WO2022119310A1 - Antibody binding specifically to aimp2 and/or aimp2 aggregate - Google Patents

Abstract

The present invention relates to antibodies binding specifically to AIMP2 and/or AIMP2 aggregates. Antibodies produced from 5B2 clone and 6C7 clone, or immunologically active fragments thereof bind specifically to AIMP2 aggregates. Antibodies produced from 3C2 clone and 7H9 clone, or immunologically active fragments thereof bind specifically to AIMP2 monomers in a normal structure. An antibody produced from 13H9 clone, or an immunologically active fragment thereof binds specifically to both an AIMP2 aggregate and an AIMP2 monomer in a normal structure. Thus, the antibodies or the immunologically active fragments thereof according to the present invention can be advantageously applied to the diagnosis and treatment of AIMP2- or AIMP2 aggregate-related diseases, especially neurodegenerative diseases.

Description

Antibodies that specifically bind AIMP2 and/or AIMP2 aggregates

The present invention was made under the support of the Ministry of Education of the Republic of Korea, project specific number 1345282452, project number NRF-2018R1D1A1B07046762, the research management specialized institution for the above project is the National Research Foundation of Korea, the research project name is “Basic Research”, and the research project name is “Lewis body formation and Research on molecular mechanisms of lesion propagation and development of diagnosis and control strategies”, organized by Sungkyunkwan University, and the research period is March 1, 2020-2021.02.28.

In addition, the present invention was made by the project specific number 1711067856 and the project number NRF-2017M3C7A1043848 under the support of the Ministry of Science and ICT of the Republic of Korea. ”, the title of the research project is “Development of a new animal model for expression of brain lesions related to basal ganglia disorder”.

This patent application claims priority to Korean Patent Application No. 10-2020-0165379 filed with the Korean Intellectual Property Office on December 1, 2020, the disclosure of which is incorporated herein by reference.

The present invention relates to antibodies that specifically bind to AIMP2 and/or AIMP2 aggregates.

Neurodegenerative brain diseases have characteristic denatured protein aggregates as a major lesion. That is, in the case of Parkinson's disease and Lewy body dementia disease, the Lewy body composed of denatured α-synuclein is shown as the main lesion, and in the case of Alzheimer's dementia, the neurofibrillar bundle composed of degenerated tau is the main lesion. indicates.

Dementia is a representative chronic progressive degenerative brain disease, and is defined as a complex of symptoms that affects both mental, cognitive and social behavioral abilities, leading to disturbances in normal daily life. , is emerging as a serious social and economical health issue for the elderly. Depending on the cause, dementia is classified into Alzheimer's disease, in which cognitive decline progresses without a clear cause, vascular dementia caused by cerebrovascular diseases such as recurrent cerebral infarction, and dementia caused by other diseases. It is known that 50-60% of all dementia patients are caused by Alzheimer's disease (as of 2014). In general, Alzheimer's disease patients show clinically distinct impairments in intellectual ability, emotional and behavioral changes, etc., and then show signs of serious cortical dysfunction such as loss of direction, memory impairment, and aphasia (Kumar V et al., Robbins and Cotran Pathologic Basis of Disease, Elsevier Saunders, 7th, 2004).

Alzheimer's disease (Alzheimer's disease) accounts for about 70% of all dementia patients (as of 2007) and occurs in 6% of the population over 65 years of age. As the main cause of cerebrovascular dementia is infarction caused by arteriosclerosis, symptoms can be improved by treatment with thrombolytic drugs, etc. However, Alzheimer's disease rarely shows prognostic signs before the age of 50, but the prevalence usually increases with age. It is a typical degenerative brain disease that worsens as brain cells are gradually destroyed. The final diagnosis of this disease is only possible through pathological examination through post-mortem autopsy. Medical Association, Neuropsychiatry, 2nd, JoongAng Munhwasa, 2007), although many researchers are actively researching it, the cause of the disease has not been fully elucidated so far. However, according to the research results so far, Alzheimer's disease is pathologically characterized by brain atrophy in the cerebral cortex or hippocampus and deposition of senile plaques or neurofibrillary tangles in the brain (Selkoe). DJ., Alzheimer's disease: Genes, proteins, and therapy, Physiol. Rev., 81, pp.741-766, 2001) It accumulates inside, and then the tau protein is aggregated in the nerve cells, resulting in synaptic damage, ultimately causing nerve dysfunction and brain cell death. (Dawbarn D et al., Neurobiology of Alzheimer's Disease, 2nd, Oxford Univ, Press, 2001; Kwangwoo Lee, Neuroscience, Beommunsa, 2005).

Parkinson's disease (PD) is the second most common age-related disease after senile dementia among neurodegenerative diseases. The number of patients is rapidly increasing. The elderly population in Korea is rapidly increasing, and the number of patients with Parkinson's disease is also continuously increasing. Although the exact cause of Parkinson's disease is not yet known, it is reported that a complex action of genetic factors and environmental factors causes Parkinson's disease. In this disease, motor function disorders such as hand tremor, rigidity syndrome, slow mobility, and postural instability appear, and emotional disorders such as emotional instability and fear are accompanied. The development of the disease is gradual, and pathologically, severe degeneration (about 70-80%) of dopaminergic neurons in the substantia nigra of the brain is observed. In these cells, lesions of abnormal deposition of proteins called Lewy bodies, which are α-synuclein aggregate lesions, are expressed, but in addition to these, several subtypes of aggregate lesions (Tau aggregate lesions, Amyloid plaque lesions) appear. It can be divided into subtypes. That is, the development of additional diagnostic markers that can appropriately classify the subtypes of Parkinson's disease in which various complex lesions appear is necessary for precise treatment tailored to the patient.

AIMP2 (Aminoacyl-tRNA synthetase complex interacting multifunctional protein-2) is a substrate protein of E3 ligase Parkin, a recessive gene for Parkinson's disease, and has been reported to be present in Lewy body inclusions in the substantia nigra of Parkinson's patients ( Corti O, et al. The p38 subunit of the aminoacyl-tRNA synthetase complex is a Parkin substrate: linking protein biosynthesis and neurodegeneration. Hum Mol Genet. 2003; 12:1427-1437). AIMP2 is a substrate of pathogenic parkin that accumulates in Parkinson's disease, and the level of AIMP2 is increased in the ventral midbrain of Parkin ^-/- mice, and the level of AIMP2 is increased in the postmortem brain of patients with Parkin mutation or sporadic Parkinson's disease. rises In addition, the accumulation and structural denaturation of AIMP2 triggers AIMP2 aggregation, and in the previous study, it was confirmed that AIMP2 was expressed in Lewy bodies containing α-synuclein as the main component in the brain of an actual Parkinson's patient. It has been reported that Klein promotes aggregation and affects the expression of Lewy bodies in Parkinson's disease. In the case of α-synuclein, Tau, and amyloid β, which are neurodegenerative disease-linked proteins, antibodies that can specifically recognize normal and aggregate forms have been developed, and diagnostic differentiation of basic, translational research and clinical brain tissue However, in the case of AIMP2, which has been recently identified as having an important function in Parkinson's disease, there are no antibodies or research results that can distinguish the normal form from the aggregate form.

Currently commercially available AIMP2 antibodies do not recognize AIMP2 aggregates. In particular, since the AIMP2 antibody of Proteintech Group, which is currently the most used, is a rabbit polyclonal antibody, it is difficult to ensure consistent quality of the product, and antibody recombination technology is not used. There has been a problem that it is difficult to apply to clinical treatment because it is impossible to manufacture humanized antibodies through the

It is an object of the present invention to provide an antibody specific for AIMP2 or a fragment having immunological activity thereof.

It is also an object of the present invention to provide an isolated nucleic acid molecule encoding the antibody or fragment having immunological activity thereof, a vector comprising the same, and a host cell transformed with the vector.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating neurodegenerative diseases.

Another object of the present invention is to provide a composition for diagnosing neurodegenerative diseases.

Another object of the present invention is to provide a kit for diagnosing neurodegenerative diseases.

Another object of the present invention is to provide an information providing method for diagnosing a neurodegenerative disease.

Another object of the present invention is to provide a method for screening a substance that inhibits or inhibits the formation of AIMP2 aggregates.

In addition, it is an object of the present invention to provide a screening method for a therapeutic agent for a neurodegenerative disease.

In order to solve the above problems, the present invention provides an antibody specific for AIMP2 or a fragment having immunological activity thereof.

In addition, the present invention provides an isolated nucleic acid molecule encoding the antibody or fragment having immunological activity thereof, a vector comprising the same, and a host cell transformed with the vector.

In addition, the present invention provides a pharmaceutical composition for preventing or treating a neurodegenerative disease comprising an antibody specific to AIMP2 or a fragment having immunological activity thereof.

In addition, the present invention provides a composition for diagnosing a neurodegenerative disease comprising an antibody specific to AIMP2 or a fragment having immunological activity thereof.

In addition, the present invention provides a neurodegenerative disease diagnostic kit comprising the composition.

In addition, the present invention provides an information providing method for diagnosing a neurodegenerative disease.

In addition, the present invention provides a method for screening a substance that inhibits or inhibits the formation of AIMP2 aggregates.

In addition, the present invention provides a screening method for a therapeutic agent for a neurodegenerative disease.

According to the present invention, the antibody or fragment having immunological activity thereof produced from the 5B2 clone and 6C7 clone of the present invention binds specifically to the AIMP2 aggregate, and the antibody produced from the 3C2 clone and the 7H9 clone or its immunological activity The fragment with the normal structure specifically binds to the AIMP2 monomer of the normal structure, and the antibody or fragment having immunological activity thereof produced in the 13H9 clone specifically binds to both the AIMP2 aggregate and the normal structure of the AIMP2 monomer, so AIMP2 or AIMP2 aggregate It can be usefully used for diagnosis and treatment of related diseases, in particular, neurodegenerative diseases.

Figure 1a shows the results of Western blot analysis of the GST-AIMP2 recombinant protein (0) and the GST-AIMP2 aggregate (7) obtained through the aggregation reaction for 7 days.

Figure 1b shows the results of analyzing the amyloid structure through Thioflavin T fluorescence analysis on the recombinant proteins, GST-AIMP2 (GST-AIMP2 monomer) and GST-AIMP2 aggregate (GST-AIMP2 fibril) of normal structure.

1c shows the results of Coomassie blue staining of the GST-AIMP2 aggregate antigen. (1: BSA, 2: size marker and 3: GST-AIMP2 aggregate)

FIG. 2a shows the results of analyzing the reactivity of the AIMP2 antibody clone with respect to the normal structure (monomer) and aggregate structure of AIMP2, and shows the results of dot blot analysis of antibodies purified from 8 types of clones.

Figure 2b shows the results of analyzing the reactivity of the AIMP2 antibody clone with respect to the normal structure (monomer) and the aggregate structure of AIMP2, and shows the results of Western blot analysis of the antibody purified from 8 types of clones.

3A is an immunofluorescence staining image obtained using an antibody (pS129-αSyn) that labels α-synuclein, a Lewy body marker, and 5B2, an AIMP2 target antibody in the form of an aggregate, for autopsy temporal lobe brain tissue of a Parkinson's patient and an age-matched normal control group. is shown.

Figure 3b shows the relative quantitative graph of the Parkinson's disease patient group compared to the normal control group of the α-synuclein-labeling antibody (pS129-αSyn) immunofluorescence signal for the image group of a.

3c is a graph showing the relative quantification of the 5B2 antibody immunofluorescence signal for the image group of a compared to the normal control group of the Parkinson's disease patient group.

FIG. 3D shows confocal immunofluorescence images using pS129-α-synuclein antibody and 5B2 antibody of autopsied temporal lobe brain tissue of a Parkinson's disease patient.

3E shows the results of immunohistochemistry (DAB staining) and distribution analysis of AIMP2 aggregates in autopsied temporal lobe brain tissues of Parkinson's disease patients and age-matched normal controls.

Figure 3f shows the results of dot blot analysis of the amount of AIMP2 aggregates in normal control plasma and Parkinson's disease patient plasma.

4A shows the results of electrophoretic separation and Coomassie blue staining of AIMP2 aggregate-specific 5B2 antibody (mouse IgG framework) and humanized humanized 5B2 antibody 5B2 purified by cloning into human IgG framework.

Figure 4b shows the results of dot blot analysis of the reaction of the 5B2 antibody (mouse IgG skeleton) to the GST-AIMP2 monomer and GST-AIMP2 aggregate.

4C shows the results of dot blot analysis of the reaction of the 5B2 humanized chimeric antibody to the GST-AIMP2 monomer and the GST-AIMP2 aggregate.

Hereinafter, the present invention will be described in detail by way of embodiments of the present invention with reference to the accompanying drawings. However, the following embodiments are presented as examples of the present invention, and when it is determined that detailed descriptions of well-known techniques or configurations known to those skilled in the art may unnecessarily obscure the gist of the present invention, the detailed description may be omitted, and , the present invention is not limited thereby. Various modifications and applications of the present invention are possible within the scope of equivalents interpreted therefrom and the description of the claims to be described later.

In addition, the terms used in this specification are terms used to properly express the preferred embodiment of the present invention, which may vary depending on the intention of a user or operator or customs in the field to which the present invention belongs. Accordingly, definitions of these terms should be made based on the content throughout this specification. Throughout the specification, when a part "includes" a certain component, it means that other components may be further included, rather than excluding other components, unless otherwise stated.

As used herein, the term "step for" or "step for" does not mean "step for".

All technical terms used in the present invention, unless otherwise defined, have the meaning as commonly understood by one of ordinary skill in the art of the present invention. In addition, although preferred methods and samples are described herein, similar or equivalent ones are also included in the scope of the present invention. The contents of all publications herein incorporated by reference are incorporated herein by reference.

Throughout this specification, common one-letter and three-letter codes for naturally occurring amino acids are used as well as generally accepted for other amino acids such as Aib (α-aminoisobutyric acid), Sar (N-methylglycine), etc. A three-character code is used. Amino acids referred to by abbreviation in the present invention are also described according to the IUPAC-IUB nomenclature as follows:

Alanine: A, Arginine: R, Asparagine: N, Aspartic Acid: D, Cysteine: C, Glutamic Acid: E, Glutamine: Q, Glycine: G, Histidine: H, Isoleucine: I, Leucine: L, Lysine: K, Methionine : M, phenylalanine: F, proline: P, serine: S, threonine: T, tryptophan: W, tyrosine: Y and valine: V.

In one aspect, the present invention relates to an antibody specific for AIMP2 or a fragment having immunological activity thereof.

In one embodiment, the antibody specific for AIMP2 may be a monoclonal antibody or a humanized antibody.

In one embodiment, the antibody or fragment having immunological activity specific for AIMP2 is CDRH (Complementarity determining regions Heavy chain) 1 comprising any one selected from the group consisting of the amino acid sequence of SEQ ID NOs: 1 to 3, the sequence A VH domain comprising a CDRH2 comprising any one selected from the group consisting of an amino acid sequence of Nos. 4 to 6, and a CDRH3 comprising any one selected from the group consisting of an amino acid sequence of SEQ ID Nos. have.

In one embodiment, the AIMP2 specific antibody or fragment having immunological activity thereof is FR1 comprising any one selected from the group consisting of the amino acid sequence of SEQ ID NOs: 20 to 22, the amino acid sequence of SEQ ID NOs: 23 to 25 VH comprising FR2 comprising any one selected from the group consisting of, FR3 comprising any one selected from the group consisting of the amino acid sequence of SEQ ID NOs: 26 to 28, and FR4 comprising the amino acid sequence of SEQ ID NOs: 29 or 30 It can contain domains.

In one embodiment, the AIMP2 specific antibody or fragment having immunological activity thereof is CDRL1 comprising any one selected from the group consisting of amino acid sequences of SEQ ID NOs: 10 to 13, amino acid sequences of SEQ ID NOs: 14 to 16 It may include a VL domain comprising CDRL2 comprising any one selected from the group consisting of, and CDRL3 comprising any one selected from the group consisting of amino acid sequences of SEQ ID NOs: 17 to 19.

In one embodiment, the AIMP2 specific antibody or fragment having immunological activity thereof is FR1 comprising any one selected from the group consisting of the amino acid sequence of SEQ ID NOs: 31 to 33, and the amino acid sequence of SEQ ID NOs: 34 to 37 FR2 containing any one selected from the group consisting of, FR3 containing any one selected from the group consisting of the amino acid sequence of SEQ ID NOs: 38 to 42, and any one selected from the group consisting of the amino acid sequence of SEQ ID NOs: 43 to 45 a VL domain comprising FR4 comprising

In one embodiment, the AIMP2 specific antibody or fragment having immunological activity thereof is a VH domain comprising any one selected from the group consisting of the amino acid sequence of SEQ ID NOs: 46 to 49; And it may include a VL domain comprising any one selected from the group consisting of the amino acid sequence of SEQ ID NOs: 50 to 54.

In one embodiment, the AIMP2 specific antibody or fragment having immunological activity thereof includes a heavy chain comprising any one selected from the group consisting of amino acid sequences of SEQ ID NOs: 55 to 58; And it may include a light chain comprising any one selected from the group consisting of the amino acid sequence of SEQ ID NOs: 59 to 63.

In one embodiment, AIMP2 may be an AIMP2 monomer or an AIMP2 aggregate.

In one embodiment, the antibody or fragment having immunological activity specific for the AIMP2 monomer comprises: a CDRH1 comprising the amino acid sequence of SEQ ID NO: 2, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 5, and an amino acid sequence of SEQ ID NO: 8 a VH domain comprising a CDRH3 comprising; and a V domain comprising a VL domain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO: 11 or 12, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 15, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 18 and a VH domain comprising the amino acid sequence of SEQ ID NO: 47 or 48; and a VL domain comprising the amino acid sequence of SEQ ID NO: 52 or 53, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 56 or 57; and a light chain comprising the amino acid sequence of SEQ ID NO: 61 or 62.

In one embodiment, the antibody or fragment having immunological activity specific for the AIMP2 monomer is an antibody produced from a 3C2 clone comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 56 and a light chain comprising the amino acid sequence of SEQ ID NO: 61 or a fragment having immunological activity thereof; Alternatively, it may be an antibody or fragment having immunological activity thereof produced from the 7H9 clone comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 57 and a light chain comprising the amino acid sequence of SEQ ID NO: 62.

In one embodiment, the antibody or fragment having immunological activity specific for AIMP2 aggregate is: CDRH1 comprising the amino acid sequence of SEQ ID NO: 1, CDRH2 comprising the amino acid sequence of SEQ ID NO: 4, and the amino acid sequence of SEQ ID NO: 7 a VH domain comprising a CDRH3 comprising; and a V domain comprising a VL domain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO: 10, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 14, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 17, a VH domain comprising the amino acid sequence of SEQ ID NO: 46; and a VL domain comprising the amino acid sequence of SEQ ID NO: 50 or 51, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 55; and a light chain comprising the amino acid sequence of SEQ ID NO: 59 or 60.

In one embodiment, the antibody or fragment having immunological activity specific for the AIMP2 monomer is an antibody produced in clone 5B2 comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 55 and a light chain comprising the amino acid sequence of SEQ ID NO: 59 or a fragment having immunological activity thereof; Alternatively, it may be an antibody or fragment having immunological activity thereof produced from a 6C7 clone comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 55 and a light chain comprising the amino acid sequence of SEQ ID NO: 60.

In one embodiment, the antibody or fragment having immunological activity specific for AIMP2 monomer and AIMP2 aggregate specific antibody or fragment having immunological activity thereof is: CDRH1 comprising the amino acid sequence of SEQ ID NO:3, SEQ ID NO:6 a VH domain comprising a CDRH2 comprising the amino acid sequence of SEQ ID NO: 9 and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 9; and a V domain comprising a VL domain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO: 13, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 16, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 19, a VH domain comprising the amino acid sequence of SEQ ID NO: 49; and a VL domain comprising the amino acid sequence of SEQ ID NO: 54, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 58; And it may be an antibody or a fragment having immunological activity thereof produced in the 13H9 clone comprising a light chain comprising the amino acid sequence of SEQ ID NO: 63.

The antibody or fragment having immunological activity thereof includes variants thereof, and includes an antibody or fragment thereof having “substantial similarity”. Said "substantial similarity" is at least about 90% sequence identity, more preferably at least about 95%, 98% when the two peptide sequences are optimally aligned, such as by the program GAP or BESTFIT using default gap weights. or share 99% sequence identity. Preferably, residue positions that are not identical differ by conservative amino acid substitutions. A “conservative amino acid substitution” is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (eg, charge or hydrophobicity). In general, conservative amino acid substitutions do not substantially change the functionality of the protein. Where two or more amino acid sequences differ from each other by conservative substitutions, the percent or degree of similarity may be up-regulated to correct for the conservative nature of the substitutions.

Such amino acid variations are made based on the relative similarity of amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, and the like. According to the analysis of the size, shape and type of amino acid side chain substituents, arginine, lysine and histidine are all positively charged residues; alanine, glycine and serine have similar sizes; It can be seen that phenylalanine, tryptophan and tyrosine have similar shapes. Therefore, based on these considerations, arginine, lysine and histidine; alanine, glycine and serine; And phenylalanine, tryptophan and tyrosine can be said to be biologically functional equivalents.

In introducing mutations, the hydropathic idex of amino acids may be considered. Each amino acid is assigned a hydrophobicity index according to its hydrophobicity and charge: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cysteine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

The hydrophobic amino acid index is very important in conferring an interactive biological function of a protein. It is a known fact that amino acids having a similar hydrophobicity index must be substituted to retain similar biological activity. When introducing a mutation with reference to the hydrophobicity index, the substitution is made between amino acids that show a difference in the hydrophobicity index, preferably within ±2, more preferably within ±1, and even more preferably within ±0.5.

On the other hand, it is also well known that substitution between amino acids having similar hydrophilicity values results in proteins having equivalent biological activity. As disclosed in US Pat. No. 4,554,101, the following hydrophilicity values are assigned to each amino acid residue: arginine (+3.0); lysine (+3.0); Aspartate (+3.0± 1); glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); Tryptophan (-3.4).

When the mutation is introduced with reference to the hydrophilicity value, the substitution is made between amino acids exhibiting a difference in the hydrophilicity value within preferably ±2, more preferably within ±1, and even more preferably within ±0.5.

Amino acid exchanges in proteins that do not entirely alter the activity of the molecule are known in the art (H. Neurath, R.L. Hill, The Proteins, Academic Press, New York, 1979). The most common exchanges are amino acid residues Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thy/Phe, Ala/ It is an exchange between Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, Asp/Gly.

Antibodies or antigen-binding fragments thereof of the present invention include antibodies or antigen-binding fragments thereof containing small changes to the above-described amino acid sequence, ie, modifications that have little effect on the tertiary structure and function of the antibody. Therefore, in some embodiments, even if it does not match the above-mentioned sequence, it may have at least 100%, 93%, 95%, 96%, 97%, or 98% or more similarity.

In one embodiment, the fragment having immunological activity of the antibody specific for AIMP2 is Fab, Fd, Fab', dAb, F(ab'), F(ab') ₂ , scFv (single chain fragment variable), Fv , single-chain antibodies, Fv dimers, complementarity determining region fragments, humanized antibodies, chimeric antibodies, and diabodies may be any one selected from the group consisting of.

The antibody is not only in the form of a whole antibody, but also includes functional fragments of antibody molecules. The whole antibody has a structure having two full-length light chains and two full-length heavy chains, and each light chain is connected to the heavy chain by a disulfide bond. A functional fragment of an antibody molecule refers to a fragment having antigen-binding function, and examples of antibody fragments include (i) a light chain variable region (VL) and a heavy chain variable region (VH) and a light chain constant region (CL) and a Fab fragment consisting of the first constant region of the heavy chain (CH1); (ii) an Fd fragment consisting of the VH and CH1 domains; (iii) an Fv fragment consisting of the VL and VH domains of a single antibody; (iv) a dAb fragment consisting of a VH domain (Ward ES et al., Nature 341:544-546 (1989)); (v) an isolated CDR region; (vi) a bivalent fragment comprising two linked Fab fragments F(ab')2 fragments: (vii) single chain Fv molecules (scFv) joined by a peptide linker joining the VH and VL domains to form an antigen binding site (viii) bispecific single chain Fv dimers (PCT/US92/09965) and (ix) diabody WO94/13804, which is a multivalent or multispecific fragment produced by gene fusion), and the like.

The antibody or fragment having immunological activity thereof of the present invention may be selected from the group consisting of an animal-derived antibody, a chimeric antibody, a humanized antibody, a human antibody, and a fragment having immunological activity thereof. The antibody may be recombinantly or synthetically produced.

An animal-derived antibody produced by immunizing an animal to be immunized with a desired antigen may cause immune rejection when administered to humans for therapeutic purposes, and a chimeric antibody has been developed to suppress such immune rejection. A chimeric antibody is one obtained by substituting a constant region of an animal-derived antibody that causes an anti-isotype reaction with that of a human antibody using a genetic engineering method. Chimeric antibodies have significantly improved anti-isotype response compared to animal-derived antibodies, but still have animal-derived amino acids in the variable region, potentially resulting in anti-idiotypic side effects. are doing A humanized antibody was developed to improve these side effects. This is produced by grafting complementarity determining regions (CDRs), which play an important role in antigen binding, into a human antibody framework among the variable regions of a chimeric antibody.

The most important thing in the CDR grafting technology for producing a humanized antibody is to select an optimized human antibody that can best accept the CDR region of an animal-derived antibody. structure analysis, molecular modeling technology, etc. are used. However, even when the CDR regions of an animal-derived antibody are transplanted into the optimized human antibody framework, there are cases in which amino acids that affect antigen binding are present while being located in the framework of the animal-derived antibody, so that antigen-binding ability is not preserved in many cases. Therefore, it can be said that the application of additional antibody engineering technology to restore antigen binding force is essential.

The antibody or fragment having immunological activity thereof may be isolated from a living body (not present in the living body) or non-naturally occurring, for example, synthetically or recombinantly produced. can

In the present invention, the term "antibody" refers to a substance produced by stimulation of an antigen in the immune system, the type is not particularly limited, and can be obtained naturally or non-naturally (eg, synthetically or recombinantly). can Antibodies are advantageous for mass expression and production because they are very stable and have a long half-life not only in vitro but also in vivo. In addition, since the antibody essentially has a dimer structure, the adhesion (avidity) is very high. A complete antibody has a structure having two full-length light chains and two full-length heavy chains, and each light chain is linked to a heavy chain by a disulfide bond. The constant region of an antibody is divided into a heavy chain constant region and a light chain constant region, and the heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ) and epsilon (ε) types, subclasses gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1) and alpha 2 (α2). The constant region of the light chain has kappa (κ) and lambda (λ) types.

As used herein, the term "heavy chain" refers to a variable region domain V _H and three constant region domains C _H1 , C _H2 and C _H3 comprising an amino acid sequence having a variable region sequence sufficient to confer specificity to an antigen. and a full-length heavy chain including a hinge and a fragment thereof. In addition, the term "light chain" refers to both full-length light chains and fragments thereof comprising a variable region domain _VL and a constant region domain _CL comprising an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen. interpreted as including

As used herein, the term "variable region or variable domain" refers to a portion of an antibody molecule that exhibits many variations in sequence while performing a function of specifically binding to an antigen, and complementarity in the variable region There are determining regions CDR1, CDR2 and CDR3. Between the CDRs, a framework region (FR) portion exists to support the CDR ring. The "complementarity determining region" is a ring-shaped region involved in antigen recognition, and the specificity of the antibody for the antigen is determined as the sequence of this region changes.

As used herein, the term “Framework” or “FR” refers to variable domain residues other than hypervariable region (HVR) residues. The FRs of a variable domain generally consist of the four FR domains FR1, FR2, FR3 and FR4. Thus, HVR and FR sequences generally appear in the following order in VH and VL/Vk:

(a) FRH1 (Framework region 1 of Heavy chain)-CDRH1 (complementarity determining region 1 of Heavy chain)-FRH2-CDRH2-FRH3-CDRH3-FRH4; and

(b) FRL1 (Framework region 1 of Light chain)-CDRL1 (complementarity determining region 1 of Light chain)-FRL2-CDRL2-FRL3-CDRL3-FRL4.

In another embodiment of the present invention, the antibody or antigen-binding fragment may be presented in the following order:

(a) FRL1 (Framework region 1 of Light chain)-CDRL1 (complementarity determining region 1 of Light chain)-FRL2-CDRL2-FRL3-CDRL3-FRL4; and

(b) FRH1 (Framework region 1 of Heavy chain)-CDRH1 (complementarity determining region 1 of Heavy chain)-FRH2-CDRH2-FRH3-CDRH3-FRH4.

As used herein, the term "scFv (single chain fragment variable)" refers to a single-chain antibody made by expressing only the variable region of an antibody through genetic recombination, and a single-chain form in which the VH region and the VL region of the antibody are connected by a short peptide chain. refers to the antibody of The term “scFv” is intended to include scFv fragments, including antigen-binding fragments, unless otherwise indicated or understood otherwise by context. This is obvious to those skilled in the art.

As used herein, the term "complementarity determining region (CDR)" refers to the amino acid sequence of the hypervariable region of the heavy chain and light chain of an immunoglobulin. The heavy and light chains may each comprise three CDRs (CDRH1, CDRH2, CDRH3 and CDRL1, CDRL2, CDRL3). The CDRs may provide key contact residues for the binding of an antibody to an antigen or epitope.

In the present invention, the terms "specifically binding" or "specifically recognized" have the same meaning as commonly known to those skilled in the art, and mean that an antigen and an antibody specifically interact to conduct an immunological reaction. .

Specific binding is at least about 1 x 10 ^-6 M or less (eg, 9 x 10 ^-7 M, 8 x 10 ^-7 M, 7 x 10 ^-7 M, 6 x 10 ^-7 M, 5 x 10 ^-7 M , 4 x 10 ^-7 M, 3 x 10 ^-7 M, 2 x 10 ^-7 M, or 1 x 10 ^-7 M), preferably 1 x 10 ^-7 M or less (eg, 9 x 10 ^-8 M , 8 x 10 ^-8 M, 7 x 10 ^-8 M, 6 x 10 ^-8 M, 5 x 10 ^-8 M, 4 x 10 ^-8 M, 3 x 10 ^-8 M, 2 x 10 ^-8 M, or 1 x 10 ^-8 M), more preferably 1 x 10 ^-8 M or less (eg, 9 x 10 ^-9 M, 8 x 10 ^-9 M, 7 x 10 ^-9 M, 6 x 10 ^-9 M , 5 x 10 ^-9 M, 4 x 10 ^-9 M, 3 x 10 ^-9 M, 2 x 10 ^-9 M, or 1 x 10 ^-9 M) of equilibrium dissociation constant (e.g., K _D less than this) indicates a tighter bond). Methods for determining whether two molecules specifically bind are well known in the art, and include, for example, equilibrium dialysis, surface plasmon resonance, and enzyme linked immunosorbent assay (ELISA). do.

As used herein, the term “affinity” refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). Unless otherwise specified, as used herein, "binding affinity" refers to an intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (eg, antibody and antigen). The affinity of a molecule Y and its partner Y can generally be expressed as the dissociation constant (Kd) Affinity can be measured by conventional methods known in the art, including those described herein.

As used herein, the term “antigen-binding fragment” refers to a fragment of the entire immunoglobulin structure, and refers to a portion of a polypeptide including an antigen-binding portion. For example, it may be scFv, (scFv) 2, scFv-Fc, Fab, Fab' or F(ab')2, but is not limited thereto. Among the antigen-binding fragments, Fab has a structure having a light chain and heavy chain variable regions, a light chain constant region and a heavy chain first constant region (C _H1 ), and has one antigen-binding site. Fab' differs from Fab in that it has a hinge region comprising one or more cysteine residues at the C-terminus of the heavy chain C _H1 domain. The F(ab')2 antibody is produced by forming a disulfide bond with a cysteine residue in the hinge region of Fab'. Fv is a minimal antibody fragment having only a heavy chain variable region and a light chain variable region, and a recombinant technique for generating an Fv fragment is well known in the art. In a double-chain Fv (two-chain Fv), the heavy chain variable region and the light chain variable region are linked by a non-covalent bond, and in single-chain Fv (single-chain Fv), the heavy chain variable region and the single chain variable region are generally shared through a peptide linker. Since they are linked by a bond or are linked directly at the C-terminus, they can form a dimer-like structure like a double-stranded Fv. The linker may be a peptide linker consisting of any amino acids from 1 to 100 or 2 to 50 amino acids, and an appropriate sequence is known in the art. The antigen-binding fragment can be obtained using a proteolytic enzyme (for example, Fab can be obtained by restriction digestion of the entire antibody with papain, and F(ab') 2 fragment can be obtained by digestion with pepsin), It can be produced through genetic recombination technology.

In the present invention, the term "hinge region" is a region included in the heavy chain of an antibody, which exists between the C _H1 and C _H2 regions, and functions to provide flexibility of the antigen-binding site in the antibody. means area. For example, the hinge may be derived from a human antibody, and specifically, may be derived from IgA, IgE, or IgG, such as IgG1, IgG2, IgG3 or IgG4.

In one aspect, the present invention relates to an isolated nucleic acid molecule encoding an antibody or immunologically active fragment thereof of the present invention, a vector comprising the same, and a host cell transformed therewith.

As used herein, the term "nucleic acid molecule" has a meaning comprehensively including DNA (gDNA and cDNA) and RNA molecules. Also included are site-modified analogues (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews , 90:543-584 (1990)).

The nucleic acid molecules of the present invention may be isolated or recombinant, and include single-stranded and double-stranded forms of DNA and RNA as well as corresponding complementary sequences. An isolated nucleic acid is a nucleic acid that has been separated from surrounding genetic sequences present in the genome of the individual from which the nucleic acid was isolated, in the case of a nucleic acid isolated from a naturally occurring source. In the case of a nucleic acid synthesized enzymatically or chemically from a template, such as a PCR product, a cDNA molecule, or an oligonucleotide, a nucleic acid resulting from such a procedure can be understood as an isolated nucleic acid molecule. An isolated nucleic acid molecule refers to a nucleic acid molecule in the form of separate fragments or as a component of a larger nucleic acid construct. Nucleic acids are operably linked when placed into a functional relationship with another nucleic acid sequence. For example, the DNA of the presequence or secretion leader is operably linked to the DNA of the polypeptide when expressed as a preprotein in the form before the polypeptide is secreted, and the promoter or enhancer is the polypeptide sequence is operably linked to a coding sequence when it affects the transcription of, or a ribosome binding site is operably linked to a coding sequence when positioned to facilitate translation. In general, operably linked means that the DNA sequences to be linked are located contiguously, and in the case of a secretory leader, they are contiguous and in the same reading frame. However, enhancers need not be located adjacent to each other. Linkage is achieved by ligation at convenient restriction enzyme sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used according to conventional methods.

An isolated nucleic acid molecule encoding an antibody or immunologically active fragment of the present invention is expressed from a coding region due to codon degeneracy or in consideration of codons preferred in the organism in which the antibody is to be expressed. Various modifications can be made to the coding region within the range that does not change the amino acid sequence of the antibody, and various modifications or modifications can be made within the range that does not affect the expression of the gene in parts other than the coding region. It will be well understood by those skilled in the art that genes are also included within the scope of the present invention. That is, as long as the nucleic acid molecule of the present invention encodes a protein having an equivalent activity, one or more nucleic acid bases may be mutated by substitution, deletion, insertion, or a combination thereof, and these are also included in the scope of the present invention. The sequence of such a nucleic acid molecule may be single-stranded or double-stranded, and may be a DNA molecule or an RNA (mRNA) molecule.

In one embodiment of the present invention, the isolated nucleic acid molecule encoding the antibody or immunologically active fragment thereof of the present invention comprises the nucleotide sequence of SEQ ID NO: 64 encoding CDRH1, the nucleotide sequence of SEQ ID NO: 65 encoding CDRH2 and the nucleotide sequence of SEQ ID NO: 66 encoding CDRH3.

In one embodiment of the present invention, the isolated nucleic acid molecule encoding the antibody or immunologically active fragment thereof of the present invention is the nucleotide sequence of SEQ ID NO: 70 encoding FR1, the nucleotide sequence of SEQ ID NO: 71 encoding FR2 sequence, the nucleotide sequence of SEQ ID NO: 72 encoding FR3 and the nucleotide sequence of SEQ ID NO: 73 encoding FR4.

In one embodiment of the present invention, the isolated nucleic acid molecule encoding the antibody of the present invention or an immunologically active fragment thereof is a nucleotide sequence of SEQ ID NO: 67 encoding CDRL1, a nucleotide sequence of SEQ ID NO: 68 encoding CDRL2 and the nucleotide sequence of SEQ ID NO: 69 encoding CDRL3.

In one embodiment of the present invention, the isolated nucleic acid molecule encoding the antibody or immunologically active fragment thereof of the present invention is the nucleotide sequence of SEQ ID NO: 74 encoding FR1, the nucleotide sequence of SEQ ID NO: 75 encoding FR2 sequence, the nucleotide sequence of SEQ ID NO: 76 encoding FR3 and the nucleotide sequence of SEQ ID NO: 77 encoding FR4.

In one embodiment of the present invention, the isolated nucleic acid molecule encoding the antibody or immunologically active fragment thereof of the present invention comprises the nucleotide sequence of SEQ ID NO: 78 encoding VH and the nucleotide sequence of SEQ ID NO: 79 encoding VL sequence may be included.

In one embodiment of the present invention, the isolated nucleic acid molecule encoding the antibody or immunologically active fragment thereof of the present invention comprises a nucleotide sequence of SEQ ID NO: 80 encoding a heavy chain and a nucleotide sequence of SEQ ID NO: 81 encoding a light chain sequence may be included. In consideration of the above-described variation having biological equivalent activity, it is construed that the nucleic acid molecule of the present invention encoding the antibody or fragment having immunological activity thereof also includes a sequence exhibiting substantial identity. The substantial identity is at least when the sequence of the present invention and any other sequences are aligned to correspond to the maximum, and the aligned sequence is analyzed using an algorithm commonly used in the art. At least 60% homology (such as 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, or 69%), more specifically at least 70% homology (such as 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%), even more specifically at least 80% homology (such as 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%), even more specifically at least 90% homology (such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%), most specifically 95% or greater homology (eg, 95%, 96%, 97%, 98%, or 99%). All integers greater than or equal to 60% and less than or equal to 100% and prime numbers therebetween are included within the scope of the present invention with respect to % homology.

Alignment methods for sequence comparison are known in the art. Various methods and algorithms for alignment are described in Smith and Waterman, Adv. Appl. Math. 2:482 (1981) ; Needleman and Wunsch, J. Mol. Bio. 48:443 (1970); Pearson and Lipman, Methods in Mol. Biol. 24: 307-31 (1988); Higgins and Sharp, Gene 73:237-44 (1988); Higgins and Sharp, CABIOS 5:151-3 (1989); Corpet et al., Nuc. Acids Res. 16:10881-90 (1988); Huang et al., Comp. Appl. BioSci. 8:155-65 (1992) and Pearson et al., Meth. Mol. Biol. 24:307-31 (1994). The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403-10(1990)) is accessible from the National Center for Biological Information (NBCI), etc. It can be used in conjunction with sequencing programs such as blastx, tblastn and tblastx. BLAST can be accessed through the BLAST page of the ncbi website. A method for comparing sequence homology using this program can be found on the BLAST help page of the ncbi website.

An isolated nucleic acid molecule encoding an antibody of the present invention or an immunologically active fragment thereof according to the present invention can be inserted into an expression vector for protein expression. Expression vectors usually contain a protein that is operatively linked, ie, placed in a functional relationship, with regulatory or regulatory sequences, selectable markers, optional fusion partners, and/or additional elements. As used herein, "operably linked" refers to a functional linkage between a nucleic acid expression control sequence (eg, a promoter, signal sequence, or array of transcriptional regulator binding sites) and another nucleic acid sequence, whereby the control sequence to regulate the transcription and/or translation of said other nucleic acid sequences. In appropriate conditions, the present invention by a method for inducing protein expression by culturing a host cell transformed with a nucleic acid, preferably an expression vector containing an isolated nucleic acid molecule encoding an antibody of the present invention or an immunologically active fragment thereof Antibodies of the invention or fragments having immunological activity thereof can be produced. A variety of suitable host cells can be used, including, but not limited to, mammalian cells, bacteria, insect cells, and yeast. Methods for introducing an exogenous nucleic acid into a host cell are known in the art and will vary depending on the host cell used. Preferably, E. coli, which has a high industrial use value due to low production cost, can be produced as a host cell.

The vector of the present invention includes, but is not limited to, a plasmid vector, a cosmid vector, a bacteriophage vector, and a viral vector. Suitable vectors include a signal sequence or leader sequence for membrane targeting or secretion in addition to expression control elements such as promoter, operator, start codon, stop codon, polyadenylation signal and enhancer, and may be prepared in various ways depending on the purpose. The promoter of the vector may be constitutive or inducible. The signal sequence includes a PhoA signal sequence and an OmpA signal sequence when the host is Escherichia sp., and an α-amylase signal sequence and a subtilisin signal when the host is a Bacillus sp. When the host is yeast, the MFα signal sequence, SUC2 signal sequence, etc., and when the host is an animal cell, an insulin signal sequence, α-interferon signal sequence, antibody molecule signal sequence, etc. can be used, It is not limited thereto. The vector may also contain a selection marker for selecting a host cell containing the vector, and in the case of a replicable expression vector, an origin of replication.

As used herein, the term "vector" refers to a carrier capable of inserting a nucleic acid sequence for introduction into a cell capable of replicating the nucleic acid sequence. Nucleic acid sequences may be exogenous or heterologous. Vectors include, but are not limited to, plasmids, cosmids, and viruses (eg, bacteriophages). One skilled in the art can construct vectors by standard recombinant techniques (Maniatis, et al., Molecular Cloning , A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1988; and Ausubel et al., In: Current Protocols in Molecular Biology, John, Wiley & Sons, Inc, NY, 1994 et al.).

In one embodiment, when constructing the vector, expression control sequences such as promoter, terminator, enhancer, etc., membrane targeting or secretion according to the type of host cell in which the antibody is to be produced. Sequences and the like can be appropriately selected and combined in various ways depending on the purpose.

In the present invention, the term “vector” includes transfer vectors and expression vectors.

As used herein, the term "delivery vector" refers to a composition of a material that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid into a cell. linear polynucleotides, polynucleotides linked to ionic or amphiphilic compounds, plasmids and viruses. More specifically, the transfer vector comprises a self-replicating plasmid or virus. It should be construed that the term may additionally include non-plasmid and non-viral compounds that promote transfer of nucleic acids into cells, such as polylysine compounds, liposomes, and the like. Viral transfer vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, and lentiviral vectors.

As used herein, the term "expression vector" refers to a vector comprising a nucleic acid sequence encoding at least a portion of a transcribed gene product. In some cases, the RNA molecule is then translated into a protein, polypeptide, or peptide. The expression vector may include various regulatory sequences. In addition to regulatory sequences that control transcription and translation, vectors and expression vectors may contain nucleic acid sequences that also serve other functions.

The recombinant vector system of the present invention can be constructed through various methods known in the art, and specific methods thereof are disclosed in Sambrook et al., Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Laboratory Press (2001). , this document is incorporated herein by reference.

The vector of the present invention may be constructed as a vector for gene cloning, a vector for protein expression, or a vector for gene delivery. In addition, the vector of the present invention can be constructed using a prokaryotic cell or a eukaryotic cell as a host.

For example, when the vector of the present invention is an expression vector and a eukaryotic cell is a host, a promoter derived from the genome of a mammalian cell (eg, metallotionine promoter, beta-actin promoter, human hegglobin promoter and human muscle creatine promoter) or promoters derived from mammalian viruses (eg, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter, tk promoter of HSV, mouse mammary tumor virus (MMTV) promoter, The LTR promoter of HIV, the promoter of Moloney virus, the promoter of Epstein Barr virus (EBV), and the promoter of Loose sarcoma virus (RSV)) can be used, and generally have a polyadenylation sequence as a transcription termination sequence.

The vector of the present invention may be fused with other sequences to facilitate purification of the antibody expressed therefrom. The sequence to be fused includes, for example, glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA) and 6x His (hexahistidine; Quiagen, USA).

In addition, when the protein expressed by the vector of the present invention is an antibody, the expressed antibody can be easily purified through a protein A column or the like without an additional sequence for purification.

On the other hand, the expression vector of the present invention may include a selectable marker gene and/or a reporter gene as a selection marker for evaluating the expression of the antibody or antigen-binding fragment thereof of the present invention, and a CAR polypeptide comprising the same.

Selectable marker genes include antibiotic resistance genes commonly used in the art, for example, ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin and tetracycline. There is a resistance gene. Reporter genes include, but are not limited to, genes such as luciferase, beta-galactosidase, chloramphenicol acetyl transferase, or green fluorescent protein.

Methods for introducing and expressing the recombinant vector of the present invention into cells are well known in the art. Vectors can be readily introduced into host cells, eg, mammalian, bacterial, yeast, or insect cells by methods known in the art. For example, a vector may be delivered into a host cell by physical, chemical, or biological means. The physical means include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Such chemical means include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. In addition, the biological means includes, but is not limited to, the use of DNA or RNA vectors, such as the above-described lentiviruses and retroviruses.

In the present invention, the term "host cell" includes eukaryotes and prokaryotes, and refers to any transformable organism capable of replicating the vector or expressing a gene encoded by the vector. A host cell may be transfected or transformed by the vector, which refers to a process in which an exogenous nucleic acid molecule is delivered or introduced into a host cell.

A host cell capable of stably and continuously cloning and expressing the vector of the present invention is known in the art and any host cell may be used. For example, suitable eukaryotic host cells of the vector include yeast (Saccharomyce cerevisiae), insect cells. , monkey kidney cells (COS7), NSO cells, SP2/0, Chinese hamster ovary (CHO) cells, W138, baby hamster kidney (BHK) cells, MDCK, myeloma cell line , HuT 78 cells and HEK-293 cells.

As used herein, the terms "transformed", "transduced" or "transfected" refer to the process by which an exogenous nucleic acid is transferred or introduced into a host cell. "Transformed", "transduced" or "transformed" An "infected" cell is a cell that has been transformed, transduced, or transfected with an exogenous nucleic acid, including the cell and progeny cells resulting from subculture thereof.

Methods for delivering the vector of the present invention into host cells include microinjection (Capecchi, M.R., Cell, 22:479 (1980)), calcium phosphate precipitation (Graham, F.L. et al. , Virology, 52:456 (1973)), electroporation (Neumann, E. et al., EMBO J., 1:841 (1982)), liposome-mediated transfection (Wong, T.K. et al., Gene) , 10:87 (1980)), DEAE-dextran treatment (Gopal, Mol. Cell Biol., 5:1188-1190 (1985)), and gene bambadment (Yang et al., Proc. Natl. Acad. Sci., 87:9568-9572 (1990)) can inject vectors into host cells.

In the present invention, the recombinant vector contained in the host cell may express the above-mentioned antibody or antigen-binding fragment, or a fusion protein comprising the same, recombined in the host cell. In this case, a large amount of the antibody or antigen-binding fragment, or fusion get protein. For example, when the expression vector includes the lac promoter, the host cell may be treated with IPTG to induce gene expression.

The culture is usually carried out under aerobic conditions such as shaking culture or rotation on a rotary machine. The culture temperature is preferably in the range of 10 to 40° C., and the culture time is generally 5 hours to 7 days. The pH of the medium is preferably maintained in the range of 3.0 to 9.0 during culture. The pH of the medium can be adjusted with inorganic or organic acids, alkaline solutions, urea, calcium carbonate, ammonia, and the like. During culture, if necessary, antibiotics such as ampicillin, streptomycin, chloramphenicol, kanamycin and tetracycline may be added for maintenance and expression of the recombinant vector. When culturing a host cell transformed with a recombinant expression vector having an inducible promoter, if necessary, a suitable inducer may be added to the medium. For example, if the expression vector contains the lac promoter, IPTG (isopropyl-beta-D-thiogalactopyranoside) may be added, and if the expression vector contains a trp promoter, indoleacrylic acid may be added to the medium.

In one embodiment, the host cell may be a bacterium or an animal cell, the animal cell line may be a CHO cell, a HEK cell or an NSO cell, and the bacterium may be E. coli.

In one aspect, the present invention relates to a pharmaceutical composition for preventing or treating neurodegenerative diseases comprising the antibody or immunologically active fragment thereof of the present invention as an active ingredient.

In one embodiment, the neurodegenerative disease is Parkinson's disease, Huntington's disease, multiple system neuronal atrophy, amyotrophic lateral sclerosis, Lou Gehrig's disease, polyglutamine ectasia, spinocerebellar ataxia, spinal and soft muscle atrophy, tauosis, dystonia , serpin deficiency, cirrhosis, type II diabetes mellitus, primary systemic amyloidosis, secondary systemic amyloidosis, pronto-transient dementia, geriatric systemic amyloidosis, familial amyloid polyneuropathy, hereditary cerebral amyloid vasculopathy, hemodialysis-associated amyloidosis, macular degeneration, dementia , Alzheimer's disease, familial AD, Lewy body dementia, radiation therapy-induced dementia, boxer's dementia, Down syndrome, Gerstmann-Straussler-Scheinker disease, inclusion body myositis, prion protein brain amyloid angiopathy, axon injury, Acute diffuse cortical suppression, alpha-synucleinic condition, Parkinsonism-dementia complex of Guam, non-Gammanian motor neuron disease with nerve fiber entanglement, silver-affinity particle disease, cortical basal degeneration, diffuse nerve fibers with calcification Tangle, frontotemporal dementia with Parkinsonism associated with chromosome 17, Hallerforden-Spatz disease, stroke, cerebral infarction, head trauma, cerebral atherosclerosis, cerebral ischemia, permanent midcerebral ischemia, peripheral nerve regeneration, superimposed post-epileptic model, spinal cord injury, Niemann-Pick disease type C, Palido-Ponto-Nigral degeneration, Pick's disease, progressive subcortical gliosis, progressive supranuclear palsy, subacute sclerosing panencephalitis, dementia with tangles only, mild cognitive impairment, post encephalitis parkinsonism, myotonic Dystrophy, tau panencephalopathy, AD-like syndrome with astrocytes, senile cardiac amyloidosis, endocrine tumors, glaucoma, ocular amyloidosis, primary retinal degeneration, optic nerve drusen, optic neuropathy, optic neuritis, and lattice dystrophy, sporadic Lou Gehrig and Creutzfeldt-Jakob disease It may be one or more selected from the group consisting of.

The pharmaceutical composition of the present invention may further include an adjuvant. The adjuvant may be used without limitation as long as it is known in the art, for example, Freund's complete adjuvant or incomplete adjuvant may be further included to increase the immunity thereof.

As used herein, the term "treatment" means, unless otherwise stated, the disease or condition to which the term applies, or one or more symptoms of the disease or condition, which reverses, ameliorates, inhibits the progression, or It means to prevent, and the term treatment as used herein refers to the act of treating.

A composition is indicated to be "pharmaceutically or physiologically acceptable" if the recipient animal can tolerate administration of the composition, or if administration of the composition to that animal is suitable. When the amount administered is physiologically important, the agent can be said to have been administered in a "therapeutically effective amount". An agent is physiologically meaningful if the presence of the agent results in a physiologically detectable change in the recipient patient.

The therapeutically effective amount of the composition of the present invention may vary depending on several factors, for example, the administration method, the target site, the condition of the patient, and the like. Therefore, when used in the human body, the dosage should be determined as an appropriate amount in consideration of both safety and efficiency. It is also possible to estimate the amount used in humans from the effective amount determined through animal experiments. These considerations in determining effective amounts are found, for example, in Hardman and Limbird, eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed. (2001), Pergamon Press; and E.W. Martin ed., Remington's Pharmaceutical Sciences, 18th ed. (1990), Mack Publishing Co.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. As used herein, the term "pharmaceutically effective amount" refers to an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment and not to cause side effects, and the effective dose level is determined by the patient's Health status, disease type, severity, drug activity, sensitivity to drug, administration method, administration time, administration route and excretion rate, treatment period, factors including drugs used in combination or concurrently, and other factors well known in the medical field can be determined according to The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, which can be easily determined by those skilled in the art.

The compositions of the present invention may also include carriers, diluents, excipients or combinations of two or more commonly used in biological agents. A pharmaceutically acceptable carrier is not particularly limited as long as it is suitable for in vivo delivery of the composition, for example, Merck Index, 13th ed., Merck & Co. Inc. Compounds described in , saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components can be mixed and used, and if necessary, other antioxidants, buffers, bacteriostats, etc. Conventional additives may be added. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to form an injectable dosage form such as an aqueous solution, suspension, emulsion, etc., pills, capsules, granules or tablets. Furthermore, it can be preferably formulated according to each disease or component using an appropriate method in the art or a method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).

The pharmaceutical composition of the present invention can be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, or sterile injection solutions according to conventional methods, respectively. have.

As used herein, the term “pharmaceutically acceptable” refers to exhibiting properties that are not toxic to cells or humans exposed to the composition.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable additive, wherein the pharmaceutically acceptable additive includes starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate. , lactose, mannitol, syrup, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, lead carnauba, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, Calcium stearate, sucrose, dextrose, sorbitol and talc and the like may be used. The pharmaceutically acceptable additive according to the present invention is preferably included in an amount of 0.1 to 90 parts by weight based on the composition, but is not limited thereto.

As used herein, the term "administration" means providing a predetermined substance to a patient by any suitable method, and parenteral administration (eg, intravenous, subcutaneous, intramuscular, intrathecal) according to a desired method. , intracerebral, intrasternal, intranasal, intrapulmonary, rectal, intraperitoneal or topical injection) or oral administration, but is not limited thereto.

The dosage of the pharmaceutical composition of the present invention depends on factors such as formulation method, administration method, patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate, reaction sensitivity, and severity of disease. These vary, and an ordinarily skilled practitioner can readily determine and prescribe a dosage effective for the desired treatment or prophylaxis. According to a preferred embodiment of the present invention, the daily dose of the pharmaceutical composition of the present invention is 0.0001-100 mg/kg.

The composition of the present invention may be administered parenterally (eg, intravenously, subcutaneously, intraperitoneally or topically) or orally according to a desired method, and the dosage may vary depending on the subject's age, weight, sex, physical condition, etc. is selected taking into account. It is self-evident that the concentration of the active ingredient included in the pharmaceutical composition can be variously selected depending on the subject, and is preferably included in the pharmaceutical composition at a concentration of 0.01 to 5,000 μg/ml. If the concentration is less than 0.01 μg/ml, pharmaceutical activity may not appear, and if it exceeds 5,000 μg/ml, it may be toxic to the human body.

The pharmaceutical composition of the present invention may be formulated in various oral or parenteral dosage forms. Formulations for oral administration include, for example, tablets, pills, hard, soft capsules, solutions, suspensions, emulsifiers, syrups, granules, and the like. crose, mannitol, sorbitol, cellulose and/or glycine), lubricants (eg silica, talc, stearic acid and its magnesium or calcium salts and/or polyethylene glycol). In addition, the tablet may contain a binder such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidine, and optionally starch, agar, alginic acid or disintegrants such as sodium salts thereof or effervescent mixtures and/or absorbents, coloring, flavoring and sweetening agents. The formulation may be prepared by conventional mixing, granulating or coating methods. In addition, representative formulations for parenteral administration are injection formulations, and water, Ringer's solution, isotonic saline, or suspension can be exemplified as a solvent for the injection formulation. The sterile, fixed oil of the injectable preparation can be used as a solvent or suspending medium, and any non-irritating fixed oil including mono- and di-glycerides can be used for this purpose. In addition, the injection preparation may use a fatty acid such as oleic acid.

In the present invention, the term "prevention" refers to any action that inhibits or delays the occurrence, spread, and recurrence of the disease or disorder by administration of the pharmaceutical composition according to the present invention.

In one aspect, the present invention relates to a composition for diagnosing a neurodegenerative disease comprising the antibody or immunologically active fragment thereof of the present invention.

In one embodiment, the presence or amount (level) of an AIMP2 monomer or AIMP2 aggregate is detected in a biological sample isolated from a subject using a composition for diagnosing a neurodegenerative disease comprising an antibody or immunologically active fragment thereof of the present invention can be measured.

As used herein, the term “detection” or “measurement” refers to quantifying the concentration of a detected or measured target.

In one embodiment, the kit may further include tools and/or reagents for collecting a biological sample from a subject or patient, as well as tools and/or reagents for preparing an AIMP2 protein or an aggregate thereof from the sample.

In one aspect, the present invention relates to a kit for diagnosing a neurodegenerative disease comprising the composition.

In the present invention, the term “neurodegenerative disease diagnosis kit” refers to a kit containing the composition for diagnosing neurodegenerative disease of the present invention. Therefore, the expression "neurodegenerative disease diagnosis kit" can be used interchangeably or mixed with the "neurodegenerative disease diagnosis composition". As used herein, the term “diagnosis” refers to determining a subject's susceptibility to a specific disease or disorder, determining whether an object currently has a specific disease or disorder, or having a specific disease or disorder. Determining a subject's prognosis (e.g., identifying a neurodegenerative disease state, determining the stage or subtype of a neurodegenerative disease, or determining the responsiveness of a neurodegenerative disease to treatment), or therametrics ( monitoring the condition of the subject to provide information on treatment efficacy).

Since the above-described composition or kit for diagnosing neurodegenerative diseases includes an antibody specific for AIMP2 or a fragment having immunological activity thereof of the present invention, basically it is suitable for various immunoassays or immunostaining. can The immunoassay or immunostaining may include radioimmunoassay, radioimmunoprecipitation, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), capture-ELISA, inhibition or competition assay, sandwich assay, flow cytometry, immunofluorescence staining and immunoaffinity tablets, but are not limited thereto. The immunoassay or immunostaining method is described in Enzyme Immunoassay, E. T. Maggio, ed., CRC Press, Boca Raton, Florida, 1980; Gaastra, W., Enzyme-linked immunosorbent assay (ELISA), in Methods in Molecular Biology, Vol. 1, Walker, J. M. ed., Humana Press, NJ, 1984; and Ed Harlow and David Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999, which is incorporated herein by reference.

For example, when the method of the present invention is performed according to a radioimmunoassay method, an antibody labeled with a radioisotope (eg, C14, I125, P32 and S35) can be used to detect AIMP2.

When the present invention is carried out in the ELISA method, a specific embodiment of the present invention comprises the steps of (i) coating a sample to be analyzed on the surface of a solid substrate; (ii) reacting the sample with an antibody that specifically binds to AIMP2 of the present invention as a primary antibody; (iii) reacting the product of step (ii) with an enzyme-conjugated secondary antibody; and (iv) measuring the activity of the enzyme.

Suitable as the solid substrate are hydrocarbon polymers (eg, polystyrene and polypropylene), glass, metal or gel, most specifically microtiter plates.

The enzyme bound to the secondary antibody includes, but is not limited to, an enzyme catalyzing a color reaction, a fluorescence reaction, a luminescence reaction, or an infrared reaction, for example, alkaline phosphatase, beta-galactosidase, hose Radish peroxidase, luciferase and cytochrome P450. When alkaline phosphatase is used as the enzyme binding to the secondary antibody, bromochloroindolyl phosphate (BCIP), nitro blue tetrazolium (NBT), naphthol-AS-B1-phosphate (naphthol-AS) as substrates -B1-phosphate) and ECF (enhanced chemifluorescence) are used, and when horse radish peroxidase is used, chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (bis -N-methylacridinium nitrate), resorufin benzyl ether, luminol, Amplex Red reagent (10-acetyl-3,7-dihydroxyphenoxazine), HYR (p-phenylenediamine-HCl and pyrocatechol), TMB (tetramethylbenzidine), ABTS (2,2-Azine-di [3-ethylbenzthiazoline sulfonate]), o-phenylenediamine (OPD) and naphthol/pyronine, glucose oxidase and t-NBT (nitroblue tetrazolium) and m-PMS ( Substrates such as phenzaine methosulfate) may be used.

When the present invention is carried out in the capture-ELISA method, a specific embodiment of the present invention comprises the steps of: (i) coating an antibody that specifically binds AIMP2 as a capturing antibody to the surface of a solid substrate; (ii) reacting the capture antibody with the sample; (iii) reacting the result of step (ii) with a detecting antibody to which a label generating signal is bound; and (iv) measuring a signal generated from the label.

The detection antibody of the present invention has a label that generates a detectable signal. The label may include chemicals (eg biotin), enzymes (alkaline phosphatase, beta-galactosidase, horse radish peroxidase and cytochrome P450), radioactive materials (eg C14, I125, P32 and S35). , a fluorescent material (eg, fluorescein), a luminescent material, a chemiluminescent material, and fluorescence resonance energy transfer (FRET). Various labels and labeling methods are described in Ed Harlow and David Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999.

In the ELISA method and the capture-ELISA method, the final enzyme activity measurement or signal measurement may be performed according to various methods known in the art. If biotin is used as a label, the signal can be easily detected with streptavidin and with luciferin when luciferase is used.

As the capture antibody and the detection antibody, two types of antibodies or fragments having immunological activity thereof that bind to different epitopes among clones of the AIMP2 specific antibody or fragment having immunological activity of the present invention may be used. .

Samples that can be applied to the kit of the present invention include, but are not limited to, cell, tissue or tissue-derived extract, lysate or purified product, blood, plasma, serum, lymph or ascites.

In one aspect, the present invention relates to an information providing method for diagnosing a neurodegenerative disease comprising detecting AIMP2 monomer or AIMP2 aggregate in a biological sample isolated from a subject using the antibody or immunologically active fragment thereof of the present invention. it's about

As used herein, the term “sample (sample)” refers to a biological sample obtained from a subject or patient. Sources of biological samples may include fresh, frozen and/or preserved organ or tissue samples or solid tissue from biopsies or aspirates; blood or any blood component; The cells may be at any point in the pregnancy or development of the subject.

In one aspect, the present invention comprises the steps of reacting a candidate compound with an AIMP2 aggregate; quantifying the AIMP2 aggregate using the antibody of the present invention or a fragment having immunological activity specific for the AIMP2 aggregate; And it relates to a method for screening a substance that inhibits or inhibits the formation of AIMP2 aggregates, comprising the step of selecting a compound having a reduced amount of AIMP2 aggregates compared to a control group not treated with the candidate compound.

In one aspect, the present invention comprises the steps of reacting a candidate compound with an AIMP2 expressing cell or AIMP2 aggregate; quantifying the expression level of AIMP2 or the amount of AIMP2 aggregates using the antibody or fragment having immunological activity of the present invention; And it relates to a screening method for a therapeutic agent for a neurodegenerative disease, comprising the step of selecting a compound having a reduced amount of AIMP2 expression or AIMP2 aggregate compared to a control group not treated with the candidate compound.

The present invention will be described in more detail through the following examples. However, the following examples are only intended to embody the contents of the present invention, and the present invention is not limited thereto.

Example 1. AIMP2 aggregate production and verification

In order to construct a single antibody clone labeling the AIMP2 aggregate, a recombinant AIMP2 aggregate was prepared as an antigen. After transforming the vector encoding the GST-AIMP2 recombinant protein into BL21, it was cultured, eluted using GST beads, and purified to obtain the GST-AIMP2 recombinant protein. The purified GST-AIMP2 recombinant protein (GST-AIMP2 monomer) was incubated in a test tube at 37° C. at 250 rpm for 7 days to form a high molecular weight aggregate (GST-AIMP2 fibril). For reference, GST-truncated AIMP2 (monomer) and AIMP2 aggregates (AIMP2 fibril) were dialyzed against the GST-AIMP2 and GST-AIMP2 aggregates, and 100ul of Glutathione Sepharose 4B (GE Heathcare #17-0756-01) and Prescission protease (GE Heathcare) #27-0843-01) was treated with 10ul and incubated overnight. The prepared GST-AIMP2 and GST-AIMP2 aggregates were analyzed by western blot using an AIMP2-specific antibody (Proteintech Group rabbit antibody), and the GST-AIMP2 aggregates were placed on an SDS-PAGE gel, followed by Coomassie blue staining. As a result, GST-AIMP2 was detected, and it was confirmed that a GST-AIMP2 aggregate (Days 7) was formed (FIG. 1a). In addition, the amyloid structure of the GST-AIMP2 aggregate was verified by thioflavin T fluorescence analysis ( FIG. 1b ).

Example 2. Hybridoma cell line production and antibody analysis

As in Example 1, ELISA analysis was performed using GST-AIMP2 of FIG. 1c , which formed an aggregate in an amyloid structure, as an antigen, and did not react to GST, and either a GST-AIMP2 normal structure (monomer) or a GST-AIMP2 aggregate Eight hybridoma clones 3C2, 5B2, 6C7, 7G2, 10F3, 12D12 and 13H9 producing an antibody binding to (fibril) were selected (Table 1).

Clone No. AIMP2 fibril (Ag) AIMP2 mono GST 3C2 1.983 1.361 0.059 5B2 1.081 0.500 0.068 6C7 1.151 0.467 0.061 7G2 1.740 1.192 0.050 7H9 1.676 1.115 0.059 10F3 0.922 0.473 0.105 12D12 0.749 0.257 0.066 13H9 1.656 1.371 0.057

After that, the culture supernatant of each hybridoma clone was collected, treated with Protein G IgG binding buffer (Thermo Fisher Scientific), and the antibody produced from each clone using Capturem Protein G Maxiprep Column (Cat. No. 635727). was purified. The isotype of the antibody of each purified clone was analyzed (Table 2).

3C2 5B2 6C7 7G2 7H9 10F3 12D12 13H9 IgG1 0.287 0.082 0.077 0.345 0.068 0.112 1.886 0.063 IgG2a 1.440 2.393 2.240 2.084 1.015 2.394 0.048 1.799 IgG2b 0.055 0.075 0.073 0.057 0.053 0.059 0.051 0.056 IgG3 0.051 0.049 0.049 0.049 0.071 0.073 0.051 0.050 IgA 0.047 0.060 0.064 0.047 0.047 0.046 0.046 0.046 IgM 0.048 0.047 0.046 0.049 0.053 0.052 0.049 0.048 Kappa 0.465 0.698 0.723 0.762 0.750 0.799 0.454 0.055 Lambda 0.060 0.056 0.056 0.052 0.062 0.058 0.055 0.813

Example 3. AIMP2 conformation-specific reactivity analysis for each clone

3-1. dot blot analysis

In order to verify the binding ability of each antibody produced for each of the eight clones selected in the above Example to the AIMP2 three-dimensional structure, dot blot analysis was performed. Specifically, the concentrations of GST, GST-AIMP2 (Monomer) and GST-AIMP2 aggregates were equally diluted with 1X PBS to 0.1 mg/ml, printed on a nitrocellulose membrane, and then lowered in a vacuum. After confirming the loading with ponceau, it was removed again and blocked with 5% skim milk. After washing the blocked membrane 3 times with TBS-T, the antibodies of each clone purified in the above example were reacted with the primary antibody for 1 hour at 4°C (as controls, rabbit AIMP2 antibody and GST antibody from Proteintech Group were used. ). It was washed 3 times with TBS-T and reacted with a secondary antibody (5000 : 1) at RT for 30 minutes. Then, the blot was developed using ECL. As a result, unlike the ELISA results, 10F3 and 12D12 also bind to GST, so it was found that they were not suitable AIMP2 antibody clones. In addition, the rabbit AIMP2 antibody of Proteintech Group used as a control antibody labeled AIMP2 normal structure and aggregate structure without selectivity, and the antibody of the 7G2 clone did not show binding ability to both the AIMP2 monomer (normal structure) and the aggregate. The antibody of the 12D12 clone was shown to recognize GST ( FIG. 2A ). In addition, the antibody clones of 5B2 and 6C7 bound relatively well to AIMP2 aggregates, and the antibody clones of 3C2, 7H9 and 13H9 showed the same level of binding signal for both the normal structure and the aggregate structure of AIMP2 (Fig. 2a).

3-2. Western blot analysis

To verify the antibody recognition of the denatured protein, GST, GST-AIMP2 (Monomer) and GST-AIMP2 aggregates (Aggregate) were separated according to size on SDS-PAGE through Western blot analysis and purified from each antibody clone. Selective reactivity was analyzed using one antibody. As a result, as in the dot blot analysis result of Example 3-1, the conventional Proteintech Group antibody not only labels the GST-AIMP2 normal structure (monomer) of about 60 kDa, but also shows the high molecular weight GST-AIMP2 aggregate structure. 5B2 clones and 6C7 clones, which showed selectivity for AIM2 aggregates in dot blot analysis, were found to selectively label high molecular weight AIMP2 aggregates. It was found that the selectivity for AIMP2 aggregates was high (Fig. 2b). In addition, the 3C2 clone and the 7H9 clone had a signal detected only in AIMP2 (60 kDa) of the normal structure, indicating that the clones showed selectivity for AIMP2 of the normal structure (monomer) rather than the aggregate (Fig. 2b). In addition, the antibody purified from the 13H9 clone was labeled regardless of the structure of AIMP2 (FIG. 2b).

As a result of combining the results of the dot blot analysis and the Western analysis, the 10F3 clone and the 12D12 clone also bound to GST, and the 7G2 clone was not a suitable AIMP2 antibody clone due to its low binding affinity to AIMP2. However, 3C2 and 7H9 clones were It was possible to distinguish between AIMP2 normal structure-specific single antibody-producing clones, 5B2 and 6C7 as AIMP2 aggregate structure-specific single antibody-producing clones, and 13H9 clones as AIMP2 structure-specific single antibody-producing clones (Table 3).

Clone Specificity isotype Light Chain Dot blot reactivity: detection of 3D structure Western blot activity Native conf. Aggregate conf. GST Native conf. Aggregate conf. GST 3C2 Monomer IgG2a Kappa O O X O X X 5B2 Aggregate IgG2a Kappa - OOO X X O X 6C7 Aggregate IgG2a Kappa O OO X O O X 7G2 NA IgG2a Kappa X X X O X X 7H9 Monomer IgG2a Kappa O O X O X X 10F3 NA IgG2a Kappa O O O O X X 12D12 NA IgG1 Kappa O O O X X X 13H9 Both IgG2a Kappa O O X O O X

Example 4. CDS amino acid sequence analysis of AIMP2 antibody

In order to secure the cDNA sequence of each antibody encoded by each antibody clone, the sequence of the antibody variable region (variable region) with reference to the existing paper ( Lena Meyer et al. A simplified workflow for monoclonal antibody sequencing, PlosOne, 2019 ) was secured. Specifically, for cDNA extracted from each hybridoma cell, the light and heavy chain variable regions were amplified by PCR using the primers in Table 4 below, cloned into a T vector, and the light chain variable region and heavy chain variable region were included. The cDNA sequence was obtained by sequencing the T vector, and the variable region sequence was analyzed using IgBlast (refer to the Igblast webpage of NCBI), and the sequence of complementarity determining region (CDR) amino acids was also analyzed.

Primer Name Forward or reverse Primer Sequence ISPCR Universal F primer 5' aagcagtggtatcaacgcagag 3' mIGK PCR R primer for kappa chain 5' acattgatgtctttggggtagaag 3' mIGL PCR R primer for lambda chain 5' atcgtacacaccagtgtggc 3' mIGHG PCR R primer for heavy chain 5' gggatccagagttccaggtc 3'

As a result, as a result of analyzing the cDNA sequences of the 5B2 clone (Table 5) and 6C7 clone (Table 6) that showed specific reactivity to the AIMP2 aggregate, the amino acid sequence of the CDR region was confirmed, and only one of the amino acids encoding the light chain was different. was analyzed (Table 5 and Table 6). Differences are indicated in Tables 5 and 6 with underlines.

In addition, by grafting the heavy chain variable region (VH) and light chain variable region (VL) of each clone with the amino acid sequences of the human IgG heavy chain constant region and the light chain constant region, respectively, a humanized chimeric antibody is prepared with a complete heavy chain and the amino acid sequence of the light chain ('heavy chain' and 'light chain' in Tables 5 and 6).

5B2 amino acid sequence analysis division order SEQ ID NO: CDRH1 GFTFSDFY One CDRH2 SRNKANDYTT 4 CDRH3 ARDAFYGYGAMDY 7 CDRL1 KSIRKY 10 CDRL2 SGS 14 CDRL3 QQHNEYPYT 17 VH MKLWLNWVFLLTLLHGIQCEVKLVESGGGLVQPGGSLRLSCATSGFTFSDFYMEWVRQPPGKRLEWIAASRNKANDYTTEYSESVKGRFIVSRDISQSILYLQMNALRAEDTAIYYCARDAFYGYGAMDYWGQGTSVTVSS 46 VL MGNQFLPGHSLDMRFQVQVLGLLLLWISGAQCDVQITQSPSYLAASPGETITINCRASKSIRKYLAWYQEKPGKTNKLLIYSGSTLQSGIPSRFSGSGSG T DFTLTISSLEPEDFAMYYCQQHNEYPYTFGGGTKLEIK 50 heavy chain MKLWLNWVFLLTLLHGIQCEVKLVESGGGLVQPGGSLRLSCATSGFTFSDFYMEWVRQPPGKRLEWIAASRNKANDYTTEYSESVKGRFIVSRDISQSILYLQMNALRAEDTAIYYCARDAFYGYGAMDYWGQGTSVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 55 light chain MGNQFLPGHSLDMRFQVQVLGLLLLWISGAQCDVQITQSPSYLAASPGETITINCRASKSIRKYLAWYQEKPGKTNKLLIYSGSTLQSGIPSRFSGSGSGTDFTLTISSLEPEDFAMYYCQQHNEYPYTFTKGGGTKLEIKRTVAAPSVFIFPPSSDEQLKSGTASVQNRRHQHQNRHQNRR 59

6C7 amino acid sequence analysis division order SEQ ID NO: CDRH1 GFTFSDFY One CDRH2 SRNKANDYTT 4 CDRH3 ARDAFYGYGAMDY 7 CDRL1 KSIRKY 10 CDRL2 SGS 14 CDRL3 QQHNEYPYT 17 VH MKLWLNWVFLLTLLHGIQCEVKLVESGGGLVQPGGSLRLSCATSGFTFSDFYMEWVRQPPGKRLEWIAASRNKANDYTTEYSESVKGRFIVSRDISQSILYLQMNALRAEDTAIYYCARDAFYGYGAMDYWGQGTSVTVSS 46 VL MRFQVQVLGLLLLWISGAQCDVQITQSPSYLAASPGETITINCRASKSIRKYLAWYQEKPGKTNKLLIYSGSTLQSGIPSRFSGSGSG I DFTLTISSLEPEDFAMYYCQQHNEYPYTFGGGTKLEIK 51 heavy chain MKLWLNWVFLLTLLHGIQCEVKLVESGGGLVQPGGSLRLSCATSGFTFSDFYMEWVRQPPGKRLEWIAASRNKANDYTTEYSESVKGRFIVSRDISQSILYLQMNALRAEDTAIYYCARDAFYGYGAMDYWGQGTSVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 55 light chain MRFQVQVLGLLLLWISGAQCDVQITQSPSYLAASPGETITINCRASKSIRKYLAWYQEKPGKTNKLLIYSGSTLQSGIPSRFSGSGSGIDFTLTISSLEPEDFAMYYCQQHNEYPYTFGGGTKLEIKRTVAAPTKSVFIFPPSDEQLKDSKSGTASVVCLLNNFYKVTKSVKTLSHQNRR 60

In the same way, as a result of analyzing the sequence of the antibody variable region of the 3C2 clone and the 7H9 clone that showed specific binding ability to AIMP2 in the normal structure, the two clones also shared the same CDR amino acid sequence, the difference being the variable region of the light chain region. appeared only in the two amino acid sequences of (Table 7 and Table 8). Differences are indicated in Tables 7 and 8 with underlines. Similarly, by grafting the heavy chain variable region (VH) and light chain variable region (VL) of each clone with the amino acid sequences of the human IgG heavy chain constant region and the light chain constant region, respectively, a complete heavy chain and It was represented by the amino acid sequence of the light chain ('heavy chain' and 'light chain' in Tables 7 and 8).

3C2 amino acid sequence analysis division order SEQ ID NO: CDRH1 GFSLSTSGMG 2 CDRH2 IWWDDDK 5 CDRH3 ARIANSWFAY 8 CDRL1 KSVSTSAYSY 11 CDRL2 LAS 15 CDRL3 QHSRELPPT 18 VH MGRLTSSFLLLIVPAYVLSQVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGVGWIRQPSGKGLEWLAHIWWDDDKYYNTALKSGLTISKDTSKNQVFLKIASVDTADTATYYCARIANSWFAYWGQGTLVTVSA 47 VL MGSCQEPKKHPLFQLSEMETDTLLLWVLLLWVPGSTGDIVLTQSPASLAVSLGQRATISCRASKSVSTSAYSYMHWYQQKPGQ P PKLLIYLASNL D SGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPPTFGAGTKLELK 52 heavy chain MGRLTSSFLLLIVPAYVLSQVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGVGWIRQPSGKGLEWLAHIWWDDDKYYNTALKSGLTISKDTSKNQVFLKIASVDTADTATYYCARIANSWFAYWGQGTLVTVSAASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 56 light chain MGSCQEPKKHPLFQLSEMETDTLLLWVLLLWVPGSTGDIVLTQSPASLAVSLGQRATISCRASKSVSTSAYSYMHWYQQKPGQPPKLLIYLASNLDSGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPPTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 61

7H9 amino acid sequence analysis division order SEQ ID NO: CDRH1 GFSLSTSGMG 2 CDRH2 IWWDDDK 5 CDRH3 ARIANSWFAY 8 CDRL1 KSVSTSGYTY 12 CDRL2 LAS 15 CDRL3 QHSRELPPT 18 VH MGDKCAVMGRLTSSFLLLIVPAYVLSQVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGVGWIRQPSGKGLEWLAHIWWDDDKYYNTALKSGLTISKDTSKNQVFLKIASVDTADTATYYCARIANSWFAYWGQGTLVTVSA 48 VL METDTLLLWVLLLWVPGSTGDIVLTQSPASLAVSLGQRATISCRASKSVSTSGYTYMHWYQQKPGQ S PKLLIYLASNL E SGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPPTFGAGTKLELK 53 heavy chain MGDKCAVMGRLTSSFLLLIVPAYVLSQVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGVGWIRQPSGKGLEWLAHIWWDDDKYYNTALKSGLTISKDTSKNQVFLKIASVDTADTATYYCARIANSWFAYWGQGTLVTVSAASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 57
light chain METDTLLLWVLLLWVPGSTGDIVLTQSPASLAVSLGQRATISCRASKSVSTSGYTYMHWYQQKPGQSPKLLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPPTFGAGTKLELKRTVAAPTKSVFIFPPSDEQLKDSKSGTASVVCLLNNFYKVTKSVKTLSHQNRHQNHQNSRVTSTEQSPKLLIYLASNLESGVPARFSGTLS 62

In addition, 13H9, an antibody clone that detects both AIMP2 and aggregates in the normal structure, showed an antibody amino acid sequence distinct from the other four clones (Table 9). Table 9 also shows the amino acid sequence of the variable region and the CDR amino acid sequence. Similarly, by grafting the heavy chain variable region (VH) and the light chain variable region (VL) with the amino acid sequences of the human IgG heavy chain constant region and the light chain constant region, respectively, to prepare a humanized chimeric antibody, the complete heavy and light chain It is represented by the amino acid sequence ('heavy chain' and 'light chain' in Table 9).

13H9 amino acid sequence analysis division order SEQ ID NO: CDRH1 DYSITSGYS 3 CDRH2 IHYRGST 6 CDRH3 ATWDYRLAY 9 CDRL1 SQHSTYT 13 CDRL2 LKKDGSH 16 CDRL3 GVGDTIKEQFVYV 19 VH MRVLILLCLFTAFPGILSDVQLQESGPDLVKPSQSLSLTCTVTDYSITSGYSWHWIRQFPGNKLEWMAYIHYRGSTNYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCATWDYRLAYWGQGTLVTVSA 49 VL MGNLSVSAEISSTCIMAWTPLFFFFVLHCSGSFSQLVLTQSSSASFSLGASAKLTCTLSSQHSTYTIEWYQQQPLKPPKYVMELKKDGSHSTGDGIPDRFSGSSSGADRYLSISNIQPEDEAIYICGVGDTIKEQFVYVFGGGTKVTVL 54 heavy chain MRVLILLCLFTAFPGILSDVQLQESGPDLVKPSQSLSLTCTVTDYSITSGYSWHWIRQFPGNKLEWMAYIHYRGSTNYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCATWDYRLAYWGQGTLVTVSAASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 58 light chain MGNLSVSAEISSTCIMAWTPLFFFFVLHCSGSFSQLVLTQSSSASFSLGASAKLTCTLSSQHSTYTIEWYQQQPLKPPKYVMELKKDGSHSTGDGIPDRFSGSSSGADRYLSISNIQPEDEAIYICGVGDTIKEQFVYVFGGGTKVTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 63

Example 5. Validation of 5B2 antibody as a marker of Parkinson's disease.

5-1. Immunofluorescence staining assay

Immunofluorescence staining analysis using an antibody (pS129-αSyn) that labels α-synuclein, a Lewy body marker, and 5B2, a target antibody for AIMP2 aggregate, for autopsy temporal lobe brain tissues of Parkinson’s disease patients and age-matched normal controls was performed (FIGS. 3a to 3d).

Brain tissue samples were transferred to 12-wells containing 1X PBS, and washed with 1X PBS. Transfer to 0.1% TritonX-100 buffer (100 ul of 10% TritonX-100, 1 ml of 10X PBS, 8.9 ml of distilled water), store at room temperature for 10 minutes, and 5% blocking solution (4.75 ml of 0.1% TritonX-100 buffer and 100% goat) 250ul of serum) for 1 hour. The primary antibody was placed in a blocking solution at a ratio of 1:500 to 1:1000 and incubated at 4 °C overnight. Washed 3 times with 1X PBS, treated with secondary antibody (1:500 in 1X PBS), and reacted at room temperature for 1 hour. After washing 3 times with 1X PBS, DAPI staining was performed for 20 seconds. It was transferred to a slide, dried for one day, mounted, and the stained tissue was observed.

As shown in Figures 3a and 3b, it was confirmed that α-synuclein and AIMP2 aggregates were found in the brain tissue of patients with Parkinson's disease at a significant intensity compared to the normal control group, and as shown in Figure 3c, the effect on Parkinson's disease It was confirmed that AIMP2 aggregates affecting

The results of confocal immunofluorescence imaging using the pS129-α-synuclein antibody and the 5B2 antibody on the autopsied temporal lobe brain tissue of a Parkinson's disease patient are shown in FIG. 3D . By confirming that the signals of α-synuclein and AIMP2 aggregate are colocalized, it can be seen that the AIMP2 aggregate, which promotes the aggregation of α-synuclein and affects the expression of Lewy bodies in Parkinson's disease, can be detected through the 5B2 antibody. there was.

5-2. DAB staining analysis

DAB staining was performed on autopsied temporal lobe brain tissues of Parkinson's disease patients and age-matched normal controls using the 5B2 antibody.

After blocking the brain tissue sample with the blocking solution shown in Table 10, the primary antibody (1:1000 in the blocking solution) was treated and incubated at 4° C., 15 rpm in a rocker overnight.

Blocking solution Final 2ml 4ml 8ml 10ml 1X PBS 1X 1.8ml 3.6ml 7.2ml 8.8ml 10% tritonX-100 0.2% 40ul 80ul 160ul 200ul 10% sodium azide 0.02% 4ul 8ul 16ul 20ull 100% goat serum 10% 200ul 400ul 800ul 1ml

PBS with TritonX-100 (1 ml 1X PBS and 10 ul TritonX-100 per well) was put in a 12-well plate and prepared. The brain tissue samples incubated overnight were washed twice with PBS with TritonX-100 for 10 minutes each. The secondary antibody of Table 11 was added and placed on a rocker at room temperature for 1 hour.

2nd antibody solution 1ml 2ml 8ml 10ml 12ml 1X PBS 930ul 1.86ml 7.44ul 9.3ml 11.16ml 100% goat serum 50ul 100ul 400ul 500ul 600ul 10% tritonX-100 20ul 40ul 160ul 200ul 240ul Biotinylated 2nd Antibody 1ul 2ul 8ul 10ul 12ul

After washing twice with PBS with TritonX-100 for 10 minutes each, it was put into the ABC kit solution and incubated at room temperature for 40 minutes. It was washed twice with PBS for 10 minutes each. Brain tissue samples were collected, put in DAB solution at once, shaken and stained, and washed three times in PBS. After putting 1X PBS and transferring the brain tissue sample to a 100 ø dish containing 100 ul to 200 ul of TritonX-100, it was transferred back to the slide glass. It was then dried overnight.

Put the brain tissue sample slides in a cage dedicated to staining, 100% ethanol for 5 minutes, 95% ethanol for 2 minutes, distilled water for 2 minutes, staining solution for 1 minute, distilled water 3 times for 2 minutes each Treatment, treatment with destaining solution twice for 5 minutes each, treatment with distilled water three times for 2 minutes each, treatment with 100% ethanol twice for 5 minutes each, and treatment with xylene for 10 minutes. The bottom part of the slide was wiped, DPX was put in, and then dried in a hood.

The observed results are shown in Fig. 3e. It was confirmed that AIMP2 aggregates were found in the brain tissue of patients with Parkinson's disease, unlike normal controls, and it was confirmed that AIMP2 aggregates affecting Parkinson's disease could be detected through the 5B2 antibody.

5-3. dot blot analysis

The amount of AIMP2 aggregates in plasma of normal controls and patients with Parkinson's disease was analyzed by dot blot analysis (FIG. 3f). As shown in FIG. 3f , it was confirmed that AIMP2 aggregates appearing in Parkinson's disease patients could be detected by the 5B2 antibody.

Example 6. Construction and Verification of Chimeric Antibodies

A humanized chimeric antibody was prepared by recombination of the light chain variable region and heavy chain variable region sequences of the 5B2 antibody obtained in Example 4 and the light chain constant region and heavy chain constant region of a human IgG antibody through cloning.

Specifically, the light chain variable region and heavy chain variable region sequences of mouse IgG of the 5B2 antibody are amplified through PCR. The amplified mouse IgG light chain variable region and heavy chain variable region sequences were cloned into VRC01 mAb Heavy and Light Chain Expression Vector, which is a plasmid encoding a human IgG framework, respectively, using quick ligation or T4 ligation, and then transferred to HEK-293T cells. speculated. The obtained colonies were cultured for 7 days, and the culture supernatant was collected to finally obtain the heavy and light chains of the humanized chimeric 5B2 antibody. The produced antibody was purified in the same manner as in Example 2.

The amino acid sequence of the humanized 5B2 chimeric antibody is shown in Table 12, and the nucleotide sequence encoding the humanized 5B2 chimeric antibody is shown in Table 13.

Humanized 5B2 Chimeric Antibody Amino Acid Sequencing division order SEQ ID NO: CDRH1 GFTFSDFY One CDRH2 SRNKANDYTT 4 CDRH3 ARDAFYGYGAMDY 7 CDRL1 KSIRKY 10 CDRL2 SGS 14 CDRL3 QQHNEYPYT 17 FR1-H EVKLVESGGGLVQPGGSLRLSCATS 20 FR2-H MEWVRQPPGKRLEWIAA 23 FR3-H EYSESVKGRFIVSRDISQSILYLQMNALRAEDTAIYYC 26 FR4-H WGQGTSVTVSS 29 FR1-L DVQITQSPSYLAASPGETITINCRAS 31 FR2-L LAWYQEKPGKTNKLLIY 34 FR3-L TLQSGIPSRFSGSGSGTDFTLTISSLEPEDFAMYYC 38 FR4-L FGGGTKLEIK 43 VH
MKLWLNWVFLLTLLHGIQCEVKLVESGGGLVQPGGSLRLSCATSGFTFSDFYMEWVRQPPGKRLEWIAASRNKANDYTTEYSESVKGRFIVSRDISQSILYLQMNALRAEDTAIYYCARDAFYGYGAMDYWGQGTSVTVSS
46 VL MGNQFLPGHSLDMRFQVQVLGLLLLWISGAQCDVQITQSPSYLAASPGETITINCRASKSIRKYLAWYQEKPGKTNKLLIYSGSTLQSGIPSRFSGSGSGTDFTLTISSLEPEDFAMYYCQQHNEYPYTFGGGTKLEIK
50 heavy chain MKLWLNWVFLLTLLHGIQCEVKLVESGGGLVQPGGSLRLSCATSGFTFSDFYMEWVRQPPGKRLEWIAASRNKANDYTTEYSESVKGRFIVSRDISQSILYLQMNALRAEDTAIYYCARDAFYGYGAMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
55 light chain MGNQFLPGHSLDMRFQVQVLGLLLLWISGAQCDVQITQSPSYLAASPGETITINCRASKSIRKYLAWYQEKPGKTNKLLIYSGSTLQSGIPSRFSGSGSGTDFTLTISSLEPEDFAMYYCQQHNEYPYTFTKGGGTKLEIKRTVAAPSVFIFPPSESDEQLKSGTASVQNRRHQHQNRR
59

Humanized 5B2 Chimeric Antibody Nucleotide Sequencing division order SEQ ID NO: CDRH1 GGGTTCACCTTCAGTGATTTCTAC 64 CDRH2 AGTAGAAACAAAGCTAATGATTATACAACA 65 CDRH3 GCAAGAGATGCCTTCTATGGTTATGGGGCTATGGACTAC 66 CDRL1 AAGAGCATTAGAAAATAT 67 CDRL2 TCTGGATCC 68 CDRL3 CAACAGCATAATGAATACCCGTACACG 69 FR1-H GAGGTGAAGCTGGTGGAATCTGGAGGAGGCTTGGTACAGCCTGGGGGTTCTTTGAGACTCTCCTGTGCAACTTCT 70 FR2-H ATGGAGTGGGTCCGCCAGCCTCCAGGGAAGAGACTGGAGTGGATTGCTGCA 71 FR3-H GAGTACAGTGAATCTGTGAAGGGTCGGTTCATCGTCTCCAGAGACATTTCCCAAAGCATCCTCTACCTTCAGATGAATGCCCTGAGAGCTGAGGACACTGCCATTTATTACTGT 72 FR4-H TGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA 73 FR1-L GATGTCCAGATAACCCAGTCTCCATCTTATCTTGCTGCATCTCCTGGAGAAACCATTACTATTAATTGCAGGGCAAGT 74 FR2-L TTAGCCTGGTATCAAGAGAAACCTGGGAAAACTAATAAGCTTCTTATCTAC 75 FR3-L ACTTTGCAATCTGGAATTCCATCAAGGTTCAGTGGCAGTGGATCTGGTACAGATTTCACTCTCACCATCAGTAGCCTGGAGCCTGAAGATTTTGCAATGTATTACTGT 76 FR4-L TTCGGAGGGGGGACCAAGCTGGAAATAAAA 77 VH GAGGTGAAGCTGGTGGAATCTGGAGGAGGCTTGGTACAGCCTGGGGGTTCTTTGAGACTCTCCTGTGCAACTTCTGGGTTCACCTTCAGTGATTTCTACATGGAGTGGGTCCGCCAGCCTCCAGGGAAGAGACTGGAGTGGATTGCTGCAAGTAGAAACAAAGCTAATGATTATACAACAGAGTACAGTGAATCTGTGAAGGGTCGGTTCATCGTCTCCAGAGACATTTCCCAAAGCATCCTCTACCTTCAGATGAATGCCCTGAGAGCTGAGGACACTGCCATTTATTACTGTGCAAGAGATGCCTTCTATGGTTATGGGGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA 78 VL GATGTCCAGATAACCCAGTCTCCATCTTATCTTGCTGCATCTCCTGGAGAAACCATTACTATTAATTGCAGGGCAAGTAAGAGCATTAGAAAATATTTAGCCTGGTATCAAGAGAAACCTGGGAAAACTAATAAGCTTCTTATCTACTCTGGATCCACTTTGCAATCTGGAATTCCATCAAGGTTCAGTGGCAGTGGATCTGGTACAGATTTCACTCTCACCATCAGTAGCCTGGAGCCTGAAGATTTTGCAATGTATTACTGTCAACAGCATAATGAATACCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA 79 heavy chain sequence AAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA 80 light chain sequence ATGGGGAATCAGTTCCTGCCAGGACACAGTTTAGATATGAGGTTCCAGGTTCAGGTTCTGGGGCTCCTTCTGCTCTGGATATCAGGTGCCCAGTGTGATGTCCAGATAACCCAGTCTCCATCTTATCTTGCTGCATCTCCTGGAGAAACCATTACTATTAATTGCAGGGCAAGTAAGAGCATTAGAAAATATTTAGCCTGGTATCAAGAGAAACCTGGGAAAACTAATAAGCTTCTTATCTACTCTGGATCCACTTTGCAATCTGGAATTCCATCAAGGTTCAGTGGCAGTGGATCTGGTACAGATTTCACTCTCACCATCAGTAGCCTGGAGCCTGAAGATTTTGCAATGTATTACTGTCAACAGCATAATGAATACCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTACCCCAGAGAAGCCAAAGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGAAACAGCCAGGAAAGCGTGACAGAGCAGGATTCCAAGGATTCCACATACAGCCTGAGCAGCACACTGACACTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACACACCAGGGACTGTCCTCCCCTGTGACAAAGAGCTTCAACAGAGGAGAATGCTGA 81

The result of electrophoresis of 5B2 antibody (mouse IgG skeleton) and humanized 5B2 chimeric antibody and staining with Coomassie blue is shown in FIG. 4A. It was confirmed that a humanized 5B2 chimeric antibody was produced.

In order to verify the binding ability of the 5B2 antibody (mouse IgG framework) and the humanized 5B2 chimeric antibody to GST-AIMP2 and aggregate structure GST-AIMP2, dot blot analysis was performed. The results are shown in Figs. 4b and 4c. As shown in FIGS. 4B and 4C , it was confirmed that the humanized 5B2 chimeric antibody also selectively binds to AIMP2 aggregates like the 5B2 antibody (mouse IgG framework).

Claims (23)

AIMP2 (Aminoacyl-tRNA synthetase complex interacting multifunctional protein-2) specific antibody or fragment having immunological activity. According to claim 1, Complementarity determining regions heavy chain (CDRH) comprising any one selected from the group consisting of the amino acid sequence of SEQ ID NOs: 1 to 3, any one selected from the group consisting of the amino acid sequence of SEQ ID NOs: 4 to 6 An antibody or fragment having immunological activity specific for AIMP2, comprising a VH domain comprising CDRH2 comprising one, and CDRH3 comprising any one selected from the group consisting of amino acid sequences of SEQ ID NOs: 7 to 9. According to claim 1, FR1 comprising any one selected from the group consisting of the amino acid sequence of SEQ ID NOs: 20 to 22, FR2 comprising any one selected from the group consisting of the amino acid sequence of SEQ ID NOs: 23 to 25, SEQ ID NO: An antibody specific for AIMP2 or immunological thereof, comprising a VH domain comprising FR3 comprising any one selected from the group consisting of the amino acid sequence of 26 to 28, and FR4 comprising the amino acid sequence of SEQ ID NO: 29 or 30 fragments with activity. The method according to claim 1, wherein the CDRL1 comprising any one selected from the group consisting of the amino acid sequence of SEQ ID NOs: 10 to 13, CDRL2 comprising any one selected from the group consisting of the amino acid sequence of SEQ ID NOs: 14 to 16, and the sequence An antibody or fragment having immunological activity specific for AIMP2, comprising a VL domain comprising CDRL3 comprising any one selected from the group consisting of the amino acid sequence of Nos. 17 to 19. According to claim 1, FR1 comprising any one selected from the group consisting of the amino acid sequence of SEQ ID NOs: 31 to 33, FR2 comprising any one selected from the group consisting of the amino acid sequence of SEQ ID NOs: 34 to 37, SEQ ID NO: AIMP2 comprising a VL domain comprising FR3 comprising any one selected from the group consisting of the amino acid sequence of 38 to 42 and FR4 comprising any one selected from the group consisting of the amino acid sequence of SEQ ID NOs: 43 to 45 A specific antibody or fragment having immunological activity thereof. According to claim 1, wherein the VH domain comprising any one selected from the group consisting of the amino acid sequence of SEQ ID NOs: 46 to 49; And a VL domain comprising any one selected from the group consisting of the amino acid sequence of SEQ ID NOs: 50 to 54, AIMP2 specific antibody or fragment having immunological activity thereof. According to claim 1, wherein the heavy chain comprising any one selected from the group consisting of the amino acid sequence of SEQ ID NOs: 55 to 58; And a light chain comprising any one selected from the group consisting of amino acid sequences of SEQ ID NOs: 59 to 63, AIMP2 specific antibody or fragment having immunological activity thereof. According to claim 1, wherein AIMP2 is an AIMP2 monomer (monomer) or AIMP2 aggregate, AIMP2 specific antibody or fragment having immunological activity thereof. The method of claim 8, wherein the antibody or fragment having immunological activity specific for the AIMP2 monomer is: (i) a VH domain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO: 2, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 5, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 8; and (ii) a V domain comprising a VL domain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO: 11 or 12, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 15, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 18 which is an antibody or fragment having immunological activity specific for AIMP2. The method of claim 8, wherein the antibody or fragment having immunological activity specific for the AIMP2 aggregate comprises: (i) a VH domain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO: 1, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 4, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 7; and (ii) a V domain comprising a VL domain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO: 10, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 14, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 17; An antibody specific for AIMP2 or a fragment having immunological activity thereof. The method according to claim 8, wherein the antibody or fragment having immunological activity specific for the antibody or fragment having immunological activity thereof is specific for AIMP2 monomer and AIMP2 aggregate: (i) a VH domain comprising a CDRH1 comprising the amino acid sequence of SEQ ID NO: 3, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 6, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 9; and (ii) a V domain comprising a VL domain comprising a CDRL1 comprising the amino acid sequence of SEQ ID NO: 13, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 16, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 19; An antibody specific for AIMP2 or a fragment having immunological activity thereof. According to claim 1, wherein the fragment having immunological activity is Fab, Fd, Fab', dAb, F(ab'), F(ab') ₂ , scFv (single chain fragment variable), Fv, single chain antibody, Fv Any one selected from the group consisting of dimers, complementarity determining region fragments, humanized antibodies, chimeric antibodies, and diabodies, AIMP2 specific antibody or fragment having immunological activity thereof. An isolated nucleic acid molecule encoding the antibody of claim 1 or a fragment having immunological activity thereof. A vector comprising the isolated nucleic acid molecule of claim 13 . A host cell transformed with the vector of claim 14 . A pharmaceutical composition for the prevention or treatment of neurodegenerative diseases comprising the antibody of claim 1 or a fragment having immunological activity as an active ingredient. 17. The method of claim 16, wherein the neurodegenerative disease is Parkinson's disease, Huntington's disease, multiple system nerve atrophy, amyotrophic lateral sclerosis, Lou Gehrig's disease, polyglutamine ectasia, spinal cerebellar ataxia, spinal and soft muscle atrophy, tauosis, muscle tone Abnormalities, serpin deficiency, cirrhosis, type II diabetes, primary systemic amyloidosis, secondary systemic amyloidosis, pronto-transient dementia, geriatric systemic amyloidosis, familial amyloid polyneuropathy, hereditary cerebral amyloid vasculopathy, hemodialysis-associated amyloidosis, macular degeneration, Dementia, Alzheimer's disease, familial AD, Lewy body dementia, radiation therapy-induced dementia, boxer's dementia, Down syndrome, Gerstmann-Strussler-Scheinker disease, inclusion body myositis, prion protein brain amyloid angiopathy, axon injury , Acute diffuse cortical suppression, alpha-synucleinic condition, Parkinsonism-dementia complex of Guam, non-Gammanian motor neuron disease with nerve fiber entanglement, silver-affinity particle disease, cortical basal degeneration, diffuse nerve with calcification Fibrous entanglement, frontotemporal dementia with Parkinsonism associated with chromosome 17, Hallerforden-Spatz disease, stroke, cerebral infarction, head trauma, cerebral atherosclerosis, cerebral ischemia, permanent midcerebral ischemia, peripheral nerve regeneration, superimposed post-epileptic model, spinal cord injury , Niemann-Pick disease type C, Palido-Ponto-Nigral degeneration, Pick's disease, progressive subcortical gliosis, progressive supranuclear palsy, subacute sclerosing panencephalitis, dementia with tangles only, mild cognitive impairment, post encephalitis parkinsonism, myotonia Sexual dystrophy, tau panencephalopathy, AD-like syndrome with astrocytes, senile cardiac amyloidosis, endocrine tumors, glaucoma, ocular amyloidosis, primary retinal degeneration, optic nerve drusen, optic neuropathy, optic neuritis, and lattice dystrophy, sporadic Lou Gehrig and Creutzfeldt- At least one selected from the group consisting of Jacob's disease, a pharmaceutical composition for the prevention or treatment of neurodegenerative diseases. A composition for diagnosing neurodegenerative diseases comprising the antibody of claim 1 or a fragment having immunological activity thereof. A neurodegenerative disease diagnostic kit comprising the composition of claim 18. An information providing method for diagnosing a neurodegenerative disease comprising detecting an AIMP2 monomer or AIMP2 aggregate in a biological sample isolated from a subject using the antibody of claim 1 or a fragment having immunological activity thereof. 21. The method of claim 20, wherein the neurodegenerative disease is Parkinson's disease, Huntington's disease, multiple system nerve atrophy, amyotrophic lateral sclerosis, Lou Gehrig's disease, polyglutamine ectasia, spinal cerebellar ataxia, spinal and soft muscle atrophy, tauosis, muscle tone Abnormalities, serpin deficiency, cirrhosis, type II diabetes, primary systemic amyloidosis, secondary systemic amyloidosis, pronto-transient dementia, geriatric systemic amyloidosis, familial amyloid polyneuropathy, hereditary cerebral amyloid vasculopathy, hemodialysis-associated amyloidosis, macular degeneration, Dementia, Alzheimer's disease, familial AD, Lewy body dementia, radiation therapy-induced dementia, boxer's dementia, Down syndrome, Gerstmann-Strussler-Scheinker disease, inclusion body myositis, prion protein brain amyloid angiopathy, axon injury , Acute diffuse cortical suppression, alpha-synucleinic condition, Parkinsonism-dementia complex of Guam, non-Gammanian motor neuron disease with nerve fiber entanglement, silver-affinity particle disease, cortical basal degeneration, diffuse nerve with calcification Fibrous entanglement, frontotemporal dementia with Parkinsonism associated with chromosome 17, Hallerforden-Spatz disease, stroke, cerebral infarction, head trauma, cerebral atherosclerosis, cerebral ischemia, permanent midcerebral ischemia, peripheral nerve regeneration, superimposed post-epileptic model, spinal cord injury , Niemann-Pick disease type C, Palido-Ponto-Nigral degeneration, Pick's disease, progressive subcortical gliosis, progressive supranuclear palsy, subacute sclerosing panencephalitis, dementia with tangles only, mild cognitive impairment, post encephalitis parkinsonism, myotonia Sexual dystrophy, tau panencephalopathy, AD-like syndrome with astrocytes, senile cardiac amyloidosis, endocrine tumors, glaucoma, ocular amyloidosis, primary retinal degeneration, optic nerve drusen, optic neuropathy, optic neuritis, and lattice dystrophy, sporadic Lou Gehrig and Creutzfeldt- At least one selected from the group consisting of Jacob's disease, an information providing method for diagnosing neurodegenerative diseases. 1) reacting the candidate compound with the AIMP2 aggregate; 2) quantifying AIMP2 aggregates using the antibody of claim 10 or 11 or a fragment having immunological activity thereof; and 3) A method for screening a substance that inhibits or inhibits the formation of AIMP2 aggregates, comprising the step of selecting a compound having a reduced amount of AIMP2 aggregates compared to a control group not treated with the candidate compound. 1) reacting the candidate compound with AIMP2 expressing cells or AIMP2 aggregates; 2) quantifying the amount of AIMP2 expression or AIMP2 aggregates using the antibody of claim 1 or a fragment having immunological activity thereof; and 3) A screening method for a therapeutic agent for a neurodegenerative disease, comprising the step of selecting a compound having a reduced amount of AIMP2 expression or AIMP2 aggregate compared to a control group not treated with the candidate compound.

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