Antibody or functional fragment thereof, and use thereof

Abstract

Provided is a tool or the like for recognizing a fused in sarcoma (FUS) aggregate. One embodiment of the present invention provides an antibody or a functional fragment thereof that specifically binds to a FUS aggregate.

Description

Antibodies or functional fragments thereof, and uses thereof

[0001] The present invention relates to an antibody or a functional fragment thereof that specifically binds to FUS aggregates, and uses thereof.

[0002] In the brains of patients with many neurodegenerative diseases, intracellular aggregates consisting of specific proteins appear in neurons and glial cells. The major constituent proteins of intracellular aggregates vary depending on the disease. For example, tau in Alzheimer's disease (AD), α-synuclein in Parkinson's disease (PD) and dementia with Lewy bodies (DLB), and TDP-43 and FUS in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) have been identified as the major constituent proteins of intracellular aggregates specific to each disease.

[0003] Among the above-mentioned proteins, tau, α-synuclein, and TDP-43 are known to be characterized by post-translational modifications such as ubiquitination and phosphorylation as intracellular aggregates that appear in the brains of patients with this disease. Furthermore, antibodies specific to these phosphorylated proteins have been developed, and these antibodies react only with intracellular aggregates and hardly react with normal, unaccumulated soluble proteins, making them useful tools for pathological diagnosis of patient brains.

[0004] Furthermore, in recent years, protein structural analysis techniques using cryo-electron microscopy have revealed the structures of tau, α-synuclein, and TDP-43 accumulated in patient brains. This analysis revealed that different aggregate structures exist for different diseases, and that diseases can be hierarchically classified based on the structure of the aggregates (Non-Patent Document 1). These protein aggregates are not formed by full-length molecules forming the core of the aggregates, but are instead divided into a core region that forms the core of the aggregate and forms a three-dimensional structure, and a fuzzy coat region that does not form a three-dimensional structure (not included in the core of the aggregate). While tau, α-synuclein, and TDP-43 aggregates accumulated in patient brains are all phosphorylated, it has been found that most of the phosphorylation sites are located in the fuzzy coat region, not in the core region of the aggregates (Non-Patent Documents 1 and 2).

[0005] Shi Y et al, Structure-based classification of tauopathies, Nature, 598, 359-363, 2021.Schweighauser M, Shi Y, Tarutani A, Kametani F, Murzin AG, Ghetti B, Matsubara T, Tomita T, Ando T, Hasegawa K, Murayama S, Yoshida M, Hasegawa M, Scheres SHW, Goedert M, Structures of α-synuclein filaments from multiple system atrophy, Nature, 585, 464-469, 2020.

[0006] The FUS (fused in sarcoma) protein is a type of RNA-binding protein present in the nucleus and is thought to be involved in DNA repair, transcriptional regulation, RNA splicing, and other processes. In 2009, the FUS gene (NM_004960.4) was identified as a new causative gene for familial ALS. In the brains of patients with neurodegenerative diseases in which accumulation of FUS aggregates is observed, FUS is localized from the nucleus to the cytoplasm, where it accumulates as ubiquitin-positive cytoplasmic inclusions (aggregates).

[0007] Tau, α-synuclein, and TDP-43 that aggregate in patient brains have been biochemically fractionated as detergent-insoluble fractions, and their post-translational modifications have been elucidated using protein chemistry. In contrast, it has not yet been shown biochemically whether FUS (FUS aggregates) that aggregate in patient brains are insoluble in detergents, and the analysis of these aggregates has not progressed sufficiently. Naturally, it is also unknown whether FUS aggregates are phosphorylated.

[0008] Furthermore, there are no antibodies specific to FUS aggregates like those of tau, α-synuclein, and TDP-43, and only antibodies that simultaneously stain FUS aggregates accumulated in the cytoplasm and normal FUS present in the nucleus exist. Therefore, it is difficult to distinguish between the two with currently available antibodies, and new antibodies that are more useful in situations such as pathological diagnosis are desired.

[0009] In view of the above-mentioned problems, one aspect of the present invention aims to provide a tool for recognizing FUS aggregates, etc.

[0010] In order to solve the above problems, the present invention includes, for example, the following aspects.

[0011] 1) An antibody or functional fragment thereof that specifically binds to FUS aggregates. 2) A phosphorylated polypeptide comprising five or more consecutive amino acid residues in the region from positions 1 to 80 of the amino acid sequence shown in SEQ ID NO: 1, and in which at least one amino acid residue selected from the group consisting of serine, threonine, and tyrosine residues is phosphorylated. 3) A kit for producing an antibody or functional fragment thereof that binds to FUS aggregates, comprising the phosphorylated polypeptide described in 2) above. 4) A method for detecting FUS aggregates, comprising a step of causing an antigen-antibody reaction between the antibody or functional fragment thereof described in 1) above and a sample. 5) A kit for detecting FUS aggregates, comprising the antibody or functional fragment thereof described in 1) above.

[0012] According to one aspect of the present invention, a tool for recognizing FUS aggregates can be provided.

[0013] 1 is a diagram showing a comparison of staining results of the dentate gyrus of the hippocampus in the brains of FTLD-FUS patients and healthy subjects using the pT7 antibody and the HPA008784 antibody, according to an example of the present invention. 2 is a diagram showing a comparison of staining results of the area around the dentate gyrus of the hippocampus in the brains of FTLD-FUS patients using the pS3 antibody, the pT7 antibody, the pT11 antibody, and the pS513 antibody, according to an example of the present invention. 3 is a diagram showing a comparison of staining results of the dentate gyrus of the hippocampus in the brains of FTLD-FUS patients using a sample containing a mixture of the pT7 antibody and the pT7 peptide or the T7 peptide, according to an example of the present invention. 4 is a diagram showing staining results of the culture supernatant of a positive single clone (#4A2_1B12) in the frontal lobe cortex of the brains of FTLD-FUS patients, according to an example of the present invention. 5 is a diagram showing staining results of the culture supernatant of a positive single clone (#5E9_1C6) in the frontal lobe cortex of the brains of FTLD-FUS patients, according to an example of the present invention. 1 shows the results of staining using the culture supernatant of a positive single clone (#6G1_2F5) in the frontal lobe cortex of the brain of an FTLD-FUS patient according to an example of the present invention. 2 shows the signal sequence and variable region sequence of an antibody peptide (clone #5E9_1C6) according to an example of the present invention. 3 shows the signal sequence and variable region sequence of an antibody peptide (clone #6G1_2F5) according to an example of the present invention. 4 shows the signal sequences and predicted positions of the actual N-termini of clone #5E9_1C6 and clone #6G1_2F5 according to an example of the present invention.

[0014] 1. Antibodies and Functional Fragments Thereof (Antibodies) An antibody according to one embodiment of the present invention is an antibody that specifically binds to FUS aggregates. As used herein, "specifically binds to FUS aggregates" means that the antibody specifically binds to FUS aggregates but does not specifically bind to other molecules. From another perspective, the antibody may bind to other molecules with lower affinity than to FUS aggregates, as measured by assays well known in the art (e.g., BIACORE®, immunoassays, etc.).

[0015] An antibody according to one embodiment of the present invention may bind to non-aggregated FUS with lower affinity than to FUS aggregates, but preferably does not substantially bind to non-aggregated FUS. That is, a particularly preferred example of an antibody specifically binds to FUS aggregates but has substantially no cross-reactivity with non-aggregated FUS.

[0016] FUS aggregates to which an antibody according to one embodiment of the present invention specifically binds refer to aggregates of FUS that have accumulated as a result of some kind of denaturation of the normal protein FUS. The type of denaturation that occurs in FUS is not particularly limited, but examples include amino acid mutations, misfolding or unfolding that may be caused by or unrelated to these mutations, etc. FUS aggregates include aggregates of the full-length FUS and aggregates of FUS fragments. FUS aggregates may comprise a core region of the aggregate and a fuzzy coat region.

[0017] The FUS aggregate may be, for example, a FUS aggregate contained in a biological sample or an isolated FUS aggregate. The FUS aggregate may be artificially constructed in vitro or artificially constructed within cells using techniques such as genetic recombination, but it may be preferable for it to be naturally generated in vivo. Examples of biological samples include body fluid samples (urine, blood, cerebrospinal fluid, lymph, etc.) and cell-containing samples (cells, tissues, organs, etc.), with cells (particularly neurons and glial cells), tissues, and parts of organs being more preferred. Preferred examples of tissues and organs include the brain (particularly the cerebrum) and spinal cord, i.e., central nervous tissue. The biological sample is preferably a human sample. For example, it has been reported that FUS aggregates accumulate in the brain tissue (particularly in the cytoplasm of neurons and glial cells) of patients with ALS or FTLD-FUS. However, due to the difficulty in isolating FUS aggregates that appear in patient brains, biochemical and protein chemical analyses have not been conducted to date.

[0018] In one example of a FUS aggregate, at least one amino acid residue corresponding to an amino acid residue selected from the group consisting of serine residues, threonine residues, and tyrosine residues in the amino acid sequence shown in SEQ ID NO: 1 is phosphorylated, or at least one amino acid residue corresponding to an amino acid residue selected from the group consisting of serine residues and threonine residues in the amino acid sequence shown in SEQ ID NO: 1 is phosphorylated. In a preferred example of the FUS aggregate, at least one amino acid residue corresponding to an amino acid residue selected from the group consisting of serine, threonine, and tyrosine residues in the region of positions 1 to 80 (more preferably, positions 70, 60, 50, 40, 30, or 20) of the amino acid sequence shown in SEQ ID NO: 1 is phosphorylated, or at least one amino acid residue corresponding to an amino acid residue selected from the group consisting of serine and threonine residues in the region of positions 1 to 80 (more preferably, positions 70, 60, 50, 40, 30, or 20) of the amino acid sequence shown in SEQ ID NO: 1 is phosphorylated. Specifically, for example, the FUS aggregate has at least one amino acid residue phosphorylated corresponding to an amino acid residue selected from the group consisting of serine residue 3, threonine residue 7, and threonine residue 11 of the amino acid sequence shown in SEQ ID NO: 1. The fact that FUS aggregates accumulated in the brains of patients are phosphorylated was first demonstrated by the present inventors (see also the Examples). Therefore, an antibody according to one embodiment of the present invention can also be considered as an antibody that specifically binds to phosphorylated FUS or a fragment thereof.

[0019] As used herein, the term "amino acid residue corresponding to a specific amino acid residue in the amino acid sequence shown in SEQ ID NO: 1" refers to 1) the specific amino acid residue itself in the amino acid sequence shown in SEQ ID NO: 1, and 2) an amino acid residue in another amino acid sequence that corresponds to the specific amino acid residue in the amino acid sequence shown in SEQ ID NO: 1. In the case of 2), it refers to amino acid residue X in another amino acid sequence that is identified by homology analysis as corresponding to amino acid residue X in the amino acid sequence shown in SEQ ID NO: 1. Examples of homology analysis methods include pairwise sequence alignment methods such as the Needleman-Wunsch method and the Smith-Waterman method, and multiple sequence alignment methods such as the ClustalW method. Those skilled in the art can use these methods to determine the "corresponding amino acid residue" in another amino acid sequence to be analyzed using the amino acid sequence shown in SEQ ID NO: 1 as a reference sequence. Examples of other amino acid sequences to be analyzed include isoforms, homologs, or variants of the reference sequence. Analysis may be performed using default settings, or parameters may be changed from the defaults as needed.

[0020] An antibody according to one embodiment of the present invention recognizes a phosphorylation site in a FUS aggregate. This allows the antibody to selectively bind to FUS aggregates over non-aggregated FUS. Examples of phosphorylation sites in FUS aggregates include the phosphorylated amino acid residues in FUS aggregates, as shown above. Preferably, the antibody recognizes at least one amino acid residue selected from the group consisting of the serine residue at position 3, the threonine residue at position 7, and the threonine residue at position 11 in the amino acid sequence set forth in SEQ ID NO: 1, and more preferably the amino acid residue corresponding to the threonine residue at position 7. An example of an epitope recognized by an antibody is a region on a FUS aggregate consisting of multiple consecutive amino acid residues including the phosphorylation site. This epitope region may consist of, for example, 20 or fewer, 15 or fewer, or 10 or fewer consecutive amino acid residues. Furthermore, this epitope region may consist of, for example, 3 or more, 4 or more, or 5 or more consecutive amino acid residues.

[0021] The epitope region is not particularly limited, but specific examples include the following: 1) A sequence comprising part or all of the consecutive amino acid residues from position 1 to position 10 in the amino acid sequence shown in SEQ ID NO: 2, with the proviso that at least one amino acid residue selected from the group consisting of serine, threonine, and tyrosine residues contained in this sequence is phosphorylated. 2) A sequence comprising part or all of the consecutive amino acid residues from position 1 to position 11 in the amino acid sequence shown in SEQ ID NO: 3, with the proviso that at least one amino acid residue selected from the group consisting of serine, threonine, and tyrosine residues contained in this sequence is phosphorylated. 3) A sequence comprising part or all of the consecutive amino acid residues from position 2 to position 12 in the amino acid sequence shown in SEQ ID NO: 4, with the proviso that at least one amino acid residue selected from the group consisting of serine, threonine, and tyrosine residues contained in this sequence is phosphorylated. 4) A sequence comprising part or all of the consecutive amino acid residues from position 2 to position 12 in the amino acid sequence shown in SEQ ID NO: 5. However, at least one amino acid residue selected from the group consisting of serine residues, threonine residues, and tyrosine residues contained in this sequence is phosphorylated.

[0022] An antibody according to one embodiment of the present invention may be a polyclonal antibody or a monoclonal antibody. This antibody may be a recombinant antibody such as a chimeric antibody, a humanized antibody, or a fully humanized antibody. An antibody according to one embodiment of the present invention may be an antibody consisting of a light chain and a heavy chain, or may be a heavy chain antibody.

[0023] (Functional fragment of antibody) The functional fragment of an antibody according to one embodiment of the present invention refers to the antigen-binding portion of the antibody that specifically binds to the FUS (fused in sarcoma) aggregates. The antigen-binding portion of the antibody is, for example, a Fab fragment, an F(ab') fragment, or the like. ２ Examples of functional antibody fragments include antibody fragments, scFv fragments, VNAR domains, and VHH domains (Nanobodies (registered trademark)). A functional antibody fragment may consist of only the antigen-binding portion of the antibody, or may be an antibody fragment in which further modifications have been made to the antigen-binding portion of the antibody.

[0024] (Example of amino acid sequences of antibodies and functional fragments thereof) An antibody and functional fragment thereof according to one embodiment of the present invention comprises at least one of a heavy chain variable region (VH) and a light chain variable region (VL), and in a typical example, comprises both a VH and a VL. The VH and VL contained in the antibody and functional fragment thereof may be the full length or a partial fragment thereof.

[0025] In a typical example, VH includes three heavy chain complementarity determining regions (CDR-H), namely, CDR-H1, CDR-H2, and CDR-H3. CDR-H1, CDR-H2, and CDR-H3 are arranged in this order from the N-terminus to the C-terminus of VH. VH may further include a heavy chain framework sequence that supports the CDR-H.

[0026] An example of an amino acid sequence for CDR-H1 is DYSMH (SEQ ID NO: 6), an example of an amino acid sequence for CDR-H2 is WINTETGEPTYADDFKG (SEQ ID NO: 7), and an example of an amino acid sequence for CDR-H3 is RARATRFAY (SEQ ID NO: 8). Another example of an amino acid sequence for CDR-H2 is WINTETGDPTYADDFKG (SEQ ID NO: 9), and another example of an amino acid sequence for CDR-H3 is RTRATRFAY (SEQ ID NO: 10). Each of the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 may have one or two amino acid mutations (e.g., substitutions, deletions, or insertions). Non-limiting examples of the amino acid mutation include an amino acid mutation in CDR-H2 or CDR-H3, particularly an amino acid mutation at the 8th amino acid in CDR-H2 (e.g., a conservative or non-conservative amino acid substitution) and an amino acid mutation at the 2nd amino acid in CDR-H3 (e.g., a conservative or non-conservative amino acid substitution).

[0027] An example of the full-length amino acid sequence of VH is shown in SEQ ID NO: 11 or SEQ ID NO: 12. The amino acid sequence of VH may also have amino acid mutations (e.g., substitutions, deletions, or insertions), and examples include those having 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more amino acid sequence identity to the amino acid sequence shown in SEQ ID NO: 11 or SEQ ID NO: 12.

[0028] In a typical example, the VL comprises three light chain complementarity determining regions (CDR-Ls), namely, CDR-L1, CDR-L2, and CDR-L3. CDR-L1, CDR-L2, and CDR-L3 are arranged in this order from the N-terminus to the C-terminus of the VL. The VL may further comprise a light chain framework sequence supporting the CDR-Ls.

[0029] An example of an amino acid sequence for CDR-L1 is KASQSVSNAVA (SEQ ID NO: 13), an example of an amino acid sequence for CDR-L2 is SASNRYT (SEQ ID NO: 14), and an example of an amino acid sequence for CDR-L3 is QQDYRFPFT (SEQ ID NO: 15). Another example of an amino acid sequence for CDR-L1 is KASQSVNNALA (SEQ ID NO: 16). The amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 may each have one or two amino acid mutations (e.g., substitutions, deletions, or insertions). Non-limiting examples of amino acid mutations include amino acid mutations in CDR-L1, particularly amino acid mutations at the 7th and / or 10th amino acids in CDR-L1 (e.g., conservative or non-conservative amino acid substitutions).

[0030] An example of the full-length amino acid sequence of VL is shown in SEQ ID NO: 17 or SEQ ID NO: 18. The amino acid sequence of VL may also have amino acid mutations (e.g., substitutions, deletions, or insertions), and examples include those having 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more amino acid sequence identity to the amino acid sequence shown in SEQ ID NO: 17 or SEQ ID NO: 18.

[0031] In the case of amino acid substitutions, the substitutions may be conservative or non-conservative. A "conservative amino acid substitution" is a substitution of an amino acid with another amino acid having a similar side chain. Groups of amino acids with similar side chains include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Substitutions with amino acids from the same group are called conservative amino acid substitutions. Conservative substitutions of amino acids in the sequence of antibodies and functional fragments thereof may not abolish the specific binding of the antibodies and functional fragments thereof to the antigen.

[0032] The antibody and functional fragment thereof according to one embodiment of the present invention may further comprise at least one of a heavy chain constant region (CH) and a light chain constant region (CL), and in a typical example, comprises both a CH and a CL. The CH and CL comprised by the antibody and functional fragment thereof may be full-length or a partial fragment thereof. In a typical example, the CH is linked to the C-terminus of the VH, and the CL is linked to the C-terminus of the VL.

[0033] The amino acid sequences of CH and CL are known. Examples of CL include, but are not limited to, human lambda or kappa CL, or lambda or kappa CL of non-human animals (e.g., mice, rats, etc.). Examples of CH include, but are not limited to, the heavy chain constant regions of human IgG (IgG1, IgG2 (IgG2a, IgG2b), IgG3, IgG4), IgA (IgA1, IgA2), IgD, IgE, or IgM), or the heavy chain constant regions of non-human animals IgG, IgA, IgD, IgE, or IgM.

[0034] The structure of an antibody or functional fragment thereof according to one embodiment of the present invention may be a known structure. For example, the structure of an antibody (immunoglobulin) is known for each subclass (isotype), such as IgG (IgG1, IgG2 (IgG2a, IgG2b), IgG3, IgG4), IgA (IgA1, IgA2), IgD, IgE, or IgM.

[0035] Examples of antibodies and functional fragments thereof according to one embodiment of the present invention include, but are not limited to, the following (A), (B), (C), and (D): (A) an antibody or a functional fragment thereof, comprising at least one of a heavy chain variable region (VH) and a light chain variable region (VL), wherein the amino acid sequences of the three heavy chain complementarity determining regions are, in order, DYSMH (SEQ ID NO: 6), WINTETGEPTYADDFKG (SEQ ID NO: 7), and RARATRFAY (SEQ ID NO: 8), and the amino acid sequences of the three light chain complementarity determining regions are, in order, KASQSVSNAVA (SEQ ID NO: 13), SASNRYT (SEQ ID NO: 14), and QQDYRFPFT (SEQ ID NO: 15), and each of the above complementarity determining regions may have one or two amino acid mutations. (B) An antibody, or a functional fragment thereof, comprising at least one of a heavy chain variable region (VH) and a light chain variable region (VL), wherein the amino acid sequences of the three complementarity determining regions of the heavy chain are, in order, DYSMH (SEQ ID NO: 6), WINTETGDPTYADDFKG (SEQ ID NO: 9), and RTRATRFAY (SEQ ID NO: 10), and the amino acid sequences of the three complementarity determining regions of the light chain are, in order, KASQSVNNALA (SEQ ID NO: 16), SASNRYT (SEQ ID NO: 14), and QQDYRFPFT (SEQ ID NO: 15), and each of the above complementarity determining regions may have one or two amino acid mutations. (C) An antibody, or a functional fragment thereof, comprising at least one of a heavy chain variable region (VH) and a light chain variable region (VL), wherein the amino acid sequences of the three complementarity determining regions of the heavy chain are, in order, DYSMH (SEQ ID NO: 6), WINTETGEPTYADDFKG (SEQ ID NO: 7), and RARATRFAY (SEQ ID NO: 8), and the amino acid sequences of the three complementarity determining regions of the light chain are, in order, KASQSVNNALA (SEQ ID NO: 16), SASNRYT (SEQ ID NO: 14), and QQDYRFPFT (SEQ ID NO: 15), and each of the above complementarity determining regions may have one or two amino acid mutations.(D) An antibody, or a functional fragment thereof, comprising at least one of a heavy chain variable region (VH) and a light chain variable region (VL), wherein the amino acid sequences of the three complementarity determining regions of the heavy chain are, in order, DYSMH (SEQ ID NO: 6), WINTETGDPTYADDFKG (SEQ ID NO: 9), and RTRATRFAY (SEQ ID NO: 10), and the amino acid sequences of the three complementarity determining regions of the light chain are, in order, KASQSVSNAVA (SEQ ID NO: 13), SASNRYT (SEQ ID NO: 14), and QQDYRFPFT (SEQ ID NO: 15), and each of the above complementarity determining regions may have one or two amino acid mutations.

[0036] (Nucleic acid constructs encoding antibodies and functional fragments thereof, etc.) A nucleic acid construct according to one embodiment of the present invention is a nucleic acid construct encoding the above-mentioned antibody or a functional fragment thereof. This nucleic acid construct, for example, encodes at least the heavy chain variable region (VH) of the above-mentioned antibody or a functional fragment thereof. This nucleic acid construct, for example, encodes at least the light chain variable region (VL) of the above-mentioned antibody or a functional fragment thereof. This nucleic acid construct, for example, encodes at least the VH and VL of the above-mentioned antibody or a functional fragment thereof. One form of the nucleic acid construct according to this embodiment is a gene encoding the above-mentioned antibody or a functional fragment thereof. Another form of the nucleic acid construct according to this embodiment is an expression cassette that expresses the above-mentioned gene. The expression cassette is preferably an expression vector. The nucleic acid construct encoding the above-mentioned antibody or a functional fragment thereof may be a single nucleic acid construct or a combination of multiple nucleic acid constructs. One embodiment of the present invention provides a host cell comprising the above-mentioned expression cassette. One embodiment of the present invention provides a method for producing the above-mentioned antibody or a functional fragment thereof by expressing the expression cassette in a host cell comprising the above-mentioned expression cassette.

[0037] The expression cassette may comprise any expression control region functional in the expression host. The type of promoter serving as the expression control region is not particularly limited. In the expression cassette, the promoter is positioned upstream of the gene. Another example of a functional sequence that may be comprised in the expression cassette is a polyA tail addition signal sequence, but is not particularly limited thereto.

[0038] In this specification, "polynucleotide" can be alternatively referred to as "nucleic acid" or "nucleic acid molecule." Unless otherwise specified, "polynucleotide" encompasses polynucleotides containing known analogs of naturally occurring nucleotides that can function in the same manner as naturally occurring nucleotides. Furthermore, "base sequence" can be alternatively referred to as "nucleic acid sequence" or "nucleotide sequence." Unless otherwise specified, "base sequence" refers to a sequence of deoxyribonucleotides or a sequence of ribonucleotides. Furthermore, polynucleotides may be single-stranded or double-stranded, and if single-stranded, they may be the sense strand or the antisense strand.

[0039] As used herein, "gene" refers to a "polynucleotide" that encodes a protein / polypeptide. As used herein, the "expression control region" of a gene refers to a "polynucleotide" that controls the expression of the gene. Examples of "expression control regions" include promoter regions and enhancer regions. As used herein, "expression cassette" refers to an expression unit that includes an expression control region that is functional in an expression host and a polynucleotide operably linked to the expression control region. In an expression cassette, the polynucleotide is preferably a gene or a gene fragment. An example of an expression cassette is one in which the expression control region and the polynucleotide are genetically linked. "Operably linked" refers to a state in which the expression of a polynucleotide is controlled by an expression control sequence. The expression cassette may be in the form of an expression vector.

[0040] 2. Novel phosphorylated polypeptide, kit containing said peptide, and method for producing antibody, etc. (Novel phosphorylated polypeptide) The novel phosphorylated polypeptide according to one embodiment of the present invention corresponds to a fragment (part) of FUS containing the phosphorylation site, and the full-length of FUS is not included in its scope. This phosphorylated polypeptide can be used, for example, as an antigen for producing an antibody according to one embodiment of the present invention or a functional fragment thereof. Note that production of an antibody or a functional fragment thereof also includes contract manufacturing based on the antigen itself or sequence information of the antigen.

[0041] The phosphorylated polypeptide is not particularly limited, but specific examples include the following: 1) A phosphorylated polypeptide comprising five or more consecutive amino acid residues in the amino acid sequence shown in SEQ ID NO: 1, in which at least one amino acid residue selected from the group consisting of serine, threonine, and tyrosine residues is phosphorylated, or at least one amino acid residue selected from the group consisting of serine and threonine residues is phosphorylated. 2) A phosphorylated polypeptide comprising five or more consecutive amino acid residues in the region from position 1 to position 80 (or positions up to position 70, up to position 60, up to position 50, up to position 40, up to position 30, or up to position 20) of the amino acid sequence shown in SEQ ID NO: 1, in which at least one amino acid residue selected from the group consisting of serine, threonine, and tyrosine residues is phosphorylated, or at least one amino acid residue selected from the group consisting of serine and threonine residues is phosphorylated. 3) A phosphorylated polypeptide comprising five or more consecutive amino acid residues including at least one amino acid residue selected from the group consisting of the serine residue at position 3, the threonine residue at position 7, and the threonine residue at position 11 of the amino acid sequence shown in SEQ ID NO: 1, wherein at least one amino acid residue selected from the group consisting of serine, threonine, and tyrosine residues is phosphorylated, or at least one amino acid residue selected from the group consisting of serine and threonine residues is phosphorylated. Alternatively, a phosphorylated polypeptide comprising five or more consecutive amino acid residues including the serine residue at position 513 of the amino acid sequence shown in SEQ ID NO: 1, wherein at least one amino acid residue selected from the group consisting of serine, threonine, and tyrosine residues is phosphorylated, or at least one amino acid residue selected from the group consisting of serine and threonine residues is phosphorylated.

[0042] A novel phosphorylated polypeptide according to one embodiment of the present invention preferably comprises 5 to 20 consecutive amino acid residues of the amino acid sequence set forth in SEQ ID NO: 1, and more preferably comprises 5 to 15 (or 14, 13, 12, 11, or 10) consecutive amino acid residues.

[0043] The phosphorylated polypeptide shown in 3) above is not particularly limited, but specific examples include the following: 3A) A polypeptide comprising a portion (5 or more consecutive amino acid residues) or all of the consecutive amino acid residues from positions 1 to 10 in the amino acid sequence shown in SEQ ID NO: 2, with the proviso that at least one amino acid residue selected from the group consisting of serine, threonine, and tyrosine residues contained in this sequence is phosphorylated. 3B) A polypeptide comprising a portion (5 or more consecutive amino acid residues) or all of the consecutive amino acid residues from positions 1 to 11 in the amino acid sequence shown in SEQ ID NO: 3, with the proviso that at least one amino acid residue selected from the group consisting of serine, threonine, and tyrosine residues contained in this sequence is phosphorylated. 3C) A polypeptide comprising a portion (5 or more consecutive amino acid residues) or all of the consecutive amino acid residues from positions 2 to 12 in the amino acid sequence shown in SEQ ID NO: 4, with the proviso that at least one amino acid residue selected from the group consisting of serine, threonine, and tyrosine residues contained in this sequence is phosphorylated. 3D) A polypeptide comprising a portion (five or more consecutive amino acid residues) or all of the consecutive amino acid residues from positions 2 to 12 in the amino acid sequence set forth in SEQ ID NO: 5, with the proviso that at least one amino acid residue selected from the group consisting of serine, threonine, and tyrosine residues contained in this sequence is phosphorylated.

[0044] The phosphorylated polypeptide may further have one or more additional amino acid residues, such as an amino acid residue added to the C-terminus or N-terminus for cross-linking with a carrier protein.

[0045] (Kit containing novel phosphorylated polypeptide, method for producing antibody, etc.) A kit for producing an antibody that binds to FUS aggregates or a functional fragment thereof according to one embodiment of the present invention includes the novel phosphorylated polypeptide described above. The novel phosphorylated polypeptide is stored in a container, for example, as a solution in a buffer solution.

[0046] The kit may further comprise one or more selected from various materials and instruments for producing antibodies or functional fragments thereof, and an instruction manual for the kit.

[0047] Materials for producing antibodies and the like include, for example, materials for preparing immunogens, such as carrier proteins such as keyhole limpet hemocyanin and various adjuvants. Also included are animals such as mice, rabbits, sheep, chickens, and camelids (alpacas, llamas, camels, etc.), which can be injected with prepared immunogens to produce antibodies and the like. For the production of monoclonal antibodies, examples include myeloma cells for producing hybridomas.

[0048] Examples of tools for producing antibodies and the like include, but are not limited to, syringes for injecting immunogens into animals; containers for storing produced antibodies and hybridomas; pipettes for injecting and recovering samples; and plates for culturing and screening hybridomas.

[0049] A specific method for producing an antibody or a functional fragment thereof that specifically recognizes a novel phosphorylated polypeptide according to one embodiment of the present invention as an antigen may be carried out in accordance with known methods for producing antibodies, etc.

[0050] [3. Use of Antibodies] (Method and Kit for Detecting FUS Aggregates) A ​​method for detecting FUS aggregates according to one embodiment of the present invention comprises a step of causing an antigen-antibody reaction by contacting a sample with an antibody or functional fragment thereof described in the above section [1. Antibodies and Functional Fragments Thereof]. The reaction conditions for the antigen-antibody reaction and the preparation of the sample may be in accordance with known methods. Furthermore, if necessary, the method may comprise a step of visualizing the results of the antigen-antibody reaction by a known method, or a step of detecting the presence or absence and amount of FUS aggregates in the sample from the results of the antigen-antibody reaction.

[0051] The sample is not particularly limited as long as it can contain FUS aggregates, but is preferably an isolated biological sample. Examples of biological samples include body fluid samples (urine, blood, cerebrospinal fluid, lymph, etc.) and cell-containing samples (cells, tissues, organs, etc.), with cells (particularly neurons and glial cells), tissues, and parts of organs being more preferred. Preferred examples of tissues and organs include the brain (particularly the cerebrum) and spinal cord, i.e., central nervous tissue. The biological sample is preferably a human sample.

[0052] A kit for detecting FUS aggregates according to one embodiment of the present invention includes an antibody or a functional fragment thereof described above in the section "1. Antibodies and Functional Fragments Thereof." The kit detects the presence or absence and amount of FUS aggregates in a sample by carrying out an antigen-antibody reaction between the antibody or the like and the sample according to the method for detecting FUS aggregates described above.

[0053] The detection kit may further include one or more selected from various materials and tools for preparing a sample, various materials and tools for visualizing the detection results, and an instruction manual for the kit. The detection kit may also be in the form of a kit commonly used in this technical field, such as an ELISA kit or an immunochromatography kit.

[0054] An example of this detection kit is a kit for the pathological diagnosis of FUS-related diseases. FUS-related diseases broadly refer to diseases accompanied by the accumulation of FUS aggregates, and particularly refer to neurodegenerative diseases accompanied by the accumulation of FUS aggregates in the cytoplasm of neurons or glial cells in central nervous tissue. Examples of such neurodegenerative diseases include familial or sporadic amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD, also known as FTLD-FUS), which are accompanied by the accumulation of FUS aggregates. Examples of samples for pathological diagnosis include brain samples from human patients suspected of being affected.

[0055] (Application to treatments, etc. targeting FUS aggregates) The antibodies and functional fragments thereof described above in the section [1. Antibodies and functional fragments thereof] specifically bind to FUS aggregates. Therefore, by utilizing this property, it is possible to construct a delivery system that delivers substances to FUS aggregates themselves or to areas where FUS aggregates have accumulated. The delivery system comprises the above-mentioned antibody or a functional fragment thereof and the substance to be delivered, preferably in which the two are directly or indirectly bound. Examples of the substance to be delivered include, but are not limited to, drugs (particularly therapeutic drugs for FUS-related diseases) and labeling tags.

[0056] (Summary) By summarizing the above embodiments, the present invention can be summarized as follows.

[0057] 1) An antibody or functional fragment thereof that specifically binds to FUS aggregates. 2) The antibody or functional fragment thereof according to 1), which recognizes a phosphorylation site in FUS aggregates. 3) The antibody or functional fragment thereof according to 1) or 2), in which the FUS aggregates are phosphorylated at least one amino acid residue corresponding to an amino acid residue selected from the group consisting of serine, threonine, and tyrosine residues in the region from positions 1 to 80 of the amino acid sequence shown in SEQ ID NO: 1. 4) comprising at least one of a heavy chain variable region (VH) and a light chain variable region (VL), wherein the amino acid sequences of the three heavy chain complementarity determining regions are, in order, DYSMH (SEQ ID NO: 6), WINTETGEPTYADDFKG (SEQ ID NO: 7), and RARATRFAY (SEQ ID NO: 8), or DYSMH (SEQ ID NO: 6), WINTETGDPTYADDFKG (SEQ ID NO: 9), and RTRATRFAY (SEQ ID NO: 10), and the amino acid sequences of the three light chain complementarity determining regions are, in order, KASQSVSNAVA (SEQ ID NO: 13), SASNRYT (SEQ ID NO: 14), and QQDYRFPFT (SEQ ID NO: 15), or KASQSVNNALA (SEQ ID NO: 16), SASNRYT (SEQ ID NO: 14), and QQDYRFPFT (SEQ ID NO: 15), The antibody or functional fragment thereof according to any one of 1) to 3), wherein each of the complementarity-determining regions may have one or two amino acid mutations. 5) A phosphorylated polypeptide comprising five or more consecutive amino acid residues in the region from positions 1 to 80 of the amino acid sequence set forth in SEQ ID NO: 1, and wherein at least one of the amino acid residues selected from the group consisting of serine, threonine, and tyrosine residues is phosphorylated. 6) The phosphorylated polypeptide according to 5), comprising five or more consecutive amino acid residues and up to 20 of the aforementioned amino acid residues. 7) A kit for producing an antibody or functional fragment thereof that binds to FUS aggregates, comprising the phosphorylated polypeptide according to 5) or 6) above. 8) A method for detecting FUS aggregates, comprising a step of causing an antigen-antibody reaction between the antibody or functional fragment thereof according to any one of 1) to 4) above and a sample. 9) A kit for detecting FUS aggregates, comprising the antibody or functional fragment thereof according to any one of 1) to 4) above.10) The kit according to 9), which is used for the pathological diagnosis of an FUS-related disease.

[0058] The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the present invention.

[0059] An example of the present invention is described below. <Production of Antibodies> Based on the amino acid sequence of the FUS protein shown in SEQ ID NO: 1, peptides shown in SEQ ID NOs: 2 to 5 (also referred to as pS3 peptide, pT7 peptide, pT11 peptide, and pS513 peptide, respectively) were designed and synthesized. The pS3 peptide, pT7 peptide, pT11 peptide, and pS513 peptide are partial sequences of the FUS protein shown in SEQ ID NO: 1, containing phosphorylated Ser (Ser3), Thr (Thr7), Thr (Thr11), and Ser (Ser513), respectively, at the N-terminus of the FUS protein, and further containing a Cys residue added to the N-terminus or C-terminus. Each peptide was cross-linked to keyhole limpet hemocyanin (KLH) via a Cys residue added to the N-terminus or C-terminus, and rabbits were immunized with each of the above KLH-crosslinked peptides as immunogens. Then, whole blood was collected and antisera were recovered to obtain rabbit polyclonal antibodies: pS3 antibody, pT7 antibody, pT11 antibody, and pS513 antibody. The processes from peptide synthesis to antibody production were outsourced to another company.

[0060] <1. Immunohistochemistry> Formalin-fixed paraffin-embedded brain blocks from healthy subjects and FTLD-FUS patients were sliced ​​to a thickness of 10 μm, and sections from the medial temporal lobe, including the hippocampus, transitional entorhinal cortex, and temporal neocortex, were immunostained with the above-mentioned pS3 antibody, pT7 antibody, pT11 antibody, and pS513 antibody to evaluate the presence or absence of aggregate formation.

[0061] Immunohistochemical staining was performed as follows. 1) First, the paraffin-embedded brain block sections were treated with 0.5% hydrogen peroxide (diluted in PBS) to remove endogenous peroxidase, and then blocked by adding PBS containing 10% bovine serum and 0.2% Triton X-100 (Tx-PBS). 2) The sections were then incubated overnight at 4°C with the primary antibodies pS3, pT7, pT11, or pS513. 3) Next, a biotin-labeled secondary antibody was added and allowed to react thoroughly, followed by the addition of an avidin-biotin-labeled enzyme complex and allowing the reaction to proceed (ABC method, Vector Laboratories, Burlingame, CA). 4) Then, diaminobenzidine (DAB) was added to the sections to allow the reaction, thereby visualizing the peroxidase reaction of the antibody. 5) Nuclear staining was then performed with hematoxylin. Between each of steps 1) to 5), the sections were washed three times with Tx-PBS for 10 minutes each.

[0062] As a control test, immunohistochemical staining was performed in the same manner as described above, except that HPA008784 antibody (Sigma-Aldrich), a commercially available FUS antibody, was used as the primary antibody. The epitope of HPA008784 antibody is the region consisting of amino acid residues 85 to 213 in the amino acid sequence shown in SEQ ID NO: 1.

[0063] The results are explained with reference to Figures 1 and 2. Figure 1 shows a comparison of the staining results of the dentate gyrus of the hippocampus in the brains of FTLD-FUS patients and healthy controls using the pT7 antibody and the HPA008784 antibody. Figure 2 shows a comparison of the staining results of the area around the dentate gyrus of the hippocampus in the brains of FTLD-FUS patients using the pS3 antibody, pT7 antibody, pT11 antibody, and pS513 antibody.

[0064] As shown in Figure 1, staining with the HPA008784 antibody made it difficult to distinguish cells containing aggregates (Figure 1, center), whereas staining with the pT7 antibody enabled the identification of cells containing aggregates (Figure 1, left). This is thought to be because the HPA008784 antibody reacts not only with FUS aggregates but also with normal FUS localized in the nucleus, whereas the pT7 antibody barely reacts with normal FUS in the nucleus and strongly stains FUS aggregates localized in the cytoplasm. In fact, when immunostaining the dentate gyrus of the hippocampus from a normal subject with the pT7 antibody, almost no staining was observed (Figure 1, right).

[0065] Furthermore, as shown in Figure 2, FUS aggregates were selectively stained with any of the pS3 antibody, pT7 antibody, pT11 antibody, and pS513 antibody, but among these, the pT7 antibody showed the best staining ability.

[0066] <2. Absorption test of pT7 antibody with peptide> In order to examine whether the above-mentioned pT7 antibody specifically recognizes phosphorylation of Thr7, an absorption test was performed on the pT7 antibody using the pT7 peptide used as the antigen when producing the pT7 antibody, and a T7 peptide which has the same amino acid sequence as the pT7 peptide but in which Thr7 is not phosphorylated.

[0067] The absorption test was performed as follows: pT7 antibody was mixed with either pT7 peptide or T7 peptide at a final concentration of 0.1 mM, and the mixture was allowed to stand at 4°C for 2 days. Each antibody-peptide mixture was diluted 2,000-fold to prepare an antibody sample, which was then reacted with paraffin sections from the brains of FTLD-FUS patients to perform immunostaining. It is expected that the antibody sample mixed with the non-phosphorylated T7 peptide will primarily contain phosphorylated antibodies (pT7 antibodies), while the antibody sample mixed with the phosphorylated pT7 peptide will primarily contain non-phosphorylated antibodies (i.e., antibodies that recognize peptides other than Thr7 phosphorylation).

[0068] The results are explained with reference to Figure 3. Figure 3 shows a comparison of the staining results of the dentate gyrus of the hippocampus in the brains of FTLD-FUS patients using pT7 antibody and antibody samples containing pT7 peptide or T7 peptide. The antibody sample containing pT7 antibody and T7 peptide strongly stained FUS aggregates (Figure 3, left), whereas the antibody sample containing pT7 antibody and pT7 peptide lost this staining ability (Figure 3, right). This demonstrates that the pT7 antibody specifically recognizes phosphorylated Thr7 and reacts with FUS aggregates, strongly suggesting that Thr7 is phosphorylated in FUS aggregates that appear in the brains of FTLD-FUS patients.

[0069] 3. Preparation of Mouse Monoclonal Antibodies Monoclonal antibodies were prepared by immunizing mice with the above-described pT7 peptide as an antigen. The antibody preparation and evaluation were carried out according to the following procedure.

[0070] 1) First, using the MBS method, pT7 peptide was conjugated to the carrier protein KLH, and mice were immunized with an emulsion of the KLH-MBS-pT7 peptide conjugate solution mixed with Freund's complete adjuvant at a volume ratio of 1:2. 2) After 17 days, the mice were boosted with the same antigen as above. Lymphocytes were collected from the mouse iliac lymph nodes and fused with myeloma cells to produce hybridomas. 3) The hybridomas obtained in 2) were seeded and cultured in 96-well plates. 4) The reactivity of the supernatants of the hybridomas cultured in 3) to the pT7 peptide was evaluated by ELISA screening. The BSA-MBS-pT7 peptide conjugate was used as the positive antigen, and the BSA-MBS-T7 peptide conjugate was used as the negative antigen. The primary antibody used was a 10-fold diluted hybridoma culture supernatant, and the secondary antibodies used were anti-mouse IgG (HRP-labeled) (JIR: 715-035-151) and anti-mouse IgG L chain (HRP-labeled) (JIR: 115-035-174), each diluted 20,000 times. ２ O ２After color development with / TMBZ, absorbance at 450 nm was measured using a plate reader. Specifically, wells where the difference in absorbance between the positive and negative plates was 0.075 or more and the absorbance of the negative plate was 0.05 or less were determined to be positive. 5) For wells determined to be positive in 4), ELISA was performed again using 10-fold diluted culture supernatant, and wells where the difference in absorbance between the positive antigen and the negative antigen was 0.05 or more were determined to be positive. 6) For the 32 clones determined to be positive in 5), the culture supernatant was used to immunostain patient brain sections, and the antibody titer was evaluated. The immunohistochemistry method was performed in accordance with the above-mentioned <1. Immunohistochemistry>. 7) For the three clones (#4A2, #5E9, and #6G1) that were positive for immunohistochemistry in 6), cells were seeded using limiting dilution in another 96-well plate, and a second ELISA screening was performed using the culture supernatant from each well. Furthermore, single colony checks were performed by microscopic observation, and cross-check ELISA was performed using serially diluted culture supernatants to evaluate specificity. Wells in which the absorbance decreased with dilution and the difference in absorbance between the positive and negative antigens in 10-fold diluted culture supernatants was 0.05 or greater were determined to be positive. 8) Of the 12 single clones in wells determined to be positive in 7), three clones were further selected (#4A2_1B12, #5E9_1C6, #6G1_2F5) taking into account the results of antibody titer evaluation based on staining of patient brain sections in 6), and the antibody isotype was determined using the Mouse Monoclonal Antibody Isotyping Test Kit (Bio-Rad).

[0071] The above-mentioned peptide synthesis, hybridoma production, ELISA, and antibody isotype determination were outsourced to other companies.

[0072] The results are explained below. The culture supernatants of the positive clones #4A2, #5E9, and #6G1 selectively stained FUS aggregates accumulated in the brains of FTLD-FUS patients. Furthermore, these culture supernatants showed no staining in control cases. On the other hand, the culture supernatant of the negative clone #1A12 showed no staining in either patient or control brains. Furthermore, the culture supernatants of the single clones #4A2_1B12, #5E9_1C6, and #6G1_2F5 of the above three positive clones were also able to selectively stain FUS aggregates accumulated in the brains of FTLD-FUS patients. The results of staining the frontal cortex of FTLD-FUS patient brains (basophilic inclusion body disease: BIBD, neuronal intermediate filament inclusion body disease: NIFID) using the culture supernatant of each single clone are shown (Figure 4: #4A2_1B12, Figure 5: #5E9_1C6, Figure 6: #6G1_2F5). These results demonstrate that multiple hybridomas producing antibodies specifically reacting with FUS aggregates were obtained. Furthermore, isotype analysis of the antibodies associated with these clones revealed them to be mouse IgG2b(k).

[0073] <4. Analysis of antibody variable regions> Of the hybridomas obtained in <3. Production of mouse monoclonal antibodies>, RNA was extracted from the positive single clones #5E9_1C6 and #6G1_2F5, and the genes for the heavy and light chain variable regions of the produced antibodies were obtained by amplifying them by RT-PCR. The sequences of the obtained fragments were analyzed to identify the structures of the antibody variable regions. Details of the analysis method are provided below.

[0074] First, hybridoma pellets (#5E9_1C6: 5.8 x 10 ６ pieces, #6G1_2F5: 1.3×10 ７Total RNA was extracted from 1000 cells (1000 cells) using TRIzol Reagent (Thermo Fisher) according to the manufacturer's protocol, followed by quality control (QC) using an Agilent 2100 Bioanalyzer (Agilent). The exact RNA concentration was also assayed using a Qubit 2.0 Fluorometer (Thermo Fisher). One-quarter of the total cells were used for RNA extraction, and sufficient RNA was confirmed.

[0075] QC results showed that the extracted total RNA was sufficient in both quantity and quality for analysis, so the genes for the heavy and light chain variable region portions of the clone-derived antibody were subsequently subjected to RT-PCR fragment acquisition and sequence analysis. The PCR fragment acquisition and sequence analysis of the variable region were performed according to the following procedure. 1) Using the extracted total RNA, full-length cDNA was obtained by reverse transcription using SMARTScribe Reverse Transcriptase (Takara Bio) and template switching. 2) The cDNA obtained in 1) was purified using Agencourt AMPure XP (Beckman Coulter) and used as a template to obtain a fragment of the target region by three-step PCR using PrimeSTAR GXL polymerase (Takara Bio). For the PCR primer set, the 5' primer was designed based on the oligo region added by the template switching method, and the 3' primer was designed based on the constant region of the isotype analyzed in the above section <3. Production of mouse monoclonal antibodies>. The PCR cycle settings are as shown in Table 1 (PCR cycle setting-1). 3) The solution of the fragments obtained in 2) was treated with Exonuclease I (New England BioLabs) to degrade the primers, and then the fragments of the target region were further enriched by nested PCR. The 5' primer was the same as in 2), and the 3' primer was designed based on the constant region of the same isotype and positioned further 5' than the primer used in 2). The PCR cycle settings are shown in Table 2 (PCR cycle settings -2). 4) The fragments were cloned into a TA vector (pGEM-T Easy, Promega) using T4 DNA Ligase (Promega). 5) From the clones obtained in 4), insert fragments were obtained by colony PCR using GoTaq Colorless Master Mix (Promega), and their lengths were confirmed using MCE-202 (Shimadzu Corporation). For each heavy chain and light chain of each sample, 12 fragments with appropriate lengths of approximately 600 bp to 800 bp were obtained. 6) The fragments obtained in 5) were treated with Exonuclease I (New England BioLabs) / Shrimp Alkaline Phosphatase (Thermo Fisher), and both end sequence information was obtained by direct sequencing. 7) The full-length variable region sequences were determined by majority vote for those with no translation frame interruptions and valid sequences for antibody genes. Specifically, the variable region sequences were determined based on the Kabat system, and the full-length heavy chain variable region and its complementarity-determining regions (CDR-H1, CDR-H2, CDR-H3) and the full-length light chain variable region and its complementarity-determining regions (CDR-L1, CDR-L2, CDR-L3) were estimated as shown in Figure 7 (#5E9_1C6) and Figure 8 (#6G1_2F5), respectively. Note that a signal sequence was immediately preceded each variable region sequence (heavy chain: MAWVWTLLFLMAAAAQSIQA, SEQ ID NO: 19; light chain: MKSQTQVFVFLLLLCVSGAHG, SEQ ID NO: 20). The actual N-terminal position was also estimated as shown in Figure 9. Sequencing was performed using the BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher) and data were acquired using a 3730xl (Thermo Fisher) instrument. For each clone, all of the 12 heavy and 12 light chain sequencing fragments that encoded valid antibody genes were used for majority voting. The remaining fragment sequences were either out of frame or out of frame with the original antibody sequence and were therefore deemed unnecessary for further analysis.The full-length sequences of the heavy chain variable regions of clone #5E9_1C6 and clone #6G1_2F5, and the full-length sequences of the light chain variable regions of clone #5E9_1C6 and clone #6G1_2F5, determined in this step, are shown in SEQ ID NOs: 11, 12, 17, and 18, respectively.

[0076] These results demonstrate that antibody structures can be determined by preparing total RNA from mouse-derived hybridomas, obtaining full-length gene fragments of the heavy and light chain variable regions of the produced antibodies, and performing DNA sequencing. Furthermore, the identified sequences also contain signal sequences, and by comparing them with the public databases mentioned above, it was possible to estimate the translation initiation position of the signal sequence and the position of the N-terminus after processing. Furthermore, it was demonstrated that the heavy and light chain variable region portions can be identified based on the determined structures using the Kabat system.

[0077] The present invention is extremely useful for the definitive pathological diagnosis of FTLD and ALS accompanied by FUS accumulation, and is expected to be in high demand.

Claims

1. An antibody or a functional fragment thereof that specifically binds to FUS (Fused in sarcoma) aggregates.

2. The antibody or functional fragment thereof described in claim 1, which recognizes a phosphorylation site in FUS aggregates.

3. The antibody or functional fragment thereof described in claim 1 or 2, wherein the FUS aggregate is phosphorylated at least one amino acid residue corresponding to an amino acid residue selected from the group consisting of serine residues, threonine residues, and tyrosine residues in the region from the 1st to the 80th amino acid residues of the amino acid sequence shown in SEQ ID NO:

1.

4. A peptide comprising at least one of a heavy chain variable region (VH) and a light chain variable region (VL), wherein the amino acid sequences of the three heavy chain complementarity determining regions are, in order, DYSMH (SEQ ID NO: 6), WINTETGEPTYADDFKG (SEQ ID NO: 7), and RARATRFAY (SEQ ID NO: 8), or DYSMH (SEQ ID NO: 6), WINTETGDPTYADDFKG (SEQ ID NO: 9), and RTRATRFAY (SEQ ID NO: 10), and the amino acid sequences of the three light chain complementarity determining regions are, in order, KASQSVSNAVA (SEQ ID NO: 13), SASNRYT (SEQ ID NO: 14), and QQDYRFPFT (SEQ ID NO: 15), or KASQSVNNALA (SEQ ID NO: 16), SASNRYT (SEQ ID NO: 14), and QQDYRFPFT (SEQ ID NO: 15), The antibody or functional fragment thereof according to any one of claims 1 to 3, wherein each of the above complementarity determining regions may have one or two amino acid mutations.

5. A phosphorylated polypeptide comprising five or more consecutive amino acid residues in the region from the 1st to the 80th amino acid residues of the amino acid sequence shown in SEQ ID NO:1, and in which at least one of the amino acid residues selected from the group consisting of serine residues, threonine residues, and tyrosine residues is phosphorylated.

6. The phosphorylated polypeptide of claim 5, comprising 5 or more and 20 or less consecutive amino acid residues.

7. A kit for producing an antibody or a functional fragment thereof that binds to FUS aggregates, comprising the phosphorylated polypeptide according to claim 5 or 6.

8. A method for detecting FUS aggregates, comprising a step of subjecting a sample to an antigen-antibody reaction with the antibody or functional fragment thereof according to any one of claims 1 to 4.

9. A kit for detecting FUS aggregates, comprising the antibody or functional fragment thereof according to any one of claims 1 to 4.

10. The kit according to claim 9, which is used for pathological diagnosis of an FUS-associated disease.

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