Bispecific antibodies against a-syn / IGF1R and uses thereof

Abstract

To provide a bispecific antibody that specifically binds to alpha-synuclein and IGF1R, a pharmaceutical composition including the bispecific antibody, and a method for producing the bispecific antibody.SOLUTION: An anti α-synuclein / anti IGF1R bispecific antibody includes an anti IGF1R antibody or an antigen-binding fragment thereof which includes: a) anti α-synuclein (anti α-syn) antibody or an antigen-binding fragment thereof; and b) (i) a heavy chain variable region (VH) including a heavy chain complementarity determining region (CDR)1(H-CDR1) including a specific sequence, H-CDR2 including a specific sequence and H-CDR3 including a specific sequence, and (ii) a light chain variable region (VL) including chain CDR1(L-CDR1) including a specific sequence light, L-CDR2 including a specific sequence, and L-CDR3 including a specific sequence.SELECTED DRAWING: None

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 α-synucleinopathies comprising said bispecific antibody, and a method for providing information for the detection of alpha-synuclein aggregates or the diagnosis of synucleinopathies comprising said bispecific antibody. [Background technology]

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

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

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

[0005] Antibodies against alpha-synuclein or fragments of alpha-synuclein to induce such antibodies have been proposed as a method of immunotherapy for synucleinopathies, but brain penetration of antibodies can be limited by the blood-brain barrier (BBB).

[0006] The lack of highly specific BBB transporters also slows 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 to deliver therapeutic and diagnostic molecules to the brain in pharmacologic effective doses without disrupting the physiology and homeostasis of the BBB. Summary of the Invention [Problem to be solved by the invention]

[0007] One example of the present invention is alpha-synuclein (a- s The present invention provides a protein complex comprising an antigen-binding site for IGF1R, and an antigen-binding site for IGF1R, or a method for producing said protein complex.

[0008] Other examples provide polynucleotides encoding the protein complexes, recombinant vectors containing the same, and recombinant cells containing the same.

[0009] Another example is a- s A bispecific antibody against yn and IGF1R and a method for producing the same are provided.

[0010] Another example is the above-mentioned a- s A pharmaceutical composition for preventing and / or treating alpha-synucleinopathies is provided, comprising a bispecific antibody against yn and IGF1R, and a pharma- ceutically acceptable excipient.

[0011] 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. [Means for solving the problem]

[0012] The present invention will now be described in further detail. One example of the present invention is alpha-synuclein (a- s A protein complex comprising an antigen-binding site for IGF1R and an antigen-binding site for alpha-synuclein (a-synuclein) obtained from said protein complex s yn) and a bispecific antibody against IGF1R (hereafter referred to as anti-a- s The present invention relates to a bispecific antibody (anti-alpha-synuclein / anti-IGF1R bispecific antibody). Thus, the bispecific antibody according to the present invention can recognize and bind to both alpha-synuclein and IGF1R as antigens.

[0013] Anti-a- s The yn / anti-IGF1R bispecific antibody is s The yn antibody or antigen-binding fragment thereof is capable of specifically recognizing and binding to alpha-synuclein, in particular to 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.

[0014] According to the present invention, the term "synucleinopathies" includes all neurodegenerative disorders characterized by pathological synuclein aggregates. Several neurodegenerative disorders are collectively grouped as synucleinopathies, including Parkinson's disease, Parkinson's disease dementia (PDD), dementia with Lewy bodies (DLB), Lewy body disease, dementia with Lewy bodies, parkinsonism with dementia, multiple system atrophy (MSA), multiple neuronal atrophy and neurodegeneration with cerebral iron deposition type I (NBIA Type I). Alpha-synuclein aggregates are also found secondarily in Alzheimer's disease (Kim et al. , Alzheimer's Research & Therapy 2014, 6:73).

[0015] Synucleinopathies are a diverse group of neurodegenerative disorders that share common pathological characteristics 。 In neuropathological experiments, characteristic lesions can be detected involving abnormal aggregation of alpha-synuclein protein in select populations of neurons and oligodendrocytes. 1 Alpha-synuclein, which was later identified as PARK1 and PARK2, is a 140 amino acid protein that is widely expressed in the neocortex, hippocampus, dentate gyrus, olfactory bulb, striatum, thalamus, and cerebellum. Alpha-synuclein is also highly expressed in hematopoietic cells, including B-, T-, and NK cells, as well as in proteinocytes and platelets. Its exact role in these cells is unknown, but it has been associated with the differentiation of megakaryocytes (platelet precursors).

[0016] In the present application, "diseases associated with alpha-synuclein aggregates" refers to a group of neurodegenerative diseases known as synucleinopathies, in which alpha-synuclein aggregates are found in lesions involving neuronal and glial cell (glia) populations and are characterized by degeneration of the dopaminergic system, altered motor skills, cognitive impairment, and the formation of Lewy bodies and / or Lewy neurites (Kim et al. , Alzheimer's Research & Therapy 2014, 6:73; McKeith et al., Neurology (1996) 47:1113-24). Such diseases include, but are not limited to, Parkinson's disease, Parkinsonian dementia, Lewy body dementia, Alzheimer-Lewy body disease, combined Alzheimer's and Parkinson's disease, multiple system atrophy, and many other neuroaxonal diseases. In one embodiment, the antibodies of the present application are effectively used to treat Parkinson's disease.

[0017] In addition, the anti-a- s The yn / anti-IGF1R bispecific antibody comprises an anti-IGF1R antibody or an antigen-binding fragment thereof, and an anti-a- s yn antibody or antigen-binding fragment thereof to cross the blood-brain barrier and exert its action in the brain, maintains efficacy for a long period of time extension May be .

[0018] Furthermore, the anti-a- s The yn / anti-IGF1R bispecific antibody binds to IGF1R on the cell surface but does not affect ligand binding or the signal transduction pathway through IGF1R. Therefore, it does not inhibit IGF1R and its ligand binding or signal transduction through IGF1R, and can be used as a shuttle means to cross the blood-brain barrier.

[0019] In particular, the anti-IGF1R antibody or antigen-binding fragment according to the invention binds to IGF1 RIt specifically 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 transmission through IGF1R, can be used for transcytosis, has the ability to pass the blood-brain barrier, and does not have ADCC (antibody-dependent cell-mediated cytotoxicity), so it does not reduce IGF1R levels in the brain even when administered repeatedly to animals and is non-toxic.

[0020] 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 inside the cells. For example, it is possible to confirm whether the anti-IGF1R antibody of the present invention is internalized inside the cells using a cell line expressing IGF1R (e.g., MCF-7, etc.). The anti-IGF1R antibodies of the present invention, such as 1564, 48G5, 54H4, 60H6, B11, and affinity variants of 1564, C04, F06, VH2, VH5, VH7, VH9, VH16, VH32, and VH35, showed higher internalization than the negative control group. This result suggests that the degree of internalization of the tested anti-IGF1R antibodies is specific to IGF1R on the cell surface. In addition, the anti-IGF1R antibody according to the present invention is in the form of an scFv, and can be prepared by binding to a therapeutic antibody in various ways. For example, scFv of anti-IGF1R antibody can be produced as a bispecific antibody in which two scFvs are bound to the C-terminus of a therapeutic antibody, such as an α-syn antibody, i.e., a bivalent bispecific antibody, or a monovalent bispecific antibody in which one scFv of the anti-IGF1R antibody is bound to the C-terminus of a therapeutic antibody, i.e., a monovalent bispecific antibody, and all of the bispecific antibodies are internalized in cells expressing IGF1R. IGF1R antibodies have high binding strength to antigens on the cell surface, which enhances the internalization effect, leading to the ability to pass through the BBB. However, if the antibody has the ability to pass through the BBB but interferes with IGF1R signaling, it may cause side effects, so it is characterized by being a non-blocking antibody against IGF1R signals while having binding strength that can function as a BBB shuttle.

[0021] The anti-IGF1R antibody or antigen-binding fragment has excellent ease of development. In this regard, it was attempted to remove post-translational modifications, such as deamidation, which occur in the CDR region of the anti-IGF1R antibody and reduce the stability and efficacy of the antibody. The substitution of amino acids that cause deamidation increases the stability and efficacy of the antibody compared to the parental antibody, without changing the binding ability to the ECD of the antigen IGF1R. When the binding ability to IGF1R of the mutants in which deamidation occurs, which are one Asn each with Q, H, and K, was analyzed by ELISA, it was confirmed that the binding ability of the mutants to IGF1R was similar to that of the parental anti-IGF1R antibody.

[0022] Additionally, the anti-IGF1R antibody according to the present invention, when linked to a bioactive substance that acts in the brain, can induce improved BBB crossing ability and efficacy compared to the bioactive substance alone.

[0023] The anti-IGF1R antibody according to one embodiment of the present invention can be used as a bispecific antibody with various therapeutic second antibodies, and in a human iPSC-derived in vitro BBB system penetration experiment, it showed 15 times higher BBB penetration ability than a single antibody composed of only a therapeutic antibody ( FIG. 16 A ). In the bispecific antibody, the anti-IGF1R antibody bound to the second antibody may be bound in a monovalent or bivalent form. For example, when a bispecific antibody having a monovalent or bivalent anti-IGF1R antibody was administered once to normal rats and the antibody amount in the blood and CSF was analyzed, the bispecific antibody having a monovalent or bivalent anti-IGF1R antibody showed a maximum 5-fold increase in the antibody amount in the blood and a maximum 5-fold increase in the antibody amount in the CSF compared to the parental anti-IGF1R antibody (1564 clone). The amount of CSF passage was increased by about 3-fold and about 4.5-fold in the brain compared to the parental anti-IGF1R antibody (1564 clone) (FIG. 16). CTherefore, the bispecific anti-IGF1R antibody improved by the above method is expected to exhibit up to about 15-fold greater CSF and about 23-fold greater brain penetration ability than a monoclonal antibody consisting of only a therapeutic second antibody.

[0024] 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 usefully used in screening for drug development, clinical trials and the like.

[0025] The anti-IGF1R antibody or antigen-binding fragment according to the present invention is an antibody or an antigen-binding fragment thereof that specifically recognizes IGF1R (Insulin-like Growth Factor 1 Receptor).

[0026] An anti-IGF1R antibody or antigen-binding fragment according to the invention "specifically binds" to its target, such as an antigen, when it binds with an affinity having a dissociation constant (KD) of ≦10-6M. ”。 An antibody specifically binds to a target with high affinity when the KD is ≦1×10−8 M or when the EC50 (effective concentration 50) is 2 nM or less. In one embodiment, the antibody or antigen-binding fragment thereof specifically binds to a target with high affinity when the KD is ≦1×10−8 M or when the EC50 (effective concentration 50) is 2 nM or less. D ≦1×10 -8 and capable of binding to IGF1R or human IGF1R. The antibodies disclosed in the present invention have been confirmed to bind to IGF1R, in particular, human IGF1R, mouse IGF1R, rat IGF1R, and monkey IGF1R.

[0027] As used herein, the term "epitope" is understood to mean an antigenic determinant, a part of an antigen that is recognized by an antibody. According to one embodiment, the binding site of the anti-IGF1R antibody according to the invention is located in the IGF1R protein, e.g., the human IGF1R protein (SEQ ID NO: 410More specifically, the binding site of the anti-IGF1R antibody according to the present invention, for example the 1564 clone antibody, to the human IGF1R protein may be the extracellular domain of SEQ ID NO: 410 The binding site 1 is Y775, P776, F778, R650, S791, and L798. and Glu779, and binding site 2 contains L641, H808, and E809. and L813, and binding site 3 is V397, D435, W434, Y460, and Thus, the epitope of the IGF1R antibody according to the present invention is a conformational epitope, which may include all or part of the three binding sites.

[0028] In the present invention, the term "antibody" refers to a substance produced in the immune system by stimulation with an antigen, and may be produced in vivo, recombinantly produced, or artificially synthesized, with no particular limitation on the type. In the present invention, the term "antibody" includes all of animal antibodies, chimeric antibodies, humanized antibodies, and human antibodies. In addition, in the present invention, the term "antibody" also includes antigen-binding fragments of antibodies that retain antigen-binding ability.

[0029] The antibody also includes a monoclonal antibody and a polyclonal antibody, and the monoclonal antibody may be an isolated antibody that specifically binds to IGF1R, which is a human antibody, a humanized antibody, or a chimeric antibody. The monoclonal antibody is an isolated antibody that specifically binds to IGF1R, which is an IgG1, IgG2, IgG3, or IgG4 type.

[0030] Antibodies according to the present invention include, but are not limited to, bispecific antibodies, minibodies, domain antibodies, antibody mimetics (or synthetic antibodies), antibody fusions (or antibody conjugates), and fragments thereof. Various antibody structures are additionally disclosed in the present invention below.

[0031] As used herein, an "antigen-binding fragment" refers to a portion of an antibody having specific binding ability to an antigen, or a polypeptide comprising the same. For example, an antigen-binding fragment may be a portion of an antibody comprising amino acid residues that interact with an antigen (e.g., an epitope) and confer specificity and / or affinity to the antibody for the antigen, or a polypeptide comprising the same. Such antigen-binding fragments typically contain one or more "complementary determining regions" (CDRs) that are complementary to one or more of the "complementary determining regions" (CDRs). ( CDR ) ), in addition to one or more “frameworks.” (FR) The CDRs are amino acid sequences that contribute to the antigen-binding specificity and affinity of the antibody, and the framework regions are amino acid sequence sites that contribute to maintaining the proper conformation of these CDRs and can promote binding between the antigen-binding region and the antigen.

[0032] In the present invention, the term "complementarity-determining regions" refers to ( CDR ) "" refers to the site in the variable region of an antibody that confers antigen-binding specificity.

[0033] The antibody may be selected from all subtypes of immunoglobulins (e.g., IgA, IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4), IgM, etc.). The IgG antibody may be of 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 bound to the other via a disulfide bond to form two heavy-light chain structures (dimers), and the two heavy-light chains are linked to each other via a disulfide bond at the Fc region of the heavy chain. The IgG antibody may be a single-target antibody that targets one antigen by including antigen-binding sites for the same antigen in both heavy-light chain structures, or a bispecific antibody that targets two antigens by including antigen-binding sites for different antigens in both heavy-light chain structures.

[0034] As used herein, an "antigen-binding fragment" of an antibody or immunoglobulin chain (heavy or light) is a portion of an antibody that lacks some amino acids compared to the full-length chain but is capable of specifically binding to an antigen. Such an antigen-binding fragment is biologically active in that it can specifically bind to a target antigen or compete with other antibodies or antigen-binding fragments for binding to a particular epitope. Specifically, the antigen-binding fragment may be an antibody fragment that contains one or more of the above complementarity determining regions, such as, but not limited to, an antibody fragment selected from the group consisting of scFv, (scFv)2, scFv-Fc, Fab, Fab', and F(ab')2. Such biologically active fragments may be produced by recombinant DNA techniques or, for example, by enzymatic or chemical cleavage of a complete antibody. Immunologically functional immunoglobulin fragments are not limited thereto.

[0035] As used herein, a "variant" of a polypeptide, such as an antigen-binding fragment, protein, or antibody, is a polypeptide that has insertions, deletions, additions, and / or substitutions at one or more amino acid residues compared 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.

[0036] In the present invention, a "derivative" of a polypeptide refers to a polypeptide that has been chemically altered through conjugation with other chemical moieties, as distinct from an insertion, deletion, addition, or substitution mutant.

[0037] The anti-IGF1R antibody or antigen-binding fragment according to the present invention does not interfere with the binding of IGF1R ligands IGF-1, IGF-2, and / or insulin to IGF1R. Specifically, the anti-IGF1R antibody or antigen-binding fragment has the advantage that it does not interfere with the binding of IGF1R ligands to the IGF1R located on the cell membrane in cells expressing IGF1R, does not inhibit signal transduction via IGF1R, and does not affect IGF1R expression on the cell surface. Thus, the anti-IGF1R antibody or antigen-binding fragment according to the present invention can be effectively used to cross the blood-brain barrier through transcytosis.

[0038] Human IGF1R can be activated by insulin-like growth factors, IGF-1 and IGF-2, and insulin (INS). Signaling through IGF1R promotes cell growth and survival through IRS adaptor proteins, which are dependent on activation of the PI3 Kinase / Akt pathway. IGF1R transmits signals to its major substrates IRS-1, IRS-2, IRS-3, and IRS-4, and to Shc proteins, resulting in activation of the Ras / Raf / MAP kinase and PI3 kinase / Akt signaling pathways. IGF1R has been found to be relatively highly expressed in the brain, compared to other transcytosis targets known to be expressed in brain endothelial cells, which have been used primarily to date to improve BBB crossing.

[0039] The anti-IGF1R antibody according to the present invention does not interfere with the binding of IGF1, IGF2 and / or insulin to IGF1R, and does not interfere with the above-mentioned signal transduction pathway via IGF1R. In addition, in one embodiment according to the present invention, it has been revealed that IGF1R shows a relatively low expression level in normal brain and peripheral tissues, such as liver, lung, and colon, when compared with other targets currently being developed for improving the BBB crossing ability of therapeutic antibodies, such as transferrin receptor and insulin receptor.

[0040] IGF1R is a target of RMT (Receptor Mediated Transcytosis) that can deliver useful substances to the inside of the brain through the blood brain barrier (BBB). However, in order to be used as a drug delivery target for passing through the blood brain barrier, it is preferable that the antibody binds to IGF1R on the cell surface without affecting ligand binding and without affecting the signal transduction pathway through IGF1R. Therefore, the anti-IGF1R antibody and its antigen-binding fragment according to the present invention can be used as a shuttle means for passing through the blood brain barrier because it does not inhibit IGF1R and its ligand binding and signal transduction through IGF1R.

[0041] The anti-IGF1R antibody or antigen-binding fragment thereof according to the present invention is capable of transcytosis and can pass through endothelial cells in the brain. Furthermore, when the antibody according to the present invention is injected into the blood vessel of a mouse, it is localized in the same location as the mouse's cerebral blood vessels. These results indicate that the antibody or antigen-binding fragment according to the present invention can be effectively used as a drug transporter that passes through the blood-brain barrier.

[0042] 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, membrane, or structure that blocks the effective passage, diffusion, or transmission of a cell, tissue, membrane, or biological molecule. Such biological barriers include nerve cells / tissues, connective tissues, muscles, membranes, or epithelial (e.g., mucosa or blood vessel) cells. A representative example is the blood-brain barrier.

[0043] In the present invention, the "blood-brain barrier" or BBB is a barrier formed by tight junctions in the endothelial cell membrane of brain capillaries that exists between the brain and spinal cord and the peripheral circulatory system. Such a barrier is very strong and also restricts the passage of low molecular weight substances with a molecular weight of about 60 Da to the brain. The blood-brain barrier of the brain, the blood-spinal cord barrier of the spinal cord, and the blood-retinal barrier of the retina are continuous capillary barriers in the central nervous system, and are usually referred to as the BBB.

[0044] In the present invention, a "blood-brain barrier transporter" is capable of transmitting said brain effector substance across such a blood-brain barrier, and said brain effector substance includes, for example, chemical compounds, proteins including peptides and polypeptides, nucleic acids, antibodies, or small molecule compounds.

[0045] The antibodies according to the present invention can be produced and selected from transgenic mice, such as those described above, by using hybridoma technology to generate antigen-specific human mAbs with the desired specificity. Such antibodies can be cloned and expressed using appropriate vectors and host cells, or the antibodies can be harvested from cultured hybridoma cells. The antibodies can also be derived from a phage-display library. The phage display technology is a method that mimics a type of immune selection, in which an antibody repertoire is displayed on the surface of filamentous bacteriophage, and then phages that bind to the desired antigen are selected. For such phage technology, see the Examples of the present invention or PCT Publication WO99 / 10494. In one embodiment, the humanized IGF1R antibodies of the present invention are selected by the phage display method.

[0046] The present invention relates to an isolated antibody or antigen-binding fragment thereof that specifically binds to IGF1R, the antibody or antigen-binding fragment thereof comprising a heavy chain complementarity determining region and a light chain complementarity determining region, and may be a polypeptide, protein, or antibody or antigen-binding fragment thereof that specifically binds to IGF1R.

[0047] Specifically, the following describes antibodies that specifically bind to IGF1R.

[0048] 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 transmission through IGF1R, is available for transcytosis, has the ability to cross the blood-brain barrier, and does not have ADCC (antibody-dependent cell-mediated cytotoxicity), so does not reduce brain IGF1R levels even when administered repeatedly to animals and is non-toxic.

[0049] 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 inside the cells. For example, it is possible to confirm whether the anti-IGF1R antibody of the present invention is internalized inside the cells using a cell line expressing IGF1R (e.g., MCF-7, etc.). The anti-IGF1R antibodies of the present invention, for example, 1564, 48G5, 54H4, 60H6, B11, and affinity variants of 1564, C04, F06, VH2, VH5, VH7, VH9, VH16, VH32, and VH35, showed higher internalization than the negative control group. This result suggests that the degree of internalization of the tested anti-IGF1R antibodies is specific to IGF1R on the cell surface. In addition, the anti-IGF1R antibody according to the present invention is in the form of an scFv, and can be prepared by binding to a therapeutic antibody in various ways. For example, scFv of anti-IGF1R antibody can be produced as a bispecific antibody in which two scFvs are bound to the C-terminus of a therapeutic antibody, for example, an α-syn antibody, i.e., a bivalent bispecific antibody, or a monovalent bispecific antibody in which one scFv of the anti-IGF1R antibody is bound to the C-terminus of a therapeutic antibody, i.e., a monovalent bispecific antibody, and all of the bispecific antibodies are internalized in cells expressing IGF1R. IGF1R antibodies have high binding strength to antigens on the cell surface, which enhances the internalization effect, leading to the ability to pass through the BBB. However, if the antibody has the ability to pass through the BBB but interferes with IGF1R signaling, it may cause side effects, so it is characterized by being a non-blocking antibody against IGF1R signals while having binding strength that can function as a BBB shuttle.

[0050] The present invention relates to an isolated antibody or antigen-binding fragment thereof that specifically binds to IGF1R, the antibody or antigen-binding fragment thereof comprising a heavy chain complementarity determining region and a light chain complementarity determining region, and may be a polypeptide, protein, or antibody or antigen-binding fragment thereof that specifically binds to IGF1R.

[0051] 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 disclosed in Table 1, or a heavy chain variable region comprising 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 disclosed in Table 2, or a light chain variable region comprising 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 include.

[0052] 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. region 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. region may include.

[0053] According to the present invention, (i) the heavy chain complementarity determining region of H-CDR1, H-CDR2 or H-CDR3 is selected from the amino acid sequences set forth in Table 1 below or comprises one or more amino acid sequences having substantial sequence identity with the selected amino acid sequence, and (ii) the light chain complementarity determining region of L-CDR1, L-CDR2 or L-CDR3 is selected from the amino acid sequences set forth in Table 2 below or comprises one or more amino acid sequences having substantial sequence identity with the selected amino acid sequence.

[0054] 「 Substantial Identity 」 By "variant" we mean that the present invention maintains the effects disclosed herein in the presence of sequence variations. In one embodiment, the variant has about 90%, 95%, or 99% identity to a heavy chain variable region disclosed. In another embodiment, the variant has about 90%, 95%, or 99% identity to a light chain variable region disclosed. For example, for variants that exhibit 90%, 95%, or 99% identity to the sequences of the antibodies or antigen-binding fragments disclosed herein, any mutations may occur in the variable regions rather than the CDRs. Framework Area occurs in.

[0055] [Table 1] Antibody clones of the present invention and CDR sequences of the heavy chain variable region TIFF0007625305000001.tif252170TIFF0007625305000002.tif255170TIFF00076253050 00003.tif255170TIFF0007625305000004.tif255170TIFF0007625305000005.tif210170

[0056] [Table 2] Antibody clones of the present invention and CDR sequences of the light chain variable region TIFF0007625305000006.tif249170TIFF0007625305000007.tif255170TIFF00076253050 00008.tif255170TIFF0007625305000009.tif255170TIFF0007625305000010.tif210170

[0057] One or more heavy chain frameworks selected from the group consisting of H-FR1, H-FR2, H-FR3, and H-FR4 according to the present invention. region can be selected from the amino acid sequences listed in Table 3 below, and one or more light chain frameworks selected from the group consisting of L-FR1, L-FR2, L-FR3, and L-FR4. region can be selected from the amino acid sequences listed in Table 4 below.

[0058] Specifically, the heavy chain variable region of the anti-IGF1R antibody according to the present invention may comprise the H-CDR1, H-CDR2 and H-CDR3 set forth in Table 1 above, or may additionally comprise the heavy chain frameworks of H-FR1, H-FR2, H-FR3 and H-FR4 in Table 3. The light chain variable region of the anti-IGF1R antibody according to the present invention may comprise the heavy chain frameworks of H-FR1, H-FR2, H-FR3 and H-FR4 in Table 3. 2 or may additionally comprise a light chain framework of L-FR1, L-FR2, L-FR3, and L-FR4 as shown in Table 4. In one example, the anti-IGF1R antibody or antigen-binding fragment thereof may comprise a heavy chain variable region comprising CDR-H1, CDR-H2, and CDR-H3 of each clone selected from the group consisting of the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 listed in Table 1, and CDR-L1, CDR-L2, and CDR-L3 of each clone selected from the group consisting of the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 listed in Table 2.

[0059] In one embodiment of the present invention, the anti-IGF1R antibody has an amino acid mutation in the Fc region, for example, an amino acid mutation such as Deam id By removing deamidation, the binding strength to the ECD of the antigen IGF1R remains unchanged, but the pK of the antibody is increased due to the decreased clearance in blood, thereby extending the half-life of the antibody. In one example, the amino acid positions where deamidation has been removed in the anti-IGF1R antibody may be N51D in the light chain LCDR2, N95aK, N95aH, N95aR, or N95aD in the LCDR3, or N54D or N54Q in the heavy chain HCDR2 in clone 1564.

[0060] [Table 3] Antibody clones of the present invention and framework sequences of the heavy chain variable region TIFF0007625305000011.tif251170TIFF0007625305000012.tif255170TIFF00076253050 00013.tif255170TIFF0007625305000014.tif255170TIFF0007625305000015.tif255170 TIFF0007625305000016.tif255170TIFF0007625305000017.tif255170TIFF00076253050 00018.tif255170TIFF0007625305000019.tif255170TIFF0007625305000020.tif113170

[0061] [Table 4] Antibody clones of the present invention and framework sequences of the light chain variable region TIFF0007625305000021.tif250170TIFF0007625305000022.tif255170TIFF00076253050 00023.tif255170TIFF0007625305000024.tif255170TIFF0007625305000025.tif255170 TIFF0007625305000026.tif255170TIFF0007625305000027.tif255170TIFF00076253050 00028.tif255170TIFF0007625305000029.tif255170TIFF0007625305000030.tif103170

[0062] The anti-IGF1R antibody according to the present invention may be an antibody comprising a light chain variable region and a heavy chain variable region, and the various heavy and light chain variable regions disclosed herein are disclosed in Tables 5 and 6, which respectively provide the SEQ ID NOs of CDR1 to CDR3 and frameworks 1 to 4 of each clone.

[0063] The heavy chain variable regions and light chain variable regions shown in Tables 5 and 6 below can be freely combined to produce various types of antibodies. Examples of combinations of the heavy chain variable regions and light chain variable regions are shown in Tables 5 and 6.

[0064] [Table 5] Heavy chain variable region of the antibody according to the present invention TIFF0007625305000031.tif250170TIFF0007625305000032.tif255170TIFF0007625305000033.tif221170

[0065] [Table 6] Light chain variable region of the antibody according to the present invention TIFF0007625305000034.tif251170TIFF0007625305000035.tif255170TIFF00076253050 00036.tif255170TIFF0007625305000037.tif255170TIFF0007625305000038.tif211170

[0066] Various heavy and light chain variable regions disclosed herein are disclosed in Tables 5 and 6. 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. An example of a combination of heavy and light chain variable regions constituting an anti-IGF1R antibody according to one embodiment of the present invention may be a combination of heavy and light chain variable regions with the same clone name described in Tables 5 and 6.

[0067] In a specific example of the present invention, a heavy chain CDR1 (H-CDR1) comprising one of the amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 9; A heavy chain CDR2 (H-CDR2) comprising one of the amino acid sequences of SEQ ID NO: 10 to SEQ ID NO: 49; A heavy chain CDR3 (H-CDR3) comprising one of the amino acid sequences of SEQ ID NO:50 to SEQ ID NO:80; A light chain CDR1 (L-CDR1) comprising one of the amino acid sequences of SEQ ID NO: 96 to SEQ ID NO: 113; SEQ ID NO: 114 to SEQ ID NO: 132 and SEQ ID NO:648 and a light chain CDR2 (L-CDR2) comprising one of the amino acid sequences: SEQ ID NO: 133 to SEQ ID NO: 161 and sequence number 650 The present invention relates to an anti-IGF1R antibody or an antigen-binding fragment thereof, which comprises at least one selected from the group consisting of light chain CDR3 (L-CDR3) comprising one of the amino acid sequences listed below.

[0068] Specifically, the antibody or antigen-binding fragment thereof according to the present invention comprises a heavy chain variable region comprising a heavy chain CDR1 (H-CDR1) comprising one of the amino acid sequences set forth in SEQ ID NOs: 1 to 9, a heavy chain CDR2 (H-CDR2) comprising one of the amino acid sequences set forth in SEQ ID NOs: 10 to 49, and a heavy chain CDR3 (H-CDR3) comprising one of the amino acid sequences set forth in SEQ ID NOs: 50 to 80; Light chain CDR1 (L-CDR1) containing one of the amino acid sequences of SEQ ID NO: 96 to SEQ ID NO: 113, and SEQ ID NO: 114 to SEQ ID NO: 132 and SEQ ID NO:648 a light chain CDR2 (L-CDR2) comprising one of the amino acid sequences And SEQ ID NO: 133 to SEQ ID NO: 161 and sequence number 650 The light chain variable region can include a light chain CDR3 (L-CDR3) comprising one of the amino acid sequences of:

[0069] 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 protein comprising the amino acid sequence of SEQ ID NO: 410. 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: 410, and the binding site 1 may comprise one or more amino acids selected from the group consisting of Y775, P776, F778, R650, S791, L798 and Glu779, the binding site 2 may comprise one or more amino acids selected from the group consisting of L641, H808, E809 and L813, and the binding site 3 may comprise one or more amino acids selected from the group consisting of V397, D435, W434, Y460 and C488. The heavy chain variable region of the antibody or antigen-binding fragment thereof according to the present invention may comprise: A heavy chain variable region framework located on the N-terminal side of H-CDR1, comprising one of the amino acid sequences of SEQ ID NOs: 81 to 84. region 1(H-FR1), A heavy chain variable region framework located between H-CDR1 and H-CDR2, comprising one of the amino acid sequences of SEQ ID NOs: 85 to 86. area 2 (H-FR2), A heavy chain variable region framework located between H-CDR2 and H-CDR3, comprising one of the amino acid sequences of SEQ ID NOs: 87 to 91. area 3 (H-FR3), and A heavy chain variable region framework located at the C-terminus of H-CDR3, comprising one of the amino acid sequences of SEQ ID NOs: 92 to 95 area 4 It may also include (H-FR4).

[0070] The light chain variable region of the antibody or antigen-binding fragment thereof according to the present invention comprises: A light chain variable region framework located on the N-terminal side of L-CDR1, comprising one of the amino acid sequences of SEQ ID NOs: 162 to 164. region 1(L-FR1), A light chain variable region framework located between L-CDR1 and L-CDR2, comprising the amino acid sequence of SEQ ID NO: 165. area 2 (L-FR2), A light chain variable region framework located between L-CDR2 and L-CDR3, comprising one of the amino acid sequences of SEQ ID NOs: 166 to 168. area 3 (L-FR3), and A light chain variable region framework located at the C-terminus of L-CDR3, comprising one of the amino acid sequences of SEQ ID NOs: 169 to 171 area 4 It may also contain (L-FR4).

[0071] The heavy chain variable regions and light chain variable regions disclosed in Tables 5 and 6 can be used as separate domain antibodies or can be freely combined with each other to form various antibodies, and can also be linked in a single chain form to form a single chain antibody such as scFv.

[0072] In the present invention, a "domain antibody" is an immunologically functional immunoglobulin fragment that contains only the variable region of the heavy chain or the variable region of the 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.

[0073] The antigen-binding fragment of the anti-IGF1R antibody according to the present invention is an antibody fragment that contains one or more complementarity determining regions. Includes , scFv, (scFv)2, scFv-Fc, Fab, Fab', F(ab')2, minibody and diabody.

[0074] Among the antigen-binding fragments, Fab has a structure that includes 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 region.

[0075] Fv is the smallest antibody fragment having only a heavy chain variable region and a light chain variable region, and includes single-chain variable fragment (scFv) and two-chain variable fragment (two-chain Fv). In a two-chain Fv, the heavy chain variable region and the light chain variable region can be linked by a non-covalent bond. In a single-chain Fv, the heavy chain variable region and the light chain variable region can be linked by a covalent bond 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 a heavy chain variable region and a light chain variable region are linked directly or via a linker, and may be one or more selected from the group consisting of an scFv in which a heavy chain variable region and a light chain variable region are linked in a single chain form, a structure similar to an scFv dimer (di-scFv), and an scFv-Fc in which a heavy chain variable region, a light chain variable region, and an Fc are linked in a single chain form.

[0076] The peptide linker may be as described above, and may be, for example, 1 to 100, for example, 2 to 50, or 5 to 25 amino acids long. The length of the peptide linker can be determined in various ways within the limits that do not affect the function of the antibody. The types of amino acids contained in the peptide linker may be, for example, one or more amino acids selected from the group consisting of Gly, Ser, and Leu, and as a specific example, it may be composed of Gly and Ser residues, or leucine (Leu) and serine (Ser). In a specific example, the peptide linker may be (G4S)n, where n is the number of (G4S) repeats and may be represented by an integer of 1 to 10, for example, 2 to 5, particularly 3 or 4. An example of the peptide linker is SEQ ID NO: 411 or 412 The peptide may be composed of the amino acids. SEQ ID NO:411: GGGGSGGGGSGGGGSGGGGS SEQ ID NO: 412: GGGSGGGGSGGGGS

[0077] 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 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.

[0078] The antigen-binding fragment can be obtained using proteolytic enzymes (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 by recombinant gene technology.

[0079] Single chain antibodies disclosed in the present invention include, but are not limited to, scFvs comprising a domain combination of heavy and light chain variable regions, or a combination of light and heavy chain variable domains comprising the CDRs.

[0080] Additionally, the anti-IGF1R antibodies and antigen-binding fragments thereof can comprise 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 the 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.

[0081] Such constant region sequences to be combined with the variable regions according to the present invention are exemplary, and the constant regions can be suitably 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.

[0082] By way of example, the variable regions according to the invention can be combined with constant regions to form the heavy and light chain sequences set out below. Exemplary combinations of the heavy and light chains are shown in Table 7. Exemplary complete antibodies are also set out in Table 16. The constant regions can be suitably selected from heavy and light chain constant regions of immunoglobulins (e.g., human immunoglobulins).

[0083] Table 7. Exemplary antibodies TIFF0007625305000039.tif81170

[0084] The antibodies described in the present invention also include bispecific and bifunctional antibodies that contain one or more CDRs or one or more variable regions as described above. Bispecific or bifunctional antibodies are artificial hybrid antibodies that recognize two different targets that may or may not be related to each other. Bispecific antibodies can be produced using a variety of methods such as fusion of hybridomas or linking of Fab' fragments.

[0085] In the present invention, a "multispecific antigen-binding protein" or "multispecific antibody" is one that targets more than one antigen or epitope and contains more than one antigen-binding site. In the present invention, a "bispecific" or "bispecific" antigen-binding protein or antibody is a hybrid antigen-binding protein or antibody that has two different antigen-binding sites. Such a bispecific antibody is a type of multispecific antigen-binding protein or multispecific antibody, and can be produced by a variety of known methods, such as fusion of hybridomas or linkage of Fab' fragments.

[0086] The anti-IGF1R antibodies and antigen-binding fragments thereof from which the multispecific antibodies, e.g., bispecific antibodies, can be produced may include all of the above-mentioned anti-IGF1R antibodies and antigen-binding fragments thereof, and may be, for example, complete antibodies, and the antigen-binding fragments may be selected from the group consisting of domain antibodies, scFv, (scFv)2, scFvFc, Fab, Fab' and F(ab')2.

[0087] The antigen-binding fragments of the anti-IGF1R antibodies can be linked with or without a linker, for example, a peptide linker. The heavy and light chain portions of the antigen-binding fragments, for example, the heavy and light chain variable regions of the scFv fragments, can also be linked with or without a peptide linker. The peptide linker can be as described above.

[0088] In the bispecific antibody, the anti-IGF1R antibody or antigen-binding fragment thereof can perform the function of transmitting the second antibody or antigen-binding fragment thereof targeting a different antigen or epitope bound to the anti-IGF1R antibody or antigen-binding fragment thereof to the brain through the blood-brain barrier. The second antibody may be, but is not limited to, an antibody that exerts its effect in the brain.

[0089] The anti-IGF1R antibodies or antigen-binding fragments thereof according to the invention can share certain regions or sequences with a different second antibody. For example, the anti-IGF1R antibodies can share constant regions or share an Fc region with the antibody or antigen-binding fragment of the second antibody.

[0090] In addition, in the present invention, the structure of the bispecific antibody includes both a bivalent form bispecific antibody in which an scFv of an anti-IGF1R antibody is linked to each Fc region of two heavy chains of a complete immunoglobulin, for example, to the end of the heavy chain, either directly or via a linker, and a monovalent bispecific antibody in which an scFv of an anti-IGF1R antibody is linked to only the end of one of the two heavy chains of a complete immunoglobulin, either directly or via a linker, although monovalent bispecific antibodies are preferred.

[0091] Specifically, in one embodiment of the present invention, a monovalent clone may have a longer half-life than a bivalent clone, and 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 to only one heavy chain end via a linker. In the complete immunoglobulin form, a domain antibody that binds to IGF1R antigen containing a linker at the C-terminus is linked to one heavy chain, and the remaining heavy chain is a heterodimer formed by applying the knob-in-hole technique, which includes two different heavy chains with neither linked to the C-terminus of the constant region.

[0092] In the bispecific antibody, the second antibody that binds to the anti-IGF1R antibody or antigen-binding fragment thereof may be a human antibody, a humanized antibody, a chimeric antibody, or an isolated antibody that specifically binds to IGF1R. 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.

[0093] The following relates to anti-syn antibodies and antigen-binding fragments thereof according to the present invention. The alpha-synuclein recognized as an antigen by the antibody provided herein may be selected from mammalian alpha-synuclein such as human alpha-synuclein, monkey alpha-synuclein (e.g., Rhesus alpha-synuclein), mouse alpha-synuclein, and rat alpha-synuclein, for example, human alpha-synuclein may be, but is not limited to, alpha-synuclein (NCBI ID: NP_000336). Unless otherwise stated herein, alpha-synuclein refers to human alpha-synuclein, and the antibody or antigen-binding fragment thereof provided herein may have specific binding ability not only to human alpha-synuclein, but also to monkey (e.g., Rhesus), rat, and / or mouse alpha-synuclein.

[0094] The antibody or antigen-binding fragment thereof binds to the C-terminal site of alpha-synuclein, and specifically, human alpha-synuclein protein may be a C-terminal region of the amino acid sequence of SEQ ID NO: 559, 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 with high affinity to alpha-synuclein aggregates.

[0095] As used herein, "affinity" refers to the strength of the interaction between an antibody or antigen-binding fragment thereof and an antigen, and can be determined by the CDR sequences of the antibody or antigen-binding fragment, and / or the physicochemical properties of the antibody or antigen-binding fragment (e.g., hydrophilicity / hydrophobicity, electrostatic properties, etc.), characteristics of the antigen such as the size, shape, and / or charge of the antigen, etc. parent sum sex Methods for determining the dissociation constant are known in the art and can usually be expressed as, but are not limited to, the dissociation constant (KD).

[0096] As used herein, the term "specifically binds to alpha-synuclein protein or alpha-synuclein aggregates" means that the affinity for alpha-synuclein protein or alpha-synuclein aggregates is relatively high compared to other antigens, and for example, the affinity for alpha-synuclein aggregates, specifically amyloid fibrils, protofibrils, and oligomers, particularly amyloid fibrils, is 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.

[0097] A humanized alpha-synuclein antibody comprising a light chain and a heavy chain according to an embodiment of the present invention, e.g., Hu11F1 1( ver.1), Hu11F11(ver.2), and Hu11F11 ABL2-4 shows higher phagocytosis promoting activity than chimeric alpha-synuclein antibodies. Hu11F11 (ver.1), Hu11F11 (ver.2), Hu11F11 (ver.3), Hu11F11 (ver.4), and ABL2-4 show higher inhibitory activity against fibril binding to nerve cell membranes than chimeric alpha-synuclein antibodies, and Hu11F11 (ver.2), Hu11F11 (ver.4), and ABL2-4 show higher inhibitory activity against the spread of alpha-synuclein secreted from alpha-synuclein overexpressing cells to other nerve cells than chimeric alpha-synuclein antibodies. The binding strength against alpha-synuclein aggregates, for example, the binding strength measured in a cell-based assay, is similar to or superior to that of chimeric alpha-synuclein antibodies.

[0098] The alpha-synuclein antibody according to the present invention inhibits cell-to-cell transmission of aggregates, which are secreted outside of neurons in the nervous system of a subject, from moving to other normal cells in the extracellular space and infecting the neurons, and also has the ability to promote phagocytosis of microglia against alpha-synuclein aggregates located in the extracellular space. Like prions, alpha-synuclein aggregates spread from one cell to another, and alpha-synuclein, especially alpha-synuclein aggregates, spread throughout the brain in normal cells, causing synucleinopathies. It is well known that alpha-synuclein aggregates are toxic to brain neurons, causing neurodegeneration and neuroinflammation. Therefore, as alpha-synuclein aggregates spread to various parts of the brain, brain cell death and brain inflammatory responses increase, which leads to the brain cell death and resulting behavioral and cognitive impairments that are observed as synucleinopathies, such as Parkinson's disease, progress.

[0099] Therefore, the alpha-synuclein antibody of the present invention can inhibit the interneuronal movement of alpha-synuclein or alpha-synuclein aggregates, thereby preventing the spread of alpha-synuclein aggregates to various regions of the brain, and can also promote the phagocytosis of microglia, thereby reducing or removing alpha-synuclein aggregates themselves present outside neurons in the target nervous system, thereby reducing the level of alpha-synuclein aggregates, which are an important cause of synucleinopathies, thereby reducing brain neuronal death and brain inflammatory responses, and can be expected to have the effect of improving, alleviating or preventing the symptoms and progression of synucleinopathies, such as Parkinson's disease.

[0100] In addition, the alpha-synuclein antibody according to the present invention has excellent activity in that it can perform both of the following two functions: (i) inhibiting the interneuronal movement of alpha-synuclein or alpha-synuclein aggregates, and (ii) reducing the level of alpha-synuclein aggregates in the brain and nervous system by promoting the phagocytosis of microglia (see the results of the cell-based assay of the present application). In particular, the alpha-synuclein antibodies currently undergoing clinical trials or published in papers have one of the activities (i) and (ii), which indicates that the alpha-synuclein antibody of the present application has an advantage in preventing or treating synucleinopathies compared to known alpha-synuclein antibodies. Therefore, the alpha-synuclein antibody according to the present invention has a better effect in removing and reducing alpha-synuclein aggregates and inhibiting their pathological action, and is therefore more effective in treating synucleinopathies or symptomatic diseases related thereto (e.g., cognitive impairment, etc.).

[0101] The antibodies or antigen-binding fragments of the present application that have high affinity for alpha-synuclein aggregates can reduce alpha-synuclein aggregate formation and lower the concentration of aggregates in the brain. Also, the antibodies or antigen-binding fragments of the present application that have high affinity for alpha-synuclein aggregates can reduce alpha-synuclein aggregate formation outside the central nervous system, ultimately altering the equilibrium between alpha-synuclein forms across the blood-brain barrier, resulting in lower concentrations of aggregates in the central nervous system.

[0102] This has a great clinical advantage since sufficient efficacy can be obtained even when the antibody is administered by a more convenient method such as, but not limited to, subcutaneous injection. Furthermore, without being limited by this theory, the antibody or antigen-binding fragment of the present application can inhibit aggregate formation by removing monomers, or can remove both monomers and aggregates.

[0103] 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). . set Conversion techniques are widely known in the art.

[0104] As used herein, "antibody" refers to an intact immunoglobulin of any isotype, or an antigen-binding fragment that can compete with the intact antibody for binding to a target antigen, including, for example, chimeric, humanized, fully human, or bispecific antibodies, or antigen-binding fragments thereof. An antibody is also a type of antigen-binding protein in its own right. An intact antibody generally contains at least two full-length heavy chains and two full-length light chains, although in some cases an antibody may contain only heavy chains.

[0105] Antibodies or antigen-binding fragments thereof may be derived from just a single source or may be chimeric; chimeric antibodies contain portions derived from two different antibodies, as described in more detail below. Antibodies or antigen-binding fragments thereof may be produced by hybridomas, recombinant DNA techniques, or enzymatic or chemical cleavage of intact antibodies. Unless otherwise stated, in this application the term antibody includes antibodies containing two full-length heavy chains and two full-length light chains, as well as derivatives, variants, fragments, and mutants thereof, examples of which are described below.

[0106] In one embodiment, the antibody is a monoclonal antibody, a bispecific antibody, a minibody, a domain antibody, an antibody mimetic (or synthetic antibody), a chimeric antibody, a humanized antibody, a human antibody, an antibody fusion (or antibody conjugate), or The present application includes various forms of antibodies, including, but not limited to, fragments thereof. In one embodiment, the antibody fragment of the antibody disclosed herein may be a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an Fv fragment, a single-body fragment (scFv), a diabody, or a single-chain antibody molecule in which the heavy chain variable region and the light chain variable region are linked via a spacer.

[0107] As used herein, "light chain" includes full-length light chains and fragments thereof having sufficient variable region sequence to provide binding specificity to an antigen or epitope. A full-length light chain includes a variable region domain, VL, and a constant region domain, CL. The variable region domain of a light chain is located at the amino-terminus of the light chain polypeptide. Types of light chains include kappa and lambda chains.

[0108] As used herein, "heavy chain" includes full-length heavy chains and fragments thereof having sufficient variable region sequence to provide binding specificity for an antigen or epitope. A full-length heavy chain includes a variable region domain, VH, and three constant region domains, CH1, CH2, and CH3. The VH domain is at the amino-terminus of the heavy chain polypeptide, the CH domain is at the carboxy-terminus, and CH3 is located proximal to the carboxy-terminus. Heavy chains include isotypes of IgG (including IgG1, IgG2, IgG3, and IgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgM, and IgE.

[0109] As used herein, an "antigen-binding fragment" refers to a portion of an antibody having specific binding ability to an antigen, or a polypeptide comprising the same. For example, an antigen-binding fragment may be a portion of an antibody that contains amino acid residues that interact with an antigen (e.g., an epitope) and confer specificity and / or affinity to the antibody for the antigen, or a polypeptide comprising the same. Such antigen-binding fragments typically contain one or more "complementary determining regions" (CDRs). ( CDR ) ), in addition to one or more "framework" regions. (FR) The CDRs are amino acid sequences that contribute to the antigen-binding specificity and affinity of the antibody, and the framework regions are amino acid sequence sites that contribute to maintaining the proper conformation of these CDRs and can promote binding between the antigen-binding region and the antigen.

[0110] As used herein, an "antigen-binding fragment" of an antibody or immunoglobulin chain (heavy or light chain) includes a portion of an antibody that lacks certain amino acids compared to the full-length chain but is still capable of specifically binding to an antigen. Such fragments are biologically active in that they are capable of specifically binding to a target antigen or of competing with other antibodies or antigen-binding fragments for binding to a particular epitope. In one aspect, such fragments include at least one CDR present in a full-length light or heavy chain, and in some embodiments, include a single chain, heavy and / or light chain, or a portion thereof. Such biologically active fragments can be produced by recombinant DNA techniques or, for example, by enzymatic or chemical cleavage of an intact antibody. Immunologically functional immunoglobulin fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, domain antibodies, and single chain antibodies (e.g., scFv, scFv-Fc, etc.). They can be derived from any mammal, including, but not limited to, human, mouse, rat, camelid, or rabbit. A functional portion of an antibody, such as one or more of the CDRs disclosed herein, can be covalently linked to a second protein or small molecule compound and used as a targeted therapeutic against a specific target.

[0111] As used herein, a "Fab fragment" is composed of one light chain and one heavy chain that contains only the CH1 and variable regions. The heavy chain of a Fab molecule cannot form disulfide bonds with other heavy chain molecules.

[0112] As used herein, an "Fc" region comprises two heavy chain fragments comprising the CH2 and CH3 domains of an antibody, which are held together by two or more disulfide bonds and hydrophobic interactions of the CH3 domains.

[0113] As used herein, a "Fab' fragment" refers to a Fab fragment that additionally contains a region located between the CH1 and CH2 domains of the heavy chain, such that interchain disulfide bonds can form between the two heavy chains of two Fab' fragment molecules to form an F(ab')2 molecule.

[0114] In the present application, the term "F(ab')2 fragment" refers to a fragment that contains two light chains and two heavy chain molecules containing a variable region, a portion of the constant region between the CH1 and CH2 domains, as described above, and an interchain disulfide bond is formed between the two heavy chain molecules. Thus, the F(ab')2 fragment is composed of two Fab' fragments, and the two Fab' fragments are associated with each other by disulfide bonds between them.

[0115] As used herein, an "Fv region" is an antibody that comprises the variable regions of the heavy and light chains, but does not include the constant regions.

[0116] In the present application, a "single chain antibody" refers to a single polypeptide chain of an antigen-binding region in which the heavy and light chain variable regions are linked by a flexible linker. For example, the single chain antibody may be one or more selected from the group consisting of scFv in which the heavy chain variable region and the light chain variable region are linked in a single chain form, and scFv-Fc in which the heavy chain variable region, the light chain variable region, and Fc are linked in a single chain form. For information on single chain antibodies, see, for example, U.S. Patent No. 5,260,203.

[0117] As used herein, a "bivalent antigen-binding protein" or a "bivalent antibody" comprises two antigen-binding sites. The two antigen-binding sites in such a bivalent antibody may have the same antigen specificity or may be bispecific antibodies that each bind to a different antigen. As used herein, a "multispecific antigen-binding protein" or a "multispecific antibody" targets more than one antigen or epitope.

[0118] As used herein, a "bispecific" or "dual-specific" antigen-binding protein or antibody is a hybrid antigen-binding protein or antibody that has two different antigen-binding sites. Such bispecific antibodies are a type of multispecific antigen-binding protein or antibody and can be produced by a variety of known methods, such as fusion of hybridomas or linking of Fab' fragments.

[0119] As used herein, a "bispecific" or "dual-specific" antigen-binding protein or antibody is a hybrid antigen-binding protein or antibody having two different antigen-binding sites. Such a bispecific antibody is a type of multispecific antigen-binding protein or antibody, and can be produced by a variety of known methods, such as fusion of hybridomas or linking of Fab' or scFv fragments. See, for example, Songsivilai and Lachmann, Clin. Exp. Immunol. 1990, 79:315-321; Kostelny et al. J. Immunol. 1992, 148:1547-1553. The two 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 according to the present application, the antibody according to the present application can be in the form of a bispecific antibody that additionally includes binding to a transporter for transport across the blood-brain barrier. One method for delivering drugs across the blood-brain barrier involves the use of delivery systems endogenous to cells, such as glucose and amino acid transporters, insulin or transferrin receptor-mediated transcytosis.

[0120] As used herein, a "conjugate" refers to a chimeric molecule of an antibody or antigen-binding fragment thereof disclosed herein with another molecule, particularly a blood-brain barrier transporter or a therapeutic agent, as described below. In a conjugate, an antibody or antigen-binding fragment thereof according to the present application is physically bound to another molecule, for example, by a covalent bond or physical forces through van der Waals or hydrophobic interactions, encapsulation, embedding, or a combination thereof. In a conjugate according to one embodiment, an antibody or antigen-binding fragment thereof according to the present application can be linked through a peptide linker.

[0121] The present application also includes one or more amino acid sequences that have substantial sequence identity with one or more amino acid sequences disclosed herein. By substantial identity, the present application means that sequence variations will maintain the effects disclosed herein. In one embodiment, about 90%, 95%, or 99% identity with a heavy chain variable region disclosed. In another embodiment, about 90%, 95%, or 99% identity with a light chain variable region disclosed. For example, in the case of variants that exhibit 90%, 95%, or 99% identity with the sequences of the antibodies or antigen-binding fragments disclosed herein, any variations will occur in the framework of the variable region rather than in the CDRs.

[0122] The antibody or antigen-binding fragment thereof that specifically binds to alpha-synuclein or an aggregate thereof according to the present invention can comprise a heavy chain variable region comprising the complementarity determining sites of CDRH1, CDRH2 and CDRH3; and a light chain variable region comprising the complementarity determining sites of CDRL1, CDRL2 and CDRL3.

[0123] In one embodiment, the anti-alpha-synuclein antibody or antigen-binding fragment thereof may comprise the following CDR sequences: A heavy chain CDR1 (H-CDR1) comprising one of the amino acid sequences of SEQ ID NO: 434 and SEQ ID NO: 439; SEQ ID NO: 435-437 and 649, and A heavy chain CDR2 (H-CDR2) comprising one of the amino acid sequences of SEQ ID NOs: 440 to 441; A heavy chain CDR3 (H-CDR3) comprising one of the amino acid sequences of SEQ ID NO: 438 and SEQ ID NO: 442; A light chain CDR1 (L-CDR1) comprising one of the amino acid sequences of SEQ ID NO: 443 and SEQ ID NO: 446; A light chain CDR2 (L-CDR2) comprising one of the amino acid sequences of SEQ ID NO: 444 and SEQ ID NO: 447, and A light chain CDR3 (L-CDR3) comprising one of the amino acid sequences of SEQ ID NO:445 and SEQ ID NO:448.

[0124] The amino acid sequences of the heavy chain CDR1 to CDR3 and the light chain CDR1 to CDR3 are summarized in Tables 8 and 9:

[0125] [Table 8] Amino acid sequences of heavy chain CDR1 to CDR3 TIFF0007625305000040.tif248170TIFF0007625305000041.tif64170

[0126] [Table 9] Amino acid sequences of light chain CDR1 to CDR3 TIFF0007625305000042.tif246170

[0127] In one embodiment, the anti-alpha-synuclein antibody or antigen-binding fragment thereof has a heavy chain CDR1 (H-CDR1) comprising the amino acid sequence of SEQ ID NO: 434, a heavy chain CDR2 (H-CDR3) comprising the amino acid sequence of SEQ ID NO: 435-437, and 649 and a light chain variable region comprising a light chain CDR1 (L-CDR1) comprising the amino acid sequence of SEQ ID NO: 443, a light chain CDR2 (L-CDR2) comprising the amino acid sequence of SEQ ID NO: 444, and a light chain CDR3 (L-CDR3) comprising the amino acid sequence of SEQ ID NO: 445.

[0128] In addition, the anti-alpha-synuclein antibody or antigen-binding fragment thereof may comprise a heavy chain variable region comprising a heavy chain CDR1 (H-CDR1) comprising the amino acid sequence of SEQ ID NO: 439, a heavy chain CDR2 (H-CDR2) comprising one of the amino acid sequences of SEQ ID NO: 440 to 441, and a heavy chain CDR3 (H-CDR3) comprising the amino acid sequence of SEQ ID NO: 442; and a light chain variable region comprising a light chain CDR1 (L-CDR1) comprising the amino acid sequence of SEQ ID NO: 446, a light chain CDR2 (L-CDR2) comprising the amino acid sequence of SEQ ID NO: 447, and a light chain CDR3 (L-CDR3) comprising the amino acid sequence of SEQ ID NO: 448.

[0129] In other embodiments, the anti-alpha-synuclein antibody or antigen-binding fragment thereof is It includes a polypeptide fragment which is a heavy chain variable region framework (H-FR1) located on the N-terminal side of H-CDR1 and includes one of the amino acid sequences of SEQ ID NOs: 449 to 450 and 468 to 473.

[0130] A heavy chain variable region framework (H-FR2) located between H-CDR1 and H-CDR2, comprising a polypeptide fragment comprising one of the amino acid sequences of SEQ ID NOs: 451 to 452 and 474 to 477; A heavy chain variable region framework (H-FR3) located between H-CDR2 and H-CDR3, comprising a polypeptide fragment comprising one of the amino acid sequences of SEQ ID NOs: 453 to 464 and 478 to 483; A heavy chain variable region framework (H-FR4) located at the C-terminus of H-CDR3, comprising a polypeptide fragment comprising one of the amino acid sequences of SEQ ID NOs: 465 to 467 and 484 to 485; A polypeptide fragment comprising a light chain variable region framework (L-FR1) located on the N-terminal side of L-CDR1 and comprising one of the amino acid sequences of SEQ ID NOs: 486 to 491 and 504 to 510, A polypeptide fragment including a light chain variable region framework (L-FR2) located between L-CDR1 and L-CDR2 and including one of the amino acid sequences of SEQ ID NOs: 492 to 494 and 511 to 514, A light chain variable region framework (L-FR3) located between L-CDR2 and L-CDR3, comprising a polypeptide fragment comprising one of the amino acid sequences of SEQ ID NOs: 495 to 500 and 515 to 521, and / or It may be a light chain variable region framework (L-FR4) located at the C-terminus of L-CDR3, and may include a polypeptide fragment including one of the amino acid sequences of SEQ ID NOs: 501 to 503 and 522.

[0131] As amino acid sequences that can be used for the framework herein, chain fu Framework region array and Light chain fu Framework region The sequence is shown in Table 10. ~13 As an example,

[0132] [Table 10] Amino acid sequences of heavy chain frameworks 1-2 TIFF0007625305000043.tif248170TIFF0007625305000044.tif53170

[0133] [Table 11] Amino acid sequences of heavy chain frameworks 3-4 TIFF0007625305000045.tif250170TIFF0007625305000046.tif54170

[0134] [Table 12] Amino acid sequences of light chain frameworks 1-2 TIFF0007625305000047.tif163170

[0135] [Table 13] Amino acid sequences of light chain frameworks 3-4 TIFF0007625305000048.tif240170

[0136] In other embodiments, the anti-alpha-synuclein antibody or antigen-binding fragment thereof comprises a heavy chain CDR1 (H-CDR1) comprising one of the amino acid sequences of SEQ ID NO: 434 and SEQ ID NO: 439, a heavy chain CDR2 (H-CDR3) comprising one of the amino acid sequences of SEQ ID NO: 435-437, and 649, and It may include a heavy chain variable region including a heavy chain CDR2 (H-CDR2) including one of the amino acid sequences of SEQ ID NOs: 440 to 441, and a heavy chain CDR3 (H-CDR3) including one of the amino acid sequences of SEQ ID NOs: 438 and 442, The heavy chain variable region comprises a heavy chain variable region framework comprising one of the amino acid sequences of SEQ ID NOs: 449 to 450 and 468 to 473. area 1(H-FR1), a heavy chain variable region framework comprising one of the amino acid sequences of SEQ ID NOs: 451 to 452 and 474 to 477 area 2 (H-FR2), a heavy chain variable region framework comprising one of the amino acid sequences of SEQ ID NOs: 453 to 464 and 478 to 483 area 3 (H-FR3), and a heavy chain variable region framework comprising one of the amino acid sequences of SEQ ID NOs: 465 to 467 and SEQ ID NOs: 484 to 485. area 4 More specifically, the heavy chain variable region can include H-FR1 comprising the amino acid sequence of SEQ ID NO: 450, H-FR2 comprising the amino acid sequence of SEQ ID NO: 451, H-FR3 comprising one of the amino acid sequences of SEQ ID NOs: 462 to 464, and H-FR4 comprising the amino acid sequence of SEQ ID NO: 467.

[0137] The anti-alpha-synuclein antibody or antigen-binding fragment thereof may also include a light chain variable region including a light chain CDR1 (L-CDR1) having one of the amino acid sequences of SEQ ID NO: 443 and SEQ ID NO: 446, a light chain CDR2 (L-CDR2) having one of the amino acid sequences of SEQ ID NO: 444 and SEQ ID NO: 447, and a light chain CDR3 (L-CDR3) having one of the amino acid sequences of SEQ ID NO: 445 and SEQ ID NO: 448; The light chain variable region may additionally comprise a light chain variable region framework comprising one of the amino acid sequences of SEQ ID NOs: 486 to 491 and 504 to 510. area 1 (L-FR1), a light chain variable region framework comprising one of the amino acid sequences of SEQ ID NOs: 492 to 494 and 511 to 514. area 2 (L-FR2), a light chain variable region framework comprising one of the amino acid sequences of SEQ ID NOs: 495 to 500 and 515 to 521. area 3 (L-FR3), and a light chain variable region framework comprising one of the amino acid sequences of SEQ ID NOs: 501 to 503 and SEQ ID NO: 522 area 4More specifically, the light chain variable region can include an L-FR1 comprising the amino acid sequence of SEQ ID NO: 491, an L-FR2 comprising the amino acid sequence of SEQ ID NO: 492, an L-FR3 comprising the amino acid sequence of SEQ ID NO: 500, and an L-FR4 comprising the amino acid sequence of SEQ ID NO: 501.

[0138] According to a specific embodiment of the present invention, the anti-alpha-synuclein antibody or antigen-binding fragment thereof comprises a heavy chain CDR1 (H-CDR1) comprising the amino acid sequence of SEQ ID NO: 434, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 435 to 437, and 649 and a heavy chain variable region comprising a heavy chain CDR2 (H-CDR2) comprising one of the amino acid sequences of SEQ ID NO: 438, and a heavy chain CDR3 (H-CDR3) comprising the amino acid sequence of SEQ ID NO: 438; and a light chain variable region comprising a light chain CDR1 (L-CDR1) comprising the amino acid sequence of SEQ ID NO: 443, a light chain CDR2 (L-CDR2) comprising the amino acid sequence of SEQ ID NO: 444, and a light chain CDR3 (L-CDR3) comprising the amino acid sequence of SEQ ID NO: 445, The heavy chain variable region is SEQ ID NO: 449 、 450 , and 471 A heavy chain variable region framework comprising one of the amino acid sequences area 1 (H-FR1), SEQ ID NO:451 、 452 , and 475 A heavy chain variable region framework comprising one of the amino acid sequences area 2 (H-FR2), a heavy chain variable region framework comprising one of the amino acid sequences of SEQ ID NOs: 453 to 464 area 3 (H-FR3), and a heavy chain variable region framework comprising one of the amino acid sequences of SEQ ID NOs: 465 to 467. area 4 (H-FR4), The light chain variable region comprises a light chain variable region framework comprising one of the amino acid sequences of SEQ ID NOs: 486 to 491. area 1 (L-FR1), a light chain variable region framework comprising one of the amino acid sequences of SEQ ID NOs: 492 to 494 area 2 (L-FR2), a light chain variable region framework comprising one of the amino acid sequences of SEQ ID NOs: 495 to 500 area 3(L-FR3), and a light chain variable region framework comprising one of the amino acid sequences of SEQ ID NOs: 501 to 503. area 4 (L-FR4).

[0139] Preferably, an anti-alpha-synuclein antibody or antigen-binding fragment thereof according to a specific embodiment of the present invention comprises a heavy chain variable region comprising heavy chain CDR1 to CDR3 comprising the amino acid sequences of SEQ ID NO: 434, SEQ ID NO: 435, and SEQ ID NO: 438, and a light chain variable region comprising light chain CDR1 to CDR3 comprising the amino acid sequences of SEQ ID NO: 443, SEQ ID NO: 444, and SEQ ID NO: 445, wherein the heavy chain variable region may comprise H-FR1 comprising the amino acid sequence of SEQ ID NO: 450, H-FR2 comprising the amino acid sequence of SEQ ID NO: 451, H-FR3 comprising one of the amino acid sequences of SEQ ID NO: 462 to 464, and H-FR4 comprising the amino acid sequence of SEQ ID NO: 467, and the light chain variable region may comprise L-FR1 comprising the amino acid sequence of SEQ ID NO: 491, L-FR2 comprising the amino acid sequence of SEQ ID NO: 492, L-FR3 comprising the amino acid sequence of SEQ ID NO: 500, and L-FR4 comprising the amino acid sequence of SEQ ID NO: 501.

[0140] According to a specific embodiment of the present invention, the anti-alpha-synuclein antibody or antigen-binding fragment thereof may include a heavy chain variable region including a heavy chain CDR1 (H-CDR1) having the amino acid sequence of SEQ ID NO: 439, a heavy chain CDR2 (H-CDR2) having one of the amino acid sequences of SEQ ID NO: 440 to 441, and a heavy chain CDR3 (H-CDR3) having the amino acid sequence of SEQ ID NO: 442; and a light chain variable region including a light chain CDR1 (L-CDR1) having the amino acid sequence of SEQ ID NO: 446, a light chain CDR2 (L-CDR2) having the amino acid sequence of SEQ ID NO: 447, and a light chain CDR3 (L-CDR3) having the amino acid sequence of SEQ ID NO: 448, The heavy chain variable region comprises a heavy chain variable region framework comprising one of the amino acid sequences of SEQ ID NOs: 468 to 473. area 1 (H-FR1), a heavy chain variable region framework comprising one of the amino acid sequences of SEQ ID NOs: 474 to 477 area 2(H-FR2), a heavy chain variable region framework comprising one of the amino acid sequences of SEQ ID NOs: 478 to 483 area 3 (H-FR3), and a heavy chain variable region framework comprising one of the amino acid sequences of SEQ ID NOs: 484 to 485. area 4 (H-FR4), The light chain variable region comprises a light chain variable region framework comprising one of the amino acid sequences of SEQ ID NOs: 504 to 510. area 1 (L-FR1), a light chain variable region framework comprising one of the amino acid sequences of SEQ ID NOs: 511 to 514. area 2 (L-FR2), a light chain variable region framework comprising one of the amino acid sequences of SEQ ID NOs: 515 to 521 area 3 (L-FR3), and a light chain variable region framework comprising the amino acid sequence of SEQ ID NO:522 area 4 (L-FR4).

[0141] In full-length light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids in length, and the heavy chains also have a "J" region of about 10 or more amino acids in length. length The "D" region of the antibody may be found in, 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.

[0142] The variable regions of immunoglobulin chains are generally identical in overall structure and contain relatively conserved framework regions (FR) connected by three hypervariable regions called "complementarity determining sites or regions or domains" or CDRs. The CDRs of the variable regions from each chain of a heavy / light chain pair are typically aligned by the framework regions to form a structure that specifically binds to a particular epitope of a target protein (alpha-synuclein). These elements of naturally occurring light and heavy chain variable regions are typically included in the following order from N-terminus to C-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The position of each of these corresponding amino acid sequences in the variable region follows the Kabat numbering system (Kabat Sequences of Proteins of Immunological Interest (1987 and 1991, NIH, Bethesda, MD) or as described in Chothia & Lesk, 1987, J. Mol. Biol. 196:901-917; Chothia et al., 1989, Nature 342:878-883).

[0143] Various heavy and light chain variable regions disclosed herein are disclosed in Tables 14 and 15. Each of these variable regions can be combined with the heavy and light chain constant regions to form the respective heavy and light chains of a complete antibody. The respective heavy and light chain sequences thus generated can also be combined to form a complete antibody structure. For example, an anti-alpha-synuclein antibody or antigen-binding fragment thereof according to the present invention can include a heavy chain variable region of one of the amino acid sequences of SEQ ID NOs: 523-534 or SEQ ID NOs: 535-541 and a light chain variable region of one of the amino acid sequences of SEQ ID NOs: 542-548 or SEQ ID NOs: 549-556, more specifically, a heavy chain variable region of one of the amino acid sequences of SEQ ID NOs: 523-534 and a light chain variable region of one of the amino acid sequences of SEQ ID NOs: 542-548, or a heavy chain variable region of one of the amino acid sequences of SEQ ID NOs: 535-541 and a light chain variable region of one of the amino acid sequences of SEQ ID NOs: 549-556.

[0144] In a specific example of the present invention, the anti-alpha-synuclein antibody or antigen-binding fragment thereof may include a heavy chain variable region of one of the amino acid sequences of SEQ ID NOs: 531 to 534 and a light chain variable region of the amino acid sequence of SEQ ID NO: 548 (e.g., Hu11F1 1( ver.1), Hu11F1 1( ver.2), Hu11F1 1( ver.3), Hu11F1 1( ver.4), or may include a heavy chain variable region having the amino acid sequence of SEQ ID NO:525 and a light chain variable region having the amino acid sequence of SEQ ID NO:546 (e.g., Hu11F1 1( ABL2-4) antibody).

[0145] The amino acid sequences of the heavy and light chain variable regions of an antibody or antigen-binding fragment according to one embodiment are illustrated in Tables 14 and 15.

[0146] Table 14: Heavy chain variable region TIFF0007625305000049.tif253170TIFF0007625305000050.tif255170

[0147] Table 15. Light chain variable region TIFF0007625305000051.tif250170TIFF0007625305000052.tif88170

[0148] Also set forth in Table 9 are exemplary antibodies, including combinations of heavy and light chain variable regions and constant regions of an antibody or antigen-binding fragment according to one embodiment.

[0149] Table 16. Exemplary anti-a-syn antibodies of the invention TIFF0007625305000053.tif251170TIFF0007625305000054.tif255170TIFF0007625305000055.tif255170TIFF0007625305000056.tif14170

[0150] The heavy and light chain variable regions disclosed herein can be combined with heavy and light chain constant regions, respectively, to form the heavy and light chains of a complete antibody. The respective heavy and light chains thus generated can be appropriately combined to form heavy-light chain combinations, which can form multimers (e.g., dimers in the case of IgG type antibodies) to form a complete antibody structure.

[0151] The constant region can be appropriately selected from immunoglobulin (e.g., human immunoglobulin) heavy chain constant region and light chain constant region. For example, the heavy chain constant region can be an IgG1 heavy chain constant region, an IgG3 heavy chain constant region, or an IgG4 heavy chain constant region, and the light chain constant region can be a kappa constant region or a lambda light chain constant region, but is not limited thereto. For antibody stability, manufacturability, antigen affinity, and / or other desired characteristics, other types of constant regions or modified constant regions can be appropriately selected and used, which is clearly known to those skilled in the art.

[0152] In other embodiments, the heavy chain variable regions and light chain variable regions disclosed in Tables 14 and 15 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.

[0153] 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.

[0154] In one example, the anti-alpha-synuclein antibody provided herein may be a monoclonal or polyclonal antibody. In another example, the anti-alpha-synuclein antibody may be a human antibody, a humanized antibody, a chimeric antibody, or an animal-derived antibody. In another example, the anti-alpha-synuclein antibody may be recombinantly or chemically synthesized.

[0155] In one embodiment, the antigen-binding fragment or antibody fragment of the antibody disclosed herein may be one or more selected from the group consisting of a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an Fv fragment, a minibody, a diabody, and a single chain antibody molecule such as an scFv.

[0156] In other embodiments, the antibodies provided herein are human or humanized and may be of various isotypes (e.g., IgA, IgG1, IgG2, IgG3, IgG4, IgE, or IgD), in particular IgG1-, IgG2-, IgG3- or IgG4-types, e.g., IgG1- or IgG2-types.

[0157] In yet other embodiments, the anti-alpha-synuclein antibody may consist only of the light chain or heavy chain described above, hi other embodiments, the anti-alpha-synuclein antibody may consist only of the light chain variable region or the heavy chain variable region.

[0158] One of ordinary skill in the art will understand that, where an antibody comprises one or more CDRs disclosed herein, each of the disclosed CDRs can be independently selected and combined with one another. Thus, antibodies can be generated having one, two, three, four, five, or six independently selected CDRs. One of ordinary skill in the art will also understand that when CDRs are selected for combination, the same type of CDR will not be used repeatedly, e.g., antibodies will not generally be made containing two CDRH2 regions.

[0159] In one embodiment, the antibody may be a monoclonal antibody or a polyclonal antibody. The antibodies disclosed herein include monoclonal antibodies that bind to alpha-synuclein. Monoclonal antibodies can be produced using any technique known in the art. For example, they can be produced by immortalizing spleen cells harvested from immunized transgenic animals. Monoclonal antibodies secreted by hybridoma cell lines can be purified using techniques known in the art.

[0160] In other embodiments, the antibody may be an animal-derived antibody (such as a mouse antibody), a chimeric antibody (such as a mouse-human chimeric antibody), a humanized antibody, or a human antibody. Antibodies can also be modified in a variety of ways for a variety of purposes. Chimeric and humanized antibodies are further provided. Chimeric antibodies are antibodies in which polypeptide fragments from different antibodies are covalently linked to form an immunologically functional light chain, heavy chain, or fragments thereof.

[0161] In such monoclonal antibodies, certain amino acid residues that typically constitute the non-antigen recognizing portions of the antibody are modified to become homologous to the corresponding residues of the corresponding isotype of a human antibody. Humanization can be accomplished, for example, by replacing at least a portion of a rodent variable region with the corresponding region of a human antibody using a variety of known methods (U.S. Patent Nos. 5,585,089 and 5,693,762; Jones et al., 1986, Nature 321:522-525; Riechmann et al., 1988, Nature 332:323-27; Verhoeyen et al., 1988, Science 239:1534-1536).

[0162] Fully human antibodies can be produced by immunizing transgenic animals (usually mice) that lack endogenous immunoglobulin production and are capable of producing human antibodies. Fully human antibodies can also be derived from phage-display libraries. The use of fully human antibodies can minimize immunogenic and allergic responses that can be elicited by administering mouse or mouse-derived mAbs to humans.

[0163] In one embodiment, the human alpha-synuclein antibody of the present application is selected by a phage display method. A recombinant method is used to convert the alpha-synuclein-specific monoclonal phage antibody selected by phage screening into a full IgG form. The sequence of each monoclonal phage antibody is obtained for recombinant production of an anti-alpha-synuclein antibody. In the obtained sequence, the heavy chain variable region sequence is grafted to the heavy chain constant region sequence, and the light chain variable region sequence is grafted to the light chain constant region sequence, and the amino acid sequence is changed to a nucleic acid sequence by a codon optimization method. The obtained nucleic acid sequence is cloned into a vector for animal cell culture, and then the vector is used to transform a host cell for protein production, such as a CHO cell, and the cell is cultured. In order to purify the antibody contained in the culture solution, the recombinant antibody is separated and purified using a purification technique such as affinity chromatography.

[0164] In other embodiments, the antibody may have the typical structure of an antibody found in nature or may have a modified structure.

[0165] An antibody having the above-mentioned typical structure may have a multimeric structure that includes a structural unit that includes two different polypeptide chains (i.e., a heavy chain and a light chain). The two different polypeptide chains may include a full-length light chain (about 25 kDa) and a full-length heavy chain (about 50-70 kDa). Each chain exhibits a characteristic folding pattern and is composed of several immunoglobulin domains, each of which consists of about 90-110 amino acids. Such domains are the basic units that make up an antibody polypeptide. The amino-terminal portion of each chain typically includes a portion called a variable region or V region, which is the portion that recognizes the antigen. The carboxy-terminal portion is more evolutionarily conserved than the amino-terminus and includes a portion called a constant region or C region. Human light chains are generally classified as kappa (κ) and lambda (λ) light chains, each of which includes one variable region and one constant region. Heavy chains are generally classified as mu (μ), delta (δ), gamma (γ), alpha (α) or epsilon (ε) chains, which are defined as IgM, IgD, IgG, IgA and IgE isotypes, respectively. IgG has multiple subtypes, including but not limited to IgG1, IgG2, IgG3 and IgG4. The heavy chain constant region typically contains one or more domains that exhibit effector function. The number of heavy chain constant region domains varies with isotype. An IgG heavy chain, for example, contains three C region domains known as CH1, CH2 and CH3, respectively. The antibodies disclosed herein may be of any one of such isotypes and subtypes. In one embodiment, the anti-alpha-synuclein antibody may be of an IgG isotype, for example, an IgG1, IgG2 or IgG4 subtype.

[0166] It is shown that the alpha-synuclein antibody of the present application specifically recognizes alpha-synuclein aggregates with high affinity and can be useful for the diagnosis or detection of such diseases. Furthermore, it is shown that the alpha-synuclein antibody of the present application that specifically recognizes alpha-synuclein aggregates can be effectively used for the treatment of synucleinopathies, particularly Parkinson's disease, by inhibiting the formation of alpha-synuclein aggregates or degrading the aggregates and inhibiting the transmission of intercellular aggregates.

[0167] Bispecific antibodies according to the invention Example is as shown in Table 17 below. [Table 17] TIFF0007625305000057.tif240170TIFF0007625305000058.tif247170TIFF0007625305000059.tif247170 TIFF0007625305000060.tif242170TIFF0007625305000061.tif245170TIFF0007625305000062.tif246170 TIFF0007625305000063.tif244170TIFF0007625305000064.tif240170TIFF0007625305000065.tif242170 TIFF0007625305000066.tif244170TIFF0007625305000067.tif243170TIFF0007625305000068.tif148170 Effect of the Invention

[0168] The antibody prepared in one embodiment of the present invention specifically binds to IGF1R with an affinity optimized for brain endothelial transcytosis, and can be usefully used to deliver therapeutic antibodies for degenerative brain diseases and brain cancer, which have limited therapeutic efficacy due to their low blood-brain barrier penetration ability. In particular, the monoclonal antibody disclosed in the present invention binds to ligands IGF-1, IGF-2, and insulin or its homologuesIt has the effect of not affecting binding to IGF1R and not inhibiting signal transmission through the IGF1R receptor, making it useful in relation to crossing the blood-brain barrier.

[0169] The antibodies disclosed in the present application can effectively remove or promote the degradation of alpha-synuclein aggregates and inhibit the intercellular transmission of alpha-synuclein, and can be useful in treating diseases associated with the accumulation of alpha-synuclein aggregates. [Brief description of the drawings]

[0170] [Figure 1] 1 shows the results of dot blot assays to determine whether an anti-a-syn antibody prepared in one embodiment of the present invention specifically recognizes aggregated native alpha-synuclein. [Diagram 2] 1 shows the results of ELISA measurement of the affinity of an anti-a-syn antibody produced in one example of the present invention. [Figure 3a] 1 shows the results of BIAcore analysis of the preferential binding specificity and affinity of an anti-a-syn antibody produced in one embodiment of the present invention for alpha-synuclein aggregates. [Figure 3b] The results of Figure 3a are presented in a table. [Figure 4a] 1 shows the results of Octet analysis of the preferential binding specificity of an anti-a-syn antibody produced in one embodiment of the present invention to alpha-synuclein aggregates. [Figure 4b] The results of Figure 4a are presented in a table. [Figure 5a] These are the results of measuring whether the anti-a-syn antibodies 3A9 and 11F11 according to an embodiment of the present invention can specifically recognize Lewy bodies and Lewy neurites in human brain tissue, respectively. The antibodies according to the present application bind to Lewy bodies (arrows) and Lewy neurites (thread-like structures at the lower left). [Figure 5b]These are the results of measuring whether the anti-a-syn antibodies 3A9 and 11F11 according to an embodiment of the present invention can specifically recognize Lewy bodies and Lewy neurites in human brain tissue, respectively. The antibodies according to the present application bind to Lewy bodies (arrows) and Lewy neurites (thread-like structures at the lower left). [Figure 6] FIG. 2 is a schematic diagram showing the epitope mapping results of an anti-a-syn antibody according to an embodiment of the present invention, which reveals that the antibody according to the present application mostly binds to the C-terminal site. [Figure 7] 1 shows the results of measuring the affinity of an anti-a-syn chimeric antibody produced in one example of the present invention to humanized 11F11 antibody by ELISA. [Figure 8] 1 shows the results of a BIAcore analysis of the preferential binding specificity and affinity of an anti-a-syn chimeric antibody and a humanized 11F11 antibody produced in one embodiment of the present invention to alpha-synuclein aggregates. [Figure 9a] The results of ELISA and dot blot analysis show that the a-syn chimeric antibody produced in one embodiment of the present invention specifically binds to alpha-synuclein that is not beta-synuclein, gamma-synuclein, or amyloid beta (amyloid beta 1-42) or tau-derived aggregates, particularly alpha-synuclein aggregates. [Figure 9b] The results of ELISA and dot blot analysis show that the a-syn chimeric antibody produced in one embodiment of the present invention specifically binds to alpha-synuclein that is not beta-synuclein, gamma-synuclein, or amyloid beta (amyloid beta 1-42) or tau-derived aggregates, particularly alpha-synuclein aggregates. [Figure 10]The results of ELISA and dot blot analysis show that the a-syn chimeric antibody produced in one embodiment of the present invention specifically binds to alpha-synuclein that is not beta-synuclein, gamma-synuclein, or amyloid beta (amyloid beta 1-42) or tau-derived aggregates, particularly alpha-synuclein aggregates. [Figure 11a] 1 shows binding data for an anti-IGF1R antibody produced in one embodiment of the present application to IGF1R protein. [Figure 11b] 1 shows binding data for an anti-IGF1R antibody produced in one embodiment of the present application to IGF1R protein. [Figure 12a] 1 shows binding data for an anti-IGF1R antibody produced in one embodiment of the present application to an IGF1R-expressing cell line. [Figure 12b] 1 shows binding data for an anti-IGF1R antibody produced in one embodiment of the present application to an IGF1R-expressing cell line. [Figure 12c] 1 shows binding data for an anti-IGF1R antibody produced in one embodiment of the present application to an IGF1R-expressing cell line. [Figure 13a] 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 13b] 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 13c] 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 14a] The data show that the anti-IGF1R antibody produced in one embodiment of the present application does not affect IGF1 or insulin-induced IGF1R signaling. [Figure 14b] The data show that the anti-IGF1R antibody produced in one embodiment of the present application does not affect IGF1 or insulin-induced IGF1R signaling. [Figure 14c] The data show that the anti-IGF1R antibody produced in one embodiment of the present application does not affect IGF1 or insulin-induced IGF1R signaling. [Figure 15a] The data show that the anti-IGF1R antibody produced in one example of the present application does not have ADCC. [Figure 15b] 1 shows data demonstrating that an anti-IGF1R antibody produced in one embodiment of the present invention does not affect IGF1R levels in the brain even after repeated administration. [Figure 15c-1] 1 is a micrograph showing that an anti-IGF1R antibody produced as an example of the present invention does not bind to normal neurons in the brain, but specifically binds to brain endothelial cells. [Figure 15c-2] 1 is a micrograph showing that an anti-IGF1R antibody produced as an example of the present invention does not bind to normal neurons in the brain, but specifically binds to brain endothelial cells. [Figure 16a] The data show that a bispecific antibody consisting of an anti-IGF1R antibody and a therapeutic antibody crosses the in vitro BBB system more easily than a monospecific antibody consisting of only a therapeutic antibody. [Figure 16b] The data show that a bispecific antibody consisting of an anti-IGF1R antibody and a therapeutic antibody crosses the in vitro BBB system more easily than a monospecific antibody consisting of only a therapeutic antibody. [Figure 16c] The data show that a bispecific antibody consisting of an anti-IGF1R antibody and a therapeutic antibody crosses the in vitro BBB system more easily than a monospecific antibody consisting of only a therapeutic antibody. [Figure 17a] The data show that bispecific antibodies consisting of anti-IGF1R antibody and therapeutic antibody and anti-IGF1R antibody are distributed more extensively in the brain and CSF when administered once to rats, compared to a monospecific antibody consisting of only therapeutic antibody. [Figure 17b]The data show that bispecific antibodies consisting of anti-IGF1R antibody and therapeutic antibody and anti-IGF1R antibody are distributed more extensively in the brain and CSF when administered once to rats, compared to a monospecific antibody consisting of only therapeutic antibody. [Figure 17c] The data show that bispecific antibodies consisting of anti-IGF1R antibody and therapeutic antibody and anti-IGF1R antibody are distributed more extensively in the brain and CSF when administered once to rats, compared to a monospecific antibody consisting of only therapeutic antibody. [Figure 17d-1] The data show that bispecific antibodies consisting of anti-IGF1R antibody and therapeutic antibody and anti-IGF1R antibody are distributed more extensively in the brain and CSF when administered once to rats, compared to a monospecific antibody consisting of only therapeutic antibody. [Figure 17d-2] The data show that bispecific antibodies consisting of anti-IGF1R antibody and therapeutic antibody and anti-IGF1R antibody are distributed more extensively in the brain and CSF when administered once to rats, compared to a monospecific antibody consisting of only therapeutic antibody. [Figure 17e] The data show that bispecific antibodies consisting of anti-IGF1R antibody and therapeutic antibody and anti-IGF1R antibody are distributed more extensively in the brain and CSF when administered once to rats, compared to a monospecific antibody consisting of only therapeutic antibody. [Figure 18a] 1 shows the results of confirming the site where deamidation occurred in an anti-IGF1R antibody prepared according to one embodiment of the present invention. [Figure 18b] These are mutants produced by substituting specific amino acids to prevent deamidation. LLIYANSN: SEQ ID NO: 651. GAWDDSLNG: SEQ ID NO: 652. AISYDNGNT: SEQ ID NO: 653. [Figure 19] This shows the results of epitope mapping of anti-IGF1R antibodies. [Figure 20a] 1 shows the results of ELISA performed to measure the binding affinity of a bispecific antibody produced in one example of the present application to each antigen. [Figure 20b] 1 shows the results of ELISA performed to measure the binding affinity of a bispecific antibody produced in one example of the present application to each antigen. [Figure 20c] These are the results of ELISA performed to compare the binding ability of chimeric antibodies and humanized antibodies to each antigen. [Figure 20d] These are the results of ELISA performed to compare the binding ability of chimeric antibodies and humanized antibodies to each antigen. [Figure 20e] 1 shows the results of evaluating the activity of a bispecific antibody produced in one example of the present application against the phagocytosis of microglia. [Figure 21a] 1 shows the results of evaluating the efficacy of a bispecific antibody prepared in one embodiment of the present application in a mouse animal model in comparison with a single antibody. [Figure 21b] 1 shows the results of evaluating the efficacy of a bispecific antibody prepared in one embodiment of the present application in a mouse animal model in comparison with a single antibody. [Figure 21c] 1 shows the results of evaluating the efficacy of a bispecific antibody prepared in one embodiment of the present application in a mouse animal model in comparison with a single antibody. [Figure 21d] 1 shows the results of evaluating the efficacy of a bispecific antibody prepared in one embodiment of the present application in a mouse animal model in comparison with a single antibody. [Figure 21e] 1 shows the results of evaluating the efficacy of a bispecific antibody prepared in one embodiment of the present application in a mouse animal model in comparison with a single antibody. [Figure 22a] The results show that Fc engineering was performed on a bispecific antibody produced in one example of the present application, thereby increasing its half-life and improving its ability to cross the BBB. [Figure 22b] The results show that Fc engineering was performed on a bispecific antibody produced in one example of the present application, thereby increasing its half-life and improving its ability to cross the BBB. [Figure 22c] The results show that Fc engineering was performed on a bispecific antibody produced in one example of the present application, thereby increasing its half-life and improving its ability to cross the BBB. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS EXAMPLES

[0171] Example 1. Production of mouse alpha-synuclein antibodies 1-1: Immunization and hybridoma production As the antigen, the full-length (140 residues) or C-terminal 21-residue truncated alpha-synuclein monomer was placed in a thermomixer C at 37°C, shaken at 1050 rpm for 14 days to aggregate, and then sonicated. L Alpha-synuclein fibrils of 140 and 119 residues each were mixed with adjuvant at a 1:1 (vol:vol) ratio and thoroughly mixed.

[0172] Amino acid sequence (SEQ ID NO:559) of Homo sapiens alpha-synuclein : MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTKEGVVHGVATVAEKTKEQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFVKKDQLGKNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEA Next, 200μL of the mixture prepared above was subcutaneously injected into 5-7 week old BALB / c female mice, and two weeks later, 200μL of the mixture prepared in the same manner was further subcutaneously injected for boosting. One week after boosting, blood was collected and immunization titeration was performed using ELISA method with the administered antigen. Next, for the third boosting, only the antigen was subcutaneously injected.

[0173] Next, the spleens of the mice that had been immunized as described above were removed, and cells were obtained from them. The spleen cells were then suspended in Hybridoma-SFM medium (Thermo Fisher Scientific, USA) supplemented with 10% FBS. To prepare hybridomas, mouse myeloma cells SP2 / 0-Ag14 and spleen cells were mixed in serum-free Hybridoma-SFM medium, and the medium was removed by centrifugation. PEG was then added to the cell pellet, and the mixture was incubated at 37°C for 1 minute to induce cell fusion.

[0174] 1-2: Single-cell cloning and antibody purification Two weeks after fusion, fusion with mouse B cells producing antibodies was confirmed using an ELISA method with antigen administered to the mouse using cell culture medium. Next, single cell cloning was performed using the hybridomas to select a total of 16 hybridomas producing monoclonal antibodies. The 9B11 clone (IgG1 kappa, respectively) was obtained using full-length (140 residues) alpha-synuclein aggregates as antigens, and the 3A9 and 11F11 clones (IgG2b kappa, IgG2b kappa, respectively) were obtained using alpha-synuclein aggregates truncated at the C-terminus 21 residues as antigens.

[0175] To purify the antibodies, each hybridoma was cultured in RPMI1640 medium containing 10% FBS, and the culture medium was replaced with serum-free SFM medium for antibody production, and then cultured for about 4 days. The cell culture supernatant was separated and centrifuged, and then filtered through a 0.22 μm filter, after which the IgG1 type was purified using a protein G column and the remaining antibodies were purified using a protein A column.

[0176] 1-3: Variable region sequence determination The variable region and CDR sequences were determined by referring to the paper by Ahn et al., Mol.Cells 2004, 18(2):237-241. After culturing the hybridoma, the cells were separated by centrifugation. The isolated hybridoma was treated with Trizol to separate RNA, which was used as a template to synthesize cDNA, and the variable region and CDR sequences were confirmed by base sequence analysis. Thus, the antibodies 3A9, 9B11, and 11F11 were obtained.

[0177] Example 2. Preparation of anti-alpha-synuclein (chimeric) antibodies 2-1: Antibody cloning and expression Using the antibody nucleotide sequences of the heavy and light chain variable regions obtained after humanization, gblocks (m.biotech), which are short nucleotide fragments, were synthesized and used to clone into an animal cell culture vector (pcDNA3.4). The gblocks were synthesized containing approximately 20bp of overlapping nucleotides before and after the variable regions, and the portion of the pcDNA3.4 vector excluding the variable regions was prepared by amplifying it by PCR and cloned using the Gibson assembly method.

[0178] 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. TM (Gibco, Cat: A29127) cells were cultured in ExpiCHO TM After adjusting the concentration to 3×10E6~4×10E6 viable cells / mL in expression medium (Gibco, Cat: A29100-01), the cells were cultured for 1 day at 8% CO2, 37℃, and 120 rpm. On the day of DNA transfection, the cells that had grown to 7×10E6~10×10E6 viable cells / mL, with a survival rate of 95% or more, were diluted with fresh medium to 6×10 6 The solution was prepared by dilution to viable cells / mL.

[0179] ExpiFectamine for transfection into prepared mother cells TM CHO transfection kit (Gibco, Cat: A29129) was used to transfect the cells with ExpiFectamine. TM CHO & plasmid DNA complex was prepared. TM SFM® (Gibco, Cat: 12309019) medium was dispensed and titrated concentrations of DNA and ExpiFectamine were added. TM After inoculating each CHO reagent, mix them together and leave them at room temperature for 5 minutes, then inoculate them onto the mother cells, inject the transfection, and start culturing. TM The enhancer and feed included in the CHO transfection kit were inoculated into the transfected cells, and after 5 days, the feed was additionally inoculated. The cells were then cultured for 10 days under conditions of 8% CO2, 37°C, and 120 rpm to complete the production.

[0180] To obtain the culture medium after the production was completed, the culture medium was transferred to a centrifuge bottle and centrifuged at 4°C and 6500 rpm for 30 minutes, and then filtered through a 0.2 μm filter to remove the suspended matter and obtain the culture medium, which was then subjected to a purification process.

[0181] 2-2: Antibody purification and sequence confirmation The culture fluid was purified using HiTrap MabSelectSure (GE Healthcare, 11-0034-94). After equilibration using equilibration buffer (50 mM Tris-HCl pH 7.2, 100 mM NaCl), the collected culture fluid was loaded onto the column. After completion of loading, intermediate washing with 50 mM sodium citrate pH 5.0 was performed, followed by elution with 50 mM sodium citrate pH 3.4. The eluate was neutralized to pH 6.0 by adding 1 M Tris-HCl pH 9.0. The eluate was then buffer exchanged and concentrated with PBS (phosphate buffered saline, pH 7.4) and stored at 4°C until use.

[0182] If additional purification is required, HiLoad 26 / 600 S The primary purified product was passed through an Uperdex 200 column using 1X PBS as a buffer, and secondary purification was performed based on the size of the eluted sample. The amino acid sequence of the purified antibody was analyzed by mass spectrometry, and it was confirmed to be identical to the variable region of a mouse-derived monoclonal antibody.

[0183] The variable regions of the 3A9, 9B11, and 11F11 antibodies confirmed by the above method were replaced with the backbone variable region of the human IgG1 isotype to prepare chimeric human IgG1 antibodies. Among the obtained chimeric antibodies, the Ch11F11 antibody is an IgG antibody and is represented by SEQ ID NO: 523 The heavy chain variable region sequence (ch11F11-VH) and SEQ ID NO: 542 The Ch3A9 antibody is an IgG antibody having a light chain variable region sequence (ch11F11-VL) of SEQ ID NO: 535 The heavy chain variable region sequence ( ch3A9 -VH) and sequence number 549 The light chain variable region sequence of ( ch3A9 -VL) combination.

[0184] Example 3: Production of humanized antibodies 3-1: Library phage preparation A mini-library was constructed in which a human framework was linked to the CDR1, CDR2, and CDR3 residues of a chimeric antibody, and mouse or human sequences were introduced into each CDR residue. Competent cells of the library were incubated with chloramphenicol (Sigma, C0857) at 34 μg / m LThe 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 2% glucose (Sigma, G5400) and 5 mM MgCl2 (Sigma, M2393), and then cultured at 37°C for about 3 hours until the OD600 value reached 0.5 to 0.7. After that, the cells were infected with helper phage and then diluted with chloramphenicol at 34 μg / m L , 5 mM MgCl2, kanamycin (Sigma, K1876) 70 μg / m L The cells were cultured in 2X YT medium supplemented with 1 mM IPTG (ELPISBIO, IPTG025) at 30°C for 16 hours to induce phage packaging. The culture was then centrifuged at 4500 rpm for 15 minutes at 4°C, and 4% PEG6000 (Fluka, 81253) and 3% NaCl (Sigma, S7653) were added to the supernatant, which was thoroughly dissolved and then reacted on ice for 1 hour. The cells were centrifuged again at 8000 rpm for 20 minutes at 4°C, and the pellet was suspended in PBS and centrifuged again at 12000 rpm for 10 minutes at 4°C to obtain the supernatant containing the library phage, which was then placed in a new tube and stored at 4°C until use.

[0185] 3-2: Phage display panning Specifically, 10 μg / m L Recombinant alpha-synuclein aggregates were added to PBS at a concentration of 100 mM, and the proteins were allowed to adsorb onto the surface of the test tube at 4°C overnight. A 3% solution of bovine serum albumin (BSA) was then added to the test tube to protect the surface from alpha-synuclein aggregates. After emptying the test tube, 10 mM alpha-synuclein dispersed in a 3% BSA solution was added to the test tube. 12CFU of the antibody phage library was placed in an immunoassay tube adsorbing alpha-synuclein monomer protein and reacted at room temperature for 1 hour (negative selection). Phages that did not bind to alpha-synuclein monomer were collected and bound to the immunoassay tube adsorbing alpha-synuclein aggregates at room temperature for 2 hours. Non-specifically bound phages were then washed off 5 to 30 times with PBS-T (0.05% Tween 20) solution, and the remaining antigen-specific phage antibodies were collected using 100 mM triethylamine solution. The collected phages were neutralized with 1M Tris buffer (pH 7.4) and then infected with ER2537 E. coli at 37°C for 1 hour, and the infected E. coli was smeared on 2X YT agar medium containing carbenicillin and cultured overnight at 37°C. The next day, the cultured E. coli was suspended in 4 ml of 2X YT carbenicillin culture medium, 15% glycerol was added, and a portion was stored at -80°C, while the remaining portion was used to produce phages for the next round of panning. This process was repeated for a total of three rounds to amplify antigen-specific antibodies. As the panning rounds progressed, the number of washings with PBS-T was increased to amplify and concentrate antigen-specific phages.

[0186] 3-3: Single clone phage antibody selection (single clone screening) In order to select monoclonal antibodies that specifically bind to alpha-synuclein aggregates from the phage pool obtained by the panning, the following experiment was carried out.

[0187] 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. The single clones were then plated at 400 μl per well. L 96 deep-well plates containing 2X YT-tetracycline / carbenicillin medium were inoculated and grown overnight, and then 10 μL of culture was added. L A new 390μ LThe cells were placed in a 96-deep well plate containing 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 overnight at 30° C. The culture medium cultured overnight was centrifuged, and the supernatant was collected.

[0188] Then, a clone expressing a single clone soluble scFv that binds to alpha-synuclein aggregates was selected using the ELISA method as follows (Steinberger. Rader and Barbas III. 2000. Phage display vectors. In: Phage Display Laboratory Manual. 1st. Cold Spring Harbor Laboratory Press. NY. USA. pp. 11.9-11.12). Specifically, the 7B7 antibody selected in Example 1-1 was placed in a 96-well plate and coated overnight at 4°C. 200 μL of 3% BSA was placed in each well and blocked at 37°C for 2 hours. Next, alpha-synuclein aggregates and monomers were loaded at a concentration of 100 ng / well, respectively, and reacted at 37°C for 2 hours. Then, the plate was washed five times with 300 μL of PBS-T. The prepared single clone supernatant was mixed with 3% BSA at 1:1 (vol:vol), and 100 μL of the mixture was loaded onto the plate bound to the aggregate and monomer, and reacted at 37°C for 2 hours. Then, the plate was washed five times with 300 μL of PBS-T, and reacted with anti-HA HRP-conjugated antibody at 37°C for 1 hour, and washed five times with PBS-T. After adding 100 μL of TMB (Tetramethylbenzidine, Sigma, T0440) to develop color, 50 μL of 1N H2SO4 was added to stop the reaction, and the absorbance was measured at 450 nm. Clones with an absorbance of 0.5 or more were considered to be positive due to binding, and clones that nonspecifically bound to BSA were excluded.

[0189] The CDR residues of the clones found in the library were analyzed in silico to select clones that had serious problems binding to the framework or did not contain T-cell epitopes, B cell epitopes, or MHCII epitopes in the framework portion excluding the CDRs.

[0190] Then, for five antibodies formed by the combination of heavy and light chain variable regions as follows, the variable regions of the humanized antibodies were replaced with the backbone variable region portion of the human IgG1 isotype to produce five IgG1 backbone humanized antibodies. Specifically, Hu11F11_(ABL2-4) is an IgG type antibody, and SEQ ID NO: 525 The heavy chain variable region sequence (Hu11F11-VH2) and SEQ ID NO: 546 and a combination of the light chain variable region sequences (Hu11F11-VL4) of SEQ ID NO: 1, where Hu11F11_(ver.1) is an IgG antibody, and 531 The heavy chain variable region sequence (Hu11F11-VH-v1) and SEQ ID NO: 548 and a combination of the light chain variable region sequences (Hu11F11-VLv3 4c) of SEQ ID NO: 1, and Hu11F11_(ver.2) is an IgG antibody, 532 The heavy chain variable region sequence (Hu11F11-VH-v2) and SEQ ID NO: 548 Hu11F11_(ver.3) is an IgG-type antibody, and the light chain variable region sequence of SEQ ID NO: 533 The heavy chain variable region sequence (Hu11F11-VH-v3) and SEQ ID NO: 548 It contains a combination of light chain variable region sequences (Hu11F11-VLv3 4c).

[0191] Hu11F11(ver.4) is the sequence 534 The heavy chain variable region sequence (Hu11F11-VH-v4) and SEQ ID NO: 548 It was confirmed that the antibody was a combination of the light chain variable region sequences (Hu11F11-VLv3 4c).

[0192] Example 4. Antigen-binding specificity and avidity analysis of alpha-synuclein antibodies 4-1: Dot blot analysis using anti-alpha-synuclein antibody A dot blot experiment was performed to analyze whether the antibody of the present invention binds to monomers or aggregates in the native state. For this purpose, 50ng or 100ng of alpha-synuclein monomer or fibril protein (prepared in the lab of Professor Lee Seung-jae, Seoul National University; Bae et al., J.Neurosci 32:13454, 2012) was spot-loaded onto a nitrocellulose membrane using a dot blot apparatus (BioRad). Monomer or fibril proteins diluted 2-fold were loaded sequentially from right to left on the membrane (12.5, 25, 50, 100ng). The membrane was blocked with 5% non-fat dry milk in TBST at room temperature for 1 hour, and the alpha-synuclein antibody prepared in Example 1 was added to TBST containing 1% bovine serum albumin at a concentration of 1mg / ml, and the membrane and the antibody were incubated at room temperature for 1 hour. After washing with TBST, the cells were incubated with a secondary antibody conjugated with HRP (horse radish peroxidase) and chemiluminescence as a substrate. e Signals were analyzed using NEN scence substrate according to the manufacturer's protocol. Results were imaged using a LAS-3000 Luminescent Image Analysis System (FUJIFILM Life Science). The results are shown in Figure 1.

[0193] As shown in Figure 1, the alpha-synuclein antibodies of the present application were found to preferentially bind only to aggregates compared to alpha-synuclein monomers. In particular, 9B11, 3A9 and 11F11 were found to bind to aggregates. The 274 antibody (Bae et al., J Neurosci. 2012 Sep 26; 32(39): 13454-13469) is a comparative antibody that binds to both monomers and aggregates.

[0194] 4-2: ELISA analysis using anti-alpha-synuclein antibody ELISA was performed to quantitatively analyze the binding strength of the antibody of the present invention to the antigen. For this purpose, the mouse anti-alpha-synuclein antibody obtained in Example 1 was used at 1 μg / ml L The plate was coated with alpha-synuclein fibril aggregates at concentrations of 10, 100, 1000, and 10,000 ng / ml, washed with PBS, and then treated with a biotin-conjugated secondary antibody and HRP-conjugated streptavidin. The plate was reacted with TMB as a substrate, and the absorbance was measured. The results are shown in Figure 2.

[0195] As shown in FIG. 2, the antibody of the present invention was found to preferentially bind to aggregates with high avidity. Analysis of the antibody through ELISA results showed that the antibody preferentially binds to aggregates at 0.1×10 -9 M~2×10 -9 Antibodies that bind to both monomers and aggregates have an affinity of about M, up to 1×10 -10 The M value was shown. The antibody according to the present invention preferentially binds to alpha-synuclein aggregates with high affinity, and binds to single bodies with lower affinity than aggregates or does not bind at all, so affinity could not be derived. These results indicate that the causative agent of neurodegenerative diseases with alpha-synuclein pathology such as Parkinson's disease can be effectively removed or the activity can be suppressed.

[0196] 4-3: BIAcore analysis using anti-alpha-synuclein antibody Quantitative analysis of monomer and aggregate antigen binding of the alpha-synuclein antibodies prepared in Example 1 was performed using BIAcore.

[0197] The instrument used was T200 (GE Healthcare, S / N: 1565888). The chip used was Protein A (GE Healthcare, Cat. 29-1275-56), the regeneration buffer was 10 mM Glycine-HCl pH 1.5 (GE Healthcare, Cat. BR-1003-54), and the running buffer, analyte dilution buffer, and sample dilution buffer were HBS-EP. The α-syn antibodies (3A9, 9B11, 11F11) prepared in Example 1 were diluted with 1X HBS-EP (GE Healthcare, Cat. BR-1006-69), and the α-syn monomer (1 mg / mL) or fibril protein (3 mg / mL) (analyte) was diluted two-fold and analyzed at a total of six concentrations (0, 0.39, 1.56, 6.25, 25, 100 nM) including 0 nM. In the case of capture, the target RU for monomers was set to 800 (theoretical), and the target RU for fibrils was set to 100 (theoretical). The capture phase had a contact time of 60 seconds and a flow rate of 30 μ The flow rate was 30 L / min and the stabilization period was 180 seconds. μ L / min, and in the dissociation phase, the dissociation time was 360 seconds and the flow rate was 30 μL In the regeneration phase, the flow rate was 30 μ The flow rate was 100 µL / min, and the regeneration time was 240 seconds (primary) and 60 seconds (secondary), twice. Fitting was performed using a 1:1 binding model, and the evaluation software used was BIACore T200 Evaluation software (GE Healthcare). The results are shown in Figure 3a and Figure 3b.

[0198] Figure 3a shows the results of a BIAcore analysis of the preferential binding specificity and affinity of a monoclonal antibody prepared according to an embodiment of the present invention to alpha-synuclein aggregates. It shows that the antibody according to the present application binds to aggregates with high affinity. These results indicate that the causative agent of a neurodegenerative disease with alpha-synuclein pathology such as Parkinson's disease can be effectively removed or its activity can be inhibited. Figure 3b shows the results of Figure 3a in a table.

[0199] As shown in the results of Figures 3a and 3b, of the four alpha-synuclein antibodies analyzed, 3A9, 9B11, and 11F11, which preferentially bind to aggregates by other methods described above, were found to bind only to aggregates by BIAcore, with a binding affinity of approximately 1–3 × 10 -9 It was revealed that it has a high affinity for M.

[0200] 4-4. Octet analysis using anti-alpha-synuclein antibodies Quantitative analysis of the monomer and aggregate antigen binding of the mouse alpha-synuclein antibodies (3A9, 9B11, 11F11) prepared in Example 1 was performed using Octet.

[0201] Specifically, the running buffer was 1X KB buffer (Cat. 18-1092) or 1X PBS buffer at 1000 rpm, and the immobilization buffer was sodium acetate, pH 5 (10 mM, Cat. 18-1068). For α-syn monomer, α-syn antigen was immobilized, and for fibrils, test antibody was immobilized. The target concentration was 20 μg / m for monomer, 20 μg / m for fibril, and 20 μg / m for fibril. L , and 0.4 μg / m for fibrils. LThe kinetics concentration was set at 50 nM for monomers and 100 nM for fibrils, diluted two-fold in sequence to set a total of seven points. The association / dissociation times were 5 min / 20 min for monomers and 5 min / 25 min for fibrils, the Biosensor was ARG2, and the fitting was 1:1 fitting. The results are shown in Figure 4. Figure 4 shows the α- s Preferential binding specificity for yn aggregates analyzed by Octet.

[0202] As shown in Figure 4a, 3A9, 9B11 and 11F11 hardly bind to monomers (red dotted box) but bind well to aggregates (the graph in the red dotted box rises). These results are similar or consistent with the results of Dot Blot, Octet and ELISA, and it was revealed that 3A9, 9B11 and 11F11, which are antibodies that preferentially bind to aggregates by the other methods among the four α-syn antibodies tested, also bind only to aggregates by Octet. Figure 4b shows the results of Figure 4a in a table. The results of Figures 4a and 4b show preferential binding to alpha-synuclein aggregates and are consistent with the results of Figure 1. It was revealed that the #274 antibody, used as a comparison group, binds well to both monomers and aggregates.

[0203] Example 5. Assay of anti-alpha-synuclein antibodies to detect Lewy bodies in human brain tissue Brain tissue sections of 10 micrometer-thick paraffin-embedded patients who died of Parkinson's disease were stained for Lewy bodies and Lewy neurites in the tissue sections using the chimeric anti-alpha-synuclein antibody obtained in Example 2 as follows. The tissue sections were treated with 90% formic acid for 3 minutes to carry out antigen retrieval, and then peroxidase activity of the tissue itself was suppressed using 1% H2O2 (50% ethanol base). To prevent non-specific binding of the tissue, 10% normal horse serum was added. Next, after washing with phosphate buffer, the adjacent sections were treated with the 3A9, 11F11 and 11F11 antibodies of the present invention at 4°C overnight. After washing with phosphate buffer, the sections were treated with anti-human IgG antibody bound to biotin at 37°C for 30 minutes, and then reacted with avidin-biotin complex at room temperature for 30 minutes (Vectastatin Elite kit; Vector Laboratories).

[0204] Then, the sections were developed with DAB containing 0.005% H2O2, and counterstained with 0.5% cresyl violet to separate the individual cells. The results are shown in Figures 5a and 5b.

[0205] 5a and 5b show the results of measuring whether the monoclonal antibodies 3A9 and 11F11 according to an embodiment of the present invention can specifically recognize Lewy bodies and Lewy neurites in human brain tissue, respectively. The antibodies according to the present application bind to Lewy bodies (arrows) and Lewy neurites (thread-like structures at the lower left).

[0206] As shown in Figures 5a and 5b, the antibody of the present application was found to effectively bind to Lewy bodies and Lewy neurites (indicated by arrows). Such results indicate that the antibody can effectively bind to alpha-synuclein aggregates, which are components of Lewy bodies in human brain tissue. This indicates that the antibody delivered to human brain can effectively and specifically bind to alpha-synuclein aggregates. This result indicates that the antibody of the present application can specifically bind to alpha-synuclein aggregates present in actual human brain tissue, and can be effectively used for the prevention and / or treatment of diseases associated with alpha-synuclein pathology.

[0207] Example 6. Epitope analysis of anti-alpha-synuclein antibodies Epitope mapping of the chimeric anti-alpha-synuclein antibodies 3A9 and 11F11 obtained in Example 2 was performed by peptide array analysis at PEPSCAN (The Netherlands). The results are shown in Figure 6. Figure 6 is a schematic diagram showing the epitope mapping results of an antibody according to one embodiment of the present invention.

[0208] As shown in FIG. 6, it was revealed that the antibody of the present application recognizes the C-terminus. It was revealed that the antibody of the present application binds mostly to the C-terminus. Alpha-synuclein antibodies that recognize the N-terminus do not recognize aggregates of other diseases such as multiple system atrophy, which belongs to synucleinopathies, commonly known as alpha-synuclein-related diseases, whereas alpha-synuclein antibodies that recognize the C-terminus have the advantage of recognizing aggregates of not only Parkinson's disease but also various other synucleinopathies. In particular, as described above, when recognizing the peptide consisting of residues 110 to 122, it showed preferential binding to aggregates.

[0209] Example 7. ELISA assay for anti-alpha-synuclein antibodies In order to quantitatively analyze the binding strength of the chimeric antibody (Ch11F11) obtained in Example 2 and the humanized antibody (Hu11F11) obtained in Example 3, sandwich ELISA was performed in a manner substantially similar to that in Example 4-2.

[0210] Specifically, each antibody was diluted 1 / 10 at concentrations of 0.04 to 400 nM and coated onto a 96-well plate, and then 2000 ng / ml L The aggregates were treated in each well. After washing with 1XPBS, the wells were treated with a capture antibody bound to biotin and streptavidin bound to HRP, and then reacted with TMB as a substrate, and the absorbance was measured. The results are shown in Figure 7.

[0211] As shown in FIG. 7, it was confirmed that the humanized antibody according to the present invention, particularly the humanized antibody derived from chimera 11F11 (humanized 11F11 antibody), exhibits binding activity equivalent to that of the chimera 11F11 clone. It was confirmed that the humanized antibodies, particularly the 11F11-derived variants, hu11F11 (ver. 1), i.e., the combination of Hu11F11-VH-v1 and Hu11F11-VLv3 4c, hu11F11 (ver. 2), i.e., the combination of Hu11F11-VH-v2 and Hu11F11-VLv3 4c, hu11F11 (ver. 3), i.e., the combination of Hu11F11-VH-v3 and Hu11F11-VLv3 4c, and hu11F11 (ver. 4), i.e., Hu11F11-VH-v4 and Hu11F11-VLv3 4c, exhibit binding strength similar to that of the chimeric 11F11 clone, and their EC 50 The EC of the chimeric 11F11 antibody was 11.5 to 15.1 nM. 50 The value was similar to 12.5 nM.

[0212] Example 8: BIAcore analysis using anti-alpha-synuclein antibodies The binding strength of the chimeric antibody obtained in Example 2 and the humanized antibody obtained in Example 3 was quantitatively analyzed using BIAcore in a manner substantially similar to that in Example 4-3. The analysis results are shown in FIG. a~cand as shown in the table below.

[0213] [Table 18] TIFF0007625305000069.tif33170

[0214] As a result, it was confirmed that the humanized antibodies described in the present application, in particular the variants of 11F11, i.e., hu11F11(ver.2) (combination of Hu11F11-VH-v2 and Hu11F11-VLv3 4c), hu11F11(ver.3) (combination of Hu11F11-VH-v3 and Hu11F11-VLv3 4c), and hu11F11(ver.4) (combination of Hu11F11-VH-v4 and Hu11F11-VLv3 4c), exhibited KD values ​​similar to those of the chimeric 11F11 clone, and the degree of binding of the humanized clone was 0.02 to 0.06 × 10 -9 The KD of M, chimeric 11F11 clone was 0.02 × 10 -9 M exhibited a low KD value, i.e., a high binding affinity to aggregates.

[0215] Example 9. Evaluation of alpha-synuclein specific binding of chimeric antibodies 9-1: ELISA analysis of beta-synuclein and gamma-synuclein After producing three chimeric antibodies, 3A9, 9B11, and 11F11, according to Example 2, the binding specificity of the antibodies to alpha-synuclein was compared with that of its homologues, beta-synuclein and gamma-synuclein, by ELISA.

[0216] For this purpose, human beta-synuclein (Uniprot: Q16143) protein (CUSABIO, Cat: CSB-EP624090HU) or human gamma-synuclein (Uniprot: O76070) protein (CUSABIO, Cat: CSB-EP021915HU) was coated onto a 96-well plate at a concentration of 100 ng / ml, washed, and then blocked with 5% BSA for 2 hours. s yn antibody ( S You CThe positive control group was a goat anti-hFc antibody bound to HRP, and the positive control group was a goat anti-rabbit antibody bound to HRP. After washing, the positive control group was treated with chimeric antibodies (3A9, 9B11, 11F11) diluted 1:10 from 400nM to 0.04nM for 2 hours, and then washed with PBS. Next, the positive control group was treated with a goat anti-rabbit antibody bound to HRP, reacted with TMB as a substrate, and the absorbance was measured at 450nm and 650nm.

[0217] The experimental results are shown in Figures 9a and 9b, which show the very low binding affinity of the chimeric antibodies (3A9, 9B11, and 11F11) according to one embodiment of the present invention to human beta-synuclein and human gamma-synuclein as analyzed by ELISA. s The yn antibody showed high binding affinity to beta- and gamma-synuclein: Figure 9a is for human beta-synuclein and Figure 9b is for human gamma-synuclein.

[0218] 9-2: Dot blot analysis for amyloid beta and tau aggregates The three chimeric antibodies 3A9, 9B11, and 11F11 of Example 2 were used to detect amyloid beta 1-42 (Uniprot: P05067; CAS number: 107761-42-2) and tau (Unip ro The specific binding ability of the antibody to alpha-synuclein aggregates was analyzed by reacting it with aggregates formed from alpha-synuclein (t:P10636-8) protein in a dot blot. The reasons are as follows.

[0219] Amyloid beta 1-42Amyloid beta and tau form aggregates, which are believed to be an important cause of degenerative neurological diseases, particularly Alzheimer's disease, and the aggregates are known to have structures such as oligomers, protofibrils, and fibrils, similar to those of alpha-synuclein aggregates. Therefore, the dot blot was performed to confirm that the chimeric antibodies 3A9, 9B11, and 11F11 recognize the specific sequence of alpha-synuclein and do not recognize the common structure of aggregates that alpha-synuclein, amyloid beta, and tau-derived aggregates have as aggregates.

[0220] The dot blot method of this example was carried out in a manner substantially similar to that of Example 4-3. 1-42 Tau protein and its corresponding aggregates were produced in the laboratory of Professor Lee Seung-jae at Seoul National University. Syn-1, 6E10, and Tau5 are alpha-synuclein and amyloid beta, respectively. 1-42 , an antibody known to bind to tau protein. The analysis results are shown in FIG.

[0221] The experimental results are shown in FIG. 10, which shows that the chimeric antibodies 3A9, 9B11, and 11F11 of the present application specifically bind only to alpha-synuclein-derived aggregates and do not specifically bind to amyloid beta. 1-42 In contrast, the results showed that amyloid beta- 1-42 The 6E10 and Tau5 antibodies, known to bind to tau, respectively, were shown to bind to the aggregates on dot blots.

[0222] The above results suggest that the chimeric antibody does not bind to alpha-synuclein homologs and aggregates derived from different proteins, suggesting that the antibody binds efficiently only to the target protein and is more effective than antibodies or drugs that bind to homologs and aggregates derived from different proteins.

[0223] Example 10: Production of bispecific antibodies 10-1: Bivalent double antibody cloning To construct a bivalent dual antibody expression vector, an antibody nucleotide sequence including a signal sequence was inserted into the MCS (multi-cloning site) 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, respectively.

[0224] In the case of a heavy chain sequence inserted into a heavy chain expression vector, anti-a- s The anti-IGF1R scFv is linked to the C-terminus of an immunoglobulin to which a heavy chain variable region encoding an anti-yn antibody and a human heavy chain constant region are linked via a linker. In the case of a light chain sequence inserted into a light chain expression vector, anti-a- s It is a form in which the light chain variable region encoding the yn antibody and the human light chain constant region are linked together.

[0225] 10-2: Monovalent double antibody cloning The monovalent doublet antibody is an anti-IGF1R scFv linked to the C-terminus by a linker. s Anti-a- in which the yn immunoglobulin heavy chain (hole) and scFv are not linked s It is a form in which an antibody light chain is joined to a heterodimer to which an yn immunoglobulin heavy chain (knob) is bound.

[0226] To increase the conjugation efficiency of heavy chain heterodimers, k The knob-in-hole technique was used, that is, 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 amino acid in the CH3 region.

[0227] 10-3: Transient expression The prepared vector was 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 transduced at a ratio of 1:1. 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 transduced at a ratio of 0.5:0.5:1.

[0228] The day before transduction, ExpiCHO TM (Gibco, Cat: A29127) cells were cultured in ExpiCHO TM After adjusting the concentration to 3×10E6~4×10E6 viable cells / mL in expression medium (Gibco, Cat: A29100-01), the cells were cultured for 1 day at 8% CO2, 37℃, and 120 rpm. On the day of DNA transfection, the cells, which had grown to 7×10E6~10×10E6 viable cells / mL and had a survival rate of 95% or more, were diluted with fresh medium to 6×106 viable cells / mL.

[0229] ExpiFectamine for transfection into prepared mother cells TM CHO transfection kit (Gibco, Cat: A29129) was used to transfect the cells with ExpiFectamine. TM CHO & plasmid DNA complex was prepared. TM SFM® (Gibco, Cat: 12309019) medium was dispensed and titrated concentrations of DNA and ExpiFectamine were added. TM After inoculating each CHO reagent, mix them together and leave them at room temperature for 5 minutes, then inoculate them onto the mother cells, inject the transfection, and start culturing. TMThe enhancer and feed included in the CHO transfection kit were inoculated into the transfected cells, and after 5 days, the feed was additionally inoculated. The cells were then cultured for 10 days under conditions of 8% CO2, 37°C, and 120 rpm to complete the production.

[0230] 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, which was then subjected to a purification process.

[0231] Example 11. IGF1R antibody production (scFV) 11-1: Production of IGF1R antibody (scFV) The monoclonal antibodies were produced using phage display / panning technology.

[0232] Specifically, the following protein was used as an antigen for phage display panning and other analyses to produce anti-IGF1R antibodies: SEQ ID NO: 1, which is an extracellular domain (ECD) of human IGF1R with the signal sequence removed. of 410 The antigen used was one with a histidine tag (His tag) attached to the C-terminus of the amino acid sequence from amino acid residues 31 to 932 (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 His tag at the C-terminus were used as antigens.

[0233] Diverse human ScFv (single-chain variable fragment) library cells (OPAL library, created by Professor Shim Hyun-bo of Ewha Women's University) 1 x 10 10The cells were inoculated into 2X YT [Tryptone (CONDA, 1612.00) 17 g, yeast extract (CONDA, 1702.00) 10 g, NaCl (Sigma, S7653) 5 g] medium containing chloramphenicol (Sigma, C0857) 34 μg / ml, 2% glucose (Sigma, G5400), and 5 mM MgCl2 (Sigma, M2393), and then cultured at 37°C for about 3 hours until the OD600 value reached 0.5 to 0.7. After that, the cells were infected with helper phage and then cultured in chloramphenicol 34 μg / ml. L , 5 mM MgCl2, kanamycin (Sigma, K1876) 70 μg / m L The cells were cultured in 2X YT medium supplemented with 1 mM IPTG (ELPISBIO, IPTG025) at 30°C for 16 hours to induce phage packaging. The culture was then centrifuged at 4500 rpm for 15 minutes at 4°C, and 4% PEG6000 (Fluka, 81253) and 3% NaCl (Sigma, S7653) were added to the supernatant, which was thoroughly dissolved and then reacted on ice for 1 hour. The cells were centrifuged again at 8000 rpm for 20 minutes at 4°C, and the pellet was suspended in PBS and centrifuged again at 12000 rpm for 10 minutes at 4°C to obtain the supernatant containing the library phage, which was then placed in a new tube and stored at 4°C until use.

[0234] 11-2: Phage display panning To screen for human IGF1R antibodies, panning was performed a total of three times as follows: The phage library used in the present invention is a synthetic human scFv library, and the phage display panning process and results are shown in Table 19 below.

[0235] [Table 19] TIFF0007625305000070.tif73170

[0236] Specifically, immunotubes M axi S orp 444202) at 5ug / m LRecombinant human IGF1R protein (R&D Systems, USA, 391-GR or Sino Biological Life Technologies, USA, 10164-H08H-50R) was added at a concentration of 1 ml L After adding the phage library and coating it on the surface of the test tube at 4°C for 16 hours, the supernatant was removed, and 4 ml of PBS solution containing 3% BSA (Bovine serum albumin) was added and reacted at 37°C for 1 hour to block non-specific binding by binding BSA to the surface where IGF1R was not adsorbed. Next, after removing the supernatant, the phage library prepared in Example 11-1 was mixed with a 1.5% BSA solution and reacted with the immunoassay tube at 37°C for 1 hour to allow IGF1R-specific phages to bind to the antigen. Next, the phage 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 collected using 100 mM triethylamine solution.

[0237] The recovered phages were neutralized with 1M 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 spread on LB agar medium containing tetracycline and carbenicillin and cultured overnight at 37°C. The following day, the cultured E. coli was transferred to 5 mL of LB agar medium containing tetracycline and carbenicillin. L The same volume of 50% glycerol was added, and one portion was stored at 80°C. L 40m L After suspension in SB-tetracycline / carbenicillin medium, 10 12 PFU of VCSM13 helper phage was added and incubated at 37°C for 1 hour with gentle stirring. Kanamycin was then added to the culture medium and incubated at 30°C for approximately 16 hours to allow only E. coli infected with the helper phage to be cultured.

[0238] The next day, the culture was centrifuged, the supernatant was removed, and a buffer containing 4% PEG8000 and 3% sodium chloride (NaCl) was added and reacted at 4℃ for about 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, which was then used for the next round of panning. The above process was repeated four times, and the number of washings with PBS-T was increased as the panning rounds progressed, allowing the antigen-specific phages to be amplified and concentrated.

[0239] 11-3: Single clone phage antibody selection (single clone screening) Clones that exhibited not only protein binding to the ECD (extracellular domain) of human IGF1R (protein binding) but also cell binding to MCF-7, an IGF1R-expressing cell line, were selected.

[0240] Specifically, the following experiment was carried out to select a monoclonal antibody that specifically binds to IGF1R from the phage pool obtained in Example 11-2.

[0241] To isolate a single clone from the enriched pool, the resulting phage pool was plated on LB-tetracycline / carbenicillin agar medium and cultured to obtain a single colony. This colony was inoculated into a 96-deep-well plate and cultured overnight, after which 10 μl of culture medium was added. LThe same method was used to inoculate the 96-deep well plate and cultured at 37°C for about 4 hours to reach a titration OD of 0.5-0.7. The culture was added with 20 MOI helper phage 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 to remove the supernatant, and ELISA was performed to select IGF1R-specific phages (Steinberger. Rader and Barbas III. 2000. Phage display vectors. In: Phage Display Laboratory Manual. 1st. Cold Spring Harbor Laboratory Press. NY. USA. pp. 11.9-11.12).

[0242] 100 ng of recombinant IGF1R was added to each well of an ELISA plate, and the plate was incubated at 4°C for about 15 hours to coat the antigen on the plate. 200 μl of PBS buffer containing 3% BSA was added per well to prevent non-specific binding. L After addition of each solution, the mixture was reacted at 37° C. for about 1 hour. The supernatant was discarded.

[0243] 100 u containing the prepared single clone phage L Add the solution to each well and react at 37℃ for 1 hour. Then add 300u of PBS-T. L To detect the 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. L After washing three times with TMB (Tetramethylbenzidine, Sigma, T0440) 100u L After adding 1N H2SO450u L The reaction was terminated by adding 0.05% CO. The absorbance was measured at 450 nm, and clones with higher absorbance than the control BSA were selected as antigen-specific antibody clones.

[0244] A total of seven clones, 1564, 48G5, 49G11, 54H4, 60A11, 60H6, and B11, were selected over two rounds of screening.

[0245] Example 12. Preparation of affinity variants of IGF1R antibodies The antibody was optimized by affinity variation of the selected clones after evaluating the ligand binding strength and BBB crossing ability. In the first attempt, NNS hand-mix primers were prepared to randomize the CDR2 of the heavy chain and the CDR3 of the light chain based on 1564 scFv, and the 1564 scFv gene containing the randomization sequence was amplified using PCR. The amplified gene was inserted into the pComb3x vector to create a library suitable for phage display, and multiple scFv clones that bind to IGF1R were selected by library panning and ELISA screening. The amino acid sequences of the variable regions of the selected clones were confirmed by gene sequencing.

[0246] In the second attempt, two mini libraries were created for the heavy and light chains by introducing germline backmutations into CDR1, CDR2, and CDR3, respectively. 96 colonies were analyzed, and 31 VL clones and 39 VH clones were confirmed to have unique sequences. Affinity variants were selected based on the productivity and antigen binding ability of the clones, and finally clones were obtained. 。

[0247] Example 13. Preparation of deamidation residue mutant antibody 13-1:Deamidation residue confirmation If deamidation occurs in CDR, the antibody may be decomposed and the binding to the antigen may weaken, resulting in reduced efficacy and sample heterogenity, which may lead to complexity in identification in future clinical approvals, etc. Therefore, we attempted to identify the location where deamidation occurs by in silico analysis and peptide mapping.

[0248] As shown in Figure 8a, in silico analysis and peptide mapping of the parental 1564 clone confirmed that deamidation did indeed occur. The samples were stored at 4°C or 40°C for one week and then analyzed, and it was confirmed that deamidation occurred in LCDR2, LCDR3, and HCDR2. The affinity variants disclosed in Example 12 were also analyzed to confirm the position of deamidation.

[0249] 13-2: Mutant antibody production The parental clone 1564 has a net charge of -0.7 in in silico analysis. Generally, if the net charge is <0 or >5.5, it is considered vulnerable to fast clearance in the body. Therefore, we thought that if the deamidation residue was replaced with positive charges such as H, R, and K, it would be possible to eliminate deamidation, increase the overall charge, and prevent fast clearance. Therefore, we produced a mutant in which the position where deamidation occurs was replaced as shown in Figure 18b.

[0250] To remove the deamidation residue, a residue-substituted mutant was produced as follows.

[0251] 1) In the amino acid sequence, Asn is changed to D or Q, which are similar to Asn. If it is confirmed that there is no change in the binding strength, the residue is replaced with Q.

[0252] 2) The net charge of the parental clone used was -0.7. Fast clearance is induced when the net charge is <0 or >5.5, so the more the charge changes to positive, the more advantageous it is. Therefore, N95a, which is a residue of deamidation in LCDR3, was replaced with H, R, and K, which carry positive charges.

[0253] Example 14. Preparation of various forms of anti-IGF1R antibodies 14-1: Preparation of anti-IGF1R minibody antibody A minibody was prepared by linking the entire scFv of the IGF1R-specific monoclonal phage antibody obtained in Examples 11 to 13 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 a nucleic acid encoding Fc.

[0254] 14-2: Preparation of bivalent anti-IGF1R antibodies The entire scFv of the IGF1R-specific monoclonal phage antibody obtained in Examples 11 to 13 was linked to the C-terminus of a therapeutic antibody in the form of IgG to produce a bivalent form. v A nucleic acid sequence encoding the amino acid sequence of was prepared, digested with restriction enzymes, and cloned into a pcDNA-based expression vector containing nucleic acid encoding the therapeutic antibody.

[0255] 14-3: Preparation of anti-IGF1R IgG (Full-IgG) antibody Among the IGF1R-specific monoclonal phage antibodies obtained in Examples 11 and 12, the gene sequences of the heavy and light chain regions of the 564 and F06 antibodies were synthesized (Genotech) to convert the sequences to full IgG1 (Full IgG) forms. The synthesized heavy and light chain genes were cloned into an expression vector.

[0256] 14-4: Preparation of monovalent anti-IGF1R scFv antibody While Example 14-1 is a minibody form in which anti-IGF1R antibodies are bound in the form of scFv to the C-terminus of each of the two heavy chain Fc, in this example, an antibody was produced in a monovalent form in which one scFv is bound only to one Fc C-terminus in the heavy chain. For this purpose, a vector was produced in which 1564, F06, C04, VH5, VH16, VH35, VH9, VH2, VH7, and VH32 of the IGF1R-specific monoclonal phage antibodies obtained in Examples 11 to 13 were bound only to one site in the Fc C-terminus, and a vector in which anti-IGF1R antibodies were not bound to the C-terminus. A knob-into-hole mutation was introduced into the Fc to produce a heteromeric form when the antibody was produced in the 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 with anti-IGF1R antibody bound to the Fc C-terminus, a vector for the heavy chain of the therapeutic antibody without anti-IGF1R antibody bound to the C-terminus, and a vector for the light chain of the therapeutic antibody.

[0257] 14-5: Expression and purification of various anti-IGF1R antibodies The vectors prepared in Examples 14-1 to 14-4 were introduced into cells as follows.

[0258] Specifically, CHO-S cells were cultured in CD-CHO (Gibco, 10743) medium at 1.5 × 10 6 cells / m L After adjusting the concentration, the cells were cultured at 8% CO2 and 37°C for 1 day. On the day of DNA transfection, 2.5-3 × 10 6 cells / m LThe cells grown in 1% DMSO were cultured at 2.1 × 10 6 cells / m L After preparing the cells at a concentration of 1000 μg / mL, the cells were cultured at 8% CO2 and 37°C for 3 hours. 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.

[0259] The vector combination was then diluted in Opti-MEM medium at 1 μg per ml of medium and added with PEI (Polysciences, 23966, stock concentration: 1 mg / ml). L ) is the culture medium m L The DNA and PEI mixture was mixed and left at room temperature for 10 minutes, then added to the flask containing the cells and incubated at 5% CO2, 37℃, 100 rpm for 4 hours, and then CD-CHO medium of the same volume as the culture volume was added and incubated at 8% CO2, 37℃, 110 rpm for 4 days.

[0260] The culture medium was passed through an equilibration buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl) to equilibrate the Mab. S elect S u R e TM (GE Healthcare, 5mL) to allow the expressed antibody to bind to the column. After that, it was eluted with 50mM Na-citrate (pH3.4), 100mM NaCl solution, and neutralized with 1M Tris-HCl (pH9.0) to a final pH of 7.2. The buffer was then exchanged for PBS (phosphate buffered saline, pH7.4). If the purity was high, it was frozen at -20℃ after preparation, and if additional purification was required, it was stored at 4℃ until further purification.

[0261] If additional purification is required, Hiload S uperdex 200 TM(GE Healthcare, Cat. No. 28-9893-36) and can be purified using a variety of other size exclusion chromatography. p After equilibration with phosphate buffered saline pH 7.4 (Gibco, Cat. No. 10010-023), the sample after the first purification was loaded onto the column. The purified sample was stored frozen at -20°C after preparation.

[0262] 14-6: Preparation of double antibodies with anti-a-syn (alpha-synuclein) The anti-IGF1R antibody according to the present invention is fused to a linker (SEQ ID NO: 411 ) to link the heavy and light chain variable regions to produce scFs v The anti-a- s The complete form of the yn antibody is a heavy chain constant region of the IGG antibody, and a linker (SEQ ID NO: 412 In this example, anti-a- s Intact form of yn antibody I g A monovalent antibody was prepared by linking one scFv molecule of anti-IGF1R antibody per one G antibody molecule, and a bivalent antibody was prepared by linking two scFv molecules of anti-IGF1R antibody per one G antibody molecule. s The sequence of the yn antibody and the sequence of the biantibody prepared according to the present invention are as shown in Table 17. Then, in the Examples, experiments were carried out on the biantibody exemplified in Table 17.

[0263] Example 15. Analysis of IGF1R specific binding ability of anti-IGF1R antibodies 15-1: Analysis of binding ability of minibody-type anti-IGF1R antibody to IGF1R (ELISA) ELISA analysis was performed to confirm whether the minibody forms of 996, 1226, 1564, and MKJP2 clones prepared in Example 14-1 bind to recombinant IGF1R in a concentration-dependent manner.

[0264] Specifically, the human recombinant IGF1R to which the antibody binds is the extracellular domain (ECD), and R&D S The antibody was purchased from Nunc Immuno Systems (6630-GR / CF). Human IGF1R was added to a 96-well ELISA plate (Nunc-Immuno Plates, NUNC, Rochester, NY) at 1 ug / ml in PBS buffer. L Dilute to 100u per well L After that, the plate was incubated at 4℃ for 16 hours for coating, and the supernatant was removed. 200u of PBS buffer containing 3% BSA (bovine serum albumin) was added per well. L The mixture was added and reacted for 2 hours to block non-specific binding.

[0265] The minibody antibodies of clones 996, 1226, 1564, and MKJP2 prepared in Example 14-1 were diluted 3-fold from the highest concentration of 20 nM to prepare 12 points, and 100 μl of each antibody was added to each well. L After that, the plate was incubated at room temperature for 1 hour, and then washed 4 times with PBS buffer containing 0.05% Tween 20. Anti-human HRP, which recognizes the human Fc present in the minibody, was diluted in blocking buffer at 1:5000 and 100 μl was added to each well. L The mixture was then transferred into a 300 ml PBS-T (Tween 20 0.05%) flask and incubated at room temperature for 1.5 hours. L After washing four times with 100% NaCl, the color was developed using TMB (Tetramethylbenzidine, Sigma, T0440). The enzyme reaction was stopped with 0.5 mol / L sulfuric acid, and the absorbance was recorded and analyzed at 450 nm using a molecular device. The experimental results are shown in Figure 1a.

[0266] The 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 with similar binding affinity, and 1226 with slightly lower binding affinity.

[0267] ELISA analysis of species cross-reactivity of 15-2::IGF1R antibody The 1564 anti-IGF1R antibody prepared by the method of Example 14-2 and the anti-IGF1R antibody obtained in Example 11-3 were analyzed for interspecies cross-linking by ELISA. For this purpose, human, monkey, mouse and rat IGF1R antigens were first added at 1 ug / ml L Dilute with 100u per well. L The plate was incubated at 4°C for 15 hours to coat the bottom of the plate. After removing the supernatant, 200 μl of PBS buffer containing 3% BSA was added. L Each well was treated with anti-IGF1R antibody at a maximum concentration of 400 nM, diluted 5-fold in PBSB (BSA 3% in PBS), and treated in each well, and reacted at 37°C for 1 hour. After washing five times with PBS buffer, anti-human Fab HRP, which recognizes the Fab portion of the bound antibody, was diluted 1:20000 and added to each well at 100 μl. L After the treatment, the cells were incubated at 37°C for 1 hour, then washed five times with PBS buffer, and TMB( t The enzyme reaction was stopped with 0.5 mol / L sulfuric acid, and the plate was read using a microplate reader (Molecular D event s The absorbance was measured at 450 nm using a 500 mAb aliquot. When performing ELISA, if there were many samples, the plate was divided into two and the experimental results are shown in Table 12 below.

[0268] Specifically, the ELISA results of the bispecific antibody against human IGF1R, the ELISA results of 1564IgG 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 are summarized in Table 12 below.

[0269] 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.

[0270] [Table 20] ELISA analysis results of antibody binding to IGF1R in various species TIFF0007625305000071.tif159170*:not available **201:scFv form of Herceptin biosimilar

[0271] 15-3: Analysis of affinity of affinity variants to IGF1R (FACS) The binding ability of the affinity variants prepared in Example 12 was analyzed by performing ELISA for IGF1R ECD protein, and the binding ability for MCF-7 was analyzed by FACS.

[0272] In the analysis of the primary clones, Table 21 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 22 shows the results of FACS analysis of binding to the MCF-7 cell line.

[0273] [Table 21] Binding ELISA of the primary selected clones to IGF1R ECD protein in the form of a bispecific antibody TIFF0007625305000072.tif99170

[0274] [Table 22] Results of FACS analysis of binding to MCF-7 cell line TIFF0007625305000073.tif62170

[0275] As a result, F06 was selected as the clone with the highest cell binding affinity compared to the parental clone (1564 clone) (affinity matured), and C04 was selected as the clone with the lowest cell binding affinity compared to the parental clone (1564 clone) (affinity reduced).

[0276] In the analysis of the secondary clones, Table 23 shows the binding ELISA results for IGF1R ECD protein in the form of bispecific antibodies of the clones prepared by the secondary preparation method.

[0277] [Table 23] ELISA results for IGF1R ECD protein of secondary selected clones TIFF0007625305000074.tif92170

[0278] Among the secondary clones, clones with significantly decreased productivity and physical properties were removed, and clones for FACS analysis were selected as shown in Table 24.

[0279] Table 24: Clones for FACS analysis TIFF0007625305000075.tif69170

[0280] Figure 12c shows the results of FACS analysis of the binding of the clones to the MCF-7 cell line, and all of the clones analyzed had reduced binding ability to MCF-7 compared to the parental clone 1564. The results show that the clones that showed reduced binding ability by ELISA also showed reduced binding ability by FACS.

[0281] The antibody clones selected were F06, C04, VH2, VH5, VH7, VH9, VH16, and VH32, and the amino acid sequences for the heavy and light chain variable regions for these antibodies are shown in Tables 5 and 6 above.

[0282] 15-4:BIAcore analysis of human IGF1R An attempt was made to analyze the binding strength between the antibody according to the present invention and human IGF1R.

[0283] The IgG form of the 1564 clone was analyzed for its binding to human IGF1R by SPR analysis. The antigen, an anti-his antibody against the His tag bound to the human IGF1R ECD, was dissolved in acetate pH 4.0 buffer at 20 μg / m L After dilution with 0.1% ethanol, the target RU was immobilized to 10,000 RU in the reference / analytic channel of a CM4 chip using the amine coupling method. 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. ss The incubation time was 5 minutes and 20 minutes, respectively. The analysis consisted of baseline 1, activation (EDC+NHS), human IGF1R loading, quenching (1M e The evaluation was performed in the following order: baseline 2, association, and dissociation. TM Analysis was performed using T200 Evaluation software (version 1.0, S / N: 04Y15X11-0149).

[0284] The analysis showed that the KD of the 1564 IgG antibody was 2.5305 × 10 -9 nM, and F06 IgG antibody is 4.7802 x 10 -7 The results were confirmed to be in the range of 0.1×10 nM, and all of them showed high binding affinity to human IGF1R. The analysis results are shown in Figure 11b. In particular, when the 1564 clone was made into an IgG form, it bound to human IGF1R with a binding affinity of 2.5305×10 -9 It was confirmed that 1564 shows a dissociation constant of nM, and that its binding strength does not change significantly depending on its form. 。

[0285] Example 16. Analysis of binding ability of anti-IGF1R antibodies to cell lines and brain endothelial cells expressing human IGF1R 16-1: FACS analysis for MCF-7 To confirm whether the minibody forms of clones 996, 1226, and 1564 prepared in Example 14-1 bind to endogenous IGF1R on the cell surface, we performed binding analysis to cell lines expressing human IGF1R and brain endothelial cells by FACS. We also confirmed the degree of binding between periplasmic extract and MCF-7 and breast cancer cell lines known to overexpress IGF1R by FACS.

[0286] Specifically, 0.43×10E6 MCF-7 cell line per sample was injected with each of the three minibodies at 20ug / ml. L After dilution with 1:500, the cells were treated with anti-human FITC and incubated at 4°C for 1 hour. After washing twice with PBS buffer, the cells were treated with anti-human FITC diluted at 1:500 and incubated at 4°C for 1 hour. After washing twice with PBS buffer, the degree of binding of anti-IGF1R minibody was measured using a FACSCalibur instrument. As a control, MCF-7 cells treated with only the secondary antibody were used. The experimental results are shown in Figure 12a.

[0287] A02, A06, A07, B01, B02, B09, B10, C04, D03, E06, F06, H04(Gly), H04(Val), VH2, VH5, VH7, VH9, VH16, VH32, and VH35 prepared in Example 12 and Example 14-2 were analyzed for their binding to MCF-7 in the same manner as described above. The 1564 clone was prepared in the same manner as in Example 14-2 and used as a parental clone for comparison, and MCF-7 cells treated with only the secondary antibody were used as a control. The analysis results are shown in Table 21 and Figure 12c.

[0288] The experimental results showed that the affinity variants and parental clones (1564 clones) in the form of scFv and bispecific antibodies in the three minibodies tested, as shown by the MFI (Mean Fluorescence Intensity) of the samples, specifically bound to endogenous IGF1R expressed on the cell surface. The results indicate that the clones obtained in the above example can be used for the intended purpose by binding to IGF1R in a form that actually exists in the body.

[0289] 16-2: FACS analysis of JIMT-1 and BT474 Except for using JIMT-1 and BT474 breast cancer cell lines instead of MCF-7 cell line used in Example 16-1, the same method was used to confirm whether the minibody forms of 996, 1226, and 1564 clones prepared in Example 14-1 bind to endogenous IGF1R on the cell surface. The experimental results are shown in Figure 12a.

[0290] The experimental results, shown by the mean fluorescence intensity (MFI) of the samples, confirmed that the scFvs in the three minibodies tested specifically bound to endogenous IGF1R on the surface of various IGF1R-expressing cell lines.

[0291] 16-3: FACS analysis of mouse brain endothelial cells The binding of the bispecific antibody form of the 1564 clone prepared by the method of Example 14-2 and the IgG form of the 1564 clone prepared by the method of Example 14-3 to mouse brain endothelial cells, bEND.3, was analyzed. In this case, a group treated with only the secondary antibody and a group treated with only the therapeutic antibody IgG (CH11F11) were used as negative control groups. FACS analysis was performed as in Examples 16-1 and 16-2. The analysis results are shown in Figure 12b.

[0292] All of the test clones tested, except for the negative control group, showed binding to bEND.3. Through these results, it was confirmed that 1564 clones of various types specifically bind to IGF1R expressed on the surface of brain endothelial cells.

[0293] Example 17. Intracellular internalization analysis of anti-IGF1R antibodies 17-1:MCF-7 internalization assay-1564, 996, 1226, MKJP2(minibody) We attempted to confirm whether the minibody forms of the 996, 1226, 1564 and MKJP2 clones prepared in Example 14-1 were internalized intracellularly in cell lines expressing IGF1R, and whether the antibodies introduced into the cells would pass through the RMT pathway without being degraded. In order for the 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.

[0294] Using MCF-7 cell line expressing IGF1R, we analyzed whether the antibody of the present invention is internalized in cells. Specifically, 30,000 MCF-7 cell lines were plated in an 8-well slide chamber and cultured for one day. The minibody antibodies of 996, 1226, 1564 and MKJP2 clones prepared in Example 14-1 were each cultured at 5 μg / ml. L Each well was treated with each of the plates at 4°C for 2 hours, washed three times with cold DMEM culture medium, and then treated with Alexa488-conjugated anti-human Fc antibody at 4°C for 1 hour.

[0295] To test for antibody complex internalization, the plate was transferred to a CO2 incubator and incubated at 37℃ for 30 minutes. 100% methanol was added to fix the culture and terminate the reaction. After fixation, the cells were washed three times with PBS. The degree of antibody internalization was imaged using a fluorescent microscope in the green filter area (Alexa488). During imaging, intracellular nuclei were stained with DAPI to confirm the position of each cell. The experimental results are shown in Figure 13a.

[0296] From the above experimental results, it was confirmed that all four antibodies tested were internalized frequently in the experiment using the MCF-7 cell line, and in particular, MKJP2 and 1564 were internalized more frequently than other clones.

[0297] 17-2:MCF-7 internalization assay-C04, F06, VH5, VH16, VH35, VH9, VH2, VH7, VH32 To analyze the cell surface IGF1R binding of 1564 variants with altered binding strength to IGF1R, FACS analysis was performed using MCF-7 cell line expressing IGF1R. IGF1R antibody made from double antibody in scFv form was treated with 2x10E5 MCF7 cells at 10ug / mL concentration for 30 minutes. After washing with PBS buffer containing 1% BSA, the cells were treated with FITC-conjugated secondary antibody to detect human antibody for 1 hour. After washing with PBS buffer, extracellular binding and internalization of variants with altered binding strength were confirmed by FACS analysis.

[0298] As shown in the results of Table 25, the double antibody containing the 1564 IGF1R antibody showed increased internalization and intensity at 37℃ 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 into cells in a binding-dependent manner.

[0299] [Table 25] TIFF0007625305000076.tif90170

[0300] 17-3: Analysis of internalization of human brain endothelial cells We analyzed whether the bivalent and monovalent forms of the 1564 clones constructed in Examples 14-2 and 14-4 were internalized into human-derived brain endothelial cells (primary human microvascular brain endothelial cells (HMBEC)). Therapeutic antibody IgG (11F11) was used as a negative control.

[0301] HMBEC (Cell Systems, cat#:ACBRI376) were plated at 90% confluency in a 12-well plate and then treated with a test antibody. The next day, the cells were fixed with 4% paraformaldehyde, rinsed with PBS, and blocked and permeabilized with a solution containing 3% BSA and TritonX for 50 minutes. After rinsing with PBS, an antibody against human Fc (Goat anti-human antibody) was administered for 2 hours and 30 minutes, and after rinsing with PBS, a secondary antibody against the primary antibody was administered for 1 hour. After rinsing with PBS, the cells were stained with Hoechst at a concentration diluted 1:1000 for 10 minutes for nuclear staining. The results were analyzed by confocal microscopy under LSM 780 NLO EC Plan-Neofluar 100X / 1.3 Oil conditions. The experimental results are shown in Figure 13b.

[0302] The bivalent and monovalent forms of the 1564 clone showed increased internalization compared to the negative control therapeutic antibody (11F11). This result suggests that the anti-IGF1R antibodies, which are bispecific antibodies of various forms linked to a therapeutic antibody, can effectively internalize the therapeutic antibody into brain endothelial cells that make up the BBB, thus ultimately increasing the ability of the therapeutic antibody to cross the BBB.

[0303] 17-4: Analysis of cellular fate in human brain endothelial cells If the antibody is internalized and then colocalizes with a lysosome-associated marker in the cell, it will be degraded in the brain endothelial cell and will not be able to cross the BBB. In contrast, if the antibody is colocalized with a marker known to be related to early endosomes or BBB crossing associated with exocytosis, it is expected that the antibody will be internalized in the brain endothelial cell and then cross the BBB via receptor-mediated transcytosis.

[0304] Among the antibodies tested in Example 17-2, 1564 bivalent form was treated with HMBEC in the same manner, and then the antibody was analyzed to determine which cellular components it colocalized with within the cells. However, after blocking and permeabilization, the following antibodies were administered simultaneously with the goat anti-human antibody that detects the administered antibody.

[0305] -Anti-Cathepsin D: Lysosomal marker -Anti-Caveolin-1: A marker for caveolin-mediated transcytosis (thought to be the main mechanism of BBB crossing) -Anti-EEA1: early endosome marker The remaining procedures were the same as in Example 17-2, except that secondary antibodies against the above-mentioned markers were used.

[0306] The analysis results are shown in Figure 13c. The 1564 clone did not colocalize with Cathepsin D at all in the form of a bispecific antibody, but instead colocalized with caveolin-1 and EEA1 at the cell membrane and inside the cell. This result indicates that after the 1564 clone is internalized, it can pass through the BBB via RMT without going through the intracellular degradation mechanism.

[0307] Example 18. Analysis of the effect of anti-IGF1R antibodies on IGF1R signal transduction 18-1: Proliferation assay of MCF-7 cell line using IGF1R Whether the anti-IGF1R antibody according to the present invention inhibits the binding between IGF1R (IGF1 receptor) and its ligand IGF1 was confirmed using the effect of IGF1 on cell proliferation.

[0308] The minibody antibodies of the 996, 1226, 1564 and MKJP2 clones prepared in Example 14-1 were each diluted 5-fold from 400 nM to prepare diluted samples, and then L Concentration of 25μ L IGF1, 25μ each L MCF-7 cell line expressing IGF1R was cultured, and on the day of the experiment, the medium was removed and the cells were subcultured. 20,000 cells (50 μg / well) were placed in each well of a 96-well plate containing IGF1 and the test antibody. L (corresponding to the above) was processed.

[0309] After culturing for 3 days at appropriate temperature and humidity, 10 μL of CCK-8 reagent was added to measure the cell growth. L The cells were treated one by one and incubated in a CO2 incubator for 4-5 hours. te The absorbance was measured at a wavelength of 450 nm.

[0310] The experimental results are shown in FIG. 14a. From the above experimental results, it was confirmed that the antibody according to the present invention does not inhibit the cell proliferation of MCF-7 cells caused by IGF1 signaling to IGF1R. C The anti-IGF1R antibody from Ione 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 pass through the BBB, but at the same time does not inhibit IGF1-induced signaling in the body, so it was confirmed that the antibody of the present invention can be used as a BBB shuttle.

[0311] 18-2: Analysis of inhibition of signaling components by IGF1R in MCF-7 cell line In order to confirm whether the anti-IGF1R antibody according to the present invention is involved in the receptor and downstream signaling components of the signaling when IGF1 bound to IGF1R-expressing cells transmits signaling into the cells, an experiment was carried out. That is, the anti-IGF1R antibody was administered to MCF-7 cell line expressing IGF1R, and the amount of total IGF1R, phosphorylated IGF1R, total Akt, and phosphorylated Akt, which are downstream factors of IGF1R, in the cells were analyzed.

[0312] After culturing MCF-7 cells, the culture medium was changed to a serum-free culture medium 20 hours before treatment with anti-IGF1R antibody. The MCF-7 cell line was treated with 100 nM of each of the minibody antibodies of the 996, 1226, 1564 and MKJP2 clones prepared in Example 4-1, and then 200 ng / ml was administered one hour later. LAfter 20 minutes, the cells were washed with PBS and lysed with M-PER supplemented with 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 then transferred to a PVDF membrane. Blocking was performed with PBST (0.1% Tween 20) containing 5% BSA for 1 hour at room temperature with gentle shaking, and then primary antibodies against IGF1R or Akt were treated overnight at 4℃ with gentle shaking. Beta actin antibody was used as a loading control. After washing, secondary antibodies were treated for 1 hour at room temperature with gentle shaking, and then washed, ECL solution was added, and the cells were analyzed by Image Quantitation. TM L AS The signal was confirmed using 4000. The experimental results are shown in Figure 14b.

[0313] 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.

[0314] 18-3: Analysis of inhibition of signaling components by IGF1R in mouse brain endothelial cells In order to confirm whether the anti-IGF1R antibody according to the present invention is involved in the receptor and downstream signaling components of IGF1 signaling delivered into mouse-derived brain endothelial cells, an experiment was carried out. That is, 11F11-1564, 3A9-1564 (CH11F11 and ch3A9, which are described in Korean Patent Publication No. 2018-0081465 as anti-a-syn monoclonal antibodies) prepared by the method of Example 14-2 and IgG form of 1564 clone prepared by the method of Example 14-3 were administered to bEND3 cell line, which was confirmed to express IGF1R, and the amount of total IGF1R, phosphorylated IGF1R, total Akt, which is a downstream factor of IGF1R, and phosphorylated Akt in the cells were analyzed.

[0315] After culturing bEND3 cells, the culture medium was changed to a serum-free culture medium 20 hours before treatment with anti-IGF1R antibody. The bispecific antibodies of 1564 and MKJP2 clones of Example 14-2 were each treated at 100 nM to the bEND cell line, and then 200 ng / ml were added one hour later. LAfter 20 minutes, the cells were washed with PBS and lysed with M-PER supplemented with 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 then transferred to a PVDF membrane. Blocking was performed with PBST (0.1% Tween 20) containing 5% BSA for 1 hour at room temperature with gentle shaking, and then primary antibodies against IGF1R or Akt were treated overnight at 4℃ with gentle shaking. Beta actin antibody was used as a loading control. After washing, secondary antibodies were treated for 1 hour at room temperature with gentle shaking, and then washed, ECL solution was added, and the cells were analyzed by Image Quantitation. TM L AS The signal was confirmed using 4000.

[0316] The experimental results are shown in FIG. 14c. 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.

[0317] Example 19. Non-toxicity analysis of anti-IGF1R antibodies 19-1: ADCC analysis of IgG-type IGF1R antibodies An experiment was conducted to confirm whether the anti-IGF1R antibody of Example 14-3 binds to the surface of IGF1R-expressing cells in the form of IgG and affects cell death through IGF1R-dependent binding. That is, an ADCC reporter bioassay kit was used to analyze whether the anti-IGF1R antibody 1564 clone binds to an IGF1R-expressing cell line, activating natural killer cells (NK cells) and adversely affecting IGF1R-expressing cells.

[0318] MCF-7 cells overexpressing IGF1R and SKBR3 cells expressing IGF1R at low levels were cultured, and 5000 cells were dispensed into a 96-well plate per well 20 hours before antibody treatment. The cultured cells were replaced with RPMI / 1640 medium containing 4% low IgG serum, and IgG form of the 1564 clone was diluted 8-fold from 133.3nM and added to each well. The stabilized ADCC effector cells were added to each well, cultured for 6 hours, and then left at room temperature for about 10 minutes. The previously prepared Bio-Glo luciferase assay reagent was added to each well, and the luminescence was measured using a PHERAster FS BMG LABTECH device to analyze the degree of ADCC induction. The experimental results are shown in Figure 15a.

[0319] From the above experimental results, it was confirmed that the antibody according to the present invention, particularly clone 1564, binds to MCF-7 cells, which are IGF1R overexpressing cell lines, and SKBR3 cells, which express IGF1R at a low level, but does not induce ADCC by effector cells.

[0320] 19-2: Analysis of IGF1R levels in the brain after repeated administration of anti-IGF1R antibody Antibodies used as BBB shuttles bind to target receptors on brain endothelial cells to enhance the passage of therapeutic antibodies, but do not alter the levels of the receptors. Down regulation of the target receptors can affect the function of the target in the brain, causing side effects.

[0321] We analyzed whether the anti-IGF1R antibody of the present invention affects the IGF1R level in the brain even after repeated administration. The Parkinson's disease therapeutic antibody prepared in Example 14-6, the bivalent bispecific antibody linked to the 1564 clone, the Parkinson's disease therapeutic antibody alone, and IgG as a negative control were repeatedly administered to Parkinson's model mice every week for three months, and the IGF1R level in the brain tissue was measured. WThe results were analyzed by ester blotting.

[0322] Brain tissues from three mice per group were perfused with PBS, homogenized, and then incubated for 10 min. μ The lysate (g) was loaded onto a 4-12% Bis-Tris gel, run, and then transferred to a PVDF membrane. The remaining procedures were the same as in Example 19-1.

[0323] The experimental results are shown in Figure 15b. The bispecific antibody composed of the 1564 clone showed similar levels of IGF1R as the Parkinson's disease therapeutic antibody alone and IgG administration groups. This result indicates that the 1564 clone does not cause serious changes in the IGF1R level in the brain even after repeated administration, and therefore the antibody is expected to have low side effects when used as a BBB shuttle.

[0324] 19-3: Analysis of intracerebral distribution of anti-IGF1R antibody after administration The BBB shuttle binds to receptors on the surface of brain endothelial cells to deliver therapeutic antibodies into the brain, but it must bind less to receptors on the surface of normal cells in the brain. If the BBB shuttle binds more to normal cells expressing antigens in the brain, less of the therapeutic antibody bound to the shuttle will reach the disease target.

[0325] In order to analyze the intracerebral distribution of the anti-IGF1R antibody of the present application, the distribution of the anti-IGF1R antibody was analyzed by immunostaining in the brain of the animal in the in vivo experiment performed in Example 21-2. SD rats administered with the bivalent form of the 1564 clone were perfused with saline using the transcardiac perfusion method. The left hemisphere was fixed in 10% neutral-buffered formalin for 24 hours. The brain was rinsed with 1X PBS and frozen in a solution containing 30% sucrose for 24 hours. The brain was placed in OCT, frozen, and stored at -80°C until sectioning. Sections were performed in the coronal direction at a thickness of 25 μm and collected in 1X PBS containing 0.1% sodium azide in a 24-well Petri dish. The tissue was blocked and permeabilized by incubating it at room temperature for 1 hour with Dako serum free protein block (X0909, DAKO) containing 0.3% Tween-20 using the free floating method. For the primary antibody, biotinylated human Fc antibody (BA3080, Vector Labs) was used at a ratio of 1:50 and incubated overnight at 4℃. After washing three times with TBS, the sections were treated with either RECA-1 (ab9774, AbCam), an antibody against endothelial cells, at a ratio of 1:2000 or NeuN (MAB377, Millipore), an antibody against neurons, at a ratio of 1:250 for 2 hours. After washing, the sections were treated with secondary antibodies conjugated with Alexa488 for 45 minutes. After washing, the sections were mounted on glass slides and nuclei were stained with ProLong Gold containing Dapi or Dako fluorescent mounting media containing Hoechst 33258. The cortex and hippocampus were analyzed using the appropriate fluorescence wavelengths using a confocal microscope. The analysis results are shown in Figure 15c.

[0326] The results showed that the bispecific antibody bound to the 1564 clone did not bind to normal neurons, but instead specifically bound to brain endothelial cells. Therefore, it is expected that the original anti-IGF1R antibody does not bind to normal cells in the brain, but only enhances the BBB-crossing ability of the therapeutic antibody, resulting in almost no side effects and improved therapeutic efficacy of the therapeutic antibody.

[0327] Example 20. In vitro BBB crossing ability analysis of anti-IGF1R antibodies 20-1.BBB penetration analysis of bivalent antibodies in sv-ARBEC-derived BBB model The in vitro BBB crossing ability of the bispecific antibodies prepared in Example 14-2 using the antibodies from Examples 11 and 12 was evaluated in a BBB model consisting of Sv-ARBEC. Sv-ARBEC was plated in a single layer on a permeable membrane, and the integrity of the system was evaluated in advance based on the resistance value (TEER) and sucrose crossing of the BBB system. At this time, the sv-ARBEC was treated with rat astrocyte culture medium (RAS-CM), which was confirmed to be useful for the integrity of the system. After the test antibodies 1564, 48G5, 54H4, 60H6, and B11 were treated on the membrane, the amount of antibody in the bottom chamber was analyzed by mass spectrometry after 90 minutes. For mass spectrometry, the signature peptide of each antibody Fc and scFv was analyzed and then used. At this time, Parkinson's disease treatment antibody alone (11F11) and the antibody plus Herceptin were treated with 11F11 and 11F11. TM A bispecific antibody conjugated to the scFv form of a biosimilar of was used as a negative control. The calibration limit of the system was determined by passing the A20.1 antibody (produced by the National Research Council), which is known not to cross the BBB. The results derived from mass spectrometry were substituted into a formula known in the prior art to determine the Papp value, which indicates the degree of BBB crossing ability in vitro.

[0328] The analysis results are shown in Figure 16a. All the test antibodies except B11 showed higher in vitro BBB crossing ability than the negative control group. In particular, the 1564 clone showed higher BBB crossing ability than the other clones. This indicates that the 1564, 48G5, 54H4, and 60H6 clones and the bispecific antibody formed by linking the clones may have higher in vivo BBB crossing ability than the single antibodies.

[0329] 20-2. Analysis of BBB penetration ability of monovalent antibodies in sv-ARBEC-derived BBB model Using the same model as 20-1, the in vitro BBB crossing ability of the monovalent antibody prepared based on Example 14-4 and the bivalent antibody prepared based on Example 14-2 was analyzed. The bivalent and monovalent antibodies prepared from the 1564 clone and the negative control Parkinson's disease therapeutic antibody (3A9) were passed through the sv-ARBEC BBB system and the crossing amount was analyzed. The analysis results are shown in Figure 16b.

[0330] The bivalent and monovalent forms of the 1564 clone all showed higher in vitro BBB crossing ability than the single antibody 3A9, and in particular, the bivalent form had a higher crossing ability than the monovalent form, indicating that the 1564 clone can improve the in vitro BBB crossing ability of therapeutic antibodies linked to it in various forms.

[0331] 20-3. Analysis of BBB penetration ability of anti-IGF1R antibodies in a human iPSC-derived BBB model The human stem cell-derived BBB model shows a higher resistance value (TEER) than rat or mouse cell-derived BBB systems and expresses all the various markers identified in the BBB. Therefore, the BBB tightness of the human stem cell-derived model is higher than that of the animal cell-derived BBB, and the model closely reproduces the human BBB.

[0332] Human amniotic fluid-derived iPSCs (AF-iPSCs) were reprogrammed into iPSCs by introducing oriP / EBNA1 episomal vectors encoding OCT4, SOX2, c-Myc, KLF4, NANOG and LIN28. The resulting colonies were treated with KO DMEM / F12, KOSR, glutamax, NEAA, and beta mercaptoethanol to produce endothelial cells in a pre-differentiation state, and then cultured in serum-free endothelial differentiation medium and 1% PDS, 20 ng / ml. L The cells were treated with bFGF to differentiate into brain endothelial cells. The acquisition of amniotic fluid cells and the establishment of a BBB system using the cells were carried out at the National Research Council. After confirming the integrity of the system by the resistance value and sucrose passage value, the passage ability was analyzed in the form of a bispecific antibody in which an anti-IGF1R antibody was bound to a Parkinson's disease therapeutic antibody in the form of an scFv. The test antibodies were the bivalent form of the 1564 clone, the monovalent form of the 1564 clone, the bivalent form of the F06 clone, and the bivalent form of the C04 clone. 11F11, a Parkinson's disease therapeutic antibody, was used as a negative control. The antibodies that passed through the BBB system were analyzed in the same manner as in Example 20-1.

[0333] The analysis results are shown in Figure 16c. All of the test clones showed higher BBB penetration ability than the negative control group, and in particular, F06 bivalent and C04 bivalent showed up to 15-fold higher BBB penetration ability than the single antibody. The results indicate that the anti-IGF1R antibody of the present application can more efficiently penetrate the human BBB system than the single antibody.

[0334] Example 21. In vivo BBB crossing ability analysis (co-localization assay) of anti-IGF1R antibodies 21-1. Colocalization with the minibody's brain vessel In order 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.

[0335] Specifically, PBS buffer or 10 mg / kg of IgG control and the minibody antibodies of clones 996, 1226 and 1564 prepared in Example 14-1 were administered once to the tail vein of male 6-8 week old BALB / c mice. After 4 hours, the mouse brain was intracardially perfused with a sufficient amount of 0.9% NaCl solution and 4% paraformaldehyde. The fixed brain was excised and sectioned at 20 μm, and then co-stained with vascular markers anti-mouse CD31 and anti-human Fc antibody to confirm the colocalization of cerebral blood vessels and IGF1R test. CD31 was imaged under a fluorescent microscope using an Alexa488-conjugated secondary antibody, and human Fc was imaged using an Alexa 594-conjugated secondary antibody.

[0336] The experimental results are shown in FIG. 17a. From the above experimental results, it was 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.), and the brain tissue was stained with a vascular marker (anti-CD31, green) and a human antibody (anti-human Fc, red). As a result, the non-blocking antibodies of the present invention showed a higher degree of colocalization than the IgG control group.

[0337] 21-2. 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) in which the 1564 clone was bound in a bivalent form via the tail vein, and the antibody levels in the CSF and brain were analyzed by mass spectrometry 24 hours later. The mass spectrometry method was the same as that in Example 20-1.

[0338] The bispecific antibody bound to the 1564 clone showed higher CSF and brain penetration than the therapeutic antibody not bound to the anti-IGF1R antibody, and this efficacy was confirmed at doses of 10 and 30 mg / kg. At a dose of 30 mg / kg, the bispecific antibody showed up to 4.5 times higher brain penetration than the monospecific antibody.

[0339] The 1564 clone was prepared in bivalent and monovalent forms as described in Examples 14-2 and 14-4, and then administered at 30 mg / kg or 60 mg / kg in the same manner as above, and the antibody levels in the CSF and brain were analyzed 24 hours later. The two forms of bispecific antibodies combining the 1564 clone showed higher CSF and brain penetration than the monospecific antibodies. In particular, the bivalent form showed higher BBB penetration than the monovalent form, with a maximum increase of approximately 5-fold in brain penetration.

[0340] The results in FIG. 17b show that clone 1564 enhances the ability of a therapeutic antibody to cross the BBB in vivo when conjugated to the therapeutic antibody in various forms.

[0341] The affinity variant of the 1564 clone constructed in Example 2 was expected to have increased serum PK compared to the parental clone. Therefore, it was expected that the affinity variant would remain in serum for a long period of time and maintain continuous BBB inflow, thereby improving its BBB passing ability. The affinity variant constructed in bivalent form in Example 14-2 or in monovalent form in 4-4 was administered at 30 mg / kg to SD rats via the tail vein, and blood was collected from the orbital venous plexus at 0, 24, and 48 hours. The test antibodies were divided into two experiments based on the backbone of the therapeutic antibody, and the bispecific antibodies of the variants used in the experiments are shown in Table 26 below.

[0342] Table 26. Bispecific antibodies used in in vivo BBB crossing assay TIFF0007625305000077.tif93170

[0343] The amount of antibodies in the blood was analyzed by ELISA. After coating a 96-well plate with goat anti-human Fc antibody, appropriately diluted samples were treated and detected with anti-human Fab HRP conjugated antibody.

[0344] The analysis results are shown in Figure 17d. As a result, in the first test group, 1564 monovalent, F06 monovalent, and C04 monovalent showed longer serum PK than the parental 1564 clone bivalent. In the second test group, except for the VH35 bivalent group, the bivalent forms of VH2, VH5, VH7, VH9, VH16, and VH32 showed increased serum PK compared to the parental 1564 bivalent.

[0345] To analyze the BBB crossing ability of the groups, CSF was extracted from the rats at 48 hours and analyzed by the same ELISA, and the analysis results are shown in Figure 17e.

[0346] In the first study group, 1564 monovalent, F06 monovalent, and C04 monovalent forms, which showed increased serum PK, showed increased CSF antibody levels compared to parental 1564 bivalent. In the second study group, bivalent VH2, VH5, VH7, VH9, VH16, and VH32, which also showed increased serum PK, showed increased CSF antibody levels compared to parental 1564 bivalent. VH35 showed shortened serum PK and CSF antibody levels compared to parental 1564 bivalent.

[0347] The results of Figures 17d and 17e show that serum PK is an important factor in the BBB crossing ability of antibodies due to continuous antibody influx into the BBB, and that the BBB crossing ability of bispecific antibodies with BBB shuttle and increased serum PK is increased. In particular, the F06 monovalent form, which has the highest CSF antibody amount, showed about 5 times higher CSF crossing ability than the parental 1564 bivalent. Since the 1564 bivalent antibody showed about 3 times higher CSF crossing ability than the monovalent antibody in Examples 18-2 and 18-3, it is expected that the F06 monovalent form will show up to about 15 times higher BBB crossing ability than the monovalent antibody.

[0348] Example 22. Deamidation of IGF1R antibody Deamidation, for example, involves attacking the side chain peptide bond of asparagine to produce a symmetrical succinimide intermediate, which is then hydrolyzed to either aspartic acid or isoaspartic acid. D Since deamination can affect the sustained activity of the protein, we sought to replace light and heavy amino acids with other amino acids to prevent deamination and at the same time ensure long-term stability.

[0349] To confirm the deamination site identified by in silico, the 1564 IGF1R antibody was incubated at 40°C and the deamination sites were analyzed by mass analysis. D As a result of checking the degree of deamination, it was confirmed that 4.6-47.3% deamination reaction occurred at the light chain CDR2 and CDR3 and heavy chain CDR2 sites as shown in Figure 18a. To prevent deamination, amino acids were substituted at each site as shown in Figure 18b, and the change in binding ability with IGF1R protein due to each amino acid substitution was confirmed. Mutants showing the same binding ability as WT 1564 were selected, and it was confirmed that mutants with three or four mutations had the same IGF1R binding ability as WT 1564. The introduction of such mutants means that the binding ability can be maintained under storage conditions in the formulation buffer and a stable form can be maintained for a long time. The analysis results are shown in Table 27.

[0350] [Table 27] TIFF0007625305000078.tif60170

[0351] Example 23. Epitope mapping of anti-IGF1R antibodies 23-1. ELISA of anti-IGF1R antibodies and boiled and native IGF1R proteins We attempted to confirm whether anti-IGF1R antibodies recognize linear or conformational epitopes. ELISA was performed using bivalent bispecific antibodies 1564, 48G5, 54H4, 60H6, and B11 against native human IGF1R ECD protein or boiled IGF1R. The ELISA method was the same as that described in Example 15. The analytical results are shown in Table 28.

[0352] [Table 28] TIFF0007625305000079.tif64170*N / A:Not available

[0353] The clones showed binding affinity to native human IGF1R ECD similar to the results in Example 15, but did not bind to boiled human IGF1R ECD, whose tertiary structure had been destroyed by heating. This indicates that the anti-IGF1R antibodies of the present application bind to a non-linear conformational epitope.

[0354] 23-2. Epitope mapping of anti-IGF1R antibodies To analyze the conformational epitope of the 1564 clone, alanine scanning was performed as follows. An IGF1R library with 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 contains mutations in which IGF1R surface residues are replaced with alanine. The prepared library was transfected into OVCAR3 cells. Cells with confirmed IGF1R expression were treated with 1564 antibody, and then fluorescently labeled with a secondary antibody labeled with DyLight650. The labeled cells were classified according to the presence or absence of IGF1R expression and the presence or absence of IGF1R binding, and then RNA deep sequencing was performed on them using Illumina HiSeq technology to analyze the frequency of each alanine mutation in the cell group. The frequency count was normalized to the results for cells expressing wild type IGF1R, and the relative frequency was calculated, and the mutations whose counts were decreased in the 1564-labeled cell group were selected. Based on these observations, it was found that the epitope of 1564 is located in the FN2 domain, and the residues that belong to this domain are Y775, P776, F778, R650, S791, and L798. The results and the sequence recognized by the 1564 clone are shown in Figure 19. Since this residue is not involved in IGF1 binding according to the prior art, the results faithfully explain the properties of 1564 in Example 23-1.

[0355] Example 24. Comparison of antigen binding strength between single and double antibodies 24-1: Binding ability of single and double antibodies to a-syn antigen When an IGF1R antibody was linked in the form of an scFv to an a-syn antibody in the form of an IgG, the effect on the binding ability of the a-syn antibody was analyzed.

[0356] 1ug / m L A-syn aggregate was coated on a 96-well plate for 18 hours at a concentration of 1000 nM, washed, and then each antibody was diluted 5-fold from 400 nM and bound to the plate. The bound antibody was conjugated with anti-human Fc-HRP, and TMB solution was added to develop color, and the degree of antibody binding was confirmed.

[0357] As shown in the results of Figure 20a, it was confirmed that the binding strength to a-syn aggregates was the same for single antibodies and double antibodies.

[0358] 24-2: Binding ability of single and double antibodies to IGF1R antigen In order to compare the degree of binding of a-syn monoclonal antibody and dual antibody to the IGF1R antigen, an experiment was carried out in the same manner as in Example 22.

[0359] As shown in the results of FIG. 20b, 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.

[0360] 24-3: Binding strength analysis of a-syn humanized antibodies An experiment was carried out in the same manner as in Example 24-1 to analyze the difference in binding ability between the chimeric bibody and the humanized bibody.

[0361] As shown in the results of Figure 20c, the humanized bibody had a binding ability to a-syn aggregates at a level similar to that of the chimeric bibody, and it was confirmed that the monovalent bibody containing one IGF1R scFv also showed a binding ability similar to that of the chimeric antibody.

[0362] An experiment was conducted in a similar manner to Example 24-2 to analyze the IGF1R binding affinity between chimeric and humanized double antibodies. As a result, as shown in Figure 20d, it was confirmed that all double antibodies showed the same binding affinity, and that single antibodies without IGF1R scFv did not bind.

[0363] 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 a-syn aggregates and IGF1R was not altered, meaning that it retained the same activity.

[0364] 24-4: Comparison of phagocytic activity of single and double antibodies Phagocytosis refers to the process by which various receptors in macrophages remove unnecessary extracellular substances. Various protein aggregates can cause immune and inflammatory reactions, which can have a detrimental effect on the human body. In particular, when an antibody is administered to remove a-syn aggregates, it is known that this is promoted by the interaction between the Fc region of the antibody and the FcrR on the cell surface. For this reason, we compared the phagocytosis activity of a single antibody and a double antibody linked to IGF1R scFv.

[0365] To compare the phagocytosis of single and double antibodies, mouse-derived BV-2 microglial cells were used. BV-2 cells were cultured in RPMI1640 medium, prepared at 2×10E6 cells / ml, and plated in a U-bottom 96-well plate at 100 μ The solution was dispensed in 10 L portions. μ g / m L a-syn aggregates and 25 μ g / m LThe antibodies were diluted in RPMI1640 medium, mixed, and left at room temperature for 20 minutes. The a-syn aggregates and antibody mixture was applied to BV-2 cells and left for 15 minutes. The cells were centrifuged at 1200 rpm to remove the a-syn aggregates in the supernatant, and washed three times with PBS, pH 2.5 buffer to remove aggregates or antibodies bound to the cell surface. The cells were fixed with 4% paraformaldehyde and washed with PBS buffer. To confirm intracellular phagocytosis of aggregates and antibodies, the cells were centrifuged at 0.5% T The cell membrane was loosened by adding riton X-100, washed with PBS buffer, and then treated with pan-a-syn antibody for 1 hour. The bound pan-a-syn antibody was anti-rabbit- A After 1 hour of treatment with lexa-488 antibody, the aggregates that had entered the cells by macrophages were confirmed by FACS analysis.

[0366] As shown in Figure 20e, normal human IgG had no effect on macrophages, but a-syn antibody treatment increased macrophages of a-syn aggregates. Comparing single and double antibodies, macrophages were observed at similar levels, confirming that the scFv-type IGF1R antibody bound to the C-terminal site of IgG did not affect the action of a-syn antibody.

[0367] Example 25. Efficacy evaluation of bispecific antibodies A bivalent bispecific antibody was prepared from chimeric 11F11 antibody and 1564 clone scFv according to Example 14-6, and 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 single antibody or human IgG or the same molar amount of bivalent bispecific antibody was intraperitoneally administered every week for three months. Five mice were used per group, and non-transgenic littermate mice were used as a control group. Then, perfusion was performed as follows.

[0368] After the last administration, the animals were anesthetized with chloral hydrate according to humane regulations and cardiac perfused with 0.9% saline for pathological analysis in the brain. One half of the perfused brain (saggital section) was then stored in 4% paraformaldehyde in phosphate buffer (pH 7.4, 4°C) until analysis, and the other half was immediately stored frozen (-70°C).

[0369] Pathological analysis was performed as follows. The paraformaldehyde-fixed brain halves were cut into 40 μm-thick serial sections using a vibratome in a free-floating manner. To confirm the expression level of alpha-synuclein in the brain of each treatment group, sections including the cortex, hippocampus, and striatum were incubated overnight at 4°C with alpha-synuclein antibodies (p129 α-syn antibody, an aggregate marker, Abcam, ab59264, or total alpha-synuclein antibody, Cell Signaling Technology, #2642). Alternatively, to analyze the activity level of astrocytes, the sections were incubated overnight at 4°C with GFAP (glial fibrillary acidic protein) (AB5804, M illipore) or to confirm the degree of brain inflammation (neuroinflammation), an antibody against IL-1β (ab9722, A bcam), respectively. Alternatively, to analyze the degree of neuronal death in hippocampus, an antibody against NeuN (Chemicon, #MAB377) was used. After incubation with the primary antibody, the sections were treated with biotin-conjugated goat anti-rabbit IgG (1:100, Vector Laboratories) and Avidin D-horseradish peroxidase (1:200, ABC Elite, Vector Laboratories), and detected with DAB (diaminobenzidine). Each immunostained section was observed under a bright-field microscope to measure the optical density. The results are shown in Figures 21a to 21e.

[0370] 25-1. Analysis of alpha-synuclein reduction ability of chimeric and bispecific antibodies FIG. 21a shows that the chimeric 11F11 antibody and the bivalent bispecific antibody of the chimeric antibody and the 1564 clone were human α- s To determine whether alpha-synuclein aggregates can be removed in a mouse animal model (TG) that overexpresses yn, the mice were administered antibodies, and then the cortex and hippomcapus in mouse brain tissue were stained with p-129 α-Syn antibody to measure the level of the aggregates. p-129 α-syn is a marker for aggregates in which the 129th residue is phosphorylated, and appears as dark brown dots or aggregates in the stained tissue.

[0371] As shown in FIG. 21a, the IgG-treated group showed a higher staining level of p-129 than the non-tg control group (#: one way ANOVA, p<0.01). In contrast, the staining level of p-129 α-syn or aggregates was significantly reduced in the groups treated with single or dual specific antibodies. In particular, the degree of reduction in the dual specific antibody-treated group was greater than that of the chimeric 11F11 antibody in hippocampus (*: one way ANOVA, p<0.05). FIG. 21b shows the same experiment as FIG. 21a, but stained with total alpha-synuclein antibody as a marker. The detection of total alpha-synuclein indicates that the antibody of the present invention has the ability to clear alpha-synuclein itself and to inhibit cell-to-cell transmission of the antibody. From another perspective, it can be interpreted as being able to inhibit the formation of monomer aggregates or to remove all monomers. The increased human alpha-synuclein in TG mice was reduced by administration of single and bispecific antibodies compared to the IgG administration group, and in particular, the efficacy of the bispecific antibody was superior to that of the single antibody in hippocampus.

[0372] The results show that the chimeric 11F11 antibody and the bispecific antibody effectively reduce alpha-synuclein and its aggregates in Parkinson's disease animal models even at a low dose of 2.5 mg / kg. In particular, the bispecific antibody has superior efficacy compared to the monospecific antibody, suggesting that the bispecific antibody can reach more of the brain and effectively treat the disease compared to the monospecific antibody due to its improved BBB crossing ability.

[0373] 25-2. Analysis of the ability of chimeric and bispecific antibodies to reduce astroglosis and inflammatory cytokine levels Gliosis is a non-specific reaction that occurs in glial cells in response to damage to the central nervous system, triggered by substances such as BBB damage, TGF-beta, or interleukins. Representative examples include astrogliosis, and GFAP protein is used as a marker. Therefore, chimeric 11F11 antibody and bispecific antibody of chimeric antibody and 1564 clone were administered to mice to analyze the effect of reducing astrogliosis and reducing the release of inflammatory cytokines that trigger it. The analysis results are shown in Figures 21c and 21d.

[0374] Figure 21c shows the results of staining mouse brain tissue with GFAP (astrogliosis) antibody as a marker after administration of the chimeric 11F11 antibody produced in one example of the present invention or the bispecific antibody of the same antibody and clone 1564 to determine whether the antibody can reduce astrogliosis in vivo. The monospecific antibody and bispecific antibody suppressed astrogliosis compared to the IgG control group, and it was confirmed that the efficacy of the bispecific antibody was particularly superior to that of the monospecific antibody in striatum.

[0375] Fig. 21d shows the results of staining mouse brain tissue with IL-1 beta antibody as a marker to determine whether the chimeric 11F11 antibody prepared in one embodiment of the present invention or the bispecific antibody of the antibody and the 1564 clone can reduce inflammatory cytokines in vivo. IL-1 beta induces inflammation, leading to the death of various nerve cells and inflammatory responses. In the hippocampus of mice administered with the antibody of the present invention, IL-1 beta was reduced in the single antibody and bispecific antibody groups compared to the IgG control group, and in particular, the bispecific antibody had a significantly greater reducing ability than the single antibody (##: One-way ANOVA, p<0.005; *: One-way ANOVA, p<0.05).

[0376] As shown in the figures, it was revealed that the antibody of the present application reduced astrogliosis and reduced the release of the inflammatory cytokine IL-1beta that triggers astrogliosis, as compared to the control group.

[0377] 25-3. Analysis of the ability of chimeric and bispecific antibodies to reduce neurodegeneration Previous literature has confirmed that alpha-synuclein induces neurotoxicity and inflammatory responses that result in brain cell death. We analyzed whether the present monospecific and bispecific antibodies could inhibit alpha-synuclein-induced brain cell death in vivo.

[0378] Staining of the cortex and hippocampus with the neuronal marker NeuN revealed that both the single and dual specific antibodies reduced the degree of 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 21e.

[0379] Example 26. Fc engineering increases half-life and improves BBB penetration FcRn is an important cell membrane receptor that increases the half-life of antibodies by drawing them into cells and circulating them to prevent them from being dissolved when they circulate in the blood vessels. It is well known that the ability to cross the BBB depends on the concentration in the blood vessels, although the activity of the transcytosis antibody is also important for the ability to cross the BBB. For this reason, in order to increase the half-life of the double antibody, we created a double antibody that has increased its binding strength with FcRn by changing the 428th amino acid in the Fc region from methionine (Met) to leucine (Leu). h uman expresses FcRn T WT and M428L biantibodies were administered to mice at a concentration of 10 mg / kg, and the half-life was increased by approximately 50% as shown in Figure 22. To reconfirm the increase in half-life, WT biantibody, bivalent M428L biantibody, and monovalent M428L biantibody were administered to monkeys and the PK profile was analyzed. As shown in Figure 22a, the blood concentration of the WT biantibody dropped rapidly after 168 hours, whereas the M428L biantibody, which has high binding affinity with FcRn, maintained a higher blood concentration than the WT. The half-life of the M428L biantibody was increased by approximately 1.5 days compared to the WT biantibody. In particular, the monovalent M428L biantibody was the most excellent in terms of clearance, and the WT biantibody showed the fastest clearance (Figure 22b).

[0380] To verify the improvement of BBB crossing due to the half-life increase, CSF was extracted 24 hours after antibody administration and the amount of antibody in the CSF was analyzed. After coating with 100ng / ml IGF1R under refrigerated conditions for 18 hours, CSF was added and the antibody bound to IGF1R was detected. As shown in Figure 22c, the M428L bibody, which had a high amount of antibody in the blood, had a high BBB crossing amount, and the monovalent M428L bibody showed an improved crossing ability compared to the bivalent M428L bibody, due to its excellent BBB crossing ability.

Claims

1. a) an anti-α-synuclein (anti-α-syn) antibody or an antigen-binding fragment thereof; and b) (i) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (CDR) 1 (H-CDR1) consisting of SEQ ID NO: 1, a H-CDR2 consisting of SEQ ID NO: 13, and a H-CDR3 consisting of SEQ ID NO: 52; and (ii) a light chain variable region (VL) comprising a light chain CDR1 (L-CDR1) consisting of SEQ ID NO: 98, a L-CDR2 consisting of SEQ ID NO: 117, and a L-CDR3 consisting of SEQ ID NO: 152 An anti-IGF1R antibody or an antigen-binding fragment thereof comprising 1. An anti-α-synuclein / anti-IGF1R bispecific antibody comprising:

2. The VH of the anti-IGF1R antibody or antigen-binding fragment thereof is Heavy chain framework region (H-FR) 1 comprising SEQ ID NO: 81, H-FR2 comprising SEQ ID NO: 86, H-FR3 comprising SEQ ID NO: 88, and H-FR4 comprising SEQ ID NO:92 including one or more of The VL of the anti-IGF1R antibody or antigen-binding fragment thereof is Light chain framework region (L-FR) 1 comprising SEQ ID NO: 162, L-FR2 comprising SEQ ID NO: 165, L-FR3 comprising SEQ ID NO: 166 or 167, and L-FR4 comprising SEQ ID NO: 170 The bispecific antibody of claim 1 , comprising one or more of the following:

3. 3. The bispecific antibody of claim 1 or 2, wherein the VH and VL of the anti-IGF1R antibody or antigen-binding fragment thereof comprise SEQ ID NOs: 221 and 340, respectively.

4. 3. The bispecific antibody of claim 1 or 2, wherein the VH and VL of the anti-IGF1R antibody or antigen-binding fragment thereof comprise SEQ ID NOs: 222 and 341, respectively.

5. The antigen-binding fragment of the anti-IGF1R antibody is a scFv, (scFv) ２ , scFv-Fc, Fab, Fab' and F(ab') ２ The bispecific antibody according to any one of claims 1 to 4, selected from the group consisting of

6. 6. The bispecific antibody of claim 5, comprising an anti-IGF1R scFv comprising, from N-terminus to C-terminus, SEQ ID NO:341, SEQ ID NO:411 and SEQ ID NO:

222.

7. The bispecific antibody of any one of claims 1 to 6, wherein the anti-IGF1R antibody or antigen-binding fragment thereof is linked to the anti-α-syn antibody or antigen-binding fragment thereof via a linker.

8. 8. The bispecific antibody of claim 7, wherein the linker is a peptide linker comprising SEQ ID NO: 411 or 412.

9. The bispecific antibody of any one of claims 1 to 8, wherein the bispecific antibody is monovalent for IGF1R and comprises an antigen-binding fragment of an anti-IGF1R antibody linked to the C-terminus of the heavy chain of an anti-α-syn antibody.

10. The bispecific antibody of claim 9, wherein the two heavy chains of the anti-α-syn antibody contain knob-in-hole mutations.

11. The bispecific antibody of any one of claims 1 to 10, wherein the anti-α-synuclein antibody or antigen-binding fragment thereof specifically binds to α-synuclein (α-syn) aggregates.

12. The anti-α-syn antibody or antigen-binding fragment thereof is H-CDR1 consisting of SEQ ID NO: 434, H-CDR2 consisting of SEQ ID NO: 435, H-CDR3 consisting of SEQ ID NO: 438, L-CDR1 consisting of SEQ ID NO: 443, L-CDR2 consisting of SEQ ID NO: 444, and L-CDR3 consisting of SEQ ID NO:445 The bispecific antibody according to any one of claims 1 to 11, comprising:

13. The anti-α-syn antibody or antigen-binding fragment thereof is H-FR1 comprising SEQ ID NO: 450, H-FR2 comprising SEQ ID NO: 451, H-FR3 comprising an amino acid sequence selected from SEQ ID NOs: 461 to 464; H-FR4 comprising SEQ ID NO: 467, L-FR1 comprising SEQ ID NO: 491, L-FR2 comprising SEQ ID NO: 492, L-FR3 comprising SEQ ID NO: 500, and L-FR4 comprising SEQ ID NO:501 The bispecific antibody of claim 12, comprising one or more of the following:

14. The anti-α-syn antibody or antigen-binding fragment thereof comprises a VH and a VL, wherein the VH and VL are each SEQ ID NOs: 531 and 548, SEQ ID NOs: 532 and 548, SEQ ID NOs: 533 and 548, or SEQ ID NOs:534 and 548 14. The bispecific antibody of claim 12 or 13, comprising:

15. The bispecific antibody of claim 14, wherein the anti-α-syn antibody or antigen-binding fragment thereof comprises a VH and a VL comprising SEQ ID NOs: 532 and 548, respectively.

16. The bispecific antibody of any one of claims 1 to 15, wherein the anti-α-syn antibody is of the IgG1, IgG2, IgG3 or IgG4 isotype subtype.

17. The bispecific antibody of claim 16, wherein the anti-α-syn antibody comprises an M428L mutation (EU numbering).

18. The anti-α-syn antibody comprises a HC and a LC, SEQ ID NOs: 560 and 566, SEQ ID NOs: 561 and 566, SEQ ID NOs: 562 and 566, or SEQ ID NOs: 563 and 566, The bispecific antibody of any one of claims 1 to 16, comprising:

19. A pharmaceutical composition comprising the bispecific antibody of any one of claims 1 to 18 and a pharma- ceutically acceptable excipient.

20. One or more nucleic acid molecules encoding the bispecific antibody of any one of claims 1 to 18.

21. 21. One or more expression vectors or host cells comprising one or more nucleic acid molecules according to claim 20.

22. Culturing the host cell of claim 21 under conditions permissive for expression of the bispecific antibody; and Isolating bispecific antibodies from cell culture A method for producing a bispecific antibody, comprising:

23. A pharmaceutical composition comprising the bispecific antibody of any one of claims 1 to 18 for treating or preventing an α-synucleinopathic disease in a subject in need thereof.

24. The pharmaceutical composition according to claim 23, wherein the α-synucleinopathy is selected from Parkinson's disease, Parkinson's disease dementia, dementia with Lewy bodies, Lewy body variant of Alzheimer's disease, combined Alzheimer's and Parkinson's disease, and multiple system atrophy.

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