R-expressing cell line. [Figure 4b] 1 shows binding data for an anti-IGF1R antibody produced in one example of the present application to an IGF1R-expressing cell line. [Figure 4c] 1 shows binding data for an anti-IGF1R antibody produced in one example of the present application to an IGF1R-expressing cell line. [Figure 5a] 1 shows data relating to the internalization of an anti-IGF1R antibody produced in one example of the present application into an IGF1R-expressing cell line and its fate within the cell. [Figure 5b] 1 shows data relating to the internalization of an anti-IGF1R antibody produced in one example of the present application into an IGF1R-expressing cell line and its fate within the cell. [Figure 5c] 1 shows data relating to the internalization of an anti-IGF1R antibody produced in one example of the present application into an IGF1R-expressing cell line and its fate within the cell. [Figure 6a] This is data showing that the anti-IGF1R antibody produced in one example of the present application does not affect IGF1 or insulin-induced IGF1R signaling. [Figure 6b] This is data showing that the anti-IGF1R antibody produced in one example of the present application does not affect IGF1 or insulin-induced IGF1R signaling. [Figure 6c] This is data showing that the anti-IGF1R antibody produced in one example of the present application does not affect IGF1 or insulin-induced IGF1R signaling. [Figure 7a]We demonstrate that bispecific antibodies consisting of an anti-IGF1R antibody and a therapeutic antibody and anti-IGF1R antibodies cross the BBB in vivo more effectively than monospecific antibodies consisting of only a therapeutic antibody. [Figure 7b] We demonstrate that bispecific antibodies consisting of an anti-IGF1R antibody and a therapeutic antibody and anti-IGF1R antibodies cross the BBB in vivo more effectively than monospecific antibodies consisting of only a therapeutic antibody. [Figure 7c] We demonstrate that bispecific antibodies consisting of an anti-IGF1R antibody and a therapeutic antibody and anti-IGF1R antibodies cross the BBB in vivo more effectively than monospecific antibodies consisting of only a therapeutic antibody. [Figure 7d] We demonstrate that bispecific antibodies consisting of an anti-IGF1R antibody and a therapeutic antibody and anti-IGF1R antibodies cross the BBB in vivo more effectively than monospecific antibodies consisting of only a therapeutic antibody. [Figure 7e] We demonstrate that bispecific antibodies consisting of an anti-IGF1R antibody and a therapeutic antibody and anti-IGF1R antibodies cross the BBB in vivo more effectively than monospecific antibodies consisting of only a therapeutic antibody. [Figure 7f] We demonstrate that bispecific antibodies consisting of an anti-IGF1R antibody and a therapeutic antibody and anti-IGF1R antibodies cross the BBB in vivo more effectively than monospecific antibodies consisting of only a therapeutic antibody. [Figure 8] This shows the results of confirming the deamidation site of the anti-IGF1R antibody. [Figure 9] 1 shows the results of epitope mapping of anti-IGF1R antibodies. [Figure 10a] 1 shows the results of ELISA performed to measure the binding affinity of bispecific antibodies produced in one example of the present application to each antigen. [Figure 10b] 1 shows the results of ELISA performed to measure the binding affinity of bispecific antibodies produced in one example of the present application to each antigen. [Figure 10c] These are the results of ELISA performed to compare the binding ability of chimeric antibodies and humanized antibodies to each antigen. [Figure 10d] These are the results of ELISA performed to compare the binding ability of chimeric antibodies and humanized antibodies to each antigen. [Figure 10e] 1 shows the results of evaluating the activity of a bispecific antibody prepared in one example of the present application on the phagocytosis of microglia. [Figure 11a] 1 shows the results of evaluating the efficacy of a bispecific antibody prepared in one example of the present application in a mouse animal model in comparison with a single antibody. [Figure 11b] 1 shows the results of evaluating the efficacy of a bispecific antibody prepared in one example of the present application in a mouse animal model in comparison with a single antibody. [Figure 11c] 1 shows the results of evaluating the efficacy of a bispecific antibody prepared in one example of the present application in a mouse animal model in comparison with a single antibody. [Figure 11d] 1 shows the results of evaluating the efficacy of a bispecific antibody prepared in one example of the present application in a mouse animal model in comparison with a single antibody. [Figure 11e] 1 shows the results of evaluating the efficacy of a bispecific antibody prepared in one example of the present application in a mouse animal model in comparison with a single antibody. [Figure 12a] This shows the results of Fc engineering performed on a bispecific antibody produced in one example of the present application, confirming an increase in half-life and improved BBB penetration. [Figure 12b] This shows the results of Fc engineering performed on a bispecific antibody produced in one example of the present application, confirming an increase in half-life and improved BBB penetration. [Figure 12c] This shows the results of Fc engineering performed on a bispecific antibody produced in one example of the present application, confirming an increase in half-life and improved BBB penetration. [Figure 13] 1 shows the results of evaluating the ability of a bispecific antibody produced in one example of the present application to reduce α-syn in a mouse animal model. [Figure 14a] Each of these figures shows the results of a comparative analysis by ELISA of the antigen binding ability of a deamidated anti-IGF1R antibody prepared in one example of the present application and a control antibody. [Figure 14b]Each of these figures shows the results of a comparative analysis by ELISA of the antigen binding ability of a deamidated anti-IGF1R antibody prepared in one example of the present application and a control antibody. [Figure 14c] Each of these figures shows the results of a comparative analysis by ELISA of the antigen binding ability of a deamidated anti-IGF1R antibody prepared in one example of the present application and a control antibody. [Figure 15a] 1 shows the results of FACS analysis of the IGF1R-specific binding ability of a deamidated bispecific antibody produced in one example of the present application. [Figure 15b] 1 shows the results of FACS analysis of the IGF1R-specific binding ability of a deamidated bispecific antibody produced in one example of the present application. [Figure 15c] 1 shows the results of FACS analysis of the IGF1R-specific binding ability of a deamidated bispecific antibody produced in one example of the present application. [Figure 16] 1 shows the results of a comparative analysis of the in vivo BBB crossing ability of a deamidated bispecific antibody prepared in one example of the present application and a control antibody. [Figure 17] 1 shows the results of a comparative analysis of the in vivo BBB crossing ability of a deamidated bispecific antibody prepared in one example of the present application and a control antibody. DETAILED DESCRIPTION OF THE INVENTION

[0129] The present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not intended to be limited by the following examples. [Example]

[0130] Example 1. Production of mouse alpha-synuclein antibodies 1-1: Immunization and hybridoma production The antigen used was either full-length (140 residues) or alpha-synuclein monomer truncated at the C-terminus by 21 residues. It was placed in a thermomixer C at 37°C, shaken at 1050 rpm for 14 days, and then sonicated to form agglomerates. The resulting 140- and 119-residue alpha-synuclein fibrils, each at a concentration of 1 mg / ml, were mixed 1:1 (vol:vol) with an adjuvant and thoroughly mixed. The amino acid sequence of human alpha-synuclein (Homo sapiens alpha-synuclein) is shown in SEQ ID NO: 173.

[0131] Next, 200 μL of the prepared mixture was subcutaneously injected into 5-7 week-old BALB / c female mice, and two weeks later, 200 μL of the mixture prepared in the same manner was injected subcutaneously as a booster. One week after the booster, blood was collected and immunization titration was performed using ELISA with the administered antigen. Then, for the third booster, only the antigen was injected subcutaneously.

[0132] Next, the spleens of the immunized mice were removed and cells were isolated. The spleen cells were then suspended in Hybridoma-SFM medium (Thermo Fisher Scientific, USA) supplemented with 10% FBS. To produce hybridomas, myeloma cells SP2 / 0-Ag14 and spleen cells were mixed in serum-free Hybridoma-SFM medium, and the mixture was centrifuged to remove the medium. PEG was then added to the cell pellet and the mixture was incubated at 37°C for 1 minute to induce cell fusion.

[0133] 1-2: Single-cell cloning and antibody purification Two weeks after fusion, fusion with antibody-producing mouse B cells was confirmed using ELISA with antigen administered to mice using cell culture medium. Next, single-cell cloning was performed using the hybridomas to select a total of 16 hybridomas producing monoclonal antibodies. Using full-length (140 residues) alpha-synuclein aggregates as antigen, we obtained clone 9B11 (IgG1 kappa), and using alpha-synuclein aggregates truncated at the C-terminal 21 residues as antigen, we obtained clones 3A9 and 11F11 (IgG2b kappa and IgG2b kappa, respectively).

[0134] To purify the antibodies, hybridomas were cultured in RPMI 1640 medium containing 10% FBS. The culture medium was then changed to serum-free SFM medium for antibody production and cultured for approximately 4 days. The cell culture supernatant was separated, centrifuged, and filtered through a 0.22 μm filter. IgG1 type antibodies were purified using a protein G column, and the remaining antibodies were purified using a protein A column.

[0135] 1-3: Variable region sequencing The variable region and CDR sequences were determined by reference to Ahn et al., Mol. Cells 2004, 18(2):237-241. Hybridomas were cultured and then centrifuged to separate the cells. The isolated hybridomas were treated with Trizol to isolate RNA, which was then used as a template to synthesize cDNA. The variable region and CDR sequences were then confirmed by sequencing.

[0136] Example 2. Production of chimeric anti-alpha-synuclein antibodies 2-1: Antibody cloning and expression Using the antibody nucleotide sequences of the heavy and light chain variable regions, short nucleotide fragments called gblocks (m.biotech) were synthesized and used to clone into an animal cell culture vector (pcDNA3.4). The gblocks were synthesized containing approximately 20 bp of overlapping nucleotides before and after the variable regions, and the portion of the pcDNA3.4 vector excluding the variable regions was amplified by PCR and prepared, followed by cloning using the Gibson assembly method.

[0137] To transfect and express the cloned antibody, the prepared vector was maxi-prepped (Qiagen) to obtain a large amount of plasmid DNA, which was then introduced into cells as follows. The day before transfection, ExpiCHO™ (Gibco, Cat: A29127) cells were adjusted to a concentration of 3 x 10E6 to 4 x 10E6 viable cells / mL in ExpiCHO™ expression medium (Gibco, Cat: A29100-01) and cultured for 1 day at 8% CO2, 37°C, and 120 rpm. On the day of DNA transfection, cells that had grown to 7 x 10E6 to 10 x 10E6 viable cells / mL with a viability of over 95% were diluted to 6 x 10E6 with fresh medium. 6 The solution was prepared by dilution with viable cells / mL.

[0138] For transfection of the prepared mother cells, ExpiFectamine™ CHO & plasmid DNA complexes were prepared using the ExpiFectamine™ CHO transfection kit (Gibco, Cat. A29129). The appropriate concentrations of DNA and ExpiFectamine™ CHO reagent were inoculated into cold OptiPRO™ SFM® (Gibco, Cat. 12309019) medium, mixed, and incubated at room temperature for 5 minutes. The mother cells were then inoculated and transfected, and culture was initiated. The day after transfection, the enhancer and feed components included in the ExpiFectamine™ CHO transfection kit were inoculated into the transfected cells. Five days later, additional feed was inoculated, and the cells were cultured for 10 days at 8% CO₂, 37°C, and 120 rpm to complete the production.

[0139] To obtain the culture medium after production, 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 the purification process.

[0140] 2-2: Antibody purification and sequence confirmation The culture medium was purified using a HiTrap MabSelectSure (GE Healthcare, 11-0034-94). After equilibration with equilibration buffer (50 mM Tris-HCl pH 7.2, 100 mM NaCl), the collected culture medium was loaded onto the column. After loading, an intermediate wash 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), concentrated, and stored at 4°C until use.

[0141] If further purification was required, the primary purified material was passed through a HiLoad 26 / 600 Superdex 200 column using 1X PBS as a buffer, and a 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 confirmed to match the variable region of a mouse-derived monoclonal antibody.

[0142] The variable regions of the 3A9, 9B11, and 11F11 antibodies identified by the above method were replaced with the backbone variable region of the human IgG1 isotype to construct chimeric human IgG1 antibodies. Among the resulting chimeric antibodies, the Ch11F11 antibody is an IgG-type antibody that contains a combination of the heavy chain variable region sequence (ch11F11-VH) of SEQ ID NO: 176 and the light chain variable region sequence (ch11F11-VL) of SEQ ID NO: 176. The CDR sequences are shown in bold underlined in Table 11 below. [Table 11]

[0143] Example 3. Production of humanized antibodies 3-1: Library phage preparation A mini-library was constructed in which a human framework was attached 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 34 μg / ml chloramphenicol (Sigma, C0857), 2% glucose (Sigma, G5400), and 5 mM MgCl (Sigma, M2393) and cultured at 37°C for approximately 3 hours until the OD600 reached 0.5 to 0.7. Then, the cells were infected with helper phage and cultured at 30°C for 16 hours in 2X YT medium supplemented with 34 μg / ml chloramphenicol, 5 mM MgCl, 70 μg / ml kanamycin (Sigma, K1876), and 1 mM IPTG (ELPISBIO, IPTG025) to induce phage packaging. The culture medium was then centrifuged at 4,500 rpm for 15 minutes at 4°C. 4% PEG6000 (Fluka, 81253) and 3% NaCl (Sigma, S7653) were added to the supernatant, thoroughly dissolved, and incubated on ice for 1 hour. This was then centrifuged again at 8,000 rpm for 20 minutes at 4°C. The pellet was suspended in PBS and centrifuged again at 12,000 rpm for 10 minutes at 4°C. The supernatant containing the library phage was then placed in a new tube and stored at 4°C until use.

[0144] 3-2: Phage display panning Specifically, recombinant alpha-synuclein aggregates at a concentration of 10 μg / ml were added to an immunotube (immunotube, Maxisorp 444202) in PBS and allowed to adsorb to the surface of the tube overnight at 4°C. Then, a 3% solution of bovine serum albumin (BSA) was added to the tube to protect the surface from alpha-synuclein aggregates. After emptying the tube, 1 x 10 α-synuclein aggregates dispersed in the 3% BSA solution were added. 12 The cfu antibody phage library was placed in an immunohistochemistry test tube containing alpha-synuclein monomer protein and incubated at room temperature for 1 hour (negative selection). Phages that did not bind to alpha-synuclein monomer were collected and allowed to bind to the immunohistochemistry test tube containing alpha-synuclein aggregates for 2 hours at room temperature. Nonspecifically bound phages were then washed 5–30 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 1 M Tris buffer (pH 7.4) and then infected with ER2537 E. coli at 37°C for 1 hour. The infected E. coli were plated 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 medium and 15% glycerol was added. A portion was stored at -80°C, and 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 washes with PBS-T was increased to amplify and concentrate antigen-specific phages.

[0145] 3-3: Single clone phage antibody screening The following experiment was carried out to select monoclonal antibodies that specifically bind to alpha-synuclein aggregates from the phage pool obtained through the panning.

[0146] To isolate single clones from the enriched pool, the phage pool was plated on LB-tetracycline / carbenicillin agar medium and cultured to obtain single colonies. Next, single clones were inoculated into a 96-deep-well plate containing 400 μl of 2X YT-tetracycline / carbenicillin medium per well and grown overnight. After that, 10 μl of the culture was transferred to a new 96-deep-well plate containing 390 μl of 2X YT-tetracycline / carbenicillin medium and cultured at 37°C for 4 hours. IPTG was added to the culture medium to a concentration of 1 mM and cultured at 30°C overnight. The overnight culture was centrifuged and the supernatant was collected.

[0147] Next, ELISA was used to select clones expressing single-clone soluble scFvs that bind to alpha-synuclein aggregates. Specifically, the 7B7 antibody screened in Example 1-1 was coated on a 96-well plate overnight at 4°C. 200 μL of 3% BSA was added to each well and blocked at 37°C for 2 hours. Alpha-synuclein aggregates and monomers were then loaded at a concentration of 100 ng / well and incubated at 37°C for 2 hours. The plate was then washed five times with 300 μL of PBS-T. The prepared single-clone supernatant was mixed with 3% BSA at a 1:1 (vol:vol) ratio, and 100 μL of this mixture was loaded onto the plate bound to the aggregates and monomers and incubated at 37°C for 2 hours. After washing five times with 300 μL of PBS-T, the plate was incubated with anti-HA HRP-conjugated antibody at 37°C for 1 hour and then washed five times with PBS-T. Color development was achieved by adding 100 μL of TMB (Tetramethylbenzidine, Sigma, T0440), and the reaction was stopped by adding 50 μL of 1N H2SO4. The absorbance was measured at 450 nm. Clones with an absorbance of 0.5 or higher were considered positive for binding, and clones that nonspecifically bound to BSA were excluded.

[0148] The CDR residues of clones found in the library were analyzed in silico to select clones that had serious problems with binding to the framework or lacked T-cell epitopes, B cell epitopes, or MHCII epitopes in the framework region excluding the CDRs.

[0149] The variable regions of the selected clones were then substituted with the backbone variable region of the human IgG1 isotype to prepare humanized antibodies with an IgG1 backbone. Specifically, hu11F11(H2L4) is an IgG antibody comprising a combination of Hu11F11-VH2 of SEQ ID NO: 146 and Hu11F11-VL4 of SEQ ID NO: 148; Hu11F11_(ver.1) is an IgG antibody comprising a combination of Hu11F11-VH-v1 of SEQ ID NO: 142 and Hu11F11-VLv3 4c of SEQ ID NO: 147; Hu11F11_(ver.2) is an IgG antibody comprising a combination of Hu11F11-VH-v2 of SEQ ID NO: 143 and Hu11F11-VLv3 4c of SEQ ID NO: 147; and Hu11F11_(ver.3) is an IgG antibody comprising a combination of Hu11F11-VH-v3 of SEQ ID NO: 144 and Hu11F11-VLv3 of SEQ ID NO: 147. It was confirmed that Hu11F11 (ver. 4) is an IgG antibody containing a combination of Hu11F11-VH-v4 of SEQ ID NO: 145 and Hu11F11-VLv3 4c of SEQ ID NO: 147.

[0150] Example 4. ELISA assay of anti-alpha-synuclein antibodies To quantitatively analyze the binding ability of the chimeric antibody (Ch11F11) obtained in Example 2 and the humanized antibody (Hu11F11) obtained in Example 3, sandwich ELISA was performed.

[0151] Specifically, each antibody was diluted 1 / 10 at concentrations ranging from 0.04 to 400 nM and coated onto a 96-well plate. Each well was then treated with 2000 ng / ml of aggregates. After washing with 1X PBS, the plate was then treated with a biotin-conjugated capture antibody and HRP-conjugated streptavidin. The plate was then reacted with TMB as a substrate, and the absorbance was measured. The results are shown in Figure 7.

[0152] As shown in Figure 1, it was confirmed that the humanized antibody according to the present invention, particularly the humanized antibody derived from chimeric 11F11 (humanized 11F11 antibody), exhibits binding activity equivalent to that of the chimeric 11F11 clone. It was confirmed that humanized antibodies, particularly 11F11-derived variants, such as hu11F11 (ver. 1), i.e., a combination of Hu11F11-VH-v1 and Hu11F11-VLv3 4c, hu11F11 (ver. 2), i.e., a combination of Hu11F11-VH-v2 and Hu11F11-VLv3 4c, hu11F11 (ver. 3), i.e., a combination of Hu11F11-VH-v3 and Hu11F11-VLv3 4c, and hu11F11 (ver. 4), i.e., Hu11F11-VH-v4 and Hu11F11-VLv3 4c, exhibit binding avidity 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.

[0153] Example 5: BIAcore analysis using anti-alpha-synuclein antibodies The binding ability of the chimeric antibody obtained in Example 2 and the humanized antibody obtained in Example 3 was quantitatively analyzed using BIAcore.

[0154] The analysis was performed using a T200 (GE Healthcare, S / N: 1565888) instrument. Protein A chips were used (GE Healthcare, Cat. 29-1275-56), the regeneration buffer was 10 mM Glycine-HCl pH 1.5 (GE Healthcare, Cat. BR-1003-54), and HBS-EP was used as the running buffer, analyte dilution buffer, and sample dilution buffer. The antibodies prepared in Examples 2 and 3 were diluted with 1X HBS-EP (GE Healthcare, Cat. BR-1006-69), and α-syn monomer (1 mg / ml) or fibril protein (3 mg / ml) (analyte) were serially diluted two-fold and analyzed at six concentrations (0, 0.39, 1.56, 6.25, 25, and 100 nM), including 0 nM. For capture, the target RU for the monomer was 800 (theoretical), and for fibrils, the target RU 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. The association phase was performed with a 120-second association time and a flow rate of 30 μl / min. The dissociation phase was performed with a 360-second dissociation time and a flow rate of 30 μl / min. The regeneration phase was performed twice, with a flow rate of 30 μl / min and a regeneration time of 240 seconds (primary) and 60 seconds (secondary). Fitting was performed using a 1:1 binding model, and evaluation was performed using BIACore T200 Evaluation software (GE Healthcare).

[0155] The results of the analysis are shown in Figures 2a to 2c and in the table below. [Table 12]

[0156] As a result, the humanized antibodies described in the present application, particularly the variants of 11F11, i.e., hu11F11(ver.2), hu11F11(ver.3), and hu11F11(ver.4), exhibited K values ​​similar to those of the chimeric 11F11 clone. D The binding level of the humanized clones was 0.02 to 0.06 × 10 -9 K of M D , 0.02 × 10 chimeric 11F11 clones -9 Low K in M D value, i.e., high binding strength to the aggregates.

[0157] Example 6. IGF1R antibody production (scFV) 6-1: Preparation of IGF1R antibody (scFV) The monoclonal antibodies were produced using phage display / panning technology. Specifically, the antigen used in phage display panning to produce anti-IGF1R antibodies and other analyses was the following protein: the extracellular domain (ECD) of human IGF1R, from which the signal sequence was removed, consisting of amino acid residues 31 to 932 of SEQ ID NO: 174, to which a histidine tag (His tag) was attached at the C-terminus (R&D Systems, USA, 391-GR). To confirm cross-species reactivity, monkey IGF1R (National Research Council Canada), mouse IGF1R (R&D systems, 6630-GR / CF), and rat IGF1R (National Research Council Canada) proteins fused with a histidine tag (His tag) at the C-terminus were used as antigens.

[0158] Diverse human-derived ScFv (Single-chain variable fragment) library cells (OPAL library, created by Professor Shim Hyun-bo of Ewha Womans University) 1 x 10 10The cells 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 34 μg / ml chloramphenicol (Sigma, C0857), 2% glucose (Sigma, G5400), and 5 mM MgCl2 (Sigma, M2393), and cultured at 37°C for approximately 3 hours until the OD600 reached 0.5 to 0.7. Then, the cells were infected with helper phage and cultured in 2X YT medium supplemented with 34 μg / ml chloramphenicol, 5 mM MgCl2, 70 μg / ml kanamycin (Sigma, K1876), and 1 mM IPTG (ELPISBIO, IPTG025) at 30°C for 16 hours to induce phage packaging. The culture medium was then centrifuged at 4,500 rpm for 15 minutes at 4°C, and the supernatant was dissolved in 4% PEG6000 (Fluka, 81253) and 3% NaCl (Sigma, S7653), followed by incubation on ice for 1 hour. This was then centrifuged again at 8,000 rpm for 20 minutes at 4°C, and the pellet was suspended in PBS and centrifuged again at 12,000 rpm for 10 minutes at 4°C to obtain the supernatant containing the library phage. This was then placed in a new tube and stored at 4°C until use.

[0159] 6-2: Phage display panning To screen for human IGF1R antibodies, a total of three rounds of panning were performed as follows: The phage library used in the present invention was a synthetic human scFv library, and the phage display panning process and results are shown in the following table. [Table 13]

[0160] Specifically, 1 ml of recombinant human IGF1R protein (R&D Systems, USA, 391-GR or Sino Biological Life Technologies, USA, 10164-H08H-50R) at a concentration of 5 μg / ml was added to an immunotube (immunotube, maxisorp 444202) and allowed to coat the surface of the tube at 4°C for 16 hours. The supernatant was then removed, and 4 ml of a PBS solution containing 3% BSA (bovine serum albumin) was added. The tube was then incubated at 37°C for 1 hour to allow BSA to bind to the surface to which IGF1R had not been adsorbed, thereby blocking nonspecific binding. Next, the supernatant was removed, and the phage library prepared in Example 11-1 was mixed with a 1.5% BSA solution and incubated in the immunotube at 37°C for 1 hour to allow IGF1R-specific phages to bind to the antigen. Next, the plate was washed once with PBS-T (phosphate buffered saline-0.05% Tween 20) solution to remove non-specifically bound phages, and then phages bound to IGF1R were recovered using 100 mM triethylamine solution.

[0161] The recovered phages were neutralized with 1 M Tris buffer (pH 7.4) and then infected with E. coli K12 ER2738 E. coli at 37°C for 1 hour. The E. coli was then smeared on LB agar medium containing tetracycline and carbenicillin and cultured overnight at 37°C. The next day, the cultured E. coli was suspended in 5 ml of SB (superbroth) medium containing tetracycline and carbenicillin, and an equal volume of 50% glycerol was added. A portion was stored at 80°C, and 50 μl of the remaining portion was suspended in 40 ml of SB-tetracycline / carbenicillin medium and cultured overnight at 37°C. 12 PFU of VCSM13 helper phage was added, and the mixture was gently stirred and cultured at 37°C for 1 hour. Kanamycin was then added to the culture medium, and the mixture was cultured at 30°C for approximately 16 hours to allow only E. coli infected with the helper phage to grow.

[0162] The next day, the culture medium was centrifuged, and the supernatant was added to a buffer containing 4% PEG8000 and 3% sodium chloride (NaCl). The mixture was incubated at 4°C for approximately 1 hour to precipitate the phages, which were then centrifuged. The supernatant was then removed, and the precipitated phages were resuspended in PBS buffer containing 1% BSA and used in the next round of panning. This process was repeated four times, with the number of washes using PBS-T increasing as the panning round progressed, allowing the amplification and concentration of antigen-specific phages.

[0163] 6-3: Single clone phage antibody screening Clones were selected that showed not only protein binding to the ECD (extracellular domain) of human IGF1R, but also cell binding to MCF-7, an IGF1R-expressing cell line.

[0164] Specifically, the following experiment was carried out to select a monoclonal antibody that specifically binds to IGF1R from the phage pool obtained through the Examples.

[0165] To isolate single clones from the enriched pool, the resulting phage pool was plated on LB-tetracycline / carbenicillin agar medium and cultured to obtain single colonies. These colonies were then inoculated into 96-deep-well plates and cultured overnight. 10 μl of the culture was then inoculated into a 96-deep-well plate in the same manner and cultured at 37°C for approximately 4 hours to reach a titered OD of 0.5-0.7. A 20 MOI helper phage was added to the culture and incubated at 37°C for 1 hour. Kanamycin was then added to the culture and cultured at 30°C overnight. The next day, the culture was centrifuged, and the supernatant was collected and subjected to ELISA to screen for IGF1R-specific phages (Steinberger, Rader, and Barbas III. 2000. Phage Display Vectors. In: Phage Display Laboratory Manual. 1 std. Cold Spring Harbor Laboratory Press, NY, USA. pp. 11.9-11.12).

[0166] 100 ng of recombinant IGF1R was added to each well of an ELISA plate and incubated at 4°C for approximately 15 hours to coat the plate with the antigen. 200 μl of PBS buffer containing 3% BSA was added to each well to prevent nonspecific binding, and the plate was incubated at 37°C for approximately 1 hour. The supernatant was discarded.

[0167] 100 μl of the prepared single clone phage solution was added to each well and incubated at 37°C for 1 hour. The wells were then washed three times with 300 μl of PBS-T. To detect phage bound to the IGF1R antigen, anti-HA HRP was diluted 1:5000 in PBS buffer containing 3% BSA and incubated at 37°C for 1 hour. After washing three times with 300 μl of PBS-T, 100 μl of TMB (Tetramethylbenzidine, Sigma, T0440) was added to develop the color, and 50 μl of 1N H2SO4 was added to terminate the reaction. Absorbance was measured at 450 nm, and clones with higher absorbance than the BSA control were selected as antigen-specific antibody clones. Two rounds of screening were performed to select clones such as 1564.

[0168] Example 7. Production of affinity variants of IGF1R antibodies We evaluated the ligand binding and BBB crossing ability of selected clones and performed affinity variation to optimize the antibody. In the first trial, we used 1564scFv as a base and created NNS hand-mix primers to randomize the heavy chain CDR2 and light chain CDR3. We then used PCR to amplify the 1564scFv gene containing the randomization sequence. The amplified gene was inserted into the pComb3x vector to create a library suitable for phage display. We then selected multiple scFv clones that bind to IGF1R through library panning and ELISA screening. The amino acid sequences of the variable regions of the selected clones were confirmed through gene sequencing.

[0169] In the second attempt, two mini-libraries were constructed for the heavy and light chains, each with germline backmutations introduced into CDR1, CDR2, and CDR3. Affinity variants were selected based on the productivity and antigen binding ability of the clones, and the final clones were selected.

[0170] Example 8. Preparation of deamidation residue mutant antibodies 8-1: Deamidation residue confirmation Deamidation involves attacking the peptide bond of an asparagine side chain to produce a symmetrical succinimide intermediate, which is then hydrolyzed to either aspartic acid or isoaspartic acid. In particular, deamidation in the CDR can lead to antibody degradation, weakening the binding to the antigen and resulting in reduced efficacy and sample heterogeneity. Sample heterogeneity can complicate identification during future clinical approval. Therefore, we used in silico analysis and peptide mapping to identify the site of deamidation and prevent deamidation to ensure stability while also achieving excellent physical properties and efficacy.

[0171] As shown in Figure 8, in silico analysis and peptide mapping of the parental 1564 clone confirmed that deamidation did occur. Samples were stored at 4°C or 40°C for one week before analysis, and deamidation was confirmed to occur in LCDR2, LCDR3, and HCDR2. The affinity variants described in Example 7 were also analyzed to confirm the location of deamidation.

[0172] 8-2: Mutant antibody production To remove the deamidation residue, a residue-substituted mutant was produced as follows.

[0173] 1) In the amino acid sequence, Asn was changed to D or Q, which are similar to Asn. If it was confirmed that there was no change in binding strength, all of the residues were replaced with Q. 2) N95a, a residue of deamidation in LCDR3, was replaced with positively charged H, R, or K. Clones that underwent this deamidation process are also called (de)(StoP) deamidated clones. 3) The residue located immediately next to the CDR where deamidation occurs was replaced. These residues are relatively small and not highly charged (e.g., glycine or serine). Therefore, by replacing the residue with another residue that is also relatively small and hydrophobic (e.g., valine or alanine), we attempted to minimize the difference in binding strength with the parental antibody (the clone before residue replacement) (Table 21). Clones to which this deamidation process has been applied are also called (de2)(StoP) deamidated clones. The method for replacing the residue next to the residue where deamidation occurs is shown in the table below.

[0174] [Table 14] Italics: deamidation residue; Underlined and bold: residue to be replaced

[0175] Example 9. Preparation of various forms of anti-IGF1R antibodies 9-1: Preparation of anti-IGF1R minibody antibody Minibodies were prepared by linking the entire scFv of the IGF1R-specific monoclonal phage antibodies obtained in Examples 6 to 8 to the C-terminus of Fc. To this end, a nucleic acid sequence encoding the amino acid sequence of the scFv disclosed herein was prepared, digested with restriction enzymes, and cloned into a pcDNA-based expression vector containing nucleic acid encoding Fc.

[0176] 9-2: Preparation of anti-IGF1R bivalent antibody A bivalent form was constructed by linking two whole scFvs of the IGF1R-specific monoclonal phage antibodies isolated in Examples 6 to 8 to the C-terminus of an IgG form of therapeutic antibody. To this end, a nucleic acid sequence encoding the amino acid sequence of the scFv disclosed herein was prepared, digested with restriction enzymes, and cloned into a pcDNA-based expression vector containing nucleic acid encoding the therapeutic antibody.

[0177] 9-3: Preparation of anti-IGF1R IgG (Full-IgG) antibody Among the IGF1R-specific monoclonal phage antibodies obtained in Examples 6 and 7, the sequences of the 1564 and F06 antibodies were converted to full IgG1 (full IgG) forms by synthesizing the heavy and light chain gene sequences (Genotech). The synthesized heavy and light chain genes were cloned into expression vectors.

[0178] 9-4: Preparation of monovalent anti-IGF1R scFv antibody While Example 9-2 showed a bivalent structure in which an anti-IGF1R antibody was attached in the form of an scFv to each of the C-termini of the two Fc heavy chains, in this example, we constructed a monovalent antibody in which one scFv was attached to only one Fc C-terminus in the heavy chain. To this end, we constructed vectors in which 1564, F06, C04, VH5, VH16, VH35, VH9, VH2, VH7, and VH32 of the IGF1R-specific monoclonal phage antibodies isolated in Examples 6-8 were attached to only one site in the Fc C-terminus, as well as vectors in which no anti-IGF1R antibody was attached to the C-terminus. A knob-into-hole mutation was introduced into the Fc to allow for the production of heteromeric forms during antibody production in cells. When transfecting CHO-S cells for antibody production, a total of three vectors were injected: a vector for the heavy chain of the therapeutic antibody to which the anti-IGF1R antibody was attached at the Fc C-terminus, a vector for the heavy chain of the therapeutic antibody to which the anti-IGF1R antibody was not attached at the C-terminus, and a vector for the light chain of the therapeutic antibody.

[0179] 9-5: Expression and purification of various anti-IGF1R antibodies The vectors prepared in Examples 9-1 to 9-4 were introduced into cells as follows. Specifically, CHO-S cells were cultured in CD-CHO (Gibco, 10743) medium at 1.5 × 10 6 After adjusting the concentration at cells / ml, the cells were cultured at 8% CO2 and 37°C for 1 day. On the day of DNA transfection, the cells were cultured at 2.5–3 × 10 6 Cells were grown at a density of 2.1 x 10 cells / ml in CD-CHO medium containing 1% DMSO. 6 After preparing the cells at a concentration of 1000 cells / ml, they were cultured for 3 hours at 37°C in 8% CO2. After centrifugation at 3000 rpm for 15 minutes, the supernatant was removed and the cells were resuspended in RPMI 1640 medium containing 2.5% FBS.

[0180] Next, the vector combination was diluted in Opti-MEM medium at 1 μg per ml, and PEI (Polysciences, 23966, stock concentration: 1 mg / ml) was diluted at 8 μg per ml of culture medium. The DNA and PEI mixture was mixed and left at room temperature for 10 minutes, then added to the flask containing the cells and cultured at 5% CO2, 37°C, 100 rpm for 4 hours. After that, an equal volume of CD-CHO medium was added and cultured at 8% CO2, 37°C, 110 rpm for 4 days.

[0181] The resulting culture medium was passed through Mab Selectsure (GE Healthcare, 5 mL) equilibrated with equilibration buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl) to allow the expressed antibodies to bind to the column. The mixture was then eluted with 50 mM Na-citrate (pH 3.4), 100 mM NaCl, and neutralized with 1 M Tris-HCl (pH 9.0) to a final pH of 7.2. The buffer was then exchanged for phosphate buffered saline (PBS, pH 7.4). If the purity was high, the mixture was formulated and stored frozen at -20°C. If further purification was required, the mixture was stored at 4°C until further purification.

[0182] If further purification is required, it can be performed using Hiload Superdex 200 (GE Healthcare, Cat. No. 28-9893-36) or various other size exclusion chromatography systems. After equilibration with equilibration buffer (1x Phosphate buffered saline pH 7.4, Gibco, Cat. No. 10010-023), the sample after primary purification was loaded onto the column. The purified sample was formulated and stored frozen at -20°C.

[0183] Example 10. Production of bispecific antibodies The heavy and light chain variable regions of the anti-IGF1R antibody according to the present invention were linked using a linker (SEQ ID NO: 134) to prepare an scFv, which was then linked to the C-terminus of the heavy chain constant region of an intact IgG anti-α-syn antibody via a linker (SEQ ID NO: 133) to prepare a bivalent antibody. Furthermore, as bivalent antibody formats, a monovalent antibody was prepared by linking one anti-IGF1R scFv molecule to one intact IgG anti-α-syn molecule, and a bivalent antibody was prepared by linking two anti-IGF1R scFv molecules.

[0184] The sequences of the anti-α-syn antibodies used to prepare the biantibodies in this example and examples of combination sequences of biantibodies prepared according to this invention are shown in Table 10. Specific methods for preparing bivalent and monovalent biantibodies are as follows.

[0185] 10-1: Bivalent double antibody cloning To construct a bivalent dual antibody expression vector, an antibody nucleotide sequence containing a signal sequence was inserted into the multicloning site (MCS) of the pcDNA3.4 (Invitrogen) vector. The dual antibody expression vector is a monocistronic vector, and a heavy chain expression vector and a light chain expression vector were constructed separately.

[0186] The heavy chain sequence inserted into the heavy chain expression vector is in a form in which the anti-IGF1R scFv is linked via a linker to the C-terminus of an immunoglobulin to which a heavy chain variable region encoding an anti-α-syn antibody and a human heavy chain constant region are linked.The light chain sequence inserted into the light chain expression vector is in a form in which a light chain variable region encoding an anti-α-syn antibody and a human light chain constant region are linked.

[0187] 10-2: Monovalent double antibody cloning Monovalent bivalent antibodies are heterodimers formed by binding an antibody light chain to an anti-α-syn immunoglobulin heavy chain (hole) to which an anti-IGF1R scFv is linked via a linker at the C-terminus, and an anti-α-syn immunoglobulin heavy chain (knob) to which no scFv is linked.

[0188] To enhance the conjugation efficiency of heavy chain heterodimers, the knob-in-hole technique was used: the hole-type heavy chain coding sequence was substituted with T366S, L368A, and Y406V in the CH3 region, and the knob-type heavy chain coding sequence was substituted with T366W in the CH3 region.

[0189] 10-3:Transient expression The prepared vectors were maxi-prepped (Qiagen) to obtain a large amount of plasmid DNA, which was then introduced into cells as follows: To produce BsAb, heavy chain expression vector DNA and light chain expression vector DNA were transfected at a 1:1 ratio. To produce monovalent BsAb, hole-type heavy chain expression vector DNA, knob-type heavy chain expression vector DNA, and light chain expression vector DNA were transfected at a 0.5:0.5:1 ratio. The day before transfection, ExpiCHO (trade name) Gibco, Cat: A29127) cells were cultured in ExpiCHO expression medium (Gibco, Cat: A29100-01) at 3 × 10 6 ~4×10 6 After adjusting the viable cell / mL concentration, the cells were cultured for 1 day at 8% CO2, 37°C, and 120 rpm. On the day of DNA transfection, 7 × 10 6 ~10×10 6 Viable cells / mL: Cells grown to a viability of 95% or more were added to 6 x 10 fresh medium. 6 The solution was prepared by dilution with viable cells / mL.

[0190] 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 complexes were prepared. Cold OptiPRO™ SFM™ (Gibco, Cat: 12309019) medium was dispensed into each well, and titrated DNA and ExpiFectamine™ CHO reagent were inoculated into each well. The mixture was mixed and left to stand at room temperature for 5 minutes, then inoculated into the mother cells for transfection and culture. The day after transfection, the enhancer and feed included in the ExpiFectamine™ CHO transfection kit were inoculated into the transfected cells. Five days later, additional feed was inoculated, and the cells were cultured for 10 days at 8% CO₂, 37°C, and 120 rpm to complete the production.

[0191] 10-4: Medium harvest 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 for the subsequent purification process.

[0192] Example 11. Analysis of IGF1R-specific binding ability of anti-IGF1R antibodies 11-1: Analysis of binding activity of minibody-type anti-IGF1R antibodies to IGF1R (ELISA) ELISA analysis was performed to confirm whether the minibody forms of the 996, 1226, 1564, and MKJP2 clones prepared in Example 9-1 bind to recombinant IGF1R in a concentration-dependent manner.

[0193] Specifically, the human recombinant IGF1R used as the antibody binding target was the extracellular domain (ECD) and was purchased from R&D Systems (6630-GR / CF). Human IGF1R was diluted to 1 μg / ml in PBS buffer and added at 100 μl per well to a 96-well ELISA plate (Nunc-Immuno Plates, NUNC, Rochester, NY). After incubation at 4°C for 16 hours, the plate was coated and the supernatant was removed. 200 μl of PBS buffer containing 3% BSA (bovine serum albumin) was added per well and incubated for 2 hours to block nonspecific binding.

[0194] The minibody antibodies of clones 996, 1226, 1564, and MKJP2 prepared in Example 9-1 were diluted 3-fold from a maximum concentration of 20 nM to create 12-point dilutions. 100 μl of each was added to each well and incubated at room temperature for 1 hour. After washing four times with PBS buffer containing 0.05% Tween 20, 100 μl of anti-human HRP, which recognizes the human Fc in the minibody, was added to each well at a 1:5000 dilution in blocking buffer and incubated at room temperature for 1.5 hours. After washing four times with 300 μl of PBS-T (0.05% Tween 20), color development was performed using TMB (Tetramethylbenzidine, Sigma, T0440). The enzyme reaction was stopped with 0.5 mol / L sulfuric acid, and the absorbance at 450 nm was recorded and analyzed using a microplate reader (Molecular Device).

[0195] Four minibody clones bound to the human IGF1R recombinant protein in a concentration-dependent manner. Specifically, MKJP2 was found to have the highest binding affinity, followed by 996 and 1564, which showed similar binding affinity, and 1226, which showed slightly lower binding affinity.

[0196] 11-2: ELISA analysis of species cross-reactivity of IGF1R antibodies The interspecies cross-linking of the 1564 anti-IGF1R antibody prepared by the method of Example 9-2 and the anti-IGF1R antibody obtained in Example 6-3 was analyzed using ELISA. Human, monkey, mouse, and rat IGF1R antigens were diluted to 1 μg / ml and added in 100 μl portions to each well. The mixture was incubated at 4°C for 15 hours to coat the bottom of the plate. After removing the supernatant, each well was treated with 200 μl of PBS buffer containing 3% BSA to block nonspecific binding. Anti-IGF1R antibodies were diluted 5-fold in PBSB (3% BSA in PBS) to a maximum concentration of 400 nM and added to each well. The wells were then incubated at 37°C for 1 hour. After washing five times with PBS buffer, 100 μl of anti-human Fab HRP, which recognizes the Fab portion of the bound antibody, was added to each well at a 1:20,000 dilution and incubated at 37°C for 1 hour. The plate was then washed five times with PBS buffer and color developed using TMB (Tetramethylbenzidine, Sigma, T0440) according to the manufacturer's protocol. The enzyme reaction was stopped with 0.5 mol / L sulfuric acid, and the absorbance was measured at 450 nm using a microplate reader (Molecular Devices). When performing ELISA, if there were many samples, the plate was divided into two and the experimental results are shown in Table 15 below.

[0197] Specifically, Table 15 summarizes the ELISA results of the bispecific antibody against human IGF1R, the ELISA results of 1564 IgG and the bispecific antibody against human IGF1R, the ELISA results of the bispecific antibody against mouse IGF1R, the ELISA results of the bispecific antibody against rat IGF1R, and the ELISA results of the bispecific antibody against monkey IGF1R.

[0198] The following experimental results demonstrate the advantage of being able to evaluate efficacy using animal models of various species, and demonstrate that the efficacy of therapeutic agents can be evaluated through disease models of various species using the antibodies of the present invention. [Table 15] *:not available **201:scFv form of Herceptin biosimilar

[0199] 11-3: Analysis of affinity of IGF1R variants (FACS) The binding ability of the affinity variants prepared in Example 7 was analyzed by ELISA against the ECD of IGF1R protein, and the binding ability to MCF-7 was analyzed by FACS.

[0200] Table 16 shows the results of ELISA analysis of the binding of the primary selected clones to IGF1R ECD protein in the form of a bispecific antibody, and Table 17 shows the results of FACS analysis of binding to the MCF-7 cell line.

[0201] [Table 16]

[0202] [Table 17]

[0203] As a result, F06 was selected as the clone with the highest cell binding affinity compared to the most parental clone (1564 clone) (affinity matured), while C04 was selected as the clone with the lowest cell binding affinity compared to the parental clone (1564 clone) (affinity reduced).

[0204] Table 18 shows the results of ELISA for the binding of the clones prepared by the secondary preparation method to IGF1R ECD protein in the form of bispecific antibodies. [Table 18]

[0205] After removing clones with poor productivity and physical properties from the secondary clones, clones for FACS analysis were selected as shown in Table 19. [Table 19]

[0206] Figure 4c shows the results of FACS analysis of the binding of these clones to the MCF-7 cell line, and all of the analyzed clones had reduced binding to MCF-7 compared to the parental clone 1564. These results indicate that the clones that showed reduced binding by ELISA also showed reduced binding by FACS.

[0207] The selected antibody clones were F06, C04, VH2, VH5, VH7, VH9, VH16, and VH32, and the amino acid sequences for the heavy chain variable regions and light chain variable regions for these antibodies are shown in Tables 4 and 5 above.

[0208] Among the antibody clones, deamidation hot spots present in the VH5, VH16, and F06 variants were removed according to Table 21 in Example 8-2 to produce mutants, which were then prepared as bispecific antibodies according to Example 10. VH5 and VH16 were prepared as bivalent bispecific antibodies with hu11F11 (ver. 2), and F06 was prepared as a monovalent bispecific antibody with hu11F11 (ver. 2). The bispecific antibodies prepared were analyzed for their binding to MCF-7 by the FACS analysis described above for three variants of VH5, VH16, and F06 (i.e., bispecific antibodies hu11f11-F06, hu11f11-VH5, and hu11F11-VH16) and deamidated mutants (i.e., bispecific antibodies hu11f11-F06(de2)(StoP), hu11f11-VH5(de2)(StoP), and hu11F11-VH16(de2)(StoP)). The components of these specific bispecific antibodies are listed in Table 10, and the deamidation site substitutions for these antibodies are listed in Table 14 of Example 8-2.

[0209] The results of the FACS analysis are shown in Table 20. All three deamidated mutants (hu11f11-F06(de2)(StoP), hu11f11-VH5(de2)(StoP), and hu11F11-VH16(de2)(StoP)) were confirmed to have no decreased binding affinity compared to the parental antibodies (VH5, VH16, and F06). [Table 20] *MFI=mean fluorescence intensity Negative control(2ndary Ab only)'s MFI:2.29

[0210] Using the bispecific antibodies prepared above, the binding affinity of the three variants (VH5, VH16, and F06) and the deamidated mutant to human IGF1R protein was analyzed by ELISA according to the method described in Example 15-2. The results are shown in the table below. It was confirmed that the binding affinity of all three mutants was not reduced compared to the parental antibody. [Table 21]

[0211] 11-4: BIAcore analysis of human IGF1R The binding strength between the antibody of the present invention and human IGF1R was analyzed.

[0212] The IgG form of clone 1564 was analyzed for its binding to human IGF1R using SPR analysis. The antigen, an anti-His antibody against the His tag bound to the human IGF1R ECD, was diluted to 20 μg / ml in acetate pH 4.0 buffer and immobilized to a target RU of 10,000 RU in the reference / analytic channel of a CM4 chip using amine coupling. During capture, PBS was used as the running buffer. The flow rate was maintained at 30 μL / min. During association / dissociation, the flow rate was 40 μL / min, and PBS was used as the running buffer. Association / dissociation lasted 5 and 20 minutes, respectively. Analysis was performed in the following order: baseline 1, activation (EDC + NHS), human IGF1R loading, quenching (1 M ethanolamine), baseline 2, association, and dissociation. Evaluation was performed using a bivalent model and analysis was performed using Biacore T200 Evaluation software (version 1.0, S / N: 04Y15X11-0149).

[0213] The analysis results showed that the KD of the 1564 IgG antibody was 2.5305 × 10 -9 nM, and F06 IgG antibody was 4.7802 x 10 -7 The results of the analysis are shown in Figure 3b. In particular, when clone 1564 was converted into an IgG form, it bound to human IGF1R with a binding affinity of 2.5305 x 10 -9 It was confirmed that 1564 exhibited a dissociation constant of nM, and that its binding ability did not change significantly depending on its form.

[0214] Example 12. Analysis of binding ability of anti-IGF1R antibodies to human IGF1R-expressing cell lines and brain endothelial cells 12-1: FACS analysis of MCF-7 To confirm whether the minibody forms of clones 996, 1226, and 1564 prepared in Example 9-1 bind to endogenous IGF1R on the cell surface, we performed binding analysis of human IGF1R-expressing cell lines and brain endothelial cells by FACS. We also examined the binding of periplasmic extract to MCF-7 and breast cancer cell lines known to overexpress IGF1R by FACS.

[0215] Specifically, 0.43 × 10 per sample 6 The MCF-7 cell line was treated with each of the three minibodies, diluted to 20 μg / ml, and incubated at 4°C for 1 hour. After washing twice with PBS buffer, the cells were treated with anti-human FITC diluted 1:500 and incubated at 4°C for 1 hour. After washing twice with PBS buffer, the extent of anti-IGF1R minibody binding was measured using a FACSCalibur instrument. MCF-7 cells treated with only the secondary antibody served as a control. The experimental results are shown in Figure 4a.

[0216] The A02, A06, A07, B01, B02, B09, B10, C04, D03, E06, F06, H04(Gly), H04(Val), VH2, VH5, VH7, VH9, VH16, VH32, and VH35 clones prepared in Examples 7 and 9-2 were analyzed for their binding to MCF-7 cells using the same method as described above. The 1564 clone, prepared in Example 14-2, was used as the parental clone for comparison, and MCF-7 cells treated with only the secondary antibody served as a control. The analysis results are shown in Figure 4c.

[0217] The experimental results, shown by the mean fluorescence intensity (MFI) of the samples, confirmed that the affinity variants and parental clones (1564 clones) in the scFv and bispecific antibody forms of the three minibodies tested specifically bind to endogenous IGF1R expressed on the cell surface. These results demonstrate that the clones obtained in this example bind to IGF1R in the form actually present in the body and can be used for their intended purposes.

[0218] 12-2: FACS analysis of JIMT-1 and BT474 Using essentially the same method as in Example 12-1, except that JIMT-1 and BT474 breast cancer cell lines were used instead of the MCF-7 cell line, we examined whether the minibody forms of clones 996, 1226, and 1564 prepared in Example 14-1 bind to endogenous IGF1R on the cell surface. The results are shown in Figure 4a.

[0219] The experimental results, shown by the MFI (Mean Fluorescence Intensity) of the sample, confirmed that the scFvs in the three tested minibodies specifically bind to endogenous IGF1R on the surface of various IGF1R-expressing cell lines.

[0220] 12-3: FACS analysis of mouse brain endothelial cells The binding of the bispecific antibody form of the 1564 clone prepared by the method in Example 9-2 and the IgG form of the 1564 clone prepared by the method in Example 14-3 to mouse brain endothelial cells, bEND.3, was analyzed. A group treated with only the secondary antibody and a group treated with the therapeutic antibody alone, IgG (CH11F11), were used as negative controls. FACS analysis was performed as in Examples 12-1 and 12-2. The analysis results are shown in Figure 4b.

[0221] All of the test clones tested, except for the negative control, showed binding to bEND.3. These results confirmed that 1564 clones with various forms specifically bound to IGF1R expressed on the surface of brain endothelial cells.

[0222] Example 13. Intracellular internalization analysis of anti-IGF1R antibodies 13-1: MCF-7 internalization assay-1564, 996, 1226, MKJP2 (mini body) We attempted to confirm whether the minibody forms of the 996, 1226, 1564, and MKJP2 clones prepared in Example 9-1 were internalized intracellularly in IGF1R-expressing cell lines and whether the antibodies introduced into the cells would pass through the RMT pathway without being degraded. For an anti-IGF1R antibody to be used as a shuttle to improve BBB crossing ability, the antibody must first be internalized into the brain endothelial cells that make up the BBB.

[0223] We analyzed the internalization of the antibodies of the present invention using the IGF1R-expressing MCF-7 cell line. Specifically, 30,000 MCF-7 cells were plated in an 8-well slide chamber and cultured for one day. Each well was treated with 5 μg / ml of the minibody antibodies 996, 1226, 1564, and MKJP2 clones prepared in Example 9-1 at 4°C for two hours, washed three times with cold DMEM medium, and then treated with an Alexa488-conjugated anti-human Fc antibody at 4°C for one hour.

[0224] To test for antibody complex internalization, the plate was transferred to a CO2 incubator and incubated at 37°C for 30 minutes. The reaction was terminated by adding 100% methanol to fix the cultures. After fixation, the cells were washed three times with PBS. The extent of antibody internalization was visualized using a fluorescent microscope in the green filter area (Alexa488). During imaging, the intracellular nuclei were stained with DAPI to confirm the location of each cell. The experimental results are shown in Figure 5a.

[0225] The results of the above experiment confirmed that all four antibodies tested were well internalized, even in experiments using the MCF-7 cell line, and that MKJP2 and 1564 were particularly well internalized compared to other clones.

[0226] 13-2:MCF-7 internalization assay-C04, F06, VH5, VH16, VH35, VH9, VH2, VH7, VH32 To analyze cell surface IGF1R binding of 1564 variants with altered IGF1R binding avidity, FACS analysis was performed using an MCF-7 cell line expressing IGF1R. An IGF1R antibody prepared as a double antibody in scFv format was treated with 2 x 10E5 MCF7 cells at a concentration of 10 μg / mL for 30 minutes. After washing with PBS buffer supplemented with 1% BSA, the cells were treated with an FITC-conjugated secondary antibody to detect human antibodies for 1 hour. After washing with PBS buffer, FACS analysis was performed to confirm the extracellular binding and internalization of the variants with altered binding avidity.

[0227] As shown in Table 22 below, the biantibody containing the 1564 IGF1R antibody showed increased internalization and intensity at 37°C compared to refrigeration, and the F06 clone, which had a high degree of cell binding, also showed increased internalization. These results suggest that the 1564 variant binds well to cells and is internalized within the cells in a binding-dependent manner. [Table 22]

[0228] 13-3: Internalization analysis of human brain endothelial cells The bivalent and monovalent forms of the 1564 clones constructed in Examples 9-2 and 9-4 were analyzed for their internalization into primary human microvascular brain endothelial cells (HMBECs). Therapeutic antibody IgG (11F11) was used as a negative control.

[0229] HMBECs (Cell Systems, cat#: ACBRI376) were plated in 12-well plates at 90% confluency and then treated with the test antibody. The following day, they were fixed with 4% paraformaldehyde, rinsed with PBS, and then blocked and permeabilized for 50 minutes with a solution containing 3% BSA and Triton X. After rinsing with PBS, a goat anti-human antibody against human Fc was applied for 2 hours and 30 minutes. After rinsing with PBS, a secondary antibody against the primary antibody was applied for 1 hour. After rinsing with PBS, the cells were stained with Hoechst at a 1:1000 dilution for 10 minutes to stain nuclei. The results were analyzed by confocal microscopy using an LSM 780 NLO EC Plan-Neofluar 100X / 1.3 Oil setting. The experimental results are shown in Figure 5b.

[0230] The bivalent and monovalent forms of clone 1564 showed increased internalization compared to the negative control therapeutic antibody (11F11). This result suggests that the anti-IGF1R antibodies described above, which are bispecific antibodies of various forms linked to therapeutic antibodies, can effectively internalize the therapeutic antibodies into brain endothelial cells that make up the BBB, thereby ultimately increasing the therapeutic antibodies' ability to cross the BBB.

[0231] 13-4: Analysis of cellular fate in human brain endothelial cells If the antibody is internalized and colocalizes with intracellular lysosome-associated markers, it will be degraded in brain endothelial cells and will not be able to cross the BBB. Conversely, if the antibody colocalizes with early endosomes associated with exocytosis or with markers known to be associated with crossing the BBB, it is predicted that the antibody will be internalized in brain endothelial cells and then cross the BBB via receptor-mediated transcytosis, exiting into the brain.

[0232] The bivalent form of 1564 of the antibodies tested in Example 13-2 was treated in the same manner with HMBEC, and the intracellular colocalization of the antibody with any cellular component was analyzed. However, after blocking and permeabilization, the following antibodies were simultaneously administered along with a goat anti-human antibody to detect the administered antibody. *Anti-Cathepsin D: lysosomal marker *Anti-Caveolin-1: A marker of caveolin-mediated transcytosis (thought to be the primary mechanism of BBB crossing) *Anti-EEA1: Early endosome marker The remaining procedures were the same as in Example 13-2, except that secondary antibodies against the above markers were used.

[0233] The analysis results are shown in Figure 5c. The 1564 clone did not colocalize with cathepsin D in the bispecific antibody form, but instead colocalized with caveolin-1 and EEA1 at the cell membrane and inside the cell. This result indicates that the 1564 clone can cross the BBB via RMT after internalization without undergoing intracellular degradation.

[0234] Example 14. Analysis of the effect of anti-IGF1R antibodies on IGF1R signal transduction 14-1: IGF1R-mediated proliferation assay of MCF-7 cell line Whether the anti-IGF1R antibody of the present invention interferes with the binding between IGF1R (IGF1 receptor) and its ligand, IGF1, was confirmed using the effect of IGF1 on cell proliferation.

[0235] The minibody antibodies of clones 996, 1226, 1564, and MKJP2 prepared in Example 9-1 were each diluted 5-fold from 400 nM to prepare diluted samples, and 25 μl of each was treated with 25 μl of IGF1 at a concentration of 20 ng / ml. MCF-7 cells expressing IGF1R were cultured and passaged on the day of the experiment, with the medium removed, and 20,000 cells (corresponding to 50 μl) were treated per well of a 96-well plate containing IGF1 and test antibodies.

[0236] After culturing for 3 days at appropriate temperature and humidity, the cells were treated with 10 μl of CCK-8 reagent and incubated in a CO2 incubator for 4-5 hours to measure cell growth. Afterwards, the cells were removed and the absorbance was measured at 450 nm wavelength using a spectrophotometer. The experimental results are shown in Figure 6a.

[0237] These experimental results confirmed that the antibody of the present invention does not inhibit MCF-7 cell proliferation caused by IGF1 signaling to IGF1R. The anti-IGF1R antibody from Imclone, used as a control, inhibited IGF1-induced MCF-7 cell proliferation in proportion to the treatment concentration. Therefore, the antibody of the present invention binds to IGF1R expressed in endothelial cells that make up the BBB and has the ability to cross the BBB, but at the same time does not inhibit IGF1-induced signaling in the body, confirming that the antibody of the present invention can be used as a BBB shuttle.

[0238] 14-2: Analysis of IGF1R-mediated signaling component inhibition in MCF-7 cell line To determine whether the anti-IGF1R antibodies of the present invention are involved in the receptor and downstream signaling components of IGF1 signaling that binds to IGF1R-expressing cells and transmits it intracellularly, we administered the anti-IGF1R antibodies to MCF-7 cells expressing IGF1R and analyzed the levels of total IGF1R, phosphorylated IGF1R, and total and phosphorylated Akt, which are downstream factors of IGF1R, in the cells.

[0239] MCF-7 cells were cultured and the culture medium was changed to serum-free medium 20 hours before treatment with anti-IGF1R antibodies. The MCF-7 cells were treated with 100 nM of each of the 996, 1226, 1564, and MKJP2 clone minibody antibodies prepared in Example 9-1. One hour later, they were treated with 200 ng / ml IGF1. After 20 minutes, the cells were washed with PBS and lysed with M-PER containing protease and a phosphatase inhibitor cocktail. Protein concentration was measured using a BCA assay kit, and 12.5 μg of protein was loaded onto an SDS-PAGE gel, electrophoresed, and transferred to a PVDF membrane. Blocking was performed with 5% BSA in PBST (0.1% Tween 20) for 1 hour at room temperature with gentle shaking, followed by overnight incubation with primary antibodies against IGF1R or Akt at 4°C with gentle shaking. β-actin antibody was used as a loading control. After washing, secondary antibodies were added for 1 hour at room temperature with gentle shaking, followed by further washing. ECL solution was added, and signals were confirmed using an Image Quant Las 4000. The experimental results are shown in Figure 6b.

[0240] From the above experimental results, it was confirmed that the antibody according to the present invention does not affect the amount of total IGF1R, phosphorylated IGF1R, or total Akt and phosphorylated Akt, which are downstream factors of IGF1R, in cells.

[0241] 14-3: Analysis of IGF1R-mediated inhibition of signaling components in mouse brain endothelial cells To determine whether the anti-IGF1R antibodies of the present invention are involved in the receptor and downstream signaling components of IGF1 signaling in mouse brain endothelial cells, we administered 11F11-1564 and 3A9-1564 (CH11F11 and ch3A9, anti-α-syn monoclonal antibodies described in Korean Patent Publication No. 2018-0081465) prepared by the method of Example 9-2 and the IgG form of the 1564 clone prepared by the method of Example 9-3 to the bEND3 cell line, which was confirmed to express IGF1R, and analyzed the levels of total IGF1R, phosphorylated IGF1R, and total and phosphorylated Akt, which are downstream factors of IGF1R, in the cells.

[0242] bEND3 cells were cultured and the culture medium was changed to serum-free medium 20 hours before treatment with anti-IGF1R antibodies. The bEND cell line was treated with 100 nM each of the bispecific antibodies from the 1564 and MKJP2 clones (Example 9-2), followed by 200 ng / ml IGF1 treatment one hour later. After 20 minutes, the cells were washed with PBS and lysed with M-PER containing a protease and phosphatase inhibitor cocktail. Protein concentration was measured using a BCA assay kit, and 12.5 μg of protein was loaded onto an SDS-PAGE gel, electrophoresed, and transferred to a PVDF membrane. Blocking was performed with 5% BSA in PBST (0.1% Tween 20) for 1 hour at room temperature with gentle shaking, followed by overnight incubation with primary antibodies against IGF1R or Akt at 4°C with gentle shaking. β-actin antibody was used as a loading control. After washing, secondary antibodies were added for 1 hour at room temperature with gentle shaking, followed by further washing. ECL solution was added, and signals were confirmed using an Image Quant Las 4000. The experimental results are shown in Figure 6c.

[0243] From the above experimental results, it was confirmed that the antibody according to the present invention does not affect the amount of total IGF1R, phosphorylated IGF1R, or total Akt and phosphorylated Akt, which are downstream factors of IGF1R, in cells.

[0244] Example 15. In vivo BBB crossing ability analysis (co-localization assay) of anti-IGF1R antibodies 15-1. Colocalization of the minibody with brain vessels To confirm whether the anti-IGF1R antibody of the present invention is distributed along the brain vasculature in vivo, the following experiment was carried out. Specifically, male 6- to 8-week-old BALB / c mice were administered a single dose of PBS buffer or 10 mg / kg of IgG control or the minibody antibodies of clones 996, 1226, and 1564 prepared in Example 9-1 via the tail vein. After 4 hours, mouse brains were intracardially perfused with a sufficient amount of 0.9% NaCl solution and 4% paraformaldehyde. Fixed brains were removed and sectioned at 20 μm. To confirm colocalization of cerebral blood vessels with the IGF1R test, costaining was performed with anti-mouse CD31 and anti-human Fc antibodies, which are vascular markers. CD31 was detected using an Alexa488-conjugated secondary antibody, and human Fc was detected using an Alexa594-conjugated secondary antibody. Images were then captured under a fluorescent microscope. The experimental results are shown in FIG. 7a.

[0245] The above experimental results confirmed that the non-blocking antibodies of the present invention have excellent BBB crossing ability. The degree of colocalization of antibodies with cerebral blood vessels was analyzed by immunostaining (Neuron (2016) Yu-Zuchero et al.). Brain tissue was stained with a vascular marker (anti-CD31, green) and a human antibody (anti-human Fc, red). The non-blocking antibodies of the present invention showed a higher degree of colocalization than the IgG control group.

[0246] 15-2. In vivo BBB crossing analysis of bispecific antibodies The in vivo BBB crossing ability of the present anti-IGF1R antibody was confirmed in normal rats. SD rats were administered a single dose of 10 mg / kg or 30 mg / kg of a Parkinson's disease treatment monoclonal antibody (11F11) or a bispecific antibody (11F11-1564) composed of the 1564 clone conjugated in a bivalent form via the tail vein. Antibody levels in the CSF and brain were analyzed by mass spectrometry 24 hours later. Specifically, sv-ARBECs were plated in a monolayer on a permeable membrane, and the integrity of the BBB system was assessed based on the resistance (TEER) and sucrose permeability. The sv-ARBECs were treated with rat astrocyte culture medium (RAS-CM), which has been shown to support system integrity. The test antibodies, 1564, 48G5, 54H4, 60H6, and B11, were applied to the membrane, and the amount of antibody in the bottom chamber was analyzed by mass spectrometry after 90 minutes. The signature peptides of each antibody's Fc and scFv were analyzed and used for mass spectrometry. A Parkinson's disease therapeutic antibody alone (11F11) and a bispecific antibody conjugated to the scFv form of a Herceptin biosimilar were used as negative controls. The A20.1 antibody (produced by the National Research Council), which is known not to cross the BBB, was passed through the system to determine the calibration limit. The mass spectrometry results were substituted into a published formula to determine the Papp value, which indicates the degree of BBB crossing ability in vitro.

[0247] Analysis showed that the bispecific antibody conjugated with clone 1564 exhibited higher CSF and brain penetration than a therapeutic antibody not conjugated with an anti-IGF1R antibody, and this efficacy was confirmed at both the 10 and 30 mg / kg doses. At the 30 mg / kg dose, the bispecific antibody exhibited up to approximately 4.5 times greater brain penetration than the monoclonal antibody. The 1564 clone was prepared in bivalent and monovalent forms as described in Examples 9-2 and 9-4, and then administered at 30 mg / kg or 60 mg / kg in the same manner as above. The antibody levels in the CSF and brain were analyzed 24 hours later. The two forms of bispecific antibodies combining the 1564 clone exhibited higher CSF and brain penetration than the monovalent forms. In particular, the bivalent form exhibited higher BBB penetration than the monovalent form, with a maximum brain penetration increase of approximately 5-fold.

[0248] The results in Figures 7b and 7c show that clone 1564, when conjugated to a therapeutic antibody in various forms, improves the ability of the therapeutic antibody to cross the BBB in vivo.

[0249] The affinity variant of the 1564 clone constructed in Example 2 was predicted to have increased serum PK compared to the parental clone. Therefore, it was expected that its BBB crossing ability would be improved by remaining in serum for a long period of time and continuously maintaining BBB influx. The affinity variants constructed in bivalent form in Example 9-2 or monovalent form in Example 9-4 were administered via the tail vein of SD rats at 30 mg / kg, and blood was collected from the ocular venous plexus at 0, 24, and 48 hours. The test antibodies were divided into two experiments depending on whether the therapeutic antibody backbone was a chimeric or humanized antibody. The bispecific antibodies of the variants used in the experiments are listed in the table below. [Table 23]

[0250] The antibody levels in the blood were analyzed by ELISA. A 96-well plate was coated with goat anti-human Fc antibody, and an appropriately diluted sample was then applied to the plate. The results are shown in Figure 7d and Figure 7e.

[0251] As a result, in the first study group, the 1564 monovalent, F06 monovalent, and C04 monovalent showed prolonged serum PK compared to the parental 1564 bivalent clone. In the second study group, the bivalent forms of VH2, VH5, VH7, VH9, VH16, and VH32, except for the VH35 bivalent group, showed increased serum PK compared to the parental 1564 bivalent.

[0252] To analyze the BBB crossing ability of this group, CSF was extracted from the rats at 48 hours and analyzed by the same ELISA. The analysis results are shown in Figure 7f.

[0253] In the first study group, the 1564 monovalent, F06 monovalent, and C04 monovalent forms, which showed increased serum PK, also showed increased CSF antibody levels compared to the parental 1564 bivalent. In the second study group, the bivalent forms VH2, VH5, VH7, VH9, VH16, and VH32, which also showed increased serum PK, showed increased CSF antibody levels compared to the parental 1564 bivalent. VH35 showed decreased serum PK and CSF antibody levels compared to the parental 1564 bivalent.

[0254] The results in Figures 7d, 7e, and 7f demonstrate that serum PK is an important factor in antibody BBB penetration due to sustained antibody influx into the BBB, and that bispecific antibodies with increased serum PK (serum PK) through BBB shuttling enhance their BBB penetration. In particular, the F06 monovalent form, which had the highest CSF antibody levels, exhibited approximately five-fold higher CSF penetration than the parental 1564 bivalent form. Since the 1564 bivalent antibody exhibited approximately three-fold increased CSF penetration compared to the monovalent form, the F06 monovalent form is expected to exhibit up to approximately 15-fold increased BBB penetration compared to the monovalent form.

[0255] Example 16. Epitope mapping of anti-IGF1R antibodies 16-1. ELISA of anti-IGF1R antibodies and boiled and native IGF1R proteins We attempted to determine whether the anti-IGF1R antibody clones contained in the bispecific antibodies of the present invention recognize linear or conformational epitopes. ELISA was performed using the bivalent bispecific antibodies 1564, 48G5, 54H4, 60H6, and B11 with native human IGF1R ECD protein or boiled IGF1R. The ELISA method was the same as described in Example 11. The analytical results are shown in the table below. [Table 24] *N / A:Not available

[0256] These clones showed similar binding affinity to native human IGF1R ECD as in Example 15, but did not bind to boiled human IGF1R ECD, which had been heated to disrupt its tertiary structure, indicating that the anti-IGF1R antibodies of the present application bind to a nonlinear conformational epitope.

[0257] 16-2. Epitope mapping of anti-IGF1R antibodies To analyze the conformational epitope of the 1564 clone, alanine scanning was performed as follows. An IGF1R library containing an N-terminal eGFP tag and a deleted C-terminal kinase domain was expressed in OVCAR3 cells, an ovarian cancer cell line confirmed to have low IGF1R expression. The IGF1R library contained mutations in which IGF1R surface residues were replaced with alanine. The prepared library was transfected into OVCAR3 cells. Cells confirmed to express IGF1R were treated with 1564 antibody and then fluorescently labeled with a DyLight650-conjugated secondary antibody. The labeled cells were sorted by IGF1R expression and by IGF1R expression and 1564 binding. RNA deep sequencing was then performed using Illumina HiSeq technology to analyze the frequency of each alanine mutation in the cell population. The frequency counts were normalized by the results for cells expressing wild-type IGF1R, and the relative frequency was calculated to select mutations that reduced the counts from the 1564-labeled cell group.

[0258] Based on these observations, the epitope of 1564 was found to be located in the FN2 domain, and the corresponding residues were found to be Y775, P776, F778, R650, S791, and L798. The results and the sequence recognized by the 1564 clone are shown in Figure 9. Since these residues are not involved in IGF1 binding according to previous literature, the results faithfully explain the properties exhibited by 1564 in Example 16-1.

[0259] Example 17. Comparison of antigen binding ability between single and double antibodies 17-1: Binding ability of single and double antibodies to α-syn antigen When an IGF1R antibody was linked in the form of an scFv to an α-syn antibody in the form of an IgG, the effect on the binding strength of the α-syn antibody was analyzed.

[0260] α-syn aggregates were coated onto a 96-well plate at a concentration of 1 μg / ml for 18 hours, washed, and then each antibody was diluted 5-fold to 400 nM and allowed to bind. The bound antibodies were conjugated with anti-human Fc-HRP and then TMB solution was added to develop color, confirming the extent of antibody binding. As shown in Figure 10a, the binding strength to α-syn aggregates was confirmed to be the same for both single and double antibodies.

[0261] 17-2: Binding ability of single and double antibodies to the IGF1R antigen To compare the binding of α-syn monoclonal antibody and dual antibody to the IGF1R antigen, we performed an experiment using the same method as before.

[0262] As shown in the results of FIG. 10b, it was confirmed that the double antibody having the IGF1R scFv antibody bound well in a concentration-dependent manner, whereas the single antibody lacking the IGF1R antibody site did not bind.

[0263] 17-3: Binding strength analysis of α-syn humanized antibodies An experiment was carried out in the same manner as in Example 17-1 to analyze the difference in binding ability between the chimeric biantibody and the humanized biantibody.

[0264] As shown in Figure 10c, the humanized bibody (combination of Hu11F11 ver2, 3, or 4 with 1564) possessed binding ability to α-syn aggregates at a level similar to that of the chimeric bibody, and it was confirmed that a monovalent bibody containing one IGF1R scFv also exhibited binding ability similar to that of the chimeric antibody.

[0265] An experiment was conducted in the same manner as in Example 17-2 to analyze the IGF1R binding affinity between chimeric and humanized double antibodies. As a result, as shown in Figure 10d, all double antibodies (combinations of Hu11F11 ver2, 3, or 4 with 1564) showed identical binding affinity, and single antibodies without IGF1R scFv did not bind.

[0266] These results suggest that when the mouse antibody was humanized to replace the site that can act as an immunogen in the human body, its binding ability to α-syn aggregates and IGF1R remained unchanged, meaning that it retains the same activity.

[0267] 17-4: Comparison of phagocytic activity between single and double antibodies Phagocytosis refers to the removal of unnecessary extracellular substances through the involvement of various receptors in macrophages. Various protein aggregates can have adverse effects on the human body by inducing immune and inflammatory responses. In particular, when antibodies are administered to remove α-syn aggregates, this is known to be promoted through the interaction between the Fc region of the antibody and the FcrR on the cell surface. For this reason, we compared the phagocytic activity of a single antibody and a double antibody linked to IGF1R scFv.

[0268] To compare the phagocytic activity of single and double antibodies, mouse-derived BV-2 microglial cells were used. BV-2 cells were cultured in RPMI 1640 medium at 2 × 10 6 The α-syn aggregates were prepared at 100 μL / ml and dispensed into a U-bottom 96-well plate. 10 μg / ml α-syn aggregates and 25 μg / ml antibody were diluted in RPMI 1640 medium, mixed, and incubated at room temperature for 20 minutes. The α-syn aggregate and antibody mixture was then applied to BV-2 cells and incubated for 15 minutes. The supernatant was centrifuged at 1200 rpm to remove α-syn aggregates, and the cells were washed three times with PBS, pH 2.5, to remove aggregates or antibodies bound to the cell surface. Cells were fixed with 4% paraformaldehyde and washed with PBS. To identify intracellularly phagocytosed aggregates and antibodies, 0.5% Triton X-100 was added to loosen the cell membrane, washed with PBS, and then treated with pan-α-syn antibody for 1 hour. The conjugated pan-α-syn antibody was treated with anti-rabbit-alexa-488 antibody for 1 hour, and then FACS analysis was performed to confirm that the aggregates had entered the cells by macrophages.

[0269] As shown in Figure 10e, normal human IgG did not affect macrophages, but treatment with α-syn antibody increased macrophages of α-syn aggregates. Comparing single and double antibodies, similar levels of macrophages were observed, confirming that the scFv form of IGF1R antibody bound to the C-terminal region of IgG did not affect the activity of α-syn antibody.

[0270] Example 18. Efficacy evaluation of bispecific antibodies A bivalent bispecific antibody was constructed using the chimeric 11F11 antibody and the 1564 clone scFv as described in Example 10. The in vivo effects of the bispecific antibody and the alpha-synuclein antibody were compared and analyzed in transgenic mice overexpressing human alpha-synuclein (mThy-1 human α-synuclein, UC San Diego). 2.5 mg / kg of a single antibody or human IgG, or the same molar amount of the bivalent bispecific antibody, were intraperitoneally administered weekly for three months. Five mice were used per group, and non-transgenic littermate mice served as controls. Perfusion was then performed as follows:

[0271] After the final administration, the animals were anesthetized with chloral hydrate according to humane regulations and then 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) at 4°C until analysis, while the other half was immediately frozen (-70°C).

[0272] Pathological analysis was performed as follows. Paraformaldehyde-fixed brain halves were cut into 40-μm-thick serial sections using a vibratome in a free-floating manner. To confirm the level of alpha-synuclein expression in the brain of each treatment group, sections including the cortex, hippocampus, and striatum were incubated overnight at 4°C with alpha-synuclein antibodies (p129 α-syn antibody, Abcam, ab59264, a marker for alpha-synuclein aggregation, or total alpha-synuclein antibody, Cell Signaling Technology, #2642). Alternatively, sections were treated with glial fibrillary acidic protein (GFAP) (AB5804, Millipore) to assess astrocyte activity, or with an antibody against IL-1β (ab9722, Abcam) to assess neuroinflammation. Alternatively, sections were treated with an antibody against NeuN (Chemicon, #MAB377) to assess hippocampal neuronal death. After incubation with the primary antibody, 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 diaminobenzidine (DAB). Optical density of each immunostained section was measured under a bright-field microscope. The results are disclosed in Figures 11a to 11e.

[0273] 18-1. Alpha-synuclein reduction ability of chimeric and bispecific antibodies Figure 11a shows the ability of the chimeric 11F11 antibody and the bivalent bispecific antibody of the chimeric antibody and the 1564 clone to clear alpha-synuclein aggregates in a mouse animal model (TG) overexpressing human alpha-Syn. After administration of the antibodies to the mice, the cortex and hippocampus of mouse brain tissue were stained with p-129 alpha-Syn antibody. p-129 alpha-syn is a marker for aggregates in which the 129th residue is phosphorylated, and is represented as dark brown dots or aggregates in the stained tissue.

[0274] Figure 11a shows that the IgG-treated group showed higher p-129 staining levels than the non-tg control group (#: one-way ANOVA, p<0.01). In contrast, the levels of p-129 α-syn or aggregate staining were significantly reduced in the groups treated with either the single antibody or the bispecific antibody. In particular, in the hippocampus, the bispecific antibody treatment showed a greater reduction than the chimeric 11F11 antibody (*: one-way ANOVA, p<0.05). Figure 11b shows the same experiment as Figure 11a, but stained with a total alpha-synuclein antibody as a marker. The detection of total alpha-synuclein indicates that the present antibodies 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 monomeric aggregates or the complete removal of monomers. The increased human alpha-synuclein in TG mice was reduced by administration of the monospecific and bispecific antibodies compared to the IgG-treated group, and the bispecific antibodies were particularly effective in the hippocampus compared to the monospecific antibodies.

[0275] These results demonstrate that the chimeric 11F11 antibody and the bispecific antibody effectively reduce alpha-synuclein and its aggregate levels in an animal model of Parkinson's disease, even at a low dose of 2.5 mg / kg. In particular, the bispecific antibody exhibited superior efficacy compared to the monospecific antibodies, suggesting that the bispecific antibody's improved BBB penetration ability allows it to reach more of the brain than the monospecific antibodies, enabling it to more effectively treat the disease.

[0276] 18-2. Analysis of the ability of chimeric and bispecific antibodies to reduce astrogliosis and inflammatory cytokine levels Gliosis is a nonspecific reaction that occurs in glial cells in response to central nervous system damage, triggered by substances such as BBB damage, TGF-beta, or interleukins. Representative examples include astrogliosis, and GFAP protein is used as a marker. Therefore, we administered the chimeric 11F11 antibody and the bispecific antibody of the chimeric antibody and the 1564 clone to mice and analyzed their effects on reducing astrogliosis and the release of inflammatory cytokines that trigger it. The analysis results are shown in Figures 21c and 21d.

[0277] Figure 11c shows the results of measuring whether the chimeric 11F11 antibody prepared in accordance with one embodiment of the present invention or the bispecific antibody of the same antibody and clone 1564 reduces astrogliosis in vivo by staining mouse brain tissue with a GFAP (astrogliosis) antibody as a marker after administration of the antibody to mice. The monospecific antibody and bispecific antibody suppressed astrogliosis compared to the IgG control group, and the bispecific antibody was found to be more effective than the monospecific antibody in the striatum in particular.

[0278] Figure 11d shows the results of measuring whether the chimeric 11F11 antibody prepared according to an example of the present invention or the bispecific antibody of the same antibody and clone 1564 reduces inflammatory cytokines in vivo by staining mouse brain tissue with an IL-1 beta antibody as a marker after administration of the antibody to mice. IL-1 beta induces inflammation, leading to the death of various neurons and an inflammatory response. In the hippocampus of mice administered with the antibody of the present invention, IL-1 beta was reduced in the single antibody and dual antibody groups compared to the IgG control group, and in particular, the dual antibody's reducing ability was significantly greater than that of the single antibody (##: One-way ANOVA, p<0.005; *: One-way ANOVA, p<0.05).

[0279] As shown in the figures, it was revealed that the antibody of the present application reduced astrogliosis and the release of the inflammatory cytokine IL-1 beta that triggers astrogliosis, compared to the control group.

[0280] 18-3. Analysis of the ability of chimeric and bispecific antibodies to reduce neurodegeneration Previous literature has confirmed that alpha-synuclein-induced neurotoxicity and inflammatory responses cause brain cell death. We analyzed whether the present monospecific and bispecific antibodies could inhibit alpha-synuclein-induced brain cell death in vivo. Staining of the cortex and hippocampus with the neuron marker NeuN revealed that both the single and dual-specific antibodies reduced brain cell death compared to the IgG control group. In particular, the dual-specific antibodies were found to have a greater inhibitory effect on brain cell death in the cortex than the single antibodies. The results are shown in Figure 11e.

[0281] Example 19. Increased half-life through Fc engineering and improved BBB crossing ability FcRn is an important cell membrane receptor that prevents antibodies from being dissolved during intravascular circulation, thereby increasing their half-life. While BBB crossing depends on the transcytosis activity of antibodies, it is well known that crossing the BBB depends on the intravascular concentration. To increase the half-life of the biantibody, we engineered a biantibody by changing the 428th amino acid in the Fc region from methionine (Met) to leucine (Leu) to enhance its binding to FcRn. We administered WT and M428L biantibodies at 10 mg / kg to human FcRn-expressing transgenic mice and compared the results. As shown in Figure 11, the half-life was increased by approximately 50%. To confirm the increased half-life, monkeys were administered WT biantibody (hu11F11(ver.2)-1564), bivalent M428L biantibody (hu11F11(ver.2)(M428L)-1564 bivalent), and monovalent M428L biantibody ((hu11F11(ver.2)(M428L)-1564 monovalent) to examine the PK. The blood concentration of the WT biantibody was analyzed. As shown in Figure 12a, the blood concentration of the WT biantibody dropped sharply after 168 hours, whereas the M428L biantibody, which has strong FcRn binding affinity, maintained a higher blood concentration than the WT biantibody. The half-life of the M428L biantibody was approximately 1.5 days longer than that of the WT biantibody. In particular, the monovalent M428L biantibody was superior in terms of clearance, and the WT biantibody showed the fastest clearance (Figure 12b).

[0282] To verify the improved BBB penetration due to the increased half-life, CSF was extracted 24 hours after antibody administration and the amount of antibody in the CSF was analyzed. After coating with 100 ng / ml IGF1R under refrigeration for 18 hours, CSF was added and antibody bound to IGF1R was detected. As shown in Figure 12c, the M428L biantibody, which had a high amount of antibody in the blood, exhibited a high BBB penetration rate. The monovalent M428L biantibody exhibited superior BBB penetration ability, demonstrating improved penetration compared to the bivalent M428L biantibody.

[0283] Example 20. Efficacy evaluation of deamidated affinity variant-based bispecific antibodies In Example 10, monovalent bispecific antibodies were prepared using the humanized 11F11 antibody hu11F11 (ver. 2) and the affinity variant F06 scFv of the 1564 clone, particularly a mutant in which a partial residue in the CDR was modified by deamidation (including hu11F11 (ver. 2)-F06 (de2) (StoP) monovalent). The in vivo efficacy of the prepared bispecific antibodies and the alpha-synuclein monospecific antibodies was compared and analyzed in transgenic mice overexpressing human alpha-synuclein (mThy-1 human α-synuclein, UC San Diego).

[0284] Specifically, 4-month-old transgenic mice were intraperitoneally injected with 20 mg / kg of IgG and hu11F11 (ver. 2), and 23.4 mg / kg of the bispecific antibody (equivalent to the molar equivalent) at 0, 72, 144, and 192 hours for 8 days. 24 hours after the final injection, the animals were anesthetized with chloral hydrate and cardiac perfused with 0.9% saline. Brains were isolated and snap-frozen at -70°C until analysis. Brain tissue was homogenized and centrifuged to remove debris. The supernatant was then collected and α-syn levels in the brain lysate were quantified by α-syn ELISA (Invitrogen #KHB0061). The results are shown in Figure 13.

[0285] As shown in Figure 13, the humanized 11F11 (ver. 2) antibody and the deamidated F06 variant-based bispecific antibody prepared in accordance with one embodiment of the present invention reduced brain α-syn compared to the IgG control group. In particular, the bispecific antibody demonstrated superior brain α-syn reduction ability compared to the α-syn monoclonal antibody.

[0286] Example 21. IGF1R-specific antigen binding assay (ELISA) of double antibodies containing (de2)(StoP) deamidated anti-IGF1R antibodies In this example, we aimed to confirm whether bivalent antibodies containing deamidated anti-IGF1R antibodies from Example 8 possess normal antigen binding activity, and to comprehensively analyze the antigen binding activity of deamidated IGF1R antibodies within various anti-IGF1R clone and bivalent antibody formats. To this end, we constructed monovalent and bivalent bivalent antibodies containing non-deamidated (wild type), (de)(StoP)-deamidated, and (de2)(StoP)-deamidated antibodies based on the anti-IGF1R clones F06, VH5, and VH16, respectively, and quantitatively analyzed and compared their binding activity to recombinant IGF1R and the presence or absence of concentration-dependent binding using sandwich ELISA. The hu11F11 (ver. 2) clone was used as the anti-alpha-synuclein antibody.

[0287] The target antigen for antibody binding, human recombinant IGF1R, was the extracellular domain (ECD) and was purchased from Sino Biological (10164-H08H). Human IGF1R was diluted to 1 μg / ml in PBS buffer and added at 100 μl per well to a 96-well ELISA plate (Nunc-Immuno Plates, NUNC, Rochester, NY). After incubation at 4°C for 16 hours, the supernatant was removed. Nonspecific binding was blocked by adding 200 μl of PBS buffer containing 1% BSA (bovine serum albumin) per well and incubating at 37°C for 2 hours.

[0288] The prepared double antibody and each control antibody (wild type and (de)(StoP) deamidated antibody) were diluted 5-fold from a maximum concentration of 400 nM to create 8 dilutions. 100 μl of each was added to each well and incubated at 37°C for 2 hours to allow binding of the antibody to the coated antigen. After the incubation, the plate was washed four times with 300 μl of PBS buffer containing 0.05% Tween 20. Anti-human Fc-HRP, which recognizes the human Fc in the double antibody, was diluted 1:2000 in blocking buffer and added at 100 μl to each well. The plate was then incubated at 37°C for 1 hour. After washing four times with 300 μl of PBS-T (0.05% Tween 20), color development was performed using TMB (Tetramethylbenzidine, Sigma, T0440). The enzyme reaction was stopped with 0.5 mol / L sulfuric acid, and the absorbance at 450 nm was recorded and analyzed using a microplate reader (molecular device). The experimental results are shown in Figures 14a to 14c and Table 25. Figure 14a shows the experimental results for the F06 monovalent antibody, F06(Stop) monovalent antibody, and F06(de2)(Stop) antibody; Figure 14b shows the experimental results for the VH5 antibody, VH5(de)(Stop) antibody, and VH5(de2)(Stop) antibody; and Figure 14c shows the experimental results for the VH16 antibody, VH16(de)(Stop) antibody, and VH16(de2)(Stop) antibody.

[0289] As shown in Figures 14a, 14b, and 14c, regardless of the type of anti-IGF1R clone and the monovalent / bivalent format, the (de2)(StoP)-deamidated antibody maintained antigen-binding activity at the same level as the non-deamidated wild-type antibody. It also exhibited superior antigen-binding activity compared to the (de)(StoP)-deamidated antibody. Table 25 below shows the experimental results for the non-deamidated (wild-type), (de)(StoP)-deamidated, and (de2)(StoP)-deamidated antibodies based on F06, VH5, and VH16. [Table 25]

[0290] Table 25 compares the sandwich ELISA results of (de)(StoP)-deamidated antibodies and (de2)(StoP)-deamidated antibodies by quantifying them. Table 25 demonstrates that (de2)(StoP)-deamidated antibodies have significantly superior antigen-binding ability compared to (de)(StoP)-deamidated antibodies, regardless of the type of anti-IGF1R antibody clone or monovalent / bivalent format.

[0291] Example 22. Cell surface IGF1R-specific antigen binding assay (FACS) of a double antibody containing (de2)(StoP) deamidated anti-IGF1R antibody The (de2)(StoP)-deamidated biantibodies tested in Example 21, as well as the wild-type and (de)(StoP)-deamidated biantibodies as controls, were used to perform FACS analysis of the cell surface IGF1R-specific antigen binding activity. MCF7 cells overexpressing IGF1R were used for the analysis.

[0292] Specifically, the double antibodies were diluted to 10 μg / ml, and then 0.5×10 per sample was used. 6 The MCF-7 cell line was treated with the secondary antibody and incubated for 2 hours at 4°C. After washing twice with PBS buffer, anti-human FITC was diluted 1:1000 and treated and incubated for 1 hour at 4°C. After washing twice with PBS buffer again, the extent of anti-IGF1R BsAb binding was measured using a FACSCalibur instrument. MCF-7 cells treated with only the secondary antibody served as a control, and the experimental results are shown in Figures 15a to 15c.

[0293] As a result of the experiment, similar to the results confirmed in Example 21, regardless of the type of anti-IGF1R clone and the monovalent / bivalent format, the (de2)(StoP)-deamidated antibody maintained the same level of antigen binding ability as the non-deamidated wild-type antibody and showed superior antigen binding ability compared to the (de)(StoP)-deamidated antibody.

[0294] Example 23. In vivo BBB crossing analysis of (de2)(StoP) deamidated anti-IGF1R antibody-based bibody The in vivo BBB crossing ability of the biantibody containing the deamidated anti-IGF1R antibody according to Example 8 was confirmed in SD (Sprague-Dawley) rats. The experimental groups and administration doses are summarized in the table below. [Table 26]

[0295] After a single injection of α-syn monoclonal antibody or α-syn / IGF1R dual antibody listed in Table 26 into the tail vein of rats, the antibody levels in the serum and cerebrospinal fluid (CSF) were analyzed by mass spectrometry 24 hours later. The specific mass spectrometry method was essentially the same as in Example 15, and the analysis results are shown in Figure 16.

[0296] In Figure 16, serum concentration refers to the antibody concentration in the blood 24 hours after administration, and was observed to be similar for the (de)(StoP)-deamidated antibody and the (de2)(StoP)-deamidated antibody. In contrast, the cerebrospinal fluid concentration, which represents the brain delivery of the antibody, was highest for the (de2)(StoP)-deamidated antibody. This indicates that the bibliographic antibody of the present invention exhibits superior BBB crossing ability compared to the deamidated anti-IGF1R antibody.

[0297] To observe the efficacy of the biantibody of the present invention over a longer period, rats were administered a single dose of the anti-alpha-synuclein monoantibody hu11F11(ver.2) and the (de2)(StoP) deamidated biantibody hu11F11(ver2)-F06 monovalent (de2)(StoP) shown in Table 26 via the tail vein, and the antibody levels in the serum, cerebrospinal fluid, and brain were analyzed by mass spectrometry up to 168 hours after administration. The mass spectrometry method was essentially the same as that described in Example 15. The analytical results are shown in Figure 17.

[0298] As shown in Figure 17, serum antibody concentrations were similar for the single antibody and the (de2)(StoP)-deamidated biantibody. However, the cerebrospinal fluid (CSF) concentration, which represents brain delivery of the antibody, showed an approximately 5.8-fold increase in the area under the curve (AUC) for the (de2)(StoP)-deamidated biantibody compared to the single antibody, and the CSF concentration at 24 hours was approximately 10-fold higher for the (de2)(StoP)-deamidated biantibody. Furthermore, the brain concentration was approximately 7.9-fold higher for the (de2)(StoP)-deamidated biantibody compared to the single antibody. These results confirm that the biantibody of the present invention exhibits excellent BBB penetration ability due to the inclusion of the (de2)(StoP)-deamidated IGR1R antibody.

Claims

1. An anti-α-syn / anti-IGF1R bispecific antibody comprising an anti-α-syn antibody or antigen-binding fragment thereof; and an anti-IGF1R antibody or antigen-binding fragment thereof, the anti-IGF1R antibody comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises a heavy chain CDR1 (H-CDR1) comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 10, a heavy chain CDR2 (H-CDR2) comprising one selected from the amino acid sequences of SEQ ID NOs: 2 to 7 and SEQ ID NOs: 11 to 18, and a heavy chain CDR3 (H-CDR3) comprising one selected from the amino acid sequences of SEQ ID NOs: 8 to 9 and SEQ ID NO: 19; a light chain variable region comprising a light chain CDR1 (L-CDR1) comprising the amino acid sequence of SEQ ID NO: 20, a light chain CDR2 (L-CDR2) comprising one selected from the amino acid sequences of SEQ ID NOs: 21 to 23, and a light chain CDR3 (L-CDR3) comprising one selected from the amino acid sequences of SEQ ID NOs: 24 to 28 and 29 to 31.

2. The bispecific antibody of claim 1, wherein the anti-α-syn antibody or antigen-binding fragment thereof specifically binds to the C-terminal region of human, monkey, rat, and mouse alpha-synuclein proteins.

3. The bispecific antibody of claim 1, wherein the anti-α-syn antibody or antigen-binding fragment thereof specifically binds to a peptide comprising at least 11 consecutive amino acids, including residues 110 to 120 from the N-terminus, or a peptide comprising at least 12 consecutive amino acids, including residues 111 to 122, in the amino acid sequence of SEQ ID NO:

173.

4. The bispecific antibody of claim 1, wherein the anti-IGF1R antibody or antigen-binding fragment thereof specifically recognizes and binds to at least one amino acid selected from the group consisting of Y775, P776, F778, R650, S791, L798, Glu779, L641, H808, E809, L813, V397, D435, W434, Y460, and C488 in a protein comprising the amino acid sequence of SEQ ID NO:

174.

5. 5. The bispecific antibody of claim 4, wherein the anti-IGF1R antibody or antigen-binding fragment thereof binds to at least one or more binding sites selected from the group consisting of binding site 1 to binding site 3 in a protein comprising the amino acid sequence of SEQ ID NO: 174, wherein binding site 1 comprises one or more amino acids selected from the group consisting of Y775, P776, F778, R650, S791, L798, and Glu779, binding site 2 comprises one or more amino acids selected from the group consisting of L641, H808, E809, and L813, and binding site 3 comprises one or more amino acids selected from the group consisting of V397, D435, W434, Y460, and C488.

6. the anti-IGF1R antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises a heavy chain CDR1 (H-CDR1) comprising the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 (H-CDR2) comprising one selected from the amino acid sequences of SEQ ID NO: 3 and SEQ ID NO: 5 to 7, and a heavy chain CDR3 (H-CDR3) comprising one selected from the amino acid sequences of SEQ ID NO: 8 to 9; 2. The bispecific antibody of claim 1 , wherein the light chain variable region comprises a light chain CDR1 (L-CDR1) comprising the amino acid sequence of SEQ ID NO: 20, a light chain CDR2 (L-CDR2) comprising one selected from the amino acid sequences of SEQ ID NOs: 22 and 23, and a light chain CDR3 (L-CDR3) comprising one selected from the amino acid sequences of SEQ ID NOs: 26 to 28.

7. 2. The bispecific antibody of claim 1, wherein the heavy chain variable region of the anti-IGF1R antibody or antigen-binding fragment thereof comprises H-FR1 comprising the amino acid sequence of SEQ ID NO: 32, H-FR2 comprising the amino acid sequence of SEQ ID NO: 33 or SEQ ID NO: 34, H-FR3 comprising the amino acid sequence of SEQ ID NO: 35, and H-FR4 comprising the amino acid sequence of SEQ ID NO: 36, and the light chain variable region comprises L-FR1 comprising the amino acid sequence of SEQ ID NO: 37, L-FR2 comprising the amino acid sequence of SEQ ID NO: 38, L-FR3 comprising the amino acid sequence of SEQ ID NO: 39 or SEQ ID NO: 40, and L-FR4 comprising the amino acid sequence of SEQ ID NO: 41 or SEQ ID NO:

42.

8. The bispecific antibody of claim 1, wherein the heavy chain variable region of the anti-IGF1R antibody comprises an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 43 to 87, and the light chain variable region comprises an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 88 to 132.

9. The bispecific antibody of claim 1, wherein the antigen-binding fragment of the anti-IGF1R antibody is selected from the group consisting of scFv, (scFv)2, scFv-Fc, Fab, Fab', and F(ab')2.

10. The bispecific antibody of claim 1, wherein the anti-α-syn antibody is a complete antibody and the anti-IGF1R antibody is an Fv-fragment of the antibody.

11. The bispecific antibody of claim 10, wherein the anti-α-syn antibody is in the IgG1, IgG2, IgG3 or IgG4 form.

12. The bispecific antibody of claim 10, which is a monovalent bispecific antibody in which one molecule of the anti-IGF1R antibody is bound to one heavy chain CH3 of an anti-α-syn antibody.

13. The heavy chain variable region of the anti-α-syn antibody contained in the bispecific antibody comprises a heavy chain CDR1 (H-CDR1) comprising the amino acid sequence of SEQ ID NO: 135, a heavy chain CDR2 (H-CDR2) comprising the amino acid sequence of SEQ ID NO: 136 or 137, and a heavy chain CDR3 (H-CDR3) comprising the amino acid sequence of SEQ ID NO: 138; 2. The bispecific antibody of claim 1 , wherein the light chain variable region comprises a light chain CDR1 (L-CDR1) comprising the amino acid sequence of SEQ ID NO: 139, a light chain CDR2 (L-CDR2) comprising the amino acid sequence of SEQ ID NO: 140, and a light chain CDR3 (L-CDR3) comprising the amino acid sequence of SEQ ID NO:

141.

14. A pharmaceutical composition for the prevention or treatment of alpha-synucleinopathy, comprising a bispecific antibody according to any one of claims 1 to 13.

15. The alpha-synucleinopathy may be Parkinson's disease (PD), Parkinson's disease dementia (PDD), dementia with Lewy bodies (DLB), Alzheimer's disease with Lewy bodies (LBV), combined Alzheimer's and Parkinson's disease, or multiple system atrophy (MSA).

15. The composition of claim 14, comprising a hydroxybenzoate (MSA).

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