Humanized antibodies to misfolded TDP-43 and methods of use

Abstract

The disclosure pertains to humanized antibodies, that specifically bind to misfolded TDP-43. Also provided are nucleic acids and vectors encoding the humanized antibodies, immunogens, compositions, cells, kits and methods of using said antibodies to treat TDP-43 proteinopathies.

Description

TITLE: HUMANIZED ANTIBODIES TO MISFOLDED TDP-43 AND METHODS OF USECROSS REFERENCE TO RELATED APPLICATION

[0001] The present application is a PCT application and claims priority from U.S. provisional application no. 63 / 713,374 filed on October 29, 2024, which is hereby incorporated by reference in its entirety.INCORPORATION OF SEQUENCE LISTING

[0002] A computer readable form of the Sequence Listing “P93249336WO01_SequenceListing” (56,104 bytes) created on October 28, 2025, is herein incorporated by reference.FIELD

[0002] The present disclosure relates to humanized TDP-43 antibodies and more specifically to humanized antibodies for detecting misfolded TDP-43 and methods of detecting misfolded TDP-43 and treating TDP-43 proteinopathies, for example also included are methods and uses for treating subjects with amyotrophic lateral sclerosis (ALS), Alzheimer’s disease, frontotemporal lobar degeneration (FTLD-TDP), primary lateral sclerosis, progressive muscular atrophy, and / or limbic-predominant age-related TDP-43 encephalopathy (LATE).BACKGROUND

[0003] Transactive response (TAR) element DNA binding protein of 43 kDa (TDP-43), is a 414 amino acid protein, and is comprised of an N-terminal ubiquitin like domain (NTD, residues 1-80), two RNA recognition motifs (RRMs) composed of residues 106-177 (RRM1), and residues 192-259 (RRM2), and a C-terminal domain (CTD, residues 274-414). The NTD flanks a domain that directs nuclear localization (NLS motifs in residues 82-98, e.g. NLS1 K82RK84 and K95VKR98). RRM2 includes a nuclear export signal (NES) from residue 239 to 250.

[0004] TDP-43 is predominantly a nuclear protein that plays a central role in RNA metabolism. TDP-43 has become a focal point of research in the amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) disease spectrum, since pathogenic inclusions within affected neurons can contain post-translationally modified TDP-43. The CTD of TDP-43 is particularly relevant to disease, as it is where nearly all familial ALS / FTD-associated mutations are found in TDP-43.

[0005] Other mutations include D169G which is located in RRM1 between beta strands 4 and 5, A90V which is a mutation in the NLS region, and the mutations K263E and N267S which are in the linker between RRM2 and the C-terminal domain.

[0006] RRM1 and RRM2 have been structurally 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 the NTD is available as PDB entry 5MRG, 2N4P and 6B1G.

[0007] 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],

[0008] TDP-43 was found to be hyperphosphorylated, ubiquitinated, and fragmented in neuronal inclusions of patients with both sporadic and familial forms of ALS and FTD [4],

[0009] Functional TDP-43 can exist as nuclear oligomers that are distinct from cytoplasmic aggregates formed upon 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 pathologic aggregation [9],

[0010] Physiological TDP-43 oligomerization is mediated by its N-terminal domain, which can adopt dynamic, solenoid-like structures, revealed by a 2.1 A crystal structure in combination nuclear magnetic resonance spectroscopy and electron microscopy [9],

[0011] Aggregates (inclusion bodies) of TDP-43 have now been found in nearly all (approx. 97%) cases of ALS and roughly half (approx. 45%) of the cases of FTD. TDP-43 is one of the main components of the cytoplasmic inclusions found in the motor neurons of ALS patients.

[0012] Precursors of TDP-43 inclusions may have concentration far below that of functional TDP-43. The low concentration of misfolded TDP-43 makes this target elusive.

[0013] Intracerebral injections of brain derived pathological TDP-43 FTLD-TDP seeds in transgenic mice expressing cytoplasmic human TDP-43 and non-transgenic mice, has led to the induction of de novo TDP-43 pathology which spread through the brain in a time dependent manner

[0010] ,

[0014] Antibodies that bind TDP-43 have been described.

[0015] WO2012174666 titled METHODS FOR THE PROGNOSTIC AND / OR DIAGNOSTIC OF NEURODEGENERATIVE DISEASE, METHODS TO IDENTIFY CANDIDATE COMPOUNDS AND COMPOUNDS FOR TREATING NEURODEGENERATIVE DISEASEdiscloses methods for diagnosing neurodegenerative diseases such as ALS and FTD through assessing the interaction between TDP-43 and NF-KB p65 using an anti-TDP-43 antibody.

[0016] W02016086320 titled TDP-43-BINDING POLYPEPTIDES USEFUL FOR THE TREATMENT OF NEURODEGENERATIVE DISEASES disclose antibodies that bind to the RRM1 domain of TDP-43 to disrupt its interaction with NF-KB for the treatment of ALS and FTD.

[0017] Tamaki, Y et al, 2018 described a misfolding specific intrabody with proteolytic signals

[0011] ,

[0018] Humanized antibodies that preferentially bind misfolded TDP-43 over natively folded TDP-43 are desirable.SUMMARY

[0019] The preceding section is provided by way of example only and is not intended to be limiting on the scope of the present disclosure and appended claims. Additional objects and advantages associated with the compositions and methods of the present disclosure will be appreciated by one of ordinary skill in the art in light of the instant claims, description, and examples. For example, the various aspects and embodiments of the disclosure may be utilized in numerous combinations, all of which are expressly contemplated by the present description. These additional advantages objects and embodiments are expressly included within the scope of the present disclosure. The publications and other materials used herein to illuminate the background of the disclosure, and in particular cases, to provide additional details respecting the practice, are incorporated by reference, and for convenience are listed in the appended reference section.

[0020] Demonstrated herein, are humanized antibodies that specifically bind misfolded TDP-43.

[0021] Accordingly, an aspect includes a humanized antibody comprising a sequence as set forth in any of SEQ ID NOs: 2-8 and / or 10-14 or a sequence with at least 80% sequence identity to any thereto wherein the CDR amino acid sequences, comprising CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, CDRL3, are as set for forth in SEQ ID Nos: 15-20, respectively; and wherein the antibody comprises at least one of the following substitutions (e.g. substituted residues) relative to SEQ ID NO: 1:L at position 4; G at position 10; Q at position 13; G at position 15; R at position 19; S at position 21; R at position 71; D at position 72; N orT at position 73; Kor S at position 75; N at position 76; L at position 78; Y at position 79; N at position 82A; R at position 83; E at position 85; Y at position 91; and / or S at position 113; and / orat least one of the following substitutions (e.g. substituted residues) relative to SEQ ID NO: 9:Q at position 3; S at position 7; L at position 11; A at position 13; S at position 14;D at position 17; R at position 18; T at position 22; S at position 63; S at position 67; D or E at position 70; S at position 77; L at position 78; P at position 80; F at position 83; K at position 103; E at position 105; and / or I at position 106.

[0022] In some embodiments, the humanized antibody comprises a heavy chain variable region comprising: i) an amino acid sequence as set forth in any one of SEQ ID NOs: 2-8; ii) an amino acid sequence with at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90% orat least 95% sequence identity to any one of SEQ ID NOs: 2-8, wherein the CDR sequences, comprising CDRH1, CDRH2, CDRH3, are as set forth in SEQ ID NOs: 15-17, respectively, or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences, comprising CDRH1, CDRH2, CDRH3, are as set forth in SEQ ID NOs: 15-17, respectively.

[0023] In some embodiments, the antibody comprises a light chain variable region comprising i) an amino acid sequence as set forth any one of SEQ ID NOs: 10-14, ii) an amino acid sequence with at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90% or at least 95% sequence identity to any one of SEQ ID NO: 10-14, wherein the CDR sequences, comprising CDRL1, CDRL2, CDRL3 are as set forth in SEQ ID NOs: 18-20, respectively or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences, comprising CDRL1, CDRL2, CDRL3 are as set forth in SEQ ID NOs: 18-20, respectively.

[0024] In some embodiments, the humanized antibody comprises a heavy chain variable region and a light chain variable region as set forth in SEQ ID NO: 6 and 12, respectively; SEQ ID NO: 6 and 13, respectively; SEQ ID NO: 7 and 14, respectively; SEQ ID NO: 8 and 12, respectively; SEQ ID NO: 8 and 13, respectively, or sequence(s) with at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90% or at least 95% sequence identity to any thereto wherein the CDR sequences, comprising CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 are maintained as set forth in SEQ ID NOs: 15-20, respectively.

[0025] In some embodiments, the humanized antibody comprises a heavy chain variable region and a light chain variable region as set forth in SEQ ID NO: 6 and 12, respectively or sequence(s) with at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90% or at least 95% sequence identity to any thereto wherein the CDR sequences are maintained as set forth in SEQ ID NOs: 15-20.

[0026] In some embodiments, the humanized antibody comprises a heavy chain variable region and a light chain variable region as set forth in SEQ ID NO: 6 and 13, respectively or sequence(s) with at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90% or at least 95% sequence identity to any thereto wherein the CDR sequences are maintained as set forth in SEQ ID NOs: 15-20.

[0027] In some embodiments, the humanized antibody comprises a heavy chain variable region and a light chain variable region as set forth in SEQ ID NO: 7 and 14, respectively or sequence(s) with at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90% or at least 95% sequence identity to any thereto wherein the CDR sequences are maintained as set forth in SEQ ID NOs: 15-20.

[0028] In some embodiments, the humanized antibody comprises a heavy chain variable region and a light chain variable region as set forth in SEQ ID NO: 8 and 12, respectively or sequence(s) with at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90% or at least 95% sequence identity to any thereto wherein the CDR sequences are maintained as set forth in SEQ ID NOs: 15-20.

[0029] In some embodiments, the humanized antibody comprises a heavy chain variable region and a light chain variable region as set forth in SEQ ID NO: 8 and 13, respectively or sequence(s) with at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90% or at least 95% sequence identity to any thereto wherein the CDR sequences are maintained as set forth in SEQ ID NOs: 15-20.

[0030] In an embodiment, the at least one substation / residue is D at position 72. In another embodiment, the at least one residue is N position 76.

[0031] In some embodiments, the humanized antibody comprises a heavy chain variable region comprising a T72D and / or a T76N substitution relative to SEQ ID NO: 1; and / or a light chain variable region comprising one or more of a V3Q substitution, a T7S substitution, a K11L substitution or a K63S substitution relative to SEQ ID NO: 9.

[0032] In some embodiments, the humanized antibody (e.g., bivalent) has a KD of at least or about 1x 10-11M, at least or about 2 x10-11M, at least or about 3 x 10-11M to misfolded TDP-43.

[0033] In some embodiments, the humanized antibody is an antibody binding fragment selected from Fab, Fab', F(ab')2, scFv, dsFv, ds-scFv, dimers, nanobodies, minibodies, diabodies, and multimers thereof.

[0034] In some embodiments, the humanized antibody binding fragment is a Fab fragment.

[0035] As shown herein, the humanized antibodies selectively binds misfolded TDP-43 over native TDP-43.

[0036] In some embodiments, the humanized antibody is a single chain antibody.

[0037] Another aspect of the disclosure is an immunoconjugate comprising the humanized antibody described herein and a detectable label.

[0038] In some embodiments, the detectable label comprises a positron emitting radionuclide, optionally for use in subject imaging such as PET imaging.

[0039] Another aspect of the disclosure is a composition comprising the humanized antibody described herein, or the immunoconjugate described herein, optionally with a diluent.

[0040] Another aspect of the disclosure is a nucleic acid molecule encoding the humanized antibody described herein. A further aspect of the disclosure is one or more nucleic acid molecules encoding the humanized antibody described herein.

[0041] A further aspect of the disclosure is a vector comprising the nucleic acid described herein.

[0042] In another aspect of the disclosure is a cell expressing the humanized antibody described herein, optionally wherein the cell is a hybridoma comprising the vector described herein.

[0043] Another aspect of the disclosure is a kit comprising the humanized antibody, the nucleic acid molecule, the vector or the cell described herein.

[0044] In yet another aspect of the disclosure is a method of treating a subject, the method comprising administering to a subject in need thereof an effective amount of the humanized antibody, the immunoconjugate, the nucleic acid or the composition described herein. A further aspect is a use of an effective amount of the humanized antibody, the immunoconjugate, the nucleic acid or the composition described herein for treating a subject in need thereof. Another aspect is a use of an effective amount of the humanized antibody, the immunoconjugate, the nucleic acid or the composition described herein in the manufacture of a medicament for treating a subject in need thereof. In yet a further aspect is an effective amount of the humanized antibody, the immunoconjugate, the nucleic acid or the composition described herein for use for treating a subject in need thereof.

[0045] In some embodiments, the subject is suspected of having, is at risk of developing, or has been diagnosed with a TDP-43 proteinopathy.

[0046] In some embodiments, the TDP-43 proteinopathy is selected from amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD-TDP), primary lateral sclerosis, progressive muscular atrophy, Alzheimer’s disease and limbic-predominant age-related TDP-43 encephalopathy (LATE). The ALS can be familial ALS (fALS) or sporadic ALS (sALS). The Alzheimer’s disease can comprise or occur alongside limbic-predominant age-related TDP-43 encephalopathy (LATE).

[0047] In some embodiments, the humanized antibody, immunoconjugate, or composition is administered in combination with another TDP-43 proteinopathy treatment.

[0048] Another aspect of the disclosure is a method of inhibiting misfolded TDP-43 cell to cell transmission, the method comprising administering the humanized antibody, the immunoconjugate, the nucleic acid or the composition described herein to cells or a subject in need thereof. A further aspect of the disclosure is use of the humanized antibody, the immunoconjugate, the nucleic acid or the composition described herein for inhibiting misfolded TDP-43 cell to cell transmission, for example between cells, or in a subject in need thereof. The cell to cell transmission can be between cells that are in vitro or in vivo.

[0049] Another aspect of the disclosure is use of the humanized antibody, the immunoconjugate, the nucleic acid or the composition described herein in the manufacture of a medicament for inhibiting misfolded TDP-43 cell to cell transmission, between cells, or in a subject in need thereof. In yet a further aspect of the disclosure is the humanized antibody, the immunoconjugate, the nucleic acid or the composition described herein for use for inhibiting misfolded TDP-43 cell to cell transmission, between cells, or in a subject in need thereof.

[0050] In some embodiments, the humanized antibody, immunoconjugate, nucleic acid or composition is administered systemically.

[0051] In some embodiments, the humanized antibody, immunoconjugate, nucleic acid or composition is administered by systemic, parenteral, intravenous, subcutaneous, intramuscular, intracranial, intraventricular, intrathecal, intraorbital, ophthalmic, intraspinal, intracisternal, intraperitoneal, intranasal, aerosol or oral administration. In some embodiments the administration is intravenous, subcutaneous or intrathecal.

[0052] Other features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detaileddescription and the specific examples while indicating embodiments of the 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 DESCRIPTION OF THE DRAWINGS

[0053] Further objects, features and advantages of the disclosure will become apparent from the following detailed description taken in conjunction with the accompanying figures showing illustrative embodiments of the disclosure, in which:

[0054] Figs. 1A-B are schematics of the sequence of parent antibody 14H1-14K2. Fig. 1A depicts SEQ ID NO: 1 and Fig. 1B depicts SEQ ID NO: 9.

[0055] Fig. 2 depicts plasmid maps for human heavy chain expression vector pANTVhGI and light chain expression vector pANTVL. The VH and VL vectors contain human genomic DNA fragments encoding the variable and constant domains, incorporating introns and polyA sequences. Expression of both chains is driven by an EF1a promoter.

[0056] Fig. 3 depicts a schematic of the Biacore scheme used to assess the binding of the humanized IgG to BSA-conjugated TDP-43 linear peptide epitope (antigen) by single cycle kinetic analysis.

[0057] Fig. 4 depicts single cycle kinetics raw sensorgrams and fitted curves with a 1:1 model (in black) for the binding of chimeric (VH0 / VL0) and humanized variants to the antigen. Kinetic analysis was carried out on a Biacore T200. Each antibody was captured on a Protein A chip before increasing concentrations of antigen were injected and a single off-rate was determined.

[0058] Figs. 5A-B depicts images of SDS-PAGE gels of chimeric (VH0 / VL0) and humanized variants. 1 pg of each sample was loaded under: (a) reduced, and; (b) non-reduced conditions on a NuPage4-12% Bis-Tris gel (ThermoFisher, Loughborough, UK). All samples were heated at 95°C for 5 min and reduction was performed with addition of NuPAGE Sample Reducing Agent (ThermoFisher, Loughborough, UK). Gels were stained with InstantBlue (Expedeon, Swavesey,UK). Mk: PAGERuler™ Plus pre-stained protein ladder (ThermoFisher, Loughborough, UK).

[0059] Fig. 6 depicts results of SE-HPLC analysis of chimeric (VH0 / VL0) and humanized variants post protein A capture purification. 10 pg of purified IgG was loaded on an AdvanceBioSEC analytical column (Agilent, Cheshire, UK) using 150 mM sodium phosphate, pH 6.9, as mobile phase.

[0060] Fig. 7 depicts an image of SDS-PAGE gel of mouse IgG2a antibody, chimeric (VH0 / VL0) fusion antibody and humanized variants. 1 pg of each sample was loaded under reduced conditions on a NuPage 4-12% Bis-Tris gel (ThermoFisher, Loughborough, UK). All samples were heated at 95°C for 5 min and reduction was performed with addition of NuPAGE Sample Reducing Agent (ThermoFisher, Loughborough, UK). Gels were stained with InstantBlue (Expedeon, Swavesey, UK). Mk: PAGERuler™ Plus pre-stained protein ladder (ThermoFisher, Loughborough, UK). For reduced samples two bands were present corresponding to heavy and light chain at 50 and 25 kDa, respectively.

[0061] Fig. 8 depicts results of Western blot of chimeric (VH0 / VL0), controls and supernatants. 7.5 pl of each sample was loaded under reduced conditions on a NuPage 4-12% Bis-Tris gel (ThermoFisher, Loughborough, UK). All samples were heated at 95°C for 5 min and reduction was performed with addition of NuPAGE Sample Reducing Agent (ThermoFisher, Loughborough, UK). Gels were transferred to a membrane using Invitrogen iBIot 2 system. Mk: PAGERuler™ Plus pre-stained protein ladder (ThermoFisher, Loughborough, UK).

[0062] Fig. 9 depicts a schematic of the Biacore protocol used to assess the binding of the humanized IgG to BSA-conjugated TDP-43 linear peptide epitope (antigen) by multi-cycle kinetic analysis.

[0063] Fig. 10 depicts Multi-cycle kinetics raw sensorgrams and fitted curves with a 1:1 model (in black) for the binding of the chimeric antibody and murine antibody to antigen. Kinetic analysis was carried out on a Biacore 8K. Each IgG was captured on a Protein A sensor chip before increasing concentrations of antigen were injected.

[0064] Figs. 11A-B depict Multi-cycle kinetics raw sensorgrams and fitted curves with a 1:1 model (in black) for the binding of the chimeric antibody and five purified lead humanized variants to antigen ((a) n=1, (b) n=2). Kinetic analysis was carried out on a Biacore 8K. Each IgG was captured on a Protein A sensor chip before increasing concentrations of antigen were injected.

[0065] Figs. 12A-B depict alignment of 14H1-14K2 humanized antibody sequences. Fig.12A depicts the humanized heavy chain variants of SEQ ID NOs: 2-9 relative to 14H1-14K2 (SEQ ID NO: 1). Fig. 12B depicts the humanized light chain variants of SEQ ID NOs: 10-14 relative to 14H1-14K2 (SEQ ID NO: 9).

[0066] Figs. 13A-R are images depicting results of immunocytochemistry of antibodies in SH-SY5Y cells.

[0067] Figs. 14A-L are images depicting results of immunocytochemistry of antibodies in HEK293T cells.DETAILED DESCRIPTION OF VARIOUS EMBODIMENTS

[0068] The following is a detailed description provided to aid those skilled in the art in practicing the present disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The terminology used in the description herein is for describing particular embodiments only and is not intended to be limiting of the disclosure. All publications, patent applications, patents, figures and other references mentioned herein are expressly incorporated by reference in their entirety.

[0069] The following non-limiting examples are illustrative of the present application:L Definitions

[0070] As used herein, the following terms may have meanings ascribed to them below, unless specified otherwise. However, it should be understood that other meanings that are known or understood by those having ordinary skill in the art are also possible, and within the scope of the present disclosure. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0071] As used herein, the term “TDP-43” (transactivation response element (TAR) DNA-binding protein 43) alternately referred to as “TDP43”, or“TDP” unless otherwise qualified, means all forms of TDP-43 including wildtype TDP-43, native TDP-43, as well as misfolded forms including mutant forms and analogs thereof from all species, particularly human TDP-43 (hTDP-43). Human TDP-43 is a protein of typically 414 amino acid residues and the amino acid sequence (e.g. Uniprot Accession number Q13148) and the nucleotide sequence (e.g. Accession number HGNC:11571) have been previously characterized.

[0072] "Wild type” as used herein refers to the primary amino acid sequence of nonmutant or naturally occurring protein.

[0073] “Native” as used herein refers to the normal three-dimensional structure of a specific protein or part thereof). Native TDP-43 is optionally referred to as "natively folded" TDP-43 "normally folded" TPD-43 and / or "healthy” TDP-43. Accordingly the term "native TDP-43 ", or “natively folded TDP-43”, herein refers to TDP-43 as natively folded after nascent translation and / or multimers including but not limited to dimeric TDP-43 and trimeric TDP-43, as folded in non-disease states (e.g. healthy cells) with a molecular structure that comprises a non-covalently associated, individual TDP-43 peptide which shows native structure under in x-ray crystallography or as reconstructed from nuclear magnetic resonance spectra. Native TDP-43 forms multimers through its NTD and TDP-43 when natively folded is typically nuclear. Misfolded aggregates of TDP-43 can be and are typically cytoplasmic.

[0074] “Misfolded” as used herein refers to a state of the secondary and tertiary structure of a polypeptide or part thereof, and indicates that the polypeptide has adopted a conformation that is not normal for that polypeptide in its properly functioning state. Although misfolding can be caused by mutations in a protein, such as amino acid deletion, substitution, or addition, wild-type sequence protein can also be misfolded in disease, and expose disease-specific epitopes for instance, as a result of microenvironmental conditions and / or amino acid modification such as nitration, oxidation, carbonylation or other modification. Misfolded TDP-43 exposes tryptophan residue at position 68 (Trp68) (e.g. Uniprot accession number Q13148) which is not antibody accessible in natively folded TDP-43. Misfolded TDP-43 can be aggregated and / or cytosolic. In the context of TDP-43, native TDP-43 forms multimers through its NTD. Misfolded multimers (e.g. disease-associated oligomers) typically oligomerize through other regions of the protein, for example its LCD and / or RRM1 domains. Accordingly, "misfolded TDP-43 polypeptide", or “misfolded TDP-43” when referring to the polypeptide herein includes TDP-43 polypeptide that has Trp68 exposed, that is oligomerized through its LCD and / or RRM1 domains, non-native dimers and trimers, as well as larger aggregates (e.g. 5 or greater subunits), which is cytosolic and / or is aggregated. Misfolded TDP-43 is prone to the formation of aggregates which results in a loss of protein function, toxicity, possession of amyloid-like features (e.g. congo red staining) and / or propagation of pathogenic aggregates.

[0075] The term “mutant TDP-43” refers to forms of TDP-43, and particularly endogenous forms of TDP-43 that occur as a result of genetic mutation that result for instance in amino acid substitution, such as those substitutions characteristic for instance of FTD or familial ALS including for example the mutations described in the bioinformatics tool described in [6],

[0076] An "epitope" as used herein means a region of a protein that is recognized by a B cell or T-cell receptor, or an antibody or a binding fragment thereof. The epitope is optionally represented herein by a linear amino acid sequence or the region of the protein recognized bythe 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 said epitope sequence.

[0077] The term “amino acid” includes all of the naturally occurring amino acids as well as modified L-amino acids as well as D-amino acids. The atoms of the amino acid can for example include different isotopes. For example, the amino acids can comprise deuterium substituted for hydrogen, nitrogen-15 substituted for nitrogen-14, and carbon-13 substituted for carbon-12 and other similar changes.

[0078] A "conservative amino acid substitution" as used herein, is one in which one amino acid residue is re-placed with another amino acid residue without abolishing the protein's desired properties. Suitable conservative amino acid substitutions can be made by substituting amino acids with similar hydrophobicity, polarity, and R-group size for one another. Examples of conservative amino acid substitutions include:

[0079] The term "antibody" as used herein is intended to include monoclonal antibodies, polyclonal antibodies, single chain, humanized and other chimeric antibodies, or fully human antibodies, as well as binding fragments thereof. Also included are vectorized antibodies or intrabodies. The antibody may be from recombinant sources and / or produced in transgenic animals. Also included are human antibodies that can be produced through using biochemical techniques or isolated from a library. Humanized or chimeric antibody may include sequences from one or more than one isotype or class.

[0080] The phrase "isolated antibody" refers to antibody produced in vivo or in vitro that has been removed from the source that produced the antibody, for example, an animal, hybridoma or other cell line (such as recombinant cells that produce antibody). The isolated antibody is optionally "purified", which means at least: 80%, 85%, 90%, 95%, 98% or 99% purity.

[0081] The term "binding fragment" as used herein to a part or portion of an antibody or antibody chain comprising fewer amino acid residues than an intact or complete antibody or antibody chain and which binds the antigen or competes with intact antibody. Exemplary binding fragments include without limitations Fab, Fab', F(ab')2, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, and multimers thereof. Fragments can be obtained via chemical or enzymatic treatment of an intact or complete anti-body or antibody chain. Fragments can also be obtained by recombinant means (vide infra). For example, F(ab')2 fragments can be generated by treating the antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments. Papain digestion can lead to 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 by recombinant expression techniques.

[0082] The term “intrabody” or “intrabodies” as used herein refers to an antibody that is expressed or can be expressed in a cell and that binds to an intracellular protein, for example an intrabody is an antibody that has been modified or adapted for intracellular localization and intracellular function. An intrabody comprises a heavy chain variable domain and a light chain variable domain and linker optionally in either variable domain orientation, e.g. heavy chain variable domain- linker - light chain variable domain or light chain variable domain- linker - heavy chain variable domain. Depending on the context, the term intrabody may refer to a nucleic acid molecule or a polypeptide molecule.

[0083] The phrase “wherein the antibody comprises at least one of the following residues relative to SEQ ID NO: X” as used herein indicates that the antibody comprises one or more the stated residues using SEQ ID NO: X to identify the corresponding positions where other amino acids outside of the substituted positions can be the same or different to the corresponding residue shown in SEQ ID NO: X. As a further example, where the antibody has one or more residues relative to SEQ ID NO: X and at least 80% sequence identity to SEQ ID NO: X, the antibody comprises the one or more residues and the overall percent identity for the sequence in question (e.g., variable domain) is at least 80% identical in sequence in SEQ ID NO: X. The one or more residues are maintained and contribute to the 20% (or less) of sequence non-identity to SEQ ID NO: X.

[0084] The term “linker” as used herein refers to a synthetic sequence (e.g, amino acid sequence in a polypeptide or nucleic acid sequence in a nucleic acid) that connects or links two sequences, e.g, that link two polypeptide domains. The linker can be a “tag linker” indicating that it is linking a detectable label or a targeting moiety linker indicating that it is linking a targetingmoiety to a polypeptide which may also comprise a linker as in the case of a heavy chain variable region linked to a light chain variable region.

[0085] The term “complementarity determining region” or “CDR” as used herein refers to particular hypervariable regions of antibodies that are commonly presumed to contribute to epitope binding. Computational methods for identifying CDR sequences include Kabat, Chothia, and IMGT. The CDRs listed in the present disclosure are identified using IMGT Blast. A person skilled in the art having regard to the sequences comprised herein would also be able to identify CDR sequences based on Kabat and Chothia etc.

[0086] The term “detectable label” as used herein refers to moieties such as peptide sequences, fluorescent proteins that can be appended or introduced into a peptide, antibody or other compound described herein and which is capable of producing, either directly or indirectly, a detectable signal. For example, the label may be radio-opaque, positron-emitting radionuclide (for example for use in PET imaging), or a radioisotope, such as 3H, 13N, 14C, 18F, 32P, 35S, 1231, 1251, 1311; a fluorescent (fluorophore) or chemiluminescent (chromophore) compound, such as fluorescein isothiocyanate, rhodamine or luciferin; an enzyme, such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase; an imaging agent; or a metal ion. The detectable label may be also detectable indirectly for example using secondary antibody.

[0087] The term "epitope selectively presented or accessible in misfolded TDP-43” as used herein refers to an epitope that is selectively presented or antibody-accessible on misfolded TDP-43 as present for example in ALS or FTD (e.g. disease associated misfolded TDP-43) whether in monomeric, dimeric or aggregated forms, 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 in misfolded TDP-43.

[0088] The term “greater affinity” as used herein refers to a degree of antibody binding where an antibody X binds to target Y more strongly (Kon) and / or with a smaller dissociation constant (Koff) than to target Z, and in this context antibody X has a greater affinity for target Y than for Z. Likewise, the term "lesser affinity" herein refers to a degree of antibody binding where an antibody X binds to target Y less strongly and / or with a larger dissociation constant than to target Z, and in this context antibody X has a lesser affinity for target Y than for Z. The affinity of binding between an antibody and its target antigen, can be expressed as KA equal to 1 / KD where KD is equal to kon / koff. The kon and koff values can be measured using surface plasmon resonance (measurable for example using a Biacore system).

[0089] Also as used herein, the term "immunogenic" refers to substances which elicit the production of antibodies, activate T-cells and other reactive immune cells directed against an antigenic portion of the immunogen.

[0090] 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. The nucleic acid can be either double stranded or single stranded, and represents the sense or antisense strand. Further, the term "nucleic acid" includes the complementary nucleic acid sequences as well as codon optimized or synonymous codon equivalents. The term "isolated nucleic acid sequences" as used herein refers to a nucleic acid 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 which naturally flank the nucleic acid (e.g. sequences located at the 5' and 3' ends of the nucleic acid) from which the nucleic acid is derived.

[0091] “Operatively linked” is intended to mean that the nucleic acid is linked to regulatory sequences in a manner which allows expression of the nucleic acid. Suitable regulatory sequences may be derived from a variety of sources, including bacterial, fungal, viral, mammalian, or insect genes. Selection of appropriate regulatory sequences is dependent on the host cell chosen and may be readily accomplished by one of ordinary skill in the art. Examples of such regulatory sequences include: a transcriptional promoter and enhancer or RNA polymerase binding sequence, a ribosomal binding sequence, including a translation initiation signal. Additionally, depending on the host cell chosen and the vector employed, other sequences, such as an origin of replication, additional DNA restriction sites, enhancers, and sequences conferring inducibility of transcription may be incorporated into the expression vector.

[0092] The term "vector" as used herein comprises any intermediary vehicle for a nucleic acid molecule which enables said nucleic acid molecule, for example, to be introduced into prokaryotic and / or eukaryotic cells and / or integrated into a genome, and include plasmids, phagemids, bacteriophages or viral vectors such as retroviral based vectors, Adeno Associatedviral vectors and the like. The term "plasmid" as used herein generally refers to a construct of extrachromosomal genetic material, usually a circular DNA duplex, which can replicate independently of chromosomal DNA.

[0093] By “at least moderately stringent hybridization conditions” it is meant that conditions are selected which promote selective hybridization between two complementary nucleic acid molecules in solution. Hybridization may occur to all or a portion of a nucleic acid sequence molecule. The hybridizing portion is typically at least 15 (e.g. 20, 25, 30, 40 or 50) nucleotides in length. Those skilled in the art will recognize that the stability of a nucleic acid duplex, or hybrids, is determined by the Tm, which in sodium containing buffers is a function of the sodium ion concentration and temperature (Tm = 81.5°C - 16.6 (Log10 [Na+]) + 0.41(%(G+C) - 600 / I), or similar equation). Accordingly, the parameters in the wash conditions that determine hybrid stability are sodium ion concentration and temperature. In order to identify molecules that are similar, but not identical, to a known nucleic acid molecule a 1% mismatch may be assumed to result in about a 1°C decrease in Tm, for example if nucleic acid molecules are sought that have a >95% identity, the final wash temperature will be reduced by about 5°C. Based on these considerations those skilled in the art will be able to readily select appropriate hybridization conditions. In preferred embodiments, stringent hybridization conditions are selected. By way of example the following conditions may be employed to achieve stringent hybridization: hybridization at 5x sodium chloride / sodium citrate (SSC) / 5x Denhardt’s solution / 1.0% SDS at Tm - 5°C based on the above equation, followed by a wash of 0.2x SSC / 0.1% SDS at 60°C. Moderately stringent hybridization conditions include a washing step in 3x SSC at 42°C. It is understood, however, that equivalent stringencies may be achieved using alternative buffers, salts and temperatures. Additional guidance regarding hybridization conditions may be found in: Current Protocols in Molecular Biology, John Wiley & Sons, N. Y., 2002, and in: Sambrook et al., Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Laboratory Press, 2001.

[0094] As used herein "specifically binds" in reference to an antibody means that the antibody recognizes its target antigen and binds its target with greater affinity than it does to a structurally different antigen and / or to an antigen with modified or mutated sequence. For example, a multivalent antibody binds its target with KD of at least 1e-6 M, at least 1e-7 M, at least 1e-8 M, at least 1e-9 M, at least 1e-10 M, at least 1e-11 M, or at least 1e10-12 M.

[0095] The term “selective” or “preferential” as used herein with respect to an antibody that selectively / preferentially binds a form of TDP-43 (e.g. native, or misfolded protein) means that the binding protein binds the form with at least 3 fold, or at least 5 fold, at least 10 fold, atleast 20 fold, at least 100 fold, at least 250 fold, or at least 500 fold or more greater affinity or reactivity. Accordingly, an antibody that is more selective for a particular conformation (e.g. misfolded protein) may preferentially binds the 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 or reactivity (e.g., measured by immunohistochemistry or immunocytochemistry of cells expressing misfolded TDP-43) compared to another form. The selective antibody may not bind one form appreciably or at all. For example, as shown herein, the humanized antibodies described herein do not detect wild-type TDP-43 at all under the conditions tested as the antibody binds a residue (Trp 68) that is exposed only in misfolded TDP-43.

[0096] The term “animal” or "subject" as used herein includes all members of the animal kingdom including mammals, optionally including or excluding humans.

[0097] The term "treating" or “treatment” as used herein and as is well understood in the art, means an approach for 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, diminishment of extent of disease, stabilized (e.g. not worsening) state of disease, inhibiting spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, diminishment of the reoccurrence of disease, and remission (whether partial or total), whether detectable or undetectable. "Treating" and "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. "Treating" and "treatment" as used herein also include prophylactic treatment, for example in a subject identified as carrying a mutation associated with familial forms, such as the familial form of ALS, or for example those carrying C9ORF72 repeat expansion or TDP-43 mutations. A subject 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 vectorized antibody or intrabody), immunoconjugate, nucleic acid, and / or composition described herein to prevent progression.

[0098] In understanding the scope of the present disclosure, the term “consisting” and its derivatives, as used herein, are intended to be close ended terms that specify the presence of stated features, elements, components, groups, integers, and / or steps, and also exclude the presence of other unstated features, elements, components, groups, integers and / or steps.

[0099] The recitation of numerical ranges by endpoints herein includes all numbers and fractions subsumed within that range (e.g. 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5). It is also to be understood that all numbers and fractions thereof are presumed to be modified by theterm "about." Further, it is to be understood that "a", "an" and "the" include plural referents unless the content clearly dictates otherwise. The term “about” means plus or minus 0.1 to 50%, 5-50%, or 10-40%, preferably 10-20%, more preferably 10% or 15%, of the number to which reference is being made.

[0100] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the description. Ranges from any lower limit to any upper limit are contemplated. The upper and lower limits of these smaller ranges which may independently be included in the smaller ranges is also encompassed within the description, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the description.

[0101] The term “about” as used herein may be used to take into account experimental error and variations that would be expected by a person having ordinary skill in the art. For example, “about” may mean plus or minus 10%, or plus or minus 5%, of the indicated value to which reference is being made.

[0102] As used herein the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise.

[0103] The phrase "and / or," as used herein in the specification and in the claims, should be understood to mean "either or both" of the elements so conjoined, e.g., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with "and / or" should be construed in the same fashion, e.g., "one or more" of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the "and / or" clause, whether related or unrelated to those elements specifically identified.

[0104] As used herein in the specification and in the claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" shall be interpreted as being inclusive, e.g., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of or "exactly one of" or, when used in the claims, "consisting of" will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted asindicating exclusive alternatives (e.g., "one or the other but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of."

[0105] As used herein, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," "composed of," and the like are to be understood to be open-ended, e.g., to mean including but not limited to. Only the transitional phrases "consisting of” and "consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively.

[0106] As used herein in the specification and in the claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from anyone or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified.II. ....., Immunoconjugates, Cells, and Nucleic Acids

[0107] Accordingly, an aspect of the disclosure includes a humanized antibody comprising a sequence as set forth in any of SEQ ID NOs: 2-8 and / or 10-14 or a sequence(s) with at least 80% sequence identity to any thereto wherein the CDR amino acid sequences, comprising CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, are as set for forth in SEQ ID Nos: 15-20, respectively and wherein the antibody comprises at least one of the following substitutions or residues relative to SEQ ID NO: 1:L at position 4; G at position 10; Q at position 13; G at position 15; R at position 19; S at position 21; R at 71; D at position 72; N or T at position 73; K or S at position 75; N at position 76; L at position 78; Y at position 79; N at position 82A; R at position 83; E at position 85; Y at position 91; or S at position 113; and / orat least one of the following substitutions or residues relative to SEQ ID NO: 9:Q at position 3; S at position 7; L at position 11; A at position 13; S at position 14; D at position 17; R at position 18; T at position 22; S at position 63; S at position 67; D or E at position 70; S at position 77; L at position 78; P at position 80; F at position 83; Kat position 103; E at position 105; or I at position 106.

[0108] An aspect of the disclosure includes a humanized antibody comprising a sequence as set forth in any of SEQ ID NOs: 2-8 and / or 10-14 or a sequence with at least 80% sequence identity to any thereto wherein the CDR amino acid sequences, comprising CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, are as set for forth in SEQ ID Nos: 15-20, respectively and wherein the heavy chain variable region and the light chain variable region each independently comprises at least one, at least two, at least three, at least four or at least five of the following substitutions / residues relative to SEQ ID NO: 1:L at position 4; G at position 10; Q at position 13; G at position 15; R at position 19;S at position 21; R at position 71; D at position 72; N orT at position 73; K or S at position 75; N at position 76; L at position 78; Y at position 79; N at position 82A; R at position 83; E at position 85; Y at position 91; or S at position 113; and / orwherein the antibody comprises at least one, at least two, at least three, at least four or at least five of the following substitutions / residues relative to SEQ ID NO: 9:Q at position 3; S at position 7; L at position 11; A at position 13; S at position 14; D at position 17; R at position 18; T at position 22; S at position 63; S at position 67; D or E at position 70; S at position 77; L at position 78; P at position 80; F at position 83; K at position 103; E at position 105; or I at position 106.

[0109] Another aspect of the disclosure includes a humanized antibody comprising a sequence as set forth in any of SEQ ID NOs: 2-8 and / or 10-14 or a sequence with at least 80% sequence identity to any thereto wherein the CDR amino acid sequences, comprising CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, are as set for forth in SEQ ID Nos: 15-20, respectively and wherein the antibody comprises the following substitutions / residues relative to SEQ ID NO: 1:L at position 4; G at position 10; Q at position 13; G at position 15; R at position 19; S at position 21; R at position 71; D at position 72; N or T at position 73; K or S at position 75; N at position 76; L at position 78; Y at position 79; N at position 82A; R at position 83; E at position 85; Y at position 91; or S at position 113; and / orthe antibody comprises the following substitutions / residues relative to SEQ ID NO: 9:Q at position 3; S at position 7; L at position 11; A at position 13; S at position 14; D at position 17; R at position 18; T at position 22; S at position 63; S at position 67; D or E at position 70; S at position 77; L at position 78; P at position 80; F at position 83; K at position 103; E at position 105; or I at position 106.

[0110] In some embodiments, the humanized antibody comprises a heavy chain variable region comprising: i) an amino acid sequence as set forth in any one of SEQ ID NOs: 2-8; ii) an amino acid sequence with at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90% or at least 95% sequence identity to any one of SEQ ID NOs: 2-8, wherein the CDR sequences, comprising CDRH1, CDRH2 and CDRH3 are as set forth in SEQ ID NOs: 15-17, respectively, or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences, comprising CDRH1, CDRH2 and CDRH3 are as set forth in SEQ ID NOs: 15-17, respectively.

[0111] In some embodiments, the antibody comprises a light chain variable region comprising i) an amino acid sequence as set forth any one of SEQ ID NOs: 10-14, ii) an amino acid sequence with at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90% or at least 95% sequence identity to any one of SEQ ID NO: 10-14, wherein the CDR sequences, comprising CDRL1, CDRL2 and CDRL3, are as set forth in SEQ ID NOs: 18-20, respectively, or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences, comprising CDRL1, CDRL2 and CDRL3 are as set forth in SEQ ID NOs: 18-20, respectively.

[0112] In some embodiments, the antibody comprises a heavy chain variable region and a light chain variable region as set out in Table 1.

[0113] In some embodiments, the antibody comprises the heavy chain variable region and / or the light chain variable region as set forth in SEQ ID NO: 6 and 12, respectively; SEQ ID NO: 6 and 13, respectively; SEQ ID NO: 7 and 14, respectively; SEQ ID NO: 8 and 12, respectively; SEQ ID NO: 8 and 13, respectively, or sequence(s) with at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90% or at least 95% sequence identity to any thereto (e.g. SEQ ID NO: 6, 7, 8, 12, 13 and / or 14) wherein the CDR sequences are maintained as set forth in SEQ ID NOs: 15-20.

[0114] In some embodiments, the antibody comprises the heavy chain variable region and / or the light chain variable region as set forth in SEQ ID NO: 6 and 12, respectively or sequence(s) with at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90% or at least 95% sequence identity to any thereto wherein the CDR sequences are maintained as set forth in SEQ ID NOs: 15-20

[0115] In some embodiments, the antibody comprises the heavy chain variable region and / or the light chain variable region as set forth in SEQ ID NO: 6 and 13, respectively orsequence(s) with at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90% or at least 95% sequence identity to any thereto wherein the CDR sequences are maintained as set forth in SEQ ID NOs: 15-20.

[0116] In some embodiments, the antibody comprises the heavy chain variable region and / or the light chain variable region as set forth in SEQ ID NO: 7 and 14, respectively or sequence(s) with at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90% or at least 95% sequence identity to any thereto wherein the CDR sequences are maintained as set forth in SEQ ID NOs: 15-20.

[0117] In some embodiments, the antibody comprises the heavy chain variable region and / or the light chain variable region as set forth in SEQ ID NO: 8 and 12, respectively or sequence(s) with at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90% or at least 95% sequence identity to any thereto wherein the CDR sequences are maintained as set forth in SEQ ID NOs: 15-20.

[0118] In some embodiments, the antibody comprises the heavy chain variable region and / or the light chain variable region as set forth in SEQ ID NO: 8 and 13, respectively, or sequence(s) with at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90% or at least 95% sequence identity to any thereto wherein the CDR sequences are maintained as set forth in SEQ ID NOs: 15-20.

[0119] In some embodiments, the amino acid sequences can have greater than 95% sequence identity. In various embodiments, the amino acid sequence has at least 96% identity to a sequence provided herein. In various embodiments, the amino acid sequence has at least 97% identity to a sequence provided herein. In various embodiments, the amino acid sequence has at least 98% identity to a sequence provided herein. In various embodiments, the amino acid sequence has at least 99% identity to a sequence provided herein.

[0120] In an embodiment, the humanized antibodies described herein comprise a heavy chain variable region comprising a conservatively substituted amino acid sequence (e.g. comprising one or more conservative substitutions in addition to the one or more humanized residue substitutions). In an embodiment, the humanized antibodies described herein comprise a light chain variable region comprising a conservatively substituted amino acid sequence (e.g. comprising one or more conservative substitutions in addition to the one or more humanized residue substitutions). For example, the heavy chain variable region and / or the light chain variableregion, optionally framework region 1, 2 and / or 3, can include 1, 2, 3, 4 or 5 conservative amino acid substitutions.

[0121] In some embodiments, the humanized antibody (bivalent antibody) has a KD of at least or about 1x 10-11M, at least or about 2 x10-11M, at least or about 3 x 10-11M to misfolded TDP-43. The KD or kinetics analysis can be measured using methods known in the art, for example, by SPR, for example using the methods provided in the examples herein.

[0122] In some embodiments, the humanized antibody is a monoclonal antibody. In some embodiments, the humanized antibody is a single chain antibody. In some embodiments, the single chain antibody is an intrabody. In an embodiment, the single chain antibody is a scFV, minibody or nanobody.

[0123] It is demonstrated herein that several of the humanized antibodies have improved stability compared to the chimeric antibody. In particular, Tm1 and Tonsetmeasured by UNcle biostability platform were improved. Accordingly, in some embodiments, the humanized antibody has a Tm1 and / or Tonsetthat is greater than the chimeric antibody variable domain defined by VH0 / VL0 (SEQ ID NO: 1 and 9). In another embodiment, the humanized antibody has a Tm1 that is greater than 60°C, greater than 61°C, greater than 62°C for example about or within 65-68°C. In some embodiments, the humanized antibody has a Tonsetthat is greater than about 52°C, greater than 55°C, for example within 52-60°C or 55-60°C. Tm1 and Tonsetcan be measured by UNcle biostability platform as described in the Examples.

[0124] In an embodiment, the at least one residue is D at position 72. In another embodiment, the at least one residue is N position 76.

[0125] In some embodiments, the humanized antibody has a heavy chain variable region comprising a T72D and / or a T76N substitution relative to SEQ ID NO: 1. In other embodiments, the humanized antibody has a light chain variable region comprising one or more of a V3Q substitution, a T7S substitution, a K11 L substitution or a K63S substitution relative to SEQ ID NO: 9.

[0126] The terms substitution, residue and mutation can be used interchangeably to refer to humanization residue changes.

[0127] As described in the Examples, the chimeric heavy chain variable domain exhibited diffuse bands when analysed by SDS-PAGE. Heavy chain variable domains of humanized antibodies that comprised D at position 72 and / or N at position 76 relative to SEQ ID NO: 1 surprisingly lost the doublet band and exhibited a single band when analysed by SDS-PAGE.

[0128] As indicated in the Examples, some humanized antibodies also had an increased expression level compared to the chimeric VH0 / VL0 antibody. For example, the selected antibodies had 1.6 - 2.1 -fold increased expression compared to the chimeric antibody in the tests conducted. As the same conditions were used to express the chimeric and humanized antibodies, the sequence substitutions may be responsible for the increased expression

[0129] Further it was demonstrated herein that VH5 / VL4, VH5 / VL3, VH6 / VL5 and VH7 / VL4 were better able to detect misfolded TDP-43 at lower concentrations (see Tables 10 and 11). Accordingly in one embodiment the humanized antibody is selected from VH5 / VL4, VH5 / VL3, VH6A / L5 and VH7A / L4 or sequences with at lest 80% sequence identity thereto.

[0130] It was also demonstrated that VH5 / VL3 and VH6 / VL5 had increased staining at both concentrations tested. Accordingly in some embodiments, the humanized antibody comprises a heavy chain variable domain SEQ ID NO: 6 or a sequence with at least 80% sequence identity to SEQ ID NO: 6, and a light chain variable of SEQ ID NO: 12 or a sequence with at least 80% sequence identity to SEQ ID NO: 12 wherein the CDRS are maintained as SEQ ID NO: 15-20; or a heavy chain variable domain of SEQ ID NO: 7 or a sequence with at least 80% sequence identity to SEQ ID NO: 7 and a light chain variable of SEQ ID NO: 14 or a sequence with at least 80% sequence identity to SEQ ID NO: 14, wherein the CDRS are maintained as SEQ ID NO: 15-20.

[0131] In some embodiments, the humanized antibody heavy chain variable region lacks the S residue at Kabat position 113 (Fig. 12A).

[0132] In some embodiments, the humanized antibody is an antibody binding fragment selected from Fab, Fab', F(ab')2, scFv, dsFv, ds-scFv, dimers, nanobodies, minibodies, diabodies, and multimers thereof.

[0133] In some embodiments, the humanized antibody binding fragment is a Fab fragment.

[0134] In some embodiments, the humanized antibody heavy chain variable region comprises human lgG1 framework sequence. In some embodiments, the light chain variable region comprises kappa framework sequence.

[0135] In some embodiments, the humanized antibody selectively binds misfolded TDP-43 over native TDP-43. In some embodiments, the antibody is a single chain antibody.

[0136] In some embodiments, the humanized antibody is an isolated humanized antibody. In some embodiments the isolated humanized antibody is purified. In some embodiments, the purified humanized antibody has at least 95%, at least 96%, at least 97% at least 98% or at least 99% purity.

[0137] Another aspect of the disclosure includes an immunoconjugate comprising the humanized antibody described herein and a detectable label, a targeting moiety, and / or a transport moiety, optionally a molecule that facilitates transport into a cell. In an embodiment, the immunoconjugate comprises an antibody described herein and a detectable label, such as a fusion tag, a transport moiety, and / or a targeting moiety. The fusion tag can be a FLAG tag or a MYC tag, and the fusion tag can be N-terminal or C-terminal and conjugated directly or indirectly via a linker. The targeting moiety can comprise a lysosomal targeting signal sequence and can be for example conjugated to a single chain antibody. The targeting moiety can be N-terminal or C-terminal. The targeting moiety can be a secretion signal peptide.

[0138] In some embodiments, the detectable label comprises a positron emitting radionuclide, optionally for use in subject imaging such as PET imaging. In such embodiments, the antibody may comprise a cell penetrating moiety such as a fused cell penetrating peptide. The cell penetrating peptide may be derived from TAT, for example YGRKKRRQRRR (SEQ ID NO: 23), CYGRKKRRQRRRC (c-Tat; SEQ ID NO: 24) or GDIMGEWGNEIFGAIAGFLGYGRKKRRQRRR (HA-Tat; SEQ ID NO: 25), or other such as RRRRRRRR (R8; SEQ ID NO; 26) and CRRRRRRRRC (cR8; SEQ ID NO; 27), RQIKIWFQNRRMKWKK (penetratin; SEQ ID NO: 28) and GWTLNSAGYLLGKINLKALAALAKKIL (transportan; SEQ ID NO: 29). The C residues at each side of the CPP sequence permit formation of a disulfide bond and cyclisation. HA refers to the influenza virus haemagglutinin protein. The cell penetrating peptide can for example comprise a radionuclide suitable for PET imaging such as carbon-11, nitrogen-13, oxygen-15, fluorine-18, gallium-68, zirconium-89, rubidium-82 and yttrium 90. Methods for attaching or incorporating the tracer molecule (e.g. radionuclide or chelator comprising radionuclide) to a polypeptide, for example an antibody, are known in the art.

[0139] Another aspect of the disclosure includes a nucleic acid molecule encoding one or both of the heavy and light chain variable domains of the humanized antibody described herein. Another aspect of the disclosure is one or more nucleic acid molecules encoding the humanized antibody described herein. For example, the heavy chain variable region and the light chainvariable region may be encoded on the same nucleic acid or encoded on separate nucleic acid molecules.

[0140] In some embodiments, the humanized antibody encoded is a single chain antibody and the heavy chain variable region and light chain variable region are linked by a linker. In some embodiments, the linker is at least 15 amino acids, 20 amino acids, optionally 15 to 20 amino acids. In one embodiment, the linker is 20 amino acids. In another embodiment, the linker is 15 amino acids. In an embodiment, the orientation of the heavy chain variable domain, linker, and the light chain variable domain is: heavy chain variable domain -linker - light chain variable domain. In another embodiment, the orientation of the heavy chain variable domain, linker, and the light chain variable domain is: light chain variable domain -linker - heavy chain variable domain.

[0141] Linker sequences that can be used include for example (Gly-Gly-Gly-Gly-Ser)3 (SEQ ID NO: 30), (Gly-Gly-Gly-Gly-Ser)4 (SEQ ID NO: 31) and GSTGGGGSGKPGSGEGGGGS (SEQ ID NO: 32). Other linkers include Glu Ser Gly Arg Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser (SEQ ID NO: 33); Glu Gly Lys Ser Ser Gly Ser Gly Ser Glu Ser Lys Ser Thr (SEQ ID NO: 34), Glu Gly Lys Ser Ser Gly Ser Gly Ser Glu Ser Lys-Ser Thr Gin (SEQ ID NO: 35), Glu Gly Lys Ser Ser Gly Ser Gly Ser Glu Ser Lys Vai Asp (SEQ ID NO: 36), Gly Ser Thr Ser Gly Ser Gly Lys Ser Ser Glu Gly Lys Gly (SEQ ID NO: 37), Lys Glu Ser Gly Ser Vai Ser Ser Glu Gin Leu Ala Gin Phe Arg Ser Leu Asp (SEQ ID NO: 38), and Glu Ser Gly Ser Vai Ser Ser Glu Glu Leu Ala Phe Arg Ser Leu Asp (SEQ ID NO: 39). Where a single chain antibody comprises a detectable label, such as a fusion tag, or targeting moiety, it may be fused directly or indirectly, for example by way of a tag linker, or targeting moiety linker optionally wherein said linker comprises or is GGGGS (SEQ ID NO: 40). A tag linker, transport moiety linker, or targeting moiety linker can be for example any linker sequence and may be the same or different from any other linker in a construct (e.g. the tag linker can be the same or different from a linker linking the heavy and light chain variable regions or heavy and light chains).

[0142] In some embodiments, the linker is at least 15 amino acids, 20 amino acids, optionally 15 to 20 amino acids. In one embodiment, the linker is 20 amino acids. In another embodiment, the linker is 15 amino acids. In an embodiment, the orientation of the heavy chain variable domain, linker, and the light chain variable domain is: heavy chain variable domain -linker - light chain variable domain. In another embodiment, the orientation of the heavy chain variable domain, linker, and the light chain variable domain is: light chain variable domain -linker - heavy chain variable domain.

[0143] In some embodiments, the nucleic acid further encodes a targeting moiety, optionally linked by a targeting moiety linker. In some embodiments, the targeting moiety comprises a lysosomal targeting signal sequence. For example, the lysosomal targeting signal sequence can be or comprise the amino acid sequence YPTL (SEQ ID NO: 41), KSIRSGYEVM (SEQ ID NO: 42), RWRKSHSSSYTPLSGSTYPEGRH (SEQ ID NO: 43). Other lysosomal targeting signal sequences that can be used include classical tyrosine-based NPXY and YXXcp sequences where X can be any amino acid residue and cp is a bulky hydrophobic residue, di-leucine-based [D / E]XXXL[L / I] or DXXLL sequences. In an embodiment, the lysosomal targeting signal sequence has the sequence KSIRSGYEVM (SEQ ID NO: 47).

[0144] In some embodiments, the nucleic acid encodes a proteinaceous (e.g. amino acid) immunoconjugate comprising a targeting moiety, such as a signal peptide and / or a cell penetrating peptide. In an embodiment, the signal peptide comprises or consists of the amino acid sequence MEWSWIFLFLLSGTAGVHS (SEQ ID NO: 48) or MRFSAQLLGLLVLWIPGSTA (SEQ ID NO: 49). For example, the signal peptide can be a heavy chain signal peptide or a light chain signal peptide. Exemplary heavy chain signal peptide sequences include / comprise MEWSWIFLFLLSGTAGVHS (native signal peptide sequence for TDP43-C1) (SEQ ID NO: 48), MNFGLRLILLVLVLKGVLC (SEQ ID NO: 50), METGLRWLLLVAVLKGVQCQ (SEQ ID NO: 51), MELGLSWIFLLAILKGVQC (SEQ ID NO: 52), MELGLRWVFLVAILEGVQC (SEQ ID NO: 53), MKHLWFFLLLVAAPRWVLS (SEQ ID NO: 54), MDWTWRILFLVAAATGAHS (SEQ ID NO: 55), MDWTWRFLFVVAAATGVQS (SEQ ID NO: 56), MEFGLSWLFLVAILKGVQC (SEQ ID NO: 57), MEFGLSWVFLVALFRGVQC (SEQ ID NO: 58) and MDLLHKNMKHLWFFLLLVAAPRWVLS (SEQ ID NO: 59). Exemplary light chain signal peptide sequences include MRFSAQLLGLLVLWIPGSTA (native signal peptide sequence for TDP43-C1) (SEQ ID NO: 49), MKLPVRLLVLMFWIPASSS (SEQ ID NO: 60), MDMRVPAQLLGLLLLWLSGARC (SEQ ID NO: 61) and MKYLLPTAAAGLLLLAAQPAMA (SEQ ID NO: 62). The single chain antibodies, and particularly when as intrabodies, optionally include a targeting moiety such as a lysosomal targeting signal sequence. The heavy chain and light chain variable regions of antibodies specific for misfolded TDP-43 can be linked using various linkers and in various orientations, with and without a lysosomal targeting signal sequence or other targeting moiety.

[0145] The nucleic acid molecules may be codon optimized.

[0146] The nucleic acid molecules may be incorporated in a known manner into an appropriate expression vector which ensures expression of the protein, for example as described in the Examples.

[0147] The present disclosure also provides variants of the nucleic acid sequences that encode the antibodies disclosed herein.

[0148] For example, the variants include nucleotide sequences that hybridize to the nucleic acid sequences encoding the antibody disclosed herein under at least moderately stringent hybridization conditions or codon degenerate or optimized sequences. The variant sequences encode for example antibodies with conservative changes outside the CDR sequences and codon optimized versions of the antibody sequences described herein, for example optimized for expression in human cells.

[0149] Another aspect of the disclosure includes a vector or expression cassette comprising the nucleic acid described herein. In some embodiments, the vector is a lentiviral vector, adeno associated virus (AAV) vector, optionally AAV serotype 9, vaccinia virus vector, Herpes simplex virus (HSV) vector, adenovirus vector, retrovirus vector, or plasmid DNA vector. Viral vectors include for example reference to the nucleic acid viral construct component or the virus particle comprising the nucleic acid viral vector.

[0150] The expression cassette can comprise for example the nucleic acid encoding the single chain antibody and linker, and regulatory sequences such as a promoter that is operatively linked to the nucleic acid. In an embodiment, the vector is an isolated vector.

[0151] Molecular cloning techniques known in the art can be used in vector construction. In an embodiment, an expression vector and the insert are digested with the appropriate restriction enzymes and ligated by T4 polymerase. The ligation reaction is transformed into competent cells. Individual colonies are picked, overnight cultures are grown, and DNA are purified for diagnostic restriction digest.

[0152] Possible expression vectors include but are not limited to cosmids, plasmids, or modified viruses (e.g., replication defective retroviruses, including lentiviral vectors, adenoviruses and adeno-associated viruses).

[0153] In one embodiment, the vector is an adeno associated virus capable of transducing neuronal cells (e.g., AAV serotype 9).

[0154] The vectors may comprise suitable regulatory sequences.

[0155] Suitable regulatory sequences may be derived from a variety of sources, including bacterial, fungal, viral, mammalian, or insect genes. Examples of such regulatory sequences include: a transcriptional promoter and enhancer or RNA polymerase binding sequence, aribosomal binding sequence, including a translation initiation signal. Additionally, depending on the cell to be transfected / infected / transduced and the vector employed, other sequences, such as an origin of replication, additional DNA restriction sites, enhancers, and sequences conferring inducibility of transcription may be incorporated into the expression vector. In an embodiment, the regulatory sequences direct or increase expression in neural tissue and / or cells. In an embodiment, the vector is a viral vector. The recombinant expression vectors may also contain a marker gene which facilitates the selection of host cells transformed, infected, or transfected with a vector for expressing an antibody described herein. The recombinant expression vectors may also contain other expression cassettes which encode for example a fusion moiety (e.g., for creating an antibody “fusion protein”) which can aid in the detection, including for example tags and labels described herein.

[0156] The nucleic acid molecules (also referred to as nucleic acids) and vectors can be used to deliver the antibody to a cell, for example cells in a subject.

[0157] A wide range of approaches to transduce the cells can be used, including viral vectors, “naked” DNA, DNA in lipid or other nanoparticles, gene gun, etc. For example, retroviral vectors such as lentiviral vectors can also be used to transduce cells with intrabodies. Other vector systems useful in practicing the present invention include the adenoviral and adeno associated virus-based vectors.

[0158] Another aspect of the disclosure includes a cell expressing an antibody described herein, optionally wherein the cell is a hybridoma comprising the vector described herein. In an embodiment, the cell is a Chinese hamster ovary (CHO) cell. In other embodiments, the cell is an insect cell (e.g. Sf9, Sf21, Hi-5 cells,) or a plant cell e.g., Nicotiana benthamiana.

[0159] Another aspect of the disclosure includes a kit comprising the antibody described herein, the nucleic acid molecule described herein, the vector described herein or the cell described herein.III. Compositions

[0160] Another aspect of the disclosure includes a composition comprising the humanized antibody described herein, the immunoconjugate described herein, the nucleic acid described herein, the expression cassette or vector described herein, or the cell described herein, optionally with a diluent.

[0161] Also provided is a composition comprising two or more humanized antibodies, immunoconjugates, nucleic acid molecules, expression cassettes, vectors or cells described herein.

[0162] In one embodiment, the composition comprises two or more humanized antibodies (e.g. two or more different antibodies).

[0163] In another embodiment, the composition comprises two or more immunoconjugates (e.g. two or more different immunoconjugates).

[0164] In another embodiment, the composition comprises two or more nucleic acid molecules (e.g. two or more different nucleic acids).

[0165] In another embodiment, the composition comprises two or more expression cassettes (e.g. two or more different expression cassettes).

[0166] In another embodiment, the composition comprises two or more vectors (e.g. two or more different vectors).

[0167] In an embodiment, the two or more is two e.g. two humanized antibodies or two immunoconjugates etc. In an embodiment, the two or more is three. In an embodiment, the two or more is four. In an embodiment, the two or more is five. In another embodiment, the two or more is six. In an embodiment, the two or more is seven.

[0168] Another aspect is a composition comprising a single chain antibody described herein. Also provided is a composition comprising two or more single chain antibodies described herein.

[0169] In an embodiment, the composition comprises a diluent. Suitable diluents for nucleic acid molecules and vectors include but are not limited to water, saline solutions and ethanol.

[0170] In an embodiment, the composition comprises lipid particles, optionally liposomes, nanoparticles or nanosomes.

[0171] Suitable diluents for polypeptides, including antibodies or fragments thereof and / or cells include but are not limited to saline solutions, pH buffered solutions and glycerol solutions or other solutions suitable for freezing polypeptides and / or cells.

[0172] In an embodiment, the composition is formulated suitably for intrathecal, intraparenchymal or intraventricular administration.

[0173] In an embodiment, the composition is formulated suitably for systemic administration.

[0174] In an embodiment, the composition comprises a pharmaceutically acceptable carrier, diluent, and / or excipient. In an embodiment, the composition is for a method described herein such as targeting misfolded TDP-43.

[0175] In an embodiment, the composition is a pharmaceutical composition, for example for a method described herein such as for treating a subject in need thereof e.g., a subject with a TDP-43 proteinopathy.IV. Methods

[0176] Another aspect includes a method of making a humanized antibody as described herein. In one embodiment, the method comprises expressing a nucleic acid molecule or vector comprising the nucleic acid molecule encoding an antibody described herein.

[0177] For example, the nucleic acid molecule can be cloned into an expression vector downstream of a promoter, optionally an EF1a promoter. In some embodiments, the nucleic acid molecule is codon optimized for example for increasing translation efficiency by a specific organism.

[0178] Another aspect of the disclosure includes a method of treating a subject.

[0179] Antibodies for example can be used to inhibit the spread of disease [12, 13 and 14]].

[0180] In an embodiment, the method comprising administering to a subject in need thereof an effective amount of the humanized antibody described herein, the immunoconjugate described herein, the nucleic acid molecule, vector or cell described herein or the composition described herein. Also provided is a use of an effective amount of the humanized antibody described herein, the immunoconjugate described herein, the nucleic acid molecule described herein, the vector described herein, the cell described herein or the composition described herein for treating a subject in need thereof. Also provided is a use of an effective amount of the humanized antibody described herein, the immunoconjugate described herein, the nucleic acid molecule described herein, the vector described herein, the cell described herein or the composition described herein in the manufacture of a medicament for treating a subject in need thereof. Further provided is an effective amount of the humanized antibody described herein, the immunoconjugate described herein, the nucleic acid molecule described herein, the vectordescribed herein or the cell described herein or the composition described herein for use in treating a subject in need thereof.

[0181] In some embodiments, the subject is suspected of having, is at risk of developing, or has been diagnosed with a TDP-43 proteinopathy.

[0182] In some embodiments, the TDP-43 proteinopathy is selected from amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD-TDP), primary lateral sclerosis, progressive muscular atrophy, Alzheimer’s disease and limbic-predominant age-related TDP-43 encephalopathy (LATE). Like ALS and other TDP-43 proteinopathies, misfolded TDP-43 can be present in Alzheimer’s disease patients. TDP-43 pathology is observed in between 20% and 50% of AD patients and in 75% of patients with severe AD.13

[0183] In some embodiments, the humanized antibody, immunoconjugate, or composition is administered in combination with another TDP-43 proteinopathy treatment.

[0184] Another aspect of the disclosure includes a method of inhibiting misfolded TDP-43 cell transmission, the method comprising administering the humanized antibody described herein, the immunoconjugate described herein, the nucleic acid described herein or the composition described herein to a subject in need thereof. A further aspect includes a use of the humanized antibody described herein, the immunoconjugate described herein, the nucleic acid described herein or the composition described herein for inhibiting misfolded TDP-43 cell transmission in a subject in need thereof. Also provided is a use of the humanized antibody described herein, the immunoconjugate described herein, the nucleic acid described herein or the composition described herein in the manufacture of a medicament for inhibiting misfolded TDP-43 cell transmission in a subject in need thereof. Further provided is the humanized antibody described herein, the immunoconjugate described herein, the nucleic acid molecule described herein or the composition described herein for use for inhibiting misfolded TDP-43 cell transmission in a subject in need thereof.

[0185] In some embodiments, the humanized antibody, immunoconjugate, nucleic acid molecule, vector, cell or composition is administered or for use by systemic administration.

[0186] In some embodiments, the humanized antibody, immunoconjugate, nucleic acid molecule, vector, cell or composition is administered or for use by parenteral, intravenous, subcutaneous, intramuscular, intracranial, intraventricular, intrathecal, intraorbital, ophthalmic, intraspinal, intracisternal, intraperitoneal, intranasal, aerosol or oral administration. In other embodiments, the administration is intravenous, subcutaneous or intrathecal.

[0187] In some embodiments, the method comprises administering to the subject in need thereof an effective amount of two or more nucleic acid molecules described herein.

[0188] In some embodiments, the method comprises administering to the subject in need thereof an effective amount of two or more expression cassettes or described herein.

[0189] In some embodiments, the method comprises administering to the subject in need thereof an effective amount of two or more humanized antibodies described herein.

[0190] In some embodiments, the method comprises administering to a subject in need thereof an effective amount of two or more compositions described herein.

[0191] It should also be understood that, in certain methods described herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited unless the context indicates otherwise.

[0192] Further, the definitions and embodiments described in particular sections are intended to be applicable to other embodiments herein described for which they are suitable as would be understood by a person skilled in the art. For example, in the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. 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.ExamplesExample 1Summary

[0193] Variable region sequences encoding the rabbit monoclonal antibody 14H1-14K2 were used to construct a chimeric antibody (VH0 / VL0) and a series of humanized antibodies. The chimeric and designed humanized variants were cloned into vectors encoding human IgG1 heavy chain and human kappa light chain. The chimeric and 35 humanized antibody variants were transiently expressed at small scale in CHO suspension cells and supernatants were tested for binding to linear misfolded TDP-43 epitope using Biacore (single-cycle kinetic analysis). Following this, five humanized antibody variants were transiently expressed at larger scale in CHO suspension cells.

[0194] Antibodies were purified from CHO supernatants using 1 mL Hitrap™ MabSelect™ PrismA columns, followed by preparative SEC using a HiLoad 16 / 60 Superdex 200 column. Chimeric and humanized antibody variants were analysed by SDS-PAGE and analytical SE-HPLC, assessed by multi-cycle kinetic analysis and using the UNcle biostability platform.Methods and ResultsDesign of humanized antibody variable regions

[0195] Seven heavy chain (VH1 to VH7) and five light chain (VL1 to VL5) sequences were designed for gene synthesis and pairing for expression in mammalian cells (Table 3). Variant sequences and alignments are shown in Table 1 and Figs. 12A-B.

[0196] Table 1: Amino acid sequences of heavy and light chain variable regions of humanized antibodies generated from 14H1-14K2 clone. CDRs according Kabat numbering are bolded and underlined.

[0197] Table 2: Amino acid sequences of heavy and light chain CDRs of humanized antibodies generated from 14H1-14K2 clone using Kabat and Chothia.

[0198] Table 3: Summary table of the humanized antibody variants to be generated including a chimeric (VH0 / VL0) and thirty five humanized variants.Analysis for T cell epitopes

[0199] The designed VH and VL sequence variants were analysed for the occurrence of potential T cell epitopes with the aid of an in silico MHC Class II peptide binding algorithm.

[0200] Analysis of protein sequences was performed with overlapping 9mer peptides, which were tested against each of the 46 MHC class II allotypes. Individual peptides, spanning the whole sequence, were given a binding score from 0 to 3 for each allele, and those scores were added together for all HLA-DR, DP, DQ alleles to provide an overall risk score (‘Position Risk Score’). Peptides were considered weak, medium or strong promiscuous MHC class II binders when their ‘Position Risk Scores’ reached 1-2, 3-5 or 6+, respectively. The ‘Total Score’ for the test protein was calculated by adding the ‘Position Risk Scores’ obtained for all individual peptides. The highest ‘Position Risk Score’ was also provided to inform the presence of strong binders and / or highly promiscuous peptides (‘Hotspot Max’). 9mer peptides fully homologous to sequences from the human proteome are typically excluded from the analysis, since germline sequences are unlikely to have immunogenic potential in healthy individuals due to T cell tolerance.Results

[0201] A number of germline MHC Class II binding ligands were identified in both the 14H1-14K2 antibody as well as the designed variants. As these epitopes are unlikely to have immunogenic potential due to T cell tolerance, these were excluded from any further analysis.

[0202] Several non-germline promiscuous weak, medium or strong affinity MHC class II binding ligands were identified in both the heavy and light chain sequences however, it wasobserved that during the humanization design process a proportion of such epitopes were either removed or showed a reduction in affinity to MHC Class II.

[0203] A subset of predicted epitopes identified in the 14H1-14K2 VL sequence were found to be associated in and close to CDR regions, in particular with VH CDR2 (with epitopes associated with W52, F52A and Y58) and VL CDR2 (with epitopes associated I48, Y49 and Y50).Construction of chimeric lgG1 and humanized variants

[0204] The VH and VL sequences of 14H1-14K2 (VH0 and VL0) together with the humanized variants were synthesized with flanking restriction enzyme sites for cloning into pANT expression vector system for human IgG 1 heavy chain and kappa light chain (Figure 2). The VH regions were cloned between the Mlu I and Hind III restriction sites, and the VL regions were cloned between the Pte I and BamH I restriction sites. All constructs were confirmed by sequencing.Small Scale Transient expression of chimeric and humanized Antibodies

[0205] Chimeric VH0 / VL0 and combinations of humanized heavy and light chain DNA constructs (a total of 35 humanized pairings, together with two control combinations, Table 4) were transiently transfected into CHO suspension cells using TransIT Pro (Merus). Supernatants were harvested 7 days post transfection. Antibody concentrations were measured on the Octet QK 384 using Protein A biosensors (Molecular Devices, Wokingham, Berkshire, UK), using an IgG1 antibody as standard (Table 4).

[0206] Table 4: Supernatant IgG expression levels (µg / mL) of the chimeric (VH0 / VL0), two controls (VH0 / VL1 and VH1 / VL0) and thirty five humanized antibody variants.2.6 Single cycle kinetic analysis of chimeric and humanized variants

[0207] In order to assess the binding of all of the variants to antigen and select lead humanized IgGs, single cycle kinetic analysis (Figure 3) was performed on supernatants from transfected cell cultures. Kinetic experiments were performed at 25°C on a Biacore T200 running Biacore T200 Control software V2.0.1 and Evaluation software V3.0 (Cytiva, Uppsala, Sweden). TDP-43 linear antigen by single cycle kinetic analysis. HBS-P+ (Cytiva, Uppsala, Sweden) was used as running buffer as well as for ligand and analyte dilutions. Supernatants containing IgG were diluted in running buffer (v / v 50:50). At the start of each cycle, antibodies were loaded onto Fc2, Fc3 and Fc4 of a Protein A sensor chip (Cytiva, Little Chalfont, UK). Ig were captured at a flow rate of 10 µl / min to give an immobilisation level (RL) of ~ 1000 RU. The surface was then allowed to stabilize.

[0208] Single cycle kinetic data was obtained using human TDP-43 antigen as the analyte injected at a flow rate of 30 µl / min to minimize any potential mass transfer effects.

[0209] Multiple repeats with the reference chimeric IgG were performed to check the stability of the surface and analyte over the kinetic cycles. The signal from the reference channel Fc1 (no IgG captured) was subtracted from that of Fc2, Fc3 and Fc4 to correct for bulk effect and differences in non-specific binding to a reference surface. The signal from each IgG blank run (IgG captured but no antigen) was subtracted to correct for differences in surface stability. The double referenced sensorgrams were fitted with the Langmuir (1:1) binding model (Equation a) where the closeness of fit of the data to the model is evaluated using the Chi square value which describes the deviation between the experimental and fitted (observed and expected) curves (Equation b). The fitting algorithm seeks to minimize the Chi square value. The fitting allows comparative ranking of variants.

[0210] Equations describing (a) the 1:1 Interaction binding kinetics, and; (b) closeness of fit assessment:s -1)e point Chi square = n = number of data pointsn* P p - number of fitted parameters

[0211] As shown in Table 5 and Figure 4, the majority of the humanized variant antibodies appeared to bind human TDP-43 antigen within two-fold of the chimeric antibody however, for certain variants, the fit is poor (as shown by high Chi2 values). Based upon expression, T cell epitope scores, and affinity data, the chimeric and five antibodies (VH5 / VL3, VH5 / VL4, VH6 / VL5, VH7 / VL3 and VH7 / VL4) were selected for larger scale expression and purification.Table 5: Single cycle kinetic parameters of chimeric (VH0 / VL0) and humanized variants (tested as cell culture supernatants) binding to human TDP-43 antigen as determined using the Biacore T200. The relative KD was calculated by dividing the KD of the humanized variant by that of the VH0 / VL0 assayed in the same experiment. VH5 / VL3, VH5 / VL4, VH6 / VL5, VH7 / VL3 and VH7 / VL4 were selected for scale up and purification.2.7 Large Scale Transient expression and purification of chimeric and lead humanized Antibodies

[0212] The chimeric VH0 / VL0 and the five selected humanized heavy and light chain DNA constructs were transiently transfected into CHO suspension cells using the MaxCyte STX® electroporation system (MaxCyte Inc., Gaithersburg, USA) with OC-400 processing assemblies. Following cell recovery, cells were diluted to 3 x106cells / mL into CD Opti-CHO medium (ThermoFisher, Loughborough, UK) containing 8 mM L-Glutamine (ThermoFisher, Loughborough, UK) and 1x Hypoxanthine-Thymidine (ThermoFisher, Loughborough, UK). Two hours after resuspension 0.5x Penicillin Streptomycin solution (ThermoFisher, Loughborough, UK) was added to the culture (5mL / L). The culture temperature was reduced 24 hours posttransfection, from 37°C to 32°C and 1 mM sodium butyrate (Sigma, Dorset, UK) was added.

[0213] Cultures were fed 24 hours and 7 days after transfection by adding 30% and 15% (of the culture volume) CHO CD Efficient Feed B (ThermoFisher, Loughborough, UK) respectively, and 3.3% and 1.65% Function Max Titre Enhancer (ThermoFisher, Loughborough, UK) respectively. CHO supernatants were harvested 13 days post transfection. Antibody concentrations were measured on the Octet QK 384 using Protein A biosensors (Molecular Devices, Wokingham, Berkshire, UK), using an IgG1 antibody as standard (Table 6). The humanized antibodies had 1.6 - 2.1-fold increased expression compared to the chimeric antibody.

[0214] Table 6: Supernatant IgG expression levels (µg / mL) of the chimeric (VH0A / L0) and five lead humanized antibodies variants following expression in CHO cells.Heavy Chain

[0215] Following culture harvest, antibody supernatants were filtered using 0.2 pM filter systems (Corning, New York, US) to remove remaining cell debris and supplemented with 10x PBS to neutralise pH. The antibodies were then purified from supernatants using 1 mL Hitrap MabSelect PrismA columns (Cytiva, Little Chalfont, UK) previously equilibrated with 10 CV(column volume) of 1x DPBS, pH 7.2-7.4. Following the sample loading, the columns were washed with 10 CV of 1x DPBS and protein eluted with 0.1 M sodium citrate, pH 3.0. Fractions were collected, and pH adjusted with 1 M Tris-HCI, pH 9.0 followed by OD280nm quantification.

[0216] PrismA purified material was then analysed by analytical SE-HPLC and, given the percentage of monomer was below 90% for all variants including the chimeric antibody (Table 7), all molecules were polished on a HiLoad 16 / 60 Superdex 200 column (Cytiva, Little Chalfont, UK) using 1x DPBS, pH 7.2-7.4 as the mobile phase. Antibody containing fractions for each variant were pooled and filter sterilised with 0.2 pM syringe filters (Sartorius, Epsom, UK) before quantification by OD280nm using an extinction coefficient (Ec (0.1%)) based on the predicted amino acid sequence.

[0217] Purified antibodies were then analysed by SDS-PAGE as shown in Figure 5A-B and analytical SE-HPLC (See Figure 6 for overlay). Table 7 shows the percentage of monomers observed for each variant as determined by analytical SE-HPLC. For reduced samples (except VH0 / VL0) two bands were present corresponding to heavy and light chain at 50 and 25 kDa, respectively (Figure 5A-B). For the chimeric, VH0 / VL0, a diffuse heavy chain band was observed.

[0218] Table 7: Percentage of monomeric antibody observed in the chimeric (VH0 / VL0) and humanized variants preparations as determined by SE-HPLC following Hitrap MabSelect PrismA purification and preparative SEC.

[0219] SDS-PAGE analysis of the purified antibodies, performed under reducing conditions, was repeated alongside the mouse lgG2a antibody as shown in Figure 7. The antibody profile for the murine antibody appears to be the same as the VH0 / VL0, with a diffuse heavy chain present in both samples.

[0220] In order to investigate this diffuse band further, Western blot analysis of supernatants containing selected humanized antibodies was performed with an anti-Fc-HRP antibody (Sigma #A0170) as shown in Figure 8. The doublet band in the heavy chain is present in VHO to VH3 but disappears from VH4 onwards. This coincides with two substitutions, both changes from threonine (T72D and T76N).2.8 Multicycle kinetics Biacore of Chimeric and lead humanized variants

[0221] In order to assess the binding of the five lead humanized variants to BSA conjugated TDP-43 linear peptide epitope (antigen), multi-cycle kinetic analysis (Figure 9) was performed on purified material. Kinetic experiments were performed at 25°C on a Biacore 8K running Biacore Insight Evaluation software (GE Healthcare, Uppsala, Sweden).

[0222] HBS-P+ (GE Healthcare, Uppsala, Sweden) was used as running buffer as well as for ligand and analyte dilutions. Purified antibodies were diluted to 2 pg / mL in running buffer and at the start of each cycle, loaded onto all of the active flow cells of a Protein A sensor chip (GE Healthcare, Little Chalfont, UK). Antibodies were captured at a flow rate of 10 µl / min to give an immobilisation level (RL) of ~ 1000 RU. The surface was then allowed to stabilise.

[0223] Multi-cycle kinetic data was obtained using BSA conjugated TDP-43 linear antigen (assuming one epitope per BSA) as the analyte injected at a flow rate of 30 µl / min to minimise any potential mass transfer effects. An eight point, two-fold dilution range from 2.0 nM to 0.0156 nM of antigen was prepared in running buffer. For each concentration, the association phases were monitored for 180 seconds and the dissociation phase was measured for 900 seconds.

[0224] Regeneration of the sensor chip surface was conducted between cycles using 10 mM Glycine-HCI, pH 1.5. Multiple repeats of a blank and of antigen were programmed into the kinetic run in order to check the stability of both the surface and analyte over the kinetic cycles.

[0225] The signal from the reference flow cell (no IgG captured) was subtracted from that of the active flow cell for each of the 8 channels to correct for bulk effect and differences in nonspecific binding to a reference surface. The signal from each IgG blank run (IgG captured but no antigen) was subtracted to correct for differences in surface stability. The double referenced sensorgrams were fitted with the Langmuir (1:1) binding model (Figure 5a) where the closeness of fit of the data to the model is evaluated using the Chi square value which describes the deviation between the experimental and fitted (observed and expected) curves (Figure 5b). The fitting algorithm seeks to minimize the Chi square value. The experiment was run initially using the chimeric antibody alongside the murine antibody. A summary of the kinetic constants determinedfrom the 1:1 model fitted curves is shown in Figure 10. Although the affinities of both antibodies are very high, the overall kinetics of 14H1-14K2 (mlgG2a) using protein A capture do look slightly different, especially in the dissociation phase, compared to the chimeric antibody and results in an approximately 10-fold improved affinity.

[0226] Table 8A: Multi-cycle kinetic parameters of chimeric (VH0 / VL0) and humanized variants (tested as purified proteins) binding to BSA-conjugated TDP-43 linear peptide epitope as determined using the Biacore 8K, n=1. The relative KD was calculated by dividing the KD of the humanized variant by that of the VH0 / VL0 assayed in the same experiment. * Kinetic constant Ka is approaching the limits that can be measured.

[0227] Table 8B: Multi-cycle kinetic parameters of chimeric (VH0 / VL0) and humanized variants (tested as purified proteins) binding to BSA conjugated TDP-43 linear antigen as determined using the Biacore 8K, n=2. The relative KD was calculated by dividing the KD of the humanized variant by that of the VH0 / VL0 assayed in the same experiment. * Kinetic constant Ka is approaching the limits that can be measured.2.9 Thermal stability analysis of chimeric and humanized variants.

[0228] Thermal ramp stability experiments (Tm and Tagg) are well established methods for ranking proteins and formulations for stability. A protein’s denaturation profile provides information about its thermal stability and represents a structural ‘fingerprint’ for assessing structural and formulation buffer modifications. A widely used measure of the thermal structural stability of a protein is the temperature at which it unfolds from the native state to a denatured state.

[0229] For many proteins, this unfolding process occurs over a narrow temperature range and the mid-point of this transition is termed ‘melting temperature’ or ‘Tm’. To determine the melting temperature of a protein, UNcle measures the fluorescence of Sypro Orange (which binds to exposed hydrophobic regions of proteins) as the protein undergoes conformational changes.

[0230] Samples for each variant were formulated in PBS and Sypro Orange at a final concentration of 0.4 mg / mL. 9 pL of each sample mixture was loaded in triplicate into UNi microcuvettes. Samples were subjected to a thermal ramp from 25 - 95 °C, with a ramp rate of 0.3 °C / minute and excitation at 473 nm. Full emission spectra were collected from 250 - 720 nm, and the area under the curve between 510 - 680 nm was used to calculate the inflection points of the transition curves (Tonsetand Tm). Monitoring of static light scattering (SLS) at 473 nm allowed the detection of protein aggregation, and Tagg (onset of aggregation) was calculated from the resulting SLS profiles. Data analysis was performed using UNcle™ software version 6.0 and is shown in Table 9. Thermal stability values determined lie in a broad range from 48.9 to 59.4°C (Tonset), 57.3 to 67.5°C (Tm1) and 68.6 to 70.7°C (Tagg) with the humanized leads generally showing improved profiles when compared to the chimeric antibody. For all humanized and chimeric antibodies tested only one transition (Tm1) was detected. All humanized antibodies appear to have broadly comparable Taggvalues when compared to the chimeric antibody. However, both Tm1 and Tonset for the humanized variants appear to be much higher (up to ~10°C) than the chimeric VH0 / VL0, suggesting that these are more stable than the chimeric antibody.

[0231] Table 9: Summary of thermal stability values for the five purified lead humanized variants and chimeric antibody, as determined using the UNcle biostability platform.Thermal stability analysis using the UNcle biostability platform showed all humanized variants had similar Taggvalues. However, Tm1 and Tonsetfor the humanized variants appear to be much higher (up to ~10°C) than chimeric VH0A / L0, suggesting that these are more stable than the chimeric antibody.Example 2: Immunocytochemistry of humanized antibodies

[0232] The following cell lines were used: HEK293T cells, SH-SY5Y cells. Seeding density= 40000 cells / well of a 24-well plate. T ransfection was performed 24hrs post seeding using Lipofectamine 3000. ICC was performed 48hrs post transfection.

[0233] The following antibodies were used:• Rabbit pan anti-Tdp43 = 1:500 to stain endogenous TDP-43 in untransfected cells• Rabbit anti-HA tag = 1:1000 to stain HA-tagged TDP-43 in transfected cells• Chimeric Human VH0A / L0 = 5ug / ml, 1ug / ml as positive control• Humanized VH5 / VL3 = 5ug / ml, 1ug / ml as a test antibody• Humanized VH5 / VL4 = 5ug / ml, 1ug / m as a test antibody• Humanized VH6 / VL5 = 5ug / ml, 1ug / ml as a test antibody• Humanized VH7 / VL3 = 5ug / ml, 1ug / ml as a test antibody• Humanized VH7 / VL4 = 5ug / ml, 1ug / ml as a test antibody• Anti-Human Alexa Fluor 568 = 1:1000• Anti-Rabbit Alexa Fluor 488 = 1:1000.

[0234] Results are depicted in Figs. 13A-R and Figs. 14A-L.

[0235] Table 10: Summary SH-SY5Y cells5jig / ml Ijjg / nnl 5 g / ml ipg / ml 5jig / rrl Ipg / ml1 VH0 / VL0 - - - ++ +2 VH5 / VL3 - - - - + ++3 VH5 / VL4 - - - - + ++4 VH6 / VL5 - - - - ++ ++6 VH7 / VL4 - - - - + +

[0236] All the candidates selectively stain the TDP-43 aggregates over wild-type (WT) TDP-43 in SH-SY5Y cells. Minimal background staining is observed with VH6 / VL5 < VH5 / VL4 < VH5 / VL3.

[0237] Table 11: Summary HEK293T cellsSpg / ml Ipg / ml 5pg / ml lpg / ml1 VHO / VLO - - + +2 VH5 / VL3 - ++ ++3 VH5 / VL4 + ++4 VH6 / VL5 - ++ ++5 VH7 / VL3 - - + +6 VH7 / VL4 + ++

[0238] All the candidates selectively stain the TDP-43 aggregates over WT TDP-43 in transfected HEK293T cells. Minimal background staining is observed with VH6 / VL5 < VH5 / VL4 < VH5 / VL3.

[0239] While the present application has been described with reference to what are presently considered to be the preferred examples, it is to be understood that the application is not limited to the disclosed examples. To the contrary, the application is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

[0240] All publications, patents and patent applications are herein incorporated 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 by reference in its entirety. Specifically, the sequences associated with each accession numbers provided herein including for example accession numbers and / or biomarker sequences (e.g. protein and / or nucleic acid) provided in the Tables or elsewhere, are incorporated by reference in its entirely.

[0241] 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 description as a whole.REFERENCES[1] Kuo PH, Chiang CH, Wnag YT, Doudeva LG, Yuan HS, The Crystal Structure of TDP-43 RRM1-DNA Complex Reveals the Specific Recognition for UG- and TG-Rich Nucleic Acids. Nucleic Acids Res., 2014, vol 42, 4712.[2] DOI: 10.2210 / pdb1wf0 / pdb (No publication).[3] Mompean, M., Romano, V., Pantoja-Uceda, D., Stuani, C., Baralle, F. E., Buratti, E., and Laurents, D. V. The TDP-43 N- Terminal Domain Structure at High Resolution. FEBS J., 2016, 283, 1242.[4] Arai, T., Hasegawa, M., Akiyama, H., Ikeda, K., Nonaka, T., Mori, H., Mann, D., Tsuchiya, K., Yoshida, M., Hashizume, Y., and Oda, T. TDP-43 is a component of ubiquitin-positive tau-negative inclusions in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Biochem. Biophys. Res. Commun., 2006, 351, 602-611.[5] Chantelle F. Sephton, Shannon K. Good, Stan Atkin, Colleen M. Dewey, Paul Mayer III, Joachim Herz, and Gang Yu J. Biol. Chem. 2010, vol.285, No.9, 6826- 6834.[6] Abel, O., Powell, J. F., Andersen, P. M., and Al-Chalabi, A. Hum Mutat, 2012, 33:1345-51.[7] Hamley, I. W. PEG-Peptide Conjugates 2014; 15, 1543-1559; dx.doi.org / 10.1021 / bm500246w.[8] Roberts, MJ et al Chemistry for peptide and protein PEGylation 64: 116-127.[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.

[0010] 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.

[0011] Tamaki, Y et al. Elimination of TDP-43 inclusions linked to amyotrophic lateral sclerosis by a misfolding-specific intrabody with dual proteolytic signals. Sci Rep 8, 6030 (2018).

[0012] Brettschneider, J et al. Sequential distribution of pTDP-43 pathology in behavioral variant frontotemporal dementia (bvFTD). Acta Neuropathol (2014) 127: 423.

[0013] Feiler, M. S. et al. TDP-43 is intercellularly transmitted across axon terminals. J Cell Biol (2015) 211: 897.

[0014] Jo, M., Lee, S., Jeon, YM. et al. The role of TDP-43 propagation in neurodegenerative diseases: integrating insights from clinical and experimental studies. Exp Mol Med 52, 1652-1662 (2020). https: / / doi.org / 10.1038 / s12276-020-00513-7.

Claims

CLAIMS1. A humanized antibody comprising a sequence as set forth in any of SEQ ID NOs: 2-8 and / or 10-14 or a sequence with at least 80% sequence identity thereto wherein the CDR amino acid sequences, comprising CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 are as set for forth in SEQ ID Nos: 15-20, respectively; and wherein the antibody comprises at least one of the following residues relative to SEQ ID NO: 1:L at position 4; G at position 10; Q at position 13; G at position 15; R at position 19; S at position 21; R at position 71; D at position 72; N or T at position 73; K or S at position 75; N at position 76; L at position 78; Y at position 79; N at position 82A; R at position 83; E at position 85; Y at position 91; and / or S at position 113; and / orat least one of the following residues relative to SEQ ID NO: 9:Q at position 3; S at position 7; L at position 11; A at position 13; S at position 14; D at position 17; R at position 18; T at position 22; S at position 63; S at position 67; D or E at position 70; S at position 77; L at position 78; P at position 80; F at position 83; K at position 103; E at position 105; and / or I at position 106.

2. The humanized antibody of claim 1, wherein the at least one residue is D at position 72, and / or wherein the at least one residue is N position 76.

3. The humanized antibody of claim 1 or 2, wherein the humanized antibody comprises a heavy chain variable region comprising: i) an amino acid sequence as set forth in any one of SEQ ID NOs: 2-8, preferably SEQ ID Nos: 6, 7 or 8 ii) an amino acid sequence with at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90% or at least 95% sequence identity to any one of SEQ ID NOs: 2-8, preferably SEQ ID Nos: 6, 7 or 8, wherein the CDR sequences are as set forth in SEQ ID NOs: 15-17), or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 15-17.

4. The humanized antibody of any one of claims 1 to 3,, wherein the antibody comprises a light chain variable region comprising i) an amino acid sequence as set forth any one of SEQ ID NOs: 10-14, preferably SEQ ID Nos: 12, 13 or 14; ii) an amino acid sequence with at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90% or at least 95% sequence identity to any one of SEQ ID NO: 10-14, preferably SEQ ID Nos: 12, 13 or 14, wherein the CDR sequences are as set forth in SEQ ID NOs: 18-20, or iii) a conservatively substituted amino acid sequence of i) wherein the CDR sequences are as set forth in SEQ ID NOs: 18-20.

5. The humanized antibody of any one of claims 1 to 4, wherein the humanized antibody comprises a heavy chain variable region and a light chain variable region as set forth in SEQ ID NO: 6 and 12, respectively; SEQ ID NO: 6 and 13, respectively; SEQ ID NO: 7 and 14, respectively; SEQ ID NO: 8 and 12, respectively; SEQ ID NO: 8 and 13, respectively, or sequence(s) with sequence with at least at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90% or at least 95% sequence identity to any thereto wherein the CDR sequences are maintained as set forth in SEQ ID NOs: 15-20; orwherein the humanized antibody comprises a heavy chain variable region and a light chain variable region as set forth in SEQ ID NO: 6 and 12, respectively; SEQ ID NO: 6 and 13, respectively; SEQ ID NO: 7 and 14, respectively; SEQ ID NO: 8 and 13, respectively, or sequence(s) with sequence with at least at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90% or at least 95% sequence identity to any thereto wherein the CDR sequences are maintained as set forth in SEQ ID NOs: 15-20.

6. The humanized antibody of any one of claims 1 to 4, wherein the humanized antibody comprises a heavy chain variable region and a light chain variable region as set forth in SEQ ID NO: 6 and 12, respectively or sequence(s) with at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90% or at least 95% sequence identity thereto wherein the CDR sequences are maintained as set forth in SEQ ID NOs: 15-20.

7. The humanized antibody of any one of claims 1 to 4, wherein the humanized antibody comprises a heavy chain variable region and a light chain variable region as set forth in SEQ ID NO: 6 and 13, respectively or sequence(s) with at least 80 at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90% or at least 95% sequence identity to any thereto wherein the CDR sequences are maintained as set forth in SEQ ID NOs: 15-20.

8. The humanized antibody of any one of claims 1 to 4, wherein the humanized antibody comprises a heavy chain variable region and a light chain variable region as set forth in SEQ ID NO: 7 and 14, respectively or sequence(s) with at least 80 at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90% or at least 95% sequence identity to any thereto wherein the CDR sequences are maintained as set forth in SEQ ID NOs: 15-20.

9. The humanized antibody of any one of claims 1 to 4, wherein the humanized antibody comprises a heavy chain variable region and a light chain variable region as set forth in SEQ ID NO: 8 and 12, respectively or sequence(s) with at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90% or at least 95% sequence identity to any thereto wherein the CDR sequences are maintained as set forth in SEQ ID NOs: 15-20.

10. The humanized antibody of any one of claims 1 to 4, wherein the humanized antibody comprises a heavy chain variable region and a light chain variable region as set forth in SEQ ID NO: 8 and 13, respectively or sequence(s) with at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90% or at least 95% sequence identity to any thereto wherein the CDR sequences are maintained as set forth in SEQ ID NOs: 15-20.

11. The humanized antibody of any one of claims 1 -4, wherein the humanized antibody comprises a heavy chain variable region comprising a T72D substitution relative to SEQ ID NO: 1; and / or a light chain variable region comprising one or more of a V3Q substitution, a T7S substitution, a K11 L substitution or a K63S substitution relative to SEQ ID NO: 9.

12. The humanized antibody of any one of claims 1 to 10, wherein the antibody when bivalent has a KD of at least or about 1x 10-11M, at least or about 2 x10-11M, at least or about 3 x 10-11M to misfolded TDP-43.

13. The humanized antibody of any one of claims 1 to 12, wherein the antibody is an antibody binding fragment selected from Fab, Fab', F(ab')2, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, and multimers thereof.

14. The humanized antibody claim 13, wherein the antibody binding fragment is a Fab fragment.

15. The humanized antibody of any one of claims 1 to 14, wherein the antibody selectively binds misfolded TDP-43 over native TDP-43.

16. The humanized antibody of any one of claims 1 to 15, wherein the antibody is a single chain antibody.

17. An immunoconjugate comprising the humanized antibody of any one of claims 1 to 16 and a detectable label.

18. The immunoconjugate of claim 17, wherein the detectable label comprises a positron emitting radionuclide, optionally for use in subject imaging such as PET imaging.

19. A composition comprising the humanized antibody of any one of claims 1 to 16, or the immunoconjugate of claim 17 or 18, optionally with a diluent.

20. A nucleic acid molecule encoding the humanized antibody of any one of claims 1 to 16.

21. A vector comprising the nucleic acid of claim 20.

22. A cell expressing the humanized antibody of any one of claims 1 to 16, optionally wherein the cell is a hybridoma or a Chinese hamster ovary cell comprising the nucleic acid of claim 20 or the vector of claim 21.

23. A kit comprising the humanized antibody of any one of claims 1 to 16, the nucleic acid molecule of claim 20, the vector of claim 21 or the cell of claim 22.

24. A use of an effective amount of the humanized antibody of any one of claims 1 to 16, the immunoconjugate of claim 17 or 18, the nucleic acid molecule of claim 20, the vector of claim 21, the cell of claim 22 or the composition of claim 19 for treating a subject in need thereof.

25. The use of claim 24, wherein the humanized antibody, immunoconjugate, nucleic acid molecule, vector, cell or composition is for use in combination with another TDP-43 proteinopathy treatment.

26. A use of inhibiting misfolded TDP-43 cell transmission, comprising use of the humanized antibody of any one of claims 1 to 16, the immunoconjugate of claim 17 or 18, the nucleic acid molecule of claim 20, the vector of claim 21, the cell of claim 22 or the composition of claim 19 to a subject in need thereof.

27. The use of any one of claims 24 to 26, wherein the humanized antibody, immunoconjugate, nucleic acid molecule, vector, cell or composition is for systemic, parenteral, intravenous, subcutaneous, intramuscular, intracranial, intraventricular, intrathecal, intraorbital, ophthalmic, intraspinal, intracisternal, intraperitoneal, intranasal, aerosol or oral administration, optionally for intravenous, subcutaneous or intrathecal administration.

28. A use of an effective amount of the humanized antibody of any one of claims 1 to 16, the immunoconjugate of claim 17 or 18, the nucleic acid molecule of claim 20, the vector of claim 21, the cell of claim 22 or the composition of claim 19 in the manufacture of a medicament for treating a subject in need thereof.

29. The use of any one of claims 24 to 28, wherein the subject in need thereof is suspected of having, is at risk of developing, or has been diagnosed with a TDP-43 proteinopathy.

30. The use of claim 29, wherein the TDP-43 proteinopathy is selected from amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD-TDP), primary lateral sclerosis, progressive muscular atrophy, Alzheimer’s disease and / or limbic-predominant age-related TDP-43 encephalopathy (LATE).

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