EA045348B1 - BISPECIFIC ANTIBODY TO α-SYN/IGF1R AND ITS APPLICATION - Google Patents

Description

Field of invention

The present invention relates to a bispecific antibody against alpha-synuclein and IGFR, to a pharmaceutical composition for the prevention and/or treatment of synucleinopathies (αsynucleinopathies), containing the specified bispecific antibody, and to a method for detecting aggregates of alpha-synuclein or providing information for the diagnosis of alpha-synucleinopathies, including said bispecific antibody.

Prior Art

Alpha-synuclein (α-synuclein, α-syn) is expressed predominantly in the presynaptic terminals of neurons and under normal conditions is a naturally unfolded monomer. Alpha-synuclein helps regulate the release of dopamine, which is a neurotransmitter that controls voluntary or involuntary movements. In particular, alpha-synuclein function is important in increased synaptic activity and aging and is an important factor in neurodegeneration.

However, in a pathological state, alpha-synuclein undergoes structural changes by binding and interacting with droplets, phospholipid bilayers, or lipid membranes to form a folded or coiled α-helical secondary structure, resulting in a fold decrease in its quantity. Agglomerates are formed containing molecules in the form of dimers, oligomers and/or fibers.

These alpha-synuclein aggregates are known to be toxic to cells and are a major component of the abnormal protein aggregates of Lewy bodies found in neurons in Parkinson's disease (PD), Parkinson's disease dementia (PDD), multiple system atrophy (MSA), dementia with small bodies Levy (DLB) and various diseases. It is also known that post-translational modifications of alpha-synuclein, such as phosphorylation or ubiquitination, are also associated with the formation of alpha-synuclein aggregates and their neurotoxicity. Alphasynuclein is known to cause the death of dopamine neurons, induce inflammatory responses in animal and cell experiments, and cause motor symptoms similar to Parkinson's disease in experimental animals. In addition, the formation of alpha-synuclein aggregates is known to be associated with the etiology of a group of neurodegenerative diseases called α-synucleinopathies, including Parkinson's disease, Parkinson's disease dementia, dementia with Lewy bodies, multiple system atrophy, and many other neuroaxonal diseases.

Antibodies to alpha-synuclein or fragments of alpha-synuclein to induce such antibodies have been proposed as immunotherapies for synuclein diseases. However, the penetration of antibodies into the brain may be limited by the blood-brain barrier (BBB).

In addition, the shortage of highly specific transporters across the BBB has delayed the development of new therapeutics and diagnostics for the treatment and diagnosis of diseases originating in the brain, including brain tumors and neurodegenerative diseases. There is a clear need for a method of delivering therapeutic and diagnostic molecules in a pharmaceutically effective dose to the brain without disrupting the physiology and homeostasis of the BBB.

Detailed Description of the Invention

Technical problem.

According to one embodiment of the present invention, there is provided a protein complex comprising an anti-alpha-synuclein (α-syn) antigen-binding region and an anti-IGF1R antigen-binding region, or a method for producing said protein complex.

According to another embodiment, there is provided a polynucleotide encoding said protein complex, a recombinant vector containing said polynucleotide, and a recombinant cell containing said recombinant vector.

According to another embodiment, a bispecific antibody against α-syn and IGF1R is provided, obtained from the specified protein complex, and a method for producing the specified bispecific antibody.

According to another embodiment, a pharmaceutical composition is provided for the prevention and/or treatment of alpha-synucleinopathies, containing the specified bispecific antibody against α-syn and IGF1R and a pharmaceutically acceptable excipient.

A method is provided for delivering drugs used to diagnose, treat, or prevent alpha-synucleinopathies to the brain using an antibody or antigen-binding fragment of the present invention.

Technical solution

Hereinafter, the present invention will be described in more detail.

One embodiment of the present invention is a protein complex comprising an antigen binding region for alpha-synuclein (α-syn) and an antigen binding region for IGF1R, and a bispecific antibody against alpha-synuclein and IGF1R (hereinafter referred to as an anti-α-syn/anti-IGF1R bispecific antibody) prepared from the specified protein complex. Thus, the bispecific antibody of the present invention can recognize and bind, as antigens, both alpha-synuclein and IGF1R.

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The anti-a-syn/anti-IGF1R bispecific antibody of the present invention contains an anti-alpha-synuclein antibody or an antigen-binding fragment thereof and specifically recognizes and binds to alpha-synuclein, especially the C-terminal region of alpha-synuclein, thereby making it useful for preventing, treatment and/or diagnosis of diseases associated with alpha-synuclein or alpha-synuclein aggregates, that is, alpha-synucleinopathies.

According to the present invention, the term synucleinopathies includes all neurodegenerative disorders characterized by pathological aggregates of synuclein. The general group of synucleinopathies includes Parkinson's disease, Parkinson's disease dementia (PDD), dementia with Lewy bodies (DLB), Lewy body disease, dementia with Lewy bodies, Parkinson's syndrome with dementia, multiple system atrophy (MSA), multiple atrophy of the nervous system and neurodegeneration type I with iron accumulation in the brain (NBIA type I). In addition, alpha-synuclein aggregates have also been found secondarily in Alzheimer's disease (Kim et al. Alzheimer's Research & Therapy 2014, 6:73).

Synucleinopathies are a heterogeneous group of neurodegenerative disorders with similar pathological features. From a neuropathological perspective, distinct lesions in the form of abnormal alpha-synuclein aggregates can be found in distinct groups of neurons and oligodendrocytes. alpha-synuclein (formerly known as PARK1 and PARK4) is a 140 amino acid protein that is widely expressed in the neocortex, hippocampus, dentate gyrus, posterior neurosphere, striatum, thalamus, and cerebellum. Alpha-synuclein expression is also high in hematopoietic cells, including monocytes such as B cells, T cells, NK cells and platelets. Its exact role in these cells is unknown, but it is associated with the differentiation of megakaryocytes (platelet precursors).

As used herein, alpha-synuclein aggregate-associated disease is a group of neurodegenerative diseases called synucleinopathies, with alpha-synuclein aggregates in pathological sites including neurons and glia, and characteristics such as degeneration of the dopamine system, changes in motor function, cognitive impairment functions and the formation of Lewy bodies and/or Lewy neurites (Kim et al. Alzheimer's Research & Therapy 2014, 6:73; McKeith et al., Neurology (1996) 47:1113-24). These diseases include, but are not limited to, Parkinson's disease, dementia in Parkinson's disease, dementia with Lewy bodies, variant Alzheimer's disease with Lewy bodies, combination of Alzheimer's disease and Parkinson's disease, multiple system atrophy and many other neuroaxonal diseases. In one embodiment, an antibody of the present invention is useful for treating Parkinson's disease.

In addition, the anti-a-syn/anti-IGF1R bispecific antibody of the present invention contains an anti-IGF1R antibody or an antigen-binding fragment thereof, whereby the anti-a-syn antibody or an antigen-binding fragment thereof can cross the blood-brain barrier to exert its effect and has a longer half-life, maintaining effectiveness for a long time.

Moreover, the anti-a-syn/ anti-IGF1R bispecific antibody of the present invention binds to IGF1R on the cell surface without affecting its ligand binding and does not affect the IGF1R signaling pathway. Because it does not inhibit the binding of IGF1R to its ligand and signaling through IGF1R, it can be used as a carrier to cross the blood-brain barrier.

In particular, the anti-IGF1R antibody or antigen-binding fragment thereof of the present invention specifically recognizes IGF1R (insulin-like growth factor type 1 receptor) and binds to IGF1R, in particular human IGF1R, murine IGF1R, rat IGF1R and monkey IGF1R. However, they do not prevent IGF1R ligands such as IGF-1, IGF-2 and/or insulin from binding to IGF1R, do not inhibit signaling through IGF1R, and can be used for transcytosis to cross the BBB. They do not exhibit antibody-dependent cell-mediated cytotoxicity (ADCC) and thus do not reduce IGF1R levels in the brain even when administered repeatedly to animals, making them non-toxic.

In particular, the anti-IGF1R antibody of the present invention binds to IGF1R located on the surface of brain endothelial cells forming the BBB and is internalized into the interior of the cell. For example, cellular internalization of an anti-IGF1R antibody of the present invention can be detected using a cell line expressing IGF1R (eg, MCF-7). Anti-IGF1R antibodies of the present invention, for example, 1564, 48G5, 54H4, 60n6, B11 and affinity variants of 1564, such as C04, F06, VH2, VH5, VH7, VH9, VH16, VH32 and VH35, have a higher degree of internalization compared to with negative control. The results obtained indicate that the degree of internalization of the anti-IGF1R antibodies analyzed is specific to IGF1R on the cell surface. In addition, the anti-IGF1R antibody of the present invention is in the form of a scFv and can be produced linked to a therapeutic antibody by various methods. For example, an anti-IGF1R scFv can be produced as part of a bispecific antibody, such as a bivalent form of a bispecific antibody where two scFvs are linked to the C-terminus of a therapeutic antibody, such as an anti-α-syn antibody, or a monovalent form of a bispecific antibody. , where one scFv is bound to the C-terminus of the therapeutic antibody. Both of these bispecific antibodies can be internalized by cells expressing IGF1R. Antibody to IGF1R has high binding affinity to antigen on the cell surface, which enhances the internalization effect and leads to the ability to pass through the BBB. However, if an antibody has the ability to cross the BBB and interfere with signaling through IGF1R, it may cause side effects. The antibody of the present invention is characterized by both binding ability suitable for passage through the BBB and the absence of blocking signaling through IGF1R.

An anti-IGF1R antibody or antigen binding fragment thereof has the excellent property of being easy to develop. In this aspect, post-translational modifications, such as deamidation, occurring in the CDR region of the anti-IGF1R antibody and reducing the stability and potency of the antibody must be eliminated. Replacing the amino acid at which deamidation occurs allows for increased stability and potency of the antibody compared to the parent antibody without changing the ability to bind to the ECD of the IGF1R antigen. By replacing the Asn of the deamidation site with other residues, including Q, H and K, one after another, mutants are obtained, and the similarity of the binding affinity of the mutants with that of the parent antibody is confirmed by the results of ELISA analysis of the binding affinity of the mutants to IGF1R.

In addition, when bound to a brain bioactive, the anti-IGF1R antibody of the present invention can improve its BBB penetration and efficacy compared to the same bioactive by itself.

The anti-IGF1R antibody according to one aspect of the present invention can be used as a bispecific antibody comprising various second therapeutic antibodies. In an in vitro BBB penetration experiment derived from human IPSCs (Fig. 16a), it was shown that compared to a single antibody consisting of the therapeutic antibody alone, it enhanced BBB penetration by 15-fold. The anti-IGF1R antibody may be coupled to a second antibody in a bispecific antibody in a monovalent or bivalent form. For example, when analyzing the amount of antibody in the blood and CSF after a single dose of bispecific antibody with anti-IGF1R antibody in monovalent form or bivalent form in normal rats, the monovalent form and bivalent form of anti-IGF1R antibodies showed up to a 5-fold increase in the amount in the blood and an increase in the amount in the CSF up to 5-fold. times compared to the parent anti-IGF1R antibody (clone 1564). They demonstrated an approximately 3-fold increase in CSF abundance and an approximately 4.5-fold increase in brain penetration compared with the parent anti-IGF1R antibody (clone 1564) (Fig. 16c). Therefore, it is expected that a bispecific antibody containing an anti-IGF1R antibody improved by the above method will demonstrate an increase in the amount of antibody in the CSF up to approximately 15-fold and increase its ability to penetrate the brain up to approximately 23-fold compared to a single antibody consisting of a second therapeutic antibodies itself.

The anti-IGF1R antibody of the present invention has been found to bind to IGF1R, in particular to mammalian IGF1R, including humans, monkeys, rats and mice, and thus may be useful for drug development screening, clinical trials and the like .

The anti-IGF1R antibody or antigen-binding fragment of the present invention is an antibody or antigen-binding fragment thereof that specifically recognizes IGF1R (insulin-like growth factor type 1 receptor).

The anti-IGF1R antibody or antigen-binding fragment of the present invention is intended to specifically bind to its target, such as an antigen, when it binds thereto with a dissociation constant (K _D ) of 10 ^-6 M or less. The antibody specifically binds to the target with high affinity when the Kd is 1x10 ^-8 M or less or when its average effective concentration (EC ₅₀ ) is 2 nM or less. In one embodiment, the antibody or antigen binding fragment thereof is capable of binding to IGF1R or human IGF1R with a KD of 1x10 ^-8 or less. The antibodies disclosed herein have been found to bind to IGF1R, particularly to human IGF1R, murine IGF1R, rat IGF1R and monkey IGF1R.

As used herein, the term epitope is an antigenic determinant, interpreted to mean the portion of the antigen recognized by the antibody. In one embodiment, the binding site of an anti-IGF1R antibody of the present invention may be an extracellular domain of an IGF1R protein, for example, a human IGF1R protein (SEQ ID NO:99). More specifically, the binding sites of the anti-IGF1R antibody of the present invention, for example, anti-human IGF1R antibody clone 1564, are binding site 1 containing Y775, P776, F778, R650, S791, L798 and Glu779, binding site 2 containing L641, H808 , Ε8θ9 and L813, and binding site

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3, containing V397, D435, W434, Y460 and C488 in a protein consisting of the amino acid sequence SEQ ID NO:99. Thus, the epitope of the anti-IGF1R antibody of the present invention may be a conformational epitope containing all or part of these three binding sites.

As used herein, the term antibody refers to a substance produced by stimulating the immune system with an antigen and may, without limitation, be produced in vivo, recombinantly produced, or artificially synthesized. In the present invention, antibodies include animal antibodies, chimeric antibodies, humanized antibodies and human antibodies. Moreover, in the present invention, the antibody also includes an antigen-binding antibody fragment having antigen-binding ability.

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

Antibodies of the present invention include, without limitation, bispecific antibodies, minibodies, domain antibodies, antibody mimetics (or synthetic antibodies), fusion antibodies (or antibody conjugates), and fragments thereof. The structure of various antibodies is further disclosed below.

As used herein, the term antigen-binding fragment refers to a portion of an antibody, or a polypeptide containing said portion, that has a specific antigen-binding affinity. For example, a portion of an antibody contains an amino acid residue that provides specificity and affinity for an antigen by interacting with the antigen. This antigen binding region typically contains one or more than one complementarity determining region (CDR). The specific antigen binding region also contains one or more framework regions (FR). CDRs are amino acid sequences that contribute to the specificity and affinity of binding to an antigen. The framework regions help maintain the appropriate conformation of these CDRs, thereby facilitating the binding of the antigen binding region to the antigen.

In the present invention, complementarity determining regions (CDRs) refer to regions that confer antigen binding specificity to the variable regions of an antibody.

The antibody may be selected from all immunoglobulin subtypes (eg, IgA, IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4), IgM, etc.). The IgG form of the antibody may be an antibody of the IgG1, IgG2, IgG3 or IgG4 subtype, for example, the IgG1 or IgG2 subtype. An IgG antibody contains two heavy chains and two light chains, each heavy chain and light chain are linked by disulfide bonds to form two heavy chain-light chain dimers, and the two formed heavy chain-light chain dimers are linked by a disulfide bond in the Fc region of the heavy chain. The IgG form of an antibody includes a single-target antibody directed to a single antigen, where both heavy chain-light chain constructs contain binding sites for the same antigen, or a bispecific antibody directed to two antigens, where the heavy chain-light chain constructs contain binding sites binding of various antigens.

The term antigen-binding fragment of a chain (heavy chain or light chain) of an antibody or immunoglobulin includes a part of the antibody that lacks certain amino acids compared to the full-length chain, but can specifically bind to an antigen. This fragment may be considered to have biological activity in that it may specifically bind to a target antigen or may compete with other antibodies or antigen binding fragments for binding to a particular epitope. Specifically, the antigen binding fragment is selected from the group consisting of antibody fragments containing one or more than one complementarity determining region, such as, but not limited to, scFv, (scFv) ₂ , scFv-Fc, Fab, Fab' and F(ab') ₂ . Such biologically active fragments can be produced by recombinant DNA technology or can be produced by enzymatic or chemical cleavage of intact antibodies. The immunologically functional fragments of immunoglobulins are not limited to these fragments.

In the present invention, for example, a variant polypeptide, such as an antigen binding fragment, protein or antibody, is a polypeptide that has an insertion, deletion, addition and/or substitution of one or more amino acid residues from other polypeptide sequences, and includes fusion polypeptides. For example, some antibodies contain conservative amino acid substitutions of one or more heavy or light chain residues, variable regions, or CDR sequences.

The term polypeptide derivative as used herein means a polypeptide that has been chemically modified by conjugation with other chemical moieties and other than insertions, deletions, additions or substitutions.

The anti-IGF1R antibody or antigen-binding fragment of the present invention does not prevent IGF1R ligands such as IGF-1, IGF-2 and/or insulin from binding to IGF1R. Specifically, an antibody or antigen-binding fragment against IGF1R provides the advantage of

- 4 045348 that they do not interfere with the binding of the IGF1R ligand to IGF1R located on the membrane of cells expressing IGF1R, and do not inhibit signaling through IGF1R and do not affect the expression of IGF1R on the cell surface. Thus, the anti-IGF1R antibody or antigen-binding fragment of the present invention can be effectively used to cross the blood-brain barrier via transcytosis.

Human IGF1R can be activated by the insulin-like growth factors IGF-1 and IGF-2 and insulin (INS). Signaling through IGF1R stimulates cell growth and survival through the adapter protein IRS, which depends on the activation of the PI3-kinase/Akt pathway. IGF1R signals to its major substrates IRS-1, IRS-2, IRS-3 and IRS-4 and Shc proteins, leading to activation of the Ras/Raf/MAP kinase and PI3 kinase/Akt signaling pathways. IGF1R has been shown to have a relatively high level of expression in the brain compared to other transcytosis targets that have been documented to be expressed in brain endothelial cells currently used to enhance BBB penetration.

The anti-IGF1R antibody of the present invention does not interfere with the binding of IGF1, IGF2 and/or insulin to IGF1R and does not interfere with signaling through the IGF1R pathway as described above. Additionally, in one embodiment of the present invention, when comparing IGF1R with other targets currently being developed to enhance the ability of therapeutic antibodies to cross the BBB, such as the transferrin receptor or the insulin receptor, it was shown to have a relatively low level of expression in normal brain and peripheral tissues such as liver, lungs or colon.

IGF1R is a target of receptor-mediated transcytosis (RTM), allowing the delivery of beneficial substances across the blood-brain barrier (BBB) into the brain. However, for its use as a target for drug delivery through the blood-brain barrier, binding to IGF1R on the cell surface is desirable without affecting its ligand binding and signaling through the IGF1R pathway. Therefore, the anti-IGF1R antibody and antigen-binding fragment thereof of the present invention do not inhibit the binding of IGF1R to its ligand and signaling through IGF1R, so that they can be used as carriers to penetrate the blood-brain barrier.

The anti-IGF1R antibody or antigen binding fragment of the present invention is capable of transcytosis and can pass through brain endothelial cells. In addition, after its introduction into the blood vessel of a mouse, the antibody of the present invention is localized in the same location as the blood vessels of the mouse brain. These results indicate that the antibody or antigen binding fragment of the present invention can be effectively used as a drug carrier that crosses the blood-brain barrier.

Thus, the anti-IGF1R antibody or antigen-binding fragment of the present invention allows a biologically active substance active in the brain to pass through the blood-brain barrier. In the present invention, a biological barrier refers to cells, tissues, membranes, or a cell, membrane or structure that prevents the effective passage, diffusion or transport of a biological molecule. These biological barriers include nerve cells/tissues, connective tissue, muscle, membranes, or epithelial cells (eg, mucous membranes or blood vessels). A typical example is the blood-brain barrier.

As used herein, the term blood-brain barrier or BBB is a barrier formed by tight junctions in the endothelial membrane of brain capillaries that separates the brain and spinal cord from the surrounding circulatory system. This barrier is so strong that it limits the passage into the brain of even those molecules whose molecular weight is small, approximately 60 Da. The brain blood-brain barrier, spinal cord vascular barrier, and retinal vascular barrier are continuous capillary barriers of the central nervous system and are commonly referred to as the BBB.

In the present invention, the blood-brain barrier carrier can pass through the blood-brain barrier and provide delivery of an antibody or an antigen-binding fragment thereof of the present invention and, for example, includes a protein, including a peptide and polypeptide, a nucleic acid, an antibody or a small molecule compound.

The production and selection of antibodies of the present invention can be carried out in transgenic mice, such as those described above, where the mice are introduced with a gene encoding antigen-specific human mAbs with the desired specificity using hybridoma technology. Such antibodies can be cloned and expressed using suitable vectors and host cells, or the antibodies can be obtained from cultured hybridoma cells. In addition, the antibody may be derived from a phage display library. Phage display technology is a method that imitates, to some extent, immune selection by selecting a repertoire of antibodies on the surface of filamentous bacteriophages and selecting from them those phages that bind to the desired antigen. Such a technique may relate to an embodiment of the present invention or PCT Publication No. WO 1999/010494. In one embodiment, the selection of a humanized anti-IGF1R antibody of the present invention is carried out by phage display.

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The present invention provides an isolated antibody or antigen binding fragment that specifically binds to IGF1R, which may be a polypeptide, protein, antibody, or antigen binding fragment thereof that specifically binds to IGF1R, comprising a heavy chain complementarity determining region and a light chain complementarity determining region.

This is described below specifically for antibodies that specifically bind to IGF1R.

The anti-IGF1R antibody or antigen-binding fragment of the present invention specifically recognizes IGF1R (insulin-like growth factor receptor type 1), recognizes and binds to IGF1R, in particular human IGF1R, mouse IGF1R, rat IGF1R and monkey IGF1R, does not interfere with the binding of IGF1R, IGF -1R, IGF-2 and/or insulin with IGF1R do not inhibit signaling through IGF1R and can be used for transcytosis to cross the blood barrier. They do not exhibit antibody-dependent cell-mediated cytotoxicity (ADCC) and do not reduce IGF1R levels in the brain even when administered repeatedly to animals.

In particular, the anti-IGF1R antibody of the present invention binds to IGF1R located on the surface of brain endothelial cells forming the BBB and is internalized into the interior of the cell. For example, cellular internalization of an anti-IGF1R antibody of the present invention can be detected using a cell line expressing IGF1R (eg, MCF-7). Anti-IGF1R antibodies of the present invention, for example, 1564, 48G5, 54H4, 60H6, B11 and affinity variants of 1564, such as C04, F06, VH2, VH5, VH7, VH9, VH16, VH32 and VH35, have a higher degree of internalization compared to with negative control. The results obtained indicate that the degree of internalization of the anti-IGF1R antibodies analyzed is specific to IGF1R on the cell surface. In addition, the anti-IGF1R antibody of the present invention is in the form of a scFv and can be produced linked to a therapeutic antibody by various methods. For example, an anti-IGF1R scFv can be produced as part of a bispecific antibody, such as a bivalent form of a bispecific antibody, where two scFvs are linked to the C terminus of a therapeutic antibody, such as an anti-α-syn antibody, or a monovalent form of a bispecific antibody, where the C The -end of the therapeutic antibody is bound to one scFv. Bispecific antibodies can be internalized by cells expressing IGF1R. Antibody to IGF1R has a high ability to bind to antigen on the surface of cells, which enhances the internalization effect and leads to the ability to pass through the BBB. However, if the antibody has the ability to cross the BBB and interferes with signaling through the IGF1R, it may cause side effects. The antibody of the present invention is characterized by both binding ability suitable for passage through the BBB and the absence of blocking signaling through IGF1R.

The present invention provides an isolated antibody or antigen binding fragment that specifically binds to IGF1R, which may be a polypeptide, protein, or antibody that specifically binds to IGF1R, comprising a heavy chain complementarity determining region and a light chain complementarity determining region.

In specific examples, the anti-IGF1R antibody or antigen binding fragment thereof may comprise:

(i) one or more than one heavy chain complementarity determining region selected from the group consisting of H-CDR1, H-CDR2 and H-CDR3 described in table. 1, or a heavy chain variable region comprising said one or more heavy chain complementarity determining regions;

(ii) one or more light chain complementarity determining region selected from the group consisting of L-CDR1, L-CDR2 and L-CDR3 described in table. 1, or a light chain variable region comprising said one or more light chain complementarity determining regions;

a combination of one or more than one heavy chain complementarity determining region (CDR) and one or more than one light chain complementarity determining region (CDR); or a combination of a heavy chain variable region and a light chain variable region.

Additionally, in said heavy chain variable region, light chain variable region, or combination of heavy chain variable region and light chain variable region, the heavy chain variable region may comprise one or more than one heavy chain framework selected from the group consisting of H-FR1 , H-FR2, H-FR3 and H-FR4, and the light chain variable region may comprise one or more light chain framework regions selected from the group consisting of L-FR1, L-FR2, L-FR3 and L- FR4.

(1) The heavy chain H-CDR1, H-CDR2 or H-CDR3 of the present invention is selected from the amino acid sequences listed in Table. 1, or contain at least one amino acid sequence substantially identical to the selected amino acid sequence. In addition, (ii) the light chain L-CDR1, L-CDR2 or L-CDR3 is selected from the amino acid sequences listed in table. 2, or contain one or more amino acid sequences substantially identical to the selected amino acid sequence.

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Sequence identity essentially means that the sequence contains changes but retains the effects disclosed in the present invention. The specified sequence is approximately 90%, 95% or 99% identical to the heavy chain variable region disclosed in one of the embodiments. This sequence is approximately 90%, 95%, or 99% identical to the light chain variable region disclosed in other embodiments. For example, in the case of a variant exhibiting approximately 90%, 95%, or 99% sequence identity to an antibody or antigen binding fragment disclosed herein, any sequence change occurs in the framework region of the variable region, but not in the CDR.

Table 1

CDR sequences of the heavy chain variable region in an antibody clone of the invention

Identifier clone CDR-H1 SEQ ID NO: CDR-H2 SEQ ID NO: CDR-H3 SEQ ID NO: 996-1 GFTFSSYDMS 1 GIYHDGSSTYYADSVKG 10 VSGTLSEPYAFFFNAMDV 50 996-2 GFTFSSYDMS 1 GIYHDGSSTYYADSVKG 10 VSGTLSEPYAFFFNAMDV 50 1226-1 GFTFSNYDM S 2 SISPDGGSKYYADSVKG eleven DGGTHWLSLFDY 51 1226-2 GFTFSNYDM S 2 SISPDGGSKYYADSVKG eleven DGGTHWLSLFDY 51 1564-1 GFTFSSYDMS 1 AISYDNGNTYYADSVKG 12 GVLTTLMNWFDY 52 1564-2 GFTFSSYDMS 1 AISYDNGNTYYADSVKG 12 GVLTTLMNWFDY 52 1564-ZR GFTFSSYDMS 1 AISYDNGNTYYADSVKG 12 GVLTTLMNWFDY 52 1564-DM GFTFSSYDMS 1 AISYDQGNTYYADSVKG 13 GVLTTLMNWFDY 52 1564-DMP GFTFSSYDMS 1 AISYDQGNTYYADSVKG 13 GVLTTLMNWFDY 52 48G5-1 GFTFSDYDM S 3 SIYPNGSSKYYADSVKG 14 AGINCTTLRCS SYD AMD V 53 48G5-2 GFTFSDYDM S 3 SIYPNGSSKYYADSVKG 14 AGINCTTLRCS SYD AMD V 53 49G11-1 GFTFSSYDMS 1 GISYSGGSTYYADSVKG 15 VGLACTPHTCSSYDAMDV 54 49G11-2 GFTFSSYDMS 1 GISYSGGSTYYADSVKG 15 VGLACTPHTCSSYDAMDV 54 54N4-1 GFTFSDYDM S 3 AISSDGSSAYYADSVKG 16 ATIYTSDAPWSSYDAMDV 55 54N4-2 GFTFSDYDM S 3 AISSDGSSAYYADSVKG 16 ATIYTSDAPWSSYDAMDV 55 60A11-1 GFTFSNYDM S 2 VISHSSSGTYYADSVKG 17 VGVACGETDCSSYDAMDV 56 60A11-2 GFTFSNYDM S 2 VISHSSSGTYYADSVKG 17 VGVACGETDCSSYDAMDV 56 60N6-1 GFTFSDYDM S 3 MIYSGSSSKYYADSVKG 18 ASIACTLQACSYDNAMDV 57 60N6-2 GFTFSDYDM S 3 MIYSGSSSKYYADSVKG 18 ASIACTLQACSYDNAMDV 57 60N6-ZR GFTFSDYDM S 3 MIYSGSSSKYYADSVKG 18 ASIACTLQACSYDNAMDV 57

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Alomseq GFTFSSYDMS 1 AIYHDGGNTYYADSVK G 19 AASPCNVHDCSYDYAMD V 58 A3_ompseq GFTFSDYDM S 3 GISYNGGSKYYADSVKG 20 VGIMCSETGCSYDNAMDV 59 A6_ompseq GFTFSDYYM S 4 GISSDGGSIYYADSVKG 21 YASPTWLHILYYSDAMDV 60 A8_ompseq GFTFSNYDM S 2 MIYSGSSSKYYADSVKG 18 ALIPCTPEGCYSYDAMDV 61 AlOompseq GFTFSGYAM S 5 AISSDGGSTYYADSVKG 22 DPWFSRWTAFDY 62 A12_ompseq GFTFSDYDM S 3 GIYPDGGNIYYADSVKG 23 GIGQCELRECS SDDGMD V 63 B5_ompseq GFTFSDYDM S 3 AIYYDSGSIYYADSVKG 24 AVSECNPLNCSYSDAMDV 64 B9_ompseq GFTFSDYDM S 3 MIYSGSSSKYYADSVKG 18 VILGCSKHSCPSSDAMDV 65 Bllompseq GFTFSDYDM S 3 AISYDNGNKYYADSVK G 25 AGVACTEHMCSSYDAMD V 66 C2_ompseq GFTFSSYDMS 1 LIYPGGGNIYYADSVKG 26 GRVPCHPGGCSYAYGMDV 67 C6_ompseq GFTFSNYAM S 6 WISSGGGSTYYADSVKG 27 LGSLFPNATASYAYGMDV 68 C7_ompseq GFTFSNYDM S 2 SISYDSGSKYYADSVKG 28 AGILCTPTHCSSYDAMDV 69 Cllompseq GFTFSDYAM S 7 SIYPDDGNTYYADSVKG 29 DGWTPDGTHFDY 70 D4_ompseq GFTFSDYDM S 3 WISHSSSGTYYADSVKG thirty VGLSCAETACSSYDAMDV 71 E6_ompseq GFTFSSYDMS 1 AISYDNGNTYYADSVKG 12 VGLDCDTTKCSSYDAMDV 72 ElOompseq GFTFSNYDM S 2 VISHSSSGTYYADSVKG 17 VGVACGETDCSSYDAMDV 56 E12_ompseq GFTFSDYDM S 3 WISHSSSGTYYADSVKG thirty VGLSCAETACSSYDAMDV 71 F6_ompseq GFTFSDYDM S 3 MIYSGSSSKYYADSVKG 18 AVRPCTDLHCS SDD AMD V 73 Fllompseq GFTFSDYDM S 3 AISYDSGSKYYADSVKG 31 VGRMCNITHCSSYDAMDV 74

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F12_ompseq GFTFSDYDM S 3 SIYYGSGNIYYADSVKG 32 DLTAPDGSSFDY 75 G9_ompseq GFTFSSYDMS 1 AISYDNGSSIYYADSVK G 33 VGLECTVEHCYSYDGMDV 76 C04-1 GFTFSSYDMS 1 AISYDNGNTYYADSVKG 12 GVLTTLMNWFDY 52 C04-2 GFTFSSYDMS 1 AISYDNGNTYYADSVKG 12 GVLTTLMNWFDY 52 C04-3P GFTFSSYDMS 1 AISYDNGNTYYADSVKG 12 GVLTTLMNWFDY 52 C04-DM GFTFSSYDMS 1 AISYDQGNTYYADSVKG 13 GVLTTLMNWFDY 52 C04-DMP GFTFSSYDMS 1 AISYDQGNTYYADSVKG 13 GVLTTLMNWFDY 52 F06-1 GFTFSSYDMS 1 AISYDNGNTYYADSVKG 12 GVLTTLMNWFDY 52 F06-2 GFTFSSYDMS 1 AISYDNGNTYYADSVKG 12 GVLTTLMNWFDY 52 F06-3P GFTFSSYDMS 1 AISYDNGNTYYADSVKG 12 GVLTTLMNWFDY 52 F06-DM GFTFSSYDMS 1 AISYDQGNTYYADSVKG 13 GVLTTLMNWFDY 52 F06-DMP GFTFSSYDMS 1 AISYDQGNTYYADSVKG 13 GVLTTLMNWFDY 52 BOI GFTFSSYDMS 1 AISWDKAQPYYADSVK G 34 GVLTTLMNWFDY 52 A07(AR) GFTFSSYDMS 1 AISWDQGNSYADSVK G 35 GVLTTLMNWFDY 52 E09 GFTFSSYDMS 1 AISWGQGKTYYADSVK G 36 GVLTTLMNWFDY 52 D03 GFTFSSYDMS 1 AISYDNGQTYYADSVKG 37 GVLTTLMNWFDY 52 A02 GFTFSSYDMS 1 AISYDNGNTYYADSVKG 12 GVLTTLMNWFDY 52 B09 GFTFSSYDMS 1 AISYDNGNTYYADSVKG 12 GVLTTLMNWFDY 52 BIO GFTFSSYDMS 1 AISYDNGNTYYADSVKG 12 GVLTTLMNWFDY 52 E06 GFTFSSYDMS 1 AISYDNGNTYYADSVKG 12 GVLTTLMNWFDY 52 H04 GFTFSSYDMS 1 AISYDNGNTYYADSVKG 12 GVLTTLMNWFDY 52 A06 GFTFSSYDMS 1 AISYDNGNTYYADSVKG 12 GVLTTLMNWFDY 52 A07(AM) GFTFSSYDMS 1 AISYDNGNTYYADSVKG 12 GVLTTLMNWFDY 52 B02 GFTFSSYDMS 1 AISYDNGNTYYADSVKG 12 GVLTTLMNWFDY 52 VH02-1 GFTFSSYAMS 8 AISGSNGNTYYADSVKG 38 GVLTTLANWFDY 77 VH02-2 GFTFSSYAMS 8 AISGSNGNTYYADSVKG 38 GVLTTLANWFDY 77 VH02-3P GFTFSSYAMS 8 AISGSNGNTYYADSVKG 38 GVLTTLANWFDY 77 VH02-DM GFTFSSYAMS 8 AISGSQGNTYYADSVKG 39 GVLTTLANWFDY 77 VH02-DMP GFTFSSYAMS 8 AISGSQGNTYYADSVKG 39 GVLTTLANWFDY 77 VH05-1 GFTFSSYDMS 1 AISGDNGSTYYADSVKG 40 GVLTTLMNWFDS 78 VH05-2 GFTFSSYDMS 1 AISGDNGSTYYADSVKG 40 GVLTTLMNWFDS 78 VH05-3P GFTFSSYDMS 1 AISGDNGSTYYADSVKG 40 GVLTTLMNWFDS 78

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VH05-DM GFTFSSYDMS 1 AISGDQGSTYYADSVKG 41 GVLTTLMNWFDS 78 VH05-DMP GFTFSSYDMS 1 AISGDQGSTYYADSVKG 41 GVLTTLMNWFDS 78 VH06-1 GFTFSSYDMS 1 AISYSNGNTYYADSVKG 42 GVLTTLANWFDY 77 VH06-2 GFTFSSYDMS 1 AISYSNGNTYYADSVKG 42 GVLTTLANWFDY 77 VH06-3P GFTFSSYDMS 1 AISYSNGNTYYADSVKG 42 GVLTTLANWFDY 77 VH06-DM GFTFSSYDMS 1 AISYSQGNTYYADSVKG 43 GVLTTLANWFDY 77 VH06-DMP GFTFSSYDMS 1 AISYSQGNTYYADSVKG 43 GVLTTLANWFDY 77 VH07-1 GFTFSSYAMS 8 AISYDNGSTYYADSVKG 44 GVLTTLMNWFDY 52 VH07-2 GFTFSSYAMS 8 AISYDNGSTYYADSVKG 44 GVLTTLMNWFDY 52 VH07-3 GFTFSSYAMS 8 AISYDNGSTYYADSVKG 44 GVLTTLMNWFDY 52 VH07-DM GFTFSSYAMS 8 AISYDQGSTYYADSVKG 45 GVLTTLMNWFDY 52 VH07-DMP GFTFSSYAMS 8 AISYDQGSTYYADSVKG 45 GVLTTLMNWFDY 52 VH09-1 GFTFSSYDMS 1 AISGDNGNTYYADSVKG 46 GVLTTLMNWFDY 52 VH09-2 GFTFSSYDMS 1 AISGDNGNTYYADSVKG 46 GVLTTLMNWFDY 52 VH09-3P GFTFSSYDMS 1 AISGDNGNTYYADSVKG 46 GVLTTLMNWFDY 52 VH09-DM GFTFSSYDMS 1 AISGDQGNTYYADSVKG 47 GVLTTLMNWFDY 52 VH09-DMP GFTFSSYDMS 1 AISGDQGNTYYADSVKG 47 GVLTTLMNWFDY 52 VH16-1 GFTFSSYDMS 1 AISGSNGNTYYADSVKG 38 GVLTTLMNWFDY 52 VH16-2 GFTFSSYDMS 1 AISGSNGNTYYADSVKG 38 GVLTTLMNWFDY 52 VH16-3P GFTFSSYDMS 1 AISGSNGNTYYADSVKG 38 GVLTTLMNWFDY 52 VH16-DM GFTFSSYDMS 1 AISGSQGNTYYADSVKG 39 GVLTTLMNWFDY 52 VH16-DMP GFTFSSYDMS 1 AISGSQGNTYYADSVKG 39 GVLTTLMNWFDY 52 VH27-1 GFTFSSYAMS 8 AISYDNGSTYYADSVKG 44 GVLTTLMNWFDS 78 VH27-2 GFTFSSYAMS 8 AISYDNGSTYYADSVKG 44 GVLTTLMNWFDS 78 VH27-3P GFTFSSYAMS 8 AISYDNGSTYYADSVKG 44 GVLTTLMNWFDS 78 VH27-DM GFTFSSYAMS 8 AISYDQGSTYYADSVKG 45 GVLTTLMNWFDS 78 VH27-DMP GFTFSSYAMS 8 AISYDQGSTYYADSVKG 45 GVLTTLMNWFDS 78 VH32-1 GFTFSSYAMS 8 AISYDNGNTYYADSVKG 12 GVLTTLANWFDY 77 VH32-2 GFTFSSYAMS 8 AISYDNGNTYYADSVKG 12 GVLTTLANWFDY 77 VH32-3 GFTFSSYAMS 8 AISYDNGNTYYADSVKG 12 GVLTTLANWFDY 77 VH32-DM GFTFSSYAMS 8 AISYDQGNTYYADSVKG 13 GVLTTLANWFDY 77 VH32-DMP GFTFSSYAMS 8 AISYDQGNTYYADSVKG 13 GVLTTLANWFDY 77 VH35-1 GFTFSSYDMS 1 AISYDNGSTYYADSVKG 44 GVLTTLMNWFDS 78 VH35-2 GFTFSSYDMS 1 AISYDNGSTYYADSVKG 44 GVLTTLMNWFDS 78 VH35-3 GFTFSSYDMS 1 AISYDNGSTYYADSVKG 44 GVLTTLMNWFDS 78 VH35-DM GFTFSSYDMS 1 AISYDQGSTYYADSVKG 45 GVLTTLMNWFDS 78 VH35-DMP GFTFSSYDMS 1 AISYDQGSTYYADSVKG 45 GVLTTLMNWFDS 78

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VH48 GFTFSSYAMS 8 AISGSNGNTYYADSVKG 38 GVLTTLANWFDS 79 VH55 GFTFSSYDMS 1 AISYDNGNTYYADSVKG 12 GVLTTLANWFDY 77 VH81 GFTFSSYDMS 1 AISGSNGSTYYADSVKG 48 GVLTTLMNWFDY 52 C12 GFTFICPILS 9 AISYDNGNTYYADSVKG 12 GVLTTLMCLRHL 80 1564-VL(N51D) GFTFSSYDMS 1 AISYDNGNTYYADSVKG 12 GVLTTLMNWFDY 52 1564- VL(N95aD) GFTFSSYDMS 1 AISYDNGNTYYADSVKG 12 GVLTTLMNWFDY 52 1564- VL(N95aH) GFTFSSYDMS 1 AISYDNGNTYYADSVKG 12 GVLTTLMNWFDY 52 1564- VL(N95aK) GFTFSSYDMS 1 AISYDNGNTYYADSVKG 12 GVLTTLMNWFDY 52 1564- VL(N95aR) GFTFSSYDMS 1 AISYDNGNTYYADSVKG 12 GVLTTLMNWFDY 52 1564-VH(N54D) GFTFSSYDMS 1 AISYDDGNTYYADSVKG 49 GVLTTLMNWFDY 52 1564-VH(N54Q) GFTFSSYDMS 1 AISYDQGNTYYADSVKG 13 GVLTTLMNWFDY 52

table 2

CDR sequences of the light chain variable region in an antibody clone of the invention

Identifier clone CDR-L1 SEQ ID NO: CDR-L2 SEQ ID NO: CDR-L3 SEQ ID NO: 996-1 TGSSSNIGSNAVN 96 SNSHRPS 114 ATWDYSLSGYV 133 996-2 TGSSSNIGSNAVN 96 SNSHRPS 114 ATWDYSLSGYV 133 1226-1 TGSSSNIGNNTVS 97 YDNHRPS 115 GSWDASLNGYV 134 1226-2 TGSSSNIGNNTVS 97 YDNHRPS 115 GSWDASLNGYV 134 1564-1 TGSSSNIGSNDVS 98 ANSNRPS 116 GAWDDSLNGYV 135 1564-2 TGSSSNIGSNDVS 98 ANSNRPS 116 GAWDDSLNGYV 135 1564-3 TGSSSNIGSNDVS 98 ANSNRPS 116 GAWDDSLNGYV 135 1564-DM TGSSSNIGSNDVS 98 AQSNRPS 117 GAWDDSLHGYV 136 1564-DMP TGSSSNIGSNDVS 98 AQSNRPS 117 GAWDDSLHGYV 136 48G5-1 SGSSSNIGNNDVS 99 DNNHRPS 118 AAWDDSLNAYV 137 48G5-2 SGSSSNIGNNDVS 99 DNNHRPS 118 AAWDDSLNAYV 137 49G11-1 TGSSSNIGSNTVY 100 SDSNRPS 119 GTWDDSLNGYV 138 49G11-2 TGSSSNIGSNTVY 100 SDSNRPS 119 GTWDDSLNGYV 138 54H4-1 SGSSSNIGSNTVT 101 ADSKRPS 120 GTWDDSLNAYV 139 54H4-2 SGSSSNIGSNTVT 101 ADSKRPS 120 GTWDDSLNAYV 139 60A11-1 SGSSSNIGSNAVT 102 DDNHRPS 121 GAWDDSLNGYV 135 60A11-2 SGSSSNIGSNAVT 102 DDNHRPS 121 GAWDDSLNGYV 135

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60H6-1 TGSSSNIGNNDVS 103 ANSHRPS 122 GSWDDSLNGYV 140 60H6-2 TGSSSNIGNNDVS 103 ANSHRPS 122 GSWDDSLNGYV 140 60H6-3 TGSSSNIGNNDVS 103 ANSHRPS 122 GSWDDSLNGYV 140 Alomseq SGSSSNIGNNAVT 104 DDSQRPS 123 GSWDDSLNGYV 140 A3_ompseq SGSSSNIGNNDVD 105 YDSQRPS 124 GTWDDSLNAYV 139 A6_ompseq SGSSSNIGNNAVT 104 DDSHRPS 125 GSWDSSLNGYV 141 A8_ompseq TGSSSNIGNNDVS 103 DDSKRPS 126 GAWDDSLNGYV 135 AlOompseq SGSSSNIGNNAVN 106 SNSKRPS 127 GTWDYSLSGYV 142 A12_ompseq TGSSSNIGNNDVN 107 SNSHRPS 114 GTWDDSLNGYV 138 B5_ompseq SGSSSNIGNNDVS 99 DDNQRPS 128 ATWDASLNGYV 143 B9_ompseq SGSSSNIGNNNVT 108 DDSQRPS 123 GSWDDSLNGYV 140 Bllompseq SGSSSNIGNNDVS 99 DDNHRPS 121 GAWDYSLSGYV 144 C2_ompseq SGSSFNIGSNDVS 109 DNSKRPS 129 GTWDDSLNGYV 138 C6_ompseq SGSSSNIGSNDVS 110 DNSQRPS 648 GSWDASLNAYV 145 C7_ompseq SGSSSNIGNNDVN 111 SNSHRPS 114 GTWDDSLNGYV 138 Cllompseq SGSSSNIGNNDVS 99 SNSHRPS 114 GSWDASLNGYV 134 D4_ompseq SGSSSNIGSNDVS 110 DDSNRPS 130 GSWDDSLNGYV 140 E6_ompseq SGSSSNIGNNDVN 111 SNSHRPS 114 GAWDYSLSAYV 146 ElOompseq SGSSSNIGSNAVT 102 DDNHRPS 121 GAWDDSLNGYV 135 E12_ompseq SGSSSNIGSNDVS 110 DDSNRPS 130 GSWDDSLNGYV 140 F6_ompseq SGSSSNIGNNDVS 99 DNNHRPS 118 AAWDDSLNAYV 137 Fllompseq TGSSSNIGSNYVS 112 DDSHRPS 125 GAWDDSLNGYV 135 F12_ompseq TGSSSNIGNNDVS 103 SDSQRPS 131 GAWDASLNGYV 147 G9_ompseq TGSSSNIGSNTVN 113 DNSQRPS 648 ASWDSLNAYV 148 C04-1 TGSSSNIGSNDVS 98 ANSNRPS 116 GAWEQWLNGYV 149 C04-2 TGSSSNIGSNDVS 98 ANSNRPS 116 GAWEQWLNGYV 149 C04-3 TGSSSNIGSNDVS 98 ANSNRPS 116 GAWEQWLNGYV 149 C04-DM TGSSSNIGSNDVS 98 AQSNRPS 117 GAWEQWLHGYV 150 C04-DMP TGSSSNIGSNDVS 98 AQSNRPS 117 GAWEQWLHGYV 150 F06-1 TGSSSNIGSNDVS 98 ANSNRPS 116 GTWAGSLNGYV 151 F06-2 TGSSSNIGSNDVS 98 ANSNRPS 116 GTWAGSLNGYV 151 F06-3 TGSSSNIGSNDVS 98 ANSNRPS 116 GTWAGSLNGYV 151 F06-DM TGSSSNIGSNDVS 98 AQSNRPS 117 GTWAGSLHGYV 152 F06-DMP TGSSSNIGSNDVS 98 AQSNRPS 117 GTWAGSLHGYV 152 BOI TGSSSNIGSNDVS 98 ANSNRPS 116 GAWDDSLNGYV 135 A07(AR) TGSSSNIGSNDVS 98 ANSNRPS 116 GAWDDSLNGYV 135 E09 TGSSSNIGSNDVS 98 ANSNRPS 116 GAWDDSLNGYV 135

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D03 TGSSSNIGSNDVS 98 ANSNRPS 116 GAWDDSLNGYV 135 A02 TGSSSNIGSNDVS 98 ANSNRPS 116 GAWDTTLNGYV 153 B09 TGSSSNIGSNDVS 98 ANSNRPS 116 GAWEESLNGYV 154 BIO TGSSSNIGSNDVS 98 ANSNRPS 116 GSWDVSLNGYV 155 E06 TGSSSNIGSNDVS 98 ANSNRPS 116 GAWDHSLNGYV 156 H04 TGSSSNIGSNDVS 98 ANSNRPS 116 GAWDQSLNGYV 157 A06 TGSSSNIGSNDVS 98 ANSNRPS 116 GASWDEWLNGYV 158 A07(AM) TGSSSNIGSNDVS 98 ANSNRPS 116 GAWDETLNGYV 159 B02 TGSSSNIGSNDVS 98 ANSNRPS 116 GTWDDSLNGYV 138 VH02-1 TGSSSNIGSNDVS 98 ANSNRPS 116 GAWDDSLNGYV 135 VH02-2 TGSSSNIGSNDVS 98 ANSNRPS 116 GAWDDSLNGYV 135 VH02-3 TGSSSNIGSNDVS 98 ANSNRPS 116 GAWDDSLNGYV 135 VH02-DM TGSSSNIGSNDVS 98 AQSNRPS 117 GAWDDSLHGYV 136 VH02-DMP TGSSSNIGSNDVS 98 AQSNRPS 117 GAWDDSLHGYV 136 VH05-1 TGSSSNIGSNDVS 98 ANSNRPS 116 GAWDDSLNGYV 135 VH05-2 TGSSSNIGSNDVS 98 ANSNRPS 116 GAWDDSLNGYV 135 VH05-3 TGSSSNIGSNDVS 98 ANSNRPS 116 GAWDDSLNGYV 135 VH05-DM TGSSSNIGSNDVS 98 AQSNRPS 117 GAWDDSLHGYV 136 VH05-DMP TGSSSNIGSNDVS 98 AQSNRPS 117 GAWDDSLHGYV 136 VH06-1 TGSSSNIGSNDVS 98 ANSNRPS 116 GAWDDSLNGYV 135 VH06-2 TGSSSNIGSNDVS 98 ANSNRPS 116 GAWDDSLNGYV 135 VH06-3 TGSSSNIGSNDVS 98 ANSNRPS 116 GAWDDSLNGYV 135 VH06-DM TGSSSNIGSNDVS 98 AQSNRPS 117 GAWDDSLHGYV 136 VH06-DMP TGSSSNIGSNDVS 98 AQSNRPS 117 GAWDDSLHGYV 136 VH07-1 TGSSSNIGSNDVS 98 ANSNRPS 116 GAWDDSLNGYV 135 VH07-2 TGSSSNIGSNDVS 98 ANSNRPS 116 GAWDDSLNGYV 135 VH07-3 TGSSSNIGSNDVS 98 ANSNRPS 116 GAWDDSLNGYV 135 VH07-DM TGSSSNIGSNDVS 98 AQSNRPS 117 GAWDDSLHGYV 136 VH07-DMP TGSSSNIGSNDVS 98 AQSNRPS 117 GAWDDSLHGYV 136 VH09-1 TGSSSNIGSNDVS 98 ANSNRPS 116 GAWDDSLNGYV 135 VH09-2 TGSSSNIGSNDVS 98 ANSNRPS 116 GAWDDSLNGYV 135 VH09-3 TGSSSNIGSNDVS 98 ANSNRPS 116 GAWDDSLNGYV 135 VH09-DM TGSSSNIGSNDVS 98 AQSNRPS 117 GAWDDSLHGYV 136 VH09-DMP TGSSSNIGSNDVS 98 AQSNRPS 117 GAWDDSLHGYV 136 VH16-1 TGSSSNIGSNDVS 98 ANSNRPS 116 GAWDDSLNGYV 135 VH16-2 TGSSSNIGSNDVS 98 ANSNRPS 116 GAWDDSLNGYV 135 VH16-3 TGSSSNIGSNDVS 98 ANSNRPS 116 GAWDDSLNGYV 135

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VH16-DM TGSSSNIGSNDVS 98 AQSNRPS 117 GAWDDSLHGYV 136 VH16-DMP TGSSSNIGSNDVS 98 AQSNRPS 117 GAWDDSLHGYV 136 VH27-1 TGSSSNIGSNDVS 98 ANSNRPS 116 GAWDDSLNGYV 135 VH27-2 TGSSSNIGSNDVS 98 ANSNRPS 116 GAWDDSLNGYV 135 VH27-3 TGSSSNIGSNDVS 98 ANSNRPS 116 GAWDDSLNGYV 135 VH27-DM TGSSSNIGSNDVS 98 AQSNRPS 117 GAWDDSLHGYV 136 VH27-DMP TGSSSNIGSNDVS 98 AQSNRPS 117 GAWDDSLHGYV 136 VH32-1 TGSSSNIGSNDVS 98 ANSNRPS 116 GAWDDSLNGYV 135 VH32-2 TGSSSNIGSNDVS 98 ANSNRPS 116 GAWDDSLNGYV 135 VH32-3 TGSSSNIGSNDVS 98 ANSNRPS 116 GAWDDSLNGYV 135 VH32-DM TGSSSNIGSNDVS 98 AQSNRPS 117 GAWDDSLHGYV 136 VH32-DMP TGSSSNIGSNDVS 98 AQSNRPS 117 GAWDDSLHGYV 136 VH35-1 TGSSSNIGSNDVS 98 ANSNRPS 116 GAWDDSLNGYV 135 VH35-2 TGSSSNIGSNDVS 98 ANSNRPS 116 GAWDDSLNGYV 135 VH35-3 TGSSSNIGSNDVS 98 ANSNRPS 116 GAWDDSLNGYV 135 VH35-DM TGSSSNIGSNDVS 98 AQSNRPS 117 GAWDDSLHGYV 136 VH35-DMP TGSSSNIGSNDVS 98 AQSNRPS 117 GAWDDSLHGYV 136 VH48 TGSSSNIGSNDVS 98 ANSNRPS 116 GAWDDSLNGYV 135 VH55 TGSSSNIGSNDVS 98 ANSNRPS 116 GAWDDSLNGYV 135 VH81 TGSSSNIGSNDVS 98 ANSNRPS 116 GAWDDSLNGYV 135 C12 TGSSSNIGSNDVS 98 ANSNRPS 116 GAWDDSLNGYV 135 1564-VL(N51D) TGSSSNIGSNDVS 98 ADSNRPS 132 GAWDDSLNGYV 135 1564-VL(N95aD) TGSSSNIGSNDVS 98 ANSNRPS 116 GAWDDSLDGYV 160 1564-VL(N95aH) TGSSSNIGSNDVS 98 ANSNRPS 116 GAWDDSLHGYV 136 1564-VL(N95aK) TGSSSNIGSNDVS 98 ANSNRPS 116 GAWDDSLKGYV 161 1564-VL(N95aR) TGSSSNIGSNDVS 98 ANSNRPS 116 GAWDDSLKGYV 161 1564-VH(N54D) TGSSSNIGSNDVS 98 ANSNRPS 116 GAWDDSLNGYV 135 1564-VH(N54Q) TGSSSNIGSNDVS 98 ANSNRPS 116 GAWDDSLNGYV 135

The heavy chain framework region selected from the group consisting of H-FR1, H-FR2, H-FR3 and HFR4 of the present invention can be selected from the amino acid sequences of the table. 3. The light chain framework region selected from the group consisting of L-FR1, L-FR2, L-FR3 and L-FR4 of the present invention can be selected from the amino acid sequences of Table. 4.

Specifically, the heavy chain variable region of the anti-IGF1R antibody of the present invention contains H-CDR1, H-CDR2 and H-CDR3, as described in Table. 1, or additionally H-FR1, H-FR2, HFR3 and H-FR4 shown in table. 3.

The light chain variable region of the anti-IGF1R antibody of the present invention contains L-CDR1, L-CDR2 and L-CDR3 shown in table. 3, or additionally L-FR1, L-FR2, L-FR3 and L-FR4 shown in table. 4. In one embodiment of the present invention, an anti-IGF1R antibody or antigen binding fragment thereof may comprise a heavy chain variable region comprising CDRH1, CDR-H2 and CDR-H3 of each clone selected from the group consisting of the amino acid sequences CDR-H1, CDR-H2 and CDR-H3 shown in table. 1, and a light chain variable region containing CDR-L1, CDR-L2 and CDR-L3 of each clone selected from the group consisting of the amino acid sequences CDR-L1, CDR-L2 and CDR-L3 shown in table. 2.

In one embodiment of the present invention, an amino acid modification, such as eliminating amino acid deamidation in the Fc region of an anti-IGF1R antibody, can improve the PK (pharmacokinetics) of the antibody by reducing its clearance in the blood without changing the ability to bind to the ECD of the IGF1R antigen and lead to an increase in half-life. In one example, the amino acid position in an anti-IGF1R antibody at which deamidation is eliminated may be N51D in L-CDR2 light chain, or N95aK, N95aH, N95aR, or N95aD in L-CDR3 light chain, or N54D or N54Q in H-CDR2 heavy clone 1564 chains.

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Table 3

Variable Heavy Chain Framework Region Sequences in an Antibody Clone of the Invention

Clone ID H-FR1 SEQ ID NO: H-FR2 SEQ ID NO: H-FR3 SEQ ID NO: H-FR4 SEQ ID NO: 996-1 EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK GLEWVS 85 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 87 WGQGTL VTVSS 92 996-2 EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK CLEWVS 86 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 87 WGQGTL VTVSS 92 1226-1 EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK GLEWVS 85 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 1226-2 EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK CLEWVS 86 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 1564-1 EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK GLEWVS 85 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 1564-2 EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK CLEWVS 86 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 1564-ZR EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK CLEWVS 86 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 1564-DM EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK CLEWVS 86 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 1564-DMP EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK CLEWVS 86 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 48G5-1 EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK GLEWVS 85 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 48G5-2 EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK CLEWVS 86 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 49G11-1 EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK GLEWVS 85 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 49G11-2 EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK CLEWVS 86 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 54N4-1 EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK GLEWVS 85 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 54N4-2 EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK CLEWVS 86 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL ATVSS 93

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60A11-1 EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK GLEWVS 85 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 60A11-2 EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK CLEWVS 86 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 60H6-1 EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK GLEWVS 85 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 60H6-2 EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK CLEWVS 86 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 60H6-3P EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK CLEWVS 86 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 Alomseq EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK GLEWVS 85 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 A3_ompseq EVQLLESGGGLV QTGGSLRLSCAA S 82 WVRQAPGK GLEWVS 85 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 A6_ompseq EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK GLEWVS 85 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 A8_ompseq EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK GLEWVS 85 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAR 89 WGQGTL VTVSS 92 AlOompseq EVQLLESGGGLA QPGGSLRLSCAA S 83 WVRQAPGK GLEWVS 85 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAR 89 WGQGTL VTVSS 92 A12_ompseq EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK GLEWVS 85 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAR 89 WGQGTL VTVSS 92 B5_ompseq EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK GLEWVS 85 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92

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B9_ompseq EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK GLEWVS 85 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 Bllompseq EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK GLEWVS 85 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 C2_ompseq EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK GLEWVS 85 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAR 89 WGQGTL VTVSS 92 C6_ompseq EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK GLEWVS 85 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 C7_ompseq EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK GLEWVS 85 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 Cllompseq EVQLLESGGGLV QTGGSLRLSCAA S 82 WVRQAPGK GLEWVS 85 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 D4_ompseq EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK GLEWVS 85 RFTISRDNSKNTLY LQMNSLRAEDAA VYYCAK 90 WGQGTL VTVSS 92 E6_ompseq EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK GLEWVS 85 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAR 89 WGQGTL VTVSS 92 ElOompseq EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK GLEWVS 85 RFTISRDNPKNTLY LQMNSLRAEDTAV YYCAK 91 WGQGTL VTVSS 92 E12_ompseq EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK GLEWVS 85 RFTISRDNSKNTLY LQMNSLRAEDAA VYYCAK 90 WGQGTL VTVSS 92 F6_ompseq EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK GLEWVS 85 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 Fllompseq EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK GLEWVS 85 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92

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F12_ompseq EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK GLEWVS 85 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTQ VTVSS 94 G9_ompseq EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK GLEWVS 85 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 C04-1 EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK GLEWVS 85 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 C04-2 EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK cLEWVS 86 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 C04-3P EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK cLEWVS 86 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 C04-DM EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK CLEWVS 86 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 C04-DMP EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK CLEWVS 86 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 F06-1 EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK GLEWVS 85 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 F06-2 EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK CLEWVS 86 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 F06-3P EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK CLEWVS 86 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 F06-DM EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK CLEWVS 86 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 F06-DMP EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK CLEWVS 86 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92

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B01 EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK GLEWVS 85 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 A07(AR) EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK GLEWVS 85 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 E09 EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK GLEWVS 85 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 D03 EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK GLEWVS 85 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 A02 EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK GLEWVS 85 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 B09 EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK GLEWVS 85 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 BIO EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK GLEWVS 85 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 E06 EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK GLEWVS 85 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 H04 EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK GLEWVS 85 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 A06 EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK GLEWVS 85 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 A07(AM) EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK GLEWVS 85 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 B02 EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK GLEWVS 85 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92

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VH02-1 EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK GLEWVS 85 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 VH02-2 EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK CLEWVS 86 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 VH02-3P EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK CLEWVS 86 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 VH02-DM EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK CLEWVS 86 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 VH02-DMP EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK CLEWVS 86 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 VH05-1 EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK GLEWVS 85 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 VH05-2 EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK CLEWVS 86 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 VH05-3P EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK CLEWVS 86 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 VH05-DM EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK CLEWVS 86 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 VH05-DMP EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK CLEWVS 86 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 VH06-1 EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK GLEWVS 85 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 VH06-2 EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK CLEWVS 86 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92

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VH06-3P EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK CLEWVS 86 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 VH06-DM EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK CLEWVS 86 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 VH06-DMP EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK CLEWVS 86 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 VH07-1 EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK GLEWVS 85 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 VH07-2 EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK CLEWVS 86 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 VH07-3 EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK CLEWVS 86 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 VH07-DM EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK CLEWVS 86 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 VH07-DMP EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK CLEWVS 86 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 VH09-1 EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK GLEWVS 85 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 VH09-2 EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK CLEWVS 86 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 VH09-3P EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK CLEWVS 86 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 VH09-DM EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK CLEWVS 86 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92

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VH09-DMP EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK CLEWVS 86 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 VH16-1 EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK GLEWVS 85 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 VH16-2 EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK CLEWVS 86 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 VH16-3P EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK CLEWVS 86 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 VH16-DM EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK CLEWVS 86 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 VH16-DMP EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK CLEWVS 86 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 VH27-1 EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK GLEWVS 85 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 VH27-2 EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK CLEWVS 86 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 VH27-3P EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK CLEWVS 86 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 VH27-DM EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK CLEWVS 86 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 VH27-DMP EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK CLEWVS 86 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 VH32-1 EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK GLEWVS 85 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92

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VH32-2 EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK CLEWVS 86 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 VH32-3 EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK CLEWVS 86 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 VH32-DM EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK CLEWVS 86 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 VH32-DMP EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK CLEWVS 86 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 VH35-1 EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK GLEWVS 85 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 VH35-2 EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK CLEWVS 86 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 VH35-3 EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK CLEWVS 86 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 VH35-DM EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK CLEWVS 86 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 VH35-DMP EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK CLEWVS 86 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 VH48 EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK GLEWVS 85 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 VH55 EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK GLEWVS 85 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 VH81 EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK GLEWVS 85 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92

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C12 EVQLLESGGGLV QPGGSLRRSCAA S 84 WVRQAPGK GLEWVS 85 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGSL VTVSS 95 1564- VL(N51D) EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK GLEWVS 85 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 1564- VL(N95aD) EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK GLEWVS 85 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 1564- VL(N95aH) EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK GLEWVS 85 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 1564- VL(N95aK) EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK GLEWVS 85 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 1564- VL(N95aR) EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK GLEWVS 85 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 1564- VH(N54D) EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK GLEWVS 85 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92 1564- VH(N54Q) EVQLLESGGGLV QPGGSLRLSCAA S 81 WVRQAPGK GLEWVS 85 RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAK 88 WGQGTL VTVSS 92

Table 4

Sequences of light chain variable region framework regions in an antibody clone of the invention

Clone ID L-FR1 SEQ ID NO: L-FR2 SEQ ID NO: L-FR3 SEQ ID NO: L-FR4 SEQ ID NO: 996-1 QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVPDRFSGSKSGTSASL AISGLRSEDEADYYC 166 FGGGT KLTVL 169 996-2 QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVPDRFSGSKSGTSASL AISGLRSEDEADYYC 166 FGCGT KLTVL 170 1226-1 QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVPDRFSGSKSGTSASL AISGLRSEDEADYYC 166 FGGGT KLTVL 169 1226-2 QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVPDRFSGSKSGTSASL AISGLRSEDEADYYC 166 FGCGT KLTVL 170

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1564-1 QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVPDRFSGSKSGTSASL AISGLRSEDEADYYC 166 FGGGT KLTVL 169 1564-2 QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVSDRFSGSKSGTSASL AISGLRSEDEADYYC 167 FGCGT KLTVL 170 1564-3 QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVPDRFSGSKSGTSASL AISGLRSEDEADYYC 166 FGCGT KLTVL 170 1564-DM QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVSDRFSGSKSGTSASL AISGLRSEDEADYYC 167 FGCGT KLTVL 170 1564-DMP QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVPDRFSGSKSGTSASL AISGLRSEDEADYYC 166 FGCGT KLTVL 170 48G5-1 QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVPDRFSGSKSGTSASL AISGLRSEDEADYYC 166 FGGGT KLTVL 169 48G5-2 QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVPDRFSGSKSGTSASL AISGLRSEDEADYYC 166 FGCGT KLTVL 170 49G11-1 QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVPDRFSGSKSGTSASL AISGLRSEDEADYYC 166 FGGGT KLTVL 169 49G11-2 QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVPDRFSGSKSGTSASL AISGLRSEDEADYYC 166 FGCGT KLTVL 170 54H4-1 QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVPDRFSGSKSGTSASL AISGLRSEDEADYYC 166 FGGGT KLTVL 169 54H4-2 QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVPDRFSGSKSGTSASL AISGLRSEDEADYYC 166 FGCGT KLTVL 170 60A11-1 QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVPDRFSGSKSGTSASL AISGLRSEDEADYYC 166 FGGGT KLTVL 169 60A11-2 QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVPDRFSGSKSGTSASL AISGLRSEDEADYYC 166 FGCGT KLTVL 170 60H6-1 QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVPDRFSGSKSGTSASL AISGLRSEDEADYYC 166 FGGGT KLTVL 169 60H6-2 QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVSDRFSGSKSGTSASL AISGLRSEDEADYYC 167 FGCGT KLTVL 170 60H6-3 QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVPDRFSGSKSGTSASL AISGLRSEDEADYYC 166 FGCGT KLTVL 170 Alomseq QSVLTQPPSAS GTPGQRATISC 163 WYQQLPG TAPKLLIY 165 GVPDRFSGSKSGTSASL AISGLRSEDEADYYC 166 FGGGT KLTVL 169 A3_ompseq QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVPDRFSGSKSGTSASL AISGLRSEDEADYYC 166 FGGGT KLTVL 169

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A6_ompseq QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVPDRFSGSKSGTSASL AISGLRSEDEADYYC 166 FGGGT KLTVL 169 A8_ompseq QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVPDRFSGSKSGTSASL AISGLRSEDEADYYC 166 FGGGT KLTVL 169 AlOompseq QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVPDRFSGSKSGTSASL AISGLRSEDEADYYC 166 FGGGT KLTVL 169 A12_ompseq QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVPDRFSGSKSGTSASL AISGLRSEDGADYYC 168 FGGGT KLTVL 169 B5_ompseq QSVLTQPPSAS GPPGQRVTISC 164 WYQQLPG TAPKLLIY 165 GVPDRFSGSKSGTSASL AISGLRSEDEADYYC 166 FGGGT KLTVL 169 B9_ompseq QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVPDRFSGSKSGTSASL AISGLRSEDEADYYC 166 FGGGT KLTVL 169 Bllompseq QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVPDRFSGSKSGTSASL AISGLRSEDEADYYC 166 FGGGT KLTVL 169 C2_ompseq QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVPDRFSGSKSGTSASL AISGLRSEDEADYYC 166 FGGGT KLTVL 169 C6_ompseq QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVPDRFSGSKSGTSASL AISGLRSEDEADYYC 166 FGGGT KLTVL 169 C7_ompseq QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVPDRFSGSKSGTSASL AISGLRSEDGADYYC 168 FGGGT KLTVL 169 Cllompseq QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVPDRFSGSKSGTSASL AISGLRSEDEADYYC 166 FGGGT KLTVL 169 D4_ompseq QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVPDRFSGSKSGTSASL AISGLRSEDEADYYC 166 FGGGT KLTVL 169 E6_ompseq QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVPDRFSGSKSGTSASL AISGLRSEDEADYYC 166 FGGGT KLTVL 169 ElOompseq QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVPDRFSGSKSGTSASL AISGLRSEDEADYYC 166 FGGGT KLTFL 171 E12_ompseq QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVPDRFSGSKSGTSASL AISGLRSEDEADYYC 166 FGGGT KLTVL 169 F6_ompseq QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVPDRFSGSKSGTSASL AISGLRSEDEADYYC 166 FGGGT KLTVL 169 Fllompseq QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVSDRFSGSKSGTSASL AISGLRSEDEADYYC 167 FGGGT KLTVL 169 F12_ompseq QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVPDRFSGSKSGTSASL AISGLRSEDEADYYC 166 FGGGT KLTVL 169

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G9_ompseq QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVPDRFSGSKSGTSASL AISGLRSEDEADYYC 166 FGGGT KLTVL 169 C04-1 QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVPDRFSGSKSGTSASL AISGLRSEDEADYYC 166 FGGGT KLTVL 169 C04-2 QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVSDRFSGSKSGTSASL AISGLRSEDEADYYC 167 FGCGT KLTVL 170 C04-3 QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVPDRFSGSKSGTSASL AISGLRSEDEADYYC 166 FGCGT KLTVL 170 C04-DM QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVSDRFSGSKSGTSASL AISGLRSEDEADYYC 167 FGCGT KLTVL 170 C04-DMP QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVPDRFSGSKSGTSASL AISGLRSEDEADYYC 166 FGCGT KLTVL 170 F06-1 QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVPDRFSGSKSGTSASL AISGLRSEDEADYYC 166 FGGGT KLTVL 169 F06-2 QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVSDRFSGSKSGTSASL AISGLRSEDEADYYC 167 FGCGT KLTVL 170 F06-3 QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVPDRFSGSKSGTSASL AISGLRSEDEADYYC 166 FGCGT KLTVL 170 F06-DM QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVSDRFSGSKSGTSASL AISGLRSEDEADYYC 167 FGCGT KLTVL 170 F06-DMP QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVPDRFSGSKSGTSASL AISGLRSEDEADYYC 166 FGCGT KLTVL 170 BOI QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVSDRFSGSKSGTSASL AISGLRSEDEADYYC 167 FGGGT KLTVL 169 A07(AR) QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVSDRFSGSKSGTSASL AISGLRSEDEADYYC 167 FGGGT KLTVL 169 E09 QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVSDRFSGSKSGTSASL AISGLRSEDEADYYC 167 FGGGT KLTVL 169 D03 QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVSDRFSGSKSGTSASL AISGLRSEDEADYYC 167 FGGGT KLTVL 169 A02 QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVSDRFSGSKSGTSASL AISGLRSEDEADYYC 167 FGGGT KLTVL 169 B09 QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVSDRFSGSKSGTSASL AISGLRSEDEADYYC 167 FGGGT KLTVL 169 BIO QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVSDRFSGSKSGTSASL AISGLRSEDEADYYC 167 FGGGT KLTVL 169

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E06 QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVSDRFSGSKSGTSASL AISGLRSEDEADYYC 167 FGGGT KLTVL 169 H04 QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVSDRFSGSKSGTSASL AISGLRSEDEADYYC 167 FGGGT KLTVL 169 A06 QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVSDRFSGSKSGTSASL AISGLRSEDEADYYC 167 FGGGT KLTVL 169 A07(AM) QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVSDRFSGSKSGTSASL AISGLRSEDEADYYC 167 FGGGT KLTVL 169 B02 QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVSDRFSGSKSGTSASL AISGLRSEDEADYYC 167 FGGGT KLTVL 169 VH02-1 QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVPDRFSGSKSGTSASL AISGLRSEDEADYYC 166 FGGGT KLTVL 169 VH02-2 QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVSDRFSGSKSGTSASL AISGLRSEDEADYYC 167 FGCGT KLTVL 170 VH02-3 QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVPDRFSGSKSGTSASL AISGLRSEDEADYYC 166 FGCGT KLTVL 170 VH02-DM QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVSDRFSGSKSGTSASL AISGLRSEDEADYYC 167 FGCGT KLTVL 170 VH02-DMP QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVPDRFSGSKSGTSASL AISGLRSEDEADYYC 166 FGCGT KLTVL 170 VH05-1 QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVPDRFSGSKSGTSASL AISGLRSEDEADYYC 166 FGGGT KLTVL 169 VH05-2 QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVSDRFSGSKSGTSASL AISGLRSEDEADYYC 167 FGCGT KLTVL 170 VH05-3 QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVPDRFSGSKSGTSASL AISGLRSEDEADYYC 166 FGCGT KLTVL 170 VH05-DM QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVSDRFSGSKSGTSASL AISGLRSEDEADYYC 167 FGCGT KLTVL 170 VH05-DMP QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVPDRFSGSKSGTSASL AISGLRSEDEADYYC 166 FGCGT KLTVL 170 VH06-1 QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVPDRFSGSKSGTSASL AISGLRSEDEADYYC 166 FGGGT KLTVL 169 VH06-2 QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVSDRFSGSKSGTSASL AISGLRSEDEADYYC 167 FGCGT KLTVL 170 VH06-3 QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVPDRFSGSKSGTSASL AISGLRSEDEADYYC 166 FGCGT KLTVL 170

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VH06-DM QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVSDRFSGSKSGTSASL AISGLRSEDEADYYC 167 FGCGT KLTVL 170 VH06-DMP QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVPDRFSGSKSGTSASL AISGLRSEDEADYYC 166 FGCGT KLTVL 170 VH07-1 QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVPDRFSGSKSGTSASL AISGLRSEDEADYYC 166 FGGGT KLTVL 169 VH07-2 QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVSDRFSGSKSGTSASL AISGLRSEDEADYYC 167 FGCGT KLTVL 170 VH07-3 QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVPDRFSGSKSGTSASL AISGLRSEDEADYYC 166 FGCGT KLTVL 170 VH07-DM QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVSDRFSGSKSGTSASL AISGLRSEDEADYYC 167 FGCGT KLTVL 170 VH07-DMP QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVPDRFSGSKSGTSASL AISGLRSEDEADYYC 166 FGCGT KLTVL 170 VH09-1 QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVPDRFSGSKSGTSASL AISGLRSEDEADYYC 166 FGGGT KLTVL 169 VH09-2 QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVSDRFSGSKSGTSASL AISGLRSEDEADYYC 167 FGCGT KLTVL 170 VH09-3 QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVPDRFSGSKSGTSASL AISGLRSEDEADYYC 166 FGCGT KLTVL 170 VH09-DM QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVSDRFSGSKSGTSASL AISGLRSEDEADYYC 167 FGCGT KLTVL 170 VH09-DMP QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVPDRFSGSKSGTSASL AISGLRSEDEADYYC 166 FGCGT KLTVL 170 VH16-1 QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVPDRFSGSKSGTSASL AISGLRSEDEADYYC 166 FGGGT KLTVL 169 VH16-2 QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVSDRFSGSKSGTSASL AISGLRSEDEADYYC 167 FGCGT KLTVL 170 VH16-3 QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVPDRFSGSKSGTSASL AISGLRSEDEADYYC 166 FGCGT KLTVL 170 VH16-DM QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVSDRFSGSKSGTSASL AISGLRSEDEADYYC 167 FGCGT KLTVL 170 VH16-DMP QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVPDRFSGSKSGTSASL AISGLRSEDEADYYC 166 FGCGT KLTVL 170 VH27-1 QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVPDRFSGSKSGTSASL AISGLRSEDEADYYC 166 FGGGT KLTVL 169

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VH27-2 QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVSDRFSGSKSGTSASL AISGLRSEDEADYYC 167 FGCGT KLTVL 170 VH27-3 QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVPDRFSGSKSGTSASL AISGLRSEDEADYYC 166 FGCGT KLTVL 170 VH27-DM QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVSDRFSGSKSGTSASL AISGLRSEDEADYYC 167 FGCGT KLTVL 170 VH27-DMP QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVPDRFSGSKSGTSASL AISGLRSEDEADYYC 166 FGCGT KLTVL 170 VH32-1 QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVPDRFSGSKSGTSASL AISGLRSEDEADYYC 166 FGGGT KLTVL 169 VH32-2 QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVSDRFSGSKSGTSASL AISGLRSEDEADYYC 167 FGCGT KLTVL 170 VH32-3 QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVPDRFSGSKSGTSASL AISGLRSEDEADYYC 166 FGCGT KLTVL 170 VH32-DM QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVSDRFSGSKSGTSASL AISGLRSEDEADYYC 167 FGCGT KLTVL 170 VH32-DMP QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVPDRFSGSKSGTSASL AISGLRSEDEADYYC 166 FGCGT KLTVL 170 VH35-1 QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVPDRFSGSKSGTSASL AISGLRSEDEADYYC 166 FGGGT KLTVL 169 VH35-2 QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVSDRFSGSKSGTSASL AISGLRSEDEADYYC 167 FGCGT KLTVL 170 VH35-3 QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVPDRFSGSKSGTSASL AISGLRSEDEADYYC 166 FGCGT KLTVL 170 VH35-DM QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVSDRFSGSKSGTSASL AISGLRSEDEADYYC 167 FGCGT KLTVL 170 VH35-DMP QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVPDRFSGSKSGTSASL AISGLRSEDEADYYC 166 FGCGT KLTVL 170 VH48 QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVPDRFSGSKSGTSASL AISGLRSEDEADYYC 166 FGGGT KLTVL 169 VH55 QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVPDRFSGSKSGTSASL AISGLRSEDEADYYC 166 FGGGT KLTVL 169 VH81 QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVPDRFSGSKSGTSASL AISGLRSEDEADYYC 166 FGGGT KLTVL 169 C12 QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVPDRFSGSKSGTSASL AISGLRSEDEADYYC 166 FGGGT KLTVL 169 1564- VL(N51D) QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVSDRFSGSKSGTSASL AISGLRSEDEADYYC 167 FGGGT KLTVL 169 1564- VL(N95aD) QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVSDRFSGSKSGTSASL AISGLRSEDEADYYC 167 FGGGT KLTVL 169 1564- VL(N95aH) QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVSDRFSGSKSGTSASL AISGLRSEDEADYYC 167 FGGGT KLTVL 169 1564- VL(N95aK) QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVSDRFSGSKSGTSASL AISGLRSEDEADYYC 167 FGGGT KLTVL 169 1564- VL(N95aR) QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVSDRFSGSKSGTSASL AISGLRSEDEADYYC 167 FGGGT KLTVL 169 1564- VH(N54D) QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVPDRFSGSKSGTSASL AISGLRSEDEADYYC 166 FGGGT KLTVL 169 1564- VH(N54Q) QSVLTQPPSAS GTPGQRVTIS 162 WYQQLPG TAPKLLIY 165 GVPDRFSGSKSGTSASL AISGLRSEDEADYYC 166 FGGGT KLTVL 169

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The anti-IGF1R antibody of the present invention may be an antibody comprising a light chain variable region and a heavy chain variable region, and various heavy and light chain variable regions disclosed herein are shown in Table. 5 and 6, CDR1 CDR3 and framework regions 1-4 of each clone are described as SEQ ID NO.

The heavy chain variable regions and light chain variable regions described in table. 5 and 6 can be freely combined to produce different types of antibodies. Examples of combinations of a heavy chain variable region and a light chain variable region are shown in Table 1. 5 and 6.

Table 5 ___________Variable region of the heavy chain of the antibody according to the invention___________

SEQ ID NO Heavy chain (VH) CDR- Sh CDR- H2 CDR- NZ N- FR1 N- FR2 N- FR3 N- FR4 172 996-1 1 10 50 81 85 87 92 173 996-2 1 10 50 81 86 87 92 174 1226-1 2 And 51 81 85 88 92 175 1226-2 2 And 51 81 86 88 92 176 1564-1 1 12 52 81 85 88 92 177 1564-2 1 12 52 81 86 88 92 178 1564-3 1 12 52 81 86 88 92

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179 1564-DM 1 13 52 81 86 88 92 180 1564-DMP 1 13 52 81 86 88 92 181 48G5-1 3 14 53 81 85 88 92 182 48G5-2 3 14 53 81 86 88 92 183 49G11-1 1 15 54 81 85 88 92 184 49G11-2 1 15 54 81 86 88 92 185 54H4-1 3 16 55 81 85 88 92 186 54H4-2 3 16 55 81 86 88 93 187 60A11-1 2 17 56 81 85 88 92 188 60A11-2 2 17 56 81 86 88 92 189 60H6-1 3 18 57 81 85 88 92 190 60H6-2 3 18 57 81 86 88 92 191 60H6-3 3 18 57 81 86 88 92 192 A1OMPSEQ 1 19 58 81 85 88 92 193 A3OMPSEQ 3 20 59 82 85 88 92 194 A6OMPSEQ 4 21 60 81 85 88 92 195 A8OMPSEQ 2 18 61 81 85 89 92 196 A10OMPSEQ 5 22 62 83 85 89 92 197 A12OMPSEQ 3 23 63 81 85 89 92 198 B5OMPSEQ 3 24 64 81 85 88 92 199 B9OMPSEQ 3 18 65 81 85 88 92 200 B11OMPSEQ 3 25 66 81 85 88 92 201 C2OMPSEQ 1 26 67 81 85 89 92 202 C6OMPSEQ 6 27 68 81 85 88 92 203 C7OMPSEQ 2 28 69 81 85 88 92 204 C11OMPSEQ 7 29 70 82 85 88 92 205 D4OMPSEQ 3 thirty 71 81 85 90 92 206 E06OMPSEQ 1 12 72 81 85 89 92 207 E10OMPSEQ 2 17 56 81 85 91 92 208 E12OMPSEQ 3 thirty 71 81 85 90 92 209 F06OMPSEQ 3 18 73 81 85 88 92

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210 F11OMPSEQ 3 31 74 81 85 88 92 211 F12OMPSEQ 3 32 75 81 85 88 94 212 G9OMPSEQ 1 33 76 81 85 88 92 213 C04-1 1 12 52 81 85 88 92 214 C04-2 1 12 52 81 86 88 92 215 C04-3 1 12 52 81 86 88 92 216 C04-DM 1 13 52 81 86 88 92 217 C04-DMP 1 13 52 81 86 88 92 218 F06-1 1 12 52 81 85 88 92 219 F06-2 1 12 52 81 86 88 92 220 F06-3 1 12 52 81 86 88 92 221 F06-DM 1 13 52 81 86 88 92 222 F06-DMP 1 13 52 81 86 88 92 223 BOI 1 34 52 81 85 88 92 224 A07(AR) 1 35 52 81 85 88 92 225 E09 1 36 52 81 85 88 92 226 D03 1 37 52 81 85 88 92 227 A02 1 12 52 81 85 88 92 228 B09 1 12 52 81 85 88 92 229 BIO 1 12 52 81 85 88 92 230 E06 1 12 52 81 85 88 92 231 H04 1 12 52 81 85 88 92 232 A06 1 12 52 81 85 88 92 233 A07(AM) 1 12 52 81 85 88 92 234 B02 1 12 52 81 85 88 92 235 VH2-1 8 38 77 81 85 88 92 236 VH2-2 8 38 77 81 86 88 92 237 VH2-3 8 38 77 81 86 88 92 238 VH2-DM 8 39 77 81 86 88 92 239 VH2-DMP 8 39 77 81 86 88 92 240 VH5-1 1 40 78 81 85 88 92

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241 VH5-2 1 40 78 81 86 88 92 242 VH5-3 1 40 78 81 86 88 92 243 VH5-DM 1 41 78 81 86 88 92 244 VH5-DMP 1 41 78 81 86 88 92 245 VH6-1 1 42 77 81 85 88 92 246 VH6-2 1 42 77 81 86 88 92 247 VH6-3 1 42 77 81 86 88 92 248 VH6-DM 1 43 77 81 86 88 92 249 VH6-DMP 1 43 77 81 86 88 92 250 VH7-1 8 44 52 81 85 88 92 251 VH7-2 8 44 52 81 86 88 92 252 VH7-3 8 44 52 81 86 88 92 253 VH7-DM 8 45 52 81 86 88 92 254 VH7-DMP 8 45 52 81 86 88 92 255 VH9-1 1 46 52 81 85 88 92 256 VH9-2 1 46 52 81 86 88 92 257 VH9-3 1 46 52 81 86 88 92 258 VH9-DM 1 47 52 81 86 88 92 259 VH9-DMP 1 47 52 81 86 88 92 260 VH16-1 1 38 52 81 85 88 92 261 VH16-2 1 38 52 81 86 88 92 262 VH16-3 1 38 52 81 86 88 92 263 VH16-DM 1 39 52 81 86 88 92 264 VH16-DMP 1 39 52 81 86 88 92 265 VH27-1 8 44 78 81 85 88 92 266 VH27-2 8 44 78 81 86 88 92 267 VH27-3 8 44 78 81 86 88 92 268 VH27-DM 8 45 78 81 86 88 92 269 VH27-DMP 8 45 78 81 86 88 92 270 VH32-1 8 12 77 81 85 88 92 271 VH32-2 8 12 77 81 86 88 92

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272 VH32-3 8 12 77 81 86 88 92 273 VH32-DM 8 13 77 81 86 88 92 274 VH32-DM 8 13 77 81 86 88 92 275 VH35-1 1 44 78 81 85 88 92 276 VH35-2 1 44 78 81 86 88 92 277 VH35-3 1 44 78 81 86 88 92 278 VH35-DM 1 45 78 81 86 88 92 279 VH35-DMP 1 45 78 81 86 88 92 280 VH48 8 38 79 81 85 88 92 281 VH55 1 12 77 81 85 88 92 282 VH81 1 48 52 81 85 88 92 283 C12 9 12 80 84 85 88 95 284 1564-VL(N51D) 1 12 52 81 85 88 92 285 1564-VL(N95aD) 1 12 52 81 85 88 92 286 1564-VL(N95aH) 1 12 52 81 85 88 92 287 1564-VL(N95aK) 1 12 52 81 85 88 92 288 1564-VL(N95aR) 1 12 52 81 85 88 92 289 1564-VH(N54D) 1 49 52 81 85 88 92 290 1564-VH(N54Q) 1 13 52 81 85 88 92

Table 6

Antibody Light Chain Variable Region of the Invention

SEQ ID NO Light chain (VH) CDR- L1 CDR- L2 CDR- L3 L- FR1 L- FR2 L- FR3 L- FR4 291 996-1 96 114 133 162 165 166 169 292 996-2 96 114 133 162 165 166 170 293 1226-1 97 115 134 162 165 166 169 294 1226-2 97 115 134 162 165 166 170 295 1564-1 98 116 135 162 165 166 169 296 1564-2 98 116 135 162 165 167 170 297 1564-3 98 116 135 162 165 166 170 298 1564-DM 98 117 136 162 165 167 170

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299 1564-DMP 98 117 136 162 165 166 170 300 48G5-1 99 118 137 162 165 166 169 301 48G5-2 99 118 137 162 165 166 170 302 49G11-1 100 119 138 162 165 166 169 303 49G11-2 100 119 138 162 165 166 170 304 54H4-1 101 120 139 162 165 166 169 305 54H4-2 101 120 139 162 165 166 170 306 60A11-1 102 121 135 162 165 166 169 307 60A11-2 102 121 135 162 165 166 170 308 60H6-1 103 122 140 162 165 166 169 309 60H6-2 103 122 140 162 165 167 170 310 60H6-3 103 122 140 162 165 166 170 311 A1OMPSEQ 104 123 140 163 165 166 169 312 A3OMPSEQ 105 124 139 162 165 166 169 313 A6OMPSEQ 104 125 141 162 165 166 169 314 A8OMPSEQ 103 126 135 162 165 166 169 315 A10OMPSEQ 106 127 142 162 165 166 169 316 A12OMPSEQ 107 114 138 162 165 168 169 317 B5OMPSEQ 99 128 143 164 165 166 169 318 B9OMPSEQ 108 123 140 162 165 166 169 319 B11OMPSEQ 99 121 144 162 165 166 169 320 C2OMPSEQ 109 129 138 162 165 166 169 321 C6OMPSEQ 110 648 145 162 165 166 169 322 C7OMPSEQ 111 114 138 162 165 166 169 323 C11OMPSEQ 99 114 134 162 165 166 169 324 D4OMPSEQ 110 130 140 162 165 166 169 325 E06OMPSEQ 111 114 146 162 165 166 169 326 E10OMPSEQ 102 121 135 162 165 166 171 327 E12OMPSEQ 110 130 140 162 165 166 169 328 F06OMPSEQ 99 118 137 162 165 166 169

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329 F11OMPSEQ 112 125 135 162 165 167 169 330 F12OMPSEQ 103 131 147 162 165 166 169 331 G9OMPSEQ 113 648 148 162 165 166 169 332 C04-1 98 116 149 162 165 166 169 333 C04-2 98 116 149 162 165 167 170 334 C04-3 98 116 149 162 165 166 170 335 C04-DM 98 117 150 162 165 167 170 336 C04-DMP 98 117 150 162 165 166 170 337 F06-1 98 116 151 162 165 166 169 338 F06-2 98 116 151 162 165 167 170 339 F06-3 98 116 151 162 165 166 170 340 F06-DM 98 117 152 162 165 167 170 341 F06-DMP 98 117 152 162 165 166 170 342 BOI 98 116 135 162 165 167 169 343 A07(AR) 98 116 135 162 165 167 169 344 E09 98 116 135 162 165 167 169 345 D03 98 116 135 162 165 167 169 346 A02 98 116 153 162 165 167 169 347 B09 98 116 154 162 165 167 169 348 BIO 98 116 155 162 165 167 169 349 E06 98 116 156 162 165 167 169 350 H04 98 116 157 162 165 167 169 351 A06 98 116 158 162 165 167 169 352 A07(AM) 98 116 159 162 165 167 169 353 B02 98 116 138 162 165 167 169 354 VH2-1 98 116 135 162 165 166 169 355 VH2-2 98 116 135 162 165 167 170 356 VH2-3 98 116 135 162 165 166 170 357 VH2-DM 98 117 136 162 165 167 170 358 VH2-DMP 98 117 136 162 165 166 170

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359 VH5-1 98 116 135 162 165 166 169 360 VH5-2 98 116 135 162 165 167 170 361 VH5-3 98 116 135 162 165 166 170 362 VH5-DM 98 117 136 162 165 167 170 363 VH5-DMP 98 117 136 162 165 166 170 364 VH6-1 98 116 135 162 165 166 169 365 VH6-2 98 116 135 162 165 167 170 366 VH6-3 98 116 135 162 165 166 170 367 VH6-DM 98 117 136 162 165 167 170 368 VH6-DMP 98 117 136 162 165 166 170 369 VH7-1 98 116 135 162 165 166 169 370 VH7-2 98 116 135 162 165 167 170 371 VH7-3 98 116 135 162 165 166 170 372 VH7-DM 98 117 136 162 165 167 170 373 VH7-DMP 98 117 136 162 165 166 170 374 VH9-1 98 116 135 162 165 166 169 375 VH9-2 98 116 135 162 165 167 170 376 VH9-3 98 116 135 162 165 166 170 377 VH9-DM 98 117 136 162 165 167 170 378 VH9-DMP 98 117 136 162 165 166 170 379 VH16-1 98 116 135 162 165 166 169 380 VH16-2 98 116 135 162 165 167 170 381 VH16-3 98 116 135 162 165 166 170 382 VH16-DM 98 117 136 162 165 167 170 383 VH16-DMP 98 117 136 162 165 166 170 384 VH27-1 98 116 135 162 165 166 169 385 VH27-2 98 116 135 162 165 167 170 386 VH27-3 98 116 135 162 165 166 170 387 VH27-DM 98 117 136 162 165 167 170 388 VH27-DMP 98 117 136 162 165 166 170

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389 VH32-1 98 116 135 162 165 166 169 390 VH32-2 98 116 135 162 165 167 170 391 VH32-3 98 116 135 162 165 166 170 392 VH32-DM 98 117 136 162 165 167 170 393 VH32-DM 98 117 136 162 165 166 170 394 VH35-1 98 116 135 162 165 166 169 395 VH35-2 98 116 135 162 165 167 170 396 VH35-3 98 116 135 162 165 166 170 397 VH35-DM 98 117 136 162 165 167 170 398 VH35-DMP 98 117 136 162 165 166 170 399 VH48 98 116 135 162 165 166 169 400 VH55 98 116 135 162 165 166 169 401 VH81 98 116 135 162 165 166 169 402 C12 98 116 135 162 165 166 169 403 1564-VL(N51D) 98 132 135 162 165 167 169 404 1564-VL(N95aD) 98 116 160 162 165 167 169 405 1564-VL(N95aH) 98 116 136 162 165 167 169 406 1564-VL(N95aK) 98 116 161 162 165 167 169 407 1564-VL(N95aR) 98 116 161 162 165 167 169 408 1564-VH(N54D) 98 116 135 162 165 166 169 409 1564-VH(N54Q) 98 116 135 162 165 166 169

The various heavy and light chain variable regions disclosed herein are presented in Table 1. 5 and 6. Each variable region can be linked to heavy and light chain constant regions to form the corresponding heavy and light chains of the intact antibody. An example of a combination of a heavy chain variable region and a light chain variable region constituting an anti-IGF1R antibody of the present invention may be a combination of a heavy chain variable region and a light chain variable region with the same clone name, as described in Table. 5 and table. 6.

In one specific example of the present invention, the anti-IGF1R antibody or antigen binding fragment thereof comprises at least one selected from the group consisting of:

Heavy chain CDR1 (H-CDR1) containing an amino acid sequence selected from SEQ ID NO:1-9,

Heavy chain CDR2 (H-CDR2) containing an amino acid sequence selected from SEQ ID NO: 10-49,

heavy chain CDR3 (H-CDR3) containing an amino acid sequence selected from SEQ ID NO:50-80;

Light chain CDR1 (L-CDR1) containing an amino acid sequence selected from SEQ ID NO:96-113,

Light chain CDR2 (L-CDR2) containing an amino acid sequence selected from SEQ ID NO:114-132, and

Light chain CDR3 (L-CDR3) containing an amino acid sequence selected from SEQ ID NO:133-161.

Specifically, the antibody or antigen binding fragment of the present invention may comprise a heavy chain variable region comprising a heavy chain CDR1 (H-CDR1) comprising an amino acid sequence selected from SEQ ID NO:1-9, a heavy chain CDR2 (H-CDR2) comprising an amino acid sequence selected from SEQ ID NO:10-49, and a heavy chain CDR3 (H-CDR3) comprising an amino acid sequence selected from SEQ ID NO:50-80, and a light chain variable region containing a light chain CDR1 (L- CDR1) containing an amino acid sequence selected from SEQ ID NO:96-113, light chain CDR2 (L-CDR2) containing an amino acid sequence selected from SEQ ID NO:114-132, and light chain CDR3 (LCDR3) containing an amino acid sequence selected from SEQ ID NO:133-161.

The anti-IGF1R antibody, or antigen-binding fragment thereof, specifically recognizes and binds to at least one amino acid selected from the group consisting of Y775, P776, F778,

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R650, S791, L798, Glu779, L641, H808, E809, L813, V397, D435, W434, Y460 and C488 in the amino acid sequence of SEQ ID NO:410. Specifically, the anti-IGF1R antibody or antigen binding fragment thereof of the present invention can bind to at least one selected from binding sites 1-3 of a protein containing the amino acid sequence of SEQ ID NO:410. Binding site 1 contains at least one amino acid selected from the group consisting of Y775, P776, F778, R650, S791, L798 and Glu779, binding site 2 contains at least one amino acid selected from the group consisting of L641, H808, E809 and L813, and binding site 3 contains at least one amino acid selected from the group consisting of V397, D435, W434, Y460 and C488.

The heavy chain variable region of an antibody or antigen binding fragment of the present invention may comprise heavy chain framework region 1 (H-FR1), located at the N-terminus of H-CDR1, containing an amino acid sequence selected from SEQ ID NO:81-84, heavy chain framework region 1 (H-FR1). chain (H-FR2) located between H-CDR1 and H-CDR2, containing an amino acid sequence selected from SEQ ID NO:85-86, heavy chain framework region (H-FR3), located between H-CDR2 and H-CDR3 containing an amino acid sequence selected from SEQ ID NO:87-91, and a heavy chain framework region (H-FR4) located at the C-terminus of H-CDR3 containing an amino acid sequence selected from SEQ ID NO:92-95.

The light chain variable region of an antibody or antigen binding fragment of the present invention may comprise light chain framework region 1 (L-FR1), located at the N-terminus of LCDR1, containing an amino acid sequence selected from SEQ ID NO:162-164, light chain framework region ( L-FR2), located between L-CDR1 and L-CDR2, containing the amino acid sequence of SEQ ID NO:165, light chain framework region (L-FR3), located between L-CDR2 and L-CDR3, containing an amino acid sequence selected from SEQ ID NO:166-168, and a light chain framework region (L-FR4) located at the C-terminus of L-CDR3 containing an amino acid sequence selected from SEQ ID NO:169-171.

Each of the heavy chain variable regions and light chain variable regions disclosed in Table. 5 and 6 can be used as separate domain antibodies, can be freely combined with each other to form different antibodies, and linked in single-chain form to produce single-chain antibodies such as scFv.

As used herein, a domain antibody is an immunologically functional fragment of an immunoglobulin containing only a heavy chain variable region or a light chain variable region. In one embodiment, two or more VH regions are covalently linked via a peptide linker to form an antibody domain. The two VH regions of this bivalent domain antibody can be directed to the same antigen or to different antigens.

The antigen binding fragments of anti-IGF1R antibodies of the present invention may be an antibody fragment selected from scFv, (scFv) ₂ , scFv-Fc, Fab, Fab', F(ab') ₂ , minibodies and diabodies, including antibody fragments containing one or more than one hypervariable region.

Among the antigen binding fragments, Fab contains a light chain variable region, a heavy chain variable region, a light chain constant region and a first heavy chain constant region (CH1) and has one antigen binding site. Fab' has a Fab hinge region containing one or more cysteine residues at the C-terminus of the heavy chain CH1 domain. An F(ab')2 antibody is prepared by binding two Fab's to form a disulfide bond between the cysteine residues of the Fab' hinge regions.

Fv is a minimal antibody fragment having only a heavy chain variable region and a light chain variable region, and includes single chain variable fragments (scFv) and double chain variable fragments (Fv). In a double-chain Fv, the heavy chain variable region and the light chain variable region may be linked by non-covalent bonds. In a single-chain Fv, the heavy chain variable region and the light chain variable region are linked covalently, directly or through a peptide linker, or linked directly at the C-terminus to form a scFv dimer-like (di-scFv) structure such as a double-chain Fv. In the present invention, a single chain Fv is a single polypeptide chain of an antigen binding region wherein the heavy and light chain variable regions are linked directly or through a linker, and may be at least one selected from the group consisting of a scFv having a single chain linked to a variable a heavy chain region and a light chain variable region, a form of structure like a scFv dimer (di-scFv), scFv-Fc, where the heavy chain variable region, the light chain variable region and the Fc are linked in the form of a single chain, and the like.

The peptide linker may be as described above and may have a length of, for example, 1 to 100 amino acids, such as 2 to 50 or 5 to 25 amino acids, and the peptide linker may have a different length within a range not affecting antibody function. The types of amino acids included in the peptide linker may include one or more amino acids selected from the group consisting of, for example, Gly, Ser and Leu, and specific examples include Gly and Ser residues or Leu and Ser residues. In one specific example, the peptide linker may be (G4S)n, where n is the number of repeats of (G4S) represented by an integer from 1 to 10, such as 2 to 5, especially 3 or 4. An example of a peptide linker may be a peptide , consisting of amino acids

SEQ ID NO:411 or 412.

SEQ ID NO:411: GGGGSGGGGSGGGGSGGGGS.

SEQ ID NO:412: GGGGSGGGGSGGGGS.

Single-stranded Fv (scFv) can be produced by fusing DNA encoding a peptide linker between DNA encoding polypeptides of two variable domains (VL and VH). The resulting polypeptide can form antigen-binding monomers or multimers (eg, dimers, trimers, or tetramers), depending on the length of the flexible linker between the two variable domains, to form their spatial structure. By combining polypeptides containing different VL and VH, multimeric scFvs that bind to different epitopes can be obtained.

Antigen-binding fragments can be produced using proteolytic enzymes (eg, restriction digestion of the full-length antibody with papain to produce Fab and digestion with pepsin to produce the F(ab') ₂ fragment) or using genetic recombinant technology.

Single chain antibodies disclosed herein include, but are not limited to, scFvs containing combinations of heavy and light chain variable domains or combinations of light and heavy chain variable domains containing CDRs.

In addition, an anti-IGF1R antibody and an antigen binding fragment thereof may comprise a heavy chain comprising a heavy chain variable region and a light chain comprising a light chain variable region. Specifically, the heavy chain variable region and the light chain variable region can be linked 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 obtain an intact antibody structure.

The constant region sequences that can be combined with the variable regions of the present invention are given by way of example and can be suitably selected from the constant regions of the heavy and light chains of immunoglobulins (eg, human immunoglobulins). For example, without limitation, 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.

As an example, the variable region of the present invention can be linked to a constant region to form the heavy chain and light chain sequences described below. in table 7 shows examples of combinations of heavy and light chains. In addition, examples of full-length antibodies are given in table. 16. The constant region may be suitably selected from a heavy chain constant region and a light chain constant region of an immunoglobulin (eg, human immunoglobulin).

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Table 7

Examples of antibodies

HC SEQ ID LC SEQ ID 1564 IgG 413 420 1226 IgG 414 421 996 IgG 415 422 48G5 IgG 416 423 54H4 IgG 417 424 60H6 IgG 418 425 Bll IgG 419 426 1564scFv 427 1226 scFv 428 996 scFv 429 48G5 scFv 430 54H4 scFv 431 60H6 scFv 432 Bll scFv 433

The antibodies described in the present invention also include bispecific antibodies and bifunctional antibodies containing one or more CDRs or one or more variable regions, as described above. Bispecific or bifunctional antibodies are artificial hybrid antibodies that recognize two different related or unrelated targets. Bispecific antibodies can be produced using a variety of methods, such as hybridoma fusion or Fab' ligation.

As used herein, a multispecific antigen binding protein or a multispecific antibody is directed at two or more than two antigens or epitopes. As used herein, a bispecific antigen binding protein or antibody or a dual specific antigen binding protein or antibody is a fusion antigen binding protein or fusion antibody having two (2) different antigen binding sites. This bispecific antibody is a type of multispecific antigen binding protein or multispecific antibody, and can be produced by various known methods, for example, methods such as hybridoma fusion or Fab' fragment binding.

A multispecific antibody, such as an anti-IGF1R antibody and an antigen-binding fragment thereof, from which a bispecific antibody can be prepared, may comprise both an anti-IGF1R antibody and an antigen-binding fragment thereof, such as a full-length antibody. The antigen binding fragment may be selected from the group consisting of domain antibodies, scFv, (scFv) ₂ , scFv-Fc, Fab, Fab' and F(ab')2.

The antigen binding fragment of an anti-IGF1R antibody may be associated with or without a linker, such as a peptide linker. In addition, the heavy and light chain portions of the antigen binding fragment, such as the heavy chain variable region and the light chain variable region of the scFv fragment, may also be linked with or without a peptide linker. The peptide linker may be as described above.

In a bispecific antibody, the anti-IGF1R antibody and its antigen-binding fragments may function to deliver a second related antibody or antigen-binding fragment directed at other antigens or epitopes across the blood-brain barrier into the brain. The second antibody may be, without limitation, a brain-active antibody.

The anti-IGF1R antibody or antigen binding fragment thereof of the present invention may have a certain common region or sequence with a different second antibody. For example, an anti-IGF1R antibody may share a constant region or Fc region with an antibody or antigen binding fragment of a second antibody.

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In addition, the bispecific antibody structure of the present invention includes a bivalent form of a bispecific antibody, wherein the anti-IFG1R antibody scFv is linked to each Fc of two heavy chains of a full-length immunoglobulin, for example, at the ends of the heavy chains, directly or through a linker, and a monovalent form of a bispecific antibody, wherein the scFv Anti-IFG1R antibodies are linked to only one end of the two full-length immunoglobulin heavy chains, either directly or through a linker, but a monovalent double antibody is preferred.

Specifically, in one embodiment of the present invention, there is a case where the half-life of the monovalent form clone is better than that of the bivalent form clone, and the structure of the monovalent form clone is a form where the anti-IGF1R domain antibody (scFv) is bound only to the end of one heavy chain of the intact immunoglobulin via linker. Specifically, the antibody is a Knob-In-Hole heterodimer containing two different heavy chains of intact immunoglobulin, where one heavy chain has an anti-IGF1R domain antibody (scFv) linked to its C-terminus and the other the heavy chain does not have an antibody domain at its C-terminus.

In a bispecific antibody, the second antibody associated with an anti-IGF1R antibody or an antigen-binding fragment thereof may be a human antibody, a humanized antibody, a chimeric antibody, or an isolated antibody that specifically binds to IGF1R. The second antibody includes, but is not limited to, full-length antibodies, bispecific antibodies, minibodies, domain antibodies, antibody mimetics (or synthetic antibodies), fusion antibodies (or antibody conjugates), and fragments thereof.

The present invention further relates to an anti-syn antibody and an antigen binding fragment thereof.

The alpha-synuclein that can be recognized by the antibody provided herein can be selected from mammalian alpha-synuclein, human alpha-synuclein, monkey alpha-synuclein (eg, rhesus alpha-synuclein), mouse alpha-synuclein, rat alpha-synuclein. synuclein and the like. For example, human alpha-synuclein may be, but is not limited to, alpha-synuclein (NCBI ID: NP_000336). Unless otherwise indicated herein, alpha-synuclein may refer to human alpha-synuclein, and the antibodies or antigen-binding fragments provided herein tend to specifically bind not only to human alpha-synuclein, but also to monkey alpha-synuclein ( for example, rhesus), rat alpha-synuclein and/or mouse alpha-synuclein.

The antibody or antigen binding fragment thereof can bind to the C-terminal region of alphasynuclein, specifically the C-terminal region containing a peptide containing at least 11 or 12 consecutive amino acids, including residues 110-120 or residues 111-122 in SEQ ID NO:559 human alpha-synuclein. It has been confirmed that the antibody or antigen binding fragment of the present invention can recognize the antigen recognition region and bind to alpha-synuclein aggregates with high affinity.

As used herein, affinity or degree of affinity represents the strength of interaction between an antibody or antigen-binding fragment thereof and an antigen and may be determined by properties of the antigen, such as size, shape and/or charge of the antigen, and CDR sequences and/or physicochemical properties (hydrophilic/hydrophobic properties, electrostatic properties, etc.) of an antibody or antigen-binding fragment. Methods for determining affinity are known in the art, and it is usually, without limitation, reported as the dissociation constant (K _D ).

As used herein, specific binding to alpha-synuclein or alpha-synuclein aggregates means that the binding affinity to the alpha-synuclein protein or alpha-synuclein aggregates is relatively high compared to other antigens and, for example, the dissociation constant (KD). may range from 0.1 x 10' ¹⁰ M to 2 x 10' ¹⁰ M or from 0.05 x 10' 1° M to 0.3 x 10' ⁹ M in relation to alpha-synuclein aggregates, specifically amyloid fibrils, protofibrils and oligomers, especially amyloid fibrils , as measured by, without limitation, Octet analysis or SPR analysis.

Humanized anti-alpha-synuclein antibodies containing a light chain and a heavy chain according to one embodiment of the present invention, for example, Hu11F11 (ver.1), Hu11F11 (ver.2) and Hu11F11_ABL2-4, demonstrate high activity in promoting phagocytic uptake compared to chimeric antibodies to alpha-synuclein. Compared with the chimeric alpha-synuclein antibody, Hu11F11 (ver.1), Hu11F11 (ver.2), u H11F11 (ver.3), Hu11F11 (ver.4) and ABL2-4 show high activity in inhibiting fibril binding with the membrane of nerve cells. Hu11F11 (ver.2), Hu11F11 (ver.4) and ABL2-4 have high activity in inhibiting the spread of alpha-synuclein secreted from alpha-synuclein-overexpressing cells to other nerve cells compared to chimeric anti-alpha-synuclein antibody and demonstrate binding affinity to alpha-synuclein aggregates, eg, similar to or superior to the activity of a chimeric alpha-synuclein antibody, in a cell-based assay.

The anti-alpha-synuclein antibody of the present invention can inhibit the transfer of alpha-synuclein aggregates secreted from a nerve cell into the nervous system of a subject to other normal tissues.

- 43 045348 ny cells in the extracellular space and infection of nerve cells (inhibit the transfer of aggregates from cell to cell) and has the ability to stimulate the phagocytic effect of microglia on alpha-synuclein aggregates in the extracellular space. Alpha-synuclein aggregates spread from one cell to another cell like prions, and alpha-synuclein, especially alpha-synuclein aggregates, spread throughout the brain, leading to synucleinopathies in normal cells. Alpha-synuclein aggregates are therefore toxic to brain neurons and are well known to lead to brain neuronal death (neurodegeneration) and neuroinflammation. Accordingly, as alpha-synuclein aggregates spread to different parts of the brain, brain cell death and neuroinflammatory responses become more pronounced, leading to brain cell death, behavioral disorders, and cognitive impairment, which are noted with the progression of synucleinopathies such as Parkinson's disease.

Accordingly, the anti-alpha-synuclein antibody of the present invention can prevent the phenomenon of distribution of alpha-synuclein aggregates into various regions of the brain, inhibiting the transmission of alpha-synuclein or alpha-synuclein aggregates between nerve cells, and reduce the level of alpha-synuclein aggregates, which are an important cause of synucleinopathies , reducing the amount or eliminating alpha-synuclein aggregates themselves in the extracellular region of nerve cells of the subject's nervous system and stimulating the phagocytic activity of microglia, which leads to a decrease in the intensity of brain nerve cell death and neuroinflammatory reactions, and is also expected to improve, relieve or prevent symptoms and progression of synucleinopathies such as Parkinson's disease.

Moreover, the anti-alpha-synuclein antibody of the present invention has excellent activity in performing both of two functions (1) inhibiting the transmission of alpha-synuclein or alpha-synuclein aggregates between nerve cells (see the results of the cellular assay disclosed herein) and ( 2) reducing the level of alpha-synuclein aggregates in the nervous system of the brain by stimulating the phagocytic activity of microglial cells. In particular, since the anti-alpha-synuclein antibodies currently undergoing clinical trials or published in the scientific literature have one of two activities (1) and (2), it is believed that the anti-alpha-synuclein antibody of the present invention has the advantage for more effective prevention or treatment of synucleinopathies compared to known alpha-synuclein antibodies. Thus, the anti-alpha-synuclein antibody of the present invention has higher efficacy in reducing and eliminating alpha-synuclein aggregates and inhibiting the action of alpha-synuclein aggregates as an etiological factor, and therefore it is more effective in synucleinopathies or related symptomatic diseases (for example, cognitive disorders).

Antibodies or antigen binding fragments of the present invention having high affinity for alpha-synuclein aggregates can reduce the formation of alpha-synuclein aggregates, thereby reducing the concentration of aggregates in the brain. In addition, an antibody or antigen-binding fragment of the present invention with high affinity for alpha-synuclein aggregates can reduce the formation of alpha-synuclein aggregates outside the central nervous system and ultimately alter the state of equilibrium between the forms of alpha-synuclein delimited by the BBB, thereby leading to decreasing the concentration of alpha-synuclein aggregates in the central nervous system.

This is a great advantage in clinical practice, since it allows sufficient effectiveness to be obtained even when administered by a more convenient route, for example, but not limited to, subcutaneous injection. In addition, the antibody or antigen binding fragment of the present invention can, without being limited by theory, inhibit the formation of aggregates by removing monomers, or eliminate both monomers and aggregates.

Antibodies or antigen-binding fragments thereof of the present invention that specifically bind to alpha-synuclein proteins or alpha-synuclein aggregates may not be naturally occurring products (they may be non-naturally occurring products, for example, products of chemical synthesis or recombinant method) . Recombination techniques are well known in the art

As used herein, antibody refers to a full-length immunoglobulin of any isotype or an antigen-binding fragment that can compete with the full-length antibody for binding to a target antigen. For example, it includes chimeric, humanized, full-length human antibodies, dual-specific antibodies, or antigen-binding fragments thereof. The antibody itself is a type of antigen-binding protein. Typically, a full-length antibody contains at least 2 full-length heavy chains and 2 full-length light chains, but in some cases the antibody may contain only heavy chains.

The antibody or antigen-binding fragment thereof may be derived from only one source or be a chimeric antibody. The chimeric antibody contains a portion derived from two types of different antibodies and is described in more detail below. An antibody or its antigen-44 045348 binding fragment can be obtained using a hybridoma, recombinant technique

DNA or enzymatic or chemical separation of the intact antibody. Unless otherwise specified herein, the term antibody includes antibodies containing 2 full-length heavy chains and 2 full-length light chains, and derivatives, variants, fragments and mutants thereof, and examples thereof are described below.

In one embodiment, an antibody includes, without limitation, a monoclonal antibody, a bispecific antibody, a minibody, a domain antibody, an antibody mimetic (or synthetic antibody), a chimeric antibody, a humanized antibody, a human antibody, a fusion antibody (or antibody conjugate), fragments thereof, and various types antibodies disclosed herein. In one embodiment, the antibody fragments disclosed herein may be Fab fragments, Fab' fragments,

F(ab') ₂ fragments, Fv fragments, single chain fragments (scFv), diabody or single chain antibody molecule obtained by joining a heavy chain variable region and a light chain variable region through a spacer.

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

As used herein, a heavy chain includes a full-length heavy chain and fragments thereof having a variable region sequence sufficient to provide binding specificity to an antigen or epitope. The full-length heavy chain contains a VH variable region domain and three (3) CH1, CH2 and CH3 constant region domains. The VH domain is present at the N-terminus of the heavy chain polypeptide, and the CH domains are present at the C-terminus, with CH3 located closest to the C-terminus. The heavy chain includes IgG (including the IgG1, IgG2a, IgG2b, IgG3, and IgG4 subtypes), IgA (including the IgA1 and IgA2 subtypes), and the IgM and IgE isotypes.

As used herein, an antigen binding fragment refers to a portion of an antibody having a specific binding affinity for an antigen and/or a polypeptide containing such a portion. For example, an antigen binding fragment may be a portion of an antibody containing an amino acid residue that imparts specificity and/or affinity for an antigen to the antibody through its interaction with the antigen (eg, an epitope), or a polypeptide containing such a portion. This antigen binding fragment typically contains one or more complementarity determining regions (CDRs) as well as one or more framework regions (FRs). CDRs are amino acid sequences that promote the specificity and affinity of binding of an antibody to an antigen, and framework regions are amino acid sequences that help maintain the proper conformation of these CDRs and promote binding of the antigen binding region to the antigen.

As used herein, an antigen-binding fragment of a chain (heavy chain or light chain) of an antibody or immunoglobulin includes a portion of the antibody that lacks certain amino acids compared to the full-length chain but can specifically bind to an antigen. This fragment may be considered to have biological activity in that it may specifically bind to a target antigen or may compete with other antibodies or antigen binding fragments for binding to a particular epitope. In one aspect, the fragment contains at least one CDR present in the full-length light chain or heavy chain, and in some embodiments it contains one chain of the heavy chain and/or light chain or a portion thereof. This biologically active fragment can be obtained by recombinant DNA techniques or can be obtained, for example, by enzymatic or chemical resolution of the intact antibody. An immunologically functional immunoglobulin fragment includes, but is not limited to, Fab, Fab', F(ab') ₂ , Fv, domain antibody, and single chain antibody (e.g., scFv, scFv-Fc, etc.) and may be of any mammalian origin, including, without limitation, human, mouse, rat, camelid or rabbit. A functional portion of an antibody, such as one or more than one CDR described herein, can be covalently linked to a secondary protein or small molecule compound and used as a therapeutic agent directed to a specific target.

As used herein, the Fab fragment consists of one light chain and one heavy chain containing only the variable region and CH1. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain.

As used herein, the Fc region contains two heavy chain fragments containing the CH2 and CH3 domains of the antibody. These 2 heavy chain fragments are linked to each other by two or more disulfide bonds and hydrophobic interaction of CH3 domains.

Herein, the Fab' fragment further contains, in addition to the Fab fragment, a region between the CH1 and CH2 domains of the heavy chain, and a disulfide bond can be formed between the two heavy chains of the two Fab' fragments.

- 45 045348

In the present description, the F(ab') ₂ fragment contains two light chains and two heavy chains containing a variable region, CH1 and part of the constant region between the CH1 and CH2 domains as described above, and an interchain disulfide bond is formed between the 2 heavy chains. Thus,

The F(ab') ₂ fragment consists of two Fab' fragments, and these two Fab' fragments are linked to each other by a disulfide bond formed between them.

As used herein, the Fv region is an antibody fragment containing a heavy chain variable region and a light chain, but not a constant region.

As used herein, a single chain antibody is a single polypeptide chain of an antigen binding region formed by connecting a heavy chain variable region and a light chain variable region through a flexible linker. For example, the single chain antibody may be at least one selected from the group consisting of scFv, wherein the heavy chain variable region and the light chain variable region are linked in single chain form, scFv-Fc, wherein the heavy chain variable region, the light chain variable region and Fc are linked in single chain form, and the like. A single chain antibody may refer, for example, to US Patent No. 5,260,203.

As used herein, a bivalent antigen binding protein or bivalent antibody contains two antigen binding sites. This bivalent antibody may contain two antigen-binding sites with specificity for the same antigen, or may be a dual-specific antibody that binds to different antigens, respectively. As used herein, a multispecific antigen binding protein or a multispecific antibody is directed at two or more than two antigens or epitopes.

As used herein, a bispecific antigen binding protein or antibody or antigen binding protein or a dual specificity antigen binding protein or antibody is a hybrid antigen binding protein or antibody having two different antigen binding sites. Such a bispecific antibody is a type of multispecific antigen binding protein or multispecific antibody and can be produced by various known methods such as hybridoma fusion or Fab' fragment fusion.

As used herein, a bispecific antigen binding protein or antibody, an antigen binding protein or a dual specific antibody is a hybrid antigen binding protein or antibody having 2 different antigen binding sites. This bispecific antibody is a type of multispecific antigen binding protein or multispecific antibody, and can be produced by various known methods, for example, methods such as hybridoma fusion or Fab' fragment binding. Possible references include, for example, Songsivilai and Lachmann, Clin. Exp. Immunol. 1990, 79:315-321, Kostelny et al., J. Immunol. 1992, 148:1547-1553 and the like. The two epitopes, different from each other, that bind two antigen binding sites present in a bispecific antigen binding protein or antibody may be located on the same target protein or on different target proteins. In one embodiment, the antibody of the present invention may be in the form of a bispecific antibody, further comprising binding to a carrier to deliver the antibody across the blood-brain barrier. One method of delivering drugs across the blood-brain barrier involves the use of delivery systems such as receptor-mediated transcytosis, for example, through the glucose transporter, amino acid transporter, insulin receptor or transferrin receptor in the cell.

As used herein, a conjugate refers to a chimeric molecule of an antibody or antigen-binding fragment thereof disclosed herein with another molecule, particularly a blood-brain barrier transporter or therapeutic agent described below. In a conjugate, an antibody or antigen-binding fragment thereof of the present invention is linked to other molecules by covalent bonding or physical forces such as van der Waals forces or hydrophobic interaction forces, encapsulation, confinement, or a combination of these methods. In the conjugate of one embodiment, the antibody or antigen binding fragment thereof of the present invention may be joined by a peptide linker.

The present invention also includes one or more amino acid sequences substantially identical to one or more amino acid sequences disclosed herein. Substantial sequence identity means that the modified sequence retains the effects disclosed in the present invention. In one embodiment, the sequence is approximately 90%, 95%, or 99% identical to the disclosed heavy chain variable region. In one embodiment, the sequence is approximately 90%, 95%, or 99% identical to the disclosed light chain variable region. For example, in the case of a variant exhibiting 90%, 95%, or 99% sequence identity to an antibody or antigen binding fragment disclosed herein, any sequence change occurs in the framework region of the variable region, but not in the CDR.

Antibodies or antigen-binding fragments thereof that specifically bind to alphasynuclein or aggregates thereof of the present invention contain a severe variable region

- 46 045348 chain containing the complementarity determining regions CDRH1, CDRH2 and CDRH3, and a light chain variable region containing the complementarity determining regions CDRL1, CDRL2 and

CDRL3.

In one embodiment, the anti-alpha-synuclein antibody or antigen-binding fragment thereof may comprise the following CDR sequences:

Heavy chain CDR1 (H-CDR1) selected from the amino acid sequences of SEQ ID NO:434 and SEQ ID NO:439,

Heavy chain CDR2 (H-CDR2) selected from the amino acid sequences of SEQ ID NOs:435-437 and 649 and SEQ ID NOs:440-441,

Heavy chain CDR3 (H-CDR3) selected from the amino acid sequences of SEQ ID NO:438 and SEQ ID NO:442,

Light chain CDR1 (L-CDR1) selected from the amino acid sequences of SEQ ID NO:443 and SEQ ID NO:446,

Light chain CDR2 (L-CDR2) selected from the amino acid sequences of SEQ ID NO:444 and SEQ ID NO:447, and

Light chain CDR3 (L-CDR3) selected from the amino acid sequences of SEQ ID NO:445 and SEQ ID NO:448.

The amino acid sequences of the heavy chain CDR1-CDR3 and the amino acid sequences of the light chain CDR1-CDR3 are set out in table. 8 and 9.

Table 8

Heavy chain amino acid sequences CDR1-CDR3

Identifier clone SEQ ID NO: VHCDR1 Amino acid follower- ness SEQ ID NO: VHCDR2 Amino acid subsequence SEQ ID NO: VHCDR3 Amino acid subsequence chllFll-VH 434 GFTFSDFYME 435 ASRNKANDYTTEYSASVKG 438 DAHGKPFAY HullFll-VHl 434 GFTFSDFYME 436 AIRNKANDYTTEYAASVKG 438 DAHGKPFAY HullFll-VH2 434 GFTFSDFYME 436 AIRNKANDYTTEYAASVKG 438 DAHGKPFAY HullFll-VH3 434 GFTFSDFYME 649 AIRNKANDYTTEYADSVKG 438 DAHGKPFAY HullFll-VH4 434 GFTFSDFYME 649 AIRNKANDYTTEYADSVKG 438 DAHGKPFAY HullFll-VHv3 434 GFTFSDFYME 437 ATRNKANDYTTEYSASVKG 438 DAHGKPFAY HullFll- VHvlmul newmu 434 GFTFSDFYME 437 ATRNKANDYTTEYSASVKG 438 DAHGKPFAY HullFll-VHv3 newmu 434 GFTFSDFYME 437 ATRNKANDYTTEYSASVKG 438 DAHGKPFAY HullFll-VH-vl 434 GFTFSDFYME 435 ASRNKANDYTTEYSASVKG 438 DAHGKPFAY HullFll-VH-v2 434 GFTFSDFYME 435 ASRNKANDYTTEYSASVKG 438 DAHGKPFAY HullFll-VH-v3 434 GFTFSDFYME 435 ASRNKANDYTTEYSASVKG 438 DAHGKPFAY HullFll-VH-v4 434 GFTFSDFYME 435 ASRNKANDYTTEYSASVKG 438 DAHGKPFAY ch3A9-VH 439 GFTFSSYAMS 440 TISNGGGYTYYPDSVKG 442 HITTVRPTKYFDY Hu3A9-VHl 439 GFTFSSYAMS 440 TISNGGGYTYYPDSVKG 442 HITTVRPTKYFDY Hu3A9-VH2 439 GFTFSSYAMS 441 TISNGGGYTYYADSVKG 442 HITTVRPTKYFDY Hu3A9-VH3 439 GFTFSSYAMS 440 TISNGGGYTYYPDSVKG 442 HITTVRPTKYFDY Hu3A9-VH4 439 GFTFSSYAMS 441 TISNGGGYTYYADSVKG 442 HITTVRPTKYFDY Hu3A9-VH-vl 439 GFTFSSYAMS 440 TISNGGGYTYYPDSVKG 442 HITTVRPTKYFDY Hu3A9-VH-v2 439 GFTFSSYAMS 440 TISNGGGYTYYPDSVKG 442 HITTVRPTKYFDY

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Table 9

Light chain amino acid sequences CDR1-CDR3

Identifier clone SEQ ID NO: VLCDR1 Amino acid subsequence SEQ ID NO: VLCDR1 Amino acid sequence SEQ ID NO: VLCDR1 Amino acid sequence chllFll-VL 443 KSSQSLLYSSNQKNYLA 444 WASTRES 445 QQYYSYPWT HullFll-VLl 443 KSSQSLLYSSNQKNYLA 444 WASTRES 445 QQYYSYPWT HullFll-VL2 443 KSSQSLLYSSNQKNYLA 444 WASTRES 445 QQYYSYPWT HullFll-VL3 443 KSSQSLLYSSNQKNYLA 444 WASTRES 445 QQYYSYPWT HullFll-VL4 443 KSSQSLLYSSNQKNYLA 444 WASTRES 445 QQYYSYPWT HullFll-VL5 443 KSSQSLLYSSNQKNYLA 444 WASTRES 445 QQYYSYPWT HullFll-VLv3 4c 443 KSSQSLLYSSNQKNYLA 444 WASTRES 445 QQYYSYPWT ch3A9-VL 446 KASQNVGTTVA 447 SASNRYT 448 QQYSNYPLT Hu3A9-VLl 446 KASQNVGTTVA 447 SASNRYT 448 QQYSNYPLT Hu3A9-VL2 446 KASQNVGTTVA 447 SASNRYT 448 QQYSNYPLT Hu3A9-VL3 446 KASQNVGTTVA 447 SASNRYT 448 QQYSNYPLT Hu3A9-VL4 446 KASQNVGTTVA 447 SASNRYT 448 QQYSNYPLT Hu3A9-VL-vl 446 KASQNVGTTVA 447 SASNRYT 448 QQYSNYPLT Hu3A9-VL-v2 446 KASQNVGTTVA 447 SASNRYT 448 QQYSNYPLT Hu3A9-VL-v2 446 KASQNVGTTVA 447 SASNRYT 448 QQYSNYPLT

In one embodiment, an anti-alpha-synuclein antibody or antigen binding fragment thereof may comprise a heavy chain variable region comprising a heavy chain CDR1 (H-CDR1) comprising the amino acid sequence of SEQ ID NO:434, a heavy chain CDR2 (H-CDR2) comprising the amino acid sequence selected from the amino acid sequences of SEQ ID NO:435-437 and 649, and a heavy chain CDR3 (H-CDR3) comprising the amino acid sequence of SEQ ID NO:438, and a heavy chain variable region containing a light chain CDR1 (L-CDR1), containing the amino acid sequence of SEQ ID NO:443, a light chain CDR2 (L-CDR2) containing the amino acid sequence of SEQ ID NO:444, and a light chain CDR3 (L-CDR3) containing the amino acid sequence of SEQ ID NO:445.

Additionally, in one embodiment, an anti-alpha-synuclein antibody or antigen-binding fragment thereof may comprise a heavy chain variable region comprising a heavy chain CDR1 (HCDR1) comprising the amino acid sequence of SEQ ID NO:439, a heavy chain CDR2 (HCDR2) comprising the amino acid sequence selected from the amino acid sequences of SEQ ID NO:440-441, and a heavy chain CDR3 (H-CDR3) containing the amino acid sequence of SEQ ID NO:442, and a heavy chain variable region containing a light chain CDR1 (L-CDR1) containing the amino acid sequence SEQ ID NO:446, light chain CDR2 (LCDR2) containing the amino acid sequence of SEQ ID NO:447, and light chain CDR3 (LCDR3) containing the amino acid sequence of SEQ ID NO:448.

In another embodiment, an anti-alpha-synuclein antibody or antigen binding fragment thereof may comprise a heavy chain framework region (H-FR1) located at the N-terminus of H-CDR1 comprising a polypeptide fragment comprising an amino acid sequence selected from SEQ ID NO:449-450 and SEQ ID NO:468-473, a heavy chain framework region (H-FR2) located between H-CDR1 and H-CDR2, containing a polypeptide fragment containing an amino acid sequence selected from SEQ ID NO:451-452 and SEQ ID NO :474-477, a heavy chain framework region (H-FR3) located between H-CDR2 and H-CDR3, containing a polypeptide fragment containing an amino acid sequence selected from SEQ ID NO:453-464 and SEQ ID NO:478-483 , a heavy chain framework region (H-FR4) located at the C-terminus of H-CDR3, containing a polypeptide fragment containing an amino acid sequence selected from SEQ ID NO:465467 and SEQ ID NO:484-485, a light chain framework region (L -FR1), located at the N-terminus of L-CDR1, containing a poly-48 045348 peptide fragment containing an amino acid sequence selected from SEQ ID NO:486491 and SEQ ID NO:504-510, light chain framework region (L-FR2) located between L-CDR1 and L-CDR2, containing a polypeptide fragment containing an amino acid sequence selected from SEQ ID

NO:492-494 and SEQ ID NO:511-514, a light chain framework region (L-FR3) located between L-CDR2 and L-CDR3, containing a polypeptide fragment containing an amino acid sequence selected from SEQ ID NO:495- 500 and SEQ ID NO:515-521, and/or a light chain framework region (L-FR4) located at the C-terminus of L-CDR3 containing a polypeptide fragment containing an amino acid sequence selected from SEQ ID NO:501503 and SEQ ID NO:522.

As amino acid sequences useful as framework regions herein, examples of heavy chain framework region sequences are given in Table. 3 and 4, and examples of light chain framework sequences are given in Table. 10 and 11.

Table 10

Amino acid sequences of framework regions 1-2 of the heavy chain

Clone SEQ ID NO: VH-FR1 sequence SEQ ID NO: Subsequence VH-FR2 chllFll-VH 449 EVQLQESGGGLVQPGGSLRLSCA T.S. 451 WVRQPPGKRLEWIA HullFll- VH1 471 EVQLVESGGGLVQPGGSLRLSCA AS 452 WVRQAPGKGLEWIA HullFll- VH2 471 EVQLVESGGGLVQPGGSLRLSCA AS 452 WVRQAPGKGLEWIA HullFll- VH3 471 EVQLVESGGGLVQPGGSLRLSCA AS 475 WVRQAPGKGLEWIA HullFll- VH4 471 EVQLVESGGGLVQPGGSLRLSCA AS 452 WVRQAPGKGLEWVA HullFll- VHv3 450 EVQLVESGGGLVQPGGSLRLSCA T.S. 451 WVRQPPGKRLEWIA HullFll- VHvlmul newmu 450 EVQLVESGGGLVQPGGSLRLSCA T.S. 451 WVRQPPGKRLEWIA

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HullFll- VHv3 newmu 450 EVQLVESGGGLVQPGGSLRLSCA T.S. 451 WVRQPPGKRLEWIA HullFll-VHvl 450 EVQLVESGGGLVQPGGSLRLSCA T.S. 451 WVRQPPGKRLEWIA HullFll-VH- v2 450 EVQLVESGGGLVQPGGSLRLSCA T.S. 451 WVRQPPGKRLEWIA HullFll-VH- v3 450 EVQLVESGGGLVQPGGSLRLSCA T.S. 451 WVRQPPGKRLEWIA HullFll-VH- v4 450 EVQLVESGGGLVQPGGSLRLSCA T.S. 451 WVRQPPGKRLEWIA ch3A9-VH 468 EVQLQESGGGLVKPGGSLKLSCA AS 474 WVRQTPEKRLEWVA Hu3A9-VHl 469 QVQLLESGGGLVQPGGSLRLSCA AS 475 WVRQAPGKGLEWVA Hu3A9-VH2 470 EVQLVQSGGGLVQPGGSLRLSCA AS 476 WVRQAPDKGLEWVA Hu3A9-VH3 471 EVQLVESGGGLVQPGGSLRLSCA AS 475 WVRQAPGKGLEWVA Hu3A9-VH4 471 EVQLVESGGGLVQPGGSLRLSCA AS 475 WVRQAPGKGLEWVA Hu3A9-VHvl 472 EVQLLESGGGLVQPGGSLRLSCA AS 477 WVRQTPEKGLEWVA Hu3A9-VH- v2 473 EVQLLESGGGLVQPGGSLRLSCA AS 477 WVRQTPEKGLEWVA

- 50 045348

Table 11

Amino acid sequences of framework regions 3-4 of the heavy chain

Clone SEQ ID NO: VH-FR3 sequence SEQ ID NO: Subsequence VH-FR4 chllFll-VH 453 RFIVSRDTSQSILYLQMNALRAED TAIYYCAR 465 WGQGTLVTVSA HullFll- VH1 454 RFTVSRDTSKNSLYLQMNSLKTE DTAVYYCAR 466 WGQGTLVTVSS HullFll- VH2 455 RFTISRDTSKNSLYLQMNSLKTED TAVYYCAR 466 WGQGTLVTVSS HullFll- VH3 456 RFTVSRDTSQNTLYLQMNSLRAE DTAVYYCAR 466 WGQGTLVTVSS HullFll- VH4 457 RFTISADTSKNTAYLQMNSLRAE DTAVYYCSR 466 WGQGTLVTVSS HullFll- VHv3 458 RFTISRDDSKSSLYLQMNSLRAED TAIYYCAR 467 WGQGTTVTVSS HullFll- VHvlmul newmu 459 RFTISRDTSQSSLYLQMNSLKTED TAVYYCAR 467 WGQGTTVTVSS HullFll- VHv3 newmu 460 RFTISRDTSQSSLYLQMNSLRAED TAIYYCAR 467 WGQGTTVTVSS HullFll-VHvl 461 RFT IS RD D SKS SL YLQMNSLRAED TAIYYCAR 467 WGQGTTVTVSS HullFll-VH- v2 462 RFTVSRDDSKSSLYLQMNSLRAE DTAIYYCAR 467 WGQGTTVTVSS HullFll-VH- v3 463 RFTISRDTSKSSLYLQMNSLRAED TAIYYCAR 467 WGQGTTVTVSS HullFll-VH- v4 464 RFTVSRDTSKSSLYLQMNSLRAE DTAIYYCAR 467 WGQGTTVTVSS ch3A9-VH 478 RFTISRDNAKNTLYLQMS SLRSED TAMYYCAR 484 WGQGTTLTVSS Hu3A9-VHl 479 RFTISRDNAKNTLYLQMNSLRSE DSAMYYCAR 485 WGQGTLVTVSS Hu3A9-VH2 480 RFTISRDNAKNTLYLQMS SLKAE DSAVYYCAR 485 WGQGTLVTVSS Hu3A9-VH3 481 RFTISRDNSKNTLYLQMNSLRAE DTAVYYCAR 485 WGQGTLVTVSS

- 51 045348

Hu3A9-VH4 482 RFTISADTSKNTAYLQMNSLRAE DTAVYYCAR 485 WGQGTLVTVSS Hu3A9-VH- vl 483 RFTISRDNSKNTLYLQMS SLRAED TAMYYCAR 484 WGQGTTLTVSS Hu3A9-VH- v2 483 RFTISRDNSKNTLYLQMS SLRAED TAMYYCAR 484 WGQGTTLTVSS

Table 12

Amino acid sequences of light chain framework regions 1-2

Clone SEQ ID NO: VL-FR1 sequence SEQ ID NO: Subsequence VL-FR2 chllFll-VL 486 DIVMTQSPSSLAVSVGEKVTMSC 492 WYQQKPGQSPKLLIY HullFll-VLl 487 DIVMTQSPDSLAVSLGERATINC 493 WYQQKPGQPPKLLIY HullFll-VL2 488 DIVMTQSPSSLAVSLGERVTITC 493 WYQQKPGQPPKLLIY HullFll-VL3 489 DIVMTQSPSSLAVSLGERATINC 493 WYQQKPGQPPKLLIY HullFll-VL4 490 DIQMTQSPSSLSSASVGDRVTITTC 494 WYQQKPGKAPKLLIY HullFll-VL5 486 DIVMTQSPSSLAVSVGEKVTMSC 493 WYQQKPGQPPKLLIY HullFll- VLv3 4c 491 DIVMTQSPSSLAVSLGERVTMSC 492 W YQQKPGQ SPKLLIY ch3A9-VL 504 DIVMTQSPKFMSTSVGDRVSITC 511 WYQQKPGQSPKLLIY Hu3A9-VLl 505 DIQMTQSPSSLSSASVGDRVTITTC 512 WYQQKPGKAPKLLIY Hu3A9-VL2 506 DIVMTQSPSTLSASVGDRVTITC 513 AWYQQKPGKAPKLLI Y Hu3A9-VL3 507 DIVMTQSPATLSVSLGERATLSC 514 W YQQKPGQ APRLLIY Hu3A9-VL4 508 DIQMTQSPSSLSSASVGDRVTITTC 512 WYQQKPGKAPKLLIY Hu3A9-VL-vl 509 DIQMTQSPSSLSSASVGDRVTITTC 511 WYQQKPGQSPKLLIY Hu3A9-VL-v2 510 DIVMTQSPSSMSTSVGDRVTITTC 511 WYQQKPGQSPKLLIY Hu3A9-VL-v2 510 DIVMTQSPSSMSTSVGDRVTITTC 511 WYQQKPGQSPKLLIY

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Table 13

Amino acid sequences of light chain framework regions 3-4

Clone SEQ ID NO: VL-FR3 sequence SEQ ID NO: Subsequence VL-FR4 chllFll-VL 495 GVPDRFTGSGSGTDFTLTISSVKA EDLAVYYC 501 FGGGTKLEIK HullFll-VLl 496 GVPDRFSGSGSGTDFTLTISSLQA EDVAVYYC 502 FGGGTKVEIK HullFll-VL2 497 GVPDRFSGSGSGTDFTLTISSVQA EDVAVYYC 501 FGGGTKLEIK HullFll-VL3 496 GVPDRFSGSGSGTDFTLTISSLQA EDVAVYYC 501 FGGGTKLEIK HullFll-VL4 498 GVPSRF SSGSGSGTDFTLTIS SLQPE DFATYYC 503 FGQGTKVEIK HullFll-VL5 499 GVPDRFSGSGSGTDFTLTISSVQA EDLAVYYC 501 FGGGTKLEIK HullFll- VLv3 4c 500 GVPDRFTGSGSGTDFTLTISSVKA EDVAVYYC 501 FGGGTKLEIK ch3A9-VL 515 GVPDRFTGSGSGTDFTLTISNMQS EDLADYFC 522 FGAGTKLELR Hu3A9-VLl 516 GVPDRF SSGSGSGTDFTLTIS SLQPE DFATYYC 522 FGAGTKLELR Hu3A9-VL2 517 GVPSRFSGSGSGTEFTLTISSLQPD DFASYYC 522 FGAGTKLELR Hu3A9-VL3 518 GIPARFSGSGSGTEFTLTISSLQSE DFAVYYC 522 FGAGTKLELR Hu3A9-VL4 519 GVPSRF SSGSGSGTDFTLTIS SLQPE DFATYYC 522 FGAGTKLELR Hu3A9-VL-vl 520 GVPDRFSGSGSGTDFTFTISSMQS EDIATYFC 522 FGAGTKLELR Hu3A9-VL-v2 521 GVPDRFSGSGSGTDFTLTISSMQS EDLADYYC 522 FGAGTKLELR Hu3A9-VL-v2 521 GVPDRFSGSGSGTDFTLTISSMQS EDLADYYC 522 FGAGTKLELR

In another embodiment, an anti-alpha-synuclein antibody or antigen binding fragment thereof may comprise a heavy chain variable region comprising a heavy chain CDR1 (H-CDR1) comprising an amino acid selected from SEQ ID NO:434 and SEQ ID NO:439, a heavy chain CDR2 ( HCDR2) containing an amino acid selected from SEQ ID NO:435-437 and 649 and SEQ ID NO:440-441, and a heavy chain CDR3 (H-CDR3) containing an amino acid selected from SEQ ID NO:438 and SEQ ID NO :442, wherein the heavy chain variable region further comprises a heavy chain framework region (H-FR1) comprising an amino acid sequence selected from SEQ ID NOs:449-450 and SEQ ID NOs:468-473, a heavy chain framework region (H-FR2 ), containing an amino acid sequence selected from SEQ ID NO:451-452 and SEQ ID NO:474-477, a heavy chain framework region (H-FR3) containing an amino acid sequence selected from SEQ ID NO:453-464 and SEQ

- 53 045348

ID NO:478-483, and a heavy chain framework region (H-FR4) containing an amino acid sequence selected from SEQ ID NO:465-467 and SEQ ID NO:484-485. More specifically, the heavy chain variable region comprises a heavy chain framework region (H-FR1) comprising the amino acid sequence of SEQ ID NO:450, a heavy chain framework region (H-FR2) comprising the amino acid sequence of SEQ ID NO:451, a heavy chain framework region (H-FR3) containing the amino acid sequence selected from SEQ ID NO:462-464, and a heavy chain framework region (H-FR4) containing the amino acid sequence of SEQ ID NO:467.

In addition, the anti-alpha-synuclein antibody or antigen binding fragment thereof may comprise a light chain variable region containing light chain CDR1 (L-CDR1) comprising an amino acid sequence selected from SEQ ID NO:443 and SEQ ID NO:446, light chain CDR2 (L-CDR2) comprising an amino acid sequence selected from SEQ ID NO:444 and SEQ ID NO:447, and a light chain CDR3 (L-CDR3) comprising an amino acid sequence selected from SEQ ID NO:445 and SEQ ID NO: 448, wherein the light chain variable region further comprises a light chain framework region (LFR1) comprising an amino acid sequence selected from SEQ ID NO:486-491 and SEQ ID NO:504-510, a light chain framework region (L-FR2) comprising an amino acid sequence selected from SEQ ID NO:492-494 and SEQ ID NO:511-514, a light chain framework region (L-FR3) comprising an amino acid sequence selected from SEQ ID NO:495-500 and SEQ ID NO:515521 , and a light chain framework region (L-FR4) containing an amino acid sequence selected from SEQ ID NO:501-503 and SEQ ID NO:522. More specifically, the light chain variable region comprises a light chain framework region (L-FR1) comprising a polypeptide fragment comprising the amino acid sequence of SEQ ID NO:491, a light chain framework region (L-FR2) comprising a polypeptide fragment comprising the amino acid sequence of SEQ ID NO:492, a light chain framework region (L-FR3) containing a polypeptide fragment containing the amino acid sequence of SEQ ID NO:500, and a light chain framework region (L-FR4) containing a polypeptide fragment containing the amino acid sequence of SEQ ID NO:501 .

An antibody or antigen binding fragment thereof according to one embodiment of the present invention may comprise a heavy chain variable region comprising a heavy chain CDR1 (HCDR1) comprising the amino acid sequence of SEQ ID NO:434, a heavy chain CDR2 (HCDR2) comprising an amino acid sequence selected from SEQ ID NO: :435-437 and 649, and a heavy chain CDR3 (H-CDR3) containing the amino acid sequence of SEQ ID NO:438, and a light chain variable region containing a light chain CDR1 (L-CDR1) containing the amino acid sequence of SEQ ID NO:443 , light chain CDR2 (L-CDR2) comprising the amino acid sequence of SEQ ID NO:444, and light chain CDR3 (L-CDR3) comprising the amino acid sequence of SEQ ID NO:445, wherein the heavy chain variable region comprises a heavy chain framework region (H -FR1) containing an amino acid sequence selected from SEQ ID NOs:449,450 and 471, heavy chain framework region (H-FR2), containing an amino acid sequence selected from SEQ ID NOs:451, 452 and 475, heavy chain framework region (H -FR3) containing an amino acid sequence selected from SEQ ID NO:453-464, and a heavy chain framework region (H-FR4) containing an amino acid sequence selected from SEQ ID NO:465-467, and wherein the light chain variable region contains a light chain framework region (L-FR1) containing an amino acid sequence selected from SEQ ID NO:486-491, a light chain framework region (L-FR2) containing an amino acid sequence selected from SEQ ID NO:492494, a light chain framework region (L-FR3) containing an amino acid sequence selected from SEQ ID NO:495-500, and a light chain framework region (L-FR4) containing an amino acid sequence selected from SEQ ID NO:501-503.

Preferably, the antibody or antigen binding fragment thereof according to one embodiment of the present invention comprises a heavy chain variable region comprising heavy chain CDR1 to CDR3 comprising the amino acid sequences of SEQ ID NOs: 434, 435 and 438, and a light chain variable region comprising light chain CDR1 to CDR3 containing the amino acid sequences of SEQ ID NO:443, 444 and 445, wherein the heavy chain variable region comprises heavy chain framework region 1 (H-FR1), containing the amino acid sequence of SEQ ID NO:450, heavy chain framework region (H-FR2), comprising the amino acid sequence of SEQ ID NO:451, a heavy chain framework region (H-FR3) comprising an amino acid sequence selected from SEQ ID NOs:462-464, and a heavy chain framework region (H-FR4) comprising the amino acid sequence of SEQ ID NO: :467, and the light chain variable region comprises light chain framework region 1 (L-FR1) containing the amino acid sequence of SEQ ID NO:491, light chain framework region (L-FR2) containing the amino acid sequence of SEQ ID NO:492, framework region a light chain (L-FR3) containing the amino acid sequence of SEQ ID NO:500, and a light chain framework region (L-FR4) containing the amino acid sequence of SEQ ID NO:501.

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An anti-alpha-synuclein antibody or antigen binding fragment thereof according to one embodiment of the present invention comprises a heavy chain variable region comprising a heavy chain CDR1 (H-CDR1) comprising the amino acid sequence of SEQ ID NO:439, a heavy chain CDR2 (H-CDR2) comprising the amino acid a sequence selected from SEQ ID NO:440-441, and a heavy chain CDR3 (H-CDR3) containing the amino acid sequence of SEQ ID NO:442, and a light chain variable region containing a light chain CDR1 (L-CDR1) containing the amino acid sequence SEQ ID NO:446, light chain CDR2 (L-CDR2) containing the amino acid sequence of SEQ ID NO:447, and light chain CDR3 (L-CDR3) containing the amino acid sequence of SEQ ID NO:448, wherein the heavy chain variable region contains a framework heavy chain region 1 (H-FR1) containing an amino acid sequence selected from SEQ ID NO:463-473, heavy chain framework region (H-FR2) containing an amino acid sequence selected from SEQ ID NO:474-477, framework region a heavy chain (H-FR3) containing an amino acid sequence selected from SEQ ID NO:478-483, and a heavy chain framework region (H-FR4) containing an amino acid sequence selected from SEQ ID NO:484-485, and wherein variable light chain region contains light chain framework region 1 (L-FR1) containing an amino acid sequence selected from SEQ ID NO:504-510, light chain framework region (L-FR2) containing an amino acid sequence selected from SEQ ID NO:511514 , a light chain framework region (L-FR3) containing the amino acid sequence selected from SEQ ID NO:515-521, and a light chain framework region (L-FR4) containing the amino acid sequence of SEQ ID NO:522.

In the full-length light chain and heavy chain forms, the variable region and constant region are connected by a J region of approximately 12 or more amino acids in length, and the heavy chain also contains a D region of approximately 10 or more amino acids in length. Possible references include, for example, Fundamental Immunology, 2nd ed., Ch. 7 (Paul, W., ed.) 1989, New York: Raven Press. Typically, a pair of light chain and heavy chain variable regions of an antibody may form an antigen binding region.

The variable region of an immunoglobulin chain typically has the same general structure and contains relatively conserved framework regions (FRs) connected by three hypervariable regions called complementarity determining regions or domains, or CDRs. The CDRs of the variable regions of each chain forming a heavy chain/light chain pair are typically aligned with framework regions to form a structure that specifically binds to a specific epitope of the target protein (alpha-synuclein). These naturally occurring light chain variable region and heavy chain variable region elements are generally included in the following order from N-terminus to C-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The position of the amino acid sequences corresponding to each of these elements in the variable region is designated according to the Kabat (Kabat Sequences of Proteins of Immunological Interest (1987 and 1991 editions, NIH, Bethesda, MD)) or Chothia numbering system & Lesk, 1987, J. Mol. Biol. 196: 901-917; Chothia et al., 1989, Nature 342: 878-883.

The various heavy chain variable regions and light chain variable regions disclosed herein are shown in Table. 14 and 15. Each of these variable regions can be coupled to a heavy chain constant region and a light chain constant region to form the heavy chain and light chain of an intact antibody. In addition, the resulting heavy chain sequence and light chain sequence can also be combined to form a full-length antibody structure. For example, an anti-alpha-synuclein antibody or antigen binding fragment thereof of the present invention may comprise a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NO:523-534 or SEQ ID NO:535-541 and a light chain variable region comprising an amino acid sequence selected from SEQ ID NO:542-548 or SEQ ID NO:549-556, and more specifically, a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NO:523-534 and a light chain variable region, comprising an amino acid sequence selected from SEQ ID NO:542-548, or a heavy chain variable region containing an amino acid sequence selected from SEQ ID NO:535-541, and a light chain variable region containing an amino acid sequence selected from SEQ ID NO: 549-556.

In one specific embodiment of the present invention, an anti-alpha-synuclein antibody or antigen binding fragment thereof may comprise a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NO:531-534 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:548 (for example, Hu11F11 (ver.1), Hu11F11 (ver.2), Hu11F11 (ver.3), Hu11F11 (ver.4) and so on), or may contain a heavy chain variable region containing the amino acid sequence SEQ ID NO: 525, and a light chain variable region containing the amino acid sequence of SEQ ID NO:546 (eg, antibody Hu11F11 (ABL2-4)).

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Examples of the amino acid sequences of the heavy chain variable region and the light chain variable region of an antibody or antigen binding fragment according to one embodiment are shown in Table. 14 and 15.

Table 14

Heavy chain variable region (VH)

Clone SEQ ID NO: Amino acid sequence chllFll-VH 523 EVQLQESGGGLVQPGGSLRLSCATSGFTFSDFYMEWVRQPPG KRLEWIAASRNKANDYTTEYSASVKGRFIVSRDTSQSILYLQM NALRAEDTAIYYCARDAHGKPFAYWGQGTLVTVSA HullFll-VHl 524 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDFYMEWVRQAPG KGLEWIAAIRNKANDYTTEYAASVKGRFTVSRDTSKNSLYLQ MNSLKTEDTAVYYCARDAHGKPFAYWGQGTL VTVSS HullFll-VH2 525 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDFYMEWVRQAPG KGLEWIAAIRNKANDYTTEYAASVKGRFTISRDTSKNSLYLQM NSLKTEDTAVYYC ARD AHGKPFAYWGQGTL VTVSS

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HullFll-VH3 526 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDFYMEWVRQAPG KGLEWIAAIRNKANDYTTEYADSVKGRFTVSRDTSQNTLYLQ MNSLRAEDTAVYYCARD AHGKPF AYWGQGTL VTVSS HullFll-VH4 527 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDFYMEWVRQAPG KGLEWVAAIRNKANDYTTEYADSVKGRFTISADTSKNTAYLQ MNSLRAEDTAVYYC SRD AHGKPF AYWGQGTL VTVS S HullFll-VHv3 528 EVQLVESGGGLVQPGGSLRLSCATSGFTFSDFYMEWVRQPPG KRLEWIAATRNKANDYTTEYSASVKGRFTISRDDSKSSLYLQM NSLRAEDT AIYYC ARD AHGKPF AYWGQGTT VTVS S HullFll- VHvlmul newmu 529 EVQLVESGGGLVQPGGSLRLSCATSGFTFSDFYMEWVRQPPG KRLEWIAATRNKANDYTTEYSASVKGRFTISRDTSQSSLYLQM NSLKTEDTAVYYCARD AHGKPF AYWGQGTTVTVSS HullFll-VHv3 newmu 530 EVQLVESGGGLVQPGGSLRLSCATSGFTFSDFYMEWVRQPPG KRLEWIAATRNKANDYTTEYSASVKGRFTISRDTSQSSLYLQM NSLRAEDT AIYYC ARD AHGKPF AYWGQGTT VTVS S HullFll-VH- vl 531 EVQLVESGGGLVQPGGSLRLSCATSGFTFSDFYMEWVRQPPG KRLEWIAASRNKANDYTTEYSASVKGRFTISRDDSKSSLYLQM NSLRAEDT AIYYC ARD AHGKPF AYWGQGTT VTVS S HullFll-VH- v2 532 EVQLVESGGGLVQPGGSLRLSCATSGFTFSDFYMEWVRQPPG KRLEWIAASRNKANDYTTEYSASVKGRFTVSRDDSKSSLYLQ MNSLRAEDT AI YYC ARD AHGKPF AYWGQGTT VTVS S HullFll-VH- v3 533 EVQLVESGGGLVQPGGSLRLSCATSGFTFSDFYMEWVRQPPG KRLEWIAASRNKANDYTTEYSASVKGRFTISRDTSKSSLYLQM NSLRAEDT AIYYC ARD AHGKPF AYWGQGTT VTVS S HullFll-VH- v4 534 EVQLVESGGGLVQPGGSLRLSCATSGFTFSDFYMEWVRQPPG KRLEWIAASRNKANDYTTEYSASVKGRFTVSRDTSKSSLYLQ MNSLRAEDT AI YYC ARD AHGKPF AYWGQGTT VTVS S ch3A9-VH 535 EVQLQESGGGLVKPGGSLKLSCAASGFFSYAMSWVRQTPE KRLEWVATISNGGGYTYYPDSVKGRFTISRDNAKNTLYLQMS SLRSEDTAMYYCARHITTVRPTKYFDYWGQGTTLTVSS

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Hu3A9-VHl 536 QVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPG KGLEWVATISNGGGYTYYPDSVKGRFTISRDNAKNTLYLQMN SLRSED SAM YYC ARHITTVRPTKYFD YWGQGTL VT VS S Hu3A9-VH2 537 EVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPD KGLEWVATISNGGGYTYYADSVKGRFTISRDNAKNTLYLQMS SLKAED S AVY YC ARHITTVRPTKYFD YWGQGTL VT VS S Hu3A9-VH3 538 EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPG KGLEWVATISNGGGYTYYPDSVKGRFTISRDNSKNTLYLQMN SLRAEDT AVY YC ARHITTVRPTKYFD YWGQGTL VTVSS Hu3A9-VH4 539 EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPG KGLEWVATISNGGGYTYYADSVKGRFTISADTSKNTAYLQMN SLRAEDT AVY YC ARHITTVRPTKYFD YWGQGTL VT VS S Hu3A9-VH-vl 540 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQTPEK GLEWVATISNGGGYTYYPDSVKGRFTISRDNSKNTLYLQMSSL RAEDT AM YYC ARHITTVRPTKYFD YWGQGTTLT VS S Hu3A9-VH-v2 541 EVQLLESGGGLVQPGGSLKLSCAASGFFSSYAMSWVRQTPEK GLEWVATISNGGGYTYYPDSVKGRFTISRDNSKNTLYLQMSSL RAEDT AM YYC ARHITTVRPTKYFD YWGQGTTLT VS S ch9Bll-VH 557 EVQLQESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPG KGLEWVARIRSKSNNYATYYADSVKDRFTISRDDSQSMLYLQ MNNLKTEDT AMYYC VRQDFD YWGQGTTLT VS S

Table 15

Light chain variable region (VL)

Clone SEQ ID NO: Amino acid sequence chllFll-VL 542 DIVMTQSPSSLAVSVGEKVTMSCKSSQSLLYSSNQKNYLAWY QQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKA EDLAVYYCQQYYSYPWTFGGGTKLEIK HullFll-VLl 543 DIVMTQSPDSLAVSLGERATINCKSSQSLLYSSNQKNYLAWYQ QKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAE

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DVAVYYCQQYYSYPWTFGGGTKVEIK HullFll-VL2 544 DIVMTQSPSSLAVSLGERVTITCKSSQSLLYSSNQKNYLAWYQ QKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSVQAE DVAVYYCQQYYSYPWTFGGGTKLEIK HullFll-VL3 545 DIVMTQSPSSLAVSLGERATINCKSSQSLLYSSNQKNYLAWYQ QKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAE DVAVYYCQQYYSYPWTFGGGTKLEIK HullFll-VL4 546 DIQMTQSPSSLSSASVGDRVTITCKSSQSLLYSSNQKNYLAWYQ QKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPED FATYYCQQYYSYPWTFGQGTKVEIK HullFll-VL5 547 DIVMTQSPSSLAVSVGEKVTMSCKSSQSLLYSSNQKNYLAWY QQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSVQA EDLAVYYCQQYYSYPWTFGGGTKLEIK HullFll-VLv3 4c 548 DIVMTQSPSSLAVSLGERVTMSCKSSQSLLYSSNQKNYLAWYQ QKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAE DVAVYYCQQYYSYPWTFGGGTKLEIK ch3A9-VL 549 DIVMTQSPKFMSTSVGDRVSITCKASQNVGTTVAWYQQKPGQ SPKLLIYSASNRYTGVPDRFTGSGSGTDFTLTISNMQSEDLADY FCQQYSNYPLTFGAGTKLELR Hu3A9-VLl 550 DIQMTQSPSSLSASVGDRVTITCKASQNVGTTVAWYQQKPGK APKLLIYSASNRYTGVPDRFSGSGSGTDFTLTISSLQPEDFATYY CQQYSNYPLTFGGGTKLEIK Hu3A9-VL2 551 DIVMTQSPSTLSASVGDRVTITCKASQNVGTTVAWYQQKPGK APKLLIYSASNRYTGVPSRFSGSGSGTEFTLTISSLQPDDFASYY CQQYSNYPLTFGQGTKVEIK Hu3A9-VL3 552 DIVMTQSPATLSVSLGERATLLSCKASQNVGTTVAWYQQKPGQ APRLLIYSASNRYTGIPARFSGSGSGTEFTLTISSLQSEDFAVYY CQQYSNYPLTFGGGTKVEIK Hu3A9-VL4 553 DIQMTQSPSSLSA L- SVGDRVTITCKASQNVGTTVAWYQQKPG KAPKLLIYSASNRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCQQYSNYPLTFGQGTKVEIK

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Hu3A9-VL-vl 554 DIQMTQSPSSLSASVGDRVTITCKASQNVGTTVAWYQQKPGQS PKLLIYSASNRYTGVPDRFSGSGSGTDFTFTISSMQSEDIATYFC QQYSNYPLTFGQGTKLEIK Hu3A9-VL-v2 555 DIVMTQSPSSMSTSVGDRVTITCKASQNVGTTVAWYQQKPGQ SPKLLIYSASNRYTGVPDRFSGSGSGTDFTLTISSMQSEDIADYY CQQYSNYPLTFGQGTKLEIK Hu3A9-VL-v2 556 DIVMTQSPSSMSTSVGDRVTITCKASQNVGTTVAWYQQKPGQ SPKLLIYSASNRYTGVPDRFSGSGSGTDFTLTISSMQSEDLADY YCQQYSNYPLTFGQGTKLEIK ch9Bll-VL 558 DIVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQ KPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAED LGVYFCSQSTHVPLTFGAGTKLEQKR

In addition, antibodies containing a combination of a heavy chain variable region with a light chain variable region and a constant region of an antibody or antigen binding fragment according to one embodiment are described in table. 16.

Table 16

Examples of antibodies according to the invention

Sample name Chain Amino acid sequence hullFll (ver.l) Heavy (SEQ ID NO:560) EVQLVESGGGLVQPGGSLRLSCATSGFTFSDFYMEWVRQPPG KRLEWIAASRNKANDYTTEYSASVKGRFTISRDDSKSSLYLQ MNSLRAEDTAIYYCARDAHGKPFAYWGQGTTVTVSSASTKG PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSN TKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS DIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVF SC SVMHEALHNHYTQKSLSLSPGK

-60045348

Light (SEQ ID NO:566) DIVMTQSPS SLAVSLGERVTMSCKS SQSLL YS SNQKNYL AWY QQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVK AEDVAVYYCQQYYSYPWTFGGGTKLEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES VTEQDSKDSTYSLS STLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC hullFll (ver. 2) Heavy (SEQ ID NO:561) EVQLVESGGGLVQPGGSLRLSCATSGFTFSDFYMEWVRQPPG KRLEWIAASRNKANDYTTEYSASVKGRFTVSRDDSKSSLYLQ MNSLRAEDTAIYYCARDAHGKPFAYWGQGTTVTVSSASTKG PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICNVNHKPSN TKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS DI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVF SC SVMHEALHNHYTQKSLSLSPGK Light (SEQ ID NO:566) DIVMTQSPS SLAVSLGERVTMSCKS SQSLL YS SNQKNYL AWY QQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVK AEDVAVYYCQQYYSYPWTFGGGTKLEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES VTEQDSKDSTYSLS STLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC

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hullFll (ver.3) Heavy (SEQ ID NO:562) EVQLVESGGGLVQPGGSLRLSCATSGFTFSDFYMEWVRQPPG KRLEWIAASRNKANDYTTEYSASVKGRFTISRDTSKSSLYLQ MNSLRAEDTAIYYCARDAHGKPFAYWGQGTTVTVSSASTKG PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSN TKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS DIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVF SC SVMHEALHNHYTQKSLSLSPGK Light (SEQ ID NO:566) DIVMTQSPSSLAVSLGERVTMSCKSSQSLLYSSNQKNYLAWY QQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVK AEDVAVYYCQQYYSYPWTFGGGTKLEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES VTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSP VTKSFNRGEC hullFll (ver. 4) Heavy (SEQ ID NO:563) EVQLVESGGGLVQPGGSLRLSCATSGFTFSDFYMEWVRQPPG KRLEWIAASRNKANDYTTEYSASVKGRFTVSRDTSKSSLYLQ MNSLRAEDTAIYYCARDAHGKPFAYWGQGTTVTVSSASTKG PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICNVNHKPSN TKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS DI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVF SC SVMHEALHNHYTQKSLSLSPGK

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Light (SEQ ID NO:566) DIVMTQSPS SLAVSLGERVTMSCKS SQSLL YS SNQKNYL AWY QQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVK AEDVAVYYCQQYYSYPWTFGGGTKLEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES VTEQDSKDSTYSLS STLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC hullFll (H2L4) Heavy (SEQ ID NO:564) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDFYMEWVRQAP GKGLEWIAAIRNKANDYTTEYAASVKGRFTISRDTSKNSLYL QMNSLKTEDTAVYYCARDAHGKPFAYWGQGTLVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HK PSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Light (SEQ ID NO:567) DIQMTQSPSSLSASVGDRVTITCKSSQSLLYSSNQKNYLAWY QQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQP EDFATYYCQQYYSYPWTFGQGTKVEIKRTVAAPSVFIFPPSDE QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT EQDSKDSTYSLSSTLTLS KADYEKHKVYACEVTHQGLSSPVT KSFNRGEC hu3A9 (VH5/L3) Heavy (SEQ ID NO:565) EVQLQESGGGLVKPGGSLKLSCAASGFFSSYAMSWVRQTPE KGLEWVATISNGGGYTYYPDSVKGRFTISRDNAKNTLYLQM NSLRSEDTAVYYCARHITTVRPTKYFDYWGQGTLVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TQTYICNVNHK PSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY PS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Light (SEQ ID NO:568) DIVMTQSPATLSVSLGERATLSCKASQNVGTTVAWYQQKPG QAPRLLIYSASNRYTGIPARFSGSGSGTEFTLTISSLQSEDFAV YYCQQYSNYPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSG TASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK DSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVTKSFNR GEC

In order for the heavy chain variable region and the light chain variable region described herein to form the heavy chain and light chain of an intact antibody, each heavy chain variable region and light chain variable region may be linked to a heavy chain constant region and a light chain constant region. Each of the heavy chain and the light chain obtained as

- 63 045348 described above can be suitably combined to form a heavy chain-light chain combination, and the resulting heavy chain-light chain combination can form a multimer (eg, a dimer in the case of an IgG antibody), resulting in the structure of an intact antibody.

The constant region may be suitably selected from constant regions of heavy and light chains of immunoglobulins (eg, human immunoglobulins). For example, without limitation, 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 constant region. Other types of constant regions or modified constant regions that will be apparent to those skilled in the art may be appropriately selected and used for antibody stability, ease of preparation, affinity for antigen, and/or other desired characteristics.

In another embodiment, each of the heavy chain variable regions and light chain variable regions disclosed in Table. 14 and 15 can be freely combined with each other to form various antibodies and linked in single-chain form to produce single-chain antibodies such as scFv.

An antibody described herein may share a certain region or sequence with another antibody disclosed herein. In one embodiment, an antibody may share a constant region with another antibody or an antigen-binding fragment thereof. In another embodiment, it may have a common Fc region.

In one embodiment, the anti-alpha-synuclein antibody of the present invention also includes a monoclonal antibody and a polyclonal antibody. In another embodiment, the anti-alpha-synuclein antibody may be a human antibody, a humanized antibody, a chimeric antibody, or an antibody of animal origin. In another embodiment, an anti-alpha-synuclein antibody can be produced by recombinant methods or chemical synthesis.

The antigen binding fragments or antibody fragments described herein may be at least one selected from the group consisting of single chain antibody molecules scFv, (scFv)2, scFv-Fc, Fab, Fab', F(ab')2, minibodies , diabodies, scFv and the like.

In another embodiment, the antibody provided herein may be a humanized antibody or a human antibody and may be of various isotypes (e.g., IgA, IgG1, IgG2, IgG3, IgG4, IgE, or IgD), especially the IgG1, IgG2, IgG3, or IgG4 types. , such as IgG1 or IgG2 types.

In another embodiment, the anti-alpha-synuclein antibody may comprise only the heavy chain or light chain described above. In another embodiment, an anti-alpha-synuclein antibody has only a heavy chain variable region or only a light chain variable region.

Those skilled in the art will appreciate that when an antibody contains one or more than one CDRs disclosed herein, each of the disclosed CDRs can be selected and combined independently of the others. In this way, antibodies containing 1, 2, 3, 4, 5 or 6 independently selected CDRs can be produced. Those skilled in the art will also appreciate that, when selecting CDRs for combination, CDRs of the same type are not reused and antibodies containing, for example, two CDR-H2 regions are not typically produced.

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

In other embodiments, the antibody may be an animal antibody (eg, mouse antibody, etc.), a chimeric antibody (eg, mouse-human chimeric antibody), a humanized antibody, or a human antibody. The antibody may also be modified in various ways for various purposes. Chimeric antibodies and humanized antibodies are also proposed. Chimeric antibodies are antibodies in which polypeptide fragments originating from different antibodies are covalently linked to form an immunologically functional light chain, heavy chain, or fragment thereof.

In such a monoclonal antibody, a specific amino acid residue, typically included in the portion of the antibody not involved in antigen recognition, is modified to be homologous to the corresponding residue of the isotype corresponding to the human antibody. Humanization can be accomplished by various known methods, for example, by replacing at least a portion of the variable region of a rodent with the corresponding region of a human antibody (U.S. Patent Nos. 5,585,089 and 5,693,762; Jones et al., Nature 1986, 321: 522-525; Riechmann et al. , Nature 1988, 332: 32327; Verhoeyen et al., Science 1988, 239: 1534-1536).

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A full-length human antibody can be produced by immunizing a transgenic animal (usually a mouse) that can produce human antibody due to the lack of endogenous immunoglobulin production. The full length human antibody may also be derived from a phage display library. The use of full-length human antibodies allows minimizing immunogenic and allergic reactions that can be caused by administering a mouse mAb or mAb of mouse origin to a person.

In one embodiment, the selection of human anti-alpha-synuclein antibodies of the present invention is carried out by phage display. Monoclonal phage antibodies specific for alpha-synuclein, selected by phage screening, are converted into full-length IgG using a recombinant method. The sequence of each monoclonal phage antibody is obtained to recombinantly produce anti-alpha-synuclein antibodies. After combining the heavy chain variable region sequence with the heavy chain constant region sequence and the light chain variable region sequence with the light chain constant region sequence in the resulting sequences, the amino acid sequences are converted into a nucleotide sequence by codon optimization method. After cloning the resulting nucleotide sequences into vectors used for culturing animal cells, host cells used to produce proteins, such as CHO cells, are transformed with the resulting vectors and cultured. To purify antibodies present in the culture medium, recombinant antibodies are isolated and purified using a purification technique such as affinity chromatography.

In another embodiment, the antibody may have the typical structure of a naturally occurring antibody or a modified structure.

An antibody having a typical structure may have a multimeric structure comprising structural units containing two different polypeptide chains (ie, a heavy chain and a light chain). These two different polypeptide chains include one full-length light chain (approximately 25 kDa) and one full-length heavy chain (approximately 50-70 kDa). Each chain has a characteristic spatial structure and consists of several immunoglobulin domains consisting of approximately 90-110 amino acids. These domains are the main structures that make up the antibody polypeptide. The N-terminal portion of each chain typically contains a portion called a variable region or V region, which is the antigen recognition portion. The C-terminal portion is evolutionarily more conserved than the N-terminal portion and contains a portion called the constant region or C region. Human light chain is generally classified as kappa (k) and lambda (λ) light chains, and they contain one variable region and one constant region. The heavy chain is usually classified as a mu (μ), delta (δ), gamma (γ), alpha (α), or epsilon (ε) chain, and these are defined as the IgM, IgD, IgG, IgA, and IgE isotypes, respectively. IgG includes numerous subtypes, including, but not limited to, IgG1, IgG2, IgG3 and IgG4. The heavy chain constant region typically contains one or more domains exhibiting effector function. The number of domains in the heavy chain constant region varies depending on the isotype. The IgG heavy chain, for example, contains three C-region domains known as CH1, CH2 and CH3, respectively. The antibody disclosed herein may be an antibody of any of these isotypes and subtypes. In one embodiment, the antibody of the present invention is an antibody of the IgG1, IgG2, IgG2a, IgG2b, IgG3 or IgG4 subtype.

The alpha-synuclein aggregate of the present invention specifically recognizes alpha-synuclein aggregates with high affinity and specificity, indicating that an anti-alpha-synuclein antibody may be useful for the diagnosis or detection of such diseases. In addition, the anti-alpha-synuclein antibody specifically recognizing alpha-synuclein aggregates of the present invention inhibits the formation of alpha-synuclein aggregates, degrades aggregates, or inhibits intercellular transport of aggregates, whereby it can be usefully used for the treatment of synucleinopathies, in particular Parkinson's disease .

Examples of bispecific antibodies of the present invention are shown in table. 17.

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Table 17

Bispecific Antibody Clone ID Bispecific antibody light chain Combined bispecific antibody heavy chain SE Q ID Explanation of the bispecific antibody heavy chain SEQ ID chi 1F11-1564-3 bivalent chllFll-VL chi 1F11-1564-3 bivalent (NS) 569 CH11F11 (IGG)- 640 (G4S)3- 412 1564-3 VL- 297 (G4S)4- 411 1564-3 VH 178 chi 1F11-1564-2 bivalent chllFll-VL chi 1F11-1564-2 bivalent (NS) 570 CH11F11 (IGG), 640 (G4S)3, 412 1564-2 VL, 296 (G4S)4, 411 1564-2 VH 177 chi 1F11-1564-3 monovalent chllFll-VL chi 1F11-1564-3 monovalent (HC 1, -depression) (1564-3 scFv) 571 CHI IF 11 (IGG) WITH MUTATION OF THE TYPE “THE HOLLOW” AT FC, 641 (G4S)3, 412 1564-3 VL, 297 (G4S)4, 411 1564-3 VH 178 chi1F11-1564 monovalent (HC 2, -protrusion) 573 CHI IF 11 (IGG) WITH PROJECTION TYPE MUTATION 573 chi 1F11-1564-2 monovalent chllFll-VL chi 1F11-1564-2 monovalent (HC 1, -depression) (1564-2 scFv) 572 CH11F11 (IGG) C MUTATION OF THE TYPE “THE HOLLOW” AT FC, 641 (G4S)3, 412 1564-2 VL, 296 (G4S)4, 411 1564-2 VH 177 chi1F11-1564 monovalent (HC 2, -protrusion) 573 CHI IF 11 (IGG) WITH BOOK TYPE MUTATION 573 ch3A9-1564-3 bivalent ch3A9-VL ch3A9-1564-3 bivalent (NS) 574 CH3A9 (IGG), 642 (G4S)3, 412 1564-3 VL, 297 (G4S)4, 411 1564-3 VH 178 ch3A9-1564-2 bivalent ch3A9-VL ch3A9-1564-2 bivalent (NS) 575 CH3A9 (IGG), 642 (G4S)3, 412

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1564-2 VL, 296 (G4S)4, 411 1564-2 VH 177 ch3A9-1564-3 monovalent ch3A9-VL ch3A9-1564-3 monovalent (HC 1, -depression) 576 CHZA9 (IGG) WITH "BALLOW" TYPE MUTATION AT FC 643 (G4S)3, 412 1564-3 VL, 297 (G4S)4, 411 1564-3 VH 178 ch3A9-1564 monovalent (HC 2, -protrusion) 578 CH3A9 (IGG) WITH BOOK TYPE MUTATION 578 ch3A9-1564-3 monovalent ch3A9-VL ch3A9-1564-2 monovalent (HC 1, -depression) 577 CH3A9 (IGG) WITH AT FC "BALL" TYPE MUTATION, 643 (G4S)3, 412 1564-2 VL, 296 (G4S)4, 411 1564-2 VH 177 ch3A9-1564 monovalent (HC 2, -protrusion) 578 CH3A9 (IGG) WITH BOOK TYPE MUTATION 578 hullFll(ver.2)-1564-3 bivalent HullFll-VLv3 4c hullFll(ver.2)-1564-3 bivalent (NS) 579 hullFll(ver.2 (IGG), 644 (G4S)3, 412 1564-3 VL, 297 (G4S)4, 411 1564-3 VH 178 hullFll(ver.2)-1564-2 bivalent HullFll-VLv3 4c hullFll(ver.2)-1564-2 bivalent (NS) 580 hullFll(ver.2 (IGG), 644 (G4S)3, 412 1564-2 VL, 296 (G4S)4, 411 1564-2 VH 177 hullFll(ver.2)-1564-3 monovalent HullFll-VLv3 4c hullFll(ver.2)-1564-3 monovalent (HC 1, -hollow) 581 hullFll(ver.2 (IGG) WITH A MUTATION OF THE "HELL" TYPE AT FC, 645 (G4S)3, 412 1564-3 VL, 297 (G4S)4, 411

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1564-3 VH 178 hullFll(ver.2)-1564 monovalent (HC 2, -protrusion) 583 hullFll(ver.2 (IGG) WITH “HUMP” TYPE MUTATION 583 hullFll(ver.2)-1564-2 monovalent HullFll-VLv3 4c hullFll(ver.2)-1564-2 monovalent (HC 1, -hollow) 582 hullFll(ver.2 (IGG) WITH MUTATION AT FC "VALLOW" TYPE, 645 (G4S)3, 412 1564-2 VL, 296 (G4S)4, 411 1564-2 VH 177 hullFll(ver.2)-1564 monovalent (HC 2, -protrusion) 583 hullFll(ver.2 (IGG) WITH “HUMP” TYPE MUTATION 583 ** human IgGl M428L mutated (CHI, СН2, СНЗ) 584 HUMAN IGG1 M428L MUTATION AT FC 584 hullFll(ver.2)(M428L )-1564-DMP bivalent HullFll-VLv3 4c hullFll(ver.2)(M428L )-1564-DMP bivalent(HC) 585 hullFll(ver.2 (IGG), M428L MUTATION, 646 (G4S)3, 412 1564-DMP VL, 299 (G4S)4, 411 1564-DMP VH 180 hullFll(ver.2)(M428L )-1564-DM bivalent HullFll-VLv3 4c hullFll(ver.2)(M428L )-1564-DM bivalen t(HC) 586 hullFll(ver.2 (IGG), M428L MUTATION, 646 (G4S)3, 412 1564-DM VL, 298 (G4S)4, 411 1564-DM VH 179 hullFll(ver.2)(M428L )-1564-DMP monovalent HullFll-VLv3 4c hullFll(ver.2)(M428L )-1564-DMP monovalent (HC 1, -depression) 587 hullFll(ver.2 (IGG), M428L, HOLLOW TYPE MUTATION, 647 (G4S)3, 412 1564-DMP VL, 299 (G4S)4, 411 1564-DMP VH 180 hullFll(ver.2)(M428L)-1564 monovalent, (NS 2, -protrusion) 589 hullFll(ver.2 (IGG), M428L, HUMP TYPE MUTATION 589 hullFll(ver.2)(M428L )-1564-DM monovalent HullFll-VLv3 4c hullFll(ver.2)(M428L )-1564-DMP 588 hullFll(ver.2 (IGG), M428L, 647

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monovalent, (HC 1, depression) MUTATION OF THE “HELLOW” TYPE, (G4S)3, 412 1564-DM VL, 298 (G4S)4, 411 1564-DM VH 179 hullFH(ver.2)(M428L )-1564 monovalent, (HC 2, -protrusion) 589 hullFll(ver.2 (IGG), M428L, HUMP TYPE MUTATION 589 hullFll(ver.2)-C04-3 monovalent HullFll-VLv3 4c hullFll(ver.2)-C04-3 monovalent (HC 1, -hollow) 590 hullFll(ver.2 (IGG), MUTATION TYPE “THE HOLLOW”, 645 (G4S)3, 412 С04-3 VL, 334 (G4S)4, 411 C04-3 VH 215 hullFll(ver.2)-C04 monovalent (HC 2, -protrusion) 583 hullFll(ver.2 (IGG), MUTATION OF “HUMP” TYPE 583 hullFll(ver.2)-C04-2 monovalent HullFll-VLv3 4c hullFll(ver.2)-C04-2 monovalent (HC 2, -protrusion) 591 hullFll(ver.2 (IGG), MUTATION OF “HUMP” TYPE 645 (G4S)3, 412 С04-2 VL, 333 (G4S)4, 411 C04-2 VH 214 hullFll(ver.2)-C04 monovalent (HC 2, -protrusion) 583 hullFll(ver.2 (IGG), MUTATION OF “HUMP” TYPE 583 hullFll(ver.2)-F06-03 monovalent hullFll(ver.2)-F06-3 monovalent (HC 1, -hollow) 592 hullFll(ver.2 (IGG), MUTATION TYPE “HULL”, 645 (G4S)3, 412 F06-3 VL, 339 (G4S)4, 411 F06-3 VH 220 hullFll(ver.2)-C04 monovalent (HC 2, -protrusion) 583 hullFll(ver.2 (IGG), MUTATION OF “HUMP” TYPE 583 hullFll(ver.2)-F06-02 monovalent HullFll-VLv3 4c hullFll(ver.2)-F06-02 monovalent (HC 1, -hollow) 593 hullFll(ver.2 (IGG), MUTATION TYPE “HULL”, 645 (G4S)3, 412

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F06-2 VL, 338 (G4S)4, 411 F06-2 VH 219 hullFll(ver.2)-C04 monovalent (HC 2, -protrusion) 583 hullFll(ver.2 (IGG), MUTATION OF “HUMP” TYPE 583 hullFll(ver.2)-VH2-3 bivalent HullFll-VLv3 4c hullFll(ver.2)-VH2-3 bivalent (HC) 594 hullFll(ver.2 (IGG), 644 (G4S)3, 412 VH2-3 VL, 356 (G4S)4, 411 VH2-3 VH 237 hullFll(ver.2)-VH2-2 bivalent HullFll-VLv3 4c hullFll(ver.2)-VH2-2 bivalent (NS)_2 595 hullFll(ver.2 (IGG), 644 (G4S)3, 412 VH2-2 VL, 355 (G4S)4, 411 VH2-2 VH 236 hullFll(ver.2)-VH5-3 bivalent HullFll-VLv3 4c hullFll(ver.2)-VH5-3 bivalent (NS) 596 hullFll(ver.2 (IGG), 644 (G4S)3, 412 VH5-3 VL, 361 (G4S)4, 411 VH5-3 VH 242 hullFll(ver.2)-VH5-2 bivalent HullFll-VLv3 4c hullFll(ver.2)-VH5-2 bivalent (NS)_2 597 hullFll(ver.2 (IGG), 644 (G4S)3, 412 VH5-2 VL, 360 (G4S)4, 411 VH5-2 VH 241 hullFll(ver.2)-VH6-3 bivalent HullFll-VLv3 4c hullFll(ver.2)-VH6-3 bivalent (HC) 598 hullFll(ver.2 (IGG), 644 (G4S)3, 412 VH6-3 VL, 366 (G4S)4, 411 VH6-3 VH 247 hullFll(ver.2)-VH6-2 bivalent HullFll-VLv3 4c hullFll(ver.2)-VH6-2 bivalent (NS)_2 599 hullFll(ver.2 (IGG), 644 (G4S)3, 412 VH6-2 VL, 365 (G4S)4, 411 VH6-2 VH 246

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hullFll(ver.2)-VH7-3 bivalent HullFll-VLv3 4c hullFll(ver.2)-VH7-3 bivalent (NS) 600 hullFll(ver.2 (IGG), 644 (G4S)3, 412 VH7-3 VL, 371 (G4S)4, 411 VH7-3 VH 252 hullFll(ver.2)-VH7-2 bivalent HullFll-VLv3 4c hullFll(ver.2)-VH7-2 bivalent (HC)_2 601 hullFll(ver.2 (IGG), 644 (G4S)3, 412 VH7-2 VL, 370 (G4S)4, 411 VH7-2 VH 251 hullFll(ver.2)-VH9-3 bivalent HullFll-VLv3 4c hullFll(ver.2)-VH9 bivalent (NS) 602 hullFll(ver.2 (IGG), 644 (G4S)3, 412 VH9-3 VL, 376 (G4S)4, 411 VH9-3 VH 257 hullFll(ver.2)-VH9-2 bivalent HullFll-VLv3 4c hullFll(ver.2)-VH9 bivalent (NS)_2 603 hullFll(ver.2 (IGG), 644 (G4S)3, 412 VH9-2 VL, 375 (G4S)4, 411 VH9-2 VH 256 hullFll(ver.2)-VH16-3 bivalent HullFll-VLv3 4c hullFll(ver.2)-VH16 bivalent (NS) 604 hullFll(ver.2 (IGG), 644 (G4S)3, 412 VH16-3 VL, 381 (G4S)4, 411 VH16-3 VH 262 hullFll(ver.2)-VH16-2 bivalent HullFll-VLv3 4c hullFll(ver.2)-VH16 bivalent (NS)_2 605 hullFll(ver.2 (IGG), 644 (G4S)3, 412 VH16-2 VL, 380 (G4S)4, 411 VH16-2 VH 262 hullFll(ver.2)-VH27 3 bivalent HullFll-VLv3 4c hullFll(ver.2)-VH27 bivalent (HC) 606 hullFll(ver.2 (IGG), 644 (G4S)3, 412 VH27-3 VL, 386 (G4S)4, 411

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VH27-3 VH 267 hullFll(ver.2)-VH27-2 bivalent HullFll-VLv3 4c hullFll(ver.2)-VH27 bivalent (HC)_2 607 hullFll(ver.2 (IGG), 644 (G4S)3, 412 VH27-2 VL, 385 (G4S)4, 411 VH27-2 VH 266 hullFll(ver.2)-VH32-3 bivalent HullFll-VLv3 4c hullFll(ver.2)-VH32 bivalent (HC) 608 hullFll(ver.2 (IGG), 644 (G4S)3, 412 VH32-3 VL, 391 (G4S)4, 411 VH32-3 VH 272 hullFll(ver.2)-VH32-2 bivalent HullFll-VLv3 4c hullFll(ver.2)-VH32 bivalent (HC)_2 609 hullFll(ver.2 (IGG), 644 (G4S)3, 412 VH32-2 VL, 390 (G4S)4, 411 VH32-2 VH 271 hullFll(ver.2)-VH35-3 bivalent HullFll-VLv3 4c hullFll(ver.2)-VH35 bivalent (HC) 610 hullFll(ver.2 (IGG), 644 (G4S)3, 412 VH35-3 VL, 396 (G4S)4, 411 VH35-3 VH 277 hullFll(ver.2)-VH35-2 bivalent HullFll-VLv3 4c hullFll(ver.2)-VH35 bivalent (NS)_2 611 hullFll(ver.2 (IGG), 644 (G4S)3, 412 VH35-2 VL, 395 (G4S)4, 411 VH35-2 VH 276 hullFll(ver.2)(M428L )-C04-DMP monovalent HullFll-VLv3 4c hullFll(ver.2)(M428L )-C04-DMP monovalent (HC 1, -depression) 612 hullFll(ver.2 (IGG), M428L, HOLLOW TYPE MUTATION, 647 (G4S)3, 412 C04-DMP VL, 336 (G4S)4, 411 C04-DMP VH 217 hullFll(ver.2)(M428L)-С04 (NS 2, -protrusion) 589 hullFll(ver.2 (IGG), M428L MUTATION, MUTATION TYPE 589

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"SPIT" hullFll(ver.2)(M428L )-C04-DM monovalent HullFll-VLv3 4c hullFll(ver.2)(M428L )-C04-DM monovalent (HC 1, -depression) 613 hullFll(ver.2 (IGG), M428L, HOLLOW TYPE MUTATION, 647 (G4S)3, 412 C04-DM VL, 335 (G4S)4, 411 C04-DM VH 216 hullFll(ver.2)(M428L)-C04 (NS 2, -protrusion) 589 hullFll(ver.2 (IGG), M428L MUTATION, HUMBLE MUTATION 589 hullFll(ver.2)(M428L )-F06-DMP monovalent HullFll-VLv3 4c hullFll(ver.2)(M428L )-F06-DMP monovalent (HC 1, -depression) 614 hullFll(ver.2 (IGG), M428L, HOLLOW TYPE MUTATION, 647 (G4S)3, 412 F06-DMP VL, 341 (G4S)4, 411 F06-DMP VH 222 hullFll(ver.2)(M428L)-F06 monovalent (HC 2, -protrusion) 589 hullFll(ver.2 (IGG),M428L MUTATION, HUMP TYPE MUTATION 589 hullFll(ver.2)(M428L )-F06-DM monovalent HullFll-VLv3 4c hullFll(ver.2)(M428L )-F06-DM monovalent (HC 1, -depression) 615 hullFll(ver.2 (IGG), M428L, HOLLOW TYPE MUTATION, 647 (G4S)3, 412 F06-DM VL, 340 (G4S)4, 411 F06-DM VH 221 hullFll(ver.2)(M428L)-F06 monovalent, deamidated, S->P (HC 2, -protrusion) 589 hullFll(ver.2 (IGG),M428L MUTATION, HUMP TYPE MUTATION 589 hullFll(ver.2)(M428L )-VH2-DMP bivalent HullFll-VLv3 4c hullFll(ver.2)(M428L )-VH2-DMP bivalent (HC) 616 hullFll(ver.2 (IGG), M428L MUTATION, 646 (G4S)3, 412 VH2-DMP VL, 358 (G4S)4, 411 VH2-DMP VH 239

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hullFll(ver.2)(M428L )-VH2-DM bivalent HullFll-VLv3 4c hullFll(ver.2)(M428L )-VH2-DM bivalent (HC) 617 hullFll(ver.2 (IGG), M428L MUTATION, 646 (G4S)3, 412 VH2-DM VL, 357 (G4S)4, 411 VH2-DM VH 238 hullFll(ver.2)(M428L )-VH5-DMP bivalent, (HC) HullFll-VLv3 4c hullFll(ver.2)(M428L )-VH5-DMP bivalent, (HC) 618 hullFll(ver.2 (IGG), M428L MUTATION, 646 (G4S)3, 412 VH5-DMP VL, 363 (G4S)4, 411 VH5-DMP VH 244 hullFll(ver.2)(M428L )-VH5-DM bivalent, (HC) HullFll-VLv3 4c hullFll(ver.2)(M428L )-VH5-DM bivalent, (HC) 619 hullFll(ver.2 (IGG), M428L MUTATION, 646 (G4S)3, 412 VH5-DM VL, 362 (G4S)4, 411 VH5-DM VH 243 hullFll(ver.2)(M428L )-VH5-DMP monovalent HullFll-VLv3 4c hullFll(ver.2)(M428L )-VH5-DMP monovalent, (HC 1, depression) 620 hullFll(ver.2 (IGG), M428L, HOLLOW TYPE MUTATION, 647 (G4S)3, 412 VH5-DMP VL, 363 (G4S)4, 411 VH5-DMP VH 244 hullFll(ver.2)(M428L )-VH5 monovalent, deamidated, S->P (HC 2, -protrusion) 589 hullFll(ver.2 (IGG),M428L MUTATION, HUMP TYPE MUTATION 589 hullFll(ver.2)(M428L )-VH5_DM monovalent HullFll-VLv3 4c hullFll(ver.2)(M428L )-VH5_DM monovalent, (HC 1, depression) 621 hullFll(ver.2 (IGG), M428L, HOLLOW TYPE MUTATION, 647 (G4S)3, 412 VH5-DM VL, 362 (G4S)4, 411 VH5-DM VH 243 hullFll(ver.2)(M428L )-VH5 monovalent, deamidated, S->P (HC 2, -protrusion) 589 hullFll(ver.2 (IGG),M428L MUTATION, MUTATION TYPE 589

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"SPIT" hullFll(ver.2)(M428L )-VH6-DMP bivalen HullFll-VLv3 4c hullFll(ver.2)(M428L )-VH-DMP6 bivalent (HC) 622 hullFll (ver. 2 (IGG), M428L MUTATION, 646 (G4S)3, 412 VH6-DMP VL, 368 (G4S)4, 411 VH6-DMP VH 249 hullFll(ver.2)(M428L )-VH6-DM bivalent HullFll-VLv3 4c hullFll(ver.2)(M428L )-VH6-DM bivalent (NS) 623 hullFll(ver.2 (IGG), M428L MUTATION, 646 (G4S)3, 412 VH6-DM bivalent VL, (G4S)4, 367 (G4S)4, 411 VH6-DM VH 248 hullFll(ver.2)(M428L )-VH7-DMP bivalent HullFll-VLv3 4c hullFll(ver.2)(M428L )-VH7-DMP bivalent (HC) 624 hullFll (ver. 2 (IGG), M428L MUTATION, 646 (G4S)3, 412 VH7-DMP VL, 373 (G4S)4, 411 VH7-DMP VH 254 hullFll(ver.2)(M428L )-VH7-DM bivalent HullFll-VLv3 4c hullFll(ver.2)(M428L )-VH7-DM bivalent, (HC) 625 hullFll (ver. 2 (IGG), M428L MUTATION, 646 (G4S)3, 412 VH7-DM VL, 372 (G4S)4, 411 VH7-DM VH 253 hullFll(ver.2)(M428L )-VH9-DMP bivalent HullFll-VLv3 4c hullFll(ver.2)(M428L )-VH9-DMP bivalent (HC) 626 hullFll (ver. 2 (IGG), M428L MUTATION, 646 (G4S)3, 412 VH9-DMP VL, 378 (G4S)4, 411 VH9-DMP VH 259 hullFll(ver.2)(M428L )-VH9-DM bivalent HullFll-VLv3 4c hullFll(ver.2)(M428L )-VH9-DM bivalent, (HC) 627 hullFll (ver. 2 (IGG), M428L MUTATION, 646 (G4S)3, 412 VH9-DM VL, 377 (G4S)4, 411 VH9-DM VH 258

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hullFll(ver.2)(M428L )-VH9 -DMP monovalent HullFll-VLv3 4c hullFll(ver.2)(M428L )-VH9- DMPmonovalent, (HC 1, -cavity) 628 hullFll(ver.2 (IGG), M428L, HOLLOW TYPE MUTATION, 647 (G4S)3, 412 VH9-DMP VL, 378 (G4S)4, 411 VH9-DMP VH 259 hullFll(ver.2)(M428L )-VH9 monovalent, deamidated, S->P (HC 2, -protrusion) 589 hullFll(ver.2 (IGG),M428L MUTATION, HUMP TYPE MUTATION 589 hullFll(ver.2)(M428L )-VH9-DM monovalent HullFll-VLv3 4c hullFll(ver.2)(M428L )-VH9-DM monovalent (HC 1, -hollow) 629 hullFll(ver.2 (IGG), M428L, HOLLOW TYPE MUTATION, 647 (G4S)3, 412 VH9-DM VL, 377 (G4S)4, 411 VH9-DM VH 258 hullFll(ver.2)(M428L )-VH9 monovalent, deamidated, S->P (HC 2, -protrusion) 589 hullFll(ver.2 (IGG),M428L MUTATION, HUMP TYPE MUTATION 589 hullFll(ver.2)(M428L )-VH16-DMP bivalent HullFll-VLv3 4c hullFll(ver.2)(M428L )-VH16-DMP bivalent, (HC) 630 hullFll(ver.2 (IGG), M428L MUTATION, 646 (G4S)3, 412 VH16-DMP VL, 383 (G4S)4, 411 VH16-DMP VH 264 hullFll(ver.2)(M428L )-VH16-DM bivalent HullFll-VLv3 4c hullFll(ver.2)(M428L )-VH16-DM bivalent (HC) 631 hullFll(ver.2 (IGG), M428L MUTATION, 646 (G4S)3, 412 VH16-DM VL, 382 (G4S)4, 411 VH16-DMVH 263 hullFll(ver.2)(M428L )-VH16-DMP monovalent HullFll-VLv3 4c hullFll(ver.2)(M428L )-VH16-DMP monovalent (HC 1, -hollow) 632 hullFll(ver.2 (IGG), M428L, HOLLOW TYPE MUTATION, 647 (G4S)3, 412 VH16-DMP VL, 383

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(G4S)4, 411 VH16-DMP VH 264 hullFll(ver.2)(M428L )-VH16 monovalent, deamidated, S->P (HC 2, -protrusion) 589 hullFll(ver.2 (IGG),M428L MUTATION, HUMP TYPE MUTATION 589 hullFll(ver.2)(M428L )-VH16-DM monovalent HullFll-VLv3 4c hullFll(ver.2)(M428L )-VH16-DM monovalent (HC 1, - depression) 633 hullFll(ver.2 (IGG), M428L, HOLLOW TYPE MUTATION, (G4S)3, VH16-DMVL, (G4S)4, VH16-DMVH 647 412 382 411 263 hullFll(ver.2)(M428L )-VH27-DMP bivalent (HC) HullFll-VLv3 4c hullFll(ver.2)(M428L )-VH16 monovalent, deamidated, S->P (HC 2, -protrusion) hullFll(ver.2)(M428L )-VH27-DMP bivalent (HC) 589 634 hullFll(ver.2 (IGG),M428L MUTATION, PROJECTION MUTATION hullFll(ver.2 (IGG), M428L MUTATION, (G4S)3, VH27-DMP VL, 589 646 412 388 (G4S)4, 411 VH27-DMP VH 269 hullFll(ver.2)(M428L )-VH27-DM bivalent (NS) HullFll-VLv3 4c hullFll(ver.2)(M428L )-VH27-DM bivalent (HC) 635 hullFll(ver.2 (IGG), M428L MUTATION, (G4S)3, VH27-DM VL, 646 412 387 (G4S)4, 411 VH27-DM VH 268 hullFll(ver.2)(M428L )-VH32-DMP bivalent HullFll-VLv3 4c hullFll(ver.2)(M428L )-VH32 -DMP bivalent (HC) 636 hullFll(ver.2 (IGG), M428L MUTATION, (G4S)3, VH32-DMP VL, 646 412 393 (G4S)4, 411 VH32-DMP VH 274 hullFll(ver.2)(M428L HullFll-VLv3 hullFll(ver.2)(M428L )-VH32-DM bivalent(HC) 637 hullFll(ver.2 (IGG), M428L MUTATION, 646 )-VH32-DM bivalent 4c (G4S)3, 412 VH32-DM VL, 392

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(G4S)4, 411 VH32-DM VH 273 hullFll(ver.2)(M428L )-VH35-DMP bivalent HullFll-VLv3 4c hullFll(ver.2)(M428L )-VH35-DMP bivalent (HC) 638 hullFll(ver.2 (IGG), M428L MUTATION, 646 (G4S)3, 412 VH35-DMP VL, 398 (G4S)4, 411 VH35-DMP VH 279 hullFll(ver.2)(M428L )-VH35-DM bivalent HullFll-VLv3 4c hullFll(ver.2)(M428L )-VH35-DMbivalen t(HC) 639 hullFll(ver.2 (IGG), M428L MUTATION, 646 (G4S)3, 412 VH35-DMVL, 397 (G4S)4, 411 VH35-DMVH 278

Invention effect

The antibody produced in one embodiment of the present invention specifically binds to IGF1R with optimized binding strength suitable for transcytosis across the brain endothelium and may be useful for the delivery of therapeutic antibodies for use in degenerative brain diseases and brain cancer, which have limited therapeutic efficacy due to low ability to penetrate the vascular barrier of the brain. In particular, the monoclonal antibody disclosed in the present invention does not affect the binding of ligands such as IGF-1, IGF-2 and insulin, and their homologues to IGF1R and acts without inhibiting signaling through IGF1R. Thus, its usefulness is related to penetration of the blood-brain barriers.

The antibodies disclosed herein effectively remove alpha-synuclein aggregates or promote their breakdown, inhibit the entry of alpha-synuclein into cells, and may therefore be useful in the treatment of diseases associated with the accumulation of alpha-synuclein aggregates.

Brief description of graphic materials

In fig. 1 shows dot blot results demonstrating that the anti-alpha-synuclein antibody produced in one embodiment of the present invention specifically binds to native alpha-synuclein in aggregated form.

In fig. 2 shows the results of an ELISA affinity analysis of an anti-alpha-synuclein antibody prepared in one embodiment of the present invention.

In fig. 3a shows the results of a BIAcore analysis of the specificity and affinity of preferential binding of an anti-alpha-synuclein antibody prepared in one embodiment of the present invention to alpha-synuclein aggregates. In fig. 3b is a table showing the results shown in FIG. 3a.

In fig. 4a shows the results of an Octet analysis of the preferential binding specificity of an anti-alpha-synuclein antibody prepared in one embodiment of the present invention to alpha-synuclein aggregates. In fig. 4b is a table showing the results shown in FIG. 4a.

In fig. 5a and 5b show the results that antibodies against alpha-synuclein 3A9 and 11F11 in one embodiment of the present invention can specifically recognize Lewy bodies and Lewy neurites in human brain tissue, respectively. Antibodies of the present invention have been shown to bind to Lewy bodies (arrows) and Lewy neurites (filamentous form at lower left).

In fig. 6 is a schematic representation of the results of epitope mapping of anti-alpha-synuclein antibodies according to one embodiment of the present invention, where the antibodies of the present invention bind primarily to the C-terminal region.

In fig. 7 shows the results of an ELISA affinity analysis of a chimeric anti-alphasynuclein antibody and a humanized 11F11 antibody produced in one embodiment of the present invention.

In fig. 8 shows the results of a BIAcore analysis of the specificity and affinity of preferential binding of the chimeric anti-alpha-synuclein antibody and the humanized 11F11 antibody produced in one embodiment of the present invention to α-syn aggregates.

In fig. 9a, 9b and 10 present dot blot and ELISA assay results demonstrating that the chimeric alpha-synuclein antibody produced in one embodiment of the present invention specifically binds to alpha-synuclein, particularly to alpha-synuclein aggregates, but not to beta-synuclein. -synuclein, gamma-synuclein, amyloid beta _1-42 or tau protein.

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In fig. 11a-11b show binding data of an anti-IGF1R antibody prepared in one embodiment of the present invention to the IGF1R protein.

In fig. 12 shows binding data of an anti-IGF1R antibody prepared in one embodiment of the present invention to cell lines expressing IGF1R.

In fig. 13 presents data regarding the internalization of an anti-IGF1R antibody produced in one embodiment of the present invention into cell lines expressing IGF1R and what happens to it in these cell lines.

In fig. 14 presents results demonstrating that an anti-IGF1R antibody produced in one embodiment of the present invention does not interfere with IGF1R or insulin signaling.

In fig. 15a presents results showing that the anti-IGF1R antibody produced in one embodiment of the present invention does not exhibit ADCC.

In fig. 15b presents results showing that repeated administration of an anti-IGF1R antibody prepared in one embodiment of the present invention does not affect the level of IGF1R in the brain.

In fig. 15c is a microscopic view demonstrating that an anti-IGF1R antibody produced in one embodiment of the present invention binds to a brain endothelial cell without binding to a normal neuron in the brain.

In fig. 16 presents results demonstrating that a bispecific antibody comprising an anti-IGF1R antibody and a therapeutic antibody can penetrate the BBB system more efficiently than the therapeutic antibody.

In fig. 17 presents results showing that anti-IGF1R antibody and a bispecific antibody comprising anti-IGF1R antibody and therapeutic antibody have a distribution in the brain and CSF greater than the therapeutic antibody alone when administered once to a rat.

In fig. 18a shows results identifying a residue susceptible to deamidation in an anti-IGF1R antibody produced in one embodiment of the present invention.

In fig. 18b shows variants obtained by substituting specific amino acids to prevent deamidation.

In fig. 19 shows the results of epitope mapping of the anti-IGF1R antibody.

In fig. 20a and 20b show the results of an ELISA assay measuring the affinity of a bispecific antibody of one embodiment of the present invention for each antigen.

In fig. 20c and 20d show the results of an ELISA assay comparing the chimeric antibody and the humanized antibody for each antigen.

In fig. Figure 20e presents the results of an assessment of microglial phagocytic activity in relation to a bispecific antibody prepared in one embodiment of the present invention.

In fig. 21a-21e show the results of evaluating the effectiveness of a bispecific antibody produced in one embodiment of the present invention compared to a single antibody in a mouse model.

In fig. 22 presents a result demonstrating the increased half-life and improved BBB penetration resulting from the design of an Fc bispecific antibody produced in one embodiment of the present invention.

Embodiment of the invention

Example 1. Preparation of mouse anti-alpha-synuclein antibody.

1-1. Immunization and production of hybridoma.

Monomeric alpha-synuclein, full-length (140 residues) or without 21 residues at the C-terminus (119 residues), was placed in a thermomixer at 37°C, aggregated with shaking at 1050 rpm for 14 days and treated with ultrasound. α-syn fibrils, 140 residues and 119 residues, 1 mg/ml, were mixed with adjuvant in a 1:1 (v/v) ratio.

Amino acid sequence of Homo sapiens alpha-synuclein (SEQ ID NO:559): MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTKEGVVHGVAT

VAEKTKEQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFVKKDQLGKNEEGAPQ

EGILEDMPVDPDNEAYEMPSEEGYQDYEPEA.

Then, 200 μl of the resulting mixture was injected subcutaneously into female BALB/c mice aged 5 to 7 weeks. After 2 weeks, another subcutaneous injection of 200 μl of the resulting mixture was performed to stimulate the production of antibodies. One week after the second injection, blood was drawn and immunization was titrated by ELISA using the administered antigen. A third injection was then performed with subcutaneous administration of the antigen itself.

The spleens were removed from immunized mice and spleen cells were obtained from them. The resulting spleen cells were suspended in Hybridoma-SFM medium (Thermo Fisher Scientific, USA), supplemented

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10%FBS. To obtain the hybridoma, spleen cells and SP2/0-Ag14 murine myeloma cells were mixed in Hybridoma-SFM medium without serum, followed by centrifugation to remove the medium.

Then PEG was added to the resulting cell pellet and incubated at 37°C for 1 minute to induce cell fusion.

1-2. Single cell cloning and antibody purification.

Two weeks after fusion induction, fusion with antibody-producing murine B cells was confirmed by ELISA using mouse-injected antigen and cell culture medium. Single cell cloning was then performed using a hybridoma, selecting 16 hybridomas that produced monoclonal antibodies. Clones 9B11 (IgG1 kappa) were generated using aggregates of full-length (140 residues) α-syn as antigen, and clones 3A9 and 11F11 (IgG2b kappa and IgG2b kappa, respectively) were generated using αsyn aggregates lacking the 21 residues at the C-terminus as antigens.

To purify antibodies, each hybridoma was cultured in RPMI1640 medium containing 10% FBS. To obtain antibodies, the culture medium was replaced with serum-free SFM medium and culture was carried out for approximately 4 days. The cell culture supernatant was separated, centrifuged, filtered through a 0.22 μm filter, and purified using a Protein G column for the IgG1 type and a Protein A column for the remaining antibodies.

1-3. Determination of the variable region sequence.

The sequences of the variable regions and CDRs were determined according to Ahn et al, Mol. Cells 2004, 18(2):237-241. Hybridomas were cultured and centrifuged to isolate only the cells. RNA was isolated from the isolated hybridomas by adding triazole and used as a template for cDNA synthesis. The sequences of the variable regions and CDRs were confirmed by sequencing.

Example 2. Preparation of (chimeric) antibodies against alpha-synuclein.

2-1. Cloning and expression of antibodies.

Using the nucleotide sequences of the heavy chain variable region and the light chain variable region of the antibody obtained after humanization, gBlock (M. Biotech) of a short nucleotide fragment was synthesized and cloned into an animal cell culture vector (pcDNA3.4). gBlock was synthesized including approximately 20 bp of overlapping sequences. before and after the variable region, and the part of the pcDNA3.4 vector without the variable region was amplified by PCR and cloned by Gibson assembly.

For transfection and expression of the cloned antibody, the resulting vector was used for Maxi-Prep (Qiagen) to obtain a large amount of plasmid DNA and then introduced into cells as follows. On the day prior to transfection, the concentration of ExpiCHO™ cells (Gibco, catalog number A29127) was adjusted to 3-4x106 viable cells per 1 ml in ExpiCHO™ expression medium (Gibco, catalog number A29100-01) and cultured at 8% CO ₂ , 37°C and 120 rpm for 1 day. On the day of DNA transfection, cells grown to 7-10 x 106 viable cells per ml with survival rates of 95% or greater were prepared by diluting them with fresh medium to 6 x 106 viable cells per ml.

For transfection of parental cells, ExpiFectamine™ CHO and plasmid DNA were complexed using the ExpiFectamine™ CHO Transfection Kit (Gibco, catalog number A29129). DNA and ExpiFectamine™ CHO reagents were prepared at appropriate concentrations by diluting them with cold OptiPRO™ SFM® Medium (Gibco, part number 12309019), added accordingly and mixed, leaving to stand at room temperature for 5 minutes. The resulting product was added to the original cells and their post-transfection cultivation was carried out. The next day after transfection, the Enhancer and Feed reagents included in the ExpiFectamine™ CHO Transfection Kit were added to the transfected cells, and after 5 days the Feed reagent was added again, after which incubation was carried out for 10 days at 8% CO ₂ , 37° C and 120 rpm to obtain transfected cells.

To obtain the culture solution, the culture medium was transferred to a centrifuge bottle for centrifugation and centrifuged at 4°C and 6500 rpm for 30 minutes, followed by filtration through a 0.2 μm filter to obtain a culture medium without suspended solids, and then the resulting culture medium the medium was used for subsequent purification.

2-2. Antibody purification and sequencing.

The culture was purified using HiTrap MabSelect SuRe (GE Healthcare, 11-0034-94). After equilibration with equilibration buffer (50 mM Tris-HCl, pH 7.2, 100 mM NaCl), the resulting culture was loaded onto the column. Upon completion of loading, the medium was washed 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 to pH 6.0. The eluate was then buffer exchanged, concentrated using PBS (phosphate buffered saline, pH 7.4) and stored at 4°C until further use.

If additional purification was necessary, a second purification was performed based on the size of the eluted sample, passing the first purified product through IX PBS buffer on a HiLoad 26/600 column

Superdex 200. The amino acid sequence of the purified antibody was analyzed by mass spectrometry and confirmed to correspond to the variable region of a mouse-derived monoclonal antibody.

The variable region backbone of the human IgG1 isotype antibody was replaced with the variable regions of antibodies 3A9, 9B11 and 11F11 determined by the method described above to obtain a chimeric human IgG1 antibody. Among the resulting chimeric antibodies, in particular, the Ch11F11 antibody is an antibody in the form of an IgG and contains a combination of the heavy chain variable region sequence SEQ ID NO:90 (ch11F11-VH) and the light chain variable region sequence SEQ ID NO:109 (ch11F11-VL) and the Ch3A9 antibody is an antibody in IgG form and contains a combination of the heavy chain variable region sequence SEQ ID NO:102 (ch11F11VH) and the light chain variable region sequence SEQ ID NO:116 (chl 1F11-VL).

Example 3. Preparation of a humanized antibody.

3-1. Preparation of phage library.

A minilibrary was constructed in which a sequence of mouse or human origin was introduced into each CDR residue by linking the human framework regions to the CDR1, CDR2 and CDR3 residues of the chimeric antibody.

Competent cells of the resulting minilibrary were added to 2X YT medium (17 g tryptone (CONDA, 1612.00), 10 g yeast extract (CONDA, 1702.00) and 5 g NaCl (Sigma, S7653)) containing 34 μg/ml chloramphenicol (Sigma, C0857) , 2% glucose (Sigma, G5400) and 5 mM MgCl ₂ (Sigma, C0857), at 30°C for 3 hours to OD600 0.5-0.7. Then the cells were infected with a helper phage and cultured in 2X YT medium containing 34 μg/ml chloramphenicol, 5 mM _MgCl2 , 70 μg/ml kanamycin (Sigma, K1876) and 1 mM IPTG (ELPISBIO, IPTG025), at 30°C for for 6 hours to induce phage packaging. The culture solution was centrifuged at 4500 rpm and 4°C for 15 minutes. 4% PEG 6000 (Fluka, 81253) and 3% NaCl (Sigma, S7653) were added to the supernatant and incubated for 1 hour on ice. The resulting product was centrifuged at 8000 rpm for 20 minutes at 4°C, then the pellet was suspended in PBS and centrifuged again at 4°C and 12000 rpm for 10 minutes to obtain a supernatant containing the phage library. The resulting supernatant was stored at 4°C until further use.

3-2. Panning with phage display.

To select antibodies that bind preferentially to alpha-synuclein aggregates rather than to its monomers, panning was performed using full-length alpha-synuclein aggregates obtained in Example 1, and a total of three pannings were performed as follows.

Bovine serum albumin (BSA) was added to the cells at a concentration of 3% in a tube at 4°C overnight, 10 μg/ml of recombinant alpha-synuclein aggregates and monomers in PBS solution were added to the cells in an immunoassay tube (Maxisorp 444202) and the surfaces were protected. to which aggregates and monomers of alpha-synuclein were not sorbed. After the tube was emptied, the phage library of antibodies, diluted in a 3% BSA solution to 1012 CFU, was placed in an immunological tube onto which alpha-synuclein aggregates and monomers were sorbed, and the interaction was carried out for 1 hour (negative selection). Then, phages not associated with alpha-synuclein aggregates and monomers were isolated and interacted for 2 hours at room temperature with sorbed alpha-synuclein aggregates and monomers. Phosphate buffered saline (0.05% Tween 20) was used to reconstitute 100 μM triethylamine solution, which was reconstituted using PBS-T solution. E. coli at 37°C for 1 hour, and infected E. coli were plated on 2X YT agar medium and cultured at 37°C overnight (pH 7.4) and infected with ER2537. The next day, the cultured E. coli were suspended in 4 ml of 2X YT culture solution containing carbenicillin, 15% glycerol was added and part was stored at -80°C, and the rest was used to obtain phages for subsequent experiments. By repeating this process for a total of 3 rounds, a pool of phages specific for the alpha-synuclein antigen was amplified and concentrated. During later rounds of panning, the number of PBS-T washes was increased to increase the number and concentration of antigen-specific phages.

3-3. Screening of individual clones.

To select monoclonal antibodies that specifically bind to alpha-synuclein aggregates from the pool of phages obtained by panning, the following experiment was performed.

To isolate monoclones from a concentrated pool, after plating the phage pool on LB agar medium with tetracycline/carbenicillin and culturing, individual colonies were isolated. Then, after adding the monoclones to a deep 96-well plate, each well containing 400 μl of 2X YT medium with tetracycline/carbenicillin, and growing overnight, 10 μl of the culture solution was added to a new deep 96-well plate, into which 390 µl of 2X YT medium with tetracycline/carbenicillin were added and cultured at 37°C for

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The overnight culture solution was centrifuged to remove the supernatant.

Clones expressing monoclonal soluble scFvs that bind to alpha-synuclein aggregates were then selected using ELISA as described below (Steinberger. Rader and Barbas III. 2000. Phage display vectors. In: Phage Display Laboratory Manual, lsted. Cold Sprin gHarbor Laboratory Press. NY. USA. pp. 11.9-11.12). Specifically, the 7B7 antibody selected in Example 1-1 was added to a 96-well plate (Nunc-Immuno Plates, NUNC, USA) and sorption was carried out at 4°C overnight. 3% BSA was added to each well in an amount of 200 μl, followed by blocking at 37°C for 2 hours. Alpha-synuclein aggregates and monomers were then added at a concentration of 100 ng per well, reacted at 37°C for 2 hours, and washed five times with 300 μl of PBS-T. The resulting supernatant of an individual clone was mixed with 3% BSA in a volume ratio of 1:1 (v/v) and 100 μl of the resulting solution was added to the plate to which the aggregates and monomers were bound, followed by interaction at 37°C for 2 hours . Cells were washed five times with 300 μl PBS-T and incubated at 37°C for 1 hour with HRP-conjugated anti-HA antibody, followed by washing five times with PBS-T. After adding 100 μl of TMB (tetramethylbenzidine, Sigma, T0440), the reaction was stopped by adding 50 μl of 1 N H ₂ SO ₄ to measure the absorbance at 450 nm. Clones with an optical density of 0.5 or more were considered positive binding reactions, and clones that bound nonspecifically to BSA were excluded.

The CDR residues of the clones found in the library were analyzed on the computer in parallel and clones were selected that resulted in severe problems with binding to framework regions, or clones that did not have a T cell epitope, a B cell epitope, and an MHCII epitope in framework regions other than the CDRs .

Then, for the five antibodies produced using the following combinations of heavy chain and light chain variable regions, the humanized antibody variable region of the human IgG1 isotype was replaced by the variable region backbone to obtain five humanized antibodies with the IgG1 backbone. Specifically, Hu11F11 (ABL2-4) is an IgG type antibody and contains a combination of the heavy chain variable region sequence SEQ ID NO:92 (Hu11F11-VH2) and the light chain variable region sequence SEQ ID NO:113 (Hu11F11-VL4). Hu11F11 (ver.1) is an IgG antibody and contains a combination of the heavy chain variable region sequence SEQ ID NO:98 (Hu11F11-VH-v1) and the light chain variable region sequence SEQ ID NO:115 (Hu11F11-VLv3 4c). Hu11F11 (ver.2) is an IgG antibody and contains a combination of the heavy chain variable region sequence SEQ ID NO:99 (Hu11F11-VH-v2) and the light chain variable region SEQ ID NO:115 (Hu11F11-VLv3 4c). Hu11F11 (ver.3) is an IgG antibody and contains the heavy chain variable region sequence SEQ ID NO:100 (Hu11F11-VH-v3) and the light chain variable region sequence SEQ ID NO:115 (Hu11F11-VLv3 4c).

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

Example 4 Antigen Binding Specificity and Affinity Analysis Using Anti-Alpha-Synuclein Antibody.

4-1. Dot blot analysis using anti-alpha-synuclein antibody.

Dot blot experiments were performed to analyze the possible binding of the antibody of the present invention to monomers or aggregates in the native state. For the experiment, 50 ng or 100 ng of monomeric or fibrillar α-syn protein (prepared by Professor Lee Seung-jae of Seoul National University; Bae et al., J. Neurosci 32: 13454, 2012) was spotted onto a nitrocellulose membrane . Double dilutions of monomeric or fibrillar protein were applied sequentially from the right edge of the membrane to the left (12.5, 25, 50, 100 ng). The membrane was blocked with 5% nonfat dry milk with TBST for 1 hour at room temperature. 1 mg/ml of the anti-α-syn antibody prepared in Example 1 was added to TBST containing 1% bovine serum albumin and incubated at room temperature for 1 hour. After washing with TBST, signals were analyzed using chemiluminescent substrate (NEN) as substrate and HRP (horseradish peroxidase)-conjugated secondary antibody following the manufacturer's instructions. The results were visualized using the LAS-3000 Luminescent Image Analysis System (FUJIFILM Life Science). The results are shown in Fig. 1.

As shown in FIG. 1, the anti-alpha-synuclein antibody of the present invention was found to bind preferentially to aggregates, but not to alpha-synuclein monomers. In particular, 9B11, 3A9 and 11F11 were associated with aggregates. Antibody 274 (Bae et al., J Neurosci. 2012 Sep 26; 32 (39): 13454-13469) was used as a reference antibody that binds to both monomers and aggregates.

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4-2. ELISA assay using mouse monoclonal antibody against α-syn.

An ELISA assay was performed to quantitatively analyze the binding affinity of an antibody of the present invention to an antigen. To do this, the antibody to alpha-synuclein obtained in example 1 was adsorbed onto a 96-well plate at a concentration of 1 μg/ml and treated with aggregates of alpha-synuclein fibrils at concentrations of 10, 100, 1000 and 10,000 ng/ml. After washing with PBS, HRP-conjugated streptavidin and biotin-conjugated secondary antibody were added and then reacted with TMB as substrate. The optical density was measured and the results are shown in FIG. 2.

As shown in FIG. 2, the antibodies of the present invention were found to bind preferentially to aggregates with high binding affinity. ELISA results showed that antibodies binding predominantly to aggregates had affinities ranging from 0.1x10'9 M to 2x10'9 M, while antibodies binding to both monomers and aggregates had a higher affinity of approximately 1x10 ' ¹⁰ M. The antibody of the present invention bound preferentially to alpha-synuclein aggregates with high affinity, but failed to obtain affinity for the monomer because it bound to the monomer with less affinity than the aggregates or did not bind to the monomer. These results indicate that the antibody in question can effectively eliminate or inhibit the action of an agent causing neurodegenerative diseases associated with alpha-synuclein etiology, such as Parkinson's disease.

4-3. BIAcore analysis using anti-alpha-synuclein antibody.

A quantitative analysis of the binding of the anti-alpha-synuclein antibody obtained in Example 1 to monomeric and aggregated antigens was carried out using BIAcore analysis.

The instrument used was a T200 (GE Healthcare, serial number 1565888). Protein A (GE Healthcare, catalog number 29-1275-56) was used as the chip. 10 mM glycine-HCl, pH 1.5 (GE Healthcare, catalog number BR-1003-54) was the reduction buffer. The working buffer, analyte dilution buffer, and sample dilution buffer were HBS-EP. Antibodies to α-syn (3A9, 9B11 and 11F11), obtained in example 1, were diluted in 1x HBS-EP (GE Healthcare, catalog number BR-1006-69), alpha-synuclein monomeric form (1 mg/ml) and fibrillar (3 mg/ml) protein were serially diluted in duplicate and a total of 6 concentrations (0, 0.39, 1.56, 6.25, 25, 100 nM) were analyzed, including 0 nM. For capture, the monomer RU number was 800 (theoretical) and the fibril RU number was -100 (theoretical). The capture phase was performed with a contact time of 60 seconds, a flow rate of 30 μL/min, and a stabilization period of 180 seconds. The association phase was performed at an association time of 120 seconds and the flow rate was 30 μL/min. The dissociation phase was performed at a dissociation time of 360 seconds and a flow rate of 30 μL/min. The regeneration phase was performed twice with regeneration times of 240 seconds (primary) and 60 seconds (secondary) and a flow rate of 30 μL/min. Fitting was performed using a 1:1 binding model, and the analytical software was BIACore T200 Evaluation Software (GE Healthcare). The results obtained are shown in Fig. 3a and 3b.

In fig. 3a shows the results of a BIAcore analysis of the specificity and affinity of preferential binding of a monoclonal antibody produced in one example of the present invention to alpha-synuclein aggregates. The antibody of the present invention has been shown to bind to aggregates with high affinity. These results indicate that the antibody in question can effectively eliminate or inhibit the action of an agent causing neurodegenerative diseases associated with alpha-synuclein etiology, such as Parkinson's disease. In fig. 3b is a table showing the results shown in FIG. 3a.

As shown in FIG. 3a and 3b, 3A9, 9B11 and 11F11, which among the four anti-alpha-synuclein antibodies analyzed by other methods described above were confirmed to preferentially bind to aggregates, only bound to aggregates with a high affinity of approximately 1x10'9 in the BIAcore analysis M up to 3x10'9 M.

4-4. Octet assay using mouse monoclonal antibody against α-syn.

A quantitative analysis of the binding of antibodies to alpha-synuclein (3A9, 9B11, 11F11) obtained in example 1 with monomeric and aggregated antigens was carried out using Octet.

Specifically, the running buffer was 1x KB buffer (cat. no. 18-1092) or 1x PBS buffer at 1000 rpm, and the immobilization buffer was sodium acetate, pH 5 (10 mM, cat. no. 18-1068). α-syn monomers. immobilized as α-syn antigen, and fibrils were immobilized for the antibodies analyzed. Target concentrations were 20 μg/mL for monomer and 0.4 μg/mL for fibrils. To evaluate the kinetics, the concentrations were sequentially reduced by half, starting from 50 nM for monomers and 100 nM for fibrils, obtaining a total of 7 points, respectively. The association/dissociation time was 5 min/20 min for monomer and 5 min/25 min for fibrils. The biosensor was ARG2, and the approximation was performed using a 1:1 approximation model. The results obtained are shown in Fig. 4. In FIG. 4a shows the results of an Octet analysis of the preferential binding specificity of an anti-alpha-synuclein antibody according to one embodiment of the present invention to α-syn aggregates.

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As shown in FIG. 4a, 3A9, 9B11, and 11F11 showed little binding to monomers (red dotted box) but bound well to aggregates (increasing portion of the graph in red dotted box). These results are similar or consistent with the results of the dot blot, Octet assay and ELISA assay, and indicate that 3A9, 9B11 and 11F11, among the four antibodies to alpha-synuclein analyzed by other methods described above, were confirmed to bind preferentially with aggregates, in the Octet analysis only aggregates were associated. In fig. 4b is a table showing the results shown in FIG. 4a. The results presented in Fig. 4a and 4b show preferential binding to alpha-synuclein aggregates and are consistent with the results presented in FIG. 1. Antibody #274, used as a control group, bound well to both monomers and aggregates.

Example 5. Detection of Lewy bodies in human brain tissue using an anti-alphasynuclein antibody.

Lewy bodies and Lewy neurites in ten micrometer thick paraffin-embedded brain sections obtained from patients who died of Parkinson's disease (Dr. Halliday, University of Sydney) were stained using the antibody used in Example 5 as follows . Tissue sections were treated with 90% formic acid for 3 minutes to unmask antigens, and then the peroxidase activity of the tissue itself was inhibited with 1% H ₂ O ₂ (based on 50% ethanol). Treatment was carried out with 10% normal horse serum to prevent nonspecific binding to tissues. Then, after washing with phosphate buffer, adjacent sections were treated with antibodies 3A9, 11F11 and 11F11 of the present invention at 4°C overnight. After washing with phosphate buffer, treatment with biotin-conjugated anti-human IgG antibody was performed at 37°C for 30 minutes and reaction to form an avidin-biotin complex at room temperature for 30 minutes (Vectastatin Elite kit; Vector Laboratories).

Staining was then performed using DAB containing 0.005% H ₂ O ₂ and counterstaining the sections with 0.5% cresyl violet to distinguish each cell. The results obtained are shown in Fig. 5a and 5b.

In fig. 8a and 8b show the results that monoclonal antibodies 3A9 and 11F11 according to one embodiment of the present invention can specifically recognize Lewy bodies and Lewy neurites in human brain tissue, respectively. They show that the antibodies of the present invention bind to Lewy bodies (arrows) and Lewy neurites (filamentous form in the lower left part).

As shown in FIG. 8a and 8b, the antibody of the present invention demonstrated effective binding to Lewy bodies and Lewy neurites (indicated by arrows). These results indicate that it is able to effectively bind to alpha-synuclein aggregates contained in Lewy bodies in human brain tissue. It has been shown that antibodies delivered to the human brain can bind efficiently and specifically to alpha-synuclein aggregates. These results indicate that the antibodies of the present invention can, in fact, specifically bind to alpha-synuclein aggregates in human brain tissue and can be effectively used to prevent and/or treat diseases associated with alpha-synuclein etiology.

Example 6: Anti-alpha-synuclein antibody epitope analysis.

Epitope mapping of antibodies 3A9 and 11F11 as chimeric antibodies obtained in example 2 was carried out by requesting peptide allelic analysis from PEPSCAN (Netherlands). The results obtained are shown in Fig. 6. In FIG. 6 is a schematic representation of the results of antibody epitope mapping according to one embodiment of the present invention.

As shown in FIG. 6, it has been demonstrated that most of the antibodies of the present invention bind to the C-terminal region. Antibodies to alpha-synuclein, which recognize the N-terminus, failed to recognize aggregates in other diseases, such as multiple system atrophy, belonging to the synucleinopathies, which are collectively called alpha-synuclein-related diseases. In contrast, anti-alpha-synuclein antibodies that recognize the C-terminal region have the advantage that they recognize aggregates in various other synucleinopathies as well as in Parkinson's disease. In particular, as described above, antibodies recognizing the region between residues 110 and 122 have been shown to bind preferentially to aggregates.

Example 7 ELISA assay using anti-alpha-synuclein antibody.

To quantitatively analyze the binding affinity of the chimeric antibodies (ch11F11) obtained in Example 2 and the humanized antibodies (Hu11F11) obtained in Example 3, a sandwich ELISA was performed using almost the same method as in Example 4-2.

Specifically, each antibody was diluted 1/10 to obtain concentrations ranging from 0.04 to 400 nM and adsorbed onto a 96-well plate, after which synuclein fibril aggregates were added to each well at a concentration of 2000 ng/ml. After washing with 1X PBS, HRP-conjugated streptavidin and secondary antibody biotin conjugate were added and then reacted with TMB as substrate. Optical density was measured. The results obtained are shown in Fig. 7.

As shown in FIG. 7, it was confirmed that the humanized antibodies of the present invention, in particular the humanized antibodies derived from chimeric 11F11 (11F11 humanized antibodies), exhibit binding affinity equivalent to the chimeric clone 11F11. It has been confirmed that humanized antibodies, especially humanized antibodies derived from chimeric 11F11, such as Hu11F11 (ver. 1) containing the combination of Hu11F11-VH-v1 and Hu11F11-VLv3 4c, Hu11F11 (ver.2) containing the combination of Hu11F11- VH-v2 and Hu11F11-VLv3 4c, hu11F11 (ver.3) containing the combination of Hu11F11-VH-v3 and Hu11F11-VLv3 4c, Hu11F11 (ver.4) containing the combination of Hu11F11-VH-v4 and Hu11F11-VLv3 4c, demonstrated affinity binding equivalent to chimeric 11F11, and their EC50 ranged from 11.5 to 15.1 nM, which is similar to the EC ₅₀ of chimeric 11F11, which was 12.5 nM.

Example 8 BIAcore analysis using anti-alpha-synuclein antibody.

To quantitatively analyze the binding affinity of the chimeric antibodies (ch11F11) obtained in Example 2 and the humanized antibodies (Hu11F11) obtained in Example 3, BIAcore analysis was performed using almost the same method as in Example 4-3. The results of the analysis are shown in Fig. 8 and in the following table.

Table 18

Clone name Kd(hM) ChllFll 0.02472 HullFll(ver.2) 0.0596 HullFll(ver.3) 0.0316 HullFll(ver.4) 0.0204

As a result, the humanized antibodies of the present invention, especially variants of clone 11F11, namely Hu11F11 (ver.2) (combination of Hu11F11-VH-v2 and Hu11F11-VLv3 4c), Hu11F11 (ver.3) (combination of Hu11F11-VH-v3 and Hu11F11-VLv3 4c) or Hu11F11 (ver.4) (combination of Hu11F11-VH-v4 and Hu11F11-VLv3 4c), showed KD values similar to those of the chimeric clone 11F11. In terms of binding affinity, the humanized clones showed a KD _of 0.02-0.06x10 ^-9 M, and the chimeric clone 11F11 showed a KD of 0.02x10 ^-9 M. That is, the humanized clones showed high binding affinity to the aggregates.

Example 9: Evaluation of Specific Binding of a Chimeric Antibody to Alpha-Synuclein.

9-1. ELISA assay with beta-synuclein and gamma-synuclein.

After obtaining three chimeric antibodies 3A9, 9B11 and 11F11 according to example 2, the binding specificity of these antibodies to alpha-synuclein was compared according to the results of ELISA analysis with betasynuclein and gamma-synuclein, which are homologues of alpha-synuclein.

To perform the assessment, each protein from human beta-synuclein (Uniprot: Q16143) (CUSABIO, catalog number CSB-EP624090HU) and human gamma-synuclein (Uniprot: 076070) (CUSABIO, catalog number CSB-EP021915HU) was adsorbed onto a 96-well plate at a concentration of 100 ng/ml, washed and subsequently blocked with 5% BSA for 2 hours. In this case, an antibody to all types of synuclein (pan-syn) (Santa Cruz, catalog number FL-140, sc-10717, rabbit), which binds to alpha-synuclein, betasynuclein and gamma-synuclein, was used as a positive control. After washing, chimeric antibodies (3A9, 9B11, 1F11) were diluted 1/10 to concentrations from 400 nM to 0.04 nM and added for 2 hours, washed with PBS and then added goat anti-hFc antibodies coupled to HRP as antibodies for detection. The absorbance was then measured at 450 nm and 650 nm after reacting with TMB as a substrate. For the positive control, goat anti-rabbit HRP-linked antibody was used as the detection antibody.

The results of the experiment are shown in Fig. 9a and 9b, which present the results of the ELISA assay demonstrating that the chimeric antibodies (33A9, 9B11, 11F11) have very low binding affinity to human beta-synuclein and human gamma-synuclein. In contrast, the pan-syn antibody used as a positive control showed high binding affinity to beta-synuclein and gamma-synuclein. Fig. 9a refers to human betasynuclein, and FIG. 9b refers to human gamma-synuclein.

9-2. Dot blot analysis with beta-amyloid and tau protein aggregates.

Three types of chimeric antibodies 3A9, 9B11 and 11F11 obtained in example 2 were reacted with aggregates of amyloid beta _1-42 (Uniprot: P05067; CAS number: 107761-42-2) and tau protein (Uniport: P10636-8). The ability of antibodies to specifically bind to alpha-synuclein aggregates was analyzed by blotting for the following reason.

Amyloid beta _1-42 and tau protein form aggregates that are believed to be important etiological factors in neurodegenerative diseases, especially Alzheimer's disease, and these aggregates have oligomers, protofibrils and fibrils, similar to alpha-synuclein aggregates. Thus, dot blot analysis confirmed that chimeric antibodies 3A9, 9B11 and 11F11 recognize a specific sequence of alpha-synuclein and do not recognize the general structure of aggregates originating from alpha-synuclein, amyloid beta and tau protein.

In this example, the dot blot method was used in much the same manner as in Example 4-3 and recombinant amyloid beta ₁ . ₄₂ , tau proteins and their aggregates were obtained by Professor Seung-jae Lee from Seoul National University. Syn-1, 6E10 and Tau5 are known antibodies that bind to alpha-synuclein, amyloid beta _1-42 and tau protein, respectively. The results of the analysis are shown in Fig. 10.

The results of the experiment are shown in Fig. 10 and demonstrate that the chimeric antibodies 3A9, 9B11 and 11F11 of the present invention bind specifically only to aggregates derived from alpha-synuclein, but not to aggregates derived from amyloid beta _1-42 and tau protein. In contrast, antibodies 6E10 and Tau5, which are known to bind amyloid _{beta1_42} and tau protein, respectively, showed binding to the corresponding aggregates in dot blot analysis.

According to the results described above, the chimeric antibodies do not bind to alphasynuclein homologues and aggregates derived from other proteins, but bind effectively only to the target protein. This suggests that they may be more effective than antibodies or drugs that bind to homologs and aggregates derived from other proteins.

Example 10. Preparation of bispecific antibodies.

10-1. Cloning of a bivalent bispecific antibody.

To construct a bivalent bispecific antibody expression vector, the antibody nucleotide sequence containing the signal sequence was introduced into the multiple cloning site (MCS) of the pcDNA3.4 vector (Invitrogen). The bispecific antibody expression vector was a monocistronic vector, and a heavy chain expression vector and a light chain expression vector were obtained accordingly.

As the heavy chain sequence introduced into the heavy chain expression vector, the anti-IGF1R scFv was linked through a linker to the C-terminus of the immunoglobulin, where the heavy chain variable region encoding the anti-alpha-synuclein antibody and the human heavy chain constant region were linked. As the light chain sequence introduced into the light chain expression vector, a light chain variable region encoding an anti-alpha-synuclein antibody and a human light chain constant region were linked.

10-2. Cloning of a monovalent bispecific antibody.

The monovalent bispecific antibody was a heterodimer containing an anti-alpha-synuclein immunoglobulin heavy chain (hollow) with an anti-IGF1R scFv bound to its C-terminus, an anti-alpha-synuclein immunoglobulin heavy chain (overhang) without a bound scFv, and a light chain conjugated with the indicated heterodimer.

To increase the efficiency of heavy chain heterodimer conjugation, the Knob-in-Hole technique was used. That is, in the CH3 part of the trench-type heavy chain coding sequence, the substitutions T366S, L368A and Y406V were made, and in the CH3 part of the knob-type heavy chain coding sequence, the T366W amino acid substitution was made.

10-3. Transient transfection.

The resulting vector was used in Maxi-Prep (Qiagen) to obtain large amounts of plasmid DNA. It was then introduced into the cells as follows. To produce monovalent BsAb, heavy chain expression vector DNA and light chain expression vector DNA were used for transfection in a 1:1 ratio. To obtain monovalent BsAb, the pit heavy chain expression vector DNA, the knob heavy chain expression vector DNA, and the light chain expression vector DNA were used for transfection in a ratio of 0.5:0.5:1.

On the day prior to transfection, the concentration of ExpiCHO™ cells (Gibco part number A29127) in ExpiCHO™ expression medium (Gibco part number A29100-01) was adjusted to 3-4x106 viable cells per ml and then incubated at 8%. CO2, 37°C and 120 rpm for 1 day. On the day of DNA transfection, cells were diluted to 7-10 x 106 viable cells per ml with 95% survival rates or better with fresh media to 6 x 106 viable cells per ml.

For transfection of parental cells, ExpiFectamine™ CHO and plasmid DNA were complexed using the ExpiFectamine™ CHO Transfection Kit (Gibco, catalog number A29129). DNA and ExpiFectamine™ CHO reagents were prepared at appropriate concentrations, added to old OptiPRO™ SFM® media (Gibco, part number 12309019) and mixed, leaving at room temperature for 5 minutes. The resulting product was added to the original cells and their post-transfection cultivation began. The day after transfection, the Enhancer and Feed reagents included in the ExpiFectamine™ CHO kit were added to the transfected cells

Transfection Kit, after 5 days the Feed reagent was added again and incubation was carried out for 10 days at 8% CO2, 37°C and 120 rpm to obtain transfected cells.

10-4. Collection of the environment.

To obtain the culture solution, upon completion of production, the culture medium was transferred to a centrifuge bottle for centrifugation and centrifuged at 4° C. and 6500 rpm for 30 minutes, followed by filtration through a 0.2 μm filter to obtain a culture medium free of suspended solids. The resulting culture medium was then used for subsequent purification.

Example 11. Preparation of anti-IGF1R antibody (scFv).

11-1. Preparation of anti-IGF1R antibody (scFv).

Monoclonal antibodies were obtained using the phage display/panning technique. Specifically, the antigens used in phage display panning and other assays were used as the following proteins. A peptide consisting of residues 31-932 of amino acid sequence SEQ ID NO:99, where the signal sequence was removed from the extracellular domain of human IGF1R, was tagged with histidine at the C-terminus and used for this example (R&D Systems, USA, 391-GR). Monkey IGF1R (National Research Council of Canada), mouse IGF1R (R&D Systems, 6630-GR/CF), and rat IGF1R (National Research Council of Canada) with a His tag at the C-terminus were used as antigen for cross-species crossover analysis. reactivity.

1x10 ¹⁰ cells of the resulting scFv (single chain variable fragment) library, which had diversity and were of human origin (obtained by SHIM Hyunbo at Ewha Womans University), were added to 2X YT medium (17 g tryptone (CONDA, 1612.00), 10 g yeast extract (CONDA , 1702.00) and 5 g NaCl (Sigma, S7653)), containing 34 μg/ml chloramphenicol (Sigma, C0857), 2% glucose (Sigma, G5400) and 5 mM MgCl ₂ (Sigma, C0857), at 30°C at 3 hours up to OD600 0.5-0.7. Then the cells were infected with a helper phage and cultured in 2X YT medium containing 34 μg/ml chloramphenicol, 5 mM _MgCl2 , 70 μg/ml kanamycin (Sigma, K1876) and 1 mM IPTG (ELPISBIO, IPTG025), at 30°C for for 16 hours to induce phage packaging. Then the culture solution was centrifuged at 4500 rpm and 4°C for 15 minutes. 4% PEG 6000 (Fluka, 81253) and 3% NaCl (Sigma, S7653) were added to the supernatant and incubated for 1 hour on ice. The resulting product was centrifuged at 8000 rpm for 20 minutes at 4°C, then the pellet was suspended in PBS and centrifuged again at 4°C and 12000 rpm for 10 minutes to obtain a supernatant containing the phage library. The resulting supernatant was stored at 4°C until further use.

11-2. Panning with phage display.

To screen for human anti-IGF1R antibody, three rounds of panning were performed as described below. The phage library was a synthetic human scFv library, and the phage display panning procedure and results obtained are shown in Table. 19.

Table 19

Stage Panning 1st round 2nd round 3rd round Antigen ECD IGF1R (biotinylated) ECD IGF1R (biotinylated) Cells MCF-7 Sorption method Indirect immobilization Indirect immobilization — Original quantity 7.0 x 10 ¹² 6.0 x 10 ¹² 5.0 x 10 ¹² Exit IGF1R or MCF-7 4.9 x 10 ⁸ 3.3 x 10 ⁵ 1.2 x 10 ⁵ Flushing PBS-T** 5 times 10 times 10 times PBS 2 times 2 times 2 times

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 immunological tube (Maxisorp 444202) and adsorbed onto the surface immunological tube at 4°C for 16 hours. The supernatant was then removed and incubated with PBS containing 3% BSA at 37°C for 1 hour to block nonspecific binding.

- 87 045348 through binding of BSA to the non-IGF1R surface. After removing the supernatant, the phage library obtained in Example 11-1, mixed with 1.5% BSA solution, was placed in an immunological tube and reacted at 37°C for 1 hour, allowing IGF1R-specific phages to contact the antigen. The resulting product was then washed with PBS-T (phosphate buffered saline with 0.05% Tween 20) to remove nonspecifically bound phages, and IGF1R bound phages were collected using 100 mM triethylamine solution.

The collected phages were neutralized with a buffer solution containing 1 M Tris (pH 7.4), transfected with E. coli K12ER2738 at 37°C for 1 hour, and infected E. coli were distributed on LB agar medium containing tetracycline and carbenicillin and cultured at 37°C C during the night. The next day, cultured E. coli were suspended in 5 ml of SB (superbroth) medium containing tetracycline and carbenicillin, and an equal volume of 50% glycerol was added. One part was stored at -80°C, and 50 μl of the product was suspended in 40 ml of SB medium (superbroth) containing tetracycline and carbenicillin, 1012 PFU of the helper phage VCSM13 was added and cultivation was carried out with stirring at 37°C for 1 hour. Kanamycin was then added to the culture solution and culture was carried out at 30°C for approximately 16 hours, culturing only E. coli infected with the helper phage.

The next day, after centrifugation of the culture solution, the supernatant was collected and added to a buffer containing 4% PEG 8000 and 3% sodium chloride (NaCl), reacted at 4°C for approximately 1 hour, phages were pelleted and centrifuged. After removal of the supernatant, the pool of pelleted phages was resuspended in PBS buffer containing 1% BSA and used for the next round of panning. During later rounds of panning, the number of PBS-T washes was increased to increase the number and concentration of antigen-specific phages.

11-3. Screening of individual clones.

Cell clones were selected that demonstrated binding affinity to the ECD (extracellular domain) of human IGF1R and MCF-7 expressing IFG1R.

Specifically, to select monoclonal antibodies that specifically bind to IGF1R from the phage pool obtained in Example 11-2, the following experiment was performed.

To isolate monoclones from a concentrated pool, a pool of phages obtained on tetracycline/carbenicillin LB agar medium was plated onto the medium and cultured to obtain individual colonies. After seeding these colonies in a deep 96-well plate and incubating overnight, 10 µl of the resulting culture solution was again added to a deep 96-well plate and incubated in the same way at 37°C for approximately 4 hours until a suitable OD (from 0 .5 to 0.7). After adding 20 MOI of helper phage to the culture solution, the resulting mixture was reacted at 37°C for 1 hour. Then kanamycin was added to the culture medium and cultivation was carried out overnight at 30°C. The next day, the culture medium was centrifuged and the supernatant was collected for an ELISA to select IGF1R-specific phages (Steinberger. Rader and Barbas III. 2000. Phage display vectors. In: Phage Display Laboratory Manual. 1 sted. Cold Spring Harbor Laboratory Press NY.USA . Pp.11.9-11.12).

100 ng of recombinant IGF1R was added to each well of the ELISA plate and reacted at 4°C for approximately 15 hours to adsorb the antigen onto the plate. To prevent nonspecific binding, 200 μl of PBS buffer containing 3% BSA was added to each well and the reaction was carried out at 37°C for approximately 1 hour. The supernatant was removed.

100 μl of a solution containing the resulting monoclonal phage was added to each well, the interaction was carried out at 37°C for 1 hour and washed 3 times with 300 μl of PBS-T. To detect phage associated with the IGF1R antigen, the anti-HA antibody with HRP was diluted 1:5000 in PBS buffer containing 3% BSA and reacted at 37°C for 1 hour. After washing 3 times with 300 µl PBS-T, 100 µl TMB (tetramethylbenzidine, Sigma, T0440) was added for staining, and 50 µl 1 N H2SO4 was added to stop the reaction. By measuring the optical density at 450 nm, clones with high optical density compared to the BSA control group were selected as antigen-specific antibody clones.

Seven (7) types of clones were selected through double screening: 1564, 48G5, 49G11, 54H4, 60A11, 60H6 and B11.

Example 12. Preparation of an affinity variant of an antibody to IGF1R.

Antibodies were optimized by varying the affinity of selected clones by assessing their ability to bind to ligand and penetrate the BBB. In the first experiment, a manual mixture of NNS primers was prepared to randomize heavy chain CDR2 and light chain CDR3 based on scFv 1564, and the scFv 1564 gene containing the randomization sequence was amplified using PCR techniques. The amplified gene products were introduced into the pComb3x vector to produce a library in a form suitable for phage display, and through panning using this library and ELISA screening, several scFv clones were selected that bound to IGF1R. The amino acid sequence of the variable region of the selected clones was determined by gene sequencing.

In the second experiment, two mini-libraries of the heavy and light chain were constructed by introducing a germline reverse mutation in CDR1, CDR2 and CDR3, respectively. Based on the analysis of 96 colonies, it was confirmed that 31 VL clones and 39 VH clones had unique sequences. As a result, clones were obtained by selecting affinity variants based on productivity and antigen binding affinity.

Example 13. Preparation of antibody variants having a residue subject to deamidation.

13-1. Determination of the residue subject to deamidation.

When deamidation occurs in the CDR region, the antibody is degraded and its binding to the antigen is weakened, which can lead to decreased efficiency and sample heterogeneity. The heterogeneity of samples leads to difficulties in its identification in clinical studies. Therefore, an attempt was made to determine the positions at which deamidation occurs using computer analysis and peptide mapping.

As shown in FIG. 8a, computer analysis and peptide mapping revealed deamidation of the original clone 1564. To do this, samples were stored at 4°C or 40°C for one week before analysis, and deamidation was confirmed to occur in L-CDR2, LCDR3 and H -CDR2. To confirm the sites of deamidation, the affinity variants disclosed in Example 12 were also analyzed.

13-2. Obtaining antibody variants.

In computer analysis, the original clone 1564 had a net charge of -0.7. Typically, with a net charge of less than 0 or greater than 5.5, the antibody undergoes rapid clearance in the body. Therefore, it is expected that replacing residues susceptible to deamidation with positive charges such as H, R, K, etc. will prevent rapid clearance by eliminating deamidation and increasing the overall charge. Thus, mutants with substitutions of deamidation sites were obtained, as shown in Fig. 18b.

To remove the residue susceptible to deamidation, a mutant having a substitution at this residue was prepared as follows.

1) In the amino acid sequence, Asn was replaced by D or Q, similar to Asn. If there was no change in the binding affinity of the mutant, all residues were replaced with Q.

2) Since the net charge of the original clone was -0.7 and the net charge resulting in rapid clearance is less than 0 or more than 5.5, it is preferable for the charge to become positive. Therefore, N95a, the L-CDR3 residue susceptible to deamidation, was replaced by H, R, and K, which have a positive charge.

Example 14. Preparation of various forms of antibodies against IGF1R.

14-1. Preparation of anti-IGF1R minibody.

The minibody was prepared by coupling the full-length scFv IGF1R-specific monoclonal phage antibody obtained in Examples 11-13 to the C-terminus of the Fc. To do this, a nucleotide sequence encoding the amino acid sequence of the scFv as disclosed in the present invention was obtained, and the resulting nucleotide sequence was digested with a restriction enzyme and cloned into a pcDNA-based expression vector containing the nucleotide sequence encoding the Fc.

14-2. Preparation of bivalent anti-IGF1R antibody.

A full-length scFv of the IGF1R-specific monoclonal phage antibody prepared in Examples 11-13 was prepared, and two full-length scFvs were coupled to each C-terminus of the therapeutic antibody in IgG form to produce a bivalent antibody. To do this, the nucleotide sequence encoding the amino acid sequence of the scFv as disclosed in the present invention, digested with a restriction enzyme, was obtained and cloned into a pcDNA-based expression vector containing the nucleotide sequence encoding the therapeutic antibody.

14-3. Obtaining an IgG antibody (Full-IgG) against IGF1R.

To convert the sequences of antibody 1564 and antibody F06 into the form of full-length IgG1 (Full IgG) IGF1R-specific monoclonal phage antibodies obtained in Examples 11 and 12, the nucleotide sequences of the heavy chain and light chain regions were synthesized (Genotec Inc.). The synthesized heavy chain and light chain genes were cloned into expression vectors.

14-4. Preparation of monovalent antibody with scFv against IGF1R.

Example 14-1 shows a minibody form where the scFv form of the anti-IGF1R antibody is linked to each C-terminus of two heavy chain Fcs. In this example, one scFv is linked to the C-terminus of only one heavy chain Fc. In the antibody form obtained in Examples 11-13, a vector was constructed where IGF1R-specific monoclonal phage antibodies 1564, F06, C04, VH5, VH16, VH35, VH9, VH2, VH7 and VH32 were linked to the C-terminus of only one Fc, and the vector without anti-IGF1R antibody associated with Skontz. When producing antibodies in cells, ridge-to-hole mutations were introduced into the Fc region to obtain a heterodimeric form. When transfected into CHO-S cells to produce an antibody, all three

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The C-terminal Fc of the therapeutic antibody, a vector corresponding to the heavy chain in which the anti-IGF1R antibody is not linked to the C-terminal Fc of the therapeutic antibody, and a vector corresponding to the light chain of the therapeutic antibody were injected into CHO-S cells.

14-5. Expression and purification of various anti-IGF1R antibodies.

The vectors obtained in examples 14-1, 14-2 14-3 and 14-4 were introduced into cells as follows.

Specifically, the concentration of CHO-S cells was adjusted to 1.5 x 10 ⁶ cells/ml in CD-CHO medium (Gibco, 10743) and then cultured at 8% CO2 and 37°C for 1 day. On the day of DNA transfection, cells grown to 2.5-3x10 ⁶ cells/ml were prepared at a concentration of 2.1x10 ⁶ cells/ml using CD-CHO medium containing 1% DMSO, and then cultured under 8% CO2 conditions. and 37°C for 3 hours. After centrifugation at 3000 rpm for 15 minutes, the supernatant was removed and resuspended in RPMI 1640 medium with 2.5% FBS.

The vector combination was then diluted in Opti-MEM medium to a concentration of 1 μg per 1 ml of medium and PEI (Polysciences, 23966, initial concentration 1 mg/ml) was diluted to 8 μg/ml of culture medium. After mixing the DNA with the PEI mixtures and leaving the resulting mixture at room temperature for 10 minutes, the mixture was poured into the vial containing the cells and incubated for 4 hours at 5% CO ₂ , 37°C, 100 rpm. Then the mixture was cultured with the addition of CD-CHO medium in a volume equal to the volume of the culture, and incubated at 8% CO ₂ , 37°C, 110 rpm for 4 days.

The resulting culture solution was passed through MabSelect SuRe (GE healthcare, 5 ml), equilibrated by passing equilibration buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl), allowing the expressed antibody to bind to the column. Then, after elution with a solution of 50 mM sodium citrate (pH 3.4) and 100 mM NaCl, neutralization was carried out using 1 M Tris-HCl (pH 9.0) such that the final pH was 7.2. After this, the buffer solution was exchanged using PBS (phosphate buffered saline, pH 7.4) and, when the degree of purity was high, the resulting solution was stored at -20°C, and if further purification was necessary, it was stored at 4°C until further purification. cleaning.

When additional purification was necessary, it was performed using HiLoad Superdex 200 (GE Healthcare, part number 28-9893-36) with the option of various size exclusion chromatography options. After equilibration with equilibration buffer (1x phosphate buffered saline, pH 7.4, Gibco, part number 10010-023), the primary purified sample was loaded onto the column. The resulting sample, which had undergone complete purification, was stored frozen at -20°C.

14-6. Preparation of bispecific antibody with anti-alpha-synuclein antibody.

The anti-IGF1R antibody in scFv form of the present invention was prepared by linking the heavy chain variable region and the light chain variable region using a linker (SEQ ID NO:411) and connecting it to the C-terminus of the heavy chain constant region of the full-length IgG form of the anti-alpha-synuclein antibody described in the following table, using a linker (SEQ ID NO:412), obtaining a bispecific antibody. In this example, a monovalent antibody was produced by binding one scFv form of anti-IGF1R antibody per molecule of anti-alpha-synuclein IgG antibody, and a bivalent antibody was produced by binding two molecules of scFv form of anti-IGF1R antibody per molecule of anti-alpha-synuclein IgG antibody, respectively. The sequences of the anti-alpha-synuclein antibody used to produce the bispecific antibody in this example and examples of bispecific antibodies produced in accordance with the present invention are described in Table. 17. Bispecific antibodies, examples of which are given in table. 17 were used for experiments in subsequent examples.

Example 15 IGF1R-Specific Binding Affinity Assay Using Anti-IGF1R Antibody.

15-1. IGF1R-specific binding affinity assay using anti-IGF1R minibody antibody (ELISA).

An ELISA assay was performed to evaluate the binding affinity and concentration-dependent binding of minibody clones 996, 1226, 1564 and MKJP2 obtained in Example 14-1 to recombinant IGF1R.

Specifically, the human recombinant IGF1R, which is the target to which the antibody binds, is an extracellular domain (ECD) and was purchased from R&D Systems (6630GR/CF). Human IGF1R was diluted to 1 μg/ml in PBS buffer, added to a 96-well ELISA plate (Nunc-Immuno Plates, NUNC, Rochester, NY) in an amount of 100 μl per well, and adsorbed at 4°C. C for 16 hours, and then the supernatant was removed.

PBS buffer containing 3% BSA (bovine serum albumin), 200 μl per well, was added and reacted for 2 hours to block nonspecific binding.

A 3-fold dilution of the minibodies of clones 996, 1226, 1564 and MKJP2 obtained in example 14-1 was carried out, starting with a maximum concentration of 20 nM, obtaining 12 points, then 100 μl was transferred

- 90 045348 into each well and left at room temperature for 1 hour. After this, the plate was washed 4 times with PBS buffer containing 0.05% Tween 20, and the interaction was carried out at room temperature for an hour, adding 100 μl of an anti-human antibody with HRP, which recognized the human Fc minibody, to each well at a dilution of 1:5000 in the blocking buffer. After washing 4 times with 300 μl PBS-T (Tween 20, 0.05%) staining was performed using TMB (tetramethylbenzidine, Sigma, T0440). The enzymatic reaction was quenched with 0.5 mol/L sulfuric acid, and the absorbance was determined and analyzed at 450 nm using a microplate reader. The results of the experiment are shown in Fig. 1a.

Four minibody clones were confirmed to bind to human recombinant IGF1R protein in a concentration-dependent manner, and specifically MKJP2 showed the highest binding potency, followed by clones 996 and 1564 showing similar binding potency, and clone 1226 showing slightly lower potency binding.

15-2. ELISA analysis of interspecies cross-reactivity of antibodies to IGF1R.

The cross-species cross-linking activity of the anti-IGF1R antibody 1564 prepared by the method described in Example 14-2 and the anti-IGF1R antibody prepared in Example 11-3 was analyzed by ELISA assay. For this purpose, human, monkey, mouse and rat IGF1R antigens were first diluted to 1 μg/ml, 100 μl was added to each well, and the interaction was carried out at 4°C for 15 hours for their sorption to the bottom of the plate. After removing the supernatant, 200 μl of PBS buffer containing 3% BSA was added to each well to block nonspecific binding. Anti-IGF1R antibodies were diluted 5-fold in PBSB (BSA, 3% in PBS) from a maximum concentration of 400 nM, added to each well, and reacted at 37°C for 1 hour. Then, after washing 5 times with PBS buffer, the anti-human Fab antibody with HRP, which recognizes the Fab part of the bound antibody, was diluted 1:20000, 100 μl was added to each well and reacted at 37°C for 1 hour. The resulting product was washed 5 times with PBS buffer and stained using TMB (tetramethylbenzidine, Sigma, T0440) following the manufacturer's method. The enzymatic reaction was quenched with 0.5 mol/L sulfuric acid, and the absorbance was measured at 450 nm using a microplate reader (Molecular Devices). When including a large number of samples in the ELISA assay, two plates were used. The results of the experiment are shown in table. 12 below.

Specifically, in table. 12 ELISA results of bispecific antibodies to human IGF1R results of ELISA IgG 1564 and bispecific antibodies to human IGF1R, mouse IGF1R, rat IGF1R and monkey IGF1R are shown in table. 12.

The experimental results set forth below demonstrate the advantage of assessing efficacy using models in different animal species and show that the effectiveness of therapeutic agents in disease models in different species can be assessed using the antibodies of the present invention.

Table 20

Results of ELISA analysis of the ability of antibodies to bind to IGF1R of various types

Experiment Antibody clone ECso (nM) Human IGF1R ELISA chllFll-1564 0.914 chllFll-48G5 1.21 chllFll-54H4 2.88 chllFll-60H6 10 chllFll-Bll 7.13 Human IGF1R ELISA 1564 IgG 0.0823 chllFll-1564 0.379 Mouse IGF1R ELISA chllFll N/A* chllFll-1564 3.02

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chllFll N/A* chi 1F11-48G5 6.2 chllFll-54H4 N/A chllFll-60H6 18.6 chllFll-Bll 148 Rat IGF1R ELISA chllFll N/A* chllFll-1564 1.05 chllFll-48G5 2.44 chllFll-54H4 14.2 chllFll-201** N/A* chllFll-1564 0.874 chllFll-60H6 38 chllFll-Bll 35.1 Monkey IGF1R ELISA chllFll N/A* chllFll-1564 2.48 chllFll-48G5 6.69 chllFll-54H4 8.83 chllFll-201** N/A* chllFll-1564 2.21 chllFll-60H6 N/A chllFll-Bll 180

* Not available, ** scFv form of Herceptin biosimilar.

15-3. IGF1R affinity variant binding (FACS) analysis.

The binding affinity of the affinity variants obtained in Example 12 was assessed by IGF1R ECD ELISA, and the binding affinity to MCF-7 was analyzed by FACS.

As an analysis of the primary clones in Table. 21 shows the results of ELISA analysis of the corresponding primary selected clones in the form of bispecific antibodies with ECD IGF1R, and table. 22 shows the results of binding affinity analysis for the MCF-7 cell line by FACS.

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As a result, clone F06 was selected as the clone with the highest cell binding ability compared to the parent clone (clone 1564) (affinity maturation), and clone C04 was selected as the clone with the least high cell binding ability by compared to the original clone 1564 (reduced affinity).

As an analysis of secondary clones in table. 23 shows the results of an ELISA binding of secondary clones in the form of bispecific antibodies to the IGF1R ECD.

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Table 23

ELISA results of secondary selected clones with ECD IGF1R

Antibody clone EC ₅ o (hM) HullFll(ver.2)-1564 0.259 Monovalent chllFll-1564 0.347 Hui 1F1 l(ver.2)-C04 0.15 Hui 1F1 l(ver.2)-F06 0.147 HullFll(ver.2)-1564 0.864 chllFll-F06 0.857 Hui 1F1 l(ver.2)-VH2 135 Hui 1F1 l(ver.2)-VH5 0.366 HullFll(ver.2)-1564 0.157 Hui 1F1 l(ver.2)-VH7 402 Hui 1F1 l(ver.2)-VH9 6.06 HullFll(ver.2)-VH16 0.236 HullFll(ver.2)-1564 0.149 Hui 1F1 l(ver.2)-VH32 121 Hui 1F1 l(ver.2)-VH35 0.167 Hui 1F1 l(ver.2)-VH27 N/A*

Clones for analysis by FACS were selected as shown in Table. 24, after clones with significantly reduced productivity and deteriorated physical properties were excluded from the secondary obtained clones.

Table 24

Clones for analysis using FACS analysis

Affinity category binding Antibody clone Explanation Binding affinity similar to parent clone 1564 C04 FACS and in vivo analysis F06 FACS and in vivo analysis VH5 FACS and in vivo analysis VH16 FACS and in vivo analysis VH35 FACS and in vivo analysis Binding affinity reduced by 50 times VH9 FACS and in vivo analysis C12 Undesirable physical properties Binding affinity reduced by 50-fold or more VH2 FACS and in vivo analysis VH6 Undesirable physical properties VH7 FACS and in vivo analysis VH27 Undesirable physical properties VH32 FACS and in vivo analysis

In fig. 12c shows the results of a FACS analysis of the binding of clones to the MCF-7 cell line, and all clones analyzed had lower binding affinity to MCF-7 than the parent clone 1564. These results indicate that the clones that showed reduced binding ability in the ELISA also showed reduced binding capacity in FACS.

Antibody clones F06, C04, VH2, VH5, VH7, VH9, VH16 and VH32 were selected, and the amino acid sequences of the heavy chain variable regions and light chain variable regions of these antibodies are shown in Table. 5 and 6 above.

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15-4. BIAcore analysis with human IGF1R.

The ability of the antibody of the present invention to bind to human

IGF1R.

The extent of binding of the IgG form of clone 1564 to human IGF1R was analyzed by SPR analysis. The anti-his antibody against the His tag associated with the ECD of human IGF1R as an antigen was diluted to 20 μg/ml in acetate buffer, pH 4.0, and then immobilized in the reference/analytical channel of the CM4 chip to 10,000 RU as the target number RU method amine combination. During capture, PBS was used as the running buffer and 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 working buffer. Association/dissociation times were 5 minutes and 20 minutes, respectively. The analysis was carried out in the following order: 1 baseline measurement, activation (EDC NHS), human IGF1R addition, quenching (1 M ethanolamine), 2 baseline measurement, association and dissociation. The obtained results were evaluated using the bivalent model and analyzed using Biacore T200 Evaluation Software (version 1.0, serial number 04Y15X11-0149).

From the analysis, it was confirmed that the KD of IgG antibody 1564 was 2.5305 x 10 ^-9 nM and the KD of IgG antibody F06 was 4.7802 x ^{10 -7} nM, both of which showed high binding ability to human IGF1R. The results of the analysis are shown in Fig. 11b. In particular, when clone 1564 was produced in IgG form, it demonstrated a dissociation constant of 2.5305 x 10 ^-9 nM for human IGF1R and allowed us to confirm that there was no significant change in binding affinity depending on the antibody form.

Example 16: Analysis of the ability of anti-IGF1R antibody to bind to a cell line expressing human IGF1R and brain endothelial cells.

16-1. FACS analysis for MCF-7.

To confirm that the minibody clones 996, 1226, and 1564 obtained in Example 14-1 bind to endogenous IGF1R on the cell surface, binding affinity analysis was performed on cell lines expressing human IGF1R and brain endothelial cells using FACS. . The extent of binding to MCF-7, a known breast cancer cell line that overexpresses IGF1R, was analyzed by FACS.

Specifically, each of the three minibodies was diluted to 20 μg/ml, 0.43x10 ⁶ MCF-7 cells were added to each sample and the interaction was carried out at 4°C for 1 hour. After washing twice with PBS buffer, human antibody with FITC, diluted 1:500, was added and the reaction was carried out at 4°C for 1 hour. After washing twice with PBS buffer, the degree of binding of anti-IGF1R minibodies was measured using a FACS Calibur instrument. MCF-7 cells treated with secondary antibodies only were used as a control. The results of the experiment are shown in Fig. 12a.

Binding affinity of A02, A06, A07, B01, B02, B09, B10, C04, D03, E06, F06, H04(Gly), H04(Val), VH2, VH5, VH7, VH9, VH16, VH32 and VH35 obtained in example 12 and example 14-2, MCF7 was analyzed in the same manner as described above. Clone 1564, obtained by the method used in Example 14-2, was used as reference source clones, and MCF-7 cells treated with secondary antibodies only were used as controls. The results of the analysis are shown in table. 22 and in fig. 12s.

The results of the above experiment were expressed as the mean fluorescence intensity (MFI) of the sample, and three scFv minibodies, bispecific antibody affinity variants, and the parent clone (clone 1564) specifically bound to endogenous IGF1R expressed on the cell surface. These results demonstrate that the clones obtained in the examples described above can be used for their intended purpose through their binding to IGF1R in a form actually present in the body.

16-2. FACS analysis for JIMT-1 and BT474.

The minibodies of clones 996, 1226 and 1564 obtained in Example 14-1 were analyzed in much the same manner, except that instead of the MCF-7 cell line used in Example 16-1, breast cancer cell lines LMT-1 and VT474. Binding to endogenous IGF1R on the cell surface was confirmed morphologically. The results of the experiment are shown in Fig. 12a.

The results of the above experiment were expressed as the mean fluorescence intensity (MFI) of the corresponding sample, and it was confirmed that the three scFv minibodies analyzed specifically bound to endogenous IGF1R on the surface of various IGF1R-expressing cell lines.

16-3. FACS analysis of mouse brain endothelial cells.

Possible binding of clone 1564 was analyzed in the form of a bispecific antibody obtained by the method used in Example 14-2, and in the form of an IgG obtained by the method used in Example

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14-3, with brain endothelial cells bEND.3. Therefore, a secondary antibody only treatment group and a therapeutic antibody only treatment group in the form of IgG (CH11F11) were used as negative controls. FACS analysis was carried out in the same way as in examples 16-1 and 16-2. The results of the analysis are shown in Fig. 12b.

All clones analyzed showed binding to bEND.3, with the exception of negative controls. These results confirm that different forms of clone 1564 bind specifically to IGF1R expressed on the surface of brain endothelial cells.

Example 17 Intracellular internalization assay of anti-IGF1R antibody.

17-1. MCF-7 internalization assay - 1564, 996, 1226, MKJP2 (minibodies).

This Example was performed to test whether the minibody clones 996, 1226, 1564, and MKJP2 obtained in Example 14-1 undergo intracellular internalization by an IGF1R-expressing cell line and whether these antibodies enter cells through the RMT pathway. without being subject to degradation. In order for an anti-IGF1R antibody to be used as a carrier to enhance the ability to penetrate the BBB, it must be internalized by the brain endothelial cells that make up the BBB.

The intracellular internalization of antibodies of the present invention was analyzed using the MCF-7 cell line expressing IGF1R. Specifically, after seeding 30,000 MCF-7 cells into an 8-well glass slide, the cells were cultured for 1 day. Minibodies of clones 996, 1226, 1564 and MKJP2, obtained in example 14-1, were added to each well with cultured cells at a concentration of 5 μg/ml at 4°C for 2 hours, washed three times with cold DMEM, and an antibody against human Fc was added , conjugated to Alexa488, at 4°C for 1 hour.

To assess the internalization of antibody complexes, the plate was transferred to a CO2 incubator and incubated at 37°C for 30 minutes. The culture was fixed by adding 100% methanol, and the reaction was stopped at the same time. After fixation, the plate was washed 3 times using PBS. Using a fluorescence microscope, the degree of antibody internalization was visualized in the area of a green filter (Alexa488). For imaging, nuclei within cells were stained using DAPI to confirm the location of each well. The results of the experiment are shown in Fig. 13a.

The results of the experiment showed that all four antibodies analyzed in this experiment using the MCF-7 cell line were well internalized. In particular, clones MKJP2 and 1564 were found to be internalized to a greater extent than other clones.

17-2. MCF-7-CO4, F06, VH5, VH16 internalization assay. VH35, VH9, VH2, VH7, VH32.

The binding of 1564 variants having altered IGF1R binding ability to cell surface IGF1R was analyzed by FACS analysis using the IGF1R expressing MCF-7 cell line. 2x105 MCF-7 cells were treated with a bispecific antibody generated using anti-IGF1R scFv antibody at a concentration of 10 μg/ml for 30 minutes. After washing with PBS buffer containing 1%BSA, a secondary antibody coupled to FITC was added to detect human antibodies for 1 hour. After washing with PBS buffer, FACS analysis confirmed the extracellular binding and internalization of various variants with altered binding affinities.

As shown in table. 25, a bispecific antibody containing anti-IGF1R antibody 1564 was found to exhibit greater internalization and greater intensity at 37°C than under refrigerated conditions. These results indicate that the 1564 variants bind well to cells and are internalized by cells in a binding-dependent manner.

Table 25

Sample Geometric mean Internalization at 37°C No processing 1.88 Only 2nd antibody 2.86 hu3A9 WT 3.4 hu3A9xl564 WT 7.72 hullFll WT 3.18 hullFl1x1564 WT 7.34 hu3A9xl564_C04 7.23

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hu3A9xl564_F06 19.8 hul 1F1 lxl564_VH5 6.1 hul 1F11X1564 VH16 5.83 hul 1F1 lxl564_VH35 7.28 hul 1F11X1564 VH9 5.01 hul 1F1 lxl564_VH2 3.19 hul 1F1 lxl564_VH7 3.84 hul 1F1 lxl564_VH32 3.24

17-3. Analysis of internalization by human brain endothelial cells.

We tested whether the bivalent form and monovalent form of clone 1564 obtained in Examples 14-2 and 14-4 were internalized by primary human brain microvascular endothelial cells (HMBEC). The therapeutic IgG antibody (11F11) was used as a negative control.

HMBEC (Cell Systems, catalog number ACBRI376) was seeded into a 12-well plate to 90% monolayer closure, followed by the addition of the antibody to be analyzed. The next day, after fixation with 4% paraformaldehyde and rinsing with PBS, blocking and permeabilization was performed using a solution containing 3% BSA and TritonX for 50 minutes. After washing with PBS, the anti-human Fc antibody (goat anti-human antibody) was incubated for 2 hours and 30 minutes, washed with PBS, and a secondary antibody against the corresponding primary antibody was added for 1 hour. After washing with PBS, cells were stained with Hoechst dye for 10 minutes at a concentration of 1:1000 to stain the nuclei. The results were analyzed under LSM 780 NLO EC Plan-Neofluar 100X /1.3 Oil on a confocal microscope. The results of the experiment are shown in Fig. 13b.

The bivalent form and monovalent form of clone 1564 demonstrated greater neutralization than the negative control therapeutic antibody (11F11). These results indicate that the anti-IGF1R antibody described above can effectively cause the therapeutic antibody to be internalized by brain endothelial cells constituting the BBB into various bispecific antibody forms containing the associated therapeutic antibody, thereby increasing the ability of the therapeutic antibody to cross the BBB.

17-4. Analysis of further transformations of antibodies in human brain endothelial cells.

If the antibody is internalized and colocalizes with a lysosomal marker inside the cell, then such antibody cannot pass through the BBB due to its destruction in brain endothelial cells. In contrast, if an antibody colocalizes with an early endosome associated with exocytosis or a known marker associated with BBB passage, the antibody is expected to cross the BBB by receptor-mediated transcytosis, i.e., be internalized by brain endothelial cells and then exists in the brain.

After the same treatment of HMBEC with bivalent form 1564, from the antibodies analyzed in Example 17-2, it was analyzed which cellular component of these cells colocalized with these antibodies. However, goat anti-human antibodies detecting processed antibodies after blocking and permeabilization were treated simultaneously with each of the following antibodies:

anti-cathepsin D antibody: lysosomal marker;

anti-caveolin-1 antibody: a marker of caveolin-mediated transcytosis (presumably the main mechanism of passage across the BBB);

anti-EEA1 antibody: a marker of early endosomes.

The remaining methods were the same as in Example 17-2, but appropriate secondary antibodies were used for markers.

The results of the analysis are shown in Fig. 13s. Clone 1564 in the bispecific antibody form did not colocalize with cathepsin D, but colocalized with caveolin-1 and EEA1 in the cell membrane and cells. These results indicate that after internalization of clone 1564, it could pass through the BBB via the RMT pathway, bypassing intracellular degradation mechanisms.

Example 18. Analysis of the effect of anti-IGF1R antibodies on signaling through IGF1R.

18-1. Proliferation assay of MCF-7 cell line using IGF1R.

The possible effect of the anti-IGF1R antibody of the present invention on the binding of IGF1R (IGF1 receptor) to its ligand was analyzed by the efficiency of cell proliferation under the influence of IGF1.

A 5-fold dilution of the minibodies of clones 996, 1226, 1564 and MKJP2 obtained in example 14-1 was carried out from 400 nM, respectively, to obtain diluted samples and then 25 µl of 20 ng was added to 25 µl of each diluted sample /ml IGF1. MCF-7 cells expressing IGF1R were cultured, passaged, removing the medium on the day of the experiment, and 20,000 cells (corresponding to 50 μl) were added to each well of a 96-well plate into which IGF1 and the antibody to be analyzed were added.

After 3 days of incubation at suitable temperature and humidity, 10 μl of the CCK8 reagent was added to assess the degree of cell growth and incubation was carried out in a CO ₂ incubator for 4-5 hours. The plate was then removed and the absorbance was measured at 450 nm using a spectrophotometer.

The results of the experiment are shown in Fig. 14A.

Based on the experimental results, it was confirmed that the antibody of the present invention did not inhibit the proliferation of MCF-7 cells caused by IGF1 signaling through IGF1R. An anti-IGF1R antibody (Imclone) used as a control group inhibited the proliferation of MCF-7 cells mediated by IGF1 signaling through IGF1R in a concentration-dependent manner. Therefore, the antibody of the present invention is an antibody having the ability to bind to IGF1R expressed on endothelial cells constituting the BBB and to penetrate the BBB, but does not inhibit IGF1 signaling in the body. Thus, it was confirmed that the antibody of the present invention can be used as a carrier across the BBB.

18-2. Analysis of inhibition of IGF1R signaling components in the MCF7 cell line.

When binding of IGF1 to cells expressing IGF1R resulted in signal transduction into those cells, the anti-IGF1R antibody of the present invention was assayed to determine the binding of IGF1 to its receptor and subsequent signal transduction components. That is, MCF-7 cell lines expressing IGF1R were treated with anti-IGF1R antibody, and total IGF1R, phosphorylated IGF1R, total Akt as a downstream factor of IGF1R, and the amount of phosphorylated Akt in the cells were then analyzed.

After culturing MCF-7 cells, the culture medium was replaced with serum-free culture medium 20 hours before anti-IGF1R antibody treatment. MCF-7 cells were treated with minibodies of clones 996, 1226, 1564 and MKJP2 obtained in Example 4-1 at a concentration of 100 nM, respectively, and after 1 hour 200 ng/ml IGF1 was added. After 20 minutes, cells were washed with PBS and then lysed with M-PER supplemented with a mixture of protease and phosphatase inhibitors. After measuring protein concentration using the BCA assay kit, 12.5 μg of protein was loaded onto an electrophoresis gel (SDS-PAGE) and then transferred to a PVDF membrane. Blocking was performed at room temperature with gentle rocking for 1 hour using PBST (0.1% Tween 20) containing 5% BSA, followed by treatment with primary antibody against IGF1R or Akt with gentle rocking at 4°C overnight. Anti-beta-actin antibody was used as a control. After washing, the secondary antibody was treated with slow shaking at room temperature for 1 hour, followed by washing. The ECL solution was added and the signals were observed using an ImageQuant LAS 4000. The experimental results are shown in FIG. 14b.

From the results of the experiment, it was confirmed that the antibody of the present invention did not affect total IGF1R, phosphorylated IGF1R, total Akt as a downstream factor of IGF1R, and the amount of phosphorylated Akt in cells.

18-3. Analysis of inhibition of IGF1R signaling components in mouse brain endothelial cells.

When binding of IGF1 to cells expressing IGF1R resulted in signal transduction into those cells, the anti-IGF1R antibody of the present invention was assayed to determine the binding of IGF1 to its receptor and subsequent signal transduction components. That is, bEND3 cells expressing IGF1R were treated with 11F11-1564, 3A9-1564 CH11F11 and ch3A9, individual anti-alpha-synuclein antibodies described in Korean Patent Publication No. 2018-0081465, obtained by the method used in Example 14-2, and total IGF1R, phosphorylated IGF1R, total Akt as a downstream factor of IGF1R, and the amount of phosphorylated Akt in the cells were then analyzed.

During incubation of bEND3 cells, the culture medium was replaced with serum-free culture medium 20 hours before anti-IGF1R antibody treatment. bEND cells were treated with bispecific antibodies of clones 1564 and MKJP2 obtained in example 14-2 at a concentration of 100 nM and after 1 hour 200 ng/ml IGF1 was added. After 20 minutes, cells were washed with PBS and then lysed with M-PER supplemented with a mixture of protease and phosphatase inhibitors. After measuring protein concentration using the BCA assay kit, 12.5 μg of protein was loaded onto an electrophoresis gel (SDS-PAGE) and then transferred to a PVDF membrane. Blocking was performed at room temperature with gentle shaking for 1 hour using PBST (0.1% Tween 20) containing 5% BSA, followed by treatment with primary antibody against IGF1R or Akt with slow shaking at 4°C. during the night. Anti-beta-actin antibody was used as a control.

After washing, the secondary antibody was treated with slow shaking at room temperature for 1 hour, followed by washing. The ECL solution was added and the signals were observed using an ImageQuant LAS 4000. The experimental results are shown in FIG. 14s.

From the results of the experiment, it was confirmed that the antibody of the present invention had no effect on total IGF1R, phosphorylated IGF1R, total Akt as a downstream factor of IGF1R, and the amount of phosphorylated Akt in cells.

Example 19 Anti-IGF1R Antibody Nontoxicity Assay.

19-1. ADCC assay for anti-IGF1R antibody in IgG form.

The anti-IGF1R antibody in IgG form of Example 14-3 was assayed to determine whether it resulted in cell death through IGF1R-dependent binding to the surface of cells expressing IGF1R. That is, the ADCC reporter bioassay kit was used to analyze natural killer (NK) cell activation and adverse effects on IGF1R-expressing cells when anti-IGF1R antibody clone 1564 binds to IGF1R-expressing cell lines.

After incubation of IGF1R-overexpressing MCF-7 cells and IGF1R-low-expressing SKBR3 cells, 5000 cells were added to each well of a 96-well plate 20 hours before antibody treatment. After cell culture, the medium was replaced with RPMI 1640 containing 4% low-IgG serum, and clone 1564 was added to each well in the form of IgG at an 8-fold dilution, starting at 133.3 nM. Stabilized ADCC effector cells were added to each well, incubated for 6 hours and then left at room temperature for approximately 10 minutes. After adding the prepared reagent for the Bio-Glo luciferase assay to each well, the degree of luminescence was measured using PHERAstarFS, BMG LABTECH equipment, analyzing the degree of ADCC induction. The results of the experiment are shown in Fig. 15a.

Based on the results of the experiment, it was confirmed that the antibody of the present invention, in particular clone 1564, bound to MCF-7 cells, which is a cell line overexpressing IGF1R, and SKBR3 cells with low expression of IGF1R, but did not induce ADCC in effector cells.

19-2. Analysis of IGF1R levels in the brain after repeated administration of anti-IGF1R antibody.

Antibodies used as carriers across the BBB bind to target receptors on brain endothelial cells, increasing the penetration of therapeutic antibodies, but should not change the level of the corresponding receptors. Down-regulation of target receptors can affect their role in the brain, causing side effects.

We analyzed whether repeated administration of the anti-IGF1R antibody of the present invention affects the levels of IGF1R in the brain. According to Example 14-6, mice with simulated Parkinson's disease were given repeated administrations of a bispecific antibody in bivalent form, where clone 1564 was coupled to a therapeutic antibody for the treatment of Parkinson's disease, a therapeutic antibody for the treatment of Parkinson's disease itself, and an IgG negative control once a week. for 3 months and then IGF1R levels in brain tissue were analyzed by Western blotting.

After brain tissue from three mice in each group was perfused with PBS and homogenized, 10 μg of lysate was added to a 4–12% Bis-Tris gel, electrophoresed, and transferred to a PVDF membrane. Otherwise the method was the same as in Example 19-1.

The results of the experiment are shown in Fig. 15b. The bispecific antibody that included clone 1564 showed similar levels of IGF1R to the Parkinson's disease therapeutic antibody alone and IgG groups. These results indicate that clone 1564 does not lead to major changes in brain IGF1R levels even after repeated administration, and therefore this antibody is expected to have few side effects when used as a BBB carrier.

19-3. Analysis of antibody distribution in the brain after administration of anti-IGF1R antibody.

The BBB transporter binds to a receptor on the surface of a brain endothelial cell and delivers the therapeutic antibody to the brain, but a small amount of antibody must bind to the receptor on the surface of a normal brain cell. If a large amount of the transporter binds to normal cells expressing an antigen in the brain across the BBB, a small amount of therapeutic antibody bound to the transporter will reach the target.

To analyze the distribution of the anti-IGF1R antibody of the present invention in the brain, the distribution of the anti-IGF1R antibody was analyzed by immunostaining the brains of animals used in the in vivo experiment conducted in Example 21-2. SD rats administered the bivalent form of clone 1564 were perfused with saline via transcardial perfusion. The left hemisphere was fixed in 10% neutral buffered formalin for 24 hours. The brains were washed using 1X PBS and frozen for 24 hours in a solution containing 30% sucrose. Brains were frozen in OCT and stored at −80°C until sectioning. 25-μm-thick coronal sections were prepared and placed in 1x PBS containing 0.1% sodium azide in a 24-well Petri dish. Tissues were blocked and permeabilized by incubation with Dako serum-free protein block (X0909, DAKO) containing 0.3% Tween-20 freely at room temperature for 1 hour. The primary antibody was treated with biotinylated anti-human Fc antibody (BA3080, Vector Labs) at a ratio of 1:50 and incubated overnight at 4°C. After washing three times with TBS, anti-endothelial cell antibody was treated with RECA-1 (ab9774, AbCam) at a ratio of 1:2000 or anti-neuronal antibody NeuN (MAB377, Millipore) at a ratio of 1:250 for 2 hours. After washing, Alexa488 binding and secondary antibody treatment for each antibody were performed for 45 minutes. After washing, sections were mounted on glass slides and nuclei were stained with ProLong Gold fluorescent slide mounting medium containing DAPI or Dako containing Hoechst 33258. The cerebral cortex and hippocampus were analyzed on a confocal microscope at the appropriate fluorescence wavelength. The results of the analysis are shown in Fig. 15s.

The results indicate that the bispecific antibody associated with clone 1564 did not bind to normal neurons, but specifically bound to brain endothelial cells. Therefore, the anti-IGF1R antibody of the present invention is expected to have few side effects due to the fact that it only increases the ability of the therapeutic antibody to cross the BBB without binding to normal brain cells, and will lead to increased therapeutic efficacy of the therapeutic antibody.

Example 20. Analysis of the ability of anti-IGF1R antibodies to penetrate the BBB in vitro.

20-1. Analysis of the ability of a bivalent antibody to penetrate the BBB in the sv-ARBEC-derived BBB model.

The antibodies obtained in Examples 11 and 12 were used to prepare the bispecific antibody in Example 14-2 and then the ability of the resulting bispecific antibody to penetrate the BBB in vitro was analyzed in the sv-ARBEC BBB model. sv-ARBEC were seeded in a single layer on a permeable membrane and the integrity of the BBB system was assessed in advance based on resistance (TEER) and the rate of sucrose passage through the BBB system. In this case, sv-ARBEC were treated with rat astrocyte culture medium (RAS-CM), which is known to promote the integrity of such a system. 90 minutes after applying the analyzed antibodies 1564, 48G5, 54H4, 60H6 and B11 in the form of bispecific antibodies to the membrane, the amount of antibodies in the lower chamber was analyzed with a mass spectrometer. For mass spectrometry, signature peptides from the Fc and scFv of each antibody were analyzed and then used. In this case, the therapeutic antibody for the treatment of Parkinson's disease (11F11) itself and a bispecific antibody, where the scFv form of the Herceptin™ biosimilar was associated with this therapeutic antibody, were used as negative controls. The calibration limits of the system were determined by passing antibody A20.1 (produced by the National Research Council), which is not known to cross the BBB. The values obtained from mass spectrometry were entered into formulas known from previously published literature, obtaining a Papp value, which reflected the degree of ability to penetrate the BBB in vitro.

The results of the analysis are shown in Fig. 16a. The analyzed antibodies, with the exception of B11, demonstrated a higher ability to penetrate the BBB than the negative control. In particular, clone 1564 demonstrated a higher ability to penetrate the BBB than the other clones. These results indicate that clones 1564, 48G5, 54H4, 60H6 and bispecific antibodies associated with clones 1564, 48G5, 54H4, 60H6 may have a higher ability to penetrate the BBB in vivo than a single antibody.

20-2. Analysis of the ability of a monovalent antibody to penetrate the BBB in the sv-ARBEC-derived BBB model.

In the same model as in example 20-1, the ability of monovalent antibodies obtained according to example 14-4 and bivalent antibodies obtained according to example 14-2 to penetrate the BBB in vitro was analyzed. The bivalent antibody and monovalent antibody produced using clone 1564 and the therapeutic antibody for Parkinson's disease (3A9) as a negative control were passed through the sv-ARBEC BBB system and then analyzed for the amount of antibodies passing through it. The results of the analysis are shown in Fig. 16b.

The bivalent form and the monovalent form of clone 1564 demonstrated greater BBB penetration ability in vitro than 3A9 antibody alone, and in particular, the bivalent form had greater BBB penetration ability than the monovalent form. These results indicate that clone 1564 can enhance the ability of therapeutic antibodies bound to it in various forms to cross the BBB in vitro.

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20-3. Analysis of the ability of anti-IGF1R antibody to penetrate the BBB in a human IPSC-derived BBB model.

The BBB model derived from human stem cells exhibits higher resistance (TEER) than BBB systems derived from rat or mouse cells and expresses all of the various markers found in the BBB. Therefore, the permeability of the BBB in the model derived from human stem cells is lower than that of the BBB derived from animal cells, and this model reproduces the human BBB well.

Vectors such as oriP/EBNA1 episomal vectors encoding OCT4, SOX2, c-Myc, KLF4, NANOG and LIN28 were introduced into human amniotic fluid-derived cells (AF-iPSCs) and reprogrammed into iPSCs. The resulting colonies were treated with KODMEM/F12, KOSR, glutamax, NEAA and beta-mercaptoethanol to obtain predifferentiated endothelial cells and then treated with serum-free endothelial differentiation medium, 1% PDS and 20 ng/ml bFGF for differentiation to obtain brain endothelial cells. The collection of amniotic fluid cells and the preparation of the BBB system using these cells was carried out by the National Research Council. After confirming the integrity of the system by resistance and sucrose passage, the anti-IGF1R antibody was analyzed for its ability to cross the BBB in the form of a bispecific antibody as a combination of a Parkinson's disease therapeutic antibody with a scFv form of the anti-IGF1R antibody. The antibodies analyzed were the bivalent form of clone 1564, the monovalent form of clone 1564, the bivalent form of clone F06 and the bivalent form of clone C04. The therapeutic antibody 11F11 was used as a negative control for the treatment of Parkinson's disease. Antibodies that passed through the BBB system were analyzed in the same way as in example 20-1.

The results of the analysis are shown in Fig. 16s. All analyzed clones demonstrated a higher ability to penetrate the BBB than the negative control group. In particular, bivalent F06 and bivalent C04 demonstrated BBB penetration abilities up to 15 times higher than that of a single antibody. The results obtained indicate that the anti-IGF1R antibodies of the present invention efficiently cross the BBB system of human origin compared to a single antibody.

Example 21. Analysis of the ability of anti-IGF1R antibodies to penetrate the BBB in vivo (colocalization assay).

21-1. Colocalization of minibodies with brain vessels.

To confirm the distribution of anti-IGF1R antibodies of the present invention throughout the brain vessels in vivo, the following experiment was performed.

Specifically, PBS buffer or 10 mg/kg control IgG and minibodies clones 996, 1226 and 1564 obtained in Example 14-1 were injected into the tail vein of 6-8 week old male BALB/c mice, respectively. After 4 hours, intracardial perfusion of the mouse brain was performed with a sufficient amount of 0.9% NaCl solution and 4% paraformaldehyde. Fixed brains were isolated, 20-μm-thick sections were prepared, and simultaneous staining was performed with anti-mouse CD31 as a vascular marker and with anti-human Fc to confirm colocalization of brain vessels and the IGF1R analyzed. An Alexa 488-conjugated secondary antibody for CD31 and an Alexa 594-conjugated secondary antibody for human Fc were used to visualize CD31 and human Fc under a fluorescence microscope.

The results of the experiment are shown in Fig. 17a.

Based on the experimental results, it was confirmed that the antibodies of the present invention, which do not block ligand binding, had excellent ability to penetrate the BBB. By staining brain tissue with vascular markers (anti-CD31 antibody, top row) and human antibodies (anti-human Fc antibody, bottom row) by analyzing the degree of colocalization of the antibody with brain blood vessels through immunostaining (Neuron (2016) Yu-Zuchero et al.) antibodies of the present invention, which do not block ligand binding, showed a higher degree of colocalization than control IgG.

21-2. Analysis of the ability of a bispecific antibody to penetrate the BBB in vivo.

An attempt was made to confirm the ability of the anti-IGF1R antibody of the present invention to cross the BBB in vivo in normal rats. PBS buffer or 10 mg/kg control IgG and therapeutic antibody for Parkinson's disease (11F11) or bivalent bispecific antibody (11F11-1564) containing clone 1564 coupled to therapeutic antibody were injected into the tail vein of SD rats, respectively. After 24 hours, the amount of antibodies in the CSF and brain was analyzed by mass spectrometry. Mass spectrometry was carried out using the same method as in example 20-1.

The bispecific antibody to which clone 1564 was associated showed greater penetration into the CSF and brain than the therapeutic antibody without anti-IGF1R antibody, and its effectiveness was confirmed at both the 10 mg/kg dose and the 30 mg/kg dose. kg. At

- 101 045348 at a dose of 30 mg/kg, the bispecific antibody demonstrated brain penetration up to approximately 4.5 times greater than that of the single antibody.

Clone 1564 was prepared in bivalent form and monovalent form according to Examples 14-2 and 14-4, and then administered at a dose of 30 mg/kg or 60 mg/kg in the same manner as described above, and after 24 hours the amount of antibodies in the CSF was analyzed and the brain. These two types of bispecific antibodies associated with clone 1564 demonstrated greater penetration into the CSF and brain than single antibodies. In particular, the bivalent form demonstrated an ability to penetrate the BBB into the brain, up to 5 times higher than the monovalent form.

The results presented in Fig. 17b demonstrate that clone 1564 enhances the ability of a therapeutic antibody to cross the BBB in the body even when bound to the therapeutic antibody in various forms.

It was expected that affinity variants of clone 1564 obtained according to example 2 would improve serum PK compared to the parent clone. Thus, it was expected that the ability to penetrate the BBB would be improved due to the longer presence of the antibody in the serum and the constant maintenance of influx across the BBB. At 0, 24 and 48 hours after intravenous administration of the affinity variants obtained in bivalent form according to Example 14-2 or in monovalent form according to Example 14-4, SD rats at a dose of 30 mg/kg underwent blood sampling from the ophthalmic vein. The antibodies analyzed were divided into two experiments according to the therapeutic antibody backbone. Bispecific antibodies of the corresponding variants used in the experiment are shown in table. 26 below.

Table 26

Bispecific antibodies used for

in vivo analysis of BBB-p dropping ability Clones with chimeric core Clones with a humanized skeleton Bivalent Ch 11 fl 1 -15 64 Bivalent Hui Ifl l(ver.2)-1564 Monovalent Chi Ifl 1-1564 Bivalent Hui Ifl l(ver.2)-VH5 Monovalent Chi Ifl 1-C04 Bivalent Hui Ifl l(ver.2)-VH16 Bivalent Chi Ifl 1-F06 Bivalent Hui Ifl l(ver.2)-VH35 Monovalent Chi Ifl 1-FOf Bivalent Hui Ifl l(ver.2)-VH9 ** Bivalent Hui Ifl l(ver.2)-VH2 ** Bivalent Hui Ifl l(ver.2)-VH7 ** Bivalent Hui Ifl l(ver.2)-VH32

Blood antibody levels were analyzed by ELISA. After the goat anti-human Fc antibody had been adsorbed onto a 96-well plate, an appropriate amount of diluted sample was added and then detected with the anti-human Fab antibody conjugated to HRP. The results of the analysis are shown in Fig. 17d.

As a result, in the first analyzed group, the monovalent form 1564, monovalent form F06 and monovalent form C04 showed a longer PK in serum than the bivalent form of the parent clone 1564. In the second analyzed group, the bivalent forms VH2, VH5, VH7, VH9, VH16 and VH32, with the exception of VH35, demonstrated superior serum PK compared to bivalent 1564.

To analyze the ability to penetrate the BBB in these groups, after 48 hours, CSF was collected from rats and analyzed by the same ELISA method. The results of the analysis are shown in Fig. 17th

In the first analysis group, monovalent form 1564, monovalent form F06 and monovalent form C04, which demonstrated better PK in serum, demonstrated higher levels of antibodies in the CSF compared to the bivalent form of the parent 1564. In the second analysis group, bivalent forms VH2, VH5, VH7, VH9 , VH16 and VH32, which also demonstrated better serum PK, demonstrated higher CSF antibody levels compared to the bivalent form of parent 1564. VH35 demonstrated shorter serum PK and lower CSF antibody levels compared to the bivalent form of parent 1564.

The results presented in Fig. 17d and 17e show that serum PK is an important factor for the ability of an antibody to cross the BBB due to the continuous influx of antibody across the BBB and that bispecific antibodies having a BBB transporter and better serum PK have a higher ability to cross the BBB . In particular, in the case of the monovalent form of F06 with the maximum level of antibody in the CSF, its ability to penetrate the BBB was approximately 5 times higher than that of the bivalent form of the parent 1564. In Examples 18-2 and 18-3, since the ability of the bivalent antibody 1564 to penetration into the CSF was approximately 3 times higher than that of a single antibody, it was expected that the ability of the monovalent form

F06 to penetrate the BBB will be approximately 15 times higher than that of a single antibody.

Example 22: Deamidation of Anti-IGF1R Antibody.

The deamidation reaction refers, for example, to the attack of a peptide bond between two sides of an asparagine to form a symmetric succinimide intermediate which, upon hydrolysis, is transformed into aspartic acid or isoaspartic acid. Because the deamidation reaction can affect the ongoing activity of the protein, substitution of light chain and heavy chain amino acids with other amino acids was performed to prevent deamidation and to ensure long-term stability.

To confirm the site of deamidation determined by computer analysis, anti-IGF1R antibody 1564 was left at 40°C and the degree of deamidation was assessed. As a result, it was confirmed that 4.6 to 47.3% of deamidation reactions occur in light chain CDR2 and CDR3 and heavy chain CDR2, as shown in FIG. 18a. To prevent deamidation, amino acid substitutions were made at each site, as shown in FIG. 18b, and changes in the ability to bind to the IGF1R protein were analyzed depending on each amino acid substitution. Mutants exhibiting the same binding affinity as wild type 1564 were selected, and the ability of these mutants, which each had 3 or 4 mutations, was confirmed to be the same as wild type 1564. The introduction of these mutations means that the buffer composition and storage conditions allow the binding affinity and stable form to be maintained for a long time. The results of the analysis are shown in table. 27.

Table 27

Experimental classification Antibody clone ECso (nM) Saturation value First 1564(WT) 2.15 1.1 N95aH 2.63 1.08 N95aR 2.3 1.17 N95aK 2.07 1.18 Second 1564(WT) 2.66 0.977 N95aD 2.07 0.896 N54D 2.89 0.933 N54Q 2.2 0.885 Third 1564(WT) 6.87 0.863 N51D 7.35 0.932

Example 23 Epitope mapping of anti-IGF1R antibodies.

23-1. ELISA analysis of anti-IGF1R antibody, heat-treated IGF1R protein and native IGF1R protein.

In this example, an attempt was made to determine which epitope the anti-IGF1R antibody recognizes: linear or conformational. ELISA was performed using bivalent bispecific antibodies 1564, 48G5, 54H4, 60H6 and B11 and the ECD protein of native human IGF1R or heated protein (heat-treated IGF1R). ELISA was carried out in the same way as shown in example 15. The results of the analysis are shown in table. 28.

Table 28

Clone name ECso (nM) relative to native IGF1R ECso against heat-treated IGF1R (nM) chllFll-1564 0.914 N/A* chllFll-48G5 1.21 N/A chllFll-54H4 2.88 N/A chllFll-60H6 10 N/A chllFll-Bll 7.13 410

* N/A - Not available.

The clones showed binding to the ECD protein of native human IGF1R, similar to that observed in example 15, but did not bind to the ECD protein of heat-treated human IGF1R, the tertiary structure of which was destroyed by heat. This means that the anti-IGF1R antibody of the present invention binds to a conformational epitope, but not to a linear epitope.

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23-2. Epitope mapping of anti-IGF1R antibody.

To analyze the conformational epitope of clone 1564, alanine scanning was performed as described below. OGFAR3 cells, an ovarian cancer cell line with confirmed low expression of IGF1R, were modified to express an IGF1R library where an eGFP tag was fused to the N terminus and the C-terminal kinase domain was removed. The IGF1R library contains mutations where residues located on the surface of IGF1R are replaced by alanine. The resulting library was transfected into OVCAR3 cells. Cells in which IGF1R expression was detected were treated with antibody 1564 and then fluorescently labeled by treatment with a secondary antibody labeled DyLight650. Labeled cells were classified by the presence or absence of IGF1R expression, IGF1R expression, and the presence or absence of 1564 binding, and deep RNA sequencing was performed using IlluminaHiSeq to analyze the frequency of each alanine mutation in the corresponding group of cells. The corresponding frequency was normalized to the result obtained for cells expressing wild-type IGF1R, and then the relative frequency was calculated to select for those mutations that were reduced in number in the group of cells labeled 1564. From these observations, it was found that the epitope of clone 1564 was located in domain FN2, and the residues belonging to it were Y775, P776, F778, R650, S791 and L798. The results obtained and the sequences recognized by clone 1564 are shown in FIG. 19. Given the lack of previously published literature implicating these residues in binding to IGF1, these results plausibly describe the properties of clone 1564 used in Example 23-1.

Example 24 Comparison of single antibody and bispecific antibody binding affinities to antigen.

24-1. Binding affinity of single antibody and bispecific antibody to alphasynuclein antigen.

When the scFv form of the anti-IGF1R antibody was coupled to an anti-alpha-synuclein IgG antibody, the effect of such binding on the binding affinity of the anti-alpha-synuclein antibody was analyzed.

Alpha-synuclein aggregates were adsorbed onto a 96-well plate at a concentration of 1 μg/ml for 18 hours and, after washing, each antibody was treated at a 5-fold dilution, starting at 400 nM. Bound antibodies were coupled to anti-human Fc antibody with HRP and then stained with TMB solution to measure the extent of antibody binding.

As shown in FIG. 20a, it was confirmed that the binding affinity of single antibody and bispecific antibody to alpha-synuclein aggregates was the same.

24-2. Binding affinity of single antibody and bispecific antibody to IGF1R antigen.

To compare the degree of binding of a single antibody to alpha-synuclein and a bispecific antibody to the IGF1R antigen, an experiment was performed in the same manner as in Example 22.

As shown in FIG. 20b, good concentration-dependent binding was confirmed for the bispecific antibody having the scFv form of the anti-IGF1R antibody, but the single antibody lacking the anti-IGF1R antibody region did not demonstrate binding.

24-3. Analysis of the binding capacity of a humanized anti-alpha-synuclein antibody.

The difference in binding affinity of the bispecific chimeric antibody and the bispecific humanized antibody was analyzed by conducting an experiment in the same manner as in Example 24-1.

As shown in FIG. 20c, the bispecific humanized antibodies had binding affinity to alpha-synuclein aggregates at a level similar to the bispecific chimeric antibody, and it was confirmed that the monovalent bispecific antibody with a single scFv to IGF1R also showed binding affinity at a level similar to the chimeric antibodies.

By analyzing the binding affinity of the bispecific chimeric antibody and the bispecific humanized antibody to IGF1R by performing the experiment in the same manner as in Example 24-2, all bispecific antibodies showed the same binding affinity, however, the single antibody without scFv to IGF1R did not demonstrate binding, as shown in fig. 20d.

These results indicate that when the antibody is humanized by replacing the region of the murine antibody that acts as an immunogen in humans, the antibody has the same activity without changing the binding affinity to alpha-synuclein and IGF1R aggregates.

24-4. Comparison of phagocytosis using a single antibody and a bispecific antibody.

Phagocytosis refers to the activity of removing extracellular substances through various macrophage receptors. Various protein aggregates induce an immune response or inflammatory response, which has an adverse effect on the human body. In particular, it is known that when an antibody is administered to remove alpha-synuclein aggregates, it is mediated by the interaction of the F region of the antibody and FcrR on the cell surface. For this reason, a comparison was made of phagocytic activity using a single antibody and a bispecific antibody coupled to scFv to IGF1R.

Mouse-derived BV-2 microglial cells were used to compare phagocytosis using a single antibody and a bispecific antibody. BV-2 cells were cultured in

- 104 045348 in RPMI1640 medium, was obtained at a concentration of 2x106 cells/ml and added in an amount of 100 μl into 96-well plates with a U-shaped bottom. 10 μg/ml alpha-synuclein aggregates and 25 μg/ml antibodies were diluted in RPMI1640 medium, mixed and left at room temperature for 20 minutes. BV-2 cells were treated with a mixture of alpha-synuclein aggregates and antibodies and left for 15 minutes. Alpha-synuclein aggregates were removed from the supernatant by centrifugation at 1200 rpm and washed three times with PBS buffer (pH 2.5) to remove aggregates or antibodies bound to the cell surface. Cells were fixed with 4% paraformaldehyde and washed with PBS buffer. To confirm the phagocytosis of aggregates and antibodies by cells, 0.5% Triton X-100 was added to destroy cell membranes, washed with PBS buffer and treated with an antibody against all forms of alphasynuclein for 1 hour. Bound antibody against all forms of alpha-synuclein was treated with anti-rabbit antibody with Alexa 488 for 1 hour and then penetration of aggregates into macrophage cells was confirmed by FACS analysis.

As shown in FIG. 20e, it was confirmed that normal human IgG had no effect on macrophages, and when treated with an antibody to alpha-synuclein, phagocytosis of alpha-synuclein aggregates was enhanced. When comparing the single antibody and the bispecific antibody, a similar level of phagocytosis and no effect of the scFv form of the anti-IGF1R antibody bound to the C-terminus of IgG on the activity of the anti-alpha-synuclein antibody were confirmed.

Example 25. Evaluation of the effectiveness of a bispecific antibody.

In accordance with Example 14-6, a bivalent bispecific antibody containing the chimeric antibody 11F11 and scFv clone 1564 was prepared, and the resulting bispecific antibody and single antibody to alpha-synuclein were analyzed for their in vivo effectiveness in transgenic mice overexpressing human alpha-synuclein (mThy -1, human α-synuclein, University of California, San Diego). 2.5 mg/kg single antibody or human IgG or the same molar amount of bivalent bispecific antibodies was administered intraperitoneally once a week for 3 months. Five mice per group were used and non-transgenic animals from the same litter were used as controls. Perfusion was then performed as follows.

Upon completion of the last administration, animals were anesthetized with chloral hydrate in accordance with animal welfare guidelines and then perfused with 0.9% saline to analyze pathological changes in the brain. One half (sagittal section) of the perfused brain was then stored in 4% paraformaldehyde (pH 7.4, 4°C) in phosphate buffer until analysis, and the other half was immediately frozen (-70°C).

Pathological analysis was performed as follows. From half the brain fixed in paraformaldehyde, continuous sections of 40 μm thickness were obtained using the free-floating section method using a vibrometer. To confirm the level of alpha-synuclein expression in the brains of each treatment group, sections including the cortex, hippocampus, and striatum were incubated with anti-alpha-synuclein antibodies (anti-p129 α-syn, aggregate marker, Abcam, ab59264, or anti-full-length alpha-synuclein antibody). synuclein) at 4°C overnight. Alternatively, sections were assayed for GFAP (AB5804, Millipore) to analyze the extent of astrocyte activity or incubated with anti-IL-1 β antibody (ab9722, Abcam) to analyze the extent of neuroinflammation, respectively. Alternatively, treatment with anti-NeuN antibody (Chemicon, #MAB377) was performed to analyze the extent of neuronal death in the hippocampus. Incubation with the primary antibody was followed by treatment with goat anti-rabbit IgG coupled to biotin (1:100, Vector Laboratories) and avidin D with horseradish peroxidase (1:200, ABC Elite, Vector Laboratories) and detection with diaminobenzidine (DAB). Bright field microscopy of each immunohistochemically stained section was performed, measuring the optical density. The results obtained are shown in Fig. 21a-21e.

25-1. Analysis of the ability of a chimeric antibody and a bispecific antibody to reduce the amount of alpha-synuclein.

In fig. 21a shows the results of staining and evaluation of the cortex and hippocampus in mouse brain tissue using the antibody to p-129 α-syn after administration of antibodies to the mice to evaluate the ability of the chimeric antibody 11F11 and the bivalent bispecific antibody containing clone 1564 and the chimeric antibody 11F11 to eliminate aggregates alpha-synuclein in a (transgenic) mouse model overexpressing human alpha-synuclein. p-129 α-syn is the form phosphorylated at residue 129, an aggregate marker, and appears as dark brown spots or aggregates in stained tissue.

According to FIG. 21a, the IgG administration group showed a higher degree of p-129 α-syn staining than the non-transgenic control group (#-one-way ANOVA, p\u003c0.01). In contrast, in the single antibody or bispecific antibody group, the degree of p-129 α-syn staining or aggregates was significantly reduced. In particular, in the hippocampus, the degree of this decrease in the bispecific antibody group was greater than in the 11F11 chimeric antibody group (* - one-way ANOVA, p\u003c0.05). In fig. 21b shows the results of the same experiment

- 105 045348 ment, as in Fig. 21a, except for staining with an antibody to full-length alpha-synuclein as a marker. The detection of all alpha-synuclein indicates that the antibody of the present invention has the ability to eliminate alpha-synuclein itself and inhibit its cell-to-cell transmission. In other aspects, it can also be interpreted as inhibiting the formation of aggregates from monomers or removing all monomers. Administration of single antibodies and bispecific antibodies reduces the degree of increase in human alpha-synuclein in transgenic mice compared with the IgG administration group. In particular, in the hippocampus, bispecific antibodies were more effective than single antibodies.

The results show that the chimeric antibody 11F11 and the bispecific antibody are effective in reducing the levels of alpha-synuclein and its aggregates in animal models of Parkinson's disease, even at a low dose of 2.5 mg/kg. In particular, the bispecific antibody is superior to the single antibody, suggesting that the bispecific antibody may reach the brain in greater quantities than the single antibody and provide more effective disease treatment due to its greater ability to penetrate the BBB. 25-2. Analysis of the ability of a chimeric antibody and a specific antibody to reduce astrogliosis and reduce inflammatory cytokine levels.

Gliosis is a nonspecific reaction that occurs in glial cells in response to damage to the central nervous system and is triggered by damage to the BBB or by substances such as TGF-beta and interleukin. In typical cases, it involves astrogliosis, and the protein GFAP is used as its marker. Therefore, the effect of reducing astrocytosis and releasing inflammatory cytokines that trigger astrocytosis was analyzed by administering the chimeric antibody 11F11 and a bispecific antibody containing clone 1564 and the chimeric antibody. The results of the analysis are shown in Fig. 21c and 21d.

In fig. 21c shows the results of staining and evaluating mouse brain tissue using GFAP (astrogliosis) as a marker after administration of antibodies to evaluate the ability of the chimeric antibody 11F11 and a bispecific antibody containing clone 1564 and the chimeric antibody obtained in one example of the present invention to reduce astrogliosis in vivo. Single antibody and bispecific antibody inhibited astrogliosis compared to the IgG control group. In particular, it was confirmed that in the striatum the effectiveness of the bispecific antibody was higher than that of the single antibody.

In fig. 21d shows the results of staining and evaluation of mouse brain tissue using anti-IL-1-beta antibody as a marker after administration of antibodies to evaluate the ability of the chimeric antibody 11F11 and a bispecific antibody containing clone 1564 and the chimeric antibody obtained in one example of the present invention, to reduce inflammatory cytokines in vivo. IL-1-beta causes inflammation, leading to the death and inflammatory response of various neurons. In the hippocampus of rats treated with antibodies of the present invention, IL-1-beta was reduced in the single antibody and bispecific antibody treatment groups compared with the IgG control group, and in particular, the bispecific antibody had a significantly greater reduction capacity than for a single antibody (## - one-way ANOVA, p\u003c0.005; * - one-way ANOVA, p\u003c0.05).

As shown in the drawings, the antibody of the present invention has been demonstrated to reduce astrogliosis and the release of the inflammatory cytokine IL-1-beta, which triggers astrogliosis, compared to control.

25-3. Analysis of the ability of a chimeric antibody and a bispecific antibody to reduce neurodegeneration.

Previously published literature has confirmed that brain cell death occurs due to neurotoxicity and an inflammatory response to alpha-synuclein. The ability of single antibodies and bispecific antibodies of the present invention to inhibit alpha-synuclein-induced brain cell death in vivo was analyzed.

NeuN staining, a marker for cortical and hippocampal neurons, found that both the single antibody and the bispecific antibody reduced brain cell death compared with the IgG control group. In particular, it was confirmed that in the cortex, the bispecific antibody had a greater ability to inhibit brain cell death than the single antibody. The results obtained are shown in Fig. 21st.

Example 26. Increased half-life by engineering Fc and increased ability to penetrate the BBB due to increased half-life.

FcRn is an important receptor on the cell membrane that increases half-life by trapping and directing antibody into cells, inhibiting the breakdown of antibody that occurs during its circulation in the blood vessels. The ability to cross the BBB is also important for the transcytotic activity of an antibody, but it is well known that the transcytotic activity of an antibody is important for its ability to cross the BBB, but antibodies cross the BBB depending on the concentration of antibodies in the blood vessels. For this reason, to increase the half-life of a bispecific antibody, bispecific antibodies were prepared by increasing the binding affinity to FcRn

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Claims (2)

by replacing methionine (Met) with leucine (Leu) at the 428th amino acid position of the Fc region. By comparing the half-life of administration of the wild-type bispecific antibody and the M428L bispecific antibody at a concentration of 10 mg/kg to transgenic mice expressing human FcRn, an increase in half-life of approximately 50% was confirmed, as shown in FIG. 22. To confirm the increase in half-life, PK profiles were analyzed when wild-type bispecific antibody, M428L bivalent bispecific antibody, and M428L monovalent bispecific antibody were administered to monkeys. In the case of a wild-type bispecific antibody as shown in FIG. 22a, its blood concentration decreased rapidly after 168 hours, while the high-affinity FcRn-binding bispecific antibody M428L maintained a higher blood concentration compared to the wild type. It was confirmed that the M428L bispecific antibody had a half-life approximately 1.5 days longer than the wild-type bispecific antibody. In particular, with regard to clearance, the monovalent bispecific antibody M428L had the best clearance, and the wild-type bispecific antibody showed the fastest clearance (Fig. 22b). To confirm improved BBB passage due to the half-life increasing effect, CSF was collected 24 hours after antibody administration and the amount of CSF antibody was analyzed. After sorption of 100 ng/ml IGF1R in a cooled state for 18 hours, CSF was added to detect antibodies that bound IGF1R. As can be seen in FIG. 22c, the passage of a large number of bispecific antibodies, present in large quantities in the blood, through the BBB and the excellent ability of the monovalent bispecific antibody M428L to penetrate the BBB, higher than that of the bivalent bispecific antibody M428L, were confirmed. CLAIM 1. Bispecific anti-a-syn/anti-IGF1R antibody containing: a) an antibody against alpha-synuclein (α-syn) or an antigen-binding fragment thereof, and b) an antibody against IGF1R or its antigen-binding fragment containing: 1) a heavy chain variable region (VH) containing heavy chain complementarity determining region (CDR) 1 (H-CDR1) containing SEQ ID NO:1, H-CDR2 containing SEQ ID NO:13, and H-CDR3 containing SEQ ID NO:52, and 2) a light chain variable region (VL) containing light chain CDR1 (L-CDR1) containing SEQ ID NO:98, L-CDR2 containing SEQ ID NO:117, and L-CDR3 containing SEQ ID NO:152. 2. Bispecific antibody according to claim 1, where: a) the VH of an antibody against IGF1R or an antigen-binding fragment thereof comprises a framework region (H-FR1) 1 containing SEQ ID NO:81, H-FR2 containing SEQ ID NO:86, H-FR3 containing SEQ ID NO:88, and H-FR4 containing the amino acid sequence of SEQ ID NO:92, or b) VL of an antibody against IGF1R or an antigen-binding fragment thereof comprises a framework region (L-FR) 1 containing SEQ ID NO:162, L-FR2 containing SEQ ID NO:165, L-FR3 containing SEQ ID NO:166, and L-FR4 containing SEQ ID NO:170. 3. The bispecific antibody according to claim 1, wherein the VH and VL antibodies against IGF1R or an antigen binding fragment thereof contain SEQ ID NO: 222 and 341, respectively. 4. Bispecific anti-a-syn/anti-IGF1R antibody containing: a) an antibody against alpha-synuclein (a-syn) or an antigen-binding fragment thereof, and b) an antibody against IGF1R or its antigen-binding fragment containing: 1) a heavy chain variable region (VH) containing heavy chain complementarity determining region (CDR) 1 (H-CDR1) containing SEQ ID NO:1, H-CDR2 containing SEQ ID NO:12, and H-CDR3 containing SEQ ID NO:52, and 2) a light chain variable region (VL) containing light chain CDR1 (L-CDR1) containing SEQ ID NO:98, L-CDR2 containing SEQ ID NO:116, and L-CDR3 containing SEQ ID NO:135. - 107 045348 5. Bispecific antibody according to claim 4, where: a) VH of an antibody against IGF1R or an antigen-binding fragment thereof contains a framework region (H-FR) 1 containing SEQ ID NO:81, H-FR2 containing SEQ ID NO:86, H-FR3 containing SEQ ID NO:88, and H-FR4 containing SEQ ID NO:92, or b) VL of an antibody against IGF1R or an antigen-binding fragment thereof contains a framework region (L-FR) 1 containing SEQ ID NO:162, L-FR2 containing SEQ ID NO:165, L-FR3 containing SEQ ID NO:167, and L-FR4 containing SEQ ID NO:170. 6. The bispecific antibody according to claim 4, wherein the VH and VL antibodies against IGF1R or an antigen binding fragment thereof contain SEQ ID NO: 177 and 296, respectively. 7. Bispecific anti-a-syn/anti-IGF1R antibody containing: a) an antibody against alpha-synuclein (α-syn) or an antigen-binding fragment thereof, and b) an antibody against IGF1R or its antigen-binding fragment containing: 1) a heavy chain variable region (VH) containing heavy chain complementarity determining region (CDR) 1 (H-CDR1) containing SEQ ID NO:1, H-CDR2 containing SEQ ID NO:12, and H-CDR3 containing SEQ ID NO:52, and 2) a light chain variable region (VL) containing light chain CDR1 (L-CDR1) containing SEQ ID NO:98, L-CDR2 containing SEQ ID NO:116, and L-CDR3 containing SEQ ID NO:151. 8. Bispecific antibody according to claim 7, where: a) VH of an antibody against IGF1R or an antigen-binding fragment thereof contains a framework region (H-FR) 1 containing SEQ ID NO:81, H-FR2 containing SEQ ID NO:86, H-FR3 located between H-CDR2 and H-CDR3 containing SEQ ID NO:88, and H-FR4 containing SEQ ID NO:92, or b) VL of an antibody against IGF1R or an antigen-binding fragment thereof contains a framework region (L-FR) 1 containing SEQ ID NO:162, L-FR2 containing SEQ ID NO:165, L-FR3 containing SEQ ID NO:167, and L-FR4 containing SEQ ID NO:170. 9. The bispecific antibody of claim 7, wherein the VH and VL antibodies against IGF1R or an antigen binding fragment thereof comprise SEQ ID NOs: 219 and 338, respectively. 10. The bispecific antibody according to any one of claims 1 to 9, wherein the anti-IGF1R antibody or antigen-binding fragment thereof specifically binds to a mammalian IGF1R protein, including human IGF1R, monkey IGF1R, rat IGF1R and mouse IGF1R. 11. Bispecific antibody according to any one of claims 1 to 10, where the binding site of the antibody against IGF1R or an antigen-binding fragment thereof contains at least one of V397, W434, D435, Y460, C488, L641, R650, Y775, P776, F778, E779 , S791, L798, H808, E809 and L813 in the protein containing SEQ ID NO:410. 12. The bispecific antibody of claim 11, wherein the anti-IGF1R antibody or antigen binding fragment thereof binds to at least one binding site selected from binding sites 1-3 in the protein comprising SEQ ID NO:410, wherein binding site 1 contains at least at least one of R650, Y775, P776, F778, E779, S791 and L798, binding site 2 contains at least one of L641, H808, E809 and L813, and binding site 3 contains at least one of V397, W434, D435, Y460 and C488. 13. The bispecific antibody according to any one of claims 1 to 12, wherein the antigen binding fragment of the anti-IGF1R antibody is a scFv. 14. The bispecific antibody of claim 13, comprising an anti-IGF1R scFv comprising, from N-terminus to C-terminus, SEQ ID NO:341, SEQ ID NO:411 and SEQ ID NO:222. 15. A bispecific antibody according to any one of claims 1 to 14, wherein the anti-α-syn antibody or an antigen-binding fragment thereof comprises H-CDR1 containing SEQ ID NO:434, H-CDR2 containing SEQ ID NO:435 or 436, H-CDR3 containing SEQ ID NO:438, L-CDR1 containing SEQ ID NO:443, L-CDR2 containing SEQ ID NO:444, and - 108 -