Cell penetrating agent and use thereof

Abstract

The present disclosure provides cell penetrating agents comprising an intercellular module and an antibody, or antigen-binding antibody fragment thereof, that specifically binds to human TDP-43, and methods of using these cell penetrating agents to treat patients suffering from TDP-43 related diseases, including amyotrophic lateral sclerosis (ALS).

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 538,599, filed on September 15, 2023, the contents of which are hereby incorporated in their entirety.

[0003] sequence list This application contains a sequence list of documents submitted electronically as an XML file named “50887-0047WO1.XML”. The XML file was created on August 28, 2024, and is 204,800 bytes in size. The material in the XML file is hereby incorporated in its entirety. Technical Field

[0004] This disclosure relates to the technical fields of immunology and medicine. Background Technology

[0005] Trans-reactive DNA-binding protein 43 (“TDP-43”) is a nuclear protein primarily involved in RNA splicing, transport, stabilization, and ultimately, regulation of gene expression. TDP-43 is a nucleic acid-binding protein containing two highly conserved nucleic acid recognition motifs and has been shown to form dimers and oligomers. While TDP-43 is universally expressed in all cell types, it has been shown to be highly expressed in neuroepithelial cells, which contain all CNS precursor cells, including neurons and glial cells. Furthermore, TDP-43 has been shown to specifically bind to numerous RNAs in neuronal cells.

[0006] TDP-43 cytoplasmic aggregates (also known as inclusion bodies) are associated with several neurodegenerative diseases, conditions, or disorders. Specifically, TDP-43 cytoplasmic aggregates and / or misfolding of TDP-43 are associated with neurodegenerative diseases including amyotrophic lateral sclerosis (“ALS”), frontotemporal dementia (“FTD” or “FTLD-TDP-43”), age-related TDP-43 encephalopathy (“LATE”) with a predominantly limbic system component, Alzheimer’s disease, multisystemic proteinopathy, and chronic traumatic encephalopathy.

[0007] ALS is the most common motor neuron disease in adults, ultimately leading to the loss of motor neurons that control voluntary muscle movement. The annual incidence of ALS is approximately 1–2 per 100,000 cases, and the prevalence is approximately 4–6 per 100,000 cases. ALS causes progressive degeneration of both upper and lower motor neurons, typically leading to death primarily attributable to respiratory failure within three to five years of diagnosis. Typical symptoms include muscle stiffness, muscle twitching, progressive muscle weakness, and muscle atrophy. About half of all people with ALS experience difficulty thinking and / or behavioral symptoms, and about 15% develop frontotemporal dementia. Pathologically, abnormal aggregation of the TDP-43 protein is observed in up to 97% of ALS patients (Nguyen, HP et al., ALS Genes in the GenomicEra and their Implications for FTD, Trends in Genetics , 34(6): 404+423(2018)).

[0008] Currently, there is no known cure for ALS. Therefore, there is an unmet need for therapies to improve the health of patients with TDP-43-related diseases (including ALS), including mortality risk and / or quality of life. Summary of the Invention

[0009] The main text discloses cell penetrants (“CPA”) comprising a cell internalization module (“CIM”) and an antibody that specifically binds to human TDP-43, compositions comprising such cell penetrants, and methods of using these cell penetrants to treat TDP-43-related diseases, including amyotrophic lateral sclerosis (ALS).

[0010] Therefore, this article provides cell penetrants comprising (i) a cell internalization module and (ii) an antibody that specifically binds to a 43kD trans-active reactive DNA-binding protein (TDP-43).

[0011] In some embodiments, CIM comprises a cell membrane internalization peptide (CMIP). In some embodiments, CIM comprises a wild-type M-lycotoxin peptide. In some embodiments, CIM comprises an M-lycotoxin derivative. In some embodiments, CIM comprises a Penetain amino acid sequence or a derivative thereof. In some embodiments, CIM comprises a Pepth amino acid sequence or a derivative thereof. In some embodiments, CIM comprises a polyarginine amino acid sequence. In some embodiments, CIM comprises more than one polyarginine amino acid sequence. In some embodiments, CIM comprises three polyarginine amino acid sequences. In some embodiments, CIM comprises a TAT amino acid sequence. In some embodiments, CIM comprises more than one TAT amino acid sequence. In some embodiments, CIM comprises three TAT amino acid sequences.

[0012] In some embodiments, the CIM comprises a macrocycle. In some embodiments, the macrocycle is formed by a covalent bond between two amino acid residues in the CIM. In some embodiments, the macrocycle is formed by a disulfide bond between two cysteine ​​residues in the CIM. In some embodiments, the CIM comprises one or more histidine residues.

[0013] In some embodiments, CIM comprises a polypeptide having an amino acid sequence selected from one of the following: SEQ ID NO: 176-184, SEQ ID NO: 192 and SEQ ID NO: 193.

[0014] In some embodiments, the CIM comprises one or more spacer regions. In some embodiments, at least one of the one or more spacer regions comprises one or more amino acid residues. In some embodiments, at least one of the one or more spacer regions comprises one or more glycine residues. In some embodiments, at least one of the one or more spacer regions comprises an amino acid sequence selected from any of SEQ ID NO: 200-203. In some embodiments, each of the one or more spacer regions comprises an amino acid sequence selected from any of SEQ ID NO: 200-203.

[0015] In some embodiments, CIM comprises a polypeptide having an amino acid sequence selected from any of SEQ ID NO: 176-193. In some embodiments, CIM is a polypeptide having an amino acid sequence selected from any of SEQ ID NO: 176-193.

[0016] In some embodiments, CIM is covalently linked to an antibody. In some embodiments, CIM is non-covalently linked to an antibody. In some embodiments, the cell penetrant includes a linker that links CIM to an antibody. In some embodiments, the linker is covalently linked to both CIM and the antibody. In some embodiments, the linker is a cleavable linker. In some embodiments, the linker is a non-cleavable linker. In some embodiments, the linker comprises a polypeptide. In some embodiments, the linker comprises one or more glycine residues. In some embodiments, the linker comprises a polypeptide containing an amino acid sequence selected from any of SEQ ID NO: 194-199. In some embodiments, the linker is a polypeptide containing an amino acid sequence selected from any of SEQ ID NO: 194-199.

[0017] In some embodiments, the antibody is attached to the C-terminus of the CIM. In some embodiments, the antibody is attached to the N-terminus of the CIM.

[0018] In some embodiments, the antibody competitively binds to TDP-43 (e.g., human TDP-43) with: an antibody containing the heavy chain variable domain of SEQ ID NO: 1 and the light chain variable domain of SEQ ID NO: 24; an antibody containing the heavy chain variable domain of SEQ ID NO: 63 and the light chain variable domain of SEQ ID NO: 65; an antibody containing the heavy chain variable domain of SEQ ID NO: 67 and the light chain variable domain of SEQ ID NO: 69; an antibody containing the heavy chain variable domain of SEQ ID NO: 71 and the light chain variable domain of SEQ ID NO: 73; an antibody containing the heavy chain variable domain of SEQ ID NO: 75 and the light chain variable domain of SEQ ID NO: 77; or an antibody containing the heavy chain variable domain of SEQ ID NO: 79 and the light chain variable domain of SEQ ID NO: 81.

[0019] In some embodiments, the antibody binds to the same epitope on TDP-43 (e.g., human TDP-43) with the following: an antibody containing the heavy chain variable domain of SEQ ID NO: 1 and the light chain variable domain of SEQ ID NO: 24; an antibody containing the heavy chain variable domain of SEQ ID NO: 63 and the light chain variable domain of SEQ ID NO: 65; an antibody containing the heavy chain variable domain of SEQ ID NO: 67 and the light chain variable domain of SEQ ID NO: 69; an antibody containing the heavy chain variable domain of SEQ ID NO: 71 and the light chain variable domain of SEQ ID NO: 73; an antibody containing the heavy chain variable domain of SEQ ID NO: 75 and the light chain variable domain of SEQ ID NO: 77; or an antibody containing the heavy chain variable domain of SEQ ID NO: 79 and the light chain variable domain of SEQ ID NO: 81.

[0020] In some embodiments, the antibody that specifically binds to TDP-43 (e.g., human TDP-43) comprises three light chain CDRs and three heavy chain CDRs of a mouse antibody, characterized by including a heavy chain variable domain of SEQ ID NO: 1 and a light chain variable domain of SEQ ID NO: 24.

[0021] In some implementations, the CDR has a definition selected from the group consisting of Kabat, Chothia, Kabat / Chothia, Composite, AbM, and Contact.

[0022] In some embodiments, the antibody comprises a humanized mature heavy chain variable domain comprising: a heavy chain CDR1 as defined by the Kabat / Chothia Composite, comprising SEQ ID NO: 49; a heavy chain CDR2 as defined by Kabat, comprising SEQ ID NO: 51; and a heavy chain CDR3 as defined by Kabat or Chothia, comprising SEQ ID NO: 52; and a humanized mature light chain variable domain comprising the three Kabat light chain CDRs of SEQ ID NO: 53-55.

[0023] In some embodiments, the humanized mature heavy chain variable domain contains a sequence that is at least 80% identical to any of SEQ ID NO: 4-23, and the humanized mature light chain variable domain contains a sequence that is at least 80% identical to any of SEQ ID NO: 27-48. In some embodiments, the humanized mature heavy chain variable domain contains a sequence that is at least 85% identical to any of SEQ ID NO: 4-23, and the humanized mature light chain variable domain contains a sequence that is at least 85% identical to any of SEQ ID NO: 27-48. In some embodiments, the humanized mature heavy chain variable domain contains a sequence that is at least 90% identical to any of SEQ ID NO: 4-23, and the humanized mature light chain variable domain contains a sequence that is at least 90% identical to any of SEQ ID NO: 27-48. In some implementations, the humanized mature heavy chain variable domain contains a sequence that is at least 95% identical to any of SEQ ID NO: 4-23, and the humanized mature light chain variable domain contains a sequence that is at least 95% identical to any of SEQ ID NO: 27-48.

[0024] In some implementations, at least one of the following positions in the humanized heavy chain variable domain is occupied by a specified amino acid: K19 is occupied by R; S35 is occupied by G; T40 is occupied by A; E42 is occupied by G; A49 is occupied by S; K43 is occupied by E; R44 is occupied by G or D; A49 is occupied by S; A74 is occupied by S; T77 is occupied by S; L78 is occupied by A or G; L80 is occupied by A or G; L82c is occupied by G; M83 is occupied by R; S84 is occupied by A; M89 is occupied by V; or F91 is occupied by Y.

[0025] In some implementations, at least one of the following positions in the humanized heavy chain variable domain is occupied by a specified amino acid: K43 is occupied by E; R44 is occupied by G or D; A49 is occupied by S; A74 is occupied by S; T77 is occupied by S; or F91 is occupied by Y.

[0026] In some implementations, at least one of the following positions in the humanized heavy chain variable domain is occupied by a specified amino acid: S35 is occupied by G; L78 is occupied by A or G; L80 is occupied by A or G; or L82c is occupied by G.

[0027] In some embodiments, F91 of the humanized heavy chain variable domain is occupied by Y; and at least one of the following positions in the humanized heavy chain variable domain is occupied by a specified amino acid: R44 is occupied by G; A49 is occupied by S; A74 is occupied by S; T77 is occupied by S; L78 is occupied by A or G; or M83 is occupied by R.

[0028] In some embodiments, at least one of the following positions in the variable domain of the humanized light chain is occupied by a specified amino acid: V3 is occupied by Q; L9 is occupied by S; D17 is occupied by Q; Q18 is occupied by P; K39 is occupied by R; K45 is occupied by R; T80 is occupied by A or S; L83 is occupied by V; L92 is occupied by G or A; V94 is occupied by I or A; A100 is occupied by G, D or R; or L104 is occupied by V.

[0029] In some implementations, at least one of the following positions in the variable domain of the humanized light chain is occupied by a specified amino acid: V3 is occupied by Q or A100 is occupied by D or R.

[0030] In some implementations, at least one of the following positions in the variable domain of the humanized light chain is occupied by a specified amino acid: L9 is occupied by S; T80 is occupied by A or S; L92 is occupied by G or A; or V94 is occupied by I or A.

[0031] In some implementations, V3 is occupied by Q; Q18 is occupied by P; A100 is occupied by D; and at least one of the following positions in the variable domain of the humanized light chain is occupied by a specified amino acid: T80 is occupied by A or L92 is occupied by A.

[0032] In some embodiments, at least one of the following positions in the humanized heavy chain variable domain is occupied by a specified amino acid: L5 is occupied by V; G44 is occupied by R; A49 is occupied by S; A74 is occupied by S; T77 is occupied by S; L78 is occupied by A or G; M89 is occupied by V, or F91 is occupied by Y; and at least one of the following positions in the humanized light chain variable domain is occupied by a specified amino acid: V3 is occupied by Q; D17 is occupied by Q; Q18 is occupied by P; K39 is occupied by R; K45 is occupied by R; T80 is occupied by A; L83 is occupied by V; L92 is occupied by A; A100 is occupied by D; or L104 is occupied by V.

[0033] In some implementations, the humanized antibody exhibits improved thermal stability compared to a reference antibody that includes a heavy chain variable domain comprising SEQ ID NO: 1 and a light chain variable domain comprising SEQ ID NO: 24.

[0034] In some embodiments, the antibody comprises a heavy chain variable domain comprising: heavy chain CDR1 as defined by Kabat / ChothiaComposite, comprising SEQ ID NO: 49 or SEQ ID NO: 50; heavy chain CDR2 as defined by Kabat, comprising SEQ ID NO: 51; heavy chain CDR3 as defined by Kabat or Chothia, comprising SEQ ID NO: 52; light chain CDR1 as defined by Kabat, comprising SEQ ID NO: 53; light chain CDR2 as defined by Kabat, comprising SEQ ID NO: 54; and light chain CDR3 as defined by Kabat, comprising one of SEQ ID NO: 55-61.

[0035] In some implementations, heavy chain CDR1 as defined by the Kabat / Chothia Composite contains SEQ ID NO: 49; heavy chain CDR2 as defined by Kabat contains SEQ ID NO: 51; heavy chain CDR3 as defined by Kabat or Chothia contains SEQ ID NO: 52; light chain CDR1 as defined by Kabat contains SEQ ID NO: 53; light chain CDR2 as defined by Kabat contains SEQ ID NO: 54; and light chain CDR3 as defined by Kabat contains SEQ ID NO: 55 or SEQ ID NO: 61.

[0036] In some embodiments, the heavy chain variable domain comprises a sequence that is at least 95% identical to any of SEQ ID NO: 4-23. In some embodiments, the heavy chain variable domain comprises a sequence that is at least 95% identical to any of SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 23. In some embodiments, the heavy chain variable domain comprises a sequence that is at least 98% identical to any of SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 23. In some embodiments, the heavy chain variable domain comprises the sequence of SEQ ID NO: 20. In some embodiments, the heavy chain variable domain comprises the sequence of SEQ ID NO: 21. In some embodiments, the heavy chain variable domain comprises the sequence of SEQ ID NO: 23.

[0037] In some embodiments, the light chain variable domain comprises a sequence that is at least 95% identical to any of SEQ ID NO: 27-48. In some embodiments, the light chain variable domain comprises a sequence that is at least 95% identical to either SEQ ID NO: 47 or SEQ ID NO: 48. In some embodiments, the light chain variable domain comprises a sequence that is at least 98% identical to either SEQ ID NO: 47 or SEQ ID NO: 48. In some embodiments, the light chain variable domain comprises SEQ ID NO: 47. In some embodiments, the light chain variable domain comprises SEQ ID NO: 48.

[0038] In some embodiments, the antibody comprises a heavy chain variable domain and a light chain variable domain, comprising: heavy chain CDR1, comprising SEQ ID NO: 84; heavy chain CDR2, comprising SEQ ID NO: 85; heavy chain CDR3, comprising SEQ ID NO: 86; light chain CDR1, comprising SEQ ID NO: 87; light chain CDR2, comprising SEQ ID NO: 88; and light chain CDR3, comprising SEQ ID NO: 89.

[0039] In some embodiments, the heavy chain variable domain contains a sequence that is at least 95% identical to SEQ ID NO: 63. In some embodiments, the heavy chain variable domain contains a sequence that is at least 98% identical to SEQ ID NO: 63. In some embodiments, the heavy chain variable domain contains the sequence of SEQ ID NO: 63.

[0040] In some embodiments, the light chain variable domain contains a sequence that is at least 95% identical to SEQ ID NO: 65. In some embodiments, the light chain variable domain contains a sequence that is at least 98% identical to SEQ ID NO: 65. In some embodiments, the light chain variable domain contains the sequence of SEQ ID NO: 65.

[0041] In some embodiments, the antibody comprises a heavy chain variable domain and a light chain variable domain, comprising: heavy chain CDR1, comprising SEQ ID NO: 90; heavy chain CDR2, comprising SEQ ID NO: 91; heavy chain CDR3, comprising SEQ ID NO: 92; light chain CDR1, comprising SEQ ID NO: 93; light chain CDR2, comprising SEQ ID NO: 94; and light chain CDR3, comprising SEQ ID NO: 95.

[0042] In some embodiments, the heavy chain variable domain contains a sequence that is at least 95% identical to SEQ ID NO: 67. In some embodiments, the heavy chain variable domain contains a sequence that is at least 98% identical to SEQ ID NO: 67. In some embodiments, the heavy chain variable domain contains the sequence of SEQ ID NO: 67.

[0043] In some embodiments, the light chain variable domain contains a sequence that is at least 95% identical to SEQ ID NO: 69. In some embodiments, the light chain variable domain contains a sequence that is at least 98% identical to SEQ ID NO: 69. In some embodiments, the light chain variable domain contains the sequence of SEQ ID NO: 69.

[0044] In some embodiments, the antibody comprises a heavy chain variable domain and a light chain variable domain, comprising: heavy chain CDR1, comprising SEQ ID NO: 96; heavy chain CDR2, comprising SEQ ID NO: 97; heavy chain CDR3, comprising SEQ ID NO: 98; light chain CDR1, comprising SEQ ID NO: 99; light chain CDR2, comprising SEQ ID NO: 100; and light chain CDR3, comprising SEQ ID NO: 101.

[0045] In some embodiments, the heavy chain variable domain contains a sequence that is at least 95% identical to SEQ ID NO: 71. In some embodiments, the heavy chain variable domain contains a sequence that is at least 98% identical to SEQ ID NO: 71. In some embodiments, the heavy chain variable domain contains the sequence of SEQ ID NO: 71.

[0046] In some embodiments, the light chain variable domain contains a sequence that is at least 95% identical to SEQ ID NO: 73. In some embodiments, the light chain variable domain contains a sequence that is at least 98% identical to SEQ ID NO: 73. In some embodiments, the light chain variable domain contains the sequence of SEQ ID NO: 73.

[0047] In some embodiments, the antibody comprises a heavy chain variable domain and a light chain variable domain, comprising: heavy chain CDR1, comprising SEQ ID NO: 102; heavy chain CDR2, comprising SEQ ID NO: 103; heavy chain CDR3, comprising SEQ ID NO: 104; light chain CDR1, comprising SEQ ID NO: 105; light chain CDR2, comprising SEQ ID NO: 106; and light chain CDR3, comprising SEQ ID NO: 107.

[0048] In some embodiments, the heavy chain variable domain contains a sequence that is at least 95% identical to SEQ ID NO: 75. In some embodiments, the heavy chain variable domain contains a sequence that is at least 98% identical to SEQ ID NO: 75. In some embodiments, the heavy chain variable domain contains the sequence of SEQ ID NO: 75.

[0049] In some embodiments, the light chain variable domain contains a sequence that is at least 95% identical to SEQ ID NO: 77. In some embodiments, the light chain variable domain contains a sequence that is at least 98% identical to SEQ ID NO: 77. In some embodiments, the light chain variable domain contains the sequence of SEQ ID NO: 77.

[0050] In some embodiments, the antibody comprises a heavy chain variable domain and a light chain variable domain, comprising: heavy chain CDR1, comprising SEQ ID NO: 108; heavy chain CDR2, comprising SEQ ID NO: 109; heavy chain CDR3, comprising SEQ ID NO: 110; light chain CDR1, comprising SEQ ID NO: 111; light chain CDR2, comprising SEQ ID NO: 112; and light chain CDR3, comprising SEQ ID NO: 113.

[0051] In some embodiments, the heavy chain variable domain contains a sequence that is at least 95% identical to SEQ ID NO: 79. In some embodiments, the heavy chain variable domain contains a sequence that is at least 98% identical to SEQ ID NO: 79. In some embodiments, the heavy chain variable domain contains the sequence of SEQ ID NO: 79.

[0052] In some embodiments, the light chain variable domain contains a sequence that is at least 95% identical to SEQ ID NO: 81. In some embodiments, the light chain variable domain contains a sequence that is at least 98% identical to SEQ ID NO: 81. In some embodiments, the light chain variable domain contains the sequence of SEQ ID NO: 81.

[0053] In some embodiments, the antibody is a humanized antibody, a chimeric antibody, or a veneered antibody. In some embodiments, the antibody is an antigen-binding antibody fragment. In some embodiments, the antigen-binding antibody fragment is a Fab fragment, a Fab'2 fragment, or a single-chain Fv.

[0054] In some implementations, the antibody is a complete antibody. In some implementations, the antibody has a human IgG1 isotype.

[0055] In some implementations, the variable structural domains of the heavy chain are fused into the constant region of the heavy chain, and the variable structural domains of the light chain are fused into the constant region of the light chain.

[0056] In some embodiments, the heavy chain constant region is a mutant form of the natural human heavy chain constant region, which binds less to the Fcγ receptor compared to the natural heavy chain constant region. In some embodiments, the heavy chain constant region has an IgG1 isotype. In some embodiments, the antibody has at least one mutation in the constant region. In some embodiments, at least one mutation reduces complement fixation or activation in the constant region. In some embodiments, at least one mutation is located at one or more of the following positions according to EU numbers: 241, 264, 265, 270, 296, 297, 318, 320, 322, 329, and 331. In some embodiments, the antibody contains alanine at positions 318, 320, and 322 according to EU numbers.

[0057] In some embodiments, the antibody selectively binds to phosphorylated TDP-43 (e.g., phosphorylated human TDP-43). In some embodiments, the antibody selectively binds to phosphorylated TDP-43 (e.g., phosphorylated human TDP-43) compared to unphosphorylated TDP-43 (e.g., unphosphorylated human TDP-43). In some embodiments, the antibody binds to phosphorylated TDP-43 (e.g., phosphorylated human TDP-43) with at least 100-fold greater affinity compared to unphosphorylated TDP-43 (e.g., unphosphorylated human TDP-43). In some embodiments, the antibody binds to phosphorylated TDP-43 (e.g., phosphorylated human TDP-43) with at least 1000-fold greater affinity compared to unphosphorylated TDP-43 (e.g., unphosphorylated human TDP-43). In some embodiments, phosphorylated TDP-43 (e.g., phosphorylated human TDP-43) comprises phosphorylation of at least one amino acid residue selected from S409 and S410. In some implementations, phosphorylated TDP-43 (e.g., phosphorylated human TDP-43) includes phosphorylation of both S409 and S410.

[0058] In some embodiments, the antibody selectively binds to cytoplasmic aggregates of TDP-43 (e.g., cytoplasmic aggregates of human TDP-43). In some embodiments, the antibody selectively binds to cytoplasmic aggregates of TDP-43 (e.g., cytoplasmic aggregates of human TDP-43) compared to nuclear TDP-43 (e.g., nuclear human TDP-43). In some embodiments, the cytoplasmic aggregates of TDP-43 comprise phosphorylated aggregates of TDP-43 (e.g., phosphorylated aggregates of human TDP-43). In some embodiments, the antibody substantially does not bind to unphosphorylated TDP-43 (e.g., unphosphorylated human TDP-43).

[0059] In some embodiments, the cell-penetrating agent comprises a polypeptide sequence that is at least 95% identical to the sequence selected from any of the following: SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172 and SEQ ID NO: 174.

[0060] In some embodiments, the cell-penetrating agent comprises a polypeptide sequence that is at least 98% identical to the sequence selected from any of the following: SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172 and SEQ ID NO: 174.

[0061] In some embodiments, the cell-penetrating agent comprises a polypeptide sequence selected from any of the following: SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172 and SEQ ID NO: 174.

[0062] In some embodiments, the cell-penetrating agent comprises a polypeptide sequence that is at least 95% identical to the sequence selected from any of the following: SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173 and SEQ ID NO: 175.

[0063] In some embodiments, the cell-penetrating agent comprises a polypeptide sequence that is at least 98% identical to the sequence selected from any of the following: SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173 and SEQ ID NO: 175.

[0064] In some embodiments, the cell-penetrating agent comprises a polypeptide sequence selected from any of the following: SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173 and SEQ ID NO: 175.

[0065] In some implementations, the antibody binds to a therapeutic agent, cytotoxic agent, cell growth inhibitor, immunomodulator, neurotrophic agent, or neuroprotective agent.

[0066] In some implementations, the heavy chain does not contain a C-terminal lysine residue.

[0067] This article also provides pharmaceutical compositions comprising any of the cell-penetrating agents and pharmaceutically acceptable carriers described herein.

[0068] This document also provides nucleic acids encoding at least a portion of any of the cell-penetrating agents described herein. In some embodiments, the nucleic acid encodes a heavy chain variable domain and / or a light chain variable domain of an antibody. In some embodiments, the nucleic acid encodes any of the CIMs described herein.

[0069] This article also provides vectors containing nucleic acids encoding mature heavy chain variable domains and light chain variable domains, said nucleic acids being operatively linked to one or more regulatory sequences to achieve expression of any of the cell penetrants described herein in mammalian cells.

[0070] In some embodiments, one or more regulatory sequences include one or more of an enhancer, a ribosome binding site, a transcription termination signal, and a promoter, optionally wherein the promoter is a eukaryotic promoter. In some embodiments, the nucleic acid is codon-optimized for expression in host cells.

[0071] This article also provides host cells transformed using any of the vectors described herein. This article also provides host cells including any of the nucleic acids described herein.

[0072] This document also provides a method for delivering an antibody specifically bound to TDP-43 into cells, comprising contacting the cells with any of the cell-penetrating agents described herein, thereby causing at least an antigen-binding fragment of the antibody to be internalized into the cells. In some embodiments, the method further comprises transferring at least an antigen-binding fragment of the antibody into the cytosol of the cells.

[0073] This article also provides a method for binding intracellular TDP-43 protein in cells, comprising contacting the cells with a cell penetrant as described in any one of claims 1 to 135, thereby causing at least an antigen-binding fragment of the antibody to be internalized and transferred into the cytosol.

[0074] This document also provides a method for binding intracellular TDP-43 protein in cells, comprising: contacting the cells with a cell penetrant as described in any one of claims 1 to 135, thereby causing at least an antigen-binding fragment of an antibody to be internalized and translocated into the cytosol; and binding at least an antigen-binding fragment of an antibody to the intracellular TDP-43 protein.

[0075] In some embodiments, the cells are mammalian cells. In some embodiments, the contact is in vitro. In some embodiments, the cells are in a subject.

[0076] This article also provides a method for inhibiting or reducing the accumulation of TDP-43 (e.g., human TDP-43) in subjects who have TDP-43-related diseases or are at risk of developing such diseases, comprising administering to the subject an effective amount of any of the cell-penetrating agents described herein, thereby inhibiting or reducing the accumulation of TDP-43 (e.g., human TDP-43) in the subject.

[0077] This article also provides a method for treating or achieving prevention of TDP-43-related disease in subjects, which includes administering a therapeutically effective amount of any of the cell-penetrating agents described herein, thereby treating or achieving prevention of TDP-43-related disease.

[0078] In some implementations, TDP-43-related diseases include amyotrophic lateral sclerosis (ALS), frontotemporal degeneration (FTLD-TDP), primary spinal lateral sclerosis, progressive muscular atrophy, and Parkinson's disease. In some implementations, TDP-43-related diseases include ALS.

[0079] This article also provides a method for detecting TDP-43 deposition (e.g., human TDP-43 deposition) in subjects who have TDP-43-related diseases or are at risk of developing such diseases, comprising administering any of the cell-penetrating agents described herein to the subject and detecting antibodies that bind to TDP-43 in the subject.

[0080] In some implementations, antibodies are administered by intravenous injection into the subject's body.

[0081] This article also provides a method for detecting TDP-43 in samples obtained from patients with TDP-43-related diseases or at risk of developing said diseases, comprising contacting the sample with any of the cell-penetrating agents described herein, and detecting antibodies against TDP-43 in the sample.

[0082] In some embodiments, the antibody is labeled. In some embodiments, the antibody is labeled with fluorescent, paramagnetic, or radioactive labels. In some embodiments, positron emission tomography (PET) or single-photon emission computed tomography (SPECT) is used to detect the radioactive label.

[0083] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference as if each individual publication, patent, patent application, or information item expressly and individually indicated to be incorporated by reference. To the extent that any incorporated publication, patent, patent application, or information item contradicts the disclosure contained in this specification, this specification is intended to supersede and / or give precedence to any such contradictory material.

[0084] When using ranges to describe values, it should be understood that the description includes disclosing all possible subranges within those ranges, as well as specific values ​​falling within those ranges, regardless of whether a specific value or a specific subrange is explicitly stated. Attached Figure Description

[0085] The following figures illustrate certain embodiments of the features and advantages of this disclosure. These embodiments are not intended to limit the scope of the appended claims in any way. Like reference numerals in the figures indicate like elements.

[0086] Figure 1 This shows the annotation format of the variable domain of the 13D3 heavy chain of the mouse antibody.

[0087] Figure 2 This shows the annotation format of the variable domain of the light chain of the murine antibody 13D3.

[0088] Figure 3 A graph showing the binding data of chimeric 13D3 antibody and its humanized form to phosphorylated human TDP-43 peptide.

[0089] Figure 4 A graph showing the binding data of chimeric 13D3 antibody and its humanized form to phosphorylated human TDP-43 peptide.

[0090] Figure 5 A graph showing the binding data of the humanized form of the 13D3 antibody hu13D2Hd5Ld2 to phosphorylated human TDP-43 peptide.

[0091] Figures 6A through 6C show immunohistochemical images of frontotemporal dementia (“FTD”) brain tissue stained with 13D3 antibody (Figures 6A and 6B) and healthy brain tissue (Figure 6C). Figure 6B is an inset of Figure 6A, showing the colocalization of 13D3 antibody with neuronal cytoplasmic aggregates associated with phosphorylated human TDP-43 FTD.

[0092] Figures 7A to 7C Immunohistochemical images of brain tissue from a TDP-43 proteopathic model stained with 13D3 antibody and inhibited by rNLS8 dox. The data show that 13D3 antibody binding to rNLS8 dox in the TDP-43 proteopathic model inhibits cytoplasmic aggregates.

[0093] Figures 8A to 8C The results show the effects of HEK cells transfected with GFP-2a-TDP43 or GFP alone. Figure 8A The staining of pTDP-43, GFP, and cell nuclei in transfected cells is shown. Figure 8B A graph showing cell counts. Figure 8C Immunohistochemical images of HEK cells are shown, demonstrating the ability of antibodies 13D3, 13C13, and 2D4 to detect mislocalized human TDP-43 overexpressed in HEK cells.

[0094] Figures 9A to 9B To show the percentage of CPA-positive cells after co-incubation with mouse 13D3 (m13D3) CPA ( Figure 9A ) and the number of CPA-positive spots per cell after incubation with m13D3 CPA ( Figure 9B (The image is shown.)

[0095] Figures 10A to 10EA graph showing the results of HEK cells transfected with different m13D3 CPAs, the results including the number of lesions per well area ( Figure 10A ), average focus intensity ( Figure 10B ), Cell count per well ( Figure 10C ), the number of TDP-43 lesions normalized by cell count ( Figure 10D ) and average lesion area ( Figure 10E ).

[0096] Figures 11A to 11D A graph showing the results of HEK cells transfected with different m13D3 CPAs, the results including the number of lesions per well area ( Figure 11A ), cell count ( Figure 11B ), lesion count ( Figure 11C ) and average lesion size ( Figure 11D ).

[0097] Figures 12A to 12D A graph showing the results of HEK cells transfected with different m13D3 CPAs, the results including the total lesion area per well ( Figure 12A ), cell count ( Figure 12B pTDP-43 lesion count ( Figure 12C ) and average lesion size ( Figure 12D ).

[0098] Figure 13 A graph showing the percentage of cell death in untransfected HEK cells (left) or GFP-2a-TDP43-transfected cells (right).

[0099] Figures 14A to 14E This is a diagram illustrating the internalization and co-localization of phosphorylated cytoplasmic aggregates of TDP-43 in HEK cells. Specifically, the diagram shows the sum of pTDP-43 lesions per well area ( Figure 14A ), average focus intensity ( Figure 14B ), cell count ( Figure 14C ), number of p-TDP-43 lesions ( ) and average lesion area ( Figure 14D ).

[0100] Figure 14E A graph showing the results of HEK cells incubated with m13D3 m-Lycotoxin [L17E] CPA or m13D3 CMIP4 CPA, the results including total lesion area ( Figures 15A to 15D ), cell count ( Figure 15A ), number of p-TDP-43 lesions ( Figure 15B ) and average lesion area ( Figure 15C ).

[0101] [[ID=4l]]Figure 15D A graph showing the results of HEK cells transfected with m13D3 CMIP4 CPA or unlabeled antibody in acetate buffer or PBS, the results including lesions per well area ( Figures 16A to 16E ), average focus intensity ( Figure l6A ), cell count ( Figure 16B ), number of p-TDP-43 lesions ( Figure 16C ) and average lesion area ( Figure 16D ).

[0102] Figure 16E Results showing HEK cells transfected with GFP-2A-TDP43 and chimeric 13D3 CPA, including colocalization imaging ( Figures 17A to l7B ), and a graph showing the percentage of p-TDP-43 co-localized with GFP-2A-TDP43 and chimeric 13D3 CPA ( Figure 17A ).

[0103] Figure 1nB A graph showing the internalization data of anti-TDP-43 CPA, including the percentage of pTDP-43 co-localized with anti-TDP-43 CPA ( Figures 18A to 18D ), the average number of 13D3 antibody spots per cell ( Figure 18A ), average spot size ( Figure 18B and average spot size (for spot intensity correction) Figure 18C ).

[0104] Figure 18D A graph showing the internalization and co-localization of phosphorylated cytoplasmic aggregates of pTDP-43, including the percentage of pTDP-43 co-localized with humanized 13D3 antibody, chimeric 13D3 antibody, and control. Figures 19A to 19D ), cell count ( Figure 19A ), area of ​​each pore spot ( Figure 19B ) and CPA spots per cell ( Figure 19C ).

[0105] Figure 19D Maps showing the internalization and co-localization of phosphorylated cytoplasmic aggregates of TPD-43 in glioblastoma cells, including total lesion area ( Figures 20A to 20C ), average lesion size ( Figure 20A ) and total lesion count ( Figure 20B ).

[0106] Figure 20C This demonstrates the internalization and co-localization of phosphorylated cytoplasmic aggregates of TDP-43 in primary rat cortical neurons. Figures 21A to 21BImages showing primary rat cortical neurons treated with various CPAs. Figure 21A A graph showing the total lesion area for each well.

[0107] Figure 21B A graph showing the results of transfected primary rat cortical neurons, the results including the total number of 13D3 spots per cell ( Figures 22A to 22F ), the total area of ​​13D3 spots in each well ( Figure 22A ), average spot size ( Figure 22B ), spot integral intensity ( Figure 22C The percentage of 13D3 co-localized with early endosomal antigen 1 (EEA1) Figure 22D ) and cell count ( Figure 22E ). Detailed Implementation

[0108] This disclosure provides systems and methods for delivering antibodies into cells. Specifically, this disclosure describes cell penetrants comprising an internalization module and a TDP-43-specifically binding agent (e.g., human TDP-43) or TDP-43 aggregates (e.g., human TDP-43 aggregates including phosphorylated aggregates of TDP-43). Pharmaceutical compositions are also provided comprising these cell penetrants and a pharmaceutically acceptable carrier, a nucleic acid and / or vector encoding these cell penetrants, and a host cell expressing the aforementioned nucleic acid and / or vector.

[0109] I. Definition The term "antibody" includes both intact antibodies and their antigen-binding fragments. Typically, fragments compete with the intact antibody from which they are derived for specific binding to a target, including individual heavy chains, light chains Fab, Fab', F(ab')2, F(ab)c, Dab, nanobodies, and Fv. Fragments can be generated via recombinant DNA technology or through enzymatic or chemical separation of intact immunoglobulins. The term "antibody" also includes bispecific or multispecific antibodies and / or humanized antibodies. Bispecific or bifunctional or multifunctional antibodies are artificial hybrid antibodies having two or more distinct heavy / light chain pairs and two or more distinct binding sites (see, for example, Songsivilai and Lachmann). Figure 22F ., 79:315-321 (1990); Kostelny Clin. Exp. Immunol , et al ., 148:1547-53 (1992)).

[0110] As used herein, a “cell penetrant” (also referred to herein as “CPA”) means an agent (e.g., a molecule and / or molecular complex) capable of entering cells (e.g., in vitro and / or in vivo mammalian cells). In some embodiments, the CPA enters the cell and is transferred to the cytosol after being internalized by the cell. In some embodiments, the CPA comprises a cell internalization module (CIM) that facilitates the internalization of the CPA by the cell. In some cases, the CPA comprises a CIM that facilitates the internalization of the CPA and its transfer to the cytosol. In some embodiments, the CPA further comprises an anti-TDP-43 antibody linked (e.g., covalently or non-covalently) to the CIM. In some embodiments, the CPA comprises a CIM covalently linked to the anti-TDP-43 antibody (e.g., via a linker between the CIM and the anti-TDP-43 antibody). In other embodiments, the CPA comprises a CIM non-covalently linked to the anti-TDP-43 antibody (e.g., via a streptavidin-biotin interaction) such that the CIM remains linked to the anti-TDP-43 antibody under relevant conditions (e.g., plasma). In some embodiments, the CPA further comprises a linker linking the CIM to an anti-TDP-43 antibody. In various cases, the linker may be cleavable or non-cleavable and / or may covalently or non-covalently link the CIM to the anti-TDP-43 antibody. In some embodiments, the CPA containing the anti-TDP-43 antibody has enhanced cell penetration compared to a reference anti-TDP-43 antibody that is not part of the CPA. In some embodiments, the CPA comprises two or more anti-TDP-43 antibodies and / or two or more CIMs (e.g., a CPA comprising a dendritic polymer linked to a plurality of CIMs and / or an anti-TDP-43 antibody). Non-limiting features and examples of cell penetrants are described herein.

[0111] As used herein, an "internalization module" (also referred to herein as "CIM") refers to a portion of the CPA that promotes internalization (and the extended anti-TDP-43 antibody). In various embodiments, the CIM may utilize one or more internalization processes, including both active and passive internalization. Exemplary processes include, but are not limited to, endocytosis (e.g., receptor-mediated endocytosis (RME), phagocytosis, pinocytosis) and membrane translocation (e.g., direct penetration and / or energy-independent internalization). In various non-limiting embodiments, the CIM comprises, for example, a cell membrane internalization peptide (CMIP), a small molecule ligand (e.g., vitamins, fatty acids, integrin-binding ligands, etc.), a portion of an antibody (e.g., the scFv portion that binds to an internalized cell surface target), or a decoy receptor ligand (e.g., a cytokine or a derivative thereof). In some embodiments, the CIM comprises a cell membrane internalization peptide (CMIP). Non-limiting features and examples of the cell internalization module are described herein.

[0112] As used herein, a “cell membrane internalization peptide” (also referred to herein as a CMIP) is a sequence of three or more natural or non-natural amino acids that, when covalently or non-covalently linked to an anti-TDP-43 antibody, results in the internalization of at least the anti-TDP-43 antibody or an active fragment of the anti-TDP-43 antibody into mammalian cells. In various embodiments, the CMIP may be internalized using one or more cell internalization processes, including both active and passive cell internalization. Exemplary processes include, but are not limited to, endocytosis and membrane translocation. In some embodiments, the CMIP interacts with cell membrane and / or cell surface antigens, resulting in the internalization of the CPA or a portion thereof (e.g., containing a portion of an anti-TDP-43 antibody or its functional portion) via endocytic vesicles. In some embodiments, the CMIP further facilitates the transfer of the CPA or a portion thereof from endocytic vesicles into the cell. In some embodiments, CMIPs further interact with the membrane of endocytic vesicles, leading to vesicle rupture and escape of the CPA or a portion thereof into the cytosol. Therefore, in some embodiments, CMIPs promote the internalization of anti-TDP-43 antibodies or a functional portion thereof into the cell, and the translocation of anti-TDP-43 antibodies or a functional portion thereof into the cytosol. Non-limiting examples of CMIPs include, for example, cationic peptides (including, for example, M-lycotoxin, TAT peptide, pentetratin, and polyarginine peptides and derivatives thereof), amphiphilic peptides (including, for example, MPG peptide, Pep-1 peptide, transporter peptides and derivatives thereof), and proline-rich peptides (including, for example, Bac7 peptide and derivatives thereof). In some embodiments, CMIPs include cell-penetrating peptides and derivatives thereof, including, for example, those by Rusteska and A. Zimmer. J. Immunol , Those described in . 202; 11:101-123. This document describes non-limiting features and examples of CMIP.

[0113] The term "epitope" refers to the site on an antigen where an antibody binds. Epitopes can be formed from consecutive amino acids or from discontinuous amino acids juxtaposed through the ternary folding of one or more proteins. Epitopes formed from consecutive amino acids (also known as linear epitopes) are generally preserved upon exposure to denaturing solvents, while epitopes formed through ternary folding (also known as conformational epitopes) are generally lost upon treatment with denaturing solvents. Epitopes typically consist of at least three, and more commonly at least five or eight to ten amino acids, exhibiting a unique spatial conformation. Methods for determining the spatial conformation of epitopes include, for example, X-ray crystallography and two-dimensional nuclear magnetic resonance (NMR). Internalization mechanisms of cell-penetrating peptides See For example, Epitope Mapping Protocols, in Methods in Molecular Biology, Vol. 66, edited by Glenn E. Morris (1996).

[0114] Antibodies that recognize the same or overlapping epitopes can be identified in a simple immunoassay that shows the ability of one antibody to compete with another for binding to a target antigen. An antibody epitope can also be defined by X-ray crystallography of the antibody binding to its antigen to identify contact residues. Alternatively, if all amino acid mutations in the antigen that reduce or eliminate the binding of one antibody reduce or eliminate the binding of another antibody, then the two antibodies have the same epitope. If some amino acid mutations that reduce or eliminate the binding of one antibody reduce or eliminate the binding of another antibody, then the two antibodies have overlapping epitopes.

[0115] The terms "humanized immunoglobulin" or "humanized antibody" refer to a chain that includes at least one humanized immunoglobulin or antibody chain. e.g At least one humanized light or heavy chain of immunoglobulin or antibody. The terms "humanized immunoglobulin chain" or "humanized antibody chain" (…) i.e "Humanized immunoglobulin light chain" or "humanized immunoglobulin heavy chain" refers to an immunoglobulin or antibody chain with a variable region. i.e The variable region (either a light chain or a heavy chain) comprises a variable framework region substantially derived from human immunoglobulins or antibodies and a complementation-determining region (CDR) substantially derived from non-human immunoglobulins or antibodies (e.g., at least one CDR, preferably two CDRs, more preferably three CDRs), and further comprises a constant region (e.g., at least one constant region or a portion thereof in the case of the light chain, and preferably three constant regions in the case of the heavy chain). The term "humanized variable region" (e.g., "humanized light chain variable region" or "humanized heavy chain variable region") refers to a variable region comprising a variable framework region substantially derived from human immunoglobulins or antibodies and a complementation-determining region (CDR) substantially derived from non-human immunoglobulins or antibodies.

[0116] Competition between antibodies is determined by measuring the specific binding of the test antibody to the reference antibody and the common antigen. i.e For example Junghans (See 50: 1495, 1990). If an excessive amount of test antibody is used... ( For example, at least 2x, 5x, 10x, 20x, or 100x) inhibits the binding of the reference antibody by at least 50%, as measured in a competitive binding assay, where the test antibody competes with the reference antibody. Some test antibodies inhibit the binding of the reference antibody by at least 75%, 90%, or 99%. Antibodies identified by competitive assays (competitive antibodies) include antibodies that bind to the same epitope as the reference antibody and antibodies that bind to adjacent epitopes that are sufficiently close to the epitope bound by the reference antibody to cause steric hindrance.

[0117] As used herein, a linker is a chemical motif that does not possess catalytic or therapeutic activity in mammalian cells and is used to covalently link two different functional molecules (e.g., an intracellular module and an anti-TDP-43 antibody). For example, a linker may be a peptide of about 3 to about 25 amino acids (e.g., about 3 to about 20 or about 3 to about 12 amino acids). In other instances, a linker may be a bond (e.g., an amide bond, an ester bond, an ether bond, and a disulfide bond). In some instances, a linker may contain a pair of affinity domains (e.g., the first domain of the pair of affinity domains may be interleukin-15, and the second domain of the pair of affinity domains may be the sushi domain of the interleukin-15 receptor α). In some instances, a linker is a glycine residue followed by a serine residue (GS). In some instances, a linker is three consecutive glycine residues (e.g., GGG). In some instances, a linker is any of SEQ ID NO: 194-199.

[0118] The term "pharmaceutically acceptable" means that the carrier, diluent, excipient, or adjuvant is compatible with the other components of the formulation and is substantially harmless to the recipient.

[0119] As used herein, “M-lycotoxin derivative” is a peptide consisting of three or more amino acids (natural or non-natural) designed based on a starting M-lycotoxin peptide. In some embodiments, “M-lycotoxin derivative” is a polypeptide having, for example, 60% to 99% sequence homology with wild-type M-lycotoxin. Other non-limiting aspects and examples of M-lycotoxin derivatives are described herein.

[0120] As used herein, the term "macromolecule" refers to a molecule with a molecular weight of at least 5 kDa and / or a hydrodynamic radius of at least 1.0 nm. As used herein, the term "macromolecule" includes biomolecules, organic polymers, and organometallic complexes.

[0121] As used herein, a "spacer" or "spacer region" refers to an amino acid that does not possess catalytic or therapeutic activity in mammalian cells. For example, a spacer may be a peptide of 1 to about 10 amino acids (e.g., 1 to about 8 amino acids, 1 to about 6 amino acids, or 1 to about 4 amino acids). In some instances, one or more spacer regions are included within an internalization module. For example, a spacer region may separate amino acids within an internalization module; for instance, a spacer may be positioned after the first amino acid of the CIM. In some instances, a spacer may be positioned before the last amino acid of the CIM. In some instances, the CIM may have two or more spacer regions (e.g., two spacer regions, three spacer sequences, four spacer regions, five spacer regions, or more). In some instances, a spacer region is a single glycine residue. In some instances, a spacer region is a pair of glycine residues. In some instances, a spacer region is three glycine residues. In some instances, a spacer region is four glycine residues, followed by a serine residue. In some instances, the spacer is any one of SEQ ID NO:200-203.

[0122] The term "TDP-43-related disease" refers to a disease or condition that is at least partly directly or indirectly characterized and / or mediated by the formation of TDP-43 aggregates and / or the mislocalization of TDP-43. This document describes non-limiting examples of TDP-43-related cancers.

[0123] The terms “patient” or “subject” include human subjects and other mammalian subjects (e.g., humans) who receive preventative or therapeutic treatment.

[0124] If the subject has at least one known risk factor ( For example, genetic, biochemical, family history, and situational exposures, which give subjects with said risk factors a statistically significant greater risk of disease than subjects without said risk factors, thus increasing the subject's risk of disease.

[0125] The term "biological sample" refers to a sample of biological material that is present in or available from a biological source (e.g., a human or mammalian subject). Such samples can be organs, organelles, tissues, tissue sections, body fluids, peripheral blood, plasma, serum, cells, molecules (such as proteins and peptides), and any part or combination thereof derived therefrom. The term "biological sample" may also cover any material derived by processing the sample. Derived material may include cells or their progeny. Processing of biological samples may involve one or more of the following: filtration, distillation, extraction, concentration, fixation, deactivation of interfering components, etc.

[0126] The term "control sample" refers to a biological sample that is unknown or suspected of including a TDP-43-affected region, or at least unknown or suspected of including a diseased region of a given type. Control samples may be obtained from individuals who do not have TDP-43-related disease. Alternatively, control samples may be obtained from patients who have TDP-43-related disease. Such samples may be obtained simultaneously with or on different occasions from biological samples believed to contain TDP-43-related disease. The biological sample and control sample may be obtained from the same tissue. Preferably, the control sample consists substantially or entirely of normal healthy regions and can be used for comparison with biological samples believed to contain regions affected by TDP-43-related disease. Preferably, the tissue in the control sample is of the same type as the tissue in the biological sample. Preferably, the TDP-43-related disease-affected cells believed to be in the biological sample are derived from the same cell type (e.g., neurons or glial cells) as the cell type in the control sample.

[0127] For the purpose of classifying amino acid substitutions as conserved or non-conserved, amino acids are grouped as follows: Group I (hydrophobic side chains): Met, Ala, Val, Leu, Ile; Group II (neutral hydrophilic side chains): Cys, Ser, Thr; Group III (acidic side chains): Asp, Glu; Group IV (basic side chains): Asn, Gln, His, Lys, Arg; Group V (residues affecting chain orientation): Gly, Pro; and Group VI (aromatic side chains): Trp, Tyr, Phe. Conservative substitution involves substitution between amino acids within the same category. Non-conservative substitution is equivalent to exchanging a member of one category for a member of another category.

[0128] The percentage of sequence identity was determined using antibody sequences that were maximized according to the Kabat numbering convention. After alignment, if the antibody region of the present invention is... ( For example, the entire mature variable region of the heavy chain or light chain is compared with the same region of the reference antibody. The percentage of sequence identity between the antibody region of the present invention and the reference antibody region is calculated by dividing the number of positions occupied by the same amino acids in the regions of the present invention and the reference antibody by the total number of alignment positions of the two regions (excluding vacancies) and multiplying by 100 to convert it into a percentage.

[0129] Unless otherwise apparent from the context, the term "about" covers non-substantial changes, such as those within the standard measurement error limits of a specified value. ( For example, the values ​​within SEM.

[0130] The phrase "substantially derived from human immunoglobulins or antibodies" means that, when compared with the amino sequence of human immunoglobulins or antibodies for comparative purposes, the region shares at least 80-90%, preferably 90-95%, and more preferably 95-99% identity with the human framework or constant region sequence. et al., Cancer Res.This allows for, for example, conserved substitutions, shared sequence substitutions, germline substitutions, and reversion mutations. The introduction of conserved substitutions, shared sequence substitutions, germline substitutions, and reversion mutations is generally referred to as the "optimization" of humanized antibodies or chains. The phrase "substantially derived from non-human immunoglobulins or antibodies" or "substantially non-human" means having an immunoglobulin or antibody sequence that is at least 80-95%, preferably 90-95%, more preferably 96%, 97%, 98%, or 99% identical to that of a non-human organism (e.g., a non-human mammal).

[0131] Therefore, all regions or residues (possibly excluding CDRs) of a humanized immunoglobulin or antibody, or a humanized immunoglobulin or antibody chain, are substantially identical to the corresponding regions or residues of one or more natural human immunoglobulin sequences. The terms "corresponding region" or "corresponding residue" refer to a region or residue on a second amino acid or nucleotide sequence that occupies the same position as on a first amino acid or nucleotide sequence when the first and second sequences are optimally aligned for comparative purposes. i.e (equivalent) regions or residues.

[0132] II. TDP-43 As described in this article, TDP-43 is a nuclear protein primarily involved in RNA splicing, transport, stabilization, and ultimately, regulation of gene expression. More specifically, TDP-43 is a multi-domain heterogeneous ribonucleoprotein (hnRNP). Proper function of TDP-43 is crucial for regulating the hundreds of mRNA transcripts it binds to. One of TDP-43's primary functions is regulating spliced ​​mRNA transcripts; however, TDP-43 also participates in various mechanisms of RNA processing and transport. For example, TDP-43 is an inhibitor of cryptic exon inclusion bodies and regulates alternative polyadenylation in >1,000 genes. TDP-43 can form ribonucleoprotein particles in various cell types, including Cajal bodies and paramalfoci in the nucleus, and is recruited to mRNA transport particles in neurons. As described in this article, various neurodegenerative diseases are associated with cytoplasmic aggregates of TDP-43 (e.g., TDP-43-related diseases).

[0133] Unless otherwise obvious from the context, references to TDP-43 refer to the natural human form of TDP-43, including any isoforms and / or post-translational modifications (e.g., phosphorylation, glycosylation, and / or acetylation). The amino acid sequence of human TDP-43 is shown below (SEQ ID NO: 82): MSEYIRVTEDENDEPIEIPSEDDGTVLLSTVTAQFPGACGLRYRNPVSQCMRGVRLVEGILHAPDAGWGNLVYVVNYPKDNKRKMDETDASSAVKVKRAVQKT SDLIVLGLPWKTTEQDLKEYFSTFGEVLMVQVKKDLKTGHSKGFGFVRFTEYETQVKVMSQRHMIDGRWCDCKLPNSKQSQDEPLRSRKVFVGRCTEDMTEDEL REFFSQYGDVMDVFIPKPFRAFAFVTFADDQIAQSLCGEDLIIKGISVHISNAEPKHNSNRQLERSGRFGGNPGGFGNQGGFGNSRGGGAGLGNNQGSNMGGGMNFGAFSINPAMMAAAQAALQSSWGMMGMLASQQNQSGPSGNNQNQGNMQREPNQAFGSGNNSYSGSNSGAAIGWGSASNAGSGSGFNGGFGSSMDSKSSGWGM TDP-43 can be phosphorylated at one or more amino acids, including serine at positions 409 and 410. In some embodiments, TDP-43 can be phosphorylated at one or more positions, including 373, 375, 379, 387, 389, 393, 395, 403, 404, 407, 409, and 410 (see, for example, Gruijs da Silva, LA, et al., Disease-linked TDP-43 hyperphosphorylation suppresses TDP-43 condensation and aggregation, The EMBO Journal, 41: e108443 (2022)).

[0134] Unless otherwise obvious from the context, references to TDP-43 or fragments thereof include natural human amino acid sequences, including isoforms, mutants, and allele variants. The ability of an antibody or antigen-binding antibody fragment to bind to TDP-43 can be determined using, for example, surface plasmon resonance.

[0135] III. Anti-TDP-43 cell penetrant This disclosure provides a cell-penetrating agent (as described herein) comprising an internalization portion and an antibody or antigen-binding fragment thereof that specifically binds to TDP-43 (e.g., human TDP-43). In some embodiments, the antibody or antigen-binding antibody fragment described herein specifically binds to phosphorylated TDP-43 (e.g., phosphorylated human TDP-43). In some embodiments, the antibody or antigen-binding antibody fragment described herein specifically binds to phosphorylated TDP-43 (e.g., phosphorylated human TDP-43), wherein one or both of serine residues at positions 409 and / or 410 of SEQ ID NO: 82 are phosphorylated. In some embodiments, the antibody or antigen-binding antibody fragment binds a 23-amino acid peptide comprising amino acids at positions 392 to 414 of SEQ ID NO: 82 (“TDP-43 (pS409 / pS410)”), wherein serine residues at positions 409 and 410 are phosphorylated.

[0136] Furthermore, this disclosure relates to cell penetrants comprising an internalized portion and an antibody specifically binding to human TDP-43, compositions comprising such cell penetrants, and methods of using these cell penetrants to treat TDP-43-related diseases, including amyotrophic lateral sclerosis (ALS).

[0137] Throughout this disclosure, references to anti-TDP-43 CPA and / or anti-TDP-43 antibodies will be made in the context of cell internalization and / or intracellular function. However, those skilled in the art will understand that such references include intact anti-TDP-43 CPA and / or anti-TDP-43 antibodies, chemically modified (e.g., oxidized, reduced) anti-TDP-43 CPA and / or anti-TDP-43 antibodies, and their partially degraded functional fragments (intact anti-TDP-43 CPA, CPA fragments, intact anti-TDP-43 antibodies and / or anti-TDP-43 antibody fragments, etc.). Therefore, references to the internalization, cytosol transfer, and target binding of anti-TDP-43 CPA or anti-TDP-43 antibodies will include references to intact anti-TDP-43 CPA and / or anti-TDP-43 antibodies, chemically modified anti-TDP-43 CPA and / or anti-TDP-43 antibodies, and their partially degraded functional fragments. For example, the methods disclosed herein may refer to the internalization and / or transfer of cytosol of anti-TDP-43 antibody, but it should be understood that such references cover the transfer of intact anti-TDP-43 CPA and / or functional fragments of anti-TDP-43 CPA, which contain anti-TDP-43 antibody or a portion thereof, and its chemically modified derivatives.

[0138] The cell-penetrating agent of this disclosure can utilize one or more of several biochemical processes to achieve the internalization of anti-TDP-43 antibodies. In various embodiments, the anti-TDP-43 CPA of this disclosure can utilize passive internalization, active internalization, or a combination thereof. In some embodiments, the anti-TDP-43 CPA utilizes active internalization (e.g., endocytosis) to achieve intracellular delivery of anti-TDP-43 antibodies.

[0139] In various embodiments, anti-TDP-43 CPA is internalized into the cell via endocytosis. Upon internalization, anti-TDP-43 CPA achieves endosome escape. In such embodiments, anti-TDP-43 CPA is thereby transferred to the cytosol, where the anti-TDP-43 antibody can bind to TDP-43 (e.g., phosphate-TDP-43). In some embodiments, endosome escape of anti-TDP-43 CPA is achieved through the interaction of the CIM with the endosome membrane, thereby leading to endosome membrane disruption. The CIM can achieve endosome escape using one or more methods. For example, the CIM may contain a cationic moiety (e.g., a positively charged amino acid, cationic polymer and / or oligomer, cationic lipid). In some such embodiments, the cationic moiety can interact with negatively charged phospholipids constituting the endosome membrane, thereby disrupting the endosome membrane and achieving endosome escape of anti-TDP-43 CPA. The anti-TDP-43 CPA of this disclosure may also contain a CIM having an amphiphilic moiety (e.g., an amphiphilic peptide). In some such embodiments, the amphiphilic portion can interact with the endosome membrane via hydrophobic interactions with membrane lipids, thereby disrupting the endosome membrane and achieving endosome escape of the anti-TDP-43 CPA. Following endosome escape of the anti-TDP-43 antibody, the antibody subsequently translocates to the cytosol, where it can bind to TDP-43 (e.g., phosphate-TDP-43). Other examples of internalization mechanisms that CPA can utilize are described in detail herein.

[0140] Therefore, this document provides cell penetrants comprising: (i) a cell internalization module (CIM) and (ii) an antibody that specifically binds to TDP-43 (e.g., human TDP-43). Various aspects of the cell penetrants disclosed herein, including examples of the cell internalization portion, anti-TDP-43 antibody, and / or optionally present linkers, are described below. Those skilled in the art will understand how the various CIMs, anti-TDP-43 antibodies, and optionally present linkers disclosed herein can be combined. Such examples are merely illustrative of the scope of this disclosure and are not limiting.

[0141] i.e As used herein, a “cell internalization module” or “CIM” is a composition that, when covalently or non-covalently linked to an anti-TDP-43 antibody molecule, results in the internalization of at least an anti-TDP-43 antibody or an active fragment thereof into the cell. Non-limiting features and examples of cell internalization modules are described herein.

[0142] In some embodiments, CIM is a peptide (e.g., an amino acid sequence). In such instances, CIM may also be referred to as a cell membrane internalization peptide or "CMIP" as further defined herein. Exemplary cell membrane internalization peptides include natural peptides and their derivatives, as well as synthetic peptides. Non-limiting examples of CMIPs include M-lycotoxin and its derivatives, TAT and its derivatives, PEPTH, polyarginine sequences, Penetratin, DPT-C9h, DPT-C9, Transportan, Xentry, Pep-1, Pep-7, Aurein 1.2, MTS, GFWFG, DPV1047, MPG, pVEC, ARF(1-22), BPrPr, MAP, p28, VT5, Bac7, C105Y, PFVYLI, and BR2.

[0143] In some implementations, the CIM is a non-peptide portion (e.g., a ligand) internalized by a cell (e.g., a mammalian cell). In such instances, the ligand can induce receptor-mediated internalization of the anti-TDP-43 antibody. Typically, ligand internalization is a receptor-mediated endocytosis process, in which the cell takes up extracellular molecules (including therapeutic agents) if the ligand binds to its homologous receptor protein on the cell surface. Receptor-mediated internalization also includes transcytosis.

[0144] CIM enables the internalization of anti-TDP-43 antibodies into mammalian cells. Generally, cell internalization is broadly classified as endocytosis. Typically, the endocytic pathway can be subdivided into two broader categories: phagocytosis and pinocytosis. During pinocytosis, the plasma membrane absorbs solutes, while during phagocytosis, much larger vesicles are internalized. Pinocytosis is often further subdivided into macropinocytosis, clathrin-dependent endocytosis (e.g., receptor-mediated endocytosis), caverin-dependent endocytosis, and clathrin / cavitin-independent endocytosis. (See, for example, Marsh, M. Endocytosis, Oxford University Press (2001); Doherty, GJ and McMahon, HT, Mechanisms of Endocytosis, Cell internalization module, 78:31.1-31.46 (2009); and Xu, Y. et al., Endocytosis and membrane receptor internalization: implication of F-BARprotein Carom, Annu. Rev. Biochem. , 22: 1439-1457 (2017), each of which is incorporated herein by reference in its entirety.

[0145] Ligand-mediated endocytosis is a mechanism by which cells internalize specific macromolecules. In some instances, the cell membrane (e.g., the plasma membrane) includes clathrin pits that protrude from the cell membrane to form small vesicles called clathrin-coated vesicles. These clathrin-coated vesicles contain receptors and bound macromolecules, i.e., ligands. The clathrin-coated vesicles then fuse with early endosomes (vesicles composed of tubular extensions located around the cell periphery). Endosomals have an acidic environment (pH 6.0–6.2) that promotes the dissociation of receptors from ligands. The ingested contents are then sorted out for recycling back to the plasma membrane or transported to lysosomes for degradation.

[0146] Peptide-based cell membrane initiation agents (CIMs) such as CMIPs internalize anti-TDP-43 antibodies via multiple mechanisms. Generally, CMIPs have been shown to utilize either endocytosis (e.g., energy-dependent internalization) or direct penetration (e.g., translocation) (energy-independent internalization) as two main internalization mechanisms, as described above. For direct penetration, various mechanisms have been described, including carpet-like models (membrane destabilization) and pore-forming models (barrel-plate). Positively charged CMIPs can interact with negatively charged membrane components (such as the phospholipid bilayer), subsequently destabilizing the membrane, and CMIPs can cross the lipid bilayer with anti-TDP-43 antibodies. Studies have shown that several CMIPs can induce different uptake mechanisms and switch between different uptake mechanisms depending on their concentration, cargo, and / or the cell line used (see, for example, Rusteska, I. and Zimmer, A., Internalization mechanisms of cell-penetrating peptides, Front Biosci , 11: 101-123, (2020)).

[0147] Once internalized into the cell, anti-TDP-43 antibodies typically need to evade the endosome pathway. Generally, the endocytic pathway in mammalian cells consists of different membrane compartments that internalize molecules from the plasma membrane (i.e., cell permeators) and recycle membrane-bound receptors back to the surface or sort internalized molecules to various degradation pathways. A key component of the endocytic pathway is the early endosome, which is the first compartment of the endocytic pathway. Early endosomes are typically located at the cell periphery and receive most types of vesicles from the cell surface. These early endosomes have characteristic microtubule-vesicle structures and a weakly acidic pH. Early endosomes are primarily sorting organelles, in which many endocytic ligands dissociate from their receptors in the acidic pH of the compartment and recycle to the cell surface. Early endosomes also undergo transcytosis via transvesicular compartment sorting to later compartments (such as late endosomes or lysosomes).

[0148] Late endosomes typically receive endocytic material in the pathway to lysosomes, usually from early endosomes in the endocytic pathway, from the trans-Golgi network (TGN) in the biosynthetic pathway, and from phagosomes in the phagocytic pathway. Late endosomes are acidic (approximately pH 5.5) and are generally considered to mediate final sorting before delivery of material to lysosomes. Lysosomes are the final compartment of the endocytic pathway. Lysosomes break down cellular waste, fats, carbohydrates, proteins, and other macromolecules into simpler compounds, which are returned to the cytoplasm as new cell building materials. Lysosomes include many different types of hydrolases that function in an acidic environment (e.g., pH approximately 4.8).

[0149] In some embodiments, CIM (as defined herein) comprises a cell membrane-internalizing peptide (e.g., CMIP4, SEQ ID NO: 176). In some embodiments, CIM comprises a wild-type M-lycotoxin peptide (SEQ ID NO: 182). In some embodiments, CIM comprises an M-lycotoxin derivative (e.g., SEQ ID NO: 183). In some embodiments, CIM comprises a Penetain amino acid sequence or a derivative thereof (e.g., SEQ ID NO: 187). In some embodiments, CIM comprises a Pepth amino acid sequence or a derivative thereof (e.g., SEQ ID NO: 184). In some embodiments, CIM comprises a polyarginine amino acid sequence (e.g., SEQ ID NO: 190, SEQ ID NO: 191, and / or SEQ ID NO: 192). In some embodiments, CIM comprises more than one polyarginine amino acid sequence (e.g., 2, 3, 4, 5, or more polyarginine amino acid sequences). In some embodiments, CIM comprises three polyarginine amino acid sequences. In some embodiments, the CIM comprises a TAT amino acid sequence (e.g., SEQ ID NO: 181, SEQ ID NO: 186, SEQ ID NO: 189, and / or SEQ ID NO: 193). In some embodiments, the CIM comprises more than one TAT amino acid sequence or a derivative thereof (e.g., 2, 3, 4, 5, or more TAT amino acid sequences or derivatives thereof). In some embodiments, the CIM comprises three TAT amino acid sequences.

[0150] In some embodiments, the CIM comprises a macrocycle. Typically, a macrocycle is a molecule or ion containing a ring of twelve or more atoms. Classic examples include crown ethers, calixarenes, porphyrins, and cyclodextrins. In some embodiments, the macrocycle is formed by a covalent bond between two amino acid residues of the CIM. In some embodiments, the macrocycle is formed by a disulfide bond between two cysteine ​​residues of the CIM. In some embodiments, the CIM contains one or more histidine residues.

[0151] In some embodiments, CIM comprises a polypeptide having an amino acid sequence selected from any of SEQ ID NO: 176-193. In some embodiments, CIM is a polypeptide having an amino acid sequence selected from any of SEQ ID NO: 176-193.

[0152] In some embodiments, the CIM is covalently linked to an antibody or an antigen-binding antibody fragment thereof. In some embodiments, the CIM is non-covalently linked to an antibody or an antigen-binding antibody fragment thereof. In some embodiments, the cell penetrant comprises a linker that links the CIM to the antibody. In some embodiments, the linker is covalently linked to both the CIM and the antibody. In some embodiments, the linker is a cleavable linker (e.g., a photocleavable linker, a chemical linker, an enzyme-cleavable linker, etc.). In some embodiments, the linker is a non-cleavable linker. In some embodiments, the linker comprises a polypeptide. In some embodiments, the linker comprises one or more glycine residues (e.g., 2, 3, 4, 5 or more glycine residues). In some examples, the linker is a glycine residue followed by a serine residue (GS). In some embodiments, the linker comprises a polypeptide comprising an amino acid sequence selected from any of SEQ ID NO: 194-199. In some embodiments, the linker is a polypeptide having an amino acid sequence selected from any of SEQ ID NO: 194-199.

[0153] In some embodiments, the antibody or antigen-binding antibody fragment of the cell penetrant is attached to the C-terminus of the CIM. In some embodiments, the antibody or antigen-binding antibody fragment of the cell penetrant is attached to the N-terminus of the CIM. In some embodiments, the antibody or antigen-binding antibody fragment of the cell penetrant is attached to the C-terminus of the CIM heavy chain. In some embodiments, the antibody or antigen-binding antibody fragment of the cell penetrant is attached to the N-terminus of the CIM heavy chain. In some embodiments, the antibody or antigen-binding antibody fragment of the cell penetrant is attached to the C-terminus of the CIM light chain. In some embodiments, the antibody or antigen-binding antibody fragment of the cell penetrant is attached to the N-terminus of the CIM light chain.

[0154] In some embodiments, the CIM includes one or more spacer regions. In some embodiments, the CIM does not include spacer regions. For example, a CIM comprising a polypeptide amino acid sequence selected from the following does not include a spacer region: SEQ ID NO: 176-184, SEQ ID NO: 192, and SEQ ID NO: 193.

[0155] In some embodiments, the CIM includes one or more spacer regions. In some embodiments, at least one of the spacer regions contains one or more amino acid residues.

[0156] As used herein, a spacer refers to an amino acid that does not possess catalytic or therapeutic activity in mammalian cells. For example, a spacer may be a peptide of 1 to approximately 10 amino acids (e.g., 1 to approximately 8 amino acids, 1 to approximately 6 amino acids, or 1 to approximately 4 amino acids). In some instances, the spacer region is contained within an internalized module sequence. For example, the spacer region may separate amino acids within the internalized module (CIM), and the spacer may be positioned after the first amino acid of the CIM. In some instances, the spacer may be positioned before the last amino acid of the CIM. In some instances, the CIM may have one or more spacer regions (e.g., two spacer regions, three spacer sequences, four spacer regions, five spacer regions, or more). In some instances, the spacer region is a single glycine residue. In some instances, the spacer region is a pair of glycine residues. In some instances, the spacer region is three glycine residues. In some instances, the spacer region is four glycine residues, followed by a serine residue. In some embodiments, at least one of the one or more spacer regions includes an amino acid sequence selected from any of SEQ ID NO: 200-203. In some embodiments, each of the one or more spacer regions includes an amino acid sequence selected from any of SEQ ID NO: 200-203.

[0157] Beilstein J Nanotechnol In various aspects of this disclosure, the anti-TDP-43 cell penetrant further comprises a linker linking a CIM to an anti-TDP-43 antibody. The linker of this disclosure can link the CIM to the anti-TDP-43 antibody via covalent or non-covalent bonding. In some embodiments, the CIM is covalently linked to the anti-TDP-43 antibody via a linker (i.e., a covalent linker). In some embodiments, the CIM is non-covalently linked to the anti-TDP-43 antibody via a linker (i.e., a non-covalent linker). In some embodiments, the CIM is covalently linked to the linker. In some embodiments, the anti-TDP-43 antibody is covalently linked to the linker. In some embodiments, the linker is covalently linked to both the CPP and the anti-TDP-43 antibody.

[0158] In some embodiments, the linker is a non-covalent linker. Non-covalent linking can be achieved using affinity pairs that interact strongly in a non-covalent manner (e.g., through hydrogen bonding, ionic bonding, van der Waals interactions, or any combination thereof). Several examples of non-covalent linking are known in the art. For example, biotin and a biotin-binding agent (e.g., streptavidin) are examples of affinity pairs. For example, non-covalent linking can be achieved between CIM and an anti-TDP-43 antibody by linking biotin to one side of a CPA (e.g., CIM) and a biotin-binding agent to the other side of the CPA (e.g., an anti-TDP-43 antibody). In some embodiments, the linker comprises a pair of affinity domains (e.g., the first domain of the pair of affinity domains may be interleukin-15, and the second domain of the pair of affinity domains may be the sushi domain of the interleukin-15 receptor α).

[0159] In some embodiments, the linker is a covalent linker. Many covalent linkers are known in the art. For example, in some embodiments, the covalent linker comprises an organic linker (e.g., an alkylene chain, polyethylene glycol chain, polyacrylamide, polyacrylic acid, polyvinyl alcohol, or polyethyleneimine chain). In some cases, the covalent linker comprises an unsubstituted or substituted alkylene chain (including, for example, a polyvinyl alcohol chain, polyacrylamide chain, or polyacrylic acid chain). In some cases, the covalent linker comprises an unsubstituted or substituted heteroalkylene chain (e.g., a polyethylene glycol chain or a polyethyleneimine chain). In various embodiments, the linker is a straight-chain linker or a branched linker. In some such embodiments, the branched linker allows for the incorporation of two or more CIMs and / or anti-TDP-43 antibodies into the CPA (e.g., a dendritic polymer linker structure).

[0160] In some embodiments, the linker comprises amino acid residues. In some embodiments, the linker comprises a polypeptide. In some exemplary embodiments, the linker may be a peptide of about 1 amino acid to about 50 amino acids (e.g., about 1 amino acid to about 40 or about 1 amino acid to about 30 amino acids). In some exemplary embodiments, the linker may be a peptide of about 1 amino acid to about 25 amino acids (e.g., about 1 amino acid to about 20 or about 1 amino acid to about 12 amino acids). In some exemplary embodiments, the linker may be a peptide of about 1 amino acid to about 10 amino acids (e.g., about 1 amino acid to about 6 or about 1 amino acid to about 7 amino acids). In some exemplary embodiments, the linker may be a peptide of about 1 amino acid to about 5 amino acids (e.g., about 1 amino acid to about 4 or about 1 amino acid to about 3 amino acids). In some exemplary embodiments, the linker may be a peptide of about 3 amino acids to about 20 amino acids (e.g., about 3 amino acids to about 15 or about 3 amino acids to about 12 amino acids). In some exemplary embodiments, the linker may be a peptide of about 3 amino acids to about 10 amino acids (e.g., about 3 amino acids to about 8 or about 3 amino acids to about 6 amino acids).

[0161] In some embodiments, the linker comprises glycine residues. In some embodiments, the linker comprises two or more glycine residues. In some embodiments, the linker comprises two or more consecutive glycine residues (e.g., two to three consecutive glycine residues, two to four consecutive glycine residues, two to five consecutive glycine residues, or two to six consecutive glycine residues). In some embodiments, the linker comprises two, three, four, five, or six consecutive glycine residues. In some embodiments, the linker comprises a serine residue. In some embodiments, the linker comprises an amino acid sequence selected from GS, GGG, GGGGS, GGGSGGGS, and GGGGSGGGGS. In some embodiments, the linker comprises the amino acid sequence GGGGS. In some embodiments, the linker comprises the amino acid sequence GGGSGGGS.

[0162] Linker The cell penetrants and methods disclosed herein facilitate the internalization and / or release of various anti-TDP-43 antibodies into cells. In some embodiments, the anti-TDP-43 antibody is a humanized antibody, a chimeric antibody, or a veneered antibody (as described herein). The complementarity-determining region (“CDR”) may be defined by different systems. For example, the CDR described herein may be selected from the group consisting of Kabat, Chothia, Kabat / Chothia Composite, AbM, and Contact.

[0163] In some embodiments, the cell-penetrating antibody comprises a humanized mature heavy chain variable domain comprising: a heavy chain CDR1 as defined by the Kabat / Chothia Composite, comprising SEQ ID NO: 49; a heavy chain CDR2 as defined by Kabat, comprising SEQ ID NO: 51; and a heavy chain CDR3 as defined by Kabat or Chothia, comprising SEQ ID NO: 52; and a humanized mature light chain variable domain comprising the three Kabat light chain CDRs of SEQ ID NO: 53-55.

[0164] In some embodiments, the humanized mature heavy chain variable domain of the antibody comprises a sequence that is at least 80% identical to any of SEQ ID NO: 4-23. In some embodiments, the humanized mature light chain variable domain comprises a sequence that is at least 80% identical to any of SEQ ID NO: 27-48. In some embodiments, the antibody or its antigen-binding fragment comprises a humanized mature heavy chain variable domain comprising a sequence that is at least 80% identical to any of SEQ ID NO: 4-23; and a humanized mature light chain variable domain comprising a sequence that is at least 80% identical to any of SEQ ID NO: 27-48.

[0165] In some embodiments, the humanized mature heavy chain variable domain of the antibody comprises a sequence that is at least 85% identical to any of SEQ ID NO: 4-23. In some embodiments, the humanized mature light chain variable domain of the antibody comprises a sequence that is at least 85% identical to any of SEQ ID NO: 27-48. In some embodiments, the antibody or its antigen-binding fragment comprises a humanized mature heavy chain variable domain comprising a sequence that is at least 85% identical to any of SEQ ID NO: 4-23; and a humanized mature light chain variable domain comprising a sequence that is at least 85% identical to any of SEQ ID NO: 27-48.

[0166] In some embodiments, the humanized mature heavy chain variable domain of the antibody comprises a sequence that is at least 90% identical to any of SEQ ID NO: 4-23. In some embodiments, the humanized mature light chain variable domain of the antibody comprises a sequence that is at least 90% identical to any of SEQ ID NO: 27-48. In some embodiments, the antibody or its antigen-binding fragment comprises a humanized mature heavy chain variable domain comprising a sequence that is at least 90% identical to any of SEQ ID NO: 4-23; and a humanized mature light chain variable domain comprising a sequence that is at least 90% identical to any of SEQ ID NO: 27-48.

[0167] In some embodiments, the humanized mature heavy chain variable domain of the antibody comprises a sequence that is at least 95% identical to any of SEQ ID NO: 4-23. In some embodiments, the humanized mature light chain variable domain of the antibody comprises a sequence that is at least 95% identical to any of SEQ ID NO: 27-48. In some embodiments, the antibody or its antigen-binding fragment comprises a humanized mature heavy chain variable domain comprising a sequence that is at least 95% identical to any of SEQ ID NO: 4-23; and a humanized mature light chain variable domain comprising a sequence that is at least 95% identical to any of SEQ ID NO: 27-48.

[0168] In some embodiments, the humanized mature heavy chain variable domain comprises a sequence of one of SEQ ID NO: 4-23. In some embodiments, the humanized mature heavy chain variable domain comprises a sequence comprising SEQ ID NO: 4. In some embodiments, the humanized mature heavy chain variable domain comprises a sequence comprising SEQ ID NO: 5. In some embodiments, the humanized mature heavy chain variable domain comprises a sequence comprising SEQ ID NO: 6. In some embodiments, the humanized mature heavy chain variable domain comprises a sequence comprising SEQ ID NO: 7. In some embodiments, the humanized mature heavy chain variable domain comprises a sequence comprising SEQ ID NO: 8. In some embodiments, the humanized mature heavy chain variable domain comprises a sequence comprising SEQ ID NO: 9. In some embodiments, the humanized mature heavy chain variable domain comprises a sequence comprising SEQ ID NO: 10. In some embodiments, the humanized mature heavy chain variable domain comprises a sequence comprising SEQ ID NO: 11. In some embodiments, the humanized mature heavy chain variable domain includes the sequence comprising SEQ ID NO: 12. In some embodiments, the humanized mature heavy chain variable domain includes the sequence comprising SEQ ID NO: 13. In some embodiments, the humanized mature heavy chain variable domain includes the sequence comprising SEQ ID NO: 14. In some embodiments, the humanized mature heavy chain variable domain includes the sequence comprising SEQ ID NO: 15. In some embodiments, the humanized mature heavy chain variable domain includes the sequence comprising SEQ ID NO: 16. In some embodiments, the humanized mature heavy chain variable domain includes the sequence comprising SEQ ID NO: 17. In some embodiments, the humanized mature heavy chain variable domain includes the sequence comprising SEQ ID NO: 18. In some embodiments, the humanized mature heavy chain variable domain includes the sequence comprising SEQ ID NO: 19. In some embodiments, the humanized mature heavy chain variable domain includes the sequence comprising SEQ ID NO: 21. In some embodiments, the humanized mature heavy chain variable domain includes the sequence comprising SEQ ID NO: 22. In some embodiments, the humanized mature heavy chain variable domain includes the sequence comprising SEQ ID NO: 23.

[0169] In some embodiments, the humanized mature light chain variable domain includes a sequence of one of SEQ ID NO: 27-48. In some embodiments, the humanized mature light chain variable domain includes a sequence including SEQ ID NO: 27. In some embodiments, the humanized mature light chain variable domain includes a sequence including SEQ ID NO: 28. In some embodiments, the humanized mature light chain variable domain includes a sequence including SEQ ID NO: 29. In some embodiments, the humanized mature light chain variable domain includes a sequence including SEQ ID NO: 30. In some embodiments, the humanized mature light chain variable domain includes a sequence including SEQ ID NO: 31. In some embodiments, the humanized mature light chain variable domain includes a sequence including SEQ ID NO: 32. In some embodiments, the humanized mature light chain variable domain includes a sequence including SEQ ID NO: 33. In some embodiments, the humanized mature light chain variable domain includes a sequence including SEQ ID NO: 34. In some embodiments, the humanized mature light chain variable domain includes the sequence comprising SEQ ID NO: 35. In some embodiments, the humanized mature light chain variable domain includes the sequence comprising SEQ ID NO: 36. In some embodiments, the humanized mature light chain variable domain includes the sequence comprising SEQ ID NO: 37. In some embodiments, the humanized mature light chain variable domain includes the sequence comprising SEQ ID NO: 38. In some embodiments, the humanized mature light chain variable domain includes the sequence comprising SEQ ID NO: 39. In some embodiments, the humanized mature light chain variable domain includes the sequence comprising SEQ ID NO: 40. In some embodiments, the humanized mature light chain variable domain includes the sequence comprising SEQ ID NO: 41. In some embodiments, the humanized mature light chain variable domain includes the sequence comprising SEQ ID NO: 42. In some embodiments, the humanized mature light chain variable domain includes the sequence comprising SEQ ID NO: 43. In some embodiments, the humanized mature light chain variable domain includes the sequence comprising SEQ ID NO: 44. In some embodiments, the humanized mature light chain variable domain includes the sequence comprising SEQ ID NO: 45. In some embodiments, the humanized mature light chain variable domain includes the sequence comprising SEQ ID NO: 46. In some embodiments, the humanized mature light chain variable domain includes the sequence comprising SEQ ID NO: 47. In some embodiments, the humanized mature light chain variable domain includes the sequence comprising SEQ ID NO: 48.

[0170] The humanized mature heavy chain of the antibody described herein may include a lysine residue at its C-terminus. However, in some embodiments, the humanized mature heavy chain (e.g., any of the mature heavy chains described herein) does not include a C-terminal lysine residue.

[0171] In some embodiments, the antibody or its antigen-binding fragment includes a humanized mature heavy chain variable domain comprising the sequence of one of SEQ ID NO: 4-23; and a humanized mature light chain variable domain comprising the sequence of one of SEQ ID NO: 27-48.

[0172] In some embodiments, the antibody or antigen-binding fragment of the cell penetrant comprises the humanized mature heavy chain variable domain of SEQ ID NO: 4 and the humanized mature light chain variable domain of any one of SEQ ID NO: 27-48. In some embodiments, the antibody or antigen-binding fragment of the cell penetrant comprises the humanized mature heavy chain variable domain of SEQ ID NO: 5 and the humanized mature light chain variable domain of any one of SEQ ID NO: 27-48. In some embodiments, the antibody or antigen-binding fragment of the cell penetrant comprises the humanized mature heavy chain variable domain of SEQ ID NO: 6 and the humanized mature light chain variable domain of any one of SEQ ID NO: 27-48. In some embodiments, the antibody or antigen-binding fragment of the cell penetrant comprises the humanized mature heavy chain variable domain of SEQ ID NO: 7 and the humanized mature light chain variable domain of any one of SEQ ID NO: 27-48. In some embodiments, the antibody of the cell penetrant or its antigen-binding fragment comprises the humanized mature heavy chain variable domain of SEQ ID NO: 8 and the humanized mature light chain variable domain of any one of SEQ ID NO: 27-48. In some embodiments, the antibody of the cell penetrant or its antigen-binding fragment comprises the humanized mature heavy chain variable domain of SEQ ID NO: 9 and the humanized mature light chain variable domain of any one of SEQ ID NO: 27-48. In some embodiments, the antibody of the cell penetrant or its antigen-binding fragment comprises the humanized mature heavy chain variable domain of SEQ ID NO: 10 and the humanized mature light chain variable domain of any one of SEQ ID NO: 27-48. In some embodiments, the antibody of the cell penetrant or its antigen-binding fragment comprises the humanized mature heavy chain variable domain of SEQ ID NO: 11 and the humanized mature light chain variable domain of any one of SEQ ID NO: 27-48. In some embodiments, the antibody of the cell penetrant or its antigen-binding fragment comprises the humanized mature heavy chain variable domain of SEQ ID NO: 12 and the humanized mature light chain variable domain of any one of SEQ ID NO: 27-48. In some embodiments, the antibody of the cell penetrant or its antigen-binding fragment comprises the humanized mature heavy chain variable domain of SEQ ID NO: 13 and the humanized mature light chain variable domain of any one of SEQ ID NO: 27-48. In some embodiments, the antibody of the cell penetrant or its antigen-binding fragment comprises the humanized mature heavy chain variable domain of SEQ ID NO: 14 and the humanized mature light chain variable domain of any one of SEQ ID NO: 27-48.In some embodiments, the antibody of the cell penetrant or its antigen-binding fragment comprises the humanized mature heavy chain variable domain of SEQ ID NO: 15 and the humanized mature light chain variable domain of any one of SEQ ID NO: 27-48. In some embodiments, the antibody of the cell penetrant or its antigen-binding fragment comprises the humanized mature heavy chain variable domain of SEQ ID NO: 16 and the humanized mature light chain variable domain of any one of SEQ ID NO: 27-48. In some embodiments, the antibody of the cell penetrant or its antigen-binding fragment comprises the humanized mature heavy chain variable domain of SEQ ID NO: 17 and the humanized mature light chain variable domain of any one of SEQ ID NO: 27-48. In some embodiments, the antibody of the cell penetrant or its antigen-binding fragment comprises the humanized mature heavy chain variable domain of SEQ ID NO: 18 and the humanized mature light chain variable domain of any one of SEQ ID NO: 27-48. In some embodiments, the antibody or antigen-binding fragment of the cell penetrant comprises the humanized mature heavy chain variable domain of SEQ ID NO: 19 and the humanized mature light chain variable domain of any one of SEQ ID NO: 27-48.

[0173] In some embodiments, the antibody of the cell penetrant or its antigen-binding fragment comprises the humanized mature heavy chain variable domain of SEQ ID NO: 20 and the humanized mature light chain variable domain of any one of SEQ ID NO: 27-48. In some embodiments, the antibody of the cell penetrant or its antigen-binding fragment comprises the humanized mature heavy chain variable domain of SEQ ID NO: 21 and the humanized mature light chain variable domain of any one of SEQ ID NO: 27-48. In some embodiments, the antibody of the cell penetrant or its antigen-binding fragment comprises the humanized mature heavy chain variable domain of SEQ ID NO: 22 and the humanized mature light chain variable domain of any one of SEQ ID NO: 27-48. In some embodiments, the antibody of the cell penetrant or its antigen-binding fragment comprises the humanized mature heavy chain variable domain of SEQ ID NO: 23 and the humanized mature light chain variable domain of any one of SEQ ID NO: 27-48.

[0174] In some embodiments, the antibody or antigen-binding fragment of the cell penetrant comprises the humanized mature heavy chain variable domain of SEQ ID NO: 20 and the humanized mature light chain variable domain of SEQ ID NO: 47. In some embodiments, the antibody or antigen-binding fragment of the cell penetrant comprises the humanized mature heavy chain variable domain of SEQ ID NO: 20 and the humanized mature light chain variable domain of SEQ ID NO: 48. In some embodiments, the antibody or antigen-binding fragment of the cell penetrant comprises the humanized mature heavy chain variable domain of SEQ ID NO: 21 and the humanized mature light chain variable domain of SEQ ID NO: 47. In some embodiments, the antibody or antigen-binding fragment of the cell penetrant comprises the humanized mature heavy chain variable domain of SEQ ID NO: 21 and the humanized mature light chain variable domain of SEQ ID NO: 48. In some embodiments, the antibody or antigen-binding fragment of the cell penetrant comprises the humanized mature heavy chain variable domain of SEQ ID NO: 23 and the humanized mature light chain variable domain of SEQ ID NO: 47. In some embodiments, the antibody or antigen-binding fragment of the cell penetrant comprises the humanized mature heavy chain variable domain of SEQ ID NO: 23 and the humanized mature light chain variable domain of SEQ ID NO: 48.

[0175] In some implementations, at least one of the following positions (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17) in the humanized heavy chain variable domain is occupied by a specified amino acid: K19 is occupied by R; S35 is occupied by G; T40 is occupied by A; E42 is occupied by G; A49 is occupied by S; K43 is occupied by E; R44 is occupied by G or D; A49 is occupied by S; A74 is occupied by S; T77 is occupied by S; L78 is occupied by A or G; L80 is occupied by A or G; L82c is occupied by G; M83 is occupied by R; S84 is occupied by A; M89 is occupied by V; or F91 is occupied by Y.

[0176] In some implementations, at least one of the following positions (e.g., 2, 3, 4, 5, or 6) in the humanized heavy chain variable domain is occupied by a specified amino acid: K43 is occupied by E; R44 is occupied by G or D; A49 is occupied by S; A74 is occupied by S; T77 is occupied by S; or F91 is occupied by Y.

[0177] In some implementations, at least one (e.g., 2, 3, or 4) of the following positions in the humanized heavy chain variable domain is occupied by a specified amino acid: S35 is occupied by G; L78 is occupied by A or G; L80 is occupied by A or G; or L82c is occupied by G.

[0178] In some implementations, F91 of the humanized heavy chain variable domain is occupied by Y; and at least one of the following positions (e.g., 2, 3, 4, 5 or 6) in the humanized heavy chain variable domain is occupied by the specified amino acid: R44 is occupied by G; A49 is occupied by S; A74 is occupied by S; T77 is occupied by S; L78 is occupied by A or G; or M83 is occupied by R.

[0179] In some implementations, at least one of the following positions (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13) in the variable domain of the humanized light chain is occupied by a specified amino acid: V3 is occupied by Q; L9 is occupied by S; D17 is occupied by Q; Q18 is occupied by P; K39 is occupied by R; K45 is occupied by R; T80 is occupied by A or S; T46 is occupied by R; L83 is occupied by V; L92 is occupied by G or A; V94 is occupied by I or A; A100 is occupied by G, D, or R; or L104 is occupied by V.

[0180] In some implementations, at least one (e.g., two) of the following positions in the variable domain of the humanized light chain is occupied by a specified amino acid: V3 is occupied by Q or A100 is occupied by D or R.

[0181] In some implementations, at least one (e.g., 2, 3, or 4) of the following positions in the humanized light chain variable domain is occupied by a specified amino acid: L9 is occupied by S; T80 is occupied by A or S; L92 is occupied by G or A; or V94 is occupied by I or A.

[0182] In some implementations, V3 is occupied by Q; Q18 is occupied by P; A100 is occupied by D; and at least one (e.g., two) of the following positions in the humanized light chain variable domain is occupied by the specified amino acid: T80 is occupied by A or L92 is occupied by A.

[0183] In some embodiments, at least one of the following positions (e.g., 2, 3, 4, 5, 6, 7, or 8) in the humanized heavy chain variable domain is occupied by a specified amino acid: L5 is occupied by V; G44 is occupied by R; A49 is occupied by S; A74 is occupied by S; T77 is occupied by S; L78 is occupied by A or G; M89 is occupied by V, or F91 is occupied by Y; and at least one of the following positions (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) in the humanized light chain variable domain is occupied by a specified amino acid: V3 is occupied by Q; D17 is occupied by Q; Q18 is occupied by P; K39 is occupied by R; K45 is occupied by R; T80 is occupied by A; L83 is occupied by V; L92 is occupied by A; A100 is occupied by D; or L104 is occupied by V.

[0184] The aforementioned substitutions may confer reduced (e.g., at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% reduction) immunogenicity to parental antibodies and / or increased (e.g., at least 1%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% increase) thermal stability to parental antibodies.

[0185] In some embodiments, the humanized antibody exhibits improved thermostability compared to a reference antibody comprising the heavy chain variable domain of SEQ ID NO: 1 and the light chain variable domain of SEQ ID NO: 24. In some embodiments, the humanized antibody exhibits improved thermostability compared to a reference antibody comprising the heavy chain variable domain of SEQ ID NO: 63 and the light chain variable domain of SEQ ID NO: 65. In some embodiments, the humanized antibody exhibits improved thermostability compared to a reference antibody comprising the heavy chain variable domain of SEQ ID NO: 67 and the light chain variable domain of SEQ ID NO: 69. In some embodiments, the humanized antibody exhibits improved thermostability compared to a reference antibody comprising the heavy chain variable domain of SEQ ID NO: 71 and the light chain variable domain of SEQ ID NO: 73. In some embodiments, the humanized antibody exhibits improved thermostability compared to a reference antibody comprising the heavy chain variable domain of SEQ ID NO: 75 and the light chain variable domain of SEQ ID NO: 77. In some implementations, the humanized antibody exhibits improved thermal stability compared to a reference antibody containing the heavy chain variable domain of SEQ ID NO: 79 and the light chain variable domain of SEQ ID NO: 81.

[0186] In some embodiments, the humanized antibody has a melting temperature of about 55°C or higher. In some embodiments, the humanized antibody has the following melting temperatures: about 56°C or higher, about 57°C or higher, about 58°C or higher, about 59°C or higher, about 60°C or higher, about 61°C or higher, about 62°C or higher, about 63°C or higher, about 64°C or higher, about 65°C or higher, about 66°C or higher, about 67°C or higher, about 68°C or higher, about 69°C or higher, about 70°C or higher, about 71°C or higher, about 72°C or higher, about 73°C or higher, about 74°C or higher, about 75°C or higher, about 76°C or higher, about 77°C or higher, about 78°C or higher, about 79°C or higher, about 80°C or higher, about 81°C or higher, about 82°C or higher, about 83°C or higher, about 84°C or higher, or about 85°C or higher. In some implementations, the humanized antibody has the following melting temperatures: at least about 55°C, at least about 56°C, at least about 57°C, at least about 58°C, at least about 59°C, at least about 60°C, at least about 61°C, at least about 62°C, at least about 63°C, at least about 64°C, at least about 65°C, at least about 66°C, at least about 67°C, at least about 68°C, at least about 69°C, at least about 70°C, at least about 71°C, at least about 72°C, at least about 73°C, at least about 74°C, at least about 75°C, at least about 76°C, at least about 77°C, at least about 78°C, at least about 79°C, at least about 80°C, at least about 81°C, at least about 82°C, at least about 83°C, at least about 84°C, or at least about 85°C.In some embodiments, the humanized antibody has the following melting temperatures: about 55°C to about 85°C, about 55°C to about 80°C, about 55°C to about 75°C, about 55°C to about 70°C, about 55°C to about 65°C, about 55°C to about 63°C, about 55°C to about 61°C, about 55°C to about 59°C, about 55°C to about 57°C, about 57°C to about 85°C, about 57°C to about 80°C, about 57°C to about 75°C, about 57°C to about 70°C, about 57°C to about 65°C, about 57°C to about 63°C, about 57°C to about 61°C, about 57°C to about 59°C, about 59°C to about 85°C, about 59°C to about 80°C, about 59°C to about 75°C, about 59°C to about 70°C, about 59°C Approximately 65°C, approximately 59°C to approximately 63°C, approximately 59°C to approximately 61°C, approximately 61°C to approximately 85°C, approximately 61°C to approximately 80°C, approximately 61°C to approximately 75°C, approximately 61°C to approximately 70°C, approximately 61°C to approximately 65°C, approximately 61°C to approximately 63°C, approximately 63°C to approximately 85°C, approximately 63°C to approximately 80°C, approximately 63°C to approximately 75°C, approximately 63°C to approximately 70°C, approximately 63°C to approximately 65°C, approximately 65°C to approximately 85°C, approximately 65°C to approximately 80°C, approximately 65°C to approximately 75°C, approximately 65°C to approximately 70°C, approximately 70°C to approximately 85°C, approximately 70°C to approximately 80°C, approximately 70°C to approximately 75°C, approximately 75°C to approximately 85°C, approximately 75°C to approximately 80°C, or approximately 80°C to approximately 85°C.

[0187] This document also provides cell-penetrating agents comprising CIM and an antibody that specifically binds to TDP-43 (e.g., human TDP-43), said antibody comprising a heavy chain variable domain comprising: a heavy chain CDR1 as defined by the Kabat / Chothia Composite, comprising SEQ ID NO: 49 or SEQ ID NO: 50; a heavy chain CDR2 as defined by Kabat, comprising SEQ ID NO: 51; a heavy chain CDR3 as defined by Kabat or Chothia, comprising SEQ ID NO: 52; a light chain CDR1 as defined by Kabat, comprising SEQ ID NO: 53; a light chain CDR2 as defined by Kabat, comprising SEQ ID NO: 54; and a light chain CDR3 as defined by Kabat, comprising one of SEQ ID NO: 55-61.

[0188] In some implementations, heavy chain CDR1 as defined by the Kabat / Chothia Composite contains SEQ ID NO: 49; heavy chain CDR2 as defined by Kabat contains SEQ ID NO: 51; heavy chain CDR3 as defined by Kabat or Chothia contains SEQ ID NO: 52; light chain CDR1 as defined by Kabat contains SEQ ID NO: 53; light chain CDR2 as defined by Kabat contains SEQ ID NO: 54; and light chain CDR3 as defined by Kabat contains SEQ ID NO: 55 or SEQ ID NO: 61.

[0189] In some embodiments, the heavy chain variable domain comprises a sequence that is at least 95% (e.g., at least 96%, at least 97%, at least 98%, at least 99%) identical to any of SEQ ID NO: 4-23. In some embodiments, the heavy chain variable domain comprises a sequence that is at least 95% (e.g., at least 96%, at least 97%, at least 98%, at least 99%) identical to any of SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 23. In some embodiments, the heavy chain variable domain comprises a sequence that is at least 98% identical to any of SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 23. In some embodiments, the heavy chain variable domain comprises the sequence of SEQ ID NO: 20. In some embodiments, the heavy chain variable domain comprises the sequence of SEQ ID NO: 21. In some embodiments, the heavy chain variable domain comprises the sequence of SEQ ID NO: 23.

[0190] In some embodiments, the light chain variable domain contains at least 95% (e.g., at least 96%, at least 97%, at least 98%, at least 99%) of the sequence identical to any of SEQ ID NO: 27-48. In some embodiments, the light chain variable domain contains at least 95% (e.g., at least 96%, at least 97%, at least 98%, at least 99%) of the sequence identical to SEQ ID NO: 47 or SEQ ID NO: 48. In some embodiments, the light chain variable domain contains at least 98% of the sequence identical to SEQ ID NO: 47 or SEQ ID NO: 48. In some embodiments, the light chain variable domain contains SEQ ID NO: 47. In some embodiments, the light chain variable domain contains SEQ ID NO: 48.

[0191] As previously described, the humanized mature light chain variable domains described herein may include a lysine residue at their C-terminus. However, in some embodiments, the humanized mature light chain variable domains (e.g., any of the mature light chain variable domains described herein) do not include a C-terminal lysine residue. For example, any of the mature light chain variable domains of SEQ ID NO: 24-48, 65, 69, 73, 77, or 81 may lack a C-terminal lysine residue.

[0192] In some embodiments, the antibody of the cell penetrant is a humanized antibody, a chimeric antibody, or a veneered antibody. In some embodiments, the antibody is an antigen-binding antibody fragment. In some embodiments, the antigen-binding antibody fragment is a Fab fragment, a Fab'2 fragment, or a single-chain Fv.

[0193] In some embodiments, the antibody in the cell penetrant is a complete antibody. In some embodiments, the antibody has a human IgG1 isotype.

[0194] In some implementations, the heavy chain variable domain is fused to the heavy chain constant region (e.g., any heavy chain constant region described herein) and the light chain variable domain is fused to the light chain constant region (e.g., any light chain constant region described herein).

[0195] In some embodiments, the heavy chain constant region is a mutant form of the natural human heavy chain constant region, which binds less to the Fcγ receptor compared to the natural heavy chain constant region. In some embodiments, the heavy chain constant region has an IgG1 isotype. In some embodiments, the antibody has at least one mutation in the constant region. In some embodiments, at least one mutation reduces complement fixation or activation in the constant region. In some embodiments, at least one mutation is located at one or more of the following positions (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11) according to EU numbers: 241, 264, 265, 270, 296, 297, 318, 320, 322, 329, and 331. In some embodiments, the antibody contains alanine at positions 318, 320, and 322 according to EU numbers.

[0196] In some embodiments, the antibody or antigen-binding antibody fragment of the cell penetrant selectively binds to phosphorylated TDP-43 (e.g., phosphorylated human TDP-43). In some embodiments, the antibody or antigen-binding antibody fragment of the cell penetrant selectively binds to phosphorylated TDP-43 (e.g., phosphorylated human TDP-43) compared to unphosphorylated TDP-43 (e.g., unphosphorylated human TDP-43).

[0197] In some embodiments, the antibody or antigen-binding antibody fragment of the cell penetrant is at a concentration higher than that of unphosphorylated TDP-43 (e.g., unphosphorylated human TDP-43) by at least (e.g., at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 100-fold, at least 150-fold, at least 200-fold, at least 250-fold, at least 300-fold, at least 350-fold, at least 400-fold, at least 450-fold, at least 500-fold, at least 550-fold, at least 600-fold, at least 650-fold, at least 700-fold, at least 750-fold, at least 800-fold, at least 850-fold, at least 900-fold, at least 950-fold, at least 1,000-fold, at least 1,050-fold). At least 1,100 times, at least 1,150 times, at least 1,200 times, at least 1,250 times, at least 1,300 times, at least 1,350 times, at least 1,400 times, at least 1,450 times, or at least 1,500 times, or about 10 times to about 1,500 times, about 10 times to 1,400 times, about 10 times to about 1,300 times, about 10 times to about 1,200 times, about 10 times to about 1,100 times, about 10 times to about 1,000 times, about 10 times to about 900 times, about 10 times to about 800 times, about 10 times to about 700 times, about 10 times to about 600 times, about 10 times to about 500 times, about 10 times to about 400 times, about 10 times to about 300 times. 0 times, about 10 times to about 200 times, about 10 times to about 100 times, about 10 times to about 50 times, about 50 times to about 1,500 times, about 50 times to 1,400 times, about 50 times to about 1,300 times, about 50 times to about 1,200 times, about 50 times to about 1,100 times, about 50 times to about 1,000 times, about 50 times to about 900 times, about 50 times to about 800 times, about 50 times to about 700 times, about 50 times to about 600 times, about 50 times to about 500 times, about 50 times to about 400 times, about 50 times to about 300 times, about 50 times to about 200 times, about 50 times to about 100 times, about 100 times to about 1,500 times, about 100 times to 1,400 times. 00 times, about 100 times to about 1,300 times, about 100 times to about 1,200 times, about 100 times to about 1,100 times, about 100 times to about 1,000 times, about 100 times to about 900 times, about 100 times to about 800 times, about 100 times to about 700 times, about 100 times to about 600 times, about 100 times to about 500 times, about 100 times to about 400 times, about 100 times to about 300 times, about 100 times to about 200 times, about 200 times to about 1,500 times, about 200 times to 1,400 times, about 200 times to about 1,300 times, about 200 times to about 1,200 times, about 200 times to about 1,100 times, about 200 times to about 1,100 times.000 times, approximately 200 times to approximately 900 times, approximately 200 times to approximately 800 times, approximately 200 times to approximately 700 times, approximately 200 times to approximately 600 times, approximately 200 times to approximately 500 times, approximately 200 times to approximately 400 times, approximately 200 times to approximately 300 times, approximately 500 times to approximately 1,500 times, approximately 500 times to 1,400 times, approximately 500 times to approximately 1,300 times, approximately 500 times to approximately 1,200 times, approximately 500 times to approximately 1,100 times, approximately 500 times to approximately 1,000 times, approximately 500 times to approximately 900 times, approximately 500 times to approximately 800 times, approximately 500 times to approximately 700 times, approximately 500 times It binds to phosphorylated TDP-43 (e.g., phosphorylated human TDP-43) with an affinity of approximately 600 times, approximately 800 times to approximately 1,500 times, approximately 800 times to approximately 1,400 times, approximately 800 times to approximately 1,200 times, approximately 800 times to approximately 1,100 times, approximately 800 times to approximately 1,000 times, approximately 800 times to approximately 900 times, approximately 1,000 times to approximately 1,500 times, approximately 1,000 times to approximately 1,400 times, approximately 1,000 times to approximately 1,300 times, approximately 1,000 times to approximately 1,200 times, or approximately 1,000 times to approximately 1,100 times.

[0198] In some embodiments, phosphorylated TDP-43 (e.g., phosphorylated human TDP-43) comprises phosphorylation of at least one amino acid residue selected from S409 and S410. In some embodiments, phosphorylated TDP-43 (e.g., phosphorylated human TDP-43) comprises phosphorylation of both S409 and S410.

[0199] In some embodiments, the antibody or fragment of the cell penetrant selectively binds to cytoplasmic aggregates of TDP-43 (e.g., cytoplasmic aggregates of human TDP-43). In some embodiments, the antibody or fragment of the cell penetrant selectively binds to cytoplasmic aggregates of TDP-43 (e.g., cytoplasmic aggregates of human TDP-43) compared to nuclear TDP-43 (e.g., nuclear human TDP-43). In some embodiments, the cytoplasmic aggregates of TDP-43 (e.g., cytoplasmic aggregates of human TDP-43) comprise phosphorylated aggregates of TDP-43. In some embodiments, the antibody or fragment of the cell penetrant substantially does not bind to unphosphorylated TDP-43 (e.g., unphosphorylated human TDP-43).

[0200] The heavy and light chain variable regions present in cell-penetrating agents may be linked to at least a portion of the human constant region. The selection of the constant region depends in part on whether antibody-conjugate-dependent cell-mediated cytotoxicity, antibody-conjugate-dependent phagocytosis, and / or complement-dependent cytotoxicity are required. For example, human isotypes IgG1 and IgG3 have complement-dependent cytotoxicity, while human isotypes IgG2 and IgG4 do not. Human IgG1 and IgG3 also induce stronger cell-mediated effector functions than human IgG2 and IgG4. The light chain constant region may be λ or κ. The numbering convention for constant regions includes EU numbers (Edelman, GM, et al., Anti-TDP-43 antibody 63:78-85 (1969)), Kabat number (Kabat, Proc. Natl. Acad. Sci. U.S.A. (National Institutes of Health, Bethesda, MD, 1991, IMGT unique numbering (Lefranc M.-P. et al., IMGT uniquenumbering for immunoglobulin and T cell receptor constant domains and Igsuperfamily C-like domains, Sequences of Proteins of Immunological Interest 29:185-203 (2005) and IMGT exon number (Lefranc, ibid.).

[0201] One or more amino acids (such as the C-terminal lysine of the heavy chain) at the amino or carboxyl terminus of the light and / or heavy chains may be deleted or derivatized in a certain proportion or in all of the molecule. Substitutions may be made in the constant region to reduce or enhance effector functions, such as complement-mediated cytotoxicity or ADCC. ( See, for example, Winter et al., U.S. Patent No. 5,624,821; Tso et al., U.S. Patent No. 5,834,597; and Lazar et al. Dev. Comp. Immunol. 103:4005 (2006)), or prolonging the half-life of human philtrum. ( See, for example, Hinton et al. Proc. Natl. Acad. Sci. USA 279:6213 (2004)). Exemplary substitutions include Gln at position 250 and / or Leu at position 428 (EU numbers are used for the constant region in this paragraph) to prolong the antibody's half-life. Substitutions at any or all of positions 234, 235, 236, and / or 237 reduce affinity for Fey receptors, particularly FcγRI receptors. (See, for example, U.S. Patent No. 6,624,821. Alanine substitutions at positions 234, 235, and 237 of human IgG1 can be used to reduce effector function. Some antibodies have alanine substitutions at positions 234, 235, and 237 of human IgG1 to reduce effector function. Optionally, positions 234, 236, and / or 237 of human IgG2 are substituted with alanine, and position 235 is substituted with glutamine. ( See, for example, U.S. Patent No. 5,624,821. In some antibodies, mutations are used in human IgG1 at one or more of positions 241, 264, 265, 270, 296, 297, 322, 329, and 331 (e.g., 2, 3, 4, 5, 6, 7, 8, or 9) numbered by EU. In some antibodies, mutations are used in human IgG1 at one or more of positions 318, 320, and 322 (e.g., 2 or 3) numbered by EU. In some antibodies, positions 234 and / or 235 are substituted with alanine and / or position 329 is substituted with glycine. In some antibodies, positions 234 and 235 are substituted with alanine. In some antibodies, the isotype is human IgG2 or IgG4.

[0202] Antibodies can be expressed as tetramers containing two light chains and two heavy chains, single heavy chains, light chains, Fab, Fab', F(ab')2, and Fv, or as single-chain antibodies in which the mature variable domains of the heavy and light chains are linked by spacers.

[0203] Human constant regions exhibit both allotropic and ethnotropic variation among individuals; that is, a constant region can differ in different individuals at one or more polymorphic sites. Ethnotropics differ from allotropics in that serum recognizing an ethnotropic binds to one or more non-polymorphic regions of other allotypes. Thus, for example, another heavy chain constant region is IgG1 Glm3 with or without a C-terminal lysine. References to human constant regions include constant regions having any natural allotype or any arrangement of residues occupying a position in a natural allotype.

[0204] J. Biol. Chem. This document discloses various components of cell penetrants, including anti-TDP-43 antibodies, linkers, and cell internalization motifs (e.g., CMIPs). Those skilled in the art will understand how to combine these features to obtain functional anti-TDP-43 cell penetrants. This document also describes cell penetrants having CMIPs, optionally present linkers, and light and / or heavy chains of anti-TDP-43 antibodies expressed as a single polypeptide (i.e., fusion protein).

[0205] In some embodiments, the cell-penetrating agent comprises a polypeptide sequence that is at least 95% identical to the sequence selected from any of the following: SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172 and SEQ ID NO: 174.

[0206] In some embodiments, the cell-penetrating agent comprises a polypeptide sequence that is at least 98% identical to the sequence selected from any of the following: SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172 and SEQ ID NO: 174.

[0207] In some embodiments, the cell-penetrating agent comprises a polypeptide sequence that is at least 98% identical to the sequence selected from any of the following: SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172 and SEQ ID NO: 174.

[0208] In some embodiments, the cell-penetrating agent comprises a polypeptide sequence that is at least 95% identical to the sequence selected from any of the following: SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173 and SEQ ID NO: 175.

[0209] In some embodiments, the cell-penetrating agent comprises a polypeptide sequence that is at least 98% identical to the sequence selected from any of the following: SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173 and SEQ ID NO: 175.

[0210] In some embodiments, the cell-penetrating agent comprises a polypeptide sequence selected from any of the following: SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173 and SEQ ID NO: 175.

[0211] In some embodiments, the cell-penetrating agent comprises a first polypeptide sequence containing an antibody-containing heavy chain and a second polypeptide containing an antibody-containing light chain. In some embodiments, the first polypeptide and / or the second polypeptide comprises a CMIP and optionally a linker. In some embodiments, the first polypeptide is selected from any of the following: SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 130, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: SEQ ID NO:170, SEQ ID NO:172, and SEQ ID NO:174; and the second polypeptide is selected from any of the following: SEQ ID NO:117, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:139, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145, SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:157, SEQ ID NO:159, SEQ ID NO:161, SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:177, SEQ ID NO:179, SEQ ID NO:161, SEQ ID NO:163, SEQ ID NO:174, SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:177, SEQ ID NO:178, SEQ ID NO:179 ... NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173 and SEQ ID NO: 175.

[0212] In some embodiments, the cell-penetrating agent comprises a first polypeptide and a second polypeptide, further wherein: the first polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 116 and the second polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 117; the first polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 118 and the second polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 119; the first polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 120 and the second polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 121; the first polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 122 and the second polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 123; the first polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 124 and the second polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 125; the first polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 126 and the second polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 127; the first polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 128 and the second polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 129; the first polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 129 ... second polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 129; the third polypeptide comprises a sequence at least 95% identical to SEQ ID NO: 129; the fourth polypeptide comprises a sequence at 127 contains at least 95% identical sequence to SEQ ID NO: 128; the first polypeptide contains at least 95% identical sequence to SEQ ID NO: 128 and the second polypeptide contains at least 95% identical sequence to SEQ ID NO: 129; the first polypeptide contains at least 95% identical sequence to SEQ ID NO: 130 and the second polypeptide contains at least 95% identical sequence to SEQ ID NO: 131; the first polypeptide contains at least 95% identical sequence to SEQ ID NO: 132 and the second polypeptide contains at least 95% identical sequence to SEQ ID NO: 133; the first polypeptide contains at least 95% identical sequence to SEQ ID NO: 134 and the second polypeptide contains at least 95% identical sequence to SEQ ID NO: 135; the first polypeptide contains at least 95% identical sequence to SEQ ID NO: 136 and the second polypeptide contains at least 95% identical sequence to SEQ ID NO: 137; the first polypeptide contains at least 95% identical sequence to SEQ ID NO: 138 and the second polypeptide contains at least 95% identical sequence to SEQ ID NO: 139. 139 contains at least 95% identical sequences; the first polypeptide contains at least 95% identical sequences to SEQ ID NO: 140 and the second polypeptide contains at least 95% identical sequences to SEQ ID NO: 141; the first polypeptide contains at least 95% identical sequences to SEQ ID NO: 142 and the second polypeptide contains at least 95% identical sequences to SEQ ID NO: 143.The first polypeptide contains at least 95% of the sequence identical to SEQ ID NO: 144 and the second polypeptide contains at least 95% of the sequence identical to SEQ ID NO: 145; the first polypeptide contains at least 95% of the sequence identical to SEQ ID NO: 146 and the second polypeptide contains at least 95% of the sequence identical to SEQ ID NO: 147; the first polypeptide contains at least 95% of the sequence identical to SEQ ID NO: 148 and the second polypeptide contains at least 95% of the sequence identical to SEQ ID NO: 149; the first polypeptide contains at least 95% of the sequence identical to SEQ ID NO: 150 and the second polypeptide contains at least 95% of the sequence identical to SEQ ID NO: 151; the first polypeptide contains at least 95% of the sequence identical to SEQ ID NO: 152 and the second polypeptide contains at least 95% of the sequence identical to SEQ ID NO: 153; the first polypeptide contains at least 95% of the sequence identical to SEQ ID NO: 154 and the second polypeptide contains at least 95% of the sequence identical to SEQ ID NO: 155; the first polypeptide contains at least 95% of the sequence identical to SEQ ID NO: 144 and the second polypeptide contains at least 95% of the sequence identical to SEQ ID NO: 155; the first polypeptide contains at least 95% of the sequence identical to SEQ ID NO: 146 and the second polypeptide contains at least 95% of the sequence identical to SEQ ID NO: 147; the first polypeptide contains at least 95% of the sequence identical to SEQ ID NO: 148 and the second polypeptide contains at least 95% of the sequence identical to SEQ ID NO: 149; the first polypeptide contains at least 95% of the sequence identical to SEQ ID NO: 154 and the second polypeptide contains at least 95% of the sequence identical to SEQ ID NO: 155; the first polypeptide contains at least 95% of The first polypeptide contains at least 95% identical sequence to SEQ ID NO: 156 and the second polypeptide contains at least 95% identical sequence to SEQ ID NO: 157; the first polypeptide contains at least 95% identical sequence to SEQ ID NO: 158 and the second polypeptide contains at least 95% identical sequence to SEQ ID NO: 159; the first polypeptide contains at least 95% identical sequence to SEQ ID NO: 160 and the second polypeptide contains at least 95% identical sequence to SEQ ID NO: 161; the first polypeptide contains at least 95% identical sequence to SEQ ID NO: 162 and the second polypeptide contains at least 95% identical sequence to SEQ ID NO: 163; the first polypeptide contains at least 95% identical sequence to SEQ ID NO: 164 and the second polypeptide contains at least 95% identical sequence to SEQ ID NO: 165; the first polypeptide contains at least 95% identical sequence to SEQ ID NO: 166 and the second polypeptide contains at least 95% identical sequence to SEQ ID NO: 167; the first polypeptide contains at least 95% identical sequence to SEQ ID NO: 167; the second polypeptide contains at least 95% identical sequence to SEQ ID NO: 166 and the third polypeptide contains at least 95% identical sequence to SEQ ID NO: 167; the second polypeptide contains at least 95% identical sequence to SEQ ID NO: 167; the third polypeptide contains at least 95% identical sequence to SEQ ID NO: 167; the fourth polypeptide contains at least 95% identical sequence to SEQ ID NO: 168 and the fifth polypeptide contains at least 95% identical sequence to SEQ ID NO: 169; the fifth polypeptide contains at least 95% identical sequence to SEQ ID NO: 169; the sixth polypeptide contains at least 95% identical sequence to SEQ ID NO: 169; the seventh polypeptide contains at least 95% identical sequence to SEQ ID NO: 169; the eighth polypeptide contains at SEQ ID NO: 168 contains at least 95% identical sequence and the second polypeptide contains at least 95% identical sequence to SEQ ID NO: 169; the first polypeptide contains at least 95% identical sequence to SEQ ID NO: 170 and the second polypeptide contains at least 95% identical sequence to SEQ ID NO: 171; the first polypeptide contains at least 95% identical sequence to SEQ ID NO: 172 and the second polypeptide contains at least 95% identical sequence to SEQ ID NO: 173.Alternatively, the first polypeptide may contain at least 95% of the same sequence as SEQ ID NO: 174, and the second polypeptide may contain at least 95% of the same sequence as SEQ ID NO: 175.

[0213] In some embodiments, the cell-penetrating agent comprises a first polypeptide and a second polypeptide, wherein: the first polypeptide comprises SEQ ID NO: 116 and the second polypeptide comprises SEQ ID NO: 117; the first polypeptide comprises SEQ ID NO: 118 and the second polypeptide comprises SEQ ID NO: 119; the first polypeptide comprises SEQ ID NO: 120 and the second polypeptide comprises SEQ ID NO: 121; the first polypeptide comprises SEQ ID NO: 122 and the second polypeptide comprises SEQ ID NO: 123; the first polypeptide comprises SEQ ID NO: 124 and the second polypeptide comprises SEQ ID NO: 125; the first polypeptide comprises SEQ ID NO: 126 and the second polypeptide comprises SEQ ID NO: 127; the first polypeptide comprises SEQ ID NO: 128 and the second polypeptide comprises SEQ ID NO: 129; the first polypeptide comprises SEQ ID NO: 130 and the second polypeptide comprises SEQ ID NO: 131; the first polypeptide comprises SEQ ID NO: 132 and the second polypeptide comprises SEQ ID NO: 133; the first polypeptide comprises SEQ ID NO: 13 ... NO: 135; The first polypeptide contains SEQ ID NO: 136 and the second polypeptide contains SEQ ID NO: 137; The first polypeptide contains SEQ ID NO: 138 and the second polypeptide contains SEQ ID NO: 139; The first polypeptide contains SEQ ID NO: 140 and the second polypeptide contains SEQ ID NO: 141; The first polypeptide contains SEQ ID NO: 142 and the second polypeptide contains SEQ ID NO: 143; The first polypeptide contains SEQ ID NO: 144 and the second polypeptide contains SEQ ID NO: 145; The first polypeptide contains SEQ ID NO: 146 and the second polypeptide contains SEQ ID NO: 147; The first polypeptide contains SEQ ID NO: 148 and the second polypeptide contains SEQ ID NO: 149; The first polypeptide contains SEQ ID NO: 150 and the second polypeptide contains SEQ ID NO: 151; The first polypeptide contains SEQ ID NO: 152 and the second polypeptide contains SEQ ID NO: 153; The first polypeptide contains SEQ ID NO: 154 and the second polypeptide contains SEQ ID NO: 155; the first polypeptide contains SEQ ID NO: 156 and the second polypeptide contains SEQ ID NO: 157; the first polypeptide contains SEQ ID NO: 158 and the second polypeptide contains SEQ ID NO: 159;The first polypeptide comprises SEQ ID NO: 160 and the second polypeptide comprises SEQ ID NO: 161; the first polypeptide comprises SEQ ID NO: 162 and the second polypeptide comprises SEQ ID NO: 163; the first polypeptide comprises SEQ ID NO: 164 and the second polypeptide comprises SEQ ID NO: 165; the first polypeptide comprises SEQ ID NO: 166 and the second polypeptide comprises SEQ ID NO: 167; the first polypeptide comprises SEQ ID NO: 168 and the second polypeptide comprises SEQ ID NO: 169; the first polypeptide comprises SEQ ID NO: 170 and the second polypeptide comprises SEQ ID NO: 171; the first polypeptide comprises SEQ ID NO: 172 and the second polypeptide comprises SEQ ID NO: 173; or the first polypeptide comprises SEQ ID NO: 174 and the second polypeptide comprises SEQ ID NO: 175.

[0214] In some embodiments, the cell-penetrating agent comprises a first polypeptide containing SEQ ID NO: 116 and a second polypeptide containing SEQ ID NO: 117. In some embodiments, the cell-penetrating agent comprises a first polypeptide containing SEQ ID NO: 118 and a second polypeptide containing SEQ ID NO: 119. In some embodiments, the cell-penetrating agent comprises a first polypeptide containing SEQ ID NO: 120 and a second polypeptide containing SEQ ID NO: 121. In some embodiments, the cell-penetrating agent comprises a first polypeptide containing SEQ ID NO: 122 and a second polypeptide containing SEQ ID NO: 123. In some embodiments, the cell-penetrating agent comprises a first polypeptide containing SEQ ID NO: 124 and a second polypeptide containing SEQ ID NO: 125. In some embodiments, the cell-penetrating agent comprises a first polypeptide containing SEQ ID NO: 126 and a second polypeptide containing SEQ ID NO: 127. In some embodiments, the cell-penetrating agent comprises a first polypeptide containing SEQ ID NO: 128 and a second polypeptide containing SEQ ID NO: 129. In some embodiments, the cell-penetrating agent comprises a first polypeptide containing SEQ ID NO: 130 and a second polypeptide containing SEQ ID NO: 131. In some embodiments, the cell-penetrating agent comprises a first polypeptide containing SEQ ID NO: 132 and a second polypeptide containing SEQ ID NO: 133. In some embodiments, the cell-penetrating agent comprises a first polypeptide containing SEQ ID NO: 134 and a second polypeptide containing SEQ ID NO: 135. In some embodiments, the cell-penetrating agent comprises a first polypeptide containing SEQ ID NO: 136 and a second polypeptide containing SEQ ID NO: 137. In some embodiments, the cell-penetrating agent comprises a first polypeptide containing SEQ ID NO: 138 and a second polypeptide containing SEQ ID NO: 139. In some embodiments, the cell-penetrating agent comprises a first polypeptide containing SEQ ID NO: 140 and a second polypeptide containing SEQ ID NO: 141. In some embodiments, the cell-penetrating agent comprises a first polypeptide containing SEQ ID NO: 142 and a second polypeptide containing SEQ ID NO: 143. In some embodiments, the cell-penetrating agent comprises a first polypeptide containing SEQ ID NO: 144 and a second polypeptide containing SEQ ID NO: 145. In some embodiments, the cell-penetrating agent comprises a first polypeptide containing SEQ ID NO: 146 and a second polypeptide containing SEQ ID NO: 147.In some embodiments, the cell-penetrating agent comprises a first polypeptide containing SEQ ID NO: 148 and a second polypeptide containing SEQ ID NO: 149. In some embodiments, the cell-penetrating agent comprises a first polypeptide containing SEQ ID NO: 150 and a second polypeptide containing SEQ ID NO: 151. In some embodiments, the cell-penetrating agent comprises a first polypeptide containing SEQ ID NO: 152 and a second polypeptide containing SEQ ID NO: 153. In some embodiments, the cell-penetrating agent comprises a first polypeptide containing SEQ ID NO: 154 and a second polypeptide containing SEQ ID NO: 155. In some embodiments, the cell-penetrating agent comprises a first polypeptide containing SEQ ID NO: 156 and a second polypeptide containing SEQ ID NO: 157. In some embodiments, the cell-penetrating agent comprises a first polypeptide containing SEQ ID NO: 158 and a second polypeptide containing SEQ ID NO: 159. In some embodiments, the cell-penetrating agent comprises a first polypeptide containing SEQ ID NO: 160 and a second polypeptide containing SEQ ID NO: 161. In some embodiments, the cell-penetrating agent comprises a first polypeptide containing SEQ ID NO: 162 and a second polypeptide containing SEQ ID NO: 163. In some embodiments, the cell-penetrating agent comprises a first polypeptide containing SEQ ID NO: 164 and a second polypeptide containing SEQ ID NO: 165. In some embodiments, the cell-penetrating agent comprises a first polypeptide containing SEQ ID NO: 166 and a second polypeptide containing SEQ ID NO: 167. In some embodiments, the cell-penetrating agent comprises a first polypeptide containing SEQ ID NO: 168 and a second polypeptide containing SEQ ID NO: 169. In some embodiments, the cell-penetrating agent comprises a first polypeptide containing SEQ ID NO: 170 and a second polypeptide containing SEQ ID NO: 171. In some embodiments, the cell-penetrating agent comprises a first polypeptide containing SEQ ID NO: 172 and a second polypeptide containing SEQ ID NO: 173. In some embodiments, the cell-penetrating agent comprises a first polypeptide containing SEQ ID NO: 174 and a second polypeptide containing SEQ ID NO: 175.

[0215] Cell-penetrating agents can also be administered in the form of nucleic acids encoding the cell-penetrating agent or antibodies present within the cell-penetrating agent. If both heavy and light chains are present, the chains are preferably linked as single-chain antibodies.

[0216] IV. Nucleic Acids, Vectors, and Host Cells This disclosure provides nucleic acids encoding at least a portion of any of the cell-penetrating agents described herein. For example, in addition to any heavy and / or light chains described herein, the nucleic acid may also encode any CIM, linker, and / or spacer described herein. In some embodiments, the nucleic acid encodes a first polypeptide comprising a heavy chain of an antibody that specifically binds to TDP-43, the first polypeptide comprising any of the following: SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172 and SEQ ID NO: 174.In some embodiments, the nucleic acid encodes a second polypeptide comprising a light chain of an antibody that specifically binds to TDP-43, the second polypeptide comprising any of the following: SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO: 163, SEQ ID NO: 154. NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173 and SEQ ID NO: 175.

[0217] In some embodiments, the nucleic acid encodes a first polypeptide comprising a heavy chain of an antibody that specifically binds to TDP-43, the first polypeptide comprising any of the following: SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 158. SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172 and SEQ ID NO: 174; and the nucleic acid encodes a second polypeptide comprising a light chain of an antibody that specifically binds to TDP-43, the second polypeptide comprising any of the following: SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155. SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173 and SEQ ID NO: 175.

[0218] In some embodiments, the nucleic acid-encoding cell-penetrating agent comprises a first polypeptide containing SEQ ID NO: 116 and a second polypeptide containing SEQ ID NO: 117. In some embodiments, the nucleic acid-encoding cell-penetrating agent comprises a first polypeptide containing SEQ ID NO: 118 and a second polypeptide containing SEQ ID NO: 119. In some embodiments, the nucleic acid-encoding cell-penetrating agent comprises a first polypeptide containing SEQ ID NO: 120 and a second polypeptide containing SEQ ID NO: 121. In some embodiments, the nucleic acid-encoding cell-penetrating agent comprises a first polypeptide containing SEQ ID NO: 122 and a second polypeptide containing SEQ ID NO: 123. In some embodiments, the nucleic acid-encoding cell-penetrating agent comprises a first polypeptide containing SEQ ID NO: 124 and a second polypeptide containing SEQ ID NO: 125. In some embodiments, the nucleic acid-encoding cell-penetrating agent comprises a first polypeptide containing SEQ ID NO: 126 and a second polypeptide containing SEQ ID NO: 127. In some embodiments, the nucleic acid-encoding cell-penetrating agent comprises a first polypeptide containing SEQ ID NO: 128 and a second polypeptide containing SEQ ID NO: 129. In some embodiments, the nucleic acid-encoding cell-penetrating agent comprises a first polypeptide containing SEQ ID NO: 130 and a second polypeptide containing SEQ ID NO: 131. In some embodiments, the nucleic acid-encoding cell-penetrating agent comprises a first polypeptide containing SEQ ID NO: 132 and a second polypeptide containing SEQ ID NO: 133. In some embodiments, the nucleic acid-encoding cell-penetrating agent comprises a first polypeptide containing SEQ ID NO: 134 and a second polypeptide containing SEQ ID NO: 135. In some embodiments, the nucleic acid-encoding cell-penetrating agent comprises a first polypeptide containing SEQ ID NO: 136 and a second polypeptide containing SEQ ID NO: 137. In some embodiments, the nucleic acid-encoding cell-penetrating agent comprises a first polypeptide containing SEQ ID NO: 138 and a second polypeptide containing SEQ ID NO: 139. In some embodiments, the nucleic acid-encoded cell-penetrating agent comprises a first polypeptide containing SEQ ID NO: 140 and a second polypeptide containing SEQ ID NO: 141. In some embodiments, the nucleic acid-encoded cell-penetrating agent comprises a first polypeptide containing SEQ ID NO: 142 and a second polypeptide containing SEQ ID NO: 143. In some embodiments, the nucleic acid-encoded cell-penetrating agent comprises a first polypeptide containing SEQ ID NO: 144 and a second polypeptide containing SEQ ID NO: 145.In some embodiments, the nucleic acid-encoding cell-penetrating agent comprises a first polypeptide containing SEQ ID NO: 146 and a second polypeptide containing SEQ ID NO: 147. In some embodiments, the nucleic acid-encoding cell-penetrating agent comprises a first polypeptide containing SEQ ID NO: 148 and a second polypeptide containing SEQ ID NO: 149. In some embodiments, the nucleic acid-encoding cell-penetrating agent comprises a first polypeptide containing SEQ ID NO: 150 and a second polypeptide containing SEQ ID NO: 151. In some embodiments, the nucleic acid-encoding cell-penetrating agent comprises a first polypeptide containing SEQ ID NO: 152 and a second polypeptide containing SEQ ID NO: 153. In some embodiments, the nucleic acid-encoding cell-penetrating agent comprises a first polypeptide containing SEQ ID NO: 154 and a second polypeptide containing SEQ ID NO: 155. In some embodiments, the nucleic acid-encoding cell-penetrating agent comprises a first polypeptide containing SEQ ID NO: 156 and a second polypeptide containing SEQ ID NO: 157. In some embodiments, the nucleic acid-encoding cell-penetrating agent comprises a first polypeptide containing SEQ ID NO: 158 and a second polypeptide containing SEQ ID NO: 159. In some embodiments, the nucleic acid-encoding cell-penetrating agent comprises a first polypeptide containing SEQ ID NO: 160 and a second polypeptide containing SEQ ID NO: 161. In some embodiments, the nucleic acid-encoding cell-penetrating agent comprises a first polypeptide containing SEQ ID NO: 162 and a second polypeptide containing SEQ ID NO: 163. In some embodiments, the nucleic acid-encoding cell-penetrating agent comprises a first polypeptide containing SEQ ID NO: 164 and a second polypeptide containing SEQ ID NO: 165. In some embodiments, the nucleic acid-encoding cell-penetrating agent comprises a first polypeptide containing SEQ ID NO: 166 and a second polypeptide containing SEQ ID NO: 167. In some embodiments, the nucleic acid-encoding cell-penetrating agent comprises a first polypeptide containing SEQ ID NO: 168 and a second polypeptide containing SEQ ID NO: 169. In some embodiments, the nucleic acid-encoded cell-penetrating agent comprises a first polypeptide containing SEQ ID NO: 170 and a second polypeptide containing SEQ ID NO: 171. In some embodiments, the nucleic acid-encoded cell-penetrating agent comprises a first polypeptide containing SEQ ID NO: 172 and a second polypeptide containing SEQ ID NO: 173. In some embodiments, the nucleic acid-encoded cell-penetrating agent comprises a first polypeptide containing SEQ ID NO: 174 and a second polypeptide containing SEQ ID NO: 175.

[0219] This disclosure further provides nucleic acids encoding either the heavy chain variable domain and / or the light chain variable domain of any of the antibodies described herein. For example, the nucleic acid may encode a heavy chain variable domain comprising any of SEQ ID NO: 4-23 and / or a light chain variable domain comprising any of SEQ ID NO: 27-48. Optionally, such nucleic acids further encode a signal peptide and may be expressed together with the signal peptide linked to a constant region. The coding sequence of the nucleic acid may be operatively linked to a regulatory sequence to ensure expression of the coding sequence (such as a promoter, enhancer, ribosome binding site, transcription termination signal, etc.). The nucleic acids encoding the heavy and light chains may exist in isolated form or may be cloned into one or more vectors. The nucleic acids may be synthesized, for example, by solid-state synthesis or by PCR of overlapping oligonucleotides. The nucleic acids encoding the heavy and light chains may, for example, be conjugated as a single continuous nucleic acid within an expression vector, or may be separate, for example, each cloned into its own expression vector. In some embodiments, the nucleic acids are codon-optimized for expression in host cells.

[0220] Several cell lines using antibody expression are known. ( Methods for generating chimeric and humanized antibodies (e.g., hybridomas). For example, the immunoglobulin variable region of the antibody can be cloned and sequenced using well-known methods. In one method, the heavy chain variable VH region is cloned using mRNA prepared from hybridoma cells via RT-PCR. A common primer is used as the 5' primer and a g2b constant region-specific 3' primer for the VH region leader peptide containing the translation start codon. Exemplary primers are described in U.S. Patent Publication US 2005 / 0009150 (hereinafter referred to as "Schenk") by Schenk et al. Sequences from multiple independently derived clones can be compared to ensure that no changes are introduced during amplification. The sequence of the VH region can also be determined or confirmed by sequencing the VH fragment obtained by the 5' RACE RT-PCR method and the 3' g2b-specific primer.

[0221] Light chain variable VL regions can be cloned in a similar manner. In one approach, a shared set of primers is designed to amplify the VL region using a 5' primer designed to hybridize with the VL region containing the translation start codon and a 3' primer specific to the Ck region downstream of the VJ conjugate region. In a second approach, a 5' RACE RT-PCR method is used to clone the VL-encoding cDNA. Exemplary primers are described in Schenk, ibid. The cloned sequence is then combined with a sequence encoding a human (or other non-human species) constant region.

[0222] This document also provides vectors comprising any of the nucleic acids described herein operatively linked to one or more regulatory sequences to achieve expression of any of the cell penetrants described herein in mammalian cells.

[0223] This document also provides vectors comprising nucleic acids encoding mature heavy chain variable domains (e.g., any of the heavy chain variable domains described herein) and light chain variable domains (e.g., any of the light chain variable domains described herein), said nucleic acids being operatively linked to one or more regulatory sequences to achieve expression of any of the antibody or antigen-binding fragments described herein in mammalian cells.

[0224] In one approach, the variable regions of the heavy and light chains are reengineered to encode splicing donor sequences downstream of the corresponding VDJ or VJ cassette and cloned into mammalian expression vectors, such as pCMV-hyl for the heavy chain and pCMV-Mcl for the light chain. These vectors encode the human Kl and Ck constant regions as exon fragments downstream of the inserted variable region cassette. After sequence validation, the heavy and light chain expression vectors are co-transfected into CHO cells to generate chimeric antibodies. Conditioned culture media are collected 48 hours post-transfection, and antibody production is determined by Western blotting or antigen binding by ELISA. The chimeric antibodies are then humanized as described above.

[0225] Chimeric antibodies, veneered antibodies, humanized antibodies, and human antibodies are typically produced through recombinant expression. Recombinant polynucleotide constructs generally include an expression control sequence operatively linked to the coding sequence of the antibody chain, including naturally related or heterologously expressed control elements such as promoters. The expression control sequence may be a promoter system within a vector capable of transforming or transfecting eukaryotic or prokaryotic host cells. Once the vector has been incorporated into a suitable host, the host is maintained under conditions suitable for high-level expression of the nucleotide sequence and for the collection and purification of cross-reactive antibodies.

[0226] Therefore, this document provides host cells transformed with any of the vectors described herein. This document also provides host cells comprising any of the nucleic acids described herein.

[0227] Expression vectors typically replicate in a host organism either as an episome or as part of the host's chromosomal DNA. Often, expression vectors contain selection markers, such as ampicillin resistance or hygromycin resistance, to allow detection of those cells transformed with the desired DNA sequence.

[0228] Exemplary anti-TDP-43 cell penetrants It is a prokaryotic host that can be used to express antibodies, especially antibody fragments. Microorganisms, such as yeast, can also be used for expression. A yeast host with a suitable vector, which, as needed, contains expression control sequences, a replication origin, and a termination sequence. Typical promoters include 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters particularly include promoters derived from alcohol dehydrogenases, isocytochrome C, and enzymes responsible for the utilization of maltose and galactose.

[0229] Mammalian cells can be used to express nucleotide segments encoding immunoglobulins or fragments thereof. See Winnacker, From Genes to Clones, (VCH Publishers, NY, 1987). Many suitable host cell lines capable of secreting complete heterologous proteins have been developed, including CHO cell lines, various COS cell lines, HeLa cells, HEK293 cells, L cells, and non-antibody-producing myeloma cells, including Sp2 / 0 and NSO. Cells can be non-human. Expression vectors for these cells may include expression control sequences such as origin of replication, promoters, and enhancers (Queen sequences). Escherichia coli Saccharomyces 89:49 (1986)), and essential processing information sites, such as ribosome binding sites, RNA splicing sites, polyadenylation sites, and transcription terminator sequences. Expression control sequences may include promoters derived from endogenous genes, cytomegalovirus, SV 40, adenovirus, bovine papillomavirus, and their analogues. et al., Immunol. Co Rev. 148: 1149 (1992). In some implementations, the promoter is a eukaryotic promoter.

[0230] Alternatively, the antibody coding sequence can be incorporated into the transgene to introduce it into the genetic body of the transgenic animal and subsequently expressed in its milk. ( See, for example, U.S. Patent Nos. 5,741,957, 5,304,489, and 5,849,992. Suitable transgenes include coding sequences of light and / or heavy chains operatively linked to promoters and enhancers from breast-specific genes such as casein or β-lactoglobulin.

[0231] Vectors containing the DNA segment of interest can be transferred into host cells using methods that depend on the type of cell host. For example, calcium chloride transfection is commonly used for prokaryotic cells, while calcium phosphate treatment, electroporation, lipid transfection, gene gun, or virus-based transfection can be used for other cell hosts. Other methods for transforming mammalian cells include the use of polybrene, protoplast fusion, liposomes, electroporation, and microinjection. To produce transgenic animals, trans-genes can be microinjected into fertilized oocytes or into the genome of embryonic stem cells, and the nucleus of such cells can be transferred into enucleated oocytes.

[0232] After introducing vectors encoding the heavy and light chains of antibodies into cell cultures, cell pools can be screened for growth productivity and product quality in serum-free media. The top-generating cell pools can then undergo FACS-based single-cell cloning to produce monoclonal cell lines. Specific productivity exceeding 50 pg or 100 pg / cell / day can be used, corresponding to product titers greater than 7.5 g / L of culture. Turbidity, filtration properties, PAGE, IEF, UV scan, HPSEC, carbohydrate-oligosaccharide localization, mass spectrometry, and binding assays such as ELISA or Biacore can also be tested on antibodies generated from single-cell clones. The selected clones can then be stored in multiple vials and cryopreserved for later use.

[0233] Once expressed, the antibody can be purified according to standard procedures in the art, including protein A capture, HPLC purification, column chromatography, gel electrophoresis, and similar methods (generally see Scopes, (Springer-Verlag, NY, 1982)).

[0234] Commercially viable methods for antibody production include codon optimization, promoter selection, transcription element selection, terminator selection, blood-free cell cloning, cell banks, copy number amplification using selectable markers, CHO terminators, or improvement of protein titers (see, for example, U.S. Patent No. 5,786,464; U.S. Patent No. 6,114,148; U.S. Patent No. 6,063,598; U.S. Patent No. 7,569,339; WO2004 / 050884; WO2008 / 012142; WO2008 / 012142; WO2005 / 019442; WO2008 / 107388; WO2009 / 027471; and U.S. Patent No. 5,888,809).

[0235] DNA can be delivered in its naked form (i.e., without colloidal or encapsulating materials). Alternatively, a variety of viral vector systems may be used, including retroviral systems (see, for example, Lawrie and Tumin, Cur. Opin. Genet. Develop. 3, 102-109 (1993)); adenoviral vectors (see, for example, Bett et al., J. Virol. 67, 5911 (1993)); adeno-associated virus vectors (see, for example, Zhou et al., J. Exp. Med. 179, 1867 (1994)); viral vectors from the pox family (including poxvirus and fowlpoxvirus); viral vectors from the alphavirus genus, such as those derived from Sindbis and Semliki forest viruses (see, for example, Dubensky et al., J. Virol. 70, 508-519 (1996)); Venezuelan equine encephalitis virus (see U.S. Patent No. 5,643,576); and rod-shaped viruses, such as vesicular stomatitis virus (see WO). 96 / 34625) and papillomavirus (Ohe et al., See 6:325-333(1995); Woo et al., WO 94 / 12629 and Xiao&Brandsma, et al., J. Immunol. 24:2630-2622(1996)).

[0236] DNA encoding an immunogen or a vector containing said immunogen can be packaged into liposomes. Suitable lipids and related analogues are described in U.S. Patent Nos. 5,208,036, 5,264,618, 5,279,833, and 5,283,185. The vector and the DNA encoding the immunogen can also be adsorbed onto or associated with a particulate carrier, examples of which include polymethyl methacrylate polymers and polylactide and poly(lactide-co-glycolic acid) (see, for example, McGee et al.). Protein Purification 1996).

[0237] V. Extraconjugate Conjugated antibodies and antigen-binding antibody fragments that specifically bind to antigens such as TDP-43 (e.g., human TDP-43) can be used to detect the presence of TDP-43; monitor and evaluate the efficacy of therapeutic agents used to treat patients diagnosed with amyotrophic lateral sclerosis (ALS), frontotemporal degeneration (FTLD-TDP), primary spinal lateral sclerosis, and progressive muscular atrophy and Parkinson's disease; inhibit or reduce TDP-43 aggregation; reduce or eliminate TDP-43 aggregates; stabilize the non-toxic conformation of TDP-43; or treat or achieve prevention of patients with ALS, frontotemporal degeneration (FTLD-TDP), primary spinal lateral sclerosis, and progressive muscular atrophy and Parkinson's disease.

[0238] The cell-penetrating agents described herein can be further bound to other therapeutic components, other proteins, other antibodies, and / or detectable markers. See WO 03 / 057838; U.S. Patent No. 8,455,622. Such therapeutic components can be any agent that can be used to treat, counteract, improve, prevent, or modify an unwanted condition or disease in a patient, such as amyotrophic lateral sclerosis (ALS), frontotemporal degeneration (FTLD-TDP), primary spinal lateral sclerosis, and progressive muscular atrophy and Parkinson's disease.

[0239] The conjugated therapeutic portion may include cytotoxic agents, cell growth inhibitors, neurotrophic agents, neuroprotective agents, radiotherapy agents, immunomodulators, or any bioactive agent that promotes or enhances antibody activity. Cytotoxic agents may be any agent that is toxic to cells. Cell growth inhibitors may be any agent that inhibits cell proliferation. Neurotrophic agents may be any agent that promotes the maintenance, growth, or differentiation of neurons, including chemical or protein agents. Neuroprotective agents may be agents that protect neurons from acute injury or degenerative processes, including chemical or protein agents. Immunomodulators may be any agent that stimulates or inhibits the development or maintenance of an immune response. Radiotherapy agents may be any molecule or compound that emits radiation. If such a therapeutic portion is conjugated to a TDP-43-specific antibody or antigen-binding antibody fragment, such as the antibodies and antigen-binding antibody fragments described herein, the conjugated therapeutic portion will have a specific affinity for cells affected by TDP-43-related diseases compared to normal cells.

[0240] Therefore, the application of these further conjugated antibodies or conjugated antigen-binding antibody fragments will directly target cells with minimal damage to surrounding normal healthy tissue. This is particularly suitable for treatment fractions that are too toxic to be administered alone. Additionally, smaller amounts of the treatment fraction can be used.

[0241] Some of these cell-penetrating agents can be linked to radioactive isotopes. Examples of radioactive isotopes include, for example, yttrium. 90 (90Y), Indium 111 (111In)131 1. 99 mTc, radioactive silver-111, radioactive silver-199 and bismuth 213 The binding of radioactive isotopes to antibodies or antigen-binding antibody fragments can be performed using conventional bifunctional chelates. For binding of radioactive silver-111 and radioactive silver-199, sulfur-based linkers can be used. See Hazra et al. Human Gene Therapy 24-25:1-7 (1994). The bonding of silver radioisotopes can involve the reduction of immunoglobulins with ascorbic acid. For radioisotopes (such as 111In and 90Y), ibritumomab tiuxetan can be used and will react with such isotopes to form 111In-ibritumomab and 90Y-ibritumomab, respectively. See Witzig, Nucleic Acids. Res. 48(Supplement 1):S91-S95 (2001).

[0242] Some of these antibodies or antigen-binding antibody fragments can be linked to other therapeutic components. These therapeutic components can be, for example, cytotoxic, cell growth-inhibiting, immunomodulatory, neurotrophic, or neuroprotective. For instance, antibodies and antigen-binding antibody fragments can be linked to toxic chemotherapeutic agents (such as maytansine and geldanamycin) or microtubule inhibitors (such as microtubule binders). ( For example, auratestatin or minor groove binders (such as calicheamicin) are used in combination. Other representative treatment modalities include those known to be used to treat, manage, or improve amyotrophic lateral sclerosis (ALS), frontotemporal degeneration (FTLD-TDP), primary spinal lateral sclerosis, and progressive muscular atrophy and Parkinson's disease.

[0243] Antibodies or antigen-binding antibody fragments can also be conjugated to detectable markers. Such antibodies and antigen-binding antibody fragments can be used, for example, to diagnose ALS, FTLD-TDP, primary lateral sclerosis, and progressive muscular atrophy and Parkinson's disease. Representative detectable markers that can be conjugated to or linked to antibody or antigen-binding antibody fragments include various enzymes such as horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; prosthetic groups such as streptavidin / biotin and avidin / biotin; fluorescent materials such as umbelliferone, luciferin, luciferin isothiocyanate, rhodamine, dichlorotriazineamine luciferin, dansyl chloride, or phycoerythrin; luminescent materials such as luminol; bioluminescent materials such as luciferase, luciferin, and jellyfish luminescent protein; and radioactive materials such as radioactive silver-111, radioactive silver-199, and bismuth. 213 ,iodine( 131 I, 125 I,123 I, 121 I), carbon ( 14 C), sulfur 5 S), tritium ( 3 H), Indium 115 In 113 In 112 In 111 In), Technetium ( 99 Tc), thallium 201 Ti, gallium 68 Ga、 67 Ga), Palladium ( 103 Pd), molybdenum ( 99 Mo), xenon ( 133 Xe), fluorine ( 18 F) 153 Sm、 177 Lu、 159 Gd, 149 Pm, 140 La、 175 Yb、 166 Ho、 90 Y、 47 Sc、 186 Re、 188 Re、 142 Pr、 105 Rh、 97 Ru、 68 Ge 57 Co、 65 ZN, 85 SR, 32 P, 153 Gd, 169 Yb、 51 CR 54 Mn, 75 Se、 113 Sn and 117 Sn; positron-emitting metals obtained using various positron emission tomography (PET) scans; non-radioactive paramagnetic metal ions; and molecules that are radioactively labeled or conjugated to specific radioactive isotopes.

[0244] Therapeutic components, other proteins, other antibodies, and / or detectable markers can be obtained directly or via intermediates. ( For example ，The linker is indirectly coupled or conjugated to the antibody or antigen-binding antibody fragment of the present invention. See, for example, Arnon et al., “Monoclonal Antibodies For Immunotargeting of Drugs in Cancer Therapy,” in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243–56 (Alan R. Liss, Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery,” in Controlled Drug Delivery (2nd ed.), Robinson et al. (eds.), pp. 623–53 (Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers of Cytotoxic Agents in Cancer Therapy: A Review,” in Monoclonal Antibodies 84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475–506 (1985); “Analysis, Results, and Future Prospective of The Therapeutic Use of Radio-labeled Antibody in Cancer Therapy,” in Monoclonal Antibodies For Cancer Detection and Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985); and Thorpe et al. J. Micro Encap. 62:119-58 (1982). Suitable linkers include, for example, cleavable and non-cleavable linkers. Different linkers may be used to release coupled therapeutic moieties, proteins, antibodies, and / or detectable labels under acidic or reducing conditions, upon exposure to specific proteases, or under other defined conditions.

[0245] In some embodiments, the cell penetrant may also be conjugated to a therapeutic agent, cytotoxic agent, cell growth inhibitor, immunomodulator, neurotrophic agent, or neuroprotective agent as described herein. For example, an antibody present in the cell penetrant may be conjugated (i.e., conjugated) to a therapeutic portion (such as a cytotoxic agent, radiotherapy agent, immunomodulator, or secondary antibody) (e.g., to form an antibody heteroconjugate). Representative therapeutic portions include agents known to be used to treat, manage, or improve symptoms of TDP-43-related diseases.

[0246] The therapeutic portion and / or detectable substance may be directly coupled or bound to any of the rodent, chimeric or humanized antibodies described herein via an intermediate (e.g., a linker) using techniques known in the art. See, for example, Arnon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy,” in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery,” in Controlled Drug Delivery (2nd ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review,” in Monoclonal Antibodies 84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); “Analysis, Results, and Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy,” in Monoclonal Antibodies For Cancer Detection And Therapy. Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985) and Thorpe et al., Immunol. Rev., 1982, 62: 119-58.

[0247] The cell-penetrating agents used in the disclosed formulations also include modified forms of murine, chimeric, or humanized 13D3 antibodies that have an extended in vivo half-life relative to the corresponding unmodified antibody. Such modifications can be prepared, for example, by glycosylation, acetylation, polyethylene glycolation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, or binding to cellular ligands or other proteins. As an example, a representative method for extending antibody half-life is described in PCT International Publication No. WO 02 / 060919.

[0248] VI. Pharmaceutical compositions and products This disclosure also provides pharmaceutical compositions and products. Therefore, pharmaceutical compositions comprising any of the cell-penetrating agents and pharmaceutically acceptable carriers described herein are provided herein.

[0249] Pharmaceutical compositions for parenteral administration are preferably sterile, substantially isotonic, and manufactured under GMP conditions. The pharmaceutical composition may be provided in unit dosage form (i.e., a single-dose dose). The pharmaceutical composition may be formulated using one or more physiologically acceptable carriers, diluents, excipients, or adjuvants. The formulation depends on the chosen route of administration. For injection, the cell-penetrating agent may be formulated in an aqueous solution, preferably in a physiologically compatible buffer such as Hank's solution, Ringer's solution, or physiological saline or acetate buffer (to reduce injection site discomfort). The solution may contain formulations such as suspending agents, stabilizers, and / or dispersants. Alternatively, the cell-penetrating agent may be in lyophilized form for preparation with a suitable medium (e.g., sterile, pyrogen-free water) prior to use.

[0250] The anti-TDP-43 cell penetrants described herein can be present in any pharmaceutically acceptable excipient or carrier. For example, the anti-TDP-43 cell penetrants described herein can be present in a buffer solution. The buffer solution may have a pH of about 6 to about 7. Typically, the formulation is sterile, for example, by aseptic filtration using a 0.2 μm or 0.22 μm filter. The formulations disclosed herein are generally stable after freezing and thawing.

[0251] In some implementation schemes, it may be necessary to... Cell Biophys. Alternatively, a pharmaceutical composition comprising any of the cell-penetrating agents described herein may be used in an in vitro method. For example, such a method may be used for non-diagnostic and / or non-therapeutic purposes. In such cases, a sample (such as cells, tissues, and / or organs) removed from a patient is exposed to a pharmaceutical composition comprising any of the cell-penetrating agents described herein.

[0252] In prophylactic applications, a cell-penetrating agent (e.g., a nucleic acid or carrier encoding any of the cell-penetrating agents described herein) or a pharmaceutical composition thereof is administered to patients suspected of or at risk of diseases such as ALS, FTLD-TDP, primary lateral sclerosis, progressive muscular atrophy, and Parkinson's disease in a regimen (dosage, frequency, and route of administration) that effectively reduces the risk of at least one sign or symptom of TDP-43-related disease, lessens its severity, or delays its onset. Specifically, the regimen preferably effectively inhibits or delays TDP-43 aggregates (e.g., human TDP-43 aggregates) in the brain, and / or inhibits or delays its toxic effects and / or inhibits / delays the development of behavioral deficits.

[0253] In therapeutic applications, a cell-penetrating agent is administered to a patient suspected of or already suffering from the disease in a regimen (dosage, frequency, and route) that effectively improves or at least inhibits further deterioration of at least one sign or symptom of the disease (e.g., ALS). Specifically, the regimen preferably effectively reduces or at least inhibits further increases in the cytoplasmic levels of TDP-43 (e.g., human TDP-43) and / or aggregates formed therefrom, associated toxicity, and / or behavioral deficits.

[0254] The regimen is considered effective in treatment or prevention if the outcome achieved by an individual patient is more favorable than the average outcome in a control group of comparable patients who were not treated using the methods disclosed herein.

[0255] VII. Treatment Plan As used herein, the terms “treat” and “treatment” mean to reduce or improve one or more symptoms or effects associated with a disease, to prevent, suppress or delay the onset of one or more symptoms or effects of a disease, to reduce the severity or frequency of one or more symptoms or effects of a disease, and / or to increase or tend toward the desired outcome as described herein.

[0256] The expected outcomes of the treatments disclosed herein vary depending on the TDP-43-related disease and patient profile, and are readily determined by those skilled in the art. Expected outcomes include improvements in the patient's health. Typically, expected outcomes include measurable indicators such as reduction or elimination of pathological ALS, FTLD-TDP, primary lateral sclerosis, and progressive muscular atrophy and Parkinson's disease.

[0257] This document provides a method for delivering an antibody specifically bound to TDP-43 into cells, comprising contacting the cells with any of the cell-penetrating agents described herein, thereby causing at least an antigen-binding fragment of the antibody to be internalized into the cells. In some embodiments, the method includes at least transferring an antigen-binding fragment of the antibody into the cytosol of the cells.

[0258] This article also provides a method for binding intracellular TDP-43 protein in cells, comprising contacting the cells with a cell-penetrating agent as described in any one of claims 1 to 135, thereby causing at least an antigen-binding fragment of the antibody to be internalized and transferred into the cytosol.

[0259] This article also provides a method for binding intracellular TDP-43 protein in cells, comprising: contacting the cells with a cell penetrant according to any one of claims 1 to 135, thereby causing at least an antigen-binding fragment of an antibody to be internalized and translocated into the cytosol; and binding at least an antigen-binding fragment of an antibody to the intracellular TDP-43 protein.

[0260] Furthermore, this article provides a method for inhibiting or reducing the accumulation of TDP-43 (e.g., human TDP-43) in subjects who have TDP-43-related diseases or are at risk of developing such diseases, comprising administering an effective amount of any of the cell-penetrating agents described herein to the subject, thereby inhibiting or reducing the accumulation of TDP-43 in the subject (i.e., the patient).

[0261] This article also provides a method for treating or achieving prevention of TDP-43-related disease in subjects, which includes administering a therapeutically effective amount of any of the cell-penetrating agents described herein, thereby treating or achieving prevention of TDP-43-related disease.

[0262] In some implementations, TDP-43-related diseases include ALS, FTLD-TDP, primary lateral sclerosis, progressive muscular atrophy, and Parkinson's disease. In some implementations, TDP-43-related diseases include ALS.

[0263] This article also provides a method for detecting TDP-43 deposition (e.g., human TDP-43 deposition) in subjects who have TDP-43-related diseases or are at risk of developing such diseases, comprising administering any of the cell-penetrating agents described herein to the subject and detecting antibodies that bind to TDP-43 in the subject.

[0264] In some embodiments, the cell-penetrating agent is administered to the subject via intravenous injection. In some embodiments, the cell-penetrating agent or antibodies contained therein are labeled. In some embodiments, the cell-penetrating agent is labeled with fluorescent, paramagnetic, or radioactive labels. In some embodiments, radioactive labels are detected using positron emission tomography (PET) or single-photon emission computed tomography (SPECT).

[0265] Cell-penetrating agents are administered in an effective regimen, meaning the dosage, route of administration, and frequency of administration for delaying the onset of at least one sign or symptom of the disease being treated, reducing its severity, inhibiting further deterioration, and / or improving the condition. If the patient already has the disease, the regimen may be called a therapeutically effective regimen. If the patient is at an elevated risk of the disease relative to the general population but has not yet experienced symptoms, the regimen may be called a preventatively effective regimen. In some cases, therapeutic or preventative efficacy may be observed in the same patient relative to a historical control or past experience. In other cases, therapeutic or preventative efficacy may be demonstrated in a preclinical or clinical trial population of treated patients relative to a control group of untreated patients.

[0266] Application can be parenteral, intravenous, oral, subcutaneous, intra-arterial, intracranial, intrathecal, intraperitoneal, local, intranasal, or intramuscular. Some cell-penetrating agents can be administered into the systemic circulation via intravenous or subcutaneous administration.

[0267] Cell-penetrating agents can be administered intravenously or subcutaneously in a dose range of approximately 0.5 mg / kg to approximately 30 mg / kg of host body weight. For example, doses may be approximately 0.5 mg / kg body weight, approximately 1.0 mg / kg, approximately 1.5 mg / kg, approximately 2.0 mg / kg, approximately 4.0 mg / kg, approximately 5.0 mg / kg, approximately 8.0 mg / kg, approximately 10 mg / kg, approximately 15 mg / kg, approximately 16 mg / kg, approximately 20 mg / kg, approximately 24 mg / kg, approximately 25 mg / kg, or approximately 30 mg / kg body weight. Dosages may also be based on body surface area, ranging from approximately 0.5 mg / m². 2 Approximately 500 mg / m 2 (e.g., 0.5, 5, 10, 50, 100, 250 or 500 mg / m²) 2 ) Administration. For intravenous administration, an amount of cell-penetrating agent formulation sufficient to achieve the individual patient's desired dose is transferred from one or more vials into one or more intravenous bags containing fluid (e.g., saline) and administered to the patient.

[0268] Cell-penetrating agents are typically administered on multiple occasions. The frequency of administration depends on factors such as the half-life of the cell-penetrating agent in circulation, the patient's condition, and the route of administration. The frequency can be daily, weekly, monthly, quarterly, or at irregular intervals in response to changes in the patient's condition or the progression of the disease being treated.

[0269] Exemplary treatment regimens require administration every two weeks, monthly, or every 3 to 6 months. The number of doses administered depends on whether the condition is acute or chronic and the condition's response to treatment. The dosing frequency can be adjusted based on the pharmacokinetic profile of the antibody agent in the patient. For example, the half-life of the cell-penetrating agent may ensure a dosing frequency of two weeks. In some embodiments disclosed herein, the cell-penetrating agent is administered to the patient for at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, 5 years, 10 years, or for the patient's lifetime.

[0270] Normal levels of TDP-43 (e.g., human TDP-43) can be represented by a sample of individuals in a general population who have not been diagnosed with a specific TDP-43-related disease (e.g., ALS) and are not considered to be at high risk of developing such a disease. ， The brain of a representative sample from a disease-free individual under 50 years of age is used to determine the level. Alternatively, if, according to the method of the invention, the PET signal in a brain region known to develop TDP-43 aggregates (e.g., human TDP-43 aggregates) is indistinguishable (within measurement accuracy) from a brain region known not to normally form such deposits, then normal levels can be identified in an individual patient. Elevated levels in an individual can be compared with normal levels. ( For example, by comparing the variance of the external mean and standard deviation, or by identifying elevated signals beyond experimental error only from brain regions associated with TDP-43 aggregates (e.g., human TDP-43 aggregates) compared to unknown regions associated with deposits. For the purpose of comparing the levels of TDP-43 aggregates (e.g., human TDP-43 aggregates) in individuals and populations, TDP-43 aggregates should preferably be measured in the same regions of the brain, including at least one region known to form TDP-43 aggregates associated with a specific disease (e.g., ALS) (e.g., in the cytoplasm).

[0271] Patients with elevated levels of TDP-43 aggregates (e.g., human TDP-43 aggregates) are candidates for initiating immunotherapy. A decrease in TDP-43 aggregate levels (e.g., human TDP-43 aggregates) after initiation of immunotherapy can initially be considered an indication that the treatment has a desired effect. The observed decrease may, for example, range from 1-100%, 1-50%, or 1-25% of baseline values. Such effects can be measured in one or more regions of the brain where deposits are known to form, or from the mean of such regions. The overall effect of treatment can be approximated by adding the percentage reduction relative to baseline to an increase in TDP-43 aggregates (e.g., human TDP-43 aggregates) that would otherwise occur in untreated patients.

[0272] Maintaining TDP-43 aggregates (e.g., human TDP-43 aggregates) at a substantially constant level or even a slight increase in TDP-43 aggregates (e.g., human TDP-43 aggregates) can also indicate a response to treatment, albeit a suboptimal one. Such responses can be compared to the time course of TDP-43 aggregate (e.g., human TDP-43 aggregate) levels in patients with an untreated specific disease (e.g., ALS) to determine whether immunotherapy has an effect in inhibiting further increases in TDP-43 aggregates (e.g., human TDP-43 aggregates).

[0273] VIII. Reagent Kit This disclosure further provides a kit (e.g., a container) containing any of the cell-penetrating agents described herein and related materials, such as instructions for use (e.g., packaging inserts). The instructions for use may contain, for example, instructions regarding the administration of the cell-penetrating agent and one or more other agents optionally present. The container for the cell-penetrating agent may be a unit dose, a bulk package (e.g., a multi-dose package), or a subunit dose.

[0274] IX. Detection Methods In some aspects, the cell-penetrating agent of this disclosure further provides a method for detecting TDP-43 in a sample. For example, in some embodiments, this disclosure provides a method for detecting TDP-43 in a sample, which includes contacting the cell-penetrating agent of this disclosure with the sample and detecting the binding of the cell-penetrating agent or antibody to TDP-43. For example, such a method may be used for... Cancer Chemother. Pharmacol., Alternatively, an in vitro method may be used. In some embodiments, the sample is a biological sample derived from a subject (e.g., a human subject). In some embodiments, the subject is a human. In some embodiments, the subject is a patient with TDP-43-related disease or at risk of having said disease. In such cases, a sample (such as cells, tissues, and / or organs) removed from the patient is exposed to the antibody or antigen-binding fragment described herein. In some embodiments, the sample comprises cells derived from the patient, and the cells are lysed prior to administration of the antibody or antigen-binding fragment described herein.

[0275] Example The following embodiments are included to illustrate the patterns disclosed herein. Certain aspects of the following embodiments are described in accordance with techniques and procedures that have been discovered or anticipated by the co-inventors of the invention to work well in the practices disclosed herein. Based on this disclosure and the general level of skill of those skilled in the art, it will be understood that the following embodiments are intended to be exemplary only and that various changes, modifications, and alterations may be made without departing from the scope of this disclosure.

[0276] Example 1. Humanized anti-TDP-43 antibody Humanized anti-TDP-43 antibody was generated from mouse monoclonal antibody 13D3. Specifically, Immunol. Rev., Show the annotation form of the heavy-chain variable structural domain and in vitro The annotation format of the light chain variable domain is shown. Both the heavy chain variable domain and light chain variable domain sequences show the native signal peptide, the variable domain, and a partially constant domain. The partially heavy chain constant domain region corresponds to the IgG2a mouse constant domain. The partially light chain constant domain corresponds to the mouse κ constant domain. References to amino acid substitutions used herein refer to the Kabat numbering system (see, e.g., Kabat EA et al.). in vitro Figure 1 (5th ed.). Bethesda, MD: National Institutes of Health (1991)).

[0277] In short, protein sequences are identified in the Protein Data Bank (PDB) database (see Deshpande et al., The RCSB Protein Data Bank: a redesigned query system and relational database based on the mmCIF schema). Figure 2 , 33:D233-D237(2005)) to search for structures that would provide a similar structural model of 13D3. The crystal structure of the antibody Fab PDB code “5BK5”, which is a human germline antibody. Based on its overall sequence similarity with 13D3 VH and VK, resolution quality (3.0 Å), 5BK5 was selected for both VH and VK structures, and it retains the same classical structure for the complementarity-determining region (CDR) loop (Scally et al., Crystal structure of anti-cirumsporozoite protein 663 germline antibody; Direct deposit to PDB (2017)).

[0278] In addition, since the 5BK5 antibody is a human lineage-derived antibody and belongs to the same classical category as human lineage IGHV3-48'03 (SEQ ID NO: 2) for the variable heavy chain domain and the same classical category as human lineage IGKV2-30*02 (SEQ ID NO: 25) for the variable light chain domain, these sequences were used as the human receptor framework.

[0279] Therefore, the framework regions of 5BK5 VH and 5BK5 VL were selected as the receptor sequences for the 13D3 CDR. 13D3 CDR models transplanted onto the corresponding human frameworks of VH and VL were established and used as a guide for further reversal mutations to improve binding specificity and reduce immunogenicity.

[0280] More specifically, the amino acid sequence consisting of the 5BK5 VH human framework and 13D3 CDR is named hu13D3VHv1d (SEQ ID NO: 20), and the amino acid sequence consisting of the 5BK5 VL human framework and 13D3 VL CDR is named hu13D3VLv1d (SEQ ID NO: 47).

[0281] The additional forms hu13D3VH and hu13D3VL were designed to enable the assessment of the contributions of various framework residues to antigen binding, thermostability, exploitability (e.g., deamination, oxidation, N-glycosylation, proteolysis, and aggregation), and immunogenicity. The substitution positions were considered based on several factors, including the position defining the classical CDR conformation (see Martin, ACR, Protein sequence and structure analysis of antibody variable domains, In: Kontermann R and Dübel S (eds.)). Sequences of Proteins of Heidelberg, Germany: Springer International Publishing AG (2010); Position within the vernier area (see Foote J. and Winter, G., Antibody framework residues affecting the conformation of the hypervariable loops). Immunological Interest Nucleic Acids Research Antibody Engineering J Mol Biol 224(2):487-99 (1992)); located at the VH / VL domain interface (see Léger OJP and Saldanha, J. Preparation of recombinant antibodies from immune rodent spleens and the design of their humanization by CDRgrafting. In: Shepherd P and Dean C (eds.) Monoclonal Antibodies: A Practical ApproachOxford, UK: Oxford University Press (2000); locations readily susceptible to post-translational modifications (such as glycosylation or pyroglutamylation); locations occupied by residues predicted to interact with the CDR, based on 13D3 CDR models transplanted onto the VH and VL frameworks; and / or locations occupied by residues rare in sequenced human antibodies, where parental mouse 13D3 residues or other residues are much more prevalent in the human antibody lineage.

[0282] The following is an overview of humanized 13D3 antibodies.

[0283] Heavy chain variable structural domain hu13D3VHvd1 (SEQ ID NO: 20) consists of CDR-H1, CDR-H2, and CDR-H3 loops of 13D3-VH transplanted onto the 5BK5 VH framework and restores all framework substitutions at key locations defining the classic Chothia class. These substitutions are part of the vernier region and are located at the VH / VL domain interface or contribute to structural stability. Hu13D3VHvd1 includes the following substitutions, which are reversion mutations of germline antibodies and are most common at the following locations: positions L5V and T77S.

[0284] hu13D3VHv2d (SEQ ID NO: 21) includes the following substitution: L78A. As indicated by the Immunoeptope Database (“IEDB”) analysis, leucine at position 78 is immunogenic. Therefore, deimmunization analysis predicts reduced immunogenicity with an alanine substitution at position 78.

[0285] Hu13D3VHv3d (SEQ ID NO: 22) includes the following substitutions: G44R, S49A, and S74A. Arginine at position 44 undergoes a G100D (glycine to aspartic acid) substitution with the variable light chain domain to form... From the beginning Contact is established, thereby strengthening the heavy chain variable domain:light chain variable domain interface. Specifically, arginine at position 44 forms a hydrogen bond and salt bridge with G100D, and also forms a hydrogen bond with F98 in the variable light chain domain. Position 49 is a vernier residue, and the substitution from serine to alanine is a reversion mutation to assess the effect on CDR conformation and binding. Finally, the substitution from serine to alanine at position 74 is a germline substitution.

[0286] Hu13D3VHv4d (SEQ ID NO: 23) includes various substitutions made in the attempted combinations of hu1353VHv1d, hu1353VHv2d and hu1353VHv3d.

[0287] Light chain variable structural domain Hu13D3VLv1d (SEQ ID NO: 47) consists of CDR-L1, CDR-L2, and CDR-L3 rings of 13D3VL transplanted onto a 5BK5 VL frame and restores all frame substitutions at key locations defining the Chothia classical class, which are part of the vernier region and located at the VH / VL structural domain interface. Hu1353VLv1d includes the following substitutions: V3Q, P15L, E17Q, L38Q, K39R, Q100D, and L104V.

[0288] Replacing glutamine with valine at position 3 reduces the immunogenicity of the light chain variable domain. Replacing proline with leucine at position 15 is a germline substitution. Replacing glutamate with glutamine at position 17 is a rare substitution. Position 17 has significant surface exposure, resulting in negatively charged plaques on the protein surface. Substitution with glutamine reduces positive plaques on the antibody surface. Because glutamine in the mouse structural model forms interchain contacts within the light chain variable domain to maintain structural conformation, glutamine is substituted for leucine at position 38. In contrast, leucine residues do not form the same contacts; therefore, this reversion mutation improves conformational stability. Replacing lysine with arginine at position 39 improves conformational stability by forming additional contacts with adjacent residues that lysine residues cannot. Position 100 in the light chain variable domain is located at the interface between the heavy chain variable domain and the light chain variable domain; however, it does not form interchain contacts with glutamine residues. Substitution at position 44 of the heavy chain variable domain with aspartic acid and simultaneous substitution with arginine produces a stronger de novo contact and thus improves the antibody's thermal stability. Substitution of leucine at position 104 with valine is predicted to decrease immunogenicity.

[0289] Hu13D3VLv2d (SEQ ID NO: 48) consists of the substitutions described above in hu13D3VLv1d, but also includes the following substitution: L92A. Since leucine at position 92 is predicted to be immunogenic, substitution with alanine at position 92 reduces the immunogenicity of the variable light chain domain.

[0290] Tables 1 and 2 below show the sequence alignments of the 13D3 variable heavy chain domain and the variable light chain domain compared to the humanized form, respectively.

[0291] Table 1.13 Sequence Alignment of D3 Humanized Variable Heavy Chain Domain

[0292]

[0293]

[0294]

[0295]

[0296]

[0297]

[0298]

[0299] Table 2. Sequence alignment of the 13D3 humanized variable light chain

[0300]

[0301]

[0302]

[0303]

[0304]

[0305] Specifically, for Vk, the human κ light chain (SEQ ID NO: 26) with NCBI accession code ABC66863 (Shringer et al., 2006) was selected. This κ light chain has the same classical category as CDR-L1 and L2, and according to IMGT convention, belongs to the hominid IGKV2-30*02 (SEQ ID NO: 25). For VH, the human Ig heavy chain AEX28899 (SEQ ID NO: 3) (GenBank: AEX28899) (Bowers et al., 2014) was selected, which also has the same classical category and belongs to the hominid IGHV3-48'03. It is a member of Kabat human heavy subgroup 3.

[0306] The AEX28899 and ABC66863 antibodies are human-derived antibodies, belonging to the same classical class and, for the heavy chain variable domain, to the human IGHV3-48'03 (SEQ ID NO: 2), and for the light chain variable domain, to the human IGHV3-48*03 (SEQ ID NO: 25). Therefore, the heavy chain variable domain sequences of AEX28899 and the light chain variable domain sequences of ABC66863 were used as the human receptor framework for the 13D3 CDR. 13D3 CDR models transplanted onto the corresponding human frameworks in VH and VL were established and used as guidance for further reversion mutations.

[0307] Humanized forms of the AEX28899 heavy chain variable domain and ABC66863 light chain variable domain sequences were used as the human receptor framework for the 13D3 CDR and were designed to enable assessment of the contributions of various framework residues to antigen binding, thermostability, exploitability (e.g., deamination, oxidation, N-glycosylation, proteolysis, and aggregation), and immunogenicity. The placement of substitutions was considered based on several factors, including the position defining the classical CDR conformation (see Martin, ACR, Protein sequence and structure analysis of antibody variable domains, In: Kontermann R and Dübel S (eds.)). Antibody Engineering Heidelberg, Germany: Springer International Publishing AG (2010); Position within the vernier region (see Foote J. and Winter, G., Antibody framework residues affecting the conformation of the hypervariable loops). J Mol Biol 224(2):487-99 (1992)); Location at the VH / VL domain interface (see Léger OJP and Saldanha, J). Preparation of recombinant antibodies from immunerodent spleens and the design of their humanization by CDR grafting. In: Shepherd P and Dean C (eds.). Monoclonal Antibodies: A Practical Approach .Oxford, UK: Oxford University Press (2000)); locations readily susceptible to post-translational modifications (such as glycosylation or pyroglutamylation); locations occupied by residues predicted to interact with the CDR, based on 13D3 CDR models transplanted onto the VH and VL frameworks; and / or locations occupied by residues rare in sequenced human antibodies, where parental mouse 13D3 residues or other residues are much more prevalent in the human antibody lineage.

[0308] The following is an overview of the VH domain substitutions in SEQ ID NO: 3: R19K: K19 in the mouse antibody structure generates a π-cation interaction with W79 in SEQ ID NO: 3; G44R: Replacing Gly with Arg at this position potentially establishes a contact with the light chain, thereby improving antibody stability; S77T: Threonine reduces immunogenicity; L78A: Leucine is predicted to be immunogenic, therefore substitution with alanine reduces the predicted immunogenicity; L78G: Glycine also reduces the predicted immunogenicity; L80A: Removal of leucine from the position reduces immunogenicity; L80G: Similar to alanine, glycine at position 80 reduces immunogenicity; L82cG: Glycine potentially reduces heavy chain immunogenicity; and R83M: A reversion mutation that can confer antibody stability. The following is an overview of the VL domain substitutions in SEQ ID NO: 26: I2V is a vernier domain residue, valine is retained to maintain the CDR conformation; L9S: Leucine is predicted to be immunogenic, and the substitution with serine reduces the predicted immunogenicity; P18Q: Glutamine in the mouse antibody model forms an H bond (LC) with K74, thereby stabilizing the interchain interaction; R46L: is an interface and vernier domain residue, therefore Leu is retained; A80S: Serine reduces the predicted immunogenicity; L92G: L92 is a CDR residue and is predicted to be immunogenic, glycine residues are predicted to reduce immunogenicity; V94I: Valine at position 94 is predicted to have a low level of immunogenicity, isoleucine is predicted to reduce immunogenicity; and V94A: Alanine is a substitute that is predicted to reduce immunogenicity.

[0309] Example 2. Characterization of humanized 13D3 antibody variants As discussed in this paper, cytoplasmic aggregates of TDP-43 are found in many neurodegenerative diseases, particularly ALS. Typically, the individual TDP-43 proteins found in cytoplasmic aggregates are phosphorylated at serine residues at positions 409 and / or 410. A binding screening assay was established to determine whether 13D3 and its humanized variants selectively bind to the phosphorylated TDP-43 found in cytoplasmic aggregates.

[0310] Approximately 50 RU of biotin-TDP43 protein was immobilized on a CM3 chip along with NeutrAvidin. Various humanized and chimeric 13D3 antibodies were flowed through the chip, and their binding to phosphorylated TDP43 protein was analyzed. Specifically, hu13D3H9L8, hu13D3H10L6, hu13D3H10L7, hu13D3H10L8, and hu13D3H9L7 were analyzed, showing similar binding to TDP43 phosphate as the chimeric 13D3 antibody. Figure 3Similarly, the binding of hu13D3Hd1Ld1, hu13D3Hd2Ld2, hu13D3Hd3Ld1, hu13D3HD4LD1, hu13D3HD1LD2, hu13D3HD2LD2, hu13D3HD3LD2, and hu13D3HD4LD2 to TDP43 phosphate was tested. Each variant showed similar binding to TDP43 phosphate as the chimeric 13D3 antibody. Figure 4 ).

[0311] In another experiment, the antibody was immobilized on a protein A chip (approximately 2000 RU), and a TDP-43 peptide with 23 amino acids phosphorylated at positions 409 and 410 (“TDP-43 peptide (pS409 / pS410)”) was flowed through the chip at concentrations between 1 nM and 100 nM (SEQ ID NO: 83). As a control, TDP-43 peptide with 23 amino acids not phosphorylated at positions 409 or 410 was also tested at concentrations between 12 nM and 1 μM. Specifically, the humanized form hu13D3H5L2 showed a 1:1 binding to various concentrations (1.2345 nM; 3.703 nM; 11.111 nM; 33.33 nM; and 100 nM) of the TDP-43 peptide (pS409 / pS410), similar to that of the 13D3 murine antibody (…). Figure 5 Furthermore, hu13D3H5L2 did not show affinity for the unphosphorylated 22 amino acid TDP-43 peptide at 12.345 nM, 37.03 nM, 111.111 nM, 333.333 nM and 1 μM (data not shown).

[0312] The thermostability of the antibody was also evaluated. Thermostability analysis was performed using differential scanning calorimetry (DSC), a method characterizing the stability of proteins or other biomolecules (e.g., antibodies or their antigen-binding fragments). 2.66 mM (0.4 mg / mL) antibody was tested in 1xPBS at pH 7.4. The testing temperature range was 25 °C to 100 °C.

[0313] Data on the binding of chimeric 13D3 and its humanized forms are summarized in Tables 3 and 4. Tables 3 and 4 also summarize thermal stability and immunogenicity data.

[0314] Table 3. Humanized TDP-43 Resistance Scale

[0315]

[0316] Table 4. Scale of Humanized 13D3 Antibody

[0317] In summary, the data shown in Tables 3 and 4 demonstrate exemplary humanized antibodies with low predicted immunogenicity scores relative to mouse 13D3 antibodies (explained in further detail below). The data further illustrate the high yields and melting temperatures of the humanized antibodies (e.g., similar to mouse 13D3 antibodies). See, for example, the antibodies h13D3Hd1-Ld1, h13D3Hd1-Ld2, and h13D3Hd2-Ld1 shown in Table 4. Furthermore, the data also demonstrate humanized antibodies with similar binding scales to mouse 13D3 antibodies.

[0318] Computer-simulated immunogenicity analysis of humanized antibodies The immunogenicity scores shown in Tables 3 and 4 are predicted immunogenicity values ​​calculated from two different software programs: the Immune Epitope Database (IEDB) and EpiQuest. The IEDB immunogenicity analysis tool is sponsored by the National Institute of Allergy and Infectious Diseases.

[0319] For IEBDB, the immunogenicity prediction method used is based on the predicted potential binding of peptides within the protein sequence to major histocompatibility class (MHC) II. The program identifies potential immunogenic regions within the protein sequence. The MHC II tool uses a broad spectrum of MHC II alleles in the human population (i.e., 26 reference alleles) to predict immunogenic regions. The software generates a series of 15-residue peptides overlapping at 10 residues. The generated 15-residue peptides are predicted to bind to the 26 reference alleles.

[0320] For each peptide, a percentile rank is generated for each of three methods (combinatorial library, SMM_align, and Sturniolo) by comparing the score of each peptide with the scores of five million randomly selected 15-residue peptides from the SWISSPROT database. The adjusted percentile rank is a frequency-adjusted percentile rank based on peptide length. A low number percentile rank indicates high affinity. The median percentile rank of the three methods is then used to generate a rank for the common method. By default, the prediction results are collapsed to display only the percentile rank and the adjusted rank. The maximum median percentile rank threshold is set to 20.

[0321] The EpiQuest T-Scanner tool classifies cytotoxic T-lymphocyte (CTL) epitopes based on their predicted immunodominance. Immunodominance of an epitope is defined as its relative strength in functional assays related to the target killing or release of the corresponding cytokine. These parameters indicate the function of the T-eptope. This procedure is designed to analyze and sort CTL peptide epitopes eluted from target cells based on their immunodominance. Typically, only a few peptide epitopes binding to MHC class I possess actual functional activity. The relative strength of a CTL (T) epitope is defined by its binding strength to MHC class I and the T-cell receptor (TCR) (in the case of MHCI). The algorithm detects the structural and compositional characteristics of the peptide epitope, which enable it to elicit a high affinity for the TCR. This analysis is haplotype-specific, and the EpiQuest T-scanner has a matrix for analyzing HLA-A2 and H2kB haplotype-binding peptides.

[0322] Example 3. Binding of anti-TDP-43 antibody to phosphorylated cytoplasmic aggregates of TDP-43 in FTP brain tissue and model systems Figures 6A through 6C show brain tissue of frontotemporal dementia (“FTD”) (Figure 6A) and healthy brain tissue (Figure 6C). Figure 6B is an inset of Figure 6A, showing the co-localization of the 13D3 antibody with neuronal cytoplasmic aggregates associated with phosphorylated TDP-43 FTD. The data indicate that the 13D3 antibody specifically binds to cytoplasmic aggregates in FTD brain tissue, but not in healthy brain tissue. Similarly, Figure 7A -C each shows that rNLS8 dox can inhibit 13D3-specific binding cytoplasmic aggregates in the TDP-43 protein disease model.

[0323] Example 4. Binding of anti-TDP-43 antibody to phosphorylated cytoplasmic aggregates and nuclear TDP-43 aggregates in transfected HEK cells. Figure 8A Confocal micrographs showing HEK cells transiently transfected with GFP-2a-TDP43 [mNLS (R82L / K83Q) DCS (C173S / C175S)] or GFP only (2a being a self-cleaving peptide that releases TDP-43 upon expression). The top image shows staining of GFP, cell nuclei (gray), and pTDP-43 (white). The bottom image shows only pTDP-43 staining, which was not present in the GFP-only control transfection. Figure 8B (Left) A graph showing cell counts in HEK cells transfected with GFP-2a-TDP43 or GFP alone. The data demonstrate that the cell counts were approximately equal between the two populations. Furthermore, Figure 8B(Right) Shows the pTDP-43 lesion count in HEK cells transfected with GFP-2a-TDP43 or GFP alone. The data indicate that TDP-43 lesions only form in HEK cells transfected with GFP-2a-TDP43. Figure 8C TDP-43 staining was performed using commercially available antibodies or antibodies disclosed herein (including 13D3, 13C13, and 2D4). Cells were treated with the antibody for 24 hours, and then incubated with 100 μg / ml antibody for another 24 hours. Cells were then washed, fixed / permeabilized, and stained with anti-pTDP-43 antibody, followed by staining with AF647-bound anti-mouse secondary antibody. The stained cells were imaged using a 40x water objective via high-content imaging (Operetta system). Quantitative analysis was performed using Harmony software.

[0324] Antibodies 13D3, 13C13, and 2D4 were used to detect mislocalized TDP-43 overexpression in HEK cells. The top row shows transfection with GFP-2a-TDP-43, where phosphorylated TDP43 does not include the nuclear localization signal (i.e., phosphorylated TDP43 is retained in the cytoplasm), and the bottom row shows transfection with the GFP-only construct. The data indicate that TDP-43 lesions only form in HEK cells transfected with GFP-2a-TDP-43.

[0325] The assay included various control antibodies, including pTDP-43 (Cosmo)+, pTDP-43 (1D3)+, total TDP-43 (PT), and 3B12 (ED)+. The control antibodies validated TDP-43 aggregation in the cytoplasm. Similarly, antibodies 13D3, 13C13, and 2D4 were tested and showed similar binding to cytoplasmic aggregates of phosphorylated TDP-43.

[0326] Example 5. Characteristics of the humanized anti-TDP-43 cell penetrant Binding data, immunogenicity, and thermal stability of the anti-TDP-43 cell penetrant were evaluated as described in Example 3. Binding data, immunogenicity scores, and thermal stability data of cell penetrants containing murine 13D3 and its humanized forms are summarized in Tables 5 and 6.

[0327] Table 5: Stability, IHC and target binding data of anti-TDP-43 CPA

[0328] Table 6: Binding data for anti-TDP-43 CPA (divalent analyte pattern)

[0329] As demonstrated by the data in Table 5, the anti-TDP-43 CPA of this disclosure is highly stable, exhibiting stability comparable to that of the corresponding antibody. For example, the anti-TDP-43 CPA in Table 5 has a melting temperature of 69.9°C to 77.5°C and is expressed in high yield. Furthermore, target binding of the anti-TDP-43 CPA was demonstrated by immunohistochemical (IHC) analysis and cytometry. Table 6 provides exemplary binding data for the anti-TDP-43 CPA, showing that the CPA binds to phosphate-TDP-43 at a sub-nanomolar KD value. Therefore, the data in Tables 5 and 6 indicate that the anti-TDP-43 CPA of this disclosure retains strong binding to phosphate-TDP-43 of the parent antibody, thermal stability, and in vitro target binding.

[0330] Example 6. Internalization and clearance of TDP-43 aggregates in cells transfected with an anti-TDP-43 cell penetration agent Figures 9 through 22 illustrate the internalization and / or target binding of the anti-TDP-43 cell penetrant of this disclosure. In Figures 9 through 22, references to “M-Lyco,” “Lycotoxin,” “ML,” etc., refer to a CPA containing L17E_M-lycotoxin linked to a 13D3 antibody (mouse, chimeric, or humanized). References to other CIMs and / or CMIPs (e.g., “CMIP,” “CMIP4,” “cTAT,” “PEPTH,” etc.) in Figures 9 through 22 refer to a CPA containing the corresponding CIM linked to a 13D3 antibody (mouse, chimeric, or humanized). Unless otherwise stated (e.g., via HC), the CIM is linked to the 13D3 antibody via the C-terminus of the light chain.

[0331] HEK cells were transiently transfected with GFP-2a-TDP43 [mNLS (R82L / K83Q) DCS (C173S / C175S)]. Twenty-four hours later, cells were incubated for four hours with 100 μg / ml of cell penetration medium containing the internalized portion and anti-TDP-43 antibody. Cells were then washed, fixed / permeabilized, and subsequently treated with AF647-conjugated anti-mouse secondary antibody. The stained cells were imaged using a 40x water objective via high-content imaging (Operetta system). Quantitative analysis was performed using Harmony software. Specifically, Figure 9AThe percentage of CPA-positive cells is shown after incubation with m13D3 CPAs containing different CIMs (i.e., TAT, M-Lycotoxin_L17E (LC), M-Lycotoxin_L17E (HC), PEPTH (HC)) or under various control conditions (i.e., unlabeled, isotyped, and mediator). HC (heavy chain) and LC (light chain) indicate the location of the CIM. The label “M-Lycotoxin” in Figure 9 refers to the CPA containing M-Lycotoxin_L17E linked to the C-terminus of the light chain (LC) or heavy chain (HC) of the m13D3 antibody.

[0332] Data showed that m13D3 CPA internalization was increased compared to naked antibody (“unlabeled”) and mediator control. Figure 9B The data show the number of CPA-positive spots per cell after incubation with m13D3 CPA. The data indicate that m13D3 CPA increased the detection of pTDP-43 lesions compared to the naked antibody and mediator controls.

[0333] Figures 10A to 10E Figure showing the results of HEK cells transfected with various m13D3 CPAs. Briefly, HEK cells were transiently transfected with GFP-2a-TDP43 [mNLS (R82L / K83Q) DCS (C173S / C175S)] or without plasmid (e.g., untransfected) as a negative control. After 24 hours, the cells were incubated for another 24 hours with 100 μg / ml antibody (e.g., CPA). The cells were then washed, fixed / permeabilized, and stained with anti-pTDP-43 antibody, followed by staining with AF647-bound anti-mouse secondary antibody. The stained cells were imaged using a 40x water objective via high-content imaging (Operetta system). Quantitative analysis was performed using Harmony software.

[0334] Figure 10A The figure shows the number of pTDP-43 lesions per well area. The label "M-Lycotoxin" in Figure 10 refers to the CPA containing M-Lycotoxin_L17E linked to the C-terminus of the light chain of the m13D3 antibody. The data indicate that cells treated with different m13D3 CPAs had a lower number of lesions per well area compared to cells treated with unlabeled m13D3 (e.g., without the internalization module). As expected, no lesions were observed in untransfected cells. Figure 10B The data show the mean focal intensity of pTDP-43 lesions. The data indicate that cells treated with different m13D3 CPAs had lower mean focal intensity compared to cells treated with unlabeled m13D3 antibody. As expected, extremely low focal intensity was observed in untransfected cells. Figure 10CThis displays a consistent cell count for each well of all test cell populations (i.e., cells transfected with or untransfected with different m13D3 CPA). Figure 10D The data show the number of pTDP-43 lesions normalized by cell count. The data indicate that cells treated with different m13D3 CPAs had a lower number of lesions per cell compared to cells treated with unlabeled m13D3. As expected, no lesions were observed in untransfected cells. Figure 10E The average lesion area of ​​p-TDP-43 lesions is shown, indicating that cells treated with different m13D3 CPAs have a lower average area intensity compared to cells treated with unlabeled m13D3; extremely low focusing intensity was observed in untransfected cells. Figure 11A The data show the number of pTDP-43 lesions per well area. The data indicate that cells treated with different m13D3 CPAs (i.e., TAT(HC), L17E M-Lycotoxin (LC), L17E M-lycotoxin (HC), and PEPTH (HC)) had a lower number of lesions per well area compared to cells treated with unlabeled m13D3. The label “M-Lycotoxin” in Figure 11 refers to the CPA containing M-Lycotoxin_L17E linked to the C-terminus of the light chain (LC) or heavy chain (HC) of the m13D3 antibody. Figure 11B This displays a consistent cell count for each well across all tested cell populations. Figure 11C The number of pTDP-43 lesions is shown as normalized by cell count, indicating that cells treated with different m13D3 CPAs had a lower number of lesions per cell compared to cells treated with unlabeled m13D3. As expected, no lesions were observed in untransfected cells. Figure 11D The data show the average lesion area of ​​pTDP-43 lesions. The data indicate that cells treated with different m13D3 CPAs had lower average area intensity compared to cells treated with unlabeled m13D3; extremely low focusing intensity was observed in untransfected cells. Figures 12A to 12D To display Figure 11A Figure 11 shows the results of cells incubated with different concentrations of m13D3 M-Lycotoxin [17E] CPA under the same experimental conditions described in -D. The label "M-Lycotoxin" in Figure 11 refers to the CPA containing M-Lycotoxin_L17E linked to the C-terminus of the light chain (LC) or heavy chain (HC) of the m13D3 antibody. Additionally, the cell internalization module is located on the heavy or light chain of the m13D3 antibody. Cells were also incubated with unlabeled m13D3, IgG isotype controls, and PBS controls. Untransfected HEK cells were used as a negative control.

[0335] Figure 12A The total lesion area of ​​pTDP-43 is shown per well. Data show a concentration-dependent decrease in total lesion area in cells treated with m13D3 M-Lycotoxin_L17ECPA. The label “M-Lycotoxin” in Figure 12 refers to the CPA containing M-Lycotoxin_L17E linked to the C-terminus of the light chain (LC) or heavy chain (HC) of the m13D3 antibody. In contrast, cells treated with 13D3 antibody, IgG isotype control, and PBS showed a higher level of lesions compared to cells treated with m13D3 m-Lycotoxin [L17E] CPA. Untransfected cells showed no lesions (data not shown). Figure 12B This shows consistent cell counts in each well across all tested cell populations, and that cell viability decreased in a concentration-independent manner in cells transfected with m13D3 M-lycotoxinCPA. Figure 12C The pTDP-43 lesion count showed that the lesion count of m13D3 M-Lycotoxin CPA increased in a concentration-dependent manner, with the number of lesions being significantly higher than that of the control. Figure 12D The pTDP-43 mean lesion size was shown, indicating that the mean lesion size of m13D3 M-Lycotoxin CPA decreased in a concentration-dependent manner, and was significantly reduced compared to the control. In summary, Figure 12A -D indicates that m13D3 M-Lycotoxin CPA interferes with lesion aggregation in a concentration-dependent manner.

[0336] Figure 13 The image shows untransfected HEK cells (left) incubated with m13D3 antibody or M-Lycotoxin m13D3 CPA for 24 hours and subjected to XTT metabolic assays to assess cell viability, or cells transfected with GFP-2a-TDP43 [mNLS (R82L / K83Q) DCS (C173S / C175S)] (right). Figure 13 The notation “M-lycotoxin” refers to the CPA containing M-Lycotoxin_L17E linked to the C-terminus of the light chain (LC) of the m13D3 antibody. Data showed no significant difference in cell death across all test populations (as measured by percentage of cell death), indicating that the CPA (m13D3 M-Lycotoxin [L17E]) does not induce significant cytotoxicity.

[0337] In summary, Figures 10 to... Figure 13 This indicates that the anti-TDP-43 cell penetrant of this disclosure is internalized by cells and can effectively bind to intracellular TDP-43.

[0338] Example 7. Internalization and clearance of phosphorylated cytoplasmic aggregates of TDP-43 using an anti-TDP-43 cell penetrant. Figures 14A to 14E To show a graph of cells incubated with different m13D3 CPAs and unlabeled m13D3 antibodies. Untransfected HEK cells were used as a negative control. Figures 14A to 14E The data in the above text Figures 11A to 11D The same experimental conditions described in the document were used to generate the product.

[0339] More specifically, Figure 14A The figure shows the sum of pTDP-43 lesions per well area. The label “ML-13D3” in Figure 14 refers to the CPA containing M-Lycotoxin_L17E linked to the C-terminus of the light chain (LC) of the m13D3 antibody. The label “CMIP1-5” in Figure 14 refers to the CPA containing CMIP1, CMIP2, CMIP3, CMIP4, or CMIP5 linked to the C-terminus of the light chain (LC) of the m13D3 antibody. The data indicate that cells treated with different m13D3 CPAs (M-Lyco_L17E, CMIP1, CMIP2, CMIP3, CMIP4, CMIP5) had a lower number of lesions per well area compared to cells treated with unlabeled m13D3. As expected, no lesions were observed in untransfected cells. Figure 14B The data show the mean focal intensity of pTDP-43 lesions. The data indicate that cells treated with different m13D3 CPAs had lower mean focal intensity compared to cells treated with unlabeled m13D3 antibody, and extremely low focal intensity was observed in untransfected cells. Figure 14C This displays a consistent cell count for each well across all tested cell populations. Figure 14D The number of p-TDP-43 lesions was displayed (normalized by cell count). Data showed that cells treated with different m13D3 CPA antibodies had a higher number of lesions per cell compared to cells treated with unlabeled m13D3 antibody. As expected, no lesions were observed in untransfected cells. Finally, Figure 14E The data show the average lesion area of ​​pTDP-43 lesions. The data indicate that cells treated with different m13D3 CPAs had lower average area intensity compared to cells treated with unlabeled m13D3; extremely low focusing intensity was observed in untransfected cells.

[0340] Figures 15A to 15D The figure shows the results of incubating cells with different concentrations of m13D3 m-Lycotoxin [L17E] CPA or m13D3 CMIP4 CPA; incubating cells with unlabeled m13D3 or IgG isotype controls; and using untransfected HEK cells as a negative control. Figures 15A to 15D The data in the above text Figures 11A to 11D The same experimental conditions described herein were used. In Figure 15, the label "13D3-ML" refers to the CPA containing M-Lycotoxin_L17E linked to the C-terminus of the light chain (LC) of the m13D3 antibody. In Figures 15 and 16, the label "13D3-CMIP4" refers to the CPA containing CMIP4 linked to the C-terminus of the light chain (LC) of the m13D3 antibody.

[0341] Figure 15A The total number of p-TDP-43 lesions was shown, indicating that the total lesion area of ​​cells treated with 13D3 m-Lyco and 13D3 CMIP4 CPA decreased in a concentration-dependent manner. In contrast, cells treated with 13D3 antibody and isotype showed a higher level of lesions. Untransfected cells did not show lesions. Figure 15B This showed consistent cell counts in each well across all tested cell populations, and cell viability decreased in a concentration-independent manner for m13D3 m-Lycotoxin CPA. Figure 15C The number of p-TDP-43 lesions normalized by cell count showed that the lesion counts of 13D3 m-Lycotoxin and 13D3 CMIP4 CPA increased in a concentration-dependent manner, with the number of lesions being significantly higher than that of the control. Figure 15D The data shows the mean lesion area of ​​p-TDP-43, indicating that the mean lesion area of ​​13D3 m-Lycotoxin and 13D3 CMIP4 CPA decreased in a concentration-dependent manner, and the mean lesion area was significantly reduced compared to the control. Therefore, Figures 15A to 15D This indicates that 13D3 m-Lycotoxin and 13D3 CMIP4 CPA interfere with lesion aggregation in a concentration-dependent manner.

[0342] To obtain Figures 16A to 16E The data provided in this study involved incubating cells with m13D3 CMIP4 CPA and unlabeled m13D3 antibody in acetate buffer or PBS. Figures 16A to 16E The data in the above text Figures 11A to 11D The same experimental conditions described in the document were used to generate the product.

[0343] More specifically, Figure 16A The pTDP-43 lesions per well area are shown, indicating that cells treated with m13D3 CMIP4 CPA had a lower number of lesions per well area compared to cells treated with unlabeled m13D3; no change in cell penetration activity was observed between PBS and acetate. Figure 16BThe mean focal intensity of pTDP-43 lesions was shown, indicating that cells treated with m13D3CMIP4 CPA had a lower mean focal intensity compared to cells treated with unlabeled m13D3; no change in cell penetrant activity was observed between PBS and acetate. Figure 16C This displays a consistent cell count for each well across all tested cell populations. Figure 16D The number of p-TDP-43 lesions normalized by cell count shows that cells treated with m13D3CMIP4 CPA had a higher number of lesions per cell compared to those treated with unlabeled m13D3; no lesions were observed in untransfected cells. Figure 16E The mean lesion area of ​​p-TDP-43 lesions is shown, indicating that cells treated with m13D3 CMIP4 CPA have a lower mean area intensity compared to cells treated with unlabeled m13D3.

[0344] Example 8. Internalization and clearance of phosphorylated cytoplasmic aggregates of TDP-43 using a novel cell internalization module and a chimeric anti-TDP-43 antibody. HEK cells were transiently transfected with GFP-2a-TDP43 [mNLS (R82L / K83Q) DCS (C173S / C175S)] (2a is a self-cleaving peptide that releases TDP-43 upon expression). After 24 hours, cells were incubated for another 24 hours with 100 μg / ml antibody (e.g., CPA). Cells were then washed, fixed / permeabilized, and stained with anti-pTDP-43 antibody, followed by staining with AF647-bound anti-mouse secondary antibody and AF594-bound anti-human secondary antibody. The stained cells were imaged using a 40x water objective via high-content imaging (Operetta system). Quantitative analysis was performed using Harmony software.

[0345] Figure 17A The image shows the use of unlabeled ch13D3 antibody ( Left ) and ch13D3 m-Lycotoxin_L17E CPA ( right Confocal micrographs of cells treated with 13D3 antibody. Lesions are shown in white, 13D3 antibody in light gray, and cell nuclei in dark gray. White arrows indicate illustrative lesions co-localized with 13D3 antibody. Figure 17A The results showed significant colocalization between pTDP43 lesions in cells treated with ch13D3 m-LycotoxinCPA and the 13D3 antibody, while very little colocalization was observed in cells treated with the ch13D3 antibody. Figure 17BA graph showing the percentage of pTDP43 colocalized with the ch13D3 antibody. The data indicate that the colocalization of cells treated with ch13D3 m-Lycotoxin CPA (approximately 50-60%) was significantly greater than that of cells treated with the ch13D3 antibody (approximately 15-20%). In summary, Figure 17A -B indicates that the anti-TDP-43 CPA of this disclosure is internalized by cells and binds to intracellular p-TDP-43.

[0346] Figures 18A to 18D To demonstrate that the anti-TDP-43 CPA of this disclosure (i.e., chimeric 13D3 CPA (ch13D3 m-Lyco CPA, ch13D3 CMIP4 with LALA mutation, ch13D3 CMIP4 with H310-H435Q mutation), unlabeled ch13D3 antibody, and hIgG isotype control) is internalized by cells and binds to intracellular p-TDP-43 (i.e., cells transfected with GFP-2a-TDP43 as described herein). The label “M-Lyco” in Figure 18 refers to the CPA containing M-Lycotoxin_L17E linked to the C-terminus of the light chain (LC) of the m13D3 antibody.

[0347] Figure 18A Provides display using confocal ( Left ) and non-confocal ( right A graph showing the percentage of p-TDP-43 co-localized with ch13D3 antibody for ch13D3 CPA, ch13D3 antibody, isotype and PBS control using microscopy. Figure 18A The colocalization of cells treated with various ch13D3 CPAs was significantly greater (approximately 50-60%) compared to colocalization of cells treated with ch13D3 antibody (approximately 15-20%). No colocalization was expressed in cells treated with isotype controls and PBS. Figure 18B Provides a graph showing the average number of 13D3 antibody spots per cell for various ch13D3 CPAs, as well as ch13D3 antibodies, isotypes, and PBS controls. Figure 18B This indicates that, compared to the control, ch13D3 CPA produces significantly more 13D3 spots per cell (approximately 2.5–3 spots per cell). Figure 18C Provides the average spot size for 13D3 antibody spots for various ch13D3 CPAs, as well as ch13D3 antibodies, isotypes, and PBS controls. Figure 18C The results showed that ch13D3 CPA resulted in significantly smaller 13D3 spots per cell compared to the control. Figure 18DProvides the average spot size for 13D3 antibody spots for various ch13D3 CPAs, as well as ch13D3 antibodies, isotypes, and PBS controls. Figure 18D The results showed that ch13D3 CPA resulted in significantly smaller 13D3 spots per cell compared to the control (corrected for spot intensity).

[0348] therefore, Figures 18A to 18D This indicates that the anti-TDP-43 CPA of this disclosure is internalized by cells and binds to intracellular p-TDP-43.

[0349] Example 9. Internalization and clearance of phosphorylated cytoplasmic aggregates of TDP-43 by anti-TDP-43 cell penetrant. Figures 19A to 19D A diagram showing the internalization and colocalization of phosphorylated cytoplasmic aggregates of pTDP-43 (i.e., cells transfected with GFP-2a-TDP43 as described herein) with novel cell internalization modules and humanized anti-TDP-43 antibodies. Figures 19A to 19D The data shown is consistent with the above. Figures 11A to 11D It was generated under the same experimental conditions described above. The label “ch13D3-ML” in Figure 19 refers to the CPA containing M-Lycotoxin_L17E linked to the C-terminus of the light chain (LC) of the m13D3 antibody.

[0350] Figure 19A Provides a graph showing the percentage of pTDP43 colocalized with the humanized 13D3 antibody in both humanized 13D3 CPA and ch13D3 and h13D3 antibody controls. Figure 19A The colocalization of cells treated with various h13D3 CPAs (approximately 60-70%) was significantly greater than that of cells treated with h13D3 antibody (approximately 40%). Figure 19B This displays a consistent cell count for each well across all tested cell populations. Figure 19C A graph showing the area of ​​pTDP43 13D3 spots colocalized with humanized 13D3 antibody in each well, as observed in cells treated with humanized 13D3 CPA and IgG isotype, ch13D3 and h13D3 antibody controls. Figure 19C The total localization area of ​​cells treated with humanized 13D3 CPA of this disclosure was significantly increased. Figure 19D A graph showing the number of 13D3 colocalization spots per cell observed in cells treated with humanized 13D3 CPA and IgG isotype, ch13D3 and h13D3 antibody controls is provided. Figure 19D The number of colocalization spots in cells treated with humanized 13D3 CPA of this disclosure was significantly increased.

[0351] In short, Figures 19A to 19D Data from the study showed that phosphorylated cytoplasmic aggregates of pTDP-43 were internalized and co-localized with novel cell internalization modules and humanized anti-TDP-43 antibodies.

[0352] Example 10. Internalization and clearance of phosphorylated cytoplasmic aggregates of TDP-43 in glioblastoma cells by an anti-TDP-43 cell penetrant. Figures 20A to 20C A diagram showing the internalization and colocalization of phosphorylated cytoplasmic aggregates of TDP-43 in glioblastoma cells with CPA including an internalization module and an anti-TDP-43 antibody (i.e., cells transfected with GFP-2a-TDP43 as described herein). Figures 20A to 20C The "M-lycotoxin" label refers to the CPA containing M-Lycotoxin_L17E linked to the C-terminus of the light chain (LC) of the m13D3 antibody. In short, U251 cells were transiently transfected with GFP-2a-TDP43 [mNLS (R82L / K83Q)DCS (C173S / C175S)]. After 24 hours, the cells were incubated for another 24 hours with 100 μg / ml antibody (e.g., CPA). The cells were then washed, fixed / permeabilized, and stained with anti-pTDP-43 antibody, followed by staining with AF647-bound anti-mouse secondary antibody. The stained cells were imaged using a 40x water objective via high-content imaging (Operetta system). Quantitative analysis was performed using Harmony software.

[0353] More specifically, Figure 20A A graph showing the total lesion area of ​​U251 glioblastoma cells treated with m13D3 m-Lycotoxin CPA or m13D3 antibody alone (e.g., unlabeled). The data indicate a significant reduction in total lesion area in glioblastoma cells treated with m13D3 m-Lycotoxin CPA compared to cells treated with m13D3 antibody. Figure 20B A graph showing the mean lesion size of U251 glioblastoma cells treated with m13D3 m-Lycotoxin CPA or m13D3 antibody alone (e.g., unlabeled). The data indicate a significant reduction in mean lesion size in glioblastoma cells treated with m13D3 m-Lycotoxin CPA compared to cells treated with m13D3 antibody. Figure 20CA graph showing the total lesion count in U251 glioblastoma cells treated with m13D3 m-Lycotoxin CPA or m13D3 antibody. The data indicate that the total lesion count in glioblastoma cells treated with m13D3 m-Lycotoxin CPA was significantly increased compared to cells treated with m13D3 antibody.

[0354] therefore, Figures 20A to 20C This indicates that the anti-TDP-43 CPA of this disclosure is internalized by glioblastoma cells that bind to intracellular p-TDP-43 and thus disrupt TDP-43 aggregation.

[0355] Example 11. Internalization and clearance of phosphorylated cytoplasmic aggregates of TDP-43 in rat cortical neurons by an anti-TDP-43 cell penetrant. Figures 21A to 21B and 22A to Figure 22F To visualize the co-localization of CPA-internalized and phosphorylated cytoplasmic aggregates, including the cell internalization module and anti-TDP-43 antibody, in primary rat cortical neurons. Briefly, primary rat cortical neurons (DIV15) were incubated with 50 μg / ml antibody for 2 hours. Cells were then washed, fixed / permeabilized, and stained with anti-EEA1 antibody, followed by staining with AF596-bound anti-rabbit secondary antibody and AF647-bound anti-mouse secondary antibody. The stained cells were imaged using a 40x water objective via high-content imaging (Operetta system). Quantitative analysis was performed using Harmony software.

[0356] Specifically, Figure 21A Images showing primary rat cortical neurons treated with IgG isotype control (top left), m13D3 (top right), m13D3 m-Lycotoxin CPA (bottom left), and m13D3CMIP4 CPA (bottom right). Figure 21A The image shows significant internalization of m13D3 CPA (depicted by white spots within the cells, bottom panel) and minimal or no internalization of the isotype control (top panel). Figure 21B The graph shows the total lesion area for each well, indicating minimal or no internalization of the allotype control and 13D3 antibody, as well as substantial internalization of m13D3 m-Lycotoxin_L17E CPA CPA and m13D3 CMIP4 CPA (labeled as 13D3-ML).

[0357] Figure 22AA graph showing the total number of 13D3 spots per cell in DIV15 rat neurons incubated with hIgG isotype control, m13D3 antibody, m13D3 m-Lycotoxin CPA, or m13D3CMIP4 CPA. Figure 22A This indicates that cells were treated with m13D3-C-terminal-LC-M-lycotoxin_L17E CPA (labeled “M-Lyco” in Figure 22) and m13D3-C-terminal-LC-M CMIP4 CPA (labeled “CMIP” in Figure 22), rather than with IgG isotype controls or m13D3 antibodies.

[0358] Figure 22B A graph showing the total area of ​​13D3 spots in each pore of DIV15 rat neurons incubated with hIgG isotype control, m13D3 antibody, m13D3 m-Lyco CPA, or m13D3 CMIP4CPA. Figure 22B This indicates that cells treated with m13D3 m-Lycotoxin CPA and m13D3 CMIP4 CPA had a higher total spot area per well compared to cells treated with IgG isotype control or m13D3 antibody.

[0359] Figure 22C A graph showing the average spot size of 13D3 spots in DIV15 rat neurons incubated with hIgG isotype control, m13D3 antibody, m13D3 m-Lycotoxin CPA, or m13D3CMIP4 CPA. Figure 22C The results showed that cells treated with m13D3 m-Lycotoxin CPA and m13D3CMIP4 CPA had larger average spot sizes compared to cells treated with IgG isotype control or m13D3 antibody.

[0360] Figure 22D A graph showing the speckle integral intensity of 13D3 spots in DIV15 rat neurons incubated with hIgG isotype control, m13D3 antibody, m13D3 m-Lycotoxin CPA, or m13D3CMIP4 CPA. Figure 22D This indicates that cells treated with m13D3 m-Lycotoxin CPA and m13D3CMIP4 CPA have higher spot integral intensities compared to cells treated with IgG isotype control or m13D3 antibody. Figure 22EA graph showing the percentage of 13D3 spots colocalized with EEA1 (early endosomal antigen 1) in DIV15 rat neurons incubated with hIgG isotype control, m13D3 antibody, m13D3 m-Lycotoxin CPA, or m13D3 CMIP4 CPA. Figure 22E The results indicate that approximately 10-20% of 13D3 spots colocalized with EEA1 in cells treated with m13D3 m-Lycotoxin CPA and m13D3 CMIP4 CPA, while no colocalization was observed in cells treated with IgG isotype control or m13D3 antibody. Figure 22F This displays a consistent cell count for each well across all tested cell populations. In summary, Figures 21A to 2 1D and Figures 22A to 22F The cell internalization module of this disclosure is shown to be internalized by primary rat cortical neurons.

[0361] References

[0362]

[0363] Sequence Appendix Variable heavy chain reference sequence SEQ ID NO: 1 m13D3-VH EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYIS T GGDSANYADNVKGRFTISRDNAKNTLYLQM NSL MSEDTAMYFCARQTYYSYGGFPYWGQGTLVTVSA SEQ ID NO: 2 IGHV3-48*03 EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYEMNWVRQAPGKGLEWVSYIS S SGSTIYYADSVKGRFTISRDNAKNSLYLQM NSL RAEDTAVYYCARYFDYWGQGTLVTVSS SEQ ID NO: 3 AEX28899 EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYEMNWVRQAPGKGLEWVSYIS S SGSTIYYADSVKGRFTISRDNAKNSLYLQM NSLRAEDTAVYYCARRNYYDS GGY GHWGQGTLVTVSS Humanized 13D3 VH design sequence SEQ ID NO: 4 h13D3VHv1 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYFMSWVRQAPGKGLEWVAYIS T GGDSANYADNVKGRFTISRDNAKNSLYLQM NSL RAEDTAVYFCARQTYYSY GGF PYWGQGTLVTVSS SEQ ID NO: 5 h13D3VHv2 EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQAPGKGLEWVAYIS T GGDSANYADNVKGRFTISRDNAKNSLYLQM NSL RAEDTAVYFCARQTYYSY GGF PYWGQGTLVTVSS SEQ ID NO: 6 h13D3VHv3 EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQAPGKGLEWVAYIS T GGDSANYADNVKGRFTISRDNAKNSLYAQM NSL RAEDTAVYFCARQTYYSY GGF PYWGQGTLVTVSS SEQ ID NO: 7 h13D3VHv4 EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQAPGKGLEWVAYIS T GGDSANYADNVKGRFTISRDNAKNTLYAQM NSL RAEDTAVYFCARQTYYSY GGF PYWGQGTLVTVSS SEQ ID NO: 8 h13D3VHv5 EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQAPGKGLEWVAYIS TGGDSANYADNVKGRFTISRDNAKNTAYAQM NSL RAEDTAVYFCARQTYYSY GGF PYWGQGTLVTVSS SEQ ID NO: 9 h13D3VHv6 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYFMSWVRQAPGKGLEWVAYIS T GGDSANYADNVKGRFTISRDNAKNTAYAQM NSL RAEDTAVYFCARQTYYSY GGF PYWGQGTLVTVSS SEQ ID NO: 10 h13D3VHv7 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYFMGWVRQAPGKGLEWVAYIS T GGDSANYADNVKGRFTISRDNAKNTAYAQM NSL RAEDTAVYFCARQTYYSY GGF PYWGQGTLVTVSS SEQ ID NO: 11 h13D3VHv8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYFMGWVRQAPGKGLEWVAYIS T GGDSANYADNVKGRFTISRDNAKNTLYGQM NSL RAEDTAVYFCARQTYYSY GGF PYWGQGTLVTVSS SEQ ID NO: 12 h13D3VHv9 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYFMGWVRQAPGKGLEWVAYIS T GGDSANYADNVKGRFTISRDNAKNTGYLQM NSL RAEDTAVYFCARQTYYSY GGF PYWGQGTLVTVSS SEQ ID NO: 13 h13D3VHv10 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYFMGWVRQAPGKGLEWVAYIST GGDSANYADNVKGRFTISRDNAKNTLYLQM NSG RAEDTAVYFCARQTYYSY GGF PYWGQGTLVTVSS SEQ ID NO: 14 h13D3VHv11 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYFMGWVRQAPGKRLEWVAYIS T GGDSANYADNVKGRFTISRDNAKNTGYLQM NSL MAEDTAVYFCARQTYYSY GGF PYWGQGTLVTVSS SEQ ID NO: 15 h13D3VHv12 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYFMGWVRQAPGERLEWVAYIS T GGDSANYADNVKGRFTISRDNAKNTGYLQM NSL RAEDTAVYFCARQTYYSY GGF PYWGQGTLVTVSS SEQ ID NO: 16 h13D3VHv13 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYFMGWVRQAPGEDLEWVAYIS T GGDSANYADNVKGRFTISRDNAKNTGYLQM NSL RAEDTAVYFCARQTYYSY GGF PYWGQGTLVTVSS SEQ ID NO: 17 h13D3VHv1b ELVQSGAEVKKPGSSVKVSCKASGFTFSNYFMSWVRQAPGQGLEWVAYISTGGDSANYADNVKGRFTITRDNSTSTLYMELSSLRSEDTAVYFCARQTYYSYGGFPYWGQGTLVTVSS SEQ ID NO: 18 h13D3VHv2b ELVQSGAEVKKPGSSVKVSCKASGFTFSNYFMSWVRQAPGQGLEWVAYISTGGDSANYADNVKGRFTITKDTSTSTLYMELSSLRSEDTAVYFCARQTYYSYGGFPYWGQGTLVTVSS SEQ ID NO: 19 h13D3VHv3b ELVQSGAEVKKPGSSVKVSCKASGFTFSNYFMSWVRQAPGQGLEWVAYISTGGDSANYADNVKGRGTITKDTSTSTLYMELSSLRSEDTAVYFCARQTYYSYGGFPYWGQGTLVTVSS SEQ ID NO: 20 h13D3VHv1d EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYFMSWVRQAPGKGLEWVSYIS T GGDSANYADNVKGRFTISRDNSKNSLYLQM NSL RAEDTAVYYCARQTYYSY GGF PYWGQGTLVTVSS SEQ ID NO: 21 h13D3VHv2d EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYFMSWVRQAPGKGLEWVSYIS T GGDSANYADNVKGRFTISRDNSKNTAYLQM NSL RAEDTAVYYCARQTYYSY GGF PYWGQGTLVTVSS SEQ ID NO: 22 h13D3VHv3d EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYFMSWVRQAPGKRLEWVAYIS T GGDSANYADNVKGRFTISRDNAKNSLYLQM NSL RAEDTAVYYCARQTYYSY GGF PYWGQGTLVTVSS SEQ ID NO: 23 h13D3VHv4d EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYFMSWVRQAPGKRLEWVSYIST GGDSANYADNVKGRFTISRDNSKNTAYLQM NSL RAEDTAVYYCARQTYYSY GGF PYWGQGTLVTVSS The underlined residues are in the positions defined below.

[0364] Position 52A*# Location 82A*# Location 82B*# Location 82C*# Location 100A* Location 100B* Location 100C*.

[0365] * Applicable to SEQ ID NO: 1, 3-16 and 20-23 # Applicable to SEQ ID NO: 2 Variable light chain reference sequence SEQ ID NO: 24 m13D3-VL DVVMTQSPLSLPVSLGDQASISCRSSQ SLVHS NGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSGSGTDFTLKISRVETEDLGVYFCSQSLHVPLTFGAGTKLELK SEQ ID NO: 25 IGKV2-30*02 DVVMTQSPLSLPVTLGQPASISCRSSQ SLVHS DGNTYLNWFQQRPGQSPRRLIYKVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTHWPYTFGQGTKLEIK SEQ ID NO: 26 ABC66863 DIVMTQSPLSLPVTLGQPASISCRSSQ SLVYS DGNTYLNWFQQRPGQSPRRLIYKVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTHRPLTFGGGTKVEIK Humanized 13D3 VL Design Sequence SEQ ID NO: 27 h13D3VLv1 DVVMTQSPLSLPVTLGQPASISCRSSQ SLVHS NGKTYLHWYQQRPGQSPRLLIYKVSDRYSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQSLHVPLTFGGGTKVEIK SEQ ID NO: 28 h13D3VLv2 DVVMTQSPLSLPVTLGQQASISCRSSQ SLVHS NGKTYLHWYQQRPGQSPRLLIYKVSDRYSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQSLHVPLTFGGGTKVEIK SEQ ID NO: 29 h13D3VLv3 DVVMTQSPSSLPVTLGQQASISCRSSQ SLVHS NGKTYLHWYQQRPGQSPRLLIYKVSDRYSGVPDRFSGSGSGTDFTLKISRVESEDVGVYFCSQSLHVPLTFGGGTKVEIK SEQ ID NO: 30 h13D3VLv4 DVVMTQSPSSLPVTLGQQASISCRSSQ SLVHS NGKTYLHWYQQRPGQSPRLLIYKVSDRYSGVPDRFSGSGSGTDFTLKISRVESEDVGVYFCSQSGHVPLTFGGGTKVEIK SEQ ID NO: 31 h13D3VLv5 DVVMTQSPSSLPVTLGQPASISCRSSQ SLVHS NGKTYLHWYQQRPGQSPRLLIYKVSDRYSGVPDRFSGSGSGTDFTLKISRVESEDVGVYFCSQSGHVPLTFGGGTKVEIK SEQ ID NO: 32 h13D3VLv6 DVVMTQSPSSLPVTLGQPASISCRSSQ SLVHS NGKTYLHWYQQRPGQSPRLLIYKVSDRYSGVPDRFSGSGSGTDFTLKISRVESEDVGVYFCSQSLHVPLTFGGGTKVEIK SEQ ID NO: 33 h13D3VLv7 DVVMTQSPSSLPVTLGQPASISCRSSQ SLVHS NGKTYLHWYQQRPGQSPRLLIYKVSDRYSGVPDRFSGSGSGTDFTLKISRVESEDVGVYFCSQSLHIPLTFGGGTKVEIK SEQ ID NO: 34 h13D3VLv8 DVVMTQSPSSLPVTLGQPASISCRSSQ SLVHS NGKTYLHWYQQRPGQSPRLLIYKVSDRYSGVPDRFSGSGSGTDFTLKISRVESEDVGVYFCSQSLHAPLTFGGGTKVEIK SEQ ID NO: 35 h13D3VLv9 DVQMTQSPSSLPVTLGQPASISCRSSQ SLVHS NGKTYLHWYQQRPGQSPRLLIYKVSDRYSGVPDRFSGSGSGTDFTLKISRVESEDVGVYFCSQSGHVPLTFGDGTKVEIK SEQ ID NO: 36 h13D3VLv10 DVQMTQSPSSLPVTLGQPASISCRSSQ SLVHS NGKTYLHWYQQRPGQSPRLLIYKVSDRYSGVPDRFSGSGSGTDFTLKISRVESEDVGVYFCSQSGHVPLTFGRGTKVEIK SEQ ID NO: 37 h13D3VLv1b EVVMTQSPATLSLSPGERATLSCRSSQ SLVHS NGKTYLHWYQQKPGQAPRLLIYKVSDRYSGVPARFSGSGSGTDFTLTISSLEPEDFAVYFCSQSLHVPLTFGGGTKVEIK SEQ ID NO: 38 h13D3VLv2b EVVMTQSPATLSLSPGERATLSCRSSQ SLVHS NGKTYLHWYQQKPGQAPRLLIYKVSDRYSGVPARFSGSGSGTEFTLTISSLEPEDFAVYFCSQSLHVPLTFGGGTKVEIK SEQ ID NO: 39 h13D3VLv3b EVVMTQSPATLSLSPGERATLSCRSSQ SLVHS NGKTYLHWYQQKPGQAPRLLIYKVSDRYSGVPARFSGSGSGTEVTLTISSLEPEDFAVYFCSQSLHVPLTFGGGTKVEIK SEQ ID NO: 40 h13D3VLv4b DVVMTQSPATLSLSPGERATLSCRSSQ SLVHS NGKTYLHWYQQKPGQAPRLLIYKVSDRYSGVPARFSGSGSGTDFTLTISSLEPEDFAVKFCSQSLHVPLTFGGGTKVEIK SEQ ID NO: 41 h13D3VLv5b DVVMTQSPATLSLSPGERATLSCRSSQ SLVHS NGKTYLHWYQQKPGQAPRLLIYKVSDRYSGVPARFSGSGSGTEFTLTISSLEPEDFAVKFCSQSLHVPLTFGGGTKVEIK SEQ ID NO: 42 h13D3VLv6b DVVMTQSPATLSLSPGERATLSCRSSQ SLVHS NGKTYLHWYQQKPGQAPRLLIYKVSDRYSGVPARFSGSGSGTEVTLTISSLEPEDFAVKFCSQSLHVPLTFGGGTKVEIK SEQ ID NO: 43 h13D3VLv7b DVVMTQSPATLSLSPGERATLSCRSSQ SLVHS NGKTYLHWYQQKPGQAPRLLIYKVSDRYSGVPARFSGSGSGTEFTLTISSLEPEDFAVYFCDQSLHVPLTFGGGTKVEIK SEQ ID NO: 44 h13D3VLv8b DVVMTQSPATLSLSPGERATLSCRSSQ SLVHSNGKTYLHWYQQKPGQAPRLLIYKVSDRYSGVPARFSGSGSGTEVTLTISSLEPEDFAVYFCDQSLHVPLTFGGGTKVEIK SEQ ID NO: 45 h13D3VLv9b DVVMTQSPATLSLSPGERATLSCRSSQ SLVHS NGKTYLHWYQQKPGQAPRLLIYKVSDRYSGVPARFSGSGSGTEFTLTISSLEPEDFAVYFCSQQLHVPLTFGGGTKVEIK SEQ ID NO: 46 h13D3VLv10b DVVMTQSPATLSLSPGERATLSCRSSQ SLVHS NGKTYLHWYQQKPGQAPRLLIYKVSDRYSGVPARFSGSGSGTEVTLTISSLEPEDFAVYFCSQQLHVPLTFGGGTKVEIK SEQ ID NO: 47 h13D3VLv1d DVQMTQSPLSLPVTLGQPASISCRSSQ SLVHS NGKTYLHWYQQRPGQSPRLLIYKVSDRYSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQSLHVPLTFGDGTKVEIK SEQ ID NO: 48 h13D3VLv2d DVQMTQSPLSLPVTLGQPASISCRSSQ SLVHS NGKTYLHWYQQRPGQSPRLLIYKVSDRYSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQSAHVPLTFGDGTKVEIK The underlined residues are in the positions defined below.

[0366] Specifically, S represents position 27A. L represents position 27B. V is at position 27C H represents position 27D S represents position 27E.

[0367] Applicable to SEQ ID NO: 24-48.

[0368] SEQ ID NO: 49 HC CDR1 #1 GFTFSNYFMS SEQ ID NO: 50 HC CDR1 #2 GFTFSNYFMG SEQ ID NO: 51 HC CDR2 #1 YISTGGDSANYADNVKG SEQ ID NO: 52 HC CDR3 #1 QTYYSYGGFPY SEQ ID NO: 53 LC CDR1 #1 RSSQSLVHSNGKTYLH SEQ ID NO: 54 LC CDR2 #1 KVSDRYS SEQ ID NO: 55 LC CDR3 #1 SQSLHVPLT SEQ ID NO: 56 LC CDR3 #2 SQSGHVPLT SEQ ID NO: 57 LC CDR3 #3 SQSLHIPLT SEQ ID NO: 58 LC CDR3 #4 SQSLHAPLT SEQ ID NO: 59 LC CDR3 #5 DQSLHVPLT SEQ ID NO: 60 LC CDR3 #6 SQQLHVPLT SEQ ID NO: 61 LC CDR3 #7 SQSAHVPLT TDP43 mouse antibody variable domain sequence SEQ ID NO: 62 1B3_JH140 VH signal peptide MNFGLSLIFLVLVLKGVLC SEQ ID NO: 63 1B3_JH140 Variable Restructuring Domain EVKLVESGGGLVQPGGSLKLSCAASGFTFSSYTMSWVRQTPEKRLELVAEISNSGGRTNYPDTVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARRRYSDYYYYAMDYWGQGTSVTVSS SEQ ID NO: 64 1B3_JH140 VL signal peptide MSSAQFLGLLLLCFQGTRC SEQ ID NO: 65 1B3_JH140 Variable Lightweight Domain DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPRTFGGGTKLEIK SEQ ID NO: 66 1C12_JH104 VH signal peptide MNFGLSLIFLVLVLKGVLC SEQ ID NO: 67 1C12_JH104 Variable Restructuring Domain EVKLVESGGGLVQPGGSLKLSCAASGFTFSSYTMSWVRQTPEKRLELVADISNSGGRTYYPDTVKGRFTISRENAKNSLYLQMSSLKSEDTAMYYCARRRYSDYYYYYAMDNWGQGTSVTVSS SEQ ID NO: 68 1C12_JH104 VL signal peptide MSSAQFLGLLLLCFQGTRC SEQ ID NO: 69 1C12_JH104 Variable Lightweight Domain DIQMTQTTSSLSASLGDRVTISCRASQDISNYLSWYQQKPDGTVKLLIYYTSRLNSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNALPRTFGGGTKLEIN SEQ ID NO: 70 11A1_LA121 VH signal peptide MEWPCIFLFLLSVTEGVHS SEQ ID NO: 71 11A1_LA121 Variable Restructuring Domain QVQLQQSGAELVRPGSSVKISCKASGYEFSRYWMNWVKQRPGQGLEWIGQIYHGDGDTNYKGKFKGKAILTADKSSSTAYMQVSSLTSEDSAVYFCVRGGYYGYAMDYWGQGTSVTVSS SEQ ID NO: 72 11A1_LA121 signal peptide MKLPVRLLVLMFWIPASSS SEQ ID NO: 73 11A1_LA121 Variable Lightweight Domain DVVMTQTPLSLPVSLGDQASISCRSSQSLLHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPYTFGGGTNLEIK SEQ ID NO: 74 11B1_LA121 VH signal peptide MEWPCIFLFLLSVTEGVHS SEQ ID NO: 75 11B1_LA121 Variable Restructuring Domain QVQLQQSGAELVRPGSSVKISCKASGYEFSRYWMNWVKQRPGQGLEWIGQIYHGDGDTNYKGKFKGKAILTADKSSSTAYMQVSSLTSEDSAVYFCVRGGYYGYAMDYWGQGTSVTVSS SEQ ID NO: 76 11B1_LA121 VL signal peptide MKLPVRLLVLMFWIPASSS SEQ ID NO: 77 11B1_LA121 Variable Lightweight Domain DVVMTQTPLSLPVSLGDQASISCRSSQSLLHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPYTFGGGTNLEIK SEQ ID NO: 78 16G5_LA121 VH signal peptide MKCSWVIFFLMAVVTGVNS SEQ ID NO: 79 16G5_LA121 Variable Restructuring Domain EVHLQQSGAELVKPGASVKLSCTGSGFNIIDTYIHWVKQRPEQGLEWIGRIDPANGNTMYASKFQDKATIIADTSSNTVYMRLGSLTSGDTAVYYCSHGDFWWGQGTTLTVSS SEQ ID NO: 80 16G5_LA121 VL signal peptide MSPAQFLFLLVLWIQETNG SEQ ID NO: 81 16G5_LA121 variable light domain DVVMTQTPLTLSIPIGQPAYISCKSSQSLLKSNGKTYLNWLFQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGIYYCVQGTHLPHTFGGGTRLEIK SEQ ID NO: 82 Human TDP-43 protein MSEYIRVTEDENDEPIEIPSEDDGTVLLSTVTAQFPGACGLRYRNPVSQCMRGVRLVEGILHAPDAGWGNLVYVVNYPKDNKRKMDETDASSAVKVKRAVQKTSDLIVLGLPWKTTEQDLKEYFSTFGEVLMVQVKKDLKTGHSKGFGFVRFTEYETQVKVMSQRHMIDGRWCDCKLPNSKQSQDEPLRSRKVFVGRCTEDMTEDELREFFSQYGDVMDVFIPKPFRAFAFVTFADDQIAQSLCGEDLIIKGISVHISNAEPKHNSNRQLERSGRFGGNPGGFGNQGGFGNSRGGGAGLGNNQGSNMGGGMNFGAFSINPAMMAAAQAALQSSWGMMGMLASQQNQSGPSGNNQNQGNMQREPNQAFGSGNNSYSGSNSGAAIGWGSASNAGSGSGFNGGFGSSMDSKSSGWGM SEQ ID NO: 83 TDP-43 peptide (pS409 / pS410) GSGSGFNGGFGSSMDSKSSGWGM SEQ ID NO: 84 1B3_JH140 variable heavy chain CDR1 SGFTFSSYTMS SEQ ID NO: 85 1B3_JH140 Variable Heavy Chain CDR2 EISNSGGRTNY SEQ ID NO: 86 1B3_JH140 Variable Heavy Chain CDR3 RRYSDYYYYAMDY SEQ ID NO: 87 1B3_JH140 Variable Light Chain CDR1 RASQDISNYLN SEQ ID NO: 88 1B3_JH140 Variable Light Chain CDR2 YTSRLHS SEQ ID NO: 89 1B3_JH140 Variable Light Chain CDR3 QQGNTLPRT SEQ ID NO: 90 1C12_JH104 Variable Heavy Chain CDR1 SGFTFSSYTMS SEQ ID NO: 91 1C12_JH104 Variable Heavy Chain CDR2 DISNSGGRTYYPDTVKG SEQ ID NO: 92 1C12_JH104 Variable Heavy Chain CDR3 RRYSDYYYYAMDN SEQ ID NO: 93 1C12_JH104 Variable Light Chain CDR1 RASQDISNYLS SEQ ID NO: 94 1C12 JH104 Variable Light Chain CDR2 YTSRLNS SEQ ID NO: 95 1C12_JH104 Variable Light Chain CDR3 QQGNALPRT SEQ ID NO: 96 11A1_LA121 Variable Heavy Chain CDR1 SGYEFSRYWMN SEQ ID NO: 97 11A1_LA121 Variable Heavy Chain CDR2 QIYHGDGDTNYKGKFKG SEQ ID NO: 98 11A1_LA121 Variable Heavy Chain CDR3 GGYYGYAMDY SEQ ID NO: 99 11A1_LA121 Variable light chain CDR1 RSSQSLLHSNGNTYLH SEQ ID NO: 100 11A1_LA121 Variable light chain CDR2 KVSNRFS SEQ ID NO: 101 11A1_LA121 Variable light chain CDR3 SQSTHVPYT SEQ ID NO: 102 11B1_LA121 Variable Heavy Chain CDR1 SGYEFSRYWMN SEQ ID NO: 103 11B1_LA121 Variable Heavy Chain CDR2 QIYHGDGDTNYKGKFKG SEQ ID NO: 104 11B1_LA121 Variable Heavy Chain CDR3 GGYYGYAMDY SEQ ID NO: 105 11B1_LA121 Variable light chain CDR1 RSSQSLLHSNGNTYLH SEQ ID NO: 106 11B1_LA121 Variable light chain CDR2 KVSNRFS SEQ ID NO: 107 11B1_LA121 Variable light chain CDR3 SQSTHVPYT SEQ ID NO: 108 16G5_LA121 Variable Heavy Chain CDR1 SGFNIIDTYIH SEQ ID NO: 109 16G5_LA121 Variable Heavy Chain CDR2 RIDPANGNTMYA SEQ ID NO: 110 16G5_LA121 Variable Heavy Chain CDR3 GDFW SEQ ID NO: 111 16G5_LA121 Variable light chain CDR1 KSSQSLLKSNGKTYLN SEQ ID NO: 112 16G5_LA121 Variable light chain CDR2 LVSKLDS SEQ ID NO: 113 16G5 LA121 Variable light chain CDR3 VQGTHLPHT SEQ ID NO: 114 m13D3 heavy chain - possesses the mouse IgG2a heavy chain constant domain; constant region underlined. EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTISRDNAKNTLYLQMNSLMSEDTAMYFCARQTYYSYGGFPYWGQGTLVTVSA AKTTAPSVYPLAPVCGDTTGSSVTLG CLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPT IKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYN STLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMP EDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO: 115 m13D3 light chain - possesses a mouse κ light chain constant domain; constant region underlined. DVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSGSGTDFTLKISRVETEDLGVYFCSQSLHVPLTFGAGTKLELK RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYP KDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC * The underlined sequences in SEQ ID NO: 114 and 115 represent constant structural domains.

[0369] SEQ ID NO: 116 13D3_Ab_LC-Ct-CMIP4 heavy chain EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTISRDNAKNTLYLQMNSLMSEDTAMYFCARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO: 117 13D3_Ab_LC-Ct-CMIP4 light chain

[0370] SEQ ID NO: 118 13D3_Ab_HC-C-terminal_TAT heavy chain

[0371] SEQ ID NO: 119 13D3_Ab_HC-C-terminal_TAT light chain DVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSGSGTDFTLKISRVETEDLGVYFCSQSLHVPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO: 120 13D3_Ab_LC-L17E_M-Lycotoxin heavy chain EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTISRDNAKNTLYLQMNSLMSEDTAMYFCARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO: 121 Light chain of 13D3_Ab_LC-L17E_M-Lycotoxin

[0372] SEQ ID NO: 122 Heavy chain of 13D3_Ab_HC-Ct-L17E_M-Lycotoxin

[0373] SEQ ID NO: 123 Light chain of 13D3_Ab_HC-Ct-L17E_M-Lycotoxin DVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSGSGTDFTLKISRVETEDLGVYFCSQSLHVPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO: 124 Heavy chain of 13D3_Ab_HC-AH-PEPTH

[0374] SEQ ID NO: 125 13D3_Ab_HC-AH-PEPTH light chain DVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSGSGTDFTLKISRVETEDLGVYFCSQSLHVPLTFGAGTKL ELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO: 126 13D3_Ab_HC-C terminal-cycR9 heavy chain

[0375] SEQ ID NO: 127 13D3_Ab_HC-C terminal-cycR9 light chain DVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSGSGTDFTLKISRVETEDLGVYFCSQSLHVPLTFGAGTKL ELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO: 128 13D3_Ab_HC-C terminal-cycTAT1 modified heavy chain

[0376] SEQ ID NO: 129 13D3_Ab_HC-C terminal-cycTAT1 modified light chain DVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSGSGTDFTLKISRVETEDLGVYFCSQSLHVPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO: 130 13D3_Ab_HC-C-terminal-TAT heavy chain

[0377] SEQ ID NO: 131 13D3_Ab_HC-C-terminal-TAT light chain DVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSGSGTDFTLKISRVETEDLGVYFCSQSLHVPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO: 132 13D3_Ab_LC-C-terminal-Penetain heavy chain EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTISRDNAKNTLYLQMNSLMSEDTAMYFCARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO: 133 13D3_Ab_LC-C-terminal - Penetain light chain

[0378] SEQ ID NO: 134 13D3_Ab_LC-C-terminal - L17E-M-Lycotoxin heavy chain EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTISRDNAKNTLYLQMNSLMSEDTAMYFCARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO: 135 13D3_Ab_LC-C-terminal-L17E-M-Lycotoxin light chain

[0379] SEQ ID NO: 136 13D3_Ab_LC-C-terminal-Pepth heavy chain EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTISRDNAKNTLYLQMNSLMSEDTAMYFCARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO: 137 13D3_Ab_LC-C-terminal - Pepth light chain

[0380] SEQ ID NO: 138 13D3_Ab_HC-C-terminal - TAT3 heavy chain

[0381] SEQ ID NO: 139 13D3_Ab_HC-C-terminal - TAT3 light chain DVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSGSGTDFTLKISRVETEDLGVYFCSQSLHVPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO: 140 13D3_Ab_LC-C-terminal_TAT3 heavy chain EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTISRDNAKNTLYLQMNSLMSEDTAMYFCARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO: 141 13D3_Ab_LC-C-terminal_TAT3 light chain

[0382] SEQ ID NO: 142 13D3_Ab_LC-C-terminal_cycTAT3 heavy chain EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTISRDNAKNTLYLQMNSLMSEDTAMYFCARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO: 143 13D3_Ab_LC-C-terminal_cycTAT3 light chain

[0383] SEQ ID NO: 144 13D3_Ab_LC-C-terminal_cycR8x3 heavy chain

[0384] SEQ ID NO: 145 13D3_Ab_LC-C-terminal_cycR8x3 light chain DVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSGSGTDFTLKISRVETEDLGVYFCSQSLHVPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO: 146 13D3_Ab_HC-C-terminal_cycTAT3 heavy chain

[0385] SEQ ID NO: 147 13D3_Ab_HC-C-terminal_cycTAT3 light chain DVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSGSGTDFTLKISRVETEDLGVYFCSQSLHVPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO: 148 13D3_Ab_LC-C-terminal_R8x3 heavy chain EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTISRDNAKNTLYLQMNSLMSEDTAMYFCARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO: 149 13D3_Ab_LC-C-terminal_R8x3 light chain

[0386] SEQ ID NO: 150 13D3_Ab_HC-C-terminal_R6H4 heavy chain

[0387] SEQ ID NO: 151 13D3_Ab_HC-C-terminal_R6H4 light chain DVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSGSGTDFTLKISRVETEDLGVYFCSQSLHVPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO: 152 13D3_Ab_HC-C-terminal_TAT-H4 heavy chain

[0388] SEQ ID NO: 153 13D3_Ab_HC-C-terminal_TAT-H4 light chain DVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSGSgalDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO: 154 h13D3H1bL1b_Ab_LC-C-terminal_CMIP4 heavy chain EVELVQSGAEVKKPGSSVKVSCKASGFTFSNYFMSWVRQAPGQGLEWVAYISTGGDSANYADNVKGRFTITRDNSTSTLYMELSSLRSEDTAVYFCARQTYYSYGGFPYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 155 h13D3H1bL1b_Ab_LC - C - terminal_CMIP4 light chain

[0389] SEQ ID NO: 156 h13D3H1bL2b_Ab_LC - C - terminal_CMIP4 heavy chain EVELVQSGAEVKKPGSSVKVSCKASGFTFSNYFMSWVRQAPGQGLEWVAYISTGGDSANYADNVKGRFTITRDNSTSTLYMELSSLRSEDTAVYFCARQTYYSYGGFPYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 157 h13D3H1bL2b_Ab_LC-C-terminal_CMIP4 light chain

[0390] SEQ ID NO: 158 h13D3H2bL1b_Ab_LC-C-terminal_CMIP4 heavy chain EVELVQSGAEVKKPGSSVKVSCKASGFTFSNYFMSWVRQAPGQGLEWVAYISTGGDSANYADNVKGRFTITKDTSTSTLYMELSSLRSEDTAVYFCARQTYYSYGGFPYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 159 h13D3H2bL1b_Ab_LC - C - terminal_CMIP4 light chain

[0391] SEQ ID NO: 160 h13D3H2bL2b_Ab_LC - C - terminal_CMIP4 heavy chain EVELVQSGAEVKKPGSSVKVSCKASGFTFSNYFMSWVRQAPGQGLEWVAYISTGGDSANYADNVKGRFTITKDTSTSTLYMELSSLRSEDTAVYFCARQTYYSYGGFPYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 161 h13D3H2bL2b_Ab_LC-C-terminal_CMIP4 light chain

[0392] SEQ ID NO: 162 h13D3H3bL1b_ Ab_LC-C-terminal_CMIP4 heavy chain EVELVQSGAEVKKPGSSVKVSCKASGFTFSNYFMSWVRQAPGQGLEWVAYISTGGDSANYADNVKGRGTITKDTSTSTLYMELSSLRSEDTAVYFCARQTYYSYGGFPYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 163 h13D3H3bL1b_ Ab_LC-C-terminal_CMIP4 light chain

[0393] SEQ ID NO: 164 h13D3H3bL2b_ Ab_LC-C-terminal_CMIP4 heavy chain EVELVQSGAEVKKPGSSVKVSCKASGFTFSNYFMSWVRQAPGQGLEWVAYISTGGDSANYADNVKGRGTITKDTSTSTLYMELSSLRSEDTAVYFCARQTYYSYGGFPYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 165 h13D3H3bL2b_ Ab_LC-C-terminal_CMIP4 light chain

[0394] SEQ ID NO: 166 m13D3_ Ab_LC-C-terminal_CMIP1 heavy chain EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTISRDNAKNTLYLQMNSLMSEDTAMYFCARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO: 167 m13D3_ Ab_LC-C-terminal_CMIP1 light chain

[0395] SEQ ID NO: 168 m13D3_ Ab_LC-C-terminal_CMIP2 heavy chain EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTISRDNAKNTLYLQMNSLMSEDTAMYFCARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO: 169 m13D3_ Ab_LC-C-terminal_CMIP2 light chain

[0396] SEQ ID NO: 170 m13D3_ Ab_LC-C-terminal_CMIP3 heavy chain EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTISRDNAKNTLYLQMNSLMSEDTAMYFCARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO: 171 m13D3_ Ab_LC-C-terminal_CMIP3 light chain

[0397] SEQ ID NO: 172 m13D3_ Ab_LC-C-terminal_CMIP4 heavy chain EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTISRDNAKNTLYLQMNSLMSEDTAMYFCARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO: 173 m13D3_ Ab_LC-C terminal_CMIP4 light chain

[0398] SEQ ID NO: 174 m13D3_ Ab_LC-C terminal_CMIP5 heavy chain EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTISRDNAKNTLYLQMNSLMSEDTAMYFCARQTYYSYGGFPYWGQ GTLVTVSAAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIK PCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIER TISKPKGSVRAPQVYVLPPPEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO: 175 m13D3_Ab_LC-C terminal_CMIP5 light chain

[0399] The following applies to SEQ ID NO: 116-175 Connector subsequences are shown in bold.

[0400] Spacer regions or amino acids are indicated by an asterisk (*) after amino acid residues (e.g., G*).

[0401] The sequence of the cell internalization module is indicated by double underscores.

[0402] SEQ ID NO: 176 CIM #1 CMIP1 IWLTALKFLGKAAAKAEAKQQLSKL SEQ ID NO: 177 CIM #2 CMIP2 IWLTALKFSGKAAAKAEAKQQLSKL SEQ ID NO: 178 CIM #3 CMIP3 IWLTASKFSGKAAAKAEAKQQLSKL SEQ ID NO: 166 CIM #4 CMIP4 IWLTALKFSGKAAAKAEAKQFLSKL SEQ ID NO: 180 CIM #5 CMIP5 IWLTALKFSGKAAAKAEAKQWLSKL SEQ ID NO: 181 CIM #6 TAT GRKKRRQRRRPPQ SEQ ID NO: 182 CIM #7 Wild-type M-lycotoxin IWLTALKFLGKHAAKHEAKQQLSKL SEQ ID NO: 183 CIM #8 L17E_M-lycotoxin IWLTALKFLGKHAAKHEAKQQLSKL SEQ ID NO: 184 CIM #9 PEPTH VKKKKIKAEIKIG SEQ ID NO: 185 CIM #10 cycR9 CG*RRRRRRRRRG*C SEQ ID NO: 186 CIM #11 cycTAT1 CG*GRKKRRQRRRPQG*C SEQ ID NO: 187 CIM #12 Penetain RQIKIWFQNRRMKWKKG* SEQ ID NO: 188 CIM #13 Tat3 GRKKRRQRRRPQGRKKRRQRRRPQGRKKRRQRRRPQG*G* SEQ ID NO: 189 CIM #14 cycTat3 CG*G*GRKKRRQRRRPQGRKKRRQRRRPQGRKKRRQRRRPQG*G*C SEQ ID NO: 190 CIM #15 R8x3 RRRRRRRRG*G*G*G*S*RRRRRRRRG*G*G*G*S*RRRRRRRRG*G* SEQ ID NO: 191 CIM #16 cycR8x3 CG*G*G*RRRRRRRRG*G*G*RRRRRRRRG*G*G*RRRRRRRRG*G*G*C SEQ ID NO: 192 CIM #17 R6H4 RRRRRRHHHH SEQ ID NO: 193 CIM #18 TatH4 GRKKRRQRRRPHHHH The following applies to SEQ ID NO: 176-193 Spacer regions or amino acids are indicated by an asterisk (*) after amino acid residues (e.g., G*).

[0403] SEQ ID NO: 194 Connector sequence #1 GGGGSGGGGS SEQ ID NO: 195 Connector sequence #2 GGGGS SEQ ID NO: 196 Connector sequence #3 GGGGS SEQ ID NO: 197 Connector sequence #4 GS SEQ ID NO: 198 Connector sequence #5 GGG SEQ ID NO: 199: Connector sequence #6 GGGSGGGS SEQ ID NO: 200 Interval #1 G SEQ ID NO: 201 Interval #2 GG SEQ ID NO: 202 Interval #3 GGG SEQ ID NO: 203 Interval #4 GGGGS

Claims

1. A cell penetrant comprising: (i) a cell internalization module (CIM) and (ii) an antibody that specifically binds to a 43 kD trans-active reactive DNA-binding protein (TDP-43).

2. The cell penetrant of claim 1, wherein the CIM comprises a cell membrane internalization peptide (CMIP).

3. The cell penetrant of claim 1 or 2, wherein the CIM comprises wild-type M-lycotoxin peptide.

4. The cell-penetrating agent of claim 1 or 2, wherein the CIM comprises an M-lycotoxin derivative.

5. The cell penetrant of claim 1 or 2, wherein the CIM comprises a Penetain amino acid sequence or a derivative thereof.

6. The cell penetrant of claim 1 or 2, wherein the CIM comprises a Pepth amino acid sequence or a derivative thereof.

7. The cell penetrant of claim 1 or 2, wherein the CIM comprises a polyarginine amino acid sequence.

8. The cell penetrant of claim 1, 2 or 7, wherein the CIM comprises more than one polyarginine amino acid sequence.

9. The cell penetrant of any one of claims 1, 2, 7 and 8, wherein the CIM comprises a polyarginine amino acid sequence.

10. The cell penetrant of claim 1 or 2, wherein the CIM comprises the TAT amino acid sequence.

11. The cell penetrant of claim 1, 2 or 10, wherein the CIM comprises more than one TAT amino acid sequence.

12. The cell penetrant of any one of claims 1, 2, 10 and 11, wherein the CIM comprises three TAT amino acid sequences.

13. The cell penetrant of any one of claims 1 to 12, wherein the CIM comprises a macrocycle.

14. The cell penetrant of claim 13, wherein the macrocycle is formed by a covalent bond between two amino acids in the CIM.

15. The cell penetrant of claim 13 or 14, wherein the macrocycle is formed by a disulfide bond between two cysteine ​​residues in the CIM.

16. The cell penetrant of any one of claims 1 to 15, wherein the CIM further comprises one or more histidine residues.

17. The cell penetrant of any one of claims 1 to 16, wherein the CIM comprises a polypeptide having an amino acid sequence selected from the following: SEQ ID NO: 176-184, SEQ ID NO: 192 and SEQ ID NO:

193.

18. The cell-penetrating agent of any one of claims 1 to 17, wherein the CIM further comprises one or more spacer regions.

19. The cell-penetrating agent of claim 18, wherein at least one of the one or more spacer regions comprises one or more amino acid residues.

20. The cell-penetrating agent of claim 18, wherein at least one of the one or more spacer regions comprises one or more glycine residues.

21. The cell-penetrating agent of claim 18, wherein at least one of the one or more spacer regions comprises an amino acid sequence selected from any one of SEQ ID NO: 200-203.

22. The cell-penetrating agent of claim 18, wherein each of the one or more spacer regions comprises an amino acid sequence selected from any of SEQ ID NO: 200-203.

23. The cell penetrant of claim 1, wherein the CIM comprises a polypeptide having an amino acid sequence selected from any of SEQ ID NO: 176-193.

24. The cell penetrant of claim 1, wherein the CIM is a polypeptide having an amino acid sequence selected from any of SEQ ID NO: 176-193.

25. The cell-penetrating agent of any one of claims 1 to 24, wherein the CIM is covalently linked to the antibody.

26. The cell-penetrating agent of any one of claims 1 to 24, wherein the CIM is non-covalently linked to the antibody.

27. The cell-penetrating agent of any one of claims 1 to 26, wherein the cell-penetrating agent further comprises a linker for linking the CIM to the antibody.

28. The cell-penetrating agent of claim 27, wherein the linker is covalently linked to both the CIM and the antibody.

29. The cell-penetrating agent of claim 27 or 28, wherein the linker is a cleavable linker.

30. The cell penetrant of claim 27 or 28, wherein the linker is a non-cleavable linker.

31. The cell-penetrating agent of any one of claims 27 to 30, wherein the linker comprises a polypeptide.

32. The cell-penetrating agent of any one of claims 27 to 31, wherein the linker comprises one or more glycine residues.

33. The cell-penetrating agent of any one of claims 27 to 32, wherein the linker comprises a polypeptide containing an amino acid sequence selected from any one of SEQ ID NO: 194-199.

34. The cell-penetrating agent according to any one of claims 27 to 29, 31 and 32, wherein the linker is a polypeptide comprising an amino acid sequence selected from any one of SEQ ID NO: 194-199.

35. The cell-penetrating agent of any one of claims 1 to 34, wherein the antibody is attached to the C-terminus of the CIM.

36. The cell-penetrating agent of any one of claims 1 to 34, wherein the antibody is attached to the N-terminus of the CIM.

37. The cell-penetrating agent of any one of claims 1 to 36, wherein the antibody comprises a heavy chain or a portion thereof.

38. The cell-penetrating agent of claim 37, wherein the CIM is covalently linked to the C-terminus of the heavy chain.

39. The cell penetrant of claim 37, wherein the CIM is covalently linked to the N-terminus of the heavy chain.

40. The cell-penetrating agent of any one of claims 1 to 39, wherein the antibody comprises a light chain or a portion thereof.

41. The cell-penetrating agent of claim 40, wherein the CIM is covalently linked to the C-terminus of the light chain.

42. The cell penetrant of claim 40, wherein the CIM is covalently linked to the N-terminus of the light chain.

43. The cell-penetrating agent of any one of claims 1 to 42, wherein the antibody competitively binds to TDP-43 with any of the following: An antibody containing the heavy chain variable domain of SEQ ID NO: 1 and the light chain variable domain of SEQ ID NO: 24; An antibody containing the heavy chain variable domain of SEQ ID NO: 63 and the light chain variable domain of SEQ ID NO: 65; An antibody containing the heavy chain variable domain of SEQ ID NO: 67 and the light chain variable domain of SEQ ID NO: 69; An antibody containing the heavy chain variable domain of SEQ ID NO: 71 and the light chain variable domain of SEQ ID NO: 73; An antibody containing the heavy chain variable domain of SEQ ID NO: 75 and the light chain variable domain of SEQ ID NO: 77; or An antibody containing the heavy chain variable domain of SEQ ID NO: 79 and the light chain variable domain of SEQ ID NO:

81.

44. The cell-penetrating agent of any one of claims 1 to 43, wherein the antibody binds to the same epitope on TDP-43 with the following: An antibody containing the heavy chain variable domain of SEQ ID NO: 1 and the light chain variable domain of SEQ ID NO: 24; An antibody containing the heavy chain variable domain of SEQ ID NO: 63 and the light chain variable domain of SEQ ID NO: 65; An antibody containing the heavy chain variable domain of SEQ ID NO: 67 and the light chain variable domain of SEQ ID NO: 69; An antibody containing the heavy chain variable domain of SEQ ID NO: 71 and the light chain variable domain of SEQ ID NO: 73; An antibody containing the heavy chain variable domain of SEQ ID NO: 75 and the light chain variable domain of SEQ ID NO: 77; or An antibody containing the heavy chain variable domain of SEQ ID NO: 79 and the light chain variable domain of SEQ ID NO:

81.

45. The cell-penetrating agent according to any one of claims 1 to 44, wherein the antibody comprises three light chain CDRs and three heavy chain CDRs of a mouse antibody, characterized in that it comprises the heavy chain variable domain of SEQ ID NO: 1 and the light chain variable domain of SEQ ID NO:

24.

46. ​​The cell penetrant of claim 45, wherein the definition of CDR is selected from the group consisting of Kabat, Chothia, Kabat / Chothia Composite, AbM, and Contact.

47. The cell-penetrating agent of claim 45 or 46, wherein the antibody comprises a humanized mature heavy chain variable domain, comprising: Heavy chain CDR1, as defined by the Kabat / Chothia Composite, includes SEQ ID NO: 49; For example, the heavy chain CDR2 as defined by Kabat contains SEQ ID NO: 51; and Heavy chain CDR3, as defined by Kabat or Chothia, contains SEQ ID NO: 52; and Humanized mature light chain variable structural domain, which contains three Kabat light chain CDRs of SEQ ID NO: 53-55.

48. The cell penetrant of claim 47, wherein the humanized mature heavy chain variable domain comprises a sequence that is at least 80% identical to any of SEQ ID NO: 4-23, and the humanized mature light chain variable domain comprises a sequence that is at least 80% identical to any of SEQ ID NO: 27-48.

49. The cell penetrant of claim 48, wherein the humanized mature heavy chain variable domain comprises a sequence that is at least 85% identical to any of SEQ ID NO: 4-23, and the humanized mature light chain variable domain comprises a sequence that is at least 85% identical to any of SEQ ID NO: 27-48.

50. The cell penetrant of claim 48 or 49, wherein the humanized mature heavy chain variable domain comprises a sequence that is at least 90% identical to any of SEQ ID NO: 4-23, and the humanized mature light chain variable domain comprises a sequence that is at least 90% identical to any of SEQ ID NO: 27-48.

51. The cell-penetrating agent of any one of claims 48 to 50, wherein the humanized mature heavy chain variable domain comprises a sequence that is at least 95% identical to any one of SEQ ID NO: 4-23, and the humanized mature light chain variable domain comprises a sequence that is at least 95% identical to any one of SEQ ID NO: 27-48.

52. The cell-penetrating agent of any one of claims 47 to 51, wherein at least one of the following positions in the humanized heavy chain variable domain is occupied by a specified amino acid: K19 is occupied by R; S35 is occupied by G; T40 is occupied by A; E42 is occupied by G; A49 is occupied by S; K43 is occupied by E; R44 is occupied by G or D; A49 is occupied by S; A74 is occupied by S; T77 is occupied by S; L78 is occupied by A or G; L80 is occupied by A or G; L82C is occupied by G; M83 is occupied by R; S84 is occupied by A; M89 is occupied by V; or F91 is occupied by Y.

53. The cell-penetrating agent of any one of claims 47 to 52, wherein at least one of the following positions in the humanized heavy chain variable domain is occupied by a specified amino acid: K43 is occupied by E; R44 is occupied by G or D; A49 is occupied by S; A74 is occupied by S; T77 is occupied by S; or F91 is occupied by Y.

54. The cell-penetrating agent of any one of claims 47 to 53, wherein at least one of the following positions in the humanized heavy chain variable domain is occupied by a specified amino acid: S35 is occupied by G; L78 is occupied by A or G; L80 is occupied by A or G; or L82C is occupied by G.

55. The cell-penetrating agent of any one of claims 47 to 54, wherein the F91 of the humanized heavy chain variable domain is occupied by Y; and at least one of the following positions in the humanized heavy chain variable domain is occupied by a specified amino acid: R44 is occupied by G; A49 is occupied by S; A74 is occupied by S; T77 is occupied by S; L78 is occupied by A or G; or M83 is occupied by R.

56. The cell-penetrating agent of any one of claims 47 to 55, wherein at least one of the following positions in the variable domain of the humanized light chain is occupied by a specified amino acid: V3 is occupied by Q; L9 is occupied by S; D17 is occupied by Q; Q18 is occupied by P; K39 is occupied by R; K45 is occupied by R; T80 is occupied by A or S; L83 is occupied by V; L92 is occupied by G or A; V94 is occupied by I or A; A100 is occupied by G, D or R; or L104 is occupied by V.

57. The cell-penetrating agent of any one of claims 47 to 56, wherein at least one of the following positions in the variable domain of the humanized light chain is occupied by a specified amino acid: V3 is occupied by Q or A100 is occupied by D or R.

58. The cell-penetrating agent of any one of claims 47 to 57, wherein at least one of the following positions in the variable domain of the humanized light chain is occupied by a specified amino acid: L9 is occupied by S; T80 is occupied by A or S; L92 is occupied by G or A; or V94 is occupied by I or A.

59. The cell penetrant of any one of claims 47 to 58, wherein V3 is occupied by Q; Q18 is occupied by P; A100 is occupied by D; and at least one of the following positions in the humanized light chain variable domain is occupied by the specified amino acid: T80 is occupied by A or L92 is occupied by A.

60. The cell-penetrating agent of any one of claims 47 to 59, wherein at least one of the following positions in the humanized heavy chain variable domain is occupied by a specified amino acid: L5 occupied by V; G44 occupied by R; A49 occupied by S; A74 occupied by S; T77 occupied by S; L78 occupied by A or G; M89 occupied by V; or F91 occupied by Y; and At least one of the following positions in the variable domain of the humanized light chain is occupied by the specified amino acid: V3 is occupied by Q; D17 is occupied by Q; Q18 is occupied by P; K39 is occupied by R; K45 is occupied by R; T80 is occupied by A; L83 is occupied by V; L92 is occupied by A; A100 is occupied by D; or L104 is occupied by V.

61. The cell-penetrating agent of claim 60, wherein the antibody comprises a heavy chain variable domain and a light chain variable domain, comprising: Heavy chain CDR1 as defined by the Kabat / Chothia Composite includes SEQ ID NO: 49 or SEQ ID NO: 50; For example, the heavy chain CDR2 as defined by Kabat contains SEQ ID NO: 51; Heavy chain CDR3, as defined by Kabat or Chothia, contains SEQ ID NO: 52; For example, the light chain CDR1 as defined by Kabat contains SEQ ID NO: 53; For example, the light chain CDR2 as defined by Kabat contains SEQ ID NO: 54; and Light chain CDR3 as defined by Kabat includes one of SEQ ID NO: 55-61.

62. The cell-penetrating agent of claim 61, wherein: The heavy chain CDR1 as defined by the Kabat / Chothia Composite includes SEQ ID NO: 49; The heavy chain CDR2 as defined by Kabat contains SEQ ID NO: 51; The heavy chain CDR3, as defined by Kabat or Chothia, contains SEQ ID NO: 52; The light chain CDR1 as defined by Kabat contains SEQ ID NO: 53; The light chain CDR2, as defined by Kabat, contains SEQ ID NO: 54; and The light chain CDR3 as defined by Kabat includes SEQ ID NO: 55 or SEQ ID NO:

61.

63. The cell-penetrating agent of claim 61 or 62, wherein the heavy chain variable domain comprises a sequence that is at least 95% identical to any of SEQ ID NO: 4-23.

64. The cell-penetrating agent of claim 63, wherein the heavy chain variable domain comprises a sequence that is at least 95% identical to any of the following: SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO:

23.

65. The cell-penetrating agent of claim 63 or 64, wherein the heavy chain variable domain comprises a sequence that is at least 98% identical to any of the following: SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO:

23.

66. The cell-penetrating agent of any one of claims 61 to 65, wherein the heavy chain variable domain comprises the sequence of SEQ ID NO:

20.

67. The cell-penetrating agent of any one of claims 61 to 65, wherein the heavy chain variable domain comprises the sequence of SEQ ID NO:

21.

68. The cell-penetrating agent of any one of claims 61 to 65, wherein the heavy chain variable domain comprises the sequence of SEQ ID NO:

23.

69. The cell-penetrating agent of any one of claims 61 to 68, wherein the light chain variable domain comprises a sequence that is at least 95% identical to any of the following: SEQ ID NO: 27-48.

70. The cell-penetrating agent of any one of claims 61 to 69, wherein the light chain variable domain comprises a sequence that is at least 95% identical to that of SEQ ID NO: 47 or SEQ ID NO:

48.

71. The cell-penetrating agent of any one of claims 61 to 70, wherein the light chain variable domain comprises a sequence that is at least 98% identical to that of SEQ ID NO: 47 or SEQ ID NO:

48.

72. The cell-penetrating agent of any one of claims 69 to 71, wherein the light chain variable domain comprises SEQ ID NO:

47.

73. The cell-penetrating agent of any one of claims 69 to 71, wherein the light chain variable domain comprises SEQ ID NO:

48.

74. The cell-penetrating agent of claim 43 or 44, wherein the antibody comprises: Heavy chain CDR1 containing SEQ ID NO: 84; Heavy chain CDR2 containing SEQ ID NO: 85; Heavy chain CDR3 containing SEQ ID NO: 86; The light chain CDR1 containing SEQ ID NO: 87; The light chain CDR2 containing SEQ ID NO: 88; and The light chain CDR3 contains SEQ ID NO:

89.

75. The cell-penetrating agent of claim 74, wherein the heavy chain variable domain comprises a sequence that is at least 95% identical to that of SEQ ID NO:

63.

76. The cell-penetrating agent of claim 74 or 75, wherein the heavy chain variable domain comprises a sequence that is at least 98% identical to that of SEQ ID NO:

63.

77. The cell-penetrating agent of any one of claims 74 to 76, wherein the heavy chain variable domain comprises the sequence of SEQ ID NO:

63.

78. The cell-penetrating agent of any one of claims 74 to 77, wherein the light chain variable domain comprises a sequence that is at least 95% identical to that of SEQ ID NO:

65.

79. The cell-penetrating agent of any one of claims 74 to 78, wherein the light chain variable domain comprises a sequence that is at least 98% identical to that of SEQ ID NO:

65.

80. The cell-penetrating agent of any one of claims 74 to 79, wherein the light chain variable domain comprises the sequence of SEQ ID NO:

65.

81. The cell-penetrating agent of claim 43 or 44, wherein the antibody comprises a heavy chain variable domain and a light chain variable domain, wherein the antibody comprises: Heavy chain CDR1 containing SEQ ID NO: 90; Heavy chain CDR2 containing SEQ ID NO: 91; Heavy chain CDR3 containing SEQ ID NO: 92; The light chain CDR1 containing SEQ ID NO: 93; The light chain CDR2 containing SEQ ID NO: 94; and It contains the light chain CDR3, which is SEQ ID NO:

95.

82. The cell penetrant of claim 81, wherein the heavy chain variable domain comprises a sequence that is at least 95% identical to that of SEQ ID NO:

67.

83. The cell penetrant of claim 81 or 82, wherein the heavy chain variable domain comprises a sequence that is at least 98% identical to SEQ ID NO:

67.

84. The cell-penetrating agent of any one of claims 81 to 83, wherein the heavy chain variable domain comprises the sequence of SEQ ID NO:

67.

85. The cell-penetrating agent of any one of claims 81 to 84, wherein the light chain variable domain comprises a sequence that is at least 95% identical to that of SEQ ID NO:

69.

86. The cell-penetrating agent of any one of claims 81 to 85, wherein the light chain variable domain comprises a sequence that is at least 98% identical to that of SEQ ID NO:

69.

87. The cell-penetrating agent of any one of claims 81 to 86, wherein the light chain variable domain comprises the sequence of SEQ ID NO:

69.

88. The cell-penetrating agent of claim 43 or 44, wherein the antibody comprises a heavy chain variable domain and a light chain variable domain, wherein the antibody comprises: Heavy chain CDR1 containing SEQ ID NO: 96; Heavy chain CDR2 containing SEQ ID NO: 97; Heavy chain CDR3 containing SEQ ID NO: 98; The light chain CDR1 containing SEQ ID NO: 99; The light chain CDR2 containing SEQ ID NO: 100; and The light chain CDR3 contains SEQ ID NO:

101.

89. The cell penetrant of claim 88, wherein the heavy chain variable domain comprises a sequence that is at least 95% identical to that of SEQ ID NO:

71.

90. The cell-penetrating agent of claim 88 or 89, wherein the heavy chain variable domain comprises a sequence that is at least 98% identical to that of SEQ ID NO:

71.

91. The cell-penetrating agent of any one of claims 88 to 90, wherein the heavy chain variable domain comprises the sequence of SEQ ID NO:

71.

92. The cell-penetrating agent of any one of claims 88 to 91, wherein the light chain variable domain comprises a sequence that is at least 95% identical to that of SEQ ID NO:

73.

93. The cell-penetrating agent of any one of claims 88 to 92, wherein the light chain variable domain comprises a sequence that is at least 98% identical to that of SEQ ID NO:

73.

94. The cell-penetrating agent of any one of claims 88 to 93, wherein the light chain variable domain comprises the sequence of SEQ ID NO:

73.

95. The cell-penetrating agent of claim 43 or 44, wherein the antibody comprises a heavy chain variable domain and a light chain variable domain, wherein the antibody comprises: Heavy chain CDR1 containing SEQ ID NO: 102; Heavy chain CDR2 containing SEQ ID NO: 103; Heavy chain CDR3 containing SEQ ID NO: 104; The light chain CDR1 containing SEQ ID NO: 105; The light chain CDR2 containing SEQ ID NO: 106; and The light chain CDR3 contains SEQ ID NO:

107.

96. The cell-penetrating agent of claim 95, wherein the heavy chain variable domain comprises a sequence that is at least 95% identical to that of SEQ ID NO:

75.

97. The cell-penetrating agent of claim 95 or 96, wherein the heavy chain variable domain comprises a sequence that is at least 98% identical to that of SEQ ID NO:

75.

98. The cell-penetrating agent of any one of claims 95 to 97, wherein the heavy chain variable domain comprises the sequence of SEQ ID NO:

75.

99. The cell-penetrating agent of any one of claims 95 to 97, wherein the light chain variable domain comprises a sequence that is at least 95% identical to that of SEQ ID NO:

77.

100. The cell-penetrating agent of any one of claims 95 to 99, wherein the light chain variable domain comprises a sequence that is at least 98% identical to that of SEQ ID NO:

77.

101. The cell-penetrating agent of any one of claims 95 to 100, wherein the light chain variable domain comprises the sequence of SEQ ID NO:

77.

102. The cell-penetrating agent of claim 43 or 44, wherein the antibody comprises a heavy chain variable domain and a light chain variable domain, wherein the antibody comprises: Heavy chain CDR1 containing SEQ ID NO: 108; Heavy chain CDR2 containing SEQ ID NO: 109; Heavy chain CDR3 containing SEQ ID NO: 110; The light chain CDR1 containing SEQ ID NO: 111; The light chain CDR2 containing SEQ ID NO: 112; and The light chain CDR3 contains SEQ ID NO:

113.

103. The cell penetrant of claim 102, wherein the heavy chain variable domain comprises a sequence that is at least 95% identical to that of SEQ ID NO:

79.

104. The cell penetrant of claim 102 or 103, wherein the heavy chain variable domain comprises a sequence that is at least 98% identical to that of SEQ ID NO:

79.

105. The cell-penetrating agent of any one of claims 102 to 104, wherein the heavy chain variable domain comprises the sequence of SEQ ID NO:

79.

106. The cell-penetrating agent of any one of claims 102 to 105, wherein the light chain variable domain comprises a sequence that is at least 95% identical to that of SEQ ID NO:

81.

107. The cell-penetrating agent of any one of claims 102 to 106, wherein the light chain variable domain comprises a sequence that is at least 98% identical to that of SEQ ID NO:

81.

108. The cell-penetrating agent of any one of claims 102 to 107, wherein the light chain variable domain comprises the sequence of SEQ ID NO:

81.

109. The cell-penetrating agent according to any one of claims 1 to 108, wherein the antibody is a humanized antibody, a chimeric antibody, or a decorative antibody.

110. The cell-penetrating agent according to any one of claims 1 to 109, wherein the antibody is an antigen-binding antibody fragment.

111. The cell penetrant of claim 110, wherein the antigen-binding antibody fragment is a Fab fragment, a Fab'2 fragment, or a single-chain Fv.

112. The cell-penetrating agent according to any one of claims 1 to 109, wherein the antibody is an intact antibody.

113. The cell-penetrating agent according to any one of claims 1 to 109 and 112, wherein the antibody has a human IgG1 isotype.

114. The cell-penetrating agent of any one of claims 1 to 109, 112 and 113, wherein the heavy chain variable domain is fused to the heavy chain constant region and the light chain variable domain is fused to the light chain constant region.

115. The cell-penetrating agent of claim 114, wherein the heavy chain constant region is a mutant form of the natural human heavy chain constant region, which binds less to the Fcγ receptor relative to the natural heavy chain constant region.

116. The cell penetrant of claim 114 or 115, wherein the heavy chain constant region has an IgG1 isotype.

117. The cell-penetrating agent of any one of claims 1 to 116, wherein the antibody has at least one mutation in the constant region.

118. The cell penetrant of claim 117, wherein the at least one mutation reduces complement fixation or activation in the constant region.

119. The cell penetrant of claim 118, wherein the at least one mutation is at one or more of the following locations numbered according to EU: 241, 264, 265, 270, 296, 297, 318, 320, 322, 329 and 331.

120. The cell-penetrating agent of claim 119, wherein the antibody has an alanine at positions 318, 320 and 322 according to EU numbers.

121. The cell-penetrating agent according to any one of claims 1 to 120, wherein the antibody selectively binds to phosphorylated TDP-43.

122. The cell penetrant of any one of claims 1 to 121, wherein the antibody selectively binds to phosphorylated TDP-43 compared to unphosphorylated TDP-43.

123. The cell penetrant of claim 121 or 122, wherein the antibody binds to phosphorylated TDP-43 with an affinity at least 100 times greater than that of unphosphorylated TDP-43.

124. The cell penetrant of any one of claims 121 to 123, wherein the antibody binds to phosphorylated TDP-43 with an affinity at least 1000 times greater than that of unphosphorylated TDP-43.

125. The cell penetrant of any one of claims 121 to 124, wherein phosphorylated TDP-43 comprises phosphorylation of at least one amino acid residue selected from S409 and S410.

126. The cell penetrant of claim 125, wherein the phosphorylated TDP-43 comprises phosphorylation of both S409 and S410.

127. The cell-penetrating agent of any one of claims 1 to 126, wherein the antibody selectively binds to the cytoplasmic aggregates of TDP-43.

128. The cell penetrant of any one of claims 1 to 127, wherein the antibody selectively binds to cytoplasmic aggregates of TDP-43 compared to nuclear TDP-43.

129. The cell penetrant of claim 127 or 128, wherein the cytoplasmic aggregates of TDP-43 comprise phosphorylated aggregates of TDP-43.

130. The cell-penetrating agent of any one of claims 1 to 129, wherein the antibody substantially does not bind to unphosphorylated TDP-43.

131. The cell-penetrating agent of claim 1, wherein the cell-penetrating agent comprises a polypeptide sequence that is at least 95% identical to a sequence selected from any of the following: SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172 and SEQ ID NO:

174.

132. The cell-penetrating agent of claim 1 or 131, wherein the cell-penetrating agent comprises a polypeptide sequence that is at least 98% identical to a sequence selected from any of the following: SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172 and SEQ ID NO:

174.

133. The cell-penetrating agent of any one of claims 1, 131, and 132, wherein the cell-penetrating agent comprises a polypeptide sequence selected from any one of the following: SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172 and SEQ ID NO:

174.

134. The cell-penetrating agent of any one of claims 1 and 131 to 133, wherein the cell-penetrating agent comprises a polypeptide sequence that is at least 95% identical to a sequence selected from any of the following: SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 16 ...4, SEQ ID NO: 155, SEQ ID NO: 159, SEQ ID NO: 159, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 159, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 15 NO: 163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173 and SEQ ID NO:

175.

135. The cell-penetrating agent of claims 1 and 131 to 134, wherein the cell-penetrating agent comprises a polypeptide sequence that is at least 98% identical to the sequence selected from any of the following: SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173 and SEQ ID NO:

175.

136. The cell-penetrating agent of claims 1 and 131 to 135, wherein the cell-penetrating agent comprises a polypeptide sequence selected from any of the following: SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO: 155, SEQ ID NO: 156 ...37, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 139, SEQ ID NO: 139, SEQ ID NO: 139, SEQ ID NO: 139, SEQ ID NO: 139, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173 and SEQ ID NO:

175.

137. The cell-penetrating agent of claim 1, wherein the cell-penetrating agent comprises a first polypeptide and a second polypeptide, further wherein: The first polypeptide contains at least 95% of the same sequence as SEQ ID NO: 116 and the second polypeptide contains at least 95% of the same sequence as SEQ ID NO: 117; The first polypeptide contains at least 95% of the same sequence as SEQ ID NO: 118 and the second polypeptide contains at least 95% of the same sequence as SEQ ID NO: 119; The first polypeptide contains at least 95% of the same sequence as SEQ ID NO: 120 and the second polypeptide contains at least 95% of the same sequence as SEQ ID NO: 121; The first polypeptide contains at least 95% of the same sequence as SEQ ID NO: 122 and the second polypeptide contains at least 95% of the same sequence as SEQ ID NO: 123; The first polypeptide contains at least 95% of the same sequence as SEQ ID NO: 124 and the second polypeptide contains at least 95% of the same sequence as SEQ ID NO: 125; The first polypeptide contains at least 95% of the same sequence as SEQ ID NO: 126 and the second polypeptide contains at least 95% of the same sequence as SEQ ID NO: 127; The first polypeptide contains at least 95% of the same sequence as SEQ ID NO: 128 and the second polypeptide contains at least 95% of the same sequence as SEQ ID NO: 129; The first polypeptide contains at least 95% of the same sequence as SEQ ID NO: 130 and the second polypeptide contains at least 95% of the same sequence as SEQ ID NO: 131; The first polypeptide contains at least 95% of the same sequence as SEQ ID NO: 132 and the second polypeptide contains at least 95% of the same sequence as SEQ ID NO: 133; The first polypeptide contains at least 95% of the same sequence as SEQ ID NO: 134 and the second polypeptide contains at least 95% of the same sequence as SEQ ID NO: 135; The first polypeptide contains at least 95% of the same sequence as SEQ ID NO: 136 and the second polypeptide contains at least 95% of the same sequence as SEQ ID NO: 137; The first polypeptide contains at least 95% of the same sequence as SEQ ID NO: 138 and the second polypeptide contains at least 95% of the same sequence as SEQ ID NO: 139; The first polypeptide contains at least 95% of the same sequence as SEQ ID NO: 140 and the second polypeptide contains at least 95% of the same sequence as SEQ ID NO: 141; The first polypeptide contains at least 95% of the same sequence as SEQ ID NO: 142 and the second polypeptide contains at least 95% of the same sequence as SEQ ID NO: 143; The first polypeptide contains at least 95% of the same sequence as SEQ ID NO: 144 and the second polypeptide contains at least 95% of the same sequence as SEQ ID NO: 145; The first polypeptide contains at least 95% of the same sequence as SEQ ID NO: 146 and the second polypeptide contains at least 95% of the same sequence as SEQ ID NO: 147; The first polypeptide contains at least 95% of the same sequence as SEQ ID NO: 148 and the second polypeptide contains at least 95% of the same sequence as SEQ ID NO: 149; The first polypeptide contains at least 95% of the same sequence as SEQ ID NO: 150 and the second polypeptide contains at least 95% of the same sequence as SEQ ID NO: 151; The first polypeptide contains at least 95% of the same sequence as SEQ ID NO: 152 and the second polypeptide contains at least 95% of the same sequence as SEQ ID NO: 153; The first polypeptide contains at least 95% of the same sequence as SEQ ID NO: 154 and the second polypeptide contains at least 95% of the same sequence as SEQ ID NO: 155; The first polypeptide contains at least 95% of the same sequence as SEQ ID NO: 156 and the second polypeptide contains at least 95% of the same sequence as SEQ ID NO: 157; The first polypeptide contains at least 95% of the same sequence as SEQ ID NO: 158 and the second polypeptide contains at least 95% of the same sequence as SEQ ID NO: 159; The first polypeptide contains at least 95% of the same sequence as SEQ ID NO: 160 and the second polypeptide contains at least 95% of the same sequence as SEQ ID NO: 161; The first polypeptide contains at least 95% of the same sequence as SEQ ID NO: 162 and the second polypeptide contains at least 95% of the same sequence as SEQ ID NO: 163; The first polypeptide contains at least 95% of the same sequence as SEQ ID NO: 164 and the second polypeptide contains at least 95% of the same sequence as SEQ ID NO: 165; The first polypeptide contains at least 95% of the same sequence as SEQ ID NO: 166 and the second polypeptide contains at least 95% of the same sequence as SEQ ID NO: 167; The first polypeptide contains at least 95% of the same sequence as SEQ ID NO: 168 and the second polypeptide contains at least 95% of the same sequence as SEQ ID NO: 169; The first polypeptide contains at least 95% of the same sequence as SEQ ID NO: 170 and the second polypeptide contains at least 95% of the same sequence as SEQ ID NO: 171; The first polypeptide contains at least 95% of the sequence identical to SEQ ID NO: 172 and the second polypeptide contains at least 95% of the sequence identical to SEQ ID NO: 173; or The first polypeptide contains at least 95% of the same sequence as SEQ ID NO: 174 and the second polypeptide contains at least 95% of the same sequence as SEQ ID NO:

175.

138. The cell-penetrating agent of claim 1, wherein the cell-penetrating agent comprises a first polypeptide and a second polypeptide, wherein: The first polypeptide comprises SEQ ID NO: 116 and the second polypeptide comprises SEQ ID NO: 117; The first polypeptide comprises SEQ ID NO: 118 and the second polypeptide comprises SEQ ID NO: 119; The first polypeptide comprises SEQ ID NO: 120 and the second polypeptide comprises SEQ ID NO: 121; The first polypeptide comprises SEQ ID NO: 122 and the second polypeptide comprises SEQ ID NO: 123; The first polypeptide comprises SEQ ID NO: 124 and the second polypeptide comprises SEQ ID NO: 125; The first polypeptide comprises SEQ ID NO: 126 and the second polypeptide comprises SEQ ID NO: 127; The first polypeptide comprises SEQ ID NO: 128 and the second polypeptide comprises SEQ ID NO: 129; The first polypeptide comprises SEQ ID NO: 130 and the second polypeptide comprises SEQ ID NO: 131; The first polypeptide comprises SEQ ID NO: 132 and the second polypeptide comprises SEQ ID NO: 133; The first polypeptide comprises SEQ ID NO: 134 and the second polypeptide comprises SEQ ID NO: 135; The first polypeptide comprises SEQ ID NO: 136 and the second polypeptide comprises SEQ ID NO: 137; The first polypeptide comprises SEQ ID NO: 138 and the second polypeptide comprises SEQ ID NO: 139; The first polypeptide comprises SEQ ID NO: 140 and the second polypeptide comprises SEQ ID NO: 141; The first polypeptide comprises SEQ ID NO: 142 and the second polypeptide comprises SEQ ID NO: 143; The first polypeptide comprises SEQ ID NO: 144 and the second polypeptide comprises SEQ ID NO: 145; The first polypeptide comprises SEQ ID NO: 146 and the second polypeptide comprises SEQ ID NO: 147; The first polypeptide comprises SEQ ID NO: 148 and the second polypeptide comprises SEQ ID NO: 149; The first polypeptide comprises SEQ ID NO: 150 and the second polypeptide comprises SEQ ID NO: 151; The first polypeptide comprises SEQ ID NO: 152 and the second polypeptide comprises SEQ ID NO: 153; The first polypeptide comprises SEQ ID NO: 154 and the second polypeptide comprises SEQ ID NO: 155; The first polypeptide comprises SEQ ID NO: 156 and the second polypeptide comprises SEQ ID NO: 157; The first polypeptide comprises SEQ ID NO: 158 and the second polypeptide comprises SEQ ID NO: 159; The first polypeptide comprises SEQ ID NO: 160 and the second polypeptide comprises SEQ ID NO: 161; The first polypeptide comprises SEQ ID NO: 162 and the second polypeptide comprises SEQ ID NO: 163; The first polypeptide comprises SEQ ID NO: 164 and the second polypeptide comprises SEQ ID NO: 165; The first polypeptide comprises SEQ ID NO: 166 and the second polypeptide comprises SEQ ID NO: 167; The first polypeptide comprises SEQ ID NO: 168 and the second polypeptide comprises SEQ ID NO: 169; The first polypeptide comprises SEQ ID NO: 170 and the second polypeptide comprises SEQ ID NO: 171; The first polypeptide comprises SEQ ID NO: 172 and the second polypeptide comprises SEQ ID NO: 173; or The first polypeptide comprises SEQ ID NO: 174 and the second polypeptide comprises SEQ ID NO:

175.

139. The cell-penetrating agent of any one of claims 1 to 138, wherein the antibody is conjugated to a therapeutic agent, a cytotoxic agent, a cell growth inhibitor, an immunomodulator, a neurotrophic agent, or a neuroprotective agent.

140. The cell-penetrating agent of any one of claims 1 to 139, wherein the heavy chain does not contain a C-terminal lysine residue.

141. A pharmaceutical composition comprising a cell-penetrating agent according to any one of claims 1 to 140 and a pharmaceutically acceptable carrier.

142. A nucleic acid encoding at least a portion of the cell penetrant of any one of claims 1 to 140.

143. The nucleic acid of claim 142, wherein the nucleic acid encodes the heavy chain variable domain and / or the light chain variable domain of the antibody.

144. The nucleic acid of claim 142 or 143, wherein the nucleic acid encodes the CIM.

145. A vector comprising the nucleic acid of any one of claims 142 to 144, said nucleic acid being operatively linked to one or more regulatory sequences to achieve expression of the cell penetrant of any one of claims 1 to 140 in mammalian cells.

146. The vector of claim 145, wherein the one or more regulatory sequences comprise one or more of an enhancer, a ribosome binding site, a transcription termination signal, and a promoter, optionally wherein the promoter is a eukaryotic promoter.

147. The vector of claim 145 or 146, wherein the nucleic acid is codon-optimized for expression in host cells.

148. A host cell transformed with the vector according to any one of claims 145 to 147.

149. A host cell comprising the nucleic acid of any one of claims 142 to 144.

150. A method of delivering an antibody specifically bound to TDP-43 into a cell, comprising contacting the cell with a cell-penetrating agent as described in any one of claims 1 to 140, thereby causing at least an antigen-binding fragment of the antibody to be internalized into the cell.

151. The method of claim 150, the method further comprising transferring at least an antigen-binding fragment of the antibody into the cytosol of the cell.

152. A method for binding intracellular TDP-43 protein in cells, the method comprising: Contacting the cell-penetrating agent of any one of claims 1 to 140 with the cells results in the internalization and translocation of at least the antigen-binding fragment of the antibody into the cytosol.

153. The method of any one of claims 150 to 152, wherein the cell is a mammalian cell.

154. The method of any one of claims 150 to 152, wherein the cells are in vitro.

155. The method of any one of claims 150 to 152, wherein the cells are in the subject.

156. A method for inhibiting or reducing TDP-43 accumulation in a subject suffering from or at risk of developing TDP-43-related disease, comprising administering to the subject an effective amount of any one of claims 1 to 140 of a cell-penetrating agent, thereby inhibiting or reducing TDP-43 accumulation in the subject.

157. A method of treating or achieving prevention of TDP-43-related disease in a subject, comprising administering a therapeutically effective amount of the cell-penetrating agent of any one of claims 1 to 140 and thereby treating or achieving prevention of said TDP-43-related disease.

158. The method of claim 157, wherein the TDP-43-related disease is amyotrophic lateral sclerosis (ALS), frontotemporal degeneration (FTLD-TDP), primary spinal lateral sclerosis, and progressive muscular atrophy and Parkinson's disease.

159. The method of claim 158, wherein the TDP-43-related disease is ALS.

160. A method for detecting TDP-43 deposits in a subject suffering from or at risk of developing TDP-43-related disease, comprising administering to the subject a cell-penetrating agent as described in any one of claims 1 to 140 and detecting antibodies in the subject that bind to TDP-43.

161. The method of claim 160, wherein the antibody is administered by intravenous injection into the body of the subject.

162. A method for detecting TDP-43 in a sample obtained from a patient with TDP-43-related disease or at risk of developing said disease, comprising contacting said sample with a cell-penetrating agent according to any one of claims 1 to 140 and detecting the binding of said antibody to TDP-43 in said sample.

163. The method of any one of claims 160 to 162, wherein the antibody is labeled.

164. The method of claim 163, wherein the antibody is labeled with fluorescent labeling, paramagnetic labeling, or radioactive labeling.

165. The method of claim 164, wherein the radioactive label is detected using positron emission tomography (PET) or single-photon emission computed tomography (SPECT).

Images