Single-chain antibody and intrabody against misfolded TDP-43, and method of use.

Abstract

This invention provides an antibody that can be expressed in cells and preferentially binds to misfolded TDP-43 rather than naturally folded TDP-43. [Solution] A nucleic acid is provided which includes a heavy chain variable region comprising complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3 that binds to Trp-68 at misfolded TDP-43, and a light chain variable region comprising complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3, wherein the heavy chain variable region and the light chain variable region include a single-chain antibody linked by a linker, and a sequence encoding the single-chain antibody.

Description

Technical Field

[0001] Related applications This application is a Patent Cooperation Treaty application claiming priority to U.S. Provisional Application No. 63 / 017,363, filed Apr. 29, 2020. The disclosure of the prior application is considered part of the disclosure of this application and is hereby incorporated by reference in its entirety.

[0002] Sequence listing inclusion A computer-readable form sequence listing, “P61456PC00 Sequence ListingST25.txt” (154,654 bytes), created on Apr. 29, 2021, is hereby incorporated by reference in its entirety.

[0003] field The present disclosure relates to TDP-43 single-chain antibodies, more specifically, intrabodies for targeting intracellular misfolded TDP-43.

Background Art

[0004] background The 43 kDa transactivation response (TAR) element DNA-binding protein (TDP-43) is a 414 amino acid protein composed of a 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 is adjacent to a domain that directs nuclear localization (NLS motif at residues 82-98, NLS1 K82RK84 and K95VKR98). RRM2 contains a nuclear export signal (NES) from residues 239 to 250.

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

[0006] TDP-43 has been shown to be hyperphosphorylated, ubiquitinated, and fragmented in neuronal inclusions in patients with both sporadic and familial ALS and FTD [4].

[0007] Functional TDP-43 can exist as a nuclear oligomer distinct from cytoplasmic aggregates formed during cellular stress. Oligomerization of functional TDP-43 is necessary for its RNA splicing function. Nuclear-mediated TDP-43 oligomerization can inhibit cytoplasmic mislocalization and the formation of pathological aggregates [9].

[0008] Physiological TDP-43 oligomerization is mediated by its N-terminal domain, which can adopt a dynamic solenoid-like structure, as revealed by the 2.1A crystal structure obtained by a combination of nuclear magnetic resonance spectroscopy and electron microscopy.[9]

[0009] TDP-43 aggregates (inclusions) are now found in almost all (approximately 97%) cases of ALS and approximately half (approximately 40%) cases of FTD. TDP-43 is one of the main components of cytoplasmic inclusions found in motor neurons and glial cells of ALS patients.

[0010] The precursor of TDP-43 inclusions can be present at much lower concentrations than functional TDP-43. The low concentration of misfolded TDP-43 makes this target difficult to capture.

[0011] Intracerebral injection of brain-derived pathogenic TDP-43 FTLD-TDP seeds in transgenic and non-transgenic mice expressing cytoplasmic human TDP-43 has resulted in the induction of de novo TDP-43 lesions that propagate to the brain in a time-dependent manner

[10] .

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

[0013] WO2012174666, titled "Methods for Prognosis and / or Diagnosis of Neurodegenerative Diseases, Candidate Compounds and Methods for Identifying Compounds for the Treatment of Neurodegenerative Diseases," discloses a method for diagnosing neurodegenerative diseases such as ALS and FTD by evaluating the interaction between TDP-43 and NF-κB p65 using an anti-TDP-43 antibody.

[0014] WO2016086320, titled "TDP-43-binding polypeptide useful for the treatment of neurodegenerative diseases," discloses an antibody for the treatment of ALS and FTD that binds to the RRM1 domain of TDP-43 and interferes with its interaction with NF-κB.

[0015] An antibody that can be expressed within cells and preferentially binds to misfolded TDP-43 rather than naturally folded TDP-43 is desired. [Overview of the project]

[0016] overview The inventors have identified a single-chain intrabody that can target cytoplasmic misfolded TDP-43 and, for example, increase its degradation when expressed in cells containing cytoplasmic misfolded TDP-43.

[0017] The single-chain intrabodies originate from antibodies that bind to the N-terminal epitope of a conformation accessible in misfolded TDP-43 but unavailable in naturally folded, non-disease-associated TDP-43. Antibodies induced against an immunogen containing the N-terminal TDP-43 sequence DAGWGNL (SEQ ID NO: 1) preferentially bound to misfolded TDP-43 aggregates. Residue W68 was found to be a crucial residue for conferring antibody specificity against misfolded TDP-43 aggregates.

[0018] One aspect comprises a single-chain antibody that binds to misfolded TDP-43 and includes a heavy chain variable region comprising complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3, and a light chain variable region comprising complementarity-determining regions CDR-L1, CDR-L2, and CDR-, wherein the heavy chain variable region and the light chain variable region are linked by a linker. The orientation of the heavy chain and light chain variable regions and the linker may be heavy chain variable region-linker-light chain variable region or light chain variable region-linker-heavy chain variable region.

[0019] In one embodiment, the single-chain antibody is an scFv, a nanobody, or a minibody.

[0020] A further aspect is the inclusion of an immunocomplex comprising the antibody described herein and a detectable label such as a positron-emitting radionuclide, a fusion tag such as a FLAG tag or myc tag, or a targeting moiety such as a lysosome or autophagy targeting sequence.

[0021] Further aspects include an isolated nucleic acid encoding a single-chain antibody as described herein, and a vector comprising the nucleic acid for, for example, delivering and / or expressing a single-chain antibody as described herein.

[0022] A further aspect comprises cells that recombinantly express the single-chain antibody described herein.

[0023] Further aspects include compositions comprising single-chain antibodies, immune complexes, isolated nucleic acids, vectors, or cells as described herein.

[0024] A method for treating a subject having TDP-43 proteinosis is further provided, comprising administering an effective amount of a nucleic acid or immune complex encoding a single-chain antibody described herein to a subject in need.

[0025] Other features and advantages of this disclosure will become apparent from the following detailed description. However, various changes and modifications within the spirit and scope of this disclosure will become apparent to those skilled in the art from this detailed description, so the detailed description and specific examples should be understood as being provided merely as illustrations, while illustrating preferred embodiments of this disclosure.

[0026] Embodiments of the present disclosure are described here in reference to the drawings: [Brief explanation of the drawing]

[0027] [Figure 1] Figure 1 shows the immunocytochemistry of cells overexpressing dNLS-TDP43 and LYS-2F7. dNLS-TDP-43 was detected using anti-HA, and LYS-2F7 was detected using anti-FLAG. The merge indicates the level of colocalization with respect to the nucleus stained with DAPI. [Figure 2] Figure 2 shows the immunocytochemistry of cells overexpressing dNLS-TDP43 and LYS-1H3-1K3. dNLS-TDP43 was detected using anti-HA, and LYS-1H3-1K3 was detected using anti-FLAG. The merge indicates the level of colocalization with respect to nuclei stained with DAPI. [Figure 3] Figure 3 shows the immunocytochemistry of cells overexpressing dNLS-TDP43 and LYS-28H3-28K1. dNLS-TDP-43 was detected using anti-HA, and LYS-28H3-28K1 was detected using anti-FLAG. The merge indicates the level of colocalization with respect to the nucleus stained with DAPI. [Figure 4] Figure 4 shows the immunocytochemistry of cells overexpressing dNLS-TDP43 and LYS-14H1-14K2. dNLS-TDP-43 was detected using anti-HA, and LYS-14H1-14K2 was detected using anti-FLAG. The merge indicates the level of colocalization with respect to the nucleus stained with DAPI. [Figure 5] Figure 5A shows the immunocytochemistry of cells overexpressing dNLSTDP-43 and YPTL-2F7. dNLS-TDP43 was detected using anti-HA, and YPTL-2F7 was detected using anti-FLAG. The merge indicates the level of colocalization with respect to the nucleus stained with DAPI. [Figure 6] Figure 6 shows the immunocytochemistry of cells overexpressing dNLS-TDP43 and YPTL-1H3-1K3. dNLS-TDP43 was detected using anti-HA, and YPTL-1H3-1K3 was detected using anti-FLAG. The merge indicates the level of colocalization with respect to nuclei stained with DAPI. [Figure 7] Figure 7 shows the immunocytochemistry of cells overexpressing dNLS-TDP43 and YPTL-28H3-28K1. dNLS-TDP43 was detected using anti-HA, and YPTL-28H3-28K1 was detected using anti-FLAG. The merge indicates the level of colocalization with respect to nuclei stained with DAPI. [Figure 8] Figure 8 shows the immunocytochemistry of cells overexpressing dNLS-TDP43 and YPTL-14H1-14K2. dNLS-TDP-43 was detected using anti-HA, and YPTL-14H1-14K2 was detected using anti-FLAG. The merge indicates the level of colocalization with respect to the nucleus stained with DAPI. [Figure 9] Figure 9 shows the immunocytochemistry of cells overexpressing dNLS-TDP43 and MYCL15H-2F7. dNLSTDP-43 was detected using anti-HA, and MYCL15H-2F7 was detected using anti-MYC. The merge indicates the level of colocalization with respect to the nucleus stained with DAPI. [Figure 10]Figure 10 shows the immunocytochemistry of cells overexpressing dNLS-TDP43 and MYCH15L-2F7. dNLS-TDP-43 was detected using anti-HA, and MYCH15L-2F7 was detected using anti-MYC. The merge indicates the level of colocalization with respect to nuclei stained with DAPI. [Figure 11] Figure 11 shows the immunocytochemistry of cells overexpressing dNLS-TDP43 and MYCH20L-2F7. dNLS-TDP-43 was detected using anti-HA, and MYCH20L-2F7 was detected using anti-MYC. The merge indicates the level of colocalization with respect to the nucleus stained with DAPI. [Figure 12] Figure 12 shows the immunocytochemistry of cells overexpressing dNLS-TDP43 and MYCL15H-1H3-1K3. dNLS-TDP43 was detected using anti-HA, and MYCL15H-1H3-1K3 was detected using anti-MYC. The merge indicates the level of colocalization with respect to nuclei stained with DAPI. [Figure 13] Figure 13 shows the immunocytochemistry of cells overexpressing dNLSTDP-43 and MYCL20H-1H3-1K3. dNLS-TDP43 was detected using anti-HA, and MYCL20H-1H3-1K3 was detected using anti-MYC. The merge indicates the level of colocalization with respect to the nucleus stained with DAPI. [Figure 14] Figure 14 shows the immunocytochemistry of cells overexpressing dNLSTDP-43 and MYCH15L-28H3-28K1. dNLSTDP-43 was detected using anti-HA, and MYCH15L-28H3-28K1 was detected using anti-MYC. The merge indicates the level of colocalization with respect to the nucleus stained with DAPI. [Figure 15] Figure 15 shows the immunocytochemistry of cells overexpressing dNLS-TDP43 and MYCL15H-14H1-14K2. dNLS-TDP-43 was detected using anti-HA, and MYCL15H-14H1-14K2 was detected using anti-MYC. The merge indicates the level of colocalization with respect to nuclei stained with DAPI. [Figure 16]Figure 16 shows the immunocytochemistry of cells overexpressing dNLS-TDP43 and MYCH15L-14H1-14K2. dNLS-TDP-43 was detected using anti-HA, and MYCH15L-14H1-14K2 was detected using anti-MYC. The merge indicates the level of colocalization with respect to the nucleus stained with DAPI. [Figure 17] Figure 17 shows the immunocytochemistry of cells overexpressing dNLS-TDP43 and MYCL20H-14H1-14K2. dNLS-TDP-43 was detected using anti-HA, and MYCL20H-14H1-14K2 was detected using anti-MYC. The merge indicates the level of colocalization with respect to nuclei stained with DAPI. [Figure 18] Figure 18 shows the immunocytochemistry of cells overexpressing dNLS-TDP43 and MYCH20L-14H1-14K2. dNLS-TDP-43 was detected using anti-HA, and MYCH20L-14H1-14K2 was detected using anti-MYC. The merge indicates the level of colocalization with respect to the nucleus stained with DAPI. [Figure 19] Figure 19 shows Western blots of cells overexpressing dNLS-TDP43 and LYS-2F7, LYS-28H3-28K1, or LYS-14H1-14K2. The expression level of dNLS-TDP43 was detected using anti-HA. The expression levels of LYS-2F7, LYS-28H3-28K1, and LYS-14H1-14K2 were detected using anti-FLAG. Actin was used as a loading control. The EV (empty vector) is a reference-negative control plasmid. [Figure 20] Figure 20 shows Western blots of cells overexpressing dNLSTDP-43 and YPTL-14H1-14K2. The expression level of dNLS-TDP43 was detected using anti-HA. The expression level of YPTL-14H1-14K2 was detected using anti-FLAG. Actin was used as a loading control. EV is the reference negative control. [Figure 21]Figure 21 shows Western blots of cells overexpressing dNLSTDP-43 and MYCL15H-2F7, MYCH15L-2F7, MYCL15H-1H3-1K3, or MYCH20L-14H1-14K2. The expression level of dNLS-TDP43 was detected using anti-HA. The expression levels of MYCL15H-2F7, MYCH15L-2F7, MYCL15H-1H3-1K3, and MYCH20L-14H1-14K2 were detected using anti-MYC. Actin was used as a loading control. EV is the reference negative control. Detailed description of the invention

[0028] Detailed explanation of this disclosure I. Definition

[0029] As used herein, the terms "TDP-43" (transactivation response element (TAR) DNA-binding protein 43), "TDP43," or, unless otherwise specified, "TDP" means, as used herein, all forms of TDP-43, including wild-type TDP-43, natural TDP-43, and misfolded forms, including mutants and analogues from all species, in particular human TDP-43 (i.e., hTDP-43). Human TDP-43 is generally a 414-amino acid protein whose amino acid sequence (e.g., Uniprot accession number Q13148) and nucleotide sequence (e.g., accession number HGNC:11571) have been previously characterized.

[0030] As used herein, "wild type" refers to the primary amino acid sequence of a non-mutated or naturally occurring protein.

[0031] As used herein, “natural” refers to the normal three-dimensional structure of a particular protein or a part thereof. Natural TDP-43 is sometimes referred to as “naturally folded” TDP-43, “normally folded” TDP-43, and / or “healthy” TDP-43. Thus, the term “natural TDP-43,” or “naturally folded TDP-43,” as used herein, refers to TDP-43 that has been naturally folded after nascent translation, and / or, but is not limited to, a multimer containing dimeric and trimer TDP-43 folded in a non-pathological state (e.g., normal cells) having a molecular structure comprising non-covalently bonded individual TDP-43 peptides that exhibit the natural structure as reconstructed from X-ray crystallography or nuclear magnetic resonance spectroscopy. Natural TDP-43 forms multimers via its NTD, and naturally folded TDP-43 is generally the nucleus. Misfold aggregates of TDP-43 can be located in the cytoplasm, and are generally cytoplasmic.

[0032] As used herein, "misfolded" refers to the secondary and tertiary structures of a polypeptide or a portion thereof, indicating that the polypeptide is conforming to a state that is abnormal with respect to its properly functioning state. Misfolding can result from mutations in a protein, such as amino acid deletions, substitutions, or additions, but wild-type sequence proteins can also be misfolded in disease, for example, as a result of microenvironmental conditions and / or amino acid modifications, such as nitration, oxidation, carbonylation, or other modifications, which can expose disease-specific epitopes. Other post-translational modifications include abnormal ubiquitination, phosphorylation, acetylation, SUMOylation, and cleavage into C-terminal fragments. Misfolded TDP43 may be aggregated and / or cytoplasmic. In the context of TDP-43, native TDP-43 forms multimers via its NTD. Misfolded multimers (e.g., disease-associated oligomers) generally oligomerize via other regions of the protein, such as its LCD and / or RRM1 domain. Therefore, “Misfolded TDP-43 polypeptide,” or “Misfolded TDP-43” as used herein to refer to polypeptides, includes cytoplasmic and / or aggregated TDP-43 polypeptides oligomerized via their LCD and / or RRM1 domains, unnatural dimers and trimers, and large aggregates (e.g., five or more subunits). Misfolded TDP-43 tends to form aggregates that result in loss of protein function, toxicity, acquisition of amyloid-like features (e.g., Congo red staining), and transmission of pathogenic aggregates.

[0033] The term “mutant TDP-43” refers to the morphology of TDP-43, in particular the intrinsic form of TDP-43 resulting from gene mutations that produce amino acid substitutions, such as those characteristic of FTD or familial ALS, including, for example, the mutations described in the bioinformatics tools described in [6].

[0034] The term "DAGWGNL(SEQ ID NO: 1)" refers to the amino acid sequence shown in SEQ ID NO: 1: aspartic acid, alanine, glycine, tryptophan, glycine, asparagine, and leucine. Similarly, GWG refers to an amino acid sequence identified by a single-letter amino acid code. Depending on the context, a reference to an amino acid sequence may refer to the sequence in TDP-43 or the isolated peptide. Sequence DAGWGNL(SEQ ID NO: 1) corresponds to residues 65-71 in the primary amino acid sequence of TDP-43.

[0035] The term "amino acid" includes all naturally occurring amino acids and modified L-amino acids and D-amino acids. The atoms of an amino acid may include, for example, different isotopes. For instance, an amino acid may consist of deuterium substituted for hydrogen, nitrogen 15 substituted for nitrogen 14, carbon 13 substituted for carbon 12, and other similar variations.

[0036] As used herein, a "conservative amino acid substitution" is a substitution in which one amino acid residue is replaced by another amino acid residue without losing the desired properties of the protein. Suitable conservative amino acid substitutions can be made by substituting amino acids having similar hydrophobicity, polarity, and R-group size with each other. Examples of conservative amino acid substitutions include:

[0037] [Table 1]

[0038] As used herein, the term “antibody” includes monoclonal antibodies, polyclonal antibodies, single-chain, humanized and other chimeric antibodies, or fully human antibodies, as well as their conjugated fragments. Vectorized antibodies or intrabodies are also included. Antibodies may be derived from recombinant sources and / or produced in transgenic animals. Human antibodies that can be produced by using biochemical techniques or isolated from libraries are also included. Humanized antibodies or chimeric antibodies may contain sequences derived from one or more isotypes or classes.

[0039] The phrase “isolated antibody” refers to an antibody produced in vivo or in vitro that has been removed from its source, e.g., an animal, hybridoma, or other cell line (e.g., recombinant cells that produce the antibody). Isolated antibodies are sometimes “purified,” which means a purity of at least 80%, 85%, 90%, 95%, 98%, or 99%.

[0040] The term “intrabody” (singular or plural), as used herein, refers to an antibody that is expressed or can be expressed within a cell and that binds to an intracellular protein. For example, an intrabody is an antibody modified or adapted for intracellular localization and intracellular function. An intrabody comprises a heavy chain variable domain, a light chain variable domain, and a linker, optionally in either variable domain orientation, for example, 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.

[0041] As used herein, the term “linker” refers to a synthetic sequence (e.g., an amino acid sequence in a polypeptide or a nucleic acid sequence in a polypeptide) that connects or ligates two sequences, for example, two polypeptide domains. A linker may be a “tag linker” indicating that it ligates a detectable label, or a “targeting moiety linker” indicating that it ligates a targeting moiety to a polypeptide, which may also include a linker, such as in the case of a heavy chain variable region ligated to a light chain variable region.

[0042] The term “complementarity-determining region” or “CDR,” as used herein, refers to a specific hypervariable region of an antibody that is generally understood to define epitope binding. Methods for identifying CDR sequences include Kabat, Chothia, and IMGT. Those skilled in the art may also identify CDR sequences based on Kabat and Chothia, etc., considering the sequences included herein.

[0043] The term “detectable label” as used herein means a portion of a peptide sequence, fluorescent protein, or other compound described herein that can be added to or introduced into the peptide, antibody, or other compound described herein and that can generate a detectable signal, either directly or indirectly.

[0044] The term “epitope selectively presented or accessible on misfolded TDP-43” means, as used herein, an epitope that is selectively presented or antibody-accessible on misfolded TDP-43 present in, for example, ALS or FTD (e.g., disease-associated misfolded TDP-43), whether monomeric, dimeric, or aggregated, but not on the molecular surface of the naturally occurring, correctly folded homodimeric form of TDP-43. As shown herein, W68 is selectively presented or accessible on misfolded TDP-43.

[0045] As used herein, the term "high affinity" means that antibody X binds to target Y more strongly (K on ) and / or with a lower dissociation constant (K off ) than to target Z. In this context, antibody X has a higher affinity for target Y than for Z. Similarly, the term "low affinity" as used herein means that antibody X binds to target Y with a lower strength and / or a higher dissociation constant than to target Z. In this context, antibody X has a lower affinity for target Y than for Z. The affinity of the binding between an antibody and its target antigen can be represented by K D , which is equal to 1 / K A , where K D is equal to k on / k off . The k on and k off values can be measured using surface plasmon resonance (e.g., measurable using a Biacore system).

[0046] The term “nucleic acid sequence,” as used herein, refers to a sequence of nucleotides or nucleotide monomers consisting of naturally occurring bases, sugars, and intersugar (skeletal) bonds. The term also includes modified or substituted sequences containing monomers or portions thereof that do not exist naturally. The nucleic acid sequences of this application may be deoxyribonucleic acid (DNA) sequences or ribonucleic acid (RNA) sequences, and may contain naturally occurring bases including adenine, guanine, cytosine, thymidine, and uracil. Sequences may contain modified bases. Examples of such modified bases include adenine, guanine, cytosine, thymidine, and uracil of aza and deaza; as well as xanthine and hypoxanthine. Nucleic acids may be double-stranded or single-stranded, representing a sense strand or an antisense strand. Furthermore, the term “nucleic acid” includes complementary nucleic acid sequences and codon-optimized or synonymous codon equivalents. The term "isolated nucleic acid sequence," as used herein, refers to a nucleic acid that is substantially free from cell material or culture medium if produced by recombinant DNA technology, or from chemical precursors or other chemical substances if chemically synthesized. An isolated nucleic acid also substantially lacks sequences naturally adjacent to the nucleic acid from which it originates (i.e., sequences located at the 5' and 3' ends of the nucleic acid).

[0047] "Operationally linked" means that the nucleic acid is linked to a regulatory sequence in a manner that enables the expression of the nucleic acid. Suitable regulatory sequences may be derived from a variety of sources, including bacterial, fungal, viral, mammalian, or insect genes. The selection of a suitable regulatory sequence depends on the selected host cell and can be easily achieved by those skilled in the art. Examples of such regulatory sequences include transcription promoters and enhancers or RNA polymerase binding sequences, and ribosome binding sequences (including translation initiation signals). Furthermore, depending on the selected host cell and the vector used, other sequences, such as origins of replication, further DNA restriction sites, enhancers, and sequences that confer transcriptional inducibility, may be incorporated into the expression vector.

[0048] The term “vector,” as used herein, comprises any intermediate vehicle for a nucleic acid molecule that enables the nucleic acid molecule to be introduced into, for example, prokaryotic and / or eukaryotic cells and / or incorporated into the genome, and includes plasmids, phagemids, bacteriophages or viral vectors, such as retrovirus-based vectors including lentiviral vectors and adeno-associated virus (AAV) vectors. The term “plasmid,” as used herein, generally refers to a construct of extrachromosomal genetic material, usually a circular double-stranded DNA, capable of independently replicating chromosomal DNA.

[0049] "At least moderately stringent hybridization conditions" means that conditions are selected that promote selective hybridization between two complementary nucleic acid molecules in solution. Hybridization may occur for all or part of the nucleic acid sequence molecule. The hybridized portion is generally at least 15 (e.g., 20, 25, 30, 40, or 50) nucleotides long. Those skilled in the art will recognize that the stability of nucleic acid double strands or hybrids in sodium-containing buffer is determined by a function of sodium ion concentration and temperature (Tm = 81.5°C - 16.6(Log10[Na+]) + 0.41(%(G+C) - 600 / l), or a similar formula). Therefore, the parameters in the washing conditions that determine the stability of the hybrid are sodium ion concentration and temperature. To identify molecules that are similar to but not identical to known nucleic acid molecules, a 1% mismatch can be assumed to result in a Tm reduction of approximately 1°C. For example, if nucleic acid molecules are to have more than 95% identity, the final washing temperature will be reduced by approximately 5°C. Based on these considerations, those skilled in the art will be able to easily select appropriate hybridization conditions. In several preferred embodiments, stringent hybridization conditions are selected. As an example, the following conditions may be used to achieve stringent hybridization: hybridization with 5x sodium chloride / sodium citrate (SSC) / 5x Denhardt's solution / 1.0% SDS at Tm-5°C based on the above formula, followed by washing with 0.2x SSC / 0.1% SDS at 60°C. Moderately stringent hybridization conditions include a washing step in 3x SSC at 42°C. However, it is understood that equivalent stringency can be achieved using alternative buffers, salts, and temperatures.Further guidelines regarding hybridization conditions can be found in *Current Protocols in Molecular Biology*, John Wiley & Sons, NY, 2002, and in *Molecular Cloning: a Laboratory Manual*, Sambrook et al., *Cold Spring Harbor Laboratory Press*, 2001.

[0050] As used herein, "to bind" or "to bind specifically" with respect to an antibody means that the antibody recognizes its target antigen and binds to that target with greater affinity than to antigens that are structurally different and / or have modified or mutant sequences. For example, a polyvalent antibody may bind to at least 1e-6, at least 1e-7, at least 1e-8, at least 1e-9 or at least 1e-10 K D It then binds to its target. An affinity of at least greater than 1e-8 is preferred. Antigen-binding fragments, such as Fab fragments containing one variable domain, can find their targets with an affinity 10 or 100 times lower than that of multivalent interactions with non-fragmented antibodies.

[0051] When the terms “selective” or “preferential” are used herein in reference to antibodies that selectively / preferentially bind to a particular form of TDP-43 (e.g., native protein or misfolded protein), it means that the binding protein binds to that form with an affinity at least 3-fold, or at least 5-fold, at least 10-fold, at least 20-fold, at least 100-fold, at least 250-fold, or at least 500-fold greater than that of another form. Thus, an antibody that is more selective to a particular conformation (e.g., misfolded protein) preferentially binds to that particular form of TDP-43 with an affinity at least 3-fold, or at least 5-fold, at least 10-fold, at least 20-fold, at least 100-fold, at least 250-fold, or at least 500-fold greater than that of another form.

[0052] The terms “animal” or “subject” as used herein include all members of the Animal Kingdom, including, as may be, mammals, and, as may be, humans.

[0053] The terms “to treat” or “treatment,” as used herein and as well as as well understood in the art, mean an approach to obtain beneficial or desired outcomes, including clinical outcomes. Beneficial or desired clinical outcomes may include, but are not limited to, reduction or improvement of one or more symptoms or conditions, whether detectable or undetectable; a reduction in the severity of the disease; a stabilized (i.e., non-worsening) state of the disease; prevention of disease transmission; delay or slowing of disease progression; improvement or mitigation of the condition; a reduction in disease relapses; and remission (whether partial or complete). “To treat” and “treatment” may also mean extending survival compared to the survival expected without treatment. “To treat” and “treatment,” as used herein, also include prophylactic treatment in subjects identified with familial variants, such as familial ALS. Subjects with TDP-43 protein disorders, such as ALS, may be treated to delay or slow disease progression. The subject may be treated with the compounds, antibodies (including vectorized antibodies or intrabodies), immunogens, immune complexes, or compositions described herein to prevent progression.

[0054] In understanding the scope of this disclosure, the term "consisting of" and its derivatives, as used herein, are limited terms that specify the presence of the mentioned features, elements, components, groups, integers and / or processes, and exclude the presence of other features, elements, components, groups, integers and / or processes that are not mentioned.

[0055] In this specification, enumerations of numerical ranges by endpoint include all numbers and fractions contained within that range (for example, 1–5 includes 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5). It is also understood that all numbers and their fractions are assumed to be modified by the term “about”. Furthermore, “a”, “an”, and “the” should be understood to include multiple referents unless explicitly indicated otherwise by the context. The term “about” means plus or minus 0.1–50%, 5–50%, or 10–40% of the number mentioned, preferably 10–20%, more preferably 10% or 15%.

[0056] Furthermore, the definitions and embodiments described in particular sections are applicable to other embodiments described herein in which they are preferred, as will be understood by those skilled in the art. For example, different aspects of the present invention are defined in more detail in the following sections. Each of the aspects thus defined may be combined with any other one or more aspects unless it is explicitly indicated otherwise. In particular, any feature indicated as preferred or advantageous may be combined with any other one or more features indicated as preferred or advantageous.

[0057] II. Antibodies, Immune Complexes, Cells, and Nucleic Acids As described in the examples, single-chain antibodies were prepared and vectorized.

[0058] Single-chain antibodies are directed to the N-terminal epitope, which is accessible in misfolded TDP-43 but unavailable in naturally folded, non-disease-associated TDP-43. Antibodies induced against an immunogen containing the N-terminal TDP-43 sequence DAGWGNL (SEQ ID NO: 1) preferentially bound to misfolded TDP-43 aggregates. Residue W68 was found to be a crucial residue for conferring antibody specificity to misfolded TDP-43 aggregates.

[0059] In one embodiment, the single-chain antibody is an intrabody.

[0060] Single-chain antibodies, when acting as intrabodies, optionally contain lysosomal targeting or autophagy targeting signals. The heavy and light chain variable regions of several antibodies specific to misfolded TDP-43 were ligated in various orientations using different linkers. Vectorized single-chain antibodies or intrabodies were expressed intracellularly alongside mutant dNLS-TDP-43, which lacked its nuclear localization signal. dNLS-TDP-43 localized in the cytoplasm, where it formed aggregates. As demonstrated herein, vectorized antibodies were able to co-localize with intracellular misfolded TDP-43 aggregates and induce their degradation. The vectorized antibodies were confirmed to be non-toxic to cells and did not interfere with normal TDP-43 function.

[0061] One aspect includes a single-chain antibody comprising a heavy-chain variable region conjugated to W68 of misfolded TDP-43 and comprising complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3, and a light-chain variable region comprising complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3, wherein the heavy-chain and light-chain variable regions are linked by a linker. The orientation of the heavy-chain and light-chain variable regions and the linker may be heavy-chain variable region-linker-light-chain variable region or light-chain variable region-linker-heavy-chain variable region. In some embodiments, the single-chain antibody further comprises a lysosome or autophagy targeting sequence.

[0062] In one embodiment, the single-chain antibody comprises the following: CDR-H1: GFTFSSYY Sequence ID 130; CDR-H2: INSNGGST Sequence ID 131; CDR-H3: VRQNYEGAY Sequence ID 132; CDR-L1: QSIVHSNGNTY Sequence ID 133; CDR-L2: KVS Sequence ID 134; and CDR-L3: FQSSHVPWT Sequence ID 135, It has a CDR sequence.

[0063] Single-chain antibodies containing the CDR sequences of SEQ ID NOs. 130-135 specifically bind to W68 in association with the misfolded TDP-43 DAGWGNL (SEQ ID NO. 1). The single-chain antibody contains the CDR sequence of antibody 2F7.

[0064] In one embodiment, the single-chain antibody comprises a heavy chain variable region comprising i) the amino acid sequence shown in SEQ ID NO: 138, ii) an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 138, where the CDR sequence is shown in SEQ ID NOs: 130-132, or iii) the conserved substitution amino acid sequence of i); and / or, the single-chain antibody comprises i) the amino acid sequence shown in SEQ ID NO: 139, ii) at least 80%, at least 85%, or at least 95% sequence identity with SEQ ID NO: 139. The light chain variable region comprises an amino acid sequence having 90% or at least 95% sequence identity, wherein the CDR sequence is one of those shown in SEQ ID NOs: 133-135, or the conserved substitution amino acid sequence of iii)i), and optionally the heavy chain variable region amino acid sequence is encoded by the nucleotide sequence or codon degeneracy shown in SEQ ID NO: 136 or an optimized version thereof, and / or the light chain variable region amino acid sequence is encoded by the nucleotide sequence or codon degeneracy shown in SEQ ID NO: 137 or an optimized version thereof.

[0065] In one embodiment, the single-chain antibody comprises the following: CDR-H1: GFSLSRYY Sequence ID 10; CDR-H2: IIPGGTT Sequence ID 11; CDR-H3: AGGPTGNSHFTL Sequence ID 12; CDR-L1: ESVYNNNH Sequence ID 13; CDR-L2: EAS Sequence ID 14; and CDR-L3: SGYKRVTTDGIA Sequence ID 15, It has a CDR sequence.

[0066] Single-chain antibodies containing CDRs of sequence numbers 10-15 specifically bind to W68 in association with the misfolded TDP-43 DAGWGNL (sequence number 1). The single-chain antibodies contain the CDR sequence of antibody 1H3-1K3.

[0067] In one embodiment, the single-chain antibody comprises a heavy-chain variable region comprising i) the amino acid sequence shown in SEQ ID NO: 98, and ii) an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 98, where the CDR sequence is shown in SEQ ID NOs: 10-12, or iii) the conserved substitution amino acid sequence of i), and / or the single-chain antibody comprises a light-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 99, and ii) an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 99, where the CDR sequence is shown in SEQ ID NOs: 13-15, or iii) the conserved substitution amino acid sequence of i), wherein the heavy-chain variable region amino acid sequence is encoded by the nucleotide sequence or codon degeneracy shown in SEQ ID NO: 76 or an optimized version thereof, and / or the light-chain variable region amino acid sequence is encoded by the nucleotide sequence or codon degeneracy shown in SEQ ID NO: 77 or an optimized version thereof.

[0068] In one embodiment, the single-chain antibody comprises the following: CDR-H1: GFSLSSYN Sequence ID 120; CDR-H2: IGTGGIT Sequence ID 121; CDR-H3: VRSSGSDWWFHI Sequence ID 122; CDR-L1: QSVYNNNN Sequence ID 123; CDR-L2: RAS Sequence ID 124; and CDR-L3: QGYFSGFITT Sequence ID 125, It has a CDR sequence.

[0069] Single-chain antibodies containing CDRs of sequence numbers 120-125 specifically bind to W68 in association with the misfolded TDP-43 DAGWGNL (sequence number 1). The single-chain antibodies contain the CDR sequence of antibody 28H3-28K1.

[0070] In one embodiment, the single-chain antibody comprises a heavy chain variable region comprising i) the amino acid sequence shown in SEQ ID NO: 128, ii) an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 128, where the CDR sequence is shown in SEQ ID NOs: 120-122, or iii) the conserved substitution amino acid sequence of i), and / or the single-chain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 129, ii) at least 80%, at least 85%, or at least 90% sequence identity with SEQ ID NO: 129 The light chain variable region comprises an amino acid sequence having % or at least 95% sequence identity, wherein the CDR sequence is one of those shown in SEQ ID NOs: 123-125, or the conserved substitution amino acid sequence of iii)i), wherein the heavy chain variable region amino acid sequence is encoded by the nucleotide sequence or codon degeneracy shown in SEQ ID NO: 126 or an optimized version thereof, and / or the light chain variable region amino acid sequence is encoded by the nucleotide sequence or codon degeneracy shown in SEQ ID NO: 127 or an optimized version thereof.

[0071] In one embodiment, the single-chain antibody comprises the following: CDR-H1: GFSFSSNYV Sequence ID 16; CDR-H2: IWFAGIVDTT Sequence ID 17; CDR-H3: ARNPVGSVNL Sequence ID 18; CDR-L1: ESVYSNNR Sequence ID 19; CDR-L2: YAS Sequence ID 20; and CDR-L3: AGWRGARTDGVD Sequence ID 21, It has a CDR sequence.

[0072] Single-chain antibodies comprising CDRs of SEQ ID NOs. 16-21 specifically bind to W68 in association with the misfolded TDP-43 DAGWGNL (SEQ ID NO. 1). In one embodiment, the single-chain antibody comprises the CDR sequence of antibody 14H1-14K2.

[0073] In one embodiment, the single-chain antibody comprises a heavy-chain variable region comprising i) the amino acid sequence shown in SEQ ID NO: 100, ii) an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with respect to SEQ ID NO: 100, where the CDR sequence is shown in SEQ ID NOs: 16-18, or iii) the conserved substitution amino acid sequence of i), and / or the single-chain antibody comprises a light-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 101, ii) an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with respect to SEQ ID NO: 101, where the CDR sequence is shown in SEQ ID NOs: 19-21, or iii) the conserved substitution amino acid sequence of i), wherein the heavy-chain variable region amino acid sequence is encoded by the nucleotide sequence or codon degeneracy shown in SEQ ID NO: 78 or an optimized version thereof, and / or the light-chain variable region amino acid sequence is encoded by the nucleotide sequence or codon degeneracy shown in SEQ ID NO: 79 or an optimized version thereof.

[0074] In one embodiment, the single-chain antibody comprises the following: CDR-H1: GFSFSSSYV Sequence ID 22; CDR-H2: SDTGINT Sequence ID 23; CDR-H3: ARRYTGDTYLGNFNL Sequence ID 24; CDR-L1: QSVYKNNY Sequence ID 25; CDR-L2: KAS Sequence ID 26; and CDR-L3: AGGWRSLNA Sequence ID 27, It has a CDR sequence.

[0075] A single-chain antibody comprising the CDRs of SEQ ID NOs. 16-21 specifically binds to W68 in association with the misfolded TDP-43 DAGWGNL (SEQ ID NO. 1). In one embodiment, the single-chain antibody comprises the CDR sequence of antibody 17.

[0076] In one embodiment, the single-chain antibody comprises a heavy-chain variable region comprising i) the amino acid sequence shown in SEQ ID NO: 102, ii) an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with respect to SEQ ID NO: 102, where the CDR sequence is shown in SEQ ID NOs: 22-24, or iii) the conserved substitution amino acid sequence of i), and / or the antibody comprises a light-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 103, ii) an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with respect to SEQ ID NO: 103, where the CDR sequence is shown in SEQ ID NOs: 25-27, or iii) the conserved substitution amino acid sequence of i), wherein the heavy-chain variable region amino acid sequence is encoded by the nucleotide sequence or codon degeneracy shown in SEQ ID NO: 80 or an optimized version thereof, and / or the light-chain variable region amino acid sequence is encoded by the nucleotide sequence or codon degeneracy shown in SEQ ID NO: 81 or an optimized version thereof.

[0077] In one embodiment, the single-chain antibody comprises the following: CDR-H1: EFSFSSRYW Sequence ID 28; CDR-H2: IYTGSIDAT Sequence ID 29; CDR-H3: VRGSDAWGLYFNL Sequence ID 30; CDR-L1: QSIHKNNY Sequence ID 31; CDR-L2: FAS Sequence ID 32; and CDR-L3: AGVYSGRIFA Sequence ID 33, It has a CDR sequence.

[0078] A single-chain antibody comprising the CDR sequences of SEQ ID NOs. 16-21 specifically binds to W68 in association with the misfolded TDP-43 DAGWGNL (SEQ ID NO. 1). In one embodiment, the single-chain antibody comprises the CDR sequence of antibody 20.

[0079] In one embodiment, the single-chain antibody comprises a heavy-chain variable region comprising i) the amino acid sequence shown in SEQ ID NO: 104, ii) an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 104, where the CDR sequence is shown in SEQ ID NOs: 28-30, or iii) the conserved substitution amino acid sequence of i), and / or the antibody comprises a light-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 105, ii) an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 105, where the CDR sequence is shown in SEQ ID NOs: 31-33, or iii) the conserved substitution amino acid sequence of i), wherein the heavy-chain variable region amino acid sequence is encoded by the nucleotide sequence or codon degeneracy shown in SEQ ID NO: 82 or an optimized version thereof, and / or the light-chain variable region amino acid sequence is encoded by the nucleotide sequence or codon degeneracy shown in SEQ ID NO: 83 or an optimized version thereof.

[0080] In one embodiment, the single-chain antibody comprises the following: CDR-H1: GFSLSSYT Sequence ID 34; CDR-H2: IYGGIGST Sequence ID 35; CDR-H3: GRGDI Sequence ID 36; CDR-L1: QSVYKNR Sequence ID 37; CDR-L2: GAS Sequence ID 38; and CDR-L3: LGNYDCSSVDCGA Sequence ID 39, It has a CDR sequence.

[0081] A single-chain antibody comprising the CDR sequence of SEQ ID NOs. 16-21 specifically binds to W68 in association with the misfolded TDP-43 DAGWGNL (SEQ ID NO. 1). In one embodiment, the single-chain antibody comprises the CDR sequence of antibody 30.

[0082] In one embodiment, the single-chain antibody comprises a heavy-chain variable region comprising i) the amino acid sequence shown in SEQ ID NO: 106, and ii) an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 106, where the CDR sequence is shown in SEQ ID NOs: 34-36, or iii) the conserved substitution amino acid sequence of i), and / or the antibody comprises a light-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 107, and ii) an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 107, where the CDR sequence is shown in SEQ ID NOs: 37-39, or iii) the conserved substitution amino acid sequence of i), wherein the heavy-chain variable region amino acid sequence is encoded by the nucleotide sequence or codon degeneracy shown in SEQ ID NO: 84 or an optimized version thereof, and / or the light-chain variable region amino acid sequence is encoded by the nucleotide sequence or codon degeneracy shown in SEQ ID NO: 85 or an optimized version thereof.

[0083] In one embodiment, the single-chain antibody comprises the following: CDR-H1: GFSFSAYY Sequence ID 40; CDR-H2: TIPIGRT Sequence ID 41; CDR-H3: AGGPTGNSHFTL Sequence ID 42; CDR-L1: ESVYNNNQ Sequence ID 43; CDR-L2: QAS Sequence ID 44; and CDR-L3: AGYKSPTTDGIA Sequence ID 45, It has a CDR sequence.

[0084] Single-chain antibodies comprising the CDRs of SEQ ID NOs. 16-21 specifically bind to W68 in association with the misfolded TDP-43 DAGWGNL (SEQ ID NO. 1). In one embodiment, the single-chain antibody comprises the CDR sequence of antibody 38.

[0085] In one embodiment, the single-chain antibody comprises a heavy-chain variable region comprising i) the amino acid sequence shown in SEQ ID NO: 108, and ii) an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 108, where the CDR sequence is shown in SEQ ID NOs: 40-42, or iii) the conserved substitution amino acid sequence of i), and / or the antibody comprises a light-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 109, and ii) an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 109, where the CDR sequence is shown in SEQ ID NOs: 43-45, or iii) the conserved substitution amino acid sequence of i), wherein the heavy-chain variable region amino acid sequence is encoded by the nucleotide sequence or codon degeneracy shown in SEQ ID NO: 86 or an optimized version thereof, and / or the light-chain variable region amino acid sequence is encoded by the nucleotide sequence or codon degeneracy shown in SEQ ID NO: 87 or an optimized version thereof.

[0086] In one embodiment, the single-chain antibody comprises the following: CDR-H1: GFSLSSYA Sequence ID 46; CDR-H2: IYNYET Sequence ID 47; CDR-H3: ARDIFRTNTNL Sequence ID 48; CDR-L1: QSVYKNNG Sequence ID 49; CDR-L2: FTS Sequence ID 50; and CDR-L3: LGGYDCSSRVCGA Sequence ID 51, It has a CDR sequence.

[0087] A single-chain antibody comprising the CDRs of SEQ ID NOs. 16-21 specifically binds to W68 in association with the misfolded TDP-43 DAGWGNL (SEQ ID NO. 1). In one embodiment, the single-chain antibody comprises the CDR sequence of antibody 36.

[0088] In one embodiment, the single-chain antibody comprises a heavy-chain variable region comprising i) the amino acid sequence shown in SEQ ID NO: 110, ii) an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 110, where the CDR sequence is shown in SEQ ID NOs: 46-48, or iii) the conserved substitution amino acid sequence of i), and / or the antibody comprises a light-chain variable region comprising the amino acid sequence shown in SEQ ID NO: 111, ii) an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 111, where the CDR sequence is shown in SEQ ID NOs: 49-51, or iii) the conserved substitution amino acid sequence of i), wherein the heavy-chain variable region amino acid sequence is encoded by the nucleotide sequence or codon degeneracy or an optimized version thereof shown in SEQ ID NO: 88, and / or the light-chain variable region amino acid sequence is encoded by the nucleotide sequence or codon degeneracy or an optimized version thereof shown in SEQ ID NO: 89.

[0089] In one embodiment, the single-chain antibody comprises the following: CDR-H1: GFTFSSYY Sequence ID 140; CDR-H2: INTNGGST Sequence ID 141; CDR-H3: VRQNYEGAY Sequence ID 142; CDR-L1: QSIVHSNGNTY Sequence ID 143; CDR-L2: KVS Sequence ID 144; and CDR-L3: FQSSHVPWT Sequence ID 145 It has a CDR sequence.

[0090] Single-chain antibodies comprising the CDRs of SEQ ID NOs. 16-21 specifically bind to W68 in association with the misfolded TDP-43 DAGWGNL (SEQ ID NO. 1). In one embodiment, the single-chain antibody comprises the CDR sequence of antibody 3F11.

[0091] In one embodiment, the antibody comprises a heavy chain variable region comprising i) the amino acid sequence shown in SEQ ID NO: 148, ii) an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 148, where the CDR sequence is shown in SEQ ID NOs: 140-142, or iii) the conserved substitution amino acid sequence of i); and / or, the antibody comprises i) the amino acid sequence shown in SEQ ID NO: 149, ii) at least 80%, at least 85%, at least The light chain variable region comprises an amino acid sequence having 90% or at least 95% sequence identity, wherein the CDR sequence is one of those shown in SEQ ID NOs: 143-145, or the conserved substitution amino acid sequence of iii)i), and optionally the heavy chain variable region amino acid sequence is encoded by the nucleotide sequence or codon degeneracy shown in SEQ ID NO: 146 or an optimized version thereof, and / or the light chain variable region amino acid sequence is encoded by the nucleotide sequence or codon degeneracy shown in SEQ ID NO: 147 or an optimized version thereof.

[0092] ii) The amino acid sequence may also have more than 95% sequence identity. In various embodiments, the amino acid sequence has at least 96% identity with the sequence provided herein. In various embodiments, the amino acid sequence has at least 97% identity with the sequence provided herein. In various embodiments, the amino acid sequence has at least 98% identity with the sequence provided herein. In various embodiments, the amino acid sequence has at least 99% identity with the sequence provided herein.

[0093] In one embodiment, the single-chain antibody comprises a heavy-chain variable region comprising a conserved substituted amino acid sequence shown in any one of SEQ ID NOs: 98, 100, 102, 104, 106, 108, 110, 128, 138, or 148. In another embodiment, the single-chain antibody comprises a heavy-chain variable region comprising a conserved substituted amino acid sequence shown in any one of SEQ ID NOs: 99, 101, 103, 105, 107, 109, 111, 129, 139, or 149. For example, the heavy-chain variable region and / or the light-chain variable region, optionally framework regions 1, 2, and / or 3, may contain 1, 2, 3, 4, or 5 conserved amino acid substitutions.

[0094] In one embodiment, the single-chain antibody comprises a signal peptide. For example, the signal peptide may be a heavy-chain signal peptide or a light-chain signal peptide. Exemplary heavy chain signal sequences include / are MNFGLRLILLVLVLKGVLC (the native signal sequence of 2F7) (SEQ ID NO: 160), METGLRWLLLVAVLKGVQCQ (SEQ ID NO: 161), MELGLSWIFLLAILKGVQC (SEQ ID NO: 162), MELGLRWVFLVAILEGVQC (SEQ ID NO: 163), MKHLWFFLLLVAAPRWVLS (SEQ ID NO: 164), MDWTWRILFLVAAATGAHS (SEQ ID NO: 165), MDWTWRFLFVVAAATGVQS (SEQ ID NO: 166), MEFGLSWLFLVAILKGVQC (SEQ ID NO: 167), MEFGLSWVFLVALFRGVQC (SEQ ID NO: 168), and MDLLHKNMKHLWFFLLLVAAPRWVLS (SEQ ID NO: 169). Exemplary light chain signal sequences include MKLPVRLLVLMFWIPASSS (the native signal sequence of 2F7) (SEQ ID NO: 170), MDMRVPAQLLGLLLLWLSGARC (SEQ ID NO: 171), and MKYLLPTAAAGLLLLAAQPAMA (SEQ ID NO: 172). A vector construct comprising a secretible single-chain antibody can be used to target secreted TDP-43 and provide a local extracellular storage site for the antibody to minimize intercellular propagation.

[0095] As described herein, the intrabodies described herein specifically and / or selectively bind to misfolded TDP-43 but do not bind to native TDP-43. Selective binding can be measured using ELISA or surface plasmon resonance assays, as described herein. Intrabodies are antibody therapies intended to function within cells, for example, by blocking toxic proteins and preventing their transmission to healthy cells.

[0096] As described in the examples, the intrabodies are derived from mouse and rabbit monoclonal antibodies.

[0097] Intrabodies can be humanized. The humanization of antibodies derived from non-human species (e.g., mouse or rabbit) is well documented in the literature. See, for example, EP-B1 0 239400 and Carter & Merchant 1997 (Curr Opin Biotechnol 8, 449-454, 1997, which is incorporated herein by reference in its entirety). Humanized antibodies are also readily available commercially (e.g., Scotgen Limited, 2 Holly Road, Twickenham, Middlesex, Great Britain).

[0098] Humanized rodent antibodies can be readily produced by CDR grafting (Riechmann et al. Nature, 332:323-327, 1988). In this approach, six CDR loops containing the antigen-binding site of a rodent monoclonal antibody are ligated to the corresponding human framework region. Because amino acids in the framework region can affect antigen recognition, CDR grafting often results in antibodies with low affinity (Foote & Winter. J Mol Biol, 224: 487-499, 1992). To maintain antibody affinity, it is often necessary to replace specific framework residues by site-directed mutagenesis or other recombination techniques, which can be supported by computer modeling of the antigen-binding site (Co et al. J Immunol, 152: 2968-2976, 1994).

[0099] Humanized antibodies can, in some cases, be obtained by resurfacing (Pedersen et al. J Mol Biol, 235: 959-973, 1994). In this approach, only the surface residues of rodent antibodies are humanized.

[0100] In one embodiment, the single-chain antibody is a chimeric antibody, such as a humanized antibody.

[0101] The linker connecting the heavy chain variable and the light chain variable should have a length of at least 5 amino acids, preferably at least or about 10 amino acids or at least or about 15 amino acids, and should be, for example, less than about 25 amino acids. A usable linker sequence is (Gly-Gly-Gly-Gly-Ser)3 (SEQ ID NO: 180). 、This includes (Gly-Gly-Gly-Gly-Ser)4 (SEQ ID NO: 181) and GSTGGGGSGKPGSGEGGGGS (SEQ ID NO: 182). Other linkers include Glu Ser Gly Arg Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser (SEQ ID NO: 183); Glu Gly Lys Ser Ser Gly Ser Gly Ser Glu Ser Lys Ser Thr (SEQ ID NO: 184), Glu Gly Lys Ser Ser Gly Ser Gly Ser Glu Ser Lys-Ser Thr Gln (SEQ ID NO: 185), Glu Gly Lys Ser Ser Gly Ser Gly Ser Glu Ser Lys Val Asp (SEQ ID NO: 186), Gly Ser Thr Ser Gly Ser Gly Lys Ser Ser Ser Glu Gly Lys Gly (SEQ ID NO: 187), Lys Glu Ser Gly Ser Val Ser Ser Glu Gln Leu Ala Gln Phe Arg Ser Leu Asp (SEQ ID NO: 188), and Glu Ser Gly Ser Val Ser Ser Glu Glu Leu Ala Phe Arg Ser Leu Asp (SEQ ID NO: 189). If a single-chain antibody comprises a detectable label such as a fusion tag or a targeting moiety, it can be fused directly or indirectly, for example, via a tag linker or a targeting moiety linker, wherein the linker comprises or is GGGGS(SEQ ID NO: 190). A further aspect is a single-chain antibody conjugated with a detectable label. In one embodiment, the detectable label is a positron-emitting radionuclide. Positron-emitting radionuclides can be used, for example, in PET imaging. In such embodiments, the single-chain antibody may comprise a cell-permeable moiety such as a fusion cell-permeable peptide.Cell-permeable peptides may be derived from TAT, e.g., YGRKKRRQRRR (SEQ ID NO: 191), CYGRKKRRQRRRC (c-Tat) (SEQ ID NO: 192), or GDIMGEWGNEIFGAIAGFLGYGRKKRRQRRR (HA-Tat) (SEQ ID NO: 193), or others, e.g., RRRRRRRR (R8) (SEQ ID NO: 194) and CRRRRRRRRC (cR8) (SEQ ID NO: 195), RQIKIWFQNRRMKWKK (Penetratin) (SEQ ID NO: 196), and GWTLNSAGYLLGKINLKALAALAKKIL (Transportan) (SEQ ID NO: 197). The C residues on each side of the CPP sequence enable the formation and cyclization of disulfide bonds. HA refers to the hemagglutinin protein of the influenza virus. Cell-permeable peptides may contain radionuclides 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 covalently linking tracer molecules to peptides are known in the art.

[0102] Accordingly, one embodiment provides an immunocomplex comprising a single-chain antibody and a detectable label, such as a fusion tag or targeting moiety, as described herein.

[0103] The fusion tag can be a FLAG tag or a MYC tag, and the tag can be N-terminus or C-terminus, and can be conjugated directly or indirectly via a linker. In another embodiment, the single-chain antibody is conjugated to a targeting portion such as a lysosomal targeting signal.

[0104] In one embodiment, the targeting portion is a lysosome or autophagy targeting signal. For example, the lysosome targeting portion may be or may include the sequences YPTL (SEQ ID NO: 173), KSIRSGYEVM (SEQ ID NO: 174), and RWRKSHSSSYTPLSGSTYPEGRH (SEQ ID NO: 175). Other lysosome targeting sequences that can be used include the classic tyrosine-based NPXY (SEQ ID NO: 176) and YXXφ (SEQ ID NO: 177) sequences (where X can be any amino acid residue and φ is a bulky hydrophobic residue), and the dileucine-based [D / E]XXXL[L / I] (SEQ ID NO: 178) or DXXLL (SEQ ID NO: 179) sequences.

[0105] In one embodiment, the single-chain antibody may comprise an antibody and linker moiety and optionally one of the lysosomal targeting moieties of SEQ ID NOs: 203, 205, 207, 209, 213, 215, 217, 219, 223, 225, 227, 229, 233, 235, 237, 239, 243, 245, 247, 249, 253, 255, 257, and / or 259 (e.g., excluding the tag and tag-linking sequence (tag linker), and including initiation methionine if none are present).

[0106] The antibody can be prepared as described in the examples, for example, by cloning the heavy chain and light chain variable regions into an expression vector and expressing them. Once the antibody is produced (e.g., purified), a signal sequence is used to instruct its secretion.

[0107] As described herein, the single-chain antibody is suitable as an intrabody for targeting misfolded TDP-43 aggregates within cells. In such a construct, the antibody is suitably expressed within the cell, and therefore no signal sequence is incorporated.

[0108] Therefore, a further aspect is the isolated nucleic acid encoding the single-chain antibody or immunocomplex described herein.

[0109] Nucleic acids encoding single-chain antibodies and immune complexes as described herein are also provided, comprising fused heavy and light chains, for example, encoding a heavy chain comprising the CDR-H1, CDR-H2, and CDR-H3 regions described herein, and a light chain comprising the CDR-L1, CDR-L2, and CDR-L3 regions described herein, and including those encoding any of the nucleic acid sequences and amino acid sequences described herein, as described in any of Tables 3, 9, 10, 11, 12, 13, and 14.

[0110] For example, the nucleic acid sequence comprises one of the sequence numbers 76-89, 126-127, 136-137, 146-147 and / or one of the sequence numbers 202, 204, 206, 208, 212, 214, 216, 218, 222, 224, 226, 228, 232, 234, 236, 238, 242, 244, 246, 248, 252, 254, 256 and / or 258, as well as optionally a lysosome targeting portion (e.g., excluding the tag and the sequence linking the tag (i.e., the tag linker), and including the start methionine codon and stop codon if they are not present).

[0111] This disclosure also provides variants of nucleic acid sequences encoding single-chain antibodies disclosed herein.

[0112] For example, the variant includes a nucleotide sequence or a codon-degenerate or optimized sequence that hybridizes to a nucleic acid sequence encoding an antibody disclosed herein under at least moderately stringent hybridization conditions. In another embodiment, the variant nucleic acid sequence has at least 70%, most preferably at least 80%, more preferably at least 90%, and most preferably at least 95% sequence identity with respect to any nucleic acid sequence comprising one of SEQ ID NOs. 76-89, SEQ ID NOs. 126-127, SEQ ID NOs. 136-137, SEQ ID NOs. 146-147 and / or SEQ ID NOs. 202, 204, 206, 208, 212, 214, 216, 218, 222, 224, 226, 228, 232, 234, 236, 238, 242, 244, 246, 248, 252, 254, 256, and / or 258 antibody and linker moieties, and optionally a lysosome targeting moiety. The variant sequence encodes, for example, antibodies having a conserved change outside the CDR sequence and a codon-optimized version of the antibody sequence described herein, optimized for expression in human cells, for example.

[0113] Another aspect is an expression cassette or vector comprising nucleic acids disclosed herein. An expression cassette may comprise, for example, a nucleic acid encoding a single-chain antibody and a linker, and a regulatory sequence such as a promoter operably linked to the nucleic acid. In one embodiment, the vector is an isolation vector.

[0114] The vector may be any vector, preferably an expression vector suitable for producing the single-chain antibodies described herein. In one embodiment, the vector is suitable for expressing, for example, a single-chain antibody (e.g., an intrabody).

[0115] The nucleic acid molecule may be incorporated in a known manner into a suitable expression vector that guarantees protein expression, for example, as described in the examples.

[0116] Molecular cloning techniques known in the art can be used to construct the vector. In one embodiment, the expression vector and insert are digested with a suitable restriction enzyme and ligated with T4 polymerase. The ligation reaction product is converted into competent cells. Individual colonies are harvested, cultured overnight, and the DNA is purified for restriction digestion for diagnostic purposes.

[0117] Possible expression vectors include, but are not limited to, cosmids, plasmids, or modified viruses (e.g., replication-deficient retroviruses, adenoviruses, and adeno-associated viruses, including lentiviral vectors).

[0118] In one embodiment, the vector is an adeno-associated virus (e.g., AAV serotype 9) that can be transduced into nerve cells.

[0119] The vector may include a suitable regulatory sequence.

[0120] Suitable regulatory sequences may originate from a variety of sources, including bacterial, fungal, viral, mammalian, or insect genes. Examples of such regulatory sequences include transcription promoters and enhancers or RNA polymerase binding sequences, and ribosome binding sequences (including translation initiation signals). Furthermore, depending on the cells being transfected / infected / transduced and the vector used, other sequences, such as origins of replication, further DNA restriction sites, enhancers, and sequences conferring transcriptional inducibility, may be incorporated into the expression vector. In some embodiments, the regulatory sequences direct or increase expression in nerve tissue and / or cells. In some embodiments, the vector is a viral vector. The recombinant expression vector may also contain marker genes to facilitate the selection of host cells transformed, infected, or transfected with the vector for expressing the antibodies described herein. The recombinant expression vector may also contain other expression cassettes encoding fusion regions (e.g., for creating antibody "fusion proteins"), such as tags and labels described herein, which can aid in detection.

[0121] Nucleic acids and vectors can be used to deliver single-chain antibodies to cells, for example, target cells.

[0122] A wide range of approaches can be used to transduce cells, including viral vectors, "naked" DNA, DNA in lipids or other nanoparticles, adjuvant-assisted DNA, and gene guns. For example, retroviral vectors such as lentiviral vectors can also be used to transduce intrabodies into cells. Other vector systems useful for carrying out the present invention include adenovirus vectors and adeno-associated virus-based vectors.

[0123] In another aspect, cells are also provided that recombinantly express the single-chain antibodies described herein (e.g., recombinant cells) and / or comprise nucleic acids, expression cassettes, or vectors encoding the single-chain antibodies described herein. In one embodiment, the cells are isolated cells that express the antibodies described herein or comprise nucleic acids, expression cassettes, or vectors disclosed herein.

[0124] In one embodiment, the cells are cells capable of producing the antibodies described herein.

[0125] IV. Composition Further aspects include compositions comprising isolated antibodies, immune complexes, nucleic acids, expression cassettes, or vectors as described herein. Compositions comprising two or more antibodies, immune complexes, nucleic acids, or vectors as described herein are also provided.

[0126] In one embodiment, the composition comprises two or more isolated antibodies (for example, two or more different antibodies).

[0127] In another embodiment, the composition comprises two or more immune complexes (for example, two or more different immune complexes).

[0128] In another embodiment, the composition comprises two or more nucleic acids (for example, two or more different nucleic acids).

[0129] In another embodiment, the composition comprises two or more expression cassettes (for example, two or more different expression cassettes).

[0130] In another embodiment, the composition comprises two or more vectors (for example, two or more different vectors).

[0131] In one embodiment, the two or more are two, for example, two antibodies or two immune complexes. In one embodiment, the two or more are three. In one embodiment, the two or more are four. In one embodiment, the two or more are five. In another embodiment, the two or more are six. In one embodiment, the two or more are seven.

[0132] Another aspect is a composition comprising the single-chain antibodies described herein. Compositions comprising two or more single-chain antibodies described herein are also provided.

[0133] In one embodiment, the composition comprises a single-chain antibody comprising a CDR of antibody 2F7.

[0134] In another embodiment, the composition comprises a single-chain antibody comprising a CDR of antibody 1H3-1K3.

[0135] In another embodiment, the composition comprises a single-chain antibody comprising a CDR of antibody 28H3-28K1.

[0136] In another embodiment, the composition comprises a single-chain antibody comprising a CDR of antibody 14H1-14K2.

[0137] In another embodiment, the composition comprises a single-chain antibody comprising the CDR of antibody 17.

[0138] In another embodiment, the composition comprises a single-chain antibody comprising a CDR of antibody 20.

[0139] In another embodiment, the composition comprises a single-chain antibody comprising a CDR of antibody 30.

[0140] In another embodiment, the composition comprises a single-chain antibody comprising the CDR of antibody 38.

[0141] In another embodiment, the composition comprises a single-chain antibody comprising the CDR of antibody 36.

[0142] In another embodiment, the composition comprises a single-chain antibody comprising a CDR of antibody 3F1.

[0143] The single-chain antibody may include a lysosome-targeting moiety.

[0144] In one embodiment, the composition comprises a single-chain antibody selected from the group consisting of LYS-2F7, LYS-28H3-28K1, LYS-14H1-14K2, YPTL-14H1-14K2, MYCH15L-2F7, MYCL15H-1H3-1K3, and MYCH20L-14H1-14K2.

[0145] In one embodiment, the composition comprises a single-chain antibody selected from the group consisting of LYS-2F7 and MYCH15L-2F7.

[0146] In one embodiment, the composition comprises a single-chain antibody selected from the group consisting of LYS-14H1-14K2, YPTL-14H1-14K2, and MYCH20L-14H1-14K2.

[0147] In another embodiment, the composition comprises two or more single-chain antibodies selected from the group consisting of LYS-2F7, LYS-28H3-28K1, LYS-14H1-14K2, YPTL-14H1-14K2, MYCH15L-2F7, MYCL15H-1H3-1K3, and MYCH20L-14H1-14K2.

[0148] In one embodiment, the two or more are two single-chain antibodies. In one embodiment, the two or more are three. In one embodiment, the two or more are four. In one embodiment, the two or more are five. In one embodiment, the two or more are six. In one embodiment, the two or more are seven.

[0149] In one embodiment, the composition comprises a diluent. Suitable diluents for nucleic acids and vectors include, but are not limited to, water, saline solution, and ethanol.

[0150] The composition may comprise lipid particles such as liposomes, nanoparticles, or nanosomes for assisting the delivery of nucleic acids and / or vectors.

[0151] Suitable diluents for polypeptides and / or cells containing antibodies or their fragments include, but are not limited to, physiological saline, pH buffer, and glycerol solution or other solutions suitable for freezing polypeptides and / or cells.

[0152] In one embodiment, the composition comprises a nucleic acid or vector as described herein. In another embodiment, the composition comprises an antibody or a portion thereof and a diluent as described herein. In one embodiment, the composition is a sterile composition.

[0153] The composition may be formulated for administration into the subarachnoid space, intracerebral parenchyma, or intraventricular space.

[0154] In one embodiment, the composition comprises a pharmaceutically acceptable carrier, diluent, and / or excipient. In one embodiment, the composition is for the methods described herein, for example, to target misfolded TDP-43.

[0155] In one embodiment, the composition comprises a pharmaceutically acceptable carrier, diluent, and / or excipient. In one embodiment, the composition is a pharmaceutical composition for the method described herein, for example, for treating a subject that requires it, such as a subject having TDP-43 proteinosis.

[0156] The aforementioned composition may comprise one or more antibodies as described herein.

[0157] VI. Method This specification includes methods for producing single-chain antibodies, nucleic acids, and vectors.

[0158] In particular, a method is provided for producing a single-chain antibody selective to W68 in association with a single-chain antibody, such as DAGWGNL (SEQ ID NO: 1). The CDR can be transplanted onto a single-chain antibody scaffold, or the heavy and light chain variable regions can be amplified and cloned into a vector.

[0159] Vectors comprising single-chain antibodies, and optionally intrabodies, can be prepared by several methods. Molecular cloning techniques known in the art can be used to construct the vectors. As described in the examples, scFv nucleic acids were constructed in various forms, such as the FLAG tag-VH-linker-VL-Lys linker-lysosome targeting sequence format. The antibody heavy chain variable domain (VH) and light chain variable domain (VL) may consist of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, respectively. The linkers for linking the VH and VL sequences and the linker for linking the lysosome targeting portion may consist of tandem repeats of 3 and 1 GGGGS, respectively. scFv nucleic acids can be synthesized by sequentially linking individual phosphoramidite monomers adjacent to restriction sites such as the 5'-NheI / HindIII-3' restriction sequence using solid-phase phosphoramidite chemistry in a DNA synthesizer. The obtained single-stranded scFv gene insert can be amplified by standard PCR to generate a double-stranded insert, which can then be cloned into an NheI / HindIII restriction enzyme digestion vector, such as the pcDNA3.1(-) vector.

[0160] Other methods can also be used. For example, mRNA can be isolated from hybridoma cell lines using an mRNA isolation kit (Qiagen, Chatsworth, California). cDNA can be synthesized by oligo-dT priming using Superscript First Strand, catalog number 12371-019 (Invitrogen, Carlsbad, California), for example, according to the manufacturer's instructions. The variable regions of the heavy chain (VH) and light chain (Vκ) can be amplified separately from the first-strand cDNA by using a mixture of universal polymerase chain reaction (PCR) primers and Platinum Pfx DNA polymerase (Invitrogen). The PCR products of the heavy chain and light chain can be cleaved with restriction enzymes such as PstI / BstEII and SacI / XhoI, respectively, and purified on an agarose gel. The cDNA inserts corresponding to VL and VH can be cloned into a pBZUT7 vector, for example, and sequenced. The VH and VL domains can be assembled by PCR and ligated together to produce full-length scFv nucleic acid. The scFv nucleic acid can then be subcloned upstream of the Myc tag. The construct can also be designed to include one or more parts, such as a signal sequence for efficient secretion, a tag such as a c-myc epitope or FLAG for easier detection, or a targeting region such as a lysosomal signal sequence or autophagy signal sequence.

[0161] The structure of the obtained single-chain antibody is (HC FR1-HC CDR1-HC FR2-HC CDR2-HC FR3-HC CDR3-HC FR4) VH -Linker-(LC FR1-LC CDR1-LC FR2-LC CDR2-LC FR3-LC CDR3-LC FR4) VL-Optionally a Lys linker -Optionally a targeting region such as a lysosome targeting sequence, where HC FR1 is the heavy chain framework 1 region, HC FR2 is the heavy chain framework 2 region, HC FR3 is the heavy chain framework 3 region, HC FR4 is the heavy chain framework 4 region, LC FR1 is the light chain framework 1 region, LC FR2 is the light chain framework 2 region, LC FR3 is the light chain framework 3 region, and LC FR4 is the light chain framework 4 region.

[0162] Furthermore, methods for detecting misfolded TDP-43 in cells or in subjects are also provided.

[0163] The labeled single-chain antibodies described herein (produced by adding a secretory signal) may also be administered to a subject to detect the location of misfolded TDP-43. This measurement may be performed, for example, by immunofluorescence or a PET tracer. The method may also include co-localization staining, such as pan-TDP-43 staining.

[0164] A method for reducing intracellular misfolded TDP-43 levels is also provided, comprising administering a nucleic acid, vector, or composition comprising the nucleic acid or vector described herein to a subject in need, for example, a subject suspected of having, at risk of developing, or diagnosed with TDP-43 proteinopathy.

[0165] For example, a method for treating a subject having TDP-43 proteinopathy is also provided, comprising administering a nucleic acid, an expression cassette, a vector, a single-chain antibody, or a composition comprising the nucleic acid, expression cassette or vector or single-chain antibody described herein, to a subject in need thereof.

[0166] In one embodiment, the TDP-43 proteinopathy is selected from amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD-TDP), primary lateral sclerosis, progressive muscular atrophy, and limbic-dominant senile TDP-43 encephalopathy (LATE).

[0167] In some embodiments, nucleic acids, vectors, or compositions are administered to a subject in combination with another TDP-43 proteinopathy treatment. Other TDP-43 proteinopathy treatments include, but are not limited to, treatments for ALS, such as riluzole (Rilutek or Tiglutik®), edaravone (Radicava®), and NuDexta® (a combination of dextromethorphan and quinidine).

[0168] The nucleic acid or vector may be included in the compositions described herein, for example, in combination with a pharmaceutically acceptable carrier, diluent and / or excipient, and may be formulated, for example, in nanoparticles or nanosomes to assist in the delivery of the nucleic acid or vector.

[0169] The compositions, antibodies, immune complexes, nucleic acids, and vectors described herein may be administered, for example, by parenteral administration, intravenous administration, subcutaneous administration, intramuscular administration, intracranial administration, intraventricular administration, subarachnoid administration, intraorbital administration, ocular administration, intraspinal administration, intracisional administration, intraperitoneal administration, intranasal administration, aerosol administration, or oral administration.

[0170] Several other embodiments involve the co-administration of the compositions, antibodies, immune complexes, nucleic acids, and vectors described herein with bioactive molecules known to facilitate transport across the blood-brain barrier.

[0171] In certain embodiments, methods for administering the compositions, antibodies, immune complexes, nucleic acids, and vectors described herein via the blood-brain barrier are also intended, such as those directed to transiently increase blood-brain barrier permeability, as described in U.S. Patent No. 7,012,061, “Method for increasing blood-brain barrier permeability” (which is incorporated herein by reference).

[0172] Viral delivery of compositions and / or nucleic acids described herein for the expression of one or more antibodies described herein in subjects or cells requiring such expression is also contemplated herein. One aspect includes a method for treating a subject having TDP-43 proteinosis, comprising administering to a subject requiring such expression, optionally in combination with another TDP-43 proteinosis treatment, an effective amount of a vectorized antibody of the disclosure herein, or a composition comprising the vectorized antibody, optionally in combination with another TDP-43 proteinosis treatment. In one embodiment, the vectorized antibody is a viral vector comprising a nucleic acid encoding the antibody described herein. In one embodiment, the method is for intracellular expression of an intrabody in a subject requiring such expression. The intrabody can, for example, inhibit intracellular misfolded TDP-43 aggregation or promote the clearance of misfolded aggregates.

[0173] For example, viral vectors such as adeno-associated viruses (AAV, e.g., AAV9) and lentiviral vectors can be used. Non-viral vectors can also be used. In certain embodiments, the nucleic acids, vectors, or compositions may be injected intraventricularly or subarachnoidally. In other embodiments, the nucleic acids, vectors, or compositions may be administered intravenously, subcutaneously, or intramuscularly, for example, using a storage facility for the sustained production of secreted single-chain antibodies.

[0174] The above disclosures describe the application in general terms. A more complete understanding can be obtained by referring to the following specific examples. These examples are described for illustrative purposes only and are not intended to limit the scope of the application. Modifications of form and substitutions of equivalents are contemplated where circumstances may suggest or provide convenience. Certain terms are used herein, but such terms are intended to be descriptive and not limiting.

[0175] The following examples, not limited to this disclosure, are illustrative: [Examples]

[0176] Example 1 Detection of misfolded TDP-43 Antibodies were produced in rabbits using an immunogen containing SEQ ID NO: 1. The antibodies were affinity-purified. The affinity-purified antibodies were tested for their ability to bind to native and misfolded TDP-43 polypeptides in cell transfection assays using immunocytochemistry.

[0177] result HEK293 cells transfected with an HA-tagged TDP-43 construct containing a triple missensantem mutation in nuclear localization signaling exhibit cytoplasmic TDP-43 aggregates detected by HA antibody. HA-positive aggregates are also evident in ΔNLS-TDP-43 W68S transfected cells. Affinity-purified polyclonal antibodies recognize aggregates in ΔNLS-TDP-43 transfected cells that contain tryptophan 68 residues (Trp68), but do not show the same reactivity when Trp68 is mutated to serine. Therefore, the anti-DAGWGNL (SEQ ID NO: 1) polyclonal rabbit antibody exhibits selectivity for misfolded TDP-43 NTDs containing Trp68 residues.

[0178] When HA-tagged wild-type TDP-43 is overexpressed without altering nuclear localization signals, it generally localizes to the nucleus, and nuclear TDP-43 does not bind to GS240 or GS243 affinity purified serum. Interestingly, GS240 and GS243 still bind to wild-type TDP-43 when present in the cytoplasm of these cells, thereby suggesting that the N-terminal ubiquitin-like domain (NTD) can be misfolded in WTTDP-43 when mislocalized to the cytoplasm.

[0179] GS240 and GS243 polyclonal antibodies exhibit minimal reactivity in the absence of TDP-43 aggregates. The reactivity of both antibodies is selective for misfolded TDP-43 in cytoplasmic aggregates and requires the presence of Trp68.

[0180] Example 2 Production of mouse monoclonal antibodies Mouse monoclonal antibodies were produced using peptides containing DAGWGNL (SEQ ID NO: 1), e.g., DAGWGNLc (SEQ ID NO: 9), linked to KLH. Hybridomas were developed and analyzed. Positive clones were identified and their isotypes were determined.

[0181] Example 3 Production of rabbit monoclonal antibodies

[0182] The DAGWGNL (SEQ ID NO: 1) peptide (peptide-KLH), conjugated to KLH via a C-terminal cysteine, was used in rabbit immunization to produce B cells that secrete monoclonal antibodies specific to the TDP-43 peptide. Based on indirect ELISA analysis of isolated B cells, B cells were selected for RNA isolation and recombinant plasmid DNA production. Functional antigen-binding recombinant mAbs were transfected and expressed to produce purified mAbs.

[0183] Example 4 Cloning and sequencing of monoclonal antibodies Mouse mAb cloning We identified and sequenced the variable regions of heavy chain immunoglobulin and light chain immunoglobulin genes in several mouse hybridoma clones.

[0184] The sequences are shown in Tables 2, 3, and 4 below, with the CDR1, CDR2, and CDR3 regions of the heavy and light chains underlined and bolded in Tables 3 and 4.

[0185] Cloning of rabbit mAbs The heavy and light chain variable regions, obtained by isolating antibody RNA and constructing recombinant plasmid DNA, were cloned into separate mammalian expression vectors containing rabbit heavy chain and kappa constant region.

[0186] The top recombinant mAb (a DNA construct containing one heavy chain and one light chain) was sequenced as shown in Tables 2, 3, and 4 below, and the CDR1, CDR2, and CDR3 regions of the heavy and light chains are underlined and bolded in Tables 3 and 4.

[0187] [Table 2]

[0188] [Table 3]

[0189] TIFF2026108674000005.tif205159

[0190] TIFF2026108674000006.tif178159

[0191] [Table 4]

[0192] TIFF2026108674000008.tif77159

[0193] Example 5 Direct binding assay The binding of antiserum, hybridoma supernatant, or purified antibody to peptides (conjugated to BSA) can be investigated by surface plasmon resonance using a Biacore® 3000 instrument (GE Healthcare).

[0194] Binding analysis is performed using antigens immobilized in high-density (at least 1000 response units (RU)) flow cells. Dilutions of selected clones are sequentially injected into surfaces for binding evaluation.

[0195] For affinity kinetics and specificity analysis, peptides comprising DAQWGNL (SEQ ID NO: 1) or GWG and conjugated to BSA are immobilized at low density (50–100 RU) in adjacent flow cells. Subsequently, 2-fold serial dilutions of selected clones (4.7 nM–75 nM) are injected sequentially into the surface at 60 μl / min for 3 minutes, followed by the dissociation phase. After double reference subtraction, the sensorgram is fitted to a Langmuir 1:1 binding model. Up to three separate analyses are performed over three consecutive days using the same sensor chip and conditions.

[0196] Binding analysis can also be performed using the Molecular Affinity Screening System (MASS-2) (Sierra Sensors GmbH, Hamburg, Germany). MASS-2 is a surface plasmon resonance (SPR) imaging biosensor that uses high-intensity laser light and high-speed optical scanning to monitor binding interactions in real time. The peptide-BSA conjugate is covalently immobilized to separate flow cells of a High Amine Capacity (HAC) sensor tip using standard amine coupling chemistry, blocking unreacted sites. Adjacent flow cells are similarly immobilized with BSA as a reference surface.

[0197] Monoclonal antibody binding kinetics Surface plasmon resonance (SPR) analysis was used to measure the binding kinetics of monoclonal antibodies to peptide epitopes. Peptides conjugated to bovine serum albumin (BSA) were immobilized at a very low density (approximately 50 RU) in the flow cell of the sensor chip. Purified mouse or rabbit monoclonal antibodies, diluted fourfold from 31.25 nM to 0.24 nM, were sequentially injected onto the surface for approximately 5 minutes, followed by dissociation in buffer and surface regeneration. Binding parameters were calculated using kinetic curve fitting and the Langmuir 1:1 interaction model. Both mouse and rabbit monoclonal antibodies showed sub-nanomolecular high affinity to the peptide epitopes, as shown in Table 5 below. Rabbit monoclonal antibodies showed higher affinity than mouse monoclonal antibodies (10 -10 Compared to the nM range, it has a much greater affinity (10 -11 The nM range was shown.

[0198] [Table 5]

[0199] Example 6 The epitope is not recognized in the naturally folded TDP N-terminal domain, but is recognized by antibodies in the denatured TDP N-terminal domain. The N-terminal domain (residues 1-80) of TDP-43 was expressed from a plasmid in E. coli and purified as previously described (Wang et al. 2018 EMBO). Native PAGE was performed using the Novex Bis-Tris system for Western blot analysis, according to the manufacturer's specifications. Denatured SDS-PAGE was also performed using the Novex Bis-Tris system, according to the manufacturer's specifications. The blots were blocked with 5% milk powder in TBST and then incubated overnight at 4°C with purified rabbit polyclonal antibody GS240. For detection with ChemiDoc MP (Biorad, USA), donkey anti-rabbit IgG HRP-labeled secondary antibody (GE Healthcare Life Sciences, USA) was used. SuperSignal West Femto (Thermo Scientific, USA) substrate was used according to the manufacturer's instructions. The antibody recognized and stained only denatured TDP-43 NTDs on the SDS-PAGE gel, but not TDP-43 NTDs on the native PAGE gel, thus confirming that the epitope is inaccessible in naturally folded TDP-43 and becomes exposed upon misfolding. Size exclusion chromatography (SEC) showed that native or denatured TDP-43 NTDs remained monomeric. Molecular weight markers are overlaid for reference.

[0200] Example 7 Immunohistochemistry of patient samples Brain and spinal cord samples were obtained from the Netherlands Brain Bank, processed, and stained. Sections (8 μM thick) from formalin-fixed, paraffin-embedded tissues were deparaffinized and then immersed in 0.3% H2O2 in phosphate-buffered saline (PBS) for 30 minutes to quench endogenous peroxidase activity. Formalin fixation forms protein crosslinks that can shield antigen sites in tissue specimens. To break these protein crosslinks, slides were pre-treated with heat and Tris-EDTA (pH 9). Primary test antibodies were incubated overnight at 4°C at dilutions of 1:4000 (rabbit polyclonal GS240 antibody, 3E8 mouse monoclonal antibody) or 1:2000 (mouse monoclonal antibodies 3F11 and 2F7). Secondary EnVision HRP conjugate goat anti-rabbit / mouse antibody (EV-GαM HRP, DAKO) was added at room temperature for 30 minutes, followed by the addition of 3,3-diaminobenzidine chromogen (DAKO) for 10 minutes. Sections were counterstained with hematoxylin.

[0201] Brain sections from patients with frontotemporal lobar degeneration (FTLD) type B were stained. Rabbit polyclonal GS240 antibody showed staining of pathogenic TDP-43 in both white matter (WM) and gray matter (GM). 3F11 and 3E8 monoclonal antibodies primarily detected pathogenic TDP-43 in GM, while 2F7 monoclonal antibody showed staining in both WM and GM. Rabbit polyclonal GS240 antibody was also tested in ALS spinal cord sections and showed positivity in motor neurons and surrounding tissue. These results indicate that the antibody can recognize the pathogenic TDP-43 epitope shown in situ in affected tissue.

[0202] Example 8 Representative staining of transfected HEK293 cells with monoclonal anti-TDP43 antibody As described in Example 1, in a cell transfection assay using immunohistochemistry, mouse monoclonal antibodies and rabbit monoclonal antibodies were tested for selective binding to misfolded TDP-43. Briefly, HEK293FT cells were transfected with a plasmid encoding HA-tagged ΔNLS-TDP43 (which forms cytoplasmic aggregates) or HA-tagged wild-type (WT) TDP-43 (expressed in the nucleus) or an empty vector. Anti-TDP-43 antibodies were diluted to 10 μg / ml for staining, and fluorescent-labeled anti-rabbit IgG or anti-mouse IgG was used as a secondary antibody for detection. Chicken anti-HA-tag antibody, followed by a fluorescent-labeled anti-chicken secondary antibody, was used for the detection of transfected TDP-43. Transfected ΔNLS-TDP43 was mislocalized to the cytoplasm, where it formed aggregates that were easily detected by staining with the anti-HA-tag antibody. The 2F7 antibody recognized the same aggregates, as confirmed by the co-localization of the two staining signals in the merged image. In contrast, transfected WT TDP-43 detected in the nucleus by the HA-tag antibody was not stained by the 2F7 antibody. As expected, cells transfected with the empty vector showed no HA-tag staining. They showed no staining with the 2F7 antibody, indicating that 2F7 could not recognize endogenous nuclear WT TDP-43 either.

[0203] Similar results were obtained with the other antibodies tested, and they are summarized in Table 6. This pattern of staining by the antibodies tested indicates their selectivity for misfolded pathogenic aggregates of TDP-43 compared to WT TDP-43.

[0204]

Table 6

[0205] Example 9 Immunocytochemistry of physiological stress granules To determine whether monoclonal antibodies against misfolded TDP-43 reacted with physiological stress granules, stressed HEK293FT cells were stained. Briefly, HEK293FT cells were stressed by exposure to 1 nM sodium arsenite for 60 minutes. The cells were then stained with a fluorescently labeled antibody against the stress granule marker G3BP1 or a 10 μg / ml monoclonal anti-TDP-43 test antibody and detected with a labeled secondary antibody. Similar results were obtained with all test antibodies except one, which are included in Table 7 below. The stressed cells showed a large number of punctate staining of G3BP1+ stress granules in the cytoplasm. In comparison, the same cells stained with the 3F11 antibody did not show the presence of cytoplasmic granules at the position of G3BP1 staining, indicating that 3F11 did not react with stress granules in these cells. The lack of binding of the tested antibodies to TDP-43 in physiological stress granules suggests that they are unlikely to interfere with the protective function of these stress granules.

[0206]

Table 7

[0207] Example 10 Characterization of the selected mAb Monoclonal antibodies were tested for the recognition of cytoplasmic aggregates formed in HEK293FT cells transfected with HA-tagged ΔNLS-TDP-43 (W68S), compared to HA-tagged ΔNLS-TDP-43 (W68S), in which tryptophan 68 (Trp68) is mutated to serine. An empty vector was used as a control. Cells were stained with either 2 μg / ml TDP-43 rabbit monoclonal antibody 28, 1 μg / ml mouse panTDP-43, or 0.5 μg / ml chicken anti-HA tag. Fluorescently labeled secondary antibodies Alexa Fluor 488-anti-rabbit, Alexa Fluor 647-anti-mouse, and Alexa Fluor 568-anti-chicken were used for detection of the conjugated primary antibody. Nuclei were stained with Hoechst 33342 dye. In cells transfected with HA-tagged ΔNLS-TDP-43, the 28 antibody stained cytoplasmic aggregates co-localized with HA-positive aggregates of misfolded ΔNLS-TDP-43. In contrast, no staining was observed in cytoplasmic aggregates formed by ΔNLS-TDP-43-W68S lacking Trp68. As expected, cells transfected with an empty vector showed no HA-tagged staining at all. They also showed no staining with 28H3-28K1, confirming that the antibody does not recognize endogenous nuclear WT TDP-43. The pan-TDP-43 antibody recognized cytoplasmic aggregates formed by both transfected TDP-43 and endogenous nuclear TDP-43. Similar results were obtained with most of the other antibodies tested, which are summarized in Table 8. As observed with the polyclonal rabbit antibody, this staining pattern with the tested monoclonal antibodies indicates their selectivity for misfolded TDP-43 NTDs containing solvent-exposed Trp68 residues. Clone 20, bound to several aggregates of ΔNLS-TDP-43-W68S, bound to physiological stress granules.

[0208] [Table 8]

[0209] Example 11 Antibody blockade of misfolded TDP-43 transmission in HEK293 cells Donor HEK293 cells were transiently transfected with HA-ΔNLSTDP-43, a TDP-43 HA-tagged nuclear localization signal-deficient mutant, to express misfolded TDP-43. 48 hours after transfection, the condition medium was collected from the donor cells and centrifuged at 1,000 g for 10 minutes to remove suspension cell debris. The clarified condition medium was incubated with 30 ug / ml of each individual TDP-43 misfolding-specific antibody or control mouse IgG1 (Biogen) at room temperature for 1 hour with constant rotation, after which naive recipient HEK293 cells were added. The antibodies tested included three mouse monoclonal antibodies against the N-terminal epitope of TDP-43 (3F11, 2F7, 3E8) and an antibody against the structural RRM1 epitope (PCT CA / 2018 / 050634, published as WO2018 / 218352) (9C5). After 48 hours of incubation, the recipient cell medium was removed, cells were washed twice with cold PBS, and lysed in 2% SDS. Protein concentrations were measured using a BCA assay. 25 ug of lysate was separated on a 10% NuPage gel (Thermo), transferred to a PVDF membrane, and then Western blotting was performed using antibodies against HA tag (Abcam, rabbit, 1:1000) or GAPDH (Thermo, mouse, 1:50K) as a loading control. HA and GAPDH immunoreactivity was detected using the ChemiDoc Imaging System, and intensity was quantified using Image Lab.

[0210] Donor cells transfected with HA-dNLS-TDP43 contained large amounts of HA-tagged TDP-43. Naive recipient cells incubated with donor cell supernatant treated with control mouse IgG1 (mIgG1) contained detectable amounts of HA-tagged TDP-43, indicating extracellular transmission of misfolded aggregates of the HA-dNLS-TDP-43 protein from donor cells to recipient cells. Recipient cells incubated with donor cell supernatant pretreated with a misfolding-specific TDP-43 antibody contained relatively small amounts of HA-tagged TDP-43, indicating inhibition of transmission by the antibody. As a negative control, recipient cells incubated with supernatant from untransfected donor cells contained no detectable amounts of HA-tagged TDP-43. For each antibody, the HA-tagged signal was first normalized relative to the GAPDH signal (HA intensity / GAPDH intensity), and the value obtained for the test antibody was divided by the value obtained for control mIgG1. Compared to control mIgG1, all antibodies tested inhibited the propagation of misfolded HA-dNLS-TDP-43 from donor cell supernatant, resulting in low levels of HA-tagged TDP-43 in recipient cells.

[0211] Example 12 Vector generation We designed various single-chain variable fragment (scFv) intrabody constructs using short heavy chain variable regions from antibodies against misfolded TDP-43. Variable heavy chains (scFv) from antibodies 2F7, 1H3-1K3, 28H3-28K1, and 14H1-14K2 that bind to an epitope containing at least W68 in association with DAGWGNL (SEQ ID NO: 1) of misfolded TDP-43 were also used. H ) region and variable light chain (V L The ) regions were linked using various amino acid linkers. H -Linker-V L or V L -Linker-V HThe scFv peptide was constructed using one of the common sequences. A FLAG or MYC tag region was ligated to the scFv to detect intrabody expression and localization, as in Example 13. The lysosome targeting region YPTL was also ligated to the scFv peptide of several intrabodies.

[0212] The constructions of 2F7, 1H3-1K3, 28H3-28K1, and 14H1-14K2 are described herein.

[0213] For each of the antibodies with the following structures, six different types of single-chain antibody constructs were generated: (a) FLAG Tag-V H -(G4S)3-Linker-V L -G4S Linker - Lysosome Targeting Tag Construct; (b)V H -(G4S)3-Linker-V L -FLAG tag-YPTL construct; (c)V via 15 amino acid linker-MYC tag construct H V connected to L ; (d) V via 15 amino acid linker-MYC tag construct L V connected to H ; (e) V via 20 amino acid linker-MYC tag construct H V connected to L and (f)V via 20 amino acid linker-MYC tag construct L V connected to H .

[0214] TDP-43 ScFv vector constructs were generated by cloning the polynucleotide sequences of each scFv construct into expression plasmids such as pcDNA3.1 vectors, e.g., pcDNA3.1(+) or pcDNA3.1(-).

[0215] The vector and scFv insert were digested with NheI and HindIII restriction enzymes. After enzymatic digestion, the vector and insert were ligated with T4 polymerase. 1 - 2 μL of the ligation reaction was transformed into TOP10 competent cells. Individual bacterial colonies were picked and overnight cultures were grown for DNA purification. After DNA purification, diagnostic restriction digests of the purified DNA with NheI and HindIII were performed. The final plasmid was sequenced.

[0216] a) N-terminal FLAG tag lysosome construct ScFv constructs comprising a lysosome targeting tag are identified as LYS - 2F7, LYS - 1H3 - 1K3, LYS - 28H3 - 28K1, and LYS - 14H1 - 14K2. The amino acid and polynucleotide sequences of the lysosome targeting constructs LYS - 2F7, LYS - 1H3 - 1K3, LYS - 28H3 - 28K1, and LYS - 14H1 - 14K2 are shown in Table 9 below. The V H and V L regions of LYS - 2F7, LYS - 1H3 - 1K3, LYS - 28H3 - 28K1, and LYS - 14H1 - 14K2 are based on the corresponding V H and V L regions from antibodies 2F7, 1H3 - 1K3, 28H3 - 28K1, and 14H1 - 14K2, respectively.

[0217]

Table 9

[0218] TIFF2026108674000014.tif147159

[0219] b) C-terminal FLAG YPTL construct YPTLs comprising the constructs in group b) have been identified as YPTL-2F7, YPTL-1H3-1K3, YPTL-28H3-28K1, and YPTL-14H1-14K2. The amino acid and polynucleotide sequences of the constructs YPTL-2F7, YPTL-1H3-1K3, YPTL-28H3-28K1, and YPTL-14H1-14K2 are shown in Table 10 below. H and V L Each region corresponds to the V from antibodies 2F7, 1H3-1K3, 28H3-28K1, and 14H1-14K2, respectively. H and V L It is based on domain.

[0220] [Table 10]

[0221] TIFF2026108674000016.tif166159

[0222] c) V L V via a 15-amino acid linker H The C-terminal MYC tag construct linked to: V L V via a 15-amino acid linker H The MYC tag constructs linked to are MYCL15H-2F7, MYCL15H-1H3-1K3, MYCL15H-28H3-28K1, and MYCL15H-14H1-14K2. These constructs are V L -15 amino acid ScFv linker sequence -V H-3 amino acid MYC linker sequence - has a common sequence of MYC tag. The amino acid and polynucleotide sequences of constructs MYCL15H-2F7, MYCL15H-1H3-1K3, MYCL15H-28H3-28K1, and MYCL15H-14H1-14K2 are shown in Table 11 below. V of MYCL15H-2F7, MYCL15H-1H3-1K3, MYCL15H-28H3-28K1, and MYCL15H-14H1-14K2 H and V L Each region corresponds to the V from antibodies 2F7, 1H3-1K3, 28H3-28K1, and 14H1-14K2, respectively. H and V L It is based on domain.

[0223] [Table 11]

[0224] TIFF2026108674000018.tif154159

[0225] d) V H V via a 15-amino acid linker L MYC structures connected to V H V via a 15-amino acid linker L The MYC structures connected to it are MYCH15L-2F7, MYCH15L-1H3-1K3, MYCH15L-28H3-28K1, and MYCH15L-14H1-14K2. All of these structures are V H -15 amino acid ScFv linker sequence -V L -3 amino acid MYC linker sequence - has a common sequence of MYC tag. The amino acid and polynucleotide sequences of constructs MYCH15L-2F7, MYCH15L-1H3-1K3, MYCH15L-28H3-28K1, and MYCH15L-14H1-14K2 are shown in Table 12 below. V of MYCH15L-2F7, MYCH15L-1H3-1K3, MYCH15L-28H3-28K1, and MYCH15L-14H1-14K2 Hand V L Each region corresponds to the V from antibodies 2F7, 1H3-1K3, 28H3-28K1, and 14H1-14K2, respectively. H and V L It is based on domain.

[0226] [Table 12]

[0227] TIFF2026108674000020.tif146159

[0228] e) V L V via a 20-amino acid linker H MYC structures connected to: The VH and VL regions of each antibody were optimized and synthesized. Each scFv fragment was amplified from the VH and VL templates, and a 15aa linker was added simultaneously. After PCR purification, the scFv fragments were cloned into the pcDNA3.1 vector using a seamless ligation protocol.

[0229] V H V via a 15-amino acid linker L The MYC structures connected to it are MYCL20H-2F7, MYCL20H-1H3-1K3, MYCL20H-28H3-28K1, and MYCL20H-14H1-14K2. All of these structures are V L -20 amino acid ScFv linker sequence -V H -3 amino acid MYC linker sequence - has a common sequence of MYC tag. The amino acid and polynucleotide sequences of constructs MYCL20H-2F7, MYCL20H-1H3-1K3, MYCL20H-28H3-28K1, and MYCL20H-14H1-14K2 are shown in Table 13 below. V of MYCL20H-2F7, MYCL20H-1H3-1K3, MYCL20H-28H3-28K1, and MYCL20H-14H1-14K2 H and V LEach region corresponds to the V from antibodies 2F7, 1H3-1K3, 28H3-28K1, and 14H1-14K2, respectively. H and V L It is based on domain.

[0230] [Table 13]

[0231] TIFF2026108674000022.tif156159

[0232] f) V H V via a 20-amino acid linker L MYC structures connected to: The VH and VL regions of each antibody were optimized and synthesized. Each scFv fragment was amplified from the VH and VL templates, and a 20aa linker was added simultaneously. After PCR purification, the scFv fragments were cloned into the pcDNA3.1 vector using a seamless ligation protocol.

[0233] V H V via a 20-amino acid linker L The MYC structures connected to it are MYCH20L-2F7, MYCH20L-1H3-1K3, MYCH20L-28H3-28K1, and MYCH20L-14H1-14K2. All of these structures are V H -20 amino acid ScFv linker sequence -V L -3 amino acid MYC linker sequence - has a common sequence of MYC tag. The amino acid and polynucleotide sequences of the constructs, MYCH20L-2F7, MYCH20L-1H3-1K3, MYCH20L-28H3-28K1, and MYCH20L-14H1-14K2 are shown in Table 14 below. V of MYCH20L-2F7, MYCH20L-1H3-1K3, MYCH20L-28H3-28K1, and MYCH20L-14H1-14K2 H and V LEach region corresponds to the V from antibodies 2F7, 1H3-1K3, 28H3-28K1, and 14H1-14K2, respectively. H and V L It is based on domain.

[0234] [Table 14]

[0235] TIFF2026108674000024.tif154159

[0236] The scFv vector construct was used for transfection and expression studies, as described in Example 13.

[0237] Example 13 Immunocytochemistry and colocalization Various constructs were expressed in HEK293 cells containing or not containing dNLSTDP-43 using the methods described below. Immunocytochemistry and co-localization analyses were also performed as described below.

[0238] The LYS-2F7, LYS-1H3-1K3, LYS-28H3-28K1, and LYS-14H1-14K2 TDP-43 intrabodies interact with dNLSTDP-43 cytoplasmic aggregates in transfected HEK293 cells (Figures 1-4). None of these intrabodies show recognizable interactions with endogenous wild-type TDP43 in the nucleus. Overexpression of TDP43 intrabodies does not affect cell viability, indicating that they do not interfere with the essential normal function of TDP43.

[0239] Similarly, overexpression of the YPTL-2F7, YPTL-1H3-1K3, YPTL-28H3-28K1, and YPTL-14H1-14K2 TDP-43 intrabodies did not affect the viability of HEK293 cells, indicating that these intrabodies do not interfere with the essential function of normal TDP43 (Figures 5-8). YPTL-2F7, YPTL-1H3-1K3, YPTL-28H3-28K1, and YPTL-14H1-14K2 all showed strong interactions with aggregated cytoplasmic dNLS-TDP-43, but not with normal nuclear TDP-43 (Figures 5-8).

[0240] Overexpressed MYCL15H-2F7, MYCH15L-2F7, MYCL15H-1H3-1K3, MYCH15L-28H3-28K1, MYCL15H-14H1-14K2, and MYCH15L-14H1-14K2 TDP-43 intrabodies colocalize with misfolded TDP43 cytoplasmic aggregates (Figures 9, 10, 12, 14, 15, and 16). None of these intrabodies show any recognizable interaction with endogenous wild-type TDP43 in the nucleus. MYCL15H-2F7, MYCH15L-2F7, MYCL15H-1H3-1K3, MYCH15L-28H3-28K1, MYCL15H-14H1-14K2, and MYCH15L-14H1-14K2 all show strong interactions with aggregated cytoplasmic dNLS-TDP-43 (Figures 9, 10, 12, 14, 15, and 16).

[0241] Overexpressed MYCL20H-1H3-1K3 and MYCL20H-14H1-14K2TDP-43 intrabodies colocalize with misfolded TDP43 cytoplasmic aggregates (Figures 13 and 17). MYCL20H-1H3-1K3 and MYCL20H-14H1-14K2TDP-43 show strong interactions with aggregated cytoplasmic dNLS-TDP-43 but not with normal nuclear TDP-43 (Figures 13 and 17).

[0242] Overexpressed MYCH20L-14H1-14K2TDP-43 intrabodies colocalize with misfolded TDP43 cytoplasmic aggregates (Figure 18). MYCH20L-2F7 showed lower expression (Figure 11). None of the intrabodies showed any recognizable interaction with endogenous wild-type TDP43 in the nucleus.

[0243] Decomposition of dNLS-TDP-43 The effect of intrabody expression on dNLS-TDP-43 degradation was evaluated as described below.

[0244] These results indicate that the intrabodies of LYS-2F7, LYS-28H3-28K1, and LYS-14H1-14K2, YPTL-14H1-14K2, MYCH15L-2F7, MYCL15H-1H3-1K3, and MYCH20L-14H1-14K2 promote dNLSTDP-43 degradation (Figures 19, 20, and 21).

[0245] material and method Cell culture and transfection The human fetal kidney 293T (HEK293T) cell line was purchased from the American Type Culture Collection (ATCC, Rockville, Maryland) and cultured with 10% fetal bovine serum (FBS) and GlutaMax®. - The samples were maintained at 37°C in Dulbecco's modified Eagle medium (DMEM) supplemented with 1 (2 mM) and antibiotics (50 U / ml penicillin and 50 mg / ml streptomycin) at 5% CO2.

[0246] HA-tagged dNLSTDP-43, a TDP43 mutant lacking functional nuclear localization signaling (NLS), was co-transfected into HEK293T cells in a 1:5 ratio with individual TDP43 single-chain variable fragment (scFv) plasmids or a control empty vector (EV) using Lipofectamine LTX reagent (ThermoFisher Scientific, Waltham, Massachusetts, USA) according to the manufacturer's instructions.

[0247] Immunocytochemistry for the detection of TDP43 intrabodies using dNLSTDP-43 72 hours after transfection, cells were washed twice with phosphate-buffered saline (PBS), fixed in 4% PFA at room temperature for 15 minutes, and then washed with 20 mM glycine at room temperature for 10 minutes with constant shaking to quench any remaining PFA. Next, cells were cultured at room temperature for 30 minutes with blocking buffer containing PBS, 1% bovine serum albumin (BSA), 10% normal goat serum, and 0.1% Triton-X-100, after which primary antibodies were added. To investigate the interaction between LYS and YPTL TDP43 intrabodies and dNLS-TDP43, mouse monoclonal anti-Flag (Sigma, St. Louis, Missouri, USA, F1804, 1:2000) was used to detect TDP43 scFv construct expression, and rabbit polyclonal anti-HA (Abcam, Cambridge, UK, ab9110, 1:1000) was used for dNLS-TDP43. To investigate the interaction between Myc TDP43 intrabodies and dNLS-TDP43, rabbit polyclonal anti-c-Myc (Abcam, ab9106, 1:1000) was used for scFv expression detection, and rat monoclonal anti-HA (Sigma, 1:2000) was used for dNLS-TDP43. After incubation of the primary antibody for 2 hours, cells were washed with PBS / 0.1% Triton-X-100 (PBST) for 10 minutes x 3 times with constant shaking, followed by incubation with Alexa Fluor® goat anti-rabbit, anti-mouse, or anti-rat secondary antibody (ThermoFisher Scientific, 1:1000) in the dark at room temperature for 30 minutes. Next, cells were washed with PBST for 10 minutes x 3 times, immersed in 5% PBS, and mounted using ProLong Gold Anti-fade Reagent with DAPI (ThermoFisher, P36931). The cells were analyzed using a confocal microscope (Leica TCS SP8 MP, Wetzlar, Germany).

[0248] Western blotting and degradation quantification 72 hours after transfection, cells were washed twice with cold PBS, dissolved in 2% SDS, and then sonicated at 30% power for 15 seconds to extract total protein. Protein content was determined by BCA assay (ThermoFisher Scientific). 10 μg of protein was separated by 4–12% NuPAGE Bis-TrisSDS-PAGE (ThermoFisher Scientific), transferred to a PVDF membrane, and blocked at room temperature for 1 hour in Tris-buffered saline (TBS) containing 5% skim milk powder and 0.1% Tween-20. The following primary antibodies were incubated overnight at 4°C: rabbit monoclonal anti-Flag for LYS and YPTL constructs (Cell Signaling Technology, Danvers, Massachusetts, USA, 147935, 1:2000); rabbit anti-c-Myc for TDP-43 scFv expression detection in MYC-tagged constructs (Abcam, 1:500); rabbit anti-HA for dNLSTDP-43 detection (Abcam, 1:4000); and mouse anti-β-actin for loading control (abm, Richmond, British Columbia, Canada, G043, 1:1000). Next, the membrane was washed with TBS / 0.1% Tween (TBST) at room temperature for 10 minutes x 3 times with continuous agitation. Subsequently, incubation with horseradish peroxidase (HRP)-conjugated goat anti-mouse antibody (Sigma, 1:5000) or donkey anti-rabbit secondary antibody (Sigma, AP182P, 1:5000) was performed at room temperature for 30 minutes. Then, the membrane was washed with TBST for 10 minutes x 3 times and luminescence was induced using SuperSignal® West Femto Maximum Sensitivity Substrate (ThermoFisher Scientific). Images were taken with ChemiDoc® MP Imager (Bio-Rad Laboratories, Hercules, California, USA).

[0249] ImageJ was used to measure the intensity of Western blot bands. To quantify the effect of the TDP43 scFv intrabody on dNLS-TDP43 degradation, HA intensity was first normalized by the corresponding β-actin for each transfection condition. Then, the normalized HA intensity for each dNLS-TDP43 / scFv cotransfection was compared to the "dNLS+EV" control.

[0250] While this application has been described with reference to what are currently considered preferred embodiments, it should be understood that this application is not limited to the disclosed embodiments. On the contrary, this application is intended to cover a variety of modifications and equivalent configurations that fall within the spirit and scope of the appended claims.

[0251] All publications, patents, and patent applications are incorporated into this Specification by reference to the same extent as each individual publication, patent, or patent application is specifically and individually indicated as being incorporated into this Specification by reference. Specifically, sequences associated with each accession number shown herein, including, for example, accession numbers and / or biomarker sequences (e.g., proteins and / or nucleic acids) shown in tables or elsewhere, are incorporated into this Specification by reference.

[0252] The claims should not be limited by preferred embodiments and examples, but should be given the broadest possible interpretation consistent with the specification as a whole.

[0253] Citation of references mentioned in the specification TIFF2026108674000025.tif161158

Claims

1. A nucleic acid comprising a sequence encoding a single-chain antibody, wherein the single-chain antibody is bound to Trp-68 at misfolded TDP-43 and comprises a heavy chain variable region comprising complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3, and a light chain variable region comprising complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3, wherein the heavy chain variable region and the light chain variable region are linked by a linker, and the amino acid sequence of the CDR comprises the following sequence: CDR-H1: GFFSSSNYV Sequence ID 16; CDR-H2: IWFAGIVDTT Sequence ID 17; CDR-H3: ARNPVGSVNL Sequence ID 18; CDR-L1: ESVYSNNR Sequence ID 19; CDR-L2: YAS Sequence ID 20; and CDR-L3: AGWRGARTDGVD Sequence ID 21, CDR-H1: GFSLSRYY Sequence ID 10; CDR-H2: IIPGGTT Sequence ID 11; CDR-H3: AGGPTGNSHFTL Sequence ID 12; CDR-L1: ESVYNNNH Sequence ID 13; CDR-L2: EAS Sequence ID 14; and CDR-L3: SGYKRVTTTDGIA Sequence ID 15, CDR-H1: GFFSSSSYV Sequence ID 22; CDR-H2: SDTGINT Sequence ID 23; CDR-H3: ARRYTGDTYLGNFNL Sequence ID 24; CDR-L1: QSVYKNNY Sequence ID 25; CDR-L2: KAS Sequence ID 26; and CDR-L3: AGGWRSLNA Sequence ID 27, CDR-H1: EFSFSSRYW Sequence ID 28; CDR-H2: IYTGSIDAT Sequence ID 29; CDR-H3: VRGSDAWGLYFNL Sequence ID 30; CDR-L1: QSIHKNNY Sequence ID 31; CDR-L2: FAS Sequence ID 32; and CDR-L3: AGVYSGRIFA Sequence ID 33, CDR-H1: GFSLSSYT Sequence ID 34; CDR-H2: IYGGIGST Sequence ID 35; CDR-H3: GRGDI SEQ ID NO: 36; CDR-L1: QSVYKNR Sequence ID 37; CDR-L2: GAS Sequence ID 38; and CDR-L3: LGNYDCSSVDCGA Sequence ID 39, CDR-H1: GFFSSAYY Sequence ID 40; CDR-H2: TIPIGRT Sequence ID 41; CDR-H3: AGGPTGNSHFTL Sequence ID 42; CDR-L1: ESVYNNNQ Sequence ID 43; CDR-L2: QAS Sequence ID No. 44; and CDR-L3: AGYKSPTTDGIA Sequence ID 45, CDR-H1: GFSLSSYA Sequence ID 46; CDR-H2: IYNYET Sequence ID 47; CDR-H3: ARDIFRTNTNL Sequence ID 48; CDR-L1: QSVYKNNG Sequence ID 49; CDR-L2: FTS Sequence ID 50; and CDR-L3: LGGYDCSSSRVCGA Sequence ID 51, CDR-H1: GFSLSSYN Sequence ID 120; CDR-H2: IGTGGIT Sequence ID 121; CDR-H3: VRSSGSDWWFHI Sequence ID 122; CDR-L1: QSVYNNNN Sequence ID 123; CDR-L2: RAS Sequence ID 124; and CDR-L3: QGYFSGFITT Sequence ID 125, CDR-H1: GFTFSSYY Sequence ID 130; CDR-H2: INSNGGST Sequence ID 131; CDR-H3: VRQNYEGAY Sequence ID 132; CDR-L1: QSIVHSNGNTY Sequence ID 133; CDR-L2: KVS Sequence ID 134; and CDR-L3: FQSSHVPWT Sequence ID 135, or CDR-H1: GFTFSSYY Sequence ID 140; CDR-H2: INTNGGST Sequence ID 141; CDR-H3: VRQNYEGAY Sequence ID 142; CDR-L1: QSIVHSNGNTY Sequence ID 143; CDR-L2: KVS Sequence ID 144; and CDR-L3: FQSSHVPWT Sequence ID 145 Preferably, the CDR sequence comprises nucleic acids including sequence numbers 10-15, 16-21, 120-125, or 130-135.

2. The nucleic acid according to claim 1, wherein the antibody comprises a heavy chain variable region comprising i) the amino acid sequence shown in SEQ ID NO: 98, and ii) an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with respect to SEQ ID NO: 98, where the CDR sequence is one shown in SEQ ID NOs: 10 to 12, or the conserved substitution amino acid sequence of iii)i); and / or the antibody comprises a light chain variable region comprising i) the amino acid sequence shown in SEQ ID NO: 99, and ii) an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with respect to SEQ ID NO: 99, where the CDR sequence is one shown in SEQ ID NOs: 13 to 15, or the conserved substitution amino acid sequence of iii)i), and optionally the heavy chain variable region amino acid sequence is encoded by the nucleotide sequence or codon degeneracy or an optimized version thereof shown in SEQ ID NO: 76, and / or the light chain variable region amino acid sequence is encoded by the nucleotide sequence or codon degeneracy or an optimized version thereof shown in SEQ ID NO:

77.

3. The antibody comprises a heavy chain variable region comprising i) the amino acid sequence shown in SEQ ID NO: 100, ii) an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 100, where the CDR sequence is one shown in SEQ ID NOs: 16-18, or iii) the conserved substituted amino acid sequence of i); and / or the antibody comprises i) the amino acid sequence shown in SEQ ID NO: 101, ii) at least 80%, at least 85%, at least 90%, or at least The nucleic acid according to claim 1, comprising a light chain variable region comprising an amino acid sequence having 95% sequence identity, wherein the CDR sequence is one of those shown in SEQ ID NOs: 19-21 or the conserved substitution amino acid sequence of iii)i), wherein the heavy chain variable region amino acid sequence is optionally encoded by the nucleotide sequence or codon degeneracy shown in SEQ ID NO: 78 or an optimized version thereof, and / or the light chain variable region amino acid sequence is encoded by the nucleotide sequence or codon degeneracy shown in SEQ ID NO: 79 or an optimized version thereof.

4. The antibody comprises a heavy chain variable region comprising i) the amino acid sequence shown in SEQ ID NO: 102, ii) an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 102, where the CDR sequence is one shown in SEQ ID NOs: 22-24, or iii) the conserved substituted amino acid sequence of i); and / or the antibody comprises i) the amino acid sequence shown in SEQ ID NO: 103, ii) at least 80%, at least 85%, at least 90%, or at least The nucleic acid according to claim 1, comprising a light chain variable region comprising an amino acid sequence having 95% sequence identity, wherein the CDR sequence is one of those shown in SEQ ID NOs: 25-27 or the conserved substitution amino acid sequence of iii)i), wherein the heavy chain variable region amino acid sequence is optionally encoded by the nucleotide sequence or codon degeneracy shown in SEQ ID NO: 80 or an optimized version thereof, and / or the light chain variable region amino acid sequence is encoded by the nucleotide sequence or codon degeneracy shown in SEQ ID NO: 81 or an optimized version thereof.

5. The antibody comprises a heavy chain variable region comprising i) the amino acid sequence shown in SEQ ID NO: 104, ii) an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 104, where the CDR sequence is one shown in SEQ ID NOs: 28-30, or iii) the conserved substituted amino acid sequence of i); and / or the antibody comprises i) the amino acid sequence shown in SEQ ID NO: 105, ii) at least 80%, at least 85%, at least 90%, or at least The nucleic acid according to claim 1, comprising a light chain variable region comprising an amino acid sequence having 95% sequence identity, wherein the CDR sequence is one of those shown in SEQ ID NOs: 31-33 or the conserved substitution amino acid sequence of iii)i), wherein the heavy chain variable region amino acid sequence is optionally encoded by the nucleotide sequence or codon degeneracy shown in SEQ ID NO: 82 or an optimized version thereof, and / or the light chain variable region amino acid sequence is encoded by the nucleotide sequence or codon degeneracy shown in SEQ ID NO: 83 or an optimized version thereof.

6. The antibody comprises a heavy chain variable region comprising i) the amino acid sequence shown in SEQ ID NO: 106, ii) an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 106, where the CDR sequence is one shown in SEQ ID NOs: 34-36, or iii) the conserved substituted amino acid sequence of i); and / or the antibody comprises i) the amino acid sequence shown in SEQ ID NO: 107, ii) at least 80%, at least 85%, at least 90%, or at least The nucleic acid according to claim 1, comprising a light chain variable region comprising an amino acid sequence having 95% sequence identity, wherein the CDR sequence is one of those shown in SEQ ID NOs: 37-39 or the conserved substitution amino acid sequence of iii)i), wherein the heavy chain variable region amino acid sequence is optionally encoded by the nucleotide sequence or codon degeneracy shown in SEQ ID NO: 84 or an optimized version thereof, and / or the light chain variable region amino acid sequence is encoded by the nucleotide sequence or codon degeneracy shown in SEQ ID NO: 85 or an optimized version thereof.

7. The antibody comprises a heavy chain variable region comprising i) the amino acid sequence shown in SEQ ID NO: 108, ii) an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 108, where the CDR sequence is one shown in SEQ ID NOs: 40-42, or iii) the conserved substituted amino acid sequence of i); and / or the antibody comprises i) the amino acid sequence shown in SEQ ID NO: 109, ii) at least 80%, at least 85%, at least 90%, or at least The nucleic acid according to claim 1, comprising a light chain variable region having an amino acid sequence having 95% sequence identity, wherein the CDR sequence is one of those shown in SEQ ID NOs: 43-45 or the conserved substitution amino acid sequence of iii)i), wherein the heavy chain variable region amino acid sequence is optionally encoded by the nucleotide sequence or codon degeneracy shown in SEQ ID NO: 86 or an optimized version thereof, and / or the light chain variable region amino acid sequence is encoded by the nucleotide sequence or codon degeneracy shown in SEQ ID NO: 87 or an optimized version thereof.

8. The antibody comprises a heavy chain variable region comprising i) the amino acid sequence shown in SEQ ID NO: 110, ii) an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 110, where the CDR sequence is the one shown in SEQ ID NOs: 46-48, or iii) the conserved substituted amino acid sequence of i); and / or the antibody comprises i) the amino acid sequence shown in SEQ ID NO: 111, ii) at least 80%, at least 85%, at least 90%, or at least The nucleic acid according to claim 1, comprising a light chain variable region comprising an amino acid sequence having 95% sequence identity, wherein the CDR sequence is one of those shown in SEQ ID NOs: 49-51 or the conserved substitution amino acid sequence of iii)i), wherein the heavy chain variable region amino acid sequence is optionally encoded by the nucleotide sequence or codon degeneracy shown in SEQ ID NO: 88 or an optimized version thereof, and / or the light chain variable region amino acid sequence is encoded by the nucleotide sequence or codon degeneracy shown in SEQ ID NO: 89 or an optimized version thereof.

9. The antibody comprises a heavy chain variable region comprising i) the amino acid sequence shown in SEQ ID NO: 128, ii) an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 128, where the CDR sequence is one shown in SEQ ID NOs: 120-122, or iii) the conserved substituted amino acid sequence of i); and / or the antibody comprises i) the amino acid sequence shown in SEQ ID NO: 129, ii) at least 80%, at least 85%, at least 90%, or at least The nucleic acid according to claim 1, comprising a light chain variable region having an amino acid sequence having 95% sequence identity, wherein the CDR sequence is one of those shown in SEQ ID NOs: 123-125 or the conserved substitution amino acid sequence of iii)i), wherein the heavy chain variable region amino acid sequence is optionally encoded by the nucleotide sequence or codon degeneracy shown in SEQ ID NO: 126 or an optimized version thereof, and / or the light chain variable region amino acid sequence is encoded by the nucleotide sequence or codon degeneracy shown in SEQ ID NO: 127 or an optimized version thereof.

10. The antibody comprises a heavy chain variable region comprising i) the amino acid sequence shown in SEQ ID NO: 138, ii) an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 138, where the CDR sequence is one shown in SEQ ID NOs: 130-132, or iii) the conserved substituted amino acid sequence of i); and / or the antibody comprises i) the amino acid sequence shown in SEQ ID NO: 139, ii) at least 80%, at least 85%, at least 90%, or at least The nucleic acid according to claim 1, comprising a light chain variable region having an amino acid sequence having 95% sequence identity, wherein the CDR sequence is one of those shown in SEQ ID NOs: 133-135 or the conserved substitution amino acid sequence of iii)i), wherein the heavy chain variable region amino acid sequence is optionally encoded by the nucleotide sequence or codon degeneracy shown in SEQ ID NO: 136 or an optimized version thereof, and / or the light chain variable region amino acid sequence is encoded by the nucleotide sequence or codon degeneracy shown in SEQ ID NO: 137 or an optimized version thereof.

11. The antibody comprises a heavy chain variable region comprising i) the amino acid sequence shown in SEQ ID NO: 148, ii) an amino acid sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 148, where the CDR sequence is one shown in SEQ ID NOs: 140-142, or iii) the conserved substituted amino acid sequence of i); and / or the antibody comprises i) the amino acid sequence shown in SEQ ID NO: 149, ii) at least 80%, at least 85%, at least 90%, or at least The nucleic acid according to claim 1, comprising a light chain variable region having an amino acid sequence having 95% sequence identity, wherein the CDR sequence is one of those shown in SEQ ID NOs: 143-145 or the conserved substitution amino acid sequence of iii)i), wherein the heavy chain variable region amino acid sequence is optionally encoded by the nucleotide sequence or codon degeneracy shown in SEQ ID NO: 146 or an optimized version thereof, and / or the light chain variable region amino acid sequence is encoded by the nucleotide sequence or codon degeneracy shown in SEQ ID NO: 147 or an optimized version thereof.

12. The nucleic acid according to any one of claims 1 to 11, wherein the orientation of the heavy chain variable domain, linker, and light chain variable domain is heavy chain variable domain-linker-light chain variable domain.

13. The nucleic acid according to any one of claims 1 to 11, wherein the orientation of the heavy chain variable domain, linker, and light chain variable domain is light chain variable domain-linker-heavy chain variable domain.

14. The nucleic acid according to any one of claims 1 to 13, wherein the linker is about 10 to about 25 amino acids, preferably 15 or 20 amino acids.

15. The linker is (Gly-Gly-Gly-Gly-Ser) ３ (Sequence ID 180), (Gly-Gly-Gly-Gly-Ser) ４ A nucleic acid according to any one of claims 1 to 14, selected from (SEQ ID NO: 181) and GSTGGGGGSGKPGSGEGGGGGS (SEQ ID NO: 182).

16. The nucleic acid according to any one of claims 1 to 15, wherein a targeting portion linker optionally further codes a targeting portion that may be linked.

17. The nucleic acid according to claim 16, wherein the targeting portion is selected from a lysosomal targeting sequence and an autophagy targeting sequence.

18. The nucleic acid according to claim 17, wherein the lysosome targeting sequence has an amino acid sequence selected from YPTL (SEQ ID NO: 173), KSIRSGYEVM (SEQ ID NO: 174), and RWRKSHSSSYTPLSGSTYPEGRH (SEQ ID NO: 175).

19. The nucleic acid according to any one of claims 1 to 18, wherein a single-chain antibody is an intrabody.

20. The nucleic acid according to any one of claims 1 to 18, further encoding a signal peptide and / or a cell-permeable peptide.

21. The nucleic acid according to any one of claims 1 to 20, wherein the single-chain antibody is an scFV, a minibody, or a nanobody.

22. The nucleic acid according to any one of claims 1 to 21, wherein the single-chain antibody is humanized.

23. The nucleic acid according to any one of claims 1 to 11, wherein the sequence encodes any antibody and linker moiety of sequence numbers 203, 205, 207, 209, 213, 215, 217, 219, 223, 225, 227, 229, 233, 235, 237, 239, 243, 245, 247, 249, 253, 255, 257, and / or 259, and optionally a lysosome targeting sequence.

24. The nucleic acid according to any one of claims 1 to 11, wherein the sequence comprises one of sequence numbers 76 to 89, 126 to 127, 136 to 137, 146 to 147, or a codon-optimized sequence thereof.

25. The nucleic acid according to any one of claims 1 to 11, wherein the sequence comprises an antibody and linker moiety of any of sequence numbers 202, 204, 206, 208, 212, 214, 216, 218, 222, 224, 226, 228, 232, 234, 236, 238, 242, 244, 246, 248, 252, 254, 256, and / or 258, and optionally a lysosome targeting sequence.

26. An expression cassette or vector comprising the nucleic acid described in any one of claims 1 to 25.

27. The vector according to claim 26, wherein the vector is a lentiviral vector or an adeno-associated virus (AAV) vector, and optionally AAV serotype 9.

28. A nucleic acid according to any one of claims 1 to 25, or a single-chain antibody encoded by an expression cassette or vector according to claim 26 or 27.

29. An immune complex comprising the antibody and a detectable label according to claim 28.

30. Cells that recombinantly express a single-chain antibody encoded by the nucleic acid according to any one of claims 1 to 25, or by the vector according to claim 26 or 27.

31. A composition comprising, optionally together with a diluent and / or a pharmaceutically acceptable carrier, a nucleic acid according to any one of claims 1 to 25, an expression cassette or vector according to claim 26 or 27, a single-chain antibody according to claim 28, an immune complex according to claim 29, or cells according to claim 30.

32. The composition according to claim 31, comprising two or more different nucleic acids, optionally together with a diluent and / or a pharmaceutically acceptable carrier.

33. The composition according to claim 31, comprising two or more different expression cassettes or vectors, optionally together with a diluent and / or a pharmaceutically acceptable carrier.

34. The composition according to claim 31, comprising two or more different single-chain antibodies, optionally together with a diluent and / or a pharmaceutically acceptable carrier.

35. The composition according to claim 31, comprising two or more different immunocomplexes, optionally together with a diluent and / or a pharmaceutically acceptable carrier.

36. The composition according to claim 31, comprising two or more different cells, optionally together with a diluent and / or a pharmaceutically acceptable carrier.

37. The composition according to any one of claims 31 to 36, comprising lipid particles, optionally liposomes, nanoparticles, or nanosomes.

38. A composition according to any one of claims 31 to 37, preferably formulated for administration into the subarachnoid space, intracerebral parenchyma, or intraventricular space.

39. A method for treating a subject, comprising administering an effective amount of a nucleic acid according to any one of claims 1 to 25, an expression cassette or vector according to claim 26 or 27, an antibody according to claim 28, or a composition according to any one of claims 31 to 38, optionally one or more nucleic acids, one or more expression cassettes or vectors, or one or more antibodies and / or compositions, to a subject in need thereof.

40. The method according to claim 39, comprising administering an effective amount of two or more nucleic acids described in any one of claims 1 to 25 to a subject in need thereof.

41. The method according to claim 39, comprising administering an effective amount of two or more expression cassettes or vectors described in claim 26 or 27 to a subject in need thereof.

42. The method according to claim 39, comprising administering an effective amount of two or more antibodies described in claim 28 to a subject in need thereof.

43. The method according to claim 39, comprising administering an effective amount of two or more compositions described in any one of claims 31 to 38 to a subject in need thereof.

44. The method according to any one of claims 39 to 43, wherein the subject is suspected of having TDP-43 proteinosis, is at risk of developing it, or has been diagnosed with it.

45. The method according to claim 44, wherein the TDP-43 proteinosis is selected from amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD-TDP), primary lateral sclerosis, progressive muscular atrophy, and limbic-dominant senile TDP-43 encephalopathy (LATE).

46. The method according to any one of claims 39 to 45, wherein the nucleic acid, expression cassette or vector, antibody, or composition is administered in combination with another treatment for TDP-43 proteinosis.

47. The method according to any one of claims 39 to 46, wherein the nucleic acid, expression cassette, vector, single-chain antibody, or composition is administered by parenteral administration, intravenous administration, subcutaneous administration, intramuscular administration, intracranial administration, intraventricular administration, subarachnoid administration, intraorbital administration, ocular administration, intraspinal administration, intracisional administration, intraperitoneal administration, intranasal administration, aerosol administration, or oral administration.

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