Epitopes in the RNA recognition motif 1 (RRM1) of TDP-43 and misfolding-selective antibodies against them

Abstract

The present disclosure relates to conformational epitopes in TDP-43, antibodies thereto, and methods for making and using immunogens and antibodies specific thereto. [Selected Figure] Figure 1B

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This is a Patent Cooperation Treaty application claiming the benefit under 35 U.S.C. § 119 of priority to U.S. Provisional Patent Application Nos. 62 / 512,647, filed May 30, 2017, 62 / 570,582, filed October 10, 2017, and 62 / 595,866, filed December 7, 2017, each of which is incorporated herein by reference in its entirety.

[0002] Incorporating a sequence listing The computer-readable medium of Sequence Listing "P51866PC00_ST25" (29,211 bytes), submitted via EFS-WEB and created on May 30, 2018, is incorporated herein by reference.

[0003] The present disclosure relates to TDP-43 epitopes and antibodies thereto, and more particularly to conformational TDP-43 epitopes predicted to be selectively accessible in misfolded TDP-43 and related antibody compositions. [Background technology]

[0004] The DNA-binding protein of the interacting receptor (TAR) element of 43 kDa (TDP-43) is a 414-amino acid protein composed of an N-terminal ubiquitin-like domain (NTD, residues 1-102), two RNA recognition motifs (RRMs) consisting of residues 106-177 (RRM1) and residues 192-259 (RRM2), and a C-terminal domain (CTD, residues 274-414). The NTD contains a nuclear localization signal (NLS, residues 82-98). RRM2 contains a nuclear export signal (NES) from residues 239-250.

[0005] TDP-43 is primarily a nuclear protein that plays a central role in RNA metabolism. TDP-43 has become a focus of research in the amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) disease spectrum, as pathogenic inclusions within affected neurons can contain post-translationally modified TDP-43. The CTD of TDP-43 is particularly relevant to the disease, as this is where nearly all familial ALS / FTD-associated mutations are found in TDP-43.

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

[0007] RRM1 and RRM2 have been structurally determined by NMR (e.g., RRM1 is available in the Protein Data Bank (PDB), a database of atomic-resolution three-dimensional structural data, as PDB entry 4IUF, RRM2 is available as PDB entry 1WF0, and NTD is available as PDB entry 2N4P).

[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 was found to be hyperphosphorylated, ubiquitinated, and fragmented in neuronal inclusions of patients with both sporadic and familial forms of ALS and FTD [4].

[0010] TDP-43 aggregates are now found in nearly all ALS cases (approximately 97%) and roughly half of FTD cases (approximately 45%). TDP-43 is one of the major components of cytoplasmic inclusions found in motor neurons of ALS patients.

[0011] Pathological precursors of TDP-43 inclusions may have concentrations far below those of functional TDP-43. Low concentrations of misfolded TDP-43 make this target difficult to find. Antibodies or drugs targeting normal TDP-43 may be lethal to cells. TDP-43 is an RNA regulatory protein essential for embryonic development [5].

[0012] Antibodies that bind to TDP-43 have been described.

[0013] WO2012 / 174666, entitled "METHODS FOR THE PROGNOSTIC AND / OR DIAGNOSTIC OF NEURODEGENERATIVE DISEASE, METHODS TO IDENTIFY CANDIDATE COMPOUNDS AND COMPOUNDS FOR TREATING NEURODEGENERATIVE DISEASE," discloses a method for diagnosing neurodegenerative diseases such as ALS and FTD through evaluating the interaction between TDP-43 and NF-κβ p65 using an anti-TDP-43 antibody.

[0014] WO2016 / 086320, entitled TDP-43-BINDING POLYPEPTIDES USEFUL FOR THE TREATMENT OF NEURODEGENERATIVE DISEASES, discloses antibodies that bind to the RRM1 domain of TDP-43 and inhibit its interaction with NF-κβ for the treatment of ALS and FTD.

[0015] Antibodies that preferentially or selectively bind to misfolded TDP-43 over natively folded TDP-43 are desirable. Summary of the Invention

[0016] Described herein is a conformational epitope in TDP-43, or an antibody thereto, comprising and / or consisting of residues TTEQ (SEQ ID NO: 1), or a portion thereof. The epitope is identified as an epitope that may be selectively exposed in misfolded species of TDP-43 in a conformation that is distinct from the conformation in the native protein.

[0017] One embodiment includes a compound, optionally a cyclic compound, comprising 1) TTE, 2) TTEQ (SEQ ID NO: 1), 3) TEQ, or a portion thereof, and a TDP-43 peptide comprising up to six consecutive residues of TDP-43, and a linker, wherein the linker is covalently attached to the N-terminal residue of the TDP-43 peptide and / or the C-terminal residue of the peptide, and wherein at least one amino acid in the TDP-43 peptide is in an alternate conformation relative to T, E, and / or Q in the corresponding linear and / or native TDP-43.

[0018] In embodiments, the TDP-43 peptide is selected from TTEQ (SEQ ID NO: 1), TTE, TEQ, KTTE (SEQ ID NO: 10), KTTEQ (SEQ ID NO: 12), TEQD (SEQ ID NO: 8), or TTEQD (SEQ ID NO: 9), and optionally, the cyclic compound is a compound selected from any one of SEQ ID NOs: 2, 3, 13, 22-39, and 42-44.

[0019] In embodiments, the cyclic compound is selected from the cyclic structures described herein, preferably the cyclic compound has a sequence selected from any one of SEQ ID NOs: 2, 3, 13, 22-39, and 42-44, more preferably SEQ ID NOs: 2, 3, 22, 23, 28, 29, 30, 31, 32, 33, 34, 35, and 42, optionally a sequence selected from any one of SEQ ID NOs: 28, 29, 30, 31, 32, 33, 34, 35, and 42, or a sequence selected from SEQ ID NOs: 2, 3, 22, 23, and 42.

[0020] In another aspect, immunogens comprising the cyclic compounds described herein are also provided.

[0021] In embodiments, the cyclic compounds are conjugated to a carrier protein or immunogenicity enhancing moiety and / or formulated with an adjuvant.

[0022] In embodiments, the carrier protein is bovine serum albumin (BSA), or the immunogenicity enhancing component is keyhole limpet hemocyanin (KLH), and / or the adjuvant is selected from aluminum phosphate, aluminum hydroxide alum, monophosphoryl lipid A, and QS21.

[0023] Another aspect provides antibodies that selectively bind to an epitope in said TDP-43 peptide in a cyclic compound described herein compared to a corresponding linear compound and / or native TDP-43 polypeptide.

[0024] In embodiments, the antibody selectively binds to cyclic compounds comprising 1) TTE, 2) TEQ, 3) TTEQ (SEQ ID NO: 1), 4) KTTE (SEQ ID NO: 10), 5) KTTEQ (SEQ ID NO: 12), 6) TEQD (SEQ ID NO: 8), and / or 7) TTEQD (SEQ ID NO: 9) compared to the corresponding linear compounds and / or native TDP-43 polypeptides.

[0025] In another embodiment, 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 the cyclic compound compared to the corresponding linear compound and / or native TDP-43 polypeptide.

[0026] In embodiments, antibodies that compete for binding to misfolded TDP-43 or cyclic peptides that contain the same or overlapping TDP-43 peptides as the antibodies described herein, preferably sharing at least 80% or more sequence identity with the heavy chain variable region and / or light chain variable region provided in Table 10, are also provided.

[0027] In embodiments, antibodies are produced or screened using the cyclic compounds or immunogens described herein.

[0028] In embodiments, the antibody comprises a set of CDRs, for example, as set forth in Table 10.

[0029] In another embodiment, the antibody comprises a heavy chain variable region comprising i) an amino acid sequence set forth in Table 10, ii) an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% sequence identity to the sequence of the heavy chain variable region, wherein the CDR sequences are as set forth in the sequence of the heavy chain variable region, or iii) a conservatively substituted amino acid sequence of i); and / or the antibody comprises a heavy chain variable region comprising i) an amino acid sequence set forth in Table 10, ii) an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% sequence identity to the sequence of the heavy chain variable region, wherein the CDR sequences are as set forth in the sequence of the heavy chain variable region. a light chain variable region comprising an amino acid sequence having at least 80%, or at least 90%, sequence identity, wherein the CDR sequences are as set forth in the sequence of the light chain variable region, or iii) a conservatively substituted amino acid sequence of i), optionally wherein the amino acid sequence of the heavy chain variable region is encoded by a nucleotide sequence set forth in Table 10, or a codon-degenerate or optimized version thereof, and / or wherein the amino acid sequence of the light chain variable region is encoded by a nucleotide sequence set forth in Table 10, or a codon-degenerate or optimized version thereof.

[0030] In another embodiment, the antibody is a monoclonal antibody, a humanized antibody, and / or a single chain antibody, or a binding fragment of any of the foregoing.

[0031] Another embodiment includes an immunoconjugate comprising an antibody described herein and a detectable label or transport moiety, optionally a molecule that facilitates transport across the blood-brain barrier and / or into cells.

[0032] A further aspect provides nucleic acids encoding the compounds, amino acid residues of immunogens, antibodies, or proteinaceous immunoconjugates described herein, or any portion thereof.

[0033] Another aspect provides a cell that expresses an antibody described herein, optionally wherein the cell is a hybridoma.

[0034] Another aspect is a composition comprising a cyclic compound, immunogen, antibody, immunoconjugate, nucleic acid, or cell described herein, optionally with a diluent.

[0035] In embodiments, the composition comprises a cyclic compound or immunogen described herein and an adjuvant.

[0036] In embodiments, the adjuvant is aluminum phosphate, aluminum hydroxide, aluminum hydroxide alum, monophosphoryl lipid A, and / or QS21.

[0037] Kits are also provided that include one or more components described herein, eg, a compound, immunogen, antibody, immunoconjugate, nucleic acid, cell, or composition described herein.

[0038] In another aspect, there is also provided a method of producing an antibody described herein, the method comprising administering to a subject an immunogen described herein, or a composition comprising the immunogen, and isolating antibodies and / or cells expressing antibodies selective or specific for a TDP43 peptide of the administered immunogen.

[0039] A further embodiment is a method for determining whether a sample suspected of containing a misfolded TDP-43 polypeptide contains a misfolded TDP-43 polypeptide, comprising: contacting the sample with an antibody described herein under conditions permissive for forming an antibody:misfolded TDP-43 polypeptide complex; detecting the presence of any complexes; The presence of the detectable complex indicates that the sample may contain a misfolded TDP-43 polypeptide.

[0040] In an embodiment, the sample comprises a brain tissue extract, spinal cord tissue, and / or CSF. In another embodiment, the sample is a human sample, optionally from a subject having or suspected of having ALS or FTD.

[0041] Other features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the present disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description. [Brief explanation of the drawings]

[0042] Embodiments of the present disclosure will now be described with reference to the following drawings.

[0043] [Figure 1A-C] Graphs depicting different metrics used to predict epitope exposure in misfolded TDP-43. Figure 1A is a graph representing epitope predictions resulting from the native structure PDB 4IUF using an increase in SASA (ΔSASA) as the criterion for epitope selection. The TTE epitope appears as a prediction for PDB structure 4IUF. Figure 1B is a graph showing epitope predictions resulting from the structure PDB 4IUF using loss of native contacts as the criterion for epitope selection. The TTEQ epitope (SEQ ID NO: 1) appears as a prediction using this metric. Figure 1C shows several metrics, including an increase in SASA (ΔSASA), an increase in the mean squared fluctuation of atomic configurations (RMSF), which represents an increase in epitope dynamics, and a decrease in Δcontacts, which is the number of native contacts. These three different metrics predict the epitopes TTE, TTE, and TTEQ (SEQ ID NO: 1), respectively. [Figure 2A-J]2A-2C are graphs showing the distribution of dihedral angles for several dihedral angles that can conformationally distinguish misfolded TTEQ (SEQ ID NO: 1) from natively folded TTEQ (SEQ ID NO: 1). The distributions for the native assemblies (dashed lines), biased assemblies (solid lines) (representing misfolded RRM1 of TDP-43), and cyclic CGGTTEQGG (SEQ ID NO: 2) (shaded histogram) scaffolds (for use in conjugation with immunogens) are shown for the following dihedral angles defined by four atoms, involving the side chain and backbone atoms of residue threonine 115 (T115): C-Cα-Cβ-Oγ1 (FIG. 2A), C-Cα-N-HN (FIG. 2B), and O-Cα-N (FIG. 2C). The dihedral angle distribution for residue T116 is shown for the angles Cβ-Cα-N-HN (Figure 2D), N-Cα-Cβ-Oγ1 (Figure 2E), and O-C-Cα-N (Figure 2F), involving the side chain and backbone atoms of residue T116. The dihedral angle distribution for residue E117 is shown for the angles Cα-Cβ-Cγ-Cδ (Figure 2G), Oε1-Cδ-Cγ-Cβ (Figure 2H). The dihedral angle distribution for residue Q118 is shown for the angles O-C-Cα-Cβ (Figure 2I), and Cα-Cβ-Cγ-Cδ (Figure 2J). This latter dihedral angle distribution is shown as an illustration of angles that do not distinguish between native, biased, and cyclic. Percent overlap values ​​are provided in Table 1A. The peak dihedral angle values ​​for the distribution are given in Table 2A. [Figure 3A-H] Equilibrium backbone Ramachandran angles (phi or φ, and psi or ψ) for residues 115T, 116T, and 117E, and 118Q in the native ensemble (dashed line), biased ensemble (solid line), and cyclic CGGTTEQGG (SEQ ID NO: 2) (shaded histograms). Ramachandran angle distributions for residue T115 are shown for the backbone angles C'-N-Cα-C' (hereafter, phi) (Figure 3A) and N-Cα-C'-N (hereafter, psi) (Figure 3B). Phi and psi are also shown for T116 (Figures 3C and D), E117 (Figures 3E and F), and Q118 (Figures 3G and H). The overlap probabilities for these Ramachandran angles are shown in Table 3A. The peak angles of the corresponding distributions are shown in Table 4A. [Figure 4A]1 is a graph showing solubility as a function of residue index for all amino acids in RRM1 of TDP-43, including TTEQ (SEQ ID NO: 1) depicted by the vertical dashed line. The epitope lies within a region of higher than average solubility along the main sequence. [Figure 4B] The SASA for residue TTEQ (SEQ ID NO: 1) is shown, where TTEQ (SEQ ID NO: 1) in the cyclic peptide cyclo(CGGTTEQGG) (SEQ ID NO: 2) is represented as a solid line with a circle design, TTEQ (SEQ ID NO: 1) in the biased, partially unfolded peptide is represented as a dashed line with a square design, and TTEQ (SEQ ID NO: 1) in the context of the native structure 4IUF is represented as a light grey line with a triangle design. [Figure 4C] The SASA for residue TTEQ (SEQ ID NO: 1) is shown, where TTEQ (SEQ ID NO: 1) in the cyclic peptide cyclo(CGTTEQG) (SEQ ID NO: 3) is represented as a solid line with a circle design, TTEQ (SEQ ID NO: 1) in the biased, partially unfolded peptide is represented as a dashed line with a square design, and TTEQ (SEQ ID NO: 1) in the context of the native structure 4IUF is represented as a light grey line with a triangle design. [Figure 5A.B] Figure 5A shows the centroid structures of the cyclic and native assemblies of the TTEQ (SEQ ID NO: 1) epitope. The black-colored conformation is the centroid of the largest cluster of cyclic peptides and therefore best represents the typical conformation of a cyclic peptide. The white-colored conformation is the centroid of the largest cluster of TTEQ (SEQ ID NO: 1) in the native assemblies (also referred to as "native peptides") and therefore should best represent the typical conformation of TTEQ (SEQ ID NO: 1) in the native assemblies. Figure 5A shows the aligned centroid structures of residues T115, T116, E117, and Q118 (TTEQ SEQ ID NO: 1) in cyclic CGGTTEQGG (SEQ ID NO: 2); the native peptide is overlapped in the figure. Figure 5B is the same as Figure 5A for the cyclic peptide structure CGTTEQG (SEQ ID NO: 3) and the native peptide structure TTEQ (SEQ ID NO: 1), both rendered in licorice representation, so that the orientation of the side chains can be seen. [Figures 6A-E]6A-6D are schematic representations of the TTE / TTEQ epitope in native and biased representations. The SASA of the epitope TTEQ (SEQ ID NO: 1) is shown in the context of native, biased, and cyclic peptides of the sequences CGGTTEQGG (SEQ ID NO: 2) and CGTTEQG (SEQ ID NO: 3). T115 and E117 are labeled. Figure 6A shows the SASA of the epitope TTEQ (SEQ ID NO: 1) in the native ensemble. Figure 6B shows the SASA of the epitope TTEQ (SEQ ID NO: 1) in the biased ensemble. Figure 6C shows the SASA of the epitope TTEQ (SEQ ID NO: 1) in the cyclic peptide cyclo(CGGTTEQGG) (SEQ ID NO: 2) ensemble. Figure 6D shows the SASA of the epitope TTEQ (SEQ ID NO: 1) in the cyclic peptide cyclo(CGTTEQG) (SEQ ID NO: 3) ensemble. These panels qualitatively demonstrate that the antigenic surface presented by the cyclic peptide is more similar to that of the biased center-of-mass structure and distinct from that of the native structure. This is supported by analyzing interactions that are present in the native ensemble but are broken in the biased ensemble. In particular, Figure 6E shows a salt bridge between the side chains of residues E117 and K137. This salt bridge is present in the native ensemble (left panel of Figure 6E) but is broken in the biased ensemble (right panel of Figure 6E). Disruption of this salt bridge facilitates exposure of the side chain of E117, making it available for antibody binding. [Figure 7A-J]7A and 7B are a series of plots comparing linear, cyclic, biased, and / or native conformations. Figures 7A and 7B are clustering plots by RMSD, with the axes corresponding to the RMSD of TTEQ (SEQ ID NO: 1) relative to the centroid structure of the cyclic peptide ensemble, the RMSD of TTEQ (SEQ ID NO: 1) relative to the centroid structure of the ensemble of native ensembles, and the RMSD of TTEQ (SEQ ID NO: 1) relative to the centroid structure of the native structural ensemble of PDB ID 4IUF. Figure 7A is a plot. Each point corresponds to a given conformation taken from either the cyclic peptide cyclo(CGGTTEQGG) (SEQ ID NO:2) equilibrium ensemble (circle as noted in the legend), the linear peptide equilibrium ensemble (+ symbol as noted in the legend), the native structural equilibrium ensemble starting from PDB ID 4IUF (inverted triangle as noted in the legend), or the biased structural ensemble (asterisk as noted in the legend). Figure 7B is similar to Figure 7A, except that the linear ensemble is not shown; the biased ensemble is now represented by a + symbol. Figure 7C is a plot showing the percentage overlap between the different ensembles as a function of the number of configurations sampled to demonstrate convergence. The numerical percentage overlap is given in Table 6A. Figures 7D and E are the same as Figures 7A and B, but show information for the cyclic peptide cyclo(CGTTEQG) (SEQ ID NO:3). Figure 7F is the same as Figure 7C, but show information for the cyclic peptide cyclo(CGTTEQG) (SEQ ID NO:3). Figure 7G shows the correlation between circular and native distributions, defined by the first finding where a portion of the distribution has a density greater than a cutoff value, thus encompassing both a given percentage of the total distribution, e.g., a density cutoff for the circular and native distributions that gives 60% of the total distribution. Then, for these sub-distributions, the correlation coefficient is

number

[0044] The generation of misfolding-specific antibodies can be achieved through the identification of targets for misfolded TDP-43 peptides that are absent or present to a lesser extent in the native structure. Misfolding-specific epitopes need not differ in primary sequence from the corresponding segments in the native structure, but should be conformationally distinct in the context of the misfolded assemblies. That is, the epitope will present a distinct conformation in terms of backbone and / or side chain conformations in the misfolded assemblies that is absent (or unfavorable) in the native structure.

[0045] Antibodies raised against regions of the native protein tend not to be selective for the misfolded protein and therefore will bind to the native functional protein as well.

[0046] As described herein, to generate antibodies that may be selective for misfolded forms of TDP-43, we sought to identify regions of the TDP-43 sequence that are prone to inhibition in the context of the native fold and that may similarly be exposed on the surface of the misfolded protein.

[0047] As described in the Examples, computer simulations using molecular dynamics with standardized force fields were used. The experimentally validated structural model of the folded structure is globally deviated from its "native" conformation, which is partially unfolded using molecular dynamics to make one or more regions of adjacent primary sequence susceptible to failure upon external challenge in an abnormal cellular environment.

[0048] We hypothesized that these weakly stable regions may be exposed in nascent misfolded proteins or in misfolded pathogenic species, and in this context, they may exist in alternate conformations rather than in the native ensemble, and therefore may constitute misfolded protein-specific epitope predictions.

[0049] Without wishing to be bound by theory, these sequence regions in the misfolded protein may be exposed in conformations that are distinct from the native-folded (e.g., on the surface) conformation, and similarly may be exposed in regions with greater exposed surface area, or in alternating side chain conformations as seen by different dihedral angle distributions, and / or in different overall conformations as measured by structural alignment, than the corresponding quantities show in native TDP-43.

[0050] As described in the Examples, the inventors identified minimal epitopes in regions predicted to be prone to disruption in the context of the native structure and therefore likely to be exposed in the partially formed native structure. The inventors designed cyclic compounds containing the identified epitopes that meet criteria for alternate conformations, such as greater exposed surface area, and / or that do not readily align to the native ensemble by root mean square deviation (RMSD), but more preferably align to the biased, partially disordered ensemble.

[0051] Antibodies raised against these epitopes are shown by immunofluorescence to recognize misfolded TDP-43 in cell culture and by immunodot blotting to bind to pathogenic TDP43 in spinal cord homogenates from ALS patients versus controls.

[0052] I. Definition As used herein, the term "TDP-43" may alternatively be referred to as "TDP43." As used herein, "TDP" refers to all forms of TDP-43, including wild-type TDP-43, naturally occurring TDP-43, and misfolded forms, including mutant forms and analogs thereof, from all species, particularly human TDP-43 (i.e., hTDP-43). Human TDP-43 is typically a 414 amino acid residue protein, and its amino acid sequence (e.g., Uniprot accession number Q13148) and nucleotide sequence (e.g., accession number HGNC:11571) have been previously characterized. TDP-43 contains RRM1 and RRM2 domains. RRM1 of TDP-43 refers to the first RNA recognition motif of the protein, consisting of amino acids 106-177. The structure of the RRM1 domain of TDP-43 has been determined and is listed in the Protein Data Bank as PDB entry 4IUF. The PDB 4IUF structure can be computationally equilibrated to obtain an equilibrium ensemble that was used for all measurements of the native conformation of the epitope in the native structure of TDP-43, referred to herein indefinitely as the "native structure of RRM1," "equilibrium native ensemble of TDP-43," "equilibrium native ensemble of RRM1 of TDP-43," or "TDP-43 native structural ensemble."

[0053] As used herein, "wild-type" refers to the predominant amino acid sequence of the non-mutated or naturally occurring protein.

[0054] As used herein, "native" refers to the normal three-dimensional structure of a specific protein or portion thereof (e.g., the atomic coordinates of the crystal structure of the native TDP-43 RRM1 domain are available in the Protein Data Bank under accession number 4IUF). Native TDP-43 is optionally referred to as "natively folded" TDP-43, "normally folded" TPD-43, and / or "healthy" TDP-43. Similarly, the native RRM1 domain of TDP-43 is optionally referred to as the "natively folded" RRM1 domain of TDP-43 or the "normally folded" RRM1 domain of TPD-43. Thus, the term "native TDP-43" or "natively folded TDP-43," as used herein, refers to nascent, post-translationally naturally folded TDP-43, including a molecular structure comprising non-covalently bound individual TDP-43 peptides that exhibits a native structure as reconstructed under X-ray crystallography or from nuclear magnetic resonance measurements, and / or dimeric TDP-43 folded in a non-disease state (e.g., normal cells). The native structure of RRM1 has an alpha / beta structure consisting of both alpha helices and beta sheets.

[0055] As used herein, "misfolded" refers to the secondary and tertiary structure of a polypeptide or a portion thereof, and indicates that the polypeptide adopts a conformation that is not normal for that polypeptide in its proper functional state. Misfolding can be caused by mutations in proteins, such as amino acid deletions, substitutions, or additions, but wild-type sequence proteins can also be misfolded in disease, exposing disease-specific epitopes, for example, as a result of microenvironmental conditions and / or amino acid modifications, such as nitration, oxidation, carbonylation, or other modifications. Other post-translational modifications include abnormal ubiquitination, phosphorylation, acetylation, sumoylation, and cleavage of C-terminal fragments into ubiquitin. Thus, when referring to a polypeptide herein, a "misfolded TDP-43 polypeptide" or "misfolded TDP-43" refers to a TDP-43 polypeptide that exhibits multiple conformations of TDP-43, which conformations contain portions of the native structure, are partially ordered, contain polymeric segments of amino acids with alternating conformations relative to native TDP-43, are partially disordered, often exhibit increased SASA, and sample a more diverse conformational ensemble than that explored in the native equilibrium ensemble.

[0056] Misfolded TDP-43 is prone to aggregate formation that leads to loss of protein function, toxicity, and propagation of pathogenic aggregates.

[0057] The term "mutant TDP-43" refers to forms of TDP-43 that arise as a result of genetic mutations that result in, for example, amino acid substitutions, such as those characteristic of FTD or familial ALS, including, for example, mutations described in the bioinformatics tool described in [6], particularly endogenous forms of TDP-43.

[0058] The term "TTEQ (SEQ ID NO: 1)" refers to the amino acid sequence: threonine, threonine, glutamic acid, glutamine, as shown in SEQ ID NO: 1. Similarly, TTE, TEQ, KTTE (SEQ ID NO: 10), KTTEQD (SEQ ID NO: 7), TTEQD (SEQ ID NO: 9), TTEQDL (SEQ ID NO: 11), and TEQD (SEQ ID NO: 8) refer to amino acid sequences identified by the single-letter amino acid code. Depending on the context, reference to an amino acid sequence may refer to a sequence in TDP-43 or an isolated peptide, such as the amino acid sequence of a cyclic compound. The sequence TTEQ (SEQ ID NO: 1) consists of residues 115-118 of the primary amino acid sequence.

[0059] As used herein, the term "TTEQ (SEQ ID NO: 1) or a related epitope and / or any portion thereof" includes, at a minimum, amino acids T115 and / or T116 and / or E117, including, for example, TTE, KTT, or TEQ. Reference to TTEQ (SEQ ID NO: 1) or a related epitope and / or any portion thereof may refer, for example, to a region on TDP-43 that is bound by an antibody generated by a cyclic compound containing the TDP-43 peptide sequence. For example, the antibody may selectively or specifically bind to T115, T116, or E117, a specific portion of T115 and / or T116 and / or E117, or any combination of the foregoing. Alternatively, it may refer to the TDP-43 peptide sequence contained in the cyclic compound used to generate the antibody.

[0060] As used herein, the term "alternate conformation than occupied by 115T, 116T, 117E, and / or 118Q in the native form" refers to having one or more different conformational properties selected from solvent accessibility (e.g., in the context of a peptide comprising TTEQ (SEQ ID NO: 1) as measured in the cyclic peptide described in the Examples, RMSD structural alignment, and one or more backbone dihedral angles or side chain dihedral angles) compared to the properties for 115T, 116T, 117E, and / or 118Q in the TDP-43 native structural ensemble, e.g., as shown in PDB 4IUF, and in figures and / or tables. Similarly, as used herein, the term "alternate conformation" refers to having one or more different conformational properties selected from solvent accessibility (e.g., in the context of a peptide comprising TTEQ (SEQ ID NO: 1) as measured in the cyclic peptide described in the Examples, RMSD structural alignment, and one or more backbone ... As shown in 4IUF, Figures 1-9, and / or Tables, comparing the properties of one or more of T, E, and / or Q, e.g., T, E, and / or Q, in native TDP-43 means having one or more different conformational properties selected from solvent accessibility (e.g., one or more backbone dihedral angles or side chain dihedral angles in the context of the corresponding linear peptide containing TTEQ (SEQ ID NO: 1) or TTE). For example, according to Figure 2, for residue 115T, the dihedrals C-CA-CB-OG1 and C-CA-N-HN distinguish both the cyclic peptide cyclo(CGGTTEQGG) (SEQ ID NO: 2) and the biased ensemble from the corresponding dihedral angles in the native form. In particular, for example, the dihedral C-CA-CB-OG1 shows a peak at 180 degrees for the cyclic and biased ensembles, which is not present in the native ensemble.

[0061] As used herein, "epitope" refers to a region of a protein recognized by a B-cell or T-cell receptor, or an antibody or binding fragment thereof. An epitope is optionally represented by a linear amino acid sequence or a region of a protein recognized by an antibody. An epitope can include one or more antigenic determinants. For example, an antibody generated against an isolated peptide corresponding to a misfolded epitope recognizes part or all of the epitope sequence.

[0062] As used herein, the term "misfolded epitope" or "conformational epitope" refers to a sequence of amino acids or antigenic determinants having a specific three-dimensional structure, where at least some aspect of the three-dimensional structure not present in the corresponding native structure is recognized by a cognate antibody. A "misfolded epitope or conformational epitope" is exposed or accessible in misfolded proteins (e.g., as present in ALS and FTD). Antibodies that selectively bind to a misfolded epitope recognize the spatial arrangement of one or more of the amino acid sequences of that conformation-specific epitope. For example, a TTEQ (SEQ ID NO: 1) conformational epitope refers to an epitope of TTEQ (SEQ ID NO: 1) that is recognized with antibody selectivity, e.g., at least 2-fold, 3-fold, 5-fold, 10-fold, 50-fold, 100-fold, 250-fold, 500-fold, or 1000-fold or more selectivity, compared to an epitope on an antibody generated using native TDP-43 or a linear peptide comprising, for example, TTEQ (SEQ ID NO: 1).

[0063] As used herein, the term "analog" includes portions, extensions, substitutions, mutations, modifications, or chemical equivalents and derivatives of the amino acid and nucleotide sequences of the present invention that function in substantially the same way as the peptides, proteins, or nucleic acid molecules described herein. Analogs of cyclic compounds, such as cyclic peptides, also include additions and deletions to the TDP-43 peptide. Nucleic acid analogs include degenerate nucleotide substitutions that encode the isolated peptides of the present invention. In addition, analog peptides and analog nucleotide sequences include their derivatives.

[0064] The term "amino acid" includes all naturally occurring amino acids as well as modified L-amino acids. The atoms of an amino acid may contain, for example, different isotopes. For example, an amino acid may contain deuterium substituted for hydrogen, nitrogen-15 substituted for nitrogen-14, and carbon-13 substituted for carbon-12, as well as other similar changes.

[0065] As used herein, a "conservative amino acid substitution" refers to a substitution in which one amino acid residue is replaced with another amino acid residue without destroying the desired properties of the protein. Suitable conservative amino acid substitutions can be made by substituting amino acids with similar hydrophobicity, polarity, and R group size. Examples of conservative amino acid substitutions include: [Table 1]

[0066] As used herein, the term "sequence identity" refers to the percentage of sequence identity between two polypeptide sequences or two nucleic acid sequences. To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of the first amino acid or nucleic acid sequence for optimal alignment with the second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical overlapping positions / total number of positions × 100%). In one embodiment, the two sequences are the same length. Determining the percent identity between two sequences can also be accomplished using a numerical algorithm. A preferred, non-limiting example of a numerical algorithm utilized for comparing two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403. BLAST nucleotide searches can be performed, for example, with the NBLAST nucleotide program parameters set for score=100, wordlength=12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the present application. BLAST protein searches can be performed, for example, with the XBLAST program parameters set for score=50, wordlength=3 to obtain amino acid sequences homologous to the protein molecules described herein.To obtain gapped alignments for comparison purposes, gapped BLAST can be utilized as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402. Alternatively, Psi-BLAST can be used to perform an iterative search to detect distant relationships between molecules (ibid.). When utilizing BLAST, gapped BLAST, and Psi-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used (see, e.g., the NCBI website). Another preferred, non-limiting example of a numerical algorithm utilized for sequence comparison is the algorithm of Myers and Miller, 1988, CABIOS 4:11-17. Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When utilizing the ALIGN program to compare amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. Techniques similar to those described above, with or without allowing gaps, can be used to determine the percent identity between two sequences. In calculating percent identity, typically only exact matches are counted.

[0067] For antibodies, the percentage of sequence homology can be determined when the antibody sequences are maximally aligned by IMGT or other (e.g., Kabat numbering convention). After alignment, if a region of a subject antibody (e.g., the entire mature variable region of a heavy or light chain) is compared to the same region of a reference antibody, the percentage of sequence identity between the subject and reference antibody regions is the number of positions occupied by the same amino acid in both the subject and reference antibody regions, divided by the total number of aligned positions in the two regions, not counting gaps, and multiplied by 100 to convert to a percentage.

[0068] As used herein, the term "antibody" is intended to include monoclonal, polyclonal, single-chain, humanized, and other chimeric antibodies, as well as binding fragments thereof. Antibodies may be derived from recombinant sources and / or produced in transgenic animals. Also included are human antibodies, which may be produced through the use of biochemical techniques or isolated from libraries. Humanized or chimeric antibodies may contain sequences from one or more isotypes or classes.

[0069] The phrase "isolated antibody" refers to an in vivo or in vitro produced antibody that has been removed from the source that produced it, e.g., an animal, hybridoma, or other cell line (such as a recombinant cell that produces the antibody). An isolated antibody is optionally "purified," meaning at least 80%, 85%, 90%, 95%, 98%, or 99% pure.

[0070] As used herein, the term "binding fragment" refers to a portion or part of an antibody or antibody chain that contains fewer amino acid residues than an intact or complete antibody or antibody chain and that binds to an antigen or competes with the 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 multimers thereof. Fragments can be obtained through chemical or enzymatic treatment of intact or complete antibodies or antibody chains. Fragments can also be obtained by recombinant means. For example, F(ab')2 fragments can be generated by treating an antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges and produce Fab' fragments. Papain digestion can result in the formation of Fab fragments. Fab, Fab', and F(ab')2, scFv, dsFv, ds-scFv, dimers, minibodies, bispecific antibodies, bispecific antibody fragments, and other fragments can also be constructed by recombinant expression techniques.

[0071] The art-recognized term "IMGT numbering" or "Immunogenetics Database numbering" refers to a system for numbering amino acid residues that are more variable than other amino acid residues (i.e., hypervariable) in the heavy and light chain variable regions of an antibody, or antigen-binding portion thereof.

[0072] The CDR sequences referred to herein are based on IGBlast identification by BLAST alignment with IMGT or NCBI germline V gene database.It has also been confirmed that the sequence corresponds to IMGT numbering.Since the full sequence of variable region is provided, CDR can be similarly identified based on other conventions such as Kabat.

[0073] When an antibody is said to bind to an epitope within specified residues, such as TTEQ (SEQ ID NO: 1), it means that the antibody selectively or specifically binds to polypeptides containing the specified residues or a portion thereof, e.g., at least one residue or at least two residues in a conformationally selective manner. Such an antibody need not contact every residue of TTEQ (SEQ ID NO: 1), and every single amino acid substitution or deletion within the epitope need not necessarily significantly or equally affect binding affinity.

[0074] As used herein, the term "detectable label" refers to a moiety, such as a peptide sequence, fluorescent protein, etc., that can be attached to or introduced into a peptide or compound described herein and that can produce a detectable signal, either directly or indirectly. For example, the label can be: 3 H, 13 N, 14 C. 18 F, 32 P, 35 S, 123 I, 125 I, 131The detectable label may be a radiopaque positron-emitting radionuclide (e.g., for use in PET imaging) or radioisotope such as 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; an imaging agent; or a metal ion. The detectable label may also be indirectly detectable, for example, using a secondary antibody.

[0075] As used herein, the term "epitope selectively presented or accessible in misfolded TDP-43" refers to an epitope that is selectively presented or accessible on misfolded TDP-43 present in ALS or FTD (e.g., disease-associated misfolded TDP-43), whether in monomeric, dimeric, or aggregated form, but that is not on the molecular surface of the native, correctly folded homodimer of TDP-43.

[0076] As used herein, the phrase "epitope consisting of TTEQ" refers to an epitope, optionally a conformational epitope, that is specifically and preferentially bound by an antibody that preferentially binds to a TTEQ (SEQ ID NO: 1) peptide compared to a mutant TTEQ (SEQ ID NO: 1) peptide in which any one or more of the residues have been mutated, for example, to alanine. Similarly, as used herein, the phrase "conformational epitope consisting of TTEQ" refers to an epitope that is specifically bound by an antibody that preferentially binds to TTEQ (SEQ ID NO: 1) in a particular conformation (e.g., a misfolded protein, a cyclic compound, or some other restricted conformation) over another conformation (e.g., a native form), optionally when at least one or more of the residues have been mutated, for example, to alanine (e.g., a cyclic conformation of a peptide comprising TTEQ (SEQ ID NO: 1) versus a cyclic conformation of a peptide in which one or more of the residues have been mutated).

[0077] The phrase "epitope consisting of TTEQ or a portion thereof" refers to an epitope that is specifically bound by an antibody that specifically or preferentially binds to the TTEQ (SEQ ID NO: 1) peptide compared to the peptide in which at least one or more of the residues, optionally 115T, 116T, 117E, and / or 118Q, are mutated to alanine or absent. Similarly, as used herein, the phrase "conformational epitope consisting of TTEQ or a portion thereof" refers to an epitope that is specifically bound by an antibody that preferentially binds to TTEQ (SEQ ID NO: 1) or a portion thereof when in a particular conformation (e.g., a misfolded protein, a cyclic, or some other restricted conformation) over another conformation (e.g., a native conformation), optionally when at least one or more of the residues, optionally 115T, 116T, 117E, and / or 118Q, are mutated to alanine or absent (e.g., a cyclic conformation of a peptide comprising TTEQ (SEQ ID NO: 1), versus a cyclic conformation of a peptide in which one or more of the residues are mutated or absent).

[0078] As used herein, the term "greater affinity" refers to the affinity of antibody X to target Z with a stronger affinity (K on ), and / or a smaller dissociation constant (K off ) where antibody X has a greater affinity for target Y than for Z. Similarly, the term "lower affinity" herein refers to the degree of antibody binding where antibody X binds to target Y weaker and / or with a larger dissociation constant than target Z, where antibody X has a lower affinity for target Y than for Z. The binding affinity between an antibody and its target antigen is expressed as K D K on / k off is equal to 1 / K D It can be expressed as KA, which is equal to k on and k off The value can be measured using surface plasmon resonance (which can be measured using, for example, a Biacore system).

[0079] Also, as used herein, the term "immunogenic" refers to a substance that elicits the production of antibodies and activates T cells and other reactive immune cells directed against the antigenic portion of the immunogen.

[0080] As used herein, "immunogen" refers to a substance that elicits an immune response and / or leads to the production of antibodies. In addition to the immunogenic compounds, conjugates, and fusions described herein, including, for example, isolated compounds conjugated to KLH, peptide mimetics can be used to elicit cross-reactive antibodies against identified epitopes, e.g., related epitopes such as TTEQ and / or TTE. To function as a useful immunogen, a TDP-43 peptide desirably incorporates a minimum of about 3, 4, 5, 6, or 7 TDP-43 residues, including at least T115 and / or T116 and / or E117, and optionally incorporates an immunogenicity enhancer such as KLH. As the number of residues in a cyclic peptide increases, the structure becomes more conformationally similar to the linear peptide. The optimal degree of similarity of the misfolded state compared to the native structure occurs at about 7 to 9 residues (see Tables 6C and 8A).

[0081] The term "corresponding linear compound" with respect to a cyclic compound refers to a compound, optionally a peptide, that contains or consists of the same sequence or chemical moieties as the cyclic compound, but in linear (non-cyclized) form.

[0082] As used herein, the term "nucleic acid sequence" refers to a sequence of nucleoside or nucleotide monomers consisting of naturally occurring bases, sugars, and intersugar (backbone) linkages. The term also includes modified or substituted sequences, including non-naturally occurring monomers or portions thereof. The nucleic acid sequences of the present application may be deoxyribonucleic acid sequences (DNA) or ribonucleic acid sequences (RNA) and may include naturally occurring bases, including adenine, guanine, cytosine, thymidine, and uracil. The sequences may also contain modified bases. Examples of such modified bases include aza- and deaza-adenine, guanine, cytosine, thymidine, and uracil, as well as xanthine and hypoxanthine. Nucleic acids can be either double-stranded or single-stranded and represent sense or antisense strands. Furthermore, the term "nucleic acid" includes complementary nucleic acid sequences as well as codon-optimized or synonymous codon equivalents. As used herein, the term "isolated nucleic acid sequence" refers to a nucleic acid that is substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or chemical precursors, or other chemicals when chemically synthesized. An isolated nucleic acid is also substantially free of sequences that naturally flank the nucleic acid from which it is derived (i.e., sequences located at the 5' and 3' ends of the nucleic acid).

[0083] "Operatively linked" is intended to mean that the nucleic acid is linked to a regulatory sequence in a manner that allows expression of the nucleic acid. Suitable regulatory sequences can be derived from a variety of sources, including bacterial, fungal, viral, mammalian, or insect genes. Selection of an appropriate regulatory sequence can be readily accomplished by one skilled in the art, depending on the host cell selected. Examples of such regulatory sequences include transcriptional promoters and enhancers, including translation initiation signals, or RNA polymerase binding sequences, ribosomal binding sequences. Furthermore, depending on the host cell selected and the vector used, other sequences, such as an origin of replication, additional DNA restriction sites, enhancers, sequences that provide for the inducibility of transcription, etc., can be incorporated into the expression vector.

[0084] As used herein, the term "vector" includes any intermediate vehicle for a nucleic acid molecule, which allows the nucleic acid molecule to be introduced into, for example, a prokaryotic and / or eukaryotic cell and / or introduced into a genome, including plasmids, phagemids, viral vectors such as bacteriophage or retroviral vectors, adeno-associated viral vectors, etc. As used herein, the term "plasmid" refers to a construct of extrachromosomal genetic material, usually a circular double-stranded DNA, that can replicate genetically independently of chromosomal DNA.

[0085] "At least moderately stringent hybridization conditions" refers to the selection of conditions that promote selective hybridization between two complementary nucleic acid molecules in solution. Hybridization can occur across all or part of the nucleic acid sequence molecule. The hybridizing portion is typically at least 15 (e.g., 20, 25, 30, 40, or 50) nucleotides in length. Those skilled in the art will understand that the stability of a nucleic acid duplex, or hybrid, is determined by its Tm, which is a function of sodium ion concentration and temperature in a sodium-containing buffer (Tm = 81.5°C - 16.6 (log 10 [Na+]) + 0.41 (% (G+C) - 600 / I), or a similar equation). Therefore, the parameters in the wash conditions that determine hybrid stability are sodium ion concentration and temperature. To identify molecules that are similar but not identical to a known nucleic acid molecule, a 1% mismatch can be expected to result in approximately a 1°C decrease in Tm; for example, if a nucleic acid molecule is desired to have >95% identity, the final wash temperature would be decreased by approximately 5°C. Based on these considerations, one of skill in the art would be able to readily select appropriate hybridization conditions. In a preferred embodiment, stringent hybridization conditions are selected. As an example, stringent hybridization can be achieved using the following conditions: hybridization in 5x sodium chloride / sodium citrate (SSC) / 5x Denhardt's solution / 1.0% SDS at Tm-5°C based on the above equation, followed by a 0.2x SSC / 0.1% SDS wash at 60°C. Moderately stringent hybridization conditions include a wash step in 3x SSC at 42°C. However, it is understood that equivalent stringency can be achieved using alternative buffers, salts, and temperatures. Additional guidance regarding hybridization conditions can be found below.Current Protocols in Molecular Biology, John Wiley&Sons, NY, 2002, and in: Sambrook et al., Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Laboratory Press, 2001.

[0086] As used herein, "specifically binds" with reference to an antibody means that the antibody recognizes and binds to its target antigen with greater affinity than to structurally different antigens and / or antigens that contain modified or mutated sequences. For example, a multivalent antibody may have a K of at least 1e-6, at least 1e-7, at least 1e-8, at least 1e-9, or at least 1e-10. D It binds to its target with an affinity of at least 1e-8. An affinity of at least 1e-8 is preferred. An antigen-binding fragment, such as a Fab fragment containing a variable domain, may find its target with an affinity that is 10- or 100-fold less than the multivalent interaction with the intact antibody.

[0087] As used herein, the term "selective" with respect to an antibody that preferentially binds to a form of TDP-43 (e.g., native or misfolded protein) means that the binding protein binds to the form with 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, at least 500-fold, or at least 1000-fold or more greater affinity. Thus, an antibody that is more selective for a particular conformation (e.g., misfolded protein, cyclic peptide) will preferentially bind to TDP-43 with at least 3-fold greater affinity compared to another form.

[0088] As used herein, the term "linker" refers to a chemical moiety that can be covalently attached to a peptide containing the TTEQ (SEQ ID NO: 1) epitope peptide and, optionally, linked to the N-terminus and C-terminus of the TTEQ (SEQ ID NO: 1) peptide to produce a cyclic compound. The linker can include a spacer, such as a cysteine ​​residue, and / or one or more functional moieties. The linker can be linked via the functional moiety to a carrier protein or an immunogen-enhancing component, such as keyhole limpet hemocyanin (KLH). The linker can be, for example, 1 to 9 amino acids.

[0089] As used herein, the term "spacer" refers to any non-immunogenic or low-immunogenic chemical moiety that can be covalently attached directly or indirectly to the N- and C-termini of a peptide to produce a cyclic compound with a longer length than the peptide itself. For example, a spacer can be linked to the N- and C-termini of a peptide consisting of TTEQ (SEQ ID NO: 1) to produce a cyclic compound with a longer backbone length than the TTEQ (SEQ ID NO: 1) sequence itself. That is, when cyclized, a peptide containing a spacer (e.g., of three amino acid residues) forms a larger closed circle than a peptide without the spacer. The spacer can include non-immunogenic moieties such as, but not limited to, repeats of G, A, or PEG when combined with a peptide such as GTTEQG (SEQ ID NO: 4), TTEQG (SEQ ID NO: 5), GTTEQ (SEQ ID NO: 6), etc. The spacer can contain or be attached to one or more functionalized moieties, such as one or more cysteine ​​(C) residues, which can be interspersed within the spacer or covalently attached to one or both ends of the spacer. A spacer is indirectly covalently linked to a peptide when a functional moiety, such as a C or D residue, is covalently attached to one or more termini of the spacer. A spacer can also contain a functional moiety in the spacer residue, such as when a biotin molecule is introduced into an amino acid residue.

[0090] As used herein, the term "functional moiety" refers to a chemical moiety that includes a "functional group," which refers to an atom or group of atoms that reacts with another group of atoms to form a chemical interaction between the two groups (so-called "complementary functional groups"). In the case of cysteine, the functional group can be -SH, which can be reacted to form a disulfide bond. Thus, the linker can be, for example, CCC. The reaction with another group of atoms can be a covalent bond or a strong non-covalent bond, as in the case of biotin-streptavidin bonds, which can have a Kd of ∼1e-14. As used herein, a strong non-covalent bond refers to an interaction with 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.

[0091] Proteins and / or other agents can be functionalized (e.g., attached) to the cyclic compound to either aid in immunogenicity or act as probes in in vitro studies. For this purpose, any functional moiety that can react (e.g., covalently or noncovalently but strongly) can be used. In one particular embodiment, the functional moiety is a cysteine ​​residue that is reacted to form a disulfide bond with an unpaired cysteine ​​on the protein of interest, which can be, for example, an immunogenicity-enhancing component such as keyhole limpet hemocyanin (KLH), or a carrier protein such as bovine serum albumin (BSA) used for in vitro immunoblotting or immunohistochemical assays.

[0092] As used herein, the term "react with" means that genetically there is an electron flow or transfer of electrostatic charge that results in the formation of a chemical interaction.

[0093] As used herein, the term "animal" or "subject" includes all members of the animal kingdom, including mammals, optionally including or excluding humans.

[0094] In understanding the scope of the present disclosure, the term "consisting of" and its derivatives, as used herein, are intended to be closed-form terms that specify the presence of stated features, elements, components, groups, integers, and / or steps, and to exclude the presence of other, unstated features, elements, components, groups, integers, and / or steps.

[0095] The recitation herein of numerical ranges by endpoints includes all numbers and fractions subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5). It is also to be understood that all numbers and fractions thereof are presumed to be modified by the term "about." Additionally, the terms "a," "an," and "the" are to be understood to include plural referents unless the context clearly dictates otherwise. The term "about" means the referenced number plus or minus 0.1 to 50%, 5 to 50%, or 10 to 40%, preferably 10 to 20%, and more preferably 10% or 15%.

[0096] Furthermore, the definitions and embodiments described in a particular section are intended to be applicable to other embodiments herein that are described as suitable as understood by those skilled in the art. For example, in the following sections, different aspects of the present invention are defined in more detail. Each aspect thus defined may be combined with any other aspect(s) unless expressly indicated to the contrary. In particular, any feature indicated as preferred or advantageous may be combined with any other feature(s) indicated as preferred or advantageous.

[0097] II. Epitopes and Binding Proteins The present inventors have identified epitopes in TDP-43, including TTEQ (SEQ ID NO: 1) at amino acids 115-118 on the TDP-43 protein, and TTE at amino acids 115-117 on the TDP-43 protein. The present inventors have further identified that these epitopes, or portions thereof, are conformational epitopes, and that TTE, TEQ, or TTEQ (SEQ ID NO: 1), or portions thereof, may be selectively accessible to antibody binding in misfolded protein species of TDP-43.

[0098] Based on one or more conformational differences identified between epitopes identified in native and biased TDP-43 assemblies, we designed conformationally restricted compounds and immunogens to produce antibodies that selectively or specifically bind to misfolded TDP-43.

[0099] Antibodies produced using the immunogen may be useful for detecting misfolded TDP-43.

[0100] As described in the Examples, cyclic compounds such as the cyclic peptides cyclo(CGGTTEQGG) (SEQ ID NO: 2), cyclo(CGTTEQG) (SEQ ID NO: 3), cyclo(CGTTEG) (SEQ ID NO: 28), and cyclo(CGTTEGG) (SEQ ID NO: 29) can capture the conformation of the epitope in misfolded TDP-43 relative to the native species. For example, the solvent-accessible surface area, RMSD structural alignment, and dihedral angle distribution of the amino acids in the cyclic 9-mer cyclo(CGGTTEQGG) (SEQ ID NO: 2) were found to be significantly different from the corresponding quantities in the native ensemble. This suggests that cyclic compounds can provide conformational epitopes that are conformationally distinct from the sequences presented in the native ensemble.

[0101] III. TTEQ (SEQ ID NO: 1) and TTE "epitope" compounds Thus, the present disclosure identifies a conformational epitope in TDP-43 consisting of amino acids TTEQ (SEQ ID NO: 1), or TTE, corresponding to amino acid residues 115-117 on TDP-43, and TEQ, corresponding to amino acids 116-118. As demonstrated in the Examples, TTEQ (SEQ ID NO: 1) and TTE were identified as regions prone to disorder in TDP-43. Residues TTE and TTEQ (SEQ ID NO: 1) appeared in computational predictions.

[0102] To identify regions that may be exposed in misfolded TDP-43, differences exist in the conformations exhibited by native and biased RRM1. For example, Table 5 shows that E117 is more exposed in the biased protein than in the native protein, and is also more exposed in the cyclic peptide cyclo(CGGTTEQGG). In the native protein, this residue participates in a salt bridge with Lys 137, which is inhibited in the biased conformation.

[0103] Some embodiments include compounds containing TDP-43 peptides that comprise or consist of related epitopes, such as TTEQ (SEQ ID NO: 1), TTE, and / or any portion of the foregoing, and when the peptide is TTEQ (SEQ ID NO: 1), the peptide is in a conformation that distinguishes it in at least one characteristic from TTE and / or TTEQ (SEQ ID NO: 1) in native TDP-43. In embodiments, the TDP-43 peptide is selected from TTE, TTEQ (SEQ ID NO: 1), KTTE (SEQ ID NO: 10), KTTEQ (SEQ ID NO: 12), TEQ, TEQD (SEQ ID NO: 8), or TTEQD (SEQ ID NO: 9). The TDP-43 peptides TTEQD (SEQ ID NO: 9), TEQD (SEQ ID NO: 8), KTTEQ (SEQ ID NO: 12), TTE, and TEQ can be used to raise antibodies encompassing epitopes collectively referred to herein as TTEQ (SEQ ID NO: 1) and related epitopes. In embodiments, the relevant epitopes include or consist of TTE, TEQD (SEQ ID NO: 8), KTTE (SEQ ID NO: 10), and epitopes comprising 1, 2, or 3 amino acids at either the N-terminus or C-terminus of TDP-43 relative to the epitope TTE.

[0104] In some embodiments, peptides, conformational peptides, including TTE, TEQ, or TTEQ (SEQ ID NO: 1) may include one or two additional residues at the N-terminus and / or C-terminus of TDP-43 of TTEQ (SEQ ID NO: 1), e.g., TTEQD (SEQ ID NO: 9) or KTTEQD (SEQ ID NO: 7). For example, three amino acids N-terminal to TTEQ (SEQ ID NO: 1) in TDP-43 is PWK, and three amino acids C-terminal to TTEQ (SEQ ID NO: 1) is DLK. In embodiments, the TDP-43 peptide is a maximum of six TDP-43 residues. In embodiments, the TDP-43 peptide is a maximum of five TDP-43 residues. In yet another embodiment, the TDP-43 peptide (e.g., in a compound such as a cyclic compound) is four TDP-43 residues, optionally TTEQ (SEQ ID NO: 1).

[0105] In embodiments, the compound further comprises a linker. The linker comprises a spacer and / or one or more functional moieties. The linker may comprise, for example, 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids, and / or equivalently functional molecules such as polyethylene glycol (PEG) moieties, and / or combinations thereof. In embodiments, the spacer amino acids are selected from non-immunogenic or low-immunogenic amino acid residues such as G and A. For example, the spacer may be GGG, GAG, G(PEG)G, PEG-PEG (also referred to as PEG2)-GG, etc. One or more functional moieties, e.g., amino acids containing a functional group, may be included to link the compound to a drug, a detectable tag, a carrier such as BSA, or an immunogenicity-enhancing component such as KLH.

[0106] In embodiments, the linker comprises GC-PEG, PEG-GC, GCG, or PEG2-CG.

[0107] In embodiments, the linker comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids.

[0108] In embodiments in which a peptide comprising TTE or TTEQ (SEQ ID NO: 1) contains one, two, or three additional residues found in TDP-43 that are N- and / or C-terminal to TTEQ (SEQ ID NO: 1), the linker in the cyclized compound is covalently attached to the N- and / or C-termini of the TDP-43 residue (e.g., if the peptide is KTTEQ (SEQ ID NO: 12), the linker is covalently attached to residues K and Q). Similarly, if the TDP-43 peptide is TTEQ (SEQ ID NO: 1), the linker is covalently attached to residues T and Q, and if the TDP-43 peptide is TTEQD (SEQ ID NO: 9), the linker is covalently attached to residues T and D.

[0109] The proteinaceous portion of the compound (or a compound in which the linker is also proteinaceous) can be prepared by chemical synthesis using techniques well known in protein chemistry, such as solid phase synthesis or synthesis in homogeneous solution.

[0110] In embodiments, the compound is a cyclic compound, for example, the cyclic compound includes a TDP-43 peptide including TTE or TTEQ (SEQ ID NO: 1). References herein to a "cyclic peptide" can refer to a fully proteinaceous compound (e.g., where the linker is 2, 3, 4, 5, 6, 7, 8, or 9 amino acids). It is understood that the properties described for the cyclic peptides determined in the examples can be incorporated into other compounds (e.g., cyclic compounds) that include non-amino acid linker molecules.

[0111] Accordingly, one embodiment provides a cyclic compound comprising the peptide TTEQ (SEQ ID NO: 1) (or a portion thereof, such as TTE) and a linker, wherein the linker is covalently attached directly or indirectly to the TTE or TTEQ (SEQ ID NO: 1)-containing peptide, and optionally wherein at least one of the T115, T116, E117, and / or Q118 residues is in an alternate conformation relative to the T, T, E, and Q residues in a native ensemble comprising TTEQ (SEQ ID NO: 1), as may appear in native TDP-43, and optionally wherein at least T, T, E, and / or Q is in either a more solvent-exposed conformation or an alternative conformation relative to the conformation occupied in a native ensemble comprising TTEQ (SEQ ID NO: 1), as may appear, for example, in a TDP-43 dimer.

[0112] In embodiments, the cyclic compound comprises a TDP-43 peptide comprising TTE or TTEQ (SEQ ID NO: 1) and up to six TDP-43 residues (e.g., one or two (or in the case of TTE, three) amino acids N- and / or C-terminal to TTE or TTEQ (SEQ ID NO: 1)), and a linker, wherein the linker is directly or indirectly covalently attached to the N- and C-terminal peptide residues of the TDP-43 peptide, and optionally at least T115, T116, E, or Q is in an alternate conformation relative to T115, T116, E, or Q in a native ensemble comprising TTEQ (SEQ ID NO: 1) and / or is in a conformation of T115, T116, E, or Q in TTEQ (SEQ ID NO: 1) in its native form, and optionally at least T115, T116, E, or Q has more surface exposure than the conformation occupied in a native ensemble comprising TTEQ (SEQ ID NO: 1).

[0113] The cyclic compound can be synthesized as a linear molecule containing a linker covalently attached to the N-terminus or C-terminus of the TDP-43 peptide, optionally a peptide containing TTEQ (SEQ ID NO: 1) or a related epitope, prior to cyclization. Alternatively, a portion of the linker is covalently attached to the N-terminus and a portion is covalently attached to the C-terminus prior to cyclization. In either case, the linear compound is cyclized, for example, via head-to-tail cyclization (e.g., amide bond cyclization).

[0114] In embodiments, the cyclic compound comprises a peptide comprising or consisting of TTEQ (SEQ ID NO: 1) and a linker, the linker being attached to the N-terminus and C-terminus of the peptide (e.g., the T and Q residues when the peptide consists of TTEQ (SEQ ID NO: 1). In embodiments, at least one of the T, E, and / or Q residues is in an alternate conformation in the cyclic compound than is occupied by at least one of the T, E, and / or Q residues in a native ensemble comprising TTEQ (SEQ ID NO: 1).

[0115] In embodiments, at least one of the T, E, and / or Q residues is in an alternate conformation in the cyclic compound than that occupied by the residues, optionally T, E, and / or Q, in the native population.

[0116] In embodiments, at least one of the T, E, and / or Q residues is in an alternate conformation in the cyclic compound than occupied by the residue in its native form.

[0117] In embodiments, the alternate conformation is the more solvent exposed conformation.

[0118] In embodiments, at least T115, optionally alone or in combination with T116, is in an alternate conformation relative to the conformation occupied in a native ensemble comprising TTEQ (SEQ ID NO: 1).

[0119] For example, the alternate conformation may include one or more different dihedral angles in residue T115 that differ from the dihedral angle in the native ensemble.

[0120] For example, the alternate conformations may include one or more different backbone dihedral angles (Ramachandran angles) that differ from the dihedral angles in the native ensemble.

[0121] In embodiments, the cyclic compound comprises a minimum average side chain / backbone dihedral angle difference between the cyclic compound and the native ensemble.

[0122] In embodiments, the cyclic compound comprises a residue selected from T, E, and Q, and one or more side chain or backbone dihedral angles differ in the cyclic compound from the corresponding dihedral angle in the context of the native ensemble by at least 30 degrees, at least 40 degrees, at least 50 degrees, at least 60 degrees, at least 70 degrees, at least 80 degrees, at least 90 degrees, at least 100 degrees, at least 110 degrees, at least 120 degrees, at least 130 degrees, at least 140 degrees, at least 150 degrees, at least 160 degrees, or at least 170 degrees.

[0123] As shown in Figures 2 and 3, the backbone and side chain dihedral angle distributions of some of T115 and T116 are substantially different in the cyclic peptide ensemble compared to the native ensemble. For example, Table 2A shows that for the simulated native ensemble and cyclic peptide, the difference in the dihedral angle C-CA-CB-OG1 of T115 is most likely about -160 degrees between the cyclic and native forms. In embodiments, the cyclic compound includes a T residue that includes a C-CA-CB-OG1 dihedral angle that is at least 30 degrees, at least 40 degrees, at least 50 degrees, at least 60 degrees, at least 70 degrees, at least 80 degrees, at least 90 degrees, at least 100 degrees, at least 110 degrees, at least 120 degrees, at least 130 degrees, at least 140 degrees, at least 150 degrees, or at least 160 degrees higher than the corresponding dihedral angle in the context of the native ensemble. Similarly, the difference in dihedral angle between the cyclic and native assemblies for the T116 dihedral N-CA-CB-OG1 is most likely about 150 degrees. Thus, in embodiments, the cyclic compound includes a T that includes a dihedral angle N-CA-CB-OG1 that differs by at least 30 degrees, at least 40 degrees, at least 50 degrees, at least 60 degrees, at least 70 degrees, at least 80 degrees, at least 90 degrees, at least 100 degrees, and up to at least 150 degrees, from the corresponding dihedral angle in the context of the linear compound. The corresponding difference in the most likely dihedral angle between the cyclic peptide and the native assemblies for the T115 dihedral C-CA-N-HN is 50 degrees. Thus, in embodiments, the cyclic compound includes a T that includes a dihedral angle for C-CA-N-HN that differs by at least 30 degrees, at least 40 degrees, or at least 50 degrees from the corresponding dihedral angle in the context of the native assemblies. The corresponding difference in the most likely dihedral angle between the cyclic peptide and the native ensemble for the T116 dihedral CB-CA-N-HN is -70 degrees. Thus, in embodiments, the cyclic compound comprises a T that comprises a dihedral angle for CB-CA-N-HN that differs by at least 30 degrees, at least 40 degrees, at least 50 degrees, at least 60 degrees, or at least 70 degrees from the corresponding dihedral angle in either the native ensemble or the native context.The angular difference may be, for example, positive or negative, (+) or (-).

[0124] According to the Ramachandran angle peak values ​​given in Table 4A, the most likely Ramachandran φ and ψ values ​​differ between the cyclic and native assemblies for residues T115, T116, E117, and Q118, as also presented in Table 4B. The differences Δφ between the cyclic and native peak φ values ​​are 50, -65, -40, and -35 degrees, respectively, and the differences Δψ between the cyclic and native peak ψ values ​​are -45, 30, 40, and 50 degrees, respectively. The overall φ and ψ values ​​differ significantly between the cyclic and native peptides. Table 3A provides the overlap of the φ and ψ value distributions in the cyclic and native assemblies. The average overlap of the φ and ψ distributions for T115, T116, E117, and Q118 is 28%, 63%, 55%, and 23%, respectively. Together, these numbers indicate the different distributions of dihedral angles for the cyclic and native assemblies.

[0125] In embodiments, the cyclic compound comprises Q comprising a Ramachandran backbone angle that differs by at least 30 degrees, at least 40 degrees, at least 50 degrees, or at least 60 degrees from the corresponding Ramachandran angle in the context of the naturally occurring compound.

[0126] The angular difference can be positive or negative, (+) or (-).

[0127] Alternate conformations can include alternate backbone orientations, e.g., different backbone orientations in which cyclic epitopes are exposed to antibodies compared to the native form.

[0128] In embodiments, T, T, E, Q, TT, TE, EQ, TTE, TEQ, and / or TTEQ (SEQ ID NO: 1) are in alternate conformations compared to those occupied by these residues in a non-misfolded protein conformation, e.g., a native ensemble.

[0129] In some embodiments, a cyclic compound containing a peptide containing TTEQ (SEQ ID NO: 1), TTE, or KTTE (SEQ ID NO: 10) can include, for example, one or more residues of TTEQ (SEQ ID NO: 1) upstream and / or downstream of one of the aforementioned TDP-43 sequences. In such cases, spacers are covalently attached to the N- and C-termini of the corresponding residues in the TDP-43 sequence.

[0130] In embodiments, the cyclic compound has a sequence selected from any one of SEQ ID NOs: 2, 3, 22, 23, 28, 29, 30, 31, 32, 33, 34, 35, and 42, or any subset thereof. In embodiments, the cyclic compound comprises a sequence selected from any one of SEQ ID NOs: 28, 29, 30, 31, 32, 33, 34, and 35. In another embodiment, the cyclic compound comprises a sequence selected from SEQ ID NOs: 2, 3, 22, 23, and 42. In yet other embodiments, the cyclic compound comprises a sequence selected from any one of SEQ ID NOs: 29, 32, and 34. In yet other embodiments, the cyclic compound comprises a sequence selected from any one of SEQ ID NOs: 2, 3, 22, and 23.

[0131] Methods for producing cyclized peptides are known in the art and include SS cyclization or amide cyclization (head-to-tail, or backbone cyclization). Methods are further described in the Examples section. For example, a peptide containing "C" residues at its N-terminus and C-terminus, such as CGTTEQGC (SEQ ID NO: 13), can be reacted by SS cyclization to produce a cyclic peptide. As described in the Examples, cyclic compounds were evaluated for their relevance to identified conformational epitopes. For example, cyclic compounds containing TTEQ (SEQ ID NO: 1) or TTE TDP-43 peptides can be used to generate antibodies selective for misfolded TDP-43.

[0132] As described herein, the epitope TTEQ (SEQ ID NO: 1) and / or portions thereof may be potential targets in strains that thrive on misfolded TDP-43, and antibodies that recognize conformational epitopes may be useful, for example, to detect such thriving strains.

[0133] Another aspect includes immunogens comprising compounds containing TTE, TTEQ (SEQ ID NO: 1), or related epitopes, optionally cyclic compounds described herein. In embodiments, the immunogen includes an immunogenicity-enhancing component such as keyhole limpet hemocyanin (KLH) and / or is formulated for co-injection with an adjuvant (e.g., alum, monophosphoryl lipid A, or QS21). Other similar components are known in the art, and additional adjuvants are described below. As further described below, the adjuvant is typically formulated in a composition with the compound. The immunogenicity-enhancing component can be attached to the compound either directly through an amide bond, disulfide bond, etc., or indirectly through a chemical linker. In another embodiment, the immunogen is a multiple antigenic peptide (MAP). For example, MAPs can be synthesized by preparing a linear compound to be cyclized, optionally cyclizing the peptide using head-to-tail cyclization, and attaching the cyclized peptide to a MAP resin through an amino acid in a linker, optionally through a C or D residue in the linker. MAPs are constructed to cyclic structures (see, e.g., Misumi et al., J. Virol. December 2001, vol. 75, no. 23, 11614-11620), and methods similar to those described herein can be used.

[0134] Immunogens containing immunogenicity-enhancing moieties can be produced by conjugating a cyclic compound containing a restricted epitope peptide and a linker containing a functional moiety such as cysteine ​​to an immunogenicity-enhancing moiety such as keyhole limpet hemocyanin (KLH) or a carrier such as bovine serum albumin (BSA), for example, using the methods described in Lateef et al. 2007, incorporated herein by reference. In embodiments, the methods described in Example 3 are used.

[0135] A further aspect is an isolated nucleic acid encoding a proteinaceous portion of a compound or immunogen described herein.

[0136] IV. Antibodies, Cells, and Nucleic Acids Therefore, compounds, particularly cyclic compounds, containing the epitope TTEQ (SEQ ID NO: 1) or related epitopes described above can be used to generate antibodies. Cyclic compounds containing TTEQ (SEQ ID NO: 1) or related epitopes, such as KTTE (SEQ ID NO: 10), TTE, TEQ, TTEQD (SEQ ID NO: 9), KTTEQD (SEQ ID NO: 7), KTTEQDL (SEQ ID NO: 14), and / or other related epitope sequences described herein can be used to generate antibodies that selectively bind to misfolded TDP-43.

[0137] Thus, for example, the compounds described herein, particularly cyclic compounds, including those containing the epitopes or cyclic compound sequences listed in Table 11, can be used to generate antibodies that specifically or selectively bind to epitopes in TDP-43 that recognize the specific conformations of these residues in misfolded TDP-43 that they contain and / or that contain one or more distinct features described herein.

[0138] Thus, certain aspects include antibodies that specifically or selectively bind to an epitope on TDP-43, the epitope comprising or consisting of TTEQ (SEQ ID NO: 1), a related epitope thereof, or a portion thereof, or any of the foregoing conformational epitopes. In embodiments, when the epitope consists of TTEQ (SEQ ID NO: 1), it is a conformational epitope.

[0139] In embodiments, the antibody is isolated.

[0140] In embodiments, the antibody does not specifically bind and / or is not selective for native TDP-43, e.g., the conformation of the relevant epitope, such as TTEQ (SEQ ID NO: 1) or TTE, as it exists in native TDP-43. Selective binding can be measured using ELISA or surface plasmon resonance measurements, as described herein.

[0141] Thus, a further aspect is an antibody that specifically or selectively binds to an epitope present on TDP-43, the epitope comprising or consisting of at least one amino acid residue primarily involved in antibody binding, the at least one amino acid being T115, T116, E, or Q embedded within the sequence TTEQ (SEQ ID NO: 1), TEQ, or KTTE (SEQ ID NO: 10), and optionally, when the epitope consists of TTEQ (SEQ ID NO: 1), it is a conformational epitope (e.g., selectively binds to the peptide in an alternate, optionally solvent-exposed conformation relative to the corresponding native ensemble, e.g., at least one amino acid of the epitope is more solvent-exposed). In embodiments, the epitope comprises or consists of at least two consecutive amino acid residues primarily involved in antibody binding, the at least two consecutive amino acids being TE or EQ embedded within TTEQ (SEQ ID NO: 1), TTE, or TEQ.

[0142] In another embodiment, the epitope is a conformational epitope and consists of TTEQ (SEQ ID NO: 1), TTE, or TEQ. In embodiments, the antibody selectively binds to TTEQ (SEQ ID NO: 1), TTE, or TEQ, or other related epitopes in cyclic compounds, optionally cyclic peptides, optionally cyclo(CGTTEQG) (SEQ ID NO: 3), or cyclic peptides having the sequence of any one of SEQ ID NOs: 2, 3, 22, 23, 28, 29, 30, 31, 32, 33, 34, 35, and 42, or any subset thereof, with respect to the corresponding linear peptide and / or native assembly.

[0143] In embodiments, the antibody specifically binds to a cyclic compound containing an epitope peptide described herein (e.g., an epitope in the cyclic compound compared to a native structural assembly) that contains at least one alternate conformational feature described herein. For example, an antibody that binds to a specific epitope conformation may also be referred to as a conformation-specific antibody. A conformation-specific antibody may differentially recognize specific misfolded TDP-43 species and may have a higher affinity for one species or a group of species compared to native species.

[0144] For example, in embodiments, an antibody specifically binds to a cyclic compound comprising residues selected from T, E, and Q, wherein at least one dihedral angle differs in the cyclic compound from the corresponding dihedral angle in the context of the native structure by at least 30 degrees, at least 40 degrees, at least 50 degrees, at least 60 degrees, at least 70 degrees, at least 80 degrees, at least 90 degrees, at least 100 degrees, at least 110 degrees, at least 120 degrees, at least 130 degrees, at least 140 degrees, or at least 150 degrees.

[0145] In embodiments, the antibody selectively binds to cyclic compounds comprising TTEQ (SEQ ID NO: 1) or portions thereof, optionally in the context of cyclo(CGGTTEQGG) (SEQ ID NO: 2) for naturally occurring assemblies comprising TTEQ (SEQ ID NO: 1), and optionally in the context of linear cyclo(CGTTEQG) (SEQ ID NO: 3) for naturally occurring assemblies comprising TTEQ (SEQ ID NO: 1). For example, in embodiments, the antibody selectively binds to TTEQ (SEQ ID NO: 1) or TTE in a cyclic conformation, and optionally has at least 2-fold, at least 3-fold, at least 5-fold, at least 8-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 100-fold, at least 500-fold, or at least 1000-fold greater selectivity (e.g., binding affinity) for TTEQ (SEQ ID NO: 1) or TTE in a cyclic conformation compared to TTEQ (SEQ ID NO: 1) or TTE in its native ensemble, as measured using the methods described herein, e.g., by ELISA, immunohistochemistry, or surface plasmon resonance.

[0146] In embodiments, the cyclic compound selectively bound to and / or used to produce antibodies is a compound in Table 11. In another embodiment, the cyclic compound is a cyclic compound selected from SEQ ID NOs: 2, 3, 22, 23, 28, 29, 30, 31, 32, 33, 34, 35, and 42, or any subset thereof. In embodiments, the cyclic compound comprises a sequence selected from any one of SEQ ID NOs: 28, 29, 30, 31, 32, 33, 34, and 35. In another embodiment, the cyclic compound comprises a sequence selected from SEQ ID NOs: 2, 3, 22, 23, and 42. In yet other embodiments, the cyclic compound comprises a sequence selected from any one of SEQ ID NOs: 29, 32, and 34. In yet other embodiments, the cyclic compound comprises a sequence selected from any one of SEQ ID NOs: 2, 3, 22, and 23.

[0147] In embodiments, the selectively conjugated cyclic compound is a compound in Table 11. In embodiments, the selectivity is at least 2-fold, at least 3-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 100-fold, at least 500-fold, or at least 1000-fold more selective for the cyclic compound and / or misfolded TDP-43 polypeptide over a species of TDP-43 selected from native TDP-43.

[0148] In yet another aspect, the disclosure provides antibodies that compete for selective binding to a cyclic peptide having a sequence selected from any one of SEQ ID NOs: 2, 3, 22, 23, 28, 29, 30, 31, 32, 33, 34, 35, and 42.

[0149] To produce monoclonal antibodies, antibody-producing cells (lymphocytes) can be obtained from a subject immunized with an immunogen as described herein and fused with myeloma cells in a standard somatic cell fusion procedure, thereby immortalizing these cells and generating hybridoma cells. Such techniques are well known in the art (e.g., the hybridoma technique originally developed by Kohler and Milstein (Nature 256:495-497 (1975)), as well as other techniques such as the human B-cell hybridoma technique (Kozbor et al., Immunol. Today 4:72 (1983)), the EBV hybridoma technique for producing human monoclonal antibodies (Cole et al., Methods Enzymol. 121:140-67 (1986)), and screening of combinatorial antibody libraries (Huse et al., Science 246:1275 (1989))). Hybridoma cells can be screened immunochemically for production of antibodies specifically reactive with the desired epitope and monoclonal antibodies can be isolated.

[0150] Specific antibodies or antibody fragments reactive with particular antigens or molecules can also be produced by screening expression libraries encoding immunoglobulin genes, or portions thereof, expressed in bacteria containing cell surface components. For example, complete Fab fragments, VH regions, 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)).

[0151] Humanization of antibodies from non-human species has been well described in the literature. See, for example, EP-B1 0 239400 and Carter & Merchant 1997 (Curr Opin Biotechnol 8, 449-454, 1997, which are incorporated herein by reference in their entirety). Humanized antibodies are also readily available commercially (e.g., Scotgen Limited, 2 Holly Road, Twickenham, Middlesex, Great Britain).

[0152] Humanized forms of rodent antibodies can be easily produced by CDR grafting (Riechmann et al. Nature, 332:323-327, 1988). In this technique, the six CDR loops comprising the antigen-binding site of a rodent monoclonal antibody are linked to corresponding human framework regions. CDR grafting often results in antibodies with reduced affinity because amino acids in the framework regions can affect antigen recognition (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 recombinant techniques, which can be assisted by computer modeling of the antigen-binding site (Co et al. J Immunol, 152:2968-2976, 1994).

[0153] Humanized forms of antibodies are optionally obtained by resurfacing (Pedersen et al. J Mol Biol, 235:959-973, 1994), a technique in which only surface residues of rodent antibodies are humanized.

[0154] Human antibodies specific to a particular antigen can be identified by a phage display strategy (Jespers et al., Bio / Technology, 12:899-903, 1994). In one approach, the heavy chain of a rodent antibody directed against a particular antigen is cloned from a repertoire of human light chains and paired with them for display as Fab fragments on filamentous phage. The phage are selected by binding to the antigen. The selected human light chains are then paired with a repertoire of human heavy chains for display on phage, and the phage are again selected by binding to the antigen. The result is a human antibody Fab fragment specific to the particular antigen. In another approach, a library of phage is produced in which members display different human antibody fragments (Fab or Fv) on their outer surface (Dower et al., WO91 / 17271 and McCafferty et al., WO92 / 01047). Phage displaying antibodies with the desired specificity are selected by affinity enrichment for a specific antigen. Human Fab or Fv fragments identified from either technique can be recloned for expression as human antibodies in mammalian cells.

[0155] Human antibodies are optionally obtained from transgenic animals (U.S. Patent Nos. 6,150,584, 6,114,598, and 5,770,429). In this technique, the heavy chain joining region (JH) gene is deleted in chimeric or germline mutant mice. The human germline immunoglobulin gene array is then transferred into such mutant mice. The resulting transgenic mice are then capable of generating a full repertoire of human antibodies in response to antigen challenge.

[0156] Humanized or human antibodies are selected from any class of immunoglobulin, including IgM, IgG, IgD, IgA, or IgE, and any isotype, including IgG1, IgG2, IgG3, and IgG4. Humanized or human antibodies can contain sequences from one or more isotypes or classes. Furthermore, these antibodies are typically produced as antigen-binding fragments, such as Fab, Fab', F(ab'), Fd, Fv, and single-domain antibody fragments, or as single-chain antibodies in which heavy and light chains are linked by a spacer. Human or humanized antibodies can also exist in monomeric or polymeric form. Humanized antibodies optionally contain one non-human chain and one humanized chain (i.e., one humanized heavy or light chain).

[0157] Furthermore, antibodies specific for the epitopes described herein are readily isolated by screening antibody phage display libraries. For example, antibody phage libraries are optionally screened using the disease-specific epitopes of the present invention to identify antibody fragments specific for the disease-specific epitopes. The identified antibody fragments are optionally used to produce recombinant antibody variants useful with different embodiments of the present invention. Antibody phage display libraries are commercially available through Xoma (Berkeley, California) methods for screening antibody phage libraries, which are well known in the art.

[0158] As shown below, several antibodies that were positive for detecting misfolded disease-associated TDP-43 in transfected cells or ALS spinal cord homogenates were sequenced.

[0159] Thus, in another embodiment, the antibody described herein comprises a light chain variable region and a heavy chain variable region, optionally fused together, 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 the CDRs comprise the following sequences: TIFF0007761373000003.tif74170

[0160] In another embodiment, the antibody described herein comprises a light chain variable region and a heavy chain variable region, optionally fused together, 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 the CDRs comprise the following sequences: TIFF0007761373000004.tif68170

[0161] In another embodiment, the antibody described herein comprises a light chain variable region and a heavy chain variable region, optionally fused together, 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 the CDRs comprise the following sequences: TIFF0007761373000005.tif74170

[0162] In another embodiment, the antibody described herein comprises a light chain variable region and a heavy chain variable region, optionally fused together, 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 the CDRs comprise the following sequences: TIFF0007761373000006.tif81170

[0163] In yet another embodiment, the antibody described herein comprises a light chain variable region and a heavy chain variable region, optionally fused together, 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 the CDRs comprise the following sequences: TIFF0007761373000007.tif81170

[0164] In yet another embodiment, the antibody described herein comprises a light chain variable region and a heavy chain variable region, optionally fused together, 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 the CDRs comprise the following sequences: TIFF0007761373000008.tif72170

[0165] As shown in Figures 13A and B, the CDRS share structural similarities. Specifically, the light chain sequences of several clones are similar.

[0166] In embodiments, the antibody is a monoclonal antibody. In embodiments, the antibody is a chimeric antibody, such as a humanized antibody, comprising the CDR sequences listed in Table 10.

[0167] In another embodiment, antibodies are also provided that comprise the CDRs and light and heavy chain variable regions of Table 10, optionally in the context of a single chain antibody.

[0168] In another aspect, the antibody comprises a heavy chain variable region comprising i) the amino acid sequence set forth in SEQ ID NO: 46, ii) an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% sequence identity to SEQ ID NO: 46, wherein the CDR sequences are as set forth in SEQ ID NOs: 67, 68, and 69, or iii) a conservatively substituted amino acid sequence of i). In another aspect, the antibody comprises a light chain variable region comprising i) the amino acid sequence set forth in SEQ ID NO: 48, ii) an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% sequence identity to SEQ ID NO: 48, wherein the CDR sequences are as set forth in SEQ ID NOs: 70, 71, and 72, or iii) a conservatively substituted amino acid sequence of i). In another embodiment, the amino acid sequence of the heavy chain variable region is encoded by the nucleotide sequence set forth in SEQ ID NO: 45, or a codon-degenerated or optimized version thereof. In another embodiment, the antibody comprises a light chain variable region amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 47, or a codon-degenerate or codon-optimized version thereof. In embodiments, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 46. In embodiments, the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 48.

[0169] In another aspect, the antibody comprises a heavy chain variable region comprising i) the amino acid sequence set forth in SEQ ID NO:50, ii) an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% sequence identity to SEQ ID NO:50, wherein the CDR sequences are as set forth in SEQ ID NOs:73, 74, and 75, or iii) a conservatively substituted amino acid sequence of i). In another aspect, the antibody comprises a light chain variable region comprising i) the amino acid sequence set forth in SEQ ID NO:52, ii) an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% sequence identity to SEQ ID NO:52, wherein the CDR sequences are as set forth in SEQ ID NOs:76, 77, and 78, or iii) a conservatively substituted amino acid sequence of i). In another embodiment, the amino acid sequence of the heavy chain variable region is encoded by the nucleotide sequence set forth in SEQ ID NO:49, or a codon-degenerated or optimized version thereof. In another embodiment, the antibody comprises a light chain variable region amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 51, or a codon-degenerate or codon-optimized version thereof. In embodiments, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 50. In embodiments, the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 52.

[0170] In another aspect, the antibody comprises a heavy chain variable region comprising i) the amino acid sequence set forth in SEQ ID NO: 54, ii) an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% sequence identity to SEQ ID NO: 54, wherein the CDR sequences are as set forth in SEQ ID NOs: 73, 79, and 80, or iii) a conservatively substituted amino acid sequence of i). In another aspect, the antibody comprises a light chain variable region comprising i) the amino acid sequence set forth in SEQ ID NO: 56, ii) an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% sequence identity to SEQ ID NO: 56, wherein the CDR sequences are as set forth in SEQ ID NOs: 76, 77, and 81, or iii) a conservatively substituted amino acid sequence of i). In another embodiment, the amino acid sequence of the heavy chain variable region is encoded by the nucleotide sequence set forth in SEQ ID NO: 53, or a codon-degenerated or optimized version thereof. In another embodiment, the antibody comprises a light chain variable region amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 55, or a codon-degenerate or codon-optimized version thereof. In embodiments, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 54. In embodiments, the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 56.

[0171] In another aspect, the antibody comprises a heavy chain variable region comprising i) the amino acid sequence set forth in SEQ ID NO:58, ii) an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% sequence identity to SEQ ID NO:58, wherein the CDR sequences are as set forth in SEQ ID NOs:67, 68, and 82, or iii) a conservatively substituted amino acid sequence of i). In another aspect, the antibody comprises a light chain variable region comprising i) the amino acid sequence set forth in SEQ ID NO:60, ii) an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% sequence identity to SEQ ID NO:60, wherein the CDR sequences are as set forth in SEQ ID NOs:83, 84, and 85, or iii) a conservatively substituted amino acid sequence of i). In another embodiment, the amino acid sequence of the heavy chain variable region is encoded by the nucleotide sequence set forth in SEQ ID NO:57, or a codon-degenerated or optimized version thereof. In another embodiment, the antibody comprises a light chain variable region amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 59, or a codon-degenerate or codon-optimized version thereof. In embodiments, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 58. In embodiments, the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 60.

[0172] In another aspect, the antibody comprises a heavy chain variable region comprising i) the amino acid sequence set forth in SEQ ID NO: 62, ii) an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% sequence identity to SEQ ID NO: 62, wherein the CDR sequences are as set forth in SEQ ID NOs: 86, 87, and 88, or iii) a conservatively substituted amino acid sequence of i). In another aspect, the antibody comprises a light chain variable region comprising i) the amino acid sequence set forth in SEQ ID NO: 64, ii) an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% sequence identity to SEQ ID NO: 64, wherein the CDR sequences are as set forth in SEQ ID NOs: 76, 77, and 92, or iii) a conservatively substituted amino acid sequence of i). In another embodiment, the amino acid sequence of the heavy chain variable region is encoded by the nucleotide sequence set forth in SEQ ID NO: 61, or a codon-degenerated or optimized version thereof. In another embodiment, the antibody comprises a light chain variable region amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 63, or a codon-degenerate or codon-optimized version thereof. In embodiments, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 62. In embodiments, the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 64.

[0173] In another aspect, the antibody comprises a heavy chain variable region comprising i) the amino acid sequence set forth in SEQ ID NO: 66, ii) an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% sequence identity to SEQ ID NO: 66, wherein the CDR sequences are as set forth in SEQ ID NOs: 89, 90, and 91, or iii) a conservatively substituted amino acid sequence of i). In another aspect, the antibody comprises a light chain variable region comprising i) the amino acid sequence set forth in SEQ ID NO: 64, ii) an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% sequence identity to SEQ ID NO: 64, wherein the CDR sequences are as set forth in SEQ ID NOs: 76, 77, and 92, or iii) a conservatively substituted amino acid sequence of i). In another embodiment, the amino acid sequence of the heavy chain variable region is encoded by the nucleotide sequence set forth in SEQ ID NO: 65, or a codon-degenerated or optimized version thereof. In another embodiment, the antibody comprises a light chain variable region amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 63, or a codon-degenerate or codon-optimized version thereof. In embodiments, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 66. In embodiments, the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 64.

[0174] Another embodiment is an antibody that specifically binds to the same epitope as an antibody having the CDR sequences listed in Table 10.

[0175] Another embodiment includes antibodies that compete for binding with human misfolded TDP43, including cyclic compounds described herein and / or antibodies described herein, such as antibodies comprising the CDR sequences listed in Table 10.

[0176] Competition between antibodies can be determined, for example, using an assay that evaluates the antibody under test for its ability to inhibit specific binding of a reference antibody to a common antigen. A test antibody competes with a reference antibody when an excess of the test antibody (e.g., at least 2-fold, 5-fold, 10-fold, or 20-fold) inhibits binding of the reference antibody by at least 50%, at least 75%, at least 80%, at least 90%, or at least 95%, as measured in a competitive binding assay.

[0177] A further aspect is an antibody conjugated to a detectable label. In embodiments, the detectable label is a positron-emitting radionuclide. Positron-emitting radionuclides can be used, for example, in PET imaging. In embodiments, the antibody is conjugated to a transport moiety that allows transport across the blood-brain barrier and / or into cells. For example, the antibody can be covalently linked to the iron transport protein melanotransferrin (p97) or fused to an antibody fragment specific for a BBB receptor such as the transferrin receptor, insulin receptor, lipoprotein receptor, basigin, Glut1, or CD98hc. Another example is fused to the BBB-penetrating single-domain antibody FC5 or a single-domain antibody directed against other BBB surface receptors. In embodiments, the antibody is conjugated to a transport moiety that facilitates entry into cells for diagnostic detection of intracellularly aggregated TDP43. For example, the antibody can be chemically linked or recombinantly fused to a cell-penetrating peptide such as transactivating transcription activator (TAT) and TAT derivatives, penetratin, or transportan.

[0178] A further aspect relates to an antibody conjugate comprising an antibody and / or a binding fragment thereof described herein and misfolded TDP-43. A further aspect is an isolated nucleic acid encoding an antibody or portion thereof described herein.

[0179] Nucleic acids encoding heavy or light chains are also provided, e.g., nucleic acids encoding heavy chains comprising the CDR-H1, CDR-H2, and / or CDR-H3 regions described herein, or encoding light chains comprising the CDR-L1, CDR-L2, and / or CDR-L3 regions described herein.

[0180] The present disclosure also provides variants of nucleic acid sequences encoding the antibodies and / or binding fragments thereof disclosed herein. For example, the variants comprise nucleotide sequences that hybridize to the nucleic acid sequences encoding the antibodies and / or binding fragments thereof disclosed herein under at least moderately stringent hybridization conditions or under codon-degenerated or sequence-optimized conditions. In another embodiment, the variant nucleic acid sequence has at least 50%, at least 60%, at least 70%, most preferably at least 80%, even more preferably at least 90%, and even most preferably at least 95% sequence identity to the nucleic acid sequence encoding any one of SEQ ID NOs: 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, and 65.

[0181] Another aspect is an expression cassette or vector comprising a nucleic acid disclosed herein. In embodiments, the vector is an isolated vector.

[0182] The vector can be any vector, including vectors suitable for producing antibodies and / or binding fragments thereof or expressing the peptide sequences described herein.

[0183] The nucleic acid molecule can be incorporated into a suitable expression vector that ensures protein expression using known methods. Possible expression vectors include, but are not limited to, cosmids, plasmids, or modified viruses (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses). The vector should be compatible with the host cell used. An expression vector is "suitable for transforming a host cell," meaning that the expression vector contains a nucleic acid molecule encoding a peptide corresponding to an epitope or antibody described herein.

[0184] In embodiments, the vector is suitable for expressing single-chain antibodies, for example, by gene therapy. The vector can be adapted for specific expression in neural tissue, for example, by using a neural-specific promoter. In embodiments, the vector contains an IRES, allowing the expression of light chain variable regions and heavy chain variable regions. Such vectors can be used to deliver antibodies in vivo.

[0185] Suitable regulatory sequences can be derived from a variety of sources, including bacterial, fungal, viral, mammalian, or insect genes.

[0186] Examples of such regulatory sequences include transcriptional promoters and enhancers or RNA polymerase binding sequences, ribosomal binding sequences, including translation initiation signals. Furthermore, depending on the host cell selected and the vector used, other sequences such as origins of replication, additional DNA restriction sites, enhancers, and sequences that provide for the inducibility of transcription may be incorporated into the expression vector.

[0187] In embodiments, the regulatory sequences direct or increase expression in neural tissues and / or cells.

[0188] In embodiments, the vector is a viral vector.

[0189] The recombinant expression vectors may also contain a marker gene that facilitates selection of host cells that have been transformed, affected, or transfected with the vector to express the antibodies or epitopic peptides described herein.

[0190] Recombinant expression vectors can also contain expression cassettes encoding fusion moieties (i.e., "fusion proteins") that provide increased expression or stability of the recombinant peptide, increased solubility of the recombinant peptide, and aid in purification of the target recombinant peptide by acting as a ligand in affinity purification, including, for example, the tags and labels described herein. Additionally, proteolytic cleavage sites can be added to the target recombinant protein to allow separation of the recombinant protein from the fusion moiety after purification of the fusion protein. Exemplary fusion expression vectors include pGEX (Amrad Corp., Melbourne, Australia), pMAL (New England Biolabs, Beverly, MA), and pRIT5 (Pharmacia, Piscataway, NJ), which fuse glutathione S-transferase (GST), maltose E-binding protein, or protein A, respectively, to the recombinant protein.

[0191] Systems for transcribing genes, for example, into neural cells and neural tissues, both in vitro and in vivo, include vectors based on viruses, most notably herpes simplex virus, adenovirus, adeno-associated virus (AAV), and retroviruses, including lentiviruses. Alternative approaches for gene delivery include the use of naked plasmid DNA and liposome-DNA complexes. Another approach is the use of AAV plasmids in which DNA is condensed with polycations, and lipids that are captured and introduced into the brain by intracerebral gene delivery (Leone et al. US Application No. 2002076394).

[0192] Thus, in another aspect, the compounds, immunogens, nucleic acids, vectors, and antibodies described herein can be formulated in vesicles such as liposomes, nanoparticles, and viral protein particles, for example, for delivery of the antibodies, compounds, immunogens, and nucleic acids described herein. Specifically, synthetic polymer vesicles, including polymersomes, can be used to administer antibodies.

[0193] In another aspect, cells expressing the antibodies or portions thereof described herein are also provided. In embodiments, the cells are isolated and / or recombinant cells that express the antibodies described herein or contain the vectors disclosed herein. In embodiments, the cells are fusion cells such as hybridomas.

[0194] Recombinant cells can be generated using any cell suitable for producing a polypeptide, e.g., for producing an antibody and / or binding fragment thereof. For example, to introduce a nucleic acid (e.g., a vector) into a cell, the cell can be transfected, transformed, or infected, depending on the vector used.

[0195] Suitable host cells include a wide variety of prokaryotic and eukaryotic host cells. For example, the proteins described herein can be expressed in bacterial cells such as E. coli, insect cells (using baculovirus), yeast cells, or mammalian cells.

[0196] In embodiments, the cell is a eukaryotic cell selected from a yeast, plant, worm, insect, avian, fish, reptile, and mammalian cell.

[0197] In another embodiment, the mammalian cell is a myeloma cell, a spleen cell, or a hybridoma cell.

[0198] In an embodiment, the cell is a neuronal cell.

[0199] Suitable yeast and fungal host cells for expressing antibodies or peptides include, but are not limited to, Saccharomyces cerevisiae, Schizosaccharomyces pombe, genera Pichia, or Kluyveromyces, and various species of the genus Aspergillus. Examples of vectors for expressing yeast S. cerevisiae include pYepSec1, pMFa, pJRY88, and pYES2 (Invitrogen Corporation, San Diego, CA). Protocols for transforming yeast and fungi are well known to those skilled in the art.

[0200] Mammalian cells that may be suitable include COS (e.g., ATCC No. CRL 1650 or 1651), BHK (e.g., ATCC No. CRL 6281), CHO (ATCC No. CCL 61), HeLa (e.g., ATCC No. CCL 2), 293 (ATCC No. 1573), and NS-1 cells, among others. Suitable expression vectors for directing expression in mammalian cells genetically include a promoter (e.g., derived from viral material such as polyoma, adenovirus 2, cytomegalovirus, and simian virus 40) and other transcriptional and translational control sequences. Examples of mammalian expression vectors include pCDM8 and pMT2PC.

[0201] In embodiments, the cells are fusion cells, such as hybridoma cells, that produce antibodies specific and / or selective for the epitopes or epitope sequences described herein, including, for example, hybridoma cells that selectively bind to TDP-43 pathological oligomers on TDP43 native structures, or that selectively bind to epitope sequences displayed in cyclic compounds relative to linear compounds, or that lack plaque binding or have negligible plaque binding.

[0202] A further aspect is a hybridoma cell line producing antibodies specific to the epitopes described herein.

[0203] V. Composition A further aspect is a composition comprising a compound, immunogen, nucleic acid, vector, or antibody described herein.

[0204] In an embodiment, the composition comprises a diluent.

[0205] Suitable diluents for nucleic acids include, but are not limited to, water, saline, and ethanol.

[0206] Suitable diluents for polypeptides, including antibodies or fragments thereof and / or cells, include, but are not limited to, saline, pH buffer solutions, and glycerol solutions, or other solutions suitable for freezing polypeptides and / or cells.

[0207] In embodiments comprising a compound or immunogen described herein, the composition comprises an adjuvant.

[0208] In embodiments, the adjuvant is selected from alum, monophosphoryl lipid A, and QS21.

[0209] Adjuvants that can be used, including intrinsic adjuvants (such as lipopolysaccharides), are typically components of killed or attenuated bacteria used in vaccines. Extrinsic adjuvants are typically immunomodulatory substances noncovalently bound to antigens and formulated to enhance the host immune response. Aluminum hydroxide, aluminum sulfate, and aluminum phosphate (collectively referred to as alum) are routinely used as adjuvants. A wide range of extrinsic adjuvants can elicit a strong immune response to immunogens. These include saponins such as Stimulons (QS21, Aquila, Worcester, Mass.), or particles derived therefrom, such as ISCOMs (immunostimulating complexes) and ISCOMATRIX, complexed to membrane protein antigens (immunostimulating complexes), pluronic polymers containing mineral oil, killed mycobacteria and mineral oil, complete Freund's adjuvant, bacterial products such as muramyl dipeptide (MDP) and lipopolysaccharide (LPS), and lipid A and liposomes.

[0210] In one embodiment, the adjuvant is aluminum hydroxide. In another embodiment, the adjuvant is aluminum phosphate. Oil-in-water emulsions include squalene, peanut oil, MF59 (WO 90 / 14387), SAF (Syntex Laboratories, Palo Alto, Calif.), and Ribi™ (Ribi Immunochem, Hamilton, Mont.). Oil-in-water emulsions may be used with immunostimulants such as muramyl peptides (e.g., N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP), -acetyl-normuramyl-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-acetylglucosaminyl-N-acetylmuramyl-L-AI-D-isoglu-L-ara-dipalmitoxypropylamide (DTP-DPP) ceramide (TM)), or other bacterial cell wall components.

[0211] The adjuvant can be administered with the immunogen as a single composition. Alternatively, the adjuvant can be administered before, simultaneously with, and / or after administration of the immunogen.

[0212] In embodiments, the composition comprises an antibody or portion thereof described herein. In another embodiment, the composition comprises an antibody or portion thereof described herein and a diluent. In embodiments, the composition is a sterile composition.

[0213] In embodiments, the composition is for the methods described herein, such as for detecting misfolded TDP-43.

[0214] VI. Kit Further aspects relate to kits comprising i) antibodies and / or binding fragments thereof, ii) nucleic acids, iii) peptides or immunogens, iv) compositions, or v) recombinant cells as described herein contained in a vial, such as a sterile vial, or other housing, and optionally reference agents and / or instructions for their use.

[0215] In an embodiment, the kit is an ELISA.

[0216] In embodiments, the kit comprises an antibody or binding fragment described herein contained in a container such as a sterile vial.

[0217] VII. Method The compounds, immunogens, and methods for producing antibodies described herein are included.

[0218] Specifically, methods are provided for generating antibodies selective for conformational epitopes of TTEQ (SEQ ID NO: 1) or related epitopes. In embodiments, the methods include administering an immunogen described herein to a subject and isolating antibodies that selectively bind to the TDP-43 peptide and / or misfolded TDP-43 of the immunogen.

[0219] Further embodiments provide methods for detecting whether a protein contains misfolded TDP-43, e.g., misfolded TDP-43 containing TTE, TTEQ (SEQ ID NO: 1), or a related conformational epitope, and / or at least one of residues T115, T116, E, or Q is in an alternate conformation than occupied by T, E, and / or Q in the non-misfolded protein conformation.

[0220] In an embodiment, the method comprises: a. contacting the sample with an antibody described herein under conditions permissive for producing an antibody:misfolded TDP-43 polypeptide complex; b. detecting the presence of any complexes; The presence of a detectable complex indicates that the sample may contain a misfolded TDP-43 polypeptide.

[0221] In another embodiment, the method comprises: (a) contacting a test sample of the subject with an antibody described herein under conditions permissive for producing an antibody-antigen complex; (b) measuring the amount of antibody-antigen complexes in the test sample; (c) comparing the amount of antibody-antigen complex in the test sample with a control, wherein detection of antibody-antigen complex in the test sample compared to the control indicates that the sample contains TDP-43, including a related epitope such as TTEQ (SEQ ID NO: 1) or TTE (e.g., misfolded TDP-43).

[0222] In embodiments, the sample is a biological sample. In embodiments, the sample comprises brain tissue, spinal cord tissue, or an extract thereof, and / or CSF. In embodiments, the sample is obtained from a human subject.

[0223] In an embodiment, the sample is from a subject with ALS. In another embodiment, the sample is from a subject with FTD.

[0224] Several methods can be used to determine whether misfolded TDP-43 polypeptides are present in a sample using the antibodies described herein, including flow cytometry, dot or slot blot, Western blot, ELISA, and immunoassays such as immunoprecipitation followed by SDS-PAGE immunocytochemistry. In embodiments, the method used comprises one or more of the steps described in Examples 7, 8, or 10.

[0225] Surface plasmon resonance can be used to assess conformational specific binding.

[0226] The labeled antibodies described herein can also be administered to a subject to detect the location of misfolded TDP-43.

[0227] Further embodiments include methods for inducing an immune response in a subject, comprising administering to the subject a compound, immunogen, and / or composition comprising a compound described herein, and optionally isolating cells and / or antibodies that specifically bind to the administered compound or immunogen. The above disclosure generally describes the present application. A more complete understanding can be obtained by reference to the following specific examples. These examples are provided for illustrative purposes only and are not intended to limit the scope of the present application. Changes and substitutions of equivalent forms are contemplated as the circumstances may suggest or may be appropriate. Although specific terms are used herein, such terms are intended in a descriptive sense and not for purposes of limitation.

[0228] The following non-limiting examples are illustrative of the present disclosure. [Example]

[0229] Example 1 TDP-43 epitope prediction Although most ALS / FTD mutations are in the C-terminus, the effect of these mutations may lead to pathological aggregation of TDP-43, and these multimeric aggregates may induce disruption of the structured domain. Therefore, the RRM1 domain was evaluated for the presence of conformation-specific epitopes present in misfolded TDP-43.

[0230] Putative misfolded epitopes in TDP-43 were predicted with the aid of a method called "collective coordinate biasing," described in WO / 2017 / 079836, SYSTEMS AND METHODS FOR PREDICTING MISFOLDED PROTEIN EPITOPES BY COLLECTIVE COORDINATE BIASING, filed November 9, 2016. As described herein, this method uses molecular dynamics-based simulations to impose global coordinate biases on a protein (or peptide aggregate) to misfold the protein (or peptide aggregate), and then predict the most likely unfolded regions of the partially unstructured protein (or peptide aggregate). Biased simulations are performed to obtain a solvent-accessible surface area (SASA) corresponding to each residue index (compared to that of the initial structure of the protein under consideration). SASA represents the surface area accessible to HO. A positive change in SASA (compared to that of the initial structure of the protein under consideration) can be considered to indicate unfolding in the region of the associated residue index. In addition to SASA, two other methods were used to identify candidate epitopes: loss of native contacts defined by non-hydrogen atoms within a cutoff length, and root mean square fluctuation (RMSF), which measures the range of deviation about the mean in a structural ensemble, where an increase in the RMSF for some amino acids indicates increased dynamics for those amino acids.

[0231] We applied this method to the natively folded RRM1 domain of TDP-43 (PDB entry 4IUF).

[0232] Simulations were performed for each initial structure using the methods described in WO / 2017 / 079836 and the CHARMM force field parameters described below. K. Vanommeslaeghe, E. Hatcher, C. Acharya, S. Kundu, S. Zhong, J. Shim, E. Darian, O. Guvench, P. Lopes, I. Vorobyov, and AD Mackerell. Charmm general force field: A force field for drug-like molecules compatible with the charmm all-atom additive biological force fields. Journal of Computational Chemistry,31(4):671-690,2010;and P.Bjelkmar,P.Larsson,MACuendet,B.Hess,and E.Lindahl.Implementation of the CHARMM force field in GROMACS:analysis of protein stability effects from correlation maps,virtual interaction sites,and water models.J.Chem.Theo.Comp.,6:459-466,2010,both of which are hereby incorporated herein by reference, with TIP3P water as solvent.

[0233] I. Conformational specific epitopes The present disclosure relates to antibodies that can be selective for misfolded TDP-43.

[0234] A prerequisite for generating misfolding-specific antibodies is the identification of targets for TDP-43 peptides that are not present (e.g., not accessible for binding) in the context of the native structure. These misfolding-specific epitopes will not differ in primary sequence from the corresponding segments in native TDP-43, but will be conformationally distinct in the context of the misfolded protein. That is, they will represent distinct conformations in the misfolded protein that are not present in the natively folded protein.

[0235] Antibodies directed against the native assembly region tend not to be selective for misfolded proteins and therefore bind to normal proteins as well. Because the concentration of normal proteins may be substantially higher than that of misfolded proteins, such antibodies may result in "target confusion" and bind primarily to normal proteins, promoting the removal of functional TDP-43 rather than selectively targeting and removing misfolded protein species. This may inhibit important RNA binding and stress response functions essential for cell survival.

[0236] To develop antibodies selective for misfolded protein forms of TDP-43, we used an ensemble coordinate algorithm to identify regions in the native protein that are likely to be inhibited upon application of an external perturbation force. Without wishing to be bound by theory, we hypothesized that inhibition in the native context may be similarly exposed on the surface of the misfolded protein. However, on the misfolded protein, these sequence regions may be exposed in a conformation distinct from that of native TDP-43. For example, on the surface, they may then be exposed in regions with a larger exposed surface area, a different dihedral angle distribution, and / or an overall different conformational geometry, as determined by structural alignment, than the corresponding quantities exhibited in either native ensemble.

[0237] Cyclic compounds comprising TTEQ (SEQ ID NO: 1) and TTE are described herein. The cyclic compounds are designed to meet one or more of the above criteria.

[0238] II. Epitope Prediction The epitopes TTEQ (SEQ ID NO: 1) and TTE appear as predicted epitopes.

[0239] The TTEQ (SEQ ID NO: 1) epitope appears as a prediction for PDB structure 4IUF when loss of native contacts is considered (Figure 1, panel B). TTE appears as an epitope for structure 4IUF when increased SASA is examined. TTE appears as an epitope for structure 4IUF when increased RMSF is examined.

[0240] For the plots of Figures 1-9 discussed herein, data are obtained from equilibrium simulations in explicit solvent (TIP3P) using the Charmm36 force field. The simulation times and number of conformations for each ensemble are as follows: Cyclic peptide ensemble: 100 ns simulation time, including a total of 5000 frames; biased ensemble: 90 ns simulation time for each unfolding trajectory; trajectory experiments were repeated 10 times, including a total of 40000 frames (equivalently, 90 * 10 = 900 ns). For simplicity, 8000 frames were evenly sampled from 40000 frames. Native 4IUF ensemble: 140 ns, including 7000 frames.

[0241] III.Dihedral angle distribution Further computational support for the identification of misfolded protein-selective epitopes is provided by both the side-chain dihedral angle distribution and the Ramachandran φ and ψ distributions for backbone dihedral angles in cyclic peptides, which are proxies for epitopes exposed in misfolded proteins. Some angles have distributions that differ substantially from the corresponding distributions in native TDP-43.

[0242] The side chain and backbone bifacial distributions were examined for four residues: T115, T116, E117, and Q118. The percent overlap of a distribution, e.g., "native" with a "cyclic" distribution, is obtained by dividing the angle into 5° components and then decreasing the cutoff in the probability amplitude from infinity until 90% of the cyclic distributions are above the cutoff and 10% remain below. This defines one or more regions of acceptable angles. The percent of native distribution within this region is then found. This recipe is non-reciprocal and genetically produces different numbers between pairs of distributions. The average overlap, e.g., the average of both native with cyclic and native with cyclic, is genetically considered.

[0243] As shown in Figure 2, for residue T115, the dihedral angles C-CA-CB-OG1, C-CA-N-HN, and OC-CA-N clearly distinguish the cyclic peptide of TTEQ (SEQ ID NO: 1) from the corresponding dihedral angles in the native ensemble. For residue T116, the dihedral angle OC-CA-N distinguishes the cyclic dihedral angle distribution from the corresponding distribution in the native ensemble. As shown in Table 1B, the dihedral overlap between the cyclic and biased ensembles is significantly increased over the overlap between the cyclic and biased ensembles for residues T115 and T116.

[0244] According to Figure 3, the backbone Ramachandran angles φ and ψ of T115 and T116 distinguish the cyclic peptides from the native population. For E117 and Q118, the cyclic peptide distributions are distinct but have overlap with the native and biased distributions, which are not significantly distinct from each other.

[0245] From the dihedral distribution shown in Figure 2, the probability that the native ensemble occupies a dihedral angle within nearly all (90%) of the cyclic peptide dihedral angles can be found. Similarly, the probability that the cyclic ensemble occupies a dihedral angle within nearly all (90%) of the native peptide dihedral angles can be found. The average of these alternative dihedral angles in Figure 2 is for residue TTEQ (SEQ ID NO: 1). T115:OC-CA-N 28%, T115:C-CA-CB-OG1 53%. All overlap probabilities are given in Table 1A. For residues T115 and T116, the overlap between the cyclic and biased ensembles is genetically greater than the overlap between the cyclic and native ensembles.

[0246] As described further below, the accumulation of relatively small differences in individual dihedral angles can lead to large and significant differences in the global conformation of the peptide, and therefore to significant deviations in the structural alignment.

[0247] Based on Figure 2, Table 1A shows the percent overlap of dihedral angle distributions for backbone and side chain angles of residues T115, T116, E117, and Q118 in the linear, cyclic peptide cyclo(CGGTTEQGG) (SEQ ID NO: 2) and native (4IUF) forms relative to each other. Column 1 is the specific dihedral angle being considered. Columns 2-7 represent the percent overlap of a considered dihedral angle of one ensemble in another ensemble. For example, column 2 shows the percent overlap between a given dihedral angle in the native ensemble and the same angle in cyclic peptide cyclo(CGGTTEQGG) (SEQ ID NO: 2).

[0248] Table 1B is derived from the numbers in Table 1A. The numbers in Table 1B show that the average overlap of the dihedral angle distribution between the cyclic and native forms is less than the average overlap of the dihedral angle distribution between the cyclic and biased forms, particularly for residues T115 and T116. This suggests that residues T115 and T116 may be the residues that confer the greatest conformational preference over the native form.

[0249] [Table 2] [Table 3]

[0250] Based on the data shown in Figure 2, Table 2A lists the peak values ​​of the dihedral angle distribution for those dihedral angles where the distribution shows a difference between the cyclic peptide cyclo(CGGTTEQGG) (SEQ ID NO:2) and the native ensemble. Column 1 is the particular dihedral angle under consideration, column 2 is the peak value of the dihedral distribution for that angle in the context of the cyclic peptide cyclo(CGGTTEQGG) (SEQ ID NO:2), column 3 is the peak value of the dihedral distribution for the peptide TTEQ (SEQ ID NO:1) in the context of the native structural ensemble, column 4 is the peak value of the dihedral distribution for the peptide TTEQ (SEQ ID NO:1) in the context of the biased structural ensemble, and column 5 is the difference in the peak value of the dihedral distribution for the cyclic and native ensembles. As shown in Table 2B, the difference is generally greater between the cyclic and native forms, followed by the cyclic and biased forms. Furthermore, averaged over a range of dihedral angles for a given residue, the magnitude of the difference (cyclic vs. native) minus (cyclic vs. bias) is largest for T115 and decreases monotonically from T115 to Q118. [Table 4] [Table 5]

[0251] IV. Ramachandran angle The backbone orientation at which the epitope is exposed to the antibody differs depending on whether the peptide is in a cyclic, biased, or native form. This discrepancy can be quantified by plotting the Ramachandran angles phi and psi (or φ and ψ) along the backbone for each residue T115, T116, E117, and Q118 in the three ensembles described above. Figure 3 plots the phi and psi angles sampled in equilibrium simulations for residues T115, T116, E117, and Q118. From panels A and B of Figure 3, it can be seen that the distribution of backbone dihedral angles for T115 and T116 in the cyclic peptide is different from the distribution of dihedral angles sampled for either native ensemble and more similar to the biased ensemble. This is further quantified in Table 3A.

[0252] The overlap of Ramachandran angle distributions, as defined above, is given in Table 3A. Table 3A shows the overlap probability of Ramachandran angles for residues T115, T116, E117, and Q118 presented in Figure 3. In particular, the average fraction of the cyclic peptide cyclo(CGGTTEQGG) (SEQ ID NO: 2) ensemble that adopts a conformation consistent with the native ensemble and the average fraction of the native ensemble that adopts a conformation consistent with the cyclic ensemble are 28%, 63%, 55%, and 23% for T115, T116, E117, and Q118, respectively. This is obtained by averaging both the psi and phi overlaps. This indicates that, for example, the orientations around T115 and Q118 in the cyclic peptide ensemble are conformationally distinct from the conformations of these residues in the native ensemble.

[0253] Table 3B shows the overlap probability averaged across both Ramachandran angles, e.g., column 2 for native cyclic and native cyclic, and similarly column 3 averages the cyclic and cyclic biases from Table 3A across phi and psi to achieve the net overlap percentage for a given residue. Table 3B shows that the Machandran angles of T115 and T116 result in the greatest conformational preference toward the biased misfolded form relative to the native form. [Table 6] [Table 7]

[0254] Table 4A provides the peak (most probable) values ​​of the Ramachandran φ, ψ angles plotted in Figure 3 for residues T115, T116, E117, and Q118. The second, third, and fourth columns show the peak Ramachandran phi / psi angle values ​​for each residue in the context of the cyclic peptide cyclo(CGGTTEQGG) (SEQ ID NO: 2), the native ensemble, and the non-native biased ensemble, respectively. The most probable Ramachandran phi and psi values ​​differ between the cyclic and native ensembles for residues T115, T116, E117, and Q118. As shown in Table 4B, the difference is slightly larger between the cyclic and native forms, and therefore between the cyclic and biased forms. When averaged over phi and psi angles, T115 shows the greatest discrimination between the above differences, so that the cyclic has a stronger overlap with the biased form than the native form, especially for residue T115. [Table 8] [Table 9]

[0255] V. Epitope Solubility and Solvent Exposure Panel A of Figure 4 plots the intrinsic solubility of each amino acid in TDP-43 RRM1. It can be seen that the epitope sequence TTEQ (SEQ ID NO: 1) is one of the more soluble regions in the protein sequence, indicating that this particular region is less susceptible to solvent accessibility biasing forces that pose an external challenge to the protein structure.

[0256] Panel B of Figure 4 and Table 5 provide the median solvent-accessible surface area (SASA) of each residue in the equilibrium ensemble of the cyclic peptide, the biased partially unfolded protein ensemble, and the native ensemble. This shows that the SASA of residue TTEQ (SEQ ID NO: 1) in the biased ensemble is increased over the native form, and similarly, the SASA of the cyclic peptide is increased over that in the biased ensemble, indicating that more surface is exposed and therefore accessible to antibody binding. The increase in exposure is most pronounced for residues T115 and E117, which show the largest increase in SASA over the native ensemble. For T115, this difference is 101 Å. 2 and for E117, this difference is 67 Å 2 is.

[0257] Figure 4C gives the intermediate SASA for each residue in Figure 4B, but now for the cyclic peptide of sequence cyclo(CGTTEQG) (SEQ ID NO: 3). Results are summarized for the cyclic peptide cyclo(CGGTTEQGG) (SEQ ID NO: 2).

[0258] Table 5 shows the SASAs of residues T115, T116, E117, and Q118 in the context of the cyclic cyclo(CGGTTEQGG) (SEQ ID NO: 2) ensemble, the biased ensemble, and the native ensemble. [Table 10]

[0259] VI. Ensembles of Cyclic Peptide Conformation Clusters Distinct from Ensembles of Either Linear or Fibrous Conformations Clear evidence that the sequence TTEQ (SEQ ID NO: 1) exhibits a different conformation in the context of a cyclic peptide than in the native ensemble can be seen by using standard structural alignment metrics between conformations and then performing clustering analysis. Equilibrium ensembles of conformations were obtained for native RRM1 (PDB 4IUF), biased RRM1, and the cyclic peptide cyclo(CGGTTEQGG) (SEQ ID NO: 2). Conformational snaps from these ensembles for residue TTEQ (SEQ ID NO: 1) were collected and structurally aligned to the centers of gravity of the largest cluster in the cyclic peptide ensemble, the largest cluster in the native ensemble, and the largest cluster of TTEQ (SEQ ID NO: 1) in the biased ensemble. Three values ​​of root mean square deviation (RMSD) were then recorded and plotted. Clustering, as used herein, was performed using the MaxCluster algorithm (http: / / www.sbg.bio.ic.ac.uk / maxcluster). The three corresponding RMSD values ​​for the cyclic, biased, and native assemblies are plotted as a three-dimensional scatter plot in Figure 7. Panel A of Figure 7 also includes results for the equilibrium assemblies of the linear peptide CGGTTEQGG (SEQ ID NO: 2). Figure 7C plots the overlap percentages between several assemblies. Two overlap numbers are particularly important. One is the overlap with the cyclic or native assemblies, including the non-native biased assemblies. The non-native biased assemblies are portions of the biased assemblies that do not contain conformations that overlap with the native assemblies. That is, any biased conformations that overlap with portions of the native assemblies are removed, and these are the biased conformations that differ from the native conformations. The higher this number, the better the epitope scaffold that selectively targets the locally unfolded state. The overlap between the cyclic peptide ensemble and the non-native biased ensemble is greater than the overlap between the linear peptide ensemble and the non-native biased ensemble. This justifies the use of cyclic scaffolds as proxies for non-native misfolded states. Cyclic peptides have substantially greater overlap with the non-native biased ensemble than with the native ensemble.

[0260] Another important number is the overlap between either the cyclic or linear structural ensemble and the native structural ensemble. This number should be low. The lower this number, the less likely antibodies raised to the epitope scaffold will target the native structure. The numerical overlap percentages are given in Table 6A. It is clear from Figure 7 and Table 6A below that the three ensembles cluster differently from each other. Specifically, the cyclic peptide structural ensemble is distinct from the native ensemble, suggesting that antibodies specific for cyclic peptide epitopes may have lower affinity for the conformational positions displayed in the linear or native ensemble. Antibodies raised to cyclic peptides may be conformationally selective, preferentially binding to misfolded protein forms over the native conformation of TDP-43. The distinction between the ensembles occurs despite overlap between some side chain and backbone dihedral angle distributions, and the many, often minor, distinctive features described above result in overall distinct conformational distributions.

[0261] Panels D, E, and F of Figure 7 depict the corresponding quantities as Figures 7A, B, and C, but now for the cyclic peptide cyclo(CGTTEQG) (SEQ ID NO: 3). The results are similar to those above: the cyclic peptide has substantially greater overlap with the non-native biased ensemble than with the native ensemble, and the cyclic peptide is a better epitope scaffold for the non-native biased state than the linear peptide.

[0262] Table 6A provides the percent overlap in the RMSD clustering scatter plot of the cyclic, biased, and native (4IUF) peptide conformations, as presented in Figure 7. Column 1 shows that the percent overlap from the cyclic to the native form is very small, only 5% for CGGTTEQGG (SEQ ID NO:2) and 4% for CGTTEQG (SEQ ID NO:3). Meanwhile, the percent overlap from the cyclic to the biased ensemble is nearly three-fold greater for CGGTTEQGG (SEQ ID NO:2), at about 14%, and about ten-fold greater for CGTTEQG (SEQ ID NO:3), at 42%. The cyclic ensemble samples non-native-like conformations approximately 95% of the time.

[0263] The overlap of cyclic peptides in the non-native biased ensemble provides the fraction of scaffolds in the average ensemble that accurately represent the misfolded non-native state. Similarly, the overlap of cyclic peptides in the native biased ensemble provides the fraction of scaffolds in the equilibrium ensemble that accurately represent the native state. The overlap of non-native biased ensemble in the cyclic peptide ensemble provides the similarity of the non-native misfolded state to the cyclic ensemble, and similarly, the overlap of native biased ensemble in the cyclic peptide ensemble provides the similarity of the native misfolded state to the cyclic ensemble. While this latter quantity is not of particular interest, the reciprocal overlap between the biased and cyclic ensembles are both metrics that quantify the appropriateness of using a cyclic scaffold. In Table 6B, to provide a goodness ratio for a cyclic peptide in targeting a non-native misfolded conformation versus a native conformation, these two metrics are averaged and divided by the overlap of the cyclic ensemble in the native form as a measure of goodness in using a given cyclic scaffold to target a non-native misfolded state versus a native form. Column 1 gives the sequence, column 2 gives the average of the overlap of the cyclic peptide in the non-native biased ensemble and the overlap of the non-native biased ensemble in the cyclic peptide ensemble, column 3 gives the overlap of the cyclic peptide in the native ensemble, column 4 gives the difference between column 2 and column 3, and column 5 gives the ratio of column 2 divided by column 3, which is defined as the goodness ratio for the cyclic peptide configuration. [Table 11] [Table 12] [Table 13] [Table 14] [Table 15]

[0264] Table 6C lists the goodness ratio (ability of a cyclic peptide to target non-native misfolded conformations versus native conformations) as defined in Table 6B for several candidate cyclic scaffolds as a function of several different cyclic radii, sorted by the goodness ratio reduction criteria. The two cyclic scaffold configurations analyzed, CGTTEQG (SEQ ID NO: 3) and CGGTTEQGG (SEQ ID NO: 2), had the highest goodness ratios and would therefore be predicted to be the best proxies for non-native misfolded structures using these evaluation criteria.

[0265] Table 6D lists the percent overlap between the cyclic and linear conformational ensembles. The ensembles differ from those seen by small to moderate percent overlap, indicating that the cyclic peptide ensembles are conformationally distinct from the linear peptide ensembles and are likely conformationally distinct from the nascent, unfolded peptide chains.

[0266] The overlap between ensembles was calculated as follows: First, the fraction (percentage) of the biased ensemble that overlaps with the circular ensemble is obtained by dividing the volume of this three-dimensional RMSD space into cubic elements of length 0.1 Å. Next, a "cutoff density" of points in the circular distribution is found such that a cube with a circular distribution density equal to or greater than the cutoff density contains 90% of the circular distribution. This defines a volume (which may be discrete) that gives a characteristic volume containing the circular distribution and eliminates any artifacts due to outliers. The fraction of points from the biased distribution that fall within this region is then found. This method allows for the overlap percentages of native circularity, biased circularity, etc. to be found.

[0267] Panels C and F of Figure 7 illustrate that the ensemble is large enough to be convergent.

[0268] Panel G of Figure 7 shows the correlation coefficients between both the cyclic-CGGTTEQGG (SEQ ID NO: 2) ensemble and the native ensemble, and between the cyclic-CGGTTEQGG (SEQ ID NO: 2) ensemble and the non-native biased ensemble, computed as follows: The correlation coefficient between two ensembles is defined by finding the portion of the distribution that has a density greater than a cutoff value, so that it encompasses a given percentage of the total distribution, e.g., a density cutoff for the cyclic and linear distributions that gives 60% of the total distribution. The correlation coefficients for these subdistributions are then calculated as

number

[0269] Panel H of Figure 7, similar to Figure 7G, shows the correlation coefficients between the cyclic (CGTTEQG) (SEQ ID NO: 3) ensemble and both the native ensemble and the non-native biased ensemble. The correlation coefficients between the native and cyclic distributions converge to about 7% when 100% of each distribution is included, and the correlation coefficients between the non-native biased and cyclic distributions converge to about 25%, or about a 3.6-fold overlap, when 100% of each distribution is included.

[0270] Panel I of Figure 7 examines the effect of single residue deletions on structural overlap, defined by averaging the overlap of the native ensemble with 90% of the cyclic (CGGTTEQGG) (SEQ ID NO: 2) ensemble and the cyclic ensemble with 90% of the native ensemble. If a single amino acid confers conformational preference over the native conformation, removing it from the structural alignment will result in significantly higher overlap between the distributions. In this study, T115 stands out as conferring the highest conformational preference for cyclic peptides.

[0271] Panel J of Figure 7 depicts the effect of a single residue deletion for the cyclic peptide cyclo(CGTTEQG) (SEQ ID NO: 3), similar to Figure 71. Again, residue T115 stands out as conferring conformational preference on the cyclic peptide.

[0272] Panel A of Figure 5 shows a schematic atomic structure of the most representative conformation of TTEQ (SEQ ID NO: 1) from the cyclic peptide ensemble of CGGTTEQGG (SEQ ID NO: 2), which constitutes the centroid of the largest cluster from the structural cyclic peptide ensemble. Similarly, the most representative conformation of the native structural ensemble, which constitutes the centroid of the largest cluster, is shown in light gray in Figure 5, optimally superimposed on the cyclic peptide shown in black by aligning them using RMSD to clarify their different orientations. Specifically, T115 has a significantly different orientation between the two centroid conformations. Panel B of Figure 5 shows the corresponding centroid conformations for the cyclic peptide and native ensemble for the cyclic sequence CGTTEQG (SEQ ID NO: 3), again optimally superimposed by aligning with respect to RMSD. Both cyclic configurations show that T115 is in an alternate conformation, and to a lesser extent, Q118 also appears in an alternate conformation.

[0273] Table 7 lists the values ​​of the Ramachandran backbone and side chain dihedral angles occupied by residues T115, T116, E117, and Q118 in the centroid structure of the cyclic CGGTTEQGG (SEQ ID NO: 2) peptide ensemble, the centroid structure of the native ensemble, and the centroid structure of the non-native biased ensemble, and the cyclic and native centroid conformations are depicted in Figure 5. The centroid structures display several dihedral angles that differ substantially between the cyclic and native conformations. Column 1 of Table 7 gives the residue and dihedral angle of interest, column 2 gives the dihedral angle value in the centroid structure of the cyclic ensemble, column 3 gives the dihedral value in the centroid of the native ensemble, column 4 gives the dihedral value in the centroid of the non-native biased ensemble, column 5 gives the magnitude of the difference between the cyclic and native dihedrals, column 6 gives the magnitude of the difference between the cyclic and non-native biased dihedrals, column 7 gives the value of column 5 averaged over the dihedrals in each amino acid, and column 8 gives the value of column 6 averaged over the dihedrals in each amino acid. It is clear that many of the cyclic dihedral angles are significantly different from the corresponding dihedral angles in the linear or native centroid, as described above for the peak values ​​of the dihedral distribution. Note, however, that the dihedral angle in the centroid structure need not be the same as the peak value of the dihedral distribution. For the example difference herein, the dihedral OG1-CB-CG2-2HG2 at residue 115T shows a 100 degree difference between the cyclic and native forms, but only a 15 degree difference between the cyclic and non-native biases. The average difference between the cyclic and native forms for 115T is 87.6 degrees, and the average difference between the cyclic and non-native biases for 115T is 51.9 degrees. Again, note that these differences are for only one conformation (the center of gravity conformation) in each of the respective ensembles. [Table 16-1] [Table 16-2] [Table 16-3]

[0274] Figure 6 again shows TTEQ (SEQ ID NO: 1) at the native, biased, and cyclic peptide centroids for CGGTTEQGG (SEQ ID NO: 2) and CGTTEQG (SEQ ID NO: 3), using a surface area representation for residue TTEQ (SEQ ID NO: 1). The surface area profile presented to the antibody differs between the centroid conformations. The conformations are shown separately but are all aligned. Comparing the centroid conformations of the native, biased, and cyclic assemblies, the exposed surface area of ​​E117 and similarly T115 increases monotonically, in agreement with the SASA depicted in Figures 4B and 4C. Therefore, antibodies raised to this region in the cyclic assemblies of TDP-43 would be unlikely to bind (e.g., with equal or similar selectivity) to TTEQ (SEQ ID NO: 1) in native TDP-43.

[0275] Figure 6E shows an example of an interaction present in the native ensemble of TDP-43 RRM1 that buries the surface area of ​​the side chain of E117 but is inhibited upon biasing of the partially unfolded protein. In the native ensemble, E117 forms a salt bridge with lysine 137 (K137), restricting the side chain of each residue and reducing the surface area exposure of E117 in the native ensemble. However, in the biased ensemble (Figure 6E, right panel), the salt bridge is broken, exposing the side chain of E117. As depicted in Figures 4B and C, other interactions within the protein are also weakened sufficiently to increase the exposure of T115.

[0276] Example 2 Clustering by RMSD for TTEs in the cyclic compounds CGTTEG and CGTTEGG A similar set of experiments was performed for TTE in the cyclic compounds CGTTEG (SEQ ID NO: 28) and CGTTEGG (SEQ ID NO: 29).

[0277] The RMSD of the TTE cyclic compounds was analyzed as in Example 1. A clustering plot by root mean square deviation (RMSD) is shown in Figure 8. The axes correspond to the RMSD of TTE relative to the centroid structure of the cyclic peptide ensemble cyclo(CGTTEG) (SEQ ID NO: 28) (Panel A) or cyclo(CGTTEGG) (SEQ ID NO: 29) (Panel B), the RMSD of TTE relative to the centroid structure of the native structural ensemble (equilibrium starting from PDB ID 4IUF), and the RMSD of TTE relative to the centroid structure of the non-native biased ensemble (biased ensemble with overlapping points from the native ensemble removed). Each point corresponds to a given conformation taken from either the cyclic peptide equilibrium ensemble (circle as noted in the legend), the non-native biased equilibrium ensemble (+ symbol as noted in the legend), or the native structural equilibrium ensemble starting from PDB ID 4IUF (inverted triangle as noted in the legend).

[0278] Figure 9 shows the percent overlap between the different ensembles as a function of the number of configurations sampled to demonstrate convergence. The cyclic ensembles correspond to the sequence cyclo(CGTTEG) (SEQ ID NO: 28) (Panel A) or cyclo(CGTTEGG) (SEQ ID NO: 29) (Panel B). As in Figure 7, the percent overlap for each pair of ensembles indicates the percent overlap of the particular ensemble with 90% of the comparison structure ensemble, as described in the text of Example 2. The numerical overlap percentages are given in Table 8A below. The overlap of the cyclic ensemble with the native ensemble was zero within the numerical accuracy of the simulation; again, low similarity to native structures is a desired feature for cyclic peptide scaffolds. [Table 17]

[0279] Table 8A shows the percentage overlap of RMSD clustering between native cyclic, non-natively biased cyclic, and non-natively biased cyclic in the cyclic forms of cyclopeptides as presented in the panels of Figure 9 for scaffolds cyclo(CGTTEG) (SEQ ID NO: 28) and cyclo(CGTTEGG) (SEQ ID NO: 29). The last column gives the ratio defined by dividing the average of columns 2 and 3 by column 4 (provided in the right-most column). The two scaffolds cyclo(CGTTEG) (SEQ ID NO: 28) and cyclo(CGTTEGG) (SEQ ID NO: 29) show the greatest differentiation between the non-natively biased and native ensembles, with a ratio of overlap equal to infinity (the cyclic ensemble has no overlap with the native ensemble).

[0280] Additional computer simulations were performed to measure the similarity between various ensembles by measuring the Jensen-Shannon distance between cyclic-native, cyclic-biased, and cyclic-(unfolded monomer) ensemble pairs [Lindorff-Larsen K, Ferkinghoff-Borg J (2009) Similarity Measures for Protein Ensembles. PLOS ONE 4(1):e4203]. Applying weights to these metrics, along with weight uncertainty, allows for the use of multiple criteria decision analysis [Hwang CL, Yoon K. Multiple attribute decision making: methods and applications a state-of-the-art survey. Vol. 186. Springer Science & Business Media; (2012)] to select viable candidates that differentiate misfolded / biased TDP-43 from native TDP-43 and prefolded TDP-43 monomers (i.e., transiently unfolded TDP-43 monomers). A 77-residue sequence in RRM1 was used to model the unfolded monomer of the RRM1 domain, starting at amino acid 103 and ending at amino acid 179. Analysis was performed and used to evaluate the similarity of the TTE and TTEQ (SEQ ID NO: 1) epitopes in the context of the biased TDP-43 RRM1 domain to various cyclic peptides, including the peptides, in the context of different scaffolds consisting of a variable number of glycine spacers. Ensemble overlap was measured for the same simulation data in terms of the number of standard deviations between two separated Gaussian distributions. All of the ensemble overlap data is shown in Table 8B. [Table 18]

[0281] Cyclic peptides containing scaffolds (linkers) with up to nine amino acids (SEQ ID NO: 35) that overlap significantly with the TTE and TTEQ (SEQ ID NO: 1) epitopes in the context of the biased TDP-43 RRM1 domain as measured by the JSD data in Table 8B, but with less overlap with either natively folded or transiently unfolded TDP-43, are given in Table 8C. [Table 19]

[0282] Example 3 Cyclic compound architecture containing conformationally restricted epitopes Peptides containing a TTE such as TTEQ (SEQ ID NO: 1) or cyclo(CGTTEQG) (SEQ ID NO: 3) or cyclo(CGTTEG) (SEQ ID NO: 28) can be cyclized head-to-tail.

[0283] Native assemblies containing TTEQ (SEQ ID NO: 1) or TTE and a linker, preferably containing two, three, or four amino acids and / or PEG units, can be synthesized using known methods, such as Fmoc-based solid-phase peptide synthesis, alone or in combination with other methods. PEG molecules can be attached to amino groups at the N-terminus using, for example, the linking chemistry described in Hamley 2014 [7] and Roberts et al. 2012 [8], each of which is incorporated herein by reference. Native assemblies can be cyclized by 1) forming a peptide bond (e.g., cyclizing the backbone) at the amino and carboxy termini of the peptide + linker, 2) covalently linking the amino or carboxy termini with side chains in the peptide + linker, or 3) covalently linking two side chains in the peptide + linker.

[0284] The bonds in the cyclic compounds may be all regular peptide bonds (homodetic cyclic peptides) or may include other types of bonds such as ester, ether, amide, or disulfide linkages (heterodetic cyclic peptides).

[0285] Peptides can be cyclized by oxidation of thiol- or mercaptan-containing residues at the N- or C-terminus, or internal to the peptide, including, for example, cysteine ​​and homocysteine. For example, two cysteine ​​residues flanking the peptide can be oxidized to form a disulfide bond. Oxidizing agents that can be used include, for example, oxygen (air), dimethyl sulfoxide, glutathione oxide, cystine, copper(II) oxide, potassium ferricyanide, thallium(III) trifluoroacetate, or other oxidizing agents, used in conjunction with methods known to those skilled in the art.

[0286] Methods and compositions related to cyclic peptide synthesis are described in U.S. Patent Publication No. 2009 / 0215172. U.S. Patent Publication No. 2010 / 0240865, U.S. Patent Publication No. 2010 / 0137559, and U.S. Patent No. 7,569,541 describe various methods for cyclization. Other examples are described in International Patent Publication No. WO 01 / 92466 and Andreu et al., 1994. Methods in Molecular Biology 35:91-169.

[0287] More specifically, cyclic peptides containing the TTEQ (SEQ ID NO: 1), TEQ, or TTE epitope can be constructed by adding a linker containing a spacer to cysteine ​​residues flanked by and / or inserted within the spacer. The peptide can be configured into a cyclic conformation by creating a disulfide linkage between non-naturally occurring cysteine ​​residues added to the N- and C-termini of the peptide. Peptides can also be synthesized into cyclic compounds by forming a peptide bond between the N- and C-terminal amino acids (e.g., head-to-tail cyclization).

[0288] Peptide synthesis was performed by CPC Scientific Inc. (Sunnyvale, CA, USA) according to standard manufacturing procedures. The structure of the cyclic peptide was designed to mimic the conformation and orientation of the amino acid backbone and side chains of TTEQ (SEQ ID NO: 1) in the misfolded TDP-43 polypeptide.

[0289] cyclo(CGTTEQG) (SEQ ID NO: 3) Cyclo(CGTTEQG) (SEQ ID NO: 3) and cyclo(CGGTTEQGG) (SEQ ID NO: 2) can be synthesized using the following method (CPC Scientific Inc., Sunnyvale, CA). The protected native assemblies were synthesized by standard conventional Fmoc-based solid-phase peptide synthesis on 2-chlorotrityl chloride resin, followed by cleavage of the resin with 30% HFIP / DCM. The protected native assemblies were cyclized to the corresponding protected cyclic peptides using EDC. HCl / HOBt / DIEA in DMF at low concentrations were used. The protected cyclic peptides were deprotected with TFA to give the crude cyclic peptides, which were purified by RP HPLC to give the pure cyclic peptides after lyophilization.

[0290] Cyclo(CGTTEQG) (SEQ ID NO: 3) and cyclo(CGGTTEQGG) (SEQ ID NO: 2) can be prepared by amide condensation of the linear peptides CGTTEQG (SEQ ID NO: 3) or CGGTTEQGG (SEQ ID NO: 2), respectively.

[0291] Cyclo(C-PEG2-TTEQG) (SEQ ID NO: 24) and cyclo(C-PEG2-TTEQGG) (SEQ ID NO: 25) can be prepared by amide condensation of the linear compounds C-PEG2-TTEQG (SEQ ID NO: 24) or C-PEG2-TTEQGG (SEQ ID NO: 25), respectively.

[0292] Cyclo(CGTTEQ-PEG2) (SEQ ID NO: 26) and cyclo(CGGTTEQ-PEG2) (SEQ ID NO: 27) can be prepared by amide condensation of the linear compounds CGTTEQ-PEG2 (SEQ ID NO: 26) or CGGTTEQ-PEG2 (SEQ ID NO: 27), respectively.

[0293] Linear (CGTTEQG) (SEQ ID NO: 3) or linear (CGGTTEQGG) (SEQ ID NO: 2) can be prepared (CPC Scientific Inc, Sunnyvale, CA). The protected native assemblies were synthesized by standard conventional Fmoc-based solid-phase peptide synthesis on Fmoc-Gly-Wang resin, and the protected peptides were then cleaved with TFA to give the crude peptides, which were purified by RP HPLC to give the pure peptides after lyophilization, which were used for conjugation to BSA.

[0294] Similarly, cyclo(CGTTEG) (SEQ ID NO:28) and cyclo(CGTTEGG) (SEQ ID NO:29) can be prepared by amide condensation of the linear peptides CGTTEG (SEQ ID NO:28) or CGTTEGG (SEQ ID NO:29), respectively.

[0295] Cyclo(C-PEG2-TTEG) (SEQ ID NO: 36) and cyclo(C-PEG2-TTEGG) (SEQ ID NO: 37) can be prepared by amide condensation of the linear compounds C-PEG2-TTEG (SEQ ID NO: 36) or C-PEG2-TTEGG (SEQ ID NO: 37), respectively.

[0296] Cyclo(CGTTE-PEG2) (SEQ ID NO: 38) and cyclo(CGGTTE-PEG2) (SEQ ID NO: 39) can be prepared by amide condensation of the linear compounds CGTTE-PEG2 (SEQ ID NO: 38) or CGGTTE-PEG2 (SEQ ID NO: 39), respectively.

[0297] Cyclic and linear (CGTTEG) (SEQ ID NO: 28) or linear (CGGTTEGG) (SEQ ID NO: 30) peptides can be prepared (CPC Scientific Inc, Sunnyvale, CA). Protected versions were synthesized by standard conventional Fmoc-based solid-phase peptide synthesis on Fmoc-Gly-Wang resin, followed by cleavage of the protected peptide with TFA to give the crude peptide, which was purified by RP HPLC to give the pure peptide after lyophilization, which was used to conjugate to BSA.

[0298] Immunogen composition The cyclic compounds cyclo(CGTTEQG) (SEQ ID NO: 3), cyclo(CGGTTEQGG) (SEQ ID NO: 2), cyclo(CGTTEG) (SEQ ID NO: 28), and cyclo(CGTTEGG) (SEQ ID NO: 29) were synthesized as described above and then conjugated to KLH (for immunization) or BSA (for screening) (CPC Scientific Inc, Sunnyvale, CA). BSA or KLH was reactivated with SMCC in PBS buffer, and then a solution of the pure peptide in PBS buffer was added to the conjugation mixture, which was stirred at room temperature for 2 hours. The conjugation mixture was then lyophilized after dialysis to give the conjugated product.

[0299] Peptides can also be conjugated to KLH (for immunization) and BSA (for screening) using a trifluoroacetic acid counterion protocol. Peptides are desalted and checked by MS and HPLC to confirm at least 95% purity.

[0300] Example 4 Antibody generation and selection A conformationally restricted compound, optionally a cyclic compound such as a cyclic peptide containing a TTE, such as TTEQ (SEQ ID NO: 1) or cyclo(CGTTEQG) (SEQ ID NO: 3), cyclo(CGGTTEQGG) (SEQ ID NO: 2), cyclo(CGTTEG) (SEQ ID NO: 28), or cyclo(CGTTEGG) (SEQ ID NO: 29), is linked to keyhole limpet hemocyanin (KLH). The linked peptide or peptides are sent to Antibodies LTD (Victoria, BC, Canada) for production of mouse monoclonal antibodies according to protocols approved by the Canadian Council on Animal Care.

[0301] Immunization Briefly, female BALB / c mice (Charles River Laboratories, Quebec) are immunized. A series of subcutaneous injections of aqueous solutions containing antigen or no adjuvant are given over a 19-day period. Mice are immunized with 100 μg of cyclic peptide-KLH per injection in sterile saline at 0.5 mg / mL per mouse. All mice are euthanized on day 19, and lymphocytes are harvested for generation of hybridoma cell lines.

[0302] Fusion / hybridoma generation Lymphocytes are isolated and fused with mouse SP2 / 0 myeloma cells in the presence of polyethylene glycol (PEG 1500). The fused cells are cultured using HAT selection. This method combines hybridoma selection and cloning into one step using semi-solid methylcellulose-based HAT selection medium. Single-cell derived hybridomas grow to 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 they reach mid-log growth (approximately 5 days).

[0303] Hybridoma analysis (screening) Tissue culture supernatants from hybridomas can be tested by indirect ELISA on the screening antigen (cyclic or linear peptide-BSA) and probed for both IgG and IgM antibodies using a secondary, goat anti-IgG / IgM (H&L)-HRP developed with TMB substrate.

[0304] Clones with an OD >0.2 in this assay are taken for the next round of testing. Positive cultures are retested on the screening antigen to confirm secretion and an unrelated antigen (human transferrin). Clones of interest are isotyped by antibody capturing ELISA to determine whether they are of the IgG or IgM isotype, and can be tested by indirect ELISA against other cyclic peptide-BSA conjugates as well as native-BSA conjugates to assess cross-reactivity.

[0305] Positive IgG-secreting clones are targeted for large-scale production.

[0306] Direct binding assay Binding of clones with linear and cyclic peptides (conjugated to BSA) can be tested by surface plasmon resonance using a Biacore™ 3000 instrument (GE Healthcare).

[0307] Binding assays are performed using high density (at least 1000 response units (RU)) antigen immobilized on flow cells. To assess binding, dilutions of selected clones are injected serially across the surface.

[0308] For affinity kinetics and specificity analysis, conformational peptides containing TTEQ (SEQ ID NO: 1) or TTE and BSA, e.g., cyclic peptides with sequences SEQ ID NO: 2, 3, 28, or 29 conjugated to BSA via amine conjugation, are immobilized at low density (50-100 RU) on adjacent flow cells. Serial two-fold dilutions (4.7 nM-75 nM) of selected clones are then injected sequentially across the surface at 60 μl / min for 3 min, followed by phase separation. After double reference subtraction, the sensorgrams are fitted to a Langmuir 1:1 binding model. Up to three separate analyses are performed on three consecutive days using the same sensor chip and the same conditions.

[0309] Binding analysis can also be performed using the Molecular Affinity Screening System (MASS-1) (Sierra Sensors GmbH, Hamburg, Germany). MASS-1 is a surface plasmon resonance (SPR) imaging analytical biosensor that uses high-intensity laser light and high-speed optical scanning to observe binding interactions in real time. Peptide-BSA conjugates are covalently immobilized to separate flow cells of a high amine capacity (HAC) sensor chip using standard amine coupling chemistry, blocking unreacted sites. Similarly, adjacent flow cells are immobilized with BSA as a reference control surface.

[0310] Tissue culture supernatants were screened for the presence of antibody binding to the cognate cyclic peptide. Each sample was diluted and injected in duplicate over the immobilized peptide and BSA reference surfaces for 2 min, followed by a 5-min phase separation with experimental buffer only. After every analysis cycle, the sensor chip surface was regenerated. Sensorgrams were double-referenced by subtracting binding from the BSA reference surface and blank experimental buffer injections and combining the response report point measured 20 s before the end of the injection in a 20-s window of data.

[0311] Isotyping Hybridoma antibodies are isotyped using antibody capture experiments. Capture plates are coated with 100 uL / well of 1:10,000 goat anti-mouse IgG / IgM (H&L) antibody in carbonate coated buffer, pH 9.6, overnight at 4°C. Primary antibody (hybridoma supernatant) is added at 100 μg / mL. Secondary antibody is added at 1:5,000. Goat anti-mouse IgGy-HRP or 1:10,000 goat anti-mouse IgM μ-HRP is added at 100 uL / well in PBS-Tween, shaken at 37°C for 1 hour. All wash steps are performed for 30 minutes with PBS-Tween. Substrate TMB, grown in the dark and stopped with an equal volume of 1 M HCl, is added at 50 uL / well.

[0312] Example 5 Monoclonal antibodies were generated using the methods of Example 4. Specifically, cyclo(CGGTTEQGG) (SEQ ID NO: 2) conjugated to KLH or cyclo(CGTTEQG) (SEQ ID NO: 3) conjugated to KLH via a cysteine ​​residue were injected into mice to generate monoclonal antibodies.

[0313] Antibody-containing hybridoma tissue culture supernatants were typed for immunoglobulin type and screened against the negative control peptide and BSA. IgG-producing clones that did not bind the negative control peptide or BSA were tested by ELISA for binding to the immunizing cyclic peptide, the corresponding linear peptide, and the related cyclic peptides cyclo(CGGTTEGGG) (SEQ ID NO: 32), cyclo(CGTTEGG) (SEQ ID NO: 29), as described below.

[0314] ELISA conditions ELISA plates were coated with 0.1 μg / well of peptide cyclo(CGGTTEQGG) (SEQ ID NO: 2) or cyclo(CGTTEQG) (SEQ ID NO: 3) or their linear versions at 100 μL / well in carbonate coating buffer (pH 9.6) overnight at 4° C. In other assays, cyclo(CGGTTEGGG) (SEQ ID NO: 32) or cyclo(CGTTEGG) (SEQ ID NO: 29) were also used to coat ELISA plates.

[0315] Plates were blocked with 3% nonfat milk in PBS for 1 hour at room temperature. 100uL / well of primary antibody hybridoma tissue culture supernatant was added and incubated at 37C for 1 hour with shaking. 100uL / well of secondary antibody goat anti-mouse IgGy-HRP at a 1:10,000 dilution was added in PBS-Tween and incubated at 37C for 1 hour with shaking. All steps were performed for 30 minutes in PBS-Tween. 50uL / well of TMB substrate was added, grown in the dark, and stopped with an equal volume of 1M HCl. Growth times were 1-2 minutes, and plates were read at 450nm.

[0316] The ELISA results are presented in Tables 9A and B, which show the binding affinity of purified monoclonal antibodies to the immunizing peptide versus the corresponding linear peptide. As shown, IgG antibodies were produced that showed preferential binding to the immunizing cyclic peptide compared to the linear peptide. [Table 20] [Table 21]

[0317] In addition, antibodies were tested for binding to the related cyclic peptides cyclo(CGGTTEGGG) (SEQ ID NO: 32) and cyclo(CGTTEGG) (SEQ ID NO: 29), which contain the TTE epitope. Fifteen of the 17 antibodies had greater affinity for the related cyclic peptide compared to the linear peptide corresponding to the immunogen, and 14 of the 17 antibodies tested had approximately two-fold or greater selectivity for the related cyclic peptide compared to the linear peptide corresponding to the immunogen.

[0318] Example 6 Characterization of misfolded TDP-43 Antibodies are tested for their ability to bind to native and misfolded TDP-43 polypeptides using surface plasmon resonance and immunohistochemistry.

[0319] Surface plasmon resonance of biological samples (Biacore) Homogenized human and mouse neurological tissue samples were weighed and then submerged in a volume of fresh ice-water TBS (supplemented with 5 mM EGTA, 5 mM EDTA (both from Sigma) and an EDTA-free protease inhibitor cocktail from Roche Diagnostics, Laval QC, Canada) to a final tissue concentration of 20% (w / v). The tissue was homogenized in this buffer using a mechanical probe homogenizer (3 × 30-second pulses with 30-second pauses, all on ice). The TBS-homogenized samples were then subjected to ultracentrifugation (70,000 × g for 90 minutes). The supernatant was collected, aliquoted, and stored at -80°C. The protein concentration of the TBS homogenate was determined using a BCA protein assay (Pierce Biotechnology Inc., Rockford IL, USA).

[0320] Surface plasmon resonance analysis was performed on CSF and neurological tissue samples from ALS and / or FTD patients and age-matched controls. Test antibodies and an IgG1 isotype control were directly immobilized at high density (approximately 10,000) on two separate flow cells of the sensor chip. Using a Biacore 3000, diluted neurological tissue samples homogenized in TBS were injected sequentially across the surface for 300 seconds, followed by 150 seconds of dissociation and surface regeneration in buffer. Binding responses were double-referenced by subtraction of the IgG1 reference surface binding, normalized to assay buffer, and samples from different groups were compared.

[0321] Example 7 Human embryonic kidney cells (HEK293FT) were transfected with HA-tagged, nuclear localization signal (TDP-43 ΔNLS We transiently transfected cells with a modified form of TDP-43 lacking the ATPase 1 (ATPase 2) gene. This form of the protein accumulates in the cytoplasm and forms misfolded aggregates. An empty vector was used as a negative control.

[0322] Cells were stained overnight at 4°C with 1 μg / ml of a polyclonal rabbit antibody against the HA tag (to detect misfolded cytoplasmic TDP-43) or 10 μg / ml of a test antibody. Bound antibody was then detected by staining with fluorescently labeled anti-rabbit (red Alexa Fluor 647) or anti-mouse (green Alexa Fluor 488) secondary antibodies for 1 hour at room temperature. Nuclear DNA was stained with Hoeschst 33342 dye (blue). Micrographs were acquired on a confocal Leica SP8 microscope in a Z-stack format with 0.3 μm steps. The included images represent Z-plane projections across the entire cell thickness. Z-stacked images (20–30 steps) of single fluorescent channels and merged signals were captured.

[0323] The results are presented in Figure 10. Panel A shows Hoechst 33342 dye stained cells revealing the cell nuclei. Figure 10B shows HA-tagged recombinant TDP-43 ΔNLSFigure 10C shows cells stained for HA-tagged recombinant TDP-43. Only the recombinant protein is detected. As expected, exogenously expressed TDP-43 lacking the NLS was found in the cytoplasm and present in misfolded aggregates. Figure 10C shows the results for a monoclonal antibody produced using cyclo(CGGTTEQGG). There was primarily cytoplasmic staining, suggesting that the test antibody does not bind to wild-type nuclear TDP-43. Figure 10D shows the results for HA-tagged recombinant TDP-43. ΔNLS細胞 Shown is a merged image where there is substantial overlap (colocalization) between the detection of plasma aggregates and test antibody staining for TDP-43.

[0324] Example 8 Human spinal cord homogenates were prepared from control and sporadic ALS patients and analyzed by dot blot with selected antibodies.

[0325] Tissue from a person without ALS was used as a control, and two different sporadic ALS tissue samples were evaluated: one obtained from a patient carrying the C90rf72 mutation and one obtained from a patient with unknown mutation status.

[0326] PVDF membranes were dotted with 10 micrograms of homogenate in duplicate, and test and control antibodies (mIgG1) were used at a 500-fold dilution of a 2 microgram / microliter stock solution. A rabbit polyclonal TDP-43 antibody (ProteinTech, Rosemont, IL) was used as a positive control.

[0327] As shown in Figure 11A, the IgG1 negative control produced low background staining for all samples, as shown in Figure 11B, and the positive control antibody produced a robust positive signal for all tested samples.

[0328] Selective staining of sALS samples was observed using the test antibodies. Antibody 1 is clone IG10, Antibody 2 is clone 2H10, Antibody 3 is clone 11F3, Antibody 4 is clone 3H5, Antibody 5 is clone 4G5, and Antibody 6 is clone 9C5. Antibody 6C5 also showed selective staining of sALS samples.

[0329] Example 9 Some of the antibodies positive for binding aggregated TDP-43 were sequenced. Transcripts of immunoglobulin genes expressed by the hybridomas were amplified with a set of proprietary primers from cDNA produced from hybridoma cells using standard RT-PCR protocols and sequenced using standard dye terminator capillary sequencing methods (Immunoprecise, Victoria BC Canada).

[0330] The complementarity-determining regions (CDRs) CDR1, CDR2, and CDR3 are highlighted, bolded, and underlined and identified according to IgBLAST (available using the National Center for Biotechnology Information tool (Ye et al., Nucleic Acids Research 2013, Vol. 41, Web Server Issue doi:10:1093|nar|gkt382)). This tool is used to search queries by BLAST against the IMGT or NCBI germline V gene databases (sequences in such databases are pre-annotated with FR / CDR boundaries). The top database sequence hits are used to map the pre-annotated FR / CDR boundary information to the query sequence. The BLAST search parameters are: Expect cutoff, 20; word size, 9; mismatch penalty, -1; dust filtering, off. [Table 22-1] [Table 22-2] [Table 22-3]

[0331] Two heavy chains were identified for antibody 2H10, labeled VH1 and VH2.

[0332] Whether one or both heavy chains produce antibodies selective for their cyclic peptide immunogens can be confirmed by expressing each heavy chain (or variable region) with the light chain (or variable region) and assessing target binding.

[0333] The heavy chain alignment of antibody 2H10-VH1 is most closely related to the heavy chains for 6C5 and 9C5 compared to 2H10-V2.

[0334] Example 10 Immunofluorescence staining of TDP-43 using antibody 9C5 Misfolded TDP-43 was recombinantly expressed using the configuration and protocol described in Example 7, with the following modifications to reduce background: Primary and secondary antibodies were spun at 10,000 RPM for 10 minutes before use, and sections were blocked with 10% NGS for 1 hour at room temperature before the addition of the primary antibody. DAPI staining was used to identify cell nuclei.

[0335] As shown in Figure 12C, the 9C5 antibody detected cytoplasmic inclusions of misfolded TDP-43. Figure 12A shows DAPI-stained cells, confirming that the detected TDP43 was cytoplasmic. Because recombinantly expressed TDP-43 primarily colocalized with the tagged HA (Figure 12D), we also stained for HA (Figure 12B). Table 11. Sequences of epitopes and cyclic compounds 1. TTEQ (SEQ ID NO: 1) 2. CGGTTEQGG, cyclo(CGGTTEQGG) (SEQ ID NO: 2) 3. CGTTEQG, cyclo(CGTTEQG) (SEQ ID NO: 3) 4. GTTEQG (SEQ ID NO: 4) 5. TTEQG (SEQ ID NO: 5) 6. GTTEQ (SEQ ID NO: 6) 7. KTTEQD (SEQ ID NO: 7) 8. TEQD (SEQ ID NO: 8) 9. TTEQD (SEQ ID NO: 9) 10. KTTE (SEQ ID NO: 10) 11. TTEQDL (SEQ ID NO: 11) 12. KTTEQ (SEQ ID NO: 12) 13. CGTTEQGC, cyclic (CGTTEQGC) (SEQ ID NO: 13) 14. KTTEQDL (SEQ ID NO: 14) 15. TEQDLK (SEQ ID NO: 15) 16. TEQDLKE (SEQ ID NO: 16) 17. TEQDLKEY (SEQ ID NO: 17) 18. TEQDLKEYF (SEQ ID NO: 18) 19. EQDL (SEQ ID NO: 19) 20. WKTTEQ (SEQ ID NO: 20) 21. TTEQDLKEYFSTFGEV (SEQ ID NO: 21) 22. CGGTTEQGGG, cyclo(CGGTTEQGGG) (SEQ ID NO: 22) 23. CGTTEQGG, cyclo(CGTTEQGG) (SEQ ID NO: 23) 24. C-PEG2-TTEQG, cyclo(C-PEG2-TTEQG), CTTEQG (SEQ ID NO: 24) 25. C-PEG2-TTEQGG, cyclo(C-PEG2-TTEQGG), CTTEQGG (SEQ ID NO: 25) 26. CGTTEQ-PEG2, cyclo(CGTTEQ-PEG2), CGTTEQ (SEQ ID NO: 26) 27. CGGTTEQ-PEG2, Cyclol (CGTTEQ-PEG2), CGTTEQ (SEQ ID NO: 27) 28. CGTTEG, cyclo(CGTTEG) (SEQ ID NO: 28) 29. CGTTEGG, cyclo(CGTTEGG), (SEQ ID NO: 29) 30. CGGTTEGG, cyclo(CGGTTEGG), (SEQ ID NO: 30) 31. CGGGTTEGG, cyclo(CGGGTTEGG), (SEQ ID NO: 31) 32. CGGTTEGGG, cyclo(CGGTTEGGG), (SEQ ID NO: 32) 33. CGGGTTEGGG, cyclo(CGGGTTEGGG), (SEQ ID NO: 33) 34. CGGGTTEGGG, cyclo(CGGGGTTEGGG), (SEQ ID NO: 34) 35. CGGGGTTEGGGG, cyclo(CGGGGTTEGGGG), (SEQ ID NO: 35) 36. C-PEG2-TTEG, cyclo(C-PEG2-TTEG), CTTEG (SEQ ID NO: 36) 37. C-PEG2-TTEGG, cyclo(C-PEG2-TTEGG), CTTEGG (SEQ ID NO: 37) 38. CGTTE-PEG2, cyclo(CGTTE-PEG2), CGTTE (SEQ ID NO: 38) 39. CGGTTE-PEG2, Cyclol (CGGTTE-PEG2), CGGTTE (SEQ ID NO: 39) 40. GGCGG (SEQ ID NO: 40) 41. GCGG (SEQ ID NO: 41) 42. CGGGTTEQGG, cyclo(CGGGTTEQGG), (SEQ ID NO: 42) 43. CGGGGTTEQGGG, cyclo(CGGGGTTEQGGG), (SEQ ID NO: 43) 44. CGGGGTTEQGGGG, cyclo(CGGGGTTEQGGGG), (SEQ ID NO: 44)

[0336] While the present application has been described with reference to what are presently considered to be the preferred examples, it should be understood that the application is not limited to the disclosed examples. To the contrary, the application is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

[0337] All publications, patents, and patent applications are incorporated by reference herein to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety. In particular, the sequences associated with each accession number provided herein, including, for example, the accession numbers and / or biomarker sequences (e.g., proteins and / or nucleic acids) provided in the Tables or elsewhere, are incorporated by reference in their entirety.

[0338] The scope of the claims should not be limited by the preferred embodiments and examples, but should be accorded the broadest interpretation consistent with the description as a whole. Citations for References Mentioned herein [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(No publication). [3] Mompean, M., Romano, V., Pantoja-Uceda, D., Stuani, C., Baralle, FE, Buratti, E., and Laurents, DVThe TDP-43 N- Terminal Domain Structure at High Resolution. FEBS J., 2016, 283, 1242. [4]Arai,T.,Hasegawa,M.,Akiyama,H.,Ikeda,K.,Nonaka,T.,Mori,H.,Mann,D.,Tsuchiya,K.,Yoshida,M.,Hashizume,Y.,and Oda,T.TDP-43 is a component of ubiquitin-positive tau- negative inclusions in frontotemporal lobar degeneration and 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 YuJ.Biol.Chem.2010,vol.285,No.9,6826-6834。 [6]Abel,O.,Powell,J.F.,Andersen,P.M.,and Al-Chalabi,A.Hum Mutat,2012,33:1345-51。 [7]Hamley,I.W.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。

Claims

1. A cyclic compound consisting of the amino acid sequence CGGTTEQGG (SEQ ID NO: 2) or CGTTEQG (SEQ ID NO: 3).

2. 10. An immunogen comprising the cyclic compound of claim 1, wherein the cyclic compound is conjugated to a carrier protein or immunogenicity-enhancing moiety and / or formulated with an adjuvant.

3. A polypeptide 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 the CDRs are the sequence: or or or A conformational antibody that selectively binds to TTEQ in misfolded TDP-43.

4. The antibody 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, and the light chain variable region comprising complementarity determining regions CDR-L1, CDR-L2, and CDR-L3, and the amino acid sequences of the CDRs are the sequences: The conformational antibody of claim 3,

5. The antibody 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, and the light chain variable region comprising complementarity determining regions CDR-L1, CDR-L2, and CDR-L3, and the amino acid sequences of the CDRs are the sequences: The conformational antibody of claim 3,

6. The antibody 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, and the light chain variable region comprising complementarity determining regions CDR-L1, CDR-L2, and CDR-L3, and the amino acid sequences of the CDRs are the sequences: The conformational antibody of claim 3,

7. The antibody 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, and the light chain variable region comprising complementarity determining regions CDR-L1, CDR-L2, and CDR-L3, and the amino acid sequences of the CDRs are the sequences: The conformational antibody of claim 3,

8. The antibody a. comprising a heavy chain variable region comprising i) the amino acid sequence set forth in SEQ ID NO: 46, or ii) a conservatively substituted amino acid sequence of i), and / or comprising i) the amino acid sequence set forth in SEQ ID NO: 48, or ii) a conservatively substituted amino acid sequence of i), b. i) comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 50, or ii) a conservatively substituted amino acid sequence of i), and / or i) comprising the amino acid sequence set forth in SEQ ID NO: 52, or ii) a conservatively substituted amino acid sequence of i), c. i) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 54, or ii) a conservatively substituted amino acid sequence of i), and / or i) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 56, or ii) a conservatively substituted amino acid sequence of i), or d. The conformational antibody of any one of claims 3 to 7, comprising a heavy chain variable region comprising i) the amino acid sequence set forth in SEQ ID NO: 58, or ii) a conservatively substituted amino acid sequence of i), and / or a light chain variable region comprising i) the amino acid sequence set forth in SEQ ID NO: 60, or ii) a conservatively substituted amino acid sequence of i).

9. The conformational antibody of any one of claims 3 to 8, wherein the antibody is a monoclonal antibody, a humanized antibody, or a single-chain antibody.

10. The antibody is selected from Fab, Fab', F(ab')2, scFv, dsFv, ds-scFv, dimers, minibodies, bispecific antibodies, and multimers thereof. The conformational antibody of any one of claims 3 to 9, which is a binding fragment thereof.

11. An immunoconjugate comprising an antibody according to any one of claims 3 to 10 and a detectable label or transport moiety.

12. A nucleic acid encoding the amino acid residues of the antibody according to any one of claims 3 to 10.

13. A cell expressing the antibody according to any one of claims 3 to 10.

14. A composition comprising the cyclic compound of claim 1, the immunogen of claim 2, the antibody of any one of claims 3 to 10, the immune complex of claim 11, the nucleic acid of claim 12, or the cell of claim 13.

15. A kit comprising the cyclic compound of claim 1, the immunogen of claim 2, the antibody of any one of claims 3 to 10, the immune complex of claim 11, the nucleic acid of claim 12, the cell of claim 13, or the composition of claim 14.

16. 1. A method for determining whether a sample suspected of containing misfolded TDP-43 polypeptide contains misfolded TDP-43 polypeptide, comprising: a. contacting the sample with the antibody of any one of claims 3 to 10 under conditions permissive for forming an antibody:misfolded TDP-43 polypeptide complex; b. detecting the presence of any complexes; The presence of a detectable complex indicates that the sample may contain a misfolded TDP-43 polypeptide.

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