Bispecific antibody against α-syn / IGF1R and its use

Abstract

To provide an anti-IGF1R antibody or an antigen-binding fragment thereof.SOLUTION: The present invention provides an anti-IGF1R antibody having a specific sequence or an antigen-binding fragment thereof, a polynucleotide encoding the anti-IGF1R antibody or the antigen-binding fragment thereof, and a pharmaceutical composition comprising the anti-IGF1R antibody or the antigen-binding fragment thereof, and a diluent, a carrier, a solubilizer, an emulsifier, a preservative or an adjuvant. The present invention also provides a method for delivering an antibody through an individual's blood-brain barrier, including administering the anti-IGF1Rantibody to the individual.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a bispecific antibody against alpha-synuclein and IGF1R, a pharmaceutical composition for preventing and / or treating synucleinopathies containing the bispecific antibody, and a method for providing information for detecting alpha-synuclein aggregates or diagnosing synucleinopathies containing the bispecific antibody.

Background Art

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

[0003] However, in pathological conditions, alpha-synuclein undergoes structural changes through binding and interaction with droplets, phospholipid bilayers or lipid membranes, etc., to form a folded or folded α-helical secondary structure, forming aggregates containing dimers, oligomers and / or fibrous forms of molecules.

[0004] Such alpha-synuclein aggregates are known to induce toxicity in cells and are the main component of Lewy bodies, which are abnormal protein aggregates found in nerve cells in Parkinson's disease (PD), Parkinson's disease dementia (PDD), multiple system atrophy (MSA), dementia with Lewy bodies (DLB), and various other diseases. Also, post-translational modifications such as phosphorylation or ubiquitination of alpha-synuclein are known to be associated with the aggregation and neurotoxicity of alpha-synuclein. Alpha-synuclein is known to kill dopamine neurons and induce an inflammatory response in animal experiments and cell experiments, and to induce movement symptoms similar to Parkinson's disease in experimental animals. Also, alpha-synuclein aggregation is known to be associated with the etiology of a group of neurodegenerative diseases called synucleinopathies, including Parkinson's disease, Parkinson's disease dementia, dementia with Lewy bodies, multiple system atrophy, and many other axonal diseases.

[0005] Antibodies against alpha-synuclein or fragments of alpha-synuclein for inducing such antibodies have been proposed as a method of immunotherapy for synucleinopathies. However, the brain penetration of antibodies can be limited by the blood-brain barrier (BBB). Also, the lack of highly specific BBB transporters has delayed the development of new therapeutic and diagnostic agents for diseases originating from the brain, including brain tumors and neurodegenerative diseases. There is clearly a need for a method to deliver therapeutic and diagnostic agent molecules to the brain at pharmaceutically effective doses without disrupting the physiology and homeostasis of the BBB.

Summary of the Invention

Problems to be Solved by the Invention

[0006] One example of the present invention provides an antibody comprising an antigen-binding site for alpha-synuclein (α-syn) and an antigen-binding site for IGF1R, or a method for producing the antibody. Another example provides a polynucleotide encoding the antibody, a recombinant vector containing the same, and a recombinant cell containing the same.

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

[0008] A method is provided for delivering a drug used for diagnosing, treating, or preventing alpha-synucleinopathy using the antibody or antigen-binding fragment according to the present invention to the brain.

[0009] One example of the present invention attempts to provide an anti-IGF1R antibody or an antigen-binding fragment thereof that specifically recognizes IGF1R (Insulin-like Growth Factor 1 Receptor), does not affect the binding of IGF1R ligands, does not suppress signal transduction through the IGF1R receptor, and enables transcytosis.

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

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

[0012] Still another example of the present invention attempts to provide a blood-brain barrier transporter capable of transmitting a physiologically active substance, for example, a physiologically active substance acting on the brain, through the IGF1R receptor through the blood-brain barrier (BBB) to the brain by the anti-IGF1R antibody or antigen-binding fragment.

[0013] In another embodiment, there is provided a protein complex that can pass through the Blood Brain Barrier (BBB) and be transmitted to the brain, in which the anti-IGF1R antibody or antigen-binding fragment is conjugated to a biologically active molecule, such as a bioactive substance that acts in the brain.

[0014] In another aspect, there is provided a method for detecting IGF1R from a biological sample, comprising providing an anti-IGF1R antibody or antigen-binding fragment according to the present invention and contacting the anti-IGF1R antibody or antigen-binding fragment with a biological sample that requires IGF1R expression detection.

[0015] There is provided a method for transmitting a bioactive substance used for the diagnosis, treatment or prevention of brain diseases using an anti-IGF1R antibody or antigen-binding fragment according to the present invention through the blood-brain barrier and into the brain.

Means for Solving the Problems

[0016] Hereinafter, the present invention will be described in more detail. As used herein, "antibody" means a complete immunoglobulin of any isotype, an antigen-binding fragment that can compete with a complete antibody for binding to a target antigen, or a combination thereof. For example, it includes chimeric, humanized, fully human antibodies, their antigen-binding fragments and combinations thereof. An antibody is also a kind of antigen-binding protein in itself. Antibodies generally include at least two full-length heavy chains and two full-length light chains, but in some cases, an antibody may contain only heavy chains. The antibodies include single-specific antibodies that specifically bind to one target and multispecific antibodies (e.g., bispecific antibodies and trispecific antibodies) that specifically bind to multiple targets.

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

[0018] As used herein, "light chain" includes full-length light chains and fragments thereof having a variable region sequence sufficient to provide binding specificity for an antigen or epitope. The full-length light chain includes the variable region domain VL and the constant region domain CL. The variable region domain of the light chain is present at the amino terminus of the light chain polypeptide. Types of light chains include kappa and lambda chains.

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

[0020] As used herein, "heavy chain" includes full-length heavy chains and fragments thereof having a variable region sequence sufficient to provide binding specificity for an antigen or epitope. The full-length heavy chain includes the variable region domain VH and three constant region domains CH1, CH2, and CH3. The VH domain is present at the amino terminus of the heavy chain polypeptide, the CH domains are present at the carboxy terminus, and CH3 is located closest to the carboxy-terminus. Heavy chains include the isotypes of IgG (including IgG1, IgG2, IgG3, and IgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgM, and IgE.

[0021] The antibody may be selected from all subtypes of immunoglobulins (e.g., IgA, IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4), IgM, etc.). The antibody in IgG form may be in IgG1, IgG2, IgG3, or IgG4 subtype, e.g., IgG1 or IgG2 subtype form. The antibody in IgG form contains two heavy chains and two light chains, and each heavy chain and light chain are bound through disulfide bonds to form two heavy chain-light chain structures (dimers), and the two formed heavy chain-light chains have a form linked through disulfide bonds at the Fc site of the heavy chain. The antibody in IgG form may be a single target antibody targeting one antigen, which contains antigen-binding sites for the same antigen on both-sided heavy chain-light chain structures, or a bispecific antibody targeting two antigens, which contains antigen-binding sites for different antigens on both-sided heavy chain-light chain structures.

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

[0023] In the present invention, a "variant" of a polypeptide such as, for example, an antigen-binding fragment, protein, or antibody is a polypeptide in which one or more amino acid residues have insertion, deletion, addition, and / or substitution as compared with other polypeptide sequences, and includes fusion polypeptides. For example, a part of an antibody contains conservative amino acid substitutions at one or more residues of the heavy chain or light chain, variable region, or CDR sequence.

[0024] In the present invention, a "derivative" of a polypeptide means a polypeptide that is chemically modified through conjugation with other chemical moieties, which is different from insertion, deletion, addition, or substitution variants.

[0025] The antibodies according to the present invention can be generated and selected from transgenic mice having the desired specificity, such as those described above, using hybridoma technology to obtain antigen-specific human mAbs. Such antibodies can be cloned and expressed using appropriate vectors and host cells, or the antibodies can be harvested from cultured hybridoma cells. Further, the antibodies can be derived from a phage-display library. Phage display technology is a method that mimics a kind of immune selection that displays an antibody repertoire on the surface of filamentous bacteriophages and then selects phages that bind to the target antigen from them. Such technology can be referred to in the examples of the present invention or PCT Publication No. WO99 / 10494. In one embodiment, the humanized antibodies of the present invention are selected through the phage display method.

[0026] The antibody or its antigen-binding fragment may be derived from a single source or may be chimeric. Chimeric antibodies contain portions derived from two different antibodies and are described in more detail below. The antibody or its antigen-binding fragment can be produced by hybridoma, recombinant DNA technology, or enzymatic or chemical cleavage of a complete antibody. Unless otherwise indicated, the term antibody in the present application includes, of course, antibodies containing two full-length heavy chains and two full-length light chains, as well as derivatives, mutants, immunologically functional immunoglobulin fragments, mutants, and combinations thereof. For example, in addition to two full-length heavy chains and two full-length light chains, one or two scFvs can be included, and these examples are as described below.

[0027] As used herein, "antigen-binding fragment" means a part of an antibody having specific binding ability to an antigen or a polypeptide containing the same. For example, the antigen-binding fragment may be a part of an antibody or a polypeptide containing the same that contains amino acid residues that interact with an antigen (e.g., an epitope) to confer specificity and / or affinity for the antigen on the antibody. Such a fragment may contain at least one CDR present within the full-length light or heavy chain, and in some embodiments, includes a single-chain heavy chain and / or light chain, or a part thereof. Such biologically active fragments can be produced by recombinant DNA technology or, for example, by enzymatic or chemical cleavage of a complete antibody.

[0028] Immunologically functional immunoglobulin fragments include, but are not limited to, Fab, Fab’, F(ab’)2, Fv, domain antibodies, and single-chain antibodies (e.g., scFv, scFv-Fc, etc.). Also, they can be derived from any mammal including, but not limited to, human, mouse, rat, camelid, or rabbit. Functional parts of an antibody such as one or more CDRs disclosed herein can be covalently linked to a second protein or small molecule compound and used as a targeted therapeutic agent for a specific target.

[0029] As used in this application, a "single-chain antibody" is a single polypeptide chain of an antigen-binding region in which the heavy-chain 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, scFv-Fc in which the heavy-chain variable region, the light-chain variable region, and Fc are linked in a single-chain form, etc. Reference may be made to, for example, U.S. Patent No. 5,260,203 for single-chain antibodies.

[0030] As used herein, "affinity" refers to the strength of the interaction between an antibody or its antigen-binding fragment and an antigen, which can be determined by the CDR sequences of the antibody or antigen-binding fragment, and / or the physicochemical properties (such as hydrophilicity / hydrophobicity, electrostatic properties, etc.) of the antibody or antigen-binding fragment, and characteristics of the antigen such as the size, shape, and / or charge of the antigen. Methods for determining such affinity are known in the art and can typically be represented by the dissociation constant (KD), but are not limited thereto.

[0031] The full-length light and heavy chains have the variable and constant regions joined by a "J" region of about 12 or more amino acids in length, and the heavy chain also contains a "D" region of about 10 or more amino acids.

[0032] See, for example, Fundamental Immunology, 2nd ed., Ch. 7 (Paul, W., ed.) 1989, New York: Raven Press. Typically, the variable regions of the antibody light / heavy chain pair form the antigen-binding site.

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

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

[0035] As used herein, a "bivalent antigen-binding protein" or "bivalent antibody" contains two antigen-binding sites. The two antigen-binding sites contained in such a bivalent antibody may have the same antigen specificity or may be a bispecific antibody that binds to different antigens respectively. As used herein, a "multispecific antigen-binding protein" or "multispecific antibody" targets two or more antigens or epitopes.

[0036] As used herein, a "bispecific" or "bispecific" antigen-binding protein or antibody is a hybrid antigen-binding protein or antibody having two different antigen-binding sites. Such bispecific antibodies are a type of multispecific antigen-binding protein or multispecific antibody and can be produced by known various methods, for example, by fusion of hybridomas or ligation of Fab' fragments 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, etc. The two different epitopes to which the two antigen-binding sites of the bispecific antigen-binding protein or antibody bind 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 take the form of a bispecific antibody that additionally contains binding to a transporter for transmission through the blood-brain barrier. One method for transmitting drugs through the blood-brain barrier involves the use of a transport system such as receptor-mediated transcytosis of cell-intrinsic glucose and amino acid transporters, insulin or transferrin.

[0037] According to the present invention, the term "synucleinopathy" includes all neurodegenerative disorders characterized by pathological synuclein aggregates. Several neurodegenerative disorders, including Parkinson's disease, Parkinson's disease dementia (PDD), dementia with Lewy bodies (DLB), Lewy body disease, dementia with Lewy bodies, Parkinsonian syndrome with dementia, multiple system atrophy (MSA), multiple nervous system atrophy, and neurodegeneration with brain iron accumulation type I (NBIA Type I), are collectively grouped as synucleinopathies. In addition, alpha-synuclein aggregation has also been found secondarily in Alzheimer's disease (Kim et al. Alzheimer's Research & Therapy 2014, 6:73).

[0038] Synucleinopathy is a diverse group of neurodegenerative disorders that share common pathological characteristics: in neuropathological experiments, specific lesions can be detected as abnormal aggregation of alpha-synuclein protein within selected populations of neurons and oligodendrocytes. Alpha-synuclein (initially identified as PARK1 and PARK4) is a 140-amino acid protein that is widely expressed in the neocortex, hippocampus, dentate gyrus, olfactory bulb, striatum, thalamus, and cerebellum. Alpha-synuclein is also highly expressed in hematopoietic cells, including protein globules and platelets, as well as B-, T-, and NK cells. The exact role of alpha-synuclein in such cells is not known, but it is thought to be associated with the differentiation of megakaryocytes (platelet precursors).

[0039] In the present invention, the "disease related to alpha-synuclein aggregates" is a group of neurodegenerative diseases called synucleinopathies, which are characterized by the discovery of alpha-synuclein aggregates in lesions including neuron and glia populations. Such diseases include, but are not limited to, Parkinson's disease, Parkinson's disease dementia, Lewy body dementia, Alzheimer's Lewy body disease, combined Alzheimer's and Parkinson's disease, multiple system atrophy, and many other axonal diseases. In one embodiment, the antibody according to the present application is effectively used for the treatment of Parkinson's disease.

[0040] In addition, the anti-α-syn / anti-IGF1R bispecific antibody according to the present invention contains an anti-IGF1R antibody or its antigen-binding fragment, enabling the anti-α-syn antibody or its antigen-binding fragment to pass through the blood-brain barrier and exert its action in the brain, and extending the half-life to maintain the drug efficacy for a long period.

[0041] Furthermore, the anti-α-syn / anti-IGF1R bispecific antibody according to the present invention binds to IGF1R on the cell surface, has the property of not affecting ligand binding and not affecting the signal transduction pathway through IGF1R. Therefore, since it does not suppress IGF1R and its ligand binding and signal transduction through IGF1R, it can be used as a shuttle means for passing through the blood-brain barrier.

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

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

[0044] For example, the anti-IGF1R antibody according to the present invention can have an scFv form and can be produced by binding to a therapeutic antibody in various ways. For example, the scFv of the anti-IGF1R antibody can be produced as a bispecific antibody in which two are bound to the C-terminus of a therapeutic antibody, such as an α-syn antibody, i.e., a bivalent form bispecific antibody, or a bispecific antibody in which one is bound, i.e., a monovalent form bispecific antibody, and all of these bispecific antibodies are internalized into cells expressing IGF1R. The high binding affinity of the IGF1R antibody for the cell surface antigen enhances the internalization effect, which will lead to the ability to cross the BBB. If the antibody interferes with IGF1R signaling while having the ability to cross the BBB, it may cause side effects. The anti-IGF1R antibody according to the present invention is a non-blocking antibody against the IGF1R signal while having a binding affinity that can function as a BBB shuttle.

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

[0046] Alternatively, at least one of the amino acids located on both sides of the deamidation site of the antibody can be substituted, preferably the amino acid immediately adjacent to the C-terminal side of the deamidation site of the antibody. For example, by substituting G located next to Asn or S located next to Asn at the deamidation site of the antibody with A or V, respectively, a deamidated antibody having a binding affinity similar to that of the parental anti-IGF1R antibody and excellent stability and BBB permeability can be produced.

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

[0048] An anti-IGF1R antibody according to an example of the present invention can be utilized as a bispecific antibody containing various therapeutic second antibodies, and in a passage experiment using a human iPSC-derived in vitro BBB system, it shows a BBB passage ability approximately 15 times higher than that of a monoclonal antibody composed of only the therapeutic antibody. The anti-IGF1R antibody bound to the second antibody in the bispecific antibody may be bound in a monovalent or bivalent form. For example, when a bispecific antibody in which the anti-IGF1R antibody is monovalent or bivalent is administered once to a normal rat and the amount of antibody in the blood and the amount of antibody in the CSF are analyzed, the bispecific antibody in which the anti-IGF1R antibody is monovalent or bivalent shows a maximum 5-fold increase in the amount of antibody in the blood and a maximum 5-fold increase in the amount of antibody in the CSF compared to the parental anti-IGF1R antibody (clone 1564). It shows an approximately 3-fold increase in CSF and an approximately 4.5-fold increase in brain passage compared to the parental anti-IGF1R antibody (clone 1564). Therefore, the bispecific antibody of the anti-IGF1R antibody improved by the above method is expected to show a maximum of approximately 15-fold CSF and approximately 23-fold brain passage ability compared to a monoclonal antibody composed of only the therapeutic second antibody.

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

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

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

[0052] As used herein, the term "epitope" is interpreted to mean an antigenic determinant, a part of an antigen recognized by an antibody. According to one embodiment, the binding site of the anti-IGF1R antibody according to the present invention may be the extracellular domain of the IGF1R protein, such as the human IGF1R protein. More specifically, the binding site of the anti-IGF1R antibody according to the present invention, such as the 1564 clone antibody, to the human IGF1R protein is on the human IGF1R protein, and binding site 1 includes Y775, P776, F778, R650, S791, L798 and Glu779, binding site 2 includes L641, H808, E809 and L813, and binding site 3 includes V397, D435, W434, Y460 and C488. Therefore, the epitope of the IGF1R antibody according to the present invention is a conformational epitope and may include all or part of the above three binding sites.

[0053] The anti-IGF1R antibody or antigen-binding fragment according to the present invention does not interfere with the binding of IGF-1, IGF-2, and / or insulin, which are ligands of IGF1R, to IGF1R. Specifically, the anti-IGF1R antibody or antigen-binding fragment does not interfere with the binding of the ligand of IGF1R to the IGF1R located on the cell membrane in cells expressing IGF1R, does not suppress signal transduction through IGF1R, and also has the advantage of not affecting the expression of IGF1R on the cell surface. Therefore, the anti-IGF1R antibody or antigen-binding fragment according to the present invention can be effectively used to pass through the blood-brain barrier through transcytosis. It has been revealed that the expression of IGF1R shows a relatively high expression level in the brain compared to other transcytosis targets known to be expressed in brain endothelial cells, which have been mainly used so far to improve the ability to pass through the BBB. In one embodiment, when IGF1R is compared with other targets currently being developed for improving the BBB permeability of therapeutic antibodies, such as the transferrin receptor and the insulin receptor, it has been revealed that IGF1R shows a relatively low expression level in peripheral tissues, such as the liver, lung, and large intestine.

[0054] IGF1R has become a target for Receptor Mediated Transcytosis (RMT) that can transmit useful substances into the brain by passing 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 preferably has the property of binding 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 they do not suppress IGF1R-ligand binding and signal transduction through IGF1R.

[0055] The anti-IGF1R antibody or antigen-binding fragment thereof according to the present invention is capable of transcytosis and can pass through the endothelial cells of the brain. Further, when the antibody according to the present invention is injected into the blood vessels of a mouse, it is located at the same position as the cerebral blood vessels of the mouse. Such 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.

[0056] Therefore, the anti-IGF1R antibody or antigen-binding fragment thereof according to the present invention enables a physiologically active substance 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 prevents the effective passage, diffusion, or transmission of cells, tissues, membranes, or biological molecules. Such biological barriers include nerve cells / tissues, connective tissues, muscles, membranes, or epithelial (e.g., mucosal or vascular) cells. A typical example is the blood-brain barrier.

[0057] In the present invention, the "blood-brain barrier" or BBB is a barrier formed by tight junctions within the capillary endothelial cell membranes of the brain that exist between the brain and the spinal cord and their surrounding circulatory systems. Such a barrier is very robust and also restricts the passage of low-molecular-weight substances with a molecular weight of about 60 Da into 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 within the central nervous system and are usually referred to as the BBB.

[0058] In the present invention, a "blood-brain barrier transporter" can pass through such a blood-brain barrier and transmit the brain-acting factor substance, and the brain-acting factor substance includes, for example, compounds, peptides, and proteins including polypeptides, nucleic acids, antibodies, or low-molecular-weight compounds.

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

[0060] One specific example is that the anti-IGF1R antibody and its antigen-binding fragment are (i) one or more heavy chain complementarity determining regions selected from the group consisting of H-CDR1, H-CDR2, and H-CDR3 listed in Table 1, or a heavy chain variable region containing the one or more heavy chain complementarity determining regions; (ii) one or more light chain complementarity determining regions selected from the group consisting of L-CDR1, L-CDR2, and L-CDR3 listed in Table 1, or a light chain variable region containing the one or more light chain complementarity determining regions; a combination of the one or more heavy chain complementarity determining regions and the one or more light chain complementarity determining regions; or a combination of the heavy chain variable region and the light chain variable region may be included.

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

[0062] In an example of the present invention, by removing the occurrence of deamidation of amino acids within the anti-IGF1R antibody, the risk of antibody degradation, which is disadvantageous for process development, storage, etc., is reduced while there is no change in the binding affinity to the ECD of IGF1R, which is the antigen. The amino acid positions where deamidation is removed in the anti-IGF1R antibody may be, for example, N51D, N51Q, S52V in the light chain LCDR2 at position 1564 (IgG), 1564 (scFv), or 1564-3, N95aK, N95aH, N95aR, N95aD, G95bA in the light chain LCDR3, or N54D, N54Q, or G55A in the heavy chain HCDR2. The removal of deamidation is not limited to only the clones mentioned above, and it is also possible for other clones by the method described in Table 14.

[0063]

Table 1

[0064]

Table 2

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

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

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

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

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

[0070] Specifically, the framework sequences of the heavy chain variable regions of the 1564 (IgG) clone contain the amino acid sequences of SEQ ID NOs: 32, 33, 35, and 36, and the remaining clones among the clones listed in Table 1 excluding the 1564 (IgG) clone contain the amino acid sequences of SEQ ID NOs: 32, 34, 35, and 36.

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

Table 3

[0072] The anti-IGF1R antibody according to the present invention may be an antibody containing a heavy chain variable region and a light chain variable region, and the various heavy and light chain variable regions disclosed in this specification are exemplified in Tables 4 and 5. The heavy chain variable regions and light chain variable regions described in Tables 4 and 5 below can be freely combined for the production of various forms of antibodies. Such respective variable regions can bind to the heavy chain and light chain constant regions to form the respective heavy and light chains of a complete antibody.

[0073]

Table 4

[0074]

Table 5

[0075] The heavy chain variable region of the anti-IGF1R antibody or its antigen-binding fragment according to the present invention can further include, more specifically, one selected from the group consisting of the amino acid sequences of SEQ ID NOs: 43 to 87. The light chain variable region of the anti-IGF1R antibody or its antigen-binding fragment according to the present invention can include one selected from the group consisting of the amino acid sequences of SEQ ID NOs: 88 to 132. Examples of the heavy chain variable region and the light chain variable region are described in Tables 4 and 5 above.

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

[0077] The heavy chain variable region and each light chain variable region described in Tables 4 and 5 above can be used as separate domain antibodies, respectively, 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.

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

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

[0080] Among the above antigen-binding fragments, Fab has a structure with a light chain variable region, a heavy chain variable region, a light chain constant region, and the first constant region (CH1) of the heavy chain 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 in Fab. An F(ab’)2 antibody is generated by forming a disulfide bond between the cysteine residues of the Fab’ hinge regions of two Fab’.

[0081] Fv is the smallest antibody fragment having only a heavy-chain variable region and a light-chain variable region, and includes single-chain Fv (scFv) and two-chain variable fragment. In the two-chain variable fragment, the heavy-chain variable region and the light-chain variable region can be linked by non-covalent bonds. In the single-chain Fv, the heavy-chain variable region and the light-chain variable region are covalently linked directly or through a peptide linker, or are directly linked at the C-terminus and can form the same structure as the scFv dimer (di-scFv) like the two-chain variable fragment. In the present invention, the single-chain Fv is a single polypeptide chain of an antigen-binding region in which the heavy-chain and light-chain variable regions are linked directly or by a linker, and 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, the same structure as the scFv dimer (di-scFv), and scFv-Fc in which the heavy-chain variable region, the light-chain variable region, and Fc are linked in a single-chain form.

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

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

[0084] The antigen-binding fragment can be obtained using proteolytic enzymes (e.g., Fab can be obtained by limited cleavage of the whole antibody with papain, and F(ab’)2 fragment can be obtained by cleavage with pepsin), and can also be produced through recombinant DNA technology. The single-chain antibodies disclosed in the present invention include, but are not limited to, scFvs containing domain combinations of heavy-chain and light-chain variable regions, or combinations of light-chain and heavy-chain variable domains containing CDRs.

[0085] The antigen-binding fragment of the anti-IGF1R antibody can be linked with or without a linker, e.g., a peptide linker. Also, the heavy-chain part and the light-chain part within the antigen-binding fragment, e.g., the heavy-chain variable region and the light-chain variable region within the scFv fragment, can be linked with or without a peptide linker. The peptide linker may be as described above.

[0086] In the bispecific antibody, the anti-IGF1R antibody and its antigen-binding fragment can perform the function of transmitting a second antibody or its antigen-binding fragment that targets different antigens or epitopes to the brain by passing through the blood-brain barrier. The second antibody may be an antibody that exerts its efficacy in the brain, but is not particularly limited, and may be the anti-alpha-synuclein antibody or its binding fragment according to the present invention.

[0087] The anti-IGF1R antibody or antigen-binding fragment thereof according to the present invention can share a specific region or sequence with a different second antibody. For example, the anti-IGF1R antibody can share the constant region of the antibody or antigen-binding fragment of the second antibody, or can share the Fc region.

[0088] In addition, the structure of the bispecific antibody in the present invention is in a bivalent form in which the scFv of the anti-IGF1R antibody is linked directly or via a linker to the Fc region of two heavy chains of a complete immunoglobulin, for example, at the end of the heavy chain, and a monovalent bispecific antibody in which the scFv of the anti-IGF1R antibody is linked directly or via a linker only to the end of one of the two heavy chains of a complete immunoglobulin. All are included, or a monovalent bispecific antibody is preferred.

[0089] Specifically, in an example of the present invention, the half-life of the monovalent form clone may be improved compared to the bivalent form clone. At this time, the structure of the monovalent form clone is in the form of a complete immunoglobulin with a domain antibody (scFv) that binds to IGF1R linked by a linker only to the end of one heavy chain. For example, in the form of a complete immunoglobulin, a domain antibody that binds to the IGF1R antigen with a linker included at the C-terminus is linked to one heavy chain, and the other heavy chain has two different heavy chains that are not linked to each other after the C-terminus of the constant region. It may be in the form of a heterodimer to which the Knob-In-hole technique is applied.

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

[0091] Hereinafter, it relates to the anti-syn antibody according to the present invention and its antigen-binding fragment.

[0092] The alpha-synuclein recognized by the antibody provided herein as an antigen may be selected from mammalian alpha-synucleins such as human alpha-synuclein, monkey alpha-synuclein (e.g., Rhesus alpha-synuclein), mouse alpha-synuclein, rat alpha-synuclein, etc. For example, human alpha-synuclein may be alpha-synuclein (NCBI ID: NP_000336), but is not limited thereto. Unless otherwise mentioned herein, alpha-synuclein may refer to human alpha-synuclein, and the antibody or its antigen-binding fragment 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.

[0093] It binds to the C-terminal region of alpha-synuclein to which the antibody or its antigen-binding fragment binds. Specifically, the human alpha-synuclein protein has the amino acid sequence of SEQ ID NO: 173, and the C-terminal region may be a C-terminal region containing a peptide composed of at least 11 or 12 consecutive amino acids including the C-terminal region, for example, residues 110 to 120 or residues 111 to 122. The antibody or its antigen-binding fragment according to the present invention has been confirmed to recognize the antigen recognition site and bind to alpha-synuclein aggregates with high affinity.

[0094] As used herein, "specifically binds to an alpha-synuclein protein or an alpha-synuclein aggregate" means that the affinity for the alpha-synuclein protein or the alpha-synuclein aggregate is relatively high compared to other antigens. For example, the affinity for alpha-synuclein aggregates, specifically amyloid fibrils, protofibrils, and oligomers, especially amyloid fibrils, is dissociation constant (K D ) 0.1 × 10 -10 M to 2 × 10 -10 M, or 0.05 × 10 -10 M to 0.3 × 10 -9 M by Octet and SPR analysis, respectively, but is not limited thereto.

[0095] The humanized alpha-synuclein antibody comprising a light chain and a heavy chain according to an example of the present invention, for example, in the case of Hu11F11 (ver.2), exhibits higher phagocytosis-promoting activity compared to the chimeric alpha-synuclein antibody. In the cases of Hu11F11 (ver.1), Hu11F11 (ver.2), Hu11F11 (ver.3), Hu11F11 (ver.4), and ABL2-4, they show high inhibitory ability against the binding of fibrils to the neuronal cell membrane compared to the chimeric alpha-synuclein antibody. In the cases of Hu11F11 (ver.2), Hu11F11 (ver.4), and ABL2-4, they show high inhibitory ability against the electrical wave of alpha-synuclein secreted from alpha-synuclein overexpressing cells to other neurons compared to the chimeric alpha-synuclein antibody. The binding ability to alpha-synuclein aggregates, for example, the binding ability measured in a cell-based assay, is similar to or has excellent activity compared to the chimeric alpha-synuclein antibody.

[0096] The alpha-synuclein antibody according to the present invention inhibits the action of alpha-synuclein aggregates secreted outside neurons in the nervous system of the subject from moving to other normal cells in the extracellular space and infecting the neurons (inhibit cell-to-cell transmission of aggregates). Also, microglia have the ability to promote phagocytosis against alpha-synuclein aggregates located in the extracellular space. Alpha-synuclein aggregates, like prions, spread from one cell to another and cause synucleinopathy as alpha-synuclein, especially alpha-synuclein aggregates, spread throughout the brain. Therefore, alpha-synuclein aggregates are known to be toxic to brain neurons and cause neurodegeneration and neuroinflammation. Therefore, as alpha-synuclein aggregates spread to various parts of the brain, cell death and neuroinflammatory responses increase, leading to the progression of synucleinopathy, such as Parkinson's disease, and the resulting impairment of behavior and cognitive function appears.

[0097] Therefore, the alpha-synuclein antibody of the present invention can suppress the intercellular movement of alpha-synuclein or alpha-synuclein aggregates, preventing the spread of alpha-synuclein aggregates to various regions of the brain. In addition, it can promote the phagocytic activity of microglia, reducing or removing the alpha-synuclein aggregates existing outside neurons in the nervous system of the subject, thereby reducing the level of alpha-synuclein aggregates, which are an important cause of synucleinopathy, reducing brain neuron death and brain inflammatory responses, and ultimately expecting to improve, alleviate or prevent the symptoms and progression of synucleinopathy, such as Parkinson's disease.

[0098] Furthermore, the alpha-synuclein antibody according to the present invention has excellent activity in that it can perform both functions related to the reduction of the level of alpha-synuclein aggregates in the brain nervous system through (i) suppressing the intercellular movement of alpha-synuclein or alpha-synuclein aggregates and (ii) promoting the phagocytic activity of microglia. In particular, the alpha-synuclein antibodies that are currently in clinical trials or published in papers have only one of the activities (i) and (ii). This indicates that the alpha-synuclein antibody of the present application has advantages in preventing or treating synucleinopathy compared to known alpha-synuclein antibodies. Therefore, the alpha-synuclein antibody according to the present invention is even more excellent in the efficacy of removing and reducing alpha-synuclein aggregates and suppressing the etiological action, and thus is more effective against synucleinopathy or related symptomatic diseases (e.g., cognitive impairment, etc.).

[0099] The antibody or antigen-binding fragment according to the present application, which has a high affinity for alpha-synuclein aggregates, can reduce alpha-synuclein aggregate formation and lower the concentration of aggregates in the brain. In addition, the antibody or antigen-binding fragment according to the present application, which has a high affinity for alpha-synuclein aggregates, can reduce alpha-synuclein aggregate formation outside the central nervous system, and ultimately, can change the equilibrium state between alpha-synuclein forms across the blood-brain barrier, resulting in the effect of lowering the concentration of aggregates in the central nervous system. This has great clinical advantages because sufficient efficacy can be obtained even when the antibody is administered by a method such as subcutaneous injection, which is, for example, not limited in this way and is more convenient.

[0100] The antibody or antigen-binding fragment according to the present application can inhibit aggregate formation through monomer removal or remove all monomers and aggregates.

[0101] The antibody or its antigen-binding fragment that specifically binds to the alpha-synuclein protein or alpha-synuclein aggregates provided in this specification may not be naturally produced (non-naturally occurring; for example, it may be chemically synthesized or recombinantly produced). Recombinant techniques as described above are widely known in the art.

[0102] The alpha-synuclein antibody or bispecific antibody containing the same according to the present invention can be used as a pharmaceutical composition for the prevention or treatment of alpha-synucleinopathy, and the alpha-synucleinopathy may include, but is not limited to, Parkinson's disease (PD), Parkinson's disease dementia (PDD), dementia with Lewy bodies (DLB), Lewy body variant of Alzheimer's disease (LBV), Combined Alzheimer's and Parkinson disease, or multiple system atrophy (MSA).

[0103] The antibody or antigen-binding fragment thereof that specifically binds to alpha-synuclein or its aggregates according to the present invention can include a heavy-chain variable region containing complementarity-determining regions of CDRH1, CDRH2, and CDRH3; and a light-chain variable region containing complementarity-determining regions of CDRL1, CDRL2, and CDRL3.

[0104] In one embodiment, the anti-alpha-synuclein antibody or its antigen-binding fragment can include the following CDR sequences: Heavy-chain CDR1 (H-CDR1) containing the amino acid sequence of SEQ ID NO: 135, Heavy-chain CDR2 (H-CDR2) containing the amino acid sequence of SEQ ID NO: 136 or 137, Heavy-chain CDR3 (H-CDR3) containing the amino acid sequence of SEQ ID NO: 138, Light-chain CDR1 (L-CDR1) containing the amino acid sequence of SEQ ID NO: 139, Light-chain CDR2 (L-CDR2) containing the amino acid sequence of SEQ ID NO: 140, and Light-chain CDR3 (L-CDR3) containing the amino acid sequence of SEQ ID NO: 141.

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

[0106] [Table 6]

[0107] [Table 7]

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

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

[0110] [Table 8]

[0111] Also, exemplary antibodies through combinations of the heavy chain variable region and the light chain variable region of an antibody or antigen-binding fragment according to one embodiment are described in Table 9. [Table 9]

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

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

[0114] The antibodies disclosed herein share a specific region or sequence with other antibodies disclosed herein. In one embodiment, the constant region of the antibody or antigen-binding fragment can be shared. In other embodiments, the Fc region can be shared.

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

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

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

[0118] The anti-alpha-synuclein antibody according to the present application may be used alone as a therapeutic antibody, but can also be used as a bispecific antibody in combination with other antibodies capable of performing the function of passing through the blood-brain barrier and being transmitted to the brain. An example of an antibody capable of performing the function of passing through the blood-brain barrier and being transmitted to the brain may be an anti-IGF1R antibody and its antigen-binding fragment. The anti-IGF1R antibody and its antigen-binding fragment capable of producing a bispecific antibody may include all of the aforementioned anti-IGF1R antibodies and their antigen-binding fragments, and may be, for example, a complete antibody. The antigen-binding fragment may be selected from the group consisting of domain antibodies, scFv, (scFv)2, scFvFc, Fab, Fab’ and F(ab’)2.

[0119] The antigen-binding fragment of the anti-IGF1R antibody can be linked with or without a linker, such as a peptide linker. Also, the heavy chain portion and the light chain portion within the antigen-binding fragment, such as the heavy chain variable region and the light chain variable region within the scFv fragment, can be linked with or without a peptide linker. The peptide linker may be as described above.

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

[0121] Examples of the heavy chains of the anti-alpha-synuclein antibodies used for the production of heavy chain combinations for the production of bispecific antibodies, including the aforementioned anti-alpha-synuclein antibody hu11F11 (ver.2), are described in Table 10 below. These heavy chain sequences are represented by SEQ ID NO: 151 to SEQ ID NO: 172, and are not intended to be limited thereto. In addition, the specific components of the bispecific antibody clones obtained by combining the heavy chain combinations using the heavy chains of the anti-alpha-synuclein antibody with the light chains are shown in Table 10 below. The bispecific antibodies presented below are described exemplarily, and even if not represented by SEQ ID NO, the composition is clear from the description of the bispecific anti-heavy chain combination. Exemplary bispecific antibodies are specifically shown in Table 10 below. [Table 10] TIFF0007710000000011.tif253170TIFF0007710000000012.tif253170TIFF0007710000000013.tif209170

[0122] The pharmaceutical composition for the prevention or treatment of alpha-synucleinopathy containing the alpha-synuclein antibody or its antigen-binding fragment according to the present invention, or a bispecific antibody containing the same, can contain the alpha-synuclein antibody or the bispecific antibody in a pharmaceutically effective amount.

[0123] As used herein, "treatment" can mean all actions related to the reduction or elimination of a disease or disease symptoms, such as reducing, alleviating, relieving, or eliminating a disease or disease symptoms, making the symptoms or pathological conditions of the disease more tolerable, or slowing the rate of deterioration of the symptoms or pathological conditions of the disease. The terms "subject" or "patient" include humans or human patients.

[0124] Also provided are pharmaceutical compositions comprising a therapeutically effective amount of an antibody, and a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and / or adjuvant. Also included, for example, is a method of treating a patient associated with alpha-synuclein by administering such a pharmaceutical composition. Pharmaceutical compositions for in vivo administration are typically provided as sterile formulations. Once formulated, the pharmaceutical composition can be stored as a solution, suspension, gel, emulsion, solid, crystal, or dehydrated or lyophilized powder in a sterile vial. Such dosage forms can be stored in a ready-to-use form, or in a form that is reconstituted immediately prior to administration (e.g., lyophilized).

[0125] The route of administration of the pharmaceutical composition can be by known methods, for example, oral; injection via intravenous, intraperitoneal, intracerebral (intraparenchymal), intraventricular, intramuscular, intraocular, intraarterial, intraportal, or intralesional routes; sustained release systems or implant devices can be used. In certain embodiments, the composition can be administered by bolus injection or continuously by infusion or implant device.

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

[0127] Antibodies produced in one example of the present invention specifically bind to IGF1R with a binding affinity optimized for transcytosis of brain endothelial cells, and can be usefully used for the delivery of therapeutic antibodies for degenerative brain diseases and brain cancer with low blood-brain barrier permeability and limited therapeutic efficacy. In particular, the antibodies disclosed in the present invention do not affect the binding of ligands IGF-1, IGF-2 and their homolog insulin to IGF1R, and do not suppress signal transduction through the IGF1R receptor, etc., and thus have utility in relation to blood-brain barrier passage. The antibodies disclosed in the present application can effectively remove or promote the degradation of alpha-synuclein aggregates, can suppress the intercellular transmission of alpha-synuclein, and can be usefully used for the treatment of diseases associated with the accumulation of alpha-synuclein aggregates. The alpha-synuclein antibody according to the present invention or a bispecific antibody containing the same can be used as a pharmaceutical composition for the prevention or treatment of alpha-synucleinopathy.

Brief Description of Drawings

[0128]

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Mode for Carrying Out the Invention

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

Examples

[0130] Example 1. Production of Mouse Alpha-Synuclein Antibody 1-1: Immunization and hybridoma production As the antigen, the full-length (140 residues) or the C-terminal 21-residue truncated alpha-synuclein monomer was placed in a thermomixer C at 37°C and shaken at 1050 rpm for 14 days for aggregation and then sonicated for use. The prepared 1 mg / ml concentration of 140-residue and 119-residue alpha-synuclein fibrils were each mixed with an adjuvant at a ratio of 1:1 (vol:vol) and thoroughly mixed. The amino acid sequence of human alpha-synuclein (Homo sapiens alpha-synuclein) is shown in SEQ ID NO: 173.

[0131] Next, 200 μL of the prepared mixture was subcutaneously injected into 5- to 7-week-old female BALB / c mice. Two weeks later, an additional 200 μL of the mixture prepared in the same manner was subcutaneously injected for boosting. One week after boosting, blood was collected and immunization titer was performed using an ELISA method with the administered antigen. Next, for the third boosting, only the antigen was subcutaneously injected.

[0132] Next, the spleens of the mice that had completed immunization as described above were removed to obtain cells. Next, the spleen cells were suspended in Hybridoma-SFM medium (Thermo Fisher Scientific, USA) supplemented with 10% FBS. To produce hybridomas, SP2 / 0-Ag14, a murine myeloma cell, was mixed with the spleen cells in Hybridoma-SFM medium without serum, and then centrifuged to remove the medium. Next, PEG was added to the cell pellet, and then cultured at 37°C for 1 minute to induce cell fusion.

[0133] 1-2: Single cell cloning and antibody purification Two weeks after fusion, the fusion with mouse B cells that produce antibodies was confirmed using an ELISA method with the antigen administered to mice using cell culture medium. Next, single cell cloning was performed using the hybridomas, and a total of 16 hybridomas that produce monoclonal antibodies were selected. Clone 9B11 (IgG1 kappa each) was obtained using the full-length (140 residues) alpha-synuclein aggregate as an antigen, and clones 3A9 and 11F11 (IgG2b kappa, IgG2b kappa respectively) were obtained using the alpha-synuclein aggregate with the C-terminal 21 residues cleaved as an antigen.

[0134] For the purification of the antibody, each hybridoma was cultured in RPMI1640 medium containing 10% FBS, and after alternating the culture medium to serum-free SFM medium for antibody production, it was cultured for about 4 days. After separating and centrifuging the cell culture supernatant, it was filtered through a 0.22 μm filter, and the IgG1 type was purified using a protein G column, and the remaining antibodies were purified using a protein A column.

[0135] 1-3: Variable region sequencing The variable region and CDR sequences were determined with reference to the paper by Ahn et al, Mol.Cells 2004, 18(2):237-241. After culturing the hybridomas, they were centrifuged to separate only the cells. Trizol was added to the separated hybridomas to isolate RNA, and after using this as a template to synthesize cDNA, the variable region and CDR sequences were confirmed through nucleotide sequence analysis.

[0136] Example 2. Production of chimeric anti-alpha-synuclein antibody 2-1: Antibody cloning and expression Using the antibody nucleotide sequences of the ensured heavy chain variable region and light chain variable region, gblocks (m.biotech), which are nucleotides of short fragments, were synthesized and cloned into a vector for animal cell culture (pcDNA3.4) using these. The gblocks were synthesized to contain approximately 20 bp of overlapping nucleotides before and after the variable region, and the portion of the pcDNA3.4 vector excluding the variable region was prepared after amplification by PCR and cloned by the Gibson assembly method.

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

[0138] For the transfection of the prepared mother cells, an ExpiFectamine (trademark) CHO transfection kit (Gibco, Cat: A29129) was used to prepare an ExpiFectamine (trademark) CHO & plasmid DNA complex. Cold OptiPRO (trademark) SFM (registered trademark) (Gibco, Cat: 12309019) medium was dispensed respectively, and after inoculating the prepared DNA and ExpiFectamine (trademark) CHO reagent at appropriate concentrations, they were mixed and left standing at room temperature for 5 minutes. After inoculating the mother cells for transfection, the culture was started. On the day after transfection, the enhancer and feed contained in the ExpiFectamine (trademark) CHO transfection kit were inoculated into the transfected cells. Five days later, after additional inoculation of the feed, the production was completed by culturing at 8% CO2, 37 °C, and 120 rpm for 10 days.

[0139] For the collection of the culture broth after production was completed, the culture broth was transferred to a centrifuge bottle and centrifuged at 4 °C and 6500 rpm for 30 minutes. Then, it was filtered through a 0.2 μm filter to obtain a culture broth free of floating matter, and the subsequent purification process was carried out.

[0140] 2-2: Antibody purification and sequence confirmation The culture broth was purified using HiTrap MabSelectSure (GE Healthcare, 11-0034-94). After equilibration with an equilibration buffer (50 mM Tris-HCl pH 7.2, 100 mM NaCl), the recovered culture broth was loaded onto the column. After loading was completed, it was washed in the middle with 50 mM Sodium Citrate pH 5.0, and then eluted using 50 mM Sodium Citrate pH 3.4. 1 M Tris-HCl pH 9.0 was added to the eluate to neutralize it to pH 6.0. Then, the eluate was buffer-exchanged and concentrated with PBS (phosphate buffered saline, pH 7.4) and stored at 4 °C until use.

[0141] When additional purification was required, the primary purified fraction was passed through a HiLoad 26 / 600 superdex 200 column with 1X PBS as the 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 a mass spectrometry method and confirmed to match the variable region of a mouse-derived monoclonal antibody.

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

Table 11

[0143] Example 3. Production of humanized antibody 3-1: Library phage preparation A mini-library was constructed in which mouse or human-derived sequences were introduced into each CDR residue while binding the human framework to the CDR1, CDR2, and CDR3 residues of the chimeric antibody. The competent cells of the library were inoculated into 2X YT medium [17 g of Tryptone (CONDA, 1612.00), 10 g of yeast extract (CONDA, 1702.00), 5 g of NaCl (Sigma, S7653)] containing 34 μg / ml of chloramphenicol (Sigma, C0857), 2% glucose (Sigma, G5400), and 5 mM MgCl2 (Sigma, M2393), and cultured at 37°C for about 3 hours until the OD600 value reached 0.5 to 0.7. After that, helper phage was infected, and then cultured at 30°C for 16 hours in 2X YT medium supplemented with 34 μg / ml of chloramphenicol, 5 mM MgCl2, 70 μg / ml of kanamycin (Sigma, K1876), and 1 mM IPTG (ELPISBIO, IPTG025) to induce phage packaging. Next, the culture solution was centrifuged at 4500 rpm and 4°C for 15 minutes. Then, 4% PEG6000 (Fluka, 81253) and 3% NaCl (Sigma, S7653) were added to the supernatant, dissolved well, and reacted on ice for 1 hour. This was centrifuged again at 8000 rpm and 4°C for 20 minutes. The pellet was suspended in PBS and then centrifuged again at 12000 rpm and 4°C for 10 minutes to obtain the supernatant containing library phage, which was put into a new tube and stored at 4°C until use.

[0144] 3-2: Phage display panning Specifically, recombinant alpha-synuclein aggregates at a concentration of 10 μg / ml were added to PBS in an immunotube (immunotube, maxisorp 444202), and the protein was adsorbed onto the surface of the test tube overnight at 4°C. Then, a 3% solution of bovine serum albumin (BSA) was added to the test tube to protect the surface where alpha-synuclein aggregates were not adsorbed. After emptying the test tube, 1 × 10 12 cfu antibody phage library was added to the immunotube on which alpha-synuclein monomer protein was adsorbed, and the reaction was carried out at room temperature for 1 hour (negative selection). Phages not bound to alpha-synuclein monomer were recovered and bound to the immunotube to which alpha-synuclein aggregates were attached at room temperature for 2 hours. Next, non-specifically bound phages were washed off 5 to 30 times with PBS-T (0.05% Tween 20) solution, and the remaining antigen-specific phage antibodies were recovered using 100 mM triethylamine solution. The recovered phages were neutralized with 1 M Tris buffer (pH 7.4), then infected with ER2537 Escherichia coli at 37°C for 1 hour, and the infected E. coli was spread 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 solution, 15% glycerol was added, part of it was stored at -80°C, and the rest was used to produce phages for the next round of panning. Such a process was repeated 3 rounds in total to amplify antigen-specific antibodies. As the panning round progressed, the number of washes with PBS-T was increased to amplify and concentrate antigen-specific phages.

[0145] 3-3: Single clone screening of phage antibody The following experiment was conducted to select a single clone antibody specifically binding to alpha-synuclein aggregates from the phage pool obtained through the above panning.

[0146] To isolate a single clone from the concentrated pool, the phage pool was spread on an LB-tetracycline / carbenicillin agar medium and then cultured to obtain single colonies. Next, the single clone was inoculated into a 96-deep well plate containing 400 μl of 2X YT-tetracycline / carbenicillin medium per well and cultured overnight. Then, 10 μl of the culture solution was placed into a new 96-deep well plate containing 390 μl of 2X YT-tetracycline / carbenicillin medium and cultured at 37 °C for 4 hours. 1 mM IPTG was added to the culture solution and cultured at 30 °C overnight. The overnight culture solution was centrifuged to obtain the supernatant.

[0147] Next, clones expressing single clone soluble scFv that binds to alpha-synuclein aggregates were selected using the ELISA method. 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 added to each well and blocked at 37 °C for 2 hours. Next, alpha-synuclein aggregates and monomers were each loaded at a concentration of 100 ng / well and then reacted at 37 °C for 2 hours. Next, it was washed 5 times with 300 μL of PBS-T. The prepared single clone supernatant was mixed with 3% BSA at a ratio of 1:1 (vol:vol), and then 100 μL of the mixed solution was loaded into the plate bound to the aggregates and monomers and reacted at 37 °C for 2 hours. Next, after washing 5 times with 300 μL of PBS-T, an anti-HA HRP-conjugated antibody was added and reacted at 37 °C for 1 hour, and then washed 5 times with PBS-T. 100 μL of TMB (Tetramethylbenzidine, Sigma, T0440) was added for color development, then 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 regarded as positive reactions by binding, and clones that bind non-specifically to BSA were excluded.

[0148] The CDR residues of the clones discovered in the library were analyzed in silico in parallel to check whether there were serious problems in the binding to the framework, and clones without T-cell epitopes, B-cell epitopes, and MHCII epitopes in the framework part excluding the CDR were selected.

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

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

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

[0152] As shown in Figure 1, it was confirmed that the humanized antibody according to the present invention, particularly the humanized antibody derived from chimeric 11F11 (humanized 11F11 antibody), exhibits binding affinity equivalent to that of the chimeric 11F11 clone. It was confirmed that humanized antibodies, particularly variants derived from 11F11, hu11F11 (ver.1), that is, the combination of Hu11F11-VH-v1 and Hu11F11-VLv3 4c, hu11F11 (ver.2), that is, the combination of Hu11F11-VH-v2 and Hu11F11-VLv3 4c, hu11F11 (ver.3), that is, the combination of Hu11F11-VH-v3 and Hu11F11-VLv3 4c, hu11F11 (ver.4), that is, Hu11F11-VH-v4 and Hu11F11-VLv3 4c show binding affinity similar to that of the chimeric 11F11 clone, and their EC 50 is 11.5 - 15.1 nM, showing a value similar to 12.5 nM, which is the EC 50 of the chimeric 11F11 antibody.

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

[0154] The equipment used for the analysis was T200 (GE Healthcare, S / N: 1565888). Protein A was used for the Chip (GE Healthcare, Cat. 29-1275-56), 10 mM Glycine-HCl pH1.5 was used for the Regeneration buffer (GE Healthcare, Cat. BR-1003-54), and HBS-EP was used as the Running buffer, analyte dilution, and sample dilution buffer. The antibodies produced in Example 2 and Example 3 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 serially diluted two-fold each to analyze at a total of 6 concentrations including 0 nM (0, 0.39, 1.56, 6.25, 25, 100 nM). For capture, the monomer had a target RU of 800 (theoretical), the fibril had a target RU of 100 (theoretical), the capture phase was performed with a contact time of 60 seconds, a flow rate of 30 μl / min, and a stabilization period of 180 seconds. In the association phase, the association time was 120 seconds and the flow rate was 30 μl / min, and in the dissociation phase, the dissociation time was 360 seconds and the flow rate was 30 μl / min. In the regeneration phase, the flow rate was 30 μl / min and the regeneration time was 240 seconds (first time), 60 seconds (second time), performed twice. Fitting was performed using a 1:1 binding model, and the evaluation software used was BIACore T200 Evaluation software (GE healthcare).

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

[0156] As a result, the humanized antibodies described in the present application, particularly variants of 11F11, namely, hu11F11 (ver.2), hu11F11 (ver.3) and hu11F11 (ver.4), showed similar K D values to the chimeric 11F11 clone. As for the degree of binding, the humanized clones had a K -9 of 0.02 - 0.06×10 D M, while the chimeric 11F11 clone had a low K -9 value of 0.02×10 D M, that is, they showed a high binding affinity for aggregates.

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

[0158] A human-derived ScFv (Single-chain variable fragment) library cell with diversity (OPAL library, produced by Professor Sim Hyun-bo of Ewha Womans University) 1×10 10After inoculating into 2X YT medium [17 g of Tryptone (CONDA, 1612.00), 10 g of yeast extract (CONDA, 1702.00), 5 g of NaCl (Sigma, S7653)] containing 34 μg / ml of chloramphenicol (Sigma, C0857), 2% glucose (Sigma, G5400), and 5 mM MgCl2 (Sigma, M2393), it was cultured at 37°C for about 3 hours until the OD600 value reached 0.5 to 0.7. Then, after infecting with helper phage, it was cultured at 30°C for 16 hours in 2X YT medium supplemented with 34 μg / ml of chloramphenicol, 5 mM MgCl2, 70 μg / ml of kanamycin (Sigma, K1876), and 1 mM IPTG (ELPISBIO, IPTG025) to induce phage packaging. Next, the culture solution was centrifuged at 4500 rpm at 4°C for 15 minutes. Then, 4% PEG6000 (Fluka, 81253) and 3% NaCl (Sigma, S7653) were added to the supernatant and dissolved well, and then reacted on ice for 1 hour. This was centrifuged again at 8000 rpm at 4°C for 20 minutes. After that, the pellet was suspended in PBS and then centrifuged again at 12000 rpm at 4°C for 10 minutes to obtain the supernatant containing library phage, which was put into a new tube and stored at 4°C until use.

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

Table 13

[0160] Specifically, 1 ml of recombinant human IGF1R protein at a concentration of 5 μg / ml (R&D Systems, USA, 391-GR or Sino Biological Life Technologies, USA, 10164-H08H-50R) was added to an immunotube (immunotube, maxisorp 444202), and the surface of the test tube was coated at 4°C for 16 hours. After removing the supernatant, 4 ml of PBS solution containing 3% BSA (Bovine serum albumin) was added and reacted at 37°C for 1 hour to bind BSA to the surface where IGF1R was not adsorbed, thereby blocking non-specific binding. 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 immunotube at 37°C for 1 hour to allow phages specific to IGF1R to bind to the antigen. Next, it was washed once with PBS-T (phosphate buffered saline - 0.05% Tween 20) solution to remove non-specifically bound phages, and then the phages bound to IGF1R were recovered using a 100 mM triethylamine solution.

[0161] After neutralizing the recovered phages with 1M Tris buffer (pH 7.4), E. coli K12 ER2738 was infected at 37°C for 1 hour, and then the E. coli was spread on an LB agar medium containing tetracycline and carbenicillin and cultured overnight at 37°C. The next day, the cultured E. coli was suspended in 5 ml of SB (superbroth) medium containing tetracycline and carbenicillin, 50% glycerol of the same volume was added, and a part was stored at 80°C. After suspending 50 μl of the remaining in 40 ml of SB-tetracycline / carbenicillin medium, 10 12 PFU of VCSM13 helper phage was added and slowly stirred, and cultured at 37°C for 1 hour. Then, after adding kanamycin to the culture solution, it was cultured at 30°C for about 16 hours so that only E. coli infected with the helper phage was cultured.

[0162] The next day, after centrifuging the culture solution, the supernatant was taken, a buffer containing 4% PEG8000 and 3% sodium chloride (NaCl) was added, and the reaction was carried out at 4°C for about 1 hour to precipitate the phage, followed by centrifugation. Next, the supernatant was removed, and the precipitated phage was resuspended in PBS buffer containing 1% BSA and used for the next round of panning. The above process was repeated 4 times, and the number of washing times with PBS-T was increased as the panning round progressed to amplify and concentrate the antigen-specific phage.

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

[0164] Specifically, the following experiments were conducted to select single clone antibodies that specifically bind to IGF1R from the phage pool obtained through the examples.

[0165] To isolate a single clone from the enriched pool, the phage pool obtained on LB-tetracycline / carbenicillin agar medium was plated and cultured to secure single colonies. After inoculating these colonies into a 96-deep well plate and culturing overnight, 10 μl of the culture solution was re-inoculated into the 96-deep well plate in the same manner and cultured at 37 °C for about 4 hours to obtain a titrated OD (0.5 - 0.7). After adding 20 MOI of helper phage to the culture solution, it was reacted at 37 °C for 1 hour. Then, kanamycin was added to the culture solution and cultured at 30 °C overnight. The next day, the culture solution was centrifuged, and the supernatant was taken for ELISA to select IGF1R-specific phages (Steinberger, Rader and Barbas III. 2000. Phage display vectors. In: Phage Display Laboratory Manual. 1st ed. Cold Spring Harbor Laboratory Press. NY, USA. pp.11.9 - 11.12).

[0166] 100 ng of recombinant IGF1R was added to each well of the ELISA plate and reacted at 4 °C for about 15 hours to coat the antigen on the plate. To prevent non-specific binding, 200 μl of PBS buffer containing 3% BSA was added to each well and reacted at 37 °C for about 1 hour. The supernatant was discarded.

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

[0168] Example 7. Production of an affinity variant of an IGF1R antibody Affinity variation was performed on the clones selected by evaluating ligand binding ability and BBB permeability to optimize the antibody. In the first attempt, an NNS hand-mix primer for randomizing CDR2 of the heavy chain and CDR3 of the light chain was prepared based on 1564 scFv, and the 1564 scFv gene containing the randomization sequence was amplified using PCR techniques. The amplified gene was inserted into the pComb3x vector to form a library suitable for phage display, and multiple scFv clones binding to IGF1R could be selected through library panning and ELISA screening. The amino acid sequences of the variable regions of the selected clones were confirmed through gene sequencing.

[0169] In the second attempt, two mini libraries were constructed for the heavy and light chains with germline backmutations introduced into CDR1, CDR2, and CDR3 respectively. Variants (affinity variants) were selected based on the productivity and antigen-binding ability of the clones, and finally clones were obtained.

[0170] Example 8. Production of Deamidation Residue Mutant Antibodies 8-1: Deamidation residue confirmation The deamidation reaction means, for example, attacking the side-chain peptide bond of asparagine to form a symmetric succinimide intermediate, which is then converted to either aspartic acid or isoaspartic acid by hydrolysis. In particular, if deamidation occurs in the CDR, the antibody may be degraded while the binding to the antigen becomes weaker, leading to a decrease in efficacy and the induction of sample heterogeneity. Sample heterogeneity induces complexity for its identification in subsequent clinical approvals and the like. Therefore, an attempt was made to identify the positions where deamidation occurs through in silico analysis and peptide mapping, prevent deamidation to ensure stability, and at the same time obtain excellent physical properties and efficacy.

[0171] As shown in Figure 8, it was confirmed by in silico analysis and peptide mapping of the parental 1564 clone that deamidation actually occurs. At this time, the samples were analyzed after storage at 4°C or 40°C for one week, and it was confirmed that deamidation occurred in LCDR2, LCDR3, and HCDR2. The positions where deamidation occurred were also confirmed by analyzing the affinity variants described in Example 7.

[0172] 8-2: Mutant antibody production Mutants were produced in which residues were substituted as follows to remove deamidation residues.

[0173] 1) In the amino acid sequence, Asn was changed to D or Q, which is similar to Asn. If it is confirmed that there is no change in the binding force, all such residues are substituted with Q. 2) N95a, which is the residue where deamidation occurs in LCDR3, was substituted with H, R, or K that carry a positive charge. Clones to which such a deamidation process is applied are also called (de)(StoP) deamidation clones. 3) Residues located immediately next to the CDRs where deamidation occurs were substituted. These residues are relatively small in size and do not have a large charge (e.g., glycine or serine). Therefore, by substituting such residues with other residues that are not relatively large in size and are hydrophobic (e.g., valine or alanine), an attempt was made to minimize the difference in binding force with the parental antibody (the clone before the residue was substituted) (Table 21). Clones to which such a deamidation process is applied are also called (de2)(StoP) deamidation clones. The method of substituting the residues next to the residues where deamidation occurs is shown in the following table.

[0174]

Table 14

[0175] Example 9. Production of various forms of anti-IGF1R antibodies 9-1: Production of anti-IGF1R antibody in minibody form The entire scFv of the IGF1R-specific monoclonal phage antibodies secured in Examples 6 to 8 was ligated to the C-terminus of Fc to produce minibodies. For this purpose, a nucleic acid sequence encoding the amino acid sequence of the scFV disclosed in the present application was produced, this sequence was cleaved with a restriction enzyme, and cloned into a pcDNA-based expression vector containing a nucleic acid encoding Fc.

[0176] 9-2: Production of anti-IGF1R bivalent antibody The entire scFv of the IGF1R-specific monoclonal phage antibodies secured in Examples 6 to 8 was ligated in two copies to the C-terminus of each of the therapeutic antibody IgG forms to produce a bivalent form. For this purpose, a nucleic acid sequence encoding the amino acid sequence of the scFV disclosed in the present application was produced, this sequence was cleaved with a restriction enzyme, and cloned into a pcDNA-based expression vector containing a nucleic acid encoding the therapeutic antibody.

[0177] 9-3: Production of anti-IGF1R IgG (Full-IgG) antibody To convert the sequences of the 1564 antibody and the F06 antibody among the IGF1R-specific monoclonal phage antibodies secured in Examples 6 and 7 into the full IgG1 form, the gene sequences of the heavy and light chain regions were synthesized (Genotech). The synthesized heavy and light chain genes were cloned into an expression vector.

[0178] 9-4: Production of anti-IGF1R scFv monovalent antibody If Example 9-2 is a bivalent form in which anti-IGF1R antibodies are bound in scFv form to the C-termini of two Fcs of the heavy chain, in this example, an antibody was produced in a monovalent form in which only one scFv was bound to the Fc C-terminus at one position in the heavy chain. For this purpose, vectors in which 1564, F06, C04, VH5, VH16, VH35, VH9, VH2, VH7, VH32 among the IGF1R-specific single clone phage antibodies secured in Examples 6 to 8 were bound only to one position in the Fc C-terminus, and vectors in which no anti-IGF1R antibody was bound to the C-terminus were produced. A knob-into-hole mutation was introduced into the Fc so that a heteromeric form would be produced during antibody production in cells. When transfecting CHO-S cells for antibody production, three vectors in total were injected: a vector for the heavy chain to which an anti-IGF1R antibody was bound to the Fc C-terminus of the therapeutic antibody, a vector for the heavy chain to which no anti-IGF1R antibody was bound to the C-terminus of the therapeutic antibody, and a vector for the light chain of the therapeutic antibody.

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

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

[0181] The obtained culture solution was passed through an equilibration buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl) and then passed through Mab selectsure (GE healthcare, 5 mL) equilibrated so that the expressed antibody binds to the column. Then, it was eluted with a 50 mM Na-citrate (pH 3.4), 100 mM NaCl solution and then neutralized with 1 M Tris-HCl (pH 9.0) so that the final pH became 7.2. Then, the buffer was exchanged with PBS (phosphate buffered saline, pH 7.4), and when the purity was high, after formulation, it was stored frozen at -20 °C, and when additional purification was required, it was stored at 4 °C until additional purification.

[0182] When additional purification was required, it was purified using Hiload superdex 200 (GE Healthcare, Cat. No.28-9893-36), and it could be purified using various other size exclusion chromatography. After equilibration using an equilibration buffer (1x Phosphate buffered saline pH 7.4, Gibco, Cat. No. 10010-023), the sample after the primary purification was loaded onto the column. The purified sample was stored frozen at -20 °C after formulation.

[0183] Example 10. Production of bispecific antibody The anti-IGF1R antibody according to the present invention was produced as an scFv by linking the heavy-chain variable region and the light-chain variable region using a linker (SEQ ID NO: 134), and linked to the C-terminus of the IgG heavy-chain constant region in the complete form of the anti-α-syn antibody through a linker (SEQ ID NO: 133) to produce a bispecific antibody. Also, as a format of the bispecific antibody, a monovalent antibody produced by linking one molecule of the scFv of the anti-IGF1R antibody per molecule of the complete form of the IgG antibody of the anti-α-syn antibody, and a bivalent antibody produced by linking two molecules of the scFv of the anti-IGF1R antibody were each produced.

[0184] Examples of the sequences of the anti-α-syn antibody used for producing the bispecific antibody in this example and the combined sequences of the bispecific antibodies produced by this invention are as described in Table 10. The specific production methods of the bivalent bispecific antibody and the monovalent bispecific antibody are as follows.

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

[0186] In the case of the heavy-chain sequence inserted into the heavy-chain expression vector, the heavy-chain variable region encoding the anti-α-syn antibody and the anti-IGF1R scFv are linked by a linker to the immunoglobulin C-terminus where the human heavy-chain constant region is linked. In the case of the light-chain sequence inserted into the light-chain expression vector, the light-chain variable region encoding the anti-α-syn antibody and the human light-chain constant region are linked.

[0187] 10-2: Monovalent bispecific antibody cloning The monovalent bispecific antibody is in a form where an antibody light chain is conjugated to a heterodimer in which an anti-IGF1R scFv is conjugated to an anti-α-syn immunoglobulin heavy chain (hole) via a linker at the C terminus and an anti-α-syn immunoglobulin heavy chain (knob) to which the scFv is not linked is bound.

[0188] To enhance the conjugation efficiency of the heavy chain heterodimer, the Knob-in-hole technique was used. That is, the coding sequence of the Hole type heavy chain was substituted with T366S, L368A, and Y406V in the CH3 region, and the coding sequence of the knob type heavy chain was substituted with the amino acid T366W in the CH3 region.

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

[0190] For transfection into the prepared mother cells, ExpiFectamine TM CHO transfection kit (Gibco, Cat: A29129) was used to prepare an ExpiFectamine TM CHO & plasmid DNA complex. Cold OptiPRO™ SFM (Gibco, Cat: 12309019) medium was aliquoted respectively, and after inoculating the prepared DNA and ExpiFectamine™ CHO reagent at titrated concentrations, they were mixed and left standing at room temperature for 5 minutes. After inoculating the mother cells for transfection, the culture was started. On the day after transfection, the enhancer and feed included in the ExpiFectamine™ CHO transfection kit were inoculated into the transfected cells. Five days later, after additional inoculation of the feed, the culture was carried out at 8% CO2, 37 °C, and 120 rpm for 10 days to complete the production.

[0191] 10-4: Medium harvest For obtaining the culture solution after the production was completed, the culture solution 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-sized filter to ensure a culture solution free of floating matter and perform the subsequent purification process.

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

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

[0194] The minibody antibodies of clones 996, 1226, 1564, and MKJP2 produced in Example 9-1 were diluted three-fold based on the highest concentration of 20 nM to create 12 points, and then 100 μl was transferred to each well. After treatment at room temperature for 1 hour, it was washed 4 times with PBS buffer containing 0.05% Tween20. Anti-human HRP that recognizes human Fc present in the minibody was diluted 1:5000 in the blocking buffer and 100 μl was transferred to each well and reacted at room temperature for 1.5 hours. Again, after washing 4 times with 300 μl of PBS-T (Tween20 0.05%), color development was carried out using TMB (Tetramethylbenzidine, Sigma, T0440). The enzyme reaction was terminated with 0.5 mol / L sulfuric acid, and the absorbance was recorded and analyzed at 450 nm using a microplate reader (molecular device).

[0195] It was confirmed that the four minibody clones bound to the human IGF1R recombinant protein in a concentration-dependent manner. Specifically, MKJP2 had the best binding ability, followed by 996 and 1564 showing similar binding abilities, and 1226 was confirmed to have a slightly inferior binding ability.

[0196] 11-2: ELISA analysis of interspecies cross-reactivity of IGF1R antibody The cross-species binding of the 1564 anti-IGF1R antibody produced by the method of Example 9-2 and the anti-IGF1R antibody obtained in Example 6-3 was analyzed through the ELISA technique. For this purpose, first, human, monkey, mouse, and rat IGF1R antigens were diluted at 1 μg / ml and 100 μl of each was placed in each well, and reacted at 4°C for 15 hours so as to be coated on the bottom of the plate. After removing the supernatant, 200 μl of PBS buffer containing 3% BSA was treated in each well to block non-specific binding. The anti-IGF1R antibody was set at a maximum concentration of 400 nM and diluted 5-fold in PBSB (3% BSA in PBS) and treated in each of the above wells, and then reacted at 37°C for 1 hour. Next, after washing 5 times with PBS buffer, anti-human Fab HRP that recognizes the Fab portion of the bound antibody was diluted 1:20000 and 100 μl was treated in each well, and then reacted at 37°C for 1 hour. Next, it was washed 5 times with PBS buffer and developed using TMB (Tetramethylbenzidine, Sigma, T0440) according to the manufacturer's method. The enzyme reaction was stopped with 0.5 mol / L sulfuric acid, and the absorbance was measured at 450 nm using a microplate reader (Molecular device). When there were many samples during the ELISA, the plate was divided into two for the experiment, and the experimental results are shown in Table 15 below.

[0197] Specifically, the ELISA results of the bispecific antibody against human IGF1R in Table 15, the ELISA results of 1564 IgG and the bispecific antibody against human IGF1R, the ELISA results of the bispecific antibody against mouse IGF1R, the ELISA results of the bispecific antibody against rat IGF1R, and the ELISA results of the bispecific antibody against monkey IGF1R were tabulated in the following table.

[0198] The following experimental results show the advantage that the efficacy can be evaluated using various species of animal models, and it can be seen that the efficacy of therapeutic agents through various species of disease models can be evaluated using the antibody according to the present invention.

Table 15

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

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

[0201]

Table 16

[0202]

Table 17

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

[0204] Analysis of the secondary clones. Table 18 shows the binding ELISA of the bispecific antibody form of the clones produced by the secondary production method to the IGF1R ECD protein.

Table 18

[0205] After excluding clones with inferior productivity and physical properties among the second-generation clones, the clones for FACS analysis were selected as shown in Table 19.

Table 19

[0206] Figure 4c shows the results of analyzing the binding of the clone to the MCF-7 cell line by FACS. The binding ability to MCF-7 decreased for all analyzed clones compared to 1564, which is the parental clone. This result indicates that the clones showing a decrease in binding ability by ELISA also show a decrease in binding ability by FACS.

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

[0208] Among the antibody clones, the deamidation hot spots present in VH5, VH16, and the F06 variant were removed according to Table 21 in Example 8-2 to produce mutants, and the variant was prepared as a bispecific antibody according to Example 10. In the cases of VH5 and VH16, they were prepared as bivalent bispecific antibodies with hu11F11 (ver.2), and in the case of F06, it was prepared as a monovalent bispecific antibody with hu11F11 (ver.2). Using the prepared bispecific antibodies, the binding affinities of three variants of VH5, VH16, and F06 (i.e., hu11f11-F06, hu11f11-VH5, hu11F11-VH16 as bispecific antibodies) and deamidated mutants (i.e., hu11f11-F06(de2)(StoP), hu11f11-VH5(de2)(StoP), hu11F11-VH16(de2)(StoP) as bispecific antibodies) against MCF-7 were analyzed by the above-mentioned FACS analysis method. The constituent components of the specific bispecific antibodies are described in Table 10 above, and the deamidation position substitutions of these antibodies are described in Table 14 of Example 8-2 above.

[0209] The results of the FACS analysis are shown in Table 20 below. It was confirmed that the binding affinities of all three deamidated mutants (hu11f11-F06(de2)(StoP), hu11f11-VH5(de2)(StoP), hu11F11-VH16(de2)(StoP)) did not decrease compared to the parental antibodies (VH5, VH16, F06).

Table 20

[0210] Using the bispecific antibody prepared as described above, the binding affinities of three variants, VH5, VH16, and F06, and the deamidated mutant for human IGF1R protein were analyzed by ELISA according to the method of Example 15-2. The results are shown in the following table. It was confirmed that the binding affinities of all three mutants did not decrease compared to the parental antibody.

Table 21

[0211] 11-4: BIAcore analysis of human IGF1R An attempt was made to analyze the binding affinity between the antibody according to the present invention and human IGF1R.

[0212] The binding degree of 1564 clone IgG form to human IGF1R was analyzed by SPR analysis. The anti-his antibody against the His tag bound to human IGF1R ECD as the antigen was diluted to 20 μg / ml in acetate pH 4.0 buffer, and then the target RU was immobilized to 10,000 RU on the reference / analytic channel of the CM4 chip by 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. Association / dissociation was 5 minutes and 20 minutes respectively. The analysis was performed in the order of baseline 1, activation (EDC + NHS), human IGF1R loading, quenching (1M Ethanolamine), baseline 2, association, and dissociation. Evaluation used a bivalent model and was analyzed using Biacore T200 Evaluation software (version 1.0, S / N: 04Y15X11-0149).

[0213] As a result of the analysis, the KD of the 1564 IgG antibody was 2.5305×10 -9 nM, and the F06 IgG antibody was 4.7802×10 -7 nM, and it was confirmed that all showed high binding affinity to human IGF1R. The result of the analysis is shown in Figure 3b. In particular, when the 1564 clone was produced in IgG form, it was confirmed that it showed a dissociation constant of 2.5305×10 -9 nM to human IGF1R, and it was confirmed that 1564 did not show a significant change in binding affinity depending on its form.

[0214] Example 12. Binding Affinity Analysis of Anti-IGF1R Antibodies to Cell Lines Expressing Human IGF1R and Brain Endothelial Cells 12-1: FACS analysis of MCF-7 To confirm whether the minibody forms of clones 996, 1226, and 1564 produced in Example 9-1 bind to endogenous IGF1R on the cell surface, binding affinity analysis to cell lines expressing human IGF1R and brain endothelial cells was performed by FACS. The binding degree between the periplasmic extract and MCF-7, a breast cancer cell line known to overexpress IGF1R, was confirmed by FACS.

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

[0216] The binding affinities of A02, A06, A07, B01, B02, B09, B10, C04, D03, E06, F06, H04(Gly), H04(Val), VH2, VH5, VH7, VH9, VH16, VH32, VH35 produced in Example 7 and Example 9-2 to MCF-7 were analyzed in the same manner as above. Clone 1564 was produced by the method of Example 14-2 and compared as the parental clone, and MCF-7 cells treated with only the secondary antibody were used as the control group. The analysis results are shown in Figure 4c.

[0217] From the experimental results, it was confirmed that the scFv and the affinity variant and parental clone (clone 1564) in the form of bispecific antibody within the three tested minibodies specifically bind to the endogenous IGF1R expressed on the cell surface, as indicated by the MFI (Mean Fluorescence Intensity) of the sample. This result shows that the clones obtained in the above examples can bind to IGF1R in the form actually existing in the body and can be used for the intended purpose.

[0218] 12-2: FACS Analysis of JIMT-1 and BT474 Except for using JIMT-1 and BT474 breast cancer cell lines instead of the MCF-7 cell line used in Example 12-1, the minibody forms of clones 996, 1226, and 1564 produced in Example 14-1 were confirmed for their binding to the endogenous IGF1R on the cell surface in a substantially similar manner. The above experimental results are shown in Figure 4a.

[0219] From the experimental results, it was confirmed that the scFv within the three tested minibodies specifically binds to the endogenous IGF1R on the surface of cell lines with diverse IGF1R expression, as indicated by the MFI (Mean Fluorescence Intensity) of the sample.

[0220] 12-3: FACS Analysis of Mouse Brain Endothelial Cells It was analyzed whether the bispecific antibody form of clone 1564 produced by the method of Example 9-2 and the IgG form of clone 1564 produced by the method of Example 14-3 bind to bEND.3, which is a mouse brain endothelial cell. At this time, the group treated with only the secondary antibody and the group treated with the therapeutic antibody alone IgG (CH11F11) were used as negative control groups. The FACS analysis method was the same as in Examples 12-1 and 12-2. The analysis results are shown in Figure 4b.

[0221] All of the tested test clones showed binding affinity to bEND.3 except for the negative control group. Through these results, it was confirmed that 1564 clones in various forms specifically bind to IGF1R expressed on the surface of brain endothelial cells.

[0222] Example 13. Intracellular internalization analysis of anti-IGF1R antibody 13-1: MCF-7 internalization assay - 1564, 996, 1226, MKJP2 (minibody) The minibody forms of the 996, 1226, 1564, and MKJP2 clones produced in Example 9-1 were analyzed to determine whether they were intracellularly internalized in cell lines expressing IGF1R and whether the antibodies introduced into the cells were degraded and passed through the RMT Pathway. In order for an anti-IGF1R antibody to be used as a shuttle to improve BBB permeability, it must be preceded by internalization within the brain endothelial cells that constitute the BBB.

[0223] The MCF-7 cell line expressing IGF1R was used to analyze whether the antibody according to the invention was internalized into cells. Specifically, 30,000 MCF-7 cell lines were plated in an 8-well slide chamber and then cultured for one day. The minibody antibodies of the 996, 1226, 1564, and MKJP2 clones produced in Example 9-1 were each treated at 5 μg / ml in each well 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.

[0224] To test the internalization of the antibody complex, the plate was transferred to a CO2 incubator and cultured at 37°C for 30 minutes. 100% methanol was added to fix the culture and simultaneously terminate the reaction. After fixation, it was washed three times with PBS. The degree of internalized antibody was imaged in the green filter region (Alexa488) on a fluorescence microscope. During imaging, DAPI was used to stain the cell nuclei to confirm the position of each cell. The experimental results are shown in Fig. 5a.

[0225] From the above experimental results, it was confirmed that internalization occurred well for all four antibodies tested even in the experiment using the MCF-7 cell line. In particular, it was found that the internalization of MKJP2 and 1564 occurred more frequently than in other clones.

[0226] 13-2: MCF-7 internalization assay - C04, F06, VH5, VH16, VH35, VH9, VH2, VH7, VH32 To analyze the cell surface IGF1R binding of the 1564 variant with altered binding affinity to IGF1R, FACS analysis was performed using an MCF-7 cell line expressing IGF1R. The IGF1R antibody produced as a bispecific antibody in scFv form was treated at a concentration of 10 μg / mL with 2×10E5 MCF7 cells for 30 minutes. After washing with a PBS buffer supplemented with 1% BSA, it was treated with a secondary antibody conjugated with FITC for detecting human antibodies for 1 hour. After washing with a PBS buffer, the extracellular binding and internalization of the variant with diverse binding affinity were confirmed through FACS analysis.

[0227] As shown in the results of Table 22 below, it was confirmed that the bispecific antibody containing 1564 IGF1R antibody had an increased internalization and an increased intensity at 37 °C compared to the refrigerated conditions, and it was also confirmed that the F06 clone with a high degree of cell binding also had an increased internalization. Such results suggest that the 1564 variant binds well to cells and is internalized into cells in a binding-dependent manner.

Table 22

[0228] 13-3: Internalization Analysis of Human Brain Endothelial Cells The bivalent and monovalent forms of the 1564 clone produced in Examples 9-2 and 9-4 were analyzed for their internalization into primary human microvascular brain endothelial cells (HMBEC). The therapeutic antibody IgG (11F11) was used as a negative control group.

[0229] HMBEC (Cell Systems, cat#: ACBRI376) was plated in a 12-well plate to 90% confluency and then the test antibody was administered. The next day, after fixation with 4% paraformaldehyde and rinsing with PBS, blocking and permeabilization were performed using a solution containing 3% BSA and Triton X 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, Hoechst was stained at a concentration diluted 1:1000 for 10 minutes for nuclear staining. The resultant was analyzed by a confocal microscope under the conditions of LSM 780 NLO EC Plan-Neofluar 100X / 1.3 Oil. The experimental results are shown in Figure 5b.

[0230] 1564 clones in bivalent and monovalent forms showed increased internalization compared to the therapeutic antibody (11F11), which is a negative control group. This result indicates that the anti-IGF1R antibody described above is a bispecific antibody in various forms conjugated with the therapeutic antibody, and can effectively internalize the therapeutic antibody into brain endothelial cells that constitute the BBB, thus ultimately suggesting an increase in the BBB permeability of the therapeutic antibody.

[0231] 13-4: Analysis of Cellular Fate within Human Brain Endothelial Cells In case the antibody co-localizes with the lysosome-related marker within the cell after being internalized, the antibody cannot pass through the BBB because it is degraded within the brain endothelial cells. In contrast, if it co-localizes with the early endosome associated with exocytosis or a marker known to be related to BBB passage, it is expected that after the antibody is internalized into the brain endothelial cells, it will pass through the BBB by receptor-mediated transcytosis and escape to the brain side.

[0232] After treating HMBECs with the bivalent form of the antibody tested in Example 13-2 in the same manner, an analysis was performed to determine which cellular components the antibody co-localizes with intracellularly. However, after blocking and permeabilization, the following antibodies were simultaneously administered together with the Goat anti-human antibody for detecting the administered antibody. *Anti-Cathepsin D: Lysosome marker *Anti-Caveolin-1: Marker for caveolin-mediated transcytosis (thought to be the main mechanism for crossing the BBB) *Anti-EEA1: Marker for early endosome The remaining method was the same as in Example 13-2, and each was treated with a secondary antibody against the above-mentioned marker.

[0233] The analysis results are shown in Figure 5c. All 1564 clones did not colocalize with Cathepsin D in the form of bispecific antibodies, but instead colocalized with caveolin-1 and EEA1 in the cell membrane and inside the cell. This result indicates that after the 1564 clones are internalized, they can cross the BBB through RMT without passing through the intracellular degradation mechanism.

[0234] Example 14. Analysis of the effect of anti-IGF1R antibody on IGF1 signaling 14-1: Proliferation Assay of MCF-7 Cell Line by IGF1R Whether the anti-IGF1R antibody according to the present invention interferes with the binding between IGF1R (IGF1 receptor) and its ligand IGF1 was confirmed using the cell proliferation efficacy of IGF1.

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

[0236] To measure the degree of cell growth by culturing for 3 days at appropriate temperature and humidity, 10 μl of CCK-8 reagent was treated and cultured in a CO2 incubator for 4 - 5 hours. Then, it was taken out and the absorbance was measured at a wavelength of 450 nm with a spectrophotometer. The experimental results are shown in Fig. 6a.

[0237] 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 by the signaling of IGF1 to IGF1R. The anti-IGF1R antibody of Imclone used as a control group inhibited the cell proliferation of MCF-7 by IGF1 in proportion to the treatment concentration. Therefore, since the antibody of the present invention binds to IGF1R expressed in endothelial cells constituting the BBB and has the ability to cross the BBB, and at the same time does not inhibit the signaling by endogenous IGF1, it was confirmed that the antibody according to the present invention can be used as a BBB shuttle.

[0238] 14-2: Inhibition Analysis of Signaling Component of MCF-7 Cell Line by IGF1R An experiment was conducted 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 that binds to IGF1R-expressing cells transmits the signaling into the cell. That is, an anti-IGF1R antibody was administered to the MCF-7 cell line expressing IGF1R, and the amounts of total IGF1R, phosphorylated IGF1R, total Akt, and phosphorylated Akt, which are downstream factors of IGF1R, in the cells were analyzed.

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

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

[0241] 14-3: Inhibition Analysis of Signaling Component of Mouse Brain Endothelial Cells by IGF1R An experiment was conducted to determine whether the anti-IGF1R antibody according to the present invention is involved in the receptor and downstream signaling components of the signaling when IGF1 transmits the signaling intracellularly to mouse-derived brain endothelial cells. That is, 11F11-1564 and 3A9-1564 (CH11F11 and ch3A9, the anti-α-syn single antibody described in Korean Patent Publication No. 2018-0081465) prepared by the method of Example 9-2 and the IgG form of the 1564 clone prepared by the method of Example 9-3 were administered to the bEND3 cell line in which IGF1R expression was confirmed, and the amounts of total IGF1R, phosphorylated IGF1R, total Akt which is a downstream factor of IGF1R, and phosphorylated Akt in the cells were analyzed thereby.

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

[0243] From the above experimental results, it was confirmed that the antibody according to the present invention does not affect the total intracellular IGF1R, phosphorylated IGF1R, total Akt, which is a downstream factor of IGF1R, and the amount of phosphorylated Akt.

[0244] Example 15. In vivo BBB permeability analysis (co-localization assay) of anti-IGF1R antibody 15-1. Colocalization of Minibody with Brain Vessel The following experiments were conducted to confirm whether the anti-IGF1R antibody of the present invention is distributed along the brain vasculature in vivo. Specifically, male BALB / c mice, 6 - 8 weeks old, were each administered a single dose via the tail vein with PBS buffer, or 10 mg / kg of IgG control, and the minibody antibodies of clones 996, 1226, and 1564 produced in Example 9-1. Four hours later, the mouse brains were intracardially perfused with a sufficient amount of 0.9% NaCl solution and 4% paraformaldehyde. The fixed brains were excised, sectioned to 20 μm, and then co-stained with the vascular markers anti-mouse CD31 and anti-human Fc antibodies to confirm the co-localization of blood vessels and IGF1R tests. CD31 was a secondary antibody conjugated with Alexa488, and human Fc was a secondary antibody conjugated with Alexa 594. Imaging was performed under a fluorescence microscope. The experimental results are shown in Figure 7a.

[0245] From the above experimental results, it was confirmed that the ligand-binding non-blocking clones according to the present invention have excellent BBB permeability. By the method of analyzing the degree of co-localization of the antibody with blood vessels by immunostaining (Neuron (2016) Yu-Zuchero et al.), the brain tissue was stained with a vascular marker (anti-CD31, green) and a human antibody (anti-human Fc, red). As a result, the ligand-binding non-blocking clones according to an example of the present application showed a higher degree of co-localization compared to the IgG control group.

[0246] 15-2. Analysis of In Vivo BBB Penetration Ability of Bispecific Antibody We attempted to confirm the in vivo BBB permeability of the anti-IGF1R antibody of the present application in normal rats. After a single intravenous injection of 10 mg / kg or 30 mg / kg of the Parkinson's disease treatment monoclonal antibody (11F11) or the bispecific antibody (11F11-1564) in which clone 1564 was conjugated in a bivalent form to SD rats, the amount of antibody in the CSF and brain was analyzed by mass spectrometry at 24 hours. The specific mass spectrometry method was to plate Sv-ARBEC in a single layer on a permeable membrane, and the integrity of the system was preliminarily evaluated based on the resistance value (TEER) of the BBB system and the degree of sucrose passage. At this time, the rat astrocyte culture medium (RAS-CM) that was confirmed to assist the integrity of the system was treated with sv-ARBEC. After treating the bivalent bispecific antibodies of the test antibodies 1564, 48G5, 54H4, 60H6, and B11 on the membrane, the amount of antibody in the bottom chamber was analyzed by mass spectrometry 90 minutes later. For mass spectrometry, it was used after the signature peptides in each antibody Fc and scFv were analyzed. At this time, the Parkinson's disease treatment monoclonal antibody alone (11F11) and the bispecific antibody in which the scFv form of the biosimilar of Herceptin was conjugated to the antibody were used as negative control groups. The A20.1 antibody (manufactured by the National Research Council), which is known not to pass through the BBB, was passed through the system to determine the detection limit line of the system. The result value derived from the mass spectrometry was substituted into the formula known from the prior literature to determine the Papp value, and this value indicates the degree of BBB permeability in vitro.

[0247] As a result of the analysis, the bispecific antibody conjugated with 1564 clones showed higher CSF and brain permeability compared to the therapeutic antibody without the anti-IGF1R antibody bound thereto, and this efficacy was confirmed at all doses of 10 and 30 mg / kg. The bispecific antibody showed brain permeability approximately 4.5-fold or more greater than that of the single antibody at a dose of 30 mg / kg. After preparing 1564 clones in bivalent and monovalent forms according to Examples 9-2 and 9-4, the antibody amounts in CSF and the brain were analyzed 24 hours after administration at 30 mg / kg or 60 mg / kg in the same manner as described above. The two forms of bispecific antibody conjugated with 1564 clones showed higher CSF and brain permeability compared to the single antibody. In particular, the bivalent form showed higher BBB permeability compared to the monovalent form, which was an increase in brain permeability of approximately 5-fold at most.

[0248] The results of FIGS. 7b and 7c show that the in vivo BBB permeability of the therapeutic antibody is also improved when 1564 clones are conjugated to the therapeutic antibody in various forms.

[0249] An affinity variant of 1564 clones produced according to Example 2 was expected to have an increased serum PK compared to the parental clone. Therefore, it was expected that its BBB permeability would be improved by remaining in the serum for a long time and continuously maintaining the BBB influx amount. After intravenously injecting the affinity variant produced in bivalent form according to Example 9-2 or in monovalent form according to 9-4 into SD rats at 30 mg / kg, blood was collected from the orbital venous plexus at 0, 24, and 48 hours. The test antibody was divided into two experiments depending on whether the backbone of the therapeutic antibody was a chimeric or humanized antibody, and the bispecific antibodies of the variants used in the experiments are as shown in the following table.

Table 23

[0250] The amount of antibody in the blood was analyzed by ELISA. After coating a 96-well plate with goat anti-human Fc antibody, an appropriately diluted sample was processed and then detected with an anti-human Fab HRP conjugated antibody. The analysis results are shown in Figures 7d and 7e.

[0251] As a result, in the primary test group, the monovalent forms of 1564, F06, and C04 showed longer serum PK than the bivalent parental 1564 clone. In the secondary 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.

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

[0253] In the primary test group, the 1564 monovalent, F06 monovalent, and C04 monovalent forms that showed increased serum PK showed increased CSF antibody amounts compared to the parental 1564 bivalent. In the secondary test group, the VH2, VH5, VH7, VH9, VH16, and VH32 bivalents that also showed increased serum PK showed increased CSF antibody amounts compared to the parental 1564 bivalent. VH35 showed shorter serum PK and CSF antibody amounts compared to the parental 1564 bivalent.

[0254] The results of FIGS. 7d, 7e and 7f indicate that PK in serum is an important factor in the ability of antibodies to cross the BBB due to the sustained influx of antibodies across the BBB, and that the ability of bispecific antibodies with a BBB shuttle to cross the BBB increases with increased serum PK. In particular, in the case of the F06 monovalent form with the highest CSF antibody level, it showed a CSF permeability approximately 5-fold higher than that of the parental 1564 bivalent form. Since the 1564 bivalent antibody has shown a CSF permeability approximately 3-fold higher than that of the single antibody in CSF, it is expected that the F06 monovalent form will show a BBB permeability increased by up to approximately 15-fold compared to the single antibody.

[0255] Example 16. Epitope mapping of anti-IGF1R antibodies 16-1. ELISA of Anti-IGF1R Antibody with Boiled and Native IGF1R Proteins It was attempted to confirm whether the anti-IGF1R antibody clones contained in the bispecific antibodies according to the present invention recognize linear or conformational epitopes. ELISA was performed on the bivalent bispecific antibodies of 1564, 48G5, 54H4, 60H6, B11 and native human IGF1R ECD protein or the protein with heat added thereto (boiled IGF1R). The ELISA method was the same as the method shown in Example 11. The analysis results are shown in the following table.

Table 24

[0256] The clones showed a binding force similar to the results in Example 15 to native human IGF1R ECD, but did not bind to boiled human IGF1R ECD with its tertiary structure destroyed by heating. This means that the anti-IGF1R antibodies of the present application bind to conformational epitopes that are not linear.

[0257] 16-2. Epitope Mapping of Anti-IGF1R Antibody To analyze the conformational epitope of the 1564 clone, alanine scanning was performed as follows. An IGF1R library with an eGFP tag fused to the N-terminus and the C-terminal kinase domain removed was expressed in OVCAR3 cells, an ovarian cancer cell line with low IGF1R expression. The IGF1R library contains mutations in which the residues on the IGF1R surface are substituted with alanine. The prepared library was transfected into OVCAR3 cells. The cells with confirmed IGF1R expression were treated with the 1564 antibody, and then treated with a secondary antibody labeled with DyLight650 for fluorescence labeling. After classifying the labeled cells according to the presence or absence of IGF1R expression and the presence or absence of binding between IGF1R and 1564, RNA deep sequencing was performed on these cells using the Illumina HiSeq technique to analyze the frequency of each alanine mutation in the cell population. After normalizing the frequency numbers with the results for cells expressing wild type IGF1R, the relative frequency was calculated, and mutations with a decreased number were selected from the cell population labeled with 1564.

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

[0259] Example 17. Comparison of Antigen-Binding Affinities of Single Antibodies and Bispecific Antibodies 17-1: Binding Affinity of Monoclonal and Bispecific Antibodies against α-syn Antigen When the IGF1R antibody was linked to the IgG-form α-syn antibody in scFv form, the effect on the binding affinity of the α-syn antibody was analyzed.

[0260] The 96-well plates were coated with α-synuclein aggregates at a concentration of 1 μg / ml for 18 hours, and after washing, each antibody was diluted 5-fold at 400 nM for binding. After binding the anti-human Fc-HRP, the TMB solution was added for color development to confirm the degree of antibody binding. As shown in the results of Figure 10a, it was confirmed that the binding ability to α-synuclein aggregates was the same in both single antibodies and bispecific antibodies.

[0261] 17-2: Binding Affinity of Monoclonal and Bispecific Antibodies against IGF1R Antigen Experiments were conducted in the same manner as before to compare the binding degree of single α-synuclein antibodies and bispecific antibodies to the IGF1R antigen.

[0262] As shown in the results of Figure 10b, it was confirmed that the bispecific antibody with the IGF1R scFv antibody bound well in a concentration-dependent manner, while the single antibody without the IGF1R antibody site did not bind.

[0263] 17-3: Binding Affinity Analysis of α-syn Humanized Antibodies Experiments were conducted in the same manner as in Example 17-1 to analyze the difference in binding ability between chimeric bispecific antibodies and humanized bispecific antibodies.

[0264] As shown in the results of Figure 10c, the humanized bispecific antibodies (combinations of Hu11F11 ver2, 3, or 4 and 1564) retained the binding ability to α-synuclein aggregates at a level similar to that of the chimeric bispecific antibodies, and it was also confirmed that the monovalent bispecific antibody with one IGF1R scFv showed a binding ability similar to that of the chimeric antibody.

[0265] Experiments were conducted in the same manner as in Example 17-2 to analyze the IGF1R binding ability between chimeric bispecific antibodies and humanized bispecific antibodies. As a result, as shown in Figure 10d, all bispecific antibodies (combinations of Hu11F11 ver2, 3, or 4 and 1564) showed the same binding ability, and it was confirmed that the single antibody without IGF1R scFv did not bind.

[0266] Such results suggest that when humanized to alternate mouse antibody sites that can act as immunogens in the human body, there was no change in the binding affinity for α-synuclein aggregates and IGF1R, meaning that they possess the same activity.

[0267] 17-4: Comparison of Phagocytic Activity between Monoclonal and Bispecific Antibodies Phagocytic action means the action of removing extracellular unnecessary substances involving various receptors of macrophages. Various protein aggregates come to induce immune and inflammatory responses and have an adverse effect on the human body. Especially when an antibody is administered for the removal of α-synuclein aggregates, it is known to be promoted through the interaction between the Fc site of the antibody and FcrR on the cell surface. For such reasons, the activity of a bispecific antibody in which a single antibody and IGF1R scFv are linked was compared with respect to phagocytic action.

[0268] To compare the phagocytic action of a single antibody and a bispecific antibody, BV-2 microglia derived from mice were used. BV-2 cells were cultured in RPMI1640 medium and prepared at 2×10 6 cells / ml and dispensed 100 μL each into a U-bottom 96 well plate. 10 μg / ml of α-synuclein aggregates and 25 μg / ml of antibody were diluted and mixed in RPMI1640 medium and then left at room temperature for 20 minutes. After treating the BV-2 cells with the α-synuclein aggregate and antibody mixture, they were left for 15 minutes. Centrifugation was performed at 1200 rpm to remove the α-synuclein aggregates in the supernatant, and the cells were washed three times with PBS, pH 2.5 buffer to remove the aggregates or antibodies bound to the cell surface. The cells were fixed with 4% paraformaldehyde and then washed with PBS buffer. To confirm the aggregates and antibodies phagocytosed into the cells, 0.5% Triton X-100 was added to loosen the cell membrane, and after washing with PBS buffer, the pan-α-syn antibody was treated for 1 hour. The bound pan-α-syn antibody was treated with an anti-rabbit-alexa-488 antibody for 1 hour, and then the aggregates that entered the cells by phagocytosis were confirmed through FACS analysis.

[0269] As shown in the results of Fig. 10e, normal human IgG did not affect phagocytosis, and it was confirmed that phagocytosis of α-syn aggregates increased when treated with the α-syn antibody. When comparing the single antibody and the bispecific antibody, it was confirmed that phagocytosis occurred at a similar level, and it was confirmed that the scFv-form IGF1R antibody bound to the IgG C-terminal region did not affect the action of the α-syn antibody.

[0270] Example 18. Evaluation of the efficacy of bispecific antibodies According to Example 10, a bivalent bispecific antibody of chimeric 11F11 antibody and 1564 clone scFv was produced, and the effects of the bispecific antibody and the α-synuclein antibody in vivo were compared and analyzed in transgenic mice (mThy-1 human α-synuclein, UC San Diego) overexpressing human α-synuclein. A single antibody or human IgG or the same molar amount of a bivalent bispecific antibody at 2.5 mg / kg was intraperitoneally administered weekly for 3 months. Five mice were used per group, and non-transgenic littermates were used as the control group. Next, perfusion was performed as follows.

[0271] After the last administration was completed, for pathological analysis of the brain, the animals were anesthetized with chloral hydrate according to humane regulations and then perfused through the heart with 0.9% physiological saline. Next, half of the perfused brain (saggital section) was stored in 4% paraformaldehyde in phosphate buffer (pH 7.4, stored at 4 °C until the time of analysis, and the other half was immediately stored in the frozen state (-70 °C).

[0272] Pathological analysis was performed as follows. Brain hemispheres fixed in paraformaldehyde were cut into 40-μm-thick serial sections in a free-floating manner using a Vibratome. To confirm the expression level of alpha-synuclein in the brain of each administration group, sections containing the cortex, hippocampus, and striatum were cultured overnight at 4°C with an alpha-synuclein antibody (p129 α-syn antibody, a marker for aggregates, abcam, ab59264 or total alpha-synuclein antibody, Cell Signaling Technology, #2642). Alternatively, to analyze the activity level of astrocytes, the sections were treated with GFAP (glial fibrillary acidic protein) (AB5804, millipore), or to confirm the degree of neuroinflammation, the sections were treated with an antibody against IL-1β (ab9722, abcam). Alternatively, to analyze the degree of neuronal cell death in the hippocampus, the sections were treated with an antibody against NeuN (Chemicon, #MAB377). After culturing with the primary antibody, biotin-conjugated goat anti-rabbit IgG (1:100, Vector Laboratories) and avidin D-horseradish peroxidase (1:200, ABC Elite, Vector Laboratories) were treated and detected with DAB (diaminobenzidine). Each immunostained section was observed under a bright-field microscope to measure the optical density. The results are disclosed in FIGS. 11a to 11e.

[0273] 18-1. Analysis of Alpha-synuclein Reduction Ability of Chimeric and Bispecific Antibodies Figure 11a shows the result of measuring whether the chimeric 11F11 antibody and the bivalent bispecific antibody of the chimeric antibody and clone 1564 can remove alpha-synuclein aggregates in a mouse animal model (TG) overexpressing human α-Syn. After antibody administration to the mice, the cortex and hippocampus in the mouse brain tissue were stained with the p-129 α-Syn antibody. p-129 α-syn is a marker for aggregates in the phosphorylated form at the 129th residue, and is shown as dark brown spots or aggregate forms in the stained tissue.

[0274] According to Figure 11a, the IgG-treated group showed a higher degree of p-129 staining compared to the non-tg control group (#: one way ANOVA, p < 0.01). In contrast, in the groups treated with the single antibody or the bispecific antibody, the staining degree of p-129 α-syn or aggregates was significantly reduced. In particular, in the hippocampus, the degree of reduction in the bispecific antibody-treated group was superior to that of the chimeric 11F11 antibody (*: one way ANOVA, p < 0.05). Figure 11b is the same experiment as Figure 11a, but shows the result of staining with the total alpha-synuclein antibody as a marker. Detection of total alpha-synuclein indicates that the antibody according to the present application has the ability to remove alpha-synuclein itself (clearing) and the ability to suppress cell-to-cell transmission of the antibody. Also, from another aspect, it can be interpreted that the formation of monomers into aggregates is suppressed, or all monomers can be removed. The increased human alpha-synuclein in TG mice was reduced by administration of the single antibody and the bispecific antibody compared to the IgG-administered group. In particular, in the hippocampus, the efficacy of the bispecific antibody was even more excellent than that of the single antibody.

[0275] The results show that the chimeric 11F11 antibody and the bispecific antibody effectively reduce alpha-synuclein and its aggregate levels in a Parkinson's disease animal model even at a low dose of 2.5 mg / kg. In particular, the bispecific antibody has superior efficacy compared to the single antibody, suggesting that the bispecific antibody can reach more of the brain compared to the single antibody based on its improved BBB permeability and can effectively treat the disease.

[0276] 18-2. Analysis of Reduction Ability of Astrogliosis and Inflammatory Cytokine Levels by Chimeric and Bispecific Antibodies Gliosis is a non-specific reaction that occurs in glial cells as a response to damage in the central nervous system, triggered by substances such as BBB damage, TGF-beta, or interleukin. Typically, it includes astrogliosis, and the GFAP protein is used as a marker. Therefore, the chimeric 11F11 antibody and the bispecific antibody of the chimeric antibody and clone 1564 were administered to mice to analyze their effects on reducing astrogliosis and reducing the release of inflammatory cytokines that trigger it. The analysis results are disclosed in FIGS. 21c and 21d.

[0277] FIG. 11c shows the results of measuring whether the chimeric 11F11 antibody produced in an example of the present invention or the bispecific antibody of the antibody and clone 1564 can reduce astrogliosis in vivo by staining the brain tissue of mice with a GFAP (astrogliosis) antibody as a marker after administering the antibody to the mice. It was confirmed that both the single antibody and the bispecific antibody suppressed astrogliosis compared to the IgG control group, and in particular, the efficacy of the bispecific antibody was superior to that of the single antibody in the striatum.

[0278] Figure 11d shows the results of measuring whether the chimeric 11F11 antibody produced in an example of the present invention or the bispecific antibody of the antibody and clone 1564 can reduce inflammatory cytokines in vivo. After administering the antibody to mice, brain tissue of the mice was stained with an IL-1 beta antibody as a marker. IL-1 beta induces inflammation and comes to induce death of various nerve cells and an inflammatory reaction. In the hippocampus of the mice administered with the antibody according to the present application, IL-1 beta decreased in the single antibody and bispecific antibody administration groups compared to the IgG control group, and in particular, the ability of the bispecific antibody to decrease was significantly superior to that of the single antibody (##: One-way ANOVA, p < 0.005; *: one way ANOVA, p < 0.05).

[0279] As shown in the above drawings, it has become clear that the antibody according to the present application reduces astrogliosis and reduces the release of the inflammatory cytokine IL-1 beta that triggers this, compared to the control group.

[0280] 18-3. Analysis of Reduction Ability of Neurodegeneration by Chimeric and Bispecific Antibodies It has been confirmed in prior literature that the death of brain cells occurs due to the neurotoxicity and inflammatory reaction of alpha-synuclein. It was analyzed whether the single antibody and bispecific antibody of the present application can suppress in vivo brain cell death caused by alpha-synuclein. As a result of staining with NeuN, a neuron marker, in the cortex and hippocampus, it became clear that the degree of brain cell death decreased in both the single antibody and bispecific antibody compared to the IgG control group. In particular, it was confirmed that the ability of the bispecific antibody to suppress brain cell death was superior to that of the single antibody in the cortex. The results are shown in Figure 11e.

[0281] Example 19. Half-life increase through Fc engineering and improvement of BBB permeability thereby FcRn is an important cell membrane receptor that increases the half-life by drawing antibodies into cells and circulating them so that they are not lysed when antibodies circulate in the blood vessels. Although the ability to cross the BBB is also important for the activity of transcytosis antibodies, it is a well-known fact that they cross the BBB depending on their concentration in the blood vessels. For this reason, a bispecific antibody was produced in which the 428th amino acid of the Fc region was changed from methionine (Met) to leucine (Leu) to increase the binding affinity with FcRn in order to increase the half-life of the bispecific antibody. As a result of administering the WT bispecific antibody and the M428L bispecific antibody at a concentration of 10 mg / kg to tg mice expressing human FcRn and comparing them, as shown in Fig. 11, a half-life increasing effect of about 50% was confirmed. To reconfirm the increase in half-life, the WT bispecific antibody (hu11F11(ver.2)-1564), the bivalent M428L bispecific antibody (hu11F11(ver.2)(M428L)-1564 bivalent), and the monovalent M428L bispecific antibody ((hu11F11(ver.2)(M428L)-1564 monovalent) were administered to monkeys and the PK profile was analyzed. As shown in Fig. 12a, in the case of the WT bispecific antibody, the blood concentration rapidly decreased after 168 hours, whereas the M428L bispecific antibody with a high binding affinity for FcRn maintained an improved blood concentration compared to WT. The half-life was confirmed to increase by about 1.5 days with the M428L bispecific antibody compared to the WT bispecific antibody. In particular, from the clearance aspect, the monovalent M428L bispecific antibody was the most excellent, and the WT bispecific antibody showed the fastest clearance (Fig. 12b).

[0282] To verify the improvement of BBB passage due to the half-life increase effect, CSF was extracted 24 hours after antibody administration, and the amount of antibody in the CSF was analyzed. After coating 100 ng / ml of IGF1R at 4°C for 18 hours, CSF was added to detect the antibody bound to IGF1R. As confirmed in Fig. 12c, it was confirmed that the amount of BBB passage of the M428L bispecific antibody with a large amount of antibody in the blood was large, and the monovalent M428L bispecific antibody was combined with excellent BBB passage ability and showed improved passage ability compared to the bivalent M428L bispecific antibody.

[0283] Example 20. Evaluation of the efficacy of deamidated Affinity variant-based bispecific antibodies Using the monovalent bispecific antibody of hu11F11 (ver. 2), a humanized 11F11 antibody according to Example 10, and F06 scFv, an affinity variant of clone 1564, especially a bispecific antibody using a mutant in which some residues in the CDR were modified for deamidation (including hu11F11(ver.2)-F06(de2)(StoP) monovalent), the in vivo efficacy of the prepared bispecific antibody and the alpha-synuclein monoclonal antibody was compared and analyzed in transgenic mice overexpressing human alpha-synuclein (mThy-1 human α-synuclein, UC San Diego).

[0284] Specifically, IgG and hu11F11 (ver. 2) at 20 mg / kg, and the bispecific antibody corresponding to the same molar number at 23.4 mg / kg were intraperitoneally administered to the transgenic mice at 4 months of age 4 times at 0, 72, 144, and 192 hours over 8 days. After the last administration, 24 hours later, the animals were anesthetized with chloral hydrate and then perfused through the heart with 0.9% saline. The brains were isolated and snap frozen at -70°C until analysis. After disrupting the brain tissue and centrifuging to remove debris, the supernatant was obtained, and the amount of α-syn in the brain lysate was quantified by ELISA for α-syn (Invitrogen #KHB0061). The results are as shown in Figure 13.

[0285] As shown in Figure 13, the humanized 11F11 (ver. 2) antibody and the deamidated F06 variant-based bispecific antibody produced in one example of the present invention were shown to reduce α-syn in the brain compared to the IgG control group. In particular, the bispecific antibody showed superior ability to reduce α-syn in the brain compared to the α-syn monoclonal antibody.

[0286] Example 21. (de2)(StoP) Analysis of IGF1R-specific antigen-binding ability of bispecific antibody containing deamidated anti-IGF1R antibody (ELISA) In this example, while confirming whether the bispecific antibody containing the deamidated anti-IGF1R antibody according to Example 8 has normal antigen-binding ability, we also attempted to comprehensively analyze the antigen-binding ability of the deamidated IGF1R antibody within various anti-IGF1R clones and bispecific antibody formats. For this purpose, based on the anti-IGF1R clones F06, VH5, and VH16, monovalent or bivalent bispecific antibodies containing the non-deamidated antibody (wild type), (de)(StoP) deamidated antibody, and (de2)(StoP) deamidated antibody were produced respectively, and the binding ability to recombinant IGF1R and the presence or absence of concentration-dependent binding were quantitatively analyzed and compared through sandwich ELISA. The hu11F11 (ver.2) clone was used as the anti-alpha-synuclein antibody.

[0287] The human recombinant IGF1R, which is the antigen to which the antibody binds, is the extracellular domain (ECD) and was purchased from Sino biological (10164-H08H). Human IGF1R was diluted to 1 μg / ml in PBS buffer and 100 μl per well was added to a 96-well ELISA plate (Nunc-Immuno Plates, NUNC, Rochester, NY). After reacting at 4°C for 16 hours for coating, the supernatant was removed. 200 μl of PBS buffer containing 1% BSA (bovine serum albumin) was added per well and reacted at 37°C for 2 hours to block non-specific binding.

[0288] The previously produced bispecific antibody and each control group antibody (wild type and (de)(StoP) deamidated antibody) were diluted 5-fold each based on a maximum concentration of 400 nM to create 8 points. After 100 μl of each was added to each well and reacted at 37°C for 2 hours, the antibody was bound to the coated antigen. After the reaction ended, it was washed 4 times with 300 μl of PBS buffer containing 0.05% Tween20. Anti-human Fc-HRP, which recognizes human Fc present in the bispecific antibody, was diluted 1:2000 in the blocking buffer and 100 μl of each was added to each well and reacted at 37°C for 1 hour. After washing 4 times again with 300 μl of PBS-T (Tween20 0.05%), TMB (Tetramethylbenzidine, Sigma, T0440) was used for color development. The enzyme reaction was stopped with 0.5 mol / L sulfuric acid, and the absorbance was recorded and analyzed at 450 nm using a microplate reader (molecular device). The above experimental results are shown in FIGS. 14a to 14c and Table 25. FIG. 14a shows the experimental results for the F06 monovalent antibody, F06(Stop) monovalent antibody, and F06(de2)(Stop) antibody. FIG. 14b shows the experimental results for the VH5 antibody, VH5(de)(Stop) antibody, and VH5(de2)(Stop) antibody. FIG. 14c shows the experimental results for the VH16 antibody, VH16(de)(Stop) antibody, and VH16(de2)(Stop) antibody.

[0289] According to FIGS. 14a, 14b, and 14c, regardless of the type of anti-IGF1R clone and the monovalent / bivalent format, the (de2)(StoP) deamidated antibody maintained the antigen-binding ability at the wild type level without deamidation. Also, it showed excellent antigen-binding ability compared to the (de)(StoP) deamidated antibody. Table 25 below shows the experimental results for the non-deamidated antibody (wild type), (de)(StoP) deamidated antibody, and (de2)(StoP) deamidated antibody based on F06, VH5, and VH16.

Table 25

[0290] Table 25 numerically compares the sandwich ELISA results of (de)(StoP)-deamidated antibody and (de2)(StoP)-deamidated antibody. According to Table 25, regardless of the type of anti-IGF1R antibody clone and monovalent / bivalent format, it was confirmed that the (de2)(StoP)-deamidated antibody has significantly better antigen-binding ability than the (de)(StoP)-deamidated antibody.

[0291] Example 22. Analysis of cell surface IGF1R-specific antigen-binding ability of bispecific antibodies containing (de2)(StoP)-deamidated anti-IGF1R antibody (FACS) Using the (de2)(StoP)-deamidated bispecific antibodies tested in Example 21, and these wild type antibodies and (de)(StoP)-deamidated bispecific antibodies as control groups, the cell surface IGF1R-specific antigen-binding ability was analyzed by FACS. MCF7 cells overexpressing IGF1R were used for the analysis.

[0292] Specifically, after diluting each of the bispecific antibodies at 10 μg / ml, 0.5×10 6 MCF-7 cell lines were treated and reacted at 4°C for 2 hours. After washing twice with PBS buffer, anti-human FITC diluted at 1:1000 was treated and reacted at 4°C for 1 hour. After washing twice again with PBS buffer, the degree of binding of anti-IGF1R BsAb was measured using a FACSCalibur instrument. MCF-7 cells treated with only the secondary antibody were used as the control group, and the experimental results are shown in FIGS. 15a to 15c.

[0293] As with the experimental results and the results confirmed in Example 21, regardless of the type of anti-IGF1R clone and the monovalent / bivalent format, the (de2)(StoP) deamidated antibody maintained an antigen-binding ability at a level equivalent to that of the wild type antibody that had not been deamidated and showed an excellent antigen-binding ability compared to the (de)(StoP) deamidated antibody.

[0294] Example 23. In vivo BBB permeability analysis of (de2)(StoP) deamidated anti-IGF1R antibody-based bispecific antibody An attempt was made to confirm the in vivo BBB permeability of a bispecific antibody containing an anti-IGF1R antibody deamidated according to Example 8 in Sprague-Dawley (SD) rats. The experimental groups and dosing volumes are tabulated below.

Table 26

[0295] After a single administration of the α-syn single antibody or α-syn / IGF1R bispecific antibody as described in Table 26 above into the tail vein of rats, the amount of antibody in their serum and cerebrospinal fluid (CSF) was analyzed by mass spectrometry at 24 hours. The specific mass spectrometry method was carried out substantially the same as in Example 15, and the analysis results are shown in Figure 16.

[0296] In Figure 16, the serum concentration means the antibody concentration in the blood 24 hours after administration, and was similarly observed for the (de)(StoP) deamidated antibody and the (de2)(StoP) deamidated antibody. On the other hand, in the case of the CSF concentration, which represents the brain delivery of the antibody, the highest level was shown for the (de2)(StoP) deamidated antibody. This indicates the fact that the bispecific antibody of the present invention shows excellent BBB permeability by the deamidated anti-IGF1R antibody.

[0297] To observe the efficacy of the bispecific antibody of the present invention for a longer period, hu11F11 (ver.2), which is an anti-alpha-synuclein single antibody shown in Table 26, and hu11F11 (ver2)-F06 monovalent (de2)(StoP), which is a (de2)(StoP) deamidated bispecific antibody, were administered once into the tail vein of rats, and then the amounts of the antibodies in serum, cerebrospinal fluid, and brain up to 168 hours were analyzed by mass spectrometry. The mass spectrometry method was performed substantially in the same manner as the method of Example 15. The analysis results are shown in Figure 17.

[0298] According to Figure 17, the serum concentration of the antibody was observed to be similar between the single antibody and the (de2)(StoP) deamidated bispecific antibody. However, in the case of the cerebrospinal fluid concentration, which represents brain delivery of the antibody, the area under the curve (AUC) increased by approximately 5.8-fold in the (de2)(StoP) deamidated bispecific antibody compared to the single antibody, and it was confirmed that the cerebrospinal fluid concentration at 24 hours was more than about 10-fold in the (de2)(StoP) deamidated bispecific antibody. Also, the brain concentration showed an approximately 7.9-fold increase in the area under the curve (AUC) in the (de2)(StoP) deamidated bispecific antibody compared to the single antibody. From this, it was confirmed that the bispecific antibody of the present invention can exhibit excellent BBB permeability by including the (de2)(StoP) deamidated IGR1R antibody.

Claims

**Claim 1** An anti-IGF1R antibody or an antigen-binding fragment thereof, comprising a heavy-chain variable region and a light-chain variable region, wherein (1) the heavy-chain variable region comprises a heavy-chain CDR1 (H-CDR1) having the amino acid sequence of SEQ ID NO: 1, a heavy-chain CDR2 (H-CDR2) having the amino acid sequence of SEQ ID NO: 5, and a heavy-chain CDR3 (H-CDR3) having the amino acid sequence of SEQ ID NO: 8, and the light-chain variable region comprises a light-chain CDR1 (L-CDR1) having the amino acid sequence of SEQ ID NO: 20, a light-chain CDR2 (L-CDR2) having the amino acid sequence of SEQ ID NO: 23, and a light-chain CDR3 (L-CDR3) having the amino acid sequence of SEQ ID NO: 27, or (2) the heavy-chain variable region comprises a heavy-chain CDR1 (H-CDR1) having the amino acid sequence of SEQ ID NO: 1, a heavy-chain CDR2 (H-CDR2) having the amino acid sequence of SEQ ID NO: 6, and a heavy-chain CDR3 (H-CDR3) having the amino acid sequence of SEQ ID NO: 9, and the light-chain variable region comprises a light-chain CDR1 (L-CDR1) having the amino acid sequence of SEQ ID NO: 20, a light-chain CDR2 (L-CDR2) having the amino acid sequence of SEQ ID NO: 23, and a light-chain CDR3 (L-CDR3) having the amino acid sequence of SEQ ID NO: 28, or (3) the heavy-chain variable region comprises a heavy-chain CDR1 (H-CDR1) having the amino acid sequence of SEQ ID NO: 1, a heavy-chain CDR2 (H-CDR2) having the amino acid sequence of SEQ ID NO: 7, and a heavy-chain CDR3 (H-CDR3) having the amino acid sequence of SEQ ID NO: 8, and the light-chain variable region comprises a light-chain CDR1 (L-CDR1) having the amino acid sequence of SEQ ID NO: 20, a light-chain CDR2 (L-CDR2) having the amino acid sequence of SEQ ID NO: 23, and a light-chain CDR3 (L-CDR3) having the amino acid sequence of SEQ ID NO: 28, An anti-IGF1R antibody or an antigen-binding fragment thereof. **Claim 2** The heavy-chain variable region of the antibody comprises the amino acid sequence of SEQ ID NO: 52, and the light-chain variable region comprises the amino acid sequence of SEQ ID NO: 97, The anti-IGF1R antibody or an antigen-binding fragment thereof according to Claim 1. **Claim 3** The heavy-chain variable region of the antibody comprises the amino acid sequence of SEQ ID NO: 53, and the light-chain variable region comprises the amino acid sequence of SEQ ID NO: 98, The anti-IGF1R antibody or an antigen-binding fragment thereof according to Claim 1. **Claim 4** The heavy-chain variable region of the antibody comprises the amino acid sequence of SEQ ID NO: 54, and the light-chain variable region comprises the amino acid sequence of SEQ ID NO: 99, The anti-IGF1R antibody or antigen-binding fragment thereof according to claim 1.

5. An antigen-binding fragment is selected from the group consisting of scFv, (scFv) ２ , scFv-Fc, Fab, Fab' and F(ab')[[]]END]] ２ selected from the group consisting of The anti-IGF1R antibody or antigen-binding fragment thereof according to claim 1.

6. A polynucleotide encoding the anti-IGF1R antibody or antigen-binding fragment thereof according to claim 1.

7. A pharmaceutical composition comprising the anti-IGF1R antibody or antigen-binding fragment thereof according to claim 1, and a diluent, carrier, solubilizer, emulsifier, preservative or adjuvant.

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