Antibody specifically binding to TDP-43 and use thereof

Abstract

The present invention relates to an antibody specifically binding to TDP-43 and use thereof. When the antibody of the present invention is used, the level of TDP-43, particularly TDP-43 aggregates, can be effectively detected. Therefore, the present invention can be a powerful tool for distinguishing TDP-43 proteinopathy from other clinically similar neurodegenerative diseases and diagnosing TDP-43 proteinopathy, serving as a biomarker for TDP-43 proteinopathy, including FTD, ALS, LATE, and the like.

Description

Antibody that specifically binds to TDP-43 and uses thereof

[0001] The present invention relates to an antibody that specifically binds to TDP-43 and its uses.

[0002] This patent application claims priority to Korean Patent Application No. 10-2024-0128416 filed with the Korean Intellectual Property Office on September 23, 2024, the disclosures of said patent application are incorporated herein by reference.

[0003]

[0004] Transactive response DNA-binding protein 43 (TDP-43), first identified in 1995 as a repressor of HIV-1 gene expression, is a highly conserved and ubiquitous RNA / DNA binding protein. TDP-43 plays a pivotal role in various cellular functions, including RNA metabolism, mRNA transport, microRNA maturation, and the regulation of stress granule formation. Depending on function, TDP-43 can move between the nucleus and the cytoplasm, but under normal physiological conditions, it is primarily located in the nucleus. Under pathological conditions, TDP-43 undergoes cleavage, hyperphosphorylation, and ubiquitination. These post-translational modifications reduce the translocation of TDP-43 to the nucleus, leading to cytoplasmic accumulation and aggregation. Diseases caused by the abnormal aggregation of TDP-43 are called TDP-43 proteinopathy, and representative diseases belonging to this category include amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD), and LATE (limbic-predominant age-related TDP-43 encephalopathy) dementia. In addition to these diseases, TDP-43 aggregates are also identified as copathologies in Alzheimer's disease (AD), Parkinson's disease (PD), chronic traumatic encephalopathy (CTE), and the rare disease inclusion body myositis (IBM). Notably, it is estimated that about one-third of Alzheimer's dementia patients suffer from LATE dementia rather than Alzheimer's dementia.Late-onset dementia is a relatively recently discovered disease with symptoms similar to Alzheimer's disease; however, pathologically, it is characterized by the accumulation of TDP-43 in the limbic system and hippocampal sclerosis instead of amyloid-beta and tau proteins. Late-onset dementia primarily develops after the age of 80, with reported incidence rates of at least 20% in those over 80, and up to 50% in some research centers. Late-onset dementia is identified as a cause of failure in clinical trials for Alzheimer's dementia treatments that primarily target amyloid-beta and tau. Currently, diagnosis of Late-onset dementia relies on post-mortem brain tissue examination, while both imaging biomarkers for TDP-43 and detection using bodily fluids remain in the research phase.

[0005] Accordingly, there is an urgent need to develop materials and methods capable of effectively detecting TDP-43.

[0006]

[0007] The inventors made diligent research efforts to develop a method for effectively detecting TDP-43. As a result, the present invention was completed by identifying an antibody capable of effectively detecting not only the TDP-43 monomer but also the TDP-43 oligomer.

[0008] Accordingly, the object of the present invention is to provide an antibody or its antigen-binding fragment that specifically binds to TDP-43.

[0009] Another object of the present invention is to provide a nucleic acid molecule comprising a nucleotide sequence encoding an antibody that specifically binds to the TDP-43 or its antigen-binding fragment.

[0010] Another objective of the present invention is to provide a recombinant vector comprising the nucleic acid molecule.

[0011] Another objective of the present invention is to provide a host cell that provides the recombinant vector.

[0012] Another objective of the present invention is to provide a composition for detecting TDP-43 comprising an antibody that specifically binds to TDP-43 or an antigen-binding fragment thereof.

[0013] Another objective of the present invention is to provide a kit for detecting TDP-43 comprising an antibody that specifically binds to TDP-43 or an antigen-binding fragment thereof.

[0014] Another objective of the present invention is to provide a method for providing information necessary for the diagnosis of a TDP-43-related disease, comprising the step of detecting TDP-43 by treating a biological sample with an antibody that specifically binds to TDP-43 or a fragment of its antigen binding.

[0015] Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims, and drawings.

[0016]

[0017] The present invention provides the inventions of 1 to 14 below.

[0018] 1. A heavy chain variable region comprising a heavy chain CDR1 (HCDR1) having an amino acid sequence represented by SEQ ID NO. 1, SEQ ID NO. 9, or SEQ ID NO. 17, an HCDR2 having an amino acid sequence represented by the following General Formula 1, and an HCDR3 having an amino acid sequence represented by SEQ ID NO. 3, SEQ ID NO. 11, or SEQ ID NO. 19; and a light chain variable region comprising an LCDR1 having an amino acid sequence represented by the following General Formula 2, an LCDR2 having an amino acid sequence of SEQ ID NO. 5, and an LCDR3 having an amino acid sequence represented by the following General Formula 3, wherein the antibody or its antigen-binding fragment specifically binding to TDP-43 comprises:

[0019] General Formula 1 (Sequence Number 50)

[0020] IISSX1GX2X3YYAX4WX5KG

[0021] General Formula 2 (Sequence No. 51)

[0022] TLSTGYSVGX6YX7X8X9

[0023] General Formula 3 (Sequence No. 52)

[0024] X 10 TX 11 HGSGX 12 SFX 13 VV

[0025] i) X1 is D or S; ii) X2 is N or S; iii) X3 is T or A; iv) X4 is N, S, or T; v) X5 is V or A; vi) X6 is E or N; vii) X7 is H or V; viii) X8 is L or I; ix) X9 is V or G; x) X 10 is that which is A or V; xi) the above X 11 is T or A; xii) the above X 12 is N or S; or xiii) the above X 13 It is H or Q.

[0026] 2. In 1, the antibody or its antigen-binding fragment specifically binding to TDP-43 comprises: an antibody or its antigen-binding fragment comprising:

[0027] (a) a heavy chain variable region comprising HCDR1 of SEQ ID NO. 1, HCDR2 of SEQ ID NO. 2, and HCDR3 of SEQ ID NO. 3; and a light chain variable region comprising LCDR1 of SEQ ID NO. 4, LCDR2 of SEQ ID NO. 5, and LCDR3 of SEQ ID NO. 6;

[0028] (b) a heavy chain variable region comprising HCDR1 of SEQ ID NO. 9, HCDR2 of SEQ ID NO. 10, and HCDR3 of SEQ ID NO. 11; and a light chain variable region comprising LCDR1 of SEQ ID NO. 12, LCDR2 of SEQ ID NO. 13, and LCDR3 of SEQ ID NO. 14; or

[0029] (c) a heavy chain variable region comprising HCDR1 of SEQ ID NO. 17, HCDR2 of SEQ ID NO. 18, and HCDR3 of SEQ ID NO. 19; and a light chain variable region comprising LCDR1 of SEQ ID NO. 20, LCDR2 of SEQ ID NO. 21, and LCDR3 of SEQ ID NO. 22.

[0030] 3. In 1 or 2, the antibody or its antigen-binding fragment specifically binding to TDP-43 comprises: an antibody or its antigen-binding fragment comprising:

[0031] i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 7 and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 8;

[0032] ii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 15 and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 16; or

[0033] iii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 23 and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 24.

[0034] 4. An antibody or its antigen-binding fragment in any one of 1 to 3, wherein the antibody or its antigen-binding fragment binds more specifically to the TDP-43 oligomer compared to the TDP-43 monomer.

[0035] 5. In any one of 1 to 4, the antibody or its antigen-binding fragment has an association rate constant (ka) for TDP-43 of 5.0 × 10 4 An antibody or its antigen-binding fragment that is (1 / Ms) or more.

[0036] 6. An antibody or its antigen-binding fragment, wherein in any one of 1 to 5, the antibody has a ka value that is at least 1.1 times higher than that of the TDP-43 monomer with respect to the TDP-43 oligomer.

[0037] 7. In any one of 1 to 6, the TDP-43 monomer can be detected at a concentration of 6.25 μg / mL or less, the TDP-43 oligomer can be detected at a concentration of 3.12 μg / mL or less, or a combination thereof.

[0038] 8. A nucleic acid molecule comprising a nucleotide sequence encoding any one of 1 to 7 or its antigen-binding fragment.

[0039] 9. A recombinant vector containing a nucleic acid molecule of 8.

[0040] 10. A host cell containing the recombinant vector of 9.

[0041] 11. A composition for detecting TDP-43 comprising any one of antibodies from 1 to 7 or an antigen-binding fragment thereof.

[0042] 12. A kit for detecting TDP-43 comprising any one of antibodies from 1 to 7 or an antigen-binding fragment thereof.

[0043] 13. A method for providing information necessary for the diagnosis of a TDP-43-related disease, comprising the step of treating a biological sample with any one of 1 to 7 antibodies or an antigen-binding fragment thereof to detect TDP-43.

[0044] 14. A method for providing information necessary for the diagnosis of a TDP-43 related disease, wherein the TDP-43 related disease is one or more selected from the group consisting of frontotemporal dementia, amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease, chronic traumatic encephalopathy, and myositis.

[0045]

[0046] In one aspect of the present invention, the present invention provides an antibody or its antigen-binding fragment that specifically binds to TDP-43, comprising: a heavy chain variable region comprising a heavy chain CDR1 (HCDR1) having an amino acid sequence represented by SEQ ID NO. 1, SEQ ID NO. 9, or SEQ ID NO. 17, an HCDR2 having an amino acid sequence represented by the following General Formula 1, and an HCDR3 having an amino acid sequence represented by SEQ ID NO. 3, SEQ ID NO. 11, or SEQ ID NO. 19; and a light chain variable region comprising an LCDR1 having an amino acid sequence represented by the following General Formula 2, an LCDR2 having an amino acid sequence of SEQ ID NO. 5, and an LCDR3 having an amino acid sequence represented by the following General Formula 3.

[0047] General formula 1

[0048] IISSX1GX2X3YYAX4WX5KG

[0049] General formula 2

[0050] TLSTGYSVGX6YX7X8X9

[0051] General formula 3

[0052] X 10 TX 11 HGSGX 12 SFX 13 VV

[0053] i) X1 is D or S; ii) X2 is N or S; iii) X3 is T or A; iv) X4 is N, S, or T; v) X5 is V or A; vi) X6 is E or N; vii) X7 is H or V; viii) X8 is L or I; ix) X9 is V or G; x) X 10 is that which is A or V; xi) the above X 11 is T or A; xii) the above X 12 is N or S; or xiii) the above X 13It is H or Q.

[0054] The inventors have made diligent research efforts to develop a method capable of effectively detecting TDP-43. As a result, they identified an antibody capable of effectively detecting TDP-43 monomers as well as TDP-43 oligomers.

[0055] In this specification, the term "antibody" refers to an antibody specific to TDP-43, comprising not only the complete antibody form but also an antigen-binding fragment of the antibody molecule.

[0056] A complete antibody has a structure consisting of two full-length light chains and two full-length heavy chains, each light chain being connected to a heavy chain by a disulfide bond. The heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types and has subclasses gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1), and alpha 2 (α2). The invariant regions of light chains have kappa and lambda types (Cellular and Molecular Immunology, Wonsiewicz, MJ, Ed., Chapter 45, pp. 41-50, WB Saunders Co. Philadelphia, PA (1991); Nisonoff, A., Introduction to Molecular Immunology, 2nd Ed., Chapter 4, pp. 45-65, Sinauer Associates, Inc., Sunderland, MA (1984)).

[0057] In this specification, the term "antigen-binding fragment" refers to a fragment possessing an antigen-binding function and includes Fab, F(ab'), F(ab')2, chemically linked F(ab')2 and Fv, etc. Among the antibody fragments, Fab has a structure having a variable region of the light chain and heavy chain, a constant region of the light chain, and a first constant region (CH1) of the heavy chain, and has one antigen-binding site. Fab' differs from Fab in that it has a hinge region containing one or more cysteine ​​residues at the C-terminus of the heavy chain CH1 domain. The F(ab')2 antibody is generated when the cysteine ​​residues in the hinge region of Fab' form a disulfide bond. Recombinant technology for generating Fv fragments as minimal antibody fragments having only a heavy chain variable region and a light chain variable region is disclosed in PCT international published patent applications WO 88 / 10649, WO 88 / 106630, WO 88 / 07085, WO 88 / 07086, and WO 88 / 09344. Two-chain Fv has a heavy chain variable region and a light chain variable region connected by non-covalent bonds, while single-chain Fv generally has a heavy chain variable region and a single chain variable region connected by covalent bonds through a peptide linker or directly connected at the C-terminus, so they can form a dimer-like structure similar to two-chain Fv. These antibody fragments can be obtained using proteolytic enzymes (for example, Fab can be obtained by restricting the whole antibody with papain and F(ab')2 fragment can be obtained by cleaving it with pepsin), or they can be produced through genetic recombination technology.

[0058] In the present invention, the antibody is preferably in the scFv form or in the complete antibody form. Additionally, the heavy chain constant region may be selected from any one isotype of gamma (γ), mu (μ), alpha (α), delta (δ), or epsilon (ε). Preferably, the constant region is gamma 1 (IgG1), gamma 3 (IgG3), and gamma 4 (IgG4). The light chain constant region may be in the kappa or lambda form, and is preferably in the kappa form.

[0059] In this specification, the term “heavy chain” refers to both the full-length heavy chain and fragments thereof, comprising a variable region domain VH and three constant region domains CH1, CH2, and CH3, which have an amino acid sequence having a sufficient variable region sequence to confer specificity to an antigen. Additionally, in this specification, the term “light chain” refers to both the full-length light chain and fragments thereof, comprising a variable region domain VL (Vk) and a constant region domain CL (Ck), which have an amino acid sequence having a sufficient variable region sequence to confer specificity to an antigen.

[0060] In this specification, the term "CDR (complementarity determining region)" refers to the amino acid sequence of the hypervariable region of the immunoglobulin heavy and light chains (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Ed., US Department of Health and Human Services, National Institutes of Health (1987)). The heavy chains (HCDR1, HCDR2, and HCDR3) and light chains (LCDR1, LCDR2, and LCDR3) each contain three CDRs. The CDRs provide the major contact residues for the antibody to bind to an antigen or epitope.

[0061] The antibodies of the present invention include, but are not limited to, monoclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, single-chain Fvs (scFV), single-chain antibodies, Fab fragments, F(ab') fragments, disulfide-conjugated Fvs (sdFV) and anti-idiotype (anti-Id) antibodies, and epitope-conjugated fragments of said antibodies. The antibodies of the present invention include minibodies, diabodies, tribodies, tetrabodies, linear antibodies, single-chain antibodies (scFV), scFv-Fc, bispecific antibodies, multispecific antibodies, glycosylated variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies as other modified arrangement forms of immunoglobulin molecules comprising antigen recognition sites of required specificity. Specific examples of modified antibodies and their antigen-binding fragments include nanobodies, AlbudAbs, DARTs (dual affinity re-targeting), BiTEs (bispecific T-cell engagers), TandAbs (tandem diabodies), DAFs (dual acting Fabs), two-in-one antibodies, SMIPs (small modular immunopharmaceuticals), FynomAbs (fynomers fused to antibodies), DVD-Igs (dual variable domain immunoglobulin), CovX-bodies (peptide modified antibodies), duobodies, and triomAbs. The list of such antibodies and their antigen-binding fragments is not limited to the above.

[0062] In this specification, the terms "Framework" or "FR" refer to variable domain residues other than hypervariable region (HVR) residues. The FR of the variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, HVR and FR sequences generally appear in the following order in VH (or VL / Vk):

[0063] (a) FRH1 (Framework region 1 of Heavy chain)-CDRH1 (complementarity determining region 1 of Heavy chain)-FRH2-CDRH2-FRH3-CDRH3-FRH4; and

[0064] (b) FRL1 (Framework region 1 of Light chain)-CDRL1 (complementarity determining region 1 of Light chain)-FRL2-CDRL2-FRL3-CDRL3-FRL4.

[0065] The term "variable region" or "variable domain" refers to a domain of the antibody heavy or light chain involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of native antibodies (VH and VL, respectively) generally have similar structures, and each domain contains four conserved framework regions (FR) and three hypervariable regions (HVR) (Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Additionally, antibodies binding to a specific antigen may be isolated using the VH or VL domain from antibodies that screen a library of VL or VH domains that are complementary to the antigen.

[0066] In another embodiment of the present invention, the antibody or its antigen-binding fragment that specifically binds to TDP-43 of the present invention is Fab or scFv. In a specific embodiment of the present invention, when the antigen-binding fragment of the antibody is scFv, the heavy chain variable region and the light chain variable region included in the scFv are connected by a linker such as (Gly-Ser)n, (Gly2-Ser)n, (Gly3-Ser)n, or (Gly4-Ser)n. Here, n is an integer from 1 to 6, specifically 3 to 4, but is not limited thereto. For example, the linker may be GGGGSGGGGSGGGGS. The light chain variable region and the heavy chain variable region of the scFv may exist in, for example, the following orientation: light chain variable region-linker-heavy chain variable region or heavy chain variable region-linker-light chain variable region.

[0067] In this specification, the term "specifically binds" or similar means that an antibody or its antigen-binding fragment, or other components such as scFvs, specifically interact with an antigen or a corresponding substance to produce an immunological response. Specific binding is at least about 1 x 10⁻⁶ -6 M or less (e.g., 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 (e.g., 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 (e.g., 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 The equilibrium dissociation constant of M) (e.g., K smaller than this). DIt can be characterized as (indicating a more specific binding). Methods for determining whether two molecules bind specifically are well known in the industry, including, for example, equilibrium dialysis, surface plasmon resonance (SPR), etc.

[0068] In this specification, the term “affinity” refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., antibody) and its binding partner (e.g., antigen). Unless otherwise specified, as used herein, “binding affinity” refers to the intrinsic binding affinity reflecting the 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule Y and its partner Y can generally be expressed as a dissociation constant (Kd). Affinity may be measured by conventional methods known in the art, including those described herein.

[0069] Additionally, as specified herein, the term “human antibody” has an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell, or an antibody derived from a non-human source utilizing a human antibody repertoire or other human antibody coding sequence. This definition of a human antibody excludes humanized antibodies containing non-human antigen-binding residues.

[0070] In this specification, the term “chimeric” antibody means an antibody in which part of the heavy chain and / or light chain is derived from a specific source or species, and the remainder of the heavy chain and / or light chain is derived from a different source or species.

[0071] In this specification, the term “humanized antibody” refers to a chimeric immunoglobulin containing a minimal sequence derived from a non-human (e.g., mouse) antibody, its immunoglobulin chain, or fragment (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of the antibody). In most cases, the humanized antibody is a human immunoglobulin (recipient antibody) in which residues of the recipient’s complementarity-determining region (CDR) are replaced by residues of the CDR of a non-human species (donor antibody), e.g., mouse, rat, or rabbit, having the desired specificity, affinity, and ability. In some cases, residues of the Fv framework region (FR) of the human immunoglobulin are replaced by corresponding non-human residues. Additionally, the humanized antibody may contain residues not found in the recipient antibody or in the imported CDR or framework sequence. Such modifications are made to further improve and optimize antibody performance. Generally, the humanized antibody will comprise at least one, and typically two, substantially all variable domains, wherein all or substantially all of the CDR region in the domain corresponds to the CDR region of a non-human immunoglobulin, and all or substantially all of the FR region has the sequence of the FR region of a human immunoglobulin. The humanized antibody comprises at least a portion of the immunoglobulin constant region (Fc region) or a substantial sequence of the human immunoglobulin constant region (Fc region).

[0072] The TDP-43 specific antibody or its antigen-binding fragment of the present invention may comprise variants of the amino acid sequence within a range capable of specifically recognizing TDP-43, as recognized by a person skilled in the art. For example, changes may be made to the amino acid sequence of the antibody to improve the binding affinity and / or other biological properties of the antibody. Such modifications include, for example, deletions, insertions, and / or substitutions of amino acid sequence residues of the antibody.

[0073] The above variants are said to have "substantial similarity," meaning that when two peptide sequences are optimally aligned, such as by a program GAP or BESTFIT using default gap weights, they share at least about 90% sequence identity, more preferably at least about 95%, 98%, or 99% sequence identity. Preferably, non-identical residue positions differ by conservative amino acid substitutions. "Conservative amino acid substitution" is when an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially alter the functionality of the protein. Where two or more amino acid sequences differ from each other due to conservative substitutions, the percentage or degree of similarity may be upregulated to compensate for the conservative nature of the substitution.

[0074] These amino acid variations are based on the relative similarities of amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, etc. Analysis of the size, shape, and type of amino acid side chain substituents reveals that arginine, lysine, and histidine are all positively charged residues; alanine, glycine, and serine have similar sizes; and 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 considered biologically functional equivalents.

[0075] In introducing mutations, the hydropathic index of the amino acids may be considered. Each amino acid is assigned a hydropathic index based on hydrophobicity and charge: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+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).

[0076] The hydrophobic amino acid index is very important in conferring interactive biological functions of proteins. It is a known fact that similar biological activity can be achieved by substituting with amino acids having similar hydrophobic indices. When introducing a variation based on the hydrophobic index, the substitution is preferably made between amino acids exhibiting a difference in hydrophobic index within ± 2, more preferably within ± 1, and even more preferably within ± 0.5.

[0077] Meanwhile, it is also well known that substitution between amino acids having similar hydrophilicity values ​​results in proteins having uniform biological activity. As disclosed in U.S. Patent No. 4,554,101, the following hydrophilicity values ​​are assigned to each amino acid residue: arginine (+3.0); lysine (+3.0); aspalate (+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).

[0078] When introducing a variation by referring to the hydrophilicity value, substitution is made between amino acids that exhibit a difference in hydrophilicity value within ± 2, more preferably within ± 1, and even more preferably within ± 0.5.

[0079] Amino acid exchanges in proteins that do not change the overall activity of the molecule are known in the art (H. Neurath, RLHill, The Proteins, Academic Press, New York, 1979). The most common exchanges are exchanges between amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly.

[0080] When the antibody or antigen-binding fragment of the present invention is prepared as an antibody-drug conjugate, said drug (payload) may be conjugated to the antibody or its antigen-binding fragment that specifically binds to the TDP-43 of the present invention by covalent bonding through a chemical linker. The term "linker" refers to any moiety that chains, links, or binds the binder (e.g., antibody or its antigen-binding fragment) to the drug described herein. Generally, a binder linker suitable for said antibody conjugate is one that is sufficiently stable to utilize the cyclic half-life of the antibody and simultaneously capable of releasing its drug after antigen-mediated internalization of the conjugate. The linker may be cleavable or non-cleavable. A cleavable linker is a linker that is cleaved by cleavage through intracellular metabolism, such as hydrolysis, reduction, or enzymatic reactions, and then internalized. A non-cleavable linker is a linker that releases the attached drug through lysosomal degradation of the antibody and then internalized. Suitable linkers include, but are not limited to, acid-degradable linkers, enzymatically cleavable linkers, reductively degradable linkers, self-sacrificial linkers, and non-cleavable linkers. Suitable linkers also include, but are not limited to, peptides, glucoronides, succinimide-thioethers, polyethylene glycol (PEG) units, hydrazones, mal-caproyl units, dipeptide units, valine-citrulline units, and para-aminobenzyl (PAB) units, or those containing these.

[0081] In another embodiment of the present invention, the antibody or its antigen-binding fragment of the present invention may be provided as a complex comprising an antibody or its antigen-binding fragment that specifically binds to the TDP-43 described above and a polypeptide. The polypeptide is not limited and may be, for example, another antibody or its antigen-binding fragment, or a target binding polypeptide. The complex is connected to each other by covalent bonds, and according to one embodiment of the present invention, the complex may be implemented in the form of a fused protein or a conjugate, and the antibody or its antigen-binding fragment and the polypeptide may be directly connected or indirectly connected through a linker (e.g., an amino acid linker).

[0082] The term "TDP-43 (TAR DNA-binding protein 43)" in this specification refers to a protein with a molecular weight of 43 kDa known through neuropathological studies. TDP-43 plays an important role in the physiological and pathological biological processes of the central nervous system by participating in RNA degradation and binding. Known functions include RNA binding and processing, regulation of gene transcription, and acting as an mRNA carrier, and it is also known as a marker of degenerative diseases.

[0083] In this specification, TDP-43 includes variants of TDP-43. The variants of TDP-43 may be peptides having amino acid sequences that maintain homology of 90% or more, more specifically 95% or more, and even more specifically 98% with respect to TDP-43. In the present invention, the TDP-43 variants may be fragments of the TDP-43 protein.

[0084] In one embodiment of the present invention, the antibody or its antigen-binding fragment that specifically binds to TDP-43 binds not only to the TDP-43 monomer but also to the oligomer of TDP-43.

[0085] In one embodiment of the present invention, the antibody or its antigen-binding fragment comprises the following:

[0086] (a) a heavy chain variable region comprising HCDR1 of SEQ ID NO. 1, HCDR2 of SEQ ID NO. 2, and HCDR3 of SEQ ID NO. 3; and a light chain variable region comprising LCDR1 of SEQ ID NO. 4, LCDR2 of SEQ ID NO. 5, and LCDR3 of SEQ ID NO. 6;

[0087] (b) a heavy chain variable region comprising HCDR1 of SEQ ID NO. 9, HCDR2 of SEQ ID NO. 10, and HCDR3 of SEQ ID NO. 11; and a light chain variable region comprising LCDR1 of SEQ ID NO. 12, LCDR2 of SEQ ID NO. 13, and LCDR3 of SEQ ID NO. 14; or

[0088] (c) a heavy chain variable region comprising HCDR1 of SEQ ID NO. 17, HCDR2 of SEQ ID NO. 18, and HCDR3 of SEQ ID NO. 19; and a light chain variable region comprising LCDR1 of SEQ ID NO. 20, LCDR2 of SEQ ID NO. 21, and LCDR3 of SEQ ID NO. 22.

[0089] In one embodiment of the present invention, the antibody or its antigen-binding fragment that specifically binds to TDP-43 comprises the following:

[0090] i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 7 and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 8;

[0091] ii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 15 and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 16; or

[0092] iii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 23 and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 24.

[0093] In one embodiment of the present invention, the antibody or its antigen-binding fragment that specifically binds to TDP-43 binds more specifically to the TDP-43 oligomer compared to the TDP-43 monomer.

[0094] The term TDP-43 oligomer in this specification refers to a soluble or insoluble form in which TDP-43 monomers are aggregated. A TDP-43 oligomer may refer to a state in which, for example, 2 to 10 TDP-43 monomers are bonded. For example, a TDP-43 oligomer may be a TDP-43 dimer, trimer, tetramer, pentamer, hexamer, heptmer, octamer, ctomer, or demer.

[0095] In one embodiment of the present invention, the antibody or its antigen-binding fragment has an association rate constant (ka) for TDP-43 of 5.0 × 10 4( It is greater than 1 / Ms). For example, the binding rate constant of the above antibody or its antigen-binding fragment to TDP-43 is 5.0 × 10 4( 1 / Ms) or more, 7.0 X 10 4( 1 / Ms) or more, 9.0 X 10 4( 1 / Ms) or more, 1.0 X 10 5( 1 / Ms) or more, 2.0 X 10 5( 1 / Ms) or more, 3.0 X 10 5( 1 / Ms) or more, 4.0 X 10 5( It may be 1 / Ms) or more, but is not limited thereto.

[0096] In one embodiment of the present invention, the antibody has a higher ka value for the TDP-43 oligomer than for the TDP-43 monomer. For example, the antibody may have a ka value that is 1.1 to 10 times, 1.1 to 9 times, 1.1 to 8 times, 1.1 to 7 times, 1.1 to 6 times, 1.1 to 5 times, 1.1 to 4 times, 1.1 to 3 times, 1 to 2 times, 2 to 10 times, 3 to 10 times, 4 to 10 times, 5 to 10 times, 6 to 10 times, 7 to 10 times, 8 to 10 times, 9 to 10 times, 2 to 5 times, or 3 to 5 times or more higher than that of the TDP-43 monomer, but is not limited thereto.

[0097] The antibody of the present invention has a high binding rate capable of rapidly binding to an antigen. Specifically, the NP47T antibody has a binding rate of ka = 1.813 × 10 for an oligomeric antigen. 5 It exhibited a binding rate of (1 / Ms), which is approximately 2.87 times faster than the ka = 6.312 × 10(1 / Ms) shown by the same antibody for monomers. The NP89T antibody also showed ka = 4.287 × 10 for oligomers. 5 (1 / Ms), for monomer, ka = 3.670 X 10 5 (1 / Ms) was expressed, showing differential binding rates depending on the structural state of the antigen. This characteristic implies that the antibody possesses excellent reactivity that can be utilized for diagnostic or therapeutic purposes, as it selectively recognizes and rapidly binds to pathological structures (e.g., protein oligomers or aggregates).

[0098] In one embodiment of the present invention, the antibody is capable of detecting TDP-43 monomers at a concentration of 6.25 μg / mL or less, detecting TDP-43 oligomers at a concentration of 3.12 μg / mL or less, or having a combination thereof.

[0099] The antibodies of the present invention are capable of binding even to antigens at low concentrations, thereby providing excellent detection sensitivity. According to dot blot analysis, the NP47T antibody showed a signal against oligomer antigens at a minimum concentration of 3.12 μg / mL, and the NP89T antibody also showed a clear response even at concentrations of 3.12 μg / mL or lower.

[0100] This low detection limit can be advantageous for this antibody to sensitively detect pathological protein aggregates in the early stages of disease and can be effectively utilized for the diagnosis of target proteins present in small amounts in vivo.

[0101]

[0102] In one aspect of the present invention, the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding the antibody or its antigen-binding fragment.

[0103] In this specification, the term “nucleic acid molecule” has a meaning that comprehensively includes DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are the basic building blocks of nucleic acid molecules, include not only natural nucleotides but also analogues in which sugar or base sites are modified (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90:543-584 (1990)).

[0104] Considering the variant having the aforementioned biological equivalence activity, the nucleic acid molecule of the present invention encoding the amino acid sequence constituting the antibody or its antigen-binding fragment is interpreted to also include a sequence exhibiting substantial identity therewith. The above substantial identity refers to a sequence that, when the sequence of the present invention described above is aligned with any other sequence to the greatest extent possible and the aligned sequence is analyzed using an algorithm commonly used in the art, exhibits at least 60% homology, more specifically 70% homology, even more specifically 80% homology, even more specifically 90% homology, and most specifically 95% 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); This is disclosed in 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), but is not limited thereto.

[0105]

[0106] In one aspect of the present invention, the present invention provides a recombinant vector comprising the nucleic acid molecule.

[0107] In this specification, the term "vector" includes plasmid vectors; cosmid vectors; and viral vectors such as bacteriophage vectors, adenovirus vectors, retrovirus vectors, and adeno-associated virus vectors as means for expressing a target gene in a host cell.

[0108] According to a preferred embodiment of the present invention, in the vector of the present invention, a nucleic acid molecule encoding a light chain variable region and a nucleic acid molecule encoding a heavy chain variable region are operatively linked with a promoter.

[0109] In this specification, the term "operationally coupled" means a functional coupling between a nucleic acid expression regulatory sequence (e.g., a promoter, a signal sequence, or an array of transcription factor binding sites) and another nucleic acid sequence, thereby allowing the regulatory sequence to regulate the transcription and / or translation of the other nucleic acid sequence.

[0110] The recombinant vector system of the present invention can be constructed through various methods known in the art, specific methods for which are disclosed in Sambrook et al., *Molecular Cloning*, *A Laboratory Manual*, *Cold Spring Harbor Laboratory Press* (2001), which is incorporated herein by reference.

[0111] The vector of the present invention can typically be constructed as a vector for cloning or as a vector for expression. Additionally, the vector of the present invention can be constructed using a prokaryotic cell or a eukaryotic cell as a host.

[0112] For example, when the vector of the present invention is an expression vector and the host is a eukaryotic cell, a promoter derived from the genome of a mammalian cell (e.g., metallothionein promoter, beta-actin promoter, human hemoglobin promoter and human muscle creatine promoter) or a promoter derived from a mammalian virus (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter, HSV TK promoter, mouse mammary tumor virus (MMTV) promoter, HIV LTR promoter, Moloney virus promoter, Epstein-Barr virus (EBV) promoter and Rhoese's sarcoma virus (RSV) promoter) may be used, and generally has a polyadenylation sequence as a transcription termination sequence.

[0113] The vector of the present invention may be fused with other sequences to facilitate the purification of antibodies expressed therefrom. Examples of fused sequences include glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA), and 6x His (hexahistidine; Quiagen, USA).

[0114] In addition, since 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, etc., without the need for an additional sequence for purification.

[0115] Meanwhile, the expression vector of the present invention includes antibiotic resistance genes commonly used in the art as selection markers, such as resistance genes for ampicillin, gentamicin, cabbageillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin, and tetracycline.

[0116]

[0117] In one aspect of the present invention, the present invention provides a host cell comprising the recombinant vector.

[0118] The above host cell is transformed by the recombinant vector described above.

[0119] Any host cell known in the art that can stably and continuously clone and express the vector of the present invention may be used, for example, suitable eukaryotic host cells for the vector include, but are not limited to, monkey kidney cells 7 (COS7), NSO cells, SP2 / 0, Chinese hamster ovary (CHO) cells, W138, baby hamster kidney (BHK) cells, MDCK, myeloma cell line, HuT 78 cells, yeast cells (Saccharomyces cerevisiae), insect cells, and HEK-293 cells.

[0120] In this specification, the terms "transformed," "transduced," or "transfected" refer to the process in which exogenous nucleic acids are delivered or introduced into a host cell. "Transformed," "transduced," or "transfected" cells are cells that have been transformed, introduced, or transfected with exogenous nucleic acids, and said cells include said cells and progeny cells resulting from their passage.

[0121] When the host cell is a prokaryotic cell, the method of delivering the vector of the present invention into a host cell can be carried out by the CaCl2 method (Cohen, SN et al., Proc. Natl. Acac. Sci. USA, 9:2110-2114 (1973)), the Hanahan method (Cohen, SN et al., Proc. Natl. Acac. Sci. USA, 9:2110-2114 (1973); and Hanahan, D., J. Mol. Biol., 166:557-580 (1983)) and the electroporation method (Dower, WJ et al., Nucleic. Acids Res., 16:6127-6145 (1988)), etc. In addition, when the host cell is a eukaryotic cell, the vector can be injected into the host cell by microinjection (Capecchi, MR, Cell, 22:479 (1980)), calcium phosphate precipitation (Graham, FL et al., Virology, 52:456 (1973)), electroporation (Neumann, E. et al., EMBO J., 1:841 (1982)), liposome-mediated transfection (Wong, TK et al., Gene, 10:87 (1980)), DEAE-dextran treatment (Gopal, Mol. Cell Biol., 5:1188-1190 (1985)), and gene bombardment (Yang et al., Proc. Natl. Acad. Sci., 87:9568-9572 (1990)).

[0122]

[0123] In one aspect of the present invention, the present invention provides a composition for detecting TDP-43 comprising an antibody that specifically binds to TDP-43 or an antigen-binding fragment thereof.

[0124] The composition for detecting TDP-43 of the present invention may further include tools, devices, or reagents that can be included in conventional diagnostic compositions for confirming the presence or absence of TDP-43 in biological samples using peptides, such as tools for collecting a sample to be tested or applying a diagnostic reagent, or reagents or devices for confirming the binding of antibodies and TDP-43.

[0125] Tools / reagents that may be included in the above detection composition include, but are not limited to, suitable carriers, labeling substances capable of generating a detectable signal, solvents, cleaning agents, buffers, stabilizers, etc. Suitable carriers may be, but are not limited to, soluble carriers, for example, physiologically acceptable buffers known in the art, e.g., PBS; insoluble carriers, for example, polymers such as polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluoropolymer, cross-linked dextran, polysaccharide, and latex plated with metal, as well as other paper, glass, metal, agarose, and combinations thereof.

[0126] Since the composition of the present invention utilizes an antibody or its antigen-binding fragment that specifically binds to the TDP-43 of the present invention described above as an active ingredient, the common details between the two are omitted to avoid excessive complexity in this specification.

[0127] Pharmaceutically acceptable carriers included in the composition of the present invention are those commonly used in formulations and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above components, the pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0128] The composition of the present invention may be prepared in a unit volume form or contained in a multi-dose container by formulation using a pharmaceutically acceptable carrier and / or excipient, according to a method that can be easily carried out by a person skilled in the art to which the invention belongs. In this case, the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or in the form of an extract, powder, suppository, powder, granule, tablet, or capsule, and may additionally include a dispersant or a stabilizer.

[0129] In one embodiment of the present invention, the composition for detecting TDP-43 of the present invention is a composition for diagnosing TDP-43-related diseases.

[0130] The above TDP-43-related diseases are one or more selected from the group consisting of LATE, frontotemporal dementia, amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease, chronic traumatic encephalopathy, and myositis. However, they are not limited thereto and may include all diseases associated with the accumulation of TDP-43. Specifically, the above frontotemporal dementia may be behavioral variant frontotemporal dementia (bvFTD), semantic dementia (SD), progressive nonfluent aphasia (PNFA), or a combination thereof.

[0131]

[0132] In one aspect of the present invention, the present invention provides a kit for detecting TDP-43 comprising an antibody that specifically binds to TDP-43 or an antigen-binding fragment thereof.

[0133] The diagnostic kit of the present invention comprises an antibody or its antigen-binding fragment that specifically binds to the TDP-43 of the present invention as described above, and diagnoses TDP-43-related diseases of the present invention; however, to avoid excessive complexity in this specification due to repetition of the content between the two, the description thereof is omitted.

[0134] Since the above-described kit contains antibodies, it can be basically manufactured to be suitable for various immunoassays or immunostainings. The above immunoassays or immunostainings include, but are not limited to, radioimmunoassays, radioimmunoprecipitation, immunoprecipitation, ELISA (enzyme-linked immunosorbent assay), sandwich ELISA, inhibition or competition assays, flow cytometry, immunofluorescence staining, and immunoaffinity purification. Methods for the above immunoassays or immunostainings include: Enzyme Immunoassay, ET Maggio, ed., CRC Press, Boca Raton, Florida, 1980; Gaastra, W., Enzyme-linked immunosorbent assay (ELISA), in Methods in Molecular Biology, Vol. 1, Walker, JM ed., Humana Press, NJ, 1984; and Ed Harlow and David Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999, are described in which the above document is incorporated herein by reference.

[0135] For example, when the method of the present invention is carried out according to a radioimmunoassay method, an antibody labeled with a radioisotope (e.g., C14, I125, P32, and S35) may be used to detect TDP-43. When the present invention is carried out by an ELISA method, a specific embodiment of the present invention comprises: (i) coating a sample to be analyzed onto the surface of a solid substrate; (ii) reacting the sample with an antibody or its antigen-binding fragment that specifically binds to TDP-43 of the present invention as a primary antibody; (iii) reacting the result of step (ii) with an enzyme-bound secondary antibody; and (iv) measuring the activity of the enzyme.

[0136] Suitable solid substrates are hydrocarbon polymers (e.g., polystyrene and polypropylene), glass, metal, or gel, most specifically microtiter plates.

[0137] The enzyme bound to the secondary antibody described above includes, but is not limited to, enzymes that catalyze chromogenic, fluorescent, luminescent, or infrared reactions, such as alkaline phosphatase, beta-galactosidase, horseradish peroxidase, luciferase, and cytochrome P 450It includes. When alkaline phosphatase is used as the enzyme binding to the secondary antibody, chromogenic reaction substrates such as bromochloroindoleyl phosphate (BCIP), nitro blue tetrazolium (NBT), naphthol-AS-B1-phosphate, and ECF (enhanced chemifluorescence) are used as substrates, and when horseradish peroxidase is used, chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (bis-N-methylacridinium nitrate), resolupin benzyl ether, luminol, Amplex Red reagent (10-acetyl-3,7-dihydroxyphenoxazine), HYR (p-phenylenediamine-HCl and pyrocatechol), TMB (tetramethylbenzidine), and ABTS Substrates such as (2,2-Azine-di[3-ethylbenzthiazoline sulfonate]), o-phenylenediamine (OPD) and naphthol / pyronine, glucose oxidase and t-NBT (nitroblue tetrazolium) and m-PMS (phenzaine methosulfate) can be used.

[0138] When the present invention is carried out in a sandwich ELISA manner, a specific embodiment of the present invention comprises: (i) coating the surface of a solid substrate with an antibody that specifically binds to TDP-43 as a capture antibody; (ii) reacting the capture antibody with a sample; (iii) reacting the result of step (ii) with a detection antibody to which a signal-generating label is attached; and (iv) measuring the signal generated from the label.

[0139] The detection antibody of the present invention has a label that generates a detectable signal. The label is a chemical substance (e.g., biotin), an enzyme (alkaline phosphatase, beta-galactosidase, horseradish peroxidase, and cytochrome P 450 ), radioactive materials (e.g., C 14 , I 125 , P 32 and S 35 These include, but are not limited to, fluorescent materials (e.g., fluorisine), luminescent materials, chemiluminescent materials, and FRET (fluorescence resonance energy transfer). Various labels and labeling methods are described in Ed Harlow and David Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999.

[0140] In the above ELISA method, the final measurement of enzyme activity or signal may be carried out according to various methods known in the art. If biotin is used as a label, the signal can be easily detected with streptavidin, and if luciferase is used, the signal can be easily detected with luciferin.

[0141] Samples applicable to the kit of the present invention include, but are not limited to, cells, tissues or tissue-derived extracts, lysates or purified substances, blood, plasma, serum, lymph, or ascites.

[0142] The antibody of the present invention may be used for in vivo or in vitro imaging. According to another aspect of the present invention, the present invention provides an imaging composition comprising the antibody of the present invention described above and a conjugate to which a label generating a detectable signal bound to the antibody is bound.

[0143] The label generating the above detectable signal is a T1 contrast agent (e.g., Gd chelate compound), a T2 contrast agent (e.g., superparamagnetic material (e.g., magnetite, Fe3O4, γ-Fe2O3, manganese ferrite, cobalt ferrite, and nickel ferrite)), a radioisotope (e.g., 11 C, 15 O, 13 N, P 32 , S 35 , 44 Sc, 45 Ti, 118 I, 136 La, 198 Tl, 200 Tl, 205 Bi and 206 Bi), fluorescent substances (fluorescein, phycoerythrin, rhodamine, lissamine, and Cy3 and Cy5), chemiluminescent groups, magnetic particles, mass labels, or electron-dense particles, including but not limited to these.

[0144] In one embodiment of the present invention, the TDP-43 (detection kit) of the present invention is a TDP-43 (related disease diagnosis kit).

[0145]

[0146] In one aspect of the present invention, the present invention provides a method for providing information necessary for the diagnosis of a TDP-43-related disease, comprising the step of detecting TDP-43 by treating a biological sample with an antibody specific to TDP-43 or a fragment of its antigen binding.

[0147] In one embodiment of the present invention, the antibody specific to TDP-43 or its antigen-binding fragment is provided in the form of a composition or a kit. Descriptions regarding the composition and the kit are omitted to avoid excessive complexity in this specification due to repetition.

[0148] In one embodiment of the present invention, the biological sample comprises, but is not limited to, cells, tissues or tissue-derived extracts, lysates or purified substances, blood, plasma, serum, lymph, or ascites.

[0149] According to one embodiment of the present invention, the detection step can be performed through any experimental method capable of confirming the binding signal of a protein and a peptide, such as ELISA or SPR techniques. For example, the TDP-43 detection step can be performed through radioimmunoassay, radioimmunoprecipitation, immunoprecipitation, ELISA (enzyme-linked immunosorbent assay), sandwich ELISA, inhibition or competition assay, flow cytometry, immunofluorescence staining, and immunoaffinity purification, but is not limited thereto.

[0150] In one embodiment of the present invention, the TDP-43-related disease is one or more selected from the group consisting of LATE, frontotemporal dementia, amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease, chronic traumatic encephalopathy, and myositis. However, it is not limited thereto and may include all diseases associated with the accumulation or oligomerization of TDP-43. Specifically, the frontotemporal dementia may be behavioral variant frontotemporal dementia, semantic dementia, non-fluent / grammatical progressive aphasia, or a combination thereof.

[0151]

[0152] The features and advantages of the present invention are summarized as follows:

[0153] (a) The present invention provides an antibody or its antigen-binding fragment that specifically binds to TDP-43.

[0154] (b) The present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding an antibody that specifically binds to the TDP-43 or its antigen-binding fragment.

[0155] (c) The present invention provides a recombinant vector comprising the nucleic acid molecule.

[0156] (d) The present invention provides a host cell that provides the recombinant vector.

[0157] (e) The present invention provides a composition for detecting TDP-43 comprising an antibody that specifically binds to TDP-43 or an antigen-binding fragment thereof.

[0158] (f) Another object of the present invention is to provide a kit for detecting TDP-43 comprising an antibody that specifically binds to TDP-43 or an antigen-binding fragment thereof.

[0159] (g) The present invention provides a method for providing information necessary for the diagnosis of a TDP-43-related disease, comprising the step of detecting TDP-43 by treating a biological sample with an antibody that specifically binds to TDP-43 or a fragment of its antigen binding.

[0160] (h) When using the antibody of the present invention, the level of TDP-43, particularly TDP-43 aggregates, can be effectively detected. Accordingly, as a biomarker for TDP-43 proteinopathy including FTD, ALS, and LATE, the present invention can be a powerful tool for distinguishing and diagnosing TDP-43 proteinopathy from other clinically similar neurodegenerative diseases.

[0161]

[0162] Figure 1 shows the results of the indirect ELISA of the anti-TDP-43 antibody.

[0163] Figure 2 shows the results of the dot blot assay of the anti-TDP-43 antibody.

[0164] Figures 3a to 3d show the SPR analysis results of NP47T(A) and NP89T(B) antibodies.

[0165] Figure 4 shows the sandwich ELISA standard curve of the anti-TDP-43 antibody.

[0166] Figure 5 shows the results of a cross-reactivity test using direct ELISA and sandwich ELISA.

[0167] Figure 6 shows plasma TDP-43 levels detected by sandwich ELISA (NP47T).

[0168]

[0169] The present invention will be described in more detail below through examples. These examples are intended solely to explain the invention more specifically, and it will be obvious to those skilled in the art that the scope of the invention is not limited by these examples according to the gist of the invention.

[0170]

[0171] Examples

[0172] Example 1: Materials and Method

[0173] 1-1. Production of TDP43 Monoclonal Antibody

[0174] Monoclonal antibodies against the human TDP-43 protein were obtained through two methods. Rabbit monoclonal antibodies were produced using the phage display method, and mouse monoclonal antibodies were produced using hybridoma cells. For animal immunization, four types of recombinant proteins (human TDP-43 full length, C-term fragment, N-term fragment, modified full length) were used, and immunization was performed three times for each antigen at intervals of 4 weeks, 2 weeks, and 2 weeks.

[0175]

[0176] 1-2. Indirect ELISA

[0177] The reactivity of the inventive antibodies (NP47T, NP54T, NP89T) against the TDP-43 protein was analyzed using the indirect ELISA described below. The streptavidin-biotin reaction was used to coat the TDP-43 protein onto microplates. Biotin was conjugated to the TDP-43 protein using a biotinylation kit (Abcam), diluted in carbonate-bicarbonate buffer, and reacted at room temperature for 2 hours to form Pierce TM Streptavidin Coated Plates (Thermo Fisher Scientific) were coated. The prepared anti-TDP-43 recombinant antibodies (NP47T, NP54T, NP89T) and one commercial antibody used as a control (clone 2E2-D3, Abnova) were diluted to the same concentration and reacted with the coated TDP-43 at room temperature for 1 hour. After washing the residue with TBST, chemiluminescence was measured using HRP-conjugated anti-IgG antibody and ECL.

[0178]

[0179] 1-3. Detection of TDP-43 species via sandwich ELISA

[0180] The antibody according to the present invention was used to measure TDP-43 in plasma. NP47T, NP54T, and NP89T were used as capture antibodies, diluted in carbonate-bicarbonate buffer, and reacted at 4°C for 17 hours to coat a 96-well microplate (Thermo Fisher Scientific). Subsequently, plate blocking was performed at room temperature for 1 hour using 1% BlockAce. Recombinant TDP-43 protein used as a standard was binary diluted in the reaction solution and reacted with the NP47T, NP54T, and NP89T coated on the plate at room temperature for 1 hour. After the reaction, residues were washed with TBST, and chemiluminescence was measured using a commercial anti-TDP-43 antibody conjugated with HRP and ECL.

[0181]

[0182] 1-4. Measurement of TDP-43 Oligomers

[0183] Plasma sample testing was performed using a sandwich ELISA with NP47T as the capture antibody. Plasma samples were diluted in the reaction solution in the same way as the standard and reacted with coated plates. The plasma samples used in the experiment consisted of 36 subjects in the dementia group and 22 subjects in the normal control (NC) group. Student's t-tests for comparison between groups were performed using GraphPad Prism 8. ROC curve analysis to verify diagnostic accuracy was conducted using MedCalc v.22.021.

[0184]

[0185] 1-5. Analysis of Antibody-Antigen Binding Affinity Using SPR

[0186] The antigen binding affinity of the monoclonal antibody was measured using SPR (BIAcore T200). The SPR analysis was performed as follows: The sensor chip was activated by injecting 0.1 M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and 0.05 M N-hydroxysuccinimide at a flow rate of 10 μl / min for 1 minute. After binding the ligand to the activated sensor chip, it was blocked with 1 M ethanolamine (pH 8.5). Subsequently, the analyte, diluted in PBS to the desired concentration, was flowed over the ligand immobilized in the channel at a flow rate of 30 μl / min to determine the association time. The dissociation time was determined by flowing running buffer (HBS-N) at the same flow rate. Both the association and dissociation times were conducted for 3 minutes. All analytes were tested at 5 concentrations, and binding strength was calculated using BIA evaluation software 3.2.

[0187]

[0188] 1-6. Dot blot experiment

[0189] After loading and fixing 2 μl of the sample onto a nitrocellulose (NC) membrane, the membrane was washed three times with TBST for 5 minutes each. The NC membrane was blocked for 1 hour using 5% BSA in TBST, then reacted with the primary antibody for 1 hour, and washed three times with TBST for 10 minutes each. The membrane was then reacted for 1 hour using an HRP-conjugated secondary antibody matching the primary antibody, washed three times with TBST for 10 minutes each, and then treated with ECL solution to check the results.

[0190]

[0191] Example 2: Generation and Characterization of TDP-43 Monoclonal Antibody

[0192] TDP-43 monoclonal antibodies were generated against four types of recombinant hTDP-43 proteins, and among the 13 clones, three antibodies (NP47T, NP54T, and NP89T) showing the highest reactivity with TDP-43 were obtained. The sequences of the three antibodies are summarized in Table 1. The reactivity of the inventive antibodies NP47T, NP54T, and NP89T was confirmed via indirect ELISA, and the reactivity was compared by treating with a commercial antibody (H00023435-M01; clone 2E2-D3) at the same concentration.

[0193] The results are shown in Figure 1.

[0194] As shown in Figure 1, all three antibodies invented showed a higher detection signal for TDP-43 at the same concentration than the monoclonal antibody H00023435-M01, and in the case of NP89T, a higher signal than the polyclonal antibody 10782-2-AP was confirmed.

[0195]

[0196] Variable region sequence of the developed antibody Antibody VH Sequence VL Sequence NP47TQSVEESGGRLVTPGTPLTLTCTVSGIDLSSNAMTWVRQAPGKGLEYIGIISSDGNTYYANWVKGRFTISKTSTTVDLRMTSPTTEDTATYFCTRGGDLWGQGTLVTVSS(Sequence No. 7)ELVLTQSPSLSASLGTTARLTCTLSTGYSVGEYHLVWLQQVPGRPPRYLLGYHTEEIKRQGSGVHSRFSGSKDDSANAGVLSISGLQPEDEADYYCATTHGSGNSFHVVFGGGTQLTVTGG(Sequence No. 8)NP54TQEQLVESGGRLVTPGTPLTLTCTVSGIDLDSYAVTWVRQAPGKGLEYIGIISSSGSAYYASWAKGRFTISKTSTTVDLKMTSPTTEDTATYFCTRGVDLWGQGTLVTVSS(Sequence No. 15)QPVLTQPPSLSASLGTTARLTCTLSTGYSVGNYVIGWYQQVPGRPPRYLLTYHTEEIKRQGSGVHSRFSGSKDDSANAGVLSISGLQSEDEADYYCVTAHGSGSSFQVVFGGGTQLTVTGG (SEQ ID NO: 16)NP89TQTVKESGGRLVTPGTPLTLTCTVSGFSLSSYAMTWVRQAPGKGLEYIGIISSSGNTYYATWAKGRLTISRTSTTVDLKMTSLTTEDTATYFCTRGGNLWGPGTLVTVSS (SEQ ID NO: 23) ELVLTQSPSLSASLGTTARLTCTLSTGYSVGNYVIGWYQQVPGRPPRYLLTYHTEEIKRQGSGVHSRFSGSKDDSANAGVLNISGLQPEDEADYYCATTHGSGNSFHVVFGGGTQLTVTGG (SEQ ID NO: 24)

[0197] Subsequently, the difference in binding affinity between the monomer and oligomer of the inventive antibody was confirmed.

[0198] The results are shown in Figure 2.

[0199] As shown in Figure 2, the reactivity to monomers and oligomers was confirmed through dot blot, and it was confirmed that the NP47T and NP54T antibodies had higher reactivity with oligomers than with monomers, while the NP89T antibody showed similar reactivity to monomers and oligomers.

[0200] Experimental results showed that the antibody NP47T detected a binding signal up to a TDP-43 oligomer concentration of 3.12 μg / mL, indicating the high selectivity and sensitivity of the antibody. The antibody NP54T detected a binding signal up to a TDP-43 oligomer concentration of 12.5 μg / mL. The antibody NP89T exhibited strong binding affinity for both oligomer and monomeric antigens, and the binding signal was maintained even when the concentration of TDP-43 monomer or oligomer was 3.12 μg / mL. Under the same concentration conditions, the binding signal for the oligomer was stronger than that for the monomer, suggesting that the antibody has a relatively high affinity for oligomers.

[0201]

[0202] The difference in binding strength of each antibody was examined using the SPR method.

[0203] The results are shown in Figures 3a to 3d.

[0204] As shown in Figures 3a to 3d, similar to the results of the dot blot, the NP47T antibody had a higher binding affinity to the oligomer than to the monomer, and the binding affinity of the NP89T antibody to the monomer and oligomer was similar.

[0205] Antibody NP47T exhibited a high binding rate to the TDP-43 oligomer and demonstrated superior binding affinity compared to the monomeric antigen. Specifically, the binding rate constant (ka) of antibody NP47T to the TDP-43 oligomer was 1.813 × 10⁻¹⁰. 5It was measured as (1 / Ms), which is 6.312 × 10⁻¹⁰, the binding rate constant that the same antibody exhibits for the TDP-43 monomer. 4 This means a binding rate approximately 2.87 times faster than (1 / Ms). In other words, the above results suggest that the antibody NP47T exhibits higher affinity and specificity for the TDP-43 oligomeric form.

[0206] In the case of antibody NP89T, very fast binding rates were observed for both monomeric and oligomeric TDP-43, with the highest binding rates specifically for the oligomeric form. The ka value of NP89T for the monomer is 3.670 × 10⁻¹⁰ 5 (1 / Ms) and the ka for the oligomer is 4.287 X 10 5 (1 / Ms) showed a tendency for the binding rate to oligomers to be slightly faster than that to monomers. This indicates that NP89T has a strong binding affinity to both antigen forms and may show a preferential binding tendency, particularly to oligomer antigens.

[0207] Thus, it was confirmed that NP47T has characteristics as an oligomer-specific antibody, and NP89T is an antibody with high broad reactivity capable of binding to both monomers and oligomers. Since the antibodies according to the present invention exhibit differential binding characteristics depending on the aggregation state of the antigen, it is possible to selectively detect pathological forms (e.g., oligomers) of disease-related proteins by utilizing this.

[0208]

[0209]

[0210] Example 3: Establishment of a TDP-43 Oligomer Detection System and Evaluation of Diagnostic Utility for Dementia

[0211] To establish a highly efficient TDP-43 oligomer detection method based on the invented antibodies, a sandwich ELISA system combining three invented antibodies and commercial antibodies was constructed, and the reactivity to TDP-43 was confirmed. Each combination was constructed based on the epitope information of the antibodies.

[0212] The results are shown in Fig. 4.

[0213] As shown in Figure 4, when the standard curves of each sandwich ELISA tested with binary dilution of TDP-43 were examined, the detection signal increased linearly as the concentration of the standard substance increased in all sandwich ELISA tests. In addition, for all three types of sandwich ELISAs, a detection signal higher than the background signal was observed at the lowest concentration of the standard substances used in the experiment. When detecting standard substances of the same concentration, the detection signal was highest in the test using NP47T as the capture antibody.

[0214]

[0215] To evaluate the cross-reactivity of the sandwich ELISA system for detecting TDP-43 oligomers with other proteins, indirect ELISA and sandwich ELISA using NP47T were performed on amyloid-beta, amylin, and alpha-synuclein in addition to the TDP-43 protein. The results are shown in Figure 5. As shown in Figure 5, it was confirmed that none of the three inventive antibodies reacted with amyloid-beta, amylin, and alpha-synuclein, and only showed reactivity with hTDP-43.

[0216]

[0217] Subsequently, to verify the diagnostic utility of the disease-diagnostic TDP-43 oligomer detection method based on the inventive antibody, the ability to detect TDP-43 oligomers in the plasma of a dementia patient group and a normal control group was evaluated. In this experiment, the NP47T sandwich ELISA, which was identified as having the highest detection signal in the detection experiment of recombinant TDP-43, was used.

[0218] The results are shown in Figure 6.

[0219] As shown in Fig. 6, the average RLU value of the semantic dementia group was 58,518, and the average RLU value of the normal control group (NC) was 15,569, indicating that a significantly higher signal was detected in the semantic dementia group compared to the normal control group (p<0.0001, Fig. 6A). To confirm diagnostic accuracy, the results of the ROC curve analysis showed that the diagnostic accuracy AUC (area under the ROC curve) was 0.9596 (p<0.001), and the sensitivity and specificity were confirmed to be 97.22% and 81.82%, respectively (Fig. 6B).

[0220]

[0221] In this study, we produced antibodies against hTDP-43 and established a method to detect TDP-43 oligomers using these antibodies. Using this method, we confirmed the ability to detect recombinant TDP-43 oligomers and verified that even low concentrations of TDP-43 oligomers in plasma can be detected. Furthermore, we confirmed high diagnostic accuracy in an evaluation of diagnostic accuracy using plasma from a group with dementia and a normal control group. These results indicate the potential of the TDP-43 oligomer detection method established in this study for diagnosing dementia and suggest its potential to diagnose various diseases caused by TDP-43 aggregation in addition to dementia.

Claims

1. A heavy chain variable region comprising a heavy chain CDR1 (HCDR1) comprising an amino acid sequence represented by SEQ ID NO. 1, SEQ ID NO. 9, or SEQ ID NO. 17, an HCDR2 comprising an amino acid sequence represented by the following General Formula 1, and an HCDR3 comprising an amino acid sequence represented by SEQ ID NO. 3, SEQ ID NO. 11, or SEQ ID NO. 19; and a light chain variable region comprising an LCDR1 comprising an amino acid sequence represented by the following General Formula 2, an LCDR2 comprising an amino acid sequence of SEQ ID NO. 5, and an LCDR3 comprising an amino acid sequence represented by the following General Formula 3, wherein the antibody or its antigen-binding fragment specifically binds to TDP-43 (TAR DNA-binding protein 43): General formula 1 IISSX1GX2X3YYAX4WX5KG General formula 2 TLSTGYSVGX6YX7X8X9 General formula 3 X 10 TX 11 HGSGX 12 SFX 13 VV i) X1 is D or S; ii) X2 is N or S; iii) X3 is T or A; iv) X4 is N, S, or T; v) X5 is V or A; vi) X6 is E or N; vii) X7 is H or V; viii) X8 is L or I; ix) X9 is V or G; x) X 10 is that which is A or V; xi) the above X 11 is T or A; xii) the above X 12 is N or S; or xiii) the above X 13 It is H or Q.

2. In claim 1, the antibody or its antigen-binding fragment is an antibody or its antigen-binding fragment that specifically binds to TDP-43, comprising: (a) a heavy chain variable region comprising HCDR1 of SEQ ID NO. 1, HCDR2 of SEQ ID NO. 2, and HCDR3 of SEQ ID NO. 3; and a light chain variable region comprising LCDR1 of SEQ ID NO. 4, LCDR2 of SEQ ID NO. 5, and LCDR3 of SEQ ID NO. 6; (b) a heavy chain variable region comprising HCDR1 of SEQ ID NO. 9, HCDR2 of SEQ ID NO. 10, and HCDR3 of SEQ ID NO. 11; and a light chain variable region comprising LCDR1 of SEQ ID NO. 12, LCDR2 of SEQ ID NO. 13, and LCDR3 of SEQ ID NO. 14; or (c) a heavy chain variable region comprising HCDR1 of SEQ ID NO. 17, HCDR2 of SEQ ID NO. 18, and HCDR3 of SEQ ID NO. 19; and a light chain variable region comprising LCDR1 of SEQ ID NO. 20, LCDR2 of SEQ ID NO. 21, and LCDR3 of SEQ ID NO.

22.

3. In claim 1, the antibody or its antigen-binding fragment is an antibody or its antigen-binding fragment that specifically binds to TDP-43, comprising: i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 7 and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 8; ii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 15 and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 16; or iii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 23 and a light chain variable region comprising the amino acid sequence of SEQ ID NO.

24.

4. The antibody or its antigen-binding fragment according to claim 1, wherein the antibody or its antigen-binding fragment binds more specifically to a TDP-43 oligomer compared to a TDP-43 monomer.

5. In paragraph 1, the antibody or its antigen-binding fragment has an association rate constant (ka) for TDP-43 of 5.0 × 10 4 An antibody or its antigen-binding fragment that is (1 / Ms) or more.

6. A nucleic acid molecule comprising a nucleotide sequence encoding the antibody or its antigen-binding fragment of any one of claims 1 to 5.

7. A recombinant vector comprising the nucleic acid molecule of claim 6.

8. A host cell containing the recombinant vector of claim 7.

9. A composition for detecting TDP-43 comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 5.

10. A kit for detecting TDP-43 comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 5.

11. A method for providing information necessary for the diagnosis of a TDP-43-related disease, comprising the step of treating a biological sample with an antibody of any one of claims 1 to 5 or an antigen-binding fragment thereof to detect TDP-43.

12. A method for providing information necessary for the diagnosis of a TDP-43 related disease according to claim 11, wherein the TDP-43 related disease is one or more selected from the group consisting of LATE (Limbic-predominant Age-related TDP-43 Encephalopathy), frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), chronic traumatic encephalopathy (CTE), and inclusion body myositis (IBM).

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