WO2025059515A2 - Cell penetrating agents and uses thereof - Google Patents
Cell penetrating agents and uses thereof Download PDFInfo
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- WO2025059515A2 WO2025059515A2 PCT/US2024/046686 US2024046686W WO2025059515A2 WO 2025059515 A2 WO2025059515 A2 WO 2025059515A2 US 2024046686 W US2024046686 W US 2024046686W WO 2025059515 A2 WO2025059515 A2 WO 2025059515A2
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22
Definitions
- TDP-43 BACKGROUND Transactive response DNA binding protein 43
- TDP-43 is a predominantly nuclear protein that is involved in RNA splicing, trafficking, 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 expressed ubiquitously in all cell types, it has been shown to be highly expressed in the neuroepithelium, which contains all CNS progenitors for neurons and glia. Moreover, TDP-43 has been shown to specifically bind numerous RNAs in neuronal cells.
- TDP-43 cytoplasmic aggregates are associated with several neurodegenerative diseases, disorders, or conditions. Specifically, TDP-43 cytoplasmic aggregates and/or misfolding of TDP-43 have been associated with neurodegenerative diseases including Amyotrophic Lateral Sclerosis (“ALS”), frontotemporal dementia (“FTD” or “FTLD- TDP-43”), limbic-predominant age-related TDP-43 encephalopathy (“LATE”), Alzheimer's disease, multisystem proteinopathy, and chronic traumatic encephalopathy.
- ALS Amyotrophic Lateral Sclerosis
- FDD frontotemporal dementia
- LATE limbic-predominant age-related TDP-43 encephalopathy
- Alzheimer's disease multisystem proteinopathy
- chronic traumatic encephalopathy a chronic traumatic encephalopathy
- ALS has an incidence of about 1-2/100,000 and a prevalence of about 4-6/100,000 cases each year. ALS results in the progressive degeneration of both upper and lower motor neurons which generally leads to death primarily due to respiratory failure in three to five years after diagnosis. Typical symptoms include stiff muscles, muscle twitches, gradual muscle weakness, and muscle wasting. Around half of all people suffering from ALS have difficulties thinking and/or behavioral symptoms and about 15% develop frontotemporal dementia. Pathologically, abnormal aggregations of TDP-43 protein are seen in up to 97% of ALS patients (Nguyen, H.P., et al., ALS Genes in the Genomic Era and their Implications for FTD, Trends in Genetics, 34(6): 404+423 (2016)).
- CCAs cell-penetrating agents
- CCM cell internalization module
- ALS Amyotrophic Lateral Sclerosis
- cell-penetrating agent that include (i) a cell internalizing module and (ii) an antibody that specifically binds to Transactive response DNA binding Protein of 43 kD (TDP-43).
- the CIM includes a Cell Membrane Internalizing Peptide (CMIP).
- CMIP Cell Membrane Internalizing Peptide
- the CIM includes a wild-type M-lycotoxin peptide.
- the CIM includes an M-lycotoxin derivative.
- the CIM includes a Penetain amino acid sequence or a derivative thereof.
- the CIM includes a Pepth amino acid sequence or a derivative thereof.
- the CIM includes a polyarginine amino acid sequence.
- the CIM includes more than one polyarginine amino acid sequence. In some embodiments, the CIM includes three Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT polyarginine amino acid sequences. In some embodiments, the CIM includes a TAT amino acid sequence. In some embodiments, the CIM includes more than one TAT amino acid sequence. In some embodiments, the CIM includes three TAT amino acid sequences. In some embodiments, the CIM includes 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.
- the CIM includes one or more histidine residues. In some embodiments, the CIM includes a polypeptide having an amino acid sequence selected from one of: SEQ ID NO: 176-184, SEQ ID NO: 192, and SEQ ID NO: 193. In some embodiments, the CIM includes one or more spacer regions. In some embodiments, at least one of the one or more spacer regions includes one or more amino acid residues. In some embodiments, at least one of the one or more spacer regions includes one or more glycine residues. In some embodiments, at least one of the one or more spacer regions includes an amino acid sequence selected from any one of SEQ ID NOs: 200-203.
- each of the one or more spacer regions includes an amino acid sequence selected from any one of SEQ ID NOs: 200-203.
- the CIM includes a polypeptide having an amino acid sequence selected from any one of SEQ ID NOs: 176-193.
- the CIM is a polypeptide having an amino acid sequence selected from any one of SEQ ID NOs: 176-193.
- the CIM is covalently linked to the antibody.
- the CIM is non-covalently linked to the antibody.
- the cell- penetrating agent includes a linker connecting the CIM to the antibody.
- the linker is covalently linked to both the CIM and the antibody.
- the linker is a cleavable linker. In some embodiments, the linker is a non-cleavable linker. In some embodiments, the linker includes a polypeptide. In some embodiments, the linker includes one or more glycine residues. In some embodiments, the linker includes a polypeptide including an amino acid sequence selected from any one of SEQ ID NOs: 194-199. In some embodiments, the linker is a polypeptide including an amino acid sequence selected from any one of SEQ ID NOs: 194-199. In some embodiments, the antibody is linked to the C-terminus of the CIM. In some embodiments, the antibody is linked to the N-terminus of the CIM. Attorney Docket Ref.
- the antibody competes for binding to TDP-43 (e.g., human TDP- 43) with: an antibody including a heavy chain variable domain of SEQ ID NO: 1 and a light chain variable domain of SEQ ID NO: 24; an antibody including a heavy chain variable domain of SEQ ID NO: 63 and a light chain variable domain of SEQ ID NO: 65; an antibody including a heavy chain variable domain of SEQ ID NO: 67 and a light chain variable domain of SEQ ID NO: 69; an antibody including a heavy chain variable domain of SEQ ID NO: 71 and a light chain variable domain of SEQ ID NO: 73; an antibody including a heavy chain variable domain of SEQ ID NO: 75 and a light chain variable domain of SEQ ID NO: 77; or an antibody including a heavy chain variable domain of SEQ ID NO: 79 and a light chain variable domain of SEQ ID NO: 81.
- TDP-43 e.g., human TDP- 43
- the antibody binds to the same epitope on TDP-43 (e.g., human TDP-43) as an antibody including a heavy chain variable domain of SEQ ID NO: 1 and a light chain variable domain of SEQ ID NO: 24; an antibody including a heavy chain variable domain of SEQ ID NO: 63 and a light chain variable domain of SEQ ID NO: 65; an antibody including a heavy chain variable domain of SEQ ID NO: 67 and a light chain variable domain of SEQ ID NO: 69; an antibody including a heavy chain variable domain of SEQ ID NO: 71 and a light chain variable domain of SEQ ID NO: 73; an antibody including a heavy chain variable domain of SEQ ID NO: 75 and a light chain variable domain of SEQ ID NO: 77; or an antibody including a heavy chain variable domain of SEQ ID NO: 79 and a light chain variable domain of SEQ ID NO: 81.
- TDP-43 e.g., human TDP-43
- the antibody that specifically binds to TDP-43 including three light chain CDRs and three heavy chain CDRs of a mouse antibody characterized by a heavy chain variable domain including SEQ ID NO: 1 and a light chain variable domain including SEQ ID NO: 24.
- the CDRs are of a definition selected from the group of Kabat, Chothia, Kabat/Chothia Composite, AbM, and Contact.
- the antibody includes a humanized mature heavy variable domain including: a heavy chain CDR1 as defined by Kabat/Chothia Composite, including SEQ ID NO: 49; a heavy chain CDR2 as defined by Kabat, including SEQ ID NO: 51; and a heavy chain CDR3 as defined by Kabat or Chothia, including SEQ ID NO: 52; and a humanized mature light chain variable domain including the three Kabat light chain CDRs of SEQ ID NOs: 53-55.
- a humanized mature heavy variable domain including: a heavy chain CDR1 as defined by Kabat/Chothia Composite, including SEQ ID NO: 49; a heavy chain CDR2 as defined by Kabat, including SEQ ID NO: 51; and a heavy chain CDR3 as defined by Kabat or Chothia, including SEQ ID NO: 52; and a humanized mature light chain variable domain including the three Kabat light chain CDRs of SEQ ID NOs: 53-55.
- the humanized mature heavy variable domain includes a sequence that is at least 80% identical to any one of SEQ ID NOs: 4-23 and the humanized mature light chain variable domain includes a sequence that is at least 80% identical to any one of SEQ ID NOs: 27-48. In some embodiments, the humanized mature heavy variable domain includes a sequence that is at least 85% to any one of SEQ ID NOs: 4-23 and the humanized mature light chain variable region includes a sequence that is at least 85% identical to any one of SEQ ID NOs: 27-48.
- the humanized mature heavy variable domain includes a sequence that is at least 90% identical, to any one of SEQ ID NOs: 4-23 and the humanized mature light chain variable region includes a sequence that is at least 90% identical, to any one of SEQ ID NOs: 27-48.
- the humanized mature heavy variable domain includes a sequence that is at least 95% identical to any one of SEQ ID NOs: 4-23 and the humanized mature light chain variable domain includes a sequence that is at least 95% identical to any one of SEQ ID NOs: 27-48.
- At least one of the following positions in the humanized heavy chain variable domain is occupied by the amino acid as specified: 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.
- At least one of the following positions in the humanized heavy chain variable domain is occupied by the amino acid as specified: 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. In some embodiments, at least one of the following positions in the humanized heavy chain variable domain is occupied by the amino acid as specified: 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.
- 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 the amino acid as specified: 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.
- 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
- M83 is occupied by R.
- At least one of the following positions in the humanized light chain variable domain is occupied by the amino acid as specified: 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.
- At least one of the following positions in the humanized light chain variable domain is occupied by the amino acid as specified: V3 is occupied by Q or A100 is occupied by D or R. In some embodiments, at least one of the following positions in the humanized light chain variable domain is occupied by the amino acid as specified: 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. In some embodiments, 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 amino acid as specified: T80 is occupied by A or L92 is occupied by A.
- At least one of the following positions in the humanized heavy chain variable domain is occupied by the amino acid as specified: 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 the amino acid as specified: 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.
- the humanized antibody has increased thermal stability as compared to a reference antibody including a heavy chain variable domain including SEQ ID NO: 1 and a light chain variable domain including SEQ ID NO: 24.
- the antibody includes a heavy chain variable domain including: a heavy chain CDR1, as defined by Kabat/Chothia Composite, including SEQ ID NO: 49 or SEQ ID NO: 50; a heavy chain CDR2, as defined by Kabat, including SEQ ID NO: 51; a heavy chain CDR3, as defined by Kabat or Chothia, including SEQ ID NO: 52; a light chain CDR1, as defined by Kabat, including SEQ ID NO: 53; a light chain CDR2, as defined by Kabat, including Attorney Docket Ref.
- the heavy chain CDR1, as defined by Kabat/Chothia Composite includes SEQ ID NO: 49; the heavy chain CDR2, as defined by Kabat, includes SEQ ID NO: 51; the heavy chain CDR3, as defined by Kabat or Chothia, includes SEQ ID NO: 52; the light chain CDR1, as defined by Kabat, includes SEQ ID NO: 53; the light chain CDR2, as defined by Kabat, includes SEQ ID NO: 54; and the light chain CDR3, as defined by Kabat, includes SEQ ID NO: 55 or SEQ ID NO: 61.
- the heavy chain variable domain includes a sequence that is at least 95% identical to any one of SEQ ID NOs: 4-23. In some embodiments, the heavy chain variable domain includes a sequence that is at least 95% identical to any one of: SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 23. In some embodiments, the heavy chain variable domain includes a sequence that is at least 98% identical to any one of: SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 23. In some embodiments, the heavy chain variable domain includes a sequence of SEQ ID NO: 20. In some embodiments, the heavy chain variable domain includes a sequence of SEQ ID NO: 21. In some embodiments, the heavy chain variable domain includes a sequence of SEQ ID NO: 23.
- the light chain variable domain includes a sequence that is at least 95% identical to any one of: SEQ ID NOs: 27-48. In some embodiments, the light chain variable domain includes a sequence that is at least 95% identical to SEQ ID NO: 47 or SEQ ID NO: 48. In some embodiments, the light chain variable domain includes a sequence that is at least 98% identical to SEQ ID NO: 47 or SEQ ID NO: 48. In some embodiments, the light chain variable domain includes SEQ ID NO: 47. In some embodiments, the light chain variable domain includes SEQ ID NO: 48.
- the antibody includes a heavy chain variable domain and a light chain variable domain including: a heavy chain CDR1 including SEQ ID NO: 84; a heavy chain CDR2 including SEQ ID NO: 85; a heavy chain CDR3 including SEQ ID NO: 86; a light chain CDR1 including SEQ ID NO: 87; a light chain CDR2 including SEQ ID NO: 88; and a light chain CDR3 including SEQ ID NO: 89.
- the heavy chain variable domain comprises a sequence that is at least 95% identical to SEQ ID NO: 63.
- the heavy chain variable domain Attorney Docket Ref.
- the heavy chain variable domain comprises a sequence of SEQ ID NO: 63.
- the light chain variable domain comprises a sequence that is at least 95% identical to SEQ ID NO: 65.
- the light chain variable domain comprises a sequence that is at least 98% identical to SEQ ID NO: 65.
- the light chain variable domain comprises a sequence of SEQ ID NO: 65.
- the antibody includes a heavy chain variable domain and a light chain variable domain including: a heavy chain CDR1 including SEQ ID NO: 90; a heavy chain CDR2 including SEQ ID NO: 91; a heavy chain CDR3 including SEQ ID NO: 92; a light chain CDR1 including SEQ ID NO: 93; a light chain CDR2 including SEQ ID NO: 94; and a light chain CDR3 including SEQ ID NO: 95.
- the heavy chain variable domain comprises a sequence that is at least 95% identical to SEQ ID NO: 67.
- the heavy chain variable domain comprises a sequence that is at least 98% identical to SEQ ID NO: 67.
- the heavy chain variable domain comprises a sequence of SEQ ID NO: 67. In some embodiments, the light chain variable domain comprises a sequence that is at least 95% identical to SEQ ID NO: 69. In some embodiments, the light chain variable domain comprises a sequence that is at least 98% identical to SEQ ID NO: 69. In some embodiments, the light chain variable domain comprises a sequence of SEQ ID NO: 69.
- the antibody includes a heavy chain variable domain and a light chain variable domain including: a heavy chain CDR1 including SEQ ID NO: 96; a heavy chain CDR2 including SEQ ID NO: 97; a heavy chain CDR3 including SEQ ID NO: 98; a light chain CDR1 including SEQ ID NO: 99; a light chain CDR2 including SEQ ID NO: 100; and a light chain CDR3 including SEQ ID NO: 101.
- the heavy chain variable domain comprises a sequence that is at least 95% identical to SEQ ID NO: 71. In some embodiments, the heavy chain variable domain comprises a sequence that is at least 98% identical to SEQ ID NO: 71.
- the heavy chain variable domain comprises a sequence of SEQ ID NO: 71.
- the light chain variable domain comprises a sequence that is at least 95% identical to SEQ ID NO: 73.
- the light chain variable domain Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT comprises a sequence that is at least 98% identical to SEQ ID NO: 73.
- the light chain variable domain comprises a sequence of SEQ ID NO: 73.
- the antibody includes a heavy chain variable domain and a light chain variable domain including: a heavy chain CDR1 including SEQ ID NO: 102; a heavy chain CDR2 including SEQ ID NO: 103; a heavy chain CDR3 including SEQ ID NO: 104; a light chain CDR1 including SEQ ID NO: 105; a light chain CDR2 including SEQ ID NO: 106; and a light chain CDR3 including SEQ ID NO: 107.
- the heavy chain variable domain comprises a sequence that is at least 95% identical to SEQ ID NO: 75.
- the heavy chain variable domain comprises a sequence that is at least 98% identical to SEQ ID NO: 75.
- the heavy chain variable domain comprises a sequence of SEQ ID NO: 75. In some embodiments, the light chain variable domain comprises a sequence that is at least 95% identical to SEQ ID NO: 77. In some embodiments, the light chain variable domain comprises a sequence that is at least 98% identical to SEQ ID NO: 77. In some embodiments, the light chain variable domain comprises a sequence of SEQ ID NO: 77.
- the heavy chain variable domain comprises a sequence of SEQ ID NO: 79. In some embodiments, the light chain variable domain comprises a sequence that is at least 95% identical to SEQ ID NO: 81. In some embodiments, the light chain variable domain comprises a sequence that is at least 98% identical to SEQ ID NO: 81. In some embodiments, the light chain variable domain comprises a sequence of SEQ ID NO: 81. 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. Attorney Docket Ref.
- the antigen-binding antibody fragment is a Fab fragment, a Fab’ 2 fragment, or a single chain Fv.
- the antibody is an intact antibody.
- the antibody has a human IgG1 isotype.
- the heavy chain variable domain is fused to a heavy chain constant region and the light chain variable domain is fused to a light chain constant region.
- the heavy chain constant region is a mutant form of a natural human heavy chain constant region which has reduced binding to an Fc ⁇ receptor relative to the natural heavy chain constant region.
- the heavy chain constant region is of IgG1 isotype.
- the antibody has at least one mutation in a constant region. In some embodiments, the at least one mutation reduces complement fixation or activation by the constant region. In some embodiments, the at least one mutation is at one or more positions of: 241, 264, 265, 270, 296, 297, 318, 320, 322, 329, and 331 by EU numbering. In some embodiments, the antibody has an alanine at positions 318, 320, and 322 by EU numbering. In some embodiments, the antibody selectively binds to phosphorylated TDP-43 (e.g., phosphorylated human TDP-43).
- phosphorylated TDP-43 e.g., phosphorylated human TDP-43
- the antibody selectively binds to phosphorylated TDP-43 (e.g., phosphorylated human TDP-43) as 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 as compared to unphosphorylated TDP-43 (e.g., unphosphorylated human TDP-43).
- the antibody binds to phosphorylated TDP-43 (e.g., phosphorylated human TDP-43) with at least 1000-fold greater affinity as compared to unphosphorylated TDP-43 (e.g., unphosphorylated human TDP-43).
- phosphorylated TDP-43 e.g., phosphorylated human TDP-43 includes phosphorylation of at least one amino acid residue selected from S409 and S410.
- phosphorylated TDP-43 e.g., phosphorylated human TDP-43) includes phosphorylation of both S409 and S410.
- 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 include phosphorylated aggregates of TDP-43 (e.g., Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT phosphorylated aggregates of human TDP-43).
- phosphorylated aggregates of TDP-43 e.g., Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT phosphorylated aggregates of human TDP-43.
- the antibody does not substantially bind unphosphorylated TDP-43 (e.g., unphosphorylated human TDP-43).
- the cell-penetrating agent comprises a polypeptide sequence that is at least 95% identical to a sequence selected from any one of: 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:
- the cell-penetrating agent comprises a polypeptide sequence that is at least 98% identical to a sequence selected from any one of: 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
- the cell-penetrating agent comprises a polypeptide sequence selected from any one of: 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.
- the cell-penetrating agent comprises a polypeptide sequence that is at least 95% identical to a sequence selected from any one of 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: Attorney Docket Ref.
- the cell-penetrating agent comprises a polypeptide sequence that is at least 98% identical to a sequence selected from any one of 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: 17
- the cell-penetrating agent comprises a polypeptide sequence selected from any one of 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:
- the antibody is conjugated to a therapeutic, cytotoxic, cytostatic, immunomodulatory, neurotrophic, or neuroprotective agent.
- the heavy chain does not comprise a C-terminal lysine residue.
- pharmaceutical compositions including any of the cell- penetrating agents described herein and a pharmaceutically acceptable carrier.
- nucleic acids encoding at least a portion of any of the cell- penetrating agent described herein.
- the nucleic acids encode the heavy chain variable domain and/or the light chain variable domain of the antibody.
- the nucleic acid encodes for any of the CIMs described herein.
- vectors including a nucleic acid encoding a mature heavy chain variable domain and a light chain variable domain operably linked to one or more regulatory sequences to effect expression in a mammalian cell of any one of the cell-penetrating agents described herein.
- the one or more regulatory sequences include one or more of a enhancer, ribosome binding site, transcription termination signal, and promoter, optionally, where the promoter is a eukaryotic promoter.
- the nucleic acid is codon- optimized for expression in a host cell.
- host cells transformed with any of the vectors described herein.
- host cells including any of the nucleic acids described herein.
- methods of delivering the antibody that specifically binds to TDP-43 into a cell comprising contacting any of the cell-penetrating agents described herein with the cell, thereby resulting in the internalization into the cell of, at a minimum, an antigen-binding fragment of the antibody.
- the method further comprises transfer of, at a minimum, an antigen-binding fragment of the antibody, to the cytosol of the cell.
- Also provided herein are methods of binding an intracellular TDP-43 protein in a cell that include contacting the cell-penetrating agent of any one of claims 1-135 with the cell, thereby resulting in the internalization of and transfer to the cytosol of, at a minimum, an antigen-binding fragment of the antibody. Also provided herein are methods of binding an intracellular TDP-43 protein in a cell that include: contacting the cell-penetrating agent of any one of claims 1-135 with the cell, thereby resulting in the internalization of and transfer to the cytosol of, at a minimum, an antigen-binding fragment of the antibody; and binding, at a minimum, an antigen-binding fragment of the antibody, to the intracellular TDP-43 protein.
- the cell is a mammalian cell. In some embodiments, the cell is in vitro. In some embodiments, the cell is in a subject. Also provided herein are methods of inhibiting or reducing aggregation of TDP-43 (e.g., human TDP-43) in a subject having or at risk of developing a TDP-43-related disease, including administering to the subject an effective amount of any of the cell-penetrating agents described herein, thereby inhibiting or reducing aggregation of TDP-43 (e.g., human TDP-43) in the subject.
- TDP-43 e.g., human TDP-43
- TDP-43-related disease is amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD-TDP), primary lateral sclerosis, and progressive muscular atrophy, and Parkinson’s disease.
- the TDP-43-related disease is ALS.
- TDP-43 deposits e.g., human TDP-43 deposits
- methods of detecting TDP-43 deposits including administering to a subject any of the cell-penetrating agents described herein, and detecting the antibody bound to TDP-43 in the subject.
- the antibody is administered by intravenous injection into the body of the subject.
- methods of detecting TDP-43 in a sample obtained from a patient having or at risk of developing a TDP-43 related disease comprising contacting the same with any of the cell-penetrating agents described herein, and detecting the binding of the antibody to the TDP-43 in the sample.
- the antibody is labeled.
- the antibody is labeled with a fluorescent label, a paramagnetic label, or a radioactive label.
- the radioactive label is detected using positron emission tomography (PET) or single-photon emission computed tomography (SPECT).
- PET positron emission tomography
- SPECT single-photon emission computed tomography
- Figure 3 is a graph showing binding data of chimeric 13D3 antibody and humanized versions thereof to phosphorylated human TDP-43 peptide.
- Figure 4 is a graph showing binding data of chimeric 13D3 antibody and humanized versions thereof to phosphorylated human TDP-43 peptide.
- Figure 5 is a graph showing binding data of a humanized version of 13D3 antibody, hu13D2Hd5Ld2, to phosphorylated human TDP-43 peptide.
- Figures 6A-C show immunohistochemical images of frontotemporal dementia (“FTD”) brain tissue (Figs. 6A and 6B) and healthy brain tissue (Fig. 6C) stained with the 13D3 antibody.
- Fig. 6B is an inset of Fig.
- FIG. 6A showing co-localization of 13D3 antibody with phosphorylated human TDP-43 FTD-associated neuronal cytoplasmic aggregates.
- Figures 7A-C are immunohistochemical images of brain tissue from a rNLS8 dox- suppressible model of TDP-43 proteinopathy stained with the 13D3 antibody. The data show that the 13D3 antibody binds cytoplasmic aggregates in a rNLS8 dox-suppressible model of TDP-43 proteinopathy.
- Figures 8A-C show the results of HEK cells transfected with GFP-2a-TDP43 or GFP only. Figure 8A shows staining for pTDP-43, GFP, and nuclei in the transfected cells.
- Figure 8B is a graph showing cell count.
- Figure 8C shows immunohistochemical images of HEK cells that show the ability of antibodies 13D3, 13C13, and 2D4 to detect overexpressed mislocalized human TDP-43 in HEK cells.
- Figures 9A-B are graphs showing the percentage of CPA-positive cells following incubation with murine 13D3 (m13D3) CPAs (Figure 9A) and the number of CPA-positive spots per cell following incubation with m13D3 CPAs ( Figure 9B).
- Figures 10A-E are graphs showing the results of transfected HEK cells with different m13D3 CPAs including the number of foci per well area ( Figure 10A), the mean focus intensity Attorney Docket Ref.
- FIG. 11A-D are graphs showing the results of transfected HEK cells with different m13D3 CPAs including the number of foci per well area (Figure 11A), cell count ( Figure 11B), foci count (Figure 11C), and mean focus size (Figure 11D).
- Figures 12A-D are graphs showing the results of transfected HEK cells with different m13D3 CPAs including total foci area per well area (Figure 12A), cell count ( Figure 12B), pTDP-43 foci count (Figure 12C), and mean focus size (Figure 12D).
- Figure 13 are graphs showing percentage cell death with either untransfected HEK cells (left) or cells transfected with GFP-2a-TDP43 (right).
- Figures 14A-E are graphs showing internalization and colocalizationof phosphorylated cytoplasmic aggregates of TDP-43 in HEK cells.
- Figures 15A-D are graphs showing the results of HEK cells incubated with either m13D3 m-Lycotoxin [L17E] CPAs or m13D3 CMIP4 CPA including total foci area ( Figure 15A), cell count ( Figure 15B), p-TDP-43 foci number ( Figure 15C), and mean focus area (Figure 15D).
- Figures 16A-E are graphs showing the results of HEK cells transfected with either m13D3 CMIP4 CPA or untagged antibody in either acetate buffer or PBS, including foci per well area (Figure 16A), mean focus intensity (Figure 16B), cell count ( Figure 16C), number of p- TDP-43 foci ( Figure 16D), and mean focus area (Figure 16E).
- Figures 17A-B show the results of HEK cells transfected with GFP-2A-TDP43 and chimeric 13D3 CPAs including colocalization imaging ( Figure 17A) and a graph showing percentage of p-TDP-43 colocalized with GFP-2A-TDP43 and chimeric 13D3 CPAs ( Figure 17B).
- Figures 18A-D are graphs showing internalization data of anti-TDP-43 CPAs including, percentage of pTDP-43 colocalized with anti-TDP-43 CPAs (Figure 18A), the average number of 13D3 antibody spots per cell (Figure 18B), average spot size (Figure 18C), and average spot size (corrected for spot intensity) (Figure 18D).
- Figures 19A-D are graphs showing internalization and colocalization of phosphorylated cytoplasmic aggregates of pTDP-43 including percentage of pTDP-43 colocalized with Attorney Docket Ref.
- FIG. 20A-C are graphs showing internalization and colocalization of phosphorylated cytoplasmic aggregates of TPD-43 in glioblastoma cells including total foci area (Figure 20A), mean foci size ( Figure 20B), and total foci count ( Figure 20C).
- Figures 21A-B show internalization and colocalization of phosphorylated cytoplasmic aggregates of TDP-43 in primary rat cortical neuron cells.
- Figure 21A shows images of primary rat cortical neuron cells treated with various CPAs.
- Figure 21B is a graph showing total of foci area per well.
- Figures 22A-F are graphs showing the results of transfected primary rat cortical neuron cells including the total number of 13D3 spots per cell (Figure 22A), total area of 13D3 spots per well (Figure 22B), mean spot size (Figure 22C), spot integrated intensity (Figure 22D), percentage of 13D3 colocalized with Early Endosome Antigen 1 (EEA1) (Figure 22E), and cell count (Figure 22F).
- DETAILED DESCRIPTION This disclosure provides systems and methods for the delivery of antibodies into a cell.
- cell-penetrating agents that comprise a cell internalization module and an antibody that specifically binds TDP-43 (e.g., human TDP-43) or aggregates of TDP-43 (e.g., aggregates of human TDP-43, including phosphorylated aggregates of TDP-43).
- TDP-43 e.g., human TDP-43
- aggregates of TDP-43 e.g., aggregates of human TDP-43, including phosphorylated aggregates of TDP-43
- pharmaceutical compositions including these cell-penetrating agents and a pharmaceutically acceptable carrier, nucleic acids and/or vectors encoding these cell-penetrating agents, and host cells expressing the aforementioned nucleic acids and/or vectors.
- antibody includes intact antibodies and antigen-binding fragments thereof.
- fragments compete with the intact antibody from which they were derived for specific binding to the target including separate heavy chains, light chains Fab, Fab’, F(ab’)2, F(ab)c, Dabs, nanobodies, and Fv. Fragments can be produced by recombinant DNA techniques, or by Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT enzymatic or chemical separation of intact immunoglobulins.
- antibody also includes a bispecific or multispecific antibody and/or a humanized antibody.
- a bispecific or bifunctional or multifunctional antibody is an artificial hybrid antibody having two or more different heavy/light chain pairs and two or more different binding sites (see, e.g., Songsivilai and Lachmann, Clin. Exp. Immunol., 79:315-321 (1990); Kostelny et al., J. Immunol., 148:1547-53 (1992)).
- a “Cell-Penetrating Agent” also referred to herein as a “CPA” refers to an agent (e.g., a molecule and/or molecular complex) capable of entering a cell (e.g., a mammalian cell in vitro and/or in vivo).
- the CPA enters the cell and is transferred to the cytosol following internalization by the cell.
- the CPA comprises a Cell Internalizing Module (CIM) that facilitates the internalization of the CPA by the cell.
- the CPA comprises a CIM that facilitates the internalization of the CPA the transfer of the CPA the cytosol.
- the CPA further comprises an anti- TDP-43 antibody that is linked (e.g., covalently or non-covalently) to the CIM.
- 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).
- 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 in relevant conditions (e.g., blood plasma).
- the CPA further comprises a linker connecting the CIM to the anti-TDP-43 antibody.
- linkers can be cleavable or non-cleavable and/or can connect the CIM to the anti-TDP-43 antibody covalently or non- covalently.
- the CPA comprising the anti-TDP-43 antibody has enhanced cell penetration compared to a reference anti-TDP-43 antibody that is not part of a CPA.
- the CPA comprises two or more anti-TDP-43 antibodies and/or two or more CIMs (e.g., the CPA comprising a dendrimer linked to a plurality of CIMs and/or anti-TDP-43 antibodies).
- CIMs e.g., the CPA comprising a dendrimer linked to a plurality of CIMs and/or anti-TDP-43 antibodies.
- a “Cell Internalizing Module” refers to a portion of the CPA that facilitates the internalization of the CPA (and by extension, the anti- TDP-43 antibody) by the cell.
- the CIM may utilize one or more cellular internalization processes, including both active and passive cellular internalization, to effect internalization. Exemplary processes include, without limitation, endocytosis (e.g., Receptor Attorney Docket Ref.
- the CIM comprises, for example, a Cell-Membrane Internalizing Peptide (CMIP), a small molecule ligand (e.g., vitamin, fatty acids, integrin binding ligand, etc.), a portion of an antibody (e.g., an scFv portion that binds an internalizing cell surface target), or a decoy receptor ligand (e.g., a cytokine or derivative thereof).
- CMIP Cell-Membrane Internalizing Peptide
- a small molecule ligand e.g., vitamin, fatty acids, integrin binding ligand, etc.
- a portion of an antibody e.g., an scFv portion that binds an internalizing cell surface target
- a decoy receptor ligand e.g., a cytokine or derivative thereof.
- the CIM comprises a Cell- Membrane Internalizing Peptide (CMIP).
- CMIP Cell- Membrane Internalizing Peptide
- CMIP Cell Membrane Internalizing Peptide
- the CMIP may utilize one or more cellular internalization processes, including both active and passive cellular internalization, to effect internalization. Exemplary processes include, without limitation, endocytosis and membrane translocation.
- the CMIP interacts with the cell membrane, and/or a cell surface antigen, leading to internalization of the CPA or a portion thereof (e.g., a portion comprising the anti-TDP-43 antibody or a functional portion thereof) via an endocytic vesicle.
- the CMIP further facilitates the transfer of the CPA or portion thereof out of the endocytic vesicle and into the cytoplasm of the cell.
- the CMIP further interacts with the membrane of the endocytic vesicle, leading to vesicle rupture and endosomal escape of the CPA or portion thereof into the cytosol.
- the CMIP facilitates both the internalization of the anti-TDP-43 antibody or functional portion thereof into the cell, as well as transfer of the anti-TDP-43 antibody or functional portion thereof into the cytosol.
- CMIPs include, for example, cationic peptides (including, for example, M-lycotoxin, TAT peptides, pentetratin, and polyarginine peptides, as well as derivatives thereof), amphipathic peptides (including, for example, MPG peptides, Pep-1 peptides, transportan peptides, as well as derivative thereof), and proline-rich peptides (including, for example, Bac7 peptides and derivatives thereof).
- CMIPs include cell-penetrating peptides and derivatives thereof, including, for example, those described in I. Ruseska and A.
- CMIPs Non-limiting features and examples of CMIPs are described herein.
- epitope refers to a site on an antigen to which an antibody binds.
- An epitope can be formed from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of one or more proteins.
- Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding (also known as conformational epitopes) are typically lost on treatment with denaturing solvents.
- An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation. Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols, in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed. (1996).
- Antibodies that recognize the same or overlapping epitopes can be identified in a simple immunoassay showing the ability of one antibody to compete with the binding of another antibody to a target antigen.
- the epitope of an antibody can also be defined X-ray crystallography of the antibody bound to its antigen to identify contact residues.
- two antibodies have the same epitope if all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other.
- Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other.
- humanized immunoglobulin refers to an immunoglobulin or antibody that includes at least one humanized immunoglobulin or antibody chain (i.e., at least one humanized light or heavy chain).
- humanized immunoglobulin chain or “humanized antibody chain” (i.e., a “humanized immunoglobulin light chain” or “humanized immunoglobulin heavy chain”) refers to an immunoglobulin or antibody chain (i.e., a light or heavy chain, respectively) having a variable region that includes a variable framework region substantially from a human immunoglobulin or antibody and complementarity determining regions (CDRs) (e.g., at least one CDR, preferably two CDRs, more preferably three CDRs) substantially from a non-human immunoglobulin or antibody, and further includes constant regions (e.g., at least one constant region or portion thereof, in the case of a light chain, and preferably three constant regions in the case of a heavy chain).
- CDRs complementarity determining regions
- variable region refers to a variable region that includes a variable framework region substantially from a human immunoglobulin or antibody and complementarity determining regions (CDRs) substantially from a non-human immunoglobulin or antibody.
- CDRs complementarity determining regions
- test antibody competes with a reference antibody if an excess of a test antibody (e.g., at least 2x, 5x, l0x, 20x or l00x) inhibits binding of the reference antibody by at least 50% as measured in a competitive binding assay. Some test antibodies inhibit binding of the reference antibody by at least 75%, 90%, or 99%.
- Antibodies identified by competition assay include antibodies binding to the same epitope as the reference antibody and antibodies binding to an adjacent epitope sufficiently proximal to the epitope bound by the reference antibody for steric hindrance to occur.
- a “linker” refers a chemical moiety that does not have catalytic or therapeutic activity in a mammalian cell that is used to covalently link two different functional molecules (e.g., a cell internalization module and an anti-TDP-43 antibody).
- a linker can be a peptide of about 3 amino acids to about 25 amino acids (e.g., about 3 amino acids to about 20, or about 3 amino acids to about 12 amino acids).
- a linker can be a bond (e.g., an amide bond, an ester bond, an ether bond, and a disulfide bond).
- a linker can comprise a pair of affinity domains (e.g., a first domain of the pair of affinity domains can be interleukin-15 and a second domain of the pair of affinity domains can be a sushi domain of interleukin-15 receptor alpha).
- a linker is a glycine residue followed by a serine residue (GS).
- a linker is three consecutive glycine residues (e.g., GGG).
- a linker is any one of SEQ ID NOs: 194-199.
- pharmaceutically acceptable means that the carrier, diluent, excipient, or auxiliary is compatible with the other ingredients of the formulation and not substantially deleterious to the recipient thereof.
- M-lycotoxin derivative is a peptide comprising three or more amino acids (naturally-occurring or non-naturally-occurring) that was designed based on a starting M- lycotoxin peptide.
- the “M-lycotoxin derivative” is a polypeptide having, Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT e.g., between 60% and 99% sequence homology to wild-type M-lycotoxin. Additional non- limiting aspects and examples of M-lycotoxin derivatives are described herein.
- the term “macromolecule” refers to a molecule having either a molecular weight of at least 5 kDa and/or a hydrodynamic radius of at least 1.0 nm.
- the term “macromolecules” includes biomolecules, organic polymers, and organometallic complexes.
- a “spacer” or “spacer region” refers to an amino acid that does not have catalytic or therapeutic activity in a mammalian cell.
- a spacer can be a peptide of 1 amino acid 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).
- one or more spacer regions are included in the cell internalizing module.
- a spacer region can separate amino acids within a cell internalizing module, e.g., a spacer can be disposed after the first amino acid of a CIM.
- a spacer can be disposed before the final amino acid of a CIM.
- a CIM can have two or more spacer regions (e.g., two spacer regions, three space sequences, four spacer regions, five spacer regions or more).
- a spacer region is a single glycine residue.
- a spacer region is a pair of glycine residues.
- a spacer region is three glycine residues.
- a spacer region is four glycine residues. In some examples, a spacer region is four glycine residues followed by a serine residue. In some examples, a spacer region is any one of SEQ ID NOs: 200-203.
- TDP-43-related disease means a disease or disorder that is characterized and/or mediated at least in part, either directly or indirectly, by formation of TDP-43 aggregates and/or mislocalization of TDP-43. Non-limiting examples of TDP-43-related diseases are described herein.
- patient or “subject” includes human and other mammalian subjects (e.g., human) that receive either prophylactic or therapeutic treatment.
- An individual is at increased risk of a disease if the subject has at least one known risk factor (e.g., genetic, biochemical, family history, and situational exposure) placing individuals with that risk factor at a statistically significant greater risk of developing the disease than individuals without the risk factor.
- the term “biological sample” refers to a sample of biological material within or obtainable from a biological source, for example a human or mammalian subject. Such samples Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT can be organs, organelles, tissues, sections of tissues, bodily fluids, peripheral blood, blood plasma, blood serum, cells, molecules such as proteins and peptides, and any parts or combinations derived therefrom.
- the term biological sample can also encompass any material derived by processing the sample. Derived material can include cells or their progeny. Processing of the biological sample may involve one or more of filtration, distillation, extraction, concentration, fixation, inactivation of interfering components, and the like.
- control sample refers to a biological sample not known or suspected to include TDP-43 affected regions, or at least not known or suspect to include diseased regions of a given type. Control samples can be obtained from individuals not afflicted with the TDP-43- related disease. Alternatively, control samples can be obtained from patients afflicted with the TDP-43-related disease. Such samples can be obtained at the same time as a biological sample thought to comprise the TDP-43-related disease or on a different occasion.
- a biological sample and a control sample can both be obtained from the same tissue.
- control samples consist essentially or entirely of normal, healthy regions and can be used in comparison to a biological sample thought to comprise TDP-43-related disease-affected regions.
- the tissue in the control sample is the same type as the tissue in the biological sample.
- the TDP-43-related disease-affected cells thought to be in the biological sample arise from the same cell type (e.g., neurons or glia) as the type of cells in the control sample.
- 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 influencing chain orientation): Gly, Pro; and Group VI (aromatic side chains): Trp, Tyr, Phe. Conservative substitutions involve substitutions between amino acids in the same class. Non-conservative substitutions constitute exchanging a member of one of these classes for a member of another.
- Percentage sequence identities are determined with antibody sequences maximally aligned by the Kabat numbering convention. After alignment, if a subject antibody region (e.g., the entire mature variable region of a heavy or light chain) is being compared with the same region of a reference antibody, the percentage sequence identity between the subject and reference antibody regions is the number of positions occupied by the same amino acid in both Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT the subject and reference antibody region divided by the total number of aligned positions of the two regions, with gaps not counted, multiplied by 100 to convert to percentage.
- the term “about” encompasses insubstantial variations, such as values within a standard margin of error of measurement (e.g., SEM) of a stated value.
- the phrase “substantially from a human immunoglobulin or antibody” means that, when aligned to a human immunoglobulin or antibody amino sequence for comparison purposes, the region shares at least 80-90%, preferably 90-95%, more preferably 95-99% identity (i.e., local sequence identity) with the human framework or constant region sequence, allowing, for example, for conservative substitutions, consensus sequence substitutions, germline substitutions, backmutations, and the like.
- a humanized antibody or chain The introduction of conservative substitutions, consensus sequence substitutions, germline substitutions, backmutations, and the like, is often referred to as “optimization” of a humanized antibody or chain.
- the phrase “substantially from a non-human immunoglobulin or antibody” or “substantially non-human” means having an immunoglobulin or antibody sequence 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.
- TDP-43 As described herein TDP-43 is a predominantly nuclear protein that is involved in RNA splicing, trafficking, stabilization, and ultimately regulation of gene expression.
- TDP-43 is a multi-domain heterogeneous ribonucleoprotein (hnRNP). Proper function of TDP-43 is essential for the regulation of hundreds of mRNA transcripts to which it binds.
- TDP-43 is also involved in different mechanisms of RNA processing and transport.
- Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT For example, TDP-43 is an inhibitor of cryptic exon inclusion and regulates the alternative polyadenylation of >1,000 genes.
- TDP-43 can form ribonucleoprotein granules in different cell types, including Cajal bodies and paraspeckles in the nucleus and is recruited to mRNA transport granules in neurons.
- Various neurodegenerative diseases are associated with cytoplasmic aggregates of TDP-43 (e.g., TDP-43-related diseases) as described herein.
- reference to TDP-43 means a natural human form of TDP-43, including any isoforms and/or post-translational modifications (e.g., phosphorylation, glycosylation, and/or acetylation).
- TDP-43 The amino acid sequence of human TDP-43 is shown below (SEQ ID NO: 82): MSEYIRVTEDENDEPIEIPSEDDGTVLLSTVTAQFPGACGLRYRNPVSQCMRGVRLVEGI LHAPDAGWGNLVYVVNYPKDNKRKMDETDASSAVKVKRAVQKTSDLIVLGLPWKTT EQDLKEYFSTFGEVLMVQVKKDLKTGHSKGFGFVRFTEYETQVKVMSQRHMIDGRWC DCKLPNSKQSQDEPLRSRKVFVGRCTEDMTEDELREFFSQYGDVMDVFIPKPFRAFAFV TFADDQIAQSLCGEDLIIKGISVHISNAEPKHNSNRQLERSGRFGGNPGGFGNQGGFGNS RGGGAGLGNNQGSNMGGGMNFGAFSINPAMMAAAQAALQSSWGMMGMLASQQNQS GPSGNNQNQGNMQREPNQAFGSGNNSYSGSNSGAAIGWGSASNAGS
- 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, e.g., Gruijs da Silva, L.A., et al., Disease-linked TDP-43 hyperphosphorylation suppresses TDP-43 condensation and aggregation, The EMBO Journal, 41: e108443 (2022)).
- reference to TDP-43, or its fragments includes the natural human amino acid sequences including isoforms, mutants, and allelic variants thereof.
- the ability of an antibody or antigen-binding antibody fragment to bind to TDP-43 can be determined using, e.g., surface plasmon resonance.
- the present disclosure provides cell-penetrating agents (as described herein) that include a cell internalization moiety and an antibody or an antigen-binding fragment thereof that specifically binds to TDP-43 (e.g., human TDP-43).
- the antibodies or Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT antigen-binding antibody fragments described herein specifically bind to phosphorylated TDP-43 (e.g., phosphorylated human TDP-43).
- the antibodies or antigen-binding antibody fragments described herein specifically bind phosphorylated TDP-43 (e.g., phosphorylated human TDP-43), where one or both of serine at position 409 and/or 410 of SEQ ID NO: 82 is phosphorylated.
- the antibodies or antigen-binding antibody fragments bind a 23 amino acid peptide (“TDP-43 (pS409/pS410)”) comprising amino acids from position 392 to position 414 of SEQ ID NO: 82, where the serines at positions 409 and 410 are phosphorylated.
- the present disclosure relates to cell-penetrating agents that include a cell internalization moiety and an antibody that specifically binds to human TDP-43, compositions comprising these cell-penetrating agents, and methods of using these cell-penetrating agents to treat TDP-43 related diseases, including Amyotrophic Lateral Sclerosis (ALS).
- ALS Amyotrophic Lateral Sclerosis
- references to the anti-TDP-43 CPA and/or anti-TDP- 43 antibody will be made in the context of cell internalization and/or intracellular function, but one of ordinary skill will understand such references to include intact anti-TDP-43 CPA and/or anti-TDP-43 antibody, chemically modified (e.g., oxidized, reduced) anti-TDP-43 CPA and/or anti-TDP-43 antibody, as well as partially degraded functional fragments thereof (intact anti- TDP-43 CPA, CPA fragment, intact anti-TDP-43 antibody, and/or anti-TDP-43 antibody fragment, etc.).
- references to internalization of, cytosolic transfer of, and target engagement by either the anti-TDP-43 CPA or the anti-TDP-43 antibody will include references to intact anti-TDP-43 CPA and/or anti-TDP-43 antibody, chemically modified anti-TDP-43 CPA and/or anti-TDP-43 antibody, and partially degraded functional fragments thereof.
- methods of the present disclosure may refer to internalization of and/or transfer to the cytosol of an anti-TDP-43 antibody, but it will be understood that such a reference encompasses the transfer of an intact anti-TDP-43 CPA and/or a functional fragment of the anti-TDP-43 CPA comprising the anti-TDP-43 antibody or a portion thereof, as well as chemically modified derivatives thereof.
- Cell penetrating agents of the present disclosure may utilize one or more of several biochemical processes to achieve internalization of the anti-TDP-43 antibody.
- the anti-TDP-43 CPAs of the present disclosure may utilize passive internalization, active internalization, or a combination thereof.
- the anti- Attorney Docket Ref the anti- Attorney Docket Ref.
- TDP-43 CPA utilizes active internalization (e.g., endocytosis) to achieve intracellular delivery of the anti-TDP-43 antibody.
- the anti-TDP-43 CPA is internalized into the cell via endocytosis. Following internalization, the anti-TDP-43 CPA achieves endosomal escape. In such embodiments, the anti-TDP-43 CPA is thereby transferred to the cytosol, where the anti- TDP-43 antibody can bind to TDP-43 (e.g., phospho-TDP-43).
- endosomal escape of the anti-TDP-43 CPA is achieved by interaction of the CIM with the membrane of the endosome, thereby leading to disruption of the endosomal membrane.
- the CIM may utilize one or more methods to achieve endosomal escape.
- the CIM may comprise a cationic portion (e.g., positively charged amino acids, a cationic polymer and/or oligomer, cationic lipid).
- the cationic portion can interact with the negatively charged phospholipids that comprise the endosomal membrane, thereby disrupting the endosomal membrane, and achieving endosomal escape of the anti-TDP-43 CPA.
- Anti-TDP-43 CPAs of the present disclosure may also comprise a CIM having an amphiphilic portion (e.g., an amphiphilic peptide).
- the amphiphilic portion can interact with the endosomal membrane via hydrophobic interactions with the membrane lipids, thereby disrupting the endosomal membrane, and achieving endosomal escape of the anti-TDP-43 CPA.
- the anti-TDP-43 antibody is then transferred to the cytosol, where it can bind to TDP-43 (e.g., phospho-TDP-43). Further examples of internalization mechanisms that can be utilized by CPAs are described in detail herein.
- cell-penetrating agents that include: (i) a cell internalizing module (CIM) and (ii) an antibody that specifically binds to TDP-43 (e.g., human TDP-43).
- CIM cell internalizing module
- TDP-43 e.g., human TDP-43
- CIM cell internalizing module
- an antibody that specifically binds to TDP-43 e.g., human TDP-43
- TDP-43 e.g., human TDP-43
- Various aspects of cell-penetrating agents of the present disclosure are described below, including examples of Cell Internalizing Moieties, anti-TDP-43 antibodies, and/or optional linkers.
- One of ordinary skill will understand how to combine the various CIM, anti-TDP-43 antibodies, and optional linkers disclosed herein. Such examples are merely illustrative of the scope of the present disclosure and are non-limiting.
- Cell Internalizing Modules Attorney Docket Ref.
- Exemplary cell membrane internalizing peptides include naturally occurring peptides, and derivatives thereof, as well as synthetic peptides.
- Non-limiting examples of CMIPs include M-lycotoxin and derivatives thereof, TAT and derivatives thereof, 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.
- a CIM is a non-peptide moiety (e.g., a ligand) that is internalized by a cell (e.g., a mammalian cell).
- a ligand can induce receptor-mediated internalization of an anti-TDP-43 antibody.
- ligand internalization is a receptor- mediated endocytic process in which cells intake extracellular molecules (including therapeutics) if the ligand binds to its cognate receptor protein on the cell’s surface.
- Receptor-mediated internalization also includes transcytosis. CIMs can effectuate the internalization of anti-TDP-43 antibody into a mammalian cell.
- endocytosis In general, the process of cellular internalization is broadly classified as endocytosis. Typically, endocytosis pathways can be subdivided into two broader categories phagocytosis and pinocytosis. During pinocytosis the plasma membrane absorbs solutes while during phagocytosis the cell internalizes much larger vesicles. Pinocytosis is generally further subdivided into macropinocytosis, clathrin-dependent endocytosis (e.g., receptor-mediated endocytosis), caveolin-dependent endocytosis, and clathrin/caveolin-independent endocytosis. (See e.g., Marsh, M.
- the cell membrane e.g., plasma membrane
- the cell membrane includes clathrin pits which protrude from the cell membrane to form small vesicles called clathrin-coated vesicles.
- These clathrin-coated vesicles contain the receptors and the bound macromolecules, i.e., ligands. Then the clathrin-coated vesicles fuse with the early endosomes (vesicles consisting of tubular extensions residing at the periphery of the cell).
- the endosomes have an acidic environment (pH 6.0-6.2) that facilitates the dissociation of receptors from the ligands.
- CMIPs Peptide based CIMs
- endocytosis e.g., energy- dependent internalization
- direct penetration e.g., translocation
- various mechanisms have been described including the carpet-like model (membrane destabilization) and the pore formation model (barrel-stave).
- CMIPs can interact with negatively charged membrane components such as the phospholipid bilayer, followed by destabilization of the membrane, and crossing of the CMIP and anti-TDP-43 antibody through the lipid bilayer.
- membrane components such as the phospholipid bilayer
- anti-TDP-43 antibody typically need to escape the endosomal pathway.
- the endocytic pathway of mammalian cells consists of distinct membrane compartments, which internalize molecules (i.e., cell penetrating agents) from the plasma membrane and recycle membrane-bound receptors back to the surface or sort internalized molecules to various degradation pathways.
- the main components of the endocytic pathway include early endosomes which are the first compartment of the endocytic pathway. Early endosomes are usually located in the periphery of the cell and receive most types of vesicles coming from the cell surface. They have a characteristic tubulo-vesicular structure and a mildly acidic pH.
- Early endosomes are principally sorting organelles where many endocytosed ligands dissociate from their receptors in the acid pH of the compartment and are recycled to the cell Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT surface. Early endosomes also sort into transcytotic pathway to later compartments (e.g., late endosomes or lysosomes) via transvesicular compartments.
- Late endosomes generally receive endocytosed material en route to lysosomes, usually from early endosomes in the endocytic pathway, from trans-Golgi network (TGN) in the biosynthetic pathway, and from phagosomes in the phagocytic pathway. They are acidic (approx. pH 5.5) and are generally thought to mediate a final sorting prior the delivery of material to lysosomes. Lysosomes are the last compartment of the endocytic pathway. Lysosomes break down cellular waste products, fats, carbohydrates, proteins, and other macromolecules into simple compounds which are returned to the cytoplasm as new cell-building materials.
- TGN trans-Golgi network
- Lysosomes include many different types of hydrolytic enzymes which function in an acidic environment (e.g., pH of approximately 4.8).
- the CIM (as defined herein) includes a Cell Membrane Internalizing Peptide (e.g., CMIP4, SEQ ID NO: 176).
- the CIM includes a wild-type M-lycotoxin peptide (SEQ ID NO: 182).
- the CIM includes an M-lycotoxin derivative (e.g., SEQ ID NO: 183).
- the CIM includes a Penetain amino acid sequence or a derivative thereof (e.g., SEQ ID NO: 187).
- the CIM includes a Pepth amino acid sequence or a derivative thereof (e.g., SEQ ID NO: 184).
- the CIM includes a polyarginine amino acid sequence (e.g., SEQ ID NO: 190, SEQ ID NO: 191, and/or SEQ ID NO: 192).
- the CIM includes more than one polyarginine amino acid sequence (e.g., 2, 3, 4, 5 or more polyarginine amino acid sequences).
- the CIM includes three polyarginine amino acid sequences.
- the CIM includes 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 includes more than one TAT amino acid sequence or derivatives thereof (e.g., 2, 3, 4, 5 or more TAT amino acid sequences or derivatives thereof). In some embodiments, the CIM includes three TAT amino acid sequences. In some embodiments, the CIM includes a macrocycle. Generally, macrocycles are molecules and ions containing a ring of twelve or more atoms. Classic examples include the crown ethers, calixarenes, porphyrins, and cyclodextrins.
- the macrocycle is formed by a covalent bond between two amino acid residues of the CIM.
- the macrocycle is formed by a disulfide bond between two cysteine residues of the CIM.
- the CIM includes one or more histidine residues.
- the CIM includes a polypeptide having an amino acid sequence selected from any one of SEQ ID NOs: 176-193.
- the CIM is a polypeptide having an amino acid sequence selected from any one of SEQ ID NOs: 176-193.
- the CIM is covalently linked to the antibody or antigen-binding antibody fragment thereof. In some embodiments, the CIM is non-covalently linked to the antibody or antigen-binding fragment antibody thereof. In some embodiments, the cell- penetrating agent includes a linker connecting 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 enzymatic- cleavable linker, etc.). In some embodiments, the linker is a non-cleavable linker.
- the linker includes a polypeptide. In some embodiments, the linker includes one or more glycine residues (e.g., 2, 3, 4, 5 or more glycine residues). In some examples, a linker is a glycine residue followed by a serine residue (GS). In some embodiments, the linker includes a polypeptide comprising an amino acid sequence selected from any one of SEQ ID NOs: 194- 199. In some embodiments, the linker is a polypeptide having an amino acid sequence selected from any one of SEQ ID NOs: 194-199. In some embodiments, the antibody or antigen-binding antibody fragment of the cell- penetrating agent is linked to the C-terminus of the CIM.
- the linker includes one or more glycine residues (e.g., 2, 3, 4, 5 or more glycine residues). In some examples, a linker is a glycine residue followed by a serine residue (GS). In some embodiments, the linker includes a poly
- the antibody or antigen-binding antibody fragment of the cell-penetrating agent is linked to the N-terminus of the CIM. In some embodiments, the antibody or antigen-binding antibody fragment of the cell- penetrating agent is linked to the C-terminus of the heavy chain of the CIM. In some embodiments, the antibody or antigen-binding antibody fragment of the cell-penetrating agent is linked to the N-terminus of the heavy chain of the CIM. In some embodiments, the antibody or antigen-binding antibody fragment of the cell-penetrating agent is linked to the C-terminus of the light chain of the CIM.
- the antibody or antigen-binding antibody fragment of the cell-penetrating agent is linked to the N-terminus of the light chain of the CIM.
- the CIM includes one or more spacer regions. In some embodiments, the CIM does not include a spacer region.
- CIMs comprising a Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT polypeptide amino acid sequence selected from: SEQ ID NO: 176-184, SEQ ID NO: 192, and SEQ ID NO: 193 do not include a spacer region.
- the CIM includes one or more spacer regions.
- At least one of the one or more spacer regions includes one or more amino acid residues.
- a spacer refers to an amino acid that does not have catalytic or therapeutic activity in a mammalian cell.
- a spacer can be a peptide of 1 amino acid 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).
- spacer regions are included in the cell internalizing module sequences.
- a spacer region can separate amino acids within a cell internalizing module (CIM), e.g., a spacer can be disposed after the first amino acid of a CIM.
- CIM cell internalizing module
- a spacer can be disposed before the final amino acid of a CIM.
- a CIM can have one or more spacer regions (e.g., two spacer regions, three space sequences, four spacer regions, five spacer regions or more).
- a spacer region is a single glycine residue.
- a spacer region is a pair of glycine residues.
- a spacer region is three glycine residues.
- a spacer region is four glycine residues.
- a spacer region is four glycine residues followed by a serine residue.
- the anti-TDP-43 cell penetrating agents further comprise a linker connecting the CIM to the anti-TDP-43 antibody.
- Linkers of the present disclosure may connect the CIM to the anti-TDP-43 antibody via either a covalent linkage or a non-covalent linkage.
- the CIM is covalently linked to the anti-TDP-43 antibody via a linker (i.e., a covalent linker).
- the CIM is non-covalently linked to the anti-TDP-43 antibody via the 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 Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT 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. In some embodiments, the linker is a non-covalent linker.
- a non-covalent linkage can be achieved using an affinity pair that interact strongly in a noncovalent manner (e.g., by hydrogen bonding, ionic bonding, van Der Waals interactions, or any combination thereof).
- an affinity pair that interact strongly in a noncovalent manner
- biotin and a biotin-binding agent e.g., streptavidin
- an affinity pair e.g., streptavidin
- biotin and a biotin-binding agent e.g., streptavidin
- a non-covalent linkage can be achieved between the CIM and the anti-TDP-43 antibody.
- a linker comprises a pair of affinity domains (e.g., a first domain of the pair of affinity domains can be interleukin-15 and a second domain of the pair of affinity domains can be a sushi domain of interleukin-15 receptor alpha).
- the linker is a covalent linker.
- the covalent linker comprises an organic linker (e.g., an alkylene chain, a polyethylene glycol chain, a polyacrylamide, a polyacrylic acid, a polyvinyl alcohol, or a polyethyleneimine chain).
- the covalent linker comprises an unsubstituted or substituted alkylene chain (including, for example, a polyvinyl alcohol chain, a polyacrylamide chain, or a 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 incorporation of two or more CIMs and/or anti-TDP-43 antibodies into a CPA (e.g., a dendrimer linker structure).
- a CPA e.g., a dendrimer linker structure
- the linker comprises an amino acid residue. In some embodiments, the linker comprises a polypeptide. In some exemplary embodiments, a linker can 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, a linker can 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, a linker can 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).
- a linker can be a peptide of about 1 amino acid to about 5 amino acids (e.g., about 1 amino acid to about 4, or Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT about 1 amino acid to about 3 amino acids).
- a linker can 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).
- a linker can 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).
- the linker comprises a glycine residue. 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.
- the linker comprises an amino acid sequence of GGGGS. In some embodiments, the linker comprises an amino acid sequence of GGGSGGGS.
- Anti-TDP-43 Antibodies Cell-penetrating agents and methods of the present disclosure facilitate the cellular internalization and/or cytosolic release of a wide range of anti-TDP-43 antibodies.
- the anti-TDP-43 antibody is a humanized antibody, a chimeric antibody, or a veneered antibody (as described herein).
- Complementarity determining regions (“CDRs”) can be defined by different systems. For example, the CDRs described herein can be selected from the group of Kabat, Chothia, Kabat/Chothia Composite, AbM, and Contact.
- the antibody of the cell-penetrating agent includes a humanized mature heavy variable domain including: a heavy chain CDR1 as defined by Kabat/Chothia Composite, including SEQ ID NO: 49; a heavy chain CDR2 as defined by Kabat, including SEQ ID NO: 51; and a heavy chain CDR3 as defined by Kabat or Chothia including SEQ ID NO: 52; and a humanized mature light chain variable domain including the three Kabat light chain CDRs of SEQ ID NOs: 53-55.
- the humanized mature heavy variable domain of the antibody includes a sequence that is at least 80% identical to any one of SEQ ID NOs: 4-23. In some Attorney Docket Ref.
- the humanized mature light chain variable domain includes a sequence that is at least 80% identical to any one of SEQ ID NOs: 27-48.
- the antibody or antigen-binding fragment thereof includes a humanized mature heavy variable domain including a sequence that is at least 80% identical to any one of SEQ ID NOs: 4-23 and a humanized mature light chain variable domain including a sequence that is at least 80% identical to any one of SEQ ID NOs: 27-48.
- the humanized mature heavy variable domain of the antibody includes a sequence that is at least 85% to any one of SEQ ID NOs: 4-23.
- the humanized mature light chain variable region of the antibody includes a sequence that is at least 85% identical to any one of SEQ ID NOs: 27-48.
- the antibody or antigen-binding fragment thereof includes a humanized mature heavy variable domain including a sequence that is at least 85% identical to any one of SEQ ID NOs: 4-23 and a humanized mature light chain variable domain including a sequence that is at least 85% identical to any one of SEQ ID NOs: 27-48.
- the humanized mature heavy variable domain of the antibody includes a sequence that is at least 90% identical, to any one of SEQ ID NOs: 4-23.
- a humanized mature light chain variable domain of the antibody includes a sequence that is at least 90% identical, to any one of SEQ ID NOs: 27-48.
- the antibody or antigen-binding fragment thereof includes a humanized mature heavy variable domain comprising a sequence that is at least 90% identical to any one of SEQ ID NOs: 4-23 and a humanized mature light chain variable domain comprising a sequence that is at least 90% identical to any one of SEQ ID NOs: 27-48.
- the humanized mature heavy variable domain of the antibody includes a sequence that is at least 95% identical to any one of SEQ ID NOs: 4-23.
- the humanized mature light chain variable domain of the antibody includes a sequence that is at least 95% identical to any one of SEQ ID NOs: 27-48.
- the antibody or antigen-binding fragment thereof includes a humanized mature heavy variable domain including a sequence that is at least 95% identical to any one of SEQ ID NOs: 4-23 and a humanized mature light chain variable domain including a sequence that is at least 95% identical to any one of SEQ ID NOs: 27-48.
- the humanized mature heavy chain variable domain includes a sequence that is one of SEQ ID NOs: 4-23.
- the humanized mature heavy chain variable domain includes a sequence comprising SEQ ID NO: 4. In some embodiments, the humanized mature heavy chain variable domain includes a sequence comprising SEQ ID NO: 5. In some embodiments, the humanized mature heavy chain variable domain includes a sequence comprising SEQ ID NO: 6. In some embodiments, the humanized mature heavy chain variable domain includes a sequence comprising SEQ ID NO: 7. In some embodiments, the humanized mature heavy chain variable domain includes a sequence comprising SEQ ID NO: 8. In some embodiments, the humanized mature heavy chain variable domain includes a sequence comprising SEQ ID NO: 9. In some embodiments, the humanized mature heavy chain variable domain includes a sequence comprising SEQ ID NO: 10.
- the humanized mature heavy chain variable domain includes a sequence comprising SEQ ID NO: 11. In some embodiments, the humanized mature heavy chain variable domain includes a sequence comprising SEQ ID NO: 12. In some embodiments, the humanized mature heavy chain variable domain includes a sequence comprising SEQ ID NO: 13. In some embodiments, the humanized mature heavy chain variable domain includes a sequence comprising SEQ ID NO: 14. In some embodiments, the humanized mature heavy chain variable domain includes a sequence comprising SEQ ID NO: 15. In some embodiments, the humanized mature heavy chain variable domain includes a sequence comprising SEQ ID NO: 16. In some embodiments, the humanized mature heavy chain variable domain includes a sequence comprising SEQ ID NO: 17.
- the humanized mature heavy chain variable domain includes a sequence comprising SEQ ID NO: 18. In some embodiments, the humanized mature heavy chain variable domain includes a sequence comprising SEQ ID NO: 19. In some embodiments, the humanized mature heavy chain variable domain includes a sequence comprising SEQ ID NO: 21. In some embodiments, the humanized mature heavy chain variable domain includes a sequence comprising SEQ ID NO: 22. In some embodiments, the humanized mature heavy chain variable domain includes a sequence comprising SEQ ID NO: 23. In some embodiments, the humanized mature light chain variable domain includes a sequence that is one of SEQ ID NOs: 27-48. In some embodiment, the humanized mature light chain variable domain includes a sequence comprising SEQ ID NO: 27.
- the humanized mature light chain variable domain includes a sequence comprising SEQ ID NO: Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT 28.
- the humanized mature light chain variable domain includes a sequence comprising SEQ ID NO: 29.
- the humanized mature light chain variable domain includes a sequence comprising SEQ ID NO: 30.
- the humanized mature light chain variable domain includes a sequence comprising SEQ ID NO: 31.
- the humanized mature light chain variable domain includes a sequence comprising SEQ ID NO: 32.
- the humanized mature light chain variable domain includes a sequence comprising SEQ ID NO: 33.
- the humanized mature light chain variable domain includes a sequence comprising SEQ ID NO: 34. In some embodiment, the humanized mature light chain variable domain includes a sequence comprising SEQ ID NO: 35. In some embodiment, the humanized mature light chain variable domain includes a sequence comprising SEQ ID NO: 36. In some embodiment, the humanized mature light chain variable domain includes a sequence comprising SEQ ID NO: 37. In some embodiment, the humanized mature light chain variable domain includes a sequence comprising SEQ ID NO: 38. In some embodiment, the humanized mature light chain variable domain includes a sequence comprising SEQ ID NO: 39. In some embodiment, the humanized mature light chain variable domain includes a sequence comprising SEQ ID NO: 40.
- the humanized mature light chain variable domain includes a sequence comprising SEQ ID NO: 41. In some embodiment, the humanized mature light chain variable domain includes a sequence comprising SEQ ID NO: 42. In some embodiment, the humanized mature light chain variable domain includes a sequence comprising SEQ ID NO: 43. In some embodiment, the humanized mature light chain variable domain includes a sequence comprising SEQ ID NO: 44. In some embodiment, the humanized mature light chain variable domain includes a sequence comprising SEQ ID NO: 45. In some embodiment, the humanized mature light chain variable domain includes a sequence comprising SEQ ID NO: 46. In some embodiment, the humanized mature light chain variable domain includes a sequence comprising SEQ ID NO: 47.
- the humanized mature light chain variable domain includes a sequence comprising SEQ ID NO: 48.
- the humanized mature heavy chain of the antibody described herein can include a lysine residue at their C-terminus. However, in some embodiments, the humanized mature heavy chain (e.g., any of the mature heavy chains described herein) does not include the C-terminal lysine residue. Attorney Docket Ref.
- the antibody or antigen-binding fragment thereof includes a humanized mature heavy variable domain including a sequence that is one of SEQ ID NOs: 4-23 and a humanized mature light chain variable domain including a sequence that is one of SEQ ID NOs: 27-48.
- the antibody or antigen-binding fragment of the cell-penetrating agent includes a humanized mature heavy chain variable domain of SEQ ID NO: 4 and a humanized mature light chain variable domain any one of SEQ ID NOs: 27-48.
- the antibody or antigen-binding fragment thereof of the cell-penetrating agent includes a humanized mature heavy chain variable domain of SEQ ID NO: 5 and a humanized mature light chain variable domain any one of SEQ ID NOs: 27-48. In some embodiments, the antibody or antigen-binding fragment thereof of the cell-penetrating agent includes a humanized mature heavy chain variable domain of SEQ ID NO: 6 and a humanized mature light chain variable domain any one of SEQ ID NOs: 27-48. In some embodiments, the antibody or antigen- binding fragment thereof of the cell-penetrating agent includes a humanized mature heavy chain variable domain of SEQ ID NO: 7 and a humanized mature light chain variable domain any one of SEQ ID NOs: 27-48.
- the antibody or antigen-binding fragment thereof of the cell-penetrating agent includes a humanized mature heavy chain variable domain of SEQ ID NO: 8 and a humanized mature light chain variable domain any one of SEQ ID NOs: 27-48. In some embodiments, the antibody or antigen-binding fragment thereof of the cell-penetrating agent includes a humanized mature heavy chain variable domain of SEQ ID NO: 9 and a humanized mature light chain variable domain any one of SEQ ID NOs: 27-48. In some embodiments, the antibody or antigen-binding fragment thereof of the cell-penetrating agent includes a humanized mature heavy chain variable domain of SEQ ID NO: 10 and a humanized mature light chain variable domain any one of SEQ ID NOs: 27-48.
- the antibody or antigen-binding fragment thereof of the cell-penetrating agent includes a humanized mature heavy chain variable domain of SEQ ID NO: 11 and a humanized mature light chain variable domain any one of SEQ ID NOs: 27-48. In some embodiments, the antibody or antigen- binding fragment thereof of the cell-penetrating agent includes a humanized mature heavy chain variable domain of SEQ ID NO: 12 and a humanized mature light chain variable domain any one of SEQ ID NOs: 27-48. In some embodiments, the antibody or antigen-binding fragment thereof of the cell-penetrating agent includes a humanized mature heavy chain variable domain of SEQ Attorney Docket Ref.
- the antibody or antigen-binding fragment thereof of the cell-penetrating agent includes a humanized mature heavy chain variable domain of SEQ ID NO: 14 and a humanized mature light chain variable domain any one of SEQ ID NOs: 27-48.
- the antibody or antigen-binding fragment thereof of the cell-penetrating agent includes a humanized mature heavy chain variable domain of SEQ ID NO: 15 and a humanized mature light chain variable domain any one of SEQ ID NOs: 27-48.
- the antibody or antigen-binding fragment thereof of the cell-penetrating agent includes a humanized mature heavy chain variable domain of SEQ ID NO: 16 and a humanized mature light chain variable domain any one of SEQ ID NOs: 27-48. In some embodiments, the antibody or antigen- binding fragment thereof of the cell-penetrating agent includes a humanized mature heavy chain variable domain of SEQ ID NO: 17 and a humanized mature light chain variable domain any one of SEQ ID NOs: 27-48. In some embodiments, the antibody or antigen-binding fragment thereof of the cell-penetrating agent includes a humanized mature heavy chain variable domain of SEQ ID NO: 18 and a humanized mature light chain variable domain any one of SEQ ID NOs: 27-48.
- the antibody or antigen-binding fragment thereof of the cell-penetrating agent includes a humanized mature heavy chain variable domain of SEQ ID NO: 19 and a humanized mature light chain variable domain any one of SEQ ID NOs: 27-48.
- the antibody or antigen-binding fragment thereof of the cell- penetrating agent includes a humanized mature heavy chain variable domain of SEQ ID NO: 20 and a humanized mature light chain variable domain any one of SEQ ID NOs: 27-48.
- the antibody or antigen-binding fragment thereof of the cell-penetrating agent includes a humanized mature heavy chain variable domain of SEQ ID NO: 21 and a humanized mature light chain variable domain including any one of SEQ ID NOs: 27-48. In some embodiments, the antibody or antigen-binding fragment thereof of the cell-penetrating agent includes a humanized mature heavy chain variable domain of SEQ ID NO: 22 and a humanized mature light chain variable domain including any one of SEQ ID NOs: 27-48.
- the antibody or antigen-binding fragment thereof of the cell-penetrating agent includes a humanized mature heavy chain variable domain of SEQ ID NO: 23 and a humanized mature light chain variable domain including any one of SEQ ID NOs: 27-48.
- the antibody or antigen-binding fragment thereof of the cell- penetrating agent includes a humanized mature heavy chain variable domain of SEQ ID NO: 20 and a humanized mature light chain variable domain of SEQ ID NO: 47.
- the antibody or antigen-binding fragment thereof of the cell-penetrating agent includes a humanized mature heavy chain variable domain of SEQ ID NO: 20 and a humanized mature light chain variable domain of SEQ ID NO: 48. In some embodiments, the antibody or antigen- binding fragment thereof of the cell-penetrating agent includes a humanized mature heavy chain variable domain of SEQ ID NO: 21 and a humanized mature light chain variable domain of SEQ ID NO: 47. In some embodiments, the antibody or antigen-binding fragment thereof of the cell- penetrating agent includes a humanized mature heavy chain variable domain of SEQ ID NO: 21 and a humanized mature light chain variable domain of SEQ ID NO: 48.
- the antibody or antigen-binding fragment thereof of the cell-penetrating agent includes a humanized mature heavy chain variable domain of SEQ ID NO: 23 and a humanized mature light chain variable domain of SEQ ID NO: 47. In some embodiments, the antibody or antigen- binding fragment thereof of the cell-penetrating agent includes a humanized mature heavy chain variable domain of SEQ ID NO: 23 and a humanized mature light chain variable domain of SEQ ID NO: 48.
- At least one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17) of the following positions in the humanized heavy chain variable domain is occupied by the amino acid as specified: 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.
- 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
- At least one (e.g., 2, 3, 4, 5, or 6) of the following positions in the humanized heavy chain variable domain is occupied by the amino acid as specified: 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.
- at least one (e.g., 2, 3, or 4) of the following positions in the humanized heavy chain variable domain is occupied by the amino acid as specified: S35 is Attorney Docket Ref.
- At least one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13) of the following positions in the humanized light chain variable domain is occupied by the amino acid as specified: 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; L46 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.
- 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
- L46 is occupied by R
- L83 is occupied by V
- L92 is occupied by G or
- At least one (e.g., two) of the following positions in the humanized light chain variable domain is occupied by the amino acid as specified: V3 is occupied by Q or A100 is occupied by D or R. In some embodiments, at least one (e.g., 2, 3, or 4) of the following positions in the humanized light chain variable domain is occupied by the amino acid as specified: 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.
- At least one (e.g., 2, 3, 4, 5, 6, 7, or 8) of the following positions in the humanized heavy chain variable domain is occupied by the amino acid as specified: 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 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) of the following positions in the humanized light chain variable domain is occupied by the amino acid as specified: 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 Attorney Docket Ref.
- No.: 50887-00047WO1 // Client Ref: 794-PCT occupied by A; L83 is occupied by V; L92 is occupied by A; A100 is occupied by D; or L104 is occupied by V.
- the aforementioned substitutions can confer reduced (e.g., at least 1% reduced, at least 5% reduced, at least 10% reduced, at least 20% reduced, at least 30% reduced, at least 40% reduced, at least 50% reduced, at least 60% reduced, at least 70% reduced, at least 80% reduced, at least 90% reduced, at least 95% reduced, or at least 99% reduced) immunogenicity and/or increased (e.g., at least 1% increased, at least 10% increased, at least 20% increased, at least 30% increased, at least 40% increased, at least 50% increased, at least 60% increased, at least 70% increased, at least 80% increased, at least 90% increased, or at least 100% increased) thermal stability of the parent antibody.
- the humanized antibody or has increased thermal stability as compared to a reference antibody including a heavy chain variable domain including SEQ ID NO: 1 and a light chain variable domain including SEQ ID NO: 24. In some embodiments, the humanized antibody has increased thermal stability as compared to a reference antibody including a heavy chain variable domain of SEQ ID NO: 63 and a light chain variable domain of SEQ ID NO: 65. In some embodiments, the humanized antibody has increased thermal stability as compared to a reference antibody including a heavy chain variable domain of SEQ ID NO: 67 and a light chain variable domain of SEQ ID NO: 69.
- the humanized antibody has increased thermal stability as compared to a reference antibody including a heavy chain variable domain of SEQ ID NO: 71 and a light chain variable domain of SEQ ID NO: 73. In some embodiments, the humanized antibody has increased thermal stability as compared to a reference antibody including a heavy chain variable domain of SEQ ID NO: 75 and a light chain variable domain of SEQ ID NO: 77. In some embodiments, the humanized antibody has increased thermal stability as compared to a reference antibody including a heavy chain variable domain of SEQ ID NO: 79 and a light chain variable domain of SEQ ID NO: 81. In some embodiments, the humanized antibody has a melting temperature of about 55 °C or greater.
- the humanized antibody has a melting temperature of about 56 °C or greater, about 57 °C or greater, about 58° C or greater, about 59 °C or greater, about 60 °C or greater, about 61 °C or greater, about 62 °C or greater, about 63 °C or greater, about 64 °C or greater, about 65 °C or greater, about 66 °C or greater, about 67 °C or greater, about 68 °C or greater, about 69 °C or greater, about 70 °C or greater, about 71 °C or greater, about 72 °C or Attorney Docket Ref.
- No.: 50887-00047WO1 // Client Ref: 794-PCT greater, about 73 °C or greater, about 74 °C or greater, about 75 °C or greater, about 76 °C or greater, about 77 °C or greater, about 78 °C or greater, about 79 °C or greater, about 80 °C or greater, about 81 °C or greater, about 82 °C or greater, about 83 °C or greater, about 84 °C or greater, or about 85 °C or greater.
- the humanized antibody has a melting temperature of 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
- the humanized antibody has a melting temperature of 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 70 °C
- cell-penetrating agents that include a CIM and an antibody that specifically binds to TDP-43 (e.g., human TDP-43), that include a heavy chain variable domain including: a heavy chain CDR1, as defined by Kabat/Chothia Composite, including SEQ ID NO: 49 or SEQ ID NO: 50; a heavy chain CDR2, as defined by Kabat, including SEQ ID NO: 51; a heavy chain CDR3, as defined by Kabat or Chothia, including SEQ ID NO: 52; a light chain Attorney Docket Ref.
- the heavy chain CDR1, as defined by Kabat/Chothia Composite includes SEQ ID NO: 49; the heavy chain CDR2, as defined by Kabat, includes SEQ ID NO: 51; the heavy chain CDR3, as defined by Kabat or Chothia, includes SEQ ID NO: 52; the light chain CDR1, as defined by Kabat, includes SEQ ID NO: 53; the light chain CDR2, as defined by Kabat, includes SEQ ID NO: 54; and the light chain CDR3, as defined by Kabat, includes SEQ ID NO: 55 or SEQ ID NO: 61.
- the heavy chain variable domain includes a sequence that is at least 95% (e.g., at least 96%, at least 97%, at least 98%, at least 99%) identical to any one of SEQ ID NOs: 4-23. In some embodiments, the heavy chain variable domain includes a sequence that is at least 95% (e.g., at least 96%, at least 97%, at least 98%, at least 99%) identical to any one of: SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 23. In some embodiments, the heavy chain variable domain includes a sequence that is at least 98% identical to any one of: SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 23.
- the heavy chain variable domain includes a sequence of SEQ ID NO: 20. In some embodiments, the heavy chain variable domain includes a sequence of SEQ ID NO: 21. In some embodiments, the heavy chain variable domain includes a sequence of SEQ ID NO: 23. In some embodiments, the light chain variable domain includes a sequence that is at least 95% (e.g., at least 96%, at least 97%, at least 98%, at least 99%) identical to any one of: SEQ ID NOs: 27-48. In some embodiments, the light chain variable domain includes a sequence that is at least 95% (e.g., at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NO: 47 or SEQ ID NO: 48.
- the light chain variable domain includes a sequence that is at least 98% identical to SEQ ID NO: 47 or SEQ ID NO: 48. In some embodiments, the light chain variable domain includes SEQ ID NO: 47. In some embodiments, the light chain variable domain includes SEQ ID NO: 48.
- the humanized mature light chain variable domains described herein can include a lysine residue at their C-terminus. However, in some embodiments, the humanized mature light chain variable domain (e.g., any of the mature light chain variable domains described herein) does not include the C-terminal lysine residue. For example, any one Attorney Docket Ref.
- the antibody of the cell-penetrating agent is a humanized antibody, a chimeric antibody, or a veneered antibody.
- the antibody is an antigen-binding antibody fragment.
- the antigen-binding antibody fragment is a Fab fragment, a Fab’2 fragment, or a single chain Fv.
- the antibody of the cell-penetrating agent is an intact antibody.
- the antibody has a human IgG1 isotype.
- the heavy chain variable domain is fused to a heavy chain constant region (e.g., any of the heavy chain constant regions described herein) and the light chain variable domain is fused to a light chain constant region (e.g., any of the light chain constant regions described herein).
- the heavy chain constant region is a mutant form of a natural human heavy chain constant region which has reduced binding to an Fc ⁇ receptor relative to the natural heavy chain constant region.
- the heavy chain constant region is of IgG1 isotype.
- the antibody has at least one mutation in a constant region.
- the at least one mutation reduces complement fixation or activation by the constant region.
- the at least one mutation is at one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11) positions of: 241, 264, 265, 270, 296, 297, 318, 320, 322, 329, and 331 by EU numbering.
- the antibody has an alanine at positions 318, 320, and 322 by EU numbering.
- the antibody or antigen-binding antibody fragment of the cell- penetrating agent selectively binds to phosphorylated TDP-43 (e.g., phosphorylated human TDP- 43).
- the antibody or antigen-binding antibody fragment of the cell- penetrating agent selectively binds to phosphorylated TDP-43 (e.g., phosphorylated human TDP- 43) as compared to unphosphorylated TDP-43 (e.g., unphosphorylated human TDP-43).
- phosphorylated TDP-43 e.g., phosphorylated human TDP- 43
- unphosphorylated TDP-43 e.g., unphosphorylated human TDP-43
- the antibody or antigen-binding antibody fragment of the cell- penetrating agent binds to phosphorylated TDP-43 (e.g., phosphorylated human TDP-43) with greater (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 Attorney Docket Ref.
- TDP-43 e.g., phosphorylated human TDP-43
- greater 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
- No.: 50887-00047WO1 // Client Ref: 794-PCT 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-fold, at least 1,150- fold, at least 1,200-fold, at least 1,250-fold, at least 1,300-fold, at least 1,350-fold, at least 1,400- fold, at least 1,450-fold, or at least 1,500-fold greater, or about 10-fold to about 1,500-fold, about 10-fold to 1,400-fold greater, about 10-fold to about 1,300-fold, about 10-fold to about 1,200- fold, about 10-fold to about 1,100-fold, about 10-fold to about 1,000-fold, about 10-fold to about 900-fold, about 10-fold to about 800-fold, about 10-fold to about 700-fold, about 10-fold to about 600-fold, about 10-fold to about 500-fold, about 10-fold to about
- phosphorylated TDP-43 e.g., phosphorylated human TDP-43
- phosphorylated TDP-43 includes phosphorylation of at least one amino acid residue selected from S409 and S410.
- phosphorylated TDP-43 e.g., phosphorylated human TDP-43 includes phosphorylation of both S409 and S410.
- the antibody, or fragment thereof, of the cell-penetrating agent selectively binds to cytoplasmic aggregates of TDP-43 (e.g., cytoplasmic aggregates of human TDP-43). In some embodiments, the antibody, or fragment thereof, of the cell-penetrating agent 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) includes phosphorylated aggregates of TDP-43.
- the antibody, or fragment thereof, of the cell-penetrating agent does not substantially bind unphosphorylated TDP-43 (e.g., unphosphorylated human TDP-43).
- the heavy and light chain variable regions present in the cell-penetrating agents can be linked to at least a portion of a human constant region.
- the choice of constant region depends, in part, whether antibody conjugate-dependent cell-mediated cytotoxicity, antibody conjugate dependent cellular phagocytosis and/or complement dependent cytotoxicity are desired.
- human isotypes IgG1 and IgG3 have complement-dependent cytotoxicity and human isotypes IgG2 and IgG4 do not.
- Light chain constant regions can be lambda or kappa. Numbering conventions for constant regions include EU numbering (Edelman, G.M. et al., Proc. Natl. Acad. Sci. U.S.A. 63:78-85 (1969)), Kabat numbering (Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1991, IMGT unique numbering (Lefranc M.-P. et al., IMGT unique numbering for immunoglobulin and T cell receptor constant domains and Ig superfamily C-like domains, Dev. Comp.
- substitutions include a Gln at position 250 and/or a Leu at position 428 (EU numbering is used in this paragraph for the constant region) for increasing the half-life of an antibody. Substitution at any or all of positions 234, 235, 236 and/or 237 reduce affinity for Fey receptors, particularly FcyRI receptor (see, e.g., U.S. Patent No. 6,624,821).
- alanine substitution at positions 234, 235, and 237 of human IgG 1 can be used for reducing effector functions.
- Some antibodies have alanine substitution at positions 234, 235 and 237 of human IgG 1 for reducing effector functions.
- positions 234, 236 and/or 237 in human IgG2 are substituted with alanine and position 235 with glutamine (see, e.g., U.S. Patent No. 5,624,821).
- a mutation at one or more (e.g., 2, 3, 4, 5, 6, 7, 8, or 9) of positions 241, 264, 265, 270, 296, 297, 322, 329, and 331 by EU numbering of human IgG 1 is used.
- a mutation at one or more (e.g., 2 or 3) of positions 318, 320, and 322 by EU numbering of human IgG1 is used.
- positions 234 and/or 235 are substituted with alanine and/or position 329 is substituted with glycine.
- positions 234 and 235 are substituted with alanine.
- the isotype is human IgG2 or IgG4.
- Antibodies can be expressed as tetramers containing two light and two heavy chains, as separate heavy chains, light chains, as Fab, Fab’, F(ab’) 2 , and Fv, or as single chain antibodies in which heavy and light chain mature variable domains are linked through a spacer.
- Human constant regions show allotypic variation and isoallotypic variation between different individuals, that is, the constant regions can differ in different individuals at one or more polymorphic positions.
- Isoallotypes differ from allotypes in that sera recognizing an isoallotype bind to a non-polymorphic region of a one or more other isotypes.
- another heavy chain constant region is of IgG1 Glm3 with or without the C-terminal lysine.
- Reference to a human constant region includes a constant region with any natural allotype or any permutation of residues occupying positions in natural allotypes.
- Exemplary Anti-TDP-43 Cell Penetrating Agents Attorney Docket Ref.
- CMIPs cell internalizing moieties
- CMIPs cell internalizing moieties
- One of ordinary skill will understand how to combine these features to arrive functional anti-TDP-43 cell- penetrating agents.
- cell-penetrating agents having the CMIP, the optional linker, and the light and/or heavy chain of the anti-TDP-43 antibody expressed as a single polypeptide (i.e., a fusion protein).
- the cell-penetrating agent comprises a polypeptide sequence that is at least 95% identical to a sequence selected from any one of: 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
- the cell-penetrating agent comprises a polypeptide sequence that is at least 98% identical to a sequence selected from any one of: 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
- the cell-penetrating agent comprises a polypeptide sequence that is at least 98% identical to a sequence selected from any one of: 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
- the cell-penetrating agent comprises a polypeptide sequence that is at least 95% identical to a sequence selected from any one of 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
- the cell-penetrating agent comprises a polypeptide sequence that is at least 98% identical to a sequence selected from any one of 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: 17
- the cell-penetrating agent comprises a polypeptide sequence selected from any one of 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:
- the cell-penetrating agent comprises a first polypeptide sequence comprising a heavy chain of the antibody and a second polypeptide comprising a light chain of the antibody.
- the first polypeptide and/or the second polypeptide comprises a CMIP and an optional linker.
- the first polypeptide is selected from any one of: 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: Attorney Docket Ref.
- the cell-penetrating agent comprises a first polypeptide and a second polypeptide, further wherein: the first polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 116 and the second polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 117; the first polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 118 and the second polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 119; the first polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 120 and the second polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 121; the first polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 122 and the second polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 123; the first polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 124
- the first polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 136 and the second polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 137; the first polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 138 and the second polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 139; the first polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 140 and the second polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 141; the first polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 142 and the second polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 143; the first polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO:
- 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
- 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:
- the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 116 and a second polypeptide comprising SEQ ID NO: 117. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 118 and a second polypeptide comprising SEQ ID NO: 119. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 120 and a second polypeptide comprising SEQ ID NO: 121. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 122 and a second polypeptide comprising SEQ ID NO: 123.
- the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 124 and a second polypeptide comprising SEQ ID NO: 125. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 126 and a second polypeptide comprising SEQ ID NO: 127. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 128 and a second polypeptide comprising SEQ ID NO: 129. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 130 and a second polypeptide comprising SEQ ID NO: 131.
- the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 132 and a second polypeptide comprising SEQ ID NO: 133.
- the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 134 and a second polypeptide comprising SEQ ID NO: 135.
- the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 136 and a second polypeptide comprising SEQ ID NO: 137.
- the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 138 and a second polypeptide comprising SEQ ID NO: 139. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 140 and a second polypeptide comprising SEQ ID NO: 141. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 142 and a second polypeptide comprising SEQ ID NO: 143. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 144 and a second polypeptide comprising SEQ ID NO: 145.
- the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 146 and a second polypeptide comprising SEQ ID NO: 147. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 148 and a second polypeptide comprising SEQ ID NO: 149. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 150 and a second polypeptide comprising SEQ ID NO: 151. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 152 and a second polypeptide comprising SEQ ID NO: 153.
- the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 154 and a second polypeptide comprising SEQ ID NO: 155. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 156 and a second polypeptide comprising SEQ ID NO: 157. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 158 and a second polypeptide comprising SEQ ID NO: 159. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 160 and a second polypeptide comprising SEQ ID NO: 161.
- the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 162 and a second polypeptide comprising SEQ ID NO: 163. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 164 and a second polypeptide comprising SEQ ID NO: 165. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 166 and a second polypeptide comprising SEQ ID NO: 167. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 168 and a second Attorney Docket Ref.
- the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 170 and a second polypeptide comprising SEQ ID NO: 171. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 172 and a second polypeptide comprising SEQ ID NO: 173. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 174 and a second polypeptide comprising SEQ ID NO: 175.
- Cell-penetrating agents can also be administered in the form of nucleic acids encoding the cell-penetrating agent or the antibody present within a cell-penetrating agent. If both heavy and light chains are present, the chains are preferably linked as a single chain antibody.
- Nucleic Acids, Vectors, and Host Cells The present disclosure provides nucleic acids encoding at least a portion of any of the cell-penetrating agents described herein.
- the nucleic acids can encode any of the CIMs, linkers, and/or spacers described herein in addition to any of the heavy chains and/or light chains described herein.
- the nucleic acids encodes a first polypeptide comprising a heavy chain of an antibody that binds specifically to TDP-43, the first polypeptide comprising any one of: 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:
- the nucleic acids encode a second polypeptide comprising a light chain of an antibody that binds specifically to TDP-43, the second polypeptide comprising any one of: 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:
- the nucleic acid encodes a first polypeptide comprising a heavy chain of an antibody that binds specifically to TDP-43, the first polypeptide comprising any one of: 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,
- the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 116 and a second polypeptide comprising SEQ ID NO: 117. In some embodiments, the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 118 and a second polypeptide comprising SEQ ID NO: 119. In some embodiments, the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 120 and a second polypeptide comprising SEQ ID NO: 121.
- the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 122 and a second polypeptide comprising SEQ ID NO: 123. In some embodiments, the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 124 and a second polypeptide comprising SEQ ID NO: 125. In some embodiments, the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 126 and a second polypeptide comprising SEQ ID NO: 127.
- the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 128 and a second polypeptide comprising SEQ ID NO: 129. In some embodiments the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 130 and a second polypeptide comprising SEQ ID NO: Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT 131. In some embodiments, the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 132 and a second polypeptide comprising SEQ ID NO: 133.
- the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 134 and a second polypeptide comprising SEQ ID NO: 135. In some embodiments the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 136 and a second polypeptide comprising SEQ ID NO: 137. In some embodiments, the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 138 and a second polypeptide comprising SEQ ID NO: 139.
- the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 140 and a second polypeptide comprising SEQ ID NO: 141. In some embodiments the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 142 and a second polypeptide comprising SEQ ID NO: 143. In some embodiments the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 144 and a second polypeptide comprising SEQ ID NO: 145.
- the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 146 and a second polypeptide comprising SEQ ID NO: 147. In some embodiments, the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 148 and a second polypeptide comprising SEQ ID NO: 149. In some embodiments, the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 150 and a second polypeptide comprising SEQ ID NO: 151.
- the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 152 and a second polypeptide comprising SEQ ID NO: 153. In some embodiments, the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 154 and a second polypeptide comprising SEQ ID NO: 155. In some embodiments the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 156 and a second polypeptide comprising SEQ ID NO: 157.
- the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 158 and a second polypeptide comprising SEQ ID NO: 159. In some embodiments, the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 160 and a second polypeptide comprising SEQ ID NO: 161. In some embodiments, the nucleic acid encodes a cell penetrating agent comprising a first Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT polypeptide comprising SEQ ID NO: 162 and a second polypeptide comprising SEQ ID NO: 163.
- the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 164 and a second polypeptide comprising SEQ ID NO: 165. In some embodiments the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 166 and a second polypeptide comprising SEQ ID NO: 167. In some embodiments, the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 168 and a second polypeptide comprising SEQ ID NO: 169.
- the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 170 and a second polypeptide comprising SEQ ID NO: 171. In some embodiments, the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 172 and a second polypeptide comprising SEQ ID NO: 173. In some embodiments, the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 174 and a second polypeptide comprising SEQ ID NO: 175.
- the present disclosure further provides nucleic acids encoding any of the heavy chain variable domains and/or light variable chain domains of any of the antibodies described herein.
- the nucleic acids can encode a heavy chain variable domain comprising any one of SEQ ID NOs: 4-23 and/or a light chain variable domain comprising any one of SEQ ID NOs: 27- 48.
- such nucleic acids further encode a signal peptide and can be expressed with the signal peptide linked to the constant region. Coding sequences of nucleic acids can be operably linked with regulatory sequences to ensure expression of the coding sequences, such as a promoter, enhancer, ribosome binding site, transcription termination signal, and the like.
- the nucleic acids encoding heavy and light chains can occur in isolated form or can be cloned into one or more vectors.
- the nucleic acids can be synthesized by, for example, solid state synthesis or PCR of overlapping oligonucleotides.
- Nucleic acids encoding heavy and light chains can be joined as one contiguous nucleic acid, e.g., within an expression vector, or can be separate, e.g., each cloned into its own expression vector.
- the nucleic acid is codon- optimized for expression in a host cell.
- a number of methods are known for producing chimeric and humanized antibodies using an antibody-expressing cell line (e.g., hybridoma).
- the immunoglobulin variable regions of antibodies can be cloned and sequenced using well known methods.
- Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT the heavy chain variable VH region is cloned by RT-PCR using mRNA prepared from hybridoma cells.
- Consensus primers are employed to the VH region leader peptide encompassing the translation initiation codon as the 5’ primer and a g2b constant regions specific 3’ primer.
- Exemplary primers are described in U.S. patent publication US 2005/0009150 by Schenk et al. (hereinafter “Schenk”).
- the sequences from multiple, independently derive clones can be compared to ensure no changes are introduced during amplification.
- the sequence of the VH region can also be determined or confirmed by sequencing a VH fragment obtained by 5’ RACE RT-PCR methodology and the 3’ g2b specific primer.
- the light chain variable VL region can be cloned in an analogous manner.
- a consensus primer set is designed for amplification of VL regions using a 5’ primer designed to hybridize to the VL region encompassing the translation initiation codon and a 3’ primer specific for the Ck region downstream of the V-J joining region.
- 5’RACE RT-PCR methodology is employed to clone a VL encoding cDNA.
- Exemplary primers are described in Schenk, supra.
- the cloned sequences are then combined with sequences encoding human (or other non-human species) constant regions.
- vectors including any of the nucleic acids described herein operably linked to one or more regulatory sequences to effect expression in a mammalian cell of any of the cell-penetrating agents described herein.
- vectors including a nucleic acid encoding a mature heavy chain variable domain (e.g., any of the heavy chain variable domains described herein) and a light chain variable domain (e.g., any of the light chain variable domains described herein) operably linked to one or more regulatory sequences to effect expression in a mammalian cell of any of the antibodies or antigen-binding fragments described herein.
- the heavy and light chain variable regions are re-engineered to encode splice donor sequences downstream of the respective VDJ or VJ junctions and are cloned into a mammalian expression vector, such as pCMV-hyl for the heavy chain and pCMV-Mcl for the light chain.
- the heavy chain and light chain expression vectors can be co-transfected into CHO cells to produce chimeric antibodies.
- Conditioned media is collected 48 hours post-transfection and assayed by Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT western blot analysis for antibody production or ELISA for antigen binding.
- the chimeric antibodies are humanized as described above. Chimeric, veneered, humanized, and human antibodies are typically produced by recombinant expression.
- Recombinant polynucleotide constructs typically include an expression control sequence operably linked to the coding sequences of antibody chains, including naturally associated or heterologous expression control elements, such as a promoter.
- the expression control sequences can be promoter systems in vectors capable of transforming or transfecting eukaryotic or prokaryotic host cells. Once the vector has been incorporated into the appropriate host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences and the collection and purification of the cross-reacting antibodies.
- host cells including any of the nucleic acids described herein.
- Expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA. Commonly, expression vectors contain selection markers, e.g., ampicillin resistance or hygromycin resistance, to permit detection of those cells transformed with the desired DNA sequences.
- E. coli is one prokaryotic host useful for expressing antibodies, particularly antibody fragments.
- Microbes, such as yeast, are also useful for expression. Saccharomyces is a yeast host with suitable vectors having expression control sequences, an origin of replication, termination sequences, and the like as desired. Typical promoters include 3-phosphoglycerate kinase and other glycolytic enzymes.
- Inducible yeast promoters include, among others, promoters from alcohol dehydrogenase, isocytochrome C, and enzymes responsible for maltose and galactose utilization.
- Mammalian cells can be used for expressing nucleotide segments encoding immunoglobulins or fragments thereof. See, Winnacker, From Genes to Clones, (VCH Publishers, NY, 1987).
- a number of suitable host cell lines capable of secreting intact heterologous proteins have been developed, and include CHO cell lines, various COS cell lines, HeLa cells, HEK293 cells, L cells, and non-antibody-producing myelomas including Sp2/0 and NS0.
- the cells can be non-human.
- Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter, an enhancer (Queen et al., Immunol. Rev. 89:49 (1986)), and necessary processing information sites, such as ribosome Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences.
- Expression control sequences can include promoters derived from endogenous genes, cytomegalovirus, SV 40, adenovirus, bovine papillomavirus, and the like. See Co et al., J. Immunol. 148: 1149 (1992).
- the promoter is a eukaryotic promoter.
- antibody coding sequences can be incorporated in transgenes for introduction into the genome of a transgenic animal and subsequent expression in the milk of the transgenic animal (see, e.g., U.S. Pat. No. 5,741,957; U.S. Pat. No. 5,304,489; and U.S. Pat. No. 5,849,992).
- Suitable transgenes include coding sequences for light and/or heavy chains operably linked with a promoter and enhancer from a mammary gland specific gene, such as casein or beta lactoglobulin.
- the vectors containing the DNA segments of interest can be transferred into the host cell by methods depending on the type of cellular host.
- transgenic animals For production of transgenic animals, trans genes can be microinjected into fertilized oocytes or can be incorporated into the genome of embryonic stem cells, and the nuclei of such cells transferred into enucleated oocytes. Having introduced vector(s) encoding antibody heavy and light chains into cell culture, cell pools can be screened for growth productivity and product quality in serum-free media.
- Top- producing cell pools can then be subjected of FACS-based single-cell cloning to generate monoclonal lines.
- Specific productivities above 50 pg or 100 pg per cell per day, which correspond to product titers of greater than 7.5 g/L culture, can be used.
- Antibodies produced by single cell clones can also be tested for turbidity, filtration properties, PAGE, IEF, UV scan, HPSEC, carbohydrate-oligosaccharide mapping, mass spectrometry, and binding assay, such as ELISA or Biacore.
- a selected clone can then be banked in multiple vials and stored frozen for subsequent use.
- antibodies can be purified according to standard procedures of the art, including protein A capture, HPLC purification, column chromatography, gel electrophoresis and the like (See generally, Scopes, Protein Purification (Springer-Verlag, NY, 1982)).
- Methodology for commercial production of antibodies can be employed, including codon optimization, selection of promoters, selection of transcription elements, selection of terminators, serum-free single cell cloning, cell banking, use of selection markers for amplification of copy number, CHO terminator, or improvement of protein titers (see, e.g., U.S. Patent No. 5,786,464; U.S. Patent No.
- the DNA can be delivered in naked form (i.e., without colloidal or encapsulating materials).
- a number of viral vector systems can be used including retro viral systems (see, e.g., Lawrie and Tumin, Cur. Opin. Genet. Develop. 3, 102-109 (1993)); adenoviral vectors (see, e.g., Bett et al, J. Virol.
- adeno-associated virus vectors see, e.g., Zhou et al., J. Exp. Med. 179, 1867 (1994)
- viral vectors from the pox family including vaccinia virus and the avian pox viruses
- viral vectors from the alpha virus genus such as those derived from Sindbis and Semliki Forest Viruses (see, e.g., Dubensky et al., J. Virol. 70, 508-519 (1996)), Venezuelan equine encephalitis virus (see U.S. Patent No.
- DNA encoding an immunogen can be packaged into liposomes. Suitable lipids and related analogs are described by U.S. Patent No. 5,208,036, U.S. Patent No. 5,264,618, U.S. Patent No.
- Vectors and DNA encoding an immunogen can also be adsorbed to or associated with particulate carriers, examples of which include polymethyl methacrylate polymers and polylactides and poly(lactide- co-glycolides) (see, e.g., McGee et al., J. Micro Encap. 1996). V.
- TDP-43 e.g., human TDP-43
- ALS amyotrophic lateral sclerosis
- FTLD- TDP frontotemporal lobar degeneration
- primary lateral sclerosis e.g., primary lateral sclerosis
- progressive muscular atrophy e.g., progressive muscular atrophy
- No.: 50887-00047WO1 // Client Ref: 794-PCT inhibiting or reducing aggregation of TDP-43; reducing or clearing TDP-43 aggregates; stabilizing non-toxic conformations of TDP-43; or treating or effecting prophylaxis of amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD-TDP), primary lateral sclerosis, and progressive muscular atrophy, and Parkinson’s disease in a patient.
- the cell-penetrating agents described herein can be further conjugated with other therapeutic moieties, other proteins, other antibodies, and/or detectable labels. See WO 03/057838; U.S. Patent No. 8,455,622.
- Such therapeutic moieties can be any agent that can be used to treat, combat, ameliorate, prevent, or improve an unwanted condition or disease in a patient, such as amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD- TDP), primary lateral sclerosis, and progressive muscular atrophy, and Parkinson’s disease.
- Conjugated therapeutic moieties can include cytotoxic agents, cytostatic agents, neurotrophic agents, neuroprotective agents, radiotherapeutic agents, immunomodulators, or any biologically active agents that facilitate or enhance the activity of the antibody.
- a cytotoxic agent can be any agent that is toxic to a cell.
- a cytostatic agent can be any agent that inhibits cell proliferation.
- a neurotrophic agent can be any agent, including chemical or proteinaceous agents, which promotes neuron maintenance, growth, or differentiation.
- a neuroprotective agent can be agent, including chemical or proteinaceous agents, which protects neurons from acute insult or degenerative processes.
- An immunomodulator can be any agent that stimulates or inhibits the development or maintenance of an immunologic response.
- a radiotherapeutic agent can be any molecule or compound that emits radiation. If such therapeutic moieties are coupled to a TDP-43 specific antibody or antigen-binding antibody fragment, such as the antibodies and antigen-binding antibody fragments described herein, the coupled therapeutic moieties will have a specific affinity for TDP-43 related disease-affected cells over normal cells.
- radioisotopes examples include, for example, yttrium 90 (90Y), indium 111 (111In), 131 1, 99 mTc, radiosilver- 111, radiosilver-199, and Bismuth 213 . Linkage of radioisotopes to antibodies or antigen-binding Attorney Docket Ref.
- ibritumomab tiuxetan For radioisotopes such as 111In and 90Y, ibritumomab tiuxetan can be used and will react with such isotopes to form 111In -ibritumomab tiuxetan and 90Y-ibritumomab tiuxetan, respectively. See Witzig, Cancer Chemother. Pharmacol., 48(Suppl l):S91-S95 (2001). Some such antibodies or antigen-binding antibody fragments can be linked to other therapeutic moieties. Such therapeutic moieties can be, for example, cytotoxic, cytostatic, immunomodulatory, neurotrophic, or neuroprotective.
- antibodies and antigen- binding antibody fragments can be conjugated with toxic chemotherapeutic drugs such as maytansine, geldanamycin, tubulin inhibitors such as tubulin binding agents (e.g., auristatins), or minor groove binding agents such as calicheamicin.
- toxic chemotherapeutic drugs such as maytansine, geldanamycin
- tubulin inhibitors such as tubulin binding agents (e.g., auristatins), or minor groove binding agents such as calicheamicin.
- Other representative therapeutic moieties include agents known to be useful for treatment, management, or amelioration of amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD-TDP), primary lateral sclerosis, and progressive muscular atrophy, and Parkinson’s disease.
- Antibodies or antigen-binding antibody fragments can also be coupled with a detectable label.
- Such antibodies and antigen-binding antibody fragments can be used, for example, for diagnosing ALS, FTLD-TDP, primary lateral sclerosis, and progressive muscular atrophy, and Parkinson’s disease.
- Representative detectable labels that may be coupled or linked to an antibody or antigen-binding antibody fragment include various enzymes, such as horseradish peroxidase, alkaline phosphatase, betagalactosidase, or acetylcholinesterase; prosthetic groups, such streptavidin/biotin and avidin/biotin; fluorescent materials, such as umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; luminescent materials, such as luminol; bioluminescent materials, such as luciferase, luciferin, and aequorin; radioactive materials, such as radiosilver-111,
- No.: 50887-00047WO1 // Client Ref: 794-PCT emission tomographies; nonradioactive paramagnetic metal ions; and molecules that are radiolabelled or conjugated to specific radioisotopes.
- Therapeutic moieties, other proteins, other antibodies, and/or detectable labels may be coupled or conjugated, directly or indirectly through an intermediate (e.g., a linker), to an antibody or antigen-binding antibody fragment of the invention. See e.g., 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.
- Suitable linkers include, for example, cleavable and noncleavable linkers. Different linkers that release the coupled therapeutic moieties, proteins, antibodies, and/or detectable labels under acidic or reducing conditions, on exposure to specific proteases, or under other defined conditions can be employed.
- the cell-penetrating agent is also conjugated to a therapeutic, cytotoxic, cytostatic, immunomodulatory, neurotrophic, or neuroprotective agent as described herein.
- antibodies present in the cell-penetrating agent can be coupled (i.e., conjugated) with a therapeutic moiety, such as a cytotoxic agent, a radiotherapeutic agent, an immunomodulator, or a second antibody (e.g., to form an antibody heteroconjugate).
- a therapeutic moiety such as a cytotoxic agent, a radiotherapeutic agent, an immunomodulator, or a second antibody (e.g., to form an antibody heteroconjugate).
- Representative therapeutic moieties include agents known to be useful for treatment, management, or amelioration of TDP-43-related diseases or symptoms of TDP-43-related diseases.
- Therapeutic moieties and/or detectable substances may be coupled or conjugated directly to any of the murine, chimeric, or humanized antibodies described herein through an intermediate (e.g., a linker) using techniques known in the art.
- Cell-penetrating agents used in the disclosed formulations also include modified forms of murine, chimeric, or humanized 13D3 antibodies, which have increased in vivo half-lives relative to the corresponding unmodified antibodies.
- modified forms may be prepared, for example, by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc.
- representative methods for antibody half-life extension are described in PCT International Publication No. WO 02/060919.
- compositions including any of the cell-penetrating agents described herein and a pharmaceutically acceptable carrier.
- Pharmaceutical compositions for parenteral administration are preferably sterile and substantially isotonic and manufactured under GMP conditions.
- Pharmaceutical compositions can be provided in unit dosage form (i.e., the dosage for a single administration).
- Pharmaceutical compositions can be formulated using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries. The formulation depends on the route of administration chosen.
- cell-penetrating agents can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank’s solution, Ringer’s solution, or physiological saline or acetate buffer (to reduce discomfort at the site of injection).
- the solution can contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
- cell-penetrating agents can be in lyophilized form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
- a suitable vehicle e.g., sterile pyrogen-free water
- the anti-TDP-43 cell penetrating agents described herein can be present in any pharmaceutically acceptable excipient or carrier.
- the anti-TDP-43 cell-penetrating agents described herein can be present in a buffer.
- the buffer can have a pH from about 6 to about 7.
- the formulations are sterile, for example, as accomplished by sterile filtration using a 0.2 ⁇ m or a 0.22 ⁇ m filter.
- the formulations disclosed herein are also generally stable upon freezing and thawing.
- the method can be for a non-diagnostic and/or non-therapeutic purposes.
- samples such as cells, tissues, and/or organs that have been removed from the patient are exposed to a pharmaceutical composition comprising any of the cell-penetrating agents described herein.
- a cell-penetrating agent e.g., or a nucleic acid or a vector encoding any of the cell-penetrating agent
- a pharmaceutical composition of the same is administered to a patient susceptible to, or otherwise at risk of a disease such as ALS, FTLD- TDP, primary lateral sclerosis, and progressive muscular atrophy, and Parkinson’s disease in regime (dose, frequency and route of administration) effective to reduce the risk, lessen the severity, or delay the onset of at least one sign or symptom of the TDP-43-related disease.
- the regime is preferably effective to inhibit or delay TDP-43 aggregates (e.g., human TDP-43 aggregates) in the brain, and/or inhibit or delay its toxic effects and/or inhibit/or delay development of behavioral deficits.
- a cell-penetrating agent is administered to a patient suspected of, or already suffering from a disease (e.g., ALS) in a regime (dose, frequency and route of administration) effective to ameliorate or at least inhibit further deterioration of at least one sign or symptom of the disease.
- the regime is preferably effective to reduce or at least inhibit further increase of cytoplasmic levels of TDP-43 (e.g., human TDP-43) and/or aggregates formed from it, associated toxicities and/or behavioral deficits.
- TDP-43 e.g., human TDP-43
- a regime is considered therapeutically or prophylactically effective if an individual treated patient achieves an outcome more favorable than the mean outcome in a control population of comparable patients not treated by methods disclosed herein.
- Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT VII.
- treatment refers to the alleviation or amelioration of one or more symptoms or effects associated with the disease, prevention, inhibition or delay of the onset of one or more symptoms or effects of the disease, lessening of the severity or frequency of one or more symptoms or effects of the disease, and/or increasing or trending toward desired outcomes as described herein. Desired outcomes of the treatments disclosed herein vary according to the TDP-43- related disease and patient profile and are readily determinable to those skilled in the art. Desired outcomes include an improvement in the patient’s health status. Generally, desired outcomes include measurable indices such as reduction or clearance of pathologic ALS, FTLD- TDP, primary lateral sclerosis, and progressive muscular atrophy, and Parkinson’s disease.
- kits for delivering an antibody that specifically binds to TDP-43 into a cell comprising contacting any of the cell-penetrating agents described herein with the cell, thereby resulting in the internalization of, at a minimum, an antigen-binding fragment of the antibody, into the cell.
- the method includes transferring, at a minimum, an antigen-binding fragment of the antibody, to the cytosol of the cell.
- methods of binding an intracellular TDP-43 protein in a cell that include contacting the cell-penetrating agent of any one of claims 1-135 with the cell, thereby resulting in the internalization of and transfer to the cytosol of, at a minimum, an antigen-binding fragment of the antibody.
- Also provided herein are methods of binding an intracellular TDP-43 protein in a cell that include: contacting the cell-penetrating agent of any one of claims 1-135 with the cell, thereby resulting in the internalization of and transfer to the cytosol of, at a minimum, an antigen-binding fragment of the antibody; and binding, at a minimum, an antigen-binding fragment of the antibody, to the intracellular TDP-43 protein.
- TDP-43 e.g., human TDP-43
- methods of inhibiting or reducing aggregation of TDP-43 including administering to the subject an effective amount of any of the cell-penetrating agents described herein, thereby inhibiting or reducing aggregation of TDP-43 in the subject (i.e., patient).
- methods of treating or effecting prophylaxis of a TDP-43- related disease in a subject including administering a therapeutically effective amount of any of Attorney Docket Ref.
- TDP-43-related disease is ALS, FTLD-TDP, primary lateral sclerosis, and progressive muscular atrophy, and Parkinson’s disease.
- the TDP-43-related disease is ALS.
- TDP-43 deposits e.g., human TDP-43 deposits
- methods of detecting TDP-43 deposits including administering to a subject any of the cell-penetrating agents described herein, and detecting the antibody bound to TDP-43 in the subject.
- the cell-penetrating agent is administered by intravenous injection into the body of the subject.
- the cell-penetrating agent or the antibody in the cell-penetrating agent is labeled.
- the cell-penetrating agent is labeled with a fluorescent label, a paramagnetic label, or a radioactive label.
- the radioactive label is detected using positron emission tomography (PET) or single-photon emission computed tomography (SPECT).
- PET positron emission tomography
- SPECT single-photon emission computed tomography
- the cell-penetrating agents are administered in an effective regime meaning a dosage, route of administration and frequency of administration that delays the onset, reduces the severity, inhibits further deterioration, and/or ameliorates at least one sign or symptom of a disorder being treated. If a patient is already suffering from a disorder, the regime can be referred to as a therapeutically effective regime. If the patient is at elevated risk of the disorder relative to the general population but is not yet experiencing symptoms, the regime can be referred to as a prophylactically effective regime.
- therapeutic or prophylactic efficacy can be observed in an individual patient relative to historical controls or past experience in the same patient. In other instances, therapeutic or prophylactic efficacy can be demonstrated in a preclinical or clinical trial in a population of treated patients relative to a control population of untreated patients.
- Administration can be parenteral, intravenous, oral, subcutaneous, intra-arterial, intracranial, intrathecal, intraperitoneal, topical, intranasal or intramuscular.
- Some cell- penetrating agents can be administered into the systemic circulation by intravenous or subcutaneous administration. Attorney Docket Ref.
- the cell-penetrating agent formulation can be administered intravenously or subcutaneously in dosage ranges from about 0.5 mg/kg to about 30 mg/kg of the host body weight.
- dosages can be about 0.5 mg/kg body weight, about 1.0 mg/kg, about 1.5 mg/kg, about 2.0 mg/kg, about 4.0 mg/kg, about 5.0 mg/kg, about 8.0 mg/kg, about 10 mg/kg, about 15 mg/kg, about 16 mg/kg, about 20 mg/kg, about 24 mg/kg, about 25 mg/kg, or about 30 mg/kg body weight.
- the dosages can also be administered according to body surface area from about 0.5 mg/m 2 to about 500 mg/m 2 , for example, 0.5, 5, 10, 50, 100, 250 or 500 mg/m 2 .
- an amount of the cell-penetrating agent formulation sufficient to achieve the desired dosage for the individual patient is transferred from one or more vials to one or more intravenous bags containing a liquid (e.g., saline) and administered to the patient.
- a liquid e.g., saline
- Cell-penetrating agents are usually administered on multiple occasions. The frequency of administration depends on the half-life of the cell-penetrating agent in circulation, the condition of the patient and the route of administration among other factors.
- the frequency can be daily, weekly, monthly, quarterly, or at irregular intervals in response to changes in the patient’s condition or progression of the disorder being treated.
- An exemplary treatment regimen entails administration once per every two weeks, once a month, or once every 3 to 6 months.
- the number of dosages administered depends on whether the disorder is acute or chronic and the response of the disorder to the treatment.
- the dosing frequency can be adjusted depending on the pharmacokinetic profile of the antibody formulation in the patient. For example, the half-life of the cell-penetrating agent may warrant a two week frequency of dosing.
- 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 life of the patient.
- Normal levels of TDP-43 e.g., human TDP-43 can be determined in the brains of a representative sample of individuals in the general population who have not been diagnosed with a particular TDP-43 related disease (e.g., ALS) and are not considered at elevated risk of developing such disease (e.g., a representative sample of disease-free individuals under 50 years of age).
- a normal level can be recognized in an individual patient if a PET signal according to the present methods in a region of the brain in which TDP-43 aggregates (e.g., human TDP-43 aggregates) are known to develop is not different (within the accuracy of measurement) from the signal from a region of the brain in which it is known that such deposits Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT do not normally develop.
- TDP-43 aggregates e.g., human TDP-43 aggregates
- An elevated level in an individual can be recognized by comparison to the normal levels (e.g., outside mean and variance of a standard deviation) or simply from an elevated signal beyond experimental error in a region of the brain associated with TDP-43 aggregates (e.g., human TDP-43 aggregates) compared with a region not known to be associated with deposits.
- TDP-43 aggregates e.g., human TDP-43 aggregates
- the TDP-43 aggregates should preferably be determined in the same region(s) of the brain, these regions including at least one region in which TDP-43 aggregates associated with a particular disease (e.g., ALS) are known to form (e.g., in the cytoplasm).
- a patient having an elevated level of TDP-43 aggregates is a candidate for commencing immunotherapy.
- a decrease in the level of TDP-43 aggregates can be first seen as an indication that the treatment is having the desired effect.
- the observed decrease can be, for example, in the range of 1-100%, 1-50%, or 1-25% of the baseline value.
- Such effects can be measured in one or more regions of the brain in which deposits are known to form or can be measured from an average of such regions.
- TDP-43 aggregates e.g., human TDP-43 aggregates
- Maintenance of TDP-43 aggregates e.g., human TDP-43 aggregates
- TDP-43 aggregates at an approximately constant level or even a small increase in TDP-43 aggregates (e.g., human TDP- 43 aggregates) can also be an indication of response to treatment albeit a suboptimal response.
- kits comprising any of the cell- penetrating agents described herein and related materials, such as instructions for use (e.g., package insert).
- the instructions for use can contain, for example, instructions for administration of the cell-penetrating agent and optionally one or more additional agents.
- the cell-penetrating agents of the present disclosure further provides methods of detecting TDP-43 in a sample.
- the present disclosure provides a method of detecting TDP-43 in a sample comprising contacting a cell- penetrating agent of the present disclosure with the sample and detecting the binding of the cell- penetrating agent or the antibody to TDP-43.
- such methods can be an ex vivo or in vitro method.
- the sample is a biological sample derived from a subject (e.g., a human subject).
- the subject is a human.
- the subject is a patient having or at risk of having a TDP-43 related disease.
- samples such as cells, tissues, and/or organs that have been removed from the patient are exposed to an antibody or antigen-binding fragments described herein.
- the sample comprises cells derived from the patient and the cells are lysed prior to administration of the antibody or antigen-binding fragments described herein.
- Example 1 Humanized Anti-TDP-43 Antibodies Humanized anti-TDP-43 antibodies were generated from murine monoclonal antibody 13D3. Figure 1 shows an annotated version of the heavy chain variable domain and Figure 2 shows an annotated version of the light chain variable domain, respectively. Both the heavy Attorney Docket Ref.
- the partial heavy chain constant domain region is a IgG2a mouse constant domain.
- the partial light chain constant domain is a mouse kappa constant domain.
- Reference to amino acid substitutions described herein refers to the Kabat numbering system (See e.g., Kabat E.A., et. al, Sequences of Proteins of Immunological Interest (5 th edition). Bethesda, MD: National Institutes of Health (1991)). Briefly, protein sequences were identified in the Protein Data Bank (PDB) database (See, Deshpande et.
- PDB Protein Data Bank
- 5BK5 antibody is a human germline derived antibody and has the same canonical classes belonging to human germline IGHV3-48’03 (SEQ ID NO: 2) for the variable heavy chain domain and IGKV2-30*02 (SEQ ID NO: 25) for the variable light chain domain these sequences were used as a human acceptor framework. Accordingly, the framework regions of 5BK5 VH and 5BK5 VL were chosen as the acceptor sequences for the CDRs of 13D3. A model of the 13D3 CDRs grafted onto the respective human frameworks for VH and VL was built and used as guidance for further backmutations to increase binding specificity and reduce immunogenicity.
- hu13D3VHv1d amino acid sequences consisting of 5BK5 VH human frameworks and 13D3 CDRs are designated hu13D3VHv1d (SEQ ID NO: 20) and the amino acid sequences consisting of 5BK5 VL human framework and 13D3 VL CDRs are designated hu13D3VLv1d (SEQ ID NO: 47).
- Additional versions of hu13D3VH and hu13D3VL were designed to enable assessment of various framework residues for their contributions to antigen binding, thermostability, developability (e.g., deamination, oxidation, N-glycosylation, proteolysis, and aggregation) and immunogenicity.
- Heavy Chain Variable Domains hu13D3VHvd1 (SEQ ID NO: 20) consists of the CDR-H1, CDR-H2, and CDR-H3 loops of 13D3-VH grafted onto the framework of 5BK5 VH and reverts all framework substitutions at positions that are key for defining the Chothia canonical classes, are part of the Vernier zone, and localize to the VH/VL domain interface or contribute to structural stability.
- Hu13D3VHvd1 includes the following substitutions which are back mutations to the germline antibody and found most frequently: position L5V and T77S.
- hu13D3VHv2d (SEQ ID NO: 21) includes the following substitution: L78A.
- Leucine at position 78 is immunogenic as indicated by Immune Epitope Database (“IEDB”) Analysis. Therefore, deimmunization analysis predicts immunogenicity reduction with an alanine substitution at position 78.
- Hu13D3VHv3d (SEQ ID NO: 22) includes the following substitutions: G44R, S49A, and S74A. Arginine at position 44 makes de novo contacts with a G100D (glycine to aspartic acid) substitution made in the variable light chain domain thereby strengthening the heavy chain variable domain: light chain variable domain interface.
- arginine at position 44 forms a hydrogen bond and a salt bridge with G100D and also forms a hydrogen bond with F98 Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT in the variable light chain domain.
- Position 49 is a Vernier zone residue and substitution from serine to alanine was a back mutation to assess the impact on CDR conformation and binding.
- a serine to alanine substitution at position 74 is a germline substitution.
- Hu13D3VHv4d (SEQ ID NO: 23) includes various substitutions made in hu1353VHv1d, hu1353VHv2d, and hu1353VHv3d tried in combination.
- Light Chain Variable Domains hu13D3VLv1d (SEQ ID NO: 47) consists of the CDR-L1, CDR- L2, and CDR-L3 loops of 13D3VL grafted onto the framework of 5BK5 VL along with reverting all framework substitutions at positions that are key for defining the Chothia canonical classes, are part of the Vernier zone, and locate to the VH/VL domain interface.
- Hu1353VLv1d includes the following substitutions: V3Q, P15L, E17Q, L38Q, K39R, Q100D, and L104V. Substitution of valine for glutamine at position 3 reduces immunogenicity of the light chain variable domain.
- Substitution of leucine for proline at position 15 is a germline substitution.
- Substitution of glutamine for glutamic acid at position 17 is a rare substitution.
- Position 17 has significant surface exposure thereby contributing to a negative charge patch at the protein surface.
- Substitution to glutamine reduces antibody surface negative patches.
- Substitution of glutamine for leucine at position 38 was made since glutamine in mouse structural models forms inter-strand contacts within the light chain variable domain to maintain structural conformation. In contrast, a leucine reside cannot form the same contacts and therefore this back mutation increases conformational stability.
- Substitution of arginine for lysine at position 39 increases conformation stability by forming additional contacts with adjacent residues that a lysine residue cannot.
- Position 100 in the light chain variable domain is a located at the heavy chain variable domain and light chain variable domain interface, however, no interchain contacts are formed with the glutamine residue. Substitution to aspartic acid along with a concurrent substitution of arginine at position 44 in the heavy chain variable domain results in stronger do novo contacts and therefore increases thermal stability of the antibody. Substitution of valine for leucine position 104 is predicted to reduce immunogenicity.
- Hu13D3VLv2d (SEQ ID NO: 48) consists of the aforementioned substitutions described above in hu13D3VLv1d, but also includes the following substitution: L92A. Leucine at position Attorney Docket Ref.
- human Ig heavy chain AEX28899 (SEQ ID NO: 3) (GenBank: AEX28899) (Bowers et al., 2014) was chosen, again with the same canonical classes and belonging to human germline IGHV3-48’03. It is a member of Kabat human heavy subgroup 3.
- AEX28899 and ABC66863 antibodies are human germline derived antibodies that have the same canonical classes and belong to human germline IGHV3-48’03 (SEQ ID NO: 2) for the heavy chain variable domain and IGHV3-48*03 (SEQ ID NO: 25) for the light chain variable domain.
- the AEX28899 heavy chain variable domain and ABC66863 light chain variable domain sequences were used as human acceptor framework for the CDRs of 13D3.
- a model of the 13D3 CDRs grafted onto the respective human frameworks for VH and VL was built and used as a guidance for further backmutations.
- Humanized versions of AEX28899 heavy chain variable domain and ABC66863 light chain variable domain sequences were used as human acceptor framework for the CDRs of 13D3 and were designed to enable assessment of various framework residues for their contributions to antigen binding, thermostability, developability (e.g., deamination, oxidation, N-glycosylation, proteolysis, and aggregation) and immunogenicity.
- positions considered for substitution were based on a variety of factors including positions that define the canonical CDR conformations (See, Martin, A.C.R., 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)); positions within the Vernier zone (See, Foote J., and Winter, G., Antibody framework residues affecting the conformation of the hypervariable loops. J Mol Biol. 224(2):487-99 (1992)); positions that localize to the VH/VL domain interface (See, Léger O.J.P. and Saldanha, J).
- VL domain substitutions in SEQ ID NO: 26 I2V is a Vernier zone residue, valine is retained to preserve CDR conformation; L9S: leucine is predicted to be immunogenic, substitution with serine lowers predicted immunogenicity; P18Q: mouse antibody model glutamine forms a H-bond with K74 (LC) thereby stabilizing interchain interaction; R46L: is an interface and Vernier zone residue and therefore Leu is retained; A80S: serine lowers predicted immunogenicity; L92G: L92 is a CDR residue and is predicted to be immunogenic, a glycine residue is predicted to reduce immunogenicity; V94I: valine at position 94 is predicted to be low level immunogenic, isoleucine is predicted to reduce immunogenicity; and V94A: alanine is an alternative substitution that is predicted to lower immunogenicity.
- cytoplasmic aggregates of TDP-43 are found in many neurodegenerative diseases and in particular, ALS.
- the individual TDP-43 proteins found in the cytoplasmic aggregates are phosphorylated at serine residues at position 409 and/or Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT 410.
- a binding screening assay was established to determine whether 13D3 and humanized variants thereof would selectively bind to phosphorylated TDP-43 found in cytoplasmic aggregates.
- About 50 RU of biotin-phosphoTDP43 proteins were immobilized on a CM3 chip with NeutrAvidin.
- TDP-43 peptide (pS409/pS410) 23 amino acid TDP-43 peptide phosphorylated at positions 409 and 410
- SEQ ID NO: 83 23 amino acid TDP-43 peptide that was not phosphorylated at positions 409 or 410 was also tested between 12 nM and 1 ⁇ M.
- humanized version hu13D3H5L2 demonstrated 1:1 binding with TDP-43 peptide (pS409/pS410) at various concentrations (1.2345 nM; 3.703 nM; 11.111 nM; 33.33 nM; and 100 nM) which was similar to the 13D3 murine antibody (Figure 5). Furthermore, hu13D3H5L2 showed no affinity for the non-phosphorylated 22 amino acid TDP-43 peptide at 12.345 nM, 37.03 nM, 111.111 nM, 333.333 nM, and 1 ⁇ M (data not shown). Thermal stability of the antibodies was also assessed.
- Thermal stability analysis was performed with Different Scanning Calorimetry which characterizes the stability of a protein or other biomolecule (e.g., an antibody or antigen-binding fragment thereof).
- Antibodies were tested 2.66 mM (0.4 mg/mL) in 1xPBS at pH 7.4. Temperature range tested was from 25°C to 100°C.
- Binding data for chimeric 13D3 and humanized versions thereof is summarized in Tables 3 and 4. Tables 3 and 4 also summarize thermal stability data and immunogenecity data.
- the program identifies potential immunogenic regions within the protein sequence.
- the MHC class II tool predicts immunogenic regions using broad spectrum alleles (i.e., 26 reference alleles) for MHC class II in the human population.
- the software generates a series of 15 residue peptides which overlap in 10 residues. Each generated 15 residue peptide is predicted for binding to the 26 reference alleles.
- a percentile rank for each of the three methods (combinatorial library, SMM_align and Sturniolo) was generated by comparing each peptide’s score against the scores of five million random 15 residue peptides selected from the SWISSPROT database. The adjusted percentile rank is the percentile rank adjusted based on the frequency of peptide lengths.
- a low number percentile rank indicates high affinity.
- the median percentile rank of the three methods were then used to generate the rank for consensus method. By default, prediction result is collapsed to show only the Percentile Rank and Adjusted Rank.
- the maximum median percentile rank threshold is set at 20.
- Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT The EpiQuest tool, T-Scanner, ranges cytotoxic T lymphocyte (CTL) epitopes according to their predicted immunodominance.
- CTL cytotoxic T lymphocyte
- the immunodominance of epitopes is defined as their relative strength in functional assays related to the target kill or release of respective cytokines. These parameters indicate functionality of the T-epitope.
- This program is designed to analyze and sort according to their immunodominance the CTL peptide epitopes eluted from target cells. Generally, only a low number of peptide epitopes that bind to MHC class I have actual functional activity. Relative strength of a CTL (T) epitope is defined by the strength of its binding to MHC class I and T cell receptor (TCR) (in the context of MHCI).
- TCR T cell receptor
- FIG. 6A-C show frontotemporal dementia (“FTD”) brain tissue (Fig. 6A) and healthy brain tissue (Fig. 6C).
- FIG. 6B is an inset of Fig. 6A showing co-localization of 13D3 antibody with phosphorylated TDP-43 FTD-associated neuronal cytoplasmic aggregates. The data demonstrate that 13D3 antibody specifically binds to cytoplasmic aggregates in FTD brain tissue, but not in healthy brain tissue.
- FTD frontotemporal dementia
- FIG. 7A-C show 13D3 specifically binding cytoplasmic aggregates in a rNLS8 dox-suppressible model of TDP-43 proteinopathy.
- Example 4. Binding of Anti-TDP-43 Antibodies to Phosphorylated Cytoplasmic Aggregates of TDP-43 and Clear TDP-43 Aggregates in Transfected HEK Cells
- Figure 8A shows confocal microscopy images of HEK cells transiently transfected with GFP-2a-TDP43 [mNLS (R82L/K83Q) DCS (C173S/C175S)] or GFP only (2a is a self-cleaving peptide that releases the TDP-43 upon expression).
- FIG. 8B (left) is a graph showing cell count for HEK cells transfected with either GFP-2a-TDP43 or GFP alone. The data demonstrate approximately equivalent cell counts between the two populations. Further, Figure 8B (right) Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT shows pTDP-43 foci counts for HEK cells transfected with GFP-2a-TDP43 or GFP alone.
- FIG. 8C shows TDP-43 staining with either commercial antibodies or antibodies of the present disclosure including 13D3, 13C13, and 2D4.
- Cells were treated with antibodies for 24 hours and 24 hours later, cells were incubated again with 100 ug/ml antibodies for another 24 hours. Cells were then washed, fixed/permeabilized, and stained with anti-pTDP-43 antibodies, followed by AF647-conjugated anti-mouse secondary antibodies. Stained cells were imaged by high content imaging (Operetta system) with 40x water objective. Quantitative analyses were carried out with Harmony software.
- Antibodies 13D3, 13C13, and 2D4 detect overexpressed mislocalized TDP-43 in HEK cells.
- the top row shows transfection with GFP-2a-TDP-43 where the phosphorylated TDP43 does not include a nuclear localization signal (i.e., phosphorylated TDP43 remains in the cytoplasm), and the bottom row shows transfection with a GFP only construct.
- the data demonstrate that TDP-43 foci are formed only in HEK cell transfected with GFP-2a-TDP-43.
- Various control antibodies were included in the assay including pTDP-43 (Cosmo)+, pTDP-43 (1D3)+, Total TDP-43 (PT), and 3B12 (ED)+.
- the control antibodies verify TDP-43 aggregation within the cytoplasm.
- Antibodies 13D3, 13C13, and 2D4 were similarly tested and demonstrated similar binding to cytoplasmic aggregates of phosphorylated TDP-43.
- Example 5 Characteristics of Humanized Anti-TDP-43 Cell Penetrating Agents Binding data, immunogenicity, and thermal stability for anti-TDP-43 cell-penetrating agents were assessed as previously described in Example 3. Binding data, immonogenciity scores, and thermal stability data for cell-penetrating agents comprising murine 13D3 and humanized versions thereof are summarized in Tables 5 and 6.
- Table 5 Stability, IHC, and Target Engagement Data for Anti-TDP-43 CPAs Yi ld T °C f' b IHC R ki T t nt Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT h 13D3Hd2-Ld1_CMIP4 183.6 71.34 5+ Yes h 13D3Hd2-Ld2_CMIP4 196.3 69.93 __ ___ Table 6: Binding Data (Bivalent Analyte Mode) for Anti-TDP-43 CPAs Cell Penetrating Agent ka1 (1/Ms) kd1 (1/s) KD1(M) KD1(nM) KD1(pM) , e highly stable, having comparable stability to the corresponding antibody.
- the anti- TDP-43 CPAs in Table 5 have melting temperatures of 69.9 °C to 77.5 °C and are expressed in high yield. Further, the anti-TDP-43 CPAs demonstrated target engagement by immunohistochemistry (IHC) analysis and a cellular assay.
- Table 6 provides binding data for exemplary anti-TDP-43 CPAs, showing that these CPAs bind phospho-TDP-43 with sub- nanomolar KD values.
- the data in Tables 5 and 6 demonstrate that anti-TDP-43 CPAs of the present disclosure retain strong the binding to phospho-TDP-43, thermal stability, and in vitro target engagement of the parent antibodies. Example 6.
- FIGS. 9-22 demonstrate internalization of and/or target engagement by anti-TDP-43 cell- penetrating agents of the present disclosure.
- references to “M-Lyco,” Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT Lycotoxin,” “ML” and the like refer to CPAs comprising L17E_M-lycotoxin linked to a 13D3 antibody (murine, chimeric, or humanized).
- FIGS. 9-22 references to “M-Lyco,” Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT Lycotoxin,” “ML” and the like refer to CPAs comprising L17E_M-lycotoxin linked to a 13D3 antibody (murine, chimeric, or humanized).
- references to other CIMs and/or CMIPs refer to CPAs comprising the corresponding CIM linked to a 13D3 antibody (murine, chimeric, or humanized). Unless otherwise specified (e.g., by HC), the CIM is linked to the 13D3 antibody via the c-terminus of the light chain.
- HEK cells were transiently transfected with GFP-2a-TDP43 [mNLS (R82L/K83Q) DCS (C173S/C175S)].
- cells were incubated with 100 ug/ml cell penetrating agents including a cell internalization moiety and an anti-TDP-43 antibody for 4 hours. The cells were then washed, fixed/permeabilized, and followed by AF647-conjugated anti-mouse secondary antibodies. Stained cells were imaged by high content imaging (Operetta system) with 40x water objective. Quantitative analyses were carried out with Harmony software.
- Figure 9A shows the percentage of CPA-positive cells following incubation with m13D3 CPAs including different CIMs (i.e., TAT, M-Lycotoxin_L17E (LC), M-Lycotoxin_L17E (HC), PEPTH (HC)) or under various control conditions (i.e., untagged, isotype, and vehicle).
- CIMs i.e., TAT, M-Lycotoxin_L17E (LC), M-Lycotoxin_L17E (HC), PEPTH (HC)
- HC dasheavy chain
- LC light chain
- Labels to “M-Lycotoxin” in FIG. 9 refer to CPAs comprising M-Lycotoxin_L17E linked at the C-terminus of either the light chain (LC) or heavy chain (HC) of the m13D3 antibody.
- HEK cells were transiently transfected with GFP-2a-TDP43 [mNLS (R82L/K83Q) DCS (C173S/C175S)] or no plasmid (e.g., untransfected) as a negative control. 24 hours later the cells were incubated with 100 ug/ml of antibodies (e.g., CPAs) for another 24 hours. Cells were then washed, fixed/permeabilized, and stained with anti-pTDP-43 antibodies, followed by AF647-conjugated anti-mouse secondary antibodies. Stained cells were imaged by high content imaging (Operetta system) with 40x water objective. Quantitative analyses were carried out with Harmony software. Attorney Docket Ref.
- FIG. 10A shows the number of pTDP-43 foci per well area.
- Labels to “M-Lycotoxin” in FIG. 10 refer to CPAs comprising M-Lycotoxin_L17E linked at the C-terminus of the light chain of the m13D3 antibody.
- the data demonstrate that cells treated with different m13D3 CPAs had a lower number of foci per well area as compared to cells treated with untagged m13D3 (e.g., no cell internalizing module). As expected, no foci were observed in untransfected cells.
- Figure 10B shows the mean focus intensity for pTDP-43 foci.
- Figure 10E shows the mean focus area for p-TDP-43 foci, demonstrating that cells treated with different m13D3 CPAs had a lower mean area intensity compared to those treated with the untagged m13D3; very low focus intensity was observed with untransfected cells
- Figure 11A shows the number of pTDP-43 foci per well area.
- the data demonstrate 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 foci per well area as compared to those treated with the untagged m13D3. Labels to “M-Lycotoxin” in FIG.
- FIG. 11 refer to CPAs comprising M-Lycotoxin_L17E linked at the C-terminus of either the light chain (LC) or heavy chain (HC) of the m13D3 antibody.
- Figure 11B shows consistent cell count per well across all cell populations tested.
- Figure 11C shows the number of pTDP-43 foci normalized by cell count which demonstrates that cells treated with different m13D3 CPAs had a lower number of foci per cell as compared to cells treated with the untagged m13D3. As expected, no foci were observed in untransfected cells.
- Figure 11D shows the mean focus area for pTDP-43 foci.
- FIG. 12A-D are graphs showing the results of cells incubated with m13D3 M- Lycotoxin [17E] CPAs at different concentrations under the same experimental conditions
- FIG. 11 refer to CPAs comprising M- Lycotoxin_L17E linked at the C-terminus of either the light chain (LC) or heavy chain (HC) of the m13D3 antibody.
- the cell internalizing module was located on either the heavy chain or light chain of the m13D3 antibody.
- Cells were also incubated with untagged m13D3, IgG isotype control, and a PBS control. Untransfected HEK cells were used as a negative control.
- Figure 12A shows pTDP-43 total foci area per well area. The data show a concentration- dependent reduction in total foci area for cells treated with the m13D3 M-Lycotoxin_L17E CPAs.
- Labels to “M-Lycotoxin” in FIG. 12 refer to CPAs comprising M-Lycotoxin_L17E linked at the C-terminus of either the light chain (LC) or heavy chain (HC) of the m13D3 antibody.
- the cells treated with the 13D3 antibody, IgG isotype control, and PBS showed higher levels of foci as compared to cells treated with the m13D3 m-Lycotoxin [L17E] CPAs. Untransfected cells had no foci (data not shown).
- Figure 12B shows consistent cell count per well across all cell populations tested and no concentration dependent reduction in cell viability for cells transfected with the m13D3 M-Lycotoxin CPAs.
- Figure 12C shows the pTDP- 43 foci count which demonstrates a concentration-dependent increase in foci count for the m13D3 M-Lycotoxin CPAs, with significantly higher number of foci compared to control.
- Figure 12D shows the pTDP-43 mean focus size, demonstrating a concentration-dependent decrease in mean focus size for the m13D3 M-Lycotoxin CPAs, with significantly lower mean focus size compared to control.
- Figures 12A-D demonstrate that the m13D3 M- Lycotoxin CPAs interfere with foci aggregation in a concentration-dependent manner.
- Figure 13 shows graphs of untransfected HEK cells (left) or transfected with GFP-2a- TDP43 [mNLS (R82L/K83Q) DCS (C173S/C175S)] (right) incubated with either m13D3 antibody or an M-Lycotoxin m13D3 CPA for 24 hours and subjected to the XTT metabolic assay to assess cell viability.
- Labels to “M-Lycotoxin” in FIG. 13 refer to CPAs comprising M- Lycotoxin_L17E linked at the C-terminus of the light chain (LC) of the m13D3 antibody.
- Figures 14A-E are graphs showing cells incubated with different m13D3 CPAs and untagged m13D3 antibody. Untransfected HEK cells were used as a negative control. The data in Figures 14A-E was generated with the same experimental conditions described in Figures 11A-D above. More specially, Figure 14A shows the sum of pTDP-43 foci per well area. Labels to “ML-13D3” in FIG. 14 refer to CPAs comprising M-Lycotoxin_L17E linked at the C-terminus of the light chain (LC) of the m13D3 antibody.
- LC light chain
- Labels to “CMIP1-5” in FIG. 14 refer to CPAs comprising CMIP1, CMIP2, CMIP3, CMIP4 or CMIP5 linked at the C-terminus of the light chain (LC) of the m13D3 antibody.
- LC light chain
- Figure 14C shows consistent cell count per well across all cell populations tested.
- Figure 14D shows the number of p-TDP-43 foci (normalized by cell count).
- the data demonstrates that cells treated with different m13D3 CPAs had a higher number of foci per cell as compared to cells treated with untagged m13D3 antibody. As expected, no foci were observed with untransfected cells.
- Figure 14E shows the mean focus area for pTDP-43 foci.
- Figures 15A-D are graphs showing the results of cells were incubated with either m13D3 m-Lycotoxin [L17E] CPAs or m13D3 CMIP4 CPA at different concentrations; cells were alternatively incubated with the untagged m13D3 or an IgG isotype control; untransfected HEK cells were used as a negative control.
- the data in Figures 15A-D was generated with the same experimental conditions described in Figures 11A-D above. Labels to “13D3-ML” in FIG.
- FIG. 15 Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT refer to CPAs comprising M-Lycotoxin_L17E linked at the C-terminus of the light chain (LC) of the m13D3 antibody.
- Labels to “13D3-CMIP4” in FIG. 15 and FIG. 16 refer to CPAs comprising CMIP4 linked at the C-terminus of the light chain (LC) of the m13D3 antibody.
- Figure 15A shows the total number of foci p-TDP-43, demonstrating a concentration- dependent reduction in total foci area for cells treated with the 13D3 m-Lyco and 13D3 CMIP4 CPAs.
- FIG. 15B shows consistent cell count per well across all cell populations tested, and no concentration dependent reduction in cell viability for the m13D3 m-Lycotoxin CPAs.
- Figure 15C shows the p-TDP-43 foci number normalized by cell count, demonstrating a concentration-dependent increase in foci count for the 13D3 m-Lycotoxin and 13D3 CMIP4 CPAs, with significantly higher number of foci compared to control.
- Figure 15D shows the p-TDP-43 mean focus area, demonstrating a concentration-dependent decrease in mean focus area for the 13D3 m-Lycotoxin and 13D3 CMIP4 CPAs, with significantly lower mean focus area compared to control.
- Figures 15A-D demonstrates that the 13D3 m- Lycotoxin and 13D3 CMIP4 CPAs interfere with foci aggregation in a concentration-dependent manner.
- cells were incubated with the m13D3 CMIP4 CPA and the untagged m13D3 antibody in either acetate buffer or PBS.
- the data in Figures 16A-E was generated with the same experimental conditions described in Figures 11A-D above.
- Figure 16A shows the pTDP-43 foci per well area, demonstrating that cells treated with the m13D3 CMIP4 CPA had a lower number of foci per well area compared to those treated with the untagged m13D3; no change in cell-penetrating activity was observed between PBS and acetate.
- Figure 16B shows the mean focus intensity for pTDP-43 foci, demonstrating that cells treated with the m13D3 CMIP4 CPA had a lower mean focus intensity compared to those treated with the untagged m13D3; no change in cell-penetrating agent activity was observed between PBS and acetate.
- Figure 16C shows consistent cell count per well across all cell populations tested.
- Figure 16D shows the number of p-TDP-43 foci normalized by cell count, demonstrating that cells treated with the m13D3 CMIP4 CPA had a higher number of foci per cell compared to those treated with the untagged m13D3; no foci were observed with untransfected cells.
- Figure 16E shows the mean focus area for p-TDP-43 foci, demonstrating Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT that cells treated with the m13D3 CMIP4 CPA had a lower mean area intensity compared to those treated with the untagged m13D3 Example 8.
- FIG. 17A shows confocal microscopy images of cells treated with the untagged ch13D3 antibody (left) and the ch13D3 m-Lycotoxin_L17E CPA (right). Foci are shown in white, the 13D3 antibody is shown in light grey, and nuclei are shown in dark grey.
- FIG 17A demonstrates significant colocalization between the pTDP43 foci and the 13D3 antibody for cells treated with the ch13D3 m-Lycotoxin CPA, with little colocalization observed for cells treated with the ch13D3 antibody.
- Figure 17B is a graph showing the percentage of pTDP43 colocalized with the ch13D3 antibody. The data demonstrate significantly greater co-localization for cells treated with the ch13D3 m- Lycotoxin CPA ( ⁇ 50-60%) as compared to the cells treated with the ch13D3 antibody ( ⁇ 15- 20%).
- Figures 17A-B demonstrates that anti-TDP-43 CPAs of the present disclosure are internalized by cells and bind to intracellular p-TDP-43.
- Figures 18A-D are graphs showing anti-TDP-43 CPAs of the present disclosure ((i.e., chimeric 13D3 CPAs (ch13D3 m-Lyco CPA, ch13D3 CMIP4 with LALA mutation, ch13D3 CMIP4 with H310-H435Q mutations), the untagged ch13D3 antibody, and an hIgG isotype control) are internalized by cells and bind to intracellular p-TDP-43 (i.e., cells transfected with GFP-2a-TDP43 as described herein).
- Labels to “M-Lyco” in FIG. 18 refer to CPAs comprising M-Lycotoxin_L17E linked at the C-terminus of the light chain (LC) of the m13D3 antibody.
- Figure 18A provides a graph showing the percentage of p-TDP-43 colocalized with the ch13D3 antibody for ch13D3 CPAs as well as ch13D3 antibody, Isotype, and PBS controls using Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT both confocal (left) and non-confocal (right) microscopy.
- FIG 18A demonstrates significantly greater co-localization for cells treated with the various ch13D3 CPAs ( ⁇ 50-60%) compared to the cells treated with the ch13D3 antibody ( ⁇ 15-20%).
- Cells treated with isotype control and PBS demonstrated no co-localization.
- Figure 18B provides a graph showing the average number of 13D3 antibody spots per cell for various ch13D3 CPAs, as well as ch13D3 antibody, isotype, and PBS controls.
- Figure 18B demonstrates that the ch13D3 CPAs result in significantly more 13D3 spots per cell ( ⁇ 2.5 – 3 spots per cell) than the controls.
- Figure 18C provides a graph showing the average spot size for the 13D3 antibody spots for various ch13D3 CPAs, as well as ch13D3 antibody, isotype, and PBS controls.
- Figure 18C demonstrates that the ch13D3 CPAs result in significantly smaller 13D3 spots per cell than the control.
- Figure 18D provides a graph showing the average spot size for the 13D3 antibody spots for various ch13D3 CPAs, as well as ch13D3 antibody, isotype, and PBS controls.
- Figure 18D demonstrates that the ch13D3 CPAs result in significantly smaller (corrected for spot intensity) 13D3 spots per cell than the control.
- Figures 18A-D demonstrates that anti-TDP-43 CPAs of the present disclosure are internalized by cells and bind to intracellular p-TDP-43.
- Example 9. Internalization and Clearance of Phosphorylated Cytoplasmic Aggregates of TDP-43 with Anti-TDP-43 Cell Penetrating Agents
- Figures 19A-D are graphs showing internalization and colocalization of phosphorylated cytoplasmic aggregates of pTDP-43 (i.e., cells transfected with GFP-2a-TDP43 as described herein) with novel cell internalizing modules and humanized anti-TDP-43 antibodies. The data shown in Figures 19A-D was generated under the same experimental conditions as described in Figures 11A-D above.
- Labels to “ch13D3-ML” in FIG. 19 refer to CPAs comprising M- Lycotoxin_L17E linked at the C-terminus of the light chain (LC) of the m13D3 antibody.
- Figure 19A provides a graph showing the percentage of pTDP43 colocalized with the humanized 13D3 antibody for humanized 13D3 CPAs as well as ch13D3 and h13D3 antibody controls.
- Figure 19A demonstrates significantly greater co-localization for cells treated with the various h13D3 CPAs ( ⁇ 60-70%) compared to the cells treated with the h13D3 antibody ( ⁇ 40%).
- Figure 19B shows consistent cell count per well across all cell populations tested.
- Figure 19C is a graph showing the 13D3 spot area per well for pTDP43 colocalized with the humanized 13D3 antibody observed in cells treated with humanized 13D3 CPAs, as well as IgG isotype, ch13D3, Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT and h13D3 antibodies controls.
- FIG 19C demonstrates significant increases in total co- localization area for cell treated with humanized 13D3 CPAs of the present disclosure.
- Figure 19D provides a graph showing the number of 13D3 co-localization spots per cell observed in cells treated with humanized 13D3 CPAs, as well as IgG isotype, ch13D3, and h13D3 antibodies controls.
- Figure 19D demonstrates significant increases in number of co-localization spots for cells treated with humanized 13D3 CPAs of the present disclosure.
- Figures 19A-D show internalization and colocalization of phosphorylated cytoplasmic aggregates of pTDP-43 with novel cell internalizing modules and humanized anti-TDP-43 antibodies.
- FIGs 20A-C are graphs showing internalization and colocalization of phosphorylated cytoplasmic aggregates of TDP-43 (i.e., cells transfected with GFP-2a-TDP43 as described herein) in glioblastoma cells with CPAs including a cell internalizing module and an anti-TDP- 43 antibody. Labels to “M-lycotoxin” in FIGs.
- 20A-C refer to CPAs comprising M- Lycotoxin_L17E linked at the C-terminus of the light chain (LC) of the m13D3 antibody.
- LC light chain
- U251 cells were transiently transfected with GFP-2a-TDP43 [mNLS (R82L/K83Q) DCS (C173S/C175S)]. 24 hours later the cells were incubated with 100 ug/ml antibodies (e.g., CPAs) for another 24 hours. The cells were then washed, fixed/permeabilized, and stained with anti- pTDP-43 antibodies, followed by AF647-conjugated anti-mouse secondary antibodies.
- Figure 20A is a graph showing the total foci area for U251 glioblastoma cells treated with either m13D3 m-Lycotoxin CPA or m13D3 antibody alone (e.g., untagged). The data demonstrate a reduction in total foci area which was significantly reduced in glioblastoma cells treated with m13D3 m-Lycotoxin CPA as compared to cells treated with the m13D3 antibody.
- Figure 20B is a graph showing the mean foci size for U251 glioblastoma cells treated with either m13D3 m-Lycotoxin CPA or m13D3 antibody alone (e.g., untagged).
- the data demonstrate a reduction in mean foci size which was significantly reduced in glioblastoma cells treated with m13D3 m-Lycotoxin CPA compared to those cells treated with Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT the m13D3 antibody.
- Figure 20C is a graph showing the total foci count for U251 glioblastoma cells treated with either m13D3 m-Lycotoxin CPA or m13D3 antibody.
- the data demonstrate an increase in total foci count which was significantly increased in glioblastoma cells treated with m13D3 m-Lycotoxin CPA as compared to those cells treated with the m13D3 antibody.
- Figures 20A-C demonstrates that anti-TDP-43 CPAs of the present disclosure are internalized by glioblastoma cells that bind to intracellular p-TDP-43 and therefore disrupt TDP- 43 aggregation.
- FIGS 21A-B and 22A-F are graphs showing that CPAs including a cell internalizing module and an anti-TDP-43 antibody are internalized and colocalization of phosphorylated cytoplasmic aggregates in primary rat cortical neurons. Briefly, primary rat cortical neurons cells (DIV15) were incubated with 50 ug/ml antibodies for 2 hours.
- Figure 21A shows images of primary rat cortical neuronal cells treated with IgG isotype control (top, left), m13D3 (top, right), m13D3 m-Lycotoxin CPA (bottom, left), and m13D3CMIP4 CPA (bottom, right).
- Figure 21A demonstrates significant internalization of the m13D3 CPAs (depicted by white spots inside the cells, bottom panels) and little to no internalization of the isotype controls (top panels).
- Figure 21B is a graph showing the total of foci area per well which demonstrates little to no internalization of the isotype control and the 13D3 antibody and substantial internalization by the m13D3 m-Lycotoxin_L17E CPA and m13D3 CMIP4 CPA (labeled as 13D3-ML).
- Figure 22A is a graph showing the total number of 13D3 spots per cell for the DIV15 rat neuronal cells incubated with hIgG isotype control, m13D3 antibody, m13D3 m-Lycotoxin CPA, or m13D3 CMIP4 CPA.
- Figure 22A demonstrates higher numbers of 13D3 spots per cell for cells treated with m13D3-Cterm-LC-M-Lycotoxin_L17E CPA (labeled as “M-Lyco” in FIG. 22) Attorney Docket Ref.
- FIG. 22 is a graph showing the total area of 13D3 spots per well for the DIV15 rat neuronal cells incubated with hIgG isotype control, m13D3 antibody, m13D3 m-Lyco CPA, or m13D3 CMIP4 CPA.
- Figure 22B demonstrates higher total spot area per well for cells treated with m13D3 m-Lycotoxin CPA and m13D3 CMIP4 CPAs, than for cells treated with either the IgG isotype control or the m13D3 antibody.
- Figure 22C is a graph showing the mean spot size for 13D3 spots in the DIV15 rat neuronal cells incubated with hIgG isotype control, m13D3 antibody, m13D3 m-Lycotoxin CPA, or m13D3 CMIP4 CPA.
- Figure 22C demonstrates higher mean spot size for cells treated with m13D3 m-Lycotoxin CPA and m13D3 CMIP4 CPAs, than for cells treated with either the IgG isotype control or the m13D3 antibody.
- Figure 22D is a graph showing spot integrated intensity for 13D3 spots in the DIV15 rat neuronal cells incubated with hIgG isotype control, m13D3 antibody, m13D3 m-Lycotoxin CPA, or m13D3 CMIP4 CPA.
- Figure 22D demonstrates higher spot integrated intensity for cells treated with m13D3 m-Lycotoxin CPA and m13D3 CMIP4 CPAs, than for cells treated with either the IgG isotype control or the m13D3 antibody.
- Figure 22E is a graph showing the percentage of 13D3 spots colocalized with EEA1 (Early Endosome Antigen 1) in the DIV15 rat neuronal cells incubated with hIgG isotype control, m13D3 antibody, m13D3 m-Lycotoxin CPA, or m13D3 CMIP4 CPA.
- EEA1 Early Endosome Antigen 1
- FIG 22E demonstrates ⁇ 10-20% of 13D3 spots colocalized with EEA1 in cells treated with m13D3 m-Lycotoxin CPA and m13D3 CMIP4 CPAs, with no co- localization observed for cells treated with either the IgG isotype control or the m13D3 antibody.
- Figure 22F shows consistent cell count per well across all cell populations tested. Collectively, Figures 21A-D and 22A-F show that cell internalizing modules of the present disclosure are internalized by primary rat cortical neurons.
- S is position 27A L is position 27B V is position 27C H is position 27D S is position 27E, respectively.
- Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT 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 CDR
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Abstract
The present disclosure provides cell-penetrating agents comprising a cell internalization 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 with TDP-43-related diseases, including Amyotrophic Lateral Sclerosis (ALS).
Description
Attorney Docket Ref. No.: 50877-0047WO1 // Client Ref: 794-PCT CELL PENETRATING AGENTS AND USES THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63/538,599, filed on September 15, 2023, the content of which are hereby incorporated by reference in its entirety. SEQUENCE LISTING This application contains a Sequence Listing that has been submitted electronically as an XML file named “50887-0047WO1.XML.” The XML file, created on August 28, 2024, is 204,800 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety. TECHNICAL FIELD This disclosure relates to the technical fields of immunology and medicine. BACKGROUND Transactive response DNA binding protein 43 (“TDP-43”) is a predominantly nuclear protein that is involved in RNA splicing, trafficking, 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 expressed ubiquitously in all cell types, it has been shown to be highly expressed in the neuroepithelium, which contains all CNS progenitors for neurons and glia. Moreover, TDP-43 has been shown to specifically bind numerous RNAs in neuronal cells. TDP-43 cytoplasmic aggregates (also known as inclusion bodies) are associated with several neurodegenerative diseases, disorders, or conditions. Specifically, TDP-43 cytoplasmic aggregates and/or misfolding of TDP-43 have been associated with neurodegenerative diseases including Amyotrophic Lateral Sclerosis (“ALS”), frontotemporal dementia (“FTD” or “FTLD- TDP-43”), limbic-predominant age-related TDP-43 encephalopathy (“LATE”), Alzheimer's disease, multisystem proteinopathy, and chronic traumatic encephalopathy.
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT ALS is the most common motor neuron disorder in adults ultimately resulting in the loss of motor neurons that control voluntary muscles. ALS has an incidence of about 1-2/100,000 and a prevalence of about 4-6/100,000 cases each year. ALS results in the progressive degeneration of both upper and lower motor neurons which generally leads to death primarily due to respiratory failure in three to five years after diagnosis. Typical symptoms include stiff muscles, muscle twitches, gradual muscle weakness, and muscle wasting. Around half of all people suffering from ALS have difficulties thinking and/or behavioral symptoms and about 15% develop frontotemporal dementia. Pathologically, abnormal aggregations of TDP-43 protein are seen in up to 97% of ALS patients (Nguyen, H.P., et al., ALS Genes in the Genomic Era and their Implications for FTD, Trends in Genetics, 34(6): 404+423 (2018)). At present, there is no known cure for ALS. Thus, there is an unmet need for therapies that improve health status, including the risk of mortality and/or enhancing the quality of life in patients with TDP-43 related diseases, including ALS. SUMMARY The present disclosure relates to cell-penetrating agents (“CPAs”) that include a cell internalization module (“CIM”) and an antibody that specifically binds to human TDP-43, compositions comprising these cell-penetrating agents, and methods of using these cell- penetrating agents to treat TDP-43 related diseases, including Amyotrophic Lateral Sclerosis (ALS). Thus, provided herein are cell-penetrating agent that include (i) a cell internalizing module and (ii) an antibody that specifically binds to Transactive response DNA binding Protein of 43 kD (TDP-43). In some embodiments, the CIM includes a Cell Membrane Internalizing Peptide (CMIP). In some embodiments, the CIM includes a wild-type M-lycotoxin peptide. In some embodiments, the CIM includes an M-lycotoxin derivative. In some embodiments, the CIM includes a Penetain amino acid sequence or a derivative thereof. In some embodiments, the CIM includes a Pepth amino acid sequence or a derivative thereof. In some embodiments, the CIM includes a polyarginine amino acid sequence. In some embodiments, the CIM includes more than one polyarginine amino acid sequence. In some embodiments, the CIM includes three
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT polyarginine amino acid sequences. In some embodiments, the CIM includes a TAT amino acid sequence. In some embodiments, the CIM includes more than one TAT amino acid sequence. In some embodiments, the CIM includes three TAT amino acid sequences. In some embodiments, the CIM includes 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 includes one or more histidine residues. In some embodiments, the CIM includes a polypeptide having an amino acid sequence selected from one of: SEQ ID NO: 176-184, SEQ ID NO: 192, and SEQ ID NO: 193. In some embodiments, the CIM includes one or more spacer regions. In some embodiments, at least one of the one or more spacer regions includes one or more amino acid residues. In some embodiments, at least one of the one or more spacer regions includes one or more glycine residues. In some embodiments, at least one of the one or more spacer regions includes an amino acid sequence selected from any one of SEQ ID NOs: 200-203. In some embodiments, each of the one or more spacer regions includes an amino acid sequence selected from any one of SEQ ID NOs: 200-203. In some embodiments, the CIM includes a polypeptide having an amino acid sequence selected from any one of SEQ ID NOs: 176-193. In some embodiments, the CIM is a polypeptide having an amino acid sequence selected from any one of SEQ ID NOs: 176-193. In some embodiments, the CIM is covalently linked to the antibody. In some embodiments, the CIM is non-covalently linked to the antibody. In some embodiments, the cell- penetrating agent includes a linker connecting 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. In some embodiments, the linker is a non-cleavable linker. In some embodiments, the linker includes a polypeptide. In some embodiments, the linker includes one or more glycine residues. In some embodiments, the linker includes a polypeptide including an amino acid sequence selected from any one of SEQ ID NOs: 194-199. In some embodiments, the linker is a polypeptide including an amino acid sequence selected from any one of SEQ ID NOs: 194-199. In some embodiments, the antibody is linked to the C-terminus of the CIM. In some embodiments, the antibody is linked to the N-terminus of the CIM.
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT In some embodiments, the antibody competes for binding to TDP-43 (e.g., human TDP- 43) with: an antibody including a heavy chain variable domain of SEQ ID NO: 1 and a light chain variable domain of SEQ ID NO: 24; an antibody including a heavy chain variable domain of SEQ ID NO: 63 and a light chain variable domain of SEQ ID NO: 65; an antibody including a heavy chain variable domain of SEQ ID NO: 67 and a light chain variable domain of SEQ ID NO: 69; an antibody including a heavy chain variable domain of SEQ ID NO: 71 and a light chain variable domain of SEQ ID NO: 73; an antibody including a heavy chain variable domain of SEQ ID NO: 75 and a light chain variable domain of SEQ ID NO: 77; or an antibody including a heavy chain variable domain of SEQ ID NO: 79 and a light chain variable domain of SEQ ID NO: 81. In some embodiments, the antibody binds to the same epitope on TDP-43 (e.g., human TDP-43) as an antibody including a heavy chain variable domain of SEQ ID NO: 1 and a light chain variable domain of SEQ ID NO: 24; an antibody including a heavy chain variable domain of SEQ ID NO: 63 and a light chain variable domain of SEQ ID NO: 65; an antibody including a heavy chain variable domain of SEQ ID NO: 67 and a light chain variable domain of SEQ ID NO: 69; an antibody including a heavy chain variable domain of SEQ ID NO: 71 and a light chain variable domain of SEQ ID NO: 73; an antibody including a heavy chain variable domain of SEQ ID NO: 75 and a light chain variable domain of SEQ ID NO: 77; or an antibody including a heavy chain variable domain of SEQ ID NO: 79 and a light chain variable domain of SEQ ID NO: 81. In some embodiments, the antibody that specifically binds to TDP-43 (e.g., human TDP- 43), including three light chain CDRs and three heavy chain CDRs of a mouse antibody characterized by a heavy chain variable domain including SEQ ID NO: 1 and a light chain variable domain including SEQ ID NO: 24. In some embodiments, the CDRs are of a definition selected from the group of Kabat, Chothia, Kabat/Chothia Composite, AbM, and Contact. In some embodiments, the antibody includes a humanized mature heavy variable domain including: a heavy chain CDR1 as defined by Kabat/Chothia Composite, including SEQ ID NO: 49; a heavy chain CDR2 as defined by Kabat, including SEQ ID NO: 51; and a heavy chain CDR3 as defined by Kabat or Chothia, including SEQ ID NO: 52; and a humanized mature light chain variable domain including the three Kabat light chain CDRs of SEQ ID NOs: 53-55.
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT In some embodiments, the humanized mature heavy variable domain includes a sequence that is at least 80% identical to any one of SEQ ID NOs: 4-23 and the humanized mature light chain variable domain includes a sequence that is at least 80% identical to any one of SEQ ID NOs: 27-48. In some embodiments, the humanized mature heavy variable domain includes a sequence that is at least 85% to any one of SEQ ID NOs: 4-23 and the humanized mature light chain variable region includes a sequence that is at least 85% identical to any one of SEQ ID NOs: 27-48. In some embodiments, the humanized mature heavy variable domain includes a sequence that is at least 90% identical, to any one of SEQ ID NOs: 4-23 and the humanized mature light chain variable region includes a sequence that is at least 90% identical, to any one of SEQ ID NOs: 27-48. In some embodiments, the humanized mature heavy variable domain includes a sequence that is at least 95% identical to any one of SEQ ID NOs: 4-23 and the humanized mature light chain variable domain includes a sequence that is at least 95% identical to any one of SEQ ID NOs: 27-48. In some embodiments, at least one of the following positions in the humanized heavy chain variable domain is occupied by the amino acid as specified: 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. In some embodiments, at least one of the following positions in the humanized heavy chain variable domain is occupied by the amino acid as specified: 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. In some embodiments, at least one of the following positions in the humanized heavy chain variable domain is occupied by the amino acid as specified: 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. 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 the amino acid as specified: 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.
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT In some embodiments, at least one of the following positions in the humanized light chain variable domain is occupied by the amino acid as specified: 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. In some embodiments, at least one of the following positions in the humanized light chain variable domain is occupied by the amino acid as specified: V3 is occupied by Q or A100 is occupied by D or R. In some embodiments, at least one of the following positions in the humanized light chain variable domain is occupied by the amino acid as specified: 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. In some embodiments, 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 amino acid as specified: T80 is occupied by A or L92 is occupied by A. In some embodiments, at least one of the following positions in the humanized heavy chain variable domain is occupied by the amino acid as specified: 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 the amino acid as specified: 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. In some embodiments, the humanized antibody has increased thermal stability as compared to a reference antibody including a heavy chain variable domain including SEQ ID NO: 1 and a light chain variable domain including SEQ ID NO: 24. In some embodiments, the antibody includes a heavy chain variable domain including: a heavy chain CDR1, as defined by Kabat/Chothia Composite, including SEQ ID NO: 49 or SEQ ID NO: 50; a heavy chain CDR2, as defined by Kabat, including SEQ ID NO: 51; a heavy chain CDR3, as defined by Kabat or Chothia, including SEQ ID NO: 52; a light chain CDR1, as defined by Kabat, including SEQ ID NO: 53; a light chain CDR2, as defined by Kabat, including
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT SEQ ID NO: 54; and a light chain CDR3, as defined by Kabat, including one of SEQ ID NOs: 55-61. In some embodiments, the heavy chain CDR1, as defined by Kabat/Chothia Composite, includes SEQ ID NO: 49; the heavy chain CDR2, as defined by Kabat, includes SEQ ID NO: 51; the heavy chain CDR3, as defined by Kabat or Chothia, includes SEQ ID NO: 52; the light chain CDR1, as defined by Kabat, includes SEQ ID NO: 53; the light chain CDR2, as defined by Kabat, includes SEQ ID NO: 54; and the light chain CDR3, as defined by Kabat, includes SEQ ID NO: 55 or SEQ ID NO: 61. In some embodiments, the heavy chain variable domain includes a sequence that is at least 95% identical to any one of SEQ ID NOs: 4-23. In some embodiments, the heavy chain variable domain includes a sequence that is at least 95% identical to any one of: SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 23. In some embodiments, the heavy chain variable domain includes a sequence that is at least 98% identical to any one of: SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 23. In some embodiments, the heavy chain variable domain includes a sequence of SEQ ID NO: 20. In some embodiments, the heavy chain variable domain includes a sequence of SEQ ID NO: 21. In some embodiments, the heavy chain variable domain includes a sequence of SEQ ID NO: 23. In some embodiments, the light chain variable domain includes a sequence that is at least 95% identical to any one of: SEQ ID NOs: 27-48. In some embodiments, the light chain variable domain includes a sequence that is at least 95% identical to SEQ ID NO: 47 or SEQ ID NO: 48. In some embodiments, the light chain variable domain includes a sequence that is at least 98% identical to SEQ ID NO: 47 or SEQ ID NO: 48. In some embodiments, the light chain variable domain includes SEQ ID NO: 47. In some embodiments, the light chain variable domain includes SEQ ID NO: 48. In some embodiment, the antibody includes a heavy chain variable domain and a light chain variable domain including: a heavy chain CDR1 including SEQ ID NO: 84; a heavy chain CDR2 including SEQ ID NO: 85; a heavy chain CDR3 including SEQ ID NO: 86; a light chain CDR1 including SEQ ID NO: 87; a light chain CDR2 including SEQ ID NO: 88; and a light chain CDR3 including SEQ ID NO: 89. In some embodiments, the heavy chain variable domain comprises a sequence that is at least 95% identical to SEQ ID NO: 63. In some embodiments, the heavy chain variable domain
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT comprises a sequence that is at least 98% identical to SEQ ID NO: 63. In some embodiments, the heavy chain variable domain comprises a sequence of SEQ ID NO: 63. In some embodiments, the light chain variable domain comprises a sequence that is at least 95% identical to SEQ ID NO: 65. In some embodiments, the light chain variable domain comprises a sequence that is at least 98% identical to SEQ ID NO: 65. In some embodiments, the light chain variable domain comprises a sequence of SEQ ID NO: 65. In some embodiments, the antibody includes a heavy chain variable domain and a light chain variable domain including: a heavy chain CDR1 including SEQ ID NO: 90; a heavy chain CDR2 including SEQ ID NO: 91; a heavy chain CDR3 including SEQ ID NO: 92; a light chain CDR1 including SEQ ID NO: 93; a light chain CDR2 including SEQ ID NO: 94; and a light chain CDR3 including SEQ ID NO: 95. In some embodiments, the heavy chain variable domain comprises a sequence that is at least 95% identical to SEQ ID NO: 67. In some embodiments, the heavy chain variable domain comprises a sequence that is at least 98% identical to SEQ ID NO: 67. In some embodiments, the heavy chain variable domain comprises a sequence of SEQ ID NO: 67. In some embodiments, the light chain variable domain comprises a sequence that is at least 95% identical to SEQ ID NO: 69. In some embodiments, the light chain variable domain comprises a sequence that is at least 98% identical to SEQ ID NO: 69. In some embodiments, the light chain variable domain comprises a sequence of SEQ ID NO: 69. In some embodiments, the antibody includes a heavy chain variable domain and a light chain variable domain including: a heavy chain CDR1 including SEQ ID NO: 96; a heavy chain CDR2 including SEQ ID NO: 97; a heavy chain CDR3 including SEQ ID NO: 98; a light chain CDR1 including SEQ ID NO: 99; a light chain CDR2 including SEQ ID NO: 100; and a light chain CDR3 including SEQ ID NO: 101. In some embodiments, the heavy chain variable domain comprises a sequence that is at least 95% identical to SEQ ID NO: 71. In some embodiments, the heavy chain variable domain comprises a sequence that is at least 98% identical to SEQ ID NO: 71. In some embodiments, the heavy chain variable domain comprises a sequence of SEQ ID NO: 71. In some embodiments, the light chain variable domain comprises a sequence that is at least 95% identical to SEQ ID NO: 73. In some embodiments, the light chain variable domain
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT comprises a sequence that is at least 98% identical to SEQ ID NO: 73. In some embodiments, the light chain variable domain comprises a sequence of SEQ ID NO: 73. In some embodiments, the antibody includes a heavy chain variable domain and a light chain variable domain including: a heavy chain CDR1 including SEQ ID NO: 102; a heavy chain CDR2 including SEQ ID NO: 103; a heavy chain CDR3 including SEQ ID NO: 104; a light chain CDR1 including SEQ ID NO: 105; a light chain CDR2 including SEQ ID NO: 106; and a light chain CDR3 including SEQ ID NO: 107. In some embodiment, the heavy chain variable domain comprises a sequence that is at least 95% identical to SEQ ID NO: 75. In some embodiments, the heavy chain variable domain comprises a sequence that is at least 98% identical to SEQ ID NO: 75. In some embodiments, the heavy chain variable domain comprises a sequence of SEQ ID NO: 75. In some embodiments, the light chain variable domain comprises a sequence that is at least 95% identical to SEQ ID NO: 77. In some embodiments, the light chain variable domain comprises a sequence that is at least 98% identical to SEQ ID NO: 77. In some embodiments, the light chain variable domain comprises a sequence of SEQ ID NO: 77. In some embodiments, the antibody includes a heavy chain variable domain and a light chain variable domain including: a heavy chain CDR1 including SEQ ID NO: 108; a heavy chain CDR2 including SEQ ID NO: 109; a heavy chain CDR3 including SEQ ID NO: 110; a light chain CDR1 including SEQ ID NO: 111; a light chain CDR2 including SEQ ID NO: 112; and a light chain CDR3 including SEQ ID NO: 113. In some embodiments, the heavy chain variable domain comprises a sequence at least 95% identical to SEQ ID NO: 79. In some embodiments, the heavy chain variable domain comprises a sequence at least 98% identical to SEQ ID NO: 79. In some embodiments, the heavy chain variable domain comprises a sequence of SEQ ID NO: 79. In some embodiments, the light chain variable domain comprises a sequence that is at least 95% identical to SEQ ID NO: 81. In some embodiments, the light chain variable domain comprises a sequence that is at least 98% identical to SEQ ID NO: 81. In some embodiments, the light chain variable domain comprises a sequence of SEQ ID NO: 81. 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.
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT In some embodiments, the antigen-binding antibody fragment is a Fab fragment, a Fab’2 fragment, or a single chain Fv. In some embodiments, the antibody is an intact antibody. In some embodiments, the antibody has a human IgG1 isotype. In some embodiments, the heavy chain variable domain is fused to a heavy chain constant region and the light chain variable domain is fused to a light chain constant region. In some embodiments, the heavy chain constant region is a mutant form of a natural human heavy chain constant region which has reduced binding to an Fcγ receptor relative to the natural heavy chain constant region. In some embodiments, the heavy chain constant region is of IgG1 isotype. In some embodiments, the antibody has at least one mutation in a constant region. In some embodiments, the at least one mutation reduces complement fixation or activation by the constant region. In some embodiments, the at least one mutation is at one or more positions of: 241, 264, 265, 270, 296, 297, 318, 320, 322, 329, and 331 by EU numbering. In some embodiments, the antibody has an alanine at positions 318, 320, and 322 by EU numbering. 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) as 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 as 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 as compared to unphosphorylated TDP-43 (e.g., unphosphorylated human TDP-43). In some embodiments, phosphorylated TDP-43 (e.g., phosphorylated human TDP-43) includes 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) includes phosphorylation of both S409 and S410. 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 include phosphorylated aggregates of TDP-43 (e.g.,
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT phosphorylated aggregates of human TDP-43). In some embodiments, the antibody does not substantially bind unphosphorylated TDP-43 (e.g., unphosphorylated human TDP-43). In some embodiments, the cell-penetrating agent comprises a polypeptide sequence that is at least 95% identical to a sequence selected from any one of: 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. In some embodiments, the cell-penetrating agent comprises a polypeptide sequence that is at least 98% identical to a sequence selected from any one of: 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 cell-penetrating agent comprises a polypeptide sequence selected from any one of: 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 cell-penetrating agent comprises a polypeptide sequence that is at least 95% identical to a sequence selected from any one of 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:
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT 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. In some embodiments, the cell-penetrating agent comprises a polypeptide sequence that is at least 98% identical to a sequence selected from any one of 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. In some embodiments, the cell-penetrating agent comprises a polypeptide sequence selected from any one of 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. In some embodiments, the antibody is conjugated to a therapeutic, cytotoxic, cytostatic, immunomodulatory, neurotrophic, or neuroprotective agent. In some embodiments, the heavy chain does not comprise a C-terminal lysine residue. Also provided herein are pharmaceutical compositions including any of the cell- penetrating agents described herein and a pharmaceutically acceptable carrier. Also provided herein are nucleic acids encoding at least a portion of any of the cell- penetrating agent described herein. In some embodiments, the nucleic acids encode the heavy chain variable domain and/or the light chain variable domain of the antibody. In some embodiments, the nucleic acid encodes for any of the CIMs described herein. Also provided herein are vectors including a nucleic acid encoding a mature heavy chain variable domain and a light chain variable domain operably linked to one or more regulatory sequences to effect expression in a mammalian cell of any one of the cell-penetrating agents described herein.
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT In some embodiments, the one or more regulatory sequences include one or more of a enhancer, ribosome binding site, transcription termination signal, and promoter, optionally, where the promoter is a eukaryotic promoter. In some embodiments, the nucleic acid is codon- optimized for expression in a host cell. Also provided herein are host cells transformed with any of the vectors described herein. Also provided herein are host cells including any of the nucleic acids described herein. Also provided herein are methods of delivering the antibody that specifically binds to TDP-43 into a cell comprising contacting any of the cell-penetrating agents described herein with the cell, thereby resulting in the internalization into the cell of, at a minimum, an antigen-binding fragment of the antibody. In some embodiments, the method further comprises transfer of, at a minimum, an antigen-binding fragment of the antibody, to the cytosol of the cell. Also provided herein are methods of binding an intracellular TDP-43 protein in a cell that include contacting the cell-penetrating agent of any one of claims 1-135 with the cell, thereby resulting in the internalization of and transfer to the cytosol of, at a minimum, an antigen-binding fragment of the antibody. Also provided herein are methods of binding an intracellular TDP-43 protein in a cell that include: contacting the cell-penetrating agent of any one of claims 1-135 with the cell, thereby resulting in the internalization of and transfer to the cytosol of, at a minimum, an antigen-binding fragment of the antibody; and binding, at a minimum, an antigen-binding fragment of the antibody, to the intracellular TDP-43 protein. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is in vitro. In some embodiments, the cell is in a subject. Also provided herein are methods of inhibiting or reducing aggregation of TDP-43 (e.g., human TDP-43) in a subject having or at risk of developing a TDP-43-related disease, including administering to the subject an effective amount of any of the cell-penetrating agents described herein, thereby inhibiting or reducing aggregation of TDP-43 (e.g., human TDP-43) in the subject. Also provided herein are methods of treating or effecting prophylaxis of a TDP-43- related disease in a subject, including administering a therapeutically effective amount of any of the cell-penetrating agents described herein thereby treating or effecting prophylaxis of the TDP- 43-associated disease.
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT In some embodiments, the TDP-43-related disease is amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD-TDP), primary lateral sclerosis, and progressive muscular atrophy, and Parkinson’s disease. In some embodiments, the TDP-43-related disease is ALS. Also provided herein are methods of detecting TDP-43 deposits (e.g., human TDP-43 deposits) in a subject having or at risk of developing a TDP-43-related disease, including administering to a subject any of the cell-penetrating agents described herein, and detecting the antibody bound to TDP-43 in the subject. In some embodiments, the antibody is administered by intravenous injection into the body of the subject. Also provided herein are methods of detecting TDP-43 in a sample obtained from a patient having or at risk of developing a TDP-43 related disease, comprising contacting the same with any of the cell-penetrating agents described herein, and detecting the binding of the antibody to the TDP-43 in the sample. In some embodiments, the antibody is labeled. In some embodiments, the antibody is labeled with a fluorescent label, a paramagnetic label, or a radioactive label. In some embodiments, the radioactive label is detected using positron emission tomography (PET) or single-photon emission computed tomography (SPECT). All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, patent application, or item of information was specifically and individually indicated to be incorporated by reference. To the extent publications, patents, patent applications, and items of information incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material. Where values are described in terms of ranges, it should be understood that the description includes the disclosure of all possible sub-ranges within such ranges, as well as specific numerical values that fall within such ranges irrespective of whether a specific numerical value or specific sub-range is expressly stated. BRIEF DESCRIPTION OF THE DRAWINGS
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT The following drawings 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 manner. Like reference symbols in the drawings indicate like elements. Figure 1 shows an annotated version of the murine antibody 13D3 heavy chain variable domain. Figure 2 shows an annotated version of the murine antibody 13D3 light chain variable domain. Figure 3 is a graph showing binding data of chimeric 13D3 antibody and humanized versions thereof to phosphorylated human TDP-43 peptide. Figure 4 is a graph showing binding data of chimeric 13D3 antibody and humanized versions thereof to phosphorylated human TDP-43 peptide. Figure 5 is a graph showing binding data of a humanized version of 13D3 antibody, hu13D2Hd5Ld2, to phosphorylated human TDP-43 peptide. Figures 6A-C show immunohistochemical images of frontotemporal dementia (“FTD”) brain tissue (Figs. 6A and 6B) and healthy brain tissue (Fig. 6C) stained with the 13D3 antibody. Fig. 6B is an inset of Fig. 6A showing co-localization of 13D3 antibody with phosphorylated human TDP-43 FTD-associated neuronal cytoplasmic aggregates. Figures 7A-C are immunohistochemical images of brain tissue from a rNLS8 dox- suppressible model of TDP-43 proteinopathy stained with the 13D3 antibody. The data show that the 13D3 antibody binds cytoplasmic aggregates in a rNLS8 dox-suppressible model of TDP-43 proteinopathy. Figures 8A-C show the results of HEK cells transfected with GFP-2a-TDP43 or GFP only. Figure 8A shows staining for pTDP-43, GFP, and nuclei in the transfected cells. Figure 8B is a graph showing cell count. Figure 8C shows immunohistochemical images of HEK cells that show the ability of antibodies 13D3, 13C13, and 2D4 to detect overexpressed mislocalized human TDP-43 in HEK cells. Figures 9A-B are graphs showing the percentage of CPA-positive cells following incubation with murine 13D3 (m13D3) CPAs (Figure 9A) and the number of CPA-positive spots per cell following incubation with m13D3 CPAs (Figure 9B). Figures 10A-E are graphs showing the results of transfected HEK cells with different m13D3 CPAs including the number of foci per well area (Figure 10A), the mean focus intensity
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT (Figure 10B), cell count per well (Figure 10C), the number of TDP-43 foci normalized by cell count (Figure 10D), and the mean focus area (Figure 10E). Figures 11A-D are graphs showing the results of transfected HEK cells with different m13D3 CPAs including the number of foci per well area (Figure 11A), cell count (Figure 11B), foci count (Figure 11C), and mean focus size (Figure 11D). Figures 12A-D are graphs showing the results of transfected HEK cells with different m13D3 CPAs including total foci area per well area (Figure 12A), cell count (Figure 12B), pTDP-43 foci count (Figure 12C), and mean focus size (Figure 12D). Figure 13 are graphs showing percentage cell death with either untransfected HEK cells (left) or cells transfected with GFP-2a-TDP43 (right). Figures 14A-E are graphs showing internalization and colocalizationof phosphorylated cytoplasmic aggregates of TDP-43 in HEK cells. Specifically, the graphs show the sum of pTDP-43 foci per well area (Figure 14A), mean focus intensity (Figure 14B), cell count (Figure 14C), number of p-TDP-43 foci (Figure 14D), and mean focus area (Figure 14E). Figures 15A-D are graphs showing the results of HEK cells incubated with either m13D3 m-Lycotoxin [L17E] CPAs or m13D3 CMIP4 CPA including total foci area (Figure 15A), cell count (Figure 15B), p-TDP-43 foci number (Figure 15C), and mean focus area (Figure 15D). Figures 16A-E are graphs showing the results of HEK cells transfected with either m13D3 CMIP4 CPA or untagged antibody in either acetate buffer or PBS, including foci per well area (Figure 16A), mean focus intensity (Figure 16B), cell count (Figure 16C), number of p- TDP-43 foci (Figure 16D), and mean focus area (Figure 16E). Figures 17A-B show the results of HEK cells transfected with GFP-2A-TDP43 and chimeric 13D3 CPAs including colocalization imaging (Figure 17A) and a graph showing percentage of p-TDP-43 colocalized with GFP-2A-TDP43 and chimeric 13D3 CPAs (Figure 17B). Figures 18A-D are graphs showing internalization data of anti-TDP-43 CPAs including, percentage of pTDP-43 colocalized with anti-TDP-43 CPAs (Figure 18A), the average number of 13D3 antibody spots per cell (Figure 18B), average spot size (Figure 18C), and average spot size (corrected for spot intensity) (Figure 18D). Figures 19A-D are graphs showing internalization and colocalization of phosphorylated cytoplasmic aggregates of pTDP-43 including percentage of pTDP-43 colocalized with
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT humanized 13D3 antibody, chimeric 13D3 antibody and controls (Figure 19A), cell count (Figure 19B), spot area per well (Figure 19C), and CPA spots per cell (Figure 19D). Figures 20A-C are graphs showing internalization and colocalization of phosphorylated cytoplasmic aggregates of TPD-43 in glioblastoma cells including total foci area (Figure 20A), mean foci size (Figure 20B), and total foci count (Figure 20C). Figures 21A-B show internalization and colocalization of phosphorylated cytoplasmic aggregates of TDP-43 in primary rat cortical neuron cells. Figure 21A shows images of primary rat cortical neuron cells treated with various CPAs. Figure 21B is a graph showing total of foci area per well. Figures 22A-F are graphs showing the results of transfected primary rat cortical neuron cells including the total number of 13D3 spots per cell (Figure 22A), total area of 13D3 spots per well (Figure 22B), mean spot size (Figure 22C), spot integrated intensity (Figure 22D), percentage of 13D3 colocalized with Early Endosome Antigen 1 (EEA1) (Figure 22E), and cell count (Figure 22F). DETAILED DESCRIPTION This disclosure provides systems and methods for the delivery of antibodies into a cell. Specifically, the present disclosure describes cell-penetrating agents that comprise a cell internalization module and an antibody that specifically binds TDP-43 (e.g., human TDP-43) or aggregates of TDP-43 (e.g., aggregates of human TDP-43, including phosphorylated aggregates of TDP-43). Also provided are pharmaceutical compositions including these cell-penetrating agents and a pharmaceutically acceptable carrier, nucleic acids and/or vectors encoding these cell-penetrating agents, and host cells expressing the aforementioned nucleic acids and/or vectors. I. Definitions The term “antibody” includes intact antibodies and antigen-binding fragments thereof. Typically, fragments compete with the intact antibody from which they were derived for specific binding to the target including separate heavy chains, light chains Fab, Fab’, F(ab’)2, F(ab)c, Dabs, nanobodies, and Fv. Fragments can be produced by recombinant DNA techniques, or by
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT enzymatic or chemical separation of intact immunoglobulins. The term “antibody” also includes a bispecific or multispecific antibody and/or a humanized antibody. A bispecific or bifunctional or multifunctional antibody is an artificial hybrid antibody having two or more different heavy/light chain pairs and two or more different binding sites (see, e.g., Songsivilai and Lachmann, Clin. Exp. Immunol., 79:315-321 (1990); Kostelny et al., J. Immunol., 148:1547-53 (1992)). As used herein a “Cell-Penetrating Agent” (also referred to herein as a “CPA”) refers to an agent (e.g., a molecule and/or molecular complex) capable of entering a cell (e.g., a mammalian cell in vitro and/or in vivo). In some embodiments, the CPA enters the cell and is transferred to the cytosol following internalization by the cell. In some embodiments, the CPA comprises a Cell Internalizing 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 the transfer of the CPA the cytosol. In some embodiments, the CPA further comprises an anti- TDP-43 antibody that is 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 in relevant conditions (e.g., blood plasma). In some embodiments, the CPA further comprises a linker connecting the CIM to the anti-TDP-43 antibody. In various cases, linkers can be cleavable or non-cleavable and/or can connect the CIM to the anti-TDP-43 antibody covalently or non- covalently. In some embodiments, the CPA comprising the anti-TDP-43 antibody has enhanced cell penetration compared to a reference anti-TDP-43 antibody that is not part of a CPA. In some embodiments, the CPA comprises two or more anti-TDP-43 antibodies and/or two or more CIMs (e.g., the CPA comprising a dendrimer linked to a plurality of CIMs and/or anti-TDP-43 antibodies). Non-limiting features and examples of cell-penetrating agents are described herein. As used herein a “Cell Internalizing Module” (also referred to herein as a “CIM”) refers to a portion of the CPA that facilitates the internalization of the CPA (and by extension, the anti- TDP-43 antibody) by the cell. In various embodiments, the CIM may utilize one or more cellular internalization processes, including both active and passive cellular internalization, to effect internalization. Exemplary processes include, without limitation, endocytosis (e.g., Receptor
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT 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 Internalizing Peptide (CMIP), a small molecule ligand (e.g., vitamin, fatty acids, integrin binding ligand, etc.), a portion of an antibody (e.g., an scFv portion that binds an internalizing cell surface target), or a decoy receptor ligand (e.g., a cytokine or derivative thereof). In some embodiments, the CIM comprises a Cell- Membrane Internalizing Peptide (CMIP). Non-limiting features and examples of cell internalizing modules are described herein. As used herein a “Cell Membrane Internalizing Peptide” (also referred to herein as a CMIP”), is a sequence of three or more naturally occurring or non-naturally-occurring amino acids that, when covalently or non-covalently linked to an anti-TDP-43 antibody, results in the internalization into a mammalian cell of, at a minimum, the anti-TDP-43 antibody or an active fragment of the anti-TDP-43 antibody. In various embodiments, the CMIP may utilize one or more cellular internalization processes, including both active and passive cellular internalization, to effect internalization. Exemplary processes include, without limitation, endocytosis and membrane translocation. In some embodiments, the CMIP interacts with the cell membrane, and/or a cell surface antigen, leading to internalization of the CPA or a portion thereof (e.g., a portion comprising the anti-TDP-43 antibody or a functional portion thereof) via an endocytic vesicle. In some embodiments, the CMIP further facilitates the transfer of the CPA or portion thereof out of the endocytic vesicle and into the cytoplasm of the cell. In some cases, the CMIP further interacts with the membrane of the endocytic vesicle, leading to vesicle rupture and endosomal escape of the CPA or portion thereof into the cytosol. Thus, in some embodiments, the CMIP facilitates both the internalization of the anti-TDP-43 antibody or functional portion thereof into the cell, as well as transfer of the anti-TDP-43 antibody or functional portion thereof into the cytosol. Non-limiting examples of CMIPs include, for example, cationic peptides (including, for example, M-lycotoxin, TAT peptides, pentetratin, and polyarginine peptides, as well as derivatives thereof), amphipathic peptides (including, for example, MPG peptides, Pep-1 peptides, transportan peptides, as well as derivative thereof), and proline-rich peptides (including, for example, Bac7 peptides and derivatives thereof). In some embodiments, CMIPs include cell-penetrating peptides and derivatives thereof, including, for example, those described in I. Ruseska and A. Zimmer, Internalization mechanisms of cell-penetrating peptides,
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT BEILSTEIN J. NANOTECHOL. 202; 11:101-123. Non-limiting features and examples of CMIPs are described herein. The term “epitope” refers to a site on an antigen to which an antibody binds. An epitope can be formed from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of one or more proteins. Epitopes formed from contiguous amino acids (also known as linear epitopes) are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding (also known as conformational epitopes) are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation. Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols, in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed. (1996). Antibodies that recognize the same or overlapping epitopes can be identified in a simple immunoassay showing the ability of one antibody to compete with the binding of another antibody to a target antigen. The epitope of an antibody can also be defined X-ray crystallography of the antibody bound to its antigen to identify contact residues. Alternatively, two antibodies have the same epitope if all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other. Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other. The term “humanized immunoglobulin” or “humanized antibody” refers to an immunoglobulin or antibody that includes at least one humanized immunoglobulin or antibody chain (i.e., at least one humanized light or heavy chain). The term “humanized immunoglobulin chain” or “humanized antibody chain” (i.e., a “humanized immunoglobulin light chain” or “humanized immunoglobulin heavy chain”) refers to an immunoglobulin or antibody chain (i.e., a light or heavy chain, respectively) having a variable region that includes a variable framework region substantially from a human immunoglobulin or antibody and complementarity determining regions (CDRs) (e.g., at least one CDR, preferably two CDRs, more preferably three CDRs) substantially from a non-human immunoglobulin or antibody, and further includes constant regions (e.g., at least one constant region or portion thereof, in the case of a light chain, and preferably three constant regions in the case of a heavy chain). The term “humanized
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT variable region” (e.g., “humanized light chain variable region” or “humanized heavy chain variable region”) refers to a variable region that includes a variable framework region substantially from a human immunoglobulin or antibody and complementarity determining regions (CDRs) substantially from a non-human immunoglobulin or antibody. Competition between antibodies is determined by an assay in which an antibody under test inhibits specific binding of a reference antibody to a common antigen (see, e.g., Junghans et al., Cancer Res. 50: 1495, 1990). A test antibody competes with a reference antibody if an excess of a test antibody (e.g., at least 2x, 5x, l0x, 20x or l00x) inhibits binding of the reference antibody by at least 50% as measured in a competitive binding assay. Some test antibodies inhibit binding of the reference antibody by at least 75%, 90%, or 99%. Antibodies identified by competition assay (competing antibodies) include antibodies binding to the same epitope as the reference antibody and antibodies binding to an adjacent epitope sufficiently proximal to the epitope bound by the reference antibody for steric hindrance to occur. As used herein a “linker” refers a chemical moiety that does not have catalytic or therapeutic activity in a mammalian cell that is used to covalently link two different functional molecules (e.g., a cell internalization module and an anti-TDP-43 antibody). For example, a linker can be a peptide of about 3 amino acids to about 25 amino acids (e.g., about 3 amino acids to about 20, or about 3 amino acids to about 12 amino acids). In other examples, a linker can be a bond (e.g., an amide bond, an ester bond, an ether bond, and a disulfide bond). In some examples, a linker can comprise a pair of affinity domains (e.g., a first domain of the pair of affinity domains can be interleukin-15 and a second domain of the pair of affinity domains can be a sushi domain of interleukin-15 receptor alpha). In some examples, a linker is a glycine residue followed by a serine residue (GS). In some examples, a linker is three consecutive glycine residues (e.g., GGG). In some examples, a linker is any one of SEQ ID NOs: 194-199. The term “pharmaceutically acceptable” means that the carrier, diluent, excipient, or auxiliary is compatible with the other ingredients of the formulation and not substantially deleterious to the recipient thereof. As used herein a “M-lycotoxin derivative” is a peptide comprising three or more amino acids (naturally-occurring or non-naturally-occurring) that was designed based on a starting M- lycotoxin peptide. In some embodiments, the “M-lycotoxin derivative” is a polypeptide having,
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT e.g., between 60% and 99% sequence homology to wild-type M-lycotoxin. Additional non- limiting aspects and examples of M-lycotoxin derivatives are described herein. As used herein, the term “macromolecule” refers to a molecule having either a molecular weight of at least 5 kDa and/or a hydrodynamic radius of at least 1.0 nm. As used herein, the term “macromolecules” includes biomolecules, organic polymers, and organometallic complexes. As used herein a “spacer” or “spacer region” refers to an amino acid that does not have catalytic or therapeutic activity in a mammalian cell. For example, a spacer can be a peptide of 1 amino acid 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 examples, one or more spacer regions are included in the cell internalizing module. For example, a spacer region can separate amino acids within a cell internalizing module, e.g., a spacer can be disposed after the first amino acid of a CIM. In some examples, a spacer can be disposed before the final amino acid of a CIM. In some examples, a CIM can have two or more spacer regions (e.g., two spacer regions, three space sequences, four spacer regions, five spacer regions or more). In some examples, a spacer region is a single glycine residue. In some examples, a spacer region is a pair of glycine residues. In some examples, a spacer region is three glycine residues. In some examples, a spacer region is four glycine residues. In some examples, a spacer region is four glycine residues followed by a serine residue. In some examples, a spacer region is any one of SEQ ID NOs: 200-203. The term “TDP-43-related disease” means a disease or disorder that is characterized and/or mediated at least in part, either directly or indirectly, by formation of TDP-43 aggregates and/or mislocalization of TDP-43. Non-limiting examples of TDP-43-related diseases are described herein. The term “patient” or “subject” includes human and other mammalian subjects (e.g., human) that receive either prophylactic or therapeutic treatment. An individual is at increased risk of a disease if the subject has at least one known risk factor (e.g., genetic, biochemical, family history, and situational exposure) placing individuals with that risk factor at a statistically significant greater risk of developing the disease than individuals without the risk factor. The term “biological sample” refers to a sample of biological material within or obtainable from a biological source, for example a human or mammalian subject. Such samples
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT can be organs, organelles, tissues, sections of tissues, bodily fluids, peripheral blood, blood plasma, blood serum, cells, molecules such as proteins and peptides, and any parts or combinations derived therefrom. The term biological sample can also encompass any material derived by processing the sample. Derived material can include cells or their progeny. Processing of the biological sample may involve one or more of filtration, distillation, extraction, concentration, fixation, inactivation of interfering components, and the like. The term “control sample” refers to a biological sample not known or suspected to include TDP-43 affected regions, or at least not known or suspect to include diseased regions of a given type. Control samples can be obtained from individuals not afflicted with the TDP-43- related disease. Alternatively, control samples can be obtained from patients afflicted with the TDP-43-related disease. Such samples can be obtained at the same time as a biological sample thought to comprise the TDP-43-related disease or on a different occasion. A biological sample and a control sample can both be obtained from the same tissue. Preferably, control samples consist essentially or entirely of normal, healthy regions and can be used in comparison to a biological sample thought to comprise TDP-43-related disease-affected regions. Preferably, the tissue in the control sample is the same type as the tissue in the biological sample. Preferably, the TDP-43-related disease-affected cells thought to be in the biological sample arise from the same cell type (e.g., neurons or glia) as the type of cells in the control sample. For purposes of classifying amino acids substitutions as conservative or nonconservative, 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 influencing chain orientation): Gly, Pro; and Group VI (aromatic side chains): Trp, Tyr, Phe. Conservative substitutions involve substitutions between amino acids in the same class. Non-conservative substitutions constitute exchanging a member of one of these classes for a member of another. Percentage sequence identities are determined with antibody sequences maximally aligned by the Kabat numbering convention. After alignment, if a subject antibody region (e.g., the entire mature variable region of a heavy or light chain) is being compared with the same region of a reference antibody, the percentage sequence identity between the subject and reference antibody regions is the number of positions occupied by the same amino acid in both
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT the subject and reference antibody region divided by the total number of aligned positions of the two regions, with gaps not counted, multiplied by 100 to convert to percentage. Unless otherwise apparent from the context, the term “about” encompasses insubstantial variations, such as values within a standard margin of error of measurement (e.g., SEM) of a stated value. The phrase “substantially from a human immunoglobulin or antibody” means that, when aligned to a human immunoglobulin or antibody amino sequence for comparison purposes, the region shares at least 80-90%, preferably 90-95%, more preferably 95-99% identity (i.e., local sequence identity) with the human framework or constant region sequence, allowing, for example, for conservative substitutions, consensus sequence substitutions, germline substitutions, backmutations, and the like. The introduction of conservative substitutions, consensus sequence substitutions, germline substitutions, backmutations, and the like, is often referred to as “optimization” of a humanized antibody or chain. The phrase “substantially from a non-human immunoglobulin or antibody” or “substantially non-human” means having an immunoglobulin or antibody sequence 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. Accordingly, all regions or residues of a humanized immunoglobulin or antibody, or of a humanized immunoglobulin or antibody chain, except possibly the CDRs, are substantially identical to the corresponding regions or residues of one or more native human immunoglobulin sequences. The term “corresponding region” or “corresponding residue” refers to a region or residue on a second amino acid or nucleotide sequence which occupies the same (i.e., equivalent) position as a region or residue on a first amino acid or nucleotide sequence, when the first and second sequences are optimally aligned for comparison purposes. II. TDP-43 As described herein TDP-43 is a predominantly nuclear protein that is involved in RNA splicing, trafficking, stabilization, and ultimately regulation of gene expression. More specifically, TDP-43 is a multi-domain heterogeneous ribonucleoprotein (hnRNP). Proper function of TDP-43 is essential for the regulation of hundreds of mRNA transcripts to which it binds. One of TDP-43’s primary functions is the regulation of splicing mRNA transcripts, however, TDP-43 is also involved in different mechanisms of RNA processing and transport.
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT For example, TDP-43 is an inhibitor of cryptic exon inclusion and regulates the alternative polyadenylation of >1,000 genes. TDP-43 can form ribonucleoprotein granules in different cell types, including Cajal bodies and paraspeckles in the nucleus and is recruited to mRNA transport granules in neurons. Various neurodegenerative diseases are associated with cytoplasmic aggregates of TDP-43 (e.g., TDP-43-related diseases) as described herein. Unless otherwise apparent from the context, reference to TDP-43 means a 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): MSEYIRVTEDENDEPIEIPSEDDGTVLLSTVTAQFPGACGLRYRNPVSQCMRGVRLVEGI LHAPDAGWGNLVYVVNYPKDNKRKMDETDASSAVKVKRAVQKTSDLIVLGLPWKTT EQDLKEYFSTFGEVLMVQVKKDLKTGHSKGFGFVRFTEYETQVKVMSQRHMIDGRWC DCKLPNSKQSQDEPLRSRKVFVGRCTEDMTEDELREFFSQYGDVMDVFIPKPFRAFAFV TFADDQIAQSLCGEDLIIKGISVHISNAEPKHNSNRQLERSGRFGGNPGGFGNQGGFGNS RGGGAGLGNNQGSNMGGGMNFGAFSINPAMMAAAQAALQSSWGMMGMLASQQNQS GPSGNNQNQGNMQREPNQAFGSGNNSYSGSNSGAAIGWGSASNAGSGSGFNGGFGSS MDSKSSGWGM TDP-43 can be phosphorylated at one or more amino acids including the serine at position 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, e.g., Gruijs da Silva, L.A., et al., Disease-linked TDP-43 hyperphosphorylation suppresses TDP-43 condensation and aggregation, The EMBO Journal, 41: e108443 (2022)). Unless otherwise apparent from context, reference to TDP-43, or its fragments includes the natural human amino acid sequences including isoforms, mutants, and allelic variants thereof. The ability of an antibody or antigen-binding antibody fragment to bind to TDP-43 can be determined using, e.g., surface plasmon resonance. III. Anti-TDP-43 Cell-Penetrating Agents The present disclosure provides cell-penetrating agents (as described herein) that include a cell internalization moiety and an antibody or an antigen-binding fragment thereof that specifically binds to TDP-43 (e.g., human TDP-43). In some embodiments, the antibodies or
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT antigen-binding antibody fragments described herein specifically bind to phosphorylated TDP-43 (e.g., phosphorylated human TDP-43). In some embodiments, the antibodies or antigen-binding antibody fragments described herein specifically bind phosphorylated TDP-43 (e.g., phosphorylated human TDP-43), where one or both of serine at position 409 and/or 410 of SEQ ID NO: 82 is phosphorylated. In some embodiments, the antibodies or antigen-binding antibody fragments, bind a 23 amino acid peptide (“TDP-43 (pS409/pS410)”) comprising amino acids from position 392 to position 414 of SEQ ID NO: 82, where the serines at positions 409 and 410 are phosphorylated. Further, the present disclosure relates to cell-penetrating agents that include a cell internalization moiety and an antibody that specifically binds to human TDP-43, compositions comprising these cell-penetrating agents, and methods of using these cell-penetrating agents to treat TDP-43 related diseases, including Amyotrophic Lateral Sclerosis (ALS). Throughout the present disclosure, references to the anti-TDP-43 CPA and/or anti-TDP- 43 antibody will be made in the context of cell internalization and/or intracellular function, but one of ordinary skill will understand such references to include intact anti-TDP-43 CPA and/or anti-TDP-43 antibody, chemically modified (e.g., oxidized, reduced) anti-TDP-43 CPA and/or anti-TDP-43 antibody, as well as partially degraded functional fragments thereof (intact anti- TDP-43 CPA, CPA fragment, intact anti-TDP-43 antibody, and/or anti-TDP-43 antibody fragment, etc.). Thus, references to internalization of, cytosolic transfer of, and target engagement by either the anti-TDP-43 CPA or the anti-TDP-43 antibody will include references to intact anti-TDP-43 CPA and/or anti-TDP-43 antibody, chemically modified anti-TDP-43 CPA and/or anti-TDP-43 antibody, and partially degraded functional fragments thereof. For example, methods of the present disclosure may refer to internalization of and/or transfer to the cytosol of an anti-TDP-43 antibody, but it will be understood that such a reference encompasses the transfer of an intact anti-TDP-43 CPA and/or a functional fragment of the anti-TDP-43 CPA comprising the anti-TDP-43 antibody or a portion thereof, as well as chemically modified derivatives thereof. Cell penetrating agents of the present disclosure may utilize one or more of several biochemical processes to achieve internalization of the anti-TDP-43 antibody. In various embodiments, the anti-TDP-43 CPAs of the present disclosure may utilize passive internalization, active internalization, or a combination thereof. In some embodiments, the anti-
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT TDP-43 CPA utilizes active internalization (e.g., endocytosis) to achieve intracellular delivery of the anti-TDP-43 antibody. In various embodiments, the anti-TDP-43 CPA is internalized into the cell via endocytosis. Following internalization, the anti-TDP-43 CPA achieves endosomal escape. In such embodiments, the anti-TDP-43 CPA is thereby transferred to the cytosol, where the anti- TDP-43 antibody can bind to TDP-43 (e.g., phospho-TDP-43). In some embodiments, endosomal escape of the anti-TDP-43 CPA is achieved by interaction of the CIM with the membrane of the endosome, thereby leading to disruption of the endosomal membrane. The CIM may utilize one or more methods to achieve endosomal escape. For example, the CIM may comprise a cationic portion (e.g., positively charged amino acids, a cationic polymer and/or oligomer, cationic lipid). In some of such embodiments, the cationic portion can interact with the negatively charged phospholipids that comprise the endosomal membrane, thereby disrupting the endosomal membrane, and achieving endosomal escape of the anti-TDP-43 CPA. Anti-TDP-43 CPAs of the present disclosure may also comprise a CIM having an amphiphilic portion (e.g., an amphiphilic peptide). In some of such embodiments, the amphiphilic portion can interact with the endosomal membrane via hydrophobic interactions with the membrane lipids, thereby disrupting the endosomal membrane, and achieving endosomal escape of the anti-TDP-43 CPA. Following endosomal escape of the anti-TDP-43 antibody, the anti-TDP-43 antibody is then transferred to the cytosol, where it can bind to TDP-43 (e.g., phospho-TDP-43). Further examples of internalization mechanisms that can be utilized by CPAs are described in detail herein. Thus, provided herein are cell-penetrating agents that include: (i) a cell internalizing module (CIM) and (ii) an antibody that specifically binds to TDP-43 (e.g., human TDP-43). Various aspects of cell-penetrating agents of the present disclosure are described below, including examples of Cell Internalizing Moieties, anti-TDP-43 antibodies, and/or optional linkers. One of ordinary skill will understand how to combine the various CIM, anti-TDP-43 antibodies, and optional linkers disclosed herein. Such examples are merely illustrative of the scope of the present disclosure and are non-limiting. Cell Internalizing Modules
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT As used herein a “cell internalizing module” or “CIM” is a composition that, when covalently or non-covalently linked to an anti-TDP-43 antibody molecule, results in the internalization into a cell of, at a minimum, the anti-TDP-43 antibody or an active fragment thereof. Non-limiting features and examples of cell internalizing modules are described herein. In some embodiments, a CIM is a peptide (e.g., an amino acid sequence). In such examples, the CIM can also be referred to as a cell membrane internalizing peptide or “CMIP” as further defined herein. Exemplary cell membrane internalizing peptides include naturally occurring peptides, and derivatives thereof, as well as synthetic peptides. Non-limiting examples of CMIPs include M-lycotoxin and derivatives thereof, TAT and derivatives thereof, 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. In some embodiments, a CIM is a non-peptide moiety (e.g., a ligand) that is internalized by a cell (e.g., a mammalian cell). In such examples, a ligand can induce receptor-mediated internalization of an anti-TDP-43 antibody. Generally, ligand internalization is a receptor- mediated endocytic process in which cells intake extracellular molecules (including therapeutics) if the ligand binds to its cognate receptor protein on the cell’s surface. Receptor-mediated internalization also includes transcytosis. CIMs can effectuate the internalization of anti-TDP-43 antibody into a mammalian cell. In general, the process of cellular internalization is broadly classified as endocytosis. Typically, endocytosis pathways can be subdivided into two broader categories phagocytosis and pinocytosis. During pinocytosis the plasma membrane absorbs solutes while during phagocytosis the cell internalizes much larger vesicles. Pinocytosis is generally further subdivided into macropinocytosis, clathrin-dependent endocytosis (e.g., receptor-mediated endocytosis), caveolin-dependent endocytosis, and clathrin/caveolin-independent endocytosis. (See e.g., Marsh, M. Endocytosis, Oxford University Press (2001); Doherty, G.J., and McMahon, H.T., Mechanisms of Endocytosis, Annu. Rev. Biochem., 78:31.1-31.46 (2009); and Xu, Y., et al., Endocytosis and membrane receptor internalization: implication of F-BAR protein Carom, Front Biosci, 22: 1439-1457 (2017), each of which is incorporated herein by reference in their entireties).
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT Ligand mediated endocytosis is the mechanism by which cells internalize specific macromolecules. In some examples, the cell membrane (e.g., plasma membrane) includes clathrin pits which protrude from the cell membrane to form small vesicles called clathrin-coated vesicles. These clathrin-coated vesicles contain the receptors and the bound macromolecules, i.e., ligands. Then the clathrin-coated vesicles fuse with the early endosomes (vesicles consisting of tubular extensions residing at the periphery of the cell). The endosomes have an acidic environment (pH 6.0-6.2) that facilitates the dissociation of receptors from the ligands. Then, the ingested content is sorted out for either recycling to the plasma membrane or transport to lysosomes for degradation. Peptide based CIMs (e.g., CMIPs) internalize anti-TDP-43 antibodies through a variety of mechanisms. In general, CMIPs have been shown to use either endocytosis (e.g., energy- dependent internalization) as described above or direct penetration (e.g., translocation) (energy- independent internalization) as the two major internalization mechanisms. For direct penetration, various mechanisms have been described including the carpet-like model (membrane destabilization) and the pore formation model (barrel-stave). Positively charged CMIPs can interact with negatively charged membrane components such as the phospholipid bilayer, followed by destabilization of the membrane, and crossing of the CMIP and anti-TDP-43 antibody through the lipid bilayer. Studies have shown that several CMIPs are able to induce and shift between different uptake mechanisms depending on their concentration, cargo, and/or the cell line used (See e.g., Ruseska, I. and Zimmer, A., Internalization mechanisms of cell- penetrating peptides, Beilstein J Nanotechnol, 11: 101-123, (2020)). Once internalized into a cell, anti-TDP-43 antibody typically need to escape the endosomal pathway. In general, the endocytic pathway of mammalian cells consists of distinct membrane compartments, which internalize molecules (i.e., cell penetrating agents) from the plasma membrane and recycle membrane-bound receptors back to the surface or sort internalized molecules to various degradation pathways. The main components of the endocytic pathway include early endosomes which are the first compartment of the endocytic pathway. Early endosomes are usually located in the periphery of the cell and receive most types of vesicles coming from the cell surface. They have a characteristic tubulo-vesicular structure and a mildly acidic pH. Early endosomes are principally sorting organelles where many endocytosed ligands dissociate from their receptors in the acid pH of the compartment and are recycled to the cell
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT surface. Early endosomes also sort into transcytotic pathway to later compartments (e.g., late endosomes or lysosomes) via transvesicular compartments. Late endosomes generally receive endocytosed material en route to lysosomes, usually from early endosomes in the endocytic pathway, from trans-Golgi network (TGN) in the biosynthetic pathway, and from phagosomes in the phagocytic pathway. They are acidic (approx. pH 5.5) and are generally thought to mediate a final sorting prior the delivery of material to lysosomes. Lysosomes are the last compartment of the endocytic pathway. Lysosomes break down cellular waste products, fats, carbohydrates, proteins, and other macromolecules into simple compounds which are returned to the cytoplasm as new cell-building materials. Lysosomes include many different types of hydrolytic enzymes which function in an acidic environment (e.g., pH of approximately 4.8). In some embodiments, the CIM (as defined herein) includes a Cell Membrane Internalizing Peptide (e.g., CMIP4, SEQ ID NO: 176). In some embodiments, the CIM includes a wild-type M-lycotoxin peptide (SEQ ID NO: 182). In some embodiments, the CIM includes an M-lycotoxin derivative (e.g., SEQ ID NO: 183). In some embodiments, the CIM includes a Penetain amino acid sequence or a derivative thereof (e.g., SEQ ID NO: 187). In some embodiments, the CIM includes a Pepth amino acid sequence or a derivative thereof (e.g., SEQ ID NO: 184). In some embodiments, the CIM includes a polyarginine amino acid sequence (e.g., SEQ ID NO: 190, SEQ ID NO: 191, and/or SEQ ID NO: 192). In some embodiments, the CIM includes more than one polyarginine amino acid sequence (e.g., 2, 3, 4, 5 or more polyarginine amino acid sequences). In some embodiments, the CIM includes three polyarginine amino acid sequences. In some embodiments, the CIM includes 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 includes more than one TAT amino acid sequence or derivatives thereof (e.g., 2, 3, 4, 5 or more TAT amino acid sequences or derivatives thereof). In some embodiments, the CIM includes three TAT amino acid sequences. In some embodiments, the CIM includes a macrocycle. Generally, macrocycles are molecules and ions containing a ring of twelve or more atoms. Classic examples include the 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
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT embodiments, the macrocycle is formed by a disulfide bond between two cysteine residues of the CIM. In some embodiments, the CIM includes one or more histidine residues. In some embodiments, the CIM includes a polypeptide having an amino acid sequence selected from any one of SEQ ID NOs: 176-193. In some embodiments, the CIM is a polypeptide having an amino acid sequence selected from any one of SEQ ID NOs: 176-193. In some embodiments, the CIM is covalently linked to the antibody or antigen-binding antibody fragment thereof. In some embodiments, the CIM is non-covalently linked to the antibody or antigen-binding fragment antibody thereof. In some embodiments, the cell- penetrating agent includes a linker connecting 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 enzymatic- cleavable linker, etc.). In some embodiments, the linker is a non-cleavable linker. In some embodiments, the linker includes a polypeptide. In some embodiments, the linker includes one or more glycine residues (e.g., 2, 3, 4, 5 or more glycine residues). In some examples, a linker is a glycine residue followed by a serine residue (GS). In some embodiments, the linker includes a polypeptide comprising an amino acid sequence selected from any one of SEQ ID NOs: 194- 199. In some embodiments, the linker is a polypeptide having an amino acid sequence selected from any one of SEQ ID NOs: 194-199. In some embodiments, the antibody or antigen-binding antibody fragment of the cell- penetrating agent is linked to the C-terminus of the CIM. In some embodiments, the antibody or antigen-binding antibody fragment of the cell-penetrating agent is linked to the N-terminus of the CIM. In some embodiments, the antibody or antigen-binding antibody fragment of the cell- penetrating agent is linked to the C-terminus of the heavy chain of the CIM. In some embodiments, the antibody or antigen-binding antibody fragment of the cell-penetrating agent is linked to the N-terminus of the heavy chain of the CIM. In some embodiments, the antibody or antigen-binding antibody fragment of the cell-penetrating agent is linked to the C-terminus of the light chain of the CIM. In some embodiments, the antibody or antigen-binding antibody fragment of the cell-penetrating agent is linked to the N-terminus of the light chain of the CIM. In some embodiments, the CIM includes one or more spacer regions. In some embodiments, the CIM does not include a spacer region. For example, CIMs comprising a
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT polypeptide amino acid sequence selected from: SEQ ID NO: 176-184, SEQ ID NO: 192, and SEQ ID NO: 193 do not include a spacer region. In some embodiments, the CIM includes one or more spacer regions. In some embodiments, at least one of the one or more spacer regions includes one or more amino acid residues. As used herein a spacer refers to an amino acid that does not have catalytic or therapeutic activity in a mammalian cell. For example, a spacer can be a peptide of 1 amino acid 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 examples, spacer regions are included in the cell internalizing module sequences. For example, a spacer region can separate amino acids within a cell internalizing module (CIM), e.g., a spacer can be disposed after the first amino acid of a CIM. In some examples, a spacer can be disposed before the final amino acid of a CIM. In some examples, a CIM can have one or more spacer regions (e.g., two spacer regions, three space sequences, four spacer regions, five spacer regions or more). In some examples, a spacer region is a single glycine residue. In some examples, a spacer region is a pair of glycine residues. In some examples, a spacer region is three glycine residues. In some examples, a spacer region is four glycine residues. In some examples, a 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 one of SEQ ID NOs: 200-203. In some embodiments, each of the one or more spacer regions includes an amino acid sequence selected from any one of SEQ ID NOs: 200-203. Linkers In various aspects of the present disclosure, the anti-TDP-43 cell penetrating agents further comprise a linker connecting the CIM to the anti-TDP-43 antibody. Linkers of the present disclosure may connect the CIM to the anti-TDP-43 antibody via either a covalent linkage or a non-covalent linkage. 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 the 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
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT 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. In some embodiments, the linker is a non-covalent linker. A non-covalent linkage can be achieved using an affinity pair that interact strongly in a noncovalent manner (e.g., by hydrogen bonding, ionic bonding, van Der Waals interactions, or any combination thereof). Numerous examples of non-covalent linkages are known in the art. For example, biotin and a biotin-binding agent (e.g., streptavidin) are one example of an affinity pair. For example, by connecting biotin to one side of the CPA (e.g., the CIM), and connecting the biotin-binding agent to the other side of the CPA (e.g., the anti-TDP-43 antibody), a non-covalent linkage can be achieved between the CIM and the anti-TDP-43 antibody. In some embodiments, a linker comprises a pair of affinity domains (e.g., a first domain of the pair of affinity domains can be interleukin-15 and a second domain of the pair of affinity domains can be a sushi domain of interleukin-15 receptor alpha). 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, a polyethylene glycol chain, a polyacrylamide, a polyacrylic acid, a polyvinyl alcohol, or a polyethyleneimine chain). In some cases, the covalent linker comprises an unsubstituted or substituted alkylene chain (including, for example, a polyvinyl alcohol chain, a polyacrylamide chain, or a 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 incorporation of two or more CIMs and/or anti-TDP-43 antibodies into a CPA (e.g., a dendrimer linker structure). In some embodiments, the linker comprises an amino acid residue. In some embodiments, the linker comprises a polypeptide. In some exemplary embodiments, a linker can 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, a linker can 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, a linker can 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, a linker can be a peptide of about 1 amino acid to about 5 amino acids (e.g., about 1 amino acid to about 4, or
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT about 1 amino acid to about 3 amino acids). In some exemplary embodiments, a linker can 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, a linker can 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). In some embodiments, the linker comprises a glycine residue. 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 an amino acid sequence of GGGGS. In some embodiments, the linker comprises an amino acid sequence of GGGSGGGS. Anti-TDP-43 Antibodies Cell-penetrating agents and methods of the present disclosure facilitate the cellular internalization and/or cytosolic release of a wide range of anti-TDP-43 antibodies. In some embodiments, the anti-TDP-43 antibody is a humanized antibody, a chimeric antibody, or a veneered antibody (as described herein). Complementarity determining regions (“CDRs”) can be defined by different systems. For example, the CDRs described herein can be selected from the group of Kabat, Chothia, Kabat/Chothia Composite, AbM, and Contact. In some embodiments, the antibody of the cell-penetrating agent includes a humanized mature heavy variable domain including: a heavy chain CDR1 as defined by Kabat/Chothia Composite, including SEQ ID NO: 49; a heavy chain CDR2 as defined by Kabat, including SEQ ID NO: 51; and a heavy chain CDR3 as defined by Kabat or Chothia including SEQ ID NO: 52; and a humanized mature light chain variable domain including the three Kabat light chain CDRs of SEQ ID NOs: 53-55. In some embodiments, the humanized mature heavy variable domain of the antibody includes a sequence that is at least 80% identical to any one of SEQ ID NOs: 4-23. In some
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT embodiments, the humanized mature light chain variable domain includes a sequence that is at least 80% identical to any one of SEQ ID NOs: 27-48. In some embodiments, the antibody or antigen-binding fragment thereof, includes a humanized mature heavy variable domain including a sequence that is at least 80% identical to any one of SEQ ID NOs: 4-23 and a humanized mature light chain variable domain including a sequence that is at least 80% identical to any one of SEQ ID NOs: 27-48. In some embodiments, the humanized mature heavy variable domain of the antibody includes a sequence that is at least 85% to any one of SEQ ID NOs: 4-23. In some embodiments, the humanized mature light chain variable region of the antibody includes a sequence that is at least 85% identical to any one of SEQ ID NOs: 27-48. In some embodiments, the antibody or antigen-binding fragment thereof, includes a humanized mature heavy variable domain including a sequence that is at least 85% identical to any one of SEQ ID NOs: 4-23 and a humanized mature light chain variable domain including a sequence that is at least 85% identical to any one of SEQ ID NOs: 27-48. In some embodiments, the humanized mature heavy variable domain of the antibody includes a sequence that is at least 90% identical, to any one of SEQ ID NOs: 4-23. In some embodiments, a humanized mature light chain variable domain of the antibody includes a sequence that is at least 90% identical, to any one of SEQ ID NOs: 27-48. In some embodiments, the antibody or antigen-binding fragment thereof, includes a humanized mature heavy variable domain comprising a sequence that is at least 90% identical to any one of SEQ ID NOs: 4-23 and a humanized mature light chain variable domain comprising a sequence that is at least 90% identical to any one of SEQ ID NOs: 27-48. In some embodiments, the humanized mature heavy variable domain of the antibody includes a sequence that is at least 95% identical to any one of SEQ ID NOs: 4-23. In some embodiments, the humanized mature light chain variable domain of the antibody includes a sequence that is at least 95% identical to any one of SEQ ID NOs: 27-48. In some embodiments, the antibody or antigen-binding fragment thereof, includes a humanized mature heavy variable domain including a sequence that is at least 95% identical to any one of SEQ ID NOs: 4-23 and a humanized mature light chain variable domain including a sequence that is at least 95% identical to any one of SEQ ID NOs: 27-48.
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT In some embodiments, the humanized mature heavy chain variable domain includes a sequence that is one of SEQ ID NOs: 4-23. In some embodiments, the humanized mature heavy chain variable domain includes a sequence comprising SEQ ID NO: 4. In some embodiments, the humanized mature heavy chain variable domain includes a sequence comprising SEQ ID NO: 5. In some embodiments, the humanized mature heavy chain variable domain includes a sequence comprising SEQ ID NO: 6. In some embodiments, the humanized mature heavy chain variable domain includes a sequence comprising SEQ ID NO: 7. In some embodiments, the humanized mature heavy chain variable domain includes a sequence comprising SEQ ID NO: 8. In some embodiments, the humanized mature heavy chain variable domain includes a sequence comprising SEQ ID NO: 9. In some embodiments, the humanized mature heavy chain variable domain includes a sequence comprising SEQ ID NO: 10. In some embodiments, the humanized mature heavy chain variable domain includes a sequence comprising SEQ ID NO: 11. In some embodiments, the humanized mature heavy chain variable domain includes a sequence comprising SEQ ID NO: 12. In some embodiments, the humanized mature heavy chain variable domain includes a sequence comprising SEQ ID NO: 13. In some embodiments, the humanized mature heavy chain variable domain includes a sequence comprising SEQ ID NO: 14. In some embodiments, the humanized mature heavy chain variable domain includes a sequence comprising SEQ ID NO: 15. In some embodiments, the humanized mature heavy chain variable domain includes a sequence comprising SEQ ID NO: 16. In some embodiments, the humanized mature heavy chain variable domain includes a sequence comprising SEQ ID NO: 17. In some embodiments, the humanized mature heavy chain variable domain includes a sequence comprising SEQ ID NO: 18. In some embodiments, the humanized mature heavy chain variable domain includes a sequence comprising SEQ ID NO: 19. In some embodiments, the humanized mature heavy chain variable domain includes a sequence comprising SEQ ID NO: 21. In some embodiments, the humanized mature heavy chain variable domain includes a sequence comprising SEQ ID NO: 22. In some embodiments, the humanized mature heavy chain variable domain includes a sequence comprising SEQ ID NO: 23. In some embodiments, the humanized mature light chain variable domain includes a sequence that is one of SEQ ID NOs: 27-48. In some embodiment, the humanized mature light chain variable domain includes a sequence comprising SEQ ID NO: 27. In some embodiment, the humanized mature light chain variable domain includes a sequence comprising SEQ ID NO:
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT 28. In some embodiment, the humanized mature light chain variable domain includes a sequence comprising SEQ ID NO: 29. In some embodiment, the humanized mature light chain variable domain includes a sequence comprising SEQ ID NO: 30. In some embodiment, the humanized mature light chain variable domain includes a sequence comprising SEQ ID NO: 31. In some embodiment, the humanized mature light chain variable domain includes a sequence comprising SEQ ID NO: 32. In some embodiment, the humanized mature light chain variable domain includes a sequence comprising SEQ ID NO: 33. In some embodiment, the humanized mature light chain variable domain includes a sequence comprising SEQ ID NO: 34. In some embodiment, the humanized mature light chain variable domain includes a sequence comprising SEQ ID NO: 35. In some embodiment, the humanized mature light chain variable domain includes a sequence comprising SEQ ID NO: 36. In some embodiment, the humanized mature light chain variable domain includes a sequence comprising SEQ ID NO: 37. In some embodiment, the humanized mature light chain variable domain includes a sequence comprising SEQ ID NO: 38. In some embodiment, the humanized mature light chain variable domain includes a sequence comprising SEQ ID NO: 39. In some embodiment, the humanized mature light chain variable domain includes a sequence comprising SEQ ID NO: 40. In some embodiment, the humanized mature light chain variable domain includes a sequence comprising SEQ ID NO: 41. In some embodiment, the humanized mature light chain variable domain includes a sequence comprising SEQ ID NO: 42. In some embodiment, the humanized mature light chain variable domain includes a sequence comprising SEQ ID NO: 43. In some embodiment, the humanized mature light chain variable domain includes a sequence comprising SEQ ID NO: 44. In some embodiment, the humanized mature light chain variable domain includes a sequence comprising SEQ ID NO: 45. In some embodiment, the humanized mature light chain variable domain includes a sequence comprising SEQ ID NO: 46. In some embodiment, the humanized mature light chain variable domain includes a sequence comprising SEQ ID NO: 47. In some embodiment, the humanized mature light chain variable domain includes a sequence comprising SEQ ID NO: 48. The humanized mature heavy chain of the antibody described herein can include a lysine residue at their C-terminus. However, in some embodiments, the humanized mature heavy chain (e.g., any of the mature heavy chains described herein) does not include the C-terminal lysine residue.
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT In some embodiments, the antibody or antigen-binding fragment thereof, includes a humanized mature heavy variable domain including a sequence that is one of SEQ ID NOs: 4-23 and a humanized mature light chain variable domain including a sequence that is one of SEQ ID NOs: 27-48. In some embodiments, the antibody or antigen-binding fragment of the cell-penetrating agent includes a humanized mature heavy chain variable domain of SEQ ID NO: 4 and a humanized mature light chain variable domain any one of SEQ ID NOs: 27-48. In some embodiments, the antibody or antigen-binding fragment thereof of the cell-penetrating agent includes a humanized mature heavy chain variable domain of SEQ ID NO: 5 and a humanized mature light chain variable domain any one of SEQ ID NOs: 27-48. In some embodiments, the antibody or antigen-binding fragment thereof of the cell-penetrating agent includes a humanized mature heavy chain variable domain of SEQ ID NO: 6 and a humanized mature light chain variable domain any one of SEQ ID NOs: 27-48. In some embodiments, the antibody or antigen- binding fragment thereof of the cell-penetrating agent includes a humanized mature heavy chain variable domain of SEQ ID NO: 7 and a humanized mature light chain variable domain any one of SEQ ID NOs: 27-48. In some embodiments, the antibody or antigen-binding fragment thereof of the cell-penetrating agent includes a humanized mature heavy chain variable domain of SEQ ID NO: 8 and a humanized mature light chain variable domain any one of SEQ ID NOs: 27-48. In some embodiments, the antibody or antigen-binding fragment thereof of the cell-penetrating agent includes a humanized mature heavy chain variable domain of SEQ ID NO: 9 and a humanized mature light chain variable domain any one of SEQ ID NOs: 27-48. In some embodiments, the antibody or antigen-binding fragment thereof of the cell-penetrating agent includes a humanized mature heavy chain variable domain of SEQ ID NO: 10 and a humanized mature light chain variable domain any one of SEQ ID NOs: 27-48. In some embodiments, the antibody or antigen-binding fragment thereof of the cell-penetrating agent includes a humanized mature heavy chain variable domain of SEQ ID NO: 11 and a humanized mature light chain variable domain any one of SEQ ID NOs: 27-48. In some embodiments, the antibody or antigen- binding fragment thereof of the cell-penetrating agent includes a humanized mature heavy chain variable domain of SEQ ID NO: 12 and a humanized mature light chain variable domain any one of SEQ ID NOs: 27-48. In some embodiments, the antibody or antigen-binding fragment thereof of the cell-penetrating agent includes a humanized mature heavy chain variable domain of SEQ
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT ID NO: 13 and a humanized mature light chain variable domain any one of SEQ ID NOs: 27-48. In some embodiments, the antibody or antigen-binding fragment thereof of the cell-penetrating agent includes a humanized mature heavy chain variable domain of SEQ ID NO: 14 and a humanized mature light chain variable domain any one of SEQ ID NOs: 27-48. In some embodiments, the antibody or antigen-binding fragment thereof of the cell-penetrating agent includes a humanized mature heavy chain variable domain of SEQ ID NO: 15 and a humanized mature light chain variable domain any one of SEQ ID NOs: 27-48. In some embodiments, the antibody or antigen-binding fragment thereof of the cell-penetrating agent includes a humanized mature heavy chain variable domain of SEQ ID NO: 16 and a humanized mature light chain variable domain any one of SEQ ID NOs: 27-48. In some embodiments, the antibody or antigen- binding fragment thereof of the cell-penetrating agent includes a humanized mature heavy chain variable domain of SEQ ID NO: 17 and a humanized mature light chain variable domain any one of SEQ ID NOs: 27-48. In some embodiments, the antibody or antigen-binding fragment thereof of the cell-penetrating agent includes a humanized mature heavy chain variable domain of SEQ ID NO: 18 and a humanized mature light chain variable domain any one of SEQ ID NOs: 27-48. In some embodiments, the antibody or antigen-binding fragment thereof of the cell-penetrating agent includes a humanized mature heavy chain variable domain of SEQ ID NO: 19 and a humanized mature light chain variable domain any one of SEQ ID NOs: 27-48. In some embodiments, the antibody or antigen-binding fragment thereof of the cell- penetrating agent includes a humanized mature heavy chain variable domain of SEQ ID NO: 20 and a humanized mature light chain variable domain any one of SEQ ID NOs: 27-48. In some embodiments, the antibody or antigen-binding fragment thereof of the cell-penetrating agent includes a humanized mature heavy chain variable domain of SEQ ID NO: 21 and a humanized mature light chain variable domain including any one of SEQ ID NOs: 27-48. In some embodiments, the antibody or antigen-binding fragment thereof of the cell-penetrating agent includes a humanized mature heavy chain variable domain of SEQ ID NO: 22 and a humanized mature light chain variable domain including any one of SEQ ID NOs: 27-48. In some embodiments, the antibody or antigen-binding fragment thereof of the cell-penetrating agent includes a humanized mature heavy chain variable domain of SEQ ID NO: 23 and a humanized mature light chain variable domain including any one of SEQ ID NOs: 27-48.
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT In some embodiments, the antibody or antigen-binding fragment thereof of the cell- penetrating agent includes a humanized mature heavy chain variable domain of SEQ ID NO: 20 and a humanized mature light chain variable domain of SEQ ID NO: 47. In some embodiments, the antibody or antigen-binding fragment thereof of the cell-penetrating agent includes a humanized mature heavy chain variable domain of SEQ ID NO: 20 and a humanized mature light chain variable domain of SEQ ID NO: 48. In some embodiments, the antibody or antigen- binding fragment thereof of the cell-penetrating agent includes a humanized mature heavy chain variable domain of SEQ ID NO: 21 and a humanized mature light chain variable domain of SEQ ID NO: 47. In some embodiments, the antibody or antigen-binding fragment thereof of the cell- penetrating agent includes a humanized mature heavy chain variable domain of SEQ ID NO: 21 and a humanized mature light chain variable domain of SEQ ID NO: 48. In some embodiments, the antibody or antigen-binding fragment thereof of the cell-penetrating agent includes a humanized mature heavy chain variable domain of SEQ ID NO: 23 and a humanized mature light chain variable domain of SEQ ID NO: 47. In some embodiments, the antibody or antigen- binding fragment thereof of the cell-penetrating agent includes a humanized mature heavy chain variable domain of SEQ ID NO: 23 and a humanized mature light chain variable domain of SEQ ID NO: 48. In some embodiments, at least one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17) of the following positions in the humanized heavy chain variable domain is occupied by the amino acid as specified: 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. In some embodiments, at least one (e.g., 2, 3, 4, 5, or 6) of the following positions in the humanized heavy chain variable domain is occupied by the amino acid as specified: 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. In some embodiments, at least one (e.g., 2, 3, or 4) of the following positions in the humanized heavy chain variable domain is occupied by the amino acid as specified: S35 is
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT occupied by G; L78 is occupied by A or G; L80 is occupied by A or G; or L82c is occupied by G. In some embodiments, F91 of the humanized heavy chain variable domain is occupied by Y; and at least one (e.g., 2, 3, 4, 5, or 6) of the following positions in the humanized heavy chain variable domain is occupied by the amino acid as specified: 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. In some embodiments, at least one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13) of the following positions in the humanized light chain variable domain is occupied by the amino acid as specified: 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; L46 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. In some embodiments, at least one (e.g., two) of the following positions in the humanized light chain variable domain is occupied by the amino acid as specified: V3 is occupied by Q or A100 is occupied by D or R. In some embodiments, at least one (e.g., 2, 3, or 4) of the following positions in the humanized light chain variable domain is occupied by the amino acid as specified: 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. In some embodiments, 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 amino acid as specified: T80 is occupied by A or L92 is occupied by A. In some embodiments, at least one (e.g., 2, 3, 4, 5, 6, 7, or 8) of the following positions in the humanized heavy chain variable domain is occupied by the amino acid as specified: 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 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) of the following positions in the humanized light chain variable domain is occupied by the amino acid as specified: 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
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT occupied by A; L83 is occupied by V; L92 is occupied by A; A100 is occupied by D; or L104 is occupied by V. The aforementioned substitutions can confer reduced (e.g., at least 1% reduced, at least 5% reduced, at least 10% reduced, at least 20% reduced, at least 30% reduced, at least 40% reduced, at least 50% reduced, at least 60% reduced, at least 70% reduced, at least 80% reduced, at least 90% reduced, at least 95% reduced, or at least 99% reduced) immunogenicity and/or increased (e.g., at least 1% increased, at least 10% increased, at least 20% increased, at least 30% increased, at least 40% increased, at least 50% increased, at least 60% increased, at least 70% increased, at least 80% increased, at least 90% increased, or at least 100% increased) thermal stability of the parent antibody. In some embodiments, the humanized antibody or has increased thermal stability as compared to a reference antibody including a heavy chain variable domain including SEQ ID NO: 1 and a light chain variable domain including SEQ ID NO: 24. In some embodiments, the humanized antibody has increased thermal stability as compared to a reference antibody including a heavy chain variable domain of SEQ ID NO: 63 and a light chain variable domain of SEQ ID NO: 65. In some embodiments, the humanized antibody has increased thermal stability as compared to a reference antibody including a heavy chain variable domain of SEQ ID NO: 67 and a light chain variable domain of SEQ ID NO: 69. In some embodiments, the humanized antibody has increased thermal stability as compared to a reference antibody including a heavy chain variable domain of SEQ ID NO: 71 and a light chain variable domain of SEQ ID NO: 73. In some embodiments, the humanized antibody has increased thermal stability as compared to a reference antibody including a heavy chain variable domain of SEQ ID NO: 75 and a light chain variable domain of SEQ ID NO: 77. In some embodiments, the humanized antibody has increased thermal stability as compared to a reference antibody including a heavy chain variable domain of SEQ ID NO: 79 and a light chain variable domain of SEQ ID NO: 81. In some embodiments, the humanized antibody has a melting temperature of about 55 °C or greater. In some embodiments, the humanized antibody has a melting temperature of about 56 °C or greater, about 57 °C or greater, about 58° C or greater, about 59 °C or greater, about 60 °C or greater, about 61 °C or greater, about 62 °C or greater, about 63 °C or greater, about 64 °C or greater, about 65 °C or greater, about 66 °C or greater, about 67 °C or greater, about 68 °C or greater, about 69 °C or greater, about 70 °C or greater, about 71 °C or greater, about 72 °C or
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT greater, about 73 °C or greater, about 74 °C or greater, about 75 °C or greater, about 76 °C or greater, about 77 °C or greater, about 78 °C or greater, about 79 °C or greater, about 80 °C or greater, about 81 °C or greater, about 82 °C or greater, about 83 °C or greater, about 84 °C or greater, or about 85 °C or greater. In some embodiments, the humanized antibody has a melting temperature of 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 a melting temperature of 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 to about 65 °C, about 59 °C to about 63 °C, about 59 °C to about 61 °C, about 61 °C to about 85 °C, about 61 °C to about 80 °C, about 61 °C to about 75 °C, about 61 °C to about 70 °C, about 61 °C to about 65 °C, about 61 °C to about 63 °C, about 63 °C to about 85 °C, about 63 °C to about 80 °C, about 63 °C to about 75 °C, about 63 °C to about 70 °C, about 63 °C to about 65 °C, about 65 °C to about 85 °C, about 65 °C to about 80 °C, about 65 °C to about 75 °C, about 65 °C to about 70 °C, about 70 °C to about 85 °C, about 70 °C to about 80 °C, about 70 °C to about 75 °C, about 75 °C to about 85 °C, about 75 °C to about 80 °C, or about 80 °C to about 85 °C. Also provided herein are cell-penetrating agents that include a CIM and an antibody that specifically binds to TDP-43 (e.g., human TDP-43), that include a heavy chain variable domain including: a heavy chain CDR1, as defined by Kabat/Chothia Composite, including SEQ ID NO: 49 or SEQ ID NO: 50; a heavy chain CDR2, as defined by Kabat, including SEQ ID NO: 51; a heavy chain CDR3, as defined by Kabat or Chothia, including SEQ ID NO: 52; a light chain
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT CDR1, as defined by Kabat, including SEQ ID NO: 53; a light chain CDR2, as defined by Kabat, including SEQ ID NO: 54; and a light chain CDR3, as defined by Kabat, including one of SEQ ID NOs: 55-61. In some embodiments, the heavy chain CDR1, as defined by Kabat/Chothia Composite, includes SEQ ID NO: 49; the heavy chain CDR2, as defined by Kabat, includes SEQ ID NO: 51; the heavy chain CDR3, as defined by Kabat or Chothia, includes SEQ ID NO: 52; the light chain CDR1, as defined by Kabat, includes SEQ ID NO: 53; the light chain CDR2, as defined by Kabat, includes SEQ ID NO: 54; and the light chain CDR3, as defined by Kabat, includes SEQ ID NO: 55 or SEQ ID NO: 61. In some embodiments, the heavy chain variable domain includes a sequence that is at least 95% (e.g., at least 96%, at least 97%, at least 98%, at least 99%) identical to any one of SEQ ID NOs: 4-23. In some embodiments, the heavy chain variable domain includes a sequence that is at least 95% (e.g., at least 96%, at least 97%, at least 98%, at least 99%) identical to any one of: SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 23. In some embodiments, the heavy chain variable domain includes a sequence that is at least 98% identical to any one of: SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 23. In some embodiments, the heavy chain variable domain includes a sequence of SEQ ID NO: 20. In some embodiments, the heavy chain variable domain includes a sequence of SEQ ID NO: 21. In some embodiments, the heavy chain variable domain includes a sequence of SEQ ID NO: 23. In some embodiments, the light chain variable domain includes a sequence that is at least 95% (e.g., at least 96%, at least 97%, at least 98%, at least 99%) identical to any one of: SEQ ID NOs: 27-48. In some embodiments, the light chain variable domain includes a sequence that is at least 95% (e.g., at least 96%, at least 97%, at least 98%, at least 99%) identical to SEQ ID NO: 47 or SEQ ID NO: 48. In some embodiments, the light chain variable domain includes a sequence that is at least 98% identical to SEQ ID NO: 47 or SEQ ID NO: 48. In some embodiments, the light chain variable domain includes SEQ ID NO: 47. In some embodiments, the light chain variable domain includes SEQ ID NO: 48. As described previously, the humanized mature light chain variable domains described herein can include a lysine residue at their C-terminus. However, in some embodiments, the humanized mature light chain variable domain (e.g., any of the mature light chain variable domains described herein) does not include the C-terminal lysine residue. For example, any one
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT of the mature light chain variable domains of SEQ ID NOs: 24-48, 65, 69, 73, 77, or 81 can lack the C-terminal lysine residue. In some embodiments, the antibody of the cell-penetrating agent 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. In some embodiments, the antibody of the cell-penetrating agent is an intact antibody. In some embodiments, the antibody has a human IgG1 isotype. In some embodiments, the heavy chain variable domain is fused to a heavy chain constant region (e.g., any of the heavy chain constant regions described herein) and the light chain variable domain is fused to a light chain constant region (e.g., any of the light chain constant regions described herein). In some embodiments, the heavy chain constant region is a mutant form of a natural human heavy chain constant region which has reduced binding to an Fcγ receptor relative to the natural heavy chain constant region. In some embodiments, the heavy chain constant region is of IgG1 isotype. In some embodiments, the antibody has at least one mutation in a constant region. In some embodiments, the at least one mutation reduces complement fixation or activation by the constant region. In some embodiments, the at least one mutation is at one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11) positions of: 241, 264, 265, 270, 296, 297, 318, 320, 322, 329, and 331 by EU numbering. In some embodiments, the antibody has an alanine at positions 318, 320, and 322 by EU numbering. In some embodiments, the antibody or antigen-binding antibody fragment of the cell- penetrating agent 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- penetrating agent selectively binds to phosphorylated TDP-43 (e.g., phosphorylated human TDP- 43) as compared to unphosphorylated TDP-43 (e.g., unphosphorylated human TDP-43). In some embodiments, the antibody or antigen-binding antibody fragment of the cell- penetrating agent binds to phosphorylated TDP-43 (e.g., phosphorylated human TDP-43) with greater (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
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT 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-fold, at least 1,150- fold, at least 1,200-fold, at least 1,250-fold, at least 1,300-fold, at least 1,350-fold, at least 1,400- fold, at least 1,450-fold, or at least 1,500-fold greater, or about 10-fold to about 1,500-fold, about 10-fold to 1,400-fold greater, about 10-fold to about 1,300-fold, about 10-fold to about 1,200- fold, about 10-fold to about 1,100-fold, about 10-fold to about 1,000-fold, about 10-fold to about 900-fold, about 10-fold to about 800-fold, about 10-fold to about 700-fold, about 10-fold to about 600-fold, about 10-fold to about 500-fold, about 10-fold to about 400-fold, about 10-fold to about 300-fold, about 10-fold to about 200-fold, about 10-fold to about 100-fold, about 10- fold to about 50-fold, about 50-fold to about 1,500-fold, about 50-fold to 1,400-fold greater, about 50-fold to about 1,300-fold, about 50-fold to about 1,200-fold, about 50-fold to about 1,100-fold, about 50-fold to about 1,000-fold, about 50-fold to about 900-fold, about 50-fold to about 800-fold, about 50-fold to about 700-fold, about 50-fold to about 600-fold, about 50-fold to about 500-fold, about 50-fold to about 400-fold, about 50-fold to about 300-fold, about 50- fold to about 200-fold, about 50-fold to about 100-fold, about 100-fold to about 1,500-fold, about 100-fold to 1,400-fold greater, about 100-fold to about 1,300-fold, about 100-fold to about 1,200-fold, about 100-fold to about 1,100-fold, about 100-fold to about 1,000-fold, about 100- fold to about 900-fold, about 100-fold to about 800-fold, about 100-fold to about 700-fold, about 100-fold to about 600-fold, about 100-fold to about 500-fold, about 100-fold to about 400-fold, about 100-fold to about 300-fold, about 100-fold to about 200-fold, about 200-fold to about 1,500-fold, about 200-fold to 1,400-fold greater, about 200-fold to about 1,300-fold, about 200- fold to about 1,200-fold, about 200-fold to about 1,100-fold, about 200-fold to about 1,000-fold, about 200-fold to about 900-fold, about 200-fold to about 800-fold, about 200-fold to about 700- fold, about 200-fold to about 600-fold, about 200-fold to about 500-fold, about 200-fold to about 400-fold, about 200-fold to about 300-fold, about 500-fold to about 1,500-fold, about 500-fold to 1,400-fold greater, about 500-fold to about 1,300-fold, about 500-fold to about 1,200-fold, about 500-fold to about 1,100-fold, about 500-fold to about 1,000-fold, about 500-fold to about 900- fold, about 500-fold to about 800-fold, about 500-fold to about 700-fold, about 500-fold to about 600-fold, about 800-fold to about 1,500-fold, about 800-fold to 1,400-fold greater, about 800- fold to about 1,300-fold, about 800-fold to about 1,200-fold, about 800-fold to about 1,100-fold, about 800-fold to about 1,000-fold, about 800-fold to about 900-fold, about 1,000-fold to about
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT 1,500-fold, about 1,000-fold to 1,400-fold greater, about 1,000-fold to about 1,300-fold, about 1,000-fold to about 1,200-fold, or about 1,000-fold to about 1,100-fold) affinity than unphosphorylated TDP-43 (e.g., unphosphorylated human TDP-43). In some embodiments, phosphorylated TDP-43 (e.g., phosphorylated human TDP-43) includes 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) includes phosphorylation of both S409 and S410. In some embodiments, the antibody, or fragment thereof, of the cell-penetrating agent selectively binds to cytoplasmic aggregates of TDP-43 (e.g., cytoplasmic aggregates of human TDP-43). In some embodiments, the antibody, or fragment thereof, of the cell-penetrating agent 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) includes phosphorylated aggregates of TDP-43. In some embodiments, the antibody, or fragment thereof, of the cell-penetrating agent does not substantially bind unphosphorylated TDP-43 (e.g., unphosphorylated human TDP-43). The heavy and light chain variable regions present in the cell-penetrating agents can be linked to at least a portion of a human constant region. The choice of constant region depends, in part, whether antibody conjugate-dependent cell-mediated cytotoxicity, antibody conjugate dependent cellular phagocytosis and/or complement dependent cytotoxicity are desired. For example, human isotypes IgG1 and IgG3 have complement-dependent cytotoxicity and human isotypes IgG2 and IgG4 do not. Human IgG1 and IgG3 also induce stronger cell mediated effector functions than human IgG2 and IgG4. Light chain constant regions can be lambda or kappa. Numbering conventions for constant regions include EU numbering (Edelman, G.M. et al., Proc. Natl. Acad. Sci. U.S.A. 63:78-85 (1969)), Kabat numbering (Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1991, IMGT unique numbering (Lefranc M.-P. et al., IMGT unique numbering for immunoglobulin and T cell receptor constant domains and Ig superfamily C-like domains, Dev. Comp. Immunol. 29:185- 203 (2005), and IMGT exon numbering (Lefranc, supra). One or several amino acids at the amino or carboxy terminus of the light and/or heavy chain, such as the C-terminal lysine of the heavy chain, may be missing or derivatized in a
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT proportion or all of the molecules. Substitutions can be made in the constant regions to reduce or increase effector function such as complement-mediated cytotoxicity or ADCC (see, e.g., Winter et al., U.S. Patent No. 5,624,821; Tso et al., US Patent No. 5,834,597; and Lazar et al., Proc. Natl. Acad. Sci. U.S.A.103:4005 (2006)), or to prolong half-life in humans (see, e.g., Hinton et al., J. Biol. Chem. 279:6213 (2004)). Exemplary substitutions include a Gln at position 250 and/or a Leu at position 428 (EU numbering is used in this paragraph for the constant region) for increasing the half-life of an antibody. Substitution at any or all of positions 234, 235, 236 and/or 237 reduce affinity for Fey receptors, particularly FcyRI receptor (see, e.g., U.S. Patent No. 6,624,821). An alanine substitution at positions 234, 235, and 237 of human IgG 1 can be used for reducing effector functions. Some antibodies have alanine substitution at positions 234, 235 and 237 of human IgG 1 for reducing effector functions. Optionally, positions 234, 236 and/or 237 in human IgG2 are substituted with alanine and position 235 with glutamine (see, e.g., U.S. Patent No. 5,624,821). In some antibodies, a mutation at one or more (e.g., 2, 3, 4, 5, 6, 7, 8, or 9) of positions 241, 264, 265, 270, 296, 297, 322, 329, and 331 by EU numbering of human IgG 1 is used. In some antibodies, a mutation at one or more (e.g., 2 or 3) of positions 318, 320, and 322 by EU numbering of human IgG1 is used. 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. Antibodies can be expressed as tetramers containing two light and two heavy chains, as separate heavy chains, light chains, as Fab, Fab’, F(ab’)2, and Fv, or as single chain antibodies in which heavy and light chain mature variable domains are linked through a spacer. Human constant regions show allotypic variation and isoallotypic variation between different individuals, that is, the constant regions can differ in different individuals at one or more polymorphic positions. Isoallotypes differ from allotypes in that sera recognizing an isoallotype bind to a non-polymorphic region of a one or more other isotypes. Thus, for example, another heavy chain constant region is of IgG1 Glm3 with or without the C-terminal lysine. Reference to a human constant region includes a constant region with any natural allotype or any permutation of residues occupying positions in natural allotypes. Exemplary Anti-TDP-43 Cell Penetrating Agents
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT Various components of cell-penetrating agents have been disclosed herein, including anti-TDP-43 antibodies, linkers and cell internalizing moieties (e.g., CMIPs). One of ordinary skill will understand how to combine these features to arrive functional anti-TDP-43 cell- penetrating agents. Also described herein are cell-penetrating agents having the CMIP, the optional linker, and the light and/or heavy chain of the anti-TDP-43 antibody expressed as a single polypeptide (i.e., a fusion protein). In some embodiments, the cell-penetrating agent comprises a polypeptide sequence that is at least 95% identical to a sequence selected from any one of: 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 cell-penetrating agent comprises a polypeptide sequence that is at least 98% identical to a sequence selected from any one of: 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 cell-penetrating agent comprises a polypeptide sequence that is at least 98% identical to a sequence selected from any one of: 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.
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT In some embodiments, the cell-penetrating agent comprises a polypeptide sequence that is at least 95% identical to a sequence selected from any one of 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. In some embodiments, the cell-penetrating agent comprises a polypeptide sequence that is at least 98% identical to a sequence selected from any one of 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. In some embodiments, the cell-penetrating agent comprises a polypeptide sequence selected from any one of 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. In some embodiments, the cell-penetrating agent comprises a first polypeptide sequence comprising a heavy chain of the antibody and a second polypeptide comprising a light chain of the antibody. In some embodiments, the first polypeptide and/or the second polypeptide comprises a CMIP and an optional linker. In some embodiments, the first polypeptide is selected from any one of: 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:
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT 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; and the second polypeptide is selected from any one of 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. In some embodiments, the cell-penetrating agent comprises a first polypeptide and a second polypeptide, further wherein: the first polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 116 and the second polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 117; the first polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 118 and the second polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 119; the first polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 120 and the second polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 121; the first polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 122 and the second polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 123; the first polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 124 and the second polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 125; the first polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 126 and the second polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 127; the first polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 128 and the second polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 129; the first polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 130 and the second polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 131; the first polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 132 and the second polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 133; the first polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 134 and the second polypeptide comprises a sequence that is
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT at least 95% identical to SEQ ID NO: 135; the first polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 136 and the second polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 137; the first polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 138 and the second polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 139; the first polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 140 and the second polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 141; the first polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 142 and the second polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 143; the first polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 144 and the second polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 145; the first polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 146 and the second polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 147; the first polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 148 and the second polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 149; the first polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 150 and the second polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 151; the first polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 152 and the second polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 153; the first polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 154 and the second polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 155; the first polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 156 and the second polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 157; the first polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 158 and the second polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 159; the first polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 160 and the second polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 161; the first polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 162 and the second polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 163; the first polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 164 and the second polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 165; the first polypeptide comprises a sequence that is at
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT least 95% identical to SEQ ID NO: 166 and the second polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 167; the first polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 168 and the second polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 169; the first polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 170 and the second polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 171; the first polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 172 and the second polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 173; or the first polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 174 and the second polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 175. 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: 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
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT 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. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 116 and a second polypeptide comprising SEQ ID NO: 117. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 118 and a second polypeptide comprising SEQ ID NO: 119. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 120 and a second polypeptide comprising SEQ ID NO: 121. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 122 and a second polypeptide comprising SEQ ID NO: 123. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 124 and a second polypeptide comprising SEQ ID NO: 125. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 126 and a second polypeptide comprising SEQ ID NO: 127. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 128 and a second polypeptide comprising SEQ ID NO: 129. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 130 and a second polypeptide comprising SEQ ID NO: 131. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 132 and a second polypeptide comprising SEQ ID NO: 133. In some
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 134 and a second polypeptide comprising SEQ ID NO: 135. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 136 and a second polypeptide comprising SEQ ID NO: 137. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 138 and a second polypeptide comprising SEQ ID NO: 139. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 140 and a second polypeptide comprising SEQ ID NO: 141. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 142 and a second polypeptide comprising SEQ ID NO: 143. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 144 and a second polypeptide comprising SEQ ID NO: 145. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 146 and a second polypeptide comprising SEQ ID NO: 147. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 148 and a second polypeptide comprising SEQ ID NO: 149. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 150 and a second polypeptide comprising SEQ ID NO: 151. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 152 and a second polypeptide comprising SEQ ID NO: 153. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 154 and a second polypeptide comprising SEQ ID NO: 155. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 156 and a second polypeptide comprising SEQ ID NO: 157. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 158 and a second polypeptide comprising SEQ ID NO: 159. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 160 and a second polypeptide comprising SEQ ID NO: 161. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 162 and a second polypeptide comprising SEQ ID NO: 163. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 164 and a second polypeptide comprising SEQ ID NO: 165. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 166 and a second polypeptide comprising SEQ ID NO: 167. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 168 and a second
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT polypeptide comprising SEQ ID NO: 169. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 170 and a second polypeptide comprising SEQ ID NO: 171. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 172 and a second polypeptide comprising SEQ ID NO: 173. In some embodiments, the cell penetrating agent comprises a first polypeptide comprising SEQ ID NO: 174 and a second polypeptide comprising SEQ ID NO: 175. Cell-penetrating agents can also be administered in the form of nucleic acids encoding the cell-penetrating agent or the antibody present within a cell-penetrating agent. If both heavy and light chains are present, the chains are preferably linked as a single chain antibody. IV. Nucleic Acids, Vectors, and Host Cells The present disclosure provides nucleic acids encoding at least a portion of any of the cell-penetrating agents described herein. For example, the nucleic acids can encode any of the CIMs, linkers, and/or spacers described herein in addition to any of the heavy chains and/or light chains described herein. In some embodiments, the nucleic acids encodes a first polypeptide comprising a heavy chain of an antibody that binds specifically to TDP-43, the first polypeptide comprising any one of: 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 acids encode a second polypeptide comprising a light chain of an antibody that binds specifically to TDP-43, the second polypeptide comprising any one of: 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.
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT In some embodiments, the nucleic acid encodes a first polypeptide comprising a heavy chain of an antibody that binds specifically to TDP-43, the first polypeptide comprising any one of: 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, and second polypeptide comprising a light chain of an antibody that binds specifically to TDP-43, the second polypeptide comprising any one of: 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. In some embodiments, the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 116 and a second polypeptide comprising SEQ ID NO: 117. In some embodiments, the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 118 and a second polypeptide comprising SEQ ID NO: 119. In some embodiments, the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 120 and a second polypeptide comprising SEQ ID NO: 121. In some embodiments, the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 122 and a second polypeptide comprising SEQ ID NO: 123. In some embodiments, the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 124 and a second polypeptide comprising SEQ ID NO: 125. In some embodiments, the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 126 and a second polypeptide comprising SEQ ID NO: 127. In some embodiments the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 128 and a second polypeptide comprising SEQ ID NO: 129. In some embodiments the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 130 and a second polypeptide comprising SEQ ID NO:
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT 131. In some embodiments, the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 132 and a second polypeptide comprising SEQ ID NO: 133. In some embodiments, the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 134 and a second polypeptide comprising SEQ ID NO: 135. In some embodiments the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 136 and a second polypeptide comprising SEQ ID NO: 137. In some embodiments, the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 138 and a second polypeptide comprising SEQ ID NO: 139. In some embodiments, the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 140 and a second polypeptide comprising SEQ ID NO: 141. In some embodiments the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 142 and a second polypeptide comprising SEQ ID NO: 143. In some embodiments the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 144 and a second polypeptide comprising SEQ ID NO: 145. In some embodiments the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 146 and a second polypeptide comprising SEQ ID NO: 147. In some embodiments, the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 148 and a second polypeptide comprising SEQ ID NO: 149. In some embodiments, the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 150 and a second polypeptide comprising SEQ ID NO: 151. In some embodiments the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 152 and a second polypeptide comprising SEQ ID NO: 153. In some embodiments, the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 154 and a second polypeptide comprising SEQ ID NO: 155. In some embodiments the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 156 and a second polypeptide comprising SEQ ID NO: 157. In some embodiments, the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 158 and a second polypeptide comprising SEQ ID NO: 159. In some embodiments, the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 160 and a second polypeptide comprising SEQ ID NO: 161. In some embodiments, the nucleic acid encodes a cell penetrating agent comprising a first
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT polypeptide comprising SEQ ID NO: 162 and a second polypeptide comprising SEQ ID NO: 163. In some embodiments, the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 164 and a second polypeptide comprising SEQ ID NO: 165. In some embodiments the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 166 and a second polypeptide comprising SEQ ID NO: 167. In some embodiments, the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 168 and a second polypeptide comprising SEQ ID NO: 169. In some embodiments, the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 170 and a second polypeptide comprising SEQ ID NO: 171. In some embodiments, the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 172 and a second polypeptide comprising SEQ ID NO: 173. In some embodiments, the nucleic acid encodes a cell penetrating agent comprising a first polypeptide comprising SEQ ID NO: 174 and a second polypeptide comprising SEQ ID NO: 175. The present disclosure further provides nucleic acids encoding any of the heavy chain variable domains and/or light variable chain domains of any of the antibodies described herein. For example, the nucleic acids can encode a heavy chain variable domain comprising any one of SEQ ID NOs: 4-23 and/or a light chain variable domain comprising any one of SEQ ID NOs: 27- 48. Optionally, such nucleic acids further encode a signal peptide and can be expressed with the signal peptide linked to the constant region. Coding sequences of nucleic acids can be operably linked with regulatory sequences to ensure expression of the coding sequences, such as a promoter, enhancer, ribosome binding site, transcription termination signal, and the like. The nucleic acids encoding heavy and light chains can occur in isolated form or can be cloned into one or more vectors. The nucleic acids can be synthesized by, for example, solid state synthesis or PCR of overlapping oligonucleotides. Nucleic acids encoding heavy and light chains can be joined as one contiguous nucleic acid, e.g., within an expression vector, or can be separate, e.g., each cloned into its own expression vector. In some embodiments, the nucleic acid is codon- optimized for expression in a host cell. A number of methods are known for producing chimeric and humanized antibodies using an antibody-expressing cell line (e.g., hybridoma). For example, the immunoglobulin variable regions of antibodies can be cloned and sequenced using well known methods. In one method,
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT the heavy chain variable VH region is cloned by RT-PCR using mRNA prepared from hybridoma cells. Consensus primers are employed to the VH region leader peptide encompassing the translation initiation codon as the 5’ primer and a g2b constant regions specific 3’ primer. Exemplary primers are described in U.S. patent publication US 2005/0009150 by Schenk et al. (hereinafter “Schenk”). The sequences from multiple, independently derive clones can be compared to ensure no changes are introduced during amplification. The sequence of the VH region can also be determined or confirmed by sequencing a VH fragment obtained by 5’ RACE RT-PCR methodology and the 3’ g2b specific primer. The light chain variable VL region can be cloned in an analogous manner. In one approach, a consensus primer set is designed for amplification of VL regions using a 5’ primer designed to hybridize to the VL region encompassing the translation initiation codon and a 3’ primer specific for the Ck region downstream of the V-J joining region. In a second approach, 5’RACE RT-PCR methodology is employed to clone a VL encoding cDNA. Exemplary primers are described in Schenk, supra. The cloned sequences are then combined with sequences encoding human (or other non-human species) constant regions. Also provided herein are vectors including any of the nucleic acids described herein operably linked to one or more regulatory sequences to effect expression in a mammalian cell of any of the cell-penetrating agents described herein. Also provided herein are vectors including a nucleic acid encoding a mature heavy chain variable domain (e.g., any of the heavy chain variable domains described herein) and a light chain variable domain (e.g., any of the light chain variable domains described herein) operably linked to one or more regulatory sequences to effect expression in a mammalian cell of any of the antibodies or antigen-binding fragments described herein. In one approach, the heavy and light chain variable regions are re-engineered to encode splice donor sequences downstream of the respective VDJ or VJ junctions and are cloned into a mammalian expression vector, such as pCMV-hyl for the heavy chain and pCMV-Mcl for the light chain. These vectors encode human γl and Ck constant regions as exonic fragments downstream of the inserted variable region cassette. Following sequence verification, the heavy chain and light chain expression vectors can be co-transfected into CHO cells to produce chimeric antibodies. Conditioned media is collected 48 hours post-transfection and assayed by
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT western blot analysis for antibody production or ELISA for antigen binding. The chimeric antibodies are humanized as described above. Chimeric, veneered, humanized, and human antibodies are typically produced by recombinant expression. Recombinant polynucleotide constructs typically include an expression control sequence operably linked to the coding sequences of antibody chains, including naturally associated or heterologous expression control elements, such as a promoter. The expression control sequences can be promoter systems in vectors capable of transforming or transfecting eukaryotic or prokaryotic host cells. Once the vector has been incorporated into the appropriate host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences and the collection and purification of the cross-reacting antibodies. Thus, provided herein are host cells transformed with any of the vectors described herein. Also provided herein are host cells including any of the nucleic acids described herein. Expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA. Commonly, expression vectors contain selection markers, e.g., ampicillin resistance or hygromycin resistance, to permit detection of those cells transformed with the desired DNA sequences. E. coli is one prokaryotic host useful for expressing antibodies, particularly antibody fragments. Microbes, such as yeast, are also useful for expression. Saccharomyces is a yeast host with suitable vectors having expression control sequences, an origin of replication, termination sequences, and the like as desired. Typical promoters include 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include, among others, promoters from alcohol dehydrogenase, isocytochrome C, and enzymes responsible for maltose and galactose utilization. Mammalian cells can be used for expressing nucleotide segments encoding immunoglobulins or fragments thereof. See, Winnacker, From Genes to Clones, (VCH Publishers, NY, 1987). A number of suitable host cell lines capable of secreting intact heterologous proteins have been developed, and include CHO cell lines, various COS cell lines, HeLa cells, HEK293 cells, L cells, and non-antibody-producing myelomas including Sp2/0 and NS0. The cells can be non-human. Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter, an enhancer (Queen et al., Immunol. Rev. 89:49 (1986)), and necessary processing information sites, such as ribosome
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences. Expression control sequences can include promoters derived from endogenous genes, cytomegalovirus, SV 40, adenovirus, bovine papillomavirus, and the like. See Co et al., J. Immunol. 148: 1149 (1992). In some embodiments, the promoter is a eukaryotic promoter. Alternatively, antibody coding sequences can be incorporated in transgenes for introduction into the genome of a transgenic animal and subsequent expression in the milk of the transgenic animal (see, e.g., U.S. Pat. No. 5,741,957; U.S. Pat. No. 5,304,489; and U.S. Pat. No. 5,849,992). Suitable transgenes include coding sequences for light and/or heavy chains operably linked with a promoter and enhancer from a mammary gland specific gene, such as casein or beta lactoglobulin. The vectors containing the DNA segments of interest can be transferred into the host cell by methods depending on the type of cellular host. For example, calcium chloride transfection is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment, electroporation, lipofection, biolistics, or viral-based transfection can be used for other cellular hosts. Other methods used to transform mammalian cells include the use of polybrene, protoplast fusion, liposomes, electroporation, and micro injection. For production of transgenic animals, trans genes can be microinjected into fertilized oocytes or can be incorporated into the genome of embryonic stem cells, and the nuclei of such cells transferred into enucleated oocytes. Having introduced vector(s) encoding antibody heavy and light chains into cell culture, cell pools can be screened for growth productivity and product quality in serum-free media. Top- producing cell pools can then be subjected of FACS-based single-cell cloning to generate monoclonal lines. Specific productivities above 50 pg or 100 pg per cell per day, which correspond to product titers of greater than 7.5 g/L culture, can be used. Antibodies produced by single cell clones can also be tested for turbidity, filtration properties, PAGE, IEF, UV scan, HPSEC, carbohydrate-oligosaccharide mapping, mass spectrometry, and binding assay, such as ELISA or Biacore. A selected clone can then be banked in multiple vials and stored frozen for subsequent use. Once expressed, antibodies can be purified according to standard procedures of the art, including protein A capture, HPLC purification, column chromatography, gel electrophoresis and the like (See generally, Scopes, Protein Purification (Springer-Verlag, NY, 1982)).
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT Methodology for commercial production of antibodies can be employed, including codon optimization, selection of promoters, selection of transcription elements, selection of terminators, serum-free single cell cloning, cell banking, use of selection markers for amplification of copy number, CHO terminator, or improvement of protein titers (see, e.g., U.S. Patent No. 5,786,464; U.S. Patent No. 6,114,148; US 6,063,598; U.S. Patent No. 7,569,339; W02004/050884; W02008/012142; W02008/012142; W02005/019442; W02008/107388; W02009/027471; and U.S. Patent No. 5,888,809). The DNA can be delivered in naked form (i.e., without colloidal or encapsulating materials). Alternatively, a number of viral vector systems can be used including retro viral systems (see, e.g., Lawrie and Tumin, Cur. Opin. Genet. Develop. 3, 102-109 (1993)); adenoviral vectors (see, e.g., Bett et al, J. Virol. 67, 5911 (1993)); adeno-associated virus vectors (see, e.g., Zhou et al., J. Exp. Med. 179, 1867 (1994)), viral vectors from the pox family including vaccinia virus and the avian pox viruses, viral vectors from the alpha virus genus such as those derived from Sindbis and Semliki Forest Viruses (see, e.g., Dubensky et al., J. Virol. 70, 508-519 (1996)), Venezuelan equine encephalitis virus (see U.S. Patent No. 5,643,576) and rhabdoviruses, such as vesicular stomatitis virus (see WO 96/34625) and papillomaviruses (Ohe et al., Human Gene Therapy 6:325-333 (1995); Woo et al., WO 94/12629 and Xiao & Brandsma, Nucleic Acids. Res. 24:2630-2622 (1996)). DNA encoding an immunogen, or a vector containing the same, can be packaged into liposomes. Suitable lipids and related analogs are described by U.S. Patent No. 5,208,036, U.S. Patent No. 5,264,618, U.S. Patent No. 5,279,833, and U.S. Patent No. 5,283,185. Vectors and DNA encoding an immunogen can also be adsorbed to or associated with particulate carriers, examples of which include polymethyl methacrylate polymers and polylactides and poly(lactide- co-glycolides) (see, e.g., McGee et al., J. Micro Encap. 1996). V. Additional Conjugates Conjugated antibodies and antigen-binding antibody fragments that specifically bind to antigens such as TDP-43 (e.g., human TDP-43), are useful in detecting the presence of TDP-43; monitoring and evaluating the efficacy of therapeutic agents being used to treat patients diagnosed with amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD- TDP), primary lateral sclerosis, and progressive muscular atrophy, and Parkinson’s disease;
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT inhibiting or reducing aggregation of TDP-43; reducing or clearing TDP-43 aggregates; stabilizing non-toxic conformations of TDP-43; or treating or effecting prophylaxis of amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD-TDP), primary lateral sclerosis, and progressive muscular atrophy, and Parkinson’s disease in a patient. The cell-penetrating agents described herein, can be further conjugated with other therapeutic moieties, other proteins, other antibodies, and/or detectable labels. See WO 03/057838; U.S. Patent No. 8,455,622. Such therapeutic moieties can be any agent that can be used to treat, combat, ameliorate, prevent, or improve an unwanted condition or disease in a patient, such as amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD- TDP), primary lateral sclerosis, and progressive muscular atrophy, and Parkinson’s disease. Conjugated therapeutic moieties can include cytotoxic agents, cytostatic agents, neurotrophic agents, neuroprotective agents, radiotherapeutic agents, immunomodulators, or any biologically active agents that facilitate or enhance the activity of the antibody. A cytotoxic agent can be any agent that is toxic to a cell. A cytostatic agent can be any agent that inhibits cell proliferation. A neurotrophic agent can be any agent, including chemical or proteinaceous agents, which promotes neuron maintenance, growth, or differentiation. A neuroprotective agent can be agent, including chemical or proteinaceous agents, which protects neurons from acute insult or degenerative processes. An immunomodulator can be any agent that stimulates or inhibits the development or maintenance of an immunologic response. A radiotherapeutic agent can be any molecule or compound that emits radiation. If such therapeutic moieties are coupled to a TDP-43 specific antibody or antigen-binding antibody fragment, such as the antibodies and antigen-binding antibody fragments described herein, the coupled therapeutic moieties will have a specific affinity for TDP-43 related disease-affected cells over normal cells. Consequently, administration of these further conjugated antibodies or conjugated antigen-binding antibody fragments directly targets cells with minimal damage to surrounding normal, healthy tissue. This can be particularly useful for therapeutic moieties that are too toxic to be administered on their own. In addition, smaller quantities of the therapeutic moieties can be used. Some such cell-penetrating agents can be linked to radioisotopes. Examples of radioisotopes include, for example, yttrium90 (90Y), indium111 (111In), 1311, 99mTc, radiosilver- 111, radiosilver-199, and Bismuth213. Linkage of radioisotopes to antibodies or antigen-binding
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT antibody fragments may be performed with conventional bifunction chelates. For radiosilver- 111 and radiosilver-199 linkage, sulfur-based linkers may be used. See Hazra et al., Cell Biophys. 24-25:1-7 (1994). Linkage of silver radioisotopes may involve reducing the immunoglobulin 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 tiuxetan and 90Y-ibritumomab tiuxetan, respectively. See Witzig, Cancer Chemother. Pharmacol., 48(Suppl l):S91-S95 (2001). Some such antibodies or antigen-binding antibody fragments can be linked to other therapeutic moieties. Such therapeutic moieties can be, for example, cytotoxic, cytostatic, immunomodulatory, neurotrophic, or neuroprotective. For example, antibodies and antigen- binding antibody fragments can be conjugated with toxic chemotherapeutic drugs such as maytansine, geldanamycin, tubulin inhibitors such as tubulin binding agents (e.g., auristatins), or minor groove binding agents such as calicheamicin. Other representative therapeutic moieties include agents known to be useful for treatment, management, or amelioration of amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD-TDP), primary lateral sclerosis, and progressive muscular atrophy, and Parkinson’s disease. Antibodies or antigen-binding antibody fragments can also be coupled with a detectable label. Such antibodies and antigen-binding antibody fragments can be used, for example, for diagnosing ALS, FTLD-TDP, primary lateral sclerosis, and progressive muscular atrophy, and Parkinson’s disease. Representative detectable labels that may be coupled or linked to an antibody or antigen-binding antibody fragment include various enzymes, such as horseradish peroxidase, alkaline phosphatase, betagalactosidase, or acetylcholinesterase; prosthetic groups, such streptavidin/biotin and avidin/biotin; fluorescent materials, such as umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; luminescent materials, such as luminol; bioluminescent materials, such as luciferase, luciferin, and aequorin; radioactive materials, such as radiosilver-111, radiosilver-199, Bismuth213, iodine (131I, 125I, 123I, 121I), carbon (14C), sulfur (5S), tritium (3H), indium (115In 113In 112In 111In), technetium (99Tc), thallium (201Ti), gallium (68Ga, 67Ga), palladium (103Pd), molybdenum (99Mo ), xenon (133Xe), fluorine (18F), 153Sm, 177Lu, 159Gd, 149Pm, 140La, 175Yb, 166Ho, 90Y, 47Sc, 186Re, 188Re, 142Pr, 105Rh, 97Ru, 68Ge, 57Co, 65ZN, 85SR, 32P, 153Gd, 169Yb, 51CR, 54Mn, 75Se, 113Sn, and 117Sn; positron emitting metals using various positron
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT emission tomographies; nonradioactive paramagnetic metal ions; and molecules that are radiolabelled or conjugated to specific radioisotopes. Therapeutic moieties, other proteins, other antibodies, and/or detectable labels may be coupled or conjugated, directly or indirectly through an intermediate (e.g., a linker), to an antibody or antigen-binding antibody fragment of the invention. See e.g., 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., Immunol. Rev., 62:119-58 (1982). Suitable linkers include, for example, cleavable and noncleavable linkers. Different linkers that release the coupled therapeutic moieties, proteins, antibodies, and/or detectable labels under acidic or reducing conditions, on exposure to specific proteases, or under other defined conditions can be employed. In some embodiments, the cell-penetrating agent is also conjugated to a therapeutic, cytotoxic, cytostatic, immunomodulatory, neurotrophic, or neuroprotective agent as described herein. For example, antibodies present in the cell-penetrating agent can be coupled (i.e., conjugated) with a therapeutic moiety, such as a cytotoxic agent, a radiotherapeutic agent, an immunomodulator, or a second antibody (e.g., to form an antibody heteroconjugate). Representative therapeutic moieties include agents known to be useful for treatment, management, or amelioration of TDP-43-related diseases or symptoms of TDP-43-related diseases. Therapeutic moieties and/or detectable substances may be coupled or conjugated directly to any of the murine, chimeric, or humanized antibodies described herein through an intermediate (e.g., a linker) using techniques known in the art. See e.g., 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);
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT 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. Cell-penetrating agents used in the disclosed formulations also include modified forms of murine, chimeric, or humanized 13D3 antibodies, which have increased in vivo half-lives relative to the corresponding unmodified antibodies. Such modified forms may be prepared, for example, by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. As one example, representative methods for antibody half-life extension are described in PCT International Publication No. WO 02/060919. VI. Pharmaceutical Compositions and Products The present disclosure also provided pharmaceutical compositions and products. Thus, provided herein are pharmaceutical compositions including any of the cell-penetrating agents described herein and a pharmaceutically acceptable carrier. Pharmaceutical compositions for parenteral administration are preferably sterile and substantially isotonic and manufactured under GMP conditions. Pharmaceutical compositions can be provided in unit dosage form (i.e., the dosage for a single administration). Pharmaceutical compositions can be formulated using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries. The formulation depends on the route of administration chosen. For injection, cell-penetrating agents can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank’s solution, Ringer’s solution, or physiological saline or acetate buffer (to reduce discomfort at the site of injection). The solution can contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, cell-penetrating agents can be in lyophilized form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT The anti-TDP-43 cell penetrating agents described herein can be present in any pharmaceutically acceptable excipient or carrier. For example, the anti-TDP-43 cell-penetrating agents described herein can be present in a buffer. The buffer can have a pH from about 6 to about 7. Typically, the formulations are sterile, for example, as accomplished by sterile filtration using a 0.2 ^m or a 0.22 ^m filter. The formulations disclosed herein are also generally stable upon freezing and thawing. In some embodiments, it can be desirable to use a pharmaceutical composition comprising any of the cell-penetrating agents described herein in an ex vivo or in vitro method. For example, the method can be for a non-diagnostic and/or non-therapeutic purposes. In such instances, samples such as cells, tissues, and/or organs that have been removed from the patient are exposed to a pharmaceutical composition comprising any of the cell-penetrating agents described herein. In prophylactic applications, a cell-penetrating agent (e.g., or a nucleic acid or a vector encoding any of the cell-penetrating agent) or a pharmaceutical composition of the same is administered to a patient susceptible to, or otherwise at risk of a disease such as ALS, FTLD- TDP, primary lateral sclerosis, and progressive muscular atrophy, and Parkinson’s disease in regime (dose, frequency and route of administration) effective to reduce the risk, lessen the severity, or delay the onset of at least one sign or symptom of the TDP-43-related disease. In particular, the regime is preferably effective to inhibit or delay TDP-43 aggregates (e.g., human TDP-43 aggregates) in the brain, and/or inhibit or delay its toxic effects and/or inhibit/or delay development of behavioral deficits. In therapeutic applications, a cell-penetrating agent is administered to a patient suspected of, or already suffering from a disease (e.g., ALS) in a regime (dose, frequency and route of administration) effective to ameliorate or at least inhibit further deterioration of at least one sign or symptom of the disease. In particular, the regime is preferably effective to reduce or at least inhibit further increase of cytoplasmic levels of TDP-43 (e.g., human TDP-43) and/or aggregates formed from it, associated toxicities and/or behavioral deficits. A regime is considered therapeutically or prophylactically effective if an individual treated patient achieves an outcome more favorable than the mean outcome in a control population of comparable patients not treated by methods disclosed herein.
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT VII. Treatment Regimens As used herein, the terms “treat” and “treatment” refer to the alleviation or amelioration of one or more symptoms or effects associated with the disease, prevention, inhibition or delay of the onset of one or more symptoms or effects of the disease, lessening of the severity or frequency of one or more symptoms or effects of the disease, and/or increasing or trending toward desired outcomes as described herein. Desired outcomes of the treatments disclosed herein vary according to the TDP-43- related disease and patient profile and are readily determinable to those skilled in the art. Desired outcomes include an improvement in the patient’s health status. Generally, desired outcomes include measurable indices such as reduction or clearance of pathologic ALS, FTLD- TDP, primary lateral sclerosis, and progressive muscular atrophy, and Parkinson’s disease. Provided herein are methods of delivering an antibody that specifically binds to TDP-43 into a cell, comprising contacting any of the cell-penetrating agents described herein with the cell, thereby resulting in the internalization of, at a minimum, an antigen-binding fragment of the antibody, into the cell. In some embodiments, the method includes transferring, at a minimum, an antigen-binding fragment of the antibody, to the cytosol of the cell. Also provided herein are methods of binding an intracellular TDP-43 protein in a cell that include contacting the cell-penetrating agent of any one of claims 1-135 with the cell, thereby resulting in the internalization of and transfer to the cytosol of, at a minimum, an antigen-binding fragment of the antibody. Also provided herein are methods of binding an intracellular TDP-43 protein in a cell that include: contacting the cell-penetrating agent of any one of claims 1-135 with the cell, thereby resulting in the internalization of and transfer to the cytosol of, at a minimum, an antigen-binding fragment of the antibody; and binding, at a minimum, an antigen-binding fragment of the antibody, to the intracellular TDP-43 protein. Also, provided herein are methods of inhibiting or reducing aggregation of TDP-43 (e.g., human TDP-43) in a subject having or at risk of developing a TDP-43-related disease, including administering to the subject an effective amount of any of the cell-penetrating agents described herein, thereby inhibiting or reducing aggregation of TDP-43 in the subject (i.e., patient). Also provided herein are methods of treating or effecting prophylaxis of a TDP-43- related disease in a subject, including administering a therapeutically effective amount of any of
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT the cell-penetrating agents described herein, thereby treating or effecting prophylaxis of the TDP-43-associated disease. In some embodiments, the TDP-43-related disease is ALS, FTLD-TDP, primary lateral sclerosis, and progressive muscular atrophy, and Parkinson’s disease. In some embodiments, the TDP-43-related disease is ALS. Also provided herein are methods of detecting TDP-43 deposits (e.g., human TDP-43 deposits) in a subject having or at risk of developing a TDP-43-related disease, including administering to a subject any of the cell-penetrating agents described herein, and detecting the antibody bound to TDP-43 in the subject. In some embodiments, the cell-penetrating agent is administered by intravenous injection into the body of the subject. In some embodiments, the cell-penetrating agent or the antibody in the cell-penetrating agent is labeled. In some embodiments, the cell-penetrating agent is labeled with a fluorescent label, a paramagnetic label, or a radioactive label. In some embodiments, the radioactive label is detected using positron emission tomography (PET) or single-photon emission computed tomography (SPECT). The cell-penetrating agents are administered in an effective regime meaning a dosage, route of administration and frequency of administration that delays the onset, reduces the severity, inhibits further deterioration, and/or ameliorates at least one sign or symptom of a disorder being treated. If a patient is already suffering from a disorder, the regime can be referred to as a therapeutically effective regime. If the patient is at elevated risk of the disorder relative to the general population but is not yet experiencing symptoms, the regime can be referred to as a prophylactically effective regime. In some instances, therapeutic or prophylactic efficacy can be observed in an individual patient relative to historical controls or past experience in the same patient. In other instances, therapeutic or prophylactic efficacy can be demonstrated in a preclinical or clinical trial in a population of treated patients relative to a control population of untreated patients. Administration can be parenteral, intravenous, oral, subcutaneous, intra-arterial, intracranial, intrathecal, intraperitoneal, topical, intranasal or intramuscular. Some cell- penetrating agents can be administered into the systemic circulation by intravenous or subcutaneous administration.
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT The cell-penetrating agent formulation can be administered intravenously or subcutaneously in dosage ranges from about 0.5 mg/kg to about 30 mg/kg of the host body weight. For example, dosages can be about 0.5 mg/kg body weight, about 1.0 mg/kg, about 1.5 mg/kg, about 2.0 mg/kg, about 4.0 mg/kg, about 5.0 mg/kg, about 8.0 mg/kg, about 10 mg/kg, about 15 mg/kg, about 16 mg/kg, about 20 mg/kg, about 24 mg/kg, about 25 mg/kg, or about 30 mg/kg body weight. The dosages can also be administered according to body surface area from about 0.5 mg/m2 to about 500 mg/m2, for example, 0.5, 5, 10, 50, 100, 250 or 500 mg/m2. For intravenous dosing, an amount of the cell-penetrating agent formulation sufficient to achieve the desired dosage for the individual patient is transferred from one or more vials to one or more intravenous bags containing a liquid (e.g., saline) and administered to the patient. Cell-penetrating agents are usually administered on multiple occasions. The frequency of administration depends on the half-life of the cell-penetrating agent in circulation, the condition of the patient and the route of administration among other factors. The frequency can be daily, weekly, monthly, quarterly, or at irregular intervals in response to changes in the patient’s condition or progression of the disorder being treated. An exemplary treatment regimen entails administration once per every two weeks, once a month, or once every 3 to 6 months. The number of dosages administered depends on whether the disorder is acute or chronic and the response of the disorder to the treatment. The dosing frequency can be adjusted depending on the pharmacokinetic profile of the antibody formulation in the patient. For example, the half-life of the cell-penetrating agent may warrant a two week frequency of dosing. 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 life of the patient. Normal levels of TDP-43 (e.g., human TDP-43) can be determined in the brains of a representative sample of individuals in the general population who have not been diagnosed with a particular TDP-43 related disease (e.g., ALS) and are not considered at elevated risk of developing such disease (e.g., a representative sample of disease-free individuals under 50 years of age). Alternatively, a normal level can be recognized in an individual patient if a PET signal according to the present methods in a region of the brain in which TDP-43 aggregates (e.g., human TDP-43 aggregates) are known to develop is not different (within the accuracy of measurement) from the signal from a region of the brain in which it is known that such deposits
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT do not normally develop. An elevated level in an individual can be recognized by comparison to the normal levels (e.g., outside mean and variance of a standard deviation) or simply from an elevated signal beyond experimental error in a region of the brain associated with TDP-43 aggregates (e.g., human TDP-43 aggregates) compared with a region not known to be associated with deposits. For purposes of comparing the levels of TDP-43 aggregates (e.g., human TDP-43 aggregates) in an individual and population, the TDP-43 aggregates should preferably be determined in the same region(s) of the brain, these regions including at least one region in which TDP-43 aggregates associated with a particular disease (e.g., ALS) are known to form (e.g., in the cytoplasm). A patient having an elevated level of TDP-43 aggregates (e.g., human TDP-43 aggregates) is a candidate for commencing immunotherapy. After commencing immunotherapy, a decrease in the level of TDP-43 aggregates (e.g., human TDP-43 aggregates) can be first seen as an indication that the treatment is having the desired effect. The observed decrease can be, for example, in the range of 1-100%, 1-50%, or 1-25% of the baseline value. Such effects can be measured in one or more regions of the brain in which deposits are known to form or can be measured from an average of such regions. The total effect of treatment can be approximated by adding the percentage reduction relative to baseline to the increase in TDP-43 aggregates (e.g., human TDP-43 aggregates) that would otherwise occur in an average untreated patient. Maintenance of TDP-43 aggregates (e.g., human TDP-43 aggregates) at an approximately constant level or even a small increase in TDP-43 aggregates (e.g., human TDP- 43 aggregates) can also be an indication of response to treatment albeit a suboptimal response. Such responses can be compared with a time course of levels of TDP-43 aggregates (e.g., human TDP-43 aggregates) in patients with a particular disease (e.g., ALS) that did not receive treatment, to determine whether the immunotherapy is having an effect in inhibiting further increases of TDP-43 aggregates (e.g., human TDP-43 aggregates). VIII. Kits The present disclosure further provides kits (e.g., containers) comprising any of the cell- penetrating agents described herein and related materials, such as instructions for use (e.g., package insert). The instructions for use can contain, for example, instructions for administration of the cell-penetrating agent and optionally one or more additional agents. The
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT containers of cell-penetrating agents may be unit doses, bulk packages (e.g., multi-dose packages), or sub-unit doses. IX. Methods of Detection In some aspects, the cell-penetrating agents of the present disclosure further provides methods of detecting TDP-43 in a sample. For example, in some embodiments, the present disclosure provides a method of detecting TDP-43 in a sample comprising contacting a cell- penetrating agent of the present disclosure with the sample and detecting the binding of the cell- penetrating agent or the antibody to TDP-43. For example, such methods can be an ex vivo or in vitro method. 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 having or at risk of having a TDP-43 related disease. In such instances, samples such as cells, tissues, and/or organs that have been removed from the patient are exposed to an antibody or antigen-binding fragments 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 fragments described herein. EXAMPLES The following examples have been included to illustrate modes disclosed herein. Certain aspects of the following examples are described in terms of techniques and procedures found or contemplated by the present co-inventors to work well in the practice disclosed herein. In light of the present disclosure and the general level of skill in the art, those of skill appreciate that the following examples are intended to be exemplary only and that numerous changes, modifications, and alterations may be employed without departing from the scope of the disclosure. Example 1. Humanized Anti-TDP-43 Antibodies Humanized anti-TDP-43 antibodies were generated from murine monoclonal antibody 13D3. Figure 1 shows an annotated version of the heavy chain variable domain and Figure 2 shows an annotated version of the light chain variable domain, respectively. Both the heavy
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT chain variable domain and the light chain variable domain sequences show the native signal peptide, the variable domain, and partial constant domains. The partial heavy chain constant domain region is a IgG2a mouse constant domain. The partial light chain constant domain is a mouse kappa constant domain. Reference to amino acid substitutions described herein refers to the Kabat numbering system (See e.g., Kabat E.A., et. al, Sequences of Proteins of Immunological Interest (5th edition). Bethesda, MD: National Institutes of Health (1991)). Briefly, protein sequences were 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, Nucleic Acids Research, 33:D233-D237 (2005)) to find structures that would provide a similar structural model of 13D3. The crystal structure of an antibody Fab PDB code “5BK5” which is a human germline antibody. 5BK5 was selected for both VH and VK structure based on its overall sequence similarity to 13D3 VH and VK, the quality of resolution (3.0 Å), and it retained the same canonical structures for the complementarity determining region (CDR) loops (Scally et al., Crystal structure of anti- cirumsporozoite protein 663 germline antibody; Direct deposit to PDB (2017)). Additionally, since 5BK5 antibody is a human germline derived antibody and has the same canonical classes belonging to human germline IGHV3-48’03 (SEQ ID NO: 2) for the variable heavy chain domain and IGKV2-30*02 (SEQ ID NO: 25) for the variable light chain domain these sequences were used as a human acceptor framework. Accordingly, the framework regions of 5BK5 VH and 5BK5 VL were chosen as the acceptor sequences for the CDRs of 13D3. A model of the 13D3 CDRs grafted onto the respective human frameworks for VH and VL was built and used as guidance for further backmutations to increase binding specificity and reduce immunogenicity. More specifically, the amino acid sequences consisting of 5BK5 VH human frameworks and 13D3 CDRs are designated hu13D3VHv1d (SEQ ID NO: 20) and the amino acid sequences consisting of 5BK5 VL human framework and 13D3 VL CDRs are designated hu13D3VLv1d (SEQ ID NO: 47). Additional versions of hu13D3VH and hu13D3VL were designed to enable assessment of various framework residues for their contributions to antigen binding, thermostability, developability (e.g., deamination, oxidation, N-glycosylation, proteolysis, and aggregation) and immunogenicity. The positions considered for substitution were based on a variety of factors
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT including positions that define the canonical CDR conformations (See, Martin, A.C.R., 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)); positions within the Vernier zone (See, Foote J., and Winter, G., Antibody framework residues affecting the conformation of the hypervariable loops. J Mol Biol. 224(2):487-99 (1992)); positions that localize to the VH/VL domain interface (See, Léger O.J.P. and Saldanha, J. Preparation of recombinant antibodies from immune rodent 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)); positions that are amenable to post-translational modifications, such as glycosylation or pyroglutamination; positions that are occupied by residues that are predicted to interact with CDRs, according to the model of 13D3 CDRs grafted onto VH and VL frameworks, and/or positions that are occupied by residues that are rare among sequenced human antibodies, where either the parental murine 13D3 residue or other residue is much more prevalent within the human antibody repertoire. The following is a summary of humanized 13D3 antibodies. Heavy Chain Variable Domains hu13D3VHvd1 (SEQ ID NO: 20) consists of the CDR-H1, CDR-H2, and CDR-H3 loops of 13D3-VH grafted onto the framework of 5BK5 VH and reverts all framework substitutions at positions that are key for defining the Chothia canonical classes, are part of the Vernier zone, and localize to the VH/VL domain interface or contribute to structural stability. Hu13D3VHvd1 includes the following substitutions which are back mutations to the germline antibody and found most frequently: position L5V and T77S. hu13D3VHv2d (SEQ ID NO: 21) includes the following substitution: L78A. Leucine at position 78 is immunogenic as indicated by Immune Epitope Database (“IEDB”) Analysis. Therefore, deimmunization analysis predicts immunogenicity reduction with an alanine substitution at position 78. Hu13D3VHv3d (SEQ ID NO: 22) includes the following substitutions: G44R, S49A, and S74A. Arginine at position 44 makes de novo contacts with a G100D (glycine to aspartic acid) substitution made in the variable light chain domain thereby strengthening the heavy chain variable domain: light chain variable domain interface. Specifically, arginine at position 44 forms a hydrogen bond and a salt bridge with G100D and also forms a hydrogen bond with F98
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT in the variable light chain domain. Position 49 is a Vernier zone residue and substitution from serine to alanine was a back mutation to assess the impact on CDR conformation and binding. Last, a serine to alanine substitution at position 74 is a germline substitution. Hu13D3VHv4d (SEQ ID NO: 23) includes various substitutions made in hu1353VHv1d, hu1353VHv2d, and hu1353VHv3d tried in combination. Light Chain Variable Domains hu13D3VLv1d (SEQ ID NO: 47) consists of the CDR-L1, CDR- L2, and CDR-L3 loops of 13D3VL grafted onto the framework of 5BK5 VL along with reverting all framework substitutions at positions that are key for defining the Chothia canonical classes, are part of the Vernier zone, and locate to the VH/VL domain interface. Hu1353VLv1d includes the following substitutions: V3Q, P15L, E17Q, L38Q, K39R, Q100D, and L104V. Substitution of valine for glutamine at position 3 reduces immunogenicity of the light chain variable domain. Substitution of leucine for proline at position 15 is a germline substitution. Substitution of glutamine for glutamic acid at position 17 is a rare substitution. Position 17 has significant surface exposure thereby contributing to a negative charge patch at the protein surface. Substitution to glutamine reduces antibody surface negative patches. Substitution of glutamine for leucine at position 38 was made since glutamine in mouse structural models forms inter-strand contacts within the light chain variable domain to maintain structural conformation. In contrast, a leucine reside cannot form the same contacts and therefore this back mutation increases conformational stability. Substitution of arginine for lysine at position 39 increases conformation stability by forming additional contacts with adjacent residues that a lysine residue cannot. Position 100 in the light chain variable domain is a located at the heavy chain variable domain and light chain variable domain interface, however, no interchain contacts are formed with the glutamine residue. Substitution to aspartic acid along with a concurrent substitution of arginine at position 44 in the heavy chain variable domain results in stronger do novo contacts and therefore increases thermal stability of the antibody. Substitution of valine for leucine position 104 is predicted to reduce immunogenicity. Hu13D3VLv2d (SEQ ID NO: 48) consists of the aforementioned substitutions described above in hu13D3VLv1d, but also includes the following substitution: L92A. Leucine at position
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT 92 is predicted to be immunogenic, therefore, substitution with alanine at position 92 reduces the immunogenicity of the variable light chain domain. Table 1 and Table 2 below show 13D3 variable heavy chain domain sequence alignment and variable light chain domain sequence alignment, respectively, compared to humanized versions. Table 1. 13D3 Humanized Variable Heavy Chain Domain Sequence Alignment 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 VH m13D3-VH (SEQ ID NO: 1) E V Q L V E S G G G L V Q P G G S L K L L L L L L L L L L L L L L L L L L L L
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT (SEQ ID NO: 23) VH 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 m13D3-VH S C A A S G F T F S S Y E M N W V R Q A A A A A A A A A A A A A A A A A A A A
VH 41 42 43 44 45 46 47 48 49 50 51 52 52A 53 54 55 56 57 58 59 P E K R L E A Y I T D A Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT h13D3VHvd1 P G K G L E W V S Y I S T G G D S A N Y h13D3VHvd2 P G K G L E W V S Y I S T G G D S A N Y Y Y
VH 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 m13D3-VH A D N V K G R F T I S R D N A K N T L Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y
VH 80 81 82 82A 82B 82C 83 84 85 86 87 88 89 90 91 92 93 94 95 96 T F N T T T T T T T T T
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT h13D3VHv10 L Q M N S G R A E D T A V Y F C A R Q T h13D3VHv11 L Q M N S L M A E D T A V Y F C A R Q T T T T T T T
VH 97 98 99 100 100A 100B 100C 100D 100E 100F 101 102 103 104 105 106 m13D3-VH Y Y S Y G G F - - - P Y W G Q G G G G G G G G G G G G G G G G G G G G
VH 107 108 109 110 111 112 113
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT h13D3VHv6 P Y W G Q G T h13D3VHv7 P Y W G Q G T
Table 2. 13D3 Humanized Variable Light Chain Sequence Alignment 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 VL m13D3-VL D V V M T Q S P L S L P V S L G D Q A S S S S S S S S S S S S S S S
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT 21 22 23 24 25 26 27 27 27 27 27 27 27 A B C D E F 28 29 30 31 32 33 34 VL m13D3-VL I S C R S S Q S L V H S _ N G K T Y L H N N H H H H H H H H H H H H
35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 VL m13D3-VL W Y Q Q K P G Q S P K L L I Y K V S D R R R R R R R R R R R R R R R
55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 VL 13D3 VL Y S G V S D R F S G S G S G T D F T L K K K K K K
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT h13D3VLv4 Y S G V P D R F S G S G S G T D F T L K h13D3VLv5 Y S G V P D R F S G S G S G T D F T L K K K K K K K K
75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 VL m13D3-VL I S R V E T E D L G V Y F C S Q S L H V W R V V V V V V A V V V V
95 96 97 98 99 101 101 102 103 104 105 106 107 VL m13D3-VL P L T F G A G T K L E L K
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT h13D3VLv9 P L T F G D G T K V E I K h13D3VLv10 P L T F G R G T K V E I K
Separately, for Vk, a human kappa light chain with NCBI accession code ABC66863 (Shringer et al, 2006) was selected (SEQ ID NO: 26). This kappa light chain has the same canonical classes for CDR-L1 & L2 and belongs to human germline IGKV2-30*02 (SEQ ID NO: 25) according to IMGT convention. For VH, human Ig heavy chain AEX28899 (SEQ ID NO: 3) (GenBank: AEX28899) (Bowers et al., 2014) was chosen, again with the same canonical classes and belonging to human germline IGHV3-48’03. It is a member of Kabat human heavy subgroup 3. AEX28899 and ABC66863 antibodies are human germline derived antibodies that have the same canonical classes and belong to human germline IGHV3-48’03 (SEQ ID NO: 2) for the heavy chain variable domain and IGHV3-48*03 (SEQ ID NO: 25) for the light chain variable domain. Therefore, the AEX28899 heavy chain variable domain and ABC66863 light chain variable domain sequences were used as human acceptor framework for the CDRs of 13D3. A model of the 13D3 CDRs grafted onto the respective human frameworks for VH and VL was built and used as a guidance for further backmutations. Humanized versions of AEX28899 heavy chain variable domain and ABC66863 light chain variable domain sequences were used as human acceptor framework for the CDRs of 13D3 and were designed to enable assessment of various framework residues for their contributions to antigen binding, thermostability, developability (e.g., deamination, oxidation, N-glycosylation, proteolysis, and aggregation) and immunogenicity. The positions considered for substitution were based on a variety of factors including positions that define the canonical CDR conformations (See, Martin, A.C.R., 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)); positions within the Vernier zone (See, Foote J., and Winter, G., Antibody framework residues affecting the conformation of the hypervariable loops. J Mol Biol. 224(2):487-99 (1992)); positions that localize to the VH/VL domain interface (See, Léger O.J.P. and Saldanha, J). Preparation of recombinant antibodies from immune rodent spleens and the design of their humanization by CDR grafting. In: Shepherd P and Dean C (eds).
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT Monoclonal Antibodies: A Practical Approach. Oxford, UK: Oxford University Press (2000)); positions that are amenable to post-translational modifications, such as glycosylation or pyroglutamination; positions that are occupied by residues that are predicted to interact with CDRs, according to the model of 13D3 CDRs grafted onto VH and VL frameworks, and/or positions that are occupied by residues that are rare among sequenced human antibodies, where either the parental murine 13D3 residue or other residue is much more prevalent within the human antibody repertoire. The following is a summary of VH domain substitutions in SEQ ID NO: 3: R19K; K19 in mouse antibody structure makes pi-cation interaction with W79 of SEQ ID NO: 3; G44R: substitution of Gly with Arg at this position potentially establishes contact with the light chain thereby increasing antibody stability; S77T: threonine reduces immunogenicity; L78A: leucine is predicted to be immunogenic, therefore substitution with alanine reduces predicted immunogenicity; L78G: glycine also reduces predicted immunogenicity; L80A: removing leucine from this position reduces immunogenicity; L80G: similar to alanine, a glycine at position 80 reduces immunogenicity; L82cG: glycine potentially reduces heavy chain immunogenicity; and R83M: is a back mutation that may confer antibody stability. The following is a summary of VL domain substitutions in SEQ ID NO: 26: I2V is a Vernier zone residue, valine is retained to preserve CDR conformation; L9S: leucine is predicted to be immunogenic, substitution with serine lowers predicted immunogenicity; P18Q: mouse antibody model glutamine forms a H-bond with K74 (LC) thereby stabilizing interchain interaction; R46L: is an interface and Vernier zone residue and therefore Leu is retained; A80S: serine lowers predicted immunogenicity; L92G: L92 is a CDR residue and is predicted to be immunogenic, a glycine residue is predicted to reduce immunogenicity; V94I: valine at position 94 is predicted to be low level immunogenic, isoleucine is predicted to reduce immunogenicity; and V94A: alanine is an alternative substitution that is predicted to lower immunogenicity. Example 2. Characterization of Humanized 13D3 Antibody Variants As discussed herein cytoplasmic aggregates of TDP-43 are found in many neurodegenerative diseases and in particular, ALS. Generally, the individual TDP-43 proteins found in the cytoplasmic aggregates are phosphorylated at serine residues at position 409 and/or
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT 410. A binding screening assay was established to determine whether 13D3 and humanized variants thereof would selectively bind to phosphorylated TDP-43 found in cytoplasmic aggregates. About 50 RU of biotin-phosphoTDP43 proteins were immobilized on a CM3 chip with NeutrAvidin. Various humanized 13D3 antibodies and chimeric 13D3 antibody were flowed over the chip and assayed for binding to phosphorylated TDP43 protein. Specifically, hu13D3H9L8, hu13D3H10L6, hu13D3H10L7, hu13D3H10L8, and hu13D3H9L7 were assayed and showed similar binding to phosphoTDP43 as chimeric 13D3 antibody (Figure 3). Similarly, hu13D3Hd1Ld1, hu13D3Hd2Ld2, hu13D3Hd3Ld1, hu13D3HD4LD1, hu13D3HD1LD2, hu13D3HD2LD2, hu13D3HD3LD2, and hu13D3HD4LD2 were tested for binding to phosphoTDP43. Each variant showed similar binding to phosphoTDP43 as chimeric 13D3 antibody (Figure 4). In another experiment, antibodies were immobilized on a Protein A Chip (~2000 RU) and a 23 amino acid TDP-43 peptide phosphorylated at positions 409 and 410 (“TDP-43 peptide (pS409/pS410)”) were flowed across the chip (SEQ ID NO: 83) at concentrations between 1 nM and 100 nM. As a control a 23 amino acid TDP-43 peptide that was not phosphorylated at positions 409 or 410 was also tested between 12 nM and 1 µM. Specifically, humanized version hu13D3H5L2 demonstrated 1:1 binding with TDP-43 peptide (pS409/pS410) at various concentrations (1.2345 nM; 3.703 nM; 11.111 nM; 33.33 nM; and 100 nM) which was similar to the 13D3 murine antibody (Figure 5). Furthermore, hu13D3H5L2 showed no affinity for the non-phosphorylated 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). Thermal stability of the antibodies was also assessed. Thermal stability analysis was performed with Different Scanning Calorimetry which characterizes the stability of a protein or other biomolecule (e.g., an antibody or antigen-binding fragment thereof). Antibodies were tested 2.66 mM (0.4 mg/mL) in 1xPBS at pH 7.4. Temperature range tested was from 25°C to 100°C. Binding data for chimeric 13D3 and humanized versions thereof is summarized in Tables 3 and 4. Tables 3 and 4 also summarize thermal stability data and immunogenecity data. Table 3. Humanzied Anti-TDP-43 Metrics
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT Yield Tm °C 1:1 binding Rmax Antibody Immunogenicity kd (1/s) KD (M) (mg/L) (f’ab) ka (1/Ms) (RU)
Table 4. Humanized 13D3 Antibody Metrics Yield Tm °C Antibody Immunogenicity ka (1/Ms) kd (1/s) KD1 (M) KD1 (nM)
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT h13D3Hd3-Ld2 350.0 76.07 2.5 1 7.56E+06 6.88E-03 9.10E-10 0.91 h13D3Hd4-Ld1 291.4 71.99 0.1 4 6.81E+06 3.64E-03 5.34E-10 0.53
that have low predicted immunogenicity scores (explained in further detail below) relative to the mouse 13D3 antibody. The data further demonstrate high yield and melting temperatures of the humanized antibodies (e.g., similar to the mouse 13D3 antibody). See for example, antibodies h13D3Hd1-Ld1, h13D3Hd1-Ld2, and h13D3Hd2-Ld1 shown in Table 4. Further, the data also demonstrate humanized antibodies with similar binding metrics to the mouse 13D3 antibody. In silico immunogenicity analysis of humanized antibodies The immunogenicity scores shown in Tables 3 and 4 are predicted values of immunogenicity calculated from two different software programs: Immune Epitope Database (IEDB) and EpiQuest. IEDB immunogenicity analysis tool is hosted by the National Institute of Allergy and Infectious Diseases. For IEDB, the immunogenicity prediction method used is based on predicted potential binding of a peptide within a protein sequence to major histocompatibility class (MHC) class II. The program identifies potential immunogenic regions within the protein sequence. The MHC class II tool predicts immunogenic regions using broad spectrum alleles (i.e., 26 reference alleles) for MHC class II in the human population. The software generates a series of 15 residue peptides which overlap in 10 residues. Each generated 15 residue peptide is predicted for binding to the 26 reference alleles. For each peptide, a percentile rank for each of the three methods (combinatorial library, SMM_align and Sturniolo) was generated by comparing each peptide’s score against the scores of five million random 15 residue peptides selected from the SWISSPROT database. The adjusted percentile rank is the percentile rank adjusted based on the frequency of peptide lengths. A low number percentile rank indicates high affinity. The median percentile rank of the three methods were then used to generate the rank for consensus method. By default, prediction result is collapsed to show only the Percentile Rank and Adjusted Rank. The maximum median percentile rank threshold is set at 20.
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT The EpiQuest tool, T-Scanner, ranges cytotoxic T lymphocyte (CTL) epitopes according to their predicted immunodominance. The immunodominance of epitopes is defined as their relative strength in functional assays related to the target kill or release of respective cytokines. These parameters indicate functionality of the T-epitope. This program is designed to analyze and sort according to their immunodominance the CTL peptide epitopes eluted from target cells. Generally, only a low number of peptide epitopes that bind to MHC class I have actual functional activity. Relative strength of a CTL (T) epitope is defined by the strength of its binding to MHC class I and T cell receptor (TCR) (in the context of MHCI). The algorithm detects the structural and compositional features of the peptide epitope that allow it to elicit a high-affinity for TCR. This analysis is haplotype-specific, and the EpiQuest T-scanner has matrix for analysis of HLAA2 and H2kB haplotypes-binding peptides. Example 3. Binding of Anti-TDP-43 Antibodies to Phosphorylated Cytoplasmic Aggregates of TDP-43 in FTP Brain Tissue and Model Systems Figures 6A-C show frontotemporal dementia (“FTD”) brain tissue (Fig. 6A) and healthy brain tissue (Fig. 6C). Fig. 6B is an inset of Fig. 6A showing co-localization of 13D3 antibody with phosphorylated TDP-43 FTD-associated neuronal cytoplasmic aggregates. The data demonstrate that 13D3 antibody specifically binds to cytoplasmic aggregates in FTD brain tissue, but not in healthy brain tissue. Similarly, each of Figures 7A-C show 13D3 specifically binding cytoplasmic aggregates in a rNLS8 dox-suppressible model of TDP-43 proteinopathy. Example 4. Binding of Anti-TDP-43 Antibodies to Phosphorylated Cytoplasmic Aggregates of TDP-43 and Clear TDP-43 Aggregates in Transfected HEK Cells Figure 8A shows confocal microscopy images of HEK cells transiently transfected with GFP-2a-TDP43 [mNLS (R82L/K83Q) DCS (C173S/C175S)] or GFP only (2a is a self-cleaving peptide that releases the TDP-43 upon expression). The top panels show staining for GFP, nuclei (grey), and pTDP-43 (white). The bottom panels show only pTDP-43 staining which is not present in the GFP alone control transfection. Figure 8B (left) is a graph showing cell count for HEK cells transfected with either GFP-2a-TDP43 or GFP alone. The data demonstrate approximately equivalent cell counts between the two populations. Further, Figure 8B (right)
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT shows pTDP-43 foci counts for HEK cells transfected with GFP-2a-TDP43 or GFP alone. The data demonstrate TDP-43 foci are formed only in the HEK cells transfected with GFP-2a- TDP43. Figure 8C shows TDP-43 staining with either commercial antibodies or antibodies of the present disclosure including 13D3, 13C13, and 2D4. Cells were treated with antibodies for 24 hours and 24 hours later, cells were incubated again with 100 ug/ml antibodies for another 24 hours. Cells were then washed, fixed/permeabilized, and stained with anti-pTDP-43 antibodies, followed by AF647-conjugated anti-mouse secondary antibodies. Stained cells were imaged by high content imaging (Operetta system) with 40x water objective. Quantitative analyses were carried out with Harmony software. Antibodies 13D3, 13C13, and 2D4 detect overexpressed mislocalized TDP-43 in HEK cells. The top row shows transfection with GFP-2a-TDP-43 where the phosphorylated TDP43 does not include a nuclear localization signal (i.e., phosphorylated TDP43 remains in the cytoplasm), and the bottom row shows transfection with a GFP only construct. The data demonstrate that TDP-43 foci are formed only in HEK cell transfected with GFP-2a-TDP-43. Various control antibodies were included in the assay including pTDP-43 (Cosmo)+, pTDP-43 (1D3)+, Total TDP-43 (PT), and 3B12 (ED)+. The control antibodies verify TDP-43 aggregation within the cytoplasm. Antibodies 13D3, 13C13, and 2D4 were similarly tested and demonstrated similar binding to cytoplasmic aggregates of phosphorylated TDP-43. Example 5. Characteristics of Humanized Anti-TDP-43 Cell Penetrating Agents Binding data, immunogenicity, and thermal stability for anti-TDP-43 cell-penetrating agents were assessed as previously described in Example 3. Binding data, immonogenciity scores, and thermal stability data for cell-penetrating agents comprising murine 13D3 and humanized versions thereof are summarized in Tables 5 and 6. Table 5: Stability, IHC, and Target Engagement Data for Anti-TDP-43 CPAs Yi ld T °C f' b IHC R ki T t nt
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT h13D3Hd2-Ld1_CMIP4 183.6 71.34 5+ Yes h13D3Hd2-Ld2_CMIP4 196.3 69.93 __ __
Table 6: Binding Data (Bivalent Analyte Mode) for Anti-TDP-43 CPAs Cell Penetrating Agent ka1 (1/Ms) kd1 (1/s) KD1(M) KD1(nM) KD1(pM)
, e highly stable, having comparable stability to the corresponding antibody. For example, the anti- TDP-43 CPAs in Table 5 have melting temperatures of 69.9 °C to 77.5 °C and are expressed in high yield. Further, the anti-TDP-43 CPAs demonstrated target engagement by immunohistochemistry (IHC) analysis and a cellular assay. Table 6 provides binding data for exemplary anti-TDP-43 CPAs, showing that these CPAs bind phospho-TDP-43 with sub- nanomolar KD values. Thus, the data in Tables 5 and 6 demonstrate that anti-TDP-43 CPAs of the present disclosure retain strong the binding to phospho-TDP-43, thermal stability, and in vitro target engagement of the parent antibodies. Example 6. Internalization and Clearance of TDP-43 Aggregates in Transfected Cells with Anti-TDP-43 Cell Penetrating Agents FIGS. 9-22 demonstrate internalization of and/or target engagement by anti-TDP-43 cell- penetrating agents of the present disclosure. Throughout FIGS. 9-22, references to “M-Lyco,”
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT Lycotoxin,” “ML” and the like refer to CPAs comprising L17E_M-lycotoxin linked to a 13D3 antibody (murine, chimeric, or humanized). Throughout FIGS. 9-22, references to other CIMs and/or CMIPs (e.g., “CMIP”, “CMIP4”, “cTAT”, “PEPTH”, etc.) refer to CPAs comprising the corresponding CIM linked to a 13D3 antibody (murine, chimeric, or humanized). Unless otherwise specified (e.g., by HC), the CIM is linked to the 13D3 antibody via the c-terminus of the light chain. HEK cells were transiently transfected with GFP-2a-TDP43 [mNLS (R82L/K83Q) DCS (C173S/C175S)]. 24 hours later, cells were incubated with 100 ug/ml cell penetrating agents including a cell internalization moiety and an anti-TDP-43 antibody for 4 hours. The cells were then washed, fixed/permeabilized, and followed by AF647-conjugated anti-mouse secondary antibodies. Stained cells were imaged by high content imaging (Operetta system) with 40x water objective. Quantitative analyses were carried out with Harmony software. Specifically, Figure 9A shows the percentage of CPA-positive cells following incubation with m13D3 CPAs including different CIMs (i.e., TAT, M-Lycotoxin_L17E (LC), M-Lycotoxin_L17E (HC), PEPTH (HC)) or under various control conditions (i.e., untagged, isotype, and vehicle). HC (heavy chain) and LC (light chain) indicate the location of the CIM. Labels to “M-Lycotoxin” in FIG. 9 refer to CPAs comprising M-Lycotoxin_L17E linked at the C-terminus of either the light chain (LC) or heavy chain (HC) of the m13D3 antibody. The data demonstrate increased internalization of the m13D3 CPAs compared to both the naked antibody (“untagged) and the vehicle controls. Figure 9B shows the number of CPA- positive spots per cell following incubation with the m13D3 CPAs. The data demonstrate increased pTDP-43 foci detection for the m13D3 CPAs compared to both the naked antibody and vehicle controls. Figures 10A-E are graphs showing the results of transfected HEK cells with various m13D3 CPAs. Briefly, HEK cells were transiently transfected with GFP-2a-TDP43 [mNLS (R82L/K83Q) DCS (C173S/C175S)] or no plasmid (e.g., untransfected) as a negative control. 24 hours later the cells were incubated with 100 ug/ml of antibodies (e.g., CPAs) for another 24 hours. Cells were then washed, fixed/permeabilized, and stained with anti-pTDP-43 antibodies, followed by AF647-conjugated anti-mouse secondary antibodies. Stained cells were imaged by high content imaging (Operetta system) with 40x water objective. Quantitative analyses were carried out with Harmony software.
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT Figure 10A shows the number of pTDP-43 foci per well area. Labels to “M-Lycotoxin” in FIG. 10 refer to CPAs comprising M-Lycotoxin_L17E linked at the C-terminus of the light chain of the m13D3 antibody. The data demonstrate that cells treated with different m13D3 CPAs had a lower number of foci per well area as compared to cells treated with untagged m13D3 (e.g., no cell internalizing module). As expected, no foci were observed in untransfected cells. Figure 10B shows the mean focus intensity for pTDP-43 foci. The data demonstrate that cells treated with different m13D3 CPAs had a lower mean focus intensity as compared to cells treated with untagged m13D3 antibody. As expected, very low focus intensity was observed with untransfected cells. Figure 10C shows consistent cell count per well across all cell populations tested (i.e., transfected with different m13D3 CPAs or untransfected cells. Figure 10D shows the number of pTDP-43 foci normalized by cell count. The data demonstrate that cells treated with different m13D3 CPAs had a lower number of foci per cell as compared to those treated with untagged m13D3. As expected, no foci were observed with untransfected cells. Figure 10E shows the mean focus area for p-TDP-43 foci, demonstrating that cells treated with different m13D3 CPAs had a lower mean area intensity compared to those treated with the untagged m13D3; very low focus intensity was observed with untransfected cells Figure 11A shows the number of pTDP-43 foci per well area. The data demonstrate 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 foci per well area as compared to those treated with the untagged m13D3. Labels to “M-Lycotoxin” in FIG. 11 refer to CPAs comprising M-Lycotoxin_L17E linked at the C-terminus of either the light chain (LC) or heavy chain (HC) of the m13D3 antibody. Figure 11B shows consistent cell count per well across all cell populations tested. Figure 11C shows the number of pTDP-43 foci normalized by cell count which demonstrates that cells treated with different m13D3 CPAs had a lower number of foci per cell as compared to cells treated with the untagged m13D3. As expected, no foci were observed in untransfected cells. Figure 11D shows the mean focus area for pTDP-43 foci. The data demonstrate that cells treated with different m13D3 CPAs had a lower mean area intensity compared to those treated with the untagged m13D3; very low focus intensity was observed with untransfected cells Figures 12A-D are graphs showing the results of cells incubated with m13D3 M- Lycotoxin [17E] CPAs at different concentrations under the same experimental conditions
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT described in Figures 11A-D. Labels to “M-Lycotoxin” in FIG. 11 refer to CPAs comprising M- Lycotoxin_L17E linked at the C-terminus of either the light chain (LC) or heavy chain (HC) of the m13D3 antibody. In addition, the cell internalizing module was located on either the heavy chain or light chain of the m13D3 antibody. Cells were also incubated with untagged m13D3, IgG isotype control, and a PBS control. Untransfected HEK cells were used as a negative control. Figure 12A shows pTDP-43 total foci area per well area. The data show a concentration- dependent reduction in total foci area for cells treated with the m13D3 M-Lycotoxin_L17E CPAs. Labels to “M-Lycotoxin” in FIG. 12 refer to CPAs comprising M-Lycotoxin_L17E linked at the C-terminus of either the light chain (LC) or heavy chain (HC) of the m13D3 antibody. In contrast, the cells treated with the 13D3 antibody, IgG isotype control, and PBS showed higher levels of foci as compared to cells treated with the m13D3 m-Lycotoxin [L17E] CPAs. Untransfected cells had no foci (data not shown). Figure 12B shows consistent cell count per well across all cell populations tested and no concentration dependent reduction in cell viability for cells transfected with the m13D3 M-Lycotoxin CPAs. Figure 12C shows the pTDP- 43 foci count which demonstrates a concentration-dependent increase in foci count for the m13D3 M-Lycotoxin CPAs, with significantly higher number of foci compared to control. Figure 12D shows the pTDP-43 mean focus size, demonstrating a concentration-dependent decrease in mean focus size for the m13D3 M-Lycotoxin CPAs, with significantly lower mean focus size compared to control. Collectively, Figures 12A-D demonstrate that the m13D3 M- Lycotoxin CPAs interfere with foci aggregation in a concentration-dependent manner. Figure 13 shows graphs of untransfected HEK cells (left) or transfected with GFP-2a- TDP43 [mNLS (R82L/K83Q) DCS (C173S/C175S)] (right) incubated with either m13D3 antibody or an M-Lycotoxin m13D3 CPA for 24 hours and subjected to the XTT metabolic assay to assess cell viability. Labels to “M-Lycotoxin” in FIG. 13 refer to CPAs comprising M- Lycotoxin_L17E linked at the C-terminus of the light chain (LC) of the m13D3 antibody. The data show no significant difference in cell death across all populations tested (as measured by percent cell death), demonstrating that the CPA (m13D3 M-Lycotoxin [L17E]) does not induce significant cytotoxicity. Collectively, Figures 10-13 demonstrate that anti-TDP-43 cell penetrating agents of the present disclosure are internalized by cells and can effectively engage intracellular TDP-43.
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT Example 7. Internalization and Clearance of Phosphorylated Cytoplasmic Aggregates of TDP-43 with Anti-TDP-43 Cell Penetrating Agents Figures 14A-E are graphs showing cells incubated with different m13D3 CPAs and untagged m13D3 antibody. Untransfected HEK cells were used as a negative control. The data in Figures 14A-E was generated with the same experimental conditions described in Figures 11A-D above. More specially, Figure 14A shows the sum of pTDP-43 foci per well area. Labels to “ML-13D3” in FIG. 14 refer to CPAs comprising M-Lycotoxin_L17E linked at the C-terminus of the light chain (LC) of the m13D3 antibody. Labels to “CMIP1-5” in FIG. 14 refer to CPAs comprising CMIP1, CMIP2, CMIP3, CMIP4 or CMIP5 linked at the C-terminus of the light chain (LC) of the m13D3 antibody. The data demonstrate that cells treated with different m13D3 CPAs (M-Lyco_L17E, CMIP1, CMIP2, CMIP3, CMIP4, CMIP5) had a lower number of foci per well area as compared to cells treated with the untagged m13D3. As expected, no foci were observed with untransfected cells. Figure 14B shows the mean focus intensity for pTDP-43 foci. The data demonstrate that cells treated with different m13D3 CPAs had a lower mean focus intensity as compared to cells treated with untagged m13D3 antibody and very low focus intensity was observed with untransfected cells. Figure 14C shows consistent cell count per well across all cell populations tested. Figure 14D shows the number of p-TDP-43 foci (normalized by cell count). The data demonstrates that cells treated with different m13D3 CPAs had a higher number of foci per cell as compared to cells treated with untagged m13D3 antibody. As expected, no foci were observed with untransfected cells. Last, Figure 14E shows the mean focus area for pTDP-43 foci. The data demonstrate that cells treated with different m13D3 CPAs had a lower mean area intensity compared to those treated with the untagged m13D3; very low focus intensity was observed with untransfected cells. Figures 15A-D are graphs showing the results of cells were incubated with either m13D3 m-Lycotoxin [L17E] CPAs or m13D3 CMIP4 CPA at different concentrations; cells were alternatively incubated with the untagged m13D3 or an IgG isotype control; untransfected HEK cells were used as a negative control. The data in Figures 15A-D was generated with the same experimental conditions described in Figures 11A-D above. Labels to “13D3-ML” in FIG. 15
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT refer to CPAs comprising M-Lycotoxin_L17E linked at the C-terminus of the light chain (LC) of the m13D3 antibody. Labels to “13D3-CMIP4” in FIG. 15 and FIG. 16 refer to CPAs comprising CMIP4 linked at the C-terminus of the light chain (LC) of the m13D3 antibody. Figure 15A shows the total number of foci p-TDP-43, demonstrating a concentration- dependent reduction in total foci area for cells treated with the 13D3 m-Lyco and 13D3 CMIP4 CPAs. In contrast, the cells treated with the 13D3 antibody and isotype showed higher levels of foci. Untransfected cells showed no foci. Figure 15B shows consistent cell count per well across all cell populations tested, and no concentration dependent reduction in cell viability for the m13D3 m-Lycotoxin CPAs. Figure 15C shows the p-TDP-43 foci number normalized by cell count, demonstrating a concentration-dependent increase in foci count for the 13D3 m-Lycotoxin and 13D3 CMIP4 CPAs, with significantly higher number of foci compared to control. Figure 15D shows the p-TDP-43 mean focus area, demonstrating a concentration-dependent decrease in mean focus area for the 13D3 m-Lycotoxin and 13D3 CMIP4 CPAs, with significantly lower mean focus area compared to control. Thus, Figures 15A-D demonstrates that the 13D3 m- Lycotoxin and 13D3 CMIP4 CPAs interfere with foci aggregation in a concentration-dependent manner. To obtain the data provided in Figures 16A-E, cells were incubated with the m13D3 CMIP4 CPA and the untagged m13D3 antibody in either acetate buffer or PBS. The data in Figures 16A-E was generated with the same experimental conditions described in Figures 11A-D above. More specifically, Figure 16A shows the pTDP-43 foci per well area, demonstrating that cells treated with the m13D3 CMIP4 CPA had a lower number of foci per well area compared to those treated with the untagged m13D3; no change in cell-penetrating activity was observed between PBS and acetate. Figure 16B shows the mean focus intensity for pTDP-43 foci, demonstrating that cells treated with the m13D3 CMIP4 CPA had a lower mean focus intensity compared to those treated with the untagged m13D3; no change in cell-penetrating agent activity was observed between PBS and acetate. Figure 16C shows consistent cell count per well across all cell populations tested. Figure 16D shows the number of p-TDP-43 foci normalized by cell count, demonstrating that cells treated with the m13D3 CMIP4 CPA had a higher number of foci per cell compared to those treated with the untagged m13D3; no foci were observed with untransfected cells. Figure 16E shows the mean focus area for p-TDP-43 foci, demonstrating
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT that cells treated with the m13D3 CMIP4 CPA had a lower mean area intensity compared to those treated with the untagged m13D3 Example 8. Internalization and Clearance of Phosphorylated Cytoplasmic Aggregates of TDP-43 with Novel Cell-Internalizing Modules and 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 the TDP-43 upon expression). 24 hours later the cells were incubated with 100 ug/ml antibodies (e.g., CPAs) for another 24 hours. The cells were then washed, fixed/permeabilized, and stained with anti-pTDP-43 antibodies, followed by AF647-conjugated anti-mouse and AF594-conjugated anti-human secondary antibodies. Stained cells were imaged by high content imaging (Operetta system) with 40x water objective. Quantitative analyses were carried out with Harmony software. Figure 17A shows confocal microscopy images of cells treated with the untagged ch13D3 antibody (left) and the ch13D3 m-Lycotoxin_L17E CPA (right). Foci are shown in white, the 13D3 antibody is shown in light grey, and nuclei are shown in dark grey. White arrows indicate illustrative foci co-localized with the 13D3 antibody. Figure 17A demonstrates significant colocalization between the pTDP43 foci and the 13D3 antibody for cells treated with the ch13D3 m-Lycotoxin CPA, with little colocalization observed for cells treated with the ch13D3 antibody. Figure 17B is a graph showing the percentage of pTDP43 colocalized with the ch13D3 antibody. The data demonstrate significantly greater co-localization for cells treated with the ch13D3 m- Lycotoxin CPA (~50-60%) as compared to the cells treated with the ch13D3 antibody (~15- 20%). Collectively, Figures 17A-B demonstrates that anti-TDP-43 CPAs of the present disclosure are internalized by cells and bind to intracellular p-TDP-43. Figures 18A-D are graphs showing anti-TDP-43 CPAs of the present disclosure ((i.e., chimeric 13D3 CPAs (ch13D3 m-Lyco CPA, ch13D3 CMIP4 with LALA mutation, ch13D3 CMIP4 with H310-H435Q mutations), the untagged ch13D3 antibody, and an hIgG isotype control) are internalized by cells and bind to intracellular p-TDP-43 (i.e., cells transfected with GFP-2a-TDP43 as described herein). Labels to “M-Lyco” in FIG. 18 refer to CPAs comprising M-Lycotoxin_L17E linked at the C-terminus of the light chain (LC) of the m13D3 antibody. Figure 18A provides a graph showing the percentage of p-TDP-43 colocalized with the ch13D3 antibody for ch13D3 CPAs as well as ch13D3 antibody, Isotype, and PBS controls using
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT both confocal (left) and non-confocal (right) microscopy. FIG 18A demonstrates significantly greater co-localization for cells treated with the various ch13D3 CPAs (~50-60%) compared to the cells treated with the ch13D3 antibody (~15-20%). Cells treated with isotype control and PBS demonstrated no co-localization. Figure 18B provides a graph showing the average number of 13D3 antibody spots per cell for various ch13D3 CPAs, as well as ch13D3 antibody, isotype, and PBS controls. Figure 18B demonstrates that the ch13D3 CPAs result in significantly more 13D3 spots per cell (~2.5 – 3 spots per cell) than the controls. Figure 18C provides a graph showing the average spot size for the 13D3 antibody spots for various ch13D3 CPAs, as well as ch13D3 antibody, isotype, and PBS controls. Figure 18C demonstrates that the ch13D3 CPAs result in significantly smaller 13D3 spots per cell than the control. Figure 18D provides a graph showing the average spot size for the 13D3 antibody spots for various ch13D3 CPAs, as well as ch13D3 antibody, isotype, and PBS controls. Figure 18D demonstrates that the ch13D3 CPAs result in significantly smaller (corrected for spot intensity) 13D3 spots per cell than the control. Thus, Figures 18A-D demonstrates that anti-TDP-43 CPAs of the present disclosure are internalized by cells and bind to intracellular p-TDP-43. Example 9. Internalization and Clearance of Phosphorylated Cytoplasmic Aggregates of TDP-43 with Anti-TDP-43 Cell Penetrating Agents Figures 19A-D are graphs showing internalization and colocalization of phosphorylated cytoplasmic aggregates of pTDP-43 (i.e., cells transfected with GFP-2a-TDP43 as described herein) with novel cell internalizing modules and humanized anti-TDP-43 antibodies. The data shown in Figures 19A-D was generated under the same experimental conditions as described in Figures 11A-D above. Labels to “ch13D3-ML” in FIG. 19 refer to CPAs comprising M- Lycotoxin_L17E linked at the C-terminus of the light chain (LC) of the m13D3 antibody. Figure 19A provides a graph showing the percentage of pTDP43 colocalized with the humanized 13D3 antibody for humanized 13D3 CPAs as well as ch13D3 and h13D3 antibody controls. Figure 19A demonstrates significantly greater co-localization for cells treated with the various h13D3 CPAs (~60-70%) compared to the cells treated with the h13D3 antibody (~40%). Figure 19B shows consistent cell count per well across all cell populations tested. Figure 19C is a graph showing the 13D3 spot area per well for pTDP43 colocalized with the humanized 13D3 antibody observed in cells treated with humanized 13D3 CPAs, as well as IgG isotype, ch13D3,
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT and h13D3 antibodies controls. FIG 19C demonstrates significant increases in total co- localization area for cell treated with humanized 13D3 CPAs of the present disclosure. Figure 19D provides a graph showing the number of 13D3 co-localization spots per cell observed in cells treated with humanized 13D3 CPAs, as well as IgG isotype, ch13D3, and h13D3 antibodies controls. Figure 19D demonstrates significant increases in number of co-localization spots for cells treated with humanized 13D3 CPAs of the present disclosure. Collectively, the data in Figures 19A-D show internalization and colocalization of phosphorylated cytoplasmic aggregates of pTDP-43 with novel cell internalizing modules and humanized anti-TDP-43 antibodies. Example 10. Internalization and Clearance of Phosphorylated Cytoplasmic Aggregates of TDP-43 in Glioblastoma Cells with Anti-TDP-43 Cell Penetrating Agents Figures 20A-C are graphs showing internalization and colocalization of phosphorylated cytoplasmic aggregates of TDP-43 (i.e., cells transfected with GFP-2a-TDP43 as described herein) in glioblastoma cells with CPAs including a cell internalizing module and an anti-TDP- 43 antibody. Labels to “M-lycotoxin” in FIGs. 20A-C refer to CPAs comprising M- Lycotoxin_L17E linked at the C-terminus of the light chain (LC) of the m13D3 antibody. Briefly, U251 cells were transiently transfected with GFP-2a-TDP43 [mNLS (R82L/K83Q) DCS (C173S/C175S)]. 24 hours later the cells were incubated with 100 ug/ml antibodies (e.g., CPAs) for another 24 hours. The cells were then washed, fixed/permeabilized, and stained with anti- pTDP-43 antibodies, followed by AF647-conjugated anti-mouse secondary antibodies. The stained cells were imaged by high content imaging (Operetta system) with 40x water objective. Quantitative analyses were carried out with Harmony software. More specifically, Figure 20A is a graph showing the total foci area for U251 glioblastoma cells treated with either m13D3 m-Lycotoxin CPA or m13D3 antibody alone (e.g., untagged). The data demonstrate a reduction in total foci area which was significantly reduced in glioblastoma cells treated with m13D3 m-Lycotoxin CPA as compared to cells treated with the m13D3 antibody. Figure 20B is a graph showing the mean foci size for U251 glioblastoma cells treated with either m13D3 m-Lycotoxin CPA or m13D3 antibody alone (e.g., untagged). The data demonstrate a reduction in mean foci size which was significantly reduced in glioblastoma cells treated with m13D3 m-Lycotoxin CPA compared to those cells treated with
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT the m13D3 antibody. Figure 20C is a graph showing the total foci count for U251 glioblastoma cells treated with either m13D3 m-Lycotoxin CPA or m13D3 antibody. The data demonstrate an increase in total foci count which was significantly increased in glioblastoma cells treated with m13D3 m-Lycotoxin CPA as compared to those cells treated with the m13D3 antibody. Thus, Figures 20A-C demonstrates that anti-TDP-43 CPAs of the present disclosure are internalized by glioblastoma cells that bind to intracellular p-TDP-43 and therefore disrupt TDP- 43 aggregation. Example 11. Internalization and Clearance of Phosphorylated Cytoplasmic Aggregates of TDP-43 in Rat Cortical Neuron Cells with Anti-TDP-43 Cell Penetrating Agents Figures 21A-B and 22A-F are graphs showing that CPAs including a cell internalizing module and an anti-TDP-43 antibody are internalized and colocalization of phosphorylated cytoplasmic aggregates in primary rat cortical neurons. Briefly, primary rat cortical neurons cells (DIV15) were incubated with 50 ug/ml antibodies for 2 hours. The cells were then washed, fixed/permeabilized, and stained with anti-EEA1 antibodies, followed by AF596-conjugated anti-rabbit and AF647-conjugated anti-mouse secondary antibodies. The stained cells were imaged by high content imaging (Operetta system) with 40x water objective. Quantitative analyses were carried out with Harmony software. Specifically, Figure 21A shows images of primary rat cortical neuronal cells treated with IgG isotype control (top, left), m13D3 (top, right), m13D3 m-Lycotoxin CPA (bottom, left), and m13D3CMIP4 CPA (bottom, right). Figure 21A demonstrates significant internalization of the m13D3 CPAs (depicted by white spots inside the cells, bottom panels) and little to no internalization of the isotype controls (top panels). Figure 21B is a graph showing the total of foci area per well which demonstrates little to no internalization of the isotype control and the 13D3 antibody and substantial internalization by the m13D3 m-Lycotoxin_L17E CPA and m13D3 CMIP4 CPA (labeled as 13D3-ML). Figure 22A is a graph showing the total number of 13D3 spots per cell for the DIV15 rat neuronal cells incubated with hIgG isotype control, m13D3 antibody, m13D3 m-Lycotoxin CPA, or m13D3 CMIP4 CPA. Figure 22A demonstrates higher numbers of 13D3 spots per cell for cells treated with m13D3-Cterm-LC-M-Lycotoxin_L17E CPA (labeled as “M-Lyco” in FIG. 22)
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT and m13D3-Cterm-LC-M CMIP4 CPA (labeled as “CMIP” in FIG. 22), than for cells treated with either the IgG isotype control or the m13D3 antibody. Figure 22B is a graph showing the total area of 13D3 spots per well for the DIV15 rat neuronal cells incubated with hIgG isotype control, m13D3 antibody, m13D3 m-Lyco CPA, or m13D3 CMIP4 CPA. Figure 22B demonstrates higher total spot area per well for cells treated with m13D3 m-Lycotoxin CPA and m13D3 CMIP4 CPAs, than for cells treated with either the IgG isotype control or the m13D3 antibody. Figure 22C is a graph showing the mean spot size for 13D3 spots in the DIV15 rat neuronal cells incubated with hIgG isotype control, m13D3 antibody, m13D3 m-Lycotoxin CPA, or m13D3 CMIP4 CPA. Figure 22C demonstrates higher mean spot size for cells treated with m13D3 m-Lycotoxin CPA and m13D3 CMIP4 CPAs, than for cells treated with either the IgG isotype control or the m13D3 antibody. Figure 22D is a graph showing spot integrated intensity for 13D3 spots in the DIV15 rat neuronal cells incubated with hIgG isotype control, m13D3 antibody, m13D3 m-Lycotoxin CPA, or m13D3 CMIP4 CPA. Figure 22D demonstrates higher spot integrated intensity for cells treated with m13D3 m-Lycotoxin CPA and m13D3 CMIP4 CPAs, than for cells treated with either the IgG isotype control or the m13D3 antibody. Figure 22E is a graph showing the percentage of 13D3 spots colocalized with EEA1 (Early Endosome Antigen 1) in the DIV15 rat neuronal cells incubated with hIgG isotype control, m13D3 antibody, m13D3 m-Lycotoxin CPA, or m13D3 CMIP4 CPA. FIG 22E demonstrates ~10-20% of 13D3 spots colocalized with EEA1 in cells treated with m13D3 m-Lycotoxin CPA and m13D3 CMIP4 CPAs, with no co- localization observed for cells treated with either the IgG isotype control or the m13D3 antibody. Figure 22F shows consistent cell count per well across all cell populations tested. Collectively, Figures 21A-D and 22A-F show that cell internalizing modules of the present disclosure are internalized by primary rat cortical neurons. References Bowers, E., et al., Decreased Mutation Frequencies among Immunoglobulin G Variable Region Genes during Viremic HIV-1 Infection, PLoS ONE, 9(1): E81913 (2014). Foote J and Winter G., Antibody framework residues affecting the conformation of the hypervariable loops. J. Mol. Biol. 224(2):487-99 (1992).
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT Kabat EA, Wu TT, Foeller C, Perry HM, Gottesman KS, Sequences of Proteins of Immunological Interest (5th edition). Bethesda, MD: National Institutes of Health (1991). Piechotta A, Parthier C, Kleinschmidt M, Gnoth K, Pillot T, Lues I, Demuth HU, Schilling S, Rahfeld JU, and Stubbs MT, Structural and functional analyses of pyroglutamate-amyloid-β- specific antibodies as a basis for Alzheimer immunotherapy. J Biol Chem. 2017 Jul 28; 292(30): 12713–12724 (2017). Wedemayer, G.J., Patten, P.A., Wang, L.H., Schultz, P.G., Stevens, R.C., Structural insights into the evolution of an antibody combining site. Science 276: 1665-1669 (1997). Cooper, L.J., Shikhman, A.R., Glass, D.D., Kangisser, D., Cunningham, M.W. and Greenspan, N.S., Role of heavy chain constant domains in antibody-antigen interaction. Apparent specificity differences among streptococcal IgG antibodies expressing identical variable domains, J. Immunol.150(6): 2231-2242 (1993) Léger OJP and Saldanha J., Preparation of recombinant antibodies from immune rodent spleens and the design of their humanisation by CDR grafting, In: Shepherd P and Dean C (eds). Monoclonal Antibodies: A Practical Approach. Oxford, UK: Oxford University Press (2000). 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). Jimenez-Gomez,G., Gomez-Perales, J.L., Ramos-Amaya,A., Gonzalez-Garcia,I., Campos- Caro,A. and Brieva, J.A., Modulated selection of IGHV gene somatic hypermutation during systemic maturation of human plasma cells, J. Leukoc. Biol. 87 (3), 523-530 (2010). Scally, SW, Bosch A, Triller G, Wardemann H, Julien J.P., Crystal structure of anti- cirumsporozoite protein 663 germline antibody. Direct deposit to PDB (2017). Shriner, A.K., et al., Analysis of the young and elderly variable gene repertoire in response to pneumococcal polysaccharides using a reconstituted SCID mouse model, Vaccine, 24(49-50): 7159-7166 (2006).
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT SEQUENCE APPENDIX Variable Heavy Reference Sequences SEQ ID NO: 1 m13D3-VH EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTI SRDNAKNTLYLQMNSLMSEDTAMYFCARQTYYSYGGFPYWGQGTLVTVSA SEQ ID NO: 2 IGHV3-48*03 EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYEMNWVRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTIS RDNAKNSLYLQMNSLRAEDTAVYYCARYFDYWGQGTLVTVSS SEQ ID NO: 3 AEX28899 EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYEMNWVRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTIS RDNAKNSLYLQMNSLRAEDTAVYYCARRNYYDSGGYGHWGQGTLVTVSS Humanized 13D3 VH design sequences SEQ ID NO: 4 h13D3VHv1 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYFMSWVRQAPGKGLEWVAYISTGGDSANYADNVKGRFTI SRDNAKNSLYLQMNSLRAEDTAVYFCARQTYYSYGGFPYWGQGTLVTVSS SEQ ID NO: 5 h13D3VHv2 EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQAPGKGLEWVAYISTGGDSANYADNVKGRFTI SRDNAKNSLYLQMNSLRAEDTAVYFCARQTYYSYGGFPYWGQGTLVTVSS SEQ ID NO: 6 h13D3VHv3 EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQAPGKGLEWVAYISTGGDSANYADNVKGRFTI SRDNAKNSLYAQMNSLRAEDTAVYFCARQTYYSYGGFPYWGQGTLVTVSS SEQ ID NO: 7 h13D3VHv4 EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQAPGKGLEWVAYISTGGDSANYADNVKGRFTI SRDNAKNTLYAQMNSLRAEDTAVYFCARQTYYSYGGFPYWGQGTLVTVSS SEQ ID NO: 8 h13D3VHv5 EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQAPGKGLEWVAYISTGGDSANYADNVKGRFTI SRDNAKNTAYAQMNSLRAEDTAVYFCARQTYYSYGGFPYWGQGTLVTVSS
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT SEQ ID NO: 9 h13D3VHv6 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYFMSWVRQAPGKGLEWVAYISTGGDSANYADNVKGRFTI SRDNAKNTAYAQMNSLRAEDTAVYFCARQTYYSYGGFPYWGQGTLVTVSS SEQ ID NO: 10 h13D3VHv7 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYFMGWVRQAPGKGLEWVAYISTGGDSANYADNVKGRFTI SRDNAKNTAYAQMNSLRAEDTAVYFCARQTYYSYGGFPYWGQGTLVTVSS SEQ ID NO: 11 h13D3VHv8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYFMGWVRQAPGKGLEWVAYISTGGDSANYADNVKGRFTI SRDNAKNTLYGQMNSLRAEDTAVYFCARQTYYSYGGFPYWGQGTLVTVSS SEQ ID NO: 12 h13D3VHv9 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYFMGWVRQAPGKGLEWVAYISTGGDSANYADNVKGRFTI SRDNAKNTGYLQMNSLRAEDTAVYFCARQTYYSYGGFPYWGQGTLVTVSS SEQ ID NO: 13 h13D3VHv10 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYFMGWVRQAPGKGLEWVAYISTGGDSANYADNVKGRFTI SRDNAKNTLYLQMNSGRAEDTAVYFCARQTYYSYGGFPYWGQGTLVTVSS SEQ ID NO: 14 h13D3VHv11 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYFMGWVRQAPGKRLEWVAYISTGGDSANYADNVKGRFTI SRDNAKNTGYLQMNSLMAEDTAVYFCARQTYYSYGGFPYWGQGTLVTVSS SEQ ID NO: 15 h13D3VHv12 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYFMGWVRQAPGERLEWVAYISTGGDSANYADNVKGRFTI SRDNAKNTGYLQMNSLRAEDTAVYFCARQTYYSYGGFPYWGQGTLVTVSS SEQ ID NO: 16 h13D3VHv13 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYFMGWVRQAPGEDLEWVAYISTGGDSANYADNVKGRFTI SRDNAKNTGYLQMNSLRAEDTAVYFCARQTYYSYGGFPYWGQGTLVTVSS SEQ ID NO: 17 h13D3VHv1b ELVQSGAEVKKPGSSVKVSCKASGFTFSNYFMSWVRQAPGQGLEWVAYISTGGDSANYADNVKGRFTITR DNSTSTLYMELSSLRSEDTAVYFCARQTYYSYGGFPYWGQGTLVTVSS
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT SEQ ID NO: 18 h13D3VHv2b ELVQSGAEVKKPGSSVKVSCKASGFTFSNYFMSWVRQAPGQGLEWVAYISTGGDSANYADNVKGRFTITK DTSTSTLYMELSSLRSEDTAVYFCARQTYYSYGGFPYWGQGTLVTVSS SEQ ID NO: 19 h13D3VHv3b ELVQSGAEVKKPGSSVKVSCKASGFTFSNYFMSWVRQAPGQGLEWVAYISTGGDSANYADNVKGRGTIT KDTSTSTLYMELSSLRSEDTAVYFCARQTYYSYGGFPYWGQGTLVTVSS SEQ ID NO: 20 h13D3VHv1d EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYFMSWVRQAPGKGLEWVSYISTGGDSANYADNVKGRFTI SRDNSKNSLYLQMNSLRAEDTAVYYCARQTYYSYGGFPYWGQGTLVTVSS SEQ ID NO: 21 h13D3VHv2d EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYFMSWVRQAPGKGLEWVSYISTGGDSANYADNVKGRFTI SRDNSKNTAYLQMNSLRAEDTAVYYCARQTYYSYGGFPYWGQGTLVTVSS SEQ ID NO: 22 h13D3VHv3d EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYFMSWVRQAPGKRLEWVAYISTGGDSANYADNVKGRFTI SRDNAKNSLYLQMNSLRAEDTAVYYCARQTYYSYGGFPYWGQGTLVTVSS SEQ ID NO: 23 h13D3VHv4d EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYFMSWVRQAPGKRLEWVSYISTGGDSANYADNVKGRFTI SRDNSKNTAYLQMNSLRAEDTAVYYCARQTYYSYGGFPYWGQGTLVTVSS Underlined residues are at the positions defined below. Position 52A*# Position 82A*# Position 82B*# Position 82C*# Position 100A* Position 100B* Position 100C*, respectively. *Applies to SEQ ID NOs: 1, 3-16 and 20-23 #Applies to SEQ ID NO: 2
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT Variable Light Reference Sequences SEQ ID NO: 24 m13D3-VL DVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSG SGTDFTLKISRVETEDLGVYFCSQSLHVPLTFGAGTKLELK SEQ ID NO: 25 IGKV2-30*02 DVVMTQSPLSLPVTLGQPASISCRSSQSLVHSDGNTYLNWFQQRPGQSPRRLIYKVSNRDSGVPDRFSGSGS GTDFTLKISRVEAEDVGVYYCMQGTHWPYTFGQGTKLEIK SEQ ID NO: 26 ABC66863 DIVMTQSPLSLPVTLGQPASISCRSSQSLVYSDGNTYLNWFQQRPGQSPRRLIYKVSNRDSGVPDRFSGSGS GTDFTLKISRVEAEDVGVYYCMQGTHRPLTFGGGTKVEIK Humanized 13D3 VL design sequences SEQ ID NO: 27 h13D3VLv1 DVVMTQSPLSLPVTLGQPASISCRSSQSLVHSNGKTYLHWYQQRPGQSPRLLIYKVSDRYSGVPDRFSGSGS GTDFTLKISRVEAEDVGVYFCSQSLHVPLTFGGGTKVEIK SEQ ID NO: 28 h13D3VLv2 DVVMTQSPLSLPVTLGQQASISCRSSQSLVHSNGKTYLHWYQQRPGQSPRLLIYKVSDRYSGVPDRFSGSG SGTDFTLKISRVEAEDVGVYFCSQSLHVPLTFGGGTKVEIK SEQ ID NO: 29 h13D3VLv3 DVVMTQSPSSLPVTLGQQASISCRSSQSLVHSNGKTYLHWYQQRPGQSPRLLIYKVSDRYSGVPDRFSGSGS GTDFTLKISRVESEDVGVYFCSQSLHVPLTFGGGTKVEIK SEQ ID NO: 30 h13D3VLv4 DVVMTQSPSSLPVTLGQQASISCRSSQSLVHSNGKTYLHWYQQRPGQSPRLLIYKVSDRYSGVPDRFSGSGS GTDFTLKISRVESEDVGVYFCSQSGHVPLTFGGGTKVEIK SEQ ID NO: 31 h13D3VLv5 DVVMTQSPSSLPVTLGQPASISCRSSQSLVHSNGKTYLHWYQQRPGQSPRLLIYKVSDRYSGVPDRFSGSGS GTDFTLKISRVESEDVGVYFCSQSGHVPLTFGGGTKVEIK SEQ ID NO: 32 h13D3VLv6 DVVMTQSPSSLPVTLGQPASISCRSSQSLVHSNGKTYLHWYQQRPGQSPRLLIYKVSDRYSGVPDRFSGSGS GTDFTLKISRVESEDVGVYFCSQSLHVPLTFGGGTKVEIK
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT SEQ ID NO: 33 h13D3VLv7 DVVMTQSPSSLPVTLGQPASISCRSSQSLVHSNGKTYLHWYQQRPGQSPRLLIYKVSDRYSGVPDRFSGSGS GTDFTLKISRVESEDVGVYFCSQSLHIPLTFGGGTKVEIK SEQ ID NO: 34 h13D3VLv8 DVVMTQSPSSLPVTLGQPASISCRSSQSLVHSNGKTYLHWYQQRPGQSPRLLIYKVSDRYSGVPDRFSGSGS GTDFTLKISRVESEDVGVYFCSQSLHAPLTFGGGTKVEIK SEQ ID NO: 35 h13D3VLv9 DVQMTQSPSSLPVTLGQPASISCRSSQSLVHSNGKTYLHWYQQRPGQSPRLLIYKVSDRYSGVPDRFSGSGS GTDFTLKISRVESEDVGVYFCSQSGHVPLTFGDGTKVEIK SEQ ID NO: 36 h13D3VLv10 DVQMTQSPSSLPVTLGQPASISCRSSQSLVHSNGKTYLHWYQQRPGQSPRLLIYKVSDRYSGVPDRFSGSGS GTDFTLKISRVESEDVGVYFCSQSGHVPLTFGRGTKVEIK SEQ ID NO: 37 h13D3VLv1b EVVMTQSPATLSLSPGERATLSCRSSQSLVHSNGKTYLHWYQQKPGQAPRLLIYKVSDRYSGVPARFSGSG SGTDFTLTISSLEPEDFAVYFCSQSLHVPLTFGGGTKVEIK SEQ ID NO: 38 h13D3VLv2b EVVMTQSPATLSLSPGERATLSCRSSQSLVHSNGKTYLHWYQQKPGQAPRLLIYKVSDRYSGVPARFSGSG SGTEFTLTISSLEPEDFAVYFCSQSLHVPLTFGGGTKVEIK SEQ ID NO: 39 h13D3VLv3b EVVMTQSPATLSLSPGERATLSCRSSQSLVHSNGKTYLHWYQQKPGQAPRLLIYKVSDRYSGVPARFSGSG SGTEVTLTISSLEPEDFAVYFCSQSLHVPLTFGGGTKVEIK SEQ ID NO: 40 h13D3VLv4b DVVMTQSPATLSLSPGERATLSCRSSQSLVHSNGKTYLHWYQQKPGQAPRLLIYKVSDRYSGVPARFSGSG SGTDFTLTISSLEPEDFAVKFCSQSLHVPLTFGGGTKVEIK SEQ ID NO: 41 h13D3VLv5b DVVMTQSPATLSLSPGERATLSCRSSQSLVHSNGKTYLHWYQQKPGQAPRLLIYKVSDRYSGVPARFSGSG SGTEFTLTISSLEPEDFAVKFCSQSLHVPLTFGGGTKVEIK
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT SEQ ID NO: 42 h13D3VLv6b DVVMTQSPATLSLSPGERATLSCRSSQSLVHSNGKTYLHWYQQKPGQAPRLLIYKVSDRYSGVPARFSGSG SGTEVTLTISSLEPEDFAVKFCSQSLHVPLTFGGGTKVEIK SEQ ID NO: 43 h13D3VLv7b DVVMTQSPATLSLSPGERATLSCRSSQSLVHSNGKTYLHWYQQKPGQAPRLLIYKVSDRYSGVPARFSGSG SGTEFTLTISSLEPEDFAVYFCDQSLHVPLTFGGGTKVEIK SEQ ID NO: 44 h13D3VLv8b DVVMTQSPATLSLSPGERATLSCRSSQSLVHSNGKTYLHWYQQKPGQAPRLLIYKVSDRYSGVPARFSGSG SGTEVTLTISSLEPEDFAVYFCDQSLHVPLTFGGGTKVEIK SEQ ID NO: 45 h13D3VLv9b DVVMTQSPATLSLSPGERATLSCRSSQSLVHSNGKTYLHWYQQKPGQAPRLLIYKVSDRYSGVPARFSGSG SGTEFTLTISSLEPEDFAVYFCSQQLHVPLTFGGGTKVEIK SEQ ID NO: 46 h13D3VLv10b DVVMTQSPATLSLSPGERATLSCRSSQSLVHSNGKTYLHWYQQKPGQAPRLLIYKVSDRYSGVPARFSGSG SGTEVTLTISSLEPEDFAVYFCSQQLHVPLTFGGGTKVEIK SEQ ID NO: 47 h13D3VLv1d DVQMTQSPLSLPVTLGQPASISCRSSQSLVHSNGKTYLHWYQQRPGQSPRLLIYKVSDRYSGVPDRFSGSGS GTDFTLKISRVEAEDVGVYFCSQSLHVPLTFGDGTKVEIK SEQ ID NO: 48 h13D3VLv2d DVQMTQSPLSLPVTLGQPASISCRSSQSLVHSNGKTYLHWYQQRPGQSPRLLIYKVSDRYSGVPDRFSGSGS GTDFTLKISRVEAEDVGVYFCSQSAHVPLTFGDGTKVEIK Underlined residues are at the positions defined below. S is position 27A L is position 27B V is position 27C H is position 27D S is position 27E, respectively. Applies to SEQ ID NOs: 24-48.
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT 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
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT SEQ ID NO: 61 LC CDR3 #7 SQSAHVPLT TDP43 mouse antibody variable domain sequences SEQ ID NO: 621B3_JH140 VH Signal peptide MNFGLSLIFLVLVLKGVLC SEQ ID NO: 631B3_JH140 Variable Heavy Domain EVKLVESGGGLVQPGGSLKLSCAASGFTFSSYTMSWVRQTPEKRLELVAEISNSGGRTNYPDTVKGRFTISR DNAKNTLYLQMSSLKSEDTAMYYCARRRYSDYYYYAMDYWGQGTSVTVSS SEQ ID NO: 641B3_JH140 VL Signal peptide MSSAQFLGLLLLCFQGTRC SEQ ID NO: 651B3_JH140 Variable Light Domain DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYS LTISNLEQEDIATYFCQQGNTLPRTFGGGTKLEIK SEQ ID NO: 661C12_JH104 VH Signal peptide MNFGLSLIFLVLVLKGVLC SEQ ID NO: 671C12_JH104 Variable Heavy Domain EVKLVESGGGLVQPGGSLKLSCAASGFTFSSYTMSWVRQTPEKRLELVADISNSGGRTYYPDTVKGRFTIS RENAKNSLYLQMSSLKSEDTAMYYCARRRYSDYYYYAMDNWGQGTSVTVSS SEQ ID NO: 681C12_JH104 VL Signal peptide MSSAQFLGLLLLCFQGTRC SEQ ID NO: 691C12_JH104 Variable Light Domain DIQMTQTTSSLSASLGDRVTISCRASQDISNYLSWYQQKPDGTVKLLIYYTSRLNSGVPSRFSGSGSGTDYSL
SEQ ID NO: 7011A1_LA121 VH Signal peptide MEWPCIFLFLLSVTEGVHS
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT SEQ ID NO: 7111A1_LA121 Variable Heavy Domain QVQLQQSGAELVRPGSSVKISCKASGYEFSRYWMNWVKQRPGQGLEWIGQIYHGDGDTNYKGKFKGKAI LTADKSSSTAYMQVSSLTSEDSAVYFCVRGGYYGYAMDYWGQGTSVTVSS SEQ ID NO: 7211A1_LA121 Signal peptide MKLPVRLLVLMFWIPASSS SEQ ID NO: 7311A1_LA121 Variable Light Domain DVVMTQTPLSLPVSLGDQASISCRSSQSLLHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGS GTDFTLKISRVEAEDLGVYFCSQSTHVPYTFGGGTNLEIK SEQ ID NO: 7411B1_LA121 VH Signal peptide MEWPCIFLFLLSVTEGVHS SEQ ID NO: 7511B1_LA121 Variable Heavy Domain QVQLQQSGAELVRPGSSVKISCKASGYEFSRYWMNWVKQRPGQGLEWIGQIYHGDGDTNYKGKFKGKAI LTADKSSSTAYMQVSSLTSEDSAVYFCVRGGYYGYAMDYWGQGTSVTVSS SEQ ID NO: 7611B1_LA121 VL Signal peptide MKLPVRLLVLMFWIPASSS SEQ ID NO: 7711B1_LA121 Variable Light Domain DVVMTQTPLSLPVSLGDQASISCRSSQSLLHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGS GTDFTLKISRVEAEDLGVYFCSQSTHVPYTFGGGTNLEIK SEQ ID NO: 7816G5_LA121 VH Signal peptide MKCSWVIFFLMAVVTGVNS SEQ ID NO: 7916G5_LA121 Variable Heavy Domain EVHLQQSGAELVKPGASVKLSCTGSGFNIIDTYIHWVKQRPEQGLEWIGRIDPANGNTMYASKFQDKATIIA DTSSNTVYMRLGSLTSGDTAVYYCSHGDFWWGQGTTLTVSS SEQ ID NO: 8016G5_LA121 VL Signal peptide MSPAQFLFLLVLWIQETNG
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT SEQ ID NO: 8116G5_LA121 Variable Light Domain DVVMTQTPLTLSIPIGQPAYISCKSSQSLLKSNGKTYLNWLFQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSG TDFTLKISRVEAEDLGIYYCVQGTHLPHTFGGGTRLEIK SEQ ID NO: 82 Human TDP-43 Protein MSEYIRVTEDENDEPIEIPSEDDGTVLLSTVTAQFPGACGLRYRNPVSQCMRGVRLVEGILHAPDAGWGNL VYVVNYPKDNKRKMDETDASSAVKVKRAVQKTSDLIVLGLPWKTTEQDLKEYFSTFGEVLMVQVKKDL KTGHSKGFGFVRFTEYETQVKVMSQRHMIDGRWCDCKLPNSKQSQDEPLRSRKVFVGRCTEDMTEDELR EFFSQYGDVMDVFIPKPFRAFAFVTFADDQIAQSLCGEDLIIKGISVHISNAEPKHNSNRQLERSGRFGGNPG GFGNQGGFGNSRGGGAGLGNNQGSNMGGGMNFGAFSINPAMMAAAQAALQSSWGMMGMLASQQNQS GPSGNNQNQGNMQREPNQAFGSGNNSYSGSNSGAAIGWGSASNAGSGSGFNGGFGSSMDSKSSGWGM SEQ ID NO: 83 TDP-43 peptide (pS409/pS410) GSGSGFNGGFGSSMDSKSSGWGM SEQ ID NO: 841B3_JH140 Variable Heavy Chain CDR1 SGFTFSSYTMS SEQ ID NO: 851B3_JH140 Variable Heavy Chain CDR2 EISNSGGRTNY SEQ ID NO: 861B3_JH140 Variable Heavy Chain CDR3 RRYSDYYYYAMDY SEQ ID NO: 871B3_JH140 Variable Light Chain CDR1 RASQDISNYLN SEQ ID NO: 881B3_JH140 Variable Light Chain CDR2 YTSRLHS SEQ ID NO: 891B3_JH140 Variable Light Chain CDR3 QQGNTLPRT SEQ ID NO: 901C12_JH104 Variable Heavy Chain CDR1 SGFTFSSYTMS
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT SEQ ID NO: 911C12_JH104 Variable Heavy Chain CDR2 DISNSGGRTYYPDTVKG SEQ ID NO: 921C12_JH104 Variable Heavy Chain CDR3 RRYSDYYYYAMDN SEQ ID NO: 931C12_JH104 Variable Light Chain CDR1 RASQDISNYLS SEQ ID NO: 941C12 JH104 Variable Light chain CDR2 YTSRLNS SEQ ID NO: 951C12_JH104 Variable Light Chain CDR3 QQGNALPRT SEQ ID NO: 9611A1_LA121 Variable Heavy Chain CDR1 SGYEFSRYWMN SEQ ID NO: 9711A1_LA121 Variable Heavy Chain CDR2 QIYHGDGDTNYKGKFKG SEQ ID NO: 9811A1_LA121 Variable Heavy Chain CDR3 GGYYGYAMDY SEQ ID NO: 9911A1_LA121 Variable Light Chain CDR1 RSSQSLLHSNGNTYLH SEQ ID NO: 10011A1_LA121 Variable Light Chain CDR2 KVSNRFS SEQ ID NO: 10111A1_LA121 Variable Light Chain CDR3 SQSTHVPYT SEQ ID NO: 10211B1_LA121 Variable Heavy Chain CDR1 SGYEFSRYWMN
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT SEQ ID NO: 10311B1_LA121 Variable Heavy Chain CDR2 QIYHGDGDTNYKGKFKG SEQ ID NO: 10411B1_LA121 Variable Heavy Chain CDR3 GGYYGYAMDY SEQ ID NO: 10511B1_LA121 Variable Light Chain CDR1 RSSQSLLHSNGNTYLH SEQ ID NO: 10611B1_LA121 Variable Light Chain CDR2 KVSNRFS SEQ ID NO: 10711B1_LA121 Variable Light Chain CDR3 SQSTHVPYT SEQ ID NO: 10816G5_LA121 Variable Heavy Chain CDR1 SGFNIIDTYIH SEQ ID NO: 10916G5_LA121 Variable Heavy Chain CDR2 RIDPANGNTMYA SEQ ID NO: 11016G5_LA121 Variable Heavy Chain CDR3 GDFW SEQ ID NO: 11116G5_LA121 Variable Light Chain CDR1 KSSQSLLKSNGKTYLN SEQ ID NO: 11216G5_LA121 Variable Light Chain CDR2 LVSKLDS SEQ ID NO: 11316G5 LA121 Variable Light Chain CDR3 VQGTHLPHT
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT SEQ ID NO: 114 m13D3 Heavy Chain – with mouse-IgG2a heavy chain constant domain; constant region is underlined EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTI SRDNAKNTLYLQMNSLMSEDTAMYFCARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTT GSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASST KVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNN VEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLP PPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERN SYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO: 115 m13D3 Light Chain – with mouse kappa light chain constant domain; constant region is underlined DVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSG SGTDFTLKISRVETEDLGVYFCSQSLHVPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNF YPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFN RNEC *Underlined sequences in SEQ ID NOs: 114 and 115 denote the constant domain. SEQ ID NO: 116 13D3_Ab_LC-Ct-CMIP4 Heavy Chain EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTI SRDNAKNTLYLQMNSLMSEDTAMYFCARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTT GSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASST KVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNN VEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLP PPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERN SYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO: 11713D3_Ab_LC-Ct-CMIP4 Light Chain DVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSG SGTDFTLKISRVETEDLGVYFCSQSLHVPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNF YPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFN RNECGGGGSGGGGSIWLTALKFSGKAAAKAEAKQFLSKL SEQ ID NO: 11813D3_Ab_HC-Cterm_TAT Heavy Chain EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTI SRDNAKNTLYLQMNSLMSEDTAMYFCARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTT GSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASST KVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNN VEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLP PPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERN SYSCSVVHEGLHNHHTTKSFSRTPGKGRKKRRQRRRPPQ SEQ ID NO: 11913D3_Ab_HC-Cterm_TAT Light Chain DVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSG SGTDFTLKISRVETEDLGVYFCSQSLHVPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNF YPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFN RNEC
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT SEQ ID NO: 12013D3_Ab_LC-L17E_M-Lycotoxin Heavy Chain EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTI SRDNAKNTLYLQMNSLMSEDTAMYFCARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTT GSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASST KVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNN VEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLP PPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERN SYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO: 12113D3_Ab_LC-L17E_M-Lycotoxin Light Chain DVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSG SGTDFTLKISRVETEDLGVYFCSQSLHVPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNF YPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFN RNECGGGGSGGGGSIWLTALKFLGKHAAKHEAKQQLSKL SEQ ID NO: 12213D3_Ab_HC-Ct-L17E_M-Lycotoxin Heavy Chain EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTI SRDNAKNTLYLQMNSLMSEDTAMYFCARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTT GSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASST KVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNN VEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLP PPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERN SYSCSVVHEGLHNHHTTKSFSRTPGKGGGGSGGGGSIWLTALKFLGKHAAKHEAKQQLSKL SEQ ID NO: 12313D3_Ab_HC-Ct-L17E_M-Lycotoxin Light Chain DVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSG SGTDFTLKISRVETEDLGVYFCSQSLHVPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNF YPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFN RNEC SEQ ID NO: 12413D3_Ab_HC-AH-PEPTH Heavy Chain EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTI SRDNAKNTLYLQMNSLMSEDTAMYFCARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTT GSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASST KVDKKIEPRGPTIKPCPPCKCPGSVKKKKIKAEIKIGAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVS EDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTIS KPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFM YSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO: 12513D3_Ab_HC-AH-PEPTH Light Chain DVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSG SGTDFTLKISRVETEDLGVYFCSQSLHVPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNF YPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFN RNEC
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT SEQ ID NO: 12613D3_Ab_HC-Cterm-cycR9 Heavy Chain EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTI SRDNAKNTLYLQMNSLMSEDTAMYFCARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTT GSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASST KVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNN VEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLP PPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERN SYSCSVVHEGLHNHHTTKSFSRTPGKGGGCG*RRRRRRRRRG*C SEQ ID NO: 12713D3_Ab_HC-Cterm-cycR9 Light Chain DVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSG SGTDFTLKISRVETEDLGVYFCSQSLHVPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNF YPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFN RNEC SEQ ID NO: 12813D3_Ab_HC-Cterm-cycTAT1 modified Heavy Chain EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTI SRDNAKNTLYLQMNSLMSEDTAMYFCARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTT GSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASST KVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNN VEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLP PPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERN SYSCSVVHEGLHNHHTTKSFSRTPGKGGGCG*GRKKRRQRRRPQG*C SEQ ID NO: 12913D3_Ab_HC-Cterm-cycTAT1 modified Light Chain DVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSG SGTDFTLKISRVETEDLGVYFCSQSLHVPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNF YPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFN RNEC SEQ ID NO: 13013D3_Ab_HC-Cterm-TAT Heavy Chain EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTI SRDNAKNTLYLQMNSLMSEDTAMYFCARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTT GSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASST KVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNN VEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLP PPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERN SYSCSVVHEGLHNHHTTKSFSRTPGKGRKKRRQRRRPPQ SEQ ID NO: 13113D3_Ab_HC-Cterm-TAT Light Chain DVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSG SGTDFTLKISRVETEDLGVYFCSQSLHVPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNF YPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFN RNEC SEQ ID NO: 13213D3_Ab_LC-Cterm-Penetain Heavy Chain EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTI SRDNAKNTLYLQMNSLMSEDTAMYFCARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTT GSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASST
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT KVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNN VEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLP PPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERN SYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO: 13313D3_Ab_LC-Cterm-Penetain Light Chain DVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSG SGTDFTLKISRVETEDLGVYFCSQSLHVPLTFGAGTKLELKADAAPTVSIFPPSSEQLTSGGASVVCFLNNFY PKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNR NECGGGGSGGGGSRQIKIWFQNRRMKWKKG* SEQ ID NO: 13413D3_Ab_LC-Cterm-L17E-M-Lycotoxin Heavy Chain EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTI SRDNAKNTLYLQMNSLMSEDTAMYFCARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTT GSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASST KVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNN VEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLP PPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERN SYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO: 13513D3_Ab_LC-Cterm-L17E-M-Lycotoxin Light Chain DVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSG SGTDFTLKISRVETEDLGVYFCSQSLHVPLTFGAGTKLELKADAAPTVSIFPPSSEQLTSGGASVVCFLNNFY PKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNR NECGGGGSGGGGSIWLTALKFLGKHAAKHEAKQQLSKL SEQ ID NO: 13613D3_Ab_LC-Cterm-Pepth Heavy Chain EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTI SRDNAKNTLYLQMNSLMSEDTAMYFCARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTT GSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASST KVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNN VEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLP PPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERN SYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO: 13713D3_Ab_LC-Cterm-Pepth Light Chain DVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSG SGTDFTLKISRVETEDLGVYFCSQSLHVPLTFGAGTKLELKADAAPTVSIFPPSSEQLTSGGASVVCFLNNFY PKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNR NECGGGGSGGGGSVKKKKIKAEIKIG* SEQ ID NO: 13813D3_Ab_HC-Cterm-TAT3 Heavy Chain EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTI SRDNAKNTLYLQMNSLMSEDTAMYFCARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTT GSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASST KVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNN VEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLP PPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERN SYSCSVVHEGLHNHHTTKSFSRTPGKGGGGSGGGGSGRKKRRQRRRPQGRKKRRQRRRPQGRKKRRQR RRPQG*G*
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT SEQ ID NO: 13913D3_Ab_HC-Cterm-TAT3 Light Chain DVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSG SGTDFTLKISRVETEDLGVYFCSQSLHVPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNF YPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFN RNEC SEQ ID NO: 14013D3_Ab_LC-Cterm_TAT3 Heavy Chain EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTI SRDNAKNTLYLQMNSLMSEDTAMYFCARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTT GSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASST KVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNN VEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLP PPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERN SYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO: 14113D3_Ab_LC-Cterm_TAT3 Light Chain DVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSG SGTDFTLKISRVETEDLGVYFCSQSLHVPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNF YPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFN RNECGGGGSGGGGSGRKKRRQRRRPQGRKKRRQRRRPQGRKKRRQRRRPQG*G* SEQ ID NO: 14213D3_Ab_LC-Cterm_cycTAT3 Heavy Chain EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTI SRDNAKNTLYLQMNSLMSEDTAMYFCARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTT GSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASST KVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNN VEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLP PPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERN SYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO: 14313D3_Ab_LC-Cterm_cycTAT3 Light Chain DVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSG SGTDFTLKISRVETEDLGVYFCSQSLHVPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNF YPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFN RNECGGGGSGGGGSCG*G*GRKKRRQRRRPQGRKKRRQRRRPQGRKKRRQRRRPQG*G*C SEQ ID NO: 14413D3_Ab_LC-Cterm_cycR8x3 Heavy Chain EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTI SRDNAKNTLYLQMNSLMSEDTAMYFCARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTT GSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASST KVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNN VEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLP PPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERN SYSCSVVHEGLHNHHTTKSFSRTPGKGGGGSGGGGSCG*G*G*RRRRRRRRG*G*G*RRRRRRRRG*G*G* RRRRRRRRG*G*G*C SEQ ID NO: 14513D3_Ab_LC-Cterm_cycR8x3 Light Chain
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT DVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSG SGTDFTLKISRVETEDLGVYFCSQSLHVPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNF YPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFN RNEC SEQ ID NO: 14613D3_Ab_HC-Cterm_cycTAT3 Heavy Chain EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTI SRDNAKNTLYLQMNSLMSEDTAMYFCARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTT GSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASST KVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNN VEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLP PPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERN SYSCSVVHEGLHNHHTTKSFSRTPGKGGGGSGGGGSCG*G*G*GRKKRRQRRRPQGRKKRRQRRRPQGR KKRRQRRRPQG*G*G*C SEQ ID NO: 14713D3_Ab_HC-Cterm_cycTAT3 Light Chain DVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSG SGTDFTLKISRVETEDLGVYFCSQSLHVPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNF YPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFN RNEC SEQ ID NO: 14813D3_Ab_LC-Cterm_R8x3 Heavy Chain EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTI SRDNAKNTLYLQMNSLMSEDTAMYFCARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTT GSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASST KVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNN VEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLP PPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERN SYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO: 14913D3_Ab_LC-Cterm_R8x3 Light Chain DVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSG SGTDFTLKISRVETEDLGVYFCSQSLHVPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNF YPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFN RNECGGGGSRRRRRRRRG*G*G*G*S*RRRRRRRRG*G*G*G*S*RRRRRRRRG*G* SEQ ID NO: 15013D3_Ab_HC-Cterm_R6H4 Heavy Chain EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTI SRDNAKNTLYLQMNSLMSEDTAMYFCARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTT GSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASST KVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNN VEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLP PPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERN SYSCSVVHEGLHNHHTTKSFSRTPGKGGGGSGGGGSRRRRRRHHHH SEQ ID NO: 15113D3_Ab_HC-Cterm_R6H4 Light Chain DVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSG SGTDFTLKISRVETEDLGVYFCSQSLHVPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNF YPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFN RNEC
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT SEQ ID NO: 15213D3_Ab_HC-Cterm_TAT-H4 Heavy Chain EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTI SRDNAKNTLYLQMNSLMSEDTAMYFCARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTT GSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASST KVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNN VEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLP PPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERN SYSCSVVHEGLHNHHTTKSFSRTPGKGGGGSGRKKRRQRRRPHHHH SEQ ID NO: 15313D3_Ab_HC-Cterm_TAT-H4 Light Chain DVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSG SgalDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFN RNEC SEQ ID NO: 154 h13D3H1bL1b_Ab_LC-Cterm_CMIP4 Heavy Chain EVELVQSGAEVKKPGSSVKVSCKASGFTFSNYFMSWVRQAPGQGLEWVAYISTGGDSANYADNVKGRFTI TRDNSTSTLYMELSSLRSEDTAVYFCARQTYYSYGGFPYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGT AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 155 h13D3H1bL1b_Ab_LC-Cterm_CMIP4 Light Chain DVVMTQSPATLSLSPGERATLSCRSSQSLVHSNGKTYLHWYQQKPGQAPRLLIYKVSDRYSGVPARFSGSG SGTDFTLTISSLEPEDFAVYFCSQSLHVPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFY PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGECGGGSGGGSIWLTALKFSGKAAAKAEAKQFLSKL SEQ ID NO: 156 h13D3H1bL2b_Ab_LC-Cterm_CMIP4 Heavy Chain EVELVQSGAEVKKPGSSVKVSCKASGFTFSNYFMSWVRQAPGQGLEWVAYISTGGDSANYADNVKGRFTI TRDNSTSTLYMELSSLRSEDTAVYFCARQTYYSYGGFPYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGT AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 157 h13D3H1bL2b_Ab_LC-Cterm_CMIP4 Light Chain DVVMTQSPATLSLSPGERATLSCRSSQSLVHSNGKTYLHWYQQKPGQAPRLLIYKVSDRYSGVPARFSGSG SGTEFTLTISSLEPEDFAVYFCSQSLHVPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFY PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGECGGGSGGGSIWLTALKFSGKAAAKAEAKQFLSKL SEQ ID NO: 158 h13D3H2bL1b_Ab_LC-Cterm_CMIP4 Heavy Chain
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT EVELVQSGAEVKKPGSSVKVSCKASGFTFSNYFMSWVRQAPGQGLEWVAYISTGGDSANYADNVKGRFTI TKDTSTSTLYMELSSLRSEDTAVYFCARQTYYSYGGFPYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGT AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 159 h13D3H2bL1b_Ab_LC-Cterm_CMIP4 Light Chain DVVMTQSPATLSLSPGERATLSCRSSQSLVHSNGKTYLHWYQQKPGQAPRLLIYKVSDRYSGVPARFSGSG SGTDFTLTISSLEPEDFAVYFCSQSLHVPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFY PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGECGGGSGGGSIWLTALKFSGKAAAKAEAKQFLSKL SEQ ID NO: 160 h13D3H2bL2b_Ab_LC-Cterm_CMIP4 Heavy Chain EVELVQSGAEVKKPGSSVKVSCKASGFTFSNYFMSWVRQAPGQGLEWVAYISTGGDSANYADNVKGRFTI TKDTSTSTLYMELSSLRSEDTAVYFCARQTYYSYGGFPYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGT AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 161 h13D3H2bL2b_Ab_LC-Cterm_CMIP4 Light Chain DVVMTQSPATLSLSPGERATLSCRSSQSLVHSNGKTYLHWYQQKPGQAPRLLIYKVSDRYSGVPARFSGSG SGTEFTLTISSLEPEDFAVYFCSQSLHVPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFY PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGECGGGSGGGSIWLTALKFSGKAAAKAEAKQFLSKL SEQ ID NO: 162 h13D3H3bL1b_ Ab_LC-Cterm_CMIP4 Heavy Chain EVELVQSGAEVKKPGSSVKVSCKASGFTFSNYFMSWVRQAPGQGLEWVAYISTGGDSANYADNVKGRGT ITKDTSTSTLYMELSSLRSEDTAVYFCARQTYYSYGGFPYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGG TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 163 h13D3H3bL1b_ Ab_LC-Cterm_CMIP4 Light Chain DVVMTQSPATLSLSPGERATLSCRSSQSLVHSNGKTYLHWYQQKPGQAPRLLIYKVSDRYSGVPARFSGSG SGTDFTLTISSLEPEDFAVYFCSQSLHVPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFY PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGECGGGSGGGSIWLTALKFSGKAAAKAEAKQFLSKL SEQ ID NO: 164 h13D3H3bL2b_ Ab_LC-Cterm_CMIP4 Heavy Chain EVELVQSGAEVKKPGSSVKVSCKASGFTFSNYFMSWVRQAPGQGLEWVAYISTGGDSANYADNVKGRGT ITKDTSTSTLYMELSSLRSEDTAVYFCARQTYYSYGGFPYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGG TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT VDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 165 h13D3H3bL2b_ Ab_LC-Cterm_CMIP4 Light Chain DVVMTQSPATLSLSPGERATLSCRSSQSLVHSNGKTYLHWYQQKPGQAPRLLIYKVSDRYSGVPARFSGSG SGTEFTLTISSLEPEDFAVYFCSQSLHVPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFY PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGECGGGSGGGSIWLTALKFSGKAAAKAEAKQFLSKL SEQ ID NO: 166 m13D3_ Ab_LC-Cterm_CMIP1 Heavy Chain EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTI SRDNAKNTLYLQMNSLMSEDTAMYFCARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTT GSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASST KVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNN VEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLP PPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERN SYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO: 167 m13D3_ Ab_LC-Cterm_CMIP1 Light Chain DVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSG SGTDFTLKISRVETEDLGVYFCSQSLHVPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNF YPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFN RNECGGGGSGGGGSIWLTALKFLGKAAAKAEAKQQLSKL SEQ ID NO: 168 m13D3_ Ab_LC-Cterm_CMIP2 Heavy Chain EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTI SRDNAKNTLYLQMNSLMSEDTAMYFCARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTT GSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASST KVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNN VEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLP PPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERN SYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO: 169 m13D3_ Ab_LC-Cterm_CMIP2 Light Chain DVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSG SGTDFTLKISRVETEDLGVYFCSQSLHVPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNF YPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFN RNECGGGGSGGGGSIWLTALKFSGKAAAKAEAKQQLSKL SEQ ID NO: 170 m13D3_ Ab_LC-Cterm_CMIP3 Heavy Chain EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTI SRDNAKNTLYLQMNSLMSEDTAMYFCARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTT GSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASST KVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNN VEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLP PPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERN SYSCSVVHEGLHNHHTTKSFSRTPGK
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT SEQ ID NO: 171 m13D3_ Ab_LC-Cterm_CMIP3 Light Chain DVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSG SGTDFTLKISRVETEDLGVYFCSQSLHVPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNF YPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFN RNECGGGGSGGGGSIWLTASKFSGKAAAKAEAKQQLSKL SEQ ID NO: 172 m13D3_ Ab_LC-Cterm_CMIP4 Heavy Chain EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTI SRDNAKNTLYLQMNSLMSEDTAMYFCARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTT GSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASST KVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNN VEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLP PPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERN SYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO: 173 m13D3_ Ab_LC-Cterm_CMIP4 Light Chain DVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSG SGTDFTLKISRVETEDLGVYFCSQSLHVPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNF YPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFN RNECGGGGSGGGGSIWLTALKFSGKAAAKAEAKQFLSKL SEQ ID NO: 174 m13D3_ Ab_LC-Cterm_CMIP5 Heavy Chain EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYFMSWVRQTPEKRLEWVAYISTGGDSANYADNVKGRFTI SRDNAKNTLYLQMNSLMSEDTAMYFCARQTYYSYGGFPYWGQGTLVTVSAAKTTAPSVYPLAPVCGDTT GSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASST KVDKKIEPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNN VEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLP PPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERN SYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO: 175 m13D3_ Ab_LC-Cterm_CMIP5 Light Chain DVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGKTYLHWYQQKPGQSPKLLIYKVSDRYSGVSDRFSGSG SGTDFTLKISRVETEDLGVYFCSQSLHVPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNF YPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFN RNECGGGGSGGGGSIWLTALKFSGKAAAKAEAKQWLSKL The following applies to SEQ ID NOs: 116-175 Linker sequences are denoted in bold font. Spacer regions or amino acids are denoted with an asterisk(*) following the amino acid residue (e.g., G*). Cell internalizing module sequences are denoted with double-underlining. SEQ ID NO: 176 CIM #1 CMIP1 IWLTALKFLGKAAAKAEAKQQLSKL SEQ ID NO: 177 CIM #2 CMIP2
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT 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 Wildtype 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
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT The following applies to SEQ ID NOs: 176-193 Spacer regions or amino acids are denoted with an asterisk(*) following the amino acid residue (e.g., G*). SEQ ID NO: 194 Linker sequence #1 GGGGSGGGGS SEQ ID NO: 195 Linker sequence #2 GGGGS SEQ ID NO: 196 Linker sequence #3 GGGGS SEQ ID NO: 197 Linker sequence #4 GS SEQ ID NO: 198 Linker sequence #5 GGG SEQ ID NO: 199: Linker sequence #6 GGGSGGGS SEQ ID NO: 200 Spacer region #1 G SEQ ID NO: 201 Spacer region #2 GG SEQ ID NO: 202 Spacer region #3 GGG SEQ ID NO: 203 Spacer region #4 GGGGS
Claims
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT WHAT IS CLAIMED IS: 1. A cell-penetrating agent comprising: (i) a cell-internalizing module (CIM) and (ii) an antibody that specifically binds to Transactive response DNA binding Protein of 43 kD (TDP-43). 2. The cell-penetrating agent of claim 1, wherein the CIM comprises a Cell Membrane Internalizing Peptide (CMIP). 3 The cell-penetrating agent of claim 1 or 2, wherein the CIM comprises a 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-penetrating agent of claim 1 or 2, wherein the CIM comprises a Penetain amino acid sequence or a derivative thereof. 6 The cell-penetrating agent of claim 1 or 2, wherein the CIM comprises a Pepth amino acid sequence or a derivative thereof. 7 The cell-penetrating agent of claim 1 or 2, wherein the CIM comprises a polyarginine amino acid sequence. 8 The cell-penetrating agent of claim 1, 2, or 7, wherein the CIM comprises more than one polyarginine amino acid sequence. 9 The cell-penetrating agent of any one of claims 1, 2, 7, and 8, wherein the CIM comprises three polyarginine amino acid sequences. 10 The cell-penetrating agent of claim 1 or 2, wherein the CIM comprises a TAT amino acid sequence.
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT 11. The cell-penetrating agent of claim 1, 2, or 10, wherein the CIM comprises more than one TAT amino acid sequence. 12. The cell-penetrating agent of any one of claims 1, 2, 10, and 11, wherein the CIM comprises three TAT amino acid sequences. 13. The cell-penetrating agent of any one of claims 1-12, wherein the CIM comprises a macrocycle. 14. The cell-penetrating agent of claim 13, wherein the macrocycle is formed through a covalent bond between two amino acids in the CIM. 15. The cell-penetrating agent of claim 13 or 14, wherein the macrocycle is formed by a disulfide bond between two cysteine residues in the CIM. 16. The cell-penetrating agent of any one of claims 1-15, wherein the CIM further comprises one or more histidine residues. 17. The cell-penetrating agent of any one of claims 1-16, wherein the CIM comprises a polypeptide having an amino acid sequence selected from one of: SEQ ID NO: 176-184, SEQ ID NO: 192, and SEQ ID NO: 193. 18. The cell-penetrating agent of any one of claims 1-17, 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 NOs: 200-203.
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT 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 one of SEQ ID NOs: 200-203. 23. The cell-penetrating agent of claim 1, wherein the CIM comprises a polypeptide having an amino acid sequence selected from any one of SEQ ID NOs: 176-193. 24. The cell-penetrating agent of claim 1, wherein the CIM is a polypeptide having an amino acid sequence selected from any one of SEQ ID NOs: 176-193. 25. The cell-penetrating agent of any one of claims 1-24, wherein the CIM is covalently linked to the antibody. 26. The cell-penetrating agent of any one of claims 1-24, wherein the CIM is non-covalently linked to the antibody. 27. The cell-penetrating agent of any one of claims 1-26, wherein the cell-penetrating agent further comprises a linker connecting 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-penetrating agent of claim 27 or 28, wherein the linker is a non-cleavable linker. 31. The cell-penetrating agent of any one of claims 27-30, wherein the linker comprises a polypeptide. 32. The cell-penetrating agent of any one of claims 27-31, wherein the linker comprises one or more glycine residues.
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT 33. The cell-penetrating agent of any one of claims 27-32, wherein the linker comprises a polypeptide comprising an amino acid sequence selected from any one of SEQ ID NOs: 194- 199. 34. The cell-penetrating agent of any one of claims 27-29, 31, and 32, wherein the linker is a polypeptide comprising an amino acid sequence selected from any one of SEQ ID NOs: 194- 199. 35. The cell-penetrating agent of any one of claims 1-34, wherein the antibody is linked to the C- terminus of the CIM. 36. The cell-penetrating agent of any one of claims 1-34, wherein the antibody is linked to the N- terminus of the CIM. 37. The cell-penetrating agent of any one of claims 1-36, 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 a C-terminus of the heavy chain. 39. The cell-penetrating agent of claim 37, wherein the CIM is covalently linked to a N-terminus of the heavy chain. 40. The cell-penetrating agent of any one of claims 1-39, 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 a C-terminus of the light chain. 42. The cell-penetrating agent of claim 40, wherein the CIM is covalently linked to a N-terminus of the light chain.
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT 43. The cell-penetrating agent of any one of claims 1-42, wherein the antibody competes for binding to TDP-43 with: an antibody comprising a heavy chain variable domain of SEQ ID NO: 1 and a light chain variable domain of SEQ ID NO: 24; an antibody comprising a heavy chain variable domain of SEQ ID NO: 63 and a light chain variable domain of SEQ ID NO: 65; an antibody comprising a heavy chain variable domain of SEQ ID NO: 67 and a light chain variable domain of SEQ ID NO: 69; an antibody comprising a heavy chain variable domain of SEQ ID NO: 71 and a light chain variable domain of SEQ ID NO: 73; an antibody comprising a heavy chain variable domain of SEQ ID NO: 75 and a light chain variable domain of SEQ ID NO: 77; or an antibody comprising a heavy chain variable domain of SEQ ID NO: 79 and a light chain variable domain of SEQ ID NO: 81. 4 The cell-penetrating agent of any one of claims 1-43, wherein the antibody binds to the same epitope on TDP-43 as: an antibody comprising a heavy chain variable domain of SEQ ID NO: 1 and a light chain variable domain of SEQ ID NO: 24; an antibody comprising a heavy chain variable domain of SEQ ID NO: 63 and a light chain variable domain of SEQ ID NO: 65; an antibody comprising a heavy chain variable domain of SEQ ID NO: 67 and a light chain variable domain of SEQ ID NO: 69; an antibody comprising a heavy chain variable domain of SEQ ID NO: 71 and a light chain variable domain of SEQ ID NO: 73; an antibody comprising a heavy chain variable domain of SEQ ID NO: 75 and a light chain variable domain of SEQ ID NO: 77; or an antibody comprising a heavy chain variable domain of SEQ ID NO: 79 and a light chain variable domain of SEQ ID NO: 81.
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT 45. The cell-penetrating agent of any one of claims 1-44, wherein the antibody comprises three light chain CDRs and three heavy chain CDRs of a mouse antibody characterized by a heavy chain variable domain comprising SEQ ID NO: 1 and a light chain variable domain comprising SEQ ID NO: 24. 46. The cell-penetrating agent of claim 45, wherein the CDRs are of a definition selected from the group 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 variable domain comprising: a heavy chain CDR1 as defined by 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 NOs: 53-55. 48. The cell-penetrating agent of claim 47, wherein the humanized mature heavy variable domain comprises a sequence that is at least 80% identical to any one of SEQ ID NOs: 4-23 and the humanized mature light chain variable domain comprises a sequence that is at least 80% identical to any one of SEQ ID NOs: 27-48. 49. The cell-penetrating agent of claim 48, wherein the humanized mature heavy variable domain comprises a sequence that is at least 85% to any one of SEQ ID NOs: 4-23 and the humanized mature light chain variable region comprises a sequence that is at least 85% identical to any one of SEQ ID NOs: 27-48. 50. The cell-penetrating agent of claim 48 or 49, wherein the humanized mature heavy variable domain comprises a sequence that is at least 90% identical, to any one of SEQ ID NOs: 4-23 and the humanized mature light chain variable region comprises a sequence that is at least 90% identical, to any one of SEQ ID NOs: 27-48.
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT 51. The cell-penetrating agent of any one of claims 48-50, wherein the humanized mature heavy variable domain comprises a sequence that is at least 95% identical to any one of SEQ ID NOs: 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 NOs: 27-48. 52. The cell-penetrating agent of any one of claims 47-51, wherein at least one of the following positions in the humanized heavy chain variable domain is occupied by the amino acid as specified: 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 47-52, wherein at least one of the following positions in the humanized heavy chain variable domain is occupied by the amino acid as specified: 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 47-53, wherein at least one of the following positions in the humanized heavy chain variable domain is occupied by the amino acid as specified: 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 47-54, wherein 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 the amino acid as specified: 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 47-55, wherein at least one of the following positions in the humanized light chain variable domain is occupied by the amino acid as
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT specified: 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 47-56, wherein at least one of the following positions in the humanized light chain variable domain is occupied by the amino acid as specified: V3 is occupied by Q or A100 is occupied by D or R. 58. The cell-penetrating agent of any one of claims 47-57, wherein at least one of the following positions in the humanized light chain variable domain is occupied by the amino acid as specified: 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-penetrating agent of any one of claims 47-58, 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 amino acid as specified: T80 is occupied by A or L92 is occupied by A. 60. The cell-penetrating agent of any one of claims 47-59, wherein at least one of the following positions in the humanized heavy chain variable domain is occupied by the amino acid as specified: 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 the amino acid as specified: 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:
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT a heavy chain CDR1, as defined by 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 NOs: 55-61. 62. The cell-penetrating agent of claim 61, wherein: the heavy chain CDR1, as defined by Kabat/Chothia Composite, comprises SEQ ID NO: 49; the heavy chain CDR2, as defined by Kabat, comprises SEQ ID NO: 51; the heavy chain CDR3, as defined by Kabat or Chothia, comprises SEQ ID NO: 52; the light chain CDR1, as defined by Kabat, comprises SEQ ID NO: 53; the light chain CDR2, as defined by Kabat, comprises SEQ ID NO: 54; and the light chain CDR3, as defined by Kabat, comprises 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 one of SEQ ID NOs: 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 one of: 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 one of: SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 23. 66. The cell-penetrating agent of any one of claims 61-65, wherein the heavy chain variable domain comprises a sequence of SEQ ID NO: 20.
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT 67. The cell-penetrating agent of any one of claims 61-65, wherein the heavy chain variable domain comprises a sequence of SEQ ID NO: 21. 68. The cell-penetrating agent of any one of claims 61-65, wherein the heavy chain variable domain comprises a sequence of SEQ ID NO: 23. 69. The cell-penetrating agent of any one of claims 61-68, wherein the light chain variable domain comprises a sequence that is at least 95% identical to any one of: SEQ ID NOs: 27-48. 70. The cell-penetrating agent of any one of claims 61-69, wherein the light chain variable domain comprises a sequence that is at least 95% identical to SEQ ID NO: 47 or SEQ ID NO: 48. 71. The cell-penetrating agent of any one of claims 61-70, wherein the light chain variable domain comprises a sequence that is at least 98% identical to SEQ ID NO: 47 or SEQ ID NO: 48. 72. The cell-penetrating agent of any one of claims 69-71, wherein the light chain variable domain comprises SEQ ID NO: 47. 73. The cell-penetrating agent of any one of claims 69-71, 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: a heavy chain CDR1 comprising SEQ ID NO: 84; a heavy chain CDR2 comprising SEQ ID NO: 85; a heavy chain CDR3 comprising SEQ ID NO: 86; a light chain CDR1 comprising SEQ ID NO: 87; a light chain CDR2 comprising SEQ ID NO: 88; and a light chain CDR3 comprising 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 SEQ ID NO: 63.
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT 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 SEQ ID NO: 63. 77. The cell-penetrating agent of any one of claims 74-76, wherein the heavy chain variable domain comprises a sequence of SEQ ID NO: 63. 78. The cell-penetrating agent of any one of claims 74-77, wherein the light chain variable domain comprises a sequence that is at least 95% identical to SEQ ID NO: 65. 79. The cell-penetrating agent of any one of claims 74-78, wherein the light chain variable domain comprises a sequence that is at least 98% identical to SEQ ID NO: 65. 80. The cell-penetrating agent of any one of claims 74-79, wherein the light chain variable domain comprises a 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: a heavy chain CDR1 comprising SEQ ID NO: 90; a heavy chain CDR2 comprising SEQ ID NO: 91; a heavy chain CDR3 comprising SEQ ID NO: 92; a light chain CDR1 comprising SEQ ID NO: 93; a light chain CDR2 comprising SEQ ID NO: 94; and a light chain CDR3 comprising SEQ ID NO: 95. 82. The cell-penetrating agent of claim 81, wherein the heavy chain variable domain comprises a sequence that is at least 95% identical to SEQ ID NO: 67. 83. The cell-penetrating agent 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.
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT 84. The cell-penetrating agent of any one of claims 81-83, wherein the heavy chain variable domain comprises a sequence of SEQ ID NO: 67. 85. The cell-penetrating agent of any one of claims 81-84, wherein the light chain variable domain comprises a sequence that is at least 95% identical to SEQ ID NO: 69. 86. The cell-penetrating agent of any one of claims 81-85, wherein the light chain variable domain comprises a sequence that is at least 98% identical to SEQ ID NO: 69. 87. The cell-penetrating agent of any one of claims 81-86, wherein the light chain variable domain comprises a 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: a heavy chain CDR1 comprising SEQ ID NO: 96; a heavy chain CDR2 comprising SEQ ID NO: 97; a heavy chain CDR3 comprising SEQ ID NO: 98; a light chain CDR1 comprising SEQ ID NO: 99; a light chain CDR2 comprising SEQ ID NO: 100; and a light chain CDR3 comprising SEQ ID NO: 101. 89. The cell-penetrating agent of claim 88, wherein the heavy chain variable domain comprises a sequence that is at least 95% identical to 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 SEQ ID NO: 71. 91. The cell-penetrating agent of any one of claims 88-90, wherein the heavy chain variable domain comprises a sequence of SEQ ID NO: 71.
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT 92. The cell-penetrating agent of any one of claims 88-91, wherein the light chain variable domain comprises a sequence that is at least 95% identical to SEQ ID NO: 73. 93. The cell-penetrating agent of any one of claims 88-92, wherein the light chain variable domain comprises a sequence that is at least 98% identical to SEQ ID NO: 73. 94. The cell-penetrating agent of any one of claims 88-93, wherein the light chain variable domain comprises a 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: a heavy chain CDR1 comprising SEQ ID NO: 102; a heavy chain CDR2 comprising SEQ ID NO: 103; a heavy chain CDR3 comprising SEQ ID NO: 104; a light chain CDR1 comprising SEQ ID NO: 105; a light chain CDR2 comprising SEQ ID NO: 106; and a light chain CDR3 comprising 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 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 SEQ ID NO: 75. 98. The cell-penetrating agent of any one of claims 95-97, wherein the heavy chain variable domain comprises a sequence of SEQ ID NO: 75. 99. The cell-penetrating agent of any one of claims 95-97, wherein the light chain variable domain comprises a sequence that is at least 95% identical to SEQ ID NO: 77.
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT 100. The cell-penetrating agent of any one of claims 95-99, wherein the light chain variable domain comprises a sequence that is at least 98% identical to SEQ ID NO: 77. 101. The cell-penetrating agent of any one of claims 95-100, wherein the light chain variable domain comprises a 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: a heavy chain CDR1 comprising SEQ ID NO: 108; a heavy chain CDR2 comprising SEQ ID NO: 109; a heavy chain CDR3 comprising SEQ ID NO: 110; a light chain CDR1 comprising SEQ ID NO: 111; a light chain CDR2 comprising SEQ ID NO: 112; and a light chain CDR3 comprising SEQ ID NO: 113. 103. The cell-penetrating agent of claim 102, wherein the heavy chain variable domain comprises a sequence at least 95% identical to SEQ ID NO: 79. 104. The cell-penetrating agent of claim 102 or 103, wherein the heavy chain variable domain comprises a sequence at least 98% identical to SEQ ID NO: 79. 105. The cell-penetrating agent of any one of claims 102-104, wherein the heavy chain variable domain comprises a sequence of SEQ ID NO: 79. 106. The cell-penetrating agent of any one of claims 102-105, wherein the light chain variable domain comprises a sequence that is at least 95% identical to SEQ ID NO: 81. 107. The cell-penetrating agent of any one of claims 102-106, wherein the light chain variable domain comprises a sequence that is at least 98% identical to SEQ ID NO: 81.
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT 108. The cell-penetrating agent of any one of claims 102-107, wherein the light chain variable domain comprises a sequence of SEQ ID NO: 81. 109. The cell-penetrating agent of any one of claims 1-108, wherein the antibody is a humanized antibody, a chimeric antibody, or a veneered antibody. 110. The cell-penetrating agent of any one of claims 1-109, wherein the antibody is an antigen- binding antibody fragment. 111. The cell-penetrating agent 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 of any one of claims 1-109, wherein the antibody is an intact antibody. 113. The cell-penetrating agent of any one of claims 1-109 and 112, wherein the antibody has a human IgG1 isotype. 114. The cell-penetrating agent of any one of claims 1-109, 112, and 113, wherein the heavy chain variable domain is fused to a heavy chain constant region and the light chain variable domain is fused to a light chain constant region. 115. The cell-penetrating agent of claim 114, wherein the heavy chain constant region is a mutant form of a natural human heavy chain constant region which has reduced binding to an Fcγ receptor relative to the natural heavy chain constant region. 116. The cell-penetrating agent of claim 114 or 115, wherein the heavy chain constant region is of IgG1 isotype. 117. The cell-penetrating agent of any one of claims 1-116, wherein the antibody has at least one mutation in a constant region. 118. The cell-penetrating agent of claim 117, wherein the at least one mutation reduces complement fixation or activation by the constant region.
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT 119. The cell-penetrating agent of claim 118, where the at least one mutation is at one or more positions of: 241, 264, 265, 270, 296, 297, 318, 320, 322, 329, and 331 by EU numbering. 120. The cell-penetrating agent of claim 119, wherein the antibody has an alanine at positions 318, 320, and 322 by EU numbering. 121. The cell-penetrating agent of any one of claims 1-120, wherein the antibody selectively binds to phosphorylated TDP-43. 122. The cell-penetrating agent of any one of claims 1-121, wherein the antibody selectively binds to phosphorylated TDP-43 as compared to unphosphorylated TDP-43. 123. The cell-penetrating agent of claim 121 or 122, wherein the antibody binds to phosphorylated TDP-43 with at least 100-fold greater affinity as compared to unphosphorylated TDP-43. 124. The cell-penetrating agent of any one of claims 121-123, wherein the antibody binds to phosphorylated TDP-43 with at least 1000-fold greater affinity as compared to unphosphorylated TDP-43. 125. The cell-penetrating agent of any one of claims 121-124, wherein phosphorylated TDP-43 comprises phosphorylation of at least one amino acid residue selected from S409 and S410. 126. The cell-penetrating agent 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 1-126, wherein the antibody selectively binds to cytoplasmic aggregates of TDP-43. 128. The cell-penetrating agent of any one of claims 1-127, wherein the antibody selectively binds to a cytoplasmic aggregate of TDP-43 compared to nuclear TDP-43.
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT 129. The cell-penetrating agent of claim 127 or 128, wherein the cytoplasmic aggregate of TDP-43 comprises phosphorylated aggregates of TDP-43. 130. The cell-penetrating agent of any one of claims 1-129, wherein the antibody does not substantially bind 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 one of: 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 one of: 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: 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
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT 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 131-133, wherein the cell-penetrating agent comprises a polypeptide sequence that is at least 95% identical to a sequence selected from any one of 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. 135. The cell-penetrating agent of claim 1 and 131-134, wherein the cell-penetrating agent comprises a polypeptide sequence that is at least 98% identical to a sequence selected from any one of 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 claim 1 and 131-135, wherein the cell-penetrating agent comprises a polypeptide sequence selected from any one of 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.
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT 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 comprises a sequence that is at least 95% identical to SEQ ID NO: 116 and the second polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 117; the first polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 118 and the second polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 119; the first polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 120 and the second polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 121; the first polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 122 and the second polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 123; the first polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 124 and the second polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 125; the first polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 126 and the second polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 127; the first polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 128 and the second polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 129; the first polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 130 and the second polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 131; the first polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 132 and the second polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 133;
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT the first polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 134 and the second polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 135; the first polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 136 and the second polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 137; the first polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 138 and the second polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 139; the first polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 140 and the second polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 141; the first polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 142 and the second polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 143; the first polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 144 and the second polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 145; the first polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 146 and the second polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 147; the first polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 148 and the second polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 149; the first polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 150 and the second polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 151; the first polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 152 and the second polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 153;
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT the first polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 154 and the second polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 155; the first polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 156 and the second polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 157; the first polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 158 and the second polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 159; the first polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 160 and the second polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 161; the first polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 162 and the second polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 163; the first polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 164 and the second polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 165; the first polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 166 and the second polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 167; the first polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 168 and the second polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 169; the first polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 170 and the second polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 171; the first polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 172 and the second polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 173; or
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT the first polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 174 and the second polypeptide comprises a sequence that is at least 95% identical to 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;
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT 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;
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT 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 1-138, wherein the antibody is conjugated to a therapeutic, cytotoxic, cytostatic, immunomodulatory, neurotrophic, or neuroprotective agent. 140. The cell-penetrating agent of any one of claims 1-139, wherein the heavy chain does not comprise a C-terminal lysine residue. 141. A pharmaceutical composition comprising a cell-penetrating agent of any one of claims 1-140 and a pharmaceutically acceptable carrier. 142. A nucleic acid encoding at least a portion of the cell-penetrating agent of any one of claims 1- 140. 143. The nucleic acid of claim 142, wherein the nucleic acid encodes for 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 for the CIM. 145. A vector comprising the nucleic acid of any one of claims 142-144 operably linked to one or more regulatory sequences to effect expression in a mammalian cell of the cell-penetrating agent of any one of claims 1-140. 146. The vector of claim 145, wherein the one or more regulatory sequences comprise one or more of an enhancer, ribosome binding site, transcription termination signal, and promoter, optionally, wherein the promoter is a eukaryotic promoter.
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT 147. The vector of claim 145 or 146, wherein the nucleic acid is codon-optimized for expression in a host cell. 148. A host cell transformed with the vector of any one of claims 145-147. 149. A host cell comprising the nucleic acid of any one of claims 142-144. 150. A method of delivering the antibody that specifically binds to TDP-43 into a cell, comprising contacting the cell-penetrating agent of any one of claims 1-140 with the cell, thereby resulting in the internalization into the cell of, at a minimum, an antigen-binding fragment of the antibody. 151. The method of claim 150, the method further comprising transfer of, at a minimum, an antigen- binding fragment of the antibody, to the cytosol of the cell. 152. A method of binding an intracellular TDP-43 protein in a cell, the method comprising: contacting the cell-penetrating agent of any one of claims 1-140 with the cell, thereby resulting in the internalization of and transfer to the cytosol of, at a minimum, an antigen-binding fragment of the antibody. 153. The method of any one of claims 150-152, wherein the cell is a mammalian cell. 154. The method of any one of claims 150-152, wherein the cell is in vitro. 155. The method of any one of claims 150-152, wherein the cell is in a subject. 156. A method of inhibiting or reducing aggregation of TDP-43 in a subject having or at risk of developing a TDP-43-related disease, comprising administering to the subject an effective amount of the cell-penetrating agent of any one of claims 1-140, thereby inhibiting or reducing aggregation of TDP-43 in the subject.
Attorney Docket Ref. No.: 50887-00047WO1 // Client Ref: 794-PCT 157. A method of treating or effecting prophylaxis of a TDP-43-related disease in a subject, comprising administering a therapeutically effective amount of the cell-penetrating agent of any one of claims 1-140 and thereby treating or effecting prophylaxis of the TDP-43-associated disease. 158. The method of claim 157, wherein the TDP-43-related disease is amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD-TDP), primary 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 of detecting TDP-43 deposits in a subject having or at risk of developing a TDP-43- related disease, comprising administering to a subject the cell-penetrating agent of any one of claims 1-140, and detecting the antibody bound to TDP-43 in the subject. 161. The method of claim 160, wherein the antibody is administered by intravenous injection into the body of the subject. 162. A method of detecting TDP-43 in a sample obtained from a patient having or at risk of developing a TDP-43 related disease, comprising contacting the sample with the cell- penetrating agent of any one of claims 1-140, and detecting the binding of the antibody to TDP-43 in the sample. 163. The method of any one of claim 160-162, wherein the antibody is labeled. 164. The method of claim 163, wherein the antibody is labeled with a fluorescent label, a paramagnetic label, or a radioactive label. 165. The method of claim 164, wherein the radioactive label is detected using positron emission tomography (PET) or single-photon emission computed tomography (SPECT).
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