WO2022020105A1 - Anti-transferrin receptor (tfr) antibody and uses thereof - Google Patents

Anti-transferrin receptor (tfr) antibody and uses thereof Download PDF

Info

Publication number
WO2022020105A1
WO2022020105A1 PCT/US2021/040984 US2021040984W WO2022020105A1 WO 2022020105 A1 WO2022020105 A1 WO 2022020105A1 US 2021040984 W US2021040984 W US 2021040984W WO 2022020105 A1 WO2022020105 A1 WO 2022020105A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
amino acid
acid sequence
antibody
light chain
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2021/040984
Other languages
English (en)
French (fr)
Inventor
Romesh R. Subramanian
Mohammed T. QATANANI
Timothy Weeden
Cody A. Desjardins
Brendan QUINN
John NAJIM
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Dyne Therapeutics Inc
Original Assignee
Dyne Therapeutics Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Dyne Therapeutics Inc filed Critical Dyne Therapeutics Inc
Priority to CN202180064690.5A priority Critical patent/CN116348138A/zh
Priority to US18/017,167 priority patent/US20230256112A1/en
Priority to IL299667A priority patent/IL299667A/en
Priority to MX2023000962A priority patent/MX2023000962A/es
Priority to KR1020237005829A priority patent/KR20230041760A/ko
Priority to EP21846109.3A priority patent/EP4185315A4/en
Priority to BR112023001005A priority patent/BR112023001005A2/pt
Priority to AU2021312708A priority patent/AU2021312708A1/en
Priority to JP2023504620A priority patent/JP2023535443A/ja
Priority to CA3186727A priority patent/CA3186727A1/en
Publication of WO2022020105A1 publication Critical patent/WO2022020105A1/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6807—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug or compound being a sugar, nucleoside, nucleotide, nucleic acid, e.g. RNA antisense
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • 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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2881—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD71
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the present application relates to novel anti-transferrin receptor (TfR) antibodies and the use of the antibodies.
  • TfR anti-transferrin receptor
  • ASCII format via EFS-Web is hereby incorporated by reference in its entirety.
  • Said ASCII copy, created on July 8, 2021, is named D082470037WO00-SEQ-DWY and is 120,479 bytes in size.
  • Transferrin Receptor is a dimeric transmembrane glycoprotein receptor involved in iron transport.
  • Two transferrin receptors have been characterized in humans, transferrin receptor 1 (TfRl) and transferrin receptor 2 (TfR2). It has been shown that TfR is overexpressed in cancer cells with higher metastatic potential.
  • TfRl has been shown to express on the endothelial cells of the blood brain barrier can be used to allow the delivery of large molecules into the brain.
  • anti-TfR antibodies selectively bind to human or non-human primate (NHP) transferrin receptor 1 (TfRl) with high specificity and affinity (e.g., subnanomolar to nanomolar range).
  • the anti-TfR antibodies described herein are useful for targeting tissues and/or (e.g., and) cells that express TfRl.
  • the anti-TfR antibodies provided herein are used for detection of TfRl in a cell or a tissue.
  • the anti-TfR antibodies provided herein are used in diagnostic, therapeutic, or research applications. In some embodiments, the anti-TfR antibodies described herein are used to deliver a molecular payload to a target cell or tissue (e.g., a cell or tissue that expresses TfRl).
  • a target cell or tissue e.g., a cell or tissue that expresses TfRl.
  • complexes comprising the anti-TfR antibodies conjugated (e.g., covalently conjugated) to a molecular payload (e.g., a diagnostic agent or a therapeutic agent) are provided.
  • the anti-TfR antibodies is used to deliver the conjugated molecular payload to a cell or a tissue that expresses TfRl (e.g., muscle or the brain) for diagnosing and/or (e.g., and) treating a disease (e.g., a muscle disease or a neurological disease).
  • the present disclosure provides data demonstrating that the anti-TfR antibodies described herein has superior activity in delivering molecular payload into a target cell (e.g., a muscle cell), compared with other known anti-TfR antibodies.
  • One aspect of the present disclosure relates to an antibody that binds to human transferrin receptor (TfR), wherein the antibody comprises:
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • VL light chain variable region
  • a heavy chain variable region (VH) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 73; and/or a light chain variable region (VL) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 74;
  • a heavy chain variable region (VH) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 73; and/or a light chain variable region (VL) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 75;
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • VL light chain variable region
  • x a heavy chain variable region (VH) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 77; and/or a light chain variable region (VL) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 80.
  • VH heavy chain variable region
  • VL light chain variable region
  • the antibody comprises:
  • VH comprising an amino acid sequence of SEQ ID NO: 69 and a VL comprising an amino acid sequence of SEQ ID NO: 70;
  • VH comprising an amino acid sequence of SEQ ID NO: 71 and a VL comprising an amino acid sequence of SEQ ID NO: 70;
  • VH comprising an amino acid sequence of SEQ ID NO: 72 and a VL comprising an amino acid sequence of SEQ ID NO: 70;
  • VH comprising an amino acid sequence of SEQ ID NO: 73 and a VL comprising an amino acid sequence of SEQ ID NO: 75;
  • VH comprising an amino acid sequence of SEQ ID NO: 77 and a VL comprising an amino acid sequence of SEQ ID NO: 78;
  • a VH comprising an amino acid sequence of SEQ ID NO: 79 and a VL comprising an amino acid sequence of SEQ ID NO: 80; or (x) a VH comprising an amino acid sequence of SEQ ID NO: 77 and a VL comprising an amino acid sequence of SEQ ID NO: 80.
  • the antibody is selected from the group consisting of a
  • the antibody is a Fab fragment.
  • the antibody comprises:
  • a heavy chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 100; and/or a light chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 90;
  • a heavy chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 103; and/or a light chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 95; or (x) a heavy chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 102; and/or a light chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 95.
  • the antibody comprises:
  • the equilibrium dissociation constant (K D ) of binding of the antibody to the transferrin receptor is in a range from 10 u M to 10 6 M.
  • the antibody does not specifically bind to the transferrin binding site of the transferrin receptor and/or the antibody does not inhibit binding of transferrin to the transferrin receptor.
  • the antibody is cross -reactive with extracellular epitopes of two or more of a human, non-human primate and rodent transferrin receptor.
  • Another aspect of the present disclosure relates to a complex comprising the antibody covalently linked to a molecular payload.
  • the molecular payload is a diagnostic agent or a therapeutic agent.
  • the molecular payload is an oligonucleotide, a polypeptide, or a small molecule.
  • the antibody and the molecular payload are linked via a linker.
  • the linker is a cleavable linker.
  • the linker comprises a valine-citrulline sequence.
  • Another aspect of the present disclosure relates to a composition comprising an antibody or a complex disclosed herein. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier.
  • Yet another aspect of the present disclosure relates to a method of delivering a molecular payload to a cell, comprising contacting the cell with a complex or composition disclosed herein.
  • the cell is a muscle cell.
  • the cell is in vitro.
  • the cell is in a subject.
  • the subject is human.
  • Another aspect of the present disclosure relates to a method of delivering a molecular payload to the muscle of a subject, comprising administering to the subject an effective amount of a complex disclosed herein.
  • the administration is intravenous.
  • Another aspect of the present disclosure relates to a method of treating a disease, comprising administering to a subject an effective amount of a complex or a composition disclosed herein, wherein the molecular payload is a therapeutic agent.
  • the disease is a muscle disease and the molecular payload is a drug for treating the muscle disease.
  • the muscle disease is a rare muscle disease or muscle atrophy.
  • FIGs. 1A-1F show binding of humanized anti-TfR Fabs to human TfRl (hTfRl) or cynomolgus monkey TfRl (cTfRl), as measured by ELISA.
  • FIG. 1A shows binding of humanized 3M12 variants to hTfRl.
  • FIG. IB shows binding of humanized 3M12 variants to cTfRl.
  • FIG. 1C shows binding of humanized 3A4 variants to hTfRl.
  • FIG. ID shows binding of humanized 3A4 variants to cTfRl.
  • FIG. IE shows binding of humanized 5H12 variants to hTfRl.
  • FIG. 2 shows the quantified cellular uptake of anti-TfR Fab conjugates into rhabdomyosarcoma (RD) cells.
  • the molecular payload in the tested conjugates are DMPK- targeting oligonucleotides and the uptake of the conjugates were facilitated by indicated anti- TfR Fabs.
  • Conjugates having a negative control Fab (anti-mouse TfR) or a positive control Fab (anti-human TfRl) are also included this assay. Cells were incubated with indicated conjugate at a concentration of 100 nM for 4 hours. Cellular uptake was measured by mean Cypher5e fluorescence.
  • FIGs. 3A-3F show binding of oligonucleotide-conjugated or unconjugated humanized anti-TfR Fabs to human TfRl (hTfRl) and cynomolgus monkey TfRl (cTfRl), as measured by ELISA.
  • FIG. 3A shows the binding of humanized 3M12 variants alone or in conjugates with a DMPK targeting oligo to hTfRl.
  • FIG. 3B shows the binding of humanized 3M12 variants alone or in conjugates with a DMPK targeting oligo to cTfRl.
  • FIG. 3C shows the binding of humanized 3 A4 variants alone or in conjugates with a DMPK targeting oligo to hTfRl.
  • FIG. 3D shows the binding of humanized 3A4 variants alone or in conjugates with a DMPK targeting oligo to cTfRl.
  • FIG. 3E shows the binding of humanized 5H12 variants alone or in conjugates with a DMPK targeting oligo to hTfRl.
  • FIG. 3F shows the binding of humanized 5H12 variants alone or in conjugates with a DMPK targeting oligo to cTfRl. The respective EC50 values are also shown.
  • FIG. 4 shows DMPK expression in RD cells treated with various concentrations of conjugates containing the indicated humanized anti-TfR Fab antibodies conjugated to a DMPK-targeting oligonucleotide (ASO300). The duration of treatment was 3 days. ASO300 delivered using transfection agents (labeled “Trans”) was used as control.
  • FIG. 5 shows the serum stability of the linker used for linking an anti-TfR antibody and a molecular payload (e.g., an oligonucleotide) in various species over time after intravenous administration.
  • a molecular payload e.g., an oligonucleotide
  • FIG. 6 shows data illustrating that conjugates containing designated anti-TfR Fabs (3M12 VH3/VK2, 3M12 VH4/VK3, and 3A4 VH3 N54S/VK4) conjugated to a DMD exon- skipping oligonucleotide resulted in enhanced exon skipping compared to the naked DMD exon skipping oligo in DMD patient myotubes.
  • designated anti-TfR Fabs 3M12 VH3/VK2, 3M12 VH4/VK3, and 3A4 VH3 N54S/VK4
  • FIGs. 7A-7E show in vivo activity of conjugates containing designated anti- TfR Fabs (control, 3M12 VH3/VK2, 3M12 VH4/VK3, and 3A4 VH3 N54S/VK4) conjugated to DMPK targeting oligonucleotide in reducing DMPK mRNA expression in mice expressing human TfRl (hTfRl knock-in mice).
  • FIG. 7A shows the experimental design (e.g., IV dosage, dosing frequency).
  • DMPK mRNA levels were measured 14 days post first dose in the tibialis anterior (FIG. 7B), gastrocnemius (FIG. 1C), heart (FIG. 7D), and diaphragm (FIG. 7E), of the mice.
  • FIG. 8 shows ELISA measurements of binding of anti-TfR Fab 3M12 VH4/Vk3 to recombinant human (circles), cynomolgus monkey (squares), mouse (upward triangles), or rat (downward triangles) TfRl protein, at a range of concentrations from 230 pM to 500 nM of the Fab. Measurement results show that the anti-TfR Fab is reactive with human and cynomolgus monkey TfRl. Binding was not observed to mouse or rat recombinant TfRl. Data is shown as relative fluorescent units normalized to baseline.
  • FIG. 9 shows results of an ELISA testing the affinity of anti-TfR Fab 3M12 VH4/Vk3 to recombinant human TfRl or TfR2 over a range of concentrations from 230 pM to 500 nM of Fab.
  • the data are presented as relative fluorescence units normalized to baseline. The results demonstrate that the Fab does not bind recombinant human TfR2.
  • FIG. 10 shows the serum stability of the linker used for linking anti-TfR Fab 3M12 VH4/Vk3 to a control antisense oligonucleotide over 72 hours incubation in PBS or in rat, mouse, cynomolgus monkey or human serum.
  • the present disclosure is based on the development of humanized anti-TfR antibodies, e.g., antibodies listed in Table 3, which showed high binding affinity and specificity to human TfR. Also provided are the use of the anti-TfR antibodies and their variants in research, diagnostic/detection, and therapeutic applications.
  • the anti-TfR antibodies described herein are used for delivering molecular payloads (e.g., oligonucleotides, peptides, small molecules) to a target cell or tissue that expresses TfR.
  • the molecular payload to be delivered is conjugated the anti-TfR antibodies and delivered to a target cell or tissue that expresses TfR via receptor internationalization.
  • tissue that express TfR and can be targeted using the anti- TfR antibodies described herein include, without limitation: brain, muscle, adrenal, appendix, bone marrow, colon, duodenum, endometrium, esophagus, fat, gall bladder, heart, kidney, liver, lung, lymph node, ovary, pancreas, placenta, prostate, salivary gland, skin, small intestine, spleen, stomach, testis, thyroid, urinary bladder.
  • such approach has beneficial effects in muscle cells and for delivering across the blood brain barrier, which have been proven challenging.
  • the present disclosure provides data demonstrating that the anti-TfR antibodies described herein has superior activity in delivering molecular payload into a target cell (e.g., a muscle cell), compared with other known anti-TfR antibodies.
  • the present disclosure also provides complexes comprising any one of the anti-TfRl antibodies covalently linked to molecular payloads.
  • the complexes are particularly useful for delivering molecular payloads that inhibit the expression or activity of target genes in muscle cells, e.g., in a subject having or suspected of having a rare muscle disease or muscle atrophy (e.g., as listed in Table 6).
  • the complexes are particularly useful for delivering drugs to the brain for treating a neurological disease (e.g., as listed in Table 7).
  • Administering means to provide a complex to a subject in a manner that is physiologically and/or (e.g., and) pharmacologically useful (e.g., to treat a condition in the subject).
  • an antibody refers to a polypeptide that includes at least one immunoglobulin variable domain or at least one antigenic determinant, e.g., paratope that specifically binds to an antigen.
  • an antibody is a full- length antibody.
  • an antibody is a chimeric antibody.
  • an antibody is a humanized antibody.
  • an antibody is a Fab fragment, a Fab’ fragment, a F(ab')2 fragment, a Fv fragment or a scFv fragment.
  • an antibody is a nanobody derived from a camelid antibody or a nanobody derived from shark antibody.
  • an antibody is a diabody.
  • an antibody comprises a framework having a human germline sequence.
  • an antibody comprises a heavy chain constant domain selected from the group consisting of IgG, IgGl, IgG2, IgG2A, IgG2B, IgG2C, IgG3, IgG4, IgAl, IgA2, IgD, IgM, and IgE constant domains.
  • an antibody comprises a heavy (H) chain variable region (abbreviated herein as VH), and/or (e.g., and) a light (L) chain variable region (abbreviated herein as VL).
  • an antibody comprises a constant domain, e.g., an Fc region.
  • An immunoglobulin constant domain refers to a heavy or light chain constant domain. Human IgG heavy chain and light chain constant domain amino acid sequences and their functional variations are known.
  • the heavy chain of an antibody described herein can be an alpha (a), delta (D), epsilon (e), gamma (g) or mu (m) heavy chain.
  • the heavy chain of an antibody described herein can comprise a human alpha (a), delta (D), epsilon (e), gamma (g) or mu (m) heavy chain.
  • an antibody described herein comprises a human gamma 1 CHI, CH2, and/or (e.g., and) CH3 domain.
  • the amino acid sequence of the VH domain comprises the amino acid sequence of a human gamma (g) heavy chain constant region, such as any known in the art.
  • human constant region sequences have been described in the art, e.g., see U.S. Pat. No. 5,693,780 and Kabat E A et ah, (1991) supra.
  • the VH domain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or at least 99% identical to any of the variable chain constant regions provided herein.
  • an antibody is modified, e.g., modified via glycosylation, phosphorylation, sumoylation, and/or (e.g., and) methylation.
  • an antibody is a glycosylated antibody, which is conjugated to one or more sugar or carbohydrate molecules.
  • the one or more sugar or carbohydrate molecule are conjugated to the antibody via N-glycosylation, O-glycosylation, C-glycosylation, glypiation (GPI anchor attachment), and/or (e.g., and) phosphoglycosylation.
  • the one or more sugar or carbohydrate molecule are monosaccharides, disaccharides, oligosaccharides, or glycans.
  • the one or more sugar or carbohydrate molecule is a branched oligosaccharide or a branched glycan.
  • the one or more sugar or carbohydrate molecule includes a mannose unit, a glucose unit, an N-acetylglucosamine unit, an N-acetylgalactosamine unit, a galactose unit, a fucose unit, or a phospholipid unit.
  • an antibody is a construct that comprises a polypeptide comprising one or more antigen binding fragments of the disclosure linked to a linker polypeptide or an immunoglobulin constant domain.
  • Linker polypeptides comprise two or more amino acid residues joined by peptide bonds and are used to link one or more antigen binding portions. Examples of linker polypeptides have been reported (see e.g., Holliger, P, et al.
  • an antibody may be part of a larger immunoadhesion molecule, formed by covalent or noncovalent association of the antibody or antibody portion with one or more other proteins or peptides.
  • immunoadhesion molecules include use of the streptavidin core region to make a tetrameric scFv molecule (Kipriyanov, S. M., et al.
  • CDR refers to the complementarity determining region within antibody variable sequences.
  • a typical antibody molecule comprises a heavy chain variable region (VH) and a light chain variable region (VL), which are usually involved in antigen binding.
  • VH and VL regions can be further subdivided into regions of hypervariability, also known as “complementarity determining regions” (“CDR”), interspersed with regions that are more conserved, which are known as “framework regions” (“FR”).
  • CDR complementarity determining regions
  • FR framework regions
  • Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the extent of the framework region and CDRs can be precisely identified using methodology known in the art, for example, by the Rabat definition, the IMGT definition, the Chothia definition, the AbM definition, and/or (e.g., and) the contact definition, all of which are well known in the art. See, e.g., Rabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242; IMGT®, the international ImMunoGeneTics information system® http://www.imgt.org, Lefranc, M.-P.
  • a CDR may refer to the CDR defined by any method known in the art. Two antibodies having the same CDR means that the two antibodies have the same amino acid sequence of that CDR as determined by the same method, for example, the IMGT definition.
  • CDR1 There are three CDRs in each of the variable regions of the heavy chain and the light chain, which are designated CDR1, CDR2 and CDR3, for each of the variable regions.
  • CDR set refers to a group of three CDRs that occur in a single variable region capable of binding the antigen. The exact boundaries of these CDRs have been defined differently according to different systems.
  • Rabat Rabat et al, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides an unambiguous residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries defining the three CDRs.
  • CDRs may be referred to as Rabat CDRs.
  • Sub-portions of CDRs may be designated as LI, L2 and L3 or HI, H2 and H3 where the "L” and the "H” designates the light chain and the heavy chains regions, respectively. These regions may be referred to as Chothia CDRs, which have boundaries that overlap with Rabat CDRs.
  • Other boundaries defining CDRs overlapping with the Rabat CDRs have been described by Padlan (FASEB J. 9:133-139 (1995)) and MacCallum (J Mol Biol 262(5):732-45 (1996)).
  • CDR boundary definitions may not strictly follow one of the above systems, but will nonetheless overlap with the Rabat CDRs, although they may be shortened or lengthened in light of prediction or experimental findings that particular residues or groups of residues or even entire CDRs do not significantly impact antigen binding.
  • the methods used herein may utilize CDRs defined according to any of these systems. Examples of CDR definition systems are provided in Table 1.
  • CDR-grafted antibody refers to antibodies which comprise heavy and light chain variable region sequences from one species but in which the sequences of one or more of the CDR regions of VH and/or (e.g., and) VL are replaced with CDR sequences of another species, such as antibodies having murine heavy and light chain variable regions in which one or more of the murine CDRs (e.g., CDR3) has been replaced with human CDR sequences.
  • Chimeric antibody refers to antibodies which comprise heavy and light chain variable region sequences from one species and constant region sequences from another species, such as antibodies having murine heavy and light chain variable regions linked to human constant regions.
  • Complementary refers to the capacity for precise pairing between two nucleotides or two sets of nucleotides. In particular, complementary is a term that characterizes an extent of hydrogen bond pairing that brings about binding between two nucleotides or two sets of nucleotides.
  • Base pairings may include both canonical Watson-Crick base pairing and non-Watson-Crick base pairing (e.g., Wobble base pairing and Hoogsteen base pairing).
  • adenosine-type bases are complementary to thymidine-type bases (T) or uracil- type bases (U), that cytosine-type bases (C) are complementary to guanosine-type bases (G), and that universal bases such as 3-nitropyrrole or 5-nitroindole can hybridize to and are considered complementary to any A, C, U, or T.
  • Inosine (I) has also been considered in the art to be a universal base and is considered complementary to any A, C, U or T.
  • a “conservative amino acid substitution” refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made.
  • Variants can be prepared according to methods for altering polypeptide sequence known to one of ordinary skill in the art such as are found in references which compile such methods, e.g. Molecular Cloning: A Laboratory Manual, J. Sambrook, et ah, eds., Fourth Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2012, or Current Protocols in Molecular Biology, F.M. Ausubel, et ah, eds., John Wiley & Sons, Inc., New York.
  • amino acids include substitutions made amongst amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.
  • Covalently linked refers to a characteristic of two or more molecules being linked together via at least one covalent bond.
  • two molecules can be covalently linked together by a single bond, e.g., a disulfide bond or disulfide bridge, that serves as a linker between the molecules.
  • two or more molecules can be covalently linked together via a molecule that serves as a linker that joins the two or more molecules together through multiple covalent bonds.
  • a linker may be a cleavable linker.
  • a linker may be a non-cleavable linker.
  • Cross-reactive As used herein and in the context of a targeting agent (e.g., antibody), the term “cross-reactive,” refers to a property of the agent being capable of specifically binding to more than one antigen of a similar type or class (e.g., antigens of multiple homologs, paralogs, or orthologs) with similar affinity or avidity.
  • an antibody that is cross-reactive against human and non-human primate antigens of a similar type or class e.g., a human transferrin receptor and non-human primate transferrin receptor
  • an antibody is cross -reactive against a human antigen and a rodent antigen of a similar type or class. In some embodiments, an antibody is cross-reactive against a rodent antigen and a non-human primate antigen of a similar type or class. In some embodiments, an antibody is cross-reactive against a human antigen, a non-human primate antigen, and a rodent antigen of a similar type or class.
  • Framework refers to the remaining sequences of a variable region minus the CDRs. Because the exact definition of a CDR sequence can be determined by different systems, the meaning of a framework sequence is subject to correspondingly different interpretations.
  • the six CDRs also divide the framework regions on the light chain and the heavy chain into four sub-regions (FR1, FR2, FR3 and FR4) on each chain, in which CDR1 is positioned between FR1 and FR2, CDR2 between FR2 and FR3, and CDR3 between FR3 and FR4.
  • a framework region represents the combined FRs within the variable region of a single, naturally occurring immunoglobulin chain.
  • a FR represents one of the four sub-regions, and FRs represents two or more of the four sub-regions constituting a framework region.
  • Human heavy chain and light chain acceptor sequences are known in the art. In one embodiment, the acceptor sequences known in the art may be used in the antibodies disclosed herein.
  • Human antibody is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences.
  • the human antibodies of the disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3.
  • the term "human antibody”, as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
  • Humanized antibody refers to antibodies which comprise heavy and light chain variable region sequences from a non-human species (e.g ., a mouse) but in which at least a portion of the VH and/or (e.g., and) VL sequence has been altered to be more "human-like", i.e., more similar to human germline variable sequences.
  • a non-human species e.g ., a mouse
  • VH and/or e.g., and VL sequence
  • One type of humanized antibody is a CDR-grafted antibody, in which human CDR sequences are introduced into non-human VH and VL sequences to replace the corresponding nonhuman CDR sequences.
  • humanized anti-TfR antibodies and antigen binding portions are provided.
  • Such antibodies may be generated by obtaining murine anti-transferrin receptor monoclonal antibodies using traditional hybridoma technology followed by humanization using in vitro genetic engineering, such as those disclosed in Kasaian et al PCT publication No. WO 2005/123126 A2.
  • Isolated antibody An "isolated antibody”, as used herein, is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds transferrin receptor is substantially free of antibodies that specifically bind antigens other than transferrin receptor).
  • An isolated antibody that specifically binds transferrin receptor complex may, however, have cross-reactivity to other antigens, such as transferrin receptor molecules from other species.
  • an isolated antibody may be substantially free of other cellular material and/or (e.g., and) chemicals.
  • Molecular payload refers to a molecule or species that functions to modulate a biological outcome.
  • a molecular payload is linked to, or otherwise associated with an anti-TfR antibody.
  • the molecular payload is a small molecule, a protein, a peptide, a nucleic acid, or an oligonucleotide.
  • the molecular payload functions to modulate the transcription of a DNA sequence, to modulate the expression of a protein, or to modulate the activity of a protein.
  • the molecular payload is an oligonucleotide that comprises a strand having a region of complementarity to a target gene.
  • Oligonucleotide refers to an oligomeric nucleic acid compound of up to 200 nucleotides in length.
  • oligonucleotides include, but are not limited to, RNAi oligonucleotides (e.g., siRNAs, shRNAs), microRNAs, gapmers, mixmers, phosphorodiamidite morpholinos, peptide nucleic acids, aptamers, guide nucleic acids (e.g., Cas9 guide RNAs), etc.
  • Oligonucleotides may be single-stranded or double-stranded.
  • an oligonucleotide may comprise one or more modified nucleotides (e.g. 2'-0-methyl sugar modifications, purine or pyrimidine modifications). In some embodiments, an oligonucleotide may comprise one or more modified intemucleotide linkage. In some embodiments, an oligonucleotide may comprise one or more phosphorothioate linkages, which may be in the Rp or Sp stereochemical conformation.
  • modified nucleotides e.g. 2'-0-methyl sugar modifications, purine or pyrimidine modifications.
  • an oligonucleotide may comprise one or more modified intemucleotide linkage.
  • an oligonucleotide may comprise one or more phosphorothioate linkages, which may be in the Rp or Sp stereochemical conformation.
  • Recombinant antibody The term "recombinant human antibody”, as used herein, is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described in more details in this disclosure), antibodies isolated from a recombinant, combinatorial human antibody library (Hoogenboom H. R.,
  • such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.
  • One embodiment of the disclosure provides fully human antibodies capable of binding human transferrin receptor which can be generated using techniques well known in the art, such as, but not limited to, using human Ig phage libraries such as those disclosed in Jermutus et al., PCT publication No. WO 2005/007699 A2.
  • Region of complementarity refers to a nucleotide sequence, e.g., of an oligonucleotide, that is sufficiently complementary to a cognate nucleotide sequence, e.g., of a target nucleic acid, such that the two nucleotide sequences are capable of annealing to one another under physiological conditions (e.g., in a cell).
  • a region of complementarity is fully complementary to a cognate nucleotide sequence of target nucleic acid.
  • a region of complementarity is partially complementary to a cognate nucleotide sequence of target nucleic acid (e.g., at least 80%, 90%, 95% or 99% complementarity). In some embodiments, a region of complementarity contains 1, 2, 3, or 4 mismatches compared with a cognate nucleotide sequence of a target nucleic acid.
  • binds refers to the ability of a molecule to bind to a binding partner with a degree of affinity or avidity that enables the molecule to be used to distinguish the binding partner from an appropriate control in a binding assay or other binding context.
  • affinity or avidity that enables the molecule to be used to distinguish the binding partner from an appropriate control in a binding assay or other binding context.
  • an antibody specifically binds to a target if the antibody has a K D for binding the target of at least about 10 4 M, 10 5 M, 10 6 M, 10 7 M, 10 8 M, 10 9 M, 10 10 M, 10 11 M, 10 12 M, 10 13 M, or less. In some embodiments, an antibody specifically binds to the transferrin receptor.
  • Subject refers to a mammal.
  • a subject is non-human primate, or rodent.
  • a subject is a human.
  • a subject is a patient, e.g., a human patient that has or is suspected of having a disease.
  • the subject is a human patient who has or is suspected of having a disease resulting from a disease-associated-repeat expansion, e.g., in a DMPK allele.
  • Transferrin receptor As used herein, the term, “transferrin receptor” (also known as TFRC, CD71, p90, TFR, or TFR1) refers to an internalizing cell surface receptor that binds transferrin to facilitate iron uptake by endocytosis.
  • a transferrin receptor may be of human (NCBI Gene ID 7037), non-human primate (e.g., NCBI Gene ID 711568 or NCBI Gene ID 102136007), or rodent (e.g., NCBI Gene ID 22042) origin.
  • multiple human transcript variants have been characterized that encoded different isoforms of the receptor (e.g., as annotated under GenBank RefSeq Accession Numbers:
  • transferrin receptor amino acid sequence corresponding to NCBI sequence NP_003225.2 (transferrin receptor protein 1 isoform 1, homo sapiens) is as follows:
  • DNEF SEQ ID NO: 105.
  • non-human primate transferrin receptor amino acid sequence corresponding to NCBI sequence NP_001244232.1(transferrin receptor protein 1, Macaca mulatta) is as follows:
  • non-human primate transferrin receptor amino acid sequence corresponding to NCBI sequence XP_005545315.1 (transferrin receptor protein 1, Macaca fascicularis) is as follows:
  • DIDNEF SEQ ID NO: 107.
  • NCBI sequence NP_001344227.1 (transferrin receptor protein 1, mus musculus) is as follows:
  • NEF SEQ ID NO: 108.
  • 2’-modified nucleoside As used herein, the terms “2’-modified nucleoside” and “2’ -modified ribonucleoside” are used interchangeably and refer to a nucleoside having a sugar moiety modified at the 2’ position. In some embodiments, the 2’ -modified nucleoside is a 2’-4’ bicyclic nucleoside, where the 2’ and 4’ positions of the sugar are bridged (e.g., via a methylene, an ethylene, or a (S)-constrained ethyl bridge).
  • the 2’- modified nucleoside is a non-bicyclic 2’-modified nucleoside, e.g., where the 2’ position of the sugar moiety is substituted.
  • Non-limiting examples of 2’-modified nucleosides include: 2’- deoxy, 2’-fluoro (2’-F), 2’-0-methyl (2’-0-Me), 2’-0-methoxyethyl (2’-MOE), 2’-0- aminopropyl (2’-0-AP), 2’-0-dimethylaminoethyl (2’-0-DMAOE), 2’-0- dimethylaminopropyl (2’-0-DMAP), 2’-0-dimethylaminoethyloxyethyl (2’-0-DMAEOE), 2’- O-N-methylacetamido (2’-0-NMA), locked nucleic acid (LNA, methylene -bridged nucleic acid), ethylene-bridged nucleic acid (ENA), and (S)
  • the 2’ -modified nucleosides described herein are high-affinity modified nucleotides and oligonucleotides comprising the 2’ -modified nucleotides have increased affinity to a target sequences, relative to an unmodified oligonucleotide. Examples of structures of 2’-modified nucleosides are provided below:
  • agents binding to transferrin receptor are capable of targeting muscle cell and/or (e.g., and) mediate the transportation of an agent across the blood brain barrier.
  • Transferrin receptors are internalizing cell surface receptors that transport transferrin across the cellular membrane and participate in the regulation and homeostasis of intracellular iron levels.
  • Some aspects of the disclosure provide transferrin receptor binding proteins, which are capable of binding to transferrin receptor.
  • Antibodies that bind, e.g. specifically bind, to a transferrin receptor may be internalized into the cell, e.g. through receptor-mediated endocytosis, upon binding to a transferrin receptor.
  • humanized antibodies that bind to transferrin receptor with high specificity and affinity.
  • the humanized anti-TfR antibody described herein specifically binds to any extracellular epitope of a transferrin receptor or an epitope that becomes exposed to an antibody.
  • the humanized anti-TfR antibodies provided herein bind specifically to transferrin receptor from human, non-human primates, mouse, rat, etc.
  • the humanized anti- TfR antibodies provided herein bind to human transferrin receptor.
  • the humanized anti-TfR antibody described herein binds to an amino acid segment of a human or non-human primate transferrin receptor, as provided in SEQ ID NOs: 105-108. In some embodiments, the humanized anti-TfR antibody described herein binds to an amino acid segment corresponding to amino acids 90-96 of a human transferrin receptor as set forth in SEQ ID NO: 105, which is not in the apical domain of the transferrin receptor. In some embodiments, the humanized anti-TfR antibodies described herein binds to TfRl but does not bind to TfR2.
  • the anti-TfR antibody described herein (e.g., 3M12 and humanized variants) bind an epitope in TfRl, wherein the epitope comprises residues in amino acids 258-291 and/or amino acids 358-381 of SEQ ID NO: 105.
  • the anti-TfR antibodies (e.g., 3M12 and humanized variants) described herein bind an epitope comprising residues in amino acids amino acids 258-291 and amino acids 358-381 of SEQ ID NO: 105.
  • the anti-TfR antibodies described herein bind an epitope comprising one or more of residues K261, S273, Y282, T362, S368, S370, and K371 of human TfRl as set forth in SEQ ID NO: 105.
  • the anti-TfR antibodies described herein bind an epitope comprising residues K261, S273, Y282, T362, S368, S370, and K371 of human TfRl as set forth in SEQ ID NO: 105.
  • an anti-TFR antibody specifically binds a TfRl (e.g., a human or non-human primate TfRl) with binding affinity (e.g., as indicated by Kd) of at least about KT 4 M, 10 5 M, 10 6 M, lO 7 M, lO 8 M, 10 9 M, lO 10 M, KT 11 M, 10 12 M, 10 13 M, or less.
  • the anti-TfR antibodies described herein binds to TfRl with a KD of sub-nanomolar range.
  • the anti-TfR antibodies described herein selectively binds to transferrin receptor 1 (TfRl) but do not bind to transferrin receptor 2 (TfR2).
  • the anti-TfR antibodies described herein binds to human TfRl and cyno TfRl (e.g., with a Kd of 10 7 M, 10 8 M, 10 9 M, 10 10 M, KT 11 M, 10 12 M, lO 13 M, or less), but does not bind to a mouse TfRl.
  • the affinity and binding kinetics of the anti-TfR antibody can be tested using any suitable method including but not limited to biosensor technology (e.g., OCTET or BIACORE).
  • binding of any one of the anti-TfR antibody described herein does not complete with or inhibit transferrin binding to the TfRl. In some embodiments, binding of any one of the anti-TfR antibody described herein does not complete with or inhibit HFE-beta-2-microglobulin binding to the TfRl.
  • the anti-TfR antibodies described herein are humanized antibodies.
  • the CDR and variable region amino acid sequences of the mouse monoclonal anti-TfR antibody from which the humanized anti-TfR antibodies described herein are derived are provided in Table 2.
  • the anti-TfR antibody of the present disclosure is a humanized variant of any one of the anti-TfR antibodies provided in Table 2.
  • the anti-TfR antibody of the present disclosure comprises a CDR-H1, a CDR- H2, a CDR-H3, a CDR-L1, a CDR-L2, and a CDR-L3 that are the same as the CDR-H1, CDR- H2, and CDR-H3 in any one of the anti-TfR antibodies provided in Table 2, and comprises a humanized heavy chain variable region and/or (e.g., and) a humanized light chain variable region.
  • Humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementarity determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity.
  • CDR complementarity determining region
  • donor antibody non-human species
  • Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • the humanized antibody may comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences, but are included to further refine and optimize antibody performance.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region or domain
  • Antibodies may have Fc regions modified as described in WO 99/58572.
  • Other forms of humanized antibodies have one or more CDRs (one, two, three, four, five, six) which are altered with respect to the original antibody, which are also termed one or more CDRs derived from one or more CDRs from the original antibody. Humanized antibodies may also involve affinity maturation.
  • the humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising one or more amino acid variations (e.g., in the VH framework region) as compared with any one of the VHs listed in Table 2, and/or (e.g., and) a humanized VL comprising one or more amino acid variations (e.g., in the VL framework region) as compared with any one of the VLs listed in Table 2.
  • the humanized anti-TfR antibody of the present disclosure comprises a humanized VH containing no more than 25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) in the framework regions as compared with the VH of any of the anti-TfR antibodies listed in Table 2 (e.g., any one of SEQ ID NOs: 17, 22, 26, 43, 61, 65, and 68).
  • amino acid variations e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation
  • the humanized anti-TfR antibody of the present disclosure comprises a humanized VL containing no more than 25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) in the framework regions as compared with the VL of any one of the anti-TfR antibodies listed in Table 2 (e.g., any one of SEQ ID NOs: 18, 44, and 62).
  • the humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising an amino acid sequence that is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regions to the VH of any of the anti-TfR antibodies listed in Table 2 (e.g., any one of SEQ ID NOs: 17, 22, 26,
  • the humanized anti-TfR antibody of the present disclosure comprises a humanized VL comprising an amino acid sequence that is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regions to the VL of any of the anti-TfR antibodies listed in Table 2 (e.g., any one of SEQ ID NOs: 18, 44, and 62).
  • the humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 1 (according to the IMGT definition system), a CDR-H2 having the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 19, or SEQ ID NO: 23 (according to the IMGT definition system), a CDR-H3 having the amino acid sequence of SEQ ID NO: 3 (according to the IMGT definition system), and containing no more than 25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) in the framework regions as compared with the VH as set forth in SEQ ID NO: 17, SEQ ID NO: 22, or SEQ ID NO: 26.
  • a humanized VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 1 (according to the IMGT definition system), a CDR-
  • the anti-TfR antibody of the present disclosure comprises a humanized VL comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 4 (according to the IMGT definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 5 (according to the IMGT definition system), and a CDR-L3 having the amino acid sequence of SEQ ID NO: 6 (according to the IMGT definition system), and containing no more than 25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) in the framework regions as compared with the VL as set forth in SEQ ID NO: 18.
  • a humanized VL comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 4 (according to the IMGT definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 5 (according to the IMGT definition system), and a
  • the humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 1 (according to the IMGT definition system), a CDR-H2 having the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 19, or SEQ ID NO: 23 (according to the IMGT definition system), a CDR-H3 having the amino acid sequence of SEQ ID NO: 3 (according to the IMGT definition system), and is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regions to the VH as set forth in SEQ ID NO: 17, SEQ ID NO: 22, or SEQ ID NO: 26.
  • a humanized VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 1 (according to the IMGT definition system), a CDR-H2 having the amino acid sequence of SEQ ID NO: 2, SEQ ID NO
  • the humanized anti-TfR antibody of the present disclosure comprises a humanized VL comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 4 (according to the IMGT definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 5 (according to the IMGT definition system), and a CDR-L3 having the amino acid sequence of SEQ ID NO: 6 (according to the IMGT definition system), and is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regions to the VL as set forth in any one of SEQ ID NO: 18.
  • a humanized VL comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 4 (according to the IMGT definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 5 (according to the IMGT definition system), and a CDR-L3 having the amino acid sequence of SEQ
  • the humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 7 (according to the Rabat definition system), a CDR-H2 having the amino acid sequence of SEQ ID NO: 8, SEQ ID NO: 20, or SEQ ID NO: 24 (according to the Rabat definition system), a CDR-H3 having the amino acid sequence of SEQ ID NO: 9 (according to the Rabat definition system), and containing no more than 25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) in the framework regions as compared with the VH as set forth in SEQ ID NO: 17, SEQ ID NO: 22, or SEQ ID NO: 26.
  • a humanized VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 7 (according to the Rabat definition system), a CDR-H2 having the
  • the humanized anti-TfR antibody of the present disclosure comprises a humanized VL comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 10 (according to the Rabat definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 11 (according to the Rabat definition system), and a CDR-L3 having the amino acid sequence of SEQ ID NO: 6 (according to the Rabat definition system), and containing no more than 25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) in the framework regions as compared with the VL as set forth in SEQ ID NO: 18.
  • a humanized VL comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 10 (according to the Rabat definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 11 (according to the Rabat definition system), and a CDR-
  • the humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 7 (according to the Kabat definition system), a CDR-H2 having the amino acid sequence of SEQ ID NO: 8, SEQ ID NO: 20, or SEQ ID NO: 24 (according to the Kabat definition system), a CDR-H3 having the amino acid sequence of SEQ ID NO: 9 (according to the Kabat definition system), and is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regions to the VH as set forth in SEQ ID NO: 17, SEQ ID NO: 22, or SEQ ID NO: 26.
  • a humanized VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 7 (according to the Kabat definition system), a CDR-H2 having the amino acid sequence of SEQ ID NO: 8, SEQ ID NO: 20, or S
  • the humanized anti-TfR antibody of the present disclosure comprises a humanized VL comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 10 (according to the Kabat definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 11 (according to the Kabat definition system), and a CDR-L3 having the amino acid sequence of SEQ ID NO: 6 (according to the Kabat definition system), and is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regions to the VL as set forth in any one of SEQ ID NO: 18.
  • a humanized VL comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 10 (according to the Kabat definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 11 (according to the Kabat definition system), and a CDR-L3 having the amino acid sequence of SEQ ID NO: 6 (
  • the humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 12 (according to the Chothia definition system), a CDR-H2 having the amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 21, or SEQ ID NO: 25 (according to the Chothia definition system), a CDR-H3 having the amino acid sequence of SEQ ID NO: 14 (according to the Chothia definition system), and containing no more than 25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) in the framework regions as compared with the VH as set forth in SEQ ID NO: 17, SEQ ID NO: 22 or SEQ ID NO: 26.
  • a humanized VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 12 (according to the Chothia definition system), a CDR-
  • the humanized anti-TfR antibody of the present disclosure comprises a humanized VL comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 15 (according to the Chothia definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 5 (according to the Chothia definition system), and a CDR-L3 having the amino acid sequence of SEQ ID NO: 16 (according to the Chothia definition system), and containing no more than 25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) in the framework regions as compared with the VL as set forth in SEQ ID NO: 18.
  • a humanized VL comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 15 (according to the Chothia definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 5 (according to the Chothia definition system), and
  • the humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 12 (according to the Chothia definition system), a CDR-H2 having the amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 21, or SEQ ID NO: 25 (according to the Chothia definition system), a CDR-H3 having the amino acid sequence of SEQ ID NO: 14 (according to the Chothia definition system), and is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regions to the VH as set forth in SEQ ID NO: SEQ ID NO: 17, SEQ ID NO: 22 or SEQ ID NO: 26.
  • a humanized VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 12 (according to the Chothia definition system), a CDR-H2 having the amino acid sequence of SEQ ID NO:
  • the anti-TfR antibody of the present disclosure comprises a humanized VL comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 15 (according to the Chothia definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 5 (according to the Chothia definition system), and a CDR-L3 having the amino acid sequence of SEQ ID NO: 16 (according to the Chothia definition system), and is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regions to the VL as set forth in any one of SEQ ID NO: 18.
  • a humanized VL comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 15 (according to the Chothia definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 5 (according to the Chothia definition system), and a CDR-L3 having the amino acid sequence of SEQ ID NO
  • the humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 27 (according to the IMGT definition system), a CDR-H2 having the amino acid sequence of SEQ ID NO: 28 (according to the IMGT definition system), a CDR-H3 having the amino acid sequence of SEQ ID NO: 29 (according to the IMGT definition system), and containing no more than 25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21,
  • the humanized anti-TfR antibody of the present disclosure comprises a humanized VL comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 30 (according to the IMGT definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 31 (according to the IMGT definition system), and a CDR-L3 having the amino acid sequence of SEQ ID NO: 32 (according to the IMGT definition system), and containing no more than 25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) in the framework regions as compared with the VL as set forth in SEQ ID NO: 44.
  • a humanized VL comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 30 (according to the IMGT definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 31 (according to the IMGT definition system),
  • the humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 27 (according to the IMGT definition system), a CDR-H2 having the amino acid sequence of SEQ ID NO: 28 (according to the IMGT definition system), a CDR-H3 having the amino acid sequence of SEQ ID NO: 29 (according to the IMGT definition system), and is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regions to the VH as set forth in SEQ ID NO: 43.
  • a humanized VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 27 (according to the IMGT definition system), a CDR-H2 having the amino acid sequence of SEQ ID NO: 28 (according to the IMGT definition system), a CDR-H3 having the amino acid sequence of SEQ ID NO: 29
  • the humanized anti-TfR antibody of the present disclosure comprises a humanized VL comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 30 (according to the IMGT definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 31 (according to the IMGT definition system), and a CDR-L3 having the amino acid sequence of SEQ ID NO: 32 (according to the IMGT definition system), and is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regions to the VL as set forth in SEQ ID NO: 44.
  • a humanized VL comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 30 (according to the IMGT definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 31 (according to the IMGT definition system), and a CDR-L3 having the amino acid sequence of SEQ ID NO
  • the humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 33 (according to the Rabat definition system), a CDR-H2 having the amino acid sequence of SEQ ID NO: 34 (according to the Rabat definition system), a CDR-H3 having the amino acid sequence of SEQ ID NO: 35 (according to the Rabat definition system), and containing no more than 25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21,
  • the humanized anti-TfR antibody of the present disclosure comprises a humanized VL comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 36 (according to the Rabat definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 37 (according to the Rabat definition system), and a CDR-L3 having the amino acid sequence of SEQ ID NO: 32 (according to the Rabat definition system), and containing no more than 25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) in the framework regions as compared with the VL as set forth in SEQ ID NO: 44.
  • a humanized VL comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 36 (according to the Rabat definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 37 (according to the Rabat definition system), and a CDR
  • the humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 33 (according to the Rabat definition system), a CDR-H2 having the amino acid sequence of SEQ ID NO: 34 (according to the Rabat definition system), a CDR-H3 having the amino acid sequence of SEQ ID NO: 35 (according to the Rabat definition system), and is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regions to the VH as set forth in SEQ ID NO: 43.
  • the humanized anti-TfR antibody of the present disclosure comprises a humanized VL comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 36 (according to the Kabat definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 37 (according to the Kabat definition system), and a CDR-L3 having the amino acid sequence of SEQ ID NO: 32 (according to the Kabat definition system), and is at least 75% (e.g., 75%,
  • the humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 38 (according to the Chothia definition system), a CDR-H2 having the amino acid sequence of SEQ ID NO: 39 (according to the Chothia definition system), a CDR-H3 having the amino acid sequence of SEQ ID NO: 40 (according to the Chothia definition system), and containing no more than 25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) in the framework regions as compared with the VH as set forth in SEQ ID NO: 43.
  • a humanized VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 38 (according to the Chothia definition system), a CDR-H2 having the amino acid sequence of SEQ ID NO: 39 (according to the Chothia definition system),
  • the humanized anti-TfR antibody of the present disclosure comprises a humanized VL comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 41 (according to the Chothia definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 31 (according to the Chothia definition system), and a CDR-L3 having the amino acid sequence of SEQ ID NO: 42 (according to the Chothia definition system), and containing no more than 25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) in the framework regions as compared with the VL as set forth in SEQ ID NO: 44.
  • a humanized VL comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 41 (according to the Chothia definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 31 (according to the Chothia definition system),
  • the humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 38 (according to the Chothia definition system), a CDR-H2 having the amino acid sequence of SEQ ID NO: 39 (according to the Chothia definition system), a CDR-H3 having the amino acid sequence of SEQ ID NO: 40 (according to the Chothia definition system), and is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regions to the VH as set forth in SEQ ID NO: 43.
  • a humanized VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 38 (according to the Chothia definition system), a CDR-H2 having the amino acid sequence of SEQ ID NO: 39 (according to the Chothia definition system), a CDR-H3 having the amino acid sequence of SEQ ID NO: 40
  • the humanized anti-TfR antibody of the present disclosure comprises a humanized VL comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 41 (according to the Chothia definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 31 (according to the Chothia definition system), and a CDR-L3 having the amino acid sequence of SEQ ID NO: 42 (according to the Chothia definition system), and is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regions to the VL as set forth in SEQ ID NO: 44.
  • a humanized VL comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 41 (according to the Chothia definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 31 (according to the Chothia definition system), and a CDR-L3 having the amino acid sequence of SEQ ID NO
  • the humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 45, SEQ ID NO: 63, or SEQ ID NO: 66 (according to the IMGT definition system), a CDR-H2 having the amino acid sequence of SEQ ID NO: 46 (according to the IMGT definition system), a CDR-H3 having the amino acid sequence of SEQ ID NO: 47 (according to the IMGT definition system), and containing no more than 25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) in the framework regions as compared with the VH as set forth in SEQ ID NO: 61, SEQ ID NO: 65, or SEQ ID NO: 68.
  • a humanized VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 45, SEQ ID NO: 63
  • the humanized anti-TfR antibody of the present disclosure comprises a humanized VL comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 48 (according to the IMGT definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 49 (according to the IMGT definition system), and a CDR-L3 having the amino acid sequence of SEQ ID NO: 50 (according to the IMGT definition system), and containing no more than 25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) in the framework regions as compared with the VL as set forth in SEQ ID NO: 62.
  • a humanized VL comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 48 (according to the IMGT definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 49 (according to the IMGT definition system
  • the humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 45, SEQ ID NO: 63, or SEQ ID NO: 66 (according to the IMGT definition system), a CDR-H2 having the amino acid sequence of SEQ ID NO: 46 (according to the IMGT definition system), a CDR-H3 having the amino acid sequence of SEQ ID NO: 47 (according to the IMGT definition system), and is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regions to the VH as set forth in SEQ ID NO: 61, SEQ ID NO: 65, SEQ ID NO: 68.
  • a humanized VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 45, SEQ ID NO: 63, or SEQ ID NO: 66 (according to the IMGT definition system
  • the humanized anti-TfR antibody of the present disclosure comprises a humanized VL comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 48 (according to the IMGT definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 49 (according to the IMGT definition system), and a CDR-L3 having the amino acid sequence of SEQ ID NO: 50 (according to the IMGT definition system), and is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regions to the VL as set forth in SEQ ID NO: 62.
  • a humanized VL comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 48 (according to the IMGT definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 49 (according to the IMGT definition system), and a CDR-L3 having the amino acid sequence of SEQ ID
  • the humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 51, SEQ ID NO: 64, or SEQ ID NO: 67 (according to the Rabat definition system), a CDR-H2 having the amino acid sequence of SEQ ID NO: 52 (according to the Rabat definition system), a CDR-H3 having the amino acid sequence of SEQ ID NO: 53 (according to the Rabat definition system), and containing no more than 25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) in the framework regions as compared with the VH as set forth in SEQ ID NO: 61, SEQ ID NO: 65, SEQ ID NO: 68.
  • a humanized VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 51, SEQ ID NO: 64, or SEQ ID NO
  • the humanized anti-TfR antibody of the present disclosure comprises a humanized VL comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 54 (according to the Rabat definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 55 (according to the Rabat definition system), and a CDR-L3 having the amino acid sequence of SEQ ID NO: 50 (according to the Rabat definition system), and containing no more than 25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) in the framework regions as compared with the VL as set forth in SEQ ID NO: 62.
  • a humanized VL comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 54 (according to the Rabat definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 55 (according to the Rabat definition system), and a C
  • the humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 51, SEQ ID NO: 64, or SEQ ID NO: 67 (according to the Rabat definition system), a CDR-H2 having the amino acid sequence of SEQ ID NO: 52 (according to the Rabat definition system), a CDR-H3 having the amino acid sequence of SEQ ID NO: 53 (according to the Rabat definition system), and is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regions to the VH as set forth in SEQ ID NO: 61, SEQ ID NO: 65, SEQ ID NO: 68.
  • a humanized VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 51, SEQ ID NO: 64, or SEQ ID NO: 67 (according to the Rabat definition system), a CDR-
  • the humanized anti-TfR antibody of the present disclosure comprises a humanized VL comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 54 (according to the Rabat definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 55 (according to the Rabat definition system), and a CDR-L3 having the amino acid sequence of SEQ ID NO: 50 (according to the Rabat definition system), and is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regions to the VL as set forth in SEQ ID NO: 62.
  • a humanized VL comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 54 (according to the Rabat definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 55 (according to the Rabat definition system), and a CDR-L3 having the amino acid sequence of SEQ ID NO: 50 (accord
  • the humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 56 (according to the Chothia definition system), a CDR-H2 having the amino acid sequence of SEQ ID NO: 57 (according to the Chothia definition system), a CDR-H3 having the amino acid sequence of SEQ ID NO: 58 (according to the Chothia definition system), and containing no more than 25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) in the framework regions as compared with the VH as set forth in SEQ ID NO: 61, SEQ ID NO: 65, SEQ ID NO: 68.
  • a humanized VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 56 (according to the Chothia definition system), a CDR-H2 having the amino acid sequence of
  • the humanized anti- TfR antibody of the present disclosure comprises a humanized VL comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 59 (according to the Chothia definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 49 (according to the Chothia definition system), and a CDR-L3 having the amino acid sequence of SEQ ID NO: 60 (according to the Chothia definition system), and containing no more than 25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) in the framework regions as compared with the VL as set forth in SEQ ID NO: 62.
  • a humanized VL comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 59 (according to the Chothia definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 49 (according to the Chothia
  • the humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 56 (according to the Chothia definition system), a CDR-H2 having the amino acid sequence of SEQ ID NO: 57 (according to the Chothia definition system), a CDR-H3 having the amino acid sequence of SEQ ID NO: 58 (according to the Chothia definition system), and is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regions to the VH as set forth in SEQ ID NO: 61, SEQ ID NO: 65, SEQ ID NO: 68.
  • a humanized VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 56 (according to the Chothia definition system), a CDR-H2 having the amino acid sequence of SEQ ID NO: 57 (according to the Chothia definition system),
  • the humanized anti-TfR antibody of the present disclosure comprises a humanized VL comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 59 (according to the Chothia definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 49 (according to the Chothia definition system), and a CDR-L3 having the amino acid sequence of SEQ ID NO: 60 (according to the Chothia definition system), and is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regions to the VL as set forth in SEQ ID NO: 62.
  • a humanized VL comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 59 (according to the Chothia definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 49 (according to the Chothia definition system), and a CDR-L3 having the amino acid sequence of S
  • the humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising the CDR-H1, CDR-H2, and CDR-H3 of any one of the anti-TfR antibodies provided in Table 2 and comprises one or more (e.g., 1, 2, 3, 4,
  • the humanized anti-TfR antibody of the present disclosure comprises a humanized VL comprising the CDR-L1, CDR-L2, and CDR-L3 of any one of the anti-TfR antibodies provided in Table 2 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) amino acid variations in the framework regions as compared with the respective humanized VL provided in Table 3.
  • the humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 69, and/or (e.g., and) a humanized VL comprising an amino acid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) to SEQ ID NO: 70.
  • the humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising the amino acid sequence of SEQ ID NO: 69 and a humanized VL comprising the amino acid sequence of SEQ ID NO: 70.
  • the humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 71, and/or (e.g., and) a humanized VL comprising an amino acid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) to SEQ ID NO: 70.
  • the humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising the amino acid sequence of SEQ ID NO: 71 and a humanized VL comprising the amino acid sequence of SEQ ID NO:
  • the humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 72, and/or (e.g., and) a humanized VL comprising an amino acid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) to SEQ ID NO: 70.
  • the humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising the amino acid sequence of SEQ ID NO: 72 and a humanized VL comprising the amino acid sequence of SEQ ID NO: 70.
  • the humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 73, and/or (e.g., and) a humanized VL comprising an amino acid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) to SEQ ID NO: 74.
  • the humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising the amino acid sequence of SEQ ID NO: 73 and a humanized VL comprising the amino acid sequence of SEQ ID NO: 74.
  • the humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 73, and/or (e.g., and) a humanized VL comprising an amino acid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) to SEQ ID NO: 75.
  • the humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising the amino acid sequence of SEQ ID NO: 73 and a humanized VL comprising the amino acid sequence of SEQ ID NO: 75.
  • the humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 76, and/or (e.g., and) a humanized VL comprising an amino acid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) to SEQ ID NO: 74.
  • the humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising the amino acid sequence of SEQ ID NO: 76 and a humanized VL comprising the amino acid sequence of SEQ ID NO: 74.
  • the humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 76, and/or (e.g., and) a humanized VL comprising an amino acid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) to SEQ ID NO: 75.
  • the humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising the amino acid sequence of SEQ ID NO: 76 and a humanized VL comprising the amino acid sequence of SEQ ID NO: 75.
  • the humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 77, and/or (e.g., and) a humanized VL comprising an amino acid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) to SEQ ID NO: 78.
  • the humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising the amino acid sequence of SEQ ID NO: 77 and a humanized VL comprising the amino acid sequence of SEQ ID NO: 78.
  • the humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 79, and/or (e.g., and) a humanized VL comprising an amino acid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) to SEQ ID NO: 80.
  • the humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising the amino acid sequence of SEQ ID NO: 79 and a humanized VL comprising the amino acid sequence of SEQ ID NO: 80.
  • the humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 77, and/or (e.g., and) a humanized VL comprising an amino acid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) to SEQ ID NO: 80.
  • the humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising the amino acid sequence of SEQ ID NO: 77 and a humanized VL comprising the amino acid sequence of SEQ ID NO: 80.
  • the humanized anti-TfR antibody described herein is a full-length IgG, which can include a heavy constant region and a light constant region from a human antibody.
  • the heavy chain of any of the anti-TfR antibodies as described herein may comprises a heavy chain constant region (CH) or a portion thereof (e.g., CHI, CH2, CH3, or a combination thereof).
  • the heavy chain constant region can of any suitable origin, e.g., human, mouse, rat, or rabbit.
  • the heavy chain constant region is from a human IgG (a gamma heavy chain), e.g., IgGl, IgG2, or IgG4.
  • An example of a human IgGl constant region is given below:
  • the heavy chain of any of the anti-TfR antibodies described herein comprises a mutant human IgGl constant region.
  • LALA mutations a mutant derived from mAb bl2 that has been mutated to replace the lower hinge residues Leu234 Leu235 with Ala234 and Ala235
  • the mutant human IgGl constant region is provided below (mutations bonded and underlined):
  • the light chain of any of the anti-TfR antibodies described herein may further comprise a light chain constant region (CL), which can be any CL known in the art.
  • CL is a kappa light chain.
  • the CL is a lambda light chain.
  • the CL is a kappa light chain, the sequence of which is provided below:
  • the humanized anti-TfR antibody described herein comprises a heavy chain comprising any one of the VH as listed in Table 3 or any variants thereof and a heavy chain constant region that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 81 or SEQ ID NO: 82.
  • the humanized anti-TfR antibody described herein comprises a heavy chain comprising any one of the VH as listed in Table 3 or any variants thereof and a heavy chain constant region that contains no more than 25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) as compared with SEQ ID NO: 81 or SEQ ID NO: 82.
  • the humanized anti-TfR antibody described herein comprises a heavy chain comprising any one of the VH as listed in Table 3 or any variants thereof and a heavy chain constant region as set forth in SEQ ID NO: 81.
  • the humanized anti-TfR antibody described herein comprises heavy chain comprising any one of the VH as listed in Table 3 or any variants thereof and a heavy chain constant region as set forth in SEQ ID NO: 82.
  • the humanized anti-TfR antibody described herein comprises a light chain comprising any one of the VL as listed in Table 3 or any variants thereof and a light chain constant region that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 83.
  • the humanized anti- TfR antibody described herein comprises a light chain comprising any one of the VL as listed in Table 3 or any variants thereof and a light chain constant region contains no more than 25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) as compared with SEQ ID NO: 83.
  • the humanized anti-TfR antibody described herein comprises a light chain comprising any one of the VL as listed in Table 3 or any variants thereof and a light chain constant region set forth in SEQ ID NO: 83.
  • Examples of IgG heavy chain and light chain amino acid sequences of the anti- TfR antibodies described are provided in Table 4 below.
  • the humanized anti-TfR antibody of the present disclosure comprises a heavy chain containing no more than 25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) as compared with the heavy chain as set forth in any one of SEQ ID NOs: 84, 86, 87, 88, 91, 92, and 94.
  • the humanized anti-TfR antibody of the present disclosure comprises a light chain containing no more than 25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16,
  • the humanized anti-TfR antibody described herein comprises a heavy chain comprising an amino acid sequence that is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to any one of SEQ ID NOs: 84, 86, 87, 88, 91, 92, and 94.
  • the humanized anti-TfR antibody described herein comprises a light chain comprising an amino acid sequence that is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to any one of SEQ ID NOs: 85, 89, 90, 93, and 95.
  • the anti-TfR antibody described herein comprises a heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 84, 86, 87, 88,
  • the anti-TfR antibody described herein comprises a light chain comprising the amino acid sequence of any one of SEQ ID NOs: 85, 89, 90, 93, and 95.
  • the humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 84, and/or (e.g., and) a light chain comprising an amino acid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) to SEQ ID NO: 85.
  • the humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 84 and a light chain comprising the amino acid sequence of SEQ ID NO: 85.
  • the humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 86, and/or (e.g., and) a light chain comprising an amino acid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) to SEQ ID NO: 85.
  • the humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 86 and a light chain comprising the amino acid sequence of SEQ ID NO: 85.
  • the humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 87, and/or (e.g., and) a light chain comprising an amino acid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) to SEQ ID NO: 85.
  • the humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 87 and a light chain comprising the amino acid sequence of SEQ ID NO: 85.
  • the humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 88, and/or (e.g., and) a light chain comprising an amino acid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) to SEQ ID NO: 89.
  • the humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 88 and a light chain comprising the amino acid sequence of SEQ ID NO: 89.
  • the humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 88, and/or (e.g., and) a light chain comprising an amino acid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) to SEQ ID NO: 90.
  • the humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 88 and a light chain comprising the amino acid sequence of SEQ ID NO: 90.
  • the humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 91, and/or (e.g., and) a light chain comprising an amino acid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) to SEQ ID NO: 89.
  • the humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 91 and a light chain comprising the amino acid sequence of SEQ ID NO: 89.
  • the humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 91, and/or (e.g., and) a light chain comprising an amino acid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) to SEQ ID NO: 90.
  • the humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 91 and a light chain comprising the amino acid sequence of SEQ ID NO: 90.
  • the humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 92, and/or (e.g., and) a light chain comprising an amino acid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) to SEQ ID NO: 93.
  • the humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 92 and a light chain comprising the amino acid sequence of SEQ ID NO: 93.
  • the humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 94, and/or (e.g., and) a light chain comprising an amino acid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) to SEQ ID NO: 95.
  • the humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 94 and a light chain comprising the amino acid sequence of SEQ ID NO: 95.
  • the humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 92, and/or (e.g., and) a light chain comprising an amino acid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) to SEQ ID NO: 95.
  • the humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 92 and a light chain comprising the amino acid sequence of SEQ ID NO: 95.
  • the anti-TfR antibody is a Fab fragment, Fab’ fragment, or F(ab')2 fragment of an intact antibody (full-length antibody).
  • Antigen binding fragment of an intact antibody (full-length antibody) can be prepared via routine methods (e.g., recombinantly or by digesting the heavy chain constant region of a full length IgG using an enzyme such as papain).
  • F(ab')2 fragments can be produced by pepsin or papain digestion of an antibody molecule, and Fab fragments that can be generated by reducing the disulfide bridges of F(ab')2 fragments.
  • a heavy chain constant region in a Fab fragment of the anti-TfRl antibody described herein comprises the amino acid sequence of:
  • the humanized anti-TfR antibody described herein comprises a heavy chain comprising any one of the VH as listed in Table 3 or any variants thereof and a heavy chain constant region that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 96.
  • the humanized anti-TfR antibody described herein comprises a heavy chain comprising any one of the VH as listed in Table 3 or any variants thereof and a heavy chain constant region that contains no more than 25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16,
  • the humanized anti-TfR antibody described herein comprises a heavy chain comprising any one of the VH as listed in Table 3 or any variants thereof and a heavy chain constant region as set forth in SEQ ID NO: 96.
  • the humanized anti-TfR antibody described herein comprises a light chain comprising any one of the VL as listed in Table 3 or any variants thereof and a light chain constant region that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 83.
  • the humanized anti- TfR antibody described herein comprises a light chain comprising any one of the VL as listed in Table 3 or any variants thereof and a light chain constant region contains no more than 25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) as compared with SEQ ID NO: 83.
  • the humanized anti-TfR antibody described herein comprises a light chain comprising any one of the VL as listed in Table 3 or any variants thereof and a light chain constant region set forth in SEQ ID NO: 83.
  • the humanized anti-TfR antibody of the present disclosure comprises a heavy chain containing no more than 25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) as compared with the heavy chain as set forth in any one of SEQ ID NOs: 97-103.
  • amino acid variations e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation
  • the humanized anti-TfR antibody of the present disclosure comprises a light chain containing no more than 25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) as compared with the light chain as set forth in any one of SEQ ID NOs: 85, 89, 90, 93, and 95.
  • 25 amino acid variations e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation
  • the humanized anti-TfR antibody described herein comprises a heavy chain comprising an amino acid sequence that is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to any one of SEQ ID NOs: 97-103.
  • the humanized anti-TfR antibody described herein comprises a light chain comprising an amino acid sequence that is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to any one of SEQ ID NOs: 85, 89, 90,
  • the anti-TfR antibody described herein comprises a heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 97-103.
  • the anti-TfR antibody described herein comprises a light chain comprising the amino acid sequence of any one of SEQ ID NOs: 85, 89, 90, 93, and 95.
  • the humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 97, and/or (e.g., and) a light chain comprising an amino acid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) to SEQ ID NO: 85.
  • the humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 97 and a light chain comprising the amino acid sequence of SEQ ID NO: 85.
  • the humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 98, and/or (e.g., and) a light chain comprising an amino acid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) to SEQ ID NO: 85.
  • the humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 98 and a light chain comprising the amino acid sequence of SEQ ID NO: 85.
  • the humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 99, and/or (e.g., and) a light chain comprising an amino acid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) to SEQ ID NO: 85.
  • the humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 99 and a light chain comprising the amino acid sequence of SEQ ID NO: 85.
  • the humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 100, and/or (e.g., and) a light chain comprising an amino acid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) to SEQ ID NO: 89.
  • the humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 100 and a light chain comprising the amino acid sequence of SEQ ID NO: 89.
  • the humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 100, and/or (e.g., and) a light chain comprising an amino acid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) to SEQ ID NO: 90.
  • the humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 100 and a light chain comprising the amino acid sequence of SEQ ID NO: 90.
  • the humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 101, and/or (e.g., and) a light chain comprising an amino acid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) to SEQ ID NO: 89.
  • the humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 101 and a light chain comprising the amino acid sequence of SEQ ID NO: 89.
  • the humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 101, and/or (e.g., and) a light chain comprising an amino acid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) to SEQ ID NO: 90.
  • the humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 101 and a light chain comprising the amino acid sequence of SEQ ID NO: 90.
  • the humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 102, and/or (e.g., and) a light chain comprising an amino acid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) to SEQ ID NO: 93.
  • the humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 102 and a light chain comprising the amino acid sequence of SEQ ID NO: 93.
  • the humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 103, and/or (e.g., and) a light chain comprising an amino acid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) to SEQ ID NO: 95.
  • the humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 103 and a light chain comprising the amino acid sequence of SEQ ID NO: 95.
  • the humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 102, and/or (e.g., and) a light chain comprising an amino acid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) to SEQ ID NO: 95.
  • the humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 102 and a light chain comprising the amino acid sequence of SEQ ID NO: 95.
  • the humanized anti-TfR receptor antibodies described herein can be in any antibody form, including, but not limited to, intact (i.e., full-length) antibodies, antigen-binding fragments thereof (such as Fab, Fab’, F(ab')2, Fv), single chain antibodies, bi-specific antibodies, or nanobodies.
  • humanized the anti- TfR antibody described herein is a scFv.
  • the humanized anti-TfR antibody described herein is a scFv-Fab (e.g., scFv fused to a portion of a constant region).
  • the anti-TfR receptor antibody described herein is a scFv fused to a constant region (e.g., human IgGl constant region as set forth in SEQ ID NO: 81 or SEQ ID NO: 82, or a portion thereof such as the Fc portion) at either the N-terminus of C-terminus.
  • a constant region e.g., human IgGl constant region as set forth in SEQ ID NO: 81 or SEQ ID NO: 82, or a portion thereof such as the Fc portion
  • conservative mutations can be introduced into antibody sequences (e.g., CDRs or framework sequences) at positions where the residues are not likely to be involved in interacting with a target antigen (e.g., transferrin receptor), for example, as determined based on a crystal structure.
  • one, two or more mutations are introduced into the Fc region of an anti-TfR antibody described herein (e.g., in a CH2 domain (residues 231-340 of human IgGl) and/or (e.g., and) CH3 domain (residues 341-447 of human IgGl) and/or (e.g., and) the hinge region, with numbering according to the Kabat numbering system (e.g., the EU index in Kabat)) to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding and/or (e.g., and) antigen-dependent cellular cytotoxicity.
  • Kabat numbering system e.g., the EU index in Kabat
  • one, two or more mutations are introduced into the hinge region of the Fc region (CHI domain) such that the number of cysteine residues in the hinge region are altered (e.g., increased or decreased) as described in, e.g., U.S. Pat. No. 5,677,425.
  • the number of cysteine residues in the hinge region of the CHI domain can be altered to, e.g., facilitate assembly of the light and heavy chains, or to alter (e.g., increase or decrease) the stability of the antibody or to facilitate linker conjugation.
  • one, two or more mutations are introduced into the Fc region of a muscle-targeting antibody described herein (e.g., in a CH2 domain (residues 231-340 of human IgGl) and/or (e.g., and) CH3 domain (residues 341-447 of human IgGl) and/or (e.g., and) the hinge region, with numbering according to the Kabat numbering system (e.g., the EU index in Kabat)) to increase or decrease the affinity of the antibody for an Fc receptor (e.g., an activated Fc receptor) on the surface of an effector cell.
  • an Fc receptor e.g., an activated Fc receptor
  • Mutations in the Fc region of an antibody that decrease or increase the affinity of an antibody for an Fc receptor and techniques for introducing such mutations into the Fc receptor or fragment thereof are known to one of skill in the art. Examples of mutations in the Fc receptor of an antibody that can be made to alter the affinity of the antibody for an Fc receptor are described in, e.g., Smith P et ah, (2012) PNAS 109: 6181-6186, U.S. Pat. No. 6,737,056, and International Publication Nos. WO 02/060919; WO 98/23289; and WO 97/34631, which are incorporated herein by reference.
  • one, two or more amino acid mutations are introduced into an IgG constant domain, or FcRn- binding fragment thereof (preferably an Fc or hinge-Fc domain fragment) to alter (e.g., decrease or increase) half-life of the antibody in vivo.
  • an IgG constant domain, or FcRn- binding fragment thereof preferably an Fc or hinge-Fc domain fragment
  • one, two or more amino acid mutations are introduced into an IgG constant domain, or FcRn- binding fragment thereof (preferably an Fc or hinge-Fc domain fragment) to decrease the half- life of the anti-anti-TfR antibody in vivo.
  • one, two or more amino acid mutations are introduced into an IgG constant domain, or FcRn-binding fragment thereof (preferably an Fc or hinge-Fc domain fragment) to increase the half-life of the antibody in vivo.
  • the antibodies can have one or more amino acid mutations (e.g., substitutions) in the second constant (CH2) domain (residues 231-340 of human IgGl) and/or (e.g., and) the third constant (CH3) domain (residues 341-447 of human IgGl), with numbering according to the EU index in Kabat (Kabat E A et ah, (1991) supra).
  • the constant region of the IgGl of an antibody described herein comprises a methionine (M) to tyrosine (Y) substitution in position 252, a serine (S) to threonine (T) substitution in position 254, and a threonine (T) to glutamic acid (E) substitution in position 256, numbered according to the EU index as in Kabat. See U.S. Pat.
  • an antibody comprises an IgG constant domain comprising one, two, three or more amino acid substitutions of amino acid residues at positions 251-257, 285-290, 308-314, 385-389, and 428-436, numbered according to the EU index as in Kabat.
  • one, two or more amino acid substitutions are introduced into an IgG constant domain Fc region to alter the effector function(s) of the anti-anti-TfR antibody.
  • the effector ligand to which affinity is altered can be, for example, an Fc receptor or the Cl component of complement. This approach is described in further detail in U.S. Pat. Nos. 5,624,821 and 5,648,260.
  • the deletion or inactivation (through point mutations or other means) of a constant region domain can reduce Fc receptor binding of the circulating antibody thereby increasing tumor localization. See, e.g., U.S. Pat. Nos.
  • one or more amino acid substitutions may be introduced into the Fc region of an antibody described herein to remove potential glycosylation sites on Fc region, which may reduce Fc receptor binding (see, e.g., Shields R F et al., (2001) J Biol Chem 276: 6591-604).
  • one or more amino in the constant region of an anti-TfR antibody described herein can be replaced with a different amino acid residue such that the antibody has altered Clq binding and/or (e.g., and) reduced or abolished complement dependent cytotoxicity (CDC).
  • CDC complement dependent cytotoxicity
  • one or more amino acid residues in the N- terminal region of the CH2 domain of an antibody described herein are altered to thereby alter the ability of the antibody to fix complement. This approach is described further in International Publication No. WO 94/29351.
  • the Fc region of an antibody described herein is modified to increase the ability of the antibody to mediate antibody dependent cellular cytotoxicity (ADCC) and/or (e.g., and) to increase the affinity of the antibody for an Fey receptor.
  • ADCC antibody dependent cellular cytotoxicity
  • the heavy and/or (e.g., and) light chain variable domain(s) sequence(s) of the antibodies provided herein can be used to generate, for example, CDR-grafted, chimeric, humanized, or composite human antibodies or antigen-binding fragments, as described elsewhere herein.
  • any variant, CDR-grafted, chimeric, humanized, or composite antibodies derived from any of the antibodies provided herein may be useful in the compositions and methods described herein and will maintain the ability to specifically bind transferrin receptor, such that the variant, CDR-grafted, chimeric, humanized, or composite antibody has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or more binding to transferrin receptor relative to the original antibody from which it is derived.
  • the antibodies provided herein comprise mutations that confer desirable properties to the antibodies.
  • the antibodies provided herein may comprise a stabilizing ‘Adair’ mutation (Angal S., et al., “A single amino acid substitution abolishes the heterogeneity of chimeric mouse/human (IgG4) antibody,” Mol Immunol 30, 105-108; 1993), where serine 228 (EU numbering; residue 241 Kabat numbering) is converted to proline resulting in an IgGl-like hinge sequence.
  • any of the antibodies may include a stabilizing ‘Adair’ mutation.
  • an antibody is modified, e.g., modified via glycosylation, phosphorylation, sumoylation, and/or (e.g., and) methylation.
  • an antibody is a glycosylated antibody, which is conjugated to one or more sugar or carbohydrate molecules.
  • the one or more sugar or carbohydrate molecule are conjugated to the antibody via N-glycosylation, O-glycosylation, C-glycosylation, glypiation (GPI anchor attachment), and/or (e.g., and) phosphoglycosylation.
  • the one or more sugar or carbohydrate molecules are monosaccharides, disaccharides, oligosaccharides, or glycans. In some embodiments, the one or more sugar or carbohydrate molecule is a branched oligosaccharide or a branched glycan. In some embodiments, the one or more sugar or carbohydrate molecule includes a mannose unit, a glucose unit, an N- acetylglucosamine unit, an N-acetylgalactosamine unit, a galactose unit, a fucose unit, or a phospholipid unit.
  • a glycosylated antibody is fully or partially glycosylated.
  • an antibody is glycosylated by chemical reactions or by enzymatic means.
  • an antibody is glycosylated in vitro or inside a cell, which may optionally be deficient in an enzyme in the N- or O- glycosylation pathway, e.g. a glycosyltransferase.
  • an antibody is functionalized with sugar or carbohydrate molecules as described in International Patent Application Publication WO2014065661, published on May 1, 2014, entitled, “ Modified antibody, antibody-conjugate and process for the preparation thereof ’.
  • any one of the anti-TfRl antibodies described herein may comprise a signal peptide in the heavy and/or (e.g., and) light chain sequence (e.g., a N- terminal signal peptide).
  • the anti-TfRl antibody described herein comprises any one of the VH and VL sequences, any one of the IgG heavy chain and light chain sequences, or any one of the Fab heavy chain and light chain sequences described herein, and further comprises a signal peptide (e.g., a N-terminal signal peptide).
  • the signal peptide comprises the amino acid sequence of MGW S CIILFLV AT AT G VHS (SEQ ID NO: 104).
  • Antibodies capable of binding TfR as described herein can be made by any method known in the art. See, for example, Harlow and Lane, (1998) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York.
  • antibodies specific to a target antigen can be made by the conventional hybridoma technology.
  • the full-length target antigen or a fragment thereof, optionally coupled to a carrier protein such as KLH, can be used to immunize a host animal for generating antibodies binding to that antigen.
  • the route and schedule of immunization of the host animal are generally in keeping with established and conventional techniques for antibody stimulation and production, as further described herein.
  • General techniques for production of mouse, humanized, and human antibodies are known in the art and are described herein. It is contemplated that any mammalian subject including humans or antibody producing cells therefrom can be manipulated to serve as the basis for production of mammalian, including human hybridoma cell lines.
  • the host animal is inoculated intraperitoneally, intramuscularly, orally, subcutaneously, intraplantar, and/or (e.g., and) intradermally with an amount of immunogen, including as described herein.
  • an antibody (monoclonal or polyclonal) of interest may be sequenced and the polynucleotide sequence may then be cloned into a vector for expression or propagation.
  • the sequence encoding the antibody of interest may be maintained in vector in a host cell and the host cell can then be expanded and frozen for future use.
  • the polynucleotide sequence may be used for genetic manipulation to "humanize” the antibody or to improve the affinity (affinity maturation), or other characteristics of the antibody.
  • the constant region may be engineered to more resemble human constant regions to avoid immune response if the antibody is used in clinical trials and treatments in humans.
  • Fully human antibodies can be obtained by using commercially available mice that have been engineered to express specific human immunoglobulin proteins.
  • Transgenic animals that are designed to produce a more desirable (e.g., fully human antibodies) or more robust immune response may also be used for generation of humanized or human antibodies. Examples of such technology are XenomouseRTM from Amgen, Inc. (Fremont, CA) and HuMAb-MouseRTM and TC MouseTM from Medarex, Inc. (Princeton, NJ) or H2L2 mice from Harbour Antibodies BV (Holland).
  • antibodies may be made recombinantly by phage display or yeast technology. See, for example, U.S. Pat. Nos.
  • Antigen-binding fragments of an intact antibody can be prepared via routine methods.
  • F(ab')2 fragments can be produced by pepsin digestion of an antibody molecule, and Fab’ fragments that can be generated by reducing the disulfide bridges of F(ab')2 fragments.
  • Genetically engineered antibodies such as humanized antibodies, chimeric antibodies, single-chain antibodies, and bi-specific antibodies, can be produced via, e.g., conventional recombinant technology.
  • DNA encoding a monoclonal antibodies specific to a target antigen can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the monoclonal antibodies).
  • the hybridoma cells serve as a preferred source of such DNA.
  • the DNA may be placed into one or more expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, human HEK293 cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
  • the DNA can then be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences, Morrison et al., (1984) Proc. Nat. Acad. Sci. 81:6851, or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non immunoglobulin polypeptide.
  • genetically engineered antibodies such as “chimeric” or “hybrid” antibodies; can be prepared that have the binding specificity of a target antigen.
  • a single-chain antibody can be prepared via recombinant technology by linking a nucleotide sequence coding for a heavy chain variable region and a nucleotide sequence coding for a light chain variable region.
  • a flexible linker is incorporated between the two variable regions.
  • Antibodies obtained following a method known in the art and described herein can be characterized using methods well known in the art. For example, one method is to identify the epitope to which the antigen binds, or “epitope mapping.” There are many methods known in the art for mapping and characterizing the location of epitopes on proteins, including solving the crystal structure of an antibody- antigen complex, competition assays, gene fragment expression assays, and synthetic peptide-based assays, as described, for example, in Chapter 11 of Harlow and Lane, Using Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1999.
  • epitope mapping can be accomplished use H/D-Ex (hydrogen deuterium exchange) coupled with proteolysis and mass spectrometry.
  • epitope mapping can be used to determine the sequence to which an antibody binds.
  • the epitope can be a linear epitope, i.e., contained in a single stretch of amino acids, or a conformational epitope formed by a three-dimensional interaction of amino acids that may not necessarily be contained in a single stretch (primary structure linear sequence).
  • Peptides of varying lengths e.g., at least 4-6 amino acids long
  • the epitope to which the antibody binds can be determined in a systematic screening by using overlapping peptides derived from the target antigen sequence and determining binding by the antibody.
  • the gene fragment expression assays the open reading frame encoding the target antigen is fragmented either randomly or by specific genetic constructions and the reactivity of the expressed fragments of the antigen with the antibody to be tested is determined.
  • the gene fragments may, for example, be produced by PCR and then transcribed and translated into protein in vitro, in the presence of radioactive amino acids. The binding of the antibody to the radioactively labeled antigen fragments is then determined by immunoprecipitation and gel electrophoresis.
  • Certain epitopes can also be identified by using large libraries of random peptide sequences displayed on the surface of phage particles (phage libraries). Alternatively, a defined library of overlapping peptide fragments can be tested for binding to the test antibody in simple binding assays. In an additional example, mutagenesis of an antigen binding domain, domain swapping experiments and alanine scanning mutagenesis can be performed to identify residues required, sufficient, and/or (e.g., and) necessary for epitope binding. Alternatively, competition assays can be performed using other antibodies known to bind to the same antigen to determine whether an antibody binds to the same epitope as the other antibodies. Competition assays are well known to those of skill in the art.
  • an anti-TfR antibody is prepared by recombinant technology as exemplified below.
  • Nucleic acids encoding the heavy and light chain of an anti-TfR antibody as described herein can be cloned into one expression vector, each nucleotide sequence being in operable linkage to a suitable promoter.
  • each of the nucleotide sequences encoding the heavy chain and light chain is in operable linkage to a distinct promoter.
  • the nucleotide sequences encoding the heavy chain and the light chain can be in operable linkage with a single promoter, such that both heavy and light chains are expressed from the same promoter.
  • an internal ribosomal entry site IRS
  • the nucleotide sequences encoding the two chains of the antibody are cloned into two vectors, which can be introduced into the same or different cells.
  • the two chains are expressed in different cells, each of them can be isolated from the host cells expressing such and the isolated heavy chains and light chains can be mixed and incubated under suitable conditions allowing for the formation of the antibody.
  • a nucleic acid sequence encoding one or all chains of an antibody can be cloned into a suitable expression vector in operable linkage with a suitable promoter using methods known in the art.
  • the nucleotide sequence and vector can be contacted, under suitable conditions, with a restriction enzyme to create complementary ends on each molecule that can pair with each other and be joined together with a ligase.
  • synthetic nucleic acid linkers can be ligated to the termini of a gene. These synthetic linkers contain nucleic acid sequences that correspond to a particular restriction site in the vector. The selection of expression vectors/promoter would depend on the type of host cells for use in producing the antibodies.
  • a variety of promoters can be used for expression of the antibodies described herein, including, but not limited to, cytomegalovirus (CMV) intermediate early promoter, a viral LTR such as the Rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR, the simian virus 40 (SV40) early promoter, E. coli lac UV promoter, and the herpes simplex tk virus promoter.
  • CMV cytomegalovirus
  • a viral LTR such as the Rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR
  • SV40 simian virus 40
  • E. coli lac UV promoter E. coli lac UV promoter
  • herpes simplex tk virus promoter E. coli lac UV promoter
  • tetR tetracycline repressor
  • Other systems include FK506 dimer, VP 16 or p65 using astradiol, RU486, diphenol murislerone, or rapamycin. Inducible systems are available from Invitrogen, Clontech and Ariad, among others.
  • Regulatable promoters that include a repressor with the operon can be used.
  • the lac repressor from E. coli can function as a transcriptional modulator to regulate transcription from lac operator-bearing mammalian cell promoters [M. Brown et al., Cell, 49:603-612 (1987)]; Gossen and Bujard (1992); [M. Gossen et al., Natl. Acad. Sci.
  • tetracycline repressor tetR
  • VP 16 transcription activator
  • tetO bearing minimal promoter derived from the human cytomegalovirus (hCMV) promoter to create a tetR-tet operator system to control gene expression in mammalian cells.
  • hCMV human cytomegalovirus
  • a tetracycline inducible switch is used.
  • the tetracycline repressor (tetR) alone, rather than the tetR-mammalian cell transcription factor fusion derivatives can function as potent trans-modulator to regulate gene expression in mammalian cells when the tetracycline operator is properly positioned downstream for the TATA element of the CMVIE promoter (Yao et al., Human Gene Therapy).
  • tetracycline inducible switch is that it does not require the use of a tetracycline repressor-mammalian cells transactivator or repressor fusion protein, which in some instances can be toxic to cells (Gossen et al., Natl. Acad. Sci. USA, 89:5547-5551 (1992); Shockett et al., Proc. Natl. Acad. Sci. USA, 92:6522-6526 (1995)), to achieve its regulatable effects.
  • the vector can contain, for example, some or all of the following: a selectable marker gene, such as the neomycin gene for selection of stable or transient transfectants in mammalian cells; enhancer/promoter sequences from the immediate early gene of human CMV for high levels of transcription; transcription termination and RNA processing signals from SV40 for mRNA stability; SV40 polyoma origins of replication and ColEl for proper episomal replication; internal ribosome binding sites (IRESes), versatile multiple cloning sites; and T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA.
  • a selectable marker gene such as the neomycin gene for selection of stable or transient transfectants in mammalian cells
  • enhancer/promoter sequences from the immediate early gene of human CMV for high levels of transcription
  • transcription termination and RNA processing signals from SV40 for mRNA stability
  • SV40 polyoma origins of replication and ColEl for proper episomal replication
  • One or more vectors comprising nucleic acids encoding any of the antibodies may be introduced into suitable host cells for producing the antibodies.
  • the host cells can be cultured under suitable conditions for expression of the antibody or any polypeptide chain thereof.
  • Such antibodies or polypeptide chains thereof can be recovered by the cultured cells (e.g., from the cells or the culture supernatant) via a conventional method, e.g., affinity purification.
  • polypeptide chains of the antibody can be incubated under suitable conditions for a suitable period of time allowing for production of the antibody.
  • methods for preparing an antibody described herein involve a recombinant expression vector that encodes both the heavy chain and the light chain of an anti-TfR antibody, as also described herein.
  • the recombinant expression vector can be introduced into a suitable host cell (e.g., a dhfr- CHO cell) by a conventional method, e.g., calcium phosphate mediated transfection.
  • a suitable host cell e.g., a dhfr- CHO cell
  • Positive transformant host cells can be selected and cultured under suitable conditions allowing for the expression of the two polypeptide chains that form the antibody, which can be recovered from the cells or from the culture medium.
  • the two chains recovered from the host cells can be incubated under suitable conditions allowing for the formation of the antibody.
  • the host cell used for expressing the anti-TfR antibodies described herein are CHO-S cells (e.g., ThermoFisher Catalog# R80007).
  • two recombinant expression vectors are provided, one encoding the heavy chain of the anti-TfR antibody and the other encoding the light chain of the anti-TfR antibody.
  • Both of the two recombinant expression vectors can be introduced into a suitable host cell (e.g., dhfr- CHO cell) by a conventional method, e.g., calcium phosphate-mediated transfection.
  • each of the expression vectors can be introduced into a suitable host cells. Positive transformants can be selected and cultured under suitable conditions allowing for the expression of the polypeptide chains of the antibody.
  • the antibody produced therein can be recovered from the host cells or from the culture medium. If necessary, the polypeptide chains can be recovered from the host cells or from the culture medium and then incubated under suitable conditions allowing for formation of the antibody.
  • each of them can be recovered from the corresponding host cells or from the corresponding culture media. The two polypeptide chains can then be incubated under suitable conditions for formation of the antibody.
  • Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells and recovery of the antibodies from the culture medium.
  • some antibodies can be isolated by affinity chromatography with a Protein A or Protein G coupled matrix.
  • any one of the anti-TfR antibodies described herein is produced by recombinant DNA technology in Chinese hamster ovary (CHO) cell suspension culture, optionally in CHO-K1 cell (e.g., CHO-K1 cells derived from European Collection of Animal Cell Culture, Cat. No. 85051005) suspension culture.
  • CHO-K1 cell e.g., CHO-K1 cells derived from European Collection of Animal Cell Culture, Cat. No. 85051005
  • an antibody provided herein may have one or more post- translational modifications.
  • N-terminal cyclization also called pyroglutamate formation (pyro-Glu)
  • pyro-Glu N-terminal cyclization
  • Glu N-terminal Glutamate
  • Gin Glutamine residues during production.
  • an antibody specified as having a sequence comprising an N-terminal glutamate or glutamine residue encompasses antibodies that have undergone pyroglutamate formation resulting from a post-translational modification.
  • pyroglutamate formation occurs in a heavy chain sequence.
  • pyroglutamate formation occurs in a light chain sequence.
  • the humanized anti-TfR antibodies described herein can be used for delivering a molecular payload to a target cell or a target tissue (e.g., a cell or tissue that expresses TfR). Accordingly, some aspects of the present disclosure provide complexes comprising any one of the humanized anti-TfR antibody described herein (e.g., humanized 3- A4, 3-M12, or 5-H12 in IgG or Fab form as provided in Table 4 and Table 5) to a molecular payload.
  • the complexes described herein may be used in various applications, e.g., diagnostic or therapeutic applications.
  • a complex comprises an anti-TfR antibody covalently linked to an oligonucleotide (e.g., an antisense oligonucleotide).
  • the complex described herein is used to modulate the activity or function of at least one gene, protein, and/or (e.g., and) nucleic acid.
  • the molecular payload present with a complex is responsible for the modulation of a gene, protein, and/or (e.g., and) nucleic acids.
  • a molecular payload may be a small molecule, protein, nucleic acid, oligonucleotide, or any molecular entity capable of modulating the activity or function of a gene, protein, and/or (e.g., and) nucleic acid in a cell.
  • a molecular payload is an oligonucleotide that targets a disease-associated repeat in muscle cells.
  • Some aspects of the disclosure provide molecular payloads, e.g., for modulating a biological outcome, e.g., the transcription of a DNA sequence, the expression of a protein, or the activity of a protein, that can be linked to any one of the anti-TfR antibodies described herein.
  • such molecular payloads are capable of targeting to a muscle cell, e.g., via specifically binding to a nucleic acid or protein in the muscle cell following delivery to the muscle cell by the linked anti-TfR antibody. It should be appreciated that various types of molecular payloads may be used in accordance with the disclosure.
  • the molecular payload may comprise, or consist of, an oligonucleotide (e.g., antisense oligonucleotide), a peptide (e.g., a peptide that binds a nucleic acid or protein associated with disease in a muscle cell), a protein (e.g., a protein that binds a nucleic acid or protein associated with disease in a muscle cell), or a small molecule (e.g., a small molecule that modulates the function of a nucleic acid or protein associated with disease in a muscle cell).
  • the molecular payload is an oligonucleotide that comprises a strand having a region of complementarity to a gene provided in Table 6.
  • the molecular payload is an agent for the treatment of a neurological disorder.
  • a “neurological disorder” as used herein refers to a disease or disorder which affects the CNS and/or (e.g., and) which has an etiology in the CNS.
  • CNS diseases or disorders include, but are not limited to, neuropathy, amyloidosis, cancer, an ocular disease or disorder, viral or microbial infection, inflammation, ischemia, neurodegenerative disease, seizure, behavioral disorders, and a lysosomal storage disease.
  • the CNS will be understood to include the eye, which is normally sequestered from the rest of the body by the blood-retina barrier.
  • neurological disorders include, but are not limited to, neurodegenerative diseases (including, but not limited to, Lewy body disease, postpoliomyelitis syndrome, Shy-Draeger syndrome, olivopontocerebellar atrophy, Parkinson's disease, multiple system atrophy, striatonigral degeneration, tauopathies (including, but not limited to, Alzheimer disease and supranuclear palsy), prion diseases (including, but not limited to, bovine spongiform encephalopathy, scrapie, Creutzfeldt- Jakob syndrome, kuru, Gerstmann-Straussler-Scheinker disease, chronic wasting disease, and fatal familial insomnia), bulbar palsy, motor neuron disease, and nervous system heterodegenerative disorders (including, but not limited to, Canavan disease, Huntington's disease, neuronal ceroid-lipofuscinosis, Alexander's disease, Tourette's syndrome, Menkes kinky hair syndrome, Cockayne syndrome, Halervorden-Spatz syndrome, la
  • At least one (e.g ., at least 2, at least 3, at least 4, at least 5, at least 10) molecular payload is linked to any one of the anti-TfR antibody described herein.
  • all molecular payloads attached to the anti- TfR antibody are the same, e.g. target the same gene.
  • all molecular payloads attached to the anti-TfR antibody are different, for example the molecular payloads may target different portions of the same target gene, or the molecular payloads may target at least two different target genes.
  • an anti-TfR antibody described herein may be attached to some molecular payloads that are the same and some molecular payloads that are different.
  • the present disclosure also provides a composition comprising a plurality of complexes, for which at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) of the complexes comprise an anti-TfR antibody linked to the same number of molecular payloads (e.g., oligonucleotides).
  • molecular payloads e.g., oligonucleotides
  • any suitable oligonucleotide may be used as a molecular payload, as described herein.
  • the oligonucleotide may be designed to cause degradation of an mRNA (e.g., the oligonucleotide may be a gapmer, an siRNA, a ribozyme or an aptamer that causes degradation).
  • the oligonucleotide may be designed to block translation of an mRNA (e.g., the oligonucleotide may be a mixmer, an siRNA or an aptamer that blocks translation).
  • an oligonucleotide may be designed to caused degradation and block translation of an mRNA.
  • an oligonucleotide may be a guide nucleic acid (e.g., guide RNA) for directing activity of an enzyme (e.g., a gene editing enzyme).
  • an enzyme e.g., a gene editing enzyme
  • Other examples of oligonucleotides are provided herein. It should be appreciated that, in some embodiments, oligonucleotides in one format (e.g., antisense oligonucleotides) may be suitably adapted to another format (e.g., siRNA oligonucleotides) by incorporating functional sequences (e.g., antisense strand sequences) from one format to the other format.
  • an oligonucleotide may comprise a region of complementarity to a target gene provided in Table 6.
  • the oligonucleotide may target IncRNA or mRNA, e.g., for degradation.
  • the oligonucleotide may target, e.g., for degradation, a nucleic acid encoding a protein involved in a mismatch repair pathway, e.g., MSH2, MutLalpha, MutSbeta, MutLalpha.
  • a protein involved in a mismatch repair pathway e.g., MSH2, MutLalpha, MutSbeta, MutLalpha.
  • proteins involved in mismatch repair pathways for which mRNAs encoding such proteins may be targeted by oligonucleotides described herein, are described in Iyer, R.R. et ak, “ DNA triplet repeat expansion and mismatch repair” Annu Rev Biochem. 2015;84:199-226.; and Schmidt M.H. and Pearson C.E., “Disease-associated repeat instability and mismatch repair” DNA Repair (Amst). 2016 Feb;38: 117-26
  • any one of the oligonucleotides can be in salt form, e.g., as sodium, potassium, or magnesium salts.
  • the 5’ or 3’ nucleoside (e.g., terminal nucleoside) of any one of the oligonucleotides described herein is conjugated to an amine group, optionally via a spacer.
  • the spacer comprises an aliphatic moiety.
  • the spacer comprises a polyethylene glycol moiety.
  • a phosphodiester linkage is present between the spacer and the 5’ or 3’ nucleoside of the oligonucleotide.
  • the 5’ or 3’ nucleoside of any one of the oligonucleotides described herein is conjugated to a compound of the formula -NH 2 -(CH 2 ) n -, wherein n is an integer from 1 to 12. In some embodiments, n is 6, 7, 8, 9, 10, 11, or 12. In some embodiments, a phosphodiester linkage is present between the compound of the formula NH 2 - (CH 2 ) n - and the 5’ or 3’ nucleoside of the oligonucleotide.
  • a compound of the formula NH 2 -(CH 2 ) 6 - is conjugated to the oligonucleotide via a reaction between 6- amino-l-hexanol (NH 2 -(CH 2 ) 6 -OH) and the 5’ phosphate of the oligonucleotide.
  • the oligonucleotide is conjugated to a targeting agent, e.g., a muscle targeting agent such as an anti-TfR antibody, e.g., via the amine group.
  • a targeting agent e.g., a muscle targeting agent such as an anti-TfR antibody, e.g., via the amine group.
  • Oligonucleotides may be of a variety of different lengths, e.g., depending on the format. In some embodiments, an oligonucleotide is 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length.
  • the oligonucleotide is 8 to 50 nucleotides in length, 8 to 40 nucleotides in length, 8 to 30 nucleotides in length, 10 to 15 nucleotides in length, 10 to 20 nucleotides in length, 15 to 25 nucleotides in length, 21 to 23 nucleotides in lengths, etc.
  • a complementary nucleic acid sequence of an oligonucleotide for purposes of the present disclosure is specifically hybridizable or specific for the target nucleic acid when binding of the sequence to the target molecule (e.g., mRNA) interferes with the normal function of the target (e.g., mRNA) to cause a loss of activity (e.g., inhibiting translation) or expression (e.g., degrading a target mRNA) and there is a sufficient degree of complementarity to avoid non-specific binding of the sequence to non-target sequences under conditions in which avoidance of non-specific binding is desired, e.g., under physiological conditions in the case of in vivo assays or therapeutic treatment, and in the case of in vitro assays, under conditions in which the assays are performed under suitable conditions of stringency.
  • the sequence to the target molecule e.g., mRNA
  • a loss of activity e.g., inhibiting translation
  • expression e.g., degrading a
  • an oligonucleotide may be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% complementary to the consecutive nucleotides of a target nucleic acid.
  • a complementary nucleotide sequence need not be 100% complementary to that of its target to be specifically hybridizable or specific for a target nucleic acid.
  • an oligonucleotide comprises region of complementarity to a target nucleic acid that is in the range of 8 to 15, 8 to 30, 8 to 40, or 10 to 50, or 5 to 50, or 5 to 40 nucleotides in length. In some embodiments, a region of complementarity of an oligonucleotide to a target nucleic acid is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
  • an oligonucleotide may contain 1, 2 or 3 base mismatches compared to the portion of the consecutive nucleotides of target nucleic acid. In some embodiments the oligonucleotide may have up to 3 mismatches over 15 bases, or up to 2 mismatches over 10 bases.
  • the oligonucleotide is complementary (e.g., at least 85% at least 90%, at least 95%, or 100%) to a target sequence of any one of the oligonucleotides provided herein. In some embodiments, such target sequence is 100% complementary to the oligonucleotide provided herein.
  • any one or more of the thymine bases (T’s) in any one of the oligonucleotides provided herein may optionally be uracil bases (U’s), and/or any one or more of the U’s may optionally be T’s.
  • oligonucleotides described herein may be modified, e.g., comprise a modified sugar moiety, a modified internucleoside linkage, a modified nucleotide and/or (e.g., and) combinations thereof.
  • oligonucleotides may exhibit one or more of the following properties: do not mediate alternative splicing; are not immune stimulatory; are nuclease resistant; have improved cell uptake compared to unmodified oligonucleotides; are not toxic to cells or mammals; have improved endosomal exit internally in a cell; minimizes TLR stimulation; or avoid pattern recognition receptors.
  • Any of the modified chemistries or formats of oligonucleotides described herein can be combined with each other. For example, one, two, three, four, five, or more different types of modifications can be included within the same oligonucleotide.
  • nucleotide modifications may be used that make an oligonucleotide into which they are incorporated more resistant to nuclease digestion than the native oligodeoxynucleotide or oligoribonucleotide molecules; these modified oligonucleotides survive intact for a longer time than unmodified oligonucleotides.
  • modified oligonucleotides include those comprising modified backbones, for example, modified internucleoside linkages such as phosphorothioates, phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages. Accordingly, oligonucleotides of the disclosure can be stabilized against nucleolytic degradation such as by the incorporation of a modification, e.g., a nucleotide modification.
  • an oligonucleotide may be of up to 50 or up to 100 nucleotides in length in which 2 to 10, 2 to 15, 2 to 16, 2 to 17, 2 to 18, 2 to 19, 2 to 20, 2 to 25, 2 to 30, 2 to 40, 2 to 45, or more nucleotides of the oligonucleotide are modified nucleotides.
  • the oligonucleotide may be of 8 to 30 nucleotides in length in which 2 to 10, 2 to 15, 2 to 16, 2 to 17, 2 to 18, 2 to 19, 2 to 20, 2 to 25, 2 to 30 nucleotides of the oligonucleotide are modified nucleotides.
  • the oligonucleotide may be of 8 to 15 nucleotides in length in which 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, 2 to 10, 2 to 11, 2 to 12, 2 to 13, 2 to 14 nucleotides of the oligonucleotide are modified nucleotides.
  • the oligonucleotides may have every nucleotide except 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides modified. Oligonucleotide modifications are described further herein c. Modified Nucleosides
  • the oligonucleotide described herein comprises at least one nucleoside modified at the 2' position of the sugar.
  • an oligonucleotide comprises at least one 2'-modified nucleoside.
  • all of the nucleosides in the oligonucleotide are 2’ -modified nucleosides.
  • the oligonucleotide described herein comprises one or more non-bicyclic 2’-modified nucleosides, e.g., 2’-deoxy, 2’-fluoro (2’-F), 2’-0-methyl (2’-0-Me), 2’-0-methoxyethyl (2’-MOE), 2’-0-aminopropyl (2’-0-AP), 2’-0-dimethylaminoethyl (2’-0- DMAOE), 2’-0-dimethylaminopropyl (2’-0-DMAP), 2’-0-dimethylaminoethyloxyethyl (2’- O-DMAEOE), or 2’-0-N-methylacetamido (2’-0-NMA) modified nucleoside.
  • 2’-deoxy, 2’-fluoro (2’-F) 2’-0-methyl (2’-0-Me), 2’-0-methoxyethyl (2’-MOE
  • the oligonucleotide described herein comprises one or more 2’- 4’ bicyclic nucleosides in which the ribose ring comprises a bridge moiety connecting two atoms in the ring, e.g., connecting the 2’-0 atom to the 4’-C atom via a methylene (LNA) bridge, an ethylene (ENA) bridge, or a (S)-constrained ethyl (cEt) bridge.
  • LNA methylene
  • ENA ethylene
  • cEt a (S)-constrained ethyl
  • ENAs are provided in International Patent Publication No. WO 2005/042777, published on May 12, 2005, and entitled ‘APP/ENA Antisense” ⁇ , Morita et al., Nucleic Acid Res., Suppl 1:241-242, 2001; Surono et al., Hum. Gene Ther., 15:749-757, 2004; Koizumi, Curr. Opin. Mol. Ther., 8:144-149, 2006 and Horie et al., Nucleic Acids Symp. Ser (Oxf), 49:171-172, 2005; the disclosures of which are incorporated herein by reference in their entireties.
  • Examples of cEt are provided in US Patents 7,101,993; 7,399,845 and 7,569,686, each of which is herein incorporated by reference in its entirety.
  • the oligonucleotide comprises a modified nucleoside disclosed in one of the following United States Patent or Patent Application Publications: US Patent 7,399,845, issued on July 15, 2008, and entitled “ 6-Modified Bicyclic Nucleic Acid Analogs”, US Patent 7,741,457, issued on June 22, 2010, and entitled “ 6-Modified Bicyclic Nucleic Acid Analogs”; US Patent 8,022,193, issued on September 20, 2011, and entitled “ 6-Modified Bicyclic Nucleic Acid Analogs”; US Patent 7,569,686, issued on August 4, 2009, and entitled “ Compounds And Methods For Synthesis Of Bicyclic Nucleic Acid Analogs”; US Patent 7,335,765, issued on February 26, 2008, and entitled ‘Wove/ Nucleoside And Oligonucleotide Analogues”; US Patent 7,314,923, issued on January 1, 2008, and entitled ‘Wove/ Nucleoside And Oligonucleotide Analogues”; US Patent 7,816,333, issued on
  • the oligonucleotide comprises at least one modified nucleoside that results in an increase in Tm of the oligonucleotide in a range of 1°C, 2 °C, 3°C, 4 °C, or 5°C compared with an oligonucleotide that does not have the at least one modified nucleoside
  • the oligonucleotide may have a plurality of modified nucleosides that result in a total increase in Tm of the oligonucleotide in a range of 2 °C, 3 °C, 4 °C, 5 °C, 6 °C, 7 °C, 8 °C, 9 °C, 10 °C, 15 °C, 20 °C, 25 °C, 30 °C, 35 °C, 40 °C, 45 °C or more compared with an oligonucleotide that does not have the modified nucleoside.
  • the oligonucleotide may comprise a mix of nucleosides of different kinds.
  • an oligonucleotide may comprise a mix of 2’-deoxyribonucleosides or ribonucleosides and 2’-fluoro modified nucleosides.
  • An oligonucleotide may comprise a mix of deoxyribonucleosides or ribonucleosides and 2’-0-Me modified nucleosides.
  • An oligonucleotide may comprise a mix of 2’-fluoro modified nucleosides and 2’-0-Me modified nucleosides.
  • An oligonucleotide may comprise a mix of 2’-4’ bicyclic nucleosides and 2’- MOE, 2’-fluoro, or 2’-0-Me modified nucleosides.
  • An oligonucleotide may comprise a mix of non-bicyclic 2’-modified nucleosides (e.g., 2’-MOE, 2’-fluoro, or 2’-0-Me) and 2’-4’ bicyclic nucleosides (e.g., LNA, ENA, cEt).
  • the oligonucleotide may comprise alternating nucleosides of different kinds.
  • an oligonucleotide may comprise alternating 2’-deoxyribonucleosides or ribonucleosides and 2’-fluoro modified nucleosides.
  • An oligonucleotide may comprise alternating deoxyribonucleosides or ribonucleosides and 2’-0-Me modified nucleosides.
  • An oligonucleotide may comprise alternating 2’-fluoro modified nucleosides and 2’-0-Me modified nucleosides.
  • An oligonucleotide may comprise alternating 2’-4’ bicyclic nucleosides and 2’-MOE, 2’-fluoro, or 2’-0-Me modified nucleosides.
  • An oligonucleotide may comprise alternating non-bicyclic 2’-modified nucleosides (e.g., 2’-MOE, 2’-fluoro, or 2’-0-Me) and 2’- 4’ bicyclic nucleosides (e.g., LNA, ENA, cEt).
  • an oligonucleotide described herein comprises a 5 - vinylphosphonate modification, one or more abasic residues, and/or one or more inverted abasic residues.
  • oligonucleotide may contain a phosphorothioate or other modified intemucleoside linkage.
  • the oligonucleotide comprises phosphorothioate intemucleoside linkages.
  • the oligonucleotide comprises phosphorothioate intemucleoside linkages between at least two nucleotides.
  • the oligonucleotide comprises phosphorothioate intemucleoside linkages between all nucleotides.
  • oligonucleotides comprise modified intemucleoside linkages at the first, second, and/or (e.g., and) third intemucleoside linkage at the 5' or 3' end of the nucleotide sequence.
  • Phosphorus-containing linkages that may be used include, but are not limited to, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates comprising 3'alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates comprising 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'; see US patent nos.
  • oligonucleotides may have heteroatom backbones, such as methylene(methylimino) or MMI backbones; amide backbones (see De Mesmaeker et al. Ace. Chem. Res. 1995, 28:366-374); morpholino backbones (see Summerton and Weller, U.S. Pat. No. 5,034,506); or peptide nucleic acid (PNA) backbones (wherein the phosphodiester backbone of the oligonucleotide is replaced with a polyamide backbone, the nucleotides being bound directly or indirectly to the aza nitrogen atoms of the polyamide backbone, see Nielsen et al., Science 1991, 254, 1497).
  • heteroatom backbones such as methylene(methylimino) or MMI backbones; amide backbones (see De Mesmaeker et al. Ace. Chem. Res. 1995, 28:366-374); morpholino backbones (see Summerton and
  • internucleotidic phosphorus atoms of oligonucleotides are chiral, and the properties of the oligonucleotides by adjusted based on the configuration of the chiral phosphorus atoms.
  • appropriate methods may be used to synthesize P-chiral oligonucleotide analogs in a stereocontrolled manner (e.g., as described in Oka N, Wada T, Stereocontrolled synthesis of oligonucleotide analogs containing chiral internucleotidic phosphorus atoms. Chem Soc Rev.
  • phosphorothioate containing oligonucleotides comprise nucleoside units that are joined together by either substantially all Sp or substantially all Rp phosphorothioate intersugar linkages are provided.
  • such phosphorothioate oligonucleotides having substantially chirally pure intersugar linkages are prepared by enzymatic or chemical synthesis, as described, for example, in US Patent 5,587,261, issued on December 12, 1996, the contents of which are incorporated herein by reference in their entirety.
  • chirally controlled oligonucleotides provide selective cleavage patterns of a target nucleic acid.
  • a chirally controlled oligonucleotide provides single site cleavage within a complementary sequence of a nucleic acid, as described, for example, in US Patent Application Publication 20170037399 Al, published on February 2, 2017, entitled “CHIRAL DESIGN”, the contents of which are incorporated herein by reference in their entirety. f. Morpholinos
  • the oligonucleotide may be a morpholino-based compound. Morpholino-based oligomeric compounds are described in Dwaine A. Braasch and David R. Corey, Biochemistry, 2002, 41(14), 4503-4510); Genesis, volume 30, issue 3, 2001; Heasman, J., Dev. Biol., 2002, 243, 209-214; Nasevicius et al., Nat. Genet., 2000, 26, 216-220; Lacerra et al., Proc. Natl. Acad. Sci., 2000, 97, 9591-9596; and U.S. Pat. No. 5,034,506, issued Jul. 23, 1991.
  • the morpholino-based oligomeric compound is a phosphorodiamidate morpholino oligomer (PMO) (e.g., as described in Iverson, Curr. Opin. Mol. Ther., 3:235-238, 2001; and Wang et al., J. Gene Med., 12:354-364, 2010; the disclosures of which are incorporated herein by reference in their entireties).
  • PMO phosphorodiamidate morpholino oligomer
  • PNAs Peptide Nucleic Acids
  • both a sugar and an internucleoside linkage (the backbone) of the nucleotide units of an oligonucleotide are replaced with novel groups.
  • the base units are maintained for hybridization with an appropriate nucleic acid target compound.
  • an oligomeric compound an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA).
  • PNA peptide nucleic acid
  • the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, for example, an aminoethylglycine backbone.
  • nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.
  • Representative publication that report the preparation of PNA compounds include, but are not limited to, US patent nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500. h. Gapmers
  • an oligonucleotide described herein is a gapmer.
  • a gapmer oligonucleotide generally has the formula 5'-X-Y-Z-3', with X and Z as flanking regions around a gap region Y.
  • flanking region X of formula 5'-X-Y-Z- 3' is also referred to as X region, flanking sequence X, 5’ wing region X, or 5’ wing segment.
  • flanking region Z of formula 5'-X-Y-Z-3' is also referred to as Z region, flanking sequence Z, 3’ wing region Z, or 3’ wing segment.
  • gap region Z of formula 5'-X-Y-Z-3' is also referred to as Z region, flanking sequence Z, 3’ wing region Z, or 3’ wing segment.
  • Y of formula 5'-X-Y-Z-3' is also referred to as Y region, Y segment, or gap-segment Y.
  • each nucleoside in the gap region Y is a 2’-deoxyribonucleoside, and neither the 5’ wing region X or the 3’ wing region Z contains any 2’-deoxyribonucleosides.
  • the Y region is a contiguous stretch of nucleotides, e.g., a region of 6 or more DNA nucleotides, which are capable of recruiting an RNAse, such as RNAse H.
  • the gapmer binds to the target nucleic acid, at which point an RNAse is recruited and can then cleave the target nucleic acid.
  • the RNAse binds to the target nucleic acid, at which point an RNAse is recruited and can then cleave the target nucleic acid.
  • Y region is flanked both 5' and 3' by regions X and Z comprising high-affinity modified nucleosides, e.g., one to six high-affinity modified nucleosides.
  • high affinity modified nucleosides include, but are not limited to, 2'-modified nucleosides (e.g., 2’-MOE, 2'O-Me, 2’-F) or 2’-4’ bicyclic nucleosides (e.g., LNA, cEt, ENA).
  • the flanking sequences X and Z may be of 1-20 nucleotides, 1-8 nucleotides, or 1-5 nucleotides in length.
  • flanking sequences X and Z may be of similar length or of dissimilar lengths.
  • the gap-segment Y may be a nucleotide sequence of 5-20 nucleotides, 5- 15 twelve nucleotides, or 6-10 nucleotides in length.
  • the gap region of the gapmer oligonucleotides may contain modified nucleotides known to be acceptable for efficient RNase H action in addition to DNA nucleotides, such as C4'-substituted nucleotides, acyclic nucleotides, and arabino- configured nucleotides.
  • the gap region comprises one or more unmodified intemucleoside linkages.
  • flanking regions each independently comprise one or more phosphorothioate intemucleoside linkages (e.g., phosphorothioate intemucleoside linkages or other linkages) between at least two, at least three, at least four, at least five or more nucleotides.
  • the gap region and two flanking regions each independently comprise modified intemucleoside linkages (e.g., phosphorothioate intemucleoside linkages or other linkages) between at least two, at least three, at least four, at least five or more nucleotides.
  • a gapmer may be produced using appropriate methods.
  • Representative U.S. patents, U.S. patent publications, and PCT publications that teach the preparation of gapmers include, but are not limited to, U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; 5,700,922; 5,898,031; 7,015,315; 7,101,993; 7,399,845; 7,432,250; 7,569,686; 7,683,036; 7,750,131; 8,580,756; 9,045,754; 9,428,534; 9,695,418; 10,017,764; 10,260,069; 9,428,534; 8,580,756; U.S. patent publication Nos. US20050074801, US20090221685; US20090286969, US
  • a gapmer is 10-40 nucleosides in length.
  • a gapmer may be 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 15-40, 15-35, 15-30, 15-25, 15-20, 20-40, 20-35, 20-30, 20-25, 25-40, 25-35, 25-30, 30-40, 30-35, or 35-40 nucleosides in length.
  • a gapmer is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleosides in length.
  • the gap region Y in a gapmer is 5-20 nucleosides in length.
  • the gap region Y may be 5-20, 5-15, 5-10, 10-20, 10-15, or 15-20 nucleosides in length.
  • the gap region Y is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleosides in length.
  • each nucleoside in the gap region Y is a 2’-deoxyribonucleoside.
  • all nucleosides in the gap region Y are 2’-deoxyribonucleosides.
  • one or more of the nucleosides in the gap region Y is a modified nucleoside (e.g., a 2’ modified nucleoside such as those described herein).
  • one or more cytosines in the gap region Y are optionally 5-methyl-cytosines.
  • each cytosine in the gap region Y is a 5-methyl-cytosines.
  • the 5’wing region of a gapmer (X in the 5'-X-Y-Z-3' formula) and the 3 ’wing region of a gapmer (Z in the 5'-X-Y-Z-3' formula) are independently 1-20 nucleosides long.
  • the 5’wing region of a gapmer (X in the 5'-X-Y-Z-3' formula) and the 3 ’wing region of the gapmer (Z in the 5'-X-Y-Z-3' formula) may be independently 1-20, 1-15, 1-10, 1-7, 1-5, 1-3, 1-2, 2-5, 2-7, 3-5, 3-7, 5-20, 5-15, 5-10, 10-20, 10-15, or 15-20 nucleosides long.
  • the 5’wing region of the gapmer (X in the 5'-X-Y-Z-3' formula) and the 3 ’wing region of the gapmer (Z in the 5'-X-Y-Z-3' formula) are independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleosides long. In some embodiments, the 5’wing region of the gapmer (X in the 5'-X-Y-Z- 3' formula) and the 3 ’wing region of the gapmer (Z in the 5'-X-Y-Z-3' formula) are of the same length.
  • the 5’wing region of the gapmer (X in the 5'-X-Y-Z-3' formula) and the 3’wing region of the gapmer (Z in the 5'-X-Y-Z-3' formula) are of different lengths. In some embodiments, the 5’wing region of the gapmer (X in the 5'-X-Y-Z-3' formula) is longer than the 3’wing region of the gapmer (Z in the 5'-X-Y-Z-3' formula). In some embodiments, the 5’wing region of the gapmer (X in the 5'-X-Y-Z-3' formula) is shorter than the 3’wing region of the gapmer (Z in the 5'-X-Y-Z-3' formula).
  • a gapmer comprises a 5'-X-Y-Z-3' of 5-10-5, 4-12-4, 3- 14-3, 2-16-2, 1-18-1, 3-10-3, 2-10-2, 1-10-1, 2-8-2, 4-6-4, 3-6-3, 2-6-2, 4-7-4, 3-7-3, 2-7-2, 4- 8-4, 3-8-3, 2-8-2, 1-8-1, 2-9-2, 1-9-1, 2-10-2, 1-10-1, 1-12-1, 1-16-1, 2-15-1, 1-15-2, 1-14-3, 3- 14-1, 2-14-2, 1-13-4, 4-13-1, 2-13-3, 3-13-2, 1-12-5, 5-12-1, 2-12-4, 4-12-2, 3-12-3, 1-11-6, 6-
  • the numbers indicate the number of nucleosides in X, Y, and Z regions in the 5'-X-Y-Z-3' gapmer.
  • one or more nucleosides in the 5 ’wing region of a gapmer (X in the 5'-X-Y-Z-3' formula) or the 3 ’wing region of a gapmer (Z in the 5'-X-Y-Z-3' formula) are modified nucleotides (e.g., high-affinity modified nucleosides).
  • the modified nucleoside e.g., high-affinity modified nucleosides
  • the 2’ -modified nucleoside is a 2’ -4’ bicyclic nucleoside or a non-bicyclic 2’ -modified nucleoside.
  • the high-affinity modified nucleoside is a 2’-4’ bicyclic nucleoside (e.g., LNA, cEt, or ENA) or a non-bicyclic 2’-modified nucleoside (e.g., 2’-fluoro (2’-F), 2’-0-methyl (2’-0-Me), 2’-0-methoxyethyl (2’- MOE), 2’-0-aminopropyl (2’-0-AP), 2’-0-dimethylaminoethyl (2’-0-DMA0E), 2’-0- dimethylaminopropyl (2’-0-DMAP), 2’-0-dimethylaminoethyloxyethyl (2’-0-DMAE0E), or 2 ’ -O-N -methylacetamido (2 ’ -O-NMA)) .
  • 2’-fluoro (2’-F 2’-0-methyl (2’-0-Me
  • MOE 2’-0-a
  • one or more nucleosides in the 5 ’wing region of a gapmer are high-affinity modified nucleosides.
  • each nucleoside in the 5’wing region of the gapmer (X in the 5'-X-Y-Z-3' formula) is a high-affinity modified nucleoside.
  • one or more nucleosides in the 3 ’wing region of a gapmer (Z in the 5'-X-Y-Z-3' formula) are high-affinity modified nucleosides.
  • each nucleoside in the 3 ’wing region of the gapmer (Z in the 5'-X-Y-Z-3' formula) is a high-affinity modified nucleoside.
  • one or more nucleosides in the 5’wing region of the gapmer (X in the 5'-X-Y-Z- 3' formula) are high-affinity modified nucleosides and one or more nucleosides in the 3 ’wing region of the gapmer (Z in the 5'-X-Y-Z-3' formula) are high-affinity modified nucleosides.
  • each nucleoside in the 5’wing region of the gapmer (X in the 5'-X-Y-Z-3' formula) is a high-affinity modified nucleoside and each nucleoside in the 3 ’wing region of the gapmer (Z in the 5'-X-Y-Z-3' formula) is high-affinity modified nucleoside.
  • the 5’wing region of a gapmer comprises the same high affinity nucleosides as the 3 ’wing region of the gapmer (Z in the 5'-X-Y-Z-3' formula).
  • the 5’wing region of the gapmer (X in the 5'-X-Y-Z-3' formula) and the 3 ’wing region of the gapmer (Z in the 5'-X-Y-Z-3' formula) may comprise one or more non-bicyclic 2’-modified nucleosides (e.g., 2’-MOE or 2’-0-Me).
  • the 5’wing region of the gapmer (X in the 5'-X-Y-Z-3' formula) and the 3’wing region of the gapmer (Z in the 5'-X-Y-Z-3' formula) may comprise one or more 2’ -4’ bicyclic nucleosides (e.g., LNA or cEt).
  • each nucleoside in the 5’wing region of the gapmer (X in the 5'-X-Y-Z-3' formula) and the 3’wing region of the gapmer (Z in the 5'-X- Y-Z-3' formula) is a non-bicyclic 2’-modified nucleosides (e.g., 2’-MOE or 2’-0-Me).
  • each nucleoside in the 5’wing region of the gapmer (X in the 5'-X-Y-Z-3' formula) and the 3’wing region of the gapmer (Z in the 5'-X-Y-Z-3' formula) is a 2’-4’ bicyclic nucleosides (e.g., LNA or cEt).
  • a gapmer comprises a 5'-X-Y-Z-3' configuration, wherein X and Z is independently 1-7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleosides in length and Y is 6-10 (e.g., 6, 7, 8, 9, or 10) nucleosides in length, wherein each nucleoside in X and Z is a non- bicyclic 2’-modified nucleosides (e.g., 2’-MOE or 2’-0-Me) and each nucleoside in Y is a 2’- deoxyribonucleoside.
  • X and Z is independently 1-7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleosides in length and Y is 6-10 (e.g., 6, 7, 8, 9, or 10) nucleosides in length, wherein each nucleoside in X and Z is a non- bicyclic 2’-modified nucleosides (e.g., 2’-MOE or 2’
  • the gapmer comprises a 5'-X-Y-Z-3' configuration, wherein X and Z is independently 1-7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleosides in length and Y is 6-10 (e.g., 6, 7, 8, 9, or 10) nucleosides in length, wherein each nucleoside in X and Z is a 2’-4’ bicyclic nucleosides (e.g., LNA or cEt) and each nucleoside in Y is a 2’- deoxyribonucleoside.
  • X and Z is independently 1-7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleosides in length and Y is 6-10 (e.g., 6, 7, 8, 9, or 10) nucleosides in length, wherein each nucleoside in X and Z is a 2’-4’ bicyclic nucleosides (e.g., LNA or cEt) and each nucleoside in Y is a
  • the 5’wing region of the gapmer (X in the 5'-X- Y-Z-3' formula) comprises different high affinity nucleosides as the 3 ’wing region of the gapmer (Z in the 5'-X-Y-Z-3' formula).
  • the 5’wing region of the gapmer (X in the 5'-X-Y-Z-3' formula) may comprise one or more non-bicyclic 2’ -modified nucleosides (e.g., 2’-MOE or 2’-0-Me) and the 3 ’wing region of the gapmer (Z in the 5'-X-Y-Z-3' formula) may comprise one or more 2’-4’ bicyclic nucleosides (e.g., LNA or cEt).
  • non-bicyclic 2’ -modified nucleosides e.g., 2’-MOE or 2’-0-Me
  • the 3 ’wing region of the gapmer (Z in the 5'-X-Y-Z-3' formula) may comprise one or more 2’-4’ bicyclic nucleosides (e.g., LNA or cEt).
  • the 3 ’wing region of the gapmer (Z in the 5'-X-Y-Z-3' formula) may comprise one or more non-bicyclic 2’-modified nucleosides (e.g., 2’-MOE or 2’-0-Me) and the 5’wing region of the gapmer (X in the 5'-X-Y-Z-3' formula) may comprise one or more 2’ -4’ bicyclic nucleosides (e.g., LNA or cEt).
  • non-bicyclic 2’-modified nucleosides e.g., 2’-MOE or 2’-0-Me
  • the 5’wing region of the gapmer (X in the 5'-X-Y-Z-3' formula) may comprise one or more 2’ -4’ bicyclic nucleosides (e.g., LNA or cEt).
  • a gapmer comprises a 5'-X-Y-Z-3' configuration, wherein X and Z is independently 1-7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleosides in length and Y is 6-10 (e.g., 6, 7, 8, 9, or 10) nucleosides in length, wherein each nucleoside in X is a non- bicyclic 2’-modified nucleosides (e.g., 2’-MOE or 2’-0-Me), each nucleoside in Z is a 2’-4’ bicyclic nucleosides (e.g., LNA or cEt), and each nucleoside in Y is a 2’-deoxyribonucleoside.
  • X and Z is independently 1-7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleosides in length and Y is 6-10 (e.g., 6, 7, 8, 9, or 10) nucleosides in length
  • each nucleoside in X is
  • the gapmer comprises a 5'-X-Y-Z-3' configuration, wherein X and Z is independently 1-7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleosides in length and Y is 6-10 (e.g., 6, 7, 8,
  • nucleosides in length wherein each nucleoside in X is a 2’-4’ bicyclic nucleosides (e.g., LNA or cEt), each nucleoside in Z is a non-bicyclic 2’-modified nucleosides (e.g., 2’- MOE or 2’-0-Me) and each nucleoside in Y is a 2’-deoxyribonucleoside.
  • each nucleoside in X is a 2’-4’ bicyclic nucleosides (e.g., LNA or cEt)
  • each nucleoside in Z is a non-bicyclic 2’-modified nucleosides (e.g., 2’- MOE or 2’-0-Me)
  • each nucleoside in Y is a 2’-deoxyribonucleoside.
  • the 5’wing region of a gapmer (X in the 5'-X-Y-Z-3' formula) comprises one or more non-bicyclic 2’-modified nucleosides (e.g., 2’-MOE or 2’-0- Me) and one or more 2’-4’ bicyclic nucleosides (e.g., LNA or cEt).
  • non-bicyclic 2’-modified nucleosides e.g., 2’-MOE or 2’-0- Me
  • 2’-4’ bicyclic nucleosides e.g., LNA or cEt
  • the 3 ’wing region of the gapmer (Z in the 5'-X-Y-Z-3' formula) comprises one or more non- bicyclic 2’-modified nucleosides (e.g., 2’-MOE or 2’-0-Me) and one or more 2’-4’ bicyclic nucleosides (e.g., LNA or cEt).
  • non- bicyclic 2’-modified nucleosides e.g., 2’-MOE or 2’-0-Me
  • 2’-4’ bicyclic nucleosides e.g., LNA or cEt
  • both the 5’wing region of the gapmer (X in the 5'-X-Y-Z-3' formula) and the 3 ’wing region of the gapmer (Z in the 5'-X-Y-Z-3' formula) comprise one or more non-bicyclic 2’-modified nucleosides (e.g., 2’-MOE or 2’-0- Me) and one or more 2’-4’ bicyclic nucleosides (e.g., LNA or cEt).
  • non-bicyclic 2’-modified nucleosides e.g., 2’-MOE or 2’-0- Me
  • 2’-4’ bicyclic nucleosides e.g., LNA or cEt
  • a gapmer comprises a 5'-X-Y-Z-3' configuration, wherein X and Z is independently 2-7 (e.g., 2, 3, 4, 5, 6, or 7) nucleosides in length and Y is 6- 10 (e.g., 6, 7, 8, 9, or 10) nucleosides in length, wherein at least one but not all (e.g., 1, 2, 3, 4,
  • positions 1, 2, 3, 4, 5, 6, or 7 in X (the 5’ most position is position 1) is a non- bicyclic 2’-modified nucleoside (e.g., 2’-MOE or 2’-0-Me), wherein the rest of the nucleosides in both X and Z are 2’ -4’ bicyclic nucleosides (e.g., LNA or cEt), and wherein each nucleoside in Y is a 2’deoxyribonucleoside.
  • a non- bicyclic 2’-modified nucleoside e.g., 2’-MOE or 2’-0-Me
  • the rest of the nucleosides in both X and Z are 2’ -4’ bicyclic nucleosides (e.g., LNA or cEt)
  • each nucleoside in Y is a 2’deoxyribonucleoside.
  • the gapmer comprises a 5'-X-Y-Z-3' configuration, wherein X and Z is independently 2-7 (e.g., 2, 3, 4, 5, 6, or 7) nucleosides in length and Y is 6-10 (e.g., 6, 7, 8, 9, or 10) nucleosides in length, wherein at least one but not all (e.g., 1, 2, 3, 4, 5, or 6) of positions 1, 2, 3, 4, 5, 6, or 7 in Z (the 5’ most position is position 1) is a non-bicyclic 2’-modified nucleoside (e.g., 2’-MOE or 2’-0-Me), wherein the rest of the nucleosides in both X and Z are 2’-4’ bicyclic nucleosides (e.g., LNA or cEt), and wherein each nucleoside in Y is a 2’deoxyribonucleoside.
  • the gapmer comprises a 5'-X-Y-Z-3' configuration, wherein
  • nucleosides in length and Y is 6-10 (e.g., 6, 7, 8, 9, or 10) nucleosides in length, wherein at least one but not all (e.g., 1, 2, 3, 4, 5, or 6) of positions 1, 2, 3, 4, 5, 6, or 7 in X and at least one of positions but not all (e.g., 1, 2, 3, 4, 5, or 6) 1, 2, 3, 4, 5, 6, or 7 in Z (the 5’ most position is position 1) is a non-bicyclic 2’-modified nucleoside (e.g., 2’-MOE or 2’-0-Me), wherein the rest of the nucleosides in both X and Z are 2’ -4’ bicyclic nucleosides (e.g., LNA or cEt), and wherein each nucleoside in Y is a 2’deoxyribonucleoside.
  • a non-bicyclic 2’-modified nucleoside e.g., 2’-MOE or 2’-0-M
  • Non-limiting examples of gapmers configurations with a mix of non-bicyclic 2’-modified nucleoside (e.g., 2’-MOE or 2’-0-Me) and 2’-4’ bicyclic nucleosides (e.g., LNA or cEt) in the 5 ’wing region of the gapmer (X in the 5'-X-Y-Z-3' formula) and/or the 3 ’wing region of the gapmer (Z in the 5'-X-Y-Z-3' formula) include: BBB-(D)n-BBBAA; KKK-(D)n- KKKAA; LLL-(D)n-LLLAA; BBB-(D)n-BBBEE; KKK-(D)n-KKKEE ; LLL-(D)n-LLLEE; BBB-(D)n-BBBAA; KKK-(D)n-KKKAA; LLL-(D)n-LLLAA; BBB-(D)n-B
  • a nucleosides comprise a 2'-modified nucleoside; “B” represents a 2’-4’ bicyclic nucleoside; “K” represents a constrained ethyl nucleoside (cEt); “L” represents an LNA nucleoside; and “E” represents a 2'- MOE modified ribonucleoside; “D” represents a 2’-deoxyribonucleoside; “n” represents the length of the gap segment (Y in the 5'-X-Y-Z-3' configuration) and is an integer between 1-20.
  • any one of the gapmers described herein comprises one or more modified nucleoside linkages (e.g., a phosphorothioate linkage) in each of the X, Y, and Z regions.
  • each intemucleoside linkage in the any one of the gapmers described herein is a phosphorothioate linkage.
  • each of the X, Y, and Z regions independently comprises a mix of phosphorothioate linkages and phosphodiester linkages.
  • each intemucleoside linkage in the gap region Y is a phosphorothioate linkage
  • the 5’wing region X comprises a mix of phosphorothioate linkages and phosphodiester linkages
  • the 3 ’wing region Z comprises a mix of phosphorothioate linkages and phosphodiester linkages.
  • an oligonucleotide described herein may be a mixmer or comprise a mixmer sequence pattern.
  • mixmers are oligonucleotides that comprise both naturally and non-naturally occurring nucleosides or comprise two different types of non- naturally occurring nucleosides typically in an alternating pattern.
  • Mixmers generally have higher binding affinity than unmodified oligonucleotides and may be used to specifically bind a target molecule, e.g., to block a binding site on the target molecule.
  • mixmers do not recruit an RNase to the target molecule and thus do not promote cleavage of the target molecule.
  • Such oligonucleotides that are incapable of recruiting RNase H have been described, for example, see W02007/112754 or W02007/112753.
  • the mixmer comprises or consists of a repeating pattern of nucleoside analogues and naturally occurring nucleosides, or one type of nucleoside analogue and a second type of nucleoside analogue.
  • a mixmer need not comprise a repeating pattern and may instead comprise any arrangement of modified nucleoside s and naturally occurring nucleoside s or any arrangement of one type of modified nucleoside and a second type of modified nucleoside.
  • the repeating pattern may, for instance be every second or every third nucleoside is a modified nucleoside, such as LNA, and the remaining nucleoside s are naturally occurring nucleosides, such as DNA, or are a 2' substituted nucleoside analogue such as 2'-MOE or 2' fluoro analogues, or any other modified nucleoside described herein. It is recognized that the repeating pattern of modified nucleoside, such as LNA units, may be combined with modified nucleoside at fixed positions — e.g. at the 5' or 3' termini.
  • a mixmer does not comprise a region of more than 5, more than 4, more than 3, or more than 2 consecutive naturally occurring nucleosides, such as DNA nucleosides.
  • the mixmer comprises at least a region consisting of at least two consecutive modified nucleoside, such as at least two consecutive LNAs.
  • the mixmer comprises at least a region consisting of at least three consecutive modified nucleoside units, such as at least three consecutive LNAs.
  • the mixmer does not comprise a region of more than 7, more than 6, more than 5, more than 4, more than 3, or more than 2 consecutive nucleoside analogues, such as LNAs.
  • LNA units may be replaced with other nucleoside analogues, such as those referred to herein.
  • Mixmers may be designed to comprise a mixture of affinity enhancing modified nucleosides, such as in non-limiting example LNA nucleosides and 2’-0-Me nucleosides.
  • a mixmer comprises modified internucleoside linkages (e.g ., phosphorothioate internucleoside linkages or other linkages) between at least two, at least three, at least four, at least five or more nucleosides.
  • a mixmer may be produced using any suitable method.
  • Representative U.S. patents, U.S. patent publications, and PCT publications that teach the preparation of mixmers include U.S. patent publication Nos. US20060128646, US20090209748, US20090298916, US20110077288, and US20120322851, and U.S. patent No. 7687617.
  • a mixmer comprises one or more morpholino nucleosides.
  • a mixmer may comprise morpholino nucleosides mixed (e.g., in an alternating manner) with one or more other nucleosides (e.g., DNA, RNA nucleosides) or modified nucleosides (e.g., LNA, 2’-0-Me nucleosides).
  • mixmers are useful for splice correcting or exon skipping, for example, as reported in Touznik A., et ah, LNA/DNA mixmer-based antisense oligonucleotides correct alternative splicing of the SMN2 gene and restore SMN protein expression in type 1 SMA fibroblasts Scientific Reports, volume 7, Article number: 3672 (2017), Chen S.
  • oligonucleotides provided herein may be in the form of small interfering RNAs (siRNA), also known as short interfering RNA or silencing RNA.
  • siRNA small interfering RNAs
  • SiRNA is a class of double- stranded RNA molecules, typically about 20-25 base pairs in length that target nucleic acids (e.g., mRNAs) for degradation via the RNA interference (RNAi) pathway in cells. Specificity of siRNA molecules may be determined by the binding of the antisense strand of the molecule to its target RNA. Effective siRNA molecules are generally less than 30 to 35 base pairs in length to prevent the triggering of non-specific RNA interference pathways in the cell via the interferon response, although longer siRNA can also be effective. In some embodiments, the siRNA molecules are 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or more base pairs in length. In some embodiments, the siRNA molecules are 8 to 30 base pairs in length, 10 to 15 base pairs in length, 10 to 20 base pairs in length, 15 to 25 base pairs in length, 19 to 21 base pairs in length, 21 to 23 base pairs in length.
  • target nucleic acids e.g.,
  • siRNA molecules that comprise a nucleotide sequence complementary to all or a portion of the target sequence, i.e. an antisense sequence, can be designed and prepared using appropriate methods (see, e.g., PCT Publication Number WO 2004/016735; and U.S. Patent Publication Nos. 2004/0077574 and 2008/0081791).
  • the siRNA molecule can be double stranded (i.e. a dsRNA molecule comprising an antisense strand and a complementary sense strand that hybridizes to form the dsRNA) or single- stranded (i.e. a ssRNA molecule comprising just an antisense strand).
  • the siRNA molecules can comprise a duplex, asymmetric duplex, hairpin or asymmetric hairpin secondary structure, having self-complementary sense and antisense strands.
  • the antisense strand of the siRNA molecule is 7, 8, 9, 10,
  • the antisense strand is 8 to 50 nucleotides in length, 8 to 40 nucleotides in length, 8 to 30 nucleotides in length, 10 to 15 nucleotides in length, 10 to 20 nucleotides in length, 15 to 25 nucleotides in length, 19 to 21 nucleotides in length, 21 to 23 nucleotides in lengths.
  • the sense strand of the siRNA molecule is 7, 8, 9, 10, 11,
  • the sense strand is 8 to 50 nucleotides in length
  • siRNA molecules comprise an antisense strand comprising a region of complementarity to a target region in a target mRNA.
  • the region of complementarity is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% complementary to a target region in a target mRNA.
  • the target region is a region of consecutive nucleotides in the target mRNA.
  • a complementary nucleotide sequence need not be 100% complementary to that of its target to be specifically hybridizable or specific for a target RNA sequence.
  • siRNA molecules comprise an antisense strand that comprises a region of complementarity to a target RNA sequence and the region of complementarity is in the range of 8 to 15, 8 to 30, 8 to 40, or 10 to 50, or 5 to 50, or 5 to 40 nucleotides in length.
  • a region of complementarity is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,
  • siRNA molecules comprise a nucleotide sequence that contains no more than 1, 2, 3, 4, or 5 base mismatches compared to the portion of the consecutive nucleotides of target RNA sequence. In some embodiments, siRNA molecules comprise a nucleotide sequence that has up to 3 mismatches over 15 bases, or up to 2 mismatches over 10 bases.
  • siRNA molecules comprise an antisense strand comprising a nucleotide sequence that is complementary (e.g., at least 85%, at least 90%, at least 95%, or 100%) to the target RNA sequence of the oligonucleotides provided herein. In some embodiments, siRNA molecules comprise an antisense strand comprising a nucleotide sequence that is at least 85%, at least 90%, at least 95%, or 100% identical to the oligonucleotides provided herein.
  • siRNA molecules comprise an antisense strand comprising at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25 or more consecutive nucleotides of the oligonucleotides provided herein.
  • Double-stranded siRNA may comprise sense and anti-sense RNA strands that are the same length or different lengths.
  • Double- stranded siRNA molecules can also be assembled from a single oligonucleotide in a stem-loop structure, wherein self-complementary sense and antisense regions of the siRNA molecule are linked by means of a nucleic acid based or non-nucleic acid-based linker(s), as well as circular single- stranded RNA having two or more loop structures and a stem comprising self-complementary sense and antisense strands, wherein the circular RNA can be processed either in vivo or in vitro to generate an active siRNA molecule capable of mediating RNAi.
  • Small hairpin RNA (shRNA) molecules thus are also contemplated herein. These molecules comprise a specific antisense sequence in addition to the reverse complement (sense) sequence, typically separated by a spacer or loop sequence. Cleavage of the spacer or loop provides a single- stranded RNA molecule and its reverse complement, such that they may anneal to form a dsRNA molecule (optionally with additional processing steps that may result in addition or removal of one, two, three or more nucleotides from the 3' end and/or (e.g., and) the 5' end of either or both strands).
  • shRNA Small hairpin RNA
  • a spacer can be of a sufficient length to permit the antisense and sense sequences to anneal and form a double- stranded structure (or stem) prior to cleavage of the spacer (and, optionally, subsequent processing steps that may result in addition or removal of one, two, three, four, or more nucleotides from the 3' end and/or (e.g., and) the 5' end of either or both strands).
  • a spacer sequence may be an unrelated nucleotide sequence that is situated between two complementary nucleotide sequence regions which, when annealed into a double- stranded nucleic acid, comprise a shRNA.
  • the overall length of the siRNA molecules can vary from about 14 to about 100 nucleotides depending on the type of siRNA molecule being designed. Generally between about 14 and about 50 of these nucleotides are complementary to the RNA target sequence, i.e. constitute the specific antisense sequence of the siRNA molecule. For example, when the siRNA is a double- or single-stranded siRNA, the length can vary from about 14 to about 50 nucleotides, whereas when the siRNA is a shRNA or circular molecule, the length can vary from about 40 nucleotides to about 100 nucleotides.
  • an siRNA molecule may comprise a 3' overhang at one end of the molecule.
  • the other end may be blunt-ended or have also an overhang (5' or 3') ⁇
  • the length of the overhangs may be the same or different.
  • the siRNA molecule of the present disclosure comprises 3' overhangs of about 1 to about 3 nucleotides on both ends of the molecule.
  • the siRNA molecule comprises 3’ overhangs of about 1 to about 3 nucleotides on the sense strand.
  • the siRNA molecule comprises 3’ overhangs of about 1 to about 3 nucleotides on the antisense strand.
  • the siRNA molecule comprises 3’ overhangs of about 1 to about 3 nucleotides on both the sense strand and the antisense strand.
  • the siRNA molecule comprises one or more modified nucleotides (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more). In some embodiments, the siRNA molecule comprises one or more modified nucleotides and/or (e.g., and) one or more modified intemucleotide linkages. In some embodiments, the modified nucleotide is a modified sugar moiety (e.g. a 2’ modified nucleotide).
  • the siRNA molecule comprises one or more 2’ modified nucleotides, e.g., a 2'-deoxy, 2'-fluoro (2’-F), 2'-0-methyl (2’-0-Me), 2'-0-methoxyethyl (2'-MOE), 2'-0-aminopropyl (2'-0-AP), 2'-0-dimethylaminoethyl (2'-0- DMAOE), 2'-0-dimethylaminopropyl (2'-0-DMAP), 2'-0-dimethylaminoethyloxyethyl (2'-0- DMAEOE), or 2'-0— N-methylacetamido (2'-0— NMA).
  • 2'-deoxy, 2'-fluoro (2’-F) 2'-0-methyl (2’-0-Me), 2'-0-methoxyethyl (2'-MOE), 2'-0-aminopropyl (2'-0-AP), 2'-0-dimethylamino
  • each nucleotide of the siRNA molecule is a modified nucleotide (e.g., a 2’ -modified nucleotide).
  • the siRNA molecule comprises one or more phosphorodiamidate morpholinos.
  • each nucleotide of the siRNA molecule is a phosphorodiamidate morpholino.
  • the siRNA molecule contains a phosphorothioate or other modified intemucleotide linkage. In some embodiments, the siRNA molecule comprises phosphorothioate internucleoside linkages. In some embodiments, the siRNA molecule comprises phosphorothioate internucleoside linkages between at least two nucleotides. In some embodiments, the siRNA molecule comprises phosphorothioate intemucleoside linkages between all nucleotides.
  • the siRNA molecule comprises modified intemucleotide linkages at the first, second, and/or (e.g., and) third intemucleoside linkage at the 5' or 3' end of the siRNA molecule.
  • the modified intemucleotide linkages are phosphorus- containing linkages.
  • phosphorus-containing linkages that may be used include, but are not limited to, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates comprising 3'alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates comprising 3 '-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5'
  • any of the modified chemistries or formats of siRNA molecules described herein can be combined with each other. For example, one, two, three, four, five, or more different types of modifications can be included within the same siRNA molecule.
  • the antisense strand comprises one or more modified nucleotides (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more). In some embodiments, the antisense strand comprises one or more modified nucleotides and/or (e.g., and) one or more modified intemucleotide linkages. In some embodiments, the modified nucleotide comprises a modified sugar moiety (e.g. a 2’ modified nucleotide).
  • the antisense strand comprises one or more 2’ modified nucleotides, e.g., a 2'-deoxy, 2'-fluoro (2’-F), 2'-0-methyl (2’-0-Me), 2'-0-methoxyethyl (2'-MOE), 2'-0-aminopropyl (2'-0-AP), 2'-0- dimethylaminoethyl (2'-0-DMAOE), 2'-0-dimethylaminopropyl (2'-0-DMAP), 2'-0- dimethylaminoethyloxyethyl (2'-0-DMAEOE), or 2'-0— N-methylacetamido (2'-0— NMA).
  • 2'-deoxy, 2'-fluoro (2’-F) 2'-0-methyl (2’-0-Me), 2'-0-methoxyethyl (2'-MOE), 2'-0-aminopropyl (2'-0-AP), 2'-0- dimethylaminoethy
  • each nucleotide of the antisense strand is a modified nucleotide (e.g., a 2’- modified nucleotide).
  • the antisense strand comprises one or more phosphorodiamidate morpholinos.
  • the antisense strand is a phosphorodiamidate morpholino oligomer (PMO).
  • antisense strand contains a phosphorothioate or other modified intemucleotide linkage. In some embodiments, the antisense strand comprises phosphorothioate internucleoside linkages. In some embodiments, the antisense strand comprises phosphorothioate internucleoside linkages between at least two nucleotides. In some embodiments, the antisense strand comprises phosphorothioate internucleoside linkages between all nucleotides.
  • the antisense strand comprises modified intemucleotide linkages at the first, second, and/or (e.g., and) third intemucleoside linkage at the 5' or 3' end of the siRNA molecule.
  • the modified intemucleotide linkages are phosphoms-containing linkages.
  • phosphorus-containing linkages that may be used include, but are not limited to, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates comprising 3'alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates comprising 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'; see US patent nos.
  • any of the modified chemistries or formats of the antisense strand described herein can be combined with each other. For example, one, two, three, four, five, or more different types of modifications can be included within the same antisense strand.
  • the sense strand comprises one or more modified nucleotides (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more). In some embodiments, the sense strand comprises one or more modified nucleotides and/or (e.g., and) one or more modified intemucleotide linkages. In some embodiments, the modified nucleotide is a modified sugar moiety (e.g. a 2’ modified nucleotide).
  • the sense strand comprises one or more 2’ modified nucleotides, e.g., a 2'-deoxy, 2'-fluoro (2’-F), 2'-0-methyl (2’-0-Me), 2'- O-methoxyethyl (2'-MOE), 2'-0-aminopropyl (2'-0-AP), 2'-0-dimethylaminoethyl (2'-0- DMAOE), 2'-0-dimethylaminopropyl (2'-0-DMAP), 2'-0-dimethylaminoethyloxyethyl (2'-0- DMAEOE), or 2'-0— N-methylacetamido (2'-0— NMA).
  • 2'-deoxy, 2'-fluoro (2’-F) 2'-0-methyl (2’-0-Me), 2'- O-methoxyethyl (2'-MOE
  • 2'-0-aminopropyl (2'-0-AP
  • each nucleotide of the sense strand is a modified nucleotide (e.g., a 2’ -modified nucleotide).
  • the sense strand comprises one or more phosphorodiamidate morpholinos.
  • the antisense strand is a phosphorodiamidate morpholino oligomer (PMO).
  • the sense strand contains a phosphorothioate or other modified intemucleotide linkage.
  • the sense strand comprises phosphorothioate intemucleoside linkages.
  • the sense strand comprises phosphorothioate intemucleoside linkages between at least two nucleotides. In some embodiments, the sense strand comprises phosphorothioate intemucleoside linkages between all nucleotides. For example, in some embodiments, the sense strand comprises modified intemucleotide linkages at the first, second, and/or (e.g., and) third intemucleoside linkage at the 5' or 3' end of the sense strand.
  • the modified intemucleotide linkages are phosphorus- containing linkages.
  • phosphorus-containing linkages that may be used include, but are not limited to, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates comprising 3'alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates comprising 3 '-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5
  • any of the modified chemistries or formats of the sense strand described herein can be combined with each other. For example, one, two, three, four, five, or more different types of modifications can be included within the same sense strand.
  • the antisense or sense strand of the siRNA molecule comprises modifications that enhance or reduce RNA-induced silencing complex (RISC) loading. In some embodiments, the antisense strand of the siRNA molecule comprises modifications that enhance RISC loading. In some embodiments, the sense strand of the siRNA molecule comprises modifications that reduce RISC loading and reduce off-target effects. In some embodiments, the antisense strand of the siRNA molecule comprises a 2'-0- methoxyethyl (2’-MOE) modification.
  • RISC RNA-induced silencing complex
  • the addition of the 2'-0-methoxyethyl (2’-MOE) group at the cleavage site improves both the specificity and silencing activity of siRNAs by facilitating the oriented RNA-induced silencing complex (RISC) loading of the modified strand, as described in Song et ah, (2017) Mol Ther Nucleic Acids 9:242-250, incorporated herein by reference in its entirety.
  • the antisense strand of the siRNA molecule comprises a 2'-OMe-phosphorodithioate modification, which increases RISC loading as described in Wu et ah, (2014) Nat Commun 5:3459, incorporated herein by reference in its entirety.
  • the sense strand of the siRNA molecule comprises a 5’- morpholino, which reduces RISC loading of the sense strand and improves antisense strand selection and RNAi activity, as described in Kumar et ah, (2019) Chem Commun (Camb) 55(35):5139-5142, incorporated herein by reference in its entirety.
  • the sense strand of the siRNA molecule is modified with a synthetic RNA-like high affinity nucleotide analogue, Locked Nucleic Acid (LNA), which reduces RISC loading of the sense strand and further enhances antisense strand incorporation into RISC, as described in Elman et ah, (2005) Nucleic Acids Res. 33(1): 439-447, incorporated herein by reference in its entirety.
  • LNA Locked Nucleic Acid
  • the sense strand of the siRNA molecule comprises a 5' unlocked nucleic acid (UNA) modification, which reduce RISC loading of the sense strand and improve silencing potency of the antisense strand, as described in Snead et al., (2013) Mol Ther Nucleic Acids 2(7):el03, incorporated herein by reference in its entirety.
  • the sense strand of the siRNA molecule comprises a 5-nitroindole modification, which decreased the RNAi potency of the sense strand and reduces off-target effects as described in Zhang et al., (2012) Chembiochem 13(13): 1940-1945, incorporated herein by reference in its entirety.
  • the sense strand comprises a 2’-0’methyl (2’-0-Me) modification, which reduces RISC loading and the off-target effects of the sense strand, as described in Zheng et al., FASEB (2013) 27(10): 4017-4026, incorporated herein by reference in its entirety.
  • the sense strand of the siRNA molecule is fully substituted with morpholino, 2’- MOE or 2’-0-Me residues, and are not recognized by RISC as described in Kole et al., (2012) Nature reviews. Drug Discovery 11(2): 125- 140, incorporated herein by reference in its entirety.
  • the antisense strand of the siRNA molecule comprises a 2’- MOE modification and the sense strand comprises an 2’-0-Me modification (see e.g., Song et al., (2017) Mol Ther Nucleic Acids 9:242-250).
  • at least one (e.g., at least 2, at least 3, at least 4, at least 5, at least 10) siRNA molecule is linked (e.g., covalently) to a muscle-targeting agent.
  • the muscle-targeting agent may comprise, or consist of, a nucleic acid (e.g., DNA or RNA), a peptide (e.g., an antibody), a lipid (e.g., a microvesicle), or a sugar moiety (e.g., a polysaccharide).
  • the muscle targeting agent is an antibody.
  • the muscle-targeting agent is an anti transferrin receptor antibody (e.g., any one of the anti-TfR antibodies provided herein).
  • the muscle-targeting agent may be linked to the 5’ end of the sense strand of the siRNA molecule.
  • the muscle-targeting agent may be linked to the 3’ end of the sense strand of the siRNA molecule. In some embodiments, the muscle-targeting agent may be linked internally to the sense strand of the siRNA molecule. In some embodiments, the muscle-targeting agent may be linked to the 5’ end of the antisense strand of the siRNA molecule. In some embodiments, the muscle-targeting agent may be linked to the 3’ end of the antisense strand of the siRNA molecule. In some embodiments, the muscle-targeting agent may be linked internally to the antisense strand of the siRNA molecule. k. microRNA (miRNAs)
  • an oligonucleotide may be a microRNA (miRNA).
  • miRNAs are small non-coding RNAs, belonging to a class of regulatory molecules that control gene expression by binding to complementary sites on a target RNA transcript.
  • miRNAs are generated from large RNA precursors (termed pri-miRNAs) that are processed in the nucleus into approximately 70 nucleotide pre-miRNAs, which fold into imperfect stem-loop structures.
  • pri-miRNAs large RNA precursors
  • pre-miRNAs typically undergo an additional processing step within the cytoplasm where mature miRNAs of 18-25 nucleotides in length are excised from one side of the pre-miRNA hairpin by an RNase III enzyme, Dicer.
  • miRNAs including pri-miRNA, pre-miRNA, mature miRNA or fragments of variants thereof that retain the biological activity of mature miRNA.
  • the size range of the miRNA can be from 21 nucleotides to 170 nucleotides. In one embodiment the size range of the miRNA is from 70 to 170 nucleotides in length. In another embodiment, mature miRNAs of from 21 to 25 nucleotides in length can be used. l. Aptamers
  • oligonucleotides provided herein may be in the form of aptamers.
  • aptamer is any nucleic acid that binds specifically to a target, such as a small molecule, protein, nucleic acid in a cell.
  • the aptamer is a DNA aptamer or an RNA aptamer.
  • a nucleic acid aptamer is a single- stranded DNA or RNA (ssDNA or ssRNA). It is to be understood that a single- stranded nucleic acid aptamer may form helices and/or (e.g., and) loop structures.
  • the nucleic acid that forms the nucleic acid aptamer may comprise naturally occurring nucleotides, modified nucleotides, naturally occurring nucleotides with hydrocarbon linkers (e.g., an alkylene) or a polyether linker (e.g., a PEG linker) inserted between one or more nucleotides, modified nucleotides with hydrocarbon or PEG linkers inserted between one or more nucleotides, or a combination of thereof.
  • hydrocarbon linkers e.g., an alkylene
  • a polyether linker e.g., a PEG linker
  • oligonucleotides provided herein may be in the form of a ribozyme.
  • a ribozyme ribonucleic acid enzyme
  • Ribozymes are molecules with catalytic activities including the ability to cleave at specific phosphodiester linkages in RNA molecules to which they have hybridized, such as mRNAs, RNA-containing substrates, IncRNAs, and ribozymes, themselves.
  • Ribozymes may assume one of several physical structures, one of which is called a "hammerhead.”
  • a hammerhead ribozyme is composed of a catalytic core containing nine conserved bases, a double-stranded stem and loop structure (stem-loop II), and two regions complementary to the target RNA flanking regions the catalytic core. The flanking regions enable the ribozyme to bind to the target RNA specifically by forming double-stranded stems I and III.
  • Cleavage occurs in cis (i.e., cleavage of the same RNA molecule that contains the hammerhead motif) or in trans (cleavage of an RNA substrate other than that containing the ribozyme) next to a specific ribonucleotide triplet by a transesterification reaction from a 3', 5'- phosphate diester to a 2', 3'-cyclic phosphate diester.
  • this catalytic activity requires the presence of specific, highly conserved sequences in the catalytic region of the ribozyme.
  • Ribozyme oligonucleotides can be prepared using well known methods (see, e.g., PCT Publications W09118624; W09413688; W09201806; and WO 92/07065; and U.S. Patents 5436143 and 5650502) or can be purchased from commercial sources (e.g., US Biochemicals) and, if desired, can incorporate nucleotide analogs to increase the resistance of the oligonucleotide to degradation by nucleases in a cell.
  • the ribozyme may be synthesized in any known manner, e.g., by use of a commercially available synthesizer produced, e.g., by Applied Biosystems, Inc.
  • the ribozyme may also be produced in recombinant vectors by conventional means. See, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory (Current edition).
  • the ribozyme RNA sequences maybe synthesized conventionally, for example, by using RNA polymerases such as T7 or SP6. n. Guide Nucleic Acids
  • oligonucleotides are guide nucleic acid, e.g., guide RNA (gRNA) molecules.
  • a guide RNA is a short synthetic RNA composed of (1) a scaffold sequence that binds to a nucleic acid programmable DNA binding protein (napDNAbp), such as Cas9, and (2) a nucleotide spacer portion that defines the DNA target sequence (e.g., genomic DNA target) to which the gRNA binds in order to bring the nucleic acid programmable DNA binding protein in proximity to the DNA target sequence.
  • napDNAbp nucleic acid programmable DNA binding protein
  • the napDNAbp is a nucleic acid-programmable protein that forms a complex with (e.g., binds or associates with) one or more RNA(s) that targets the nucleic acid- programmable protein to a target DNA sequence (e.g., a target genomic DNA sequence).
  • a nucleic acid -programmable nuclease when in a complex with an RNA, may be referred to as a nuclease:RNA complex.
  • Guide RNAs can exist as a complex of two or more RNAs, or as a single RNA molecule.
  • gRNAs Guide RNAs
  • sgRNAs single-guide RNAs
  • gRNAs guide RNAs
  • gRNAs that exist as a single RNA species comprise two domains: (1) a domain that shares homology to a target nucleic acid (i.e., directs binding of a Cas9 complex to the target); and (2) a domain that binds a Cas9 protein.
  • domain (2) corresponds to a sequence known as a tracrRNA and comprises a stem-loop structure.
  • domain (2) is identical or homologous to a tracrRNA as provided in Jinek et ah, Science 337:816-821 (2012), the entire contents of which is incorporated herein by reference.
  • a gRNA comprises two or more of domains (1) and (2), and may be referred to as an extended gRNA.
  • an extended gRNA will bind two or more Cas9 proteins and bind a target nucleic acid at two or more distinct regions, as described herein.
  • the gRNA comprises a nucleotide sequence that complements a target site, which mediates binding of the nuclease/RNA complex to said target site, providing the sequence specificity of the nuclease:RNA complex.
  • the RNA- programmable nuclease is the (CRISPR-associated system) Cas9 endonuclease, for example, Cas9 (Csnl) from Streptococcus pyogenes (see, e.g., “Complete genome sequence of an Ml strain of Streptococcus pyogenes.” Ferretti J.J., McShan W.M., Ajdic D.J., Savic D.J., Savic
  • molecular payloads may comprise multimers (e.g., concatemers) of 2 or more oligonucleotides connected by a linker.
  • the oligonucleotide loading of a complex/conjugate can be increased beyond the available linking sites on a targeting agent (e.g., available thiol sites or amine sites on an antibody) or otherwise tuned to achieve a particular payload loading content.
  • Oligonucleotides in a multimer can be the same or different (e.g., targeting different genes or different sites on the same gene or products thereof).
  • multimers comprise 2 or more oligonucleotides linked together by a cleavable linker. However, in some embodiments, multimers comprise 2 or more oligonucleotides linked together by a non-cleavable linker. In some embodiments, a multimer comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more oligonucleotides linked together. In some embodiments, a multimer comprises 2 to 5, 2 to 10 or 4 to 20 oligonucleotides linked together. [000252] In some embodiments, a multimer comprises 2 or more oligonucleotides linked end-to-end (in a linear arrangement).
  • a multimer comprises 2 or more oligonucleotides linked end-to-end via an oligonucleotide based linker (e.g., poly-dT linker, an abasic linker).
  • a multimer comprises a 5’ end of one oligonucleotide linked to a 3’ end of another oligonucleotide.
  • a multimer comprises a 3’ end of one oligonucleotide linked to a 3’ end of another oligonucleotide.
  • a multimer comprises a 5’ end of one oligonucleotide linked to a 5’ end of another oligonucleotide.
  • multimers can comprise a branched structure comprising multiple oligonucleotides linked together by a branching linker.
  • an oligonucleotide (e.g., an antisense oligonucleotide including a morpholino) of the present disclosure target splicing.
  • the oligonucleotide targets splicing by inducing exon skipping and restoring the reading frame within a gene.
  • the oligonucleotide may induce skipping of an exon encoding a frameshift mutation and/or (e.g., and) an exon that encodes a premature stop codon.
  • an oligonucleotide may induce exon skipping by blocking spliceosome recognition of a splice site.
  • exon skipping results in a truncated but functional protein compared to the reference protein (e.g., truncated but functional DMD protein as described below).
  • the oligonucleotide promotes inclusion of a particular exon (e.g., exon 7 of the SMN2 gene described below).
  • an oligonucleotide may induce inclusion of an exon by targeting a splice site inhibitory sequence.
  • RNA splicing has been implicated in muscle diseases, including Duchenne muscular dystrophy (DMD) and spinal muscular atrophy (SMA).
  • an oligonucleotide of the present disclosure promotes skipping of one or more DMD exons (e.g., exon 8, exon 43, exon 44, exon 45, exon 50, exon 51, exon 52, exon 53, and/or (e.g., and) exon 55) and results in a functional truncated protein. See, e.g., U.S. Patent No. 8,486,907 published on July 16, 2013 and U.S. 20140275212 published on September 18, 2014.
  • an oligonucleotide of the present disclosure promotes inclusion of SMN2 exon 7.
  • an oligonucleotide is an antisense oligonucleotide that targets SMN2 splice site inhibitory sequences (see, e.g., US Patent Number 7,838,657, which was published on November 23, 2010).
  • Any suitable small molecule may be used as a molecular payload, as described herein.
  • a protein is an enzyme (e.g ., an acid alpha- glucosidase, e.g., as encoded by the GAA gene).
  • an enzyme e.g ., an acid alpha- glucosidase, e.g., as encoded by the GAA gene.
  • These peptides or proteins may be produced, synthesized, and/or (e.g., and) derivatized using several methodologies, e.g. phage displayed peptide libraries, one-bead one-compound peptide libraries, or positional scanning synthetic peptide combinatorial libraries. Exemplary methodologies have been characterized in the art and are incorporated by reference (Gray, B.P. and Brown, K.C. “Combinatorial Peptide Libraries: Mining for Cell-Binding Peptides” Chem Rev. 2014, 114:2, 1020-1081.;
  • a gene expression construct may be a vector or a cDNA fragment.
  • a gene expression construct may be messenger RNA (mRNA).
  • mRNA messenger RNA
  • a mRNA used herein may be a modified mRNA, e.g., as described in US Patent 8,710,200, issued on April 24, 2014, entitled “ Engineered nucleic acids encoding a modified erythropoietin and their expression” .
  • a mRNA may comprise a 5 '-methyl cap.
  • a mRNA may comprise a polyA tail, optionally of up to 160 nucleotides in length.
  • a gene expression construct may encode a sequence of a protein that is deficient in a muscle disease. In some embodiments, the gene expression construct may be expressed, e.g., overexpressed, within the nucleus of a muscle cell. In some embodiments, the gene expression construct encodes a gene that is deficient in a muscle disease. In some embodiments, the gene expression construct encodes a protein that comprises at least one zinc finger. In some embodiments, the gene expression construct encodes a protein that binds to a gene in Table 6.
  • the gene expression construct encodes a protein that leads to a reduction in the expression of a protein (e.g., mutant protein) encoded by a gene in Table 6.
  • the gene expression construct encodes a gene editing enzyme. Additional examples of nucleic acid constructs that may be used as molecular payloads are provided in International Patent Application Publication WO2017152149A1, published on September 19, 2017, entitled, “CLOSED-ENDED LINEAR DUPLEX DNA FOR NON- VIRAL GENE TRANSFER”; US Patent 8,853,377B2, issued on October 7, 2014, entitled, “MRNA FOR USE IN TREATMENT OF HUMAN GENETIC DISEASES”; and US Patent US8822663B2, issued on September 2, 2014, ENGINEERED NUCLEIC ACIDS AND METHODS OF USE THEREOF,” the contents of each of which are incorporated herein by reference in their entireties.
  • any suitable detectable label or diagnostic agent can be used as the molecular payload of the present disclosure.
  • a “diagnostic agent” refers to an agent that is used for diagnostic purpose, e.g., by detecting another molecule in a cell or a tissue.
  • the diagnostic agent is an agent that targets (e.g., binds) a biomarker known to be associated with a disease (e.g., a nucleic acid biomarker, protein biomarker, or a metabolite biomarker) in a subject and produces a detectable signal, which can be used to determine the presence/absence of the biomarker, thus to diagnose a disease.
  • the diagnostic agent may be, without limitation, an antibody or an antisense nucleic acid.
  • the diagnostic agent contains a detectable label.
  • a detectable label refers to a moiety that has at least one element, isotope, or a structural or functional group incorporated that enables detection of a molecule, e.g., a protein or polypeptide, or other entity, to which the diagnostic agent binds.
  • a detectable label falls into any one (or more) of five classes: a) an agent which contains isotopic moieties, which may be radioactive or heavy isotopes, including, but not limited to, 2H, 3H, 13C, 14C, 15N, 18F, 3 IP, 32P, 35S, 67Ga, 76Br, 99mTc (Tc-99m), lllln, 1231, 1251, 1311, 153Gd, 169Yb, and 186Re; b) an agent which contains an immune moiety, which may be an antibody or antigen, which may be bound to an enzyme (e.g., such as horseradish peroxidase); c) an agent comprising a colored, luminescent, phosphorescent, or fluorescent moiety (e.g., such as the fluorescent label fluorescein isothiocyanate (FITC); d) an agent which has one or more photo affinity moieties; and e) an agent which is a lig
  • a detectable label comprises a radioactive isotope. In some embodiments, a detectable label comprises a fluorescent moiety. In some embodiments, the detectable label comprises a dye, e.g., a fluorescent dye, e.g., fluorescein isothiocyanate, Texas red, rhodamine, Cy3, Cy5,
  • the detectable label comprises biotin.
  • the detectable molecule is a fluorescent polypeptide (e.g., GFP or a derivative thereof such as enhanced GFP (EGFP)) or a lucif erase (e.g., a firefly, Renilla, or Gaussia luciferase).
  • a detectable label may react with a suitable substrate (e.g., a luciferin) to generate a detectable signal.
  • suitable substrate e.g., a luciferin
  • fluorescent proteins include GFP and derivatives thereof, proteins comprising chromophores that emit light of different colors such as red, yellow, and cyan fluorescent proteins, etc.
  • Exemplary fluorescent proteins include, e.g., Sirius, Azurite, EBFP2, TagBFP, mTurquoise, ECFP, Cerulean, TagCFP, mTFPl, mUkGl, mAGl, AcGFPl, TagGFP2, EGFP, mWasabi, EmGFP, TagYPF, EYFP, Topaz, SYFP2, Venus, Citrine, mKO, mK02, mOrange, mOrange2, TagRFP, TagRFP-T, mStrawberry, mRuby, mCherry, mRaspberry, mKate2, mPlum, mNeptune, T- Sapphire, mAmetrine, mKeima.
  • a detectable label comprises a dark quencher, e.g., a substance that absorbs excitation energy from a fluorophore and dissipates the energy as heat.
  • Complexes described herein generally comprise a linker that connects any one of the anti-TfR antibodies described herein to a molecular payload.
  • a linker comprises at least one covalent bond.
  • a linker may be a single bond, e.g., a disulfide bond or disulfide bridge, that connects an anti-TfR antibody to a molecular payload.
  • a linker may connect any one of the anti-TfR antibodies described herein to a molecular payload through multiple covalent bonds.
  • a linker may be a cleavable linker.
  • a linker may be a non-cleavable linker.
  • a linker is generally stable in vitro and in vivo, and may be stable in certain cellular environments. Additionally, generally a linker does not negatively impact the functional properties of either the anti-TfR antibody or the molecular payload. Examples and methods of synthesis of linkers are known in the art (see, e.g. Kline, T. et al. “Methods to Make Homogenous Antibody Drug Conjugates.” Pharmaceutical Research, 2015, 32:11, 3480- 3493.; Jain, N. et al. “Current ADC Linker Chemistry” Pharm Res. 2015, 32:11, 3526-3540.; McCombs, J.R. and Owen, S.C. “Antibody Drug Conjugates: Design and Selection of Linker, Payload and Conjugation Chemistry” AAPS J. 2015, 17:2, 339-351.).
  • a precursor to a linker typically will contain two different reactive species that allow for attachment to both the anti-TfR antibody and a molecular payload.
  • the two different reactive species may be a nucleophile and/or (e.g., and) an electrophile.
  • a linker is connected to an anti-TfR antibody via conjugation to a lysine residue or a cysteine residue of the anti-TfR antibody.
  • a linker is connected to a cysteine residue of an anti-TfR antibody via a maleimide-containing linker, wherein optionally the maleimide-containing linker comprises a maleimidocaproyl or maleimidomethyl cyclohexane- 1-carboxylate group.
  • a linker is connected to a cysteine residue of an anti-TfR antibody or thiol functionalized molecular payload via a 3-arylpropionitrile functional group.
  • a linker is connected to a lysine residue of an anti-TfR antibody.
  • a linker is connected to an anti-TfR antibody and/or (e.g., and) a molecular payload via an amide bond, a carbamate bond, a hydrazide, a trizaole, a thioether, or a disulfide bond.
  • a cleavable linker may be a protease-sensitive linker, a pH-sensitive linker, or a glutathione- sensitive linker. These linkers are generally cleavable only intracellularly and are preferably stable in extracellular environments, e.g. extracellular to a muscle cell.
  • Protease-sensitive linkers are cleavable by protease enzymatic activity. These linkers typically comprise peptide sequences and may be 2-10 amino acids, about 2-5 amino acids, about 5-10 amino acids, about 10 amino acids, about 5 amino acids, about 3 amino acids, or about 2 amino acids in length.
  • a peptide sequence may comprise naturally-occurring amino acids, e.g. cysteine, alanine, or non-naturally-occurring or modified amino acids.
  • Non-naturally occurring amino acids include b-amino acids, homo amino acids, proline derivatives, 3-substituted alanine derivatives, linear core amino acids, N- methyl amino acids, and others known in the art.
  • a protease- sensitive linker comprises a valine-citmlline or alanine-citrulline dipeptide sequence.
  • a protease- sensitive linker can be cleaved by a lysosomal protease, e.g. cathepsin B, and/or (e.g., and) an endosomal protease.
  • a pH-sensitive linker is a covalent linkage that readily degrades in high or low pH environments.
  • a pH-sensitive linker may be cleaved at a pH in a range of 4 to 6.
  • a pH-sensitive linker comprises a hydrazone or cyclic acetal.
  • a pH-sensitive linker is cleaved within an endosome or a lysosome.
  • a glutathione-sensitive linker comprises a disulfide moiety.
  • a glutathione-sensitive linker is cleaved by a disulfide exchange reaction with a glutathione species inside a cell.
  • the disulfide moiety further comprises at least one amino acid, e.g. a cysteine residue.
  • the linker is a Val-cit linker (e.g., as described in US Patent 6,214,345, incorporated herein by reference).
  • the val-cit linker before conjugation, has a structure of:
  • the val-cit linker after conjugation, has a structure of:
  • the Val-cit linker is attached to a reactive chemical moiety (e.g., SPAAC for click chemistry conjugation).
  • a reactive chemical moiety e.g., SPAAC for click chemistry conjugation
  • the val-cit linker attached to a reactive chemical moiety has the structure of: wherein n is any number from 0-10. In some embodiments, n is 3.
  • the val-cit linker attached to a reactive chemical moiety is conjugated (e.g., via a different chemical moiety) to a molecular payload (e.g., an oligonucleotide).
  • a reactive chemical moiety e.g., SPAAC for click chemistry conjugation
  • a molecular payload e.g., an oligonucleotide
  • the val-cit linker attached to a reactive chemical moiety (e.g., SPAAC for click chemistry conjugation) and conjugated to a molecular payload has the structure of (before click chemistry conjugation): wherein n is any number from 0-10. In some embodiments, n is 3.
  • the val-cit linker after conjugation to a molecular payload (e.g., an oligonucleotide), the val-cit linker has a structure of: wherein n is any number from 0-10, and wherein m is any number from 0-10. In some embodiments, n is 3 and m is 4. ii. Non-Cleavable Linkers
  • non-cleavable linkers may be used. Generally, a non- cleavable linker cannot be readily degraded in a cellular or physiological environment.
  • a non-cleavable linker comprises an optionally substituted alkyl group, wherein the substitutions may include halogens, hydroxyl groups, oxygen species, and other common substitutions.
  • a linker may comprise an optionally substituted alkyl, an optionally substituted alkylene, an optionally substituted arylene, a heteroarylene, a peptide sequence comprising at least one non-natural amino acid, a truncated glycan, a sugar or sugars that cannot be enzymatically degraded, an azide, an alkyne-azide, a peptide sequence comprising a LPXT sequence, a thioether, a biotin, a biphenyl, repeating units of polyethylene glycol or equivalent compounds, acid esters, acid amides, sulfamides, and/or (e.g., and) an alkoxy-amine linker.
  • sortase-mediated ligation will be utilized to covalently link an anti-TfR antibody comprising a LPXT sequence to a molecular payload comprising a (G) n sequence (see, e.g. Proft T. Sortase-mediated protein ligation: an emerging biotechnology tool for protein modification and immobilization ⁇ Biotechnol Lett. 2010,
  • a linker may comprise a substituted alkylene, an optionally substituted alkenylene, an optionally substituted alkynylene, an optionally substituted cycloalkylene, an optionally substituted cycloalkenylene, an optionally substituted arylene, an optionally substituted heteroarylene further comprising at least one heteroatom selected from N, O, and S,; an optionally substituted heterocyclylene further comprising at least one heteroatom selected from N, O, and S,; an imino, an optionally substituted nitrogen species, an optionally substituted oxygen species O, an optionally substituted sulfur species, or a poly(alkylene oxide), e.g. polyethylene oxide or polypropylene oxide iii. Linker conjugation
  • a linker is connected to an anti-TfR antibody and/or (e.g., and) molecular payload via a phosphate, thioether, ether, carbon-carbon, carbamate, or amide bond.
  • a linker is connected to an oligonucleotide through a phosphate or phosphorothioate group, e.g. a terminal phosphate of an oligonucleotide backbone.
  • a linker is connected to an anti-TfR antibody, through a lysine or cysteine residue present on the anti-TfR antibody.
  • a linker is connected to an anti-TfR antibody and/or (e.g., and) molecular payload by a cycloaddition reaction between an azide and an alkyne to form a triazole, wherein the azide and the alkyne may be located on the anti-TfR antibody, molecular payload, or the linker.
  • an alkyne may be a cyclic alkyne, e.g., a cyclooctyne.
  • an alkyne may be bicyclononyne (also known as bicyclo[6.1.0]nonyne or BCN) or substituted bicyclononyne.
  • a cyclooctane is as described in International Patent Application Publication WO2011136645, published on November 3, 2011, entitled, “ Fused Cyclooctyne Compounds And Their Use In Metal-free Click Reactions”.
  • an azide may be a sugar or carbohydrate molecule that comprises an azide.
  • an azide may be 6-azido-6- deoxygalactose or 6-azido-N-acetylgalactosamine.
  • a sugar or carbohydrate molecule that comprises an azide is as described in International Patent Application Publication W02016170186, published on October 27, 2016, entitled, “ Process For The Modification Of A Glycoprotein Using A Glycosyltransferase That Is Or Is Derived From A b(l,4)-N-Acetylgalactosaminyltransferase”.
  • a cycloaddition reaction between an azide and an alkyne to form a triazole wherein the azide and the alkyne may be located on the anti-TfR antibody, molecular payload, or the linker is as described in International Patent Application Publication WO2014065661, published on May 1, 2014, entitled, “ Modified antibody, antibody-conjugate and process for the preparation thereof or International Patent Application Publication W02016170186, published on October 27, 2016, entitled, “ Process For The Modification Of A Glycoprotein Using A Glycosyltransferase That Is Or Is Derived From A b(1, 4)-N-Acetylgalactosaminyltransj erase” .
  • a linker further comprises a spacer, e.g., a polyethylene glycol spacer or an acyl/carbomoyl sulfamide spacer, e.g., a HydraSpaceTM spacer.
  • a spacer is as described in Verkade, J.M.M. et ah, “A Polar Sulfamide Spacer Significantly Enhances the Manufacturability, Stability, and Therapeutic Index of Antibody- Drug Conjugates” , Antibodies, 2018, 7, 12.
  • a linker is connected to an anti-TfR antibody and/or (e.g., and) molecular payload by the Diels-Alder reaction between a dienophile and a diene/hetero-diene, wherein the dienophile and the diene/hetero-diene may be located on the anti-TfR antibody, molecular payload, or the linker.
  • a linker is connected to an anti-TfR antibody and/or (e.g., and) molecular payload by other pericyclic reactions, e.g. ene reaction.
  • a linker is connected to an anti-TfR antibody and/or (e.g., and) molecular payload by an amide, thioamide, or sulfonamide bond reaction.
  • a linker is connected to an anti-TfR antibody and/or (e.g., and) molecular payload by a condensation reaction to form an oxime, hydrazone, or semicarbazide group existing between the linker and the anti-TfR antibody and/or (e.g., and) molecular payload.
  • a linker is connected to an anti-TfR antibody and/or (e.g., and) molecular payload by a conjugate addition reaction between a nucleophile, e.g. an amine or a hydroxyl group, and an electrophile, e.g. a carboxylic acid or an aldehyde.
  • a nucleophile e.g. an amine or a hydroxyl group
  • an electrophile e.g. a carboxylic acid or an aldehyde.
  • a nucleophile may exist on a linker and an electrophile may exist on an anti-TfR antibody or molecular payload prior to a reaction between a linker and an anti-TfR antibody or molecular payload.
  • an electrophile may exist on a linker and a nucleophile may exist on an anti-TfR antibody or molecular payload prior to a reaction between a linker and an anti-TfR antibody or molecular payload.
  • an electrophile may be an azide, pentafluorophenyl, a silicon centers, a carbonyl, a carboxylic acid, an anhydride, an isocyanate, a thioisocyanate, a succinimidyl ester, a sulfosuccinimidyl ester, a maleimide, an alkyl halide, an alkyl pseudohalide, an epoxide, an episulfide, an aziridine, an aryl, an activated phosphorus center, and/or (e.g., and) an activated sulfur center.
  • a nucleophile may be an optionally substituted alkene, an optionally substituted alkyne, an optionally substituted aryl, an optionally substituted heterocyclyl, a hydroxyl group, an amino group, an alkylamino group, an anilido group, or a thiol group.
  • the val-cit linker attached to a reactive chemical moiety e.g., SPAAC for click chemistry conjugation
  • SPAAC for click chemistry conjugation
  • the val-cit linker attached to a reactive chemical moiety is conjugated to an anti-TfR antibody having a structure of: wherein m is any number from 0-10. In some embodiments, m is 4.
  • the val-cit linker attached to a reactive chemical moiety e.g., SPAAC for click chemistry conjugation
  • conjugated to an anti-TfR antibody has a structure of: wherein n is any number from 0-10, wherein m is any number from 0-10. In some embodiments, n is 3 and/or (e.g., and) m is 4.
  • the val-cit linker that links the antibody and the molecular payload has a structure of: wherein n is any number from 0-10, wherein m is any number from 0-10. In some embodiments, n is 3 and/or (e.g., and) m is 4. In some embodiments, n is 3 and/or (e.g., and) m is 4.
  • X is NH (e.g., NH from an amine group of a lysine), S (e.g., S from a thiol group of a cysteine), or O (e.g., O from a hydroxyl group of a serine, threonine, or tyrosine) of the antibody.
  • NH e.g., NH from an amine group of a lysine
  • S e.g., S from a thiol group of a cysteine
  • O e.g., O from a hydroxyl group of a serine, threonine, or tyrosine
  • the complex described herein has a structure of: wherein n is any number from 0-10, wherein m is any number from 0-10. In some embodiments, n is 3 and/or (e.g., and) m is 4.
  • LI is linked to a 5’ phosphate of the oligonucleotide.
  • LI is optional (e.g., need not be present).
  • any one of the complexes described herein has a structure of: HN antibody wherein n is 0-15 (e.g., 3) and m is 0-15 (e.g., 4).
  • complexes comprising any one the anti-TfR antibodies described herein covalently linked to any of the molecular payloads (e.g., an oligonucleotide) described herein.
  • the anti-TfR antibody e.g., any one of the anti-TfR antibody provided in Table 2
  • a molecular payload e.g., an oligonucleotide
  • Any of the linkers described herein may be used.
  • the linker is linked to the 5' end, the 3' end, or internally of the oligonucleotide.
  • the linker is linked to the anti-TfR antibody via a thiol-reactive linkage (e.g., via a cysteine in the anti-TfR antibody).
  • the linker e.g., a Val-cit linker
  • the antibody e.g., an anti-TfR antibody described herein
  • an amine group e.g., via a lysine in the antibody.
  • n is a number between 0-10
  • m is a number between 0-10
  • the linker is linked to the antibody via an amine group (e.g., on a lysine residue), and/or (e.g., and) wherein the linker is linked to the oligonucleotide (e.g., at the 5’ end, 3’ end, or internally).
  • the linker is linked to the antibody via a lysine, the linker is linked to the oligonucleotide at the 5’ end, n is 3, and m is 4.
  • the molecular payload is an oligonucleotide comprising a sense strand and an antisense strand, and the linker is linked to the sense strand or the antisense strand at the 5’ end or the 3’ end.
  • antibodies can be linked to molecular payloads with different stoichiometries, a property that may be referred to as a drug to antibody ratios (DAR) with the “drug” being the molecular payload.
  • DAR drug to antibody ratios
  • three molecular payloads 3).
  • an average DAR of complexes in such a mixture may be in a range of 1 to 3, 1 to 4, 1 to 5 or more.
  • DAR may be increased by conjugating molecular payloads to different sites on an antibody and/or (e.g., and) by conjugating multimers to one or more sites on antibody.
  • a DAR of 2 may be achieved by conjugating a single molecular payload to two different sites on an antibody or by conjugating a dimer molecular payload to a single site of an antibody.
  • the complex described herein comprises an anti-TfR antibody described herein (e.g., antibodies in Tables 2-5) covalently linked to a molecular payload.
  • the complex described herein comprises an anti-TfR antibody described herein (e.g., antibodies in Tables 2-5) covalently linked to molecular payload via a linker (e.g., a Val-cit linker).
  • the linker e.g., a Val-cit linker
  • the linker is linked to the antibody (e.g., an anti-TfR antibody described herein) via a thiol-reactive linkage (e.g., via a cysteine in the antibody).
  • the linker e.g., a Val-cit linker
  • the antibody e.g., an anti-TfR antibody described herein
  • an amine group e.g., via a lysine in the antibody
  • the molecular payload is an oligonucleotide comprising a region of complementarity of at least 15 nucleotides to any one of the gene target sequences described herein.
  • the complex described herein comprises an anti-TfR antibody covalently linked to a molecular payload, wherein the anti-TfR antibody comprises a CDR-H1, a CDR-H2, and a CDR-H3 that are the same as the CDR-H1, CDR-H2, and CDR- H3 shown in Table 2; and a CDR-L1, a CDR-L2, and a CDR-L3 that are the same as the CDR- Ll, CDR-L2, and CDR-L3 shown in Table 2.
  • the complex described herein comprises an anti-TfR antibody covalently linked to a molecular payload, wherein the anti-TfR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 69, SEQ ID NO: 71, or SEQ ID NO: 72, and a VL comprising the amino acid sequence of SEQ ID NO: 70.
  • the complex described herein comprises an anti-TfR antibody covalently linked to a molecular payload, wherein the anti-TfR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 73 or SEQ ID NO: 76, and a VL comprising the amino acid sequence of SEQ ID NO: 74.
  • the complex described herein comprises an anti-TfR antibody covalently linked to a molecular payload, wherein the anti-TfR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 73 or SEQ ID NO: 76, and a VL comprising the amino acid sequence of SEQ ID NO: 75.
  • the complex described herein comprises an anti-TfR antibody covalently linked to a molecular payload, wherein the anti-TfR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 77, and a VL comprising the amino acid sequence of SEQ ID NO: 78.
  • the complex described herein comprises an anti-TfR antibody covalently linked to a molecular payload, wherein the anti-TfR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 77 or SEQ ID NO: 79, and a VL comprising the amino acid sequence of SEQ ID NO: 80.
  • the complex described herein comprises an anti-TfR antibody covalently linked to a molecular payload, wherein the anti-TfR antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 84, SEQ ID NO: 86 or SEQ ID NO: 87 and a light chain comprising the amino acid sequence of SEQ ID NO: 85.
  • the complex described herein comprises an anti-TfR antibody covalently linked to a molecular payload, wherein the anti-TfR antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 88 or SEQ ID NO: 91, and a light chain comprising the amino acid sequence of SEQ ID NO: 89.
  • the complex described herein comprises an anti-TfR antibody covalently linked to a molecular payload, wherein the anti-TfR antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 88 or SEQ ID NO: 91, and a light chain comprising the amino acid sequence of SEQ ID NO: 90.
  • the complex described herein comprises an anti-TfR antibody covalently linked to a molecular payload, wherein the anti-TfR antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 92 or SEQ ID NO: 94, and a light chain comprising the amino acid sequence of SEQ ID NO: 95.
  • the complex described herein comprises an anti-TfR antibody covalently linked to a molecular payload, wherein the anti-TfR antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 92, and a light chain comprising the amino acid sequence of SEQ ID NO: 93.
  • the complex described herein comprises an anti-TfR antibody covalently linked to a molecular payload, wherein the anti-TfR antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 97, SEQ ID NO: 98, or SEQ ID NO: 99 and a VL comprising the amino acid sequence of SEQ ID NO: 85.
  • the complex described herein comprises an anti-TfR antibody covalently linked to a molecular payload, wherein the anti-TfR antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 100 or SEQ ID NO: 101 and a light chain comprising the amino acid sequence of SEQ ID NO: 89.
  • the complex described herein comprises an anti-TfR antibody covalently linked to a molecular payload, wherein the anti-TfR antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 100 or SEQ ID NO: 101 and a light chain comprising the amino acid sequence of SEQ ID NO: 90.
  • the complex described herein comprises an anti-TfR antibody covalently linked to a molecular payload, wherein the anti-TfR antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 102 and a light chain comprising the amino acid sequence of SEQ ID NO: 93.
  • the complex described herein comprises an anti-TfR antibody covalently linked to a molecular payload, wherein the anti-TfR antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 102 or SEQ ID NO: 103 and a light chain comprising the amino acid sequence of SEQ ID NO: 95.
  • the complex described herein comprises an anti-TfR antibody covalently linked via a lysine to the 5’ end of an oligonucleotide, wherein the anti- TfR antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 84 and a light chain comprising the amino acid sequence of SEQ ID NO: 85; wherein the complex has the structure of: wherein n is 3 and m is 4.
  • the complex described herein comprises an anti-TfR antibody covalently linked via a lysine to the 5’ end of an oligonucleotide, wherein the anti- TfR antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 86 and a light chain comprising the amino acid sequence of SEQ ID NO: 85; wherein the complex has the structure of: wherein n is 3 and m is 4.
  • the complex described herein comprises an anti-TfR antibody covalently linked via a lysine to the 5’ end of an oligonucleotide, wherein the anti- TfR antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 87 and a light chain comprising the amino acid sequence of SEQ ID NO: 85; wherein the complex has the structure of: wherein n is 3 and m is 4.
  • the complex described herein comprises an anti-TfR antibody covalently linked via a lysine to the 5’ end of an oligonucleotide, wherein the anti- TfR antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 88 and a light chain comprising the amino acid sequence of SEQ ID NO: 89; wherein the complex has the structure of: wherein n is 3 and m is 4.
  • the complex described herein comprises an anti-TfR antibody covalently linked via a lysine to the 5’ end of an oligonucleotide, wherein the anti- TfR antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 88 and a light chain comprising the amino acid sequence of SEQ ID NO: 90; wherein the complex has the structure of: wherein n is 3 and m is 4.
  • the complex described herein comprises an anti-TfR antibody covalently linked via a lysine to the 5’ end of an oligonucleotide, wherein the anti- TfR antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 91 and a light chain comprising the amino acid sequence of n SEQ ID NO: 89; wherein the complex has the structure of: wherein n is 3 and m is 4.
  • the complex described herein comprises an anti-TfR antibody covalently linked via a lysine to the 5’ end of an oligonucleotide, wherein the anti- TfR antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 91 and a light chain comprising the amino acid sequence of SEQ ID NO: 90; wherein the complex has the structure of: wherein n is 3 and m is 4.
  • the complex described herein comprises an anti-TfR antibody covalently linked via a lysine to the 5’ end of an oligonucleotide, wherein the anti- TfR antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 92 and a light chain comprising the amino acid sequence of SEQ ID NO: 93; wherein the complex has the structure of: wherein n is 3 and m is 4.
  • the complex described herein comprises an anti-TfR antibody covalently linked via a lysine to the 5’ end of an oligonucleotide, wherein the anti- TfR antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 94 and a light chain comprising the amino acid sequence of SEQ ID NO: 95; wherein the complex has the structure of: wherein n is 3 and m is 4.
  • the complex described herein comprises an anti-TfR antibody covalently linked via a lysine to the 5’ end of an oligonucleotide, wherein the anti- TfR antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 92 and a light chain comprising the amino acid sequence of SEQ ID NO: 95; wherein the complex has the structure of: wherein n is 3 and m is 4.
  • the complex described herein comprises an anti-TfR Fab covalently linked via a lysine to the 5’ end of an oligonucleotide, wherein the anti-TfR Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 69 and a VL comprising the amino acid sequence of SEQ ID NO: 70; wherein the complex has the structure of: wherein n is 3 and m is 4.
  • the complex described herein comprises an anti-TfR Fab covalently linked via a lysine to the 5’ end of an oligonucleotide, wherein the anti-TfR Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 71 and a VL comprising the amino acid sequence of SEQ ID NO: 70; wherein the complex has the structure of: wherein n is 3 and m is 4.
  • the complex described herein comprises an anti-TfR Fab covalently linked via a lysine to the 5’ end of an oligonucleotide, wherein the anti-TfR Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 72 and a VL comprising the amino acid sequence of SEQ ID NO: 70; wherein the complex has the structure of: wherein n is 3 and m is 4.
  • the complex described herein comprises an anti-TfR Fab covalently linked via a lysine to the 5’ end of an oligonucleotide, wherein the anti-TfR Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 73 and a VL comprising the amino acid sequence of SEQ ID NO: 74; wherein the complex has the structure of: wherein n is 3 and m is 4.
  • the complex described herein comprises an anti-TfR Fab covalently linked via a lysine to the 5’ end of an oligonucleotide, wherein the anti-TfR Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 73 and a VL comprising the amino acid sequence of SEQ ID NO: 75; wherein the complex has the structure of: wherein n is 3 and m is 4.
  • the complex described herein comprises an anti-TfR Fab covalently linked via a lysine to the 5’ end of an oligonucleotide, wherein the anti-TfR Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 76 and a VL comprising the amino acid sequence of SEQ ID NO: 74; wherein the complex has the structure of: wherein n is 3 and m is 4.
  • the complex described herein comprises an anti-TfR Fab covalently linked via a lysine to the 5’ end of an oligonucleotide, wherein the anti-TfR Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 76 and a VL comprising the amino acid sequence of SEQ ID NO: 75; wherein the complex has the structure of: wherein n is 3 and m is 4.
  • the complex described herein comprises an anti-TfR Fab covalently linked via a lysine to the 5’ end of an oligonucleotide, wherein the anti-TfR Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 77 and a VL comprising the amino acid sequence of SEQ ID NO: 78; wherein the complex has the structure of: wherein n is 3 and m is 4.
  • the complex described herein comprises an anti-TfR Fab covalently linked via a lysine to the 5’ end of an oligonucleotide, wherein the anti-TfR Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 79 and a VL comprising the amino acid sequence of SEQ ID NO: 80; wherein the complex has the structure of: wherein n is 3 and m is 4.
  • the complex described herein comprises an anti-TfR Fab covalently linked via a lysine to the 5’ end of an oligonucleotide, wherein the anti-TfR Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 77 and a VL comprising the amino acid sequence of SEQ ID NO: 80; wherein the complex has the structure of: wherein n is 3 and m is 4.
  • the complex described herein comprises an anti-TfR Fab covalently linked via a lysine to the 5’ end of an oligonucleotide, wherein the anti-TfR Fab comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 97 and a light chain comprising the amino acid sequence of SEQ ID NO: 85; wherein the complex has the structure of: wherein n is 3 and m is 4.
  • the complex described herein comprises an anti-TfR Fab covalently linked via a lysine to the 5’ end of an oligonucleotide, wherein the anti-TfR Fab comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 98 and a light chain comprising the amino acid sequence of SEQ ID NO: 85; wherein the complex has the structure of: wherein n is 3 and m is 4.
  • the complex described herein comprises an anti-TfR Fab covalently linked via a lysine to the 5’ end of an oligonucleotide, wherein the anti-TfR Fab comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 99 and a light chain comprising the amino acid sequence of SEQ ID NO: 85; wherein the complex has the structure of: wherein n is 3 and m is 4.
  • the complex described herein comprises an anti-TfR Fab covalently linked via a lysine to the 5’ end of an oligonucleotide, wherein the anti-TfR Fab comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 100 and a light chain comprising the amino acid sequence of i SEQ ID NO: 89; wherein the complex has the structure of: wherein n is 3 and m is 4.
  • the complex described herein comprises an anti-TfR Fab covalently linked via a lysine to the 5’ end of an oligonucleotide, wherein the anti-TfR Fab comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 100 and a light chain comprising the amino acid sequence of SEQ ID NO: 90; wherein the complex has the structure of: wherein n is 3 and m is 4.
  • the complex described herein comprises an anti-TfR Fab covalently linked via a lysine to the 5’ end of an oligonucleotide, wherein the anti-TfR Fab comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 101 and a light chain comprising the amino acid sequence of SEQ ID NO: 89; wherein the complex has the structure of: wherein n is 3 and m is 4.
  • the complex described herein comprises an anti-TfR Fab covalently linked via a lysine to the 5’ end of an oligonucleotide, wherein the anti-TfR Fab comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 101 and a light chain comprising the amino acid sequence of SEQ ID NO: 90; wherein the complex has the structure of: wherein n is 3 and m is 4.
  • the complex described herein comprises an anti-TfR Fab covalently linked via a lysine to the 5’ end of an oligonucleotide, wherein the anti-TfR Fab comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 102 and a light chain comprising the amino acid sequence of SEQ ID NO: 93; wherein the complex has the structure of: wherein n is 3 and m is 4.
  • the complex described herein comprises an anti-TfR Fab covalently linked via a lysine to the 5’ end of an oligonucleotide, wherein the anti-TfR Fab comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 103 and a light chain comprising the amino acid sequence of SEQ ID NO: 95; wherein the complex has the structure of: wherein n is 3 and m is 4.
  • the complex described herein comprises an anti-TfR Fab covalently linked via a lysine to the 5’ end of an oligonucleotide, wherein the anti-TfR Fab comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 102 and a light chain comprising the amino acid sequence of SEQ ID NO: 95; wherein the complex has the structure of: wherein n is 3 and m is 4.
  • LI is any one of the spacers described herein.
  • LI is: wherein the piperazine moiety links to the oligonucleotide, wherein L2 is wherein the piperazine moiety links to the oligonucleotide.
  • LI is linked to a 5’ phosphate of the oligonucleotide.
  • LI is optional (e.g., need not be present).
  • the anti-TfR antibodies or complexes provided herein may be formulated in any suitable manner.
  • the antibodies or complexes provided herein are formulated in a manner suitable for pharmaceutical use.
  • the antibodies or complexes can be delivered to a subject using a formulation that minimizes degradation, facilitates delivery and/or (e.g., and) uptake, or provides another beneficial property to the complexes in the formulation.
  • compositions comprising the antibodies or complexes and pharmaceutically acceptable carriers. Such compositions can be suitably formulated such that when administered to a subject, either into the immediate environment of a target cell or systemically, a sufficient amount of the complexes enter target muscle cells.
  • compositions may include separately one or more components of complexes provided herein (e.g., anti-TfR antibodies, linkers, molecular payloads, or precursor molecules of any one of them).
  • antibodies or complexes are formulated in water or in an aqueous solution (e.g., water with pH adjustments).
  • antibodies or complexes are formulated in basic buffered aqueous solutions (e.g., PBS).
  • formulations as disclosed herein comprise an excipient.
  • an excipient confers to a composition improved stability, improved absorption, improved solubility and/or (e.g., and) therapeutic enhancement of the active ingredient.
  • an excipient is a buffering agent (e.g., sodium citrate, sodium phosphate, a tris base, or sodium hydroxide) or a vehicle (e.g., a buffered solution, petrolatum, dimethyl sulfoxide, or mineral oil).
  • a buffering agent e.g., sodium citrate, sodium phosphate, a tris base, or sodium hydroxide
  • a vehicle e.g., a buffered solution, petrolatum, dimethyl sulfoxide, or mineral oil.
  • a complex or component thereof e.g., oligonucleotide or antibody
  • a composition comprising a complex, or component thereof, described herein may be a lyoprotectant (e.g., mannitol, lactose, polyethylene glycol, or polyvinyl pyrolidone), or a collapse temperature modifier (e.g., dextran, ficoll, or gelatin).
  • a lyoprotectant e.g., mannitol, lactose, polyethylene glycol, or polyvinyl pyrolidone
  • a collapse temperature modifier e.g., dextran, ficoll, or gelatin
  • a pharmaceutical composition is formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, administration.
  • the route of administration is intravenous or subcutaneous.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • formulations include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride in the composition.
  • Sterile injectable solutions can be prepared by incorporating the complexes in a required amount in a selected solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • a composition may contain at least about 0.1% of the complex, or component thereof, or more, although the percentage of the active ingredient(s) may be between about 1 % and about 80% or more of the weight or volume of the total composition.
  • Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.
  • the anti-TfR antibodies described herein is used for delivering a molecular payload (e.g., a diagnostic or therapeutic agent) to a target cell or tissue that expresses a transferrin receptor.
  • a molecular payload e.g., a diagnostic or therapeutic agent
  • the target cell is a muscle cell.
  • the target tissue is muscle.
  • the target tissue is brain.
  • the anti-TfR antibody may be conjugated (e.g., covalently conjugated) to the molecular payload to form a complex a. Diagnostic and Detection Methods
  • antibodies or antigen-binding fragments are, for example, suited for use in immunoassays in which they can be utilized in liquid phase or bound to a solid phase carrier.
  • immunoassays which can utilize the antibody or antigen -binding fragments are competitive and non-competitive immunoassays in either a direct or indirect format.
  • immunoassays examples include the Enzyme Linked Immunoassay (ELISA), radioimmunoassay (RIA), the sandwich (immunometric assay), flow cytometry, the western blot assay, immunoprecipitation assays, immunohistochemistry, immuno-microscopy, lateral flow immuno-chromatographic assays, and proteomics arrays.
  • the antigens and antibodies or antigen-binding fragments can be bound to many different solid supports (e.g., carriers, membrane, columns, proteomics array, etc.).
  • solid support materials include glass, polystyrene, polyvinyl chloride, polyvinylidene difluoride, polypropylene, polyethylene, polycarbonate, dextran, nylon, amyloses, natural and modified celluloses, such as nitrocellulose, polyacrylamides, agaroses, and magnetite.
  • the nature of the support can be either fixed or suspended in a solution (e.g., beads).
  • any one of the anti-TfR antibodies provided herein is useful for detecting the presence of transferrin receptor in a biological sample.
  • a biological sample comprises a cell or tissue, such as blood, CSF, and BBB- containing tissue.
  • the biological sample can be in vitro (e.g., cultured) or in vivo (e.g., in a subject).
  • the present disclosure also contemplates the use of any one of the anti-TfR antibodies described herein in research use (e.g., as a reagent for immuno assays such as western blotting, immunostaining, ELISA, and/or (e.g., and) FACS).
  • an anti-TfR antibody for use in a method of diagnosis or detection is provided.
  • a method of detecting the presence of transferrin receptor in a biological sample is provided.
  • the method comprises contacting the biological sample with an anti-TfR antibody as described herein under conditions permissive for binding of the anti-TfR antibody to the transferrin receptor, and detecting whether a complex is formed between the anti-TfR antibody and the transferrin receptor.
  • Such method may be an in vitro or in vivo method.
  • an anti- TfR antibody is used to select subjects eligible for therapy with an anti-TfR antibody, e.g. where transferrin receptor is a biomarker for selection of patients.
  • disorders that may be diagnosed using an anti-TfR antibody described herein include disorders involving immature red blood cells, due to the fact that transferrin receptor is expressed in reticulocytes and is therefore detectable by any of the antibodies of the invention.
  • disorders include anemia and other disorders arising from reduced levels of reticulocytes, or congenital polycythemia or neoplastic polycythemia vera, where raised red blood cell counts due to hyperproliferation of, e.g., reticulocytes, results in thickening of blood and concomitant physiological symptoms.
  • labeled anti-TfR antibodies are used.
  • Labels include, but are not limited to, labels or moieties that are detected directly (such as fluorescent, chromophoric, electron-dense, chemiluminescent, and radioactive labels), as well as moieties, such as enzymes or ligands, that are detected indirectly, e.g., through an enzymatic reaction or molecular interaction.
  • Exemplary labels include, but are not limited to, the radioisotopes 32P, 14C, 1251, 3H, and 131, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luceriferases, e.g., firefly luciferase and bacterial luciferase (U.S. Pat. No.
  • luciferin 2,3-dihydrophthalazinediones
  • horseradish peroxidase HRP
  • alkaline phosphatase b-galactosidase
  • glucoamylase lysozyme
  • saccharide oxidases e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase
  • heterocyclic oxidases such as uricase and xanthine oxidase, coupled with an enzyme that employs hydrogen peroxide to oxidize a dye precursor such as HRP, lactoperoxidase, or microperoxidase, biotin/avidin, spin labels, bacteriophage labels, stable free radicals, and the like.
  • the detectable label is an agent suitable for detecting transferrin receptor in a cell in vitro, which can be a radioactive molecule, a radiopharmaceutical, or an iron oxide particle.
  • Radioactive molecules suitable for in vivo imaging include, but are not limited to, 122 I, 123 I, 124 I, 125 I, 131 I, 18 F, 75 Br, 76 Br, 77 Br, 211 At, 225 Ac, 177 Lu, 153 Sm, 186 Re, 188 Re, 67 Cu, 213 Bi, 212 Bi, 212 Pb, and 67 Ga.
  • radiopharmaceuticals suitable for in vivo imaging include ni In Oxyquinoline, 131 I Sodium iodide, “mTc Mebrofenin, and “mTc Red Blood Cells, 123 I Sodium iodide, "mTc Exametazime, "mTc Macroaggregate Albumin, “mTc Medronate, “mTc Mertiatide, “mTc Oxidronate, “mTc Pentetate, “mTc Pertechnetate, "mTc Sestamibi, “mTc Sulfur Colloid, “mTc Tetrofosmin, Thallium-201, or Xenon-133.
  • the anti-TfR antibody described herein can be used to deliver a detectable label to a target cell or tissue (e.g., muscle cell or across the blood brain barrier to the brain) for visualization of the cell or tissue (e.g., by fluorescent microscopy or by magnetic resonance imaging (MRI). Any of the detectable labels described herein can be used for this purpose.
  • a target cell or tissue e.g., muscle cell or across the blood brain barrier to the brain
  • MRI magnetic resonance imaging
  • the anti-TfR antibody used in a diagnostic or detection method lacks effector function or has reduced effector function.
  • the anti-TfR antibody used in a diagnostic/detection method is engineered to have no or reduced effector function (e.g., by using a Fab, modifying the Ig backbone, introducing one or more Fc mutations reducing or eliminating effector function, and/or (e.g., and) modifying the glycosylation state of the antibody).
  • ELISA enzyme linked immunosorbent assay
  • a suitable amount of anti-TfR antibodies, conjugated with a label can be administered to a subject in need of the examination. Presence of the labeled antibody can be detected based on the signal released from the label by routine methods. Assays for evaluating uptake of systemically administered antibody and other biological activity of the antibody are known to those skilled in the art.
  • an anti-TfR antibody can be used to study bioactivity of transferrin receptor and/or (e.g., and) detect the presence of transferrin receptor intracellularly.
  • a suitable amount of anti-TfR antibody can be brought in contact with a sample (e.g. a new cell type that is not previously identified as transferrin receptor producing cells) suspected of producing transferrin receptor.
  • the antibody and the sample may be incubated under suitable conditions for a suitable period to allow for binding of the antibody to the transferrin receptor antigen.
  • Such an interaction can then be detected via routine methods, e.g., ELISA, histological staining or FACS.
  • the anti-TfR antibodies described herein can be used for delivering molecular payloads that are therapeutic agents (e.g., oligonucleotides, peptides/proteins, nucleic acid constructs, etc.).
  • the present disclosure also provides complexes comprising the anti-TfR antibodies covalently linked to a molecular payload for use in treating diseases.
  • complexes comprising an anti-TfR antibody covalently linked to a molecular payload as described herein are effective in treating a muscle disease (e.g., a rare muscle disease or muscle atrophy).
  • complexes are effective in treating a rare muscle disease provided in Table 6.
  • a muscle disease is associated with a disease allele, for example, a disease allele for a particular muscle disease may comprise a genetic alteration in a corresponding gene listed in Table 6.
  • complexes are effective in treating muscle atrophy associated with the activity of one or more genes listed in Table 6 under the “Muscle Atrophy Gene Targets” section.
  • muscle atrophy is due to a chronic illness, including AIDS, congestive heart failure, cancer, chronic obstructive pulmonary disease, and renal failure, or muscle disuse.
  • complexes comprising an anti-TfR antibody covalently linked to a molecular payload as described herein are effective in treating a neurological disease.
  • neurological diseases include, but are not limited to, neuropathy, amyloidosis, cancer, an ocular disease or disorder, viral or microbial infection, inflammation, ischemia, neurodegenerative disease, seizure, behavioral disorders, and a lysosomal storage disease.
  • the CNS will be understood to include the eye, which is normally sequestered from the rest of the body by the blood-retina barrier.
  • neurological disorders include, but are not limited to, neurodegenerative diseases (including, but not limited to, Lewy body disease, postpoliomyelitis syndrome, Shy-Draeger syndrome, olivopontocerebellar atrophy, Parkinson's disease, multiple system atrophy, striatonigral degeneration, tauopathies (including, but not limited to, Alzheimer disease and supranuclear palsy), prion diseases (including, but not limited to, bovine spongiform encephalopathy, scrapie, Creutzfeldt- Jakob syndrome, kuru, Gerstmann-Straussler-Scheinker disease, chronic wasting disease, and fatal familial insomnia), bulbar palsy, motor neuron disease, and nervous system heterodegenerative disorders (including, but not limited to, Canavan disease, Huntington's disease, neuronal ceroid-lipofuscinosis, Alexander's disease, Tourette's syndrome, Menkes kinky hair syndrome, Cockayne syndrome, Halervorden-Spatz syndrome, la
  • the complex comprises an anti-TfR antibody described herein conjugated to a drug for treating a neurological disease (e.g., the drugs listed in Table 7).
  • a subject may be a human subject, a non-human primate subject, a rodent subject, or any suitable mammalian subject.
  • a subject may have a muscle disease provided in Table 6.
  • a subject may have muscle atrophy, or be at risk of developing muscle atrophy.
  • An aspect of the disclosure includes a method involving administering to a subject an effective amount of a complex as described herein.
  • an effective amount of a pharmaceutical composition that comprises a complex comprising an anti-TfR antibody covalently linked to a molecular payload can be administered to a subject in need of treatment.
  • a pharmaceutical composition comprising a complex as described herein may be administered by a suitable route, which may include intravenous administration, e.g., as a bolus or by continuous infusion over a period of time.
  • intravenous administration may be performed by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra- articular, intrasynovial, or intrathecal routes.
  • a pharmaceutical composition may be in solid form, aqueous form, or a liquid form.
  • an aqueous or liquid form may be nebulized or lyophilized.
  • a nebulized or lyophilized form may be reconstituted with an aqueous or liquid solution.
  • compositions for intravenous administration may contain various carriers such as vegetable oils, dimethylactamide, dimethyformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (glycerol, propylene glycol, liquid polyethylene glycol, and the like).
  • water soluble antibodies can be administered by the drip method, whereby a pharmaceutical formulation containing the antibody and a physiologically acceptable excipients is infused.
  • Physiologically acceptable excipients may include, for example, 5% dextrose, 0.9% saline, Ringer’s solution or other suitable excipients.
  • Intramuscular preparations e.g., a sterile formulation of a suitable soluble salt form of the antibody
  • a pharmaceutical excipient such as Water-for- Injection, 0.9% saline, or 5% glucose solution.
  • a pharmaceutical composition that comprises a complex comprising an anti-TfR antibody covalently linked to a molecular payload is administered via site- specific or local delivery techniques.
  • these techniques include implantable depot sources of the complex, local delivery catheters, site specific carriers, direct injection, or direct application.
  • a pharmaceutical composition that comprises a complex comprising an anti-TfR antibody covalently linked to a molecular payload is administered at an effective concentration that confers therapeutic effect on a subject.
  • Effective amounts vary, as recognized by those skilled in the art, depending on the severity of the disease, unique characteristics of the subject being treated, e.g. age, physical conditions, health, or weight, the duration of the treatment, the nature of any concurrent therapies, the route of administration and related factors. These related factors are known to those in the art and may be addressed with no more than routine experimentation.
  • an effective concentration is the maximum dose that is considered to be safe for the patient. In some embodiments, an effective concentration will be the lowest possible concentration that provides maximum efficacy.
  • Empirical considerations e.g. the half-life of the complex in a subject, generally will contribute to determination of the concentration of pharmaceutical composition that is used for treatment.
  • the frequency of administration may be empirically determined and adjusted to maximize the efficacy of the treatment.
  • an initial candidate dosage may be about 1 to 100 mg/kg, or more, depending on the factors described above, e.g. safety or efficacy.
  • a treatment will be administered once.
  • a treatment will be administered daily, biweekly, weekly, bimonthly, monthly, or at any time interval that provide maximum efficacy while minimizing safety risks to the subject.
  • the efficacy and the treatment and safety risks may be monitored throughout the course of treatment
  • the efficacy of treatment may be assessed using any suitable methods.
  • the efficacy of treatment may be assessed by evaluation of observation of symptoms associated with a muscle disease and/or (e.g., and) muscle atrophy.
  • a pharmaceutical composition that comprises a complex comprising an anti-TfR antibody covalently linked to a molecular payload described herein is administered to a subject at an effective concentration sufficient to inhibit activity or expression of a target gene by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% relative to a control, e.g. baseline level of gene expression prior to treatment.
  • a single dose or administration of a pharmaceutical composition that comprises a complex comprising an anti-TfR antibody covalently linked to a molecular payload described herein to a subject is sufficient to inhibit activity or expression of a target gene for at least 1-5, 1-10, 5-15, 10-20, 15-30, 20-40, 25-50, or more days.
  • a single dose or administration of a pharmaceutical composition that comprises a complex comprising an anti-TfR antibody covalently linked to a molecular payload described herein to a subject is sufficient to inhibit activity or expression of a target gene for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks.
  • a single dose or administration of a pharmaceutical composition that comprises a complex comprising an anti-TfR antibody covalently linked to a molecular payload described herein to a subject is sufficient to inhibit activity or expression of a target gene for at least 1, 2, 3, 4, 5, or 6 months.
  • a pharmaceutical composition may comprise more than one complex comprising an anti-TfR antibody covalently linked to a molecular payload.
  • a pharmaceutical composition may further comprise any other suitable therapeutic agent for treatment of a subject, e.g. a human subject having a muscle disease (e.g., a muscle disease provided in Table 6).
  • the other therapeutic agents may enhance or supplement the effectiveness of the complexes described herein.
  • the other therapeutic agents may function to treat a different symptom or disease than the complexes described herein.
  • Kits for Therapeutic and Diagnostic Applications [000382] The present disclosure also provides kits for the therapeutic or diagnostic applications as disclosed herein. Such kits can include one or more containers comprising an anti-TfR antibody, e.g., any of those described herein.
  • the kit can comprise instructions for use in accordance with any of the methods described herein.
  • the included instructions can comprise a description of administration of the anti-TfR antibody to treat, delay the onset, or alleviate a target disease as those described herein.
  • the kit may further comprise a description of selecting an individual suitable for treatment based on identifying whether that individual has the target disease.
  • the instructions comprise a description of administering an antibody to an individual at risk of the target disease.
  • the instructions relating to the use of an anti-TfR antibody generally include information as to dosage, dosing schedule, and route of administration for the intended treatment.
  • the containers may be unit doses, bulk packages (e.g., multi-dose packages) or sub unit doses.
  • Instructions supplied in the kits of the invention are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.
  • the label or package insert indicates that the composition is used for treating, delaying the onset and/or (e.g., and) alleviating a disease or disorder treatable by modulating immune responses, such as autoimmune diseases.
  • Instructions may be provided for practicing any of the methods described herein.
  • kits of this invention are in suitable packaging.
  • suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like.
  • kits for use in combination with a specific device such as an inhaler, nasal administration device (e.g., an atomizer) or an infusion device such as a minipump.
  • a kit may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
  • the container may also have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
  • At least one active agent in the composition is an anti-TfR antibody as those described herein.
  • Kits may optionally provide additional components such as buffers and interpretive information.
  • the kit comprises a container and a label or package insert(s) on or associated with the container.
  • the invention provides articles of manufacture comprising contents of the kits described above.
  • kits for use in detecting transferrin receptor in a sample may comprise any of the anti-TfR antibodies described herein.
  • the anti-TfR antibody can be conjugated with a detectable label as those described herein.
  • conjugated or “attached” means two entities are associated, preferably with sufficient affinity that the therapeutic/diagnostic benefit of the association between the two entities is realized.
  • the association between the two entities can be either direct or via a linker, such as a polymer linker.
  • Conjugated or attached can include covalent or noncovalent bonding as well as other forms of association, such as entrapment, e.g., of one entity on or within the other, or of either or both entities on or within a third entity, such as a micelle.
  • the kit may comprise a secondary antibody capable of binding to anti-TfR antibody.
  • the kit may further comprise instructions for using the anti-TfR antibody for detecting transferrin receptor.
  • the anti-TfR antibodies shown in Table 2 were subjected to humanization and mutagenesis to reduce manufacturability liabilities.
  • the humanized variants were screened and tested for their binding properties and biological actives.
  • Humanized variants of anti-TfRl heavy and light chain variable regions (5 variants each) were designed using Composite Human Technology. Genes encoding Fabs having these heavy and light chain variable regions were synthesized, and vectors were constructed to express each humanized heavy and light chain variant. Subsequently, each vector was expressed on a small scale and the resultant humanized anti-TfRl Fabs were analyzed.
  • Humanized Fabs were selected for further testing based upon several criteria including Biacore assays of antibody affinity for the target antigen, relative expression, percent homology to human germline sequence, and the number of MHC class II predicted T cell epitopes (determined using iTopeTM MCH class II in silico analysis). [000392] Potential liabilities were identified within the parental sequence of some antibodies by introducing amino acid substitutions in the heavy chain and light chain variable regions. These substitutions were chosen based on relative expression levels, iTopeTM score and relative KD from Biacore single cycle kinetics analysis. The humanized variants were tested and variants were selected initially based upon affinity for the target antigen.
  • the selected humanized Fabs were further screened based on a series of biophysical assessments of stability and susceptibility to aggregation and degradation of each analyzed variant, shown in Table 8 and Table 9.
  • the selected Fabs were analyzed for their properties binding to TfRl by kinetic analysis. The results of these analyses are shown in Table 10.
  • the selected humanized Fabs were conjugated to a DMPK-targeting oligonucleotide ASO300.
  • the selected Fabs are thermally stable, as indicated by the comparable binding affinity to human and cyno TfRl after been exposed to high temperature (40 °C) for 9 days, compared to before the exposure (see Table 10).
  • ELISAs were conducted. High binding, black, flat bottom, 96 well plates (Coming# 3925) were first coated with 100 pL/well of recombinant huTfRl at 1 pg/mL in PBS and incubated at 4°C overnight. Wells were emptied and residual liquid was removed. Blocking was conducted by adding 200 pL of 1%BSA (w/w) in PBS to each well. Blocking was allowed to proceed for 2 hours at room temperature on a shaker at 300 rpm. After blocking, liquid was removed and wells were washed three times with 300 pL of TBST.
  • Anti-TfRl antibodies were then added in 0.5% BSA/TBST in triplicate in an 8 point serial dilution (dilution range 5 pg/mL - 5 ng/mL). A positive control and isotype controls were also included on the ELISA plate. The plate was incubated at room temperature on an orbital shaker for 60 minutes at 300 rpm, and the plate was washed three times with 300 pL of TBST. Anti-(H+L)IgG-A488 (1:500) (Invitrogen #A11013) was diluted in 0.5% BSA in TBST, and 100 pL was added to each well. The plate was then allowed to incubate at room temperature for 60 minutes at 300 rpm on orbital shaker.
  • results of these two sets of ELISA analyses for binding of the humanized anti- TfR Fabs to hTfRl and cTfRl demonstrate that humanized 3M12 Fabs show consistent binding to both hTfRl and cTfRl, and that humanized 3A4 Fabs show decreased binding to cTfRl relative to hTfRl.
  • Antibody-oligonucleotide conjugates were prepared using six humanized anti- TfR Fabs, each of which were conjugated to a DMPK targeting oligonucleotide ASO300. Conjugation efficiency and down-stream purification were characterized, and various properties of the product conjugates were measured. The results demonstrate that conjugation efficiency was robust across all 10 variants tested, and that the purification process (hydrophobic interaction chromatography followed by hydroxyapatite resin chromatography) were effective. The purified conjugates showed a >97% purity as analyzed by size exclusion chromatography.
  • the term ‘unconjugated’ indicates that the antibody was not conjugated to an oligonucleotide.
  • Conjugates containing humanized anti-TfR Fabs 3M12(VH3/Vk2), 3M-12 (VH4/Vk3), and 3A4(VH3-N54S/Vk4) were conjugated to a DMPK-targeting antisense oligonucleotide ASO300 and were tested in rhabdomyosarcoma (RD) cells for knockdown of DMPK transcript expression.
  • Antibodies were conjugated to ASO300 via the linker shown in Formula (C).
  • RD cells were cultured in a growth medium of DMEM with glutamine, supplemented with 10% FBS and penicillin/streptomycin until nearly confluent. Cells were then seeded into a 96 well plate at 20K cells per well and were allowed to recover for 24 hours. Cells were then treated with the conjugates for 3 days. Total RNA was collected from cells, cDNA was synthesized and DMPK expression was measured by qPCR.
  • Results in FIG. 4 show that DMPK expression level was reduced in cells treated with each indicated conjugate, relative to expression in PBS-treated cells, indicating that the humanized anti-TfR Fabs are able to mediate the uptake of the DMPK-targeting oligonucleotide by the RD cells and that the internalized DMPK-targeting oligonucleotide are effective in knocking down DMPK mRNA level.
  • Oligonucleotides which were linked to antibodies in examples were conjugated via a cleavable linker shown in Formula (C). It is important that the linker maintain stability in serum and provide release kinetics that favor sufficient payload accumulation in the targeted muscle cell. This serum stability is important for systemic intravenous administration, stability of the conjugated oligonucleotide in the bloodstream, delivery to muscle tissue and internalization of the therapeutic payload in the muscle cells.
  • the linker has been confirmed to facilitate precise conjugation of multiple types of payloads (including ASOs, siRNAs and PMOs) to Fabs. This flexibility enabled rational selection of the appropriate type of payload to address the genetic basis of each muscle disease. Additionally, the linker and conjugation chemistry allowed the optimization of the ratio of payload molecules attached to each Fab for each type of payload, and enabled rapid design, production and screening of molecules to enable use in various muscle disease applications.
  • FIG. 5 shows serum stability of the linker in vivo , which was comparable across multiple species over the course of 72 hours after intravenous dosing. At least 75% stability was measured in each case at 72 hours after dosing.
  • Example 4 Exon-skipping activity of anti-TfR conjugates in DMD patient myotubes [000404]
  • the exon-skipping activities of anti-TfR conjugates containing an anti-TfR Fab (3M12 VH3/VK2, 3M12 VH4/VK3, and 3A4 VH3 N54S/VK4) conjugated to a DMD exon51- skipping oligonucleotide were evaluated.
  • Immortalized human myoblasts bearing an exon 52 deletion were thawed and seeded at a density of le6 cell/flask in Promocell Skeletal Cell Growth Media (with 5% FBS and lx Pen-Strep) and allowed to grow to confluency.
  • cDNA synthesis was performed on 75 ng of total RNA, and mutation specific PCRs were performed to evaluate the degree of exon 51 skipping in each cell type. Mutation- specific PCR products were run on a 4% agarose gel and visualized using SYBR gold. Densitometry was used to calculate the relative amounts of the skipped and unskipped amplicon and exon skipping was determined as a ratio of the exon 51 skipped amplicon divided by the total amount of amplicon present:
  • the term ‘unconjugated’ indicates that the oligonucleotide was not conjugated to an antibody.
  • DMPK levels are reduced, the nuclear foci are diminished, releasing splicing proteins, allowing restoration of normal mRNA processing, and potentially stopping or reversing disease progression.
  • mice Male and female C57BL/6 mice where one TfRl allele was replaced with a human TFR1 allele were administered between the ages of 5 and 15 weeks according to the dosing schedule outlined in Table 11 and in FIG. 7A. Mice were sacrificed 14 days after the first injection and selected muscles collected as indicated in Table 12.
  • SEQ ID NO: 35 amino acids C89-F760)/anti-TfR mixture prepared was mixed with 2 pL of DSS d0/dl2 (2mg/mL;DMF) before 180 minutes incubation time at room temperature. After incubation, reaction was stopped by adding lpL of Ammonium Bicarbonate (20 mM final concentration) before 1 hour incubation time at room temperature. Then, the solution was dried using a speedvac before FLO 8M urea suspension (20pL). After mixing, 2 pi of DTT (500 mM) were added to the solution. The mixture was then incubated 1 hour at 37°C.
  • the trypsin buffer contains 50mM Ambic pH 8.5, 5% acetonitrile;
  • the Chymotrypsin buffer contains Tris HC1 lOOmM, CaCh lOmM pH 7.8;
  • the ASP-N buffer contains Phopshate buffer 50MM pH 7.8;
  • the elastase buffer contains Tris HC150mM pH 8.0 and the thermolysin buffer contains Tris HC150mM, CaCh 0.5mM pH 9.0.
  • Example 7 Characterization of binding activities of anti-TfR Fab 3M12 VH4/Vk3 [000419] In vitro studies were performed to test the specificity of anti-TfR Fab 3M12 VH4/Vk3 for human and cynomolgus monkey TfRl binding and to confirm its selectivity for human TfRl vs TfR2. The binding affinity of anti-TfR Fab 3M12 VH4/Vk3 to TfRl from various species was determined using an enzyme-linked immunosorbent assay (ELISA). Serial dilutions of the Fab were added to plates precoated with recombinant human, cynomolgus monkey, mouse, or rat TfRl.
  • ELISA enzyme-linked immunosorbent assay
  • binding of the Fab was quantified by addition of a fluorescently tagged anti-(H-i-L) IgG secondary antibody and measurement of fluorescence intensity at 495nm excitation and 520nm emission.
  • the Fab showed strong binding affinity to human and cynomolgus monkey TfRl, and no detectable binding of mouse or rat TfRl was observed (FIG. 8).
  • SPR Surface plasmon resonance
  • Anti-TfR Fab VH4/Vk3 was conjugated to a control antisense oligonucleotide (ASO) via a linker as shown in Formula (C) and the resulting conjugate was tested for stability of the linker conjugating the Fab to the ASO.
  • Serum stability was measured by incubating fluorescently labeled conjugate in PBS or in rat, mouse, cynomolgus monkey, or human serum and measuring relative fluorescence intensity over time, with higher fluorescence indicating more conjugate remaining intact.
  • FIG. 10 shows serum stability was similar across multiple species and remained high after 72 hours.
  • Anti-TfR Fab 3M12 VH4/Vk3 was conjugated to a dystrophin (DMD) exon51- skipping antisense oligonucleotide (ASO) targeting an exonic splicing enhancer (ESE) sequence in DMD exon 51.
  • DMD dystrophin
  • ASO exon51- skipping antisense oligonucleotide
  • ESE exonic splicing enhancer
  • the exon51 skipping oligonucleotide is a phosphorodiamidate morpholino oligomer (PMO) of 30 nucleotides in length.
  • PMO phosphorodiamidate morpholino oligomer
  • Conjugate doses are listed as mg/kg of anti-TfR Fab 3M12 VH4/Vk3-ASO conjugate.
  • c ASO doses are listed as mg/kg ASO equivalent of the anti-TfR Fab 3M12 VH4/Vk3-ASO dose.
  • Tissue ASO accumulation was also quantified using a hybridization ELISA with a probe complementary to the ASO sequence.
  • a standard curve was generated and ASO levels (in ng/g) were derived from a linear regression of the standard curve.
  • the ASO was distributed to all tissues evaluated at a higher level following the administration of the anti-TfR Fab VH4/Vk3-ASO conjugate as compared to the administration of unconjugated ASO.
  • Intravenous administration of unconjugated ASO resulted in levels of ASO that were close to background levels in all tissues evaluated at 2 and 4 weeks after the first does was administered.
  • the term ‘unconjugated’ indicates that the oligonucleotide was not conjugated to an antibody.
  • Conjugate doses are listed as mg/kg of anti-TfR Fab 3M12 VH4/Vk3 ASO conjugate.
  • c ASO doses are listed as mg/kg ASO or ASO equivalent of the anti-TfR Fab 3M12 VH4/Vk3-ASO conjugate dose.
  • VH heavy chain variable region
  • VL light chain variable region
  • a heavy chain variable region (VH) comprising an amino acid sequence at least 85% identical to SEQ ID NO: 71; and/or a light chain variable region (VL) comprising an amino acid sequence at least 85% identical to SEQ ID NO: 70;
  • a heavy chain variable region (VH) comprising an amino acid sequence at least 85% identical to SEQ ID NO: 72; and/or a light chain variable region (VL) comprising an amino acid sequence at least 85% identical to SEQ ID NO: 70;
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • VL light chain variable region
  • x a heavy chain variable region (VH) comprising an amino acid sequence at least 85% identical to SEQ ID NO: 77; and/or a light chain variable region (VL) comprising an amino acid sequence at least 85% identical to SEQ ID NO: 80.
  • VH heavy chain variable region
  • VL light chain variable region
  • the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 72 and a VL comprising an amino acid of SEQ ID NO: 70.
  • a heavy chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 91; and/or a light chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 89;
  • a heavy chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 94; and/or a light chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 95; or (x) a heavy chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 92; and/or a light chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 95.
  • a heavy chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 101; and/or a light chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 89;
  • a heavy chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 102; and/or a light chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 93;
  • a heavy chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 103; and/or a light chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 95; or
  • (x) a heavy chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 102; and/or a light chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 95.
  • (x) a heavy chain comprising the amino acid sequence of SEQ ID NO: 102; and/or a light chain comprising the amino acid sequence of SEQ ID NO: 95.
  • a vector comprising the nucleic acid of embodiment 22.
  • a cell comprising the vector of embodiment 23.
  • a method producing an anti-TfR antibody comprising culturing the cell of embodiment 24 under conditions suitable for the expression of the antibody.
  • a complex comprising the antibody of any one of embodiments 1-21 covalently linked to a molecular payload.
  • composition comprising the antibody of any one of embodiments 1-21, the nucleic acid of embodiment 22, the vector of embodiment 23, or the complex of any one of embodiments 26-31.
  • composition of embodiment 32 further comprising a pharmaceutically acceptable carrier.
  • a method of detecting a transferrin receptor in a biological sample comprising contacting the antibody of any one of embodiments 1-21 with the biological sample and measuring binding of the antibody to the biological sample. 35. The method of embodiment 34, wherein the antibody is covalently linked to a diagnostic agent.
  • a method of delivering a molecular payload to a cell comprising contacting the complex of any one of embodiments 26-31 with the cell.
  • a method of delivering a molecular payload to the brain or the muscle of a subject comprising administering to the subject an effective amount of the complex of any one of embodiments 26-31.
  • a method of treating a disease comprising administering to a subject an effective amount of the complex of any one of embodiments 26-31, wherein the molecular payload is a therapeutic agent.
  • TfR human transferrin receptor
  • a heavy chain variable region (VH) comprising an amino acid sequence at least 85% identical to SEQ ID NO: 76; and/or a light chain variable region (VL) comprising an amino acid sequence at least 85% identical to SEQ ID NO: 75;
  • a heavy chain variable region (VH) comprising an amino acid sequence at least 85% identical to SEQ ID NO: 69; and/or a light chain variable region (VL) comprising an amino acid sequence at least 85% identical to SEQ ID NO: 70;
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • VL light chain variable region
  • x a heavy chain variable region (VH) comprising an amino acid sequence at least 85% identical to SEQ ID NO: 77; and/or a light chain variable region (VL) comprising an amino acid sequence at least 85% identical to SEQ ID NO: 80.
  • VH heavy chain variable region
  • VL light chain variable region
  • TfR human transferrin receptor
  • sequences presented in the sequence listing may be referred to in describing the structure of an oligonucleotide or other nucleic acid.
  • the actual oligonucleotide or other nucleic acid may have one or more alternative nucleotides (e.g., an RNA counterpart of a DNA nucleotide or a DNA counterpart of an RNA nucleotide) and/or (e.g., and) one or more modified nucleotides and/or (e.g., and) one or more modified intemucleotide linkages and/or (e.g., and) one or more other modification compared with the specified sequence while retaining essentially same or similar complementary properties as the specified sequence.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Cell Biology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Physical Education & Sports Medicine (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Neurology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Medicinal Preparation (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
PCT/US2021/040984 2020-07-23 2021-07-09 Anti-transferrin receptor (tfr) antibody and uses thereof Ceased WO2022020105A1 (en)

Priority Applications (10)

Application Number Priority Date Filing Date Title
CN202180064690.5A CN116348138A (zh) 2020-07-23 2021-07-09 抗转铁蛋白受体（tfr）抗体及其用途
US18/017,167 US20230256112A1 (en) 2020-07-23 2021-07-09 Anti-transferrin receptor (tfr) antibody and uses thereof
IL299667A IL299667A (en) 2020-07-23 2021-07-09 Anti-transferrin (TFR) antibody and uses thereof
MX2023000962A MX2023000962A (es) 2020-07-23 2021-07-09 Anticuerpo del receptor anti-transferrina (tfr) y usos del mismo.
KR1020237005829A KR20230041760A (ko) 2020-07-23 2021-07-09 항-트랜스페린 수용체 (tfr) 항체 및 그의 용도
EP21846109.3A EP4185315A4 (en) 2020-07-23 2021-07-09 ANTI-TRANSFERRIN RECEPTOR (TFR) ANTIBODIES AND USES THEREOF
BR112023001005A BR112023001005A2 (pt) 2020-07-23 2021-07-09 Anticorpo anti-receptor de transferrina (tfr) e usos do mesmo
AU2021312708A AU2021312708A1 (en) 2020-07-23 2021-07-09 Anti-transferrin receptor (TFR) antibody and uses thereof
JP2023504620A JP2023535443A (ja) 2020-07-23 2021-07-09 抗トランスフェリン受容体（ＴｆＲ）抗体およびその使用
CA3186727A CA3186727A1 (en) 2020-07-23 2021-07-09 Anti-transferrin receptor (tfr) antibody and uses thereof

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
US202063055721P 2020-07-23 2020-07-23
US63/055,721 2020-07-23
US202063069071P 2020-08-23 2020-08-23
US63/069,071 2020-08-23
US202163143825P 2021-01-30 2021-01-30
US63/143,825 2021-01-30

Publications (1)

Publication Number Publication Date
WO2022020105A1 true WO2022020105A1 (en) 2022-01-27

Family

ID=79729848

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2021/040984 Ceased WO2022020105A1 (en) 2020-07-23 2021-07-09 Anti-transferrin receptor (tfr) antibody and uses thereof

Country Status (11)

Country Link
US (1) US20230256112A1 (enExample)
EP (1) EP4185315A4 (enExample)
JP (1) JP2023535443A (enExample)
KR (1) KR20230041760A (enExample)
CN (1) CN116348138A (enExample)
AU (1) AU2021312708A1 (enExample)
BR (1) BR112023001005A2 (enExample)
CA (1) CA3186727A1 (enExample)
IL (1) IL299667A (enExample)
MX (1) MX2023000962A (enExample)
WO (1) WO2022020105A1 (enExample)

Cited By (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11369689B2 (en) 2018-08-02 2022-06-28 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating dystrophinopathies
US11390682B2 (en) 2018-08-02 2022-07-19 Dyne Therapeutics, Inc. Methods of intravenouisly delivering anti-transferrin antibody/oligonucleotide complexes to subjects having muscular dystrophy
US11633498B2 (en) 2021-07-09 2023-04-25 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating myotonic dystrophy
US11638761B2 (en) 2021-07-09 2023-05-02 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating Facioscapulohumeral muscular dystrophy
US11648318B2 (en) 2021-07-09 2023-05-16 Dyne Therapeutics, Inc. Anti-transferrin receptor (TFR) antibody and uses thereof
US11771776B2 (en) 2021-07-09 2023-10-03 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating dystrophinopathies
US11827702B2 (en) 2021-09-01 2023-11-28 Biogen Ma Inc. Anti-transferrin receptor antibodies and uses thereof
WO2024026474A1 (en) * 2022-07-29 2024-02-01 Regeneron Pharmaceuticals, Inc. Compositions and methods for transferrin receptor (tfr)-mediated delivery to the brain and muscle
US11911484B2 (en) 2018-08-02 2024-02-27 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating myotonic dystrophy
US11931421B2 (en) 2022-04-15 2024-03-19 Dyne Therapeutics, Inc. Muscle targeting complexes and formulations for treating myotonic dystrophy
WO2024064756A1 (en) * 2022-09-20 2024-03-28 Dana-Farber Cancer Institute, Inc. Receptor-mediated endocytosis for targeted internalization and degradation of membrane proteins and cargos
US11969475B2 (en) 2021-07-09 2024-04-30 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating facioscapulohumeral muscular dystrophy
US12018087B2 (en) 2018-08-02 2024-06-25 Dyne Therapeutics, Inc. Muscle-targeting complexes comprising an anti-transferrin receptor antibody linked to an oligonucleotide and methods of delivering oligonucleotide to a subject
US12097263B2 (en) 2018-08-02 2024-09-24 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating myotonic dystrophy
US12128109B2 (en) 2021-07-09 2024-10-29 Dyne Therapeutics, Inc. Muscle targeting complexes and formulations for treating dystrophinopathies
WO2025024334A1 (en) 2023-07-21 2025-01-30 Marrow Therapeutics, Inc. Hematopoietic cell targeting conjugates and related methods
WO2025090898A1 (en) * 2023-10-26 2025-05-01 Biogen Ma Inc. Anti-transferrin receptor antibodies and uses thereof cross reference to related applications
EP4185329A4 (en) * 2020-07-23 2025-06-04 Dyne Therapeutics, Inc. Muscle-targeting complexes and uses thereof
US12329825B1 (en) 2018-08-02 2025-06-17 Dyne Therapeutics, Inc. Muscle targeting complexes comprising an anti-transferrin receptor antibody linked to an oligonucleotide and method of use thereof to induce exon skipping of exon 44 of dystrophin in a subject
EP4185330A4 (en) * 2020-07-23 2025-06-25 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating dystrophinopathies
US12370264B1 (en) 2018-08-02 2025-07-29 Dyne Therapeutics, Inc. Complexes comprising an anti-transferrin receptor antibody linked to an oligonucleotide and method of delivering oligonucleotide to a subject

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2025140522A1 (en) * 2023-12-29 2025-07-03 Nona Biosciences (Suzhou) Co., Ltd. Anti-tfr1 antibodies, preparation methods and uses thereof
CN119192378B (zh) * 2024-10-16 2025-10-17 中国人民解放军军事科学院军事医学研究院 一种pH敏感型人源抗转铁蛋白受体1抗体
CN119161483A (zh) * 2024-10-16 2024-12-20 中国人民解放军军事科学院军事医学研究院 人源抗转铁蛋白受体1抗体及其应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130028891A1 (en) * 2010-04-13 2013-01-31 The Regents Of The University Of California Unconjugated Anti-TfR Antibodies and Compositions Thereof for the Treatment of Cancer
US20190153083A1 (en) * 2016-04-12 2019-05-23 Universite Francois Rabelais Novel peptide structures and use thereof in the treatment of toxoplasmosis
WO2019229658A1 (en) * 2018-05-30 2019-12-05 Novartis Ag Entpd2 antibodies, combination therapies, and methods of using the antibodies and combination therapies

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
HRP20240107T1 (hr) * 2013-05-20 2024-04-12 F. Hoffmann - La Roche Ag Protutijela na transferinski receptor i postupci uporabe
MY187033A (en) * 2015-06-24 2021-08-27 Japan Chem Res Anti-human transferrin receptor antibody permeating blood-brain barrier
US20210324101A1 (en) * 2018-08-02 2021-10-21 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating hypertrophic cardiomyopathy
EP3829635A4 (en) * 2018-08-02 2022-05-11 Dyne Therapeutics, Inc. MUSCLE TARGETING COMPLEXES AND THEIR USES FOR THE TREATMENT OF MUSCLE ATROPHY
IL319265A (en) * 2018-12-21 2025-04-01 Avidity Biosciences Inc Anti-transferrin receptor antibodies and their uses

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130028891A1 (en) * 2010-04-13 2013-01-31 The Regents Of The University Of California Unconjugated Anti-TfR Antibodies and Compositions Thereof for the Treatment of Cancer
US20190153083A1 (en) * 2016-04-12 2019-05-23 Universite Francois Rabelais Novel peptide structures and use thereof in the treatment of toxoplasmosis
WO2019229658A1 (en) * 2018-05-30 2019-12-05 Novartis Ag Entpd2 antibodies, combination therapies, and methods of using the antibodies and combination therapies

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP4185315A4 *

Cited By (58)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US12018087B2 (en) 2018-08-02 2024-06-25 Dyne Therapeutics, Inc. Muscle-targeting complexes comprising an anti-transferrin receptor antibody linked to an oligonucleotide and methods of delivering oligonucleotide to a subject
US12325753B2 (en) 2018-08-02 2025-06-10 Dyne Therapeutics, Inc. Method of using an anti-transferrin receptor antibody to deliver an oligonucleotide to a subject having facioscapulohumeral muscular dystrophy
US11497815B2 (en) 2018-08-02 2022-11-15 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating dystrophinopathies
US11518816B2 (en) 2018-08-02 2022-12-06 Dyne Therapeutics, Inc. Methods of delivering an oligonucleotide to a subject having facioscapulohumeral muscular dystrophy
US12496352B2 (en) 2018-08-02 2025-12-16 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating myotonic dystrophy
US11633496B2 (en) 2018-08-02 2023-04-25 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating dystrophinopathies
US12478687B2 (en) 2018-08-02 2025-11-25 Dyne Therapeutics, Inc. United states
US12460011B2 (en) 2018-08-02 2025-11-04 Dyne Therapeutics, Inc. Complexes comprising an anti-transferrin receptor antibody linked to an oligonucleotide and method of delivering an oligonucleotide to a subject
US12428487B2 (en) 2018-08-02 2025-09-30 Dyne Therapeutics, Inc. Complexes comprising an anti-transferrin receptor antibody linked to an oligonicleotide and method of delivering oligonucleotide to a subject
US12370264B1 (en) 2018-08-02 2025-07-29 Dyne Therapeutics, Inc. Complexes comprising an anti-transferrin receptor antibody linked to an oligonucleotide and method of delivering oligonucleotide to a subject
US12357703B2 (en) 2018-08-02 2025-07-15 Dyne Therapeutics, Inc. Muscle-targeting complexes comprising an anti-transferin receptor antibody linked to an oligonucleotide and method of use thereof to induce exon skipping
US12329825B1 (en) 2018-08-02 2025-06-17 Dyne Therapeutics, Inc. Muscle targeting complexes comprising an anti-transferrin receptor antibody linked to an oligonucleotide and method of use thereof to induce exon skipping of exon 44 of dystrophin in a subject
US11787869B2 (en) 2018-08-02 2023-10-17 Dyne Therapeutics, Inc. Methods of using muscle targeting complexes to deliver an oligonucleotide to a subject having facioscapulohumeral muscular dystrophy or a disease associated with muscle weakness
US11795233B2 (en) 2018-08-02 2023-10-24 Dyne Therapeutics, Inc. Muscle-targeting complex comprising an anti-transferrin receptor antibody linked to an oligonucleotide
US11795234B2 (en) 2018-08-02 2023-10-24 Dyne Therapeutics, Inc. Methods of producing muscle-targeting complexes comprising an anti-transferrin receptor antibody linked to an oligonucleotide
US11369689B2 (en) 2018-08-02 2022-06-28 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating dystrophinopathies
US11833217B2 (en) 2018-08-02 2023-12-05 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating dystrophinopathies
US12319743B2 (en) 2018-08-02 2025-06-03 Dyne Therapeutics, Inc. Complexes comprising an anti-transferrin receptor antibody linked to an oligonucleotide and method of delivering oligonucleotide to a subject
US12280122B2 (en) 2018-08-02 2025-04-22 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating myotonic dystrophy
US12263225B2 (en) 2018-08-02 2025-04-01 Dyne Therapeutics, Inc. Muscle-targeting complexes comprising an anti-transferrin receptor antibody linked to an oligonucleotide and methods of use thereof to target dystrophin and to treat Duchenne muscular dystrophy
US11911484B2 (en) 2018-08-02 2024-02-27 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating myotonic dystrophy
US12097263B2 (en) 2018-08-02 2024-09-24 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating myotonic dystrophy
US12173079B2 (en) 2018-08-02 2024-12-24 Dyne Therapeutics, Inc. Muscle-targeting complexes comprising an anti-transferrin receptor antibody linked to an oligonucleotide
US12173078B2 (en) 2018-08-02 2024-12-24 Dyne Therapeutics, Inc. Complexes comprising an anti-transferrin receptor antibody linked to an oligonucleotide
US11390682B2 (en) 2018-08-02 2022-07-19 Dyne Therapeutics, Inc. Methods of intravenouisly delivering anti-transferrin antibody/oligonucleotide complexes to subjects having muscular dystrophy
US12012460B2 (en) 2018-08-02 2024-06-18 Dyne Therapeutics, Inc. Muscle-targeting complexes comprising an anti-transferrin receptor antibody linked to an oligonucleotide
US12005124B2 (en) 2018-08-02 2024-06-11 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating dystrophinopathies
EP4185329A4 (en) * 2020-07-23 2025-06-04 Dyne Therapeutics, Inc. Muscle-targeting complexes and uses thereof
EP4185330A4 (en) * 2020-07-23 2025-06-25 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating dystrophinopathies
US11839660B2 (en) 2021-07-09 2023-12-12 Dyne Therapeutics, Inc. Anti-transferrin receptor antibody and uses thereof
US11759525B1 (en) 2021-07-09 2023-09-19 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating facioscapulohumeral muscular dystrophy
US12128109B2 (en) 2021-07-09 2024-10-29 Dyne Therapeutics, Inc. Muscle targeting complexes and formulations for treating dystrophinopathies
US12144868B2 (en) 2021-07-09 2024-11-19 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating dystrophinopathies
US12144867B2 (en) 2021-07-09 2024-11-19 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating dystrophinopathies
US12102687B2 (en) 2021-07-09 2024-10-01 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating myotonic dystrophy
US11633498B2 (en) 2021-07-09 2023-04-25 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating myotonic dystrophy
US11638761B2 (en) 2021-07-09 2023-05-02 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating Facioscapulohumeral muscular dystrophy
US12239717B2 (en) 2021-07-09 2025-03-04 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating dystrophinopathies
US11648318B2 (en) 2021-07-09 2023-05-16 Dyne Therapeutics, Inc. Anti-transferrin receptor (TFR) antibody and uses thereof
US12440575B2 (en) 2021-07-09 2025-10-14 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating dystrophinopathies
US11986537B2 (en) 2021-07-09 2024-05-21 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating dystrophinopathies
US12239716B2 (en) 2021-07-09 2025-03-04 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating dystrophinopathies
US11672872B2 (en) 2021-07-09 2023-06-13 Dyne Therapeutics, Inc. Anti-transferrin receptor antibody and uses thereof
US11844843B2 (en) 2021-07-09 2023-12-19 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating facioscapulohumeral muscular dystrophy
US12329824B1 (en) 2021-07-09 2025-06-17 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating dystrophinopathies
US12403203B2 (en) 2021-07-09 2025-09-02 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating dystrophinopathies
US11771776B2 (en) 2021-07-09 2023-10-03 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating dystrophinopathies
US11969475B2 (en) 2021-07-09 2024-04-30 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating facioscapulohumeral muscular dystrophy
US12397062B2 (en) 2021-07-09 2025-08-26 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating dystrophinopathies
US11679161B2 (en) 2021-07-09 2023-06-20 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating facioscapulohumeral muscular dystrophy
US11827702B2 (en) 2021-09-01 2023-11-28 Biogen Ma Inc. Anti-transferrin receptor antibodies and uses thereof
US12440574B2 (en) 2022-04-15 2025-10-14 Dyne Therapeutics, Inc. Muscle targeting complexes and formulations for treating myotonic dystrophy
US11931421B2 (en) 2022-04-15 2024-03-19 Dyne Therapeutics, Inc. Muscle targeting complexes and formulations for treating myotonic dystrophy
WO2024026474A1 (en) * 2022-07-29 2024-02-01 Regeneron Pharmaceuticals, Inc. Compositions and methods for transferrin receptor (tfr)-mediated delivery to the brain and muscle
WO2024064754A1 (en) * 2022-09-20 2024-03-28 Dana-Farber Cancer Institute, Inc. Receptor-mediated endocytosis for targeted degradation and delivery of therapeutic agents
WO2024064756A1 (en) * 2022-09-20 2024-03-28 Dana-Farber Cancer Institute, Inc. Receptor-mediated endocytosis for targeted internalization and degradation of membrane proteins and cargos
WO2025024334A1 (en) 2023-07-21 2025-01-30 Marrow Therapeutics, Inc. Hematopoietic cell targeting conjugates and related methods
WO2025090898A1 (en) * 2023-10-26 2025-05-01 Biogen Ma Inc. Anti-transferrin receptor antibodies and uses thereof cross reference to related applications

Also Published As

Publication number Publication date
AU2021312708A1 (en) 2023-03-16
CN116348138A (zh) 2023-06-27
JP2023535443A (ja) 2023-08-17
US20230256112A1 (en) 2023-08-17
KR20230041760A (ko) 2023-03-24
MX2023000962A (es) 2023-04-19
IL299667A (en) 2023-03-01
BR112023001005A2 (pt) 2023-03-28
EP4185315A4 (en) 2024-07-31
CA3186727A1 (en) 2022-01-27
EP4185315A1 (en) 2023-05-31

Similar Documents

Publication Publication Date Title
US11839660B2 (en) Anti-transferrin receptor antibody and uses thereof
US20230256112A1 (en) Anti-transferrin receptor (tfr) antibody and uses thereof
US20230113823A1 (en) Anti-transferrin receptor (tfr) antibody and uses thereof
US20230103793A1 (en) Anti-transferrin receptor (tfr) antibody and uses thereof
US12403203B2 (en) Muscle targeting complexes and uses thereof for treating dystrophinopathies
US20230285586A1 (en) Muscle targeting complexes and uses thereof for treating dystrophinopathies
US20230346966A1 (en) Muscle-targeting complexes and uses thereof in treating muscle atrophy
US12440575B2 (en) Muscle targeting complexes and uses thereof for treating dystrophinopathies
AU2021318851A1 (en) Muscle targeting complexes and uses thereof for treating myotonic dystrophy
AU2021313057A1 (en) Muscle targeting complexes and uses thereof for treating facioscapulohumeral muscular dystrophy
EP4093773A1 (en) Muscle-targeting complexes and uses thereof in treating muscle atrophy
WO2021142260A1 (en) Muscle targeting complexes and uses thereof for modulation of acvr1

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21846109

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 202317003369

Country of ref document: IN

ENP Entry into the national phase

Ref document number: 3186727

Country of ref document: CA

ENP Entry into the national phase

Ref document number: 2023504620

Country of ref document: JP

Kind code of ref document: A

REG Reference to national code

Ref country code: BR

Ref legal event code: B01A

Ref document number: 112023001005

Country of ref document: BR

ENP Entry into the national phase

Ref document number: 20237005829

Country of ref document: KR

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: 2021846109

Country of ref document: EP

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2021846109

Country of ref document: EP

Effective date: 20230223

ENP Entry into the national phase

Ref document number: 2021312708

Country of ref document: AU

Date of ref document: 20210709

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 112023001005

Country of ref document: BR

Kind code of ref document: A2

Effective date: 20230118