Antibody against misfolded TDP-43 and method of use thereof

Abstract

The present disclosure relates to antibodies that specifically bind to W68 in the context of DAGWGNL (SEQ ID NO: 1). Also provided are isolated peptides, isolated nucleic acids, immunogens, compositions, kits, and methods of using the reagents to detect misfolded TDP-43.

Description

[Technical Field]

[0001] Related applications This application is a Patent Cooperation Treaty application claiming the benefit of 35 U.S.SC § 119 based on the priority of U.S. Provisional Application No. 62 / 779,904 filed on 14 December 2018 and U.S. Provisional Application No. 62 / 923,789 filed on 21 October 2019, each of which is incorporated herein by reference in its entirety.

[0002] Sequence listing inclusion The computer-readable sequence listing "PP54811PC00 PC00_ST25" (58,887 bytes), submitted via EFS-WEB and created on December 12, 2019, is incorporated herein by reference.

[0003] field This disclosure relates to a TDP-43 antibody, more specifically, an antibody for detecting misfolded TDP-43, and a method for detecting misfolded TDP-43. [Background technology]

[0004] background The 43kDa transactivation response (TAR) element DNA-binding protein (TDP-43) is a 414-amino acid protein consisting of an N-terminal ubiquitin-like domain (NTD, residues 1-80), two RNA recognition motifs (RRMs) comprising residues 106-177 (RRM1) and 192-259 (RRM2), and a C-terminal domain (CTD, residues 274-414). The NTD is adjacent to a domain that directs nuclear localization (NLS motifs at residues 82-98, NLS1 K82RK84 and K95VKR98). RRM2 contains a nuclear export signal (NES) from residues 239 to 250.

[0005] TDP-43 is a nuclear protein that plays a central role in RNA metabolism. Because pathogenic inclusions in affected neurons can contain post-translational modified TDP-43, TDP-43 has become a focus of research in the amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) disease spectrums. CTD of TDP-43 is particularly associated with the disease because almost all familial ALS / FTD-associated mutations are found at the TDP-43 site.

[0006] Other mutations include D169G located in RRM1 between beta strands 4 and 5, A90V which is a mutation in the NLS region, and K263E and N267S which are present in the linker between RRM2 and the C-terminal domain.

[0007] RRM1 and RRM2 have been structurally determined by NMR. For example, RRM1 is available as PDB entry 4IUF in the Protein Databank (PDB), a database of atomic-resolution three-dimensional structural data, while RRM2 is available as PDB entry 1WF0, and NTD is available as PDB entries 5MRG, 2N4P, and 6B1G.

[0008] The structure of 4IUF was reported by Kuo et al. [1]. The structure of 1WF0 was reported by He et al. [2]. The structure of 2N4P was reported by Mompean et al. [3].

[0009] TDP-43 has been shown to be hyperphosphorylated, ubiquitinated, and fragmented in neuronal inclusions in patients with both sporadic and familial ALS and FTD [4].

[0010] Functional TDP-43 can exist as a nuclear oligomer distinct from cytoplasmic aggregates formed during cellular stress. Oligomerization of functional TDP-43 is necessary for its RNA splicing function. Nuclear-mediated TDP-43 oligomerization can inhibit cytoplasmic mislocalization and the formation of pathological aggregates [9].

[0011] Physiological TDP-43 oligomerization is mediated by its N-terminal domain, which can adopt a dynamic solenoid-like structure, as revealed by the 2.1A crystal structure obtained by a combination of nuclear magnetic resonance spectroscopy and electron microscopy.[9]

[0012] TDP-43 aggregates (inclusions) are now found in almost all (approximately 97%) cases of ALS and approximately half (approximately 45%) cases of FTD. TDP-43 is one of the main components of cytoplasmic inclusions found in motor neurons of ALS patients.

[0013] The precursor of TDP-43 inclusions can be present at much lower concentrations than functional TDP-43. The low concentration of misfolded TDP-43 makes this target difficult to capture.

[0014] Intracerebral injection of brain-derived pathogenic TDP-43 FTLD-TDP seeds in transgenic and non-transgenic mice expressing cytoplasmic human TDP-43 has resulted in the induction of de novo TDP-43 lesions that propagate to the brain in a time-dependent manner

[10] .

[0015] Antibodies that bind to TDP-43 have been described to date.

[0016] WO2012174666, titled "Methods for Prognosis and / or Diagnosis of Neurodegenerative Diseases, Candidate Compounds and Methods for Identifying Compounds for the Treatment of Neurodegenerative Diseases," discloses a method for diagnosing neurodegenerative diseases such as ALS and FTD by evaluating the interaction between TDP-43 and NF-κB p65 using an anti-TDP-43 antibody.

[0017] WO2016086320, entitled "TDP-43 Binding Polypeptides Useful for Treating Neurodegenerative Diseases," discloses antibodies that bind to the RRM1 domain of TDP-43 and interfere with its interaction with NF-kB for the treatment of ALS and FTD.

[0018] Antibodies that preferentially bind to misfolded TDP-43 rather than native-folded TDP-43 are desired. SUMMARY OF THE INVENTION

[0019] overview The inventors have identified antibodies that preferentially bind to TDP-43 aggregates rather than native-folded nuclear or cytoplasmic TPD-43. The inventors have identified epitopes that are differentially accessible in misfolded TDP-43 and produced antibodies that recognize said epitopes in misfolded TDP-43 aggregates. As shown herein, said epitopes are available for binding in misfolded TDP-43 (solvent-accessible) but not in native-folded non-disease-related TDP-43. In particular, the inventors have determined that W68 is an important residue for conferring antibody specificity to misfolded TDP-43 aggregates. Such antibodies generally recognize aggregated TDP-43 that is not associated with stress granules and can inhibit the intercellular propagation of pathogenic TDP-43.

[0020] Thus, one aspect includes an isolated peptide comprising all or a portion of DAGWGNL (SEQ ID NO: 1), wherein said portion is at least 5 amino acids and comprises GWG.

[0021] In another embodiment, said portion is at least 6 contiguous amino acids of DAGWGNL (SEQ ID NO: 1).

[0022] In one embodiment, the peptide comprises up to 21 residues, and W68 is preferably located in the middle of the three divisions of the peptide.

[0023] Another aspect is an immunogen comprising a peptide, wherein the peptide comprises all or part of DAGWGNL(SEQ ID NO: 1), where the part comprises at least five amino acids, and optionally at least six amino acids, of DAGWGNL(SEQ ID NO: 1).

[0024] In one embodiment, the immunogen comprises a plurality of peptides, each peptide comprising at least five amino acids, and optionally at least six amino acids, of DAGWGNL (SEQ ID NO: 1), wherein the plurality of peptides are synthesized as a multiantigenic peptide (MAP).

[0025] In another embodiment, the peptide is coupled with a carrier protein or an immunogenicity-enhancing component.

[0026] In another embodiment, the carrier protein is bovine serum albumin (BSA), or the immunogenicity-enhancing component is keyhole limpet hemocyanin (KLH).

[0027] In one embodiment, the immunogen is used to produce an antibody that selectively binds to misfolded TDP-43 and / or an antibody that specifically binds to at least W68 in relation to DAGWGNL (SEQ ID NO: 1). In one embodiment, the antibody preferentially binds to misfolded TDP-43.

[0028] A further aspect involves an antibody that binds to TDP-43 and preferentially binds to misfolded TDP-43 compared to natural TDP-43.

[0029] In one embodiment, the antibody specifically binds to at least W68 in relation to DAGWGNL (SEQ ID NO: 1).

[0030] In one embodiment, the antibody is produced or screened using a peptide or immunogen described herein.

[0031] In one embodiment, the antibody is a monoclonal antibody.

[0032] In one embodiment, the antibody is a humanized antibody.

[0033] In one embodiment, the antibody is a single-chain antibody.

[0034] In one embodiment, the antibody is a binding fragment selected from Fab, Fab', F(ab')2, scFv, dsFv, ds-scFv, dimers, nanobodies, minibodies, diabodies, and their polymers.

[0035] In one embodiment, the antibody is purified by affinity.

[0036] Further aspects include an immune complex comprising the antibody and detectable label described herein.

[0037] Further aspects include isolated nucleic acids encoding amino acid residues of the peptides, immunogens, or antibodies described herein, as well as vectors comprising such nucleic acids for, for example, delivering and / or expressing the peptides, immunogens, or antibodies described herein.

[0038] A further aspect comprises cells that recombinantly express the peptides, immunogens, or antibodies described herein.

[0039] In one embodiment, the cells expressing antibodies are hybridomas.

[0040] Further aspects include compositions comprising isolated peptides, immunogens, antibodies, immune complexes, isolated nucleic acids, or cells as described herein.

[0041] In one embodiment, the composition, which comprises a peptide or immunogen, further comprises an adjuvant.

[0042] In one embodiment, the adjuvant is an incomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide, alum, monophosphoryl lipid A and / or QS21.

[0043] Kits are also provided that include isolated peptides, immunogens, antibodies, immune complexes, isolated nucleic acids, cells, and / or compositions, as well as compartments for housing the reagents.

[0044] In another embodiment, the kit further includes instructions for use in an ELISA for the method described herein.

[0045] A further aspect includes a method for producing an antibody, comprising administering an isolated peptide described herein (e.g., at least five amino acids, optionally at least six amino acids, of DAGWGNL (SEQ ID NO: 1)), an immunogen, or a composition to a non-human subject, or immunizing the non-human subject with such composition.

[0046] In one embodiment, the method further comprises isolating an antibody that specifically binds to W68 in relation to DAGWGNL (SEQ ID NO: 1).

[0047] In one embodiment, the method further comprises forming an antibody-producing hybridoma.

[0048] Another aspect involves antibodies produced by the methods described herein.

[0049] A further aspect is a method for determining whether a sample contains misfolded TDP-43, comprising contacting the sample with an antibody described herein under conditions that allow the formation of an antibody:misfolded TDP-43 complex, and detecting the presence of any complex, wherein the presence of a detectable complex indicates that the sample may contain a misfolded TDP-43 polypeptide.

[0050] In one embodiment, the sample is a biological sample obtained from a subject.

[0051] In one embodiment, the sample comprises blood, serum, plasma, and / or solid tissue.

[0052] In one embodiment, the sample is a human sample.

[0053] In one embodiment, the subject has or is suspected of having amyotrophic lateral sclerosis (ALS) or frontotemporal dementia (FTD).

[0054] Further provided are methods for treating a subject, comprising administering an effective amount of an antibody, immune complex, nucleic acid, or composition described herein to a subject in need thereof.

[0055] Other features and advantages of this disclosure will become apparent from the following detailed description. However, various changes and modifications within the spirit and scope of this disclosure will become apparent to those skilled in the art from this detailed description, so the detailed description and specific examples should be understood as being provided merely as illustrations, while illustrating preferred embodiments of this disclosure. [Brief explanation of the drawing]

[0056] Embodiments of the present disclosure are described here in reference to the drawings:

[0057] [Figure 1]Figures 1A–1L show HEK-293 cells transfected with a TDP-43 construct having a triple missensant tandem mutation in nuclear localization signaling, wild-type TDP-43, or an empty vector. TDP-43 in these cells was detected using DAGWGNL(SEQ ID NO: 1) peptide affinity-purified rabbit polyclonal anti-TDP-43 antibody (GS240) produced against an immunogen containing the DAGWGNL(SEQ ID NO: 1) peptide described herein, or an anti-HA tagged antibody that reacts with the TDP-43-HA fusion construct. [Figure 2] Figures 2A–2L show HEK-293 cells transfected with a TDP-43-HA construct having a triple missensant tandem mutation in nuclear localization signaling, wild-type TDP-43-HA, or an empty vector. TDP-43 was detected in the cells using a purified polyclonal anti-TDP-43 antibody (GS243) produced against an immunogen containing the DAGWGNL (SEQ ID NO: 1) peptide described herein, or an anti-HA tagged antibody. [Figure 3] Figures 3A-3E are graphs showing the binding kinetics of mouse (3A and 3B) and rabbit (3C, 3D, and 3E) monoclonal antibodies. [Figure 4] Figures 4A and 4B show antibody recognition of the epitope in the denatured TDP43N-terminal domain, which is not the case in the naturally folded TDP43N-terminal domain. [Figure 5] Figures 5A and 5B show staining of sections derived from FTD brain (5A) and ALS spinal cord (5B) with rabbit polyclonal GS240 antibody, while Figures 5C-5E show staining of sections derived from FTD brain with mouse monoclonal antibody. [Figure 6] Figure 6 shows a series of images illustrating the co-localization of mouse monoclonal Ab 2F7 staining using cytoplasmic TDP43 aggregates in ΔNLS-TDP43-HA transfected cells, although this is not the case in WT nuclear TDP-43-HA. [Figure 7]Figures 7A and 7B show images demonstrating that the 3F11 mouse monoclonal antibody against the disease-associated N-terminal TDP43 epitope does not react with physiological stress granules. [Figure 8] Figure 8 shows a series of images illustrating the co-localization of rabbit monoclonal Ab 28H3 staining using cytoplasmic TDP43 aggregates in ΔNLS-TDP-43 transfected cells, although this is not the case in ΔNLS-TDP-43-W68S transfected cells. [Figure 9] Figure 9 shows antibody blockade of misfolded TDP-43 transmission in HEK293 cells. Detailed description of the invention

[0058] Detailed explanation of this disclosure As described in the examples, the inventors identified an epitope specifically accessible in misfolded TDP-43. The inventors produced an antibody that specifically binds to the epitope. Furthermore, the inventors determined the importance of W68 in relation to DAGWGNL (SEQ ID NO: 1). Antibodies produced against this peptide sequence have been shown to preferentially bind to misfolded TDP-43 compared to natural TDP-43.

[0059] I. Definition As used herein, the terms "TDP-43" (transactivation response element (TAR) DNA-binding protein 43), "TDP43," or, unless otherwise specified, "TDP" means, as used herein, all forms of TDP-43, including wild-type TDP-43, natural TDP-43, and misfolded forms, including mutants and analogues from all species, in particular human TDP-43 (i.e., hTDP-43). Human TDP-43 is generally a 414-amino acid protein whose amino acid sequence (e.g., Uniprot accession number Q13148) and nucleotide sequence (e.g., accession number HGNC:11571) have been previously characterized.

[0060] As used herein, "wild type" refers to the primary amino acid sequence of a non-mutated or naturally occurring protein.

[0061] As used herein, “natural” refers to the normal three-dimensional structure of a particular protein or a part thereof. Natural TDP-43 is sometimes referred to as “naturally folded” TDP-43, “normally folded” TDP-43, and / or “healthy” TDP-43. Thus, the term “natural TDP-43,” or “naturally folded TDP-43,” as used herein, refers to TDP-43 that has been naturally folded after nascent translation, and / or, but is not limited to, a multimer containing dimeric and trimer TDP-43 folded in a non-pathological state (e.g., normal cells) having a molecular structure comprising non-covalently bonded individual TDP-43 peptides that exhibit the natural structure as reconstructed from X-ray crystallography or nuclear magnetic resonance spectroscopy. Natural TDP-43 forms multimers via its NTD, and when naturally folded, TDP-43 is generally the nucleus. Misfold aggregates of TDP-43 can be located in the cytoplasm, and are generally cytoplasmic.

[0062] As used herein, "misfolded" refers to the secondary and tertiary structures of a polypeptide or a portion thereof, indicating that the polypeptide is conforming to a state that is abnormal with respect to its properly functioning state. Misfolding can result from mutations in a protein, such as amino acid deletions, substitutions, or additions, but wild-type sequence proteins can also be misfolded in disease, for example, as a result of microenvironmental conditions and / or amino acid modifications, such as nitration, oxidation, carbonylation, or other modifications, which can expose disease-specific epitopes. Other post-translational modifications include abnormal ubiquitination, phosphorylation, acetylation, SUMOylation, and cleavage into C-terminal fragments. Misfolded TDP43 may be aggregated and / or cytoplasmic. In the context of TDP-43, native TDP-43 forms multimers via its NTD. Misfolded multimers (e.g., disease-associated oligomers) generally oligomerize via other regions of the protein, such as its LCD and / or RRM1 domain. Therefore, “Misfolded TDP-43 polypeptide,” or “Misfolded TDP-43” as used herein to refer to polypeptides, includes cytoplasmic and / or aggregated TDP-43 polypeptides oligomerized via their LCD and / or RRM1 domains, unnatural dimers and trimers, and large aggregates (e.g., five or more subunits). Misfolded TDP-43 tends to form aggregates that result in loss of protein function, toxicity, acquisition of amyloid-like features (e.g., Congo red staining), and transmission of pathogenic aggregates.

[0063] The term “mutant TDP-43” refers to the morphology of TDP-43, in particular the intrinsic form of TDP-43 resulting from gene mutations that produce amino acid substitutions, such as those characteristic of FTD or familial ALS, including, for example, the mutations described in the bioinformatics tools described in [6].

[0064] The term "DAGWGNL(SEQ ID NO: 1)" refers to the amino acid sequence shown in SEQ ID NO: 1: aspartic acid, alanine, glycine, tryptophan, glycine, asparagine, and leucine. Similarly, GWG refers to an amino acid sequence identified by a single-letter amino acid code. Depending on the context, a reference to an amino acid sequence may refer to the sequence in TDP-43 or the isolated peptide. Sequence DAGWGNL(SEQ ID NO: 1) corresponds to residues 65-71 in the primary amino acid sequence of TDP-43.

[0065] As used herein, “epitope” means a region of a protein recognized by a B cell or T cell receptor, or by an antibody or its binding fragment. An epitope may, as used herein, be represented by a linear amino acid sequence or region of a protein recognized by an antibody. An epitope may comprise one or more antigenic determinants. For example, an antibody prepared against an isolated peptide corresponding to a misfolded epitope recognizes part or all of the epitope sequence. As shown in the examples, the antibody may require Trp68 for binding. An immunogen comprising at least five, and possibly at least six, residues of SEQ ID NO: 1 may be used to produce an antibody that preferentially binds to misfolded TDP-43, for example, to W68 in relation to SEQ ID NO: 1 or DAGWGNL (SEQ ID NO: 1). References to "DAGWGNL (SEQ ID NO: 1) or related epitope" mean SEQ ID NO: 1 or a portion thereof in any linear peptide or region on TDP-43 that is conjugated by an antibody produced by an immunogen comprising a TDP-43 peptide sequence such as DAGWG (SEQ ID NO: 2), DAGWGN (SEQ ID NO: 3), AGWGN (SEQ ID NO: 4), AGWGNL (SEQ ID NO: 5), and GWGNL (SEQ ID NO: 6).

[0066] The term “analog” as used herein includes portions, extensions, substitutions, variants, modifications, or chemical equivalents of the amino acid and nucleotide sequences of the present invention, and their derivatives, that perform substantially the same function in substantially the same manner as the peptides, proteins, or nucleic acid molecules described herein. Peptide analogs also include additions and deletions to the TDP-43 peptide. Nucleic acid analogs include denatured nucleotide substitutions that encode the isolated peptides of the present invention. Furthermore, analog peptides and analog nucleotide sequences include their derivatives.

[0067] The term "amino acid" includes all naturally occurring amino acids and modified L-amino acids and D-amino acids. The atoms of an amino acid may include, for example, different isotopes. For instance, an amino acid may consist of deuterium substituted for hydrogen, nitrogen 15 substituted for nitrogen 14, carbon 13 substituted for carbon 12, and other similar variations.

[0068] As used herein, a "conservative amino acid substitution" is a substitution in which one amino acid residue is replaced by another amino acid residue without losing the desired properties of the protein. Suitable conservative amino acid substitutions can be made by substituting amino acids having similar hydrophobicity, polarity, and R-group size with each other. Examples of conservative amino acid substitutions include:

[0069] [Table 1]

[0070] As used herein, the term “antibody” includes monoclonal antibodies, polyclonal antibodies, single-chain, humanized and other chimeric antibodies, or fully human antibodies, as well as their conjugated fragments. Vectorized antibodies or intrabodies are also included. Antibodies may be derived from recombinant sources and / or produced in transgenic animals. Human antibodies that can be produced by using biochemical techniques or isolated from libraries are also included. Humanized antibodies or chimeric antibodies may contain sequences derived from one or more isotypes or classes.

[0071] The phrase “isolated antibody” refers to an antibody produced in vivo or in vitro that has been removed from its source, e.g., an animal, hybridoma, or other cell line (e.g., recombinant cells that produce the antibody). Isolated antibodies are sometimes “purified,” which means a purity of at least 80%, 85%, 90%, 95%, 98%, or 99%.

[0072] The term “binding fragment,” as used herein, refers to a part or portion of an antibody or antibody chain that comprises fewer amino acid residues than an intact or complete antibody or antibody chain and that binds to an antigen or competes with an intact antibody. Exemplary binding fragments include, but are not limited to, Fab, Fab', F(ab')2, scFv, dsFv, ds-scFv, dimers, nanobodies, minibodies, diabodies, and their polymers. Fragments can be obtained by chemical or enzymatic treatment of an intact or complete antibody or antibody chain. Fragments can also be obtained by recombinant means (see below). For example, an F(ab')2 fragment can be produced by treating an antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide crosslinks to produce a Fab' fragment. Papain digestion can result in the formation of a Fab fragment. Fab, Fab' and F(ab')2, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments, and other fragments can also be constructed by recombinant expression techniques. When an antibody is said to bind to an epitope within a specific residue, such as DAGWGNL (SEQ ID NO: 1), it means that the antibody selectively or specifically binds to a polypeptide containing at least one or at least two residues in relation to a specific residue or a portion thereof, e.g., a specific residue such as (SEQ ID NO: 1). Such an antibody does not necessarily contact all residues of DAGWGNL (SEQ ID NO: 1), and not all single amino acid substitutions or deletions within the epitope necessarily have a significant or equal impact on binding affinity.

[0073] As used herein, the term "complementary determining region" or "CDR" refers to certain hypervariable regions of an antibody that are generally presumed to contribute to epitope binding. Computational methods for identifying CDR sequences include Kabat, Chothia, and IMGT. The CDRs recited in this disclosure have been identified using IMGT Blast. One of ordinary skill in the art could also identify CDR sequences based on Kabat and Chothia, among others, in view of the sequences included herein.

[0074] As used herein, the term "detectable label" refers to a moiety, such as a peptide sequence, fluorescent protein, that can be added to or introduced into a peptide, antibody, or other compound described herein and that can generate a signal that is detectable either directly or indirectly. For example, a label can be a radiopaque positron emitting radionuclide (e.g., for use in PET imaging), or a radioisotope, such as ,

[0075] H, 13 N, 14 C, 18 F, 32 P, 35 S, 123 I, 125 I, 131 I; a fluorescent (fluorophore) or chemiluminescent (chromophore) compound, such as fluorescein isothiocyanate, rhodamine, or luciferin; an enzyme, such as alkaline phosphatase, beta-galactosidase, or horseradish peroxidase; a contrast agent; or a metal ion. A detectable label can be, for example, one that is detectable indirectly using a secondary antibody.

[0075] The term “epitope selectively presented or accessible on misfolded TDP-43” means, as used herein, an epitope that is selectively presented or antibody-accessible on misfolded TDP-43 present in, for example, ALS or FTD (e.g., disease-associated misfolded TDP-43), whether monomeric, dimeric, or aggregated, but not on the molecular surface of the naturally occurring, correctly folded homodimeric form of TDP-43. As shown herein, W68 is selectively presented or accessible on misfolded TDP-43.

[0076] When used herein, the term "high affinity" means that antibody X is more potent than target Z (K on ) and / or a small dissociation constant (K off ) refers to the degree of antibody binding to target Y, and in this context, antibody X has a greater affinity for target Y than Z. Similarly, the term “low affinity” refers, as used herein, to the degree of antibody binding to target Y, where antibody X binds to target Y with weaker strength and / or a larger dissociation constant than target Z, and in this context, antibody X has a lower affinity for target Y than Z. The affinity of binding between an antibody and its target antigen is 1 / K D K equal to A It can be expressed as, where K D is k on / k off It is equal to k. on and k off The value can be measured using surface plasmon resonance (for example, using a Biacore system).

[0077] Furthermore, as used herein, the term "immunogenic" refers to a substance that induces antibody production and activates T cells and other reactive immune cells directed toward the antigenic portion of the immunogen.

[0078] As used herein, “immunogen” means a substance that induces an immune response and / or causes antibody production. In addition to immunogenic compounds, the complexes and fusions described herein may include, for example, isolated compounds conjugated to KLH, identified epitopes, e.g., DAGWGNL and / or related epitopes, e.g., DAGWG (SEQ ID NO: 2), DAGWGN (SEQ ID NO: 3), AGWGN (SEQ ID NO: 4), AGWGNL (SEQ ID NO: 5), and GWGNL (SEQ ID NO: 6), which induce cross-reactive antibodies against these epitopes. To serve as a useful immunogen, the TDP-43 peptide preferably incorporates at least about 5, 6, or 7 TDP-43 residues and up to about 15, 17, 19, 20, or 21 TDP-43 amino acids, and optionally incorporates immunogenicity enhancers such as KLH, for example, via a linker or scaffold used in multiantigenic peptides (MAPs).

[0079] As used herein, the term “nucleic acid sequence” refers to a sequence of nucleosides or nucleotide monomers consisting of naturally occurring bases, sugars, and intersugar (skeletal) bonds. The term also includes modified or substituted sequences containing monomers or portions thereof that do not exist naturally. The nucleic acid sequences of this application may be deoxyribonucleic acid sequences (DNA) or ribonucleic acid sequences (RNA), and may contain naturally occurring bases including adenine, guanine, cytosine, thymidine, and uracil. Sequences may also contain modified bases. Examples of such modified bases include adenine, guanine, cytosine, thymidine, and uracil in aza and deaza; as well as xanthine and hypoxanthine. Nucleic acids may be double-stranded or single-stranded, representing a sense strand or an antisense strand. Furthermore, the term “nucleic acid” includes complementary nucleic acid sequences and codon-optimized or synonymous codon equivalents. The term "isolated nucleic acid sequence," as used herein, refers to a nucleic acid that is substantially free from cell material or culture medium if produced by recombinant DNA technology, or from chemical precursors or other chemical substances if chemically synthesized. An isolated nucleic acid also substantially lacks sequences naturally adjacent to the nucleic acid from which it originates (i.e., sequences located at the 5' and 3' ends of the nucleic acid).

[0080] "Operationally linked" means that the nucleic acid is linked to a regulatory sequence in a manner that enables the expression of the nucleic acid. Suitable regulatory sequences may be derived from a variety of sources, including bacterial, fungal, viral, mammalian, or insect genes. The selection of a suitable regulatory sequence depends on the selected host cell and can be easily achieved by those skilled in the art. Examples of such regulatory sequences include transcription promoters and enhancers or RNA polymerase binding sequences, and ribosome binding sequences (including translation initiation signals). Furthermore, depending on the selected host cell and the vector used, other sequences, such as origins of replication, further DNA restriction sites, enhancers, and sequences that confer transcriptional inducibility, may be incorporated into the expression vector.

[0081] The term “vector,” as used herein, comprises any intermediate vehicle for a nucleic acid molecule that enables the nucleic acid molecule to be introduced into, for example, prokaryotic and / or eukaryotic cells and / or incorporated into the genome, and includes plasmids, phagemids, bacteriophages, or viral vectors, such as retrovirus-based vectors and adeno-associated virus vectors. The term “plasmid,” as used herein, generally refers to a construct of extrachromosomal genetic material, usually a circular double-stranded DNA, capable of independently replicating chromosomal DNA.

[0082] "At least moderately stringent hybridization conditions" means that conditions are selected that promote selective hybridization between two complementary nucleic acid molecules in solution. Hybridization may occur for all or part of the nucleic acid sequence molecule. The hybridized portion is generally at least 15 (e.g., 20, 25, 30, 40, or 50) nucleotides long. Those skilled in the art will recognize that the stability of nucleic acid double strands or hybrids in sodium-containing buffer is determined by a function of sodium ion concentration and temperature (Tm = 81.5°C - 16.6(Log10[Na+]) + 0.41(%(G+C) - 600 / l), or a similar formula). Therefore, the parameters in the washing conditions that determine the stability of the hybrid are sodium ion concentration and temperature. To identify molecules that are similar to but not identical to known nucleic acid molecules, a 1% mismatch can be assumed to result in a Tm reduction of approximately 1°C. For example, if nucleic acid molecules are to have more than 95% identity, the final washing temperature will be reduced by approximately 5°C. Based on these considerations, those skilled in the art will be able to easily select appropriate hybridization conditions. In several preferred embodiments, stringent hybridization conditions are selected. As an example, the following conditions may be used to achieve stringent hybridization: hybridization with 5x sodium chloride / sodium citrate (SSC) / 5x Denhardt's solution / 1.0% SDS at Tm-5°C based on the above formula, followed by washing with 0.2x SSC / 0.1% SDS at 60°C. Moderately stringent hybridization conditions include a washing step in 3x SSC at 42°C. However, it is understood that equivalent stringency can be achieved using alternative buffers, salts, and temperatures.Further guidelines regarding hybridization conditions can be found in *Current Protocols in Molecular Biology*, John Wiley & Sons, NY, 2002, and in *Molecular Cloning: a Laboratory Manual*, Sambrook et al., *Cold Spring Harbor Laboratory Press*, 2001.

[0083] As used herein, "specifically binds" with respect to an antibody means that the antibody recognizes its target antigen and binds to that target with greater affinity than to antigens that are structurally different and / or have modified or mutant sequences. For example, a polyvalent antibody may bind to at least 1e-6, at least 1e-7, at least 1e-8, at least 1e-9 or at least 1e-10 K D It then binds to its target. An affinity of at least greater than 1e-8 is preferred. Antigen-binding fragments, such as Fab fragments containing one variable domain, can find their targets with an affinity 10 or 100 times lower than that of multivalent interactions with non-fragmented antibodies.

[0084] When the terms “selective” or “preferential” are used herein in reference to antibodies that selectively / preferentially bind to a particular form of TDP-43 (e.g., native protein or misfolded protein), it means that the binding protein binds to that form with an affinity at least 3-fold, or at least 5-fold, at least 10-fold, at least 20-fold, at least 100-fold, at least 250-fold, or at least 500-fold greater than that of another form. Thus, an antibody that is more selective to a particular conformation (e.g., misfolded protein) preferentially binds to that particular form of TDP-43 with an affinity at least 3-fold, or at least 5-fold, at least 10-fold, at least 20-fold, at least 100-fold, at least 250-fold, or at least 500-fold greater than that of another form.

[0085] The term "antibody that binds to the TDP-43 sequence DAGWGNL (SEQ ID NO: 1) in misfolded TDP-43" means, as used herein, an antibody such as a binding fragment that specifically or preferentially binds to the sequence or any portion thereof, but does not bind to unrelated sequences, in the context of misfolded TDP-43 compared to natural TDP-43.

[0086] The term "linker," as used herein, means a chemical moiety that can be covalently bonded to the entirety of the peptide comprising DAGWG (SEQ ID NO: 2), DAGWGN (SEQ ID NO: 3), AGWGN (SEQ ID NO: 4), AGWGNL (SEQ ID NO: 5), and GWGNL (SEQ ID NO: 6), or the entirety of the SEQ ID NO: 1 epitope peptide. The linker may comprise a glycine residue and / or a PEG moiety, as well as one or more functionalizable moieties, such as cysteine ​​residues. The linker may be linked via the functionalizable moieties to a carrier protein or immunoenhancing component, such as keyhole limpet hemocyanin (KLH). The linker may consist of, for example, 1 to 9 amino acids, or the functionalizable moiety alone.

[0087] The term “functionalizable portion,” as used herein, refers to a chemical entity having a “functional group,” which is a group of atoms or a single atom that reacts with another group of atoms or a single atom (a so-called “complementary functional group”) to form a chemical interaction between the two groups or atoms. In the case of cysteine, the functional group may be -SH, which can react to form a disulfide bond. The reaction with another group of atoms may be a covalent bond or a strong non-covalent bond, for example, in the case of a biotin-streptavidin bond, which may have a Kd of about 1e-14. A strong non-covalent bond, as used herein, means an interaction at a Kd of at least 1e-9, at least 1e-10, at least 1e-11, at least 1e-12, at least 1e-13, or at least 1e-14.

[0088] Proteins and / or other agents may be functionalized (e.g., conjugated) to peptides for either to enhance immunogenicity or to serve as probes in in vitro studies. For this purpose, any functionalizable moiety that can react (e.g., covalently or non-covalently but strongly) may be used. In one particular embodiment, the functionalizable moiety may be an immunogenicity-enhancing component such as keyhole limpet hemocyanin (KLH) used for in vitro immunoblotting or immunohistochemical assays, or a cysteine ​​residue that reacts to form a disulfide bond with an unpaired cysteine ​​on the protein of interest, such as bovine serum albumin (BSA).

[0089] The terms “animal” or “subject” as used herein include all members of the Animal Kingdom, including, as may be, mammals, and, as may be, humans.

[0090] The terms “to treat” or “treatment,” as used herein and as well as as well understood in the art, mean an approach to obtain beneficial or desired outcomes, including clinical outcomes. Beneficial or desired clinical outcomes may include, but are not limited to, reduction or improvement of one or more symptoms or conditions, whether detectable or undetectable; a reduction in the severity of the disease; a stabilized (i.e., non-worsening) state of the disease; prevention of disease transmission; delay or slowing of disease progression; improvement or mitigation of the condition; a reduction in disease relapses; and remission (whether partial or complete). “To treat” and “treatment” may also mean extending survival compared to the survival expected without treatment. “To treat” and “treatment,” as used herein, also include prophylactic treatment in subjects identified with familial variants, such as familial ALS. Subjects with TDP-43 protein disorders, such as ALS, may be treated to delay or slow disease progression. The subject may be treated with the compounds, antibodies (including vectorized antibodies or intrabodies), immunogens, immune complexes, or compositions described herein to prevent progression.

[0091] In understanding the scope of this disclosure, the term "consisting of" and its derivatives, as used herein, are limited terms that specify the presence of the mentioned features, elements, components, groups, integers and / or processes, and exclude the presence of other features, elements, components, groups, integers and / or processes that are not mentioned.

[0092] In this specification, enumerations of numerical ranges by endpoint include all numbers and fractions contained within that range (for example, 1–5 includes 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5). It is also understood that all numbers and their fractions are assumed to be modified by the term “about”. Furthermore, “a”, “an”, and “the” should be understood to include multiple referents unless explicitly indicated otherwise by the context. The term “about” means plus or minus 0.1–50%, 5–50%, or 10–40% of the number mentioned, preferably 10–20%, more preferably 10% or 15%.

[0093] Furthermore, the definitions and embodiments described in particular sections are applicable to other embodiments described herein in which they are preferred, as will be understood by those skilled in the art. For example, different aspects of the present invention are defined in more detail in the following sections. Each of the aspects thus defined may be combined with any other one or more aspects unless it is explicitly indicated otherwise. In particular, any feature indicated as preferred or advantageous may be combined with any other one or more features indicated as preferred or advantageous.

[0094] II. Peptides comprising all or part of DAGWGNL (SEQ ID NO: 1) and related immunogens This disclosure identifies an epitope on misfolded TDP-43, and antibodies produced using the corresponding peptide preferentially recognize misfolded TDP-43. As shown in the examples, the epitope, in particular W68, is inaccessible or not very accessible in naturally folded TDP-43. The inventors produced antibodies using an immunogen comprising the TDP-43 peptide DAGWGNL (SEQ ID NO: 1), which corresponds to amino acid residues 65-71 on TDP-43. Antibodies were produced using the immunogen, which does not clearly react with TDP-43 mutated with respect to this residue, as shown below.

[0095] Therefore, one aspect includes an isolated peptide comprising all or part of DAGWGNL, wherein the part comprises at least five amino acids and contains GWG.

[0096] In one embodiment, the part is at least six adjacent amino acids of SEQ ID NO: 1.

[0097] The peptide may comprise a further TDP-43 sequence, for example, up to 11 amino acids, up to 13 amino acids, up to 15 amino acids, up to 17 or 19 amino acids, or up to 21 amino acids. Preferably, the sequence is located centrally or near the center of W68. For example, if the peptide is 21 amino acids long, W68 may consist of residues 8, 9, 10, 11, or 12. In one embodiment, the peptide is EGILHAPDAGWGNLVYVVNYP (SEQ ID NO: 7) or a portion thereof comprising at least GWG.

[0098] In one embodiment, the peptide comprises or is a continuous sequence derived from the N-terminal ubiquitin-like domain of TDP-43, corresponding to residues 1 to 80 of TDP-43.

[0099] The isolated peptide may also include a linker to a non-TDP-43 sequence, for example, a continuous TDP-43 sequence, comprising, for example, 1 to 9 glycine and / or PEG moieties and / or Cys residues at the N-terminus and / or C-terminus.

[0100] Peptides may also be prepared by chemical synthesis using techniques well known in protein chemistry, such as solid-phase synthesis or synthesis in homogeneous solution.

[0101] In relation to DAGWGNL (SEQ ID NO: 1) as described herein, epitope W68 may be a potential target in misfolded TDP-43 transmission strains, and antibodies that recognize the epitope may be useful, for example, for detecting such transmission strains.

[0102] Another aspect includes an immunogen comprising a peptide comprising DAGWGNL (SEQ ID NO: 1), and optionally at least five residues, and optionally at least six residues, of DAGWG (SEQ ID NO: 2), DAGWGN (SEQ ID NO: 3), AGWGN (SEQ ID NO: 4), AGWGNL (SEQ ID NO: 5), and GWGNL (SEQ ID NO: 6). In one embodiment, the peptide of the immunogen comprises DAGWGNL. The immunogen may also comprise peptides having further TDP-43 or non-TDP-43 residues as described herein. In one embodiment, the peptide contained in the immunogen comprises, or is, a continuous sequence derived from the N-terminal ubiquitin-like domain of TDP-43, corresponding to residues 1 to 80 of TDP-43.

[0103] As described in the examples, immunogens can be prepared by chemically synthesizing peptides such as DAGWGNL (SEQ ID NO: 1), DAGWG (SEQ ID NO: 2), DAGWGN (SEQ ID NO: 3), AGWGN (SEQ ID NO: 4), AGWGNL (SEQ ID NO: 5), or GWGNL (SEQ ID NO: 6), which optionally have a C-terminal cysteine ​​residue or an N-terminal cysteine ​​residue (e.g., cDAGWGNL (SEQ ID NO: 8) or DAGWGNLc (SEQ ID NO: 9)), using techniques well known in protein chemistry, such as solid-phase synthesis or synthesis in homogeneous solution. The peptides may be N-terminally acetylated or C-terminally amidated. The peptides may be conjugated to immunogenicity enhancers, for example, via a C-terminal cysteine ​​residue or an N-terminal cysteine ​​residue or other functionalizable moieties, or otherwise modified to increase immunogenicity.

[0104] In one embodiment, the immunogen comprises a plurality of peptides, each peptide comprising all or part of DAGWGNL(SEQ ID NO: 1), the portion comprising at least five, and possibly at least six, residues of DAGWGNL(SEQ ID NO: 1), where the plurality of peptides are synthesized as a multiantigenic peptide (MAP), which is a branched polylysine dendrimer. The plurality of epitope peptides are bound, for example, to one or both of the amino terminus and side chains of lysine.

[0105] In one embodiment, the peptide is conjugated with a carrier protein or an immunogenicity enhancer. The immunogenicity enhancer may be conjugated with the compound directly, for example, via an amide bond, a disulfide bond, or indirectly via a linker.

[0106] Immunogens having immunogenicity-enhancing components can be produced, for example, by using the method described in Lateef et al 2007 (which is incorporated herein by reference) to conjugate a linker comprising the peptide and a functionalizable moiety such as cysteine ​​with an immunogenicity-enhancing component such as keyhole limpet hemocyanin (KLH) or a carrier such as bovine serum albumin (BSA). In one embodiment, the method described in Example 1 is used.

[0107] Further aspects include antibodies that preferentially bind to misfolded TDP-43, for example, cytoplasmic and / or aggregated misfolded TDP-43 produced by the immunogen described herein.

[0108] In one embodiment, the produced antibody specifically binds to at least W68 in relation to DAGWGNL (SEQ ID NO: 1).

[0109] III. Antibodies, Immune Complexes, Cells, and Nucleic Acids An isolated peptide comprising DAGWGNL (SEQ ID NO: 1) or a related epitope and the immunogen described above can be used to produce an antibody that preferentially binds to misfolded TDP-43 compared to native TDP-43.

[0110] Therefore, one aspect involves antibodies that bind to misfolded TDP-43 compared to natural TDP-43.

[0111] In one embodiment, the antibody binds to the TDP-43 sequence DAGWGNL (SEQ ID NO: 1), its associated epitope, or a portion thereof, wherein the antibody preferentially binds to misfolded TDP-43 compared to natural TDP-43.

[0112] In one embodiment, the antibody specifically binds to W68, its associated epitope, or a portion thereof in relation to DAGWGNL (SEQ ID NO: 1), wherein the antibody preferentially binds to misfolded TDP-43 compared to natural TDP-43.

[0113] In one embodiment, the antibody does not specifically bind to and / or is not selective for natural TDP-43. Selective binding can be measured using ELISA or surface plasmon resonance assay as described herein.

[0114] In one embodiment, the antibody is isolated.

[0115] A further aspect is an antibody that specifically or selectively binds to an epitope present on TDP-43, wherein the epitope comprises or consists of at least one amino acid residue primarily involved in binding to the antibody, where the at least one amino acid is W68 in relation to DAGWGNL (SEQ ID NO: 1). In one embodiment, the epitope comprises or consists of at least three consecutive amino acid residues primarily involved in binding to the antibody, where the at least three consecutive amino acids are GWG contained within DAGWGNL (SEQ ID NO: 1).

[0116] To produce monoclonal antibodies, antibody-producing cells (lymphocytes) are recovered from a subject immunized with the immunogen described herein and fused with myeloma cells using a standard somatic cell fusion procedure, thereby immortalizing these cells and obtaining hybridoma cells. Such techniques are well known in the art (e.g., the hybridoma technique first developed by Kohler and Milstein (Nature 256:495-497 (1975))), as are other techniques, such as human B-cell hybridoma technique (Kozbor et al., Immunol.Today 4:72 (1983)), EBV hybridoma technique for producing human monoclonal antibodies (Cole et al., Methods Enzymol, 121:140-67 (1986)), and combinatorial antibody library screening (Huse et al., Science 246:1275 (1989)). Hybridoma cells can be immunochemically screened to produce antibodies that are specifically reactive with a desired epitope, and monoclonal antibodies can be isolated.

[0117] Specific antibodies or antibody fragments that are reactive to a particular antigen or molecule may be produced by screening immunoglobulin genes, or expression libraries encoding portions thereof, expressed in bacteria having cell surface components. For example, complete Fab fragments, VH and FV regions can be expressed in bacteria using phage expression libraries (see, e.g., Ward et al., Nature 41:544-546 (1989); Huse et al., Science 246:1275-1281 (1989); and McCafferty et al., Nature 348:552-554 (1990)).

[0118] The humanization of antibodies derived from non-human species (e.g., mouse or rabbit) is well documented in the literature. See, for example, EP-B1 0 239400 and Carter & Merchant 1997 (Curr Opin Biotechnol 8, 449-454, 1997, the entire content of which is incorporated herein by reference). Humanized antibodies are also readily available commercially (e.g., Scotgen Limited, 2 Holly Road, Twickenham, Middlesex, Great Britain).

[0119] Humanized rodent antibodies can be readily produced by CDR grafting (Riechmann et al. Nature, 332:323-327, 1988). In this approach, six CDR loops containing the antigen-binding site of a rodent monoclonal antibody are ligated to the corresponding human framework region. Because amino acids in the framework region can affect antigen recognition, CDR grafting often results in antibodies with low affinity (Foote & Winter. J Mol Biol, 224: 487-499, 1992). To maintain antibody affinity, it is often necessary to replace specific framework residues by site-directed mutagenesis or other recombination techniques, which can be supported by computer modeling of the antigen-binding site (Co et al. J Immunol, 152: 2968-2976, 1994).

[0120] Humanized antibodies can, in some cases, be obtained by resurfacing (Pedersen et al. J Mol Biol, 235: 959-973, 1994). In this approach, only the surface residues of rodent antibodies are humanized.

[0121] Human antibodies specific to particular antigens may be identified by phage display strategies (Jespers et al. Bio / Technology, 12: 899-903, 1994). In one approach, heavy chains of rodent antibodies directed towards specific antigens are cloned and paired with a repertoire of human light chains for presentation as Fab fragments on filamentous phages. The phages are selected by binding to the antigen. The selected human light chains are then paired with a repertoire of human heavy chains for presentation on the phage, and the phages are again selected by binding to the antigen. The result is a human antibody Fab fragment specific to a particular antigen. In another approach, a library of phages is constructed in which members display different human antibody fragments (Fab or Fv) on their outer surfaces (Dower et al., WO 91 / 17271 and McCafferty et al., WO92 / 01047). Phage display antibodies with the desired specificity are selected by affinity enrichment against the specific antigen. Human Fab or Fv fragments identified by either approach may be recloned for expression as human antibodies in mammalian cells.

[0122] Human antibodies can, in some cases, be obtained from transgenic animals (U.S. Patents 6,150,584; 6,114,598; and 5,770,429). In this approach, the heavy chain linkage region (JH) gene is deleted in chimeric or germline mutant mice. A human germline immunoglobulin gene array is then introduced into such mutant mice. The resulting transgenic mice can then produce a complete repertoire of human antibodies upon antigen challenge.

[0123] Humanized antibodies or human antibodies are selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA, or IgE, and any isotype, including IgG1, IgG2, IgG3, and IgG4. Humanized antibodies or human antibodies may contain sequences derived from one or more isotypes or classes. Furthermore, these antibodies are generally produced as antigen-binding fragments, e.g., Fab, Fab', F(ab')2, Fd, Fv, and single-domain antibody fragments, or as single-chain antibodies in which heavy and light chains are linked by spacers. Human antibodies or humanized antibodies may also exist in monomeric or polymeric form. Humanized antibodies may optionally consist of one non-human chain and one humanized chain (i.e., one humanized heavy or light chain).

[0124] Furthermore, antibodies specific to the epitopes described herein can be readily isolated by screening antibody phage display libraries. For example, antibody phage libraries are screened by using the disease-specific epitopes of the present invention to identify antibody fragments against disease-specific epitopes. The identified antibody fragments are optionally used to produce a variety of useful recombinant antibodies having multiple different embodiments of the present invention. Antibody phage display libraries are commercially available, for example, through Xoma, Inc. (Berkeley, California). Methods for screening antibody phage libraries are well known in the art.

[0125] Accordingly, in one embodiment, the antibody described herein comprises optionally fused light chain variable regions and heavy chain variable regions, the heavy chain variable region comprising complementarity-determining regions CDR-H1, CDR-H2 and CDR-H3, the light chain variable region comprising complementarity-determining regions CDR-L1, CDR-L2 and CDR-L3, and the amino acid sequence of CDR-H3 comprising the sequence: AGGPTGNSFTL (SEQ ID NO: 12), ARNPVGSVNL (SEQ ID NO: 18), ARRYTGDTYLGNFNL (SEQ ID NO: 24), GRGDI (SEQ ID NO: 36), ARDIFRTNTNL (SEQ ID NO: 48), VRSSGSDWWFHI (SEQ ID NO: 122), or VRQNYEGAY (SEQ ID NO: 132). In one embodiment, the sequence of CDR-H3 comprises the sequence AGGPTGNSFTL (SEQ ID NO: 12). In one embodiment, the sequence of CDR-H3 includes the sequence ARNPVGSVNL (sequence number 18). In one embodiment, the sequence of CDR-H3 includes the sequence ARRYTGDTYLGNFNL (sequence number 24). In one embodiment, the sequence of CDR-H3 includes the sequence GRGDI (sequence number 36). In one embodiment, the sequence of CDR-H3 includes the sequence ARDIFRTNTNL (sequence number 48). In one embodiment, the sequence of CDR-H3 includes the sequence VRSSGSDWWFHI (sequence number 122). In one embodiment, the sequence of CDR-H3 includes the sequence VRQNYEGAY (sequence number 132).

[0126] Therefore, in one embodiment, the antibody described herein comprises a light chain variable region and a heavy chain variable region, the heavy chain variable region comprising complementarity-determining regions CDR-H1, CDR-H2 and CDR-H3, the light chain variable region comprising complementarity-determining regions CDR-L1, CDR-L2 and CDR-L3, and the amino acid sequences of CDR-H3 and CDR-L3 are: AGGPTGNSHFTL (SEQ ID NO: 12) and SGYKRVTTDGIA (SEQ ID NO: 15); ARNPVGSVNL (SEQ ID NO: 18) and AGWRGARTDGVD (SEQ ID NO: 21); AR It comprises RYTGDTYLGNFNL (SEQ ID NO: 24) and AGGWRSLNA (SEQ ID NO: 27); GRGDI (SEQ ID NO: 36) and LGNYDCSSVDCGA (SEQ ID NO: 39); AGGPTGNSFTL (SEQ ID NO: 42) and AGYKSPTTDGIA (SEQ ID NO: 45); ARDIFRTNTNL (SEQ ID NO: 48) and LGGYDCSSRVCGA (SEQ ID NO: 51); VRSSGSDWWFHI (SEQ ID NO: 122) and QGYFSGFITT (SEQ ID NO: 125); or VRQNYEGAY (SEQ ID NO: 132) and FQSSHVPWT (SEQ ID NO: 135).

[0127] In one embodiment, the amino acid sequences of CDR-H3 and CDR-L3 include the sequences: AGGPTGNSFTL (SEQ ID NO: 12) and SGYKRVTTDGIA (SEQ ID NO: 15). In one embodiment, the amino acid sequences of CDR-H3 and CDR-L3 include the sequences: ARNPVGSVNL (SEQ ID NO: 18) and AGWRGARTDGVD (SEQ ID NO: 21). In one embodiment, the amino acid sequences of CDR-H3 and CDR-L3 include the sequences: ARRYTGDTYLGNFNL (SEQ ID NO: 24) and AGGWRSLNA (SEQ ID NO: 27). In one embodiment, the amino acid sequences of CDR-H3 and CDR-L3 include the sequences: GRGDI (SEQ ID NO: 36) and LGNYDCSSVDCGA (SEQ ID NO: 39). In one embodiment, the amino acid sequences of CDR-H3 and CDR-L3 include the sequences: AGGPTGNSFTL (SEQ ID NO: 42) and AGGKSPTTDGIA (SEQ ID NO: 45). In one embodiment, the amino acid sequences of CDR-H3 and CDR-L3 include the sequences: ARDIFRTNTNL (SEQ ID NO: 48) and LGGYDCSSRVCGA (SEQ ID NO: 51). In one embodiment, the amino acid sequences of CDR-H3 and CDR-L3 include the sequences: VRSSGSDWWFHI (SEQ ID NO: 122) and QGYFSGFITT (SEQ ID NO: 125). In one embodiment, the amino acid sequences of CDR-H3 and CDR-L3 include the sequences: VRQNYEGAY (SEQ ID NO: 132) and FQSSHVPWT (SEQ ID NO: 135).

[0128] In one aspect, the present disclosure relates to an antibody comprising optionally fused light chain variable regions and heavy chain variable regions, wherein the heavy chain variable region comprises complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3, and the light chain variable region comprises complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3, and the amino acid sequence of the CDR comprises the following sequence: CDR-H1: GFSLSRYY Sequence ID 10; CDR-H2: IIPGGTT Sequence ID 11; CDR-H3: AGGPTGNSHFTL Sequence ID 12; CDR-L1: ESVYNNNH Sequence ID 13; CDR-L2: EAS Sequence ID 14; and CDR-L3: SGYKRVTTDGIA Sequence ID 15, We provide antibodies.

[0129] In one aspect, the present disclosure relates to an antibody comprising optionally fused light chain variable regions and heavy chain variable regions, wherein the heavy chain variable region comprises complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3, and the light chain variable region comprises complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3, and the amino acid sequence of the CDR comprises the following sequence: CDR-H1: GFSFSSNYV Sequence ID 16; CDR-H2: IWFAGIVDTT Sequence ID 17; CDR-H3: ARNPVGSVNL Sequence ID 18; CDR-L1: ESVYSNNR Sequence ID 19; CDR-L2: YAS Sequence ID 20; and CDR-L3: AGWRGARTDGVD Sequence ID 21, We provide antibodies.

[0130] In one aspect, the present disclosure relates to an antibody comprising optionally fused light chain variable regions and heavy chain variable regions, wherein the heavy chain variable region comprises complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3, and the light chain variable region comprises complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3, and the amino acid sequence of the CDR comprises the following sequence: CDR-H1: GFSFSSSYV Sequence ID 22; CDR-H2: SDTGINT Sequence ID 23; CDR-H3: ARRYTGDTYLGNFNL Sequence ID 24; CDR-L1: QSVYKNNY Sequence ID 25; CDR-L2: KAS Sequence ID 26; and CDR-L3: AGGWRSLNA Sequence ID 27, We provide antibodies.

[0131] In one aspect, the present disclosure relates to an antibody comprising optionally fused light chain variable regions and heavy chain variable regions, wherein the heavy chain variable region comprises complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3, and the light chain variable region comprises complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3, and the amino acid sequence of the CDR comprises the following sequence: CDR-H1: EFSFSSRYW Sequence ID 28; CDR-H2: IYTGSIDAT Sequence ID 29; CDR-H3: VRGSDAWGLYFNL Sequence ID 30; CDR-L1: QSIHKNNY Sequence ID 31; CDR-L2: FAS Sequence ID 32; and CDR-L3: AGVYSGRIFA Sequence ID 33, We provide antibodies.

[0132] In one aspect, the present disclosure relates to an antibody comprising optionally fused light chain variable regions and heavy chain variable regions, wherein the heavy chain variable region comprises complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3, and the light chain variable region comprises complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3, and the amino acid sequence of the CDR comprises the following sequence: CDR-H1: GFSLSSYT Sequence ID 34; CDR-H2: IYGGIGST Sequence ID 35; CDR-H3: GRGDI Sequence ID 36; CDR-L1: QSVYKNR Sequence ID 37; CDR-L2: GAS Sequence ID 38; and CDR-L3: LGNYDCSSVDCGA Sequence ID 39, We provide antibodies.

[0133] In one aspect, the present disclosure relates to an antibody comprising optionally fused light chain variable regions and heavy chain variable regions, wherein the heavy chain variable region comprises complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3, and the light chain variable region comprises complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3, and the amino acid sequence of the CDR comprises the following sequence: CDR-H1: GFSFSAYY Sequence ID 40; CDR-H2: TIPIGRT Sequence ID 41; CDR-H3: AGGPTGNSHFTL Sequence ID 42; CDR-L1: ESVYNNNQ Sequence ID 43; CDR-L2: QAS Sequence ID 44; and CDR-L3: AGYKSPTTDGIA Sequence ID 45, We provide antibodies.

[0134] In one aspect, the present disclosure relates to an antibody comprising optionally fused light chain variable regions and heavy chain variable regions, wherein the heavy chain variable region comprises complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3, and the light chain variable region comprises complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3, and the amino acid sequence of the CDR comprises the following sequence: CDR-H1: GFSLSSYA Sequence ID 46; CDR-H2: IYNYET Sequence ID 47; CDR-H3: ARDIFRTNTNL Sequence ID 48; CDR-L1: QSVYKNNG Sequence ID 49; CDR-L2: FTS Sequence ID 50; and CDR-L3: LGGYDCSSRVCGA Sequence ID 51, We provide antibodies.

[0135] In one aspect, the present disclosure relates to an antibody comprising optionally fused light chain variable regions and heavy chain variable regions, wherein the heavy chain variable region comprises complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3, and the light chain variable region comprises complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3, and the amino acid sequence of the CDR comprises the following sequence: CDR-H1: GFSLSSYN Sequence ID 120; CDR-H2: IGTGGIT Sequence ID 121; CDR-H3: VRSSGSDWWFHI Sequence ID 122; CDR-L1: QSVYNNNN Sequence ID 123; CDR-L2: RAS Sequence ID 124; and CDR-L3: QGYFSGFITT Sequence ID 125, We provide antibodies.

[0136] In one aspect, the present disclosure relates to an antibody comprising optionally fused light chain variable regions and heavy chain variable regions, wherein the heavy chain variable region comprises complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3, and the light chain variable region comprises complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3, and the amino acid sequence of the CDR comprises the following sequence: CDR-H1: GFTFSSYY Sequence ID 130; CDR-H2: INSNGGST Sequence ID 131; CDR-H3: VRQNYEGAY Sequence ID 132; CDR-L1: QSIVHSNGNTY Sequence ID 133; CDR-L2: KVS Sequence ID 134; and CDR-L3: FQSSHVPWT Sequence ID 135, We provide antibodies.

[0137] In one aspect, the present disclosure relates to an antibody comprising optionally fused light chain variable regions and heavy chain variable regions, wherein the heavy chain variable region comprises complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3, and the light chain variable region comprises complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3, and the amino acid sequence of the CDR comprises the following sequence: CDR-H1: GFTFSSYY Sequence ID 140; CDR-H2: INTNGGST Sequence ID 141; CDR-H3: VRQNYEGAY Sequence ID 142; CDR-L1: QSIVHSNGNTY Sequence ID 143; CDR-L2: KVS Sequence ID 144; and CDR-L3: FQSSHVPWT Sequence ID 145, We provide antibodies.

[0138] In one embodiment, the antibody comprises a heavy chain variable region comprising i) the amino acid sequence shown in SEQ ID NO: 98, and ii) an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% sequence identity with respect to SEQ ID NO: 98, wherein the CDR sequence is shown in SEQ ID NOs: 10-12, or iii) the conserved substitution amino acid sequence of i); and / or the antibody comprises a light chain variable region comprising i) the amino acid sequence shown in SEQ ID NO: 99, and ii) an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% sequence identity with respect to SEQ ID NO: 99, wherein the CDR sequence is shown in SEQ ID NOs: 13-15, or iii) the conserved substitution amino acid sequence of i); optionally, the heavy chain variable region amino acid sequence is encoded by the nucleotide sequence or codon degeneracy shown in SEQ ID NO: 76 or an optimized version thereof, and / or the light chain variable region amino acid sequence is encoded by the nucleotide sequence or codon degeneracy shown in SEQ ID NO: 77 or an optimized version thereof.

[0139] In one embodiment, the antibody comprises a heavy chain variable region comprising i) the amino acid sequence shown in SEQ ID NO: 100, ii) an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% sequence identity with respect to SEQ ID NO: 100, where the CDR sequence is shown in SEQ ID NOs: 16-18, or iii) the conserved substitution amino acid sequence of i); and / or, the antibody comprises i) the amino acid sequence shown in SEQ ID NO: 101, ii) at least 50%, at least 60%, and The light chain variable region comprises an amino acid sequence having 70%, at least 80%, or at least 90% sequence identity, wherein the CDR sequence is one of those shown in SEQ ID NOs: 19-21, or the conserved substitution amino acid sequence of iii)i), and optionally the heavy chain variable region amino acid sequence is encoded by the nucleotide sequence or codon degeneracy shown in SEQ ID NO: 78 or an optimized version thereof, and / or the light chain variable region amino acid sequence is encoded by the nucleotide sequence or codon degeneracy shown in SEQ ID NO: 79 or an optimized version thereof.

[0140] In one embodiment, the antibody comprises a heavy chain variable region comprising i) the amino acid sequence shown in SEQ ID NO: 102, ii) an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% sequence identity with respect to SEQ ID NO: 102, where the CDR sequence is shown in SEQ ID NOs: 22-24, or iii) the conserved substitution amino acid sequence of i); and / or the antibody comprises i) the amino acid sequence shown in SEQ ID NO: 103, ii) at least 50%, at least 60%, at least The heavy chain variable region comprises an amino acid sequence having 70%, at least 80%, or at least 90% sequence identity, wherein the CDR sequence is one of those shown in SEQ ID NOs. 25-27, or the conserved substitution amino acid sequence of iii)i), and optionally the heavy chain variable region amino acid sequence is encoded by the nucleotide sequence or codon degeneracy shown in SEQ ID NO. 80 or an optimized version thereof, and / or the light chain variable region amino acid sequence is encoded by the nucleotide sequence or codon degeneracy shown in SEQ ID NO. 81 or an optimized version thereof.

[0141] In one embodiment, the antibody comprises a heavy chain variable region comprising i) the amino acid sequence shown in SEQ ID NO: 104, ii) an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% sequence identity with respect to SEQ ID NO: 104, where the CDR sequence is shown in SEQ ID NOs: 28-30, or iii) the conserved substitution amino acid sequence of i); and / or, the antibody comprises i) the amino acid sequence shown in SEQ ID NO: 105, ii) at least 50%, at least 60%, and The light chain variable region comprises an amino acid sequence having 70%, at least 80%, or at least 90% sequence identity, wherein the CDR sequence is one of those shown in SEQ ID NOs: 31-33, or the conserved substitution amino acid sequence of iii)i), and optionally the heavy chain variable region amino acid sequence is encoded by the nucleotide sequence or codon degeneracy shown in SEQ ID NO: 82 or an optimized version thereof, and / or the light chain variable region amino acid sequence is encoded by the nucleotide sequence or codon degeneracy shown in SEQ ID NO: 83 or an optimized version thereof.

[0142] In one embodiment, the antibody comprises a heavy chain variable region comprising i) the amino acid sequence shown in SEQ ID NO: 106, ii) an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% sequence identity with respect to SEQ ID NO: 106, where the CDR sequence is shown in SEQ ID NOs: 34-36, or iii) the conserved substitution amino acid sequence of i); and / or the antibody comprises i) the amino acid sequence shown in SEQ ID NO: 107, ii) at least 50%, at least 60%, at least The light chain variable region comprises an amino acid sequence having 70%, at least 80%, or at least 90% sequence identity, wherein the CDR sequence is one of those shown in SEQ ID NOs. 37-39, or the conserved substitution amino acid sequence of iii)i), and optionally the heavy chain variable region amino acid sequence is encoded by the nucleotide sequence or codon degeneracy shown in SEQ ID NO. 84 or an optimized version thereof, and / or the light chain variable region amino acid sequence is encoded by the nucleotide sequence or codon degeneracy shown in SEQ ID NO. 85 or an optimized version thereof.

[0143] In one embodiment, the antibody comprises a heavy chain variable region comprising i) the amino acid sequence shown in SEQ ID NO: 108, ii) an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% sequence identity with respect to SEQ ID NO: 108, where the CDR sequence is shown in SEQ ID NOs: 40-42, or iii) the conserved substitution amino acid sequence of i); and / or the antibody comprises i) the amino acid sequence shown in SEQ ID NO: 109, ii) at least 50%, at least 60%, at least The light chain variable region comprises an amino acid sequence having 70%, at least 80%, or at least 90% sequence identity, wherein the CDR sequence is one of those shown in SEQ ID NOs. 43-45, or the conserved substitution amino acid sequence of iii)i), and optionally the heavy chain variable region amino acid sequence is encoded by the nucleotide sequence or codon degeneracy shown in SEQ ID NO. 86 or an optimized version thereof, and / or the light chain variable region amino acid sequence is encoded by the nucleotide sequence or codon degeneracy shown in SEQ ID NO. 87 or an optimized version thereof.

[0144] In one embodiment, the antibody comprises a heavy chain variable region comprising i) the amino acid sequence shown in SEQ ID NO: 110, ii) an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% sequence identity with respect to SEQ ID NO: 110, where the CDR sequence is shown in SEQ ID NOs: 46-48, or iii) the conserved substitution amino acid sequence of i); and / or, the antibody comprises i) the amino acid sequence shown in SEQ ID NO: 111, ii) at least 50%, at least 60%, and The heavy chain variable region comprises an amino acid sequence having 70%, at least 80%, or at least 90% sequence identity, wherein the CDR sequence is one of those shown in SEQ ID NOs. 49-51, or the conserved substitution amino acid sequence of iii)i), and optionally the heavy chain variable region amino acid sequence is encoded by the nucleotide sequence or codon degeneracy shown in SEQ ID NO. 88 or an optimized version thereof, and / or the light chain variable region amino acid sequence is encoded by the nucleotide sequence or codon degeneracy shown in SEQ ID NO. 89 or an optimized version thereof.

[0145] In one embodiment, the antibody comprises a heavy chain variable region comprising i) the amino acid sequence shown in SEQ ID NO: 128, ii) an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% sequence identity with respect to SEQ ID NO: 128, where the CDR sequence is shown in SEQ ID NOs: 120-122, or iii) the conserved substitution amino acid sequence of i); and / or the antibody comprises i) the amino acid sequence shown in SEQ ID NO: 129, ii) at least 50%, at least 60%, at least 70% sequence identity with respect to SEQ ID NO: 129 The light chain variable region comprises an amino acid sequence having 0%, at least 80%, or at least 90% sequence identity, wherein the CDR sequence is one of those shown in SEQ ID NOs: 123-125, or the conserved substitution amino acid sequence of iii)i), and optionally the heavy chain variable region amino acid sequence is encoded by the nucleotide sequence or codon degeneracy shown in SEQ ID NO: 126 or an optimized version thereof, and / or the light chain variable region amino acid sequence is encoded by the nucleotide sequence or codon degeneracy shown in SEQ ID NO: 127 or an optimized version thereof.

[0146] In one embodiment, the antibody comprises a heavy chain variable region comprising i) the amino acid sequence shown in SEQ ID NO: 138, ii) an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% sequence identity with respect to SEQ ID NO: 138, where the CDR sequence is shown in SEQ ID NOs: 130-132, or iii) the conserved substitution amino acid sequence of i); and / or, the antibody comprises i) the amino acid sequence shown in SEQ ID NO: 129, ii) at least 50%, at least 60%, at least The light chain variable region comprises an amino acid sequence having 70%, at least 80%, or at least 90% sequence identity, wherein the CDR sequence is one of those shown in SEQ ID NOs: 133-135, or the conserved substitution amino acid sequence of iii)i), and optionally the heavy chain variable region amino acid sequence is encoded by the nucleotide sequence or codon degeneracy shown in SEQ ID NO: 136 or an optimized version thereof, and / or the light chain variable region amino acid sequence is encoded by the nucleotide sequence or codon degeneracy shown in SEQ ID NO: 137 or an optimized version thereof.

[0147] In one embodiment, the antibody comprises a heavy chain variable region comprising i) the amino acid sequence shown in SEQ ID NO: 148, ii) an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% sequence identity with respect to SEQ ID NO: 148, where the CDR sequence is shown in SEQ ID NOs: 140-142, or iii) the conserved substitution amino acid sequence of i); and / or, the antibody comprises i) the amino acid sequence shown in SEQ ID NO: 149, ii) at least 50%, at least 60%, at least The light chain variable region comprises an amino acid sequence having 70%, at least 80%, or at least 90% sequence identity, wherein the CDR sequence is one of those shown in SEQ ID NOs: 143-145, or the conserved substitution amino acid sequence of iii)i), and optionally the heavy chain variable region amino acid sequence is encoded by the nucleotide sequence or codon degeneracy shown in SEQ ID NO: 146 or an optimized version thereof, and / or the light chain variable region amino acid sequence is encoded by the nucleotide sequence or codon degeneracy shown in SEQ ID NO: 147 or an optimized version thereof.

[0148] In one embodiment, the antibody comprises a heavy chain variable region comprising a conserved substituted amino acid sequence shown in any one of SEQ ID NOs: 98, 100, 102, 104, 106, 108, 110, 128, 138, or 148. In another embodiment, the antibody comprises a heavy chain variable region comprising a conserved substituted amino acid sequence shown in any one of SEQ ID NOs: 99, 101, 103, 105, 107, 109, 111, 129, 139, or 149. For example, the heavy chain variable region and / or the light chain variable region, optionally framework regions 1, 2, and / or 3, may contain 1, 2, 3, 4, or 5 conserved amino acid substitutions.

[0149] In one embodiment, the antibody is a monoclonal antibody. In another embodiment, the antibody is a chimeric antibody, such as a humanized antibody. In yet another embodiment, the antibody is a single-chain antibody.

[0150] In one embodiment, the antibody is purified by affinity.

[0151] In one embodiment, the antibody is produced or screened using an isolated peptide or immunogen described herein.

[0152] Another aspect involves antibodies that compete with the antibodies described herein, and optionally the antibodies comprising the CDR set described herein, for binding to human misfolded TDP43.

[0153] Antibody competition can be determined, for example, using an assay that evaluates the ability of the test antibody to inhibit the specific binding of the reference antibody to a common antigen. The test antibody competes with the reference antibody if, as measured in a competitive binding assay, an excess of the test antibody (e.g., at least 2x, 5x, 10x, or 20x) inhibits the binding of the reference antibody by at least 50%, at least 75%, at least 80%, at least 90%, or at least 95%.

[0154] A further aspect is an antibody conjugated with a detectable label. In one embodiment, the detectable label is a positron-emitting radionuclide. Positron-emitting radionuclides can be used, for example, in PET imaging.

[0155] Accordingly, one embodiment provides an immunocomplex comprising the antibody and detectable label described herein.

[0156] Further aspects relate to antibody conjugates comprising the antibody and / or its binding fragment and misfolded TDP-43 as described herein. Further aspects relate to isolated nucleic acids encoding the antibody or a portion thereof as described herein.

[0157] Nucleic acids encoding heavy chains or light chains are also provided, for example, nucleic acids encoding heavy chains comprising the CDR-H1, CDR-H2 and / or CDR-H3 regions described herein, or nucleic acids encoding light chains comprising the CDR-L1, CDR-L2 and / or CDR-L3 regions described herein and more specifically in Table 3.

[0158] For example, the nucleic acid sequence includes one of the following: SEQ ID NOs. 76-89, SEQ ID NOs. 126-127, SEQ ID NOs. 136-137, and / or SEQ ID NOs. 146-147.

[0159] This disclosure also provides variants of nucleic acid sequences encoding the antibodies disclosed herein.

[0160] For example, the variant includes a nucleotide sequence that hybridizes to a nucleic acid sequence encoding an antibody disclosed herein under at least moderately stringent hybridization conditions, or a codon-degenerate or optimized sequence. In another embodiment, the variant nucleic acid sequence has at least 50%, at least 60%, at least 70%, most preferably at least 80%, more preferably at least 90%, and most preferably at least 95% sequence identity with respect to a nucleic acid sequence comprising any one of SEQ ID NOs. 76-89, SEQ ID NOs. 126-127, SEQ ID NOs. 136-137 and / or SEQ ID NOs. 146-147.

[0161] Further aspects include providing isolated nucleic acids encoding amino acid residues of isolated peptides, immunogens, or antibodies described herein.

[0162] Another aspect is an expression cassette or vector comprising nucleic acids disclosed herein. In one embodiment, the vector is an isolation vector.

[0163] The vector may be any vector suitable for producing an antibody and / or its binding fragment, or for expressing a peptide sequence as described herein.

[0164] Nucleic acid molecules may be incorporated in known ways into a suitable expression vector that ensures protein expression. Possible expression vectors include, but are not limited to, cosmids, plasmids, or modified viruses (e.g., replication-deficient retroviruses, adenoviruses, and adeno-associated viruses). The vector should be compatible with the host cell in which it is used. An expression vector is "suitable for transformation of host cells," meaning that the expression vector contains a nucleic acid molecule encoding a peptide corresponding to an epitope / peptide or antibody as described herein.

[0165] In one embodiment, the vector is suitable for expressing, for example, a single-chain antibody (e.g., an intrabody). Suitable regulatory sequences may be derived from a variety of sources, including bacterial, fungal, viral, mammalian, or insect genes. Examples of such regulatory sequences include transcription promoters and enhancers or RNA polymerase binding sequences, and ribosome binding sequences (including translation initiation signals). Furthermore, depending on the selected host cell and the vector used, other sequences, such as origins of replication, further DNA restriction sites, enhancers, and sequences conferring transcriptional inducibility, may be incorporated into the expression vector. In one embodiment, the regulatory sequences direct or increase expression in nerve tissue and / or cells. In one embodiment, the vector is a viral vector. The recombinant expression vector may also contain marker genes to facilitate the selection of host cells transformed, infected, or transfected with the vector for expressing the antibodies or epitope peptides described herein. Recombinant expression vectors may contain an expression cassette encoding a fusion moiety (i.e., a "fusion protein") that results in increased expression or stability of the recombinant peptide; increased solubility of the recombinant peptide; and assistance in the purification of the target recombinant peptide by acting as a ligand in affinity purification, including, for example, the tags and labels described herein. Furthermore, proteolytic cleavage sites may be added to the target recombinant protein to enable the separation of the recombinant protein from the fusion moiety following the purification of the fusion protein. Typical fusion expression vectors include pGEX (Amrad Corp., Melbourne, Australia), pMAL (New England Biolabs, Beverly, Massachusetts), and pRIT5 (Pharmacia, Piscataway, New Jersey), which fuse glutathione S-transferase (GST), maltose E-binding protein, or protein A to the recombinant protein, respectively.

[0166] In another aspect, cells expressing the antibodies described herein are also provided. In one embodiment, the cells are isolated cells and / or recombinant cells expressing the antibodies described herein or comprising the vectors disclosed herein. In one embodiment, the cells are fusion cells, for example, hybridomas.

[0167] The recombinant cells can be prepared using any cells suitable for producing polypeptides, for example, antibodies and / or their binding fragments. For example, the cells may be transfected, transformed, or infected, depending on the vector used, in order to introduce nucleic acids (e.g., vectors) into the cells.

[0168] Suitable host cells include a wide variety of prokaryotic and eukaryotic host cells. For example, the proteins described herein may be expressed in bacterial cells such as Escherichia coli, insect cells (using baculoviruses), yeast cells, or mammalian cells.

[0169] In one embodiment, the cells are eukaryotic cells selected from cells of yeast, plants, worms, insects, birds, fish, reptiles, and mammals.

[0170] In another embodiment, the mammalian cells are myeloma cells, spleen cells, or hybridoma cells.

[0171] In one embodiment, the cells are nerve cells.

[0172] Suitable yeast and fungal host cells for expressing antibodies or peptides include, but are not limited to, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia or Cliveromyces genera, and various species of Aspergillus. Examples of vectors for expression in yeast S. cerevisiae include pYepSec1, pMFa, pJRY88, and pYES2 (Invitrogen Corporation, San Diego, California). Protocols for transforming yeast and fungi are well known to those skilled in the art.

[0173] Suitable mammalian cells include, in particular, COS (e.g., ATCC numbers CRL1650 or 1651), BHK (e.g., ATCC number CRL6281), CHO (ATCC number CCL61), HeLa (e.g., ATCC number CCL2), 293 (ATCC number 1573), and NS-1 cells. Suitable expression vectors for directing expression in mammalian cells generally include promoters (e.g., derived from viral materials such as polyoma, adenovirus 2, cytomegalovirus, and simian virus 40), as well as other transcriptional and translational regulatory sequences. Examples of mammalian expression vectors include pCDM8 and pMT2PC.

[0174] In one embodiment, the cells are fusion cells, for example, hybridoma cells, which produce antibodies specific to and / or selective to the epitopes or epitope sequences described herein, including, for example, those that selectively bind to misfolded TDP-43.

[0175] A further aspect is the hybridoma cell line that produces antibodies specific to the epitopes described herein.

[0176] IV. Composition Further aspects include compositions comprising the isolated peptides, immunogens, antibodies, or immune complexes described herein. Compositions comprising two or more of the isolated peptides, immunogens, antibodies, or immune complexes described herein are also provided.

[0177] In one embodiment, the composition comprises a diluent. Suitable diluents for nucleic acids and vectors include, but are not limited to, water, saline solution, and ethanol.

[0178] Suitable diluents for polypeptides and / or cells containing antibodies or their fragments include, but are not limited to, physiological saline, pH buffer, and glycerol solution or other solutions suitable for freezing polypeptides and / or cells.

[0179] In some embodiments comprising peptides, compounds, or immunogens described herein, the composition comprises an adjuvant.

[0180] In one embodiment, the adjuvant is selected from alum, monophosphoryl lipid A, and QS21.

[0181] Adjuvants that can be used include, for example, endogenous adjuvants (such as lipopolysaccharides), which are typically components of dead or attenuated bacteria used as vaccines. Exogenous adjuvants are generally immunomodulators that are non-covalently bound to antigens and are prescribed to enhance the host immune response. Aluminum hydroxide, aluminum sulfate, and aluminum phosphate (collectively known as alum) are conventionally used as adjuvants. A wide range of exogenous adjuvants can induce a potent immune response to immunogens. These include saponins, such as Stimulon (QS21, Aquila, Worcester, Massachusetts) or particles made therefrom, such as ISCOM and (immunostimulatory complex) and ISCOMATRIX (immunostimulatory complex) which are complexed with membrane protein antigens, Pluronic polymers with mineral oil, dead mycobacteria and mineral oil, Freund's complete adjuvant, bacterial products, such as muramyl dipeptide (MDP) and lipopolysaccharide (LPS), as well as lipid A and liposomes.

[0182] In one embodiment, the adjuvant is aluminum hydroxide. In another embodiment, the adjuvant is aluminum phosphate. Examples of oil-in-water emulsions include squalene; peanut oil; MF59 (WO90 / 14387); SAF (Syntex Laboratories, Palo Alto, California); and Ribi (Ribi Immunochem, Hamilton, Montana). Oil-in-water emulsions may be used with immunostimulants, such as muramyl peptides (e.g., N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetylmuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutamyl-L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE), N-acetylglucosamyl-N-acetylmuramyl-L-Al-D-isoglu-L-Ala-dipalmitoxypropylamide (DTP-DPP) Teramide®), or other bacterial cell wall components.

[0183] The adjuvant may be administered together with the immunogen as a single composition. Alternatively, the adjuvant may be administered before, simultaneously with, and / or after the administration of the immunogen.

[0184] In one embodiment, the composition comprises the antibody or a portion thereof as described herein. In another embodiment, the composition comprises the antibody or a portion thereof and a diluent as described herein. In one embodiment, the composition is a sterile composition.

[0185] In some embodiments, the composition is sterile.

[0186] In one embodiment, the composition is for the purposes of the methods described herein, such as detecting misfolded TDP-43.

[0187] In one embodiment, the composition comprises a pharmaceutically acceptable carrier, diluent, and / or excipient. In one embodiment, the composition is a pharmaceutical composition for the method described herein, for example, for treating a subject that requires it, such as a subject having TDP-43 proteinosis.

[0188] The aforementioned composition may comprise one or more antibodies as described herein.

[0189] V. Kits and Packages Further aspects relate to kits or packages comprising i) isolated peptides, ii) immunogens, iii) antibodies, iv) immune complexes, v) isolated nucleic acids, or vi) compositions, and optionally reference agents and / or instructions for their use, contained in vials or other containers such as sterile vials.

[0190] In one embodiment, the kit is an ELISA. In another embodiment, the kit is a multiplex assay or planar array kit for carrying out the method described herein, similar to, for example, those available through MesoScale, Quanterix, or Singulex.

[0191] In one embodiment, the kit comprises the antibody described herein contained in a container such as a sterile vial.

[0192] In one embodiment, the kit includes instructions for use for ELISA or the method described herein.

[0193] VI. Method This specification includes methods for producing isolated peptides, immunogens, and antibodies.

[0194] In particular, a method is provided for producing antibodies selective to W68 or related epitopes in relation to DAGWGNL (SEQ ID NO: 1). In one embodiment, the method comprises administering an isolated peptide, immunogen, or composition described herein to a non-human subject, and isolating an antibody that selectively binds to the TDP-43 peptide of the immunogen and / or misfolded TDP-43. For example, isolating the antibody may involve one or more methods described herein.

[0195] In one embodiment, the method comprises isolating an antibody that selectively binds to the TDP-43 peptide of an immunogen and / or misfolded TDP-43 from an expression library encoding an immunoglobulin gene or a portion thereof. The expression library may be a phage display library.

[0196] Further aspects include methods for inducing an immune response in a non-human subject, comprising administering to the subject a compound, immunogen and / or composition comprising the compound described herein; and optionally, isolating cells and / or antibodies that specifically bind to the administered compound or immunogen.

[0197] In one embodiment, the method further comprises isolating an antibody that specifically binds to W68 or an associated epitope in relation to DAGWGNL (SEQ ID NO: 1).

[0198] In one embodiment, the method further comprises forming an antibody-producing hybridoma. For example, as discussed above, monoclonal antibodies can be produced using the method described herein.

[0199] Further aspects include providing antibodies produced by the methods described herein.

[0200] A further aspect is the provision of a method for detecting whether a sample contains misfolded TDP-43.

[0201] In one embodiment, the method is a. Contacting the sample with the antibody described herein under conditions that allow for the production of an antibody:misfolded TDP-43 polypeptide complex; and b. Detecting the presence of any complex It includes, The presence of a detectable complex indicates that the sample may contain a misfolded TDP-43 polypeptide.

[0202] In another embodiment, the method is (a) Contacting the test sample in question with the antibody described herein under conditions that allow for the production of an antibody-antigen complex; (b) Measuring the amount of antibody-antigen complex in the test sample; and (c) Compare the amount of antibody-antigen complex in the test sample with that of the control. It includes, Here, detecting the antibody-antigen complex in the test sample compared to the control indicates that the sample contains misfolded TDP-43.

[0203] The measurement may be performed, for example, by immunofluorescence. The method may also include colocalization staining, such as pan-TDP-43 staining.

[0204] In one embodiment, the sample is a biological sample. In one embodiment, the sample comprises blood, serum, plasma, brain tissue, spinal cord tissue or extracts thereof and / or CSF. The sample may also be a fraction. For example, the sample may comprise extracellular vesicles derived from brain, CSF, and / or blood. In one embodiment, the sample is obtained from a human subject.

[0205] In one embodiment, the sample is derived from a subject having ALS. In another embodiment, the sample is derived from a subject having FTD. In one embodiment, the sample is derived from limbic-dominant senile TDP-43 encephalopathy (LATE).

[0206] Numerous immunoassays, including flow cytometry, dot or slot blotting, Western blotting, ELISA, and SDS-PAGE immunocytochemistry following immunoprecipitation, can be used to determine whether misfolded TDP-43 polypeptides are present in a sample using the antibodies described herein. Other immuno-based methods that can be used include singleplex and multiplex immunoassay platforms.

[0207] A singleplex bead-based platform can be used in which individual immune complexes are isolated and detected on paramagnetic beads. A suitable bead-based singleplex platform is available from Quanterix.

[0208] Suitable multiplex immunoassay platforms for these purposes include bead or particle-based platforms and planar array platforms.

[0209] Bead-based or particle-based platforms can be used in which the capture antibody is immobilized on particles, such as fluorescent dye beads or paramagnetic beads, and the analyte is detected with a second detection antibody. Suitable bead-based multiplex platforms include, for example, Luminex® from AbCam, FirePlex® from AbCam, and those available from Quanterix.

[0210] A planar array platform can be used in which the capture antibody is immobilized in a microarray format on a solid surface, such as a membrane, a glass surface, or on individual wells of a 96-well or 384-well plate, and the analyte is detected with a second detection antibody. For spatial separation of the microarray platform, spot coordinates can be used to identify the detected analytes. Suitable planar array platforms include, for example, those available from Quaternix or Mesoscale.

[0211] Following a bead-based or particle-based assay using a fluorescently labeled detection antibody, single-molecule counting may be performed, in which the detection antibody is eluted from the immunocomplex and quantified by detecting and counting individual fluorescent molecules using capillary fluid dynamics and lasers. A suitable technique is, for example, one available from Singulex.

[0212] Surface plasmon resonance can be used to evaluate conformation-specific coupling.

[0213] The labeled antibodies described herein may also be administered to a subject to detect the location of misfolded TDP-43.

[0214] A method for inhibiting misfolded TDP-43 intercellular transmission is also provided, comprising administering an antibody described herein to a subject in need, for example, a subject suspected of having, at risk of developing, or diagnosed with TDP-43 proteinopathy.

[0215] A method for treating TDP-43 proteinosis is also provided, comprising administering an effective amount of the antibody, immune complex, or composition comprising the antibody of the immune complex described herein to a subject in need.

[0216] In one embodiment, the TDP-43 proteinopathy is selected from amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD-TDP), primary lateral sclerosis, progressive muscular atrophy, and limbic-dominant senile TDP-43 encephalopathy (LATE).

[0217] A further aspect is a method of treating a subject, comprising administering to a subject in need, optionally in combination with another TDP-43 proteinopathy treatment, an effective amount of the antibodies or immune complexes disclosed herein, or a composition comprising such antibodies or immune complexes. Other TDP-43 proteinopathy treatments include, but are not limited to, treatments for ALS, such as riluzole (Rilutek or Tiglutik®), edaravone (Radicava®), and NuDexta® (a combination of dextromethorphan and quinidine).

[0218] The antibodies may be included in the compositions described herein, for example, in combination with pharmaceutically acceptable carriers, diluents and / or excipients, and may be formulated, for example, in vesicles to improve delivery. Combinations of antibodies (e.g., two or more antibodies) and / or immune complexes may also be used.

[0219] The compositions, antibodies, immunogens, and immune complexes described herein may be administered, for example, by parenteral administration, intravenous administration, subcutaneous administration, intramuscular administration, intracranial administration, intraventricular administration, subarachnoid administration, intraorbital administration, ocular administration, intraspinal administration, intracisional administration, intraperitoneal administration, intranasal administration, aerosol administration, or oral administration.

[0220] In certain embodiments, the composition is administered systemically.

[0221] Several other embodiments involve the co-administration of the compositions, antibodies, and immune complexes described herein with bioactive molecules known to facilitate transport across the blood-brain barrier.

[0222] In certain embodiments, methods for administering the compositions, antibodies, and immune complexes described herein via the blood-brain barrier are also envisioned, such as those directed to transiently increase blood-brain barrier permeability, as described in U.S. Patent No. 7,012,061, “Method for increasing blood-brain barrier permeability” (which is incorporated herein by reference).

[0223] Viral delivery of compositions and / or nucleic acids described herein for the expression of one or more antibodies described herein in subjects or cells requiring such expression is also contemplated herein. One aspect includes a method of treating a subject requiring such expression, comprising administering to a subject requiring such expression an effective amount of a vectorized antibody of the disclosure herein, or a composition comprising the vectorized antibody, optionally in combination with another TDP-43 proteinosis treatment. In one embodiment, the vectorized antibody is a viral vector comprising a nucleic acid encoding the antibody described herein. In one embodiment, the method is for intracellular expression of an intrabody in a subject requiring such expression. The intrabody may, for example, inhibit intracellular misfolded TDP-43 aggregation or promote the clearance of misfolded aggregates.

[0224] The vectorized antibody may be present in the composition. Non-viral vectors, such as adeno-associated viruses (AAVs, e.g., AAV9) and lentiviral vectors, may also be used. In certain embodiments, the nucleic acids and / or vectors may be injected intraventricularly or subarachnoidally.

[0225] The present invention is as follows: [1] An antibody that binds to the TDP-43 sequence DAGWGNL (SEQ ID NO: 1) in misfolded TDP-43 compared to natural TDP-43. [2] The antibody according to [1] above, wherein the antibody specifically binds to at least W68 in relation to DAGWGNL (SEQ ID NO: 1). [3] The antibody according to [1] or [2] above, wherein the antibody preferentially binds to misfolded TDP-43 that is not associated with stress granules. [4] The antibody according to any one of [1] to [3] above, wherein the antibody binds to DAGWG (SEQ ID NO: 2), AGWGN (SEQ ID NO: 3), GWGNL (SEQ ID NO: 4), and / or DAGWGNL (SEQ ID NO: 5). [5] The antibody according to any one of [1] to [4] above, wherein the antibody is at least 2-fold, 3-fold, at least 5-fold, at least 10-fold, or at least 20-fold more selective for misfolded TDP-43 compared to natural TDP-43. [6] The antibody is i. An isolated peptide comprising all or part of DAGWGNL (SEQ ID NO: 1) (where the part comprises at least five adjacent amino acids and contains GWG, and optionally the part is DAGWG (SEQ ID NO: 2), AGWGN (SEQ ID NO: 3), or GWGNL (SEQ ID NO: 4); or ii. An immunogen comprising a peptide (the peptide comprises all or part of DAGWGNL, where the part comprises at least five adjacent amino acids of SEQ ID NO: 1, and optionally, i) the immunogen comprises a plurality of peptides, each peptide comprising all or part of DAGWGNL (SEQ ID NO: 1), where the plurality of peptides are synthesized as a multiantigenic peptide (MAP), or ii) the peptide is coupled to a carrier protein or an immunogenicity enhancing component, where optionally the carrier protein is bovine serum albumin (BSA), or the immunogenicity enhancing component is keyhole limpet hemocyanin (KLH), and optionally the immunogen is used to produce an antibody that specifically binds to at least W68 in relation to DAGWGNL (SEQ ID NO: 1), preferably, where the antibody preferentially binds to misfolded TDP-43), An antibody according to any of the above [1] to [5], which is produced and / or screened using [1] to [5]. [7] The antibody according to any one of [1] to [6] above, wherein the antibody is a monoclonal antibody. [8] The antibody according to any one of [1] to [7] above, wherein the antibody is a humanized antibody. [9] The antibody according to any of [1] to [8] above, wherein the antibody is a single-chain antibody.

[10] The antibody according to any one of [1] to [9] above, wherein the antibody is a binding fragment selected from Fab, Fab', F(ab')2, scFv, dsFv, ds-scFv, dimer, nanobody, minibody, diabody, and their polymers.

[11] The antibody described in any of [1] to

[10] above, wherein the antibody is affinity-purified.

[12] An antibody according to any one of [1] to

[11] above, comprising a light chain variable region and a heavy chain variable region, wherein the heavy chain variable region comprises complementarity-determining regions CDR-H1, CDR-H2 and CDR-H3, and the light chain variable region comprises complementarity-determining regions CDR-L1, CDR-L2 and CDR-L3, and the amino acid sequence of CDR-H3 comprises sequence:AGGPTGNSHFTL (SEQ ID NO: 12), ARNPVGSVNL (SEQ ID NO: 18), ARRYTGDTYLGNFNL (SEQ ID NO: 24), GRGDI (SEQ ID NO: 36), ARDIFRTNTNL (SEQ ID NO: 48), VRSSGSDWWFHI (SEQ ID NO: 122), or VRQNYEGAY (SEQ ID NO: 132).

[13] comprising a light chain variable region and a heavy chain variable region, wherein the heavy chain variable region comprises complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3, and the light chain variable region comprises complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3, and the amino acid sequences of CDR-H3 and CDR-L3 comprise the following sequences: CDR-H3 comprises AGGPTGNSHFTL (SEQ ID NO: 12), and CDR-L3 comprises SGYKRVTTDGIA (SEQ ID NO: 15); CDR-H3 comprises ARNPVGSVNL (SEQ ID NO: 18), and CDR-L3 comprises AGWRGARTDGVD (SEQ ID NO: 21); CDR-H3 contains ARRYTGDTYLGNFNL (SEQ ID NO: 24), and CDR-L3 contains AGGWRSLNA (SEQ ID NO: 27); CDR-H3 comprises GRGDI (SEQ ID NO: 36), and CDR-L3 comprises LGNYDCSSVDCGA (SEQ ID NO: 39); CDR-H3 contains AGGPTGNSHFTL (SEQ ID NO: 42), and CDR-L3 contains AGYKSPTTDGIA (SEQ ID NO: 45); CDR-H3 contains ARDIFRTNTNL (sequence number 48), and CDR-L3 contains LGGYDCSSRVCGA (sequence number 51); CDR-H3 comprises VRSSGSDWWFHI (SEQ ID NO: 122), and CDR-L3 comprises QGYFSGFITT (SEQ ID NO: 125); or CDR-H3 includes VRQNYEGAY (sequence number 132), and CDR-L3 includes FQSSHVPWT (sequence number 135). The antibody described in any of the above [1] to

[12] .

[14] comprising a light chain variable region and a heavy chain variable region, wherein the heavy chain variable region comprises complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3, and the light chain variable region comprises complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3, and the amino acid sequence of the CDR comprises the following sequence: CDR-H1: GFSLSRYY Sequence ID 10; CDR-H2: IIPGGTT Sequence ID 11; CDR-H3: AGGPTGNSHFTL Sequence ID 12; CDR-L1: ESVYNNNH Sequence ID 13; CDR-L2: EAS Sequence ID 14; and CDR-L3: SGYKRVTTDGIA Sequence ID 15, CDR-H1: GFSFSSNYV Sequence ID 16; CDR-H2: IWFAGIVDTT Sequence ID 17; CDR-H3: ARNPVGSVNL Sequence ID 18; CDR-L1: ESVYSNNR Sequence ID 19; CDR-L2: YAS Sequence ID 20; and CDR-L3: AGWRGARTDGVD Sequence ID 21, CDR-H1: GFSFSSSYV Sequence ID 22; CDR-H2: SDTGINT Sequence ID 23; CDR-H3: ARRYTGDTYLGNFNL Sequence ID 24; CDR-L1: QSVYKNNY Sequence ID 25; CDR-L2: KAS Sequence ID 26; and CDR-L3: AGGWRSLNA Sequence ID 27, CDR-H1: EFSFSSRYW Sequence ID 28; CDR-H2: IYTGSIDAT Sequence ID 29; CDR-H3: VRGSDAWGLYFNL Sequence ID 30; CDR-L1: QSIHKNNY Sequence ID 31; CDR-L2: FAS Sequence ID 32; and CDR-L3: AGVYSGRIFA Sequence ID 33, CDR-H1: GFSLSSYT Sequence ID 34; CDR-H2: IYGGIGST Sequence ID 35; CDR-H3: GRGDI Sequence ID 36; CDR-L1: QSVYKNR Sequence ID 37; CDR-L2: GAS Sequence ID 38; and CDR-L3: LGNYDCSSVDCGA Sequence ID 39, CDR-H1: GFSFSAYY Sequence ID 40; CDR-H2: TIPIGRT Sequence ID 41; CDR-H3: AGGPTGNSHFTL Sequence ID 42; CDR-L1: ESVYNNNQ Sequence ID 43; CDR-L2: QAS Sequence ID 44; and CDR-L3: AGYKSPTTDGIA Sequence ID 45, CDR-H1: GFSLSSYA Sequence ID 46; CDR-H2: IYNYET Sequence ID 47; CDR-H3: ARDIFRTNTNL Sequence ID 48; CDR-L1: QSVYKNNG Sequence ID 49; CDR-L2: FTS Sequence ID 50; and CDR-L3: LGGYDCSSRVCGA Sequence ID 51, CDR-H1: GFSLSSYN Sequence ID 120; CDR-H2: IGTGGIT Sequence ID 121; CDR-H3: VRSSGSDWWFHI Sequence ID 122; CDR-L1: QSVYNNNN Sequence ID 123; CDR-L2: RAS Sequence ID 124; and CDR-L3: QGYFSGFITT Sequence ID 125, CDR-H1: GFTFSSYY Sequence ID 130; CDR-H2: INSNGGST Sequence ID 131; CDR-H3: VRQNYEGAY Sequence ID 132; CDR-L1: QSIVHSNGNTY Sequence ID 133; CDR-L2: KVS Sequence ID 134; and CDR-L3: FQSSHVPWT Sequence ID 135, or CDR-H1: GFTFSSYY Sequence ID 140; CDR-H2: INTNGGST Sequence ID 141; CDR-H3: VRQNYEGAY Sequence ID 142; CDR-L1: QSIVHSNGNTY Sequence ID 143; CDR-L2: KVS Sequence ID 144; and CDR-L3: FQSSHVPWT Sequence ID 145, The antibody described in any of the above [1] to

[13] .

[15] The antibody comprises a heavy chain variable region comprising i) the amino acid sequence shown in SEQ ID NO: 98, ii) an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% sequence identity with respect to SEQ ID NO: 98, where the CDR sequence is the sequence shown in SEQ ID NOs: 10-12, or iii) the conserved substitution amino acid sequence of i); and / or the antibody comprises i) the amino acid sequence shown in SEQ ID NO: 99, ii) at least 50%, at least 60%, at least 70%, at least 80%, or An antibody according to any of [1] to

[14] above, comprising a light chain variable region having an amino acid sequence having at least 90% sequence identity, wherein the CDR sequence is one of those shown in SEQ ID NOs: 13-15 or the conserved substitution amino acid sequence of iii)i), wherein the heavy chain variable region amino acid sequence is optionally encoded by the nucleotide sequence or codon degeneracy shown in SEQ ID NO: 76 or an optimized version thereof, and / or the light chain variable region amino acid sequence is encoded by the nucleotide sequence or codon degeneracy shown in SEQ ID NO: 77 or an optimized version thereof.

[16] The antibody comprises a heavy chain variable region comprising i) the amino acid sequence shown in SEQ ID NO: 100, ii) an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% sequence identity with respect to SEQ ID NO: 100, where the CDR sequence is shown in SEQ ID NOs: 16-18, or iii) the conserved substitution amino acid sequence of i); and / or the antibody comprises i) the amino acid sequence shown in SEQ ID NO: 101, ii) at least 50%, at least 60%, at least 70%, at least 80%, or The antibody according to any of [1] to

[14] above, comprising a light chain variable region having at least 90% sequence identity, wherein the CDR sequence is one of those shown in SEQ ID NOs: 19-21 or the conserved substitution amino acid sequence of iii)i), and optionally the heavy chain variable region amino acid sequence is encoded by the nucleotide sequence or codon degeneracy shown in SEQ ID NO: 78 or an optimized version thereof, and / or the light chain variable region amino acid sequence is encoded by the nucleotide sequence or codon degeneracy shown in SEQ ID NO: 79 or an optimized version thereof.

[17] The antibody comprises a heavy chain variable region comprising i) the amino acid sequence shown in SEQ ID NO: 102, ii) an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% sequence identity with respect to SEQ ID NO: 102, where the CDR sequence is shown in SEQ ID NOs: 22-24, or iii) the conserved substitution amino acid sequence of i); and / or the antibody comprises i) the amino acid sequence shown in SEQ ID NO: 103, ii) at least 50%, at least 60%, at least 70%, or at least 80% sequence identity with respect to SEQ ID NO: 103. The antibody according to any of [1] to

[14] above, comprising a light chain variable region comprising an amino acid sequence having at least 90% sequence identity, wherein the CDR sequence is one of those shown in SEQ ID NOs. 25 to 27, or the conserved substitution amino acid sequence of iii)i), and optionally the heavy chain variable region amino acid sequence is encoded by the nucleotide sequence or codon degeneracy shown in SEQ ID NOs. 80 or an optimized version thereof, and / or the light chain variable region amino acid sequence is encoded by the nucleotide sequence or codon degeneracy shown in SEQ ID NOs. 81 or an optimized version thereof.

[18] The antibody comprises a heavy chain variable region comprising i) the amino acid sequence shown in SEQ ID NO: 104, ii) an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% sequence identity with respect to SEQ ID NO: 104, where the CDR sequence is shown in SEQ ID NOs: 28-30, or iii) the conserved substitution amino acid sequence of i); and / or the antibody comprises i) the amino acid sequence shown in SEQ ID NO: 105, ii) at least 50%, at least 60%, at least 70%, or at least 80% sequence identity with respect to SEQ ID NO: 105 The antibody according to any of [1] to

[14] above, comprising a light chain variable region comprising an amino acid sequence having at least 90% sequence identity, wherein the CDR sequence is one of those shown in SEQ ID NOs: 31-33 or the conserved substitution amino acid sequence of iii)i), wherein the heavy chain variable region amino acid sequence is optionally encoded by the nucleotide sequence or codon degeneracy shown in SEQ ID NO: 82 or an optimized version thereof, and / or the light chain variable region amino acid sequence is encoded by the nucleotide sequence or codon degeneracy shown in SEQ ID NO: 83 or an optimized version thereof.

[19] The antibody comprises a heavy chain variable region comprising i) the amino acid sequence shown in SEQ ID NO: 106, ii) an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% sequence identity with respect to SEQ ID NO: 106, where the CDR sequence is shown in SEQ ID NOs: 34-36, or iii) the conserved substitution amino acid sequence of i); and / or the antibody comprises i) the amino acid sequence shown in SEQ ID NO: 107, ii) at least 50%, at least 60%, at least 70%, or at least 80% sequence identity with respect to SEQ ID NO: 107 The antibody according to any of [1] to

[14] above, comprising a light chain variable region comprising an amino acid sequence having at least 90% sequence identity, wherein the CDR sequence is one of those shown in SEQ ID NOs: 37-39 or the conserved substitution amino acid sequence of iii)i), wherein the heavy chain variable region amino acid sequence is optionally encoded by the nucleotide sequence or codon degeneracy shown in SEQ ID NO: 84 or an optimized version thereof, and / or the light chain variable region amino acid sequence is encoded by the nucleotide sequence or codon degeneracy shown in SEQ ID NO: 85 or an optimized version thereof.

[20] The antibody comprises a heavy chain variable region comprising i) the amino acid sequence shown in SEQ ID NO: 108, ii) an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% sequence identity with respect to SEQ ID NO: 108, where the CDR sequence is shown in SEQ ID NOs: 40-42, or iii) the conserved substitution amino acid sequence of i); and / or the antibody comprises i) the amino acid sequence shown in SEQ ID NO: 109, ii) at least 50%, at least 60%, at least 70%, or at least 80% sequence identity with respect to SEQ ID NO: 109 The antibody according to any of [1] to

[14] above, comprising a light chain variable region comprising an amino acid sequence having at least 90% sequence identity, wherein the CDR sequence is one of those shown in SEQ ID NOs. 43 to 45, or the conserved substitution amino acid sequence of iii)i), and optionally the heavy chain variable region amino acid sequence is encoded by the nucleotide sequence or codon degeneracy shown in SEQ ID NO. 86 or an optimized version thereof, and / or the light chain variable region amino acid sequence is encoded by the nucleotide sequence or codon degeneracy shown in SEQ ID NO. 87 or an optimized version thereof.

[21] The antibody comprises a heavy chain variable region comprising i) the amino acid sequence shown in SEQ ID NO: 110, ii) an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% sequence identity with respect to SEQ ID NO: 110, where the CDR sequence is shown in SEQ ID NOs: 46-48, or iii) the conserved substitution amino acid sequence of i); and / or the antibody comprises i) the amino acid sequence shown in SEQ ID NO: 111, ii) at least 50%, at least 60%, at least 70%, or at least 80% sequence identity with respect to SEQ ID NO: 111. The antibody according to any of [1] to

[14] above, comprising a light chain variable region comprising an amino acid sequence having at least 90% sequence identity, wherein the CDR sequence is one of those shown in SEQ ID NOs: 49 to 51, or the conserved substitution amino acid sequence of iii)i), and optionally the heavy chain variable region amino acid sequence is encoded by the nucleotide sequence or codon degeneracy or an optimized version thereof shown in SEQ ID NO: 88, and / or the light chain variable region amino acid sequence is encoded by the nucleotide sequence or codon degeneracy or an optimized version thereof shown in SEQ ID NO: 89.

[22] The antibody comprises a heavy chain variable region comprising i) the amino acid sequence shown in SEQ ID NO: 128, ii) an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% sequence identity with SEQ ID NO: 128, where the CDR sequence is shown in SEQ ID NOs: 120-122, or iii) the conserved substitution amino acid sequence of i); and / or the antibody comprises i) the amino acid sequence shown in SEQ ID NO: 129, ii) at least 50%, at least 60%, at least 70%, or at least 80% sequence identity with SEQ ID NO: 129 The antibody according to any of [1] to

[14] above, comprising a light chain variable region having at least 90% sequence identity, wherein the CDR sequence is one of those shown in SEQ ID NOs: 123 to 125, or the conserved substitution amino acid sequence of iii)i), and optionally the heavy chain variable region amino acid sequence is encoded by the nucleotide sequence or codon degeneracy or an optimized version thereof shown in SEQ ID NO: 126, and / or the light chain variable region amino acid sequence is encoded by the nucleotide sequence or codon degeneracy or an optimized version thereof shown in SEQ ID NO: 127.

[23] The antibody comprises a heavy chain variable region comprising i) the amino acid sequence shown in SEQ ID NO: 138, ii) an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% sequence identity with SEQ ID NO: 138, where the CDR sequence is shown in SEQ ID NOs: 130-132, or iii) the conserved substitution amino acid sequence of i); and / or the antibody comprises i) the amino acid sequence shown in SEQ ID NO: 139, ii) at least 50%, at least 60%, at least 70%, or at least 80% sequence identity with SEQ ID NO: 139 The antibody according to any of [1] to

[14] above, wherein the heavy chain variable region comprises an amino acid sequence having at least 90% sequence identity, where the CDR sequence is one of those shown in SEQ ID NOs: 133 to 135, or the conserved substitution amino acid sequence of iii)i), and optionally the heavy chain variable region amino acid sequence is encoded by the nucleotide sequence or codon degeneracy shown in SEQ ID NO: 136 or an optimized version thereof, and / or the light chain variable region amino acid sequence is encoded by the nucleotide sequence or codon degeneracy shown in SEQ ID NO: 137 or an optimized version thereof.

[24] The antibody comprises a heavy chain variable region comprising i) the amino acid sequence shown in SEQ ID NO: 148, ii) an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% sequence identity with SEQ ID NO: 148, where the CDR sequence is shown in SEQ ID NOs: 140-142, or iii) the conserved substitution amino acid sequence of i); and / or the antibody comprises i) the amino acid sequence shown in SEQ ID NO: 149, ii) at least 50%, at least 60%, at least 70%, or at least 80% sequence identity with SEQ ID NO: 149 The antibody according to any of [1] to

[14] above, wherein the heavy chain variable region comprises an amino acid sequence having at least 90% sequence identity, where the CDR sequence is one of those shown in SEQ ID NOs: 143-145, or the conserved substitution amino acid sequence of iii)i), and optionally the heavy chain variable region amino acid sequence is encoded by the nucleotide sequence or codon degeneracy or an optimized version thereof shown in SEQ ID NO: 146, and / or the light chain variable region amino acid sequence is encoded by the nucleotide sequence or codon degeneracy or an optimized version thereof shown in SEQ ID NO: 147.

[25] An immune complex comprising the antibody described in any of [1] to

[24] above and a detectable label.

[26] An isolated peptide comprising all or part of DAGWGNL (SEQ ID NO: 1), wherein the part comprises at least five adjacent amino acids and contains GWG.

[27] The isolated peptide according to

[26] , wherein the portion thereof is DAGWG (SEQ ID NO: 2), AGWGN (SEQ ID NO: 3), or GWGNL (SEQ ID NO: 4).

[28] An immunogen comprising a peptide, wherein the peptide comprises all or part of DAGWGNL, wherein the part is at least five adjacent amino acids of SEQ ID NO: 1.

[29] The immunogen described in

[28] above, comprising a plurality of peptides, each peptide comprising all or part of DAGWGNL (SEQ ID NO: 1), wherein the plurality of peptides are synthesized as a multiantigenic peptide (MAP).

[30] The immunogen according to

[29] , wherein the peptide is coupled to a carrier protein or an immunogenicity enhancing component, wherein the carrier protein is bovine serum albumin (BSA) or the immunogenicity enhancing component is keyhole limpet hemocyanin (KLH).

[31] The immunogen according to any one of

[28] to

[30] above, wherein the immunogen is used to produce an antibody that specifically binds to misfolded TDP-43, specifically binds to DAGWGNL (SEQ ID NO: 1), and / or specifically binds to at least W68 in relation to DAGWGNL (SEQ ID NO: 1).

[32] The immunogen according to

[31] above, wherein the antibody preferentially binds to misfolded TDP-43.

[33] In the case thereof, an isolated nucleic acid comprising an isolated peptide as described in

[26] or

[27] above, an immunogen as described in any of

[28] to

[32] above, or an antibody as described in any of [1] to

[24] above, which may be contained in the vector.

[34] Cells that recombinantly express the peptide described in

[26] or

[27] above, the immunogen described in any of

[28] to

[32] above, or the antibody described in any of [1] to

[24] above, wherein the cell is a hybridoma when it expresses the antibody.

[35] A composition comprising, optionally, a diluent and a pharmaceutically acceptable carrier, the isolated peptide described in

[26] or

[27] above, the immunogen described in any of

[28] to

[32] above, the antibody described in any of [1] to

[24] above, the immune complex described in

[25] above, the isolated nucleic acid described in

[33] above, or the cell described in

[34] above.

[36] A composition comprising two or more antibodies described in any of [1] to

[24] above or two or more immune complexes described in

[25] above.

[37] The composition according to

[35] , further comprising an adjuvant, comprising the immunogen.

[38] The composition according to

[37] , wherein the adjuvant is aluminum phosphate, aluminum hydroxide, alum, monophosphoryl lipid A and / or QS21.

[39] A method for treating a subject, comprising administering an effective amount of an antibody according to any of [1] to

[24] above, an immune complex according to

[25] above, a nucleic acid according to

[33] above, or a composition according to any of

[36] to

[38] above to a subject in need thereof.

[40] The method according to

[39] above, wherein the subject is suspected of having TDP-43 proteinosis, is at risk of developing it, or has been diagnosed with it.

[41] The method according to

[40] , wherein the TDP-43 protein disorder is selected from amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD-TDP), primary lateral sclerosis, progressive muscular atrophy, and limbic-dominant senile TDP-43 encephalopathy (LATE).

[42] The method according to any one of

[39] to

[41] above, wherein the antibody, immune complex, or composition is administered in combination with the treatment of another TDP-43 proteinosis.

[43] A method for inhibiting TDP-43 cell propagation, comprising administering an antibody according to any of [1] to

[24] above, an immune complex according to

[25] above, a nucleic acid according to

[33] above, or a composition according to any of

[36] to

[38] above to a subject requiring such action.

[44] The method according to any one of the above

[39] to

[43] , wherein the antibody, immune complex, nucleic acid, or composition is administered by parenteral administration, intravenous administration, subcutaneous administration, intramuscular administration, intracranial administration, intraventricular administration, subarachnoid administration, intraorbital administration, ocular administration, intraspinal administration, intracisional administration, intraperitoneal administration, intranasal administration, aerosol administration, or oral administration.

[45] The method according to any one of the above

[39] to

[44] , wherein the antibody, immune complex, nucleic acid, or composition is administered co-administered with a bioactive molecule known to promote transport across the blood-brain barrier.

[46] i. The isolated peptide described in

[26] or

[27] above; ii. Any of the immunogens listed in

[28] to

[32] above; iii. Any antibody described in any of the above [1] to

[24] ; iv. The immune complexes described in

[25] above; v. Isolated nucleic acids as described in

[33] above; vi. The cells described above

[34] ; and / or vii. The compositions described in any of the above

[35] to

[38] ; and viii. Compartments such as vials for containing the isolated peptides, immunogens, antibodies, immune complexes, isolated nucleic acids, cells, and / or compositions, and ix. A kit comprising, depending on the circumstances, instructions for use in an ELISA or the method described herein.

[47] A method for producing an antibody according to any of [1] to

[24] above, comprising administering to a non-human subject an immunogen according to any of

[28] to

[32] above or a composition comprising an immunogen according to any of

[28] to

[32] above and optionally a pharmaceutically acceptable carrier, and isolating an antibody and / or cells expressing an antibody that is selective to the administered isolated peptide, immunogen, or composition.

[48] ​​A method for preparing an antibody, comprising immunizing a non-human subject with an immunogen described in any of

[28] to

[32] above, or a composition comprising an immunogen described in any of

[28] to

[32] above.

[49] The method according to

[48] , further comprising isolating an antibody that specifically binds to W68 in relation to DAGWGNL (SEQ ID NO: 1).

[50] The method according to

[49] above, further comprising forming an antibody-producing hybridoma.

[51] An antibody produced by any of the methods described in

[48] to

[50] above.

[52] A method for determining whether a sample suspected to contain cytoplasm / aggregates and / or misfolded TDP-43 contains cytoplasm / aggregates or misfolded TDP-43, a. Contacting the sample with the antibody described in any of [1] to

[24] above under conditions that allow for the formation of an antibody:misfolded TDP-43 complex; and b. Detecting the presence of any complex It includes, Herein, the presence of a detectable complex indicates that the sample may contain cytoplasmic / aggregated and / or misfolded TDP-43 polypeptides, method.

[53] The method according to

[52] above, wherein the sample is a biological sample obtained from the subject.

[54] The method according to

[53] above, wherein the sample comprises a fraction of any of the aforementioned, which optionally comprises blood, serum, plasma and / or solid tissue, and optionally brain, CSF and / or extracellular vesicles derived from blood.

[55] The method according to

[53] or

[54] above, wherein the sample is, optionally, a human sample from a subject having or suspected to have amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), primary lateral sclerosis, or limbic-dominant senile TDP-43 encephalopathy (LATE).

[56] An antibody comprising a light chain variable region and a heavy chain variable region, wherein the heavy chain variable region comprises complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3, and the light chain variable region comprises complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3, and the amino acid sequence of the CDR comprises the following sequence: CDR-H1: GFSLSRYY Sequence ID 10; CDR-H2: IIPGGTT Sequence ID 11; CDR-H3: AGGPTGNSHFTL Sequence ID 12; CDR-L1: ESVYNNNH Sequence ID 13; CDR-L2: EAS Sequence ID 14; and CDR-L3: SGYKRVTTDGIA Sequence ID 15, CDR-H1: GFSFSSNYV Sequence ID 16; CDR-H2: IWFAGIVDTT Sequence ID 17; CDR-H3: ARNPVGSVNL Sequence ID 18; CDR-L1: ESVYSNNR Sequence ID 19; CDR-L2: YAS Sequence ID 20; and CDR-L3: AGWRGARTDGVD Sequence ID 21, CDR-H1: GFSFSSSYV Sequence ID 22; CDR-H2: SDTGINT Sequence ID 23; CDR-H3: ARRYTGDTYLGNFNL Sequence ID 24; CDR-L1: QSVYKNNY Sequence ID 25; CDR-L2: KAS Sequence ID 26; and CDR-L3: AGGWRSLNA Sequence ID 27, CDR-H1: GFSLSSYT Sequence ID 34; CDR-H2: IYGGIGST Sequence ID 35; CDR-H3: GRGDI Sequence ID 36; CDR-L1: QSVYKNR Sequence ID 37; CDR-L2: GAS Sequence ID 38; and CDR-L3: LGNYDCSSVDCGA Sequence ID 39, CDR-H1: GFSFSAYY Sequence ID 40; CDR-H2: TIPIGRT Sequence ID 41; CDR-H3: AGGPTGNSHFTL Sequence ID 42; CDR-L1: ESVYNNNQ Sequence ID 43; CDR-L2: QAS Sequence ID 44; and CDR-L3: AGYKSPTTDGIA Sequence ID 45, CDR-H1: GFSLSSYA Sequence ID 46; CDR-H2: IYNYET Sequence ID 47; CDR-H3: ARDIFRTNTNL Sequence ID 48; CDR-L1: QSVYKNNG Sequence ID 49; CDR-L2: FTS Sequence ID 50; and CDR-L3: LGGYDCSSRVCGA Sequence ID 51, CDR-H1: GFSLSSYN Sequence ID 120; CDR-H2: IGTGGIT Sequence ID 121; CDR-H3: VRSSGSDWWFHI Sequence ID 122; CDR-L1: QSVYNNNN Sequence ID 123; CDR-L2: RAS Sequence ID 124; and CDR-L3: QGYFSGFITT Sequence ID 125, CDR-H1: GFTFSSYY Sequence ID 130; CDR-H2: INSNGGST Sequence ID 131; CDR-H3: VRQNYEGAY Sequence ID 132; CDR-L1: QSIVHSNGNTY Sequence ID 133; CDR-L2: KVS Sequence ID 134; and CDR-L3: FQSSHVPWT Sequence ID 135, or CDR-H1: GFTFSSYY Sequence ID 140; CDR-H2: INTNGGST Sequence ID 141; CDR-H3: VRQNYEGAY Sequence ID 142; CDR-L1: QSIVHSNGNTY Sequence ID 143; CDR-L2: KVS Sequence ID 144; and CDR-L3: FQSSHVPWT Sequence ID 145, antibody. The above disclosures describe the application in general terms. A more complete understanding can be obtained by referring to the following specific examples. These examples are described for illustrative purposes only and are not intended to limit the scope of the application. Modifications of form and substitutions of equivalents are contemplated where circumstances may suggest or provide convenience. Certain terms are used herein, but such terms are intended to be descriptive and not limiting.

[0226] The following examples, not limited to this disclosure, are illustrative: [Examples]

[0227] Example 1 TDP-43 Epitope TDP-43 NTD comprises a ubiquitin-like domain. TDP-43 NTD Trp68 is uniquely present in TDP-43 when aligned with other protein-containing ubiquitin-like domains (e.g., human ubiquitin, Axyn 1, human, and Dvl-2) [9]. It was determined that Trp68 is not solvent-exposed in PDB structures such as PDBID 2N4P and 6B1G. It was hypothesized that Trp68 (i.e., its position in the human sequence represented at NM_007375.3) would be solvent-exposed (antibody-accessible) in misfolded TDP-43. A seven-amino acid peptide centered on tryptophan 68 was selected, and an immunogen was designed as described in Example 2.

[0228] Example 2 Construction of immunogens The peptide DAGWGNLc (SEQ ID NO: 9) was synthesized according to a standard protocol (GenScript USA Inc., Piscataway, New Jersey). The N-terminus of the peptide was acetylated.

[0229] Next, the synthesized peptide was conjugated via a C-terminal cys residue to keyhole limpet hemocyanin (KLH) (for immunization) or BSA (for screening) to produce the immunogen used in Example 3.

[0230] Example 3 Production and selection of polyclonal antibodies Polyclonal antibodies were produced using an immunogen containing the peptide DAGWGNL (SEQ ID NO: 1), which is linked to KLH via a C-terminal cysteine.

[0231] immunization Two New Zealand rabbits (named GS240 and GS243) were immunized with the KLH-conjugate DAGWGNLc (SEQ ID NO: 9) peptide described in Example 2 (primary immunization and two booster injections per animal). After the third immunization, blood was collected from each rabbit and antiserum was isolated. A preservative (0.02% sodium azide final concentration) was added to each isolated antiserum (unpurified antiserum). Pre-immunized serum was also obtained from each animal and combined with the preservative (0.02% sodium azide).

[0232] Affinity Purification The IgG fraction of each rabbit antiserum was isolated by sulfate precipitation. The precipitate was then subjected to affinity column precipitation using resin prepared with DAGWGNL peptide. After incubation of the antiserum in the column, the antibody was eluted using a stepwise pH gradient in PBS. The final concentration was determined by BCA assay, and titration was performed by ELISA. The affinity-purified antibody from GS240 had a concentration of approximately 0.5 mg / mL, and the affinity-purified antibody from GS243 had a concentration of approximately 0.6 mg / mL.

[0233] ELISA conditions: ELISA plates were coated overnight at 4°C with 4 μg / well of DAGWGNL (SEQ ID NO: 1) peptide in 100 μL / well of PBS (pH 7.4).

[0234] The secondary antibody used was an anti-rabbit IgG Fc monoclonal secondary antibody conjugated to HRP (GenScript, catalog number A01856).

[0235] The ELISA results are shown in Tables 1A and 1B, which illustrate the serial dilution binding of unpurified antiserum (1A) and affinity-purified antibodies (1B) prepared against immunized peptides.

[0236] [Table 2]

[0237] [Table 3]

[0238] Example 4 Detection of misfolded TDP-43 Affinity-purified antibodies were tested for their ability to bind to native and misfolded TDP-43 polypeptides in cell transfection assays using immunocytochemistry.

[0239] method The cell culture and immunohistochemical methods used were based on the methods previously described in Pokrishevsky E, Grad LI, Cashman NR. (2016) TDP-43 or FUS-induced misfolded human wild-type SOD1 can propagate intercellularly in a prion-like fashion. Sci Rep. 2016;6:22155. doi: 10.1038 / srep22155 (which is incorporated herein by reference).

[0240] Briefly, human embryonic kidney cells (HEK293FT; ATCC, Manassas, Virginia) were cultured on borosilicate coverslips pre-coated with 0.01% poly-D-lysine in fully modified Dulbecco's Eagle medium (DMEM) containing 10% FBS, 100 U / mL penicillin, 100 μg / mL streptomycin, and 2 mM L-glutamine (ThermoFisher Scientific, Massachusetts, USA). The cells were transfected with different TDP-43 DNA plasmid constructs containing a chimeric reporter protein using Lipofectamine® LTX (ThermoFisher Scientific, Massachusetts, USA) according to the manufacturer's instructions. The constructs used were an empty vector, HA-tagged human TDP-43 with a triple missensant tandem mutation in the nuclear localization signal (ΔNLS-TDP-43), human TDP43 with the original NLS sequence (WT-TDP43), and ΔNLS-TDP43 (W68S) in which tryptophan 68 was mutated to serine.

[0241] 48 hours after transfection, cells were fixed in 4% paraformaldehyde at 37°C for 1 hour, rapidly washed in cold PBS, permeabilized in 0.01% Triton X in PBS for 10 minutes, and then blocked in 10% normal goat serum (NGS) at room temperature for 1 hour. For staining, the affinity-purified rabbit antibodies described in Example 3 were diluted in 10% NGS in PBS to approximately 0.5 μg / mL for antiserum GS240 and approximately 0.60 μg / mL for antiserum GS243, with 1 μg / mL rat anti-HA tag used as counterstain (Roche Diagnostics, Indiana). Cells were incubated overnight with both antibodies at 4°C, then washed three times in PBS, and incubated with secondary antibodies conjugated with Alexa Fluor®-568 or 647 fluorescent dye (Life Technologies, Carlsbad, California; 1:1000 dilution) at room temperature in the dark for 1 hour. DNA was counterstained for 1 minute with 2 μg / ml bis-benzimide H33342 trihydrochloride (Hoechst 33342). After washing four times in PBS, the cells were placed on glass slides coated with Fluoromount-G (Southern Biotech, Birmingham, Alabama). Confocal images of individual sections were taken using a Leica TCS SP8® microscope (Leica Canada) with LAS-X® software.

[0242] result: The results are shown in Figures 1A-1L and 2A-2L.

[0243] In Figures 1A, 1E, 1I, 2A, 2E, and 2I, HEK293 cells transfected with an HA-tagged TDP-43 construct containing a triple missensant tandem mutation in nuclear localization signaling show cytoplasmic TDP-43 aggregates detected by HA antibodies. HA-positive aggregates are also evident in ΔNLS-TDP-43 W68S transfected cells (Figures 1B, 1F, 1J, 2B, 2F, and 2J). Affinity-purified polyclonal antibodies GS240 (Figure 1) and GS243 (Figure 2) recognize aggregates in ΔNLS-TDP-43 transfected cells containing tryptophan 68 residues (Trp68) (Figures 1A, 1E, 1I, 2A, 2E, and 2I), but do not show the same reactivity when Trp68 is mutated to serine (Figures 1B, 1F, 1J, 2B, 2F, and 2J). Therefore, the anti-DAGWGNL (SEQ ID NO: 1) polyclonal rabbit antibody exhibits selectivity for misfolded TDP-43NTD containing the Trp68 residue.

[0244] When HA-tagged wild-type TDP-43 is overexpressed without altering nuclear localization signaling, it generally localizes to the nucleus, and nuclear TDP-43 does not bind to GS240 or GS243 affinity purified serum (Figure 1C, 1G, 1K, 2C, 2G, and 2K). Interestingly, GS240 and GS243 still bind to wild-type TDP-43 when present in the cytoplasm of these cells (Figure 1K and 2K), thereby suggesting that the N-terminal ubiquitin-like domain (NTD) may be misfolded in WT-TDP43 when mislocalized to the cytoplasm.

[0245] GS240 and GS243 polyclonal antibodies exhibit minimal reactivity in the absence of TDP-43 aggregates (as observed in background staining and empty vector transfection in Figures 1L and 2L). The reactivity of both antibodies is selective for misfolded TDP-43 in cytoplasmic aggregates and requires the presence of Trp68.

[0246] Example 5 Production of mouse monoclonal antibodies Peptides containing DAGWGNL (SEQ ID NO: 1), such as cDAGWGNL (SEQ ID NO: 8) or DAGWGNLc (SEQ ID NO: 9), linked to KLH, can be used to produce monoclonal antibodies.

[0247] immunization Simply put, mice are immunized by a series of subcutaneous injections of aqueous solutions over a long period, then euthanized, and lymphocytes are harvested to create hybridoma cell lines.

[0248] The emergence of a hybrid / fusion creature. Lymphocytes are isolated and fused with mouse SP2 / 0 myeloma cells in the presence of polyethylene glycol (PEG1500). The fused cells are cultured using HAT selection. This method combines hybridoma selection and cloning into a single step using a semi-solid methylcellulose-based HAT selection medium. Single-cell hybridomas grow and form monoclonal colonies on the semi-solid medium. Approximately 10 days after the fusion event, the resulting hybridoma clones are transferred to 96-well tissue culture plates and grown in HT-containing medium until logarithmic metaphase is reached (approximately 5 days).

[0249] Hybridoma analysis (screening) Hybridoma-derived tissue culture supernatants can be tested by indirect ELISA against screening antigens, allowing for the detection of both IgG and IgM antibodies using goat anti-IgG / IgM(H&L)-HRP secondary assays, and further development with TMB substrates.

[0250] Positive cultures are retested against a screening antigen to confirm secretion and against an unrelated antigen (human transferrin). The clones of interest can be isotyped by antibody capture ELISA to determine whether they are IgG or IgM isotypes, and can also be tested by indirect ELISA against other peptide-BSA conjugates lacking try68, for example.

[0251] The positive IgG-secreting clones will be used for large-scale production.

[0252] Isotyping Hybridoma antibodies are isotyped using an antibody capture experiment. Capture plates are coated overnight at 4°C with 1:10,000 goat anti-mouse IgG / IgM (H&L) antibody in 100 μL / well carbonate coating buffer pH 9.6. Primary antibody (hybridoma supernatant) is added at 100 μg / mL. Secondary antibody is added at 1:5,000. Goat anti-mouse IgGγ-HRP or 1:10,000 goat anti-mouse IgMμ-HRP is added at 100 μL / well in PBS-Tween, shaking for 1 hour at 37°C. The entire washing process is performed in PBS-Tween for 30 minutes. Substrate TMB is added at 50 μL / well, developed in the dark, and stopped with an equal volume of 1M HCl.

[0253] Antibody-containing hybridoma tissue culture supernatants are classified according to immunoglobulin type and screened for negative control peptides and BSA. IgG-producing clones that do not bind to the negative control peptide or BSA are tested for binding to peptide DAGWGNL (SEQ ID NO: 1) by ELISA.

[0254] Example 6 Production of rabbit monoclonal antibodies The DAGWGNL (SEQ ID NO: 1) peptide (peptide-KLH), conjugated to KLH via a C-terminal cysteine, was used in rabbit immunization to produce B cells that secrete monoclonal antibodies specific to the TDP-43 peptide. Based on indirect ELISA analysis of isolated B cells, B cells were selected for RNA isolation and recombinant plasmid DNA production. Functional antigen-binding recombinant mAbs were transfected and expressed to produce purified mAbs.

[0255] immunization : Two sets of rabbits were immunized simultaneously: one set was immunized using a rapid rabbit immunization method (28 days), and the other set was immunized using a standard rabbit immunization method (78 days).

[0256] Rapid rabbit immunization: Two New Zealand White (NZB) rabbits were immunized by subcutaneous injection (SC) of 200 μg of peptide-KLH. On days 7 and 14 post-immunization, the rabbits received first and second booster injections (SC) of 100 μg of peptide-KLH. On day 21, test bleeding was performed, and serum was titrated by indirect ELISA probing for IgG. On day 28, the rabbits were bled for heparinized whole blood collection and downstream development of rabbit monoclonal antibodies. An appropriate titer is >0.300 OD at a 1:64,000 dilution.

[0257] Standard rabbit immunization: Two New Zealand White (NZB) rabbits were immunized by subcutaneous injection (SC) of 250 μg of peptide-KLH. First and second booster immunizations (SC) of 250 μg of peptide-KLH were administered to the rabbits on days 28 and 47 post-immunization. On day 58, test bleeding was performed and serum was titrated by indirect ELISA probing for IgG. On day 66, a third booster immunization (SC) of 250 μg of peptide-KLH was administered. On day 78, the rabbits were bled for heparinized whole blood collection and downstream development of rabbit monoclonal antibodies. An appropriate titer is >0.300 OD at a 1:64,000 dilution. The number of days may vary by + / - 1-2 days.

[0258] B cell isolation, enrichment, and screening - 14 days In vitro culture of B cells - Day 7: Rabbit-derived B cells were isolated from heparinized whole blood and cultured. Biopanning was performed using TDP43 peptide. Antigen-specific B cells were then seeded for further culture and screening.

[0259] Antibody Analysis (Screening) - Day 7: B cell culture supernatants from plates were tested against BSA-conjugate TDP43 linear peptide by indirect ELISA. This was explored with a secondary antibody against rabbit IgG antibody. The most responsive antigen-specific B cells were transferred to new plates. Subsequently, the clonal supernatant was titrated against BSA-conjugate TDP43 linear peptide by indirect ELISA to capture the top clone selection.

[0260] Example 7 Cloning and sequencing of monoclonal antibodies Mouse mAb cloning We identified and sequenced the variable regions of heavy chain immunoglobulin and light chain immunoglobulin genes in several mouse hybridoma clones.

[0261] method Total RNA was isolated from hybridoma cells and reverse transcribed to cDNA using either isotype-specific antisense primers or universal primers. Heavy and light chain antibody fragments were amplified by rapid amplification of cDNA ends (RACE). The amplified antibody fragments were cloned separately into standard cloning vectors. Colony PCR was performed to screen for clones with accurate-sized inserts. Five clones were selected from each hybridoma cell line and sequenced for both the heavy and light chains. Sequence alignment was performed using the five clones to confidently determine the heavy and light chain sequences of each monoclonal antibody.

[0262] The sequences are shown in Tables 2, 3, and 4 below, with the CDR1, CDR2, and CDR3 regions of the heavy and light chains underlined and bolded in Tables 3 and 4.

[0263] Cloning of rabbit mAbs Cloning - Day 14: For the top clone, antibody RNA was isolated and recombinant plasmid DNA was prepared. For each top clone, the heavy chain and light chain variable regions were cloned into separate mammalian expression vectors containing the rabbit heavy chain and kappa constant region.

[0264] Expression - Day 14: Heavy and light chain vectors were co-transfected into mammalian cells for small-scale transfection. The supernatant was screened by indirect ELISA for BSA-conjugated TDP43 peptide to confirm the creation of functional recombinant mAbs.

[0265] Purification: The top recombinant mAb (a DNA construct containing one heavy chain and one light chain) was sequenced as shown in Tables 2, 3, and 4 below, with the CDR1, CDR2, and CDR3 regions of the heavy and light chains underlined and bolded in Tables 3 and 4. Further mAbs were sequenced and contained sequence numbers 52-75, 90-97, and 112-119.

[0266] [Table 4]

[0267] [Table 5] TIFF0007829171000006.tif160129TIFF0007829171000007.tif157129TIFF0007829171000008.tif62128

[0268] [Table 6] TIFF0007829171000010.tif110129

[0269] Example 8 Direct binding assay The binding of antiserum, hybridoma supernatant, or purified antibody to peptides (conjugated to BSA) can be investigated by surface plasmon resonance using a Biacore® 3000 instrument (GE Healthcare).

[0270] Binding analysis is performed using antigens immobilized in high-density (at least 1000 response units (RU)) flow cells. Dilutions of selected clones are sequentially injected into surfaces for binding evaluation.

[0271] For affinity kinetics and specificity analysis, peptides comprising DAQWGNL (SEQ ID NO: 1) or GWG and conjugated to BSA are immobilized at low density (50–100 RU) in adjacent flow cells. Subsequently, 2-fold serial dilutions of selected clones (4.7 nM–75 nM) are injected sequentially into the surface at 60 μl / min for 3 minutes, followed by the dissociation phase. After double reference subtraction, the sensorgram is fitted to a Langmuir 1:1 binding model. Up to three separate analyses are performed over three consecutive days using the same sensor chip and conditions.

[0272] Binding analysis can also be performed using the Molecular Affinity Screening System (MASS-2) (Sierra Sensors GmbH, Hamburg, Germany). MASS-2 is a surface plasmon resonance (SPR) imaging biosensor that uses high-intensity laser light and high-speed optical scanning to monitor binding interactions in real time. The peptide-BSA conjugate is covalently immobilized to separate flow cells of a High Amine Capacity (HAC) sensor tip using standard amine coupling chemistry, blocking unreacted sites. Adjacent flow cells are similarly immobilized with BSA as a reference surface.

[0273] Monoclonal antibody binding kinetics Surface plasmon resonance (SPR) analysis was used to measure the binding kinetics of monoclonal antibodies to peptide epitopes. Peptides conjugated to bovine serum albumin (BSA) were immobilized at a very low density (approximately 50 RU) in the flow cell of the sensor chip. Purified mouse or rabbit monoclonal antibodies, diluted fourfold from 31.25 nM to 0.24 nM, were sequentially injected onto the surface for approximately 5 minutes, followed by dissociation in buffer and surface regeneration. Binding parameters were calculated using kinetic curve fitting and the Langmuir 1:1 interaction model. Both mouse and rabbit monoclonal antibodies showed sub-nanomolecular high affinity to the peptide epitopes, as shown in Figures 3A-3E and Table 5 below. Rabbit monoclonal antibodies showed high affinity to the mouse monoclonal antibodies (10 -10 Compared to the nM range, it has a much greater affinity (10 -11 The nM range was shown.

[0274] [Table 7]

[0275] Example 9 The epitope is not recognized in the naturally folded TDP N-terminal domain, but is recognized by antibodies in the denatured TDP N-terminal domain. The N-terminal domain (residues 1-80) of TDP-43 was expressed from a plasmid in E. coli and purified as previously described (Wang et al. 2018 EMBO). The purified TDP-43 NTD was dialyzed against PBS, concentrated to 0.5 mg / ml, and stored at -80°C. Native PAGE was performed using the Novex Bis-Tris system for Western blot analysis according to the manufacturer's specifications. Protein samples were mixed with NativePAGE® Sample Buffer. Precast NativePAGE® 4-16% Bis-Tris gels were run at 150V constant voltage for 60 minutes at 4°C, followed by 250V for 30 minutes. Denatured SDS-PAGE was performed using the Novex Bis-Tris system according to the manufacturer's specifications. Protein samples were mixed with NuPAGE® LDS Sample Buffer. Precast NuPAGE® 4-12% Bis-Tris gels were run at room temperature under a constant voltage of 200V for 35 minutes. Proteins were then blotted onto PVDF (Thermo Fisher Scientific, USA) membranes using an XCell II Blot Module (Invitrogen, USA) according to the manufacturer's protocol. The blots were blocked with 5% milk powder in TBST and then incubated overnight at 4°C with purified rabbit polyclonal antibody GS240. For detection with ChemiDoc MP (Biorad, USA), donkey anti-rabbit IgG HRP-labeled secondary antibody (GE Healthcare Life Sciences, USA) was used. SuperSignal West Femto (Thermo Scientific, USA) substrates were used according to the manufacturer's instructions. As shown in Figure 4A, the antibody recognized and stained only denatured TDP-43 NTDs on the SDS-PAGE gel, but not on native TDP-43 NTDs on the PAGE gel, thus confirming that the epitope is inaccessible in naturally folded TDP-43 and becomes exposed upon misfolding (lanes 1, 2, and 3 contain 0.6, 0.3, and 0.15 μg / lane, respectively).Size exclusion chromatography (SEC) (Figure 4B) shows that the native and denatured forms of TDP-43 NTD remain monomeric. Molecular weight markers are overlaid for reference. SEC was performed using a high-performance liquid chromatography instrument on a Superdex 75 (10 / 300) HPLC column (GE Healthcare Life Sciences, USA). TDP-43 NTD (0.5 mg / ml) was denatured by incubation at 37°C for 10 minutes in 6M guanidine-HCl, 50 mM Tris-HCl buffer (pH 7.5), 150 mM NaCl, and 20 mM DTT. The resulting protein sample (100 μl) was loaded onto a column pre-equilibriumized with the same buffer and eluted at 0.5 ml / min. The native form was loaded onto a column pre-equilibriumized with 1x PBS buffer containing 5 mM DTT and eluted at 0.5 ml / min.

[0276] Example 10 Immunohistochemistry of patient samples Brain and spinal cord samples were obtained from the Netherlands Brain Bank and processed and stained at the Netherlands Institute for Neuroscience (Amsterdam, Netherlands). Sections (8 μM thick) from formalin-fixed, paraffin-embedded tissues were placed on Superfrost plus tissue slides (Menzel-Glaser, Germany) and dried overnight at 37°C. The sections were deparaffinized and then immersed in 0.3% H2O2 in phosphate-buffered saline (PBS) for 30 minutes to quench endogenous peroxidase activity. Formalin fixation forms protein crosslinks that can shield antigen sites in tissue specimens. To break these protein crosslinks, the slides were pre-treated with heat and Tris-EDTA (pH 9). The primary test antibody was diluted with antibody diluent (Sigma) and incubated overnight at 4°C at dilutions of 1:4000 (rabbit polyclonal GS240 antibody, 3E8 mouse monoclonal antibody) or 1:2000 (mouse monoclonal antibodies 3F11 and 2F7). Secondary EnVision HRP conjugate goat anti-rabbit / mouse antibody (EV-GαM HRP, DAKO) was added at room temperature for 30 minutes, followed by the addition of 3,3-diaminobenzidine chromogen (DAKO) for 10 minutes. The sections were counterstained with hematoxylin to visualize the cell nuclei, dehydrated, and mounted using Quick-D mounting medium (Klinipath).

[0277] Staining of brain sections from patients with frontotemporal lobar degeneration (FTLD) type B is shown in Figures 5A, 5C, 5D, and 5E. As shown in Figure 5A, rabbit polyclonal GS240 antibody stains pathogenic TDP-43 in both white matter (WM) and gray matter (GM). 3F11 (Figure 5C) and 3E8 (Figure 5E) monoclonal antibodies primarily detect pathogenic TDP-43 in GM, while 2F7 monoclonal antibody (Figure 5D) stains in both WM and GM. Rabbit polyclonal GS240 antibody was also tested in ALS spinal cord sections (Figure 5B) and showed positivity in motor neurons and surrounding tissue. These results indicate that the antibody can recognize the pathogenic TDP-43 epitope shown in situ in affected tissue.

[0278] Example 11 Representative staining of transfected HEK293 cells with monoclonal anti-TDP43 antibody As described in Example 4, mouse monoclonal antibodies and rabbit monoclonal antibodies were tested for selective binding to misfolded TDP-43 in a cell transfection assay using immunohistochemistry. Briefly, HEK293FT cells were transfected with plasmids or empty vectors encoding either HA-tagged ΔNLS-TDP43 (forming cytoplasmic aggregates) or HA-tagged wild-type (WT) TDP-43 (expressed in the nucleus). Anti-TDP-43 antibody was diluted to 10 ug / ml for staining, and fluorescently labeled anti-rabbit IgG or anti-mouse IgG was used as a secondary antibody for detection. Chicken anti-HA-tagged antibody, followed by a fluorescently labeled anti-chicken secondary antibody, was used for the detection of transfected TDP-43. Representative results obtained with 2F7 mouse monoclonal antibody are shown in Figure 6. Transfected ΔNLS-TDP43 mislocalizes in the cytoplasm, where it forms aggregates readily detectable by staining with anti-HA-tagged antibody. The 2F7 antibody recognized the same aggregates, as confirmed by the colocalization of two staining signals in the merged image. In contrast, transfected WT TDP43, which is detected in the nucleus by the HA-tagged antibody, was not stained by the 2F7 antibody. As expected, cells transfected with an empty vector showed no HA-tagged staining at all. They also showed no staining at all with the 2F7 antibody, indicating that 2F7 is also unable to recognize endogenous nuclear WT TDP-43.

[0279] Similar results were obtained with the other antibodies tested, which are summarized in Table 6. This staining pattern with the tested antibodies demonstrates their selectivity for misfolded pathogenic aggregates of TDP-43 compared to WT TDP-43.

[0280] [Table 8]

[0281] Example 12 Immunocytochemistry of physiological stress granules To determine whether a monoclonal antibody against misfolded TDP-43 reacted with physiological stress granules, stressed HEK293FT cells were stained. Briefly, HEK293FT cells were stressed by 60 minutes of exposure to 1 nM sodium arsenite. The cells were then stained with a fluorescently labeled antibody against the stress granule marker G3BP1 or a 10 ug / ml monoclonal anti-TDP-43 test antibody, followed by detection with a labeled secondary antibody. Representative results obtained with the mouse monoclonal antibody 3F11 are shown in Figures 7A and 7B. Similar results were obtained with the other antibodies tested, with the exception of one, which are listed in Table 7 below. Stressed cells showed a large amount of punctate staining of G3BP1+ stress granules in the cytoplasm (Figure 7A). In comparison, the same cells stained with the 3F11 antibody (Figure 7B) did not show the presence of cytoplasmic granules at the G3BP1 staining site, indicating that 3F11 did not react with stress granules in these cells. The absence of binding of the tested antibodies to TDP-43 in physiological stress granules suggests that they are unlikely to interfere with the protective function of these stress granules.

[0282] [Table 9]

[0283] Example 13 Characterization of the selected mAb Monoclonal antibodies were tested as described in Example 4 for the recognition of cytoplasmic aggregates formed in HEK293FT cells transfected with HA-tagged ΔNLS-TDP-43 (W68S), in comparison with HA-tagged ΔNLS-TDP-43 (W68S), in which tryptophan 68 (Trp68) is mutated to serine. An empty vector was used as a control. In the representative example shown in Figure 8, cells were stained with either 2 μg / ml TDP-43 rabbit monoclonal antibody 28H3-28K1, 1 μg / ml mouse pan-TDP43, or 0.5 μg / ml chicken anti-HA tag. Fluorescently labeled secondary antibodies Alexa Fluor 488-anti-rabbit, Alexa Fluor 647-anti-mouse, and Alexa Fluor 568-anti-chicken were used for the detection of the conjugated primary antibody. Nuclei were stained with Hoechst 33342 dye. In cells transfected with HA-tagged ΔNLS-TDP-43, the 28H3-28K1 antibody stained cytoplasmic aggregates co-localized with HA-positive aggregates of misfolded ΔNLS-TDP-43. In contrast, no staining was observed in cytoplasmic aggregates formed by ΔNLS-TDP-43-W68S lacking Trp68. As expected, cells transfected with an empty vector showed no HA-tagged staining at all. They also showed no staining with 28H3-28K1, confirming that the antibody does not recognize endogenous nuclear WT TDP-43. The pan-TDP-43 antibody recognized cytoplasmic aggregates formed by both transfected TDP-43 and endogenous nuclear TDP-43.

[0284] Similar results were obtained with most of the other antibodies tested, which are summarized in Table 8. As observed with the polyclonal rabbit antibody, this staining pattern with the tested monoclonal antibodies indicates their selectivity for misfolded TDP-43 NTDs containing solvent-exposed Trp68 residues. Clone 20, bound to several aggregates of ΔNLS-TDP-43-W68S, bound to physiological stress granules.

[0285] [Table 10]

[0286] Example 14 Antibody blockade of misfolded TDP-43 transmission in HEK293 cells Donor HEK293 cells were transiently transfected with HA-ΔNLS-TDP43, a TDP-43 HA-tagged nuclear localization signal-deficient mutant, to express misfolded TDP-43. 48 hours after transfection, the condition medium was collected from the donor cells and centrifuged at 1,000 g for 10 minutes to remove suspension cell debris. The clarified condition medium was incubated with 30 ug / ml of each individual TDP-43 misfolding-specific antibody or control mouse IgG1 (Biogen) at room temperature for 1 hour with constant rotation, after which naive recipient HEK293 cells were added. The antibodies tested included three mouse monoclonal antibodies (3F11, 2F7, 3E8) against the N-terminal epitope of TDP-43 and two antibodies (9C5, 5B7) against the structural RRM1 epitope (PCT CA / 2018 / 050634, published as WO2018 / 218352). After 48 hours of incubation, the recipient cell medium was removed, the cells were washed twice with cold PBS, and dissolved in 2% SDS. Protein concentrations were measured using a BCA assay. 25 ug of lysate was separated on a 10% NuPage gel (Thermo), transferred to a PVDF membrane, and then Western blotting was performed using antibodies against HA tag (Abcam, rabbit, 1:1000) or GAPDH (Thermo, mouse, 1:50K) as a loading control. HA and GAPDH immunoreactivity were detected using the ChemiDoc Imaging System, and their intensity was quantified using Image Lab.

[0287] Donor cells transfected with HA-dNLS-TDP43 contained large amounts of HA-tagged TDP-43. Naive recipient cells incubated with donor cell supernatant treated with control mouse IgG1 (mIgG1) contained detectable amounts of HA-tagged TDP-43, indicating extracellular transmission of misfolded aggregates of the HA-dNLS-TDP-43 protein from donor cells to recipient cells. Recipient cells incubated with donor cell supernatant pretreated with a misfolding-specific TDP-43 antibody contained relatively small amounts of HA-tagged TDP-43, indicating inhibition of transmission by the antibody. As a negative control, recipient cells incubated with supernatant from non-transfected donor cells contained no detectable amounts of HA-tagged TDP-43. Quantitative analysis by Western blot is shown in Figure 9. For each antibody, the HA-tagged signal was first normalized relative to the GAPDH signal (HA intensity / GAPDH intensity), and the value obtained for the test antibody was divided by the value obtained for control mIgG1. Compared to control mIgG1, all antibodies tested inhibited the propagation of misfolded HA-dNLS-TDP-43 from donor cell supernatant, resulting in low levels of HA-tagged TDP-43 in recipient cells.

[0288] Example 15 Development of antibodies selective for misfold disease-related TDP-43 Misfolded TDP-43 isoforms are associated with neurotoxicity and prion-like intercellular transmission in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTLD). Tryptophan (Trp68) in the TDP-43 N-terminal domain (NTD) has been found to be involved in cross-dissemination of SOD1 misfolding transmission, despite being inaccessible in naturally folded NTDs [9]. We hypothesized that if TDP-43 mislocalizes / aggregates in the cytoplasm, NTD Trp68 would become exposed.

[0289] Design / Method Rabbits were immunized with an unfolded NTD linear peptide epitope containing Trp68 to produce polyclonal and monoclonal antibodies (pAbs, mAbs). Mice were immunized with the same epitope to produce mAbs. Monoclonal antibody affinity for the immunized peptide was determined by surface plasmon resonance (SPR). NTDs were expressed in E. coli, and the properly folded monomer state was confirmed by size exclusion chromatography, followed by denaturation and testing using native gel electrophoresis and immunoblotting. Antibody specificity was confirmed by immunohistochemistry (IHC) of patient samples and by immunocytochemistry (ICC) of HEK293 cells transfected with a TDP-43 triple tandem mutation (ΔNLS) in the nuclear localization sequence. The ability of antibodies to inhibit intercellular propagation of misfolded TDP-43 was evaluated in vitro.

[0290] result SPR of mAbs revealed picomolar affinity to epitopes. Recombinant NTDs showed pAb immunoreactivity only under denaturation conditions. IHC of ALS / FTLD CNS sections was reactive to the antibody, but not in normal CNS. ICC revealed immunoreactivity to mislocalized / aggregated ΔNLS-TDP-43, but not to nucleus wild-type TDP-43. The antibody was also unable to recognize TDP-43 in physiological stress granules in HEK293 cells. A ΔNLS-TDP-43 construct with Trp68 mutated to serine did not show immunoreactivity in transfected cells, indicating that Trp68 is immunodominant in immunizing peptides. Antibodies tested in cell culture assays were able to inhibit intercellular propagation of misfolded TDP-43, a process thought to be central to the pathogenesis (References 10, 11, 12).

[0291] conclusion We have developed a family of antibodies highly sensitive to solvent exposure of NTD Trp68 that are selective for misfolded / aggregated disease-associated TDP-43 while preserving physiologically important molecular species. These antibodies may prove useful as biomarkers and immunotherapies for TDP-43-related diseases.

[0292] While this application has been described with reference to what are currently considered preferred embodiments, it should be understood that this application is not limited to the disclosed embodiments. On the contrary, this application is intended to cover a variety of modifications and equivalent configurations that fall within the spirit and scope of the appended claims.

[0293] All publications, patents, and patent applications are incorporated into this Specification by reference to the same extent as each individual publication, patent, or patent application is specifically and individually indicated as being incorporated into this Specification by reference. Specifically, sequences associated with each accession number shown herein, including, for example, accession numbers and / or biomarker sequences (e.g., proteins and / or nucleic acids) shown in tables or elsewhere, are incorporated into this Specification by reference.

[0294] The claims should not be limited by preferred embodiments and examples, but should be given the broadest possible interpretation consistent with the specification as a whole.

[0295] Citation of references mentioned in the specification [1] Kuo PH, Chiang CH, Wnag YT, Doudeva LG, Yuan HS, The Crystal Structure of TDP-43 RRM1-DNA Complex Reveals the Specific Recognition for UG- and TG-Rich Nucleic Acids. Nucleic Acids Res., 2014, vol 42, 4712. [2] DOI: 10.2210 / pdb1wf0 / pdb (Unpublished). [3] Mompean , M. , Romano , V. , Pantoja-Uceda , D. , Stuani , C. , Baralle , FE , Buratti , E. , and Laurents , DV The TDP-43 N- Terminal Domain Structure at High Resolution . FEBS J., 2016, 283, 1242. [4] Arai, T., M. Hasegawa, H. Akiyama, K. Ikeda, T. Nonaka, H. Mori, D. Mann, K. Tsuchiya, M. Yoshida, Y. Hashizume, and T. Oda amyotrophic lateral sclerosis. Biochem. Biophys. Res. Commun., 2006, 351, 602–611. [5] Chantelle F. Sephton, Shannon K. Good, Stan Atkin, Colleen M. Dewey, Paul Mayer III, Joachim Herz, and Gang Yu J. Biol. Chem. 2010, vol.285, no.9, 6826-6834. [6] Abel, O., Powell, JF,Andersen, PM, and Al-Chalabi, A. Hum Mutat, 2012, 33:1345-51. [7] Hamley, IW PEG-Peptide Conjugates 2014; 15, 1543–1559; dx.doi.org / 10.1021 / bm500246w. [8] Roberts, MJ et al Chemistry for peptide and protein PEGylation 64: 116-127. [9] Afroz, T et al. Functional and dynamic polymerization of the ALS-linked protein TDP-43 antagonizes its pathologic aggregation. Nat Comm (2017) 8:45.

[10] Porta, S et al. Patient-derived frontotemporal lobar degeneration brain extracts induce formation and spreading of TDP-43 pathology in vivo. Nature Comm (2018) 9:4220.

[11] Brettschneider, J et al. Sequential distribution of pTDP-43 pathology in behavioral variant frontotemporal dementia (bvFTD). Acta Neuropathol (2014) 127: 423.

[12] Feiler, M.S. et al. TDP-43 is intercellularly transmitted across axon terminals. J Cell Biol (2015) 211: 897.

Claims

1. An antibody that selectively binds to the TDP-43 sequence DAGWGNL (SEQ ID NO: 1) in misfolded TDP-43 compared to natural TDP-43, comprising a light chain variable region and a heavy chain variable region, wherein the heavy chain variable region comprises complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3, and the light chain variable region comprises complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3, and the amino acid sequence of the CDR comprises the following sequence: CDR-H1: GFFSSSNYV Sequence ID 16; CDR-H2: IWFAGIVDTT Sequence ID 17; CDR-H3: ARNPVGSVNL Sequence ID 18; CDR-L1: ESVYSNNR Sequence ID 19; CDR-L2: YAS Sequence ID 20; and CDR-L3: AGWRGARTDGVD Sequence ID 21, CDR-H1: GFSLSRYY Sequence ID 10; CDR-H2: IIPGGTT Sequence ID 11; CDR-H3: AGGPTGNSHFTL Sequence ID 12; CDR-L1: ESVYNNNH Sequence ID 13; CDR-L2: EAS Sequence ID 14; and CDR-L3: SGYKRVTTTDGIA Sequence ID 15, CDR-H1: GFFSSSSYV Sequence ID 22; CDR-H2: SDTGINT Sequence ID 23; CDR-H3: ARRYTGDTYLGNFNL Sequence ID 24; CDR-L1: QSVYKNNY Sequence ID 25; CDR-L2: KAS Sequence ID 26; and CDR-L3: AGGWRSLNA Sequence ID 27, CDR-H1: GFSLSSYT Sequence ID 34; CDR-H2: IYGGIGST Sequence ID 35; CDR-H3: GRGDI SEQ ID NO: 36; CDR-L1: QSVYKNR Sequence ID 37; CDR-L2: GAS Sequence ID 38; and CDR-L3: LGNYDCSSVDCGA Sequence ID 39, CDR-H1: GFFSSAYY Sequence ID 40; CDR-H2: TIPIGRT Sequence ID 41; CDR-H3: AGGPTGNSHFTL Sequence ID 42; CDR-L1: ESVYNNNQ Sequence ID 43; CDR-L2: QAS Sequence ID No. 44; and CDR-L3: AGYKSPTTDGIA Sequence ID 45, CDR-H1: GFSLSSYA Sequence ID 46; CDR-H2: IYNYET Sequence ID 47; CDR-H3: ARDIFRTNTNL Sequence ID 48; CDR-L1: QSVYKNNG Sequence ID 49; CDR-L2: FTS Sequence ID 50; and CDR-L3: LGGYDCSSSRVCGA Sequence ID 51, CDR-H1: GFSLSSYN Sequence ID 120; CDR-H2: IGTGGIT Sequence ID 121; CDR-H3: VRSSGSDWWFHI Sequence ID 122; CDR-L1: QSVYNNNN Sequence ID 123; CDR-L2: RAS Sequence ID 124; and CDR-L3: QGYFSGFITT Sequence ID 125, CDR-H1: GFTFSSYY Sequence ID 130; CDR-H2: INSNGGST Sequence ID 131; CDR-H3: VRQNYEGAY Sequence ID 132; CDR-L1: QSIVHSNGNTY Sequence ID 133; CDR-L2: KVS Sequence ID 134; and CDR-L3: FQSSHVPWT Sequence ID 135, or CDR-H1: GFTFSSYY Sequence ID 140; CDR-H2: INTNGGST Sequence ID 141; CDR-H3: VRQNYEGAY Sequence ID 142; CDR-L1: QSIVHSNGNTY Sequence ID 143; CDR-L2: KVS Sequence ID 144; and CDR-L3: FQSSHVPWT Sequence ID 145, antibody.

2. The antibody according to claim 1, wherein the antibody binds to DAGWG (SEQ ID NO: 2), AGWGN (SEQ ID NO: 4), GWGNL (SEQ ID NO: 6), and / or AGWGNL (SEQ ID NO: 5).

3. The antibody according to claim 1 or 2, wherein the antibody is at least 2 times, 3 times, at least 5 times, at least 10 times, or at least 20 times more selective for misfolded TDP-43 compared to natural TDP-43.

4. The aforementioned antibody i. An isolated peptide comprising all or part of DAGWGNL (SEQ ID NO: 1), wherein the part comprises at least five adjacent amino acids and GWG, and optionally the part is DAGWG (SEQ ID NO: 2), AGWGN (SEQ ID NO: 4), or GWGNL (SEQ ID NO: 6); or ii. An immunogen comprising a peptide, wherein the peptide comprises all or part of DAGWGNL, the part comprising at least five adjacent amino acids of SEQ ID NO: 1, and optionally i) the immunogen comprises a plurality of peptides, each peptide comprising all or part of DAGWGNL (SEQ ID NO: 1), the plurality of peptides being synthesized as a multiantigenic peptide (MAP), or ii) the peptides being coupled to a carrier protein, optionally the carrier protein being bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH). An antibody according to any one of claims 1 to 3, which is produced and / or screened using

5. The antibody according to any one of claims 1 to 4, wherein the antibody is a monoclonal antibody.

6. The antibody according to any one of claims 1 to 5, wherein the antibody is a humanized antibody.

7. The antibody according to any one of claims 1 to 6, wherein the antibody is a single-chain antibody.

8. The antibody according to any one of claims 1 to 7, wherein the antibody is a binding fragment selected from Fab, Fab', F(ab')2, scFv, dsFv, ds-scFv, dimer, minibody, diabody, and polymers thereof.

9. The antibody according to any one of claims 1 to 8, wherein the antibody is affinity-purified.

10. The antibody comprises a heavy chain variable region comprising i) the amino acid sequence shown in SEQ ID NO: 100, ii) an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% sequence identity with respect to SEQ ID NO: 100, where the CDR sequence is shown in SEQ ID NOs: 16-18, or iii) a conserved substitution amino acid sequence of i), where the CDR sequence is shown in SEQ ID NOs: 16-18; and / or the antibody comprises i) the amino acid sequence shown in SEQ ID NO: 101, ii) at least 50%, at least 60%, at least 70%, or at least 90% sequence identity with respect to SEQ ID NO: 101 The antibody according to any one of claims 1 to 9, comprising a light chain variable region comprising an amino acid sequence having at least 80% or at least 90% sequence identity, wherein the CDR sequence is shown in SEQ ID NOs: 19 to 21, or the conserved substitution amino acid sequence of iii)i), wherein the CDR sequence is shown in SEQ ID NOs: 19 to 21, wherein the heavy chain variable region amino acid sequence is optionally encoded by the nucleotide sequence or codon-degenerate version shown in SEQ ID NO: 78, and / or the light chain variable region amino acid sequence is encoded by the nucleotide sequence or codon-degenerate version shown in SEQ ID NO:

79.

11. The antibody comprises a heavy chain variable region comprising: i) the amino acid sequence shown in SEQ ID NO: 98; ii) an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% sequence identity with respect to SEQ ID NO: 98, where the CDR sequence is shown in SEQ ID NOs: 10-12; or iii) a conserved substitution amino acid sequence of i), where the CDR sequence is shown in SEQ ID NOs: 10-12; and / or the antibody comprises a heavy chain variable region comprising: i) the amino acid sequence shown in SEQ ID NO: 99; ii) at least 50%, at least 60%, at least 70%, at least The antibody according to any one of claims 1 to 9, comprising a light chain variable region having an amino acid sequence having 80% or at least 90% sequence identity, wherein the CDR sequence is shown in SEQ ID NOs: 13 to 15, or the conserved substitution amino acid sequence of iii)i), wherein the CDR sequence is shown in SEQ ID NOs: 13 to 15, wherein the heavy chain variable region amino acid sequence is optionally encoded by the nucleotide sequence or codon-degenerate version shown in SEQ ID NO: 76, and / or the light chain variable region amino acid sequence is encoded by the nucleotide sequence or codon-degenerate version shown in SEQ ID NO:

77.

12. The antibody comprises a heavy chain variable region comprising i) the amino acid sequence shown in SEQ ID NO: 102, ii) an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% sequence identity with respect to SEQ ID NO: 102, where the CDR sequence is shown in SEQ ID NOs: 22-24, or iii) a conserved substitution amino acid sequence of i), where the CDR sequence is shown in SEQ ID NOs: 22-24; and / or the antibody comprises i) the amino acid sequence shown in SEQ ID NO: 103, ii) at least 50%, at least 60%, at least 70%, or at least 90% sequence identity with respect to SEQ ID NO: 103 The antibody according to any one of claims 1 to 9, comprising a light chain variable region comprising an amino acid sequence having at least 80% or at least 90% sequence identity, wherein the CDR sequence is shown in SEQ ID NOs. 25 to 27, or the conserved substitution amino acid sequence of iii)i), wherein the CDR sequence is shown in SEQ ID NOs. 25 to 27, wherein the heavy chain variable region amino acid sequence is optionally encoded by the nucleotide sequence or codon-degenerate version shown in SEQ ID NO. 80, and / or the light chain variable region amino acid sequence is encoded by the nucleotide sequence or codon-degenerate version shown in SEQ ID NO.

81.

13. The antibody comprises a heavy chain variable region comprising i) the amino acid sequence shown in SEQ ID NO: 106, ii) an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% sequence identity with respect to SEQ ID NO: 106, where the CDR sequence is shown in SEQ ID NOs: 34-36, or iii) a conserved substitution amino acid sequence of i), where the CDR sequence is shown in SEQ ID NOs: 34-36; and / or the antibody comprises i) the amino acid sequence shown in SEQ ID NO: 107, ii) at least 50%, at least 60%, at least 70%, or at least 90% sequence identity with respect to SEQ ID NO: 107 The antibody according to any one of claims 1 to 9, comprising a light chain variable region comprising an amino acid sequence having at least 80% or at least 90% sequence identity, wherein the CDR sequence is shown in SEQ ID NOs: 37 to 39, or the conserved substitution amino acid sequence of iii)i), wherein the CDR sequence is shown in SEQ ID NOs: 37 to 39, wherein the heavy chain variable region amino acid sequence is optionally encoded by the nucleotide sequence or codon-degenerate version shown in SEQ ID NO: 84, and / or the light chain variable region amino acid sequence is encoded by the nucleotide sequence or codon-degenerate version shown in SEQ ID NO:

85.

14. The antibody comprises a heavy chain variable region comprising i) the amino acid sequence shown in SEQ ID NO: 108, ii) an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% sequence identity with respect to SEQ ID NO: 108, where the CDR sequence is shown in SEQ ID NOs: 40-42, or iii) a conserved substitution amino acid sequence of i), where the CDR sequence is shown in SEQ ID NOs: 40-42; and / or the antibody comprises i) the amino acid sequence shown in SEQ ID NO: 109, ii) at least 50%, at least 60%, at least 70%, or less than SEQ ID NO: 109 The antibody according to any one of claims 1 to 9, comprising a light chain variable region comprising an amino acid sequence having at least 80% or at least 90% sequence identity, wherein the CDR sequence is shown in SEQ ID NOs: 43 to 45, or the conserved substitution amino acid sequence of iii)i), wherein the CDR sequence is shown in SEQ ID NOs: 43 to 45, wherein the heavy chain variable region amino acid sequence is optionally encoded by the nucleotide sequence or codon-degenerate version shown in SEQ ID NO: 86, and / or the light chain variable region amino acid sequence is encoded by the nucleotide sequence or codon-degenerate version shown in SEQ ID NO:

87.

15. The antibody comprises a heavy chain variable region comprising i) the amino acid sequence shown in SEQ ID NO: 110, ii) an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% sequence identity with respect to SEQ ID NO: 110, where the CDR sequence is shown in SEQ ID NOs: 46-48, or iii) a conserved substitution amino acid sequence of i), where the CDR sequence is shown in SEQ ID NOs: 46-48; and / or the antibody comprises i) the amino acid sequence shown in SEQ ID NO: 111, ii) at least 50%, at least 60%, at least 70%, or at least 90% sequence identity with respect to SEQ ID NO: 111 The antibody according to any one of claims 1 to 9, comprising a light chain variable region comprising an amino acid sequence having at least 80% or at least 90% sequence identity, wherein the CDR sequence is shown in SEQ ID NOs: 49 to 51, or the conserved substitution amino acid sequence of iii)i), wherein the CDR sequence is shown in SEQ ID NOs: 49 to 51, wherein the heavy chain variable region amino acid sequence is optionally encoded by the nucleotide sequence or codon-degenerate version shown in SEQ ID NO: 88, and / or the light chain variable region amino acid sequence is encoded by the nucleotide sequence or codon-degenerate version shown in SEQ ID NO:

89.

16. The antibody comprises a heavy chain variable region comprising: i) the amino acid sequence shown in SEQ ID NO: 128; ii) an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% sequence identity with SEQ ID NO: 128, where the CDR sequence is shown in SEQ ID NOs: 120-122; or iii) a conserved substitution amino acid sequence of i), where the CDR sequence is shown in SEQ ID NOs: 120-122; and / or the antibody comprises a heavy chain variable region comprising: i) the amino acid sequence shown in SEQ ID NO: 129; ii) at least 50%, at least 60%, at least 70%, or at least 90% sequence identity with SEQ ID NO:

129. The antibody according to any one of claims 1 to 9, comprising a light chain variable region comprising an amino acid sequence having 80% or at least 90% sequence identity, wherein the CDR sequence is shown in SEQ ID NOs: 123 to 125, or the conserved substitution amino acid sequence of iii)i), wherein the CDR sequence is shown in SEQ ID NOs: 123 to 125, wherein the heavy chain variable region amino acid sequence is optionally encoded by the nucleotide sequence or codon-degenerate version shown in SEQ ID NO: 126, and / or the light chain variable region amino acid sequence is encoded by the nucleotide sequence or codon-degenerate version shown in SEQ ID NO:

127.

17. The antibody comprises a heavy chain variable region comprising: i) the amino acid sequence shown in SEQ ID NO: 138; ii) an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% sequence identity with SEQ ID NO: 138, where the CDR sequence is shown in SEQ ID NOs: 130-132; or iii) a conserved substitution amino acid sequence of i), where the CDR sequence is shown in SEQ ID NOs: 130-132; and / or the antibody comprises a heavy chain variable region comprising: i) the amino acid sequence shown in SEQ ID NO: 139; ii) at least 50%, at least 60%, at least 70%, or at least 90% sequence identity with SEQ ID NO:

139. The antibody according to any one of claims 1 to 9, comprising a light chain variable region comprising an amino acid sequence having 80% or at least 90% sequence identity, wherein the CDR sequence is shown in SEQ ID NOs: 133 to 135, or the conserved substitution amino acid sequence of iii)i), wherein the CDR sequence is shown in SEQ ID NOs: 133 to 135, wherein the heavy chain variable region amino acid sequence is optionally encoded by the nucleotide sequence or codon-degenerate version shown in SEQ ID NO: 136, and / or the light chain variable region amino acid sequence is encoded by the nucleotide sequence or codon-degenerate version shown in SEQ ID NO:

137.

18. The antibody comprises a heavy chain variable region comprising: i) the amino acid sequence shown in SEQ ID NO: 148; ii) an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% sequence identity with SEQ ID NO: 148, where the CDR sequence is shown in SEQ ID NOs: 140-142; or iii) a conserved substitution amino acid sequence of i), where the CDR sequence is shown in SEQ ID NOs: 140-142; and / or the antibody comprises a heavy chain variable region comprising: i) the amino acid sequence shown in SEQ ID NO: 149; ii) at least 50%, at least 60%, at least 70%, or at least 90% sequence identity with SEQ ID NO:

149. The antibody according to any one of claims 1 to 9, comprising a light chain variable region comprising an amino acid sequence having 80% or at least 90% sequence identity, wherein the CDR sequence is shown in SEQ ID NOs: 143 to 145, or the conserved substitution amino acid sequence of iii)i), wherein the CDR sequence is shown in SEQ ID NOs: 143 to 145, wherein the heavy chain variable region amino acid sequence is optionally encoded by the nucleotide sequence or codon-degenerate version shown in SEQ ID NO: 146, and / or the light chain variable region amino acid sequence is encoded by the nucleotide sequence or codon-degenerate version shown in SEQ ID NO:

147.

19. An immune complex comprising the antibody and a detectable label according to any one of claims 1 to 18.

20. An isolated nucleic acid contained in a vector, encoding an amino acid residue of the antibody according to any one of claims 1 to 18.

21. Cells that recombinantly express the antibody according to any one of claims 1 to 18.

22. A composition comprising, in some cases, a diluent and a pharmaceutically acceptable carrier, the antibody according to any one of claims 1 to 18.

23. A composition comprising two or more antibodies according to any one of claims 1 to 18.

24. The composition according to claim 22 or 23, comprising an effective amount of the antibody according to any one of claims 1 to 18 for use in treating a target subject of interest.

25. The composition according to claim 24, wherein the subject is suspected of having TDP-43 proteinosis, is at risk of developing it, or has been diagnosed with it.

26. The composition according to claim 25, wherein the TDP-43 proteinopathy is selected from amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD-TDP), primary lateral sclerosis, progressive muscular atrophy, and limbic-dominant senile TDP-43 encephalopathy (LATE).

27. The composition according to any one of claims 24 to 26 for administration in combination with the treatment of another TDP-43 proteinosis.

28. A composition according to any one of claims 22 to 27 for use in inhibiting TDP-43 cell propagation in a target area where it is needed.

29. A composition according to any one of claims 22 to 28 for parenteral administration, intravenous administration, subcutaneous administration, intramuscular administration, intracranial administration, intraventricular administration, subarachnoid administration, intraorbital administration, ocular administration, intraspinal administration, intracisional administration, intraperitoneal administration, intranasal administration, aerosol administration, or oral administration.

30. A composition according to any one of claims 22 to 29 for co-administration with a bioactive molecule known to promote transport across the blood-brain barrier.

31. i. The antibody according to any one of claims 1 to 18; and / or ii. The composition according to any one of claims 22 to 30; and iii. Compartments such as vials for containing the antibody and / or composition, and iv. A kit comprising, depending on the circumstances, instructions for use in ELISA or the method described herein.

32. A method for determining whether a sample suspected to contain cytoplasmic TDP-43 / aggregated TDP-43 and / or misfolded TDP-43 contains cytoplasmic TDP-43 / aggregated TDP-43 or misfolded TDP-43, a. Contacting the sample with the antibody described in any one of claims 1 to 18 under conditions that allow the formation of an antibody:misfolded TDP-43 complex; and b. Antibody: Detects the presence of a misfolded TDP-43 complex. It includes, Herein, the presence of a detectable complex indicates that the sample may contain cytoplasmic TDP-43 polypeptide / aggregated TDP-43 polypeptide and / or misfolded TDP-43 polypeptide, in a method.

33. The method according to claim 32, wherein the sample is a biological sample obtained from the subject.

34. The method according to claim 33, wherein the sample comprises a fraction of any of the aforementioned, which comprises blood, serum, plasma and / or solid tissue, and optionally brain, CSF and / or extracellular vesicles derived from blood.

35. The method according to claim 33 or 34, wherein the sample is, in some cases, a human sample derived from a subject having or suspected of having amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), primary lateral sclerosis, or limbic-dominant senile TDP-43 encephalopathy (LATE).

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