Antibodies to misfolded TDP-43 and methods of use

Abstract

To provide an antibody for detecting misfolded TDP-43 and a method for detecting misfolded TDP-43.SOLUTION: The disclosure provides antibodies that preferentially bind to TDP-43 aggregates but not to natively folded nuclear or cytosolic TPD-43, where the antibodies bind to a specific sequence in the TDP-43 sequence in misfolded TDP-43 compared with native TDP-43. Also provided are, isolated epitopic peptides differentially accessible in misfolded TDP-43, isolated nucleic acids, immunogens, compositions, kits, and methods of using the reagents to detect misfolded TDP-43.SELECTED DRAWING: Figure 9

Description

[Technical Field]

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

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

[0003] Field The present disclosure relates to TDP-43 antibodies, and more particularly to antibodies for detecting misfolded TDP-43 and methods for detecting misfolded TDP-43. [Background technology]

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

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

[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, present in the linker between RRM2 and the C-terminal domain.

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

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

[0009] TDP-43 was found to be hyperphosphorylated, ubiquitinated, and fragmented in neuronal inclusions in patients with both sporadic and familial forms of ALS and FTD [4].

[0010] Functional TDP-43 can exist as nuclear oligomers distinct from cytoplasmic aggregates that form during cellular stress. Functional TDP-43 oligomerization is required for its RNA splicing function. NTD-driven TDP-43 oligomerization in the nucleus can inhibit cytoplasmic mislocalization and the formation of pathological aggregates [9].

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

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

[0013] Precursors of TDP-43 inclusions can have much lower concentrations than functional TDP-43, and the low concentrations of misfolded TDP-43 make this target elusive.

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

[10] .

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

[0016] WO2012174666, entitled "Method for prognosis and / or diagnosis of neurodegenerative diseases, method for identifying candidate compounds and compounds for treating neurodegenerative diseases," discloses a method for diagnosing neurodegenerative diseases, such as ALS and FTD, through evaluation of the interaction between TDP-43 and NF-kB p65 using anti-TDP-43 antibodies.

[0017] WO2016086320, entitled "TDP-43-binding polypeptides useful for the treatment of neurodegenerative diseases," discloses antibodies that bind to the RRM1 domain of TDP-43 and disrupt its interaction with NF-kB for the treatment of ALS and FTD.

[0018] Antibodies that preferentially bind to misfolded TDP-43 over natively folded TDP-43 are desired. Summary of the Invention

[0019] overview The present inventors have identified antibodies that preferentially bind to TDP-43 aggregates, but not to natively folded nuclear or cytoplasmic TDP-43. The inventors identified differentially accessible epitopes in misfolded TDP-43 and generated antibodies that recognize the epitopes in misfolded TDP-43 aggregates. As shown herein, the epitopes are available for binding in misfolded TDP-43 (solvent-accessible) but not in natively folded, non-disease-associated TDP-43. In particular, the inventors determined that W68 is a critical residue for conferring antibody specificity to misfolded TDP-43 aggregates. Such antibodies can recognize aggregated TDP-43, which is not generally associated with stress granules, and inhibit cell-to-cell spread of pathogenic TDP-43.

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

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

[0022] In one embodiment, the peptide is a maximum of 21 residues, and optionally W68 is preferably located in the middle of the third division of the peptide.

[0023] Another aspect includes an immunogen comprising a peptide, wherein the peptide comprises all or a portion of DAGWGNL (SEQ ID NO: 1), wherein the portion is at least 5 amino acids, and optionally at least 6 amino acids, of DAGWGNL (SEQ ID NO: 1).

[0024] In one embodiment, the immunogen comprises a plurality of peptides, each peptide comprising at least 5 amino acids, and optionally at least 6 amino acids, of DAGWGNL (SEQ ID NO: 1), wherein the plurality of peptides are synthesized as a multiple antigenic peptide (MAP).

[0025] In another embodiment, the peptide is conjugated to a carrier protein or immunogenicity enhancing moiety.

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

[0027] In one embodiment, the immunogen is used to generate antibodies that selectively bind to misfolded TDP-43 and / or that specifically bind to at least W68 in the context of DAGWGNL (SEQ ID NO: 1). In one embodiment, the antibodies preferentially bind to misfolded TDP-43.

[0028] Further aspects include antibodies that bind to TDP-43 and that bind preferentially to misfolded TDP-43 relative to native TDP-43.

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

[0030] In certain embodiments, the antibodies are generated or screened using the peptides or immunogens described herein.

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

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

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

[0034] In certain embodiments, the antibody is a binding fragment selected from a Fab, a Fab', a F(ab')2, a scFv, a dsFv, a ds-scFv, a dimer, a nanobody, a minibody, a diabody, and multimers thereof.

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

[0036] A further aspect comprises an immunoconjugate comprising an antibody described herein and a detectable label.

[0037] Further aspects include isolated nucleic acids encoding amino acid residues of the peptides, immunogens, or antibodies described herein, as well as vectors comprising said nucleic acids, e.g., for delivery and / or expression of the peptides, immunogens, or antibodies described herein.

[0038] A further aspect comprises a cell recombinantly expressing a peptide, immunogen or antibody described herein.

[0039] In one embodiment, the cell, when expressing an antibody, is a hybridoma.

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

[0041] In certain embodiments, the composition, when comprising a peptide or immunogen, further comprises an adjuvant.

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

[0043] Kits comprising isolated peptides, immunogens, antibodies, immunoconjugates, isolated nucleic acids, cells, and / or compositions and compartments for housing said reagents are also provided.

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

[0045] A further aspect includes a method of producing an antibody comprising administering to or immunizing a non-human subject with an isolated peptide (e.g., at least 5 amino acids, optionally at least 6 amino acids, of DAGWGNL (SEQ ID NO: 1)), immunogen, or composition described herein.

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

[0047] In certain embodiments, the method further comprises forming antibody-producing hybridomas.

[0048] Another aspect includes an antibody produced by the methods described herein.

[0049] Further aspects include methods for determining whether a sample contains misfolded TDP-43, comprising contacting the sample with an antibody described herein under conditions that allow antibody:misfolded TDP-43 complexes to form, and detecting the presence of any complexes, wherein the presence of a detectable complex indicates that the sample may contain a misfolded TDP-43 polypeptide.

[0050] In certain embodiments, the sample is a biological sample obtained from a subject.

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

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

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

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

[0055] 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]

[0056] Embodiments of the present disclosure will now be described in connection with the drawings in which:

[0057] [Figure 1]1A-2L show HEK-293 cells transfected with a TDP-43 construct carrying a triple missense tandem mutation in the nuclear localization signal, wild-type TDP-43, or an empty vector. TDP-43 in the cells was detected using a DAGWGNL (SEQ ID NO: 1) peptide affinity-purified rabbit polyclonal anti-TDP-43 antibody (GS240) raised against an immunogen comprising the DAGWGNL (SEQ ID NO: 1) peptide described herein, or an anti-HA tag antibody reactive with the TDP-43-HA fusion construct. [Figure 2] Figures 2A-2L show HEK-293 cells transfected with a TDP-43-HA construct carrying a triple missense tandem mutation in the nuclear localization signal, wild-type TDP-43-HA, or an empty vector. TDP-43 was detected in the cells using a purified polyclonal anti-TDP-43 antibody (GS243) raised against an immunogen comprising the DAGWGNL (SEQ ID NO: 1) peptide described herein, or an anti-HA tag antibody. [Figure 3A] 3A-3E are graphs showing the binding kinetics of mouse (3A and 3B) and rabbit (3C, 3D and 3E) monoclonal antibodies. [Figure 3B] 3A-3E are graphs showing the binding kinetics of mouse (3A and 3B) and rabbit (3C, 3D and 3E) monoclonal antibodies. [Figure 3C] 3A-3E are graphs showing the binding kinetics of mouse (3A and 3B) and rabbit (3C, 3D and 3E) monoclonal antibodies. [Figure 3D] 3A-3E are graphs showing the binding kinetics of mouse (3A and 3B) and rabbit (3C, 3D and 3E) monoclonal antibodies. [Figure 3E] 3A-3E are graphs showing the binding kinetics of mouse (3A and 3B) and rabbit (3C, 3D and 3E) monoclonal antibodies. [Figure 4]Figures 4A and 4B show the recognition of epitopes by antibodies in the denatured TDP43 N-terminal domain, but not in the natively folded TDP43 N-terminal domain. [Figure 5A] Figures 5A and 5B are images showing staining of sections from FTD brain (5A) and ALS spinal cord (5B) with rabbit polyclonal GS240 antibody, and Figures 5C-5E are images showing staining of sections from FTD brain with mouse monoclonal antibody. [Figure 5B] Figures 5A and 5B are images showing staining of sections from FTD brain (5A) and ALS spinal cord (5B) with rabbit polyclonal GS240 antibody, and Figures 5C-5E are images showing staining of sections from FTD brain with mouse monoclonal antibody. [Figure 5C] Figures 5A and 5B are images showing staining of sections from FTD brain (5A) and ALS spinal cord (5B) with rabbit polyclonal GS240 antibody, and Figures 5C-5E are images showing staining of sections from FTD brain with mouse monoclonal antibody. [Figure 5D] Figures 5A and 5B are images showing staining of sections from FTD brain (5A) and ALS spinal cord (5B) with rabbit polyclonal GS240 antibody, and Figures 5C-5E are images showing staining of sections from FTD brain with mouse monoclonal antibody. [Figure 5E] Figures 5A and 5B are images showing staining of sections from FTD brain (5A) and ALS spinal cord (5B) with rabbit polyclonal GS240 antibody, and Figures 5C-5E are images showing staining of sections from FTD brain with mouse monoclonal antibody. [Figure 6] Figure 6 is a series of images showing colocalization of mouse monoclonal Ab 2F7 staining with cytoplasmic TDP43 aggregates in ΔNLS-TDP43-HA transfected cells, but not in WT nuclear TDP-43-HA. [Figure 7] 7A and 7B are images showing that the 3F11 mouse monoclonal antibody against a disease-associated N-terminal TDP43 epitope does not react with physiological stress granules. [Figure 8] Figure 8 is a series of images showing colocalization of rabbit monoclonal Ab 28H3 staining with cytoplasmic TDP43 aggregates in ΔNLS-TDP-43-transfected cells, but not in ΔNLS-TDP-43-W68S-transfected cells. [Figure 9] FIG. 9 shows antibody blockade of misfolded TDP-43 propagation in HEK293 cells. DETAILED DESCRIPTION OF THE INVENTION

[0058] Detailed Description of the Disclosure As described in the Examples, the present inventors have identified a specifically accessible epitope in misfolded TDP-43. The present inventors have generated antibodies that specifically bind to the epitope. Furthermore, the present inventors have determined the importance of W68 in relation to DAGWGNL (SEQ ID NO: 1). Antibodies generated against this peptide sequence have been shown to preferentially bind to misfolded TDP-43 compared to native TDP-43.

[0059] I. Definition As used herein, the term "TDP-43" (transactivation response element (TAR) DNA-binding protein 43), or "TDP43," or, unless otherwise limited, "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 generally a 414 amino acid residue protein, and its amino acid sequence (e.g., Uniprot Accession No. Q13148) and nucleotide sequence (e.g., Accession No. HGNC:11571) have been previously characterized.

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

[0061] As used herein, "native" refers to the normal three-dimensional structure of a particular protein or a portion thereof. Native TDP-43 is sometimes referred to as "naturally folded" TDP-43, "normally folded" TDP-43, and / or "healthy" TDP-43. Thus, the term "native TDP-43" or "naturally folded TDP-43" refers herein to TDP-43 that is naturally folded after de novo translation and / or a multimer, including, but not limited to, dimeric TDP-43 and trimeric TDP-43, that is folded in a non-pathological state (e.g., normal cells) and has a molecular structure comprising non-covalently linked individual TDP-43 peptides that exhibit a native structure reconstructed from X-ray crystallography or nuclear magnetic resonance spectroscopy. Native TDP-43 forms multimers through its NTD, and naturally folded TDP-43 is generally nuclear. Misfolded aggregates of TDP-43 can be, and generally are, cytoplasmic.

[0062] As used herein, "misfolded" refers to the secondary and tertiary structure of a polypeptide or a portion thereof, indicating that the polypeptide adopts a conformation that is not normal for that polypeptide in its properly functioning state. Misfolding can occur due to mutations in the protein, 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 aberrant ubiquitination, phosphorylation, acetylation, sumoylation, and cleavage into C-terminal fragments. Misfolded TDP43 can be aggregated and / or cytoplasmic. In the context of TDP-43, native TDP-43 forms multimers via its NTD. Misfolded multimers (e.g., disease-associated oligomers) generally oligomerize via other regions of the protein, such as its LCD and / or RRM1 domain. Thus, "misfolded TDP-43 polypeptide," or "misfolded TDP-43" as used herein to refer to a polypeptide, includes cytoplasmic and / or aggregated TDP-43 polypeptides that are oligomerized via their LCD and / or RRM1 domains, non-natural dimers and trimers, and large aggregates (e.g., five or more subunits). Misfolded TDP-43 tends to form aggregates that result in loss of protein function, toxicity, acquisition of amyloid-like characteristics (e.g., Congo red staining), and propagation of pathogenic aggregates.

[0063] The term "mutant TDP-43" refers to forms of TDP-43, particularly endogenous forms of TDP-43 that result from genetic mutations that result in amino acid substitutions, such as those characteristic of FTD or familial ALS, including, for example, mutations described in the bioinformatics tool described in [6].

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

[0065] As used herein, "epitope" 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 sometimes referred to herein as a linear amino acid sequence or region of a protein recognized by an antibody. An epitope can comprise 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. As shown in the Examples, an antibody may require Trp68 for binding. An immunogen comprising at least five, and optionally at least six, residues of SEQ ID NO: 1 can be used to generate antibodies that preferentially bind to misfolded TDP-43, e.g., bind to W68 in the context of SEQ ID NO: 1 or DAGWGNL (SEQ ID NO: 1). Reference to "DAGWGNL (SEQ ID NO: 1) or related epitope" means SEQ ID NO: 1 or a portion thereof, in any linear peptide or region on TDP-43 that is bound by antibodies produced by an immunogen comprising a TDP-43 peptide sequence such as, for example, DAGWG (SEQ ID NO: 2), DAGWGN (SEQ ID NO: 3), AGWGN (SEQ ID NO: 4), AGWGNL (SEQ ID NO: 5), and GWGNL (SEQ ID NO: 6).

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

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

[0068] As used herein, a "conservative amino acid substitution" is one in which one amino acid residue is replaced with another amino acid residue without eliminating 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 for each other. Examples of conservative amino acid substitutions include the following:

[0069] [Table 1]

[0070] The term "antibody," as used herein, is intended to include monoclonal, polyclonal, single-chain, humanized and other chimeric, or fully human antibodies, as well as binding fragments thereof. Also included are vectored antibodies or intrabodies. Antibodies may be derived from recombinant sources and / or produced in transgenic animals. Also included are human antibodies, which may be produced using biochemical techniques or isolated from libraries. Humanized or chimeric antibodies may contain sequences from one or more isotypes or classes.

[0071] The phrase "isolated antibody" refers to an antibody produced in vivo or in vitro that has been removed from its source, e.g., an animal, hybridoma, or other cell line (e.g., a recombinant cell that produces the antibody). An isolated antibody is optionally "purified," meaning at least 80%, 85%, 90%, 95%, 98%, or 99% pure.

[0072] The term "binding fragment," as used herein, refers to a portion or part of an antibody or antibody chain that comprises fewer amino acid residues than an intact or complete antibody or antibody chain and that binds to an antigen or competes with 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 by chemical or enzymatic treatment of intact or complete antibodies or antibody chains. Fragments can also be obtained by recombinant means (see below). 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 to 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, diabodies, bispecific antibody fragments, and other fragments can also be constructed using recombinant expression techniques. When an antibody is said to bind to an epitope within a particular residue, such as DAGWGNL (SEQ ID NO: 1), what is meant is that the antibody selectively or specifically binds to a polypeptide containing the particular residue or a portion thereof, e.g., at least one residue or at least two residues in relation to a particular residue, such as (SEQ ID NO: 1). Such antibodies do not necessarily contact every residue of DAGWGNL (SEQ ID NO: 1), and not every single amino acid substitution or deletion within the epitope necessarily affects binding affinity significantly or equally.

[0073] The term " complementarity determining region " or " CDR " as used herein refers to the specific hypervariable region of an antibody that is generally predicted to contribute to epitope binding.Calculation methods for identifying CDR sequences include Kabat, Chothia, and IMGT.The CDRs listed in this disclosure are identified using IMGT Blast.Those skilled in the art can also identify CDR sequences based on Kabat, Chothia, etc., taking into account the sequences contained herein.

[0074] The term "detectable label," as used herein, refers to a moiety, such as a peptide sequence, fluorescent protein, etc., that can be attached to or introduced into a peptide, antibody, or other compound described herein, and that is capable of generating a detectable signal, either directly or indirectly. For example, the label can be a radio-opaque positron-emitting radionuclide (e.g., for use in PET imaging), or a radioisotope, e.g., 3 H, 13 N, 14 C. 18 F, 32 P, 35 S, 123 I, 125 I, 131 The detectable label may be a fluorescent (fluorophore) or chemiluminescent (chromophore) compound, such as fluorescein isothiocyanate, rhodamine, or luciferin; an enzyme, such as alkaline phosphatase, beta-galactosidase, or horseradish peroxidase; a contrast agent; or a metal ion. The detectable label may be indirectly detectable, for example, using a secondary antibody.

[0075] The term "epitope selectively presented or accessible on misfolded TDP-43" as used herein refers to an epitope that is selectively presented or accessible to antibodies on misfolded TDP-43, whether in a monomeric, dimeric, or aggregated form, such as in ALS or FTD (e.g., disease-related misfolded TDP-43), but not on the molecular surface of the native, correctly folded homodimeric form of TDP-43. As shown herein, W68 is selectively presented or accessible on misfolded TDP-43.

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

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

[0078] As used herein, "immunogen" refers to a substance that induces an immune response and / or induces the production of antibodies. In addition to immunogenic compounds, conjugates and fusions described herein may be used, including, for example, isolated compounds conjugated to KLH, peptide mimetics that induce cross-reactive antibodies against identified epitopes, such as DAGWGNL and / or related epitopes, such as DAGWG (SEQ ID NO: 2), DAGWGN (SEQ ID NO: 3), AGWGN (SEQ ID NO: 4), AGWGNL (SEQ ID NO: 5), and GWGNL (SEQ ID NO: 6). To serve as a useful immunogen, a TDP-43 peptide desirably incorporates at least about 5, 6, or 7 TDP-43 residues and up to about 15, 17, 19, 20, or 21 TDP-43 amino acids, optionally incorporating an immunogenicity enhancer such as KLH, for example, via a linker or scaffold used in multiple antigenic peptides (MAPs).

[0079] The term "nucleic acid sequence," as used herein, 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 comprising 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; and 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. The term "isolated nucleic acid sequence," as used herein, 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).

[0080] "Operably linked" means 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. The selection of an appropriate regulatory sequence depends on the host cell selected and can be easily accomplished by one skilled in the art. Examples of such regulatory sequences include transcription 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 confer inducibility of transcription, may be incorporated into the expression vector.

[0081] The term "vector," as used herein, comprises 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 integrated into the genome, and includes plasmids, phagemids, bacteriophages, or viral vectors, e.g., retrovirus-based vectors, adeno-associated virus vectors, etc. The term "plasmid," as used herein, generally refers to a construct of extrachromosomal genetic material, usually a circular double-stranded DNA, which is capable of replicating independently of chromosomal DNA.

[0082] "At least moderately stringent hybridization conditions" refers to conditions selected that promote selective hybridization between two complementary nucleic acid molecules in solution. Hybridization can occur to all or part of a nucleic acid sequence molecule. The hybridizing portion is generally at least 15 (e.g., 20, 25, 30, 40, or 50) nucleotides in length. Those skilled in the art will recognize that the stability of a nucleic acid duplex or hybrid in a sodium-containing buffer is determined by a function of sodium ion concentration and temperature (Tm = 81.5°C - 16.6 (Log10[Na+]) + 0.41 (%(G+C) - 600 / l), or a similar formula). Therefore, the parameters in the 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 a decrease in Tm of approximately 1°C; for example, if the nucleic acid molecules are to have greater than 95% identity, the final wash temperature will decrease by approximately 5°C. Based on these considerations, those skilled in the art will be able to easily select appropriate hybridization conditions. In preferred embodiments, stringent hybridization conditions are selected. As an example, the following conditions can be used to achieve stringent hybridization: hybridization in 5x sodium chloride / sodium citrate (SSC) / 5x Denhardt's solution / 1.0% SDS at Tm-5°C based on the above formula, followed by a wash in 0.2x SSC / 0.1% SDS 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.Further guidance on hybridization conditions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, NY, 2002, and Sambrook et al., Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Laboratory Press, 2001.

[0083] As used herein, "specifically binds" with respect to an antibody means that the antibody recognizes and binds to its target antigen with greater affinity than to a structurally different antigen and / or an antigen with a modified or mutated sequence. 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 greater than 1e-8. An affinity of at least greater than 1e-8 is preferred. An antigen-binding fragment, such as a Fab fragment comprising one variable domain, may find its target with an affinity that is 10- or 100-fold less than the multivalent interaction with the unfragmented antibody.

[0084] The terms "selective" or "preferentially," as used herein with reference to an antibody that selectively / preferentially binds to a form of TDP-43 (e.g., native protein or misfolded protein), mean that the binding protein binds to that 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, or at least 500-fold or more greater affinity. Thus, an antibody that is more selective for a particular conformation (e.g., misfolded protein) will preferentially bind to a particular form of TDP-43 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, or at least 500-fold or more greater affinity compared to another form.

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

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

[0087] The term "functionalizable moiety," as used herein, refers to a chemical entity having a "functional group," which, as used herein, refers to a group or single atom that reacts with another group or single atom (a so-called "complementary functional group") to form a chemical interaction between the two groups or atoms. In the case of cysteine, the functional group can be -SH, which can react to form a disulfide bond. The reaction with another group can be a covalent bond or a strong non-covalent bond, e.g., in the case of a biotin-streptavidin bond, it can have a Kd of about 1e-14. A strong non-covalent bond, as used herein, means 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.

[0088] Proteins and / or other agents can be functionalized (e.g., coupled / conjugated) to peptides to either promote immunogenicity or serve as probes in in vitro testing. For this purpose, any functionalizable moiety that can react (e.g., form a covalent or noncovalent but strong bond) may be used. In one specific embodiment, the functionalizable moiety is a cysteine ​​residue that is reacted to form a disulfide bond with an unpaired cysteine ​​on the protein of interest, which may 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 immunohistochemistry assays.

[0089] The term "animal" or "subject" as used herein includes all members of the animal kingdom, including or excluding mammals, optionally including humans.

[0090] The terms "treating" or "treatment," as used herein and as well understood in the art, refer to an approach to obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, whether detectable or undetectable, reduction in the extent of disease, a stabilized (i.e., non-worsening) state of disease, prevention of disease spread, delay or slowing of disease progression, improvement or alleviation of pathology, reduction in disease recurrence, and remission (whether partial or complete). "Treating" and "treatment" can also mean prolonging survival compared to expected survival in the absence of treatment. "Treating" and "treatment," as used herein, also include prophylactic treatment in subjects identified as having a mutation associated with a familial form of ALS, for example, a familial form. Subjects with a TDP-43 proteinopathy, such as ALS, can be treated to delay or slow disease progression. Subjects can be treated with a compound, antibody (including vectored antibody or intrabody), immunogen, immunoconjugate, or composition described herein to prevent progression.

[0091] In understanding the scope of the present disclosure, the term "consisting of" and its derivatives, as used herein, shall be considered limiting terms specifying the presence of stated features, elements, components, groups, integers and / or steps, and excluding the presence of other unstated features, elements, components, groups, integers and / or steps.

[0092] 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 understood that all numbers and fractions thereof are presumed to be modified by the term "about." Additionally, "a," "an," and "the" should be understood to include plural referents unless the content 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%.

[0093] Furthermore, definitions and embodiments described in a particular section are intended to be applicable to other embodiments described herein where they are 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 so defined may be combined with any other aspect or aspects, unless expressly indicated otherwise. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

[0094] II. Peptides Comprising All or Part of DAGWGNL (SEQ ID NO: 1) and Their Related Immunogens The present disclosure identifies epitopes on misfolded TDP-43, and antibodies generated using corresponding peptides preferentially recognize misfolded TDP-43. As shown in the Examples, the epitope, particularly W68, is inaccessible or less accessible in natively folded TDP-43. The inventors generated antibodies using an immunogen comprising the TDP-43 peptide DAGWGNL (SEQ ID NO: 1), which corresponds to amino acid residues 65-71 on TDP-43. As shown below, antibodies were generated using the immunogen that did not appreciably react with TDP-43 mutated with respect to this residue.

[0095] Thus, one aspect includes an isolated peptide comprising all or a portion of DAGWGNL, wherein the portion is at least 5 amino acids and comprises GWG.

[0096] In one embodiment, the portion is at least 6 contiguous amino acids of SEQ ID NO:1.

[0097] The peptide may comprise an additional TDP-43 contiguous sequence, for example, up to 11 amino acids, up to 13 amino acids, up to 15 amino acids, up to 17, 19, or up to 21 amino acids. Preferably, the sequence is centered or approximately centered on W68. For example, if the peptide is 21 amino acids long, W68 may be residue 8, 9, 10, 11, or 12. In one embodiment, the peptide is EGILHAPDAGWGNLVYVVNYP (SEQ ID NO: 7), or a portion thereof comprising at least GWG.

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

[0099] The isolated peptide can also comprise a linker comprising, for example, 1 to 9 glycine and / or PEG moieties and / or Cys residues N-terminal and / or C-terminal to a non-TDP-43 sequence, for example, a TDP-43 contiguous sequence.

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

[0101] The epitope W68 in the context of DAGWGNL (SEQ ID NO: 1) described herein may be a potential target in misfolded TDP-43 transmission strains, and antibodies recognizing said epitope may be useful, for example, for the detection of such transmission strains.

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

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

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

[0105] In certain embodiments, the peptide is conjugated to a carrier protein or immunogenicity enhancing moiety, which may be conjugated to the compound either directly, for example, via an amide bond, a disulfide bond, or indirectly via a linker.

[0106] Immunogens with immunogenicity-enhancing moieties can be produced by conjugating the peptides and a linker comprising a functionalizable moiety, such as cysteine, to an immunogenicity-enhancing moiety, such as keyhole limpet hemocyanin (KLH), or a carrier, such as bovine serum albumin (BSA), using, for example, the method described in Example 1, which is incorporated herein by reference.

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

[0108] In one embodiment, the antibodies produced specifically bind to at least W68 in the context of DAGWGNL (SEQ ID NO: 1).

[0109] III. Antibodies, immune complexes, cells and nucleic acids Isolated peptides comprising DAGWGNL (SEQ ID NO: 1) or related epitopes and immunogens described above can be used to generate antibodies that preferentially bind to misfolded TDP-43 compared to native TDP-43.

[0110] Thus, one aspect includes an antibody that binds to misfolded TDP-43 compared to native TDP-43.

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

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

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

[0114] In some embodiments, the antibody is isolated.

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

[0116] To produce monoclonal antibodies, antibody-producing cells (lymphocytes) can be collected from a subject immunized with an immunogen described herein and fused with myeloma cells by standard somatic cell fusion procedures, thereby immortalizing these cells and resulting in hybridoma cells. Such techniques are well known in the art (e.g., the hybridoma technique first developed by Kohler and Milstein (Nature 256:495-497 (1975)), as 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 to produce antibodies specifically reactive with a desired epitope, and monoclonal antibodies can be isolated.

[0117] Specific antibodies, or antibody fragments, reactive with particular antigens or molecules may be generated by screening expression libraries encoding immunoglobulin genes, or portions thereof, expressed in bacteria bearing 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)).

[0118] Humanization of antibodies from non-human species (e.g., from mice or rabbits) 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, the entire contents of which are incorporated herein by reference). Humanized antibodies are also readily available commercially (e.g., Scotgen Limited, 2 Holly Road, Twickenham, Middlesex, Great Britain).

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

[0120] Humanized forms of antibodies are sometimes obtained by resurfacing (Pedersen et al. J Mol Biol, 235: 959-973, 1994), an approach in which only surface residues of a rodent antibody are humanized.

[0121] 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 specific antigen is cloned and paired with a repertoire of human light chains for display as Fab fragments on filamentous phage. The phage are selected for 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 for binding to the antigen. The result is a human antibody Fab fragment specific to a particular antigen. In another approach, a library of phage is generated, each member of which displays a different human antibody fragment (Fab or Fv) on its outer surface (Dower et al., WO 91 / 17271 and McCafferty et al., WO92 / 01047). Phage-displayed antibodies with the desired specificity are selected by affinity enrichment against a specific antigen. Human Fab or Fv fragments identified from either approach may be recloned for expression as human antibodies in mammalian cells.

[0122] Human antibodies are sometimes obtained from transgenic animals (U.S. Patent Nos. 6,150,584; 6,114,598; and 5,770,429). In this approach, the heavy-chain joining region (JH) gene is deleted in chimeric or germline mutant mice. The human germline immunoglobulin gene array is then introduced into these mutant mice. The resulting transgenic mice are then capable of producing a full repertoire of human antibodies upon antigen challenge.

[0123] Humanized or human antibodies can be selected from any immunoglobulin class, including IgM, IgG, IgD, IgA, or IgE, and any isotype, including IgG1, IgG2, IgG3, and IgG4. Humanized or human antibodies can contain sequences from more than one isotype or class. Furthermore, these antibodies are typically produced as antigen-binding fragments, such as Fab, Fab', F(ab')2, Fd, Fv, and single-domain antibody fragments, or as single-chain antibodies in which the heavy and light chains are linked by a spacer. Human or humanized antibodies can also exist in monomeric or polymeric form. Humanized antibodies optionally comprise one non-human chain and one humanized chain (i.e., one humanized heavy or light chain).

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

[0125] Thus, in certain embodiments, the antibodies described herein comprise 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 sequence of CDR-H3 comprises the sequence: AGGPTGNSHFTL (SEQ ID NO: 12), ARNPVGSVNL (SEQ ID NO: 18), ARRYTGDTYLGNFNL (SEQ ID NO: 24), GRGDI (SEQ ID NO: 36), ARDIFRTNTNL (SEQ ID NO: 48), VRSSGSDWWFHI (SEQ ID NO: 122), or VRQNYEGAY (SEQ ID NO: 132). In one embodiment, the sequence of CDR-H3 comprises the sequence AGGPTGNSHFTL (SEQ ID NO: 12). In one embodiment, the CDR-H3 sequence comprises the sequence ARNPVGSVNL (SEQ ID NO: 18). In one embodiment, the CDR-H3 sequence comprises the sequence ARRYTGDTYLGNFNL (SEQ ID NO: 24). In one embodiment, the CDR-H3 sequence comprises the sequence GRGDI (SEQ ID NO: 36). In one embodiment, the CDR-H3 sequence comprises the sequence ARDIFRTNTNL (SEQ ID NO: 48). In one embodiment, the CDR-H3 sequence comprises the sequence VRSSGSDWWFHI (SEQ ID NO: 122). In one embodiment, the CDR-H3 sequence comprises the sequence VRQNYEGAY (SEQ ID NO: 132).

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

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

[0128] In one aspect, the disclosure provides an antibody comprising 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: CDR-H1: GFSLSRYY SEQ ID NO: 10; CDR-H2: IIPGGTT SEQ ID NO: 11; CDR-H3: AGGPTGNSHFTL SEQ ID NO: 12; CDR-L1: ESVYNNNH SEQ ID NO: 13; CDR-L2: EAS SEQ ID NO: 14; and CDR-L3: SGYKRVTTDGIA SEQ ID NO: 15, An antibody is provided.

[0129] In one aspect, the disclosure provides an antibody comprising 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: CDR-H1: GFSFSSNYV SEQ ID NO: 16; CDR-H2: IWFAGIVDTT SEQ ID NO: 17; CDR-H3: ARNPVGSVNL SEQ ID NO: 18; CDR-L1: ESVYSNNR SEQ ID NO: 19; CDR-L2: YAS SEQ ID NO: 20; and CDR-L3: AGWRGARTDGVD SEQ ID NO: 21, An antibody is provided.

[0130] In one aspect, the disclosure provides an antibody comprising 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: CDR-H1: GFSFSSSYV SEQ ID NO: 22; CDR-H2: SDTGINT SEQ ID NO:23; CDR-H3: ARRYTGDTYLGNFNL SEQ ID NO: 24; CDR-L1: QSVYKNNY SEQ ID NO: 25; CDR-L2: KAS SEQ ID NO: 26; and CDR-L3: AGGWRSLNA SEQ ID NO: 27, An antibody is provided.

[0131] In one aspect, the disclosure provides an antibody comprising 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: CDR-H1: EFSFSSRYW SEQ ID NO: 28; CDR-H2: IYTGSIDAT SEQ ID NO: 29; CDR-H3: VRGSDAWGLYFNL SEQ ID NO: 30; CDR-L1: QSIHKNNY SEQ ID NO: 31; CDR-L2: FAS SEQ ID NO: 32; and CDR-L3: AGVYSGRIFA SEQ ID NO: 33, An antibody is provided.

[0132] In one aspect, the disclosure provides an antibody comprising 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: CDR-H1: GFSLSSYT SEQ ID NO: 34; CDR-H2: IYGGIGST SEQ ID NO: 35; CDR-H3: GRGDI SEQ ID NO: 36; CDR-L1: QSVYKNR SEQ ID NO: 37; CDR-L2: GAS SEQ ID NO: 38; and CDR-L3: LGNYDCSSVDCGA SEQ ID NO: 39, An antibody is provided.

[0133] In one aspect, the disclosure provides an antibody comprising 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: CDR-H1: GFSFSAYY SEQ ID NO: 40; CDR-H2: TIPIGRT SEQ ID NO: 41; CDR-H3: AGGPTGNSHFTL SEQ ID NO: 42; CDR-L1: ESVYNNNQ SEQ ID NO: 43; CDR-L2: QAS SEQ ID NO: 44; and CDR-L3: AGYKSPTTDGIA SEQ ID NO: 45, An antibody is provided.

[0134] In one aspect, the disclosure provides an antibody comprising 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: CDR-H1: GFSLSSYA SEQ ID NO: 46; CDR-H2: IYNYET SEQ ID NO: 47; CDR-H3: ARDIFRTNTNL SEQ ID NO: 48; CDR-L1: QSVYKNNG SEQ ID NO: 49; CDR-L2: FTS SEQ ID NO: 50; and CDR-L3: LGGYDCSSRVCGA SEQ ID NO: 51, An antibody is provided.

[0135] In one aspect, the disclosure provides an antibody comprising 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: CDR-H1: GFSLSSYN SEQ ID NO: 120; CDR-H2: IGTGGIT SEQ ID NO: 121; CDR-H3: VRSSGSDWWFHI SEQ ID NO: 122; CDR-L1: QSVYNNNN SEQ ID NO: 123; CDR-L2: RAS SEQ ID NO: 124; and CDR-L3: QGYFSGFITT SEQ ID NO: 125, An antibody is provided.

[0136] In one aspect, the disclosure provides an antibody comprising 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: CDR-H1: GFTFSSYY SEQ ID NO: 130; CDR-H2: INSNGGST SEQ ID NO: 131; CDR-H3: VRQNYEGAY SEQ ID NO: 132; CDR-L1: QSIVHSNGNTY SEQ ID NO: 133; CDR-L2: KVS SEQ ID NO: 134; and CDR-L3: FQSSHVPWT SEQ ID NO: 135, An antibody is provided.

[0137] In one aspect, the disclosure provides an antibody comprising 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: CDR-H1: GFTFSSYY SEQ ID NO: 140; CDR-H2: INTNGGST SEQ ID NO: 141; CDR-H3: VRQNYEGAY SEQ ID NO: 142; CDR-L1: QSIVHSNGNTY SEQ ID NO: 143; CDR-L2: KVS SEQ ID NO: 144; and CDR-L3: FQSSHVPWT SEQ ID NO: 145, An antibody is provided.

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

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

[0140] In some embodiments, the antibody comprises a heavy chain variable region comprising i) the amino acid sequence set forth in SEQ ID NO: 102, ii) an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% sequence identity to SEQ ID NO: 102, wherein the CDR sequences are set forth in SEQ ID NOs: 22-24, or iii) a conservatively substituted amino acid sequence of i); and / or the antibody comprises i) the amino acid sequence set forth in SEQ ID NO: 103, 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: 103. a light chain variable region comprising an amino acid sequence having 70%, at least 80%, or at least 90% sequence identity thereto, wherein the CDR sequences are set forth in SEQ ID NOs:25-27, or iii) a conservatively substituted amino acid sequence of i), wherein optionally the heavy chain variable region amino acid sequence is encoded by the nucleotide sequence set forth in SEQ ID NO:80 or a codon-degenerate or optimized version thereof, and / or the light chain variable region amino acid sequence is encoded by the nucleotide sequence set forth in SEQ ID NO:81 or a codon-degenerate or optimized version thereof.

[0141] In one embodiment, the antibody comprises a heavy chain variable region comprising i) the amino acid sequence set forth in SEQ ID NO: 104, ii) an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% sequence identity to SEQ ID NO: 104, wherein the CDR sequences are set forth in SEQ ID NOs: 28-30, or iii) a conservatively substituted amino acid sequence of i); and / or the antibody comprises i) the amino acid sequence set forth in SEQ ID NO: 105, 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: 105. and / or a light chain variable region comprising an amino acid sequence having at least 70%, at least 80%, or at least 90% sequence identity with the heavy chain variable region of SEQ ID NO: 1, wherein the CDR sequences are set forth in SEQ ID NOs: 31-33, or iii) a conservatively substituted amino acid sequence of i), wherein optionally the heavy chain variable region amino acid sequence is encoded by the nucleotide sequence set forth in SEQ ID NO: 82 or a codon-degenerate or optimized version thereof, and / or the light chain variable region amino acid sequence is encoded by the nucleotide sequence set forth in SEQ ID NO: 83 or a codon-degenerate or optimized version thereof.

[0142] In some embodiments, the antibody comprises a heavy chain variable region comprising i) the amino acid sequence set forth in SEQ ID NO: 106, ii) an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% sequence identity to SEQ ID NO: 106, wherein the CDR sequences are set forth in SEQ ID NOs: 34-36, or iii) a conservatively substituted amino acid sequence of i); and / or the antibody comprises i) the amino acid sequence set forth in SEQ ID NO: 107, 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: 107. a light chain variable region comprising an amino acid sequence having 70%, at least 80%, or at least 90% sequence identity thereto, wherein the CDR sequences are set forth in SEQ ID NOs: 37-39, or iii) a conservatively substituted amino acid sequence of i), wherein optionally the heavy chain variable region amino acid sequence is encoded by the nucleotide sequence set forth in SEQ ID NO: 84 or a codon-degenerate or optimized version thereof, and / or the light chain variable region amino acid sequence is encoded by the nucleotide sequence set forth in SEQ ID NO: 85 or a codon-degenerate or optimized version thereof.

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

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

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

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

[0147] In some embodiments, the antibody comprises a heavy chain variable region comprising i) the amino acid sequence set forth in SEQ ID NO: 148, ii) an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% sequence identity to SEQ ID NO: 148, wherein the CDR sequences are set forth in SEQ ID NOs: 140-142, or iii) a conservatively substituted amino acid sequence of i); and / or the antibody comprises i) the amino acid sequence set forth in SEQ ID NO: 149, 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: 149. a light chain variable region comprising an amino acid sequence having 70%, at least 80%, or at least 90% sequence identity thereto, wherein the CDR sequences are set forth in SEQ ID NOs: 143-145, or iii) a conservatively substituted amino acid sequence of i), wherein optionally the heavy chain variable region amino acid sequence is encoded by the nucleotide sequence set forth in SEQ ID NO: 146 or a codon-degenerate or optimized version thereof, and / or the light chain variable region amino acid sequence is encoded by the nucleotide sequence set forth in SEQ ID NO: 147 or a codon-degenerate or optimized version thereof.

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

[0149] In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a chimeric antibody, e.g., a humanized antibody. In some embodiments, the antibody is a single-chain antibody.

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

[0151] In certain embodiments, the antibodies are generated or screened using the isolated peptides or immunogens described herein.

[0152] Another aspect includes an antibody that competes with an antibody described herein, optionally an antibody comprising a set of CDRs described herein, for binding to human misfolded TDP43.

[0153] Competition between antibodies can be determined, for example, using an assay in which a test antibody is evaluated for its ability to inhibit specific binding of a reference antibody to a common antigen. A test antibody competes with a reference antibody if 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.

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

[0155] Accordingly, certain embodiments provide an immunoconjugate comprising an antibody described herein and a detectable label.

[0156] A further aspect relates to an antibody conjugate comprising an antibody and / or 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.

[0157] 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 and more particularly in Table 3.

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

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

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

[0161] A further aspect provides an isolated nucleic acid encoding amino acid residues of an isolated peptide, immunogen, or antibody described herein.

[0162] Another aspect is an expression cassette or vector comprising a nucleic acid disclosed herein. In certain embodiments, the vector is an isolated vector.

[0163] The vector can be any vector, including vectors suitable for producing antibodies and / or binding fragments thereof or expressing the peptide sequences described herein.

[0164] The nucleic acid molecule may be incorporated in a known manner into a suitable expression vector that ensures protein expression. Possible expression vectors include, but are not limited to, cosmids, plasmids, or modified viruses (e.g., replication-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," which means that the expression vector contains a nucleic acid molecule encoding an epitope / peptide or peptide corresponding to an antibody described herein.

[0165] In some embodiments, the vector is suitable for expressing, for example, a single-chain antibody (e.g., an intrabody). Suitable regulatory sequences can be derived from a variety of sources, including bacterial, fungal, viral, mammalian, or insect genes. 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 confer inducibility of transcription, may be incorporated into the expression vector. In some embodiments, the regulatory sequences direct or enhance expression in neural tissues and / or cells. In some embodiments, the vector is a viral vector. The recombinant expression vector may also contain a marker gene that facilitates the selection of host cells transformed, infected, or transfected with the vector to express the antibodies or epitope peptides described herein. The recombinant expression vector may contain an expression cassette encoding a fusion moiety (i.e., a "fusion protein") that provides increased expression or stability of the recombinant peptide; increased solubility of the recombinant peptide; and aids in the purification of the target recombinant peptide by acting as a ligand in affinity purification, including, for example, the tags and labels described herein. Additionally, a proteolytic cleavage site may be added to the target recombinant protein to allow separation of the recombinant protein from the fusion moiety following purification of the fusion protein. Exemplary fusion expression vectors include pGEX (Amrad Corp., Melbourne, Australia), pMAL (New England Biolabs, Beverly, Massachusetts), and pRIT5 (Pharmacia, Piscataway, New Jersey), which fuse glutathione S-transferase (GST), maltose E-binding protein, or protein A, respectively, to the recombinant protein.

[0166] In another aspect, a cell expressing an antibody described herein is also provided. In some embodiments, the cell is an isolated cell and / or a recombinant cell that expresses an antibody described herein or comprises a vector disclosed herein. In some embodiments, the cell is a fusion cell, e.g., a hybridoma.

[0167] The recombinant cells can be made using any cells suitable for producing a polypeptide, e.g., for producing an antibody and / or a binding fragment thereof. For example, to introduce a nucleic acid (e.g., a vector) into a cell, the cell may be transfected, transformed, or infected, depending on the vector used.

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

[0169] In certain embodiments, the cell is a eukaryotic cell selected from a yeast, plant, worm, insect, bird, fish, reptile, and mammalian cell.

[0170] In another embodiment, the mammalian cell is a myeloma cell, a spleen cell, or a hybridoma cell.

[0171] In one embodiment, the cell is a neuronal cell.

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

[0173] Mammalian cells that may be suitable include, among others, COS (e.g., ATCC No. CRL1650 or 1651), BHK (e.g., ATCC No. CRL6281), CHO (ATCC No. CCL61), HeLa (e.g., ATCC No. CCL2), 293 (ATCC No. 1573), and NS-1 cells. Suitable expression vectors for directing expression in mammalian cells generally include a promoter (e.g., derived from viral material such as polyoma, adenovirus 2, cytomegalovirus, and simian virus 40) and other transcriptional and translational regulatory sequences. Examples of mammalian expression vectors include pCDM8 and pMT2PC.

[0174] In some embodiments, the cells are fusion cells, e.g., hybridoma cells, which produce antibodies specific and / or selective for an epitope or epitope sequence described herein, including, e.g., those that selectively bind to misfolded TDP-43.

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

[0176] IV. Composition A further aspect is a composition comprising an isolated peptide, immunogen, antibody, or immunoconjugate described herein. Also provided are compositions comprising two or more of the isolated peptides, immunogens, antibodies, or immunoconjugates described herein.

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

[0178] Suitable diluents for polypeptides and / or cells, including antibodies or fragments thereof, include, but are not limited to, saline, pH buffers, and glycerol solutions or other solutions suitable for freezing the polypeptides and / or cells.

[0179] In certain embodiments, the composition comprising a peptide, compound, or immunogen described herein comprises an adjuvant.

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

[0181] Adjuvants that can be used include, for example, intrinsic adjuvants (such as lipopolysaccharides), which are typically components of killed or attenuated bacteria used in vaccines. Extrinsic adjuvants are immunomodulators that are typically non-covalently bound to antigens and formulated to enhance the host immune response. Aluminum hydroxide, aluminum sulfate, and aluminum phosphate (collectively commonly referred to as alum) are commonly used as adjuvants. A wide range of exogenous adjuvants can induce a strong immune response to immunogens. These include saponins, such as Stimulon (QS21, Aquila, Worcester, Massachusetts) or particles made therefrom, such as ISCOMs and ISCOMATRIX complexed with membrane protein antigens, pluronic polymers with mineral oil, killed mycobacteria and mineral oil, Freund's complete adjuvant, bacterial products such as muramyl dipeptide (MDP) and lipopolysaccharide (LPS), and lipid A, and liposomes.

[0182] 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.). The oil-in-water emulsions may also 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-Al-D-isoglu-L-Ala-dipalmitoxypropylamide (DTP-DPP) Teramide™), or other bacterial cell wall components.

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

[0184] In one embodiment, 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 one embodiment, the composition is a sterile composition.

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

[0186] In some embodiments, the composition is for use in a method described herein, such as detecting misfolded TDP-43.

[0187] In some embodiments, the composition comprises a pharmaceutically acceptable carrier, diluent, and / or excipient. In some embodiments, the composition is a pharmaceutical composition for a method described herein, such as treating a subject in need thereof, e.g., a subject with a TDP-43 proteinopathy.

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

[0189] V. Kits and Packages A further aspect relates to a kit or package comprising i) an isolated peptide, ii) an immunogen, iii) an antibody, iv) an immune complex, v) an isolated nucleic acid, or vi) a composition, and optionally a reference agent and / or instructions for its use, contained in a vial, such as a sterile vial, or other container.

[0190] In some embodiments, the kit is an ELISA, hi some embodiments, the kit is a multiplex assay or planar array kit similar to those available, for example, through MesoScale, Quanterix, or Singulex, or for carrying out the methods described herein.

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

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

[0193] VI. Method Included are methods of producing the isolated peptides, immunogens and antibodies described herein.

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

[0195] In one embodiment, the method comprises isolating antibodies that selectively bind to an immunogen and / or a TDP-43 peptide of misfolded TDP-43 from an expression library encoding immunoglobulin genes, or portions thereof. Optionally, the expression library is a phage display library.

[0196] A further aspect includes a method of inducing an immune response in a non-human subject, comprising administering to said 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.

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

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

[0199] A further aspect provides an antibody produced by the methods described herein.

[0200] A further aspect provides a method for detecting whether a sample comprises misfolded TDP-43.

[0201] In one embodiment, the method further comprises: a. contacting the sample with an antibody described herein under conditions permissive to produce an antibody:misfolded TDP-43 polypeptide complex; and b. Detecting the presence of any complexes comprising wherein the presence of a detectable complex indicates that the sample may contain a misfolded TDP-43 polypeptide.

[0202] In another embodiment, the method further comprises: (a) contacting the subject's test sample with an antibody described herein under conditions permissive to produce an antibody-antigen complex; (b) determining the amount of antibody-antigen complexes in the test sample; and (c) comparing the amount of antibody-antigen complex in the test sample with a control. comprising wherein detecting an antibody-antigen complex in the test sample relative to a control indicates that the sample comprises misfolded TDP-43.

[0203] The measuring may be, for example, by immunofluorescence. The method may also include co-localization staining, for example, pan-TDP-43 staining.

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

[0205] In one embodiment, the sample is from a subject with ALS. In another embodiment, the sample is from a subject with FTD. In one embodiment, the sample is from a subject with limbic-predominant late-life TDP-43 encephalopathy (LATE).

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

[0207] Singleplex bead-based platforms can be used in which individual immune complexes are isolated on paramagnetic beads and detected. Suitable bead-based singleplex platforms include those available from Quanterix.

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

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

[0210] Planar array platform can be used, in which capture antibody is immobilized on solid surface, for example, membrane, glass surface, or on each well of, for example, 96-well plate or 384-well plate, in microarray format, and analyte is detected by second detection antibody.Because of the spatial separation of microarray platform, spot coordinates can be used to identify the detected analyte.Suitable planar array platform includes, for example, those available from Quaternix or Mesoscale.

[0211] Bead- or particle-based assays utilizing fluorescently labeled detection antibodies may be followed by single-molecule counting, in which the detection antibodies are eluted from the immune complexes and quantified by detecting and counting individual fluorescent molecules using capillary fluidics and lasers. Suitable techniques include, for example, those available from Singulex.

[0212] Surface plasmon resonance can be used to assess conformation-specific binding.

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

[0214] Also provided is a method of inhibiting misfolded TDP-43 cell-to-cell spread, comprising administering an antibody described herein to a subject in need thereof, e.g., a subject suspected of having, at risk of developing, or diagnosed with a TDP-43 proteinopathy.

[0215] Also provided are methods for treating a TDP-43 proteinopathy, comprising administering to a subject in need thereof an effective amount of the antibodies, immunoconjugates, or compositions comprising said antibodies of the immunoconjugates disclosed herein.

[0216] In certain embodiments, the TDP-43 proteinopathy is selected from amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD-TDP), primary lateral sclerosis, progressive muscular atrophy, and limbic-predominant late-life TDP-43 encephalopathy (LATE).

[0217] A further aspect is a method of treating a subject in need thereof, comprising administering to a subject an effective amount of the antibody or immunoconjugate disclosed herein, or a composition comprising said antibody or immunoconjugate, optionally in combination with another TDP-43 proteinopathy treatment, including, but not limited to, treatments for ALS, such as riluzole (Rilutek or Tiglutik™), edaravone (Radicava™), and Nuedexta™ (a combination of dextromethorphan and quinidine).

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

[0219] The compositions, antibodies, immunogens, and immunoconjugates described herein can be administered, for example, parenterally, intravenously, subcutaneously, intramuscularly, intracranially, intraventricularly, intrathecally, intraorbitally, ocularly, intraspinally, intracisternally, intraperitoneally, intranasally, by aerosol, or orally.

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

[0221] Other embodiments contemplate the co-administration of the compositions, antibodies, and immunoconjugates described herein with biologically active molecules known to facilitate transport across the blood-brain barrier.

[0222] In certain embodiments, methods of administering the compositions, antibodies, and immunoconjugates described herein across the blood-brain barrier, such as those directed to transiently increasing the permeability of the blood-brain barrier, are also contemplated, such as those described in U.S. Pat. No. 7,012,061, "Methods for Increasing the Permeability of the Blood-Brain Barrier," which is incorporated herein by reference.

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

[0224] The vectorized antibody can be in a composition.Non-viral vectors, such as adeno-associated virus (AAV, e.g., AAV9) and lentiviral vectors, can also be used.In certain embodiments, the nucleic acid and / or vector can be injected intracerebroventricularly or intrathecally.

[0225] 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 set forth for illustrative purposes only and are not intended to limit the scope of the present application. Modifications in form and substitution of equivalents are contemplated wherever circumstances may suggest or render expedient. Although specific terms are used herein, such terms are intended to be in a descriptive sense and not for purposes of limitation.

[0226] The following non-limiting examples are illustrative of the present disclosure: [Example]

[0227] Example 1 TDP-43 epitope The TDP-43 NTD comprises a ubiquitin-like domain. TDP-43 NTD Trp68 is uniquely present in TDP-43 when aligned with other ubiquitin-like domain-containing proteins (e.g., human ubiquitin, Axin1, human Dvl-2) [9]. Trp68 was determined to be not solvent-exposed in PDB structures such as PDBID 2N4P and 6B1G. We hypothesized that Trp68 (i.e., the position in the human sequence represented in NM_007375.3) would be solvent-exposed (accessible to antibodies) in misfolded TDP-43. A seven-amino acid peptide centered on tryptophan 68 was selected, and an immunogen was designed as described in Example 2.

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

[0229] The synthesized peptides were then conjugated via the C-terminal cys residue to keyhole limpet hemocyanin (KLH) (for immunization) or BSA (for screening) to produce the immunogens used in Example 3.

[0230] Example 3 Polyclonal antibody generation and selection Polyclonal antibodies were produced using an immunogen comprising the peptide DAGWGNL (SEQ ID NO: 1) linked to KLH via a C-terminal cysteine.

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

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

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

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

[0235] The ELISA results are shown in Tables 1A and 1B, which show the binding of serial dilutions of crude antisera (1A) and affinity-purified antibodies raised against the immunizing peptide (1B).

[0236] [Table 2]

[0237] [Table 3]

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

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

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

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

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

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

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

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

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

[0247] immunization Briefly, mice are immunized by a series of subcutaneous injections of an aqueous solution over an extended period of time, after which the mice are euthanized and lymphocytes are harvested for generation of hybridoma cell lines.

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

[0249] Hybridoma analysis (screening) Tissue culture supernatants from hybridomas can be tested by indirect ELISA against the screening antigen, probing for both IgG and IgM antibodies using a goat anti-IgG / IgM (H&L)-HRP secondary and developed with TMB substrate.

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

[0251] Positive IgG-secreting clones are subjected to large-scale production.

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

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

[0254] Example 6 Rabbit monoclonal antibody production The DAGWGNL (SEQ ID NO: 1) peptide conjugated to KLH via the C-terminal cysteine ​​(peptide-KLH) was used to immunize rabbits to generate B cells secreting monoclonal antibodies specific to the TDP-43 peptide. Based on indirect ELISA testing of isolated B cells, B cells were selected for RNA isolation and recombinant plasmid DNA production. Functional antigen-binding recombinant mAbs were transfected and expressed to generate purified mAbs.

[0255] immunization : Two sets of rabbits were immunized simultaneously: one set with rapid rabbit (28 day) immunization and one set with standard rabbit (78 day) immunization.

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

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

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

[0259] Antibody analysis (screening) - Day 7: B cell culture supernatants from the plates were tested against BSA-conjugated TDP43 linear peptides by indirect ELISA, probed with a secondary antibody against rabbit IgG antibodies. The most responsive antigen-specific B cells were transferred to new plates. Clonal supernatants were then titrated by indirect ELISA against BSA-conjugated TDP43 linear peptides and captured to identify top clones.

[0260] Example 7 Monoclonal antibody cloning and sequencing Cloning of mouse mAb The variable regions of the heavy and light chain immunoglobulin genes for several murine hybridoma clones were identified and sequenced.

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

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

[0263] Cloning of rabbit mAb Cloning - Day 14: Top clones underwent antibody RNA isolation and recombinant plasmid DNA production. For each top clone, the heavy and light chain variable regions were cloned into separate mammalian expression vectors containing rabbit heavy and kappa constant regions.

[0264] Expression - 14 days: The heavy and light chain vectors were co-transfected into mammalian cells for small-scale transfection. Supernatants were screened by indirect ELISA against a BSA-conjugated TDP43 peptide to confirm that functional recombinant mAbs were generated.

[0265] Purification: The top recombinant mAbs (DNA constructs containing one heavy and one light chain) were sequenced as shown in Tables 2, 3, and 4 below, with the CDR1, CDR2, and CDR3 regions of the heavy and light chains underlined and in bold in Tables 3 and 4. Additional mAbs were sequenced and had SEQ ID NOs: 52-75, 90-97, and 112-119.

[0266] [Table 4]

[0267] [Table 5] TIFF2025146838000007.tif160129TIFF2025146838000008.tif157129TIFF2025146838000009.tif62128

[0268] [Table 6] TIFF2025146838000011.tif110129

[0269] Example 8 Direct binding assay Binding of antisera, hybridoma supernatants or purified antibodies to the peptides (conjugated to BSA) can be examined by surface plasmon resonance using a Biacore™ 3000 instrument (GE Healthcare).

[0270] Binding assays are performed using antigen immobilized on high density (at least 1000 response units (RU)) flow cells. Dilutions of selected clones are injected sequentially over the surface to assess binding.

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

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

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

[0274] [Table 7]

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

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

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

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

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

[0280] [Table 8]

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

[0282] [Table 9]

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

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

[0285] [Table 10]

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

[0287] Donor cells transfected with HA-dNLS-TDP43 contained large amounts of HA-tagged TDP-43. Naive recipient cells incubated with the supernatant of donor cells treated with control mouse IgG1 (mIgG1) contained detectable amounts of HA-tagged TDP-43, indicating extracellular transmission of misfolded aggregates of HA-dNLS-TDP-43 protein from donor cells to recipient cells. Recipient cells incubated with the supernatant of donor cells pretreated with a misfolding-specific TDP-43 antibody contained relatively low amounts of HA-tagged TDP-43, indicating inhibition of transmission by the antibody. As a negative control, recipient cells incubated with the supernatant from untransfected donor cells did not contain any detectable HA-tagged TDP-43. Quantitative analysis of the Western blots is shown in Figure 9. For each antibody, the HA-tagged signal was first normalized to the GAPDH signal (HA intensity / GAPDH intensity), and the value obtained for the test antibody was divided by the value obtained with the control mIgG1. Compared to the control mIgG1, all tested antibodies inhibited the spread of misfolded HA-dNLS-TDP-43 from donor cell supernatants, resulting in low levels of HA-tagged TDP-43 in recipient cells.

[0288] Example 15 Generation of antibodies selective for misfolded disease-associated TDP-43 Misfolded TDP-43 species are implicated in neurotoxicity and prion-like cell-to-cell propagation in amyotrophic lateral sclerosis (ALS) and frontotemporal lobe dementia (FTLD). Tryptophan (Trp68) in the TDP-43 N-terminal domain (NTD) was found to be involved in cross-seeding of SOD1 misfolding despite being inaccessible in the natively folded NTD [9]. We hypothesized that NTD Trp68 becomes exposed when TDP-43 mislocalizes / aggregates in the cytoplasm.

[0289] Design / Method To generate polyclonal and monoclonal antibodies (pAbs, mAbs), rabbits were immunized with a misfolded NTD linear peptide epitope containing Trp68. To generate mAbs, mice were immunized with the same epitope. Monoclonal antibody affinity to the immunizing peptide was determined by surface plasmon resonance (SPR). NTD was expressed in Escherichia coli, and the properly folded monomeric state was confirmed by size-exclusion chromatography, followed by denaturing and native gel electrophoresis and immunoblotting. Antibody specificity was confirmed by immunohistochemistry (IHC) on patient samples and immunocytochemistry (ICC) on HEK293 cells transfected with a TDP-43 triple tandem mutation in the nuclear localization sequence (ΔNLS). The ability of the antibodies to inhibit cell-to-cell spread of misfolded TDP-43 was assessed in vitro.

[0290] result SPR of the mAb revealed picomolar affinity for the epitope. Recombinant NTD showed pAb immunoreactivity only under denaturing conditions. IHC of ALS / FTLD CNS sections, but not normal CNS, was reactive for the antibody. ICC revealed immunoreactivity for mislocalized / aggregated ΔNLS-TDP-43, but not for nuclear wild-type TDP-43. The antibody also failed to recognize TDP-43 in physiological stress granules in HEK293 cells. A ΔNLS-TDP-43 construct in which Trp68 was mutated to serine showed no immunoreactivity in transfected cells, indicating that Trp68 is immunodominant in the immunizing peptide. The antibody, tested in cell culture assays, was able to inhibit cell-to-cell spread of misfolded TDP-43, a process thought to be central to pathogenesis (Refs. 10, 11, 12).

[0291] conclusion We developed a family of antibodies sensitive to the solvent exposure of NTD Trp68 that are selective for misfolded / aggregated disease-associated TDP-43 while sparing physiologically important molecular species. These antibodies may find utility in biomarker and immunotherapy applications for TDP-43-associated diseases.

[0292] While the present application has been described with reference to what are presently considered to be the preferred embodiments, it is to be understood that the present application is not limited to the disclosed embodiments. To the contrary, the present application is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

[0293] All publications, patents, and patent applications are incorporated herein by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated herein by reference in its entirety. Specifically, for example, the sequences associated with each accession number set forth herein, including the accession number and / or biomarker sequences (e.g., proteins and / or nucleic acids) set forth in the tables or elsewhere, are incorporated herein by reference in their entirety.

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

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

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

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

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

Claims

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

2. The isolated peptide according to claim 1, wherein the portion thereof is DAGWG (SEQ ID NO: 2), AGWGN (SEQ ID NO: 4), or GWGNL (SEQ ID NO: 6).

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

1.

4. The immunogen according to claim 3, comprising a plurality of peptides, each peptide comprising all or part of DAGWGNL (SEQ ID NO: 1).

5. The immunogen according to claim 4, wherein the plurality of peptides are synthesized as multiantigenic peptides (MAPs).

6. The immunogen according to any one of claims 3 to 5, wherein the peptide is coupled to a carrier protein or keyhole limpet hemocyanin (KLH), and the carrier protein is optionally bovine serum albumin (BSA).

7. The immunogen according to claim 6, wherein the carrier protein is bovine serum albumin (BSA).

8. The immunogen according to any one of claims 3 to 7, for use in producing an antibody that specifically binds to misfolded TDP-43, specifically binds to DAGWGNL (SEQ ID NO: 1), and / or specifically binds to at least W68 in relation to DAGWGNL (SEQ ID NO: 1).

9. The immunogen according to claim 8, wherein the antibody preferentially binds to misfolded TDP-43.

10. An isolated nucleic acid encoding an amino acid residue of an isolated peptide according to claim 1 or 2, or an immunogen according to any one of claims 3 to 9.

11. The isolated nucleic acid according to claim 10, which is contained in a vector.

12. Cells that recombinantly express the peptide according to claim 1 or 2, or the immunogen according to any one of claims 3 to 9.

13. A composition comprising an isolated peptide according to claim 1 or 2, an immunogen according to any one of claims 3 to 9, an isolated nucleic acid according to claim 10 or 11, or a cell according to claim 12.

14. The composition according to claim 13, further comprising a diluent or a pharmaceutically acceptable carrier.

15. The composition according to claim 13 or 14, further comprising the immunogen, which further comprises an adjuvant.

16. The composition according to claim 15, wherein the adjuvant is aluminum phosphate, aluminum hydroxide, alum, monophosphoryl lipid A and / or QS21.

17. A composition according to any one of claims 13 to 16 for use in the treatment of the target.

18. The composition according to claim 17, wherein the subject is suspected of having TDP-43 proteinosis, is at risk of developing it, or has been diagnosed with it.

19. The composition according to claim 18, wherein the TDP-43 proteinosis is selected from amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD-TDP), primary lateral sclerosis, progressive muscular atrophy, and limbic-dominant senile TDP-43 encephalopathy (LATE).

20. The composition according to any one of claims 17 to 19, for administration in combination with the treatment of another TDP-43 proteinosis.

21. The composition according to any one of claims 13 to 16, for use in inhibiting TDP-43 cell propagation in a target where it is needed.

22. The composition according to any one of claims 17 to 21, wherein the antibody, immune complex, nucleic acid, or composition is for administration by parenteral administration, intravenous administration, subcutaneous administration, intramuscular administration, intracranial administration, intraventricular administration, subarachnoid administration, intraorbital administration, ocular administration, intraspinal administration, intracisional administration, intraperitoneal administration, intranasal administration, aerosol administration, or oral administration.

23. The composition according to any one of claims 17 to 22, which is administered simultaneously with a bioactive molecule known to promote transport across the blood-brain barrier.

24. i. The isolated peptide according to either claim 1 or 2; ii. The immunogen according to any one of claims 3 to 9; iii. Isolated nucleic acid according to claim 10 or 11; iv. The cells according to claim 12; and / or v. The composition according to any one of claims 13 to 16; and vi. Compartments such as vials for containing the isolated peptide, immunogen, isolated nucleic acid, cells, and / or composition, and vii. A kit, which may include instructions for use in ELISA or the method described herein.

25. A method for preparing an antibody, comprising immunizing a non-human subject with an immunogen according to any one of claims 3 to 9, or a composition comprising an immunogen according to any one of claims 3 to 9.

26. The method according to claim 25, further comprising isolating an antibody that specifically binds to W68 in relation to DAGWGNL (SEQ ID NO: 1).

27. The method according to claim 26, further comprising forming an antibody-producing hybridoma.

28. An antibody produced by the method described in any one of claims 25 to 27.

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