Transferrin receptor-binding molecule conjugates for delivery of oligonucleotides to cells

Abstract

Provided herein is a conjugate comprising an anti-TfR antibody antigen-binding domain that binds to the transferrin receptor and an oligonucleotide(s). The conjugate can be used to deliver the oligonucleotide to cells that express the transferrin receptor. Delivering the oligonucleotide to cells can be used to regulate the expression of a target gene or sequence within the cell. A TfR binder-oligonucleotide conjugate for delivering the oligonucleotide to the CNS or cells expressing the transferrin receptor (TfR) is described, comprising an oligonucleotide linked to an anti-TfR antibody antigen-binding domain.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 357,959, filed July 1, 2022, which is incorporated herein by reference.

[0002] Sequence Listing The sequence listing set forth in file DNL-041-01-WO_SeqListing.txt is 177 kilobytes in size, was created on June 30, 2023, and is incorporated herein by reference.

[0003] The presently disclosed subject matter relates to TfR binding agent-oligonucleotide conjugates that bind to transferrin receptors on target cells and modulate expression of target genes or sequences in those cells, and methods for their use. [Background technology]

[0004] In vivo delivery of nucleic acid-based molecules, such as antisense oligonucleotides or RNAi agents, often requires specific targeting to reach specific tissues or cell types. Delivery to non-hepatic tissues, in particular, remains an obstacle, limiting the use of such therapies. Delivery of oligonucleotides to the central nervous system (CNS) poses unique challenges due to the blood-brain barrier (BBB). One means of delivering oligonucleotides to the CNS is via intrathecal delivery. However, intrathecal delivery is invasive, carries a higher risk of side effects, and often results in uneven distribution.

[0005] The transferrin receptor is a potential target for the delivery of cancer diagnostic and therapeutic agents. This type II transmembrane glycoprotein is responsible for cellular iron transport and is found at low levels on the surface of many normal cell types.

[0006] What is needed are therapeutic modalities that can target the transferrin receptor to deliver cargo to cells via the transferrin receptor. Summary of the Invention [Means for solving the problem]

[0007] A TfR-binding agent-oligonucleotide conjugate for delivering oligonucleotides to the CNS or cells expressing the transferrin receptor (TfR) is described, comprising an oligonucleotide linked to an anti-TfR antibody antigen-binding domain. The anti-TfR antibody antigen-binding domain can be, but is not limited to, an antibody, a single-chain antibody, a Fab, a F(ab')2, a single-chain Fab (scFab), an Fv fragment, a single-chain variable fragment (scFv), a bivalent scFv, a heavy-chain-only antibody variable domain (nanobody, e.g., VHH or vNAR), or a nanobody. In some embodiments, the anti-TfR antibody antigen-binding domain comprises or consists of an scFv. In some embodiments, the anti-TfR antibody antigen-binding domain comprises or consists of a Fab. In some embodiments, the anti-TfR antibody antigen-binding domain comprises or consists of an scFab. The anti-TfR antibody antigen-binding domain can be derived from any known antibody that specifically binds to TfR. In some embodiments, the anti-TfR antibody antigen-binding domain comprises a VH domain comprising a CDR having the sequence of SEQ ID NO: 12-14 and a VL domain comprising a CDR having the sequence of SEQ ID NO: 15-17. In some embodiments, the anti-TfR antibody antigen-binding domain comprises a VH domain comprising a CDR having the sequence of SEQ ID NO: 21-23 and a VL domain comprising a CDR having the sequence of SEQ ID NO: 24-26. In some embodiments, the anti-TfR antibody antigen-binding domain comprises a VH domain comprising a CDR having the sequence of SEQ ID NO: 114-116 and a VL domain comprising a CDR having the sequence of SEQ ID NO: 117-119. In some embodiments, the anti-TfR antibody antigen-binding domain comprises a VH domain comprising a CDR having the sequence of SEQ ID NO: 126-128 and a VL domain comprising a CDR having the sequence of SEQ ID NO: 129-131. In some embodiments, the anti-TfR antibody antigen-binding domain comprises a VH domain comprising a CDR having the sequence of SEQ ID NO: 134-136 and a VL domain comprising a CDR having the sequence of SEQ ID NO: 137-139. In some embodiments, the anti-TfR antibody antigen-binding domain comprises a VH domain comprising CDRs having the sequences of SEQ ID NOs: 154-156 and a VL domain comprising CDRs having the sequences of SEQ ID NOs: 157-159.In some embodiments, the anti-TfR antibody antigen-binding domain comprises a VH domain comprising a CDR having the sequence of SEQ ID NO: 161-163 and a VL domain comprising a CDR having the sequence of SEQ ID NO: 164-166. In some embodiments, the antigen-binding domain of the anti-TfR antibody specifically binds to human TfR. In some embodiments, the TfR binding region binds to the apical domain of TfR. An oligonucleotide can be linked directly or indirectly to the anti-TfR antibody antigen-binding domain. For indirect linkage, the oligonucleotide can be linked to the anti-TfR antibody antigen-binding domain via a chemical linker and / or a peptide. The peptide can be, but is not limited to, an Fc polypeptide, an Fc dimer, or albumin. The oligonucleotide can be, but is not limited to, an antisense oligonucleotide (ASO) or an RNA interference oligonucleotide. The anti-TfR antibody antigen-binding domain can have substitutions or modifications that facilitate conjugation of the oligonucleotide. The peptide (eg, Fc polypeptide, Fc dimer, or albumin), if present, may have substitutions or modifications that facilitate conjugation of oligonucleotides.

[0008] In some embodiments, the TfR-binding agent-oligonucleotide conjugate comprises an oligonucleotide linked to an anti-TfR Fab or scFab. In some embodiments, the TfR-binding agent-oligonucleotide conjugate comprises an oligonucleotide linked to a monomeric anti-TfR Fab or scFab (monoFab). MonoFab indicates that the TfR-binding agent-oligonucleotide conjugate comprises a single Fab or scFab (i.e., the TfR-binding agent-oligonucleotide conjugate does not contain a second antibody antigen-binding domain). In some embodiments, the monoFab is linked to an Fc polypeptide or Fc dimer. In some embodiments, the monoFab is linked to an Fc polypeptide or Fc dimer, and the oligonucleotide is linked to the Fc polypeptide, Fc dimer. The Fc polypeptide, Fc dimer, Fab, or scFab may have substitutions or modifications that facilitate conjugation of the oligonucleotide.

[0009] In some embodiments, the TfR-binding agent-oligonucleotide conjugate comprises: PF-P' | Formula (I) (L-(O) y ) n comprising or consisting of: P comprises an anti-TfR antibody antigen-binding domain; F is optionally present or absent, and if present comprises a peptide, an Fc polypeptide, an Fc dimer, or albumin; L is optionally present or absent and, if present, is a linking group; P' is optionally present or absent and, if present, comprises an anti-TfR antibody antigen-binding domain, a non-binding Fab, a non-binding variable region (NBVR), or an antibody binding domain that does not specifically bind transferrin; O is an oligonucleotide, y is an integer greater than or equal to 1 (e.g., 1, 2, 3, or 4); n is an integer greater than or equal to 1 (e.g., 1, 2, 3, 4, 5, 6, 7, or 8).

[0010] PF-P' may be referred to as a TfR-binding agent. In some embodiments, PF-P' comprises an anti-TfR antibody. In some embodiments, PF comprises a monovalent anti-TfR antibody. When P' is present and F comprises an Fc dimer, P or the heavy chain component of P can form a single polypeptide chain with one Fc polypeptide of the Fc dimer and P', or the heavy chain component of P' can form a single polypeptide chain with the other Fc polypeptide of the Fc dimer. When n is 2 or more, y is independently 1 or more (e.g., 1, 2, 3, or 4) for each (L-(O)y). In some embodiments, the oligonucleotide comprises an ASO.

[0011] In some embodiments, P is an anti-TfR Fab or scFab, F is an Fc dimer, and P' is absent. In some embodiments, the oligonucleotide comprises an ASO.

[0012] In some embodiments, P is an anti-TfR Fab or scFab, F is an Fc dimer, and P' is a non-binding Fab or NBVR. In some embodiments, the oligonucleotide comprises an ASO.

[0013] In some embodiments, P is an anti-TfR scFv, VHH, or nanobody, F is an Fc dimer, and P' is absent. In some embodiments, the oligonucleotide comprises an ASO.

[0014] In some embodiments, P is an anti-TfR scFv, VHH, or nanobody, F is an Fc dimer, and P' is a non-binding Fab or NBVR. In some embodiments, the oligonucleotide comprises an ASO.

[0015] In some embodiments, P is an anti-TfR scFv, VHH, or nanobody, F is albumin, and P' is absent. In some embodiments, the oligonucleotide comprises an ASO.

[0016] In some embodiments, the TfR-binding agent-oligonucleotide conjugate comprises: A protein, antibody Fc constant domain dimers, a first Fab that specifically binds to the transferrin receptor (TfR); and a protein comprising a modification for covalent conjugation; and an oligonucleotide conjugated at the modification site.

[0017] The antibody Fc constant domain dimer comprises a first Fc polypeptide and a second Fc polypeptide. In some embodiments, the first Fab comprises a VH domain comprising a CDR having a sequence of SEQ ID NO: 12-14 and a VL domain comprising a CDR having a sequence of SEQ ID NO: 15-17. In some embodiments, the first Fab comprises a VH domain comprising a CDR having a sequence of SEQ ID NO: 21-23 and a VL domain comprising a CDR having a sequence of SEQ ID NO: 24-26. In some embodiments, the first Fab comprises a VH domain comprising a CDR having a sequence of SEQ ID NO: 114-116 and a VL domain comprising a CDR having a sequence of SEQ ID NO: 117-119. In some embodiments, the first Fab comprises a VH domain comprising a CDR having a sequence of SEQ ID NO: 126-128 and a VL domain comprising a CDR having a sequence of SEQ ID NO: 129-131. In some embodiments, the first Fab comprises a VH domain comprising a CDR having a sequence of SEQ ID NO: 134-139 and a VL domain comprising a CDR having a sequence of SEQ ID NO: 137-139. In some embodiments, the first Fab comprises a VH domain comprising a CDR having a sequence of SEQ ID NO: 154-156 and a VL domain comprising a CDR having a sequence of SEQ ID NO: 157-159. In some embodiments, the first Fab comprises a VH domain comprising a CDR having a sequence of SEQ ID NO: 161-163 and a VL domain comprising a CDR having a sequence of SEQ ID NO: 164-166. The first Fab may be linked to a first Fc polypeptide or a second Fc polypeptide to form a Fab-Fc fusion. In some embodiments, the TfR-binding agent-oligonucleotide conjugate further comprises a second Fab. The second Fab may be, but is not limited to, a Fab that specifically binds to TfR, a non-binding Fab, or a non-binding variable region (NBVR). The second Fab can be linked to the first Fc polypeptide or the second Fc polypeptide to form a Fab-Fc fusion. In some embodiments, the TfR binding agent-oligonucleotide conjugate comprises a second Fab, wherein the first Fab is linked to the first Fc polypeptide and the second Fab is linked to the second Fc polypeptide.In some embodiments, the TfR-binding agent-oligonucleotide conjugate comprises a second Fab, wherein the first Fab is linked to a second Fc polypeptide and the second Fab is linked to the first Fc polypeptide. In some embodiments, the oligonucleotide is conjugated to the antibody via a linker "L".

[0018] In certain embodiments, the TfR-binding agent-oligonucleotide conjugate comprises an antibody-oligonucleotide conjugate comprising: An antibody that binds to the transferrin receptor (TfR), comprising a heavy chain CDR of SEQ ID NO: 12 to 14, 21 to 23, 114 to 116, 126 to 128, 134 to 136, 154 to 156, or 161 to 163, and a light chain CDR of SEQ ID NO: 15 to 17, 24 to 26, 117 to 119, 129 to 131, 137 to 139, 157 to 159, or 164 to 166; and Oligonucleotides conjugated to cysteine modifications on the constant domain of an antibody.

[0019] In some embodiments, the oligonucleotide is conjugated to the antibody via a linker "L."

[0020] In certain embodiments, the TfR-binding agent-oligonucleotide conjugate comprises an antibody-oligonucleotide conjugate comprising: An antibody that binds to the transferrin receptor (TfR), comprising a heavy chain CDR of SEQ ID NO: 12 to 14, 21 to 23, 114 to 116, 126 to 128, 134 to 136, 154 to 156, or 161 to 163, and a light chain CDR of SEQ ID NO: 15 to 17, 24 to 26, 117 to 119, 129 to 131, 137 to 139, 157 to 159, or 164 to 166; and Oligonucleotides conjugated to cysteine modifications on the constant domain of an antibody.

[0021] In certain embodiments, the TfR-binding agent-oligonucleotide conjugates described herein have the following structure: [ka] In certain embodiments, the subject matter described herein relates to a method of modulating expression of a target gene in muscle cells or CNS cells of a patient, the method comprising administering to the patient a conjugate described herein or a pharmaceutical composition comprising a conjugate. These and other embodiments are fully described herein. [Brief explanation of the drawings]

[0022] [Figure 1] Shown are huIgG, intact drug, % intact drug, and total ASO in the CNS, i.e., cortex, spinal cord, for TfR mono-Fab conjugates 24 hours after a single dose. [Figure 2A] huIgG and intact drug in the CNS, i.e., cortex, spinal cord, for TfR mono-Fab conjugates 72 hours after the last dose in a multiple dose study. [Figure 2B] 1 shows total ASO and Malat1 knockdown in the CNS, i.e., cortex, spinal cord, for TfR mono-Fab conjugates 72 hours after the final dose in a multiple dose study. [Figure 3] Shown are huIgG, intact drug, % intact drug, and total ASO in the periphery for TfR mono-Fab conjugates 24 hours after a single dose. [Figure 4] Shown are huIgG, intact drug, and total ASO for TfR mono-Fab conjugates 72 hours after the last dose in a multiple dose study. [Figure 5] Malat1 is shown for TfR mono-Fab conjugates 72 hours after the last dose in a multiple dose study. [Figure 6] 1 shows Malat1 knockdown in the CNS and periphery for anti-TfR bivalent antibody conjugates. [Figure 7]1 shows plasma clearance of TfR monoFab:ASO conjugates. [Figure 8] huIgG concentrations in brain, spinal cord, and peripheral tissues of TfR-monoFab:ASO conjugates are shown. [Figure 9] Figure 1 shows ASO concentrations in brain, spinal cord, and peripheral tissues for TfR-mono Fab:ASO conjugates. Unconjugated ASO is the first bar in each tissue (brain and SC not shown). TfR-mono Fab is the middle bar in each tissue. TfR-mono Fab2 is the third bar in each tissue. [Figure 10] Shows ASO concentrations in the brain 72 hours after Tfr albumin:ASO conjugate. [Figure 11] Shows ASO concentrations in kidney and liver after 72 hours of Tfr albumin:ASO conjugate. [Figure 12] 1 shows plasma clearance of Tfr albumin:ASO conjugates. [Figure 13] Illustrated are exemplary TfR-binding agent-oligonucleotide conjugates with (a) anti-TfR Fab / non-binding Fab antibody (top left), (b) anti-TfR mono-Fab antibody (top right), and (c) anti-TfR scFv-albumin (bottom). (a), (b), and (c) are shown with ASO bound. (c) is shown with an optional 6xHis tag. DETAILED DESCRIPTION OF THE INVENTION

[0023] overview Oligonucleotide therapy for disorders caused by genetic abnormalities or increased protein accumulation is becoming more and more common approach to regulate gene expression and treat disorders.Delivery of oligonucleotide to cells remains a challenge.This paper discloses a TfR binder-oligonucleotide conjugate that utilizes transferrin receptor to deliver oligonucleotide to target cells.

[0024] In certain embodiments, the described TfR binder-oligonucleotide conjugates can cross the blood-brain barrier (BBB). Generally, the BBB represents a barrier to delivering systemically administered oligonucleotides to their relevant sites of action in the CNS. Intrathecal (IT) delivery, in which drugs are administered directly into the cerebrospinal fluid (CSF) space, allows bypassing the BBB. However, one limitation of this approach is that direct delivery of these oligonucleotide therapeutics to the CSF via IT approach does not achieve effective distribution within the CNS.

[0025] In certain embodiments, the TfR binder-oligonucleotide conjugate can deliver the conjugated oligonucleotide to the CNS or cells that express transferrin receptor.The cells can be, but are not limited to, muscle cells or cancer cells.The muscle cells can be, but are not limited to, skeletal muscle cells or cardiac cells.

[0026] Described herein are TfR-binding agent-oligonucleotide conjugates and methods of use thereof. In some embodiments, the TfR-binding agent comprises a monovalent antibody (a mono-Fab, i.e., an antibody having a single anti-TfR antibody antigen-binding domain, e.g., a single Fab arm or scFv). In some embodiments, the TfR-binding agent comprises a bispecific, bivalent antibody (i.e., an antibody having a single anti-TfR antibody antigen-binding domain and a single non-binding Fab or NBVR). In some embodiments, the TfR-binding agent comprises an anti-TfR scFab, svFc, VHH, vNAR, or nanobody linked to albumin. In some embodiments, the TfR-binding agent comprises a bivalent anti-TfR antibody (e.g., anti-TfR(Fab)2). In some embodiments, the TfR-binding agent-oligonucleotide conjugate comprises an oligonucleotide covalently linked to an anti-TfR antibody.

[0027] In certain embodiments, the oligonucleotide is conjugated at a cysteine modification on the constant domain of an antibody, Fc polypeptide, or Fc dimer.

[0028] The antigen-binding domain of an anti-TfR antibody for use in forming a TfR binder can be derived from an antibody known to have affinity for the transferrin receptor. Derived indicates that the antigen-binding domain of the anti-TfR antibody comprises an antibody, an antigen-binding fragment of an antibody, or an antigen-binding region having the CDR sequences of an antibody. Examples of antibodies or protein molecules that can be used to conjugate oligonucleotides include those described in WO2014 / 033074, WO2016 / 081640, and WO2020 / 132584, each of which is incorporated herein by reference in its entirety.

[0029] The oligonucleotide may also be referred to as the cargo delivered to the target cell by the TfR-binding agent-oligonucleotide conjugate. The oligonucleotide may be, but is not limited to, an antisense oligonucleotide ("ASO") or an RNAi agent (e.g., siRNA or shRNA).

[0030] In further embodiments, provided herein are therapeutic methods and methods of use that use the conjugates described herein to target oligonucleotides (e.g., ASOs or RNAi agents) to transferrin receptor-expressing cells (e.g., for delivery of the oligonucleotide to the cells).

[0031] definition As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0032] As used herein, when used to modify a quantity specified by a numerical value or range, the terms "about" and "approximately" indicate numerical values and reasonable deviations from the value known to one of ordinary skill in the art, e.g., ±20%, ±10%, or ±5%, within the intended meaning of the recited value.

[0033] The term "non-targeting Fab fragment" refers to a Fab fragment that does not specifically bind to an antigen through the heavy or light chain variable domain of the antigen, or that does not specifically bind to an antigen expressed in a given mammal, e.g., a primate (e.g., human and non-human primates) or rodent (e.g., mouse), or in a particular tissue within such a mammal, through the heavy or light chain variable domain of the antigen.

[0034] "Transferrin receptor" or "TfR" refers to transferrin receptor protein 1. The human transferrin receptor 1 polypeptide sequence is set forth in SEQ ID NO: 2. Transferrin receptor protein 1 sequences from other species are also known (e.g., chimpanzee, accession number XP_003310238.1; rhesus monkey, NP_001244232.1; dog, NP_001003111.1; cow, NP_001193506.1; mouse, NP_035768.1; rat, NP_073203.1; and chicken, NP_990587.1). The term "transferrin receptor" also encompasses allelic variants of exemplary reference sequences, e.g., human sequences, encoded by genes at the transferrin receptor protein 1 chromosomal locus. Full-length transferrin receptor protein includes a short N-terminal intracellular region, a transmembrane region, and a large extracellular domain. The extracellular domain is characterized by three domains: a protease-like domain, a helical domain, and an apical domain. The apical domain comprises residues 189-383 of human TfR. The apical domain sequence of human transferrin receptor 1 is shown in SEQ ID NO:3.

[0035] The term "constant domain" refers to the light chain constant region domain polypeptide (CL) and the CH1, CH2 and CH3 domain polypeptides from the heavy chain.

[0036] The terms "CH1 domain," "CH3 domain," and "CH2 domain" refer to immunoglobulin constant region domain polypeptides. In the context of an IgG antibody, a CH3 domain polypeptide refers to the segment of amino acids from about 341 to about 447 as numbered according to the EU numbering scheme, a CH2 domain polypeptide refers to the segment of amino acids from about 231 to about 340 as numbered according to the EU numbering scheme, and a CH1 domain polypeptide refers to the segment of amino acids from about 118 to about 215 as numbered according to the EU numbering scheme. CH1, CH2, and CH3 domain polypeptides may also be numbered according to the IMGT (ImMunoGeneTics) numbering scheme, where the CH1 domain is numbered 1-98, the CH2 domain is numbered 1-110, and the CH3 domain is numbered 1-107 according to the IMGT Scientific chart numbering (IMGT website). The CH2 and CH3 domains are part of the Fc polypeptide of an immunoglobulin. In the context of an IgG antibody, the Fc polypeptide refers to the segment of amino acids from about 231 to about 447, numbered according to the EU numbering scheme.

[0037] The term "variable domain" refers to a light chain variable domain polypeptide (VL) and a heavy chain variable domain polypeptide (VH). The VL contains three complementarity-determining regions (CDR) regions, CDR-L1, CDR-L2, and CDR-L3, and the VH contains three CDR regions (CDR-H1, CDR-H2, and CDR-H3). The CDR regions together form the antibody binding site that binds to an antigen.

[0038] The term "Fc polypeptide" refers to the C-terminal region of a naturally occurring immunoglobulin heavy chain polypeptide characterized by an Ig fold as a structural domain. An Fc polypeptide typically contains a constant region sequence comprising at least a CH2 domain and / or a CH3 domain, and may also contain at least a portion of a hinge region. Exemplary hinge region sequences, or portions thereof, are set forth in SEQ ID NOS: 4-6.

[0039] The term "Fc polypeptide dimer" refers to a dimer of two Fc polypeptides. In some embodiments, an Fc polypeptide dimer can bind to an Fc receptor (e.g., FcγR). In an Fc polypeptide dimer, two Fc polypeptides dimerize through interaction between two CH3 antibody constant domains. In some embodiments, two Fc polypeptides may also dimerize through one or more disulfide bonds formed between the hinge domains of two dimerized Fc domain monomers. An Fc polypeptide dimer can be a heterodimer or a homodimer. An Fc polypeptide dimer can comprise two wild-type Fc polypeptides, a wild-type Fc polypeptide and a modified Fc polypeptide, or two modified Fc polypeptides. In the case of an Fc polypeptide dimer comprising two modified Fc polypeptides, the two modified Fc polypeptides can be the same or different.

[0040] The antibody antigen-binding domain includes the antigen-binding domain of an immunoglobulin or a peptide having a structure similar to that of an immunoglobulin. The immunoglobulin may be, but is not limited to, an IgG, IgM, IgE, IgA, IgD, or a heavy chain antibody. The antibody antigen-binding domain may be, but is not limited to, a Fab, scFab, Fv fragment, scFv, or a heavy chain-only antibody variable domain (e.g., a nanobody, e.g., a VHH or vNAR).

[0041] The term "CL domain" refers to a light chain immunoglobulin constant domain. In the context of an IgG antibody, a kappa CL domain polypeptide refers to the segment of amino acids from about 108 to about 214, numbered according to the EU numbering scheme. Alternatively, kappa and lambda CL domains may also be numbered according to the IMGT (ImMunoGeneTics) numbering scheme, in which kappa CL domain numbering is 1-107 and lambda CL domain numbering is 1-106, according to the IMGT Scientific chart numbering (IMGT website).

[0042] The term "Fab" or "Fab fragment" refers to a monovalent fragment consisting of the VL, VH, CL, and CH1 domains. The term "Fab" refers to a monovalent antigen-binding fragment consisting of the light chain variable region (VL) and light chain constant region (CL) (together with the antibody light chain), and the heavy chain variable region and heavy chain CH1 constant region (together with the antibody Fd fragment). Fab or Fab fragments may or may not include all or part of the antibody hinge region.

[0043] The term "single-chain Fab" or "scFab" refers to an antigen-binding fragment consisting of an Fab in which an Fd fragment and a light chain are linked via a peptide linker. The linker can connect the N-terminus of the Fd fragment to the C-terminus of the light chain, or the N-terminus of the light chain to the C-terminus of the Fd fragment.

[0044] The term "Fv fragment" refers to the V fragments that together form a binding site for an antigen. H and V L It refers to an antigen-binding fragment consisting of

[0045] The term "single-chain variable fragment" or "scFv" refers to an antigen-binding fragment consisting of a heavy chain variable region and a light chain variable region linked via a peptide linker. H N-terminus of V L and the C-terminus of or V L N-terminus of V H The scFv lacks a constant region. Modified scFvs and methods for modifying scFvs to bind to target proteins are described in WO2022 / 258841, which is incorporated herein by reference.

[0046] The term "nanobody" refers to an antibody fragment consisting of a single monomeric variable antibody domain. Nanobodies derived from camelid heavy chain antibodies may be referred to as "VHH" fragments. Nanobodies derived from cartilaginous fish heavy chain antibodies may be referred to as "vNARs." Modified VHH fragments and methods for modifying VHH fragments to bind to target proteins, including TfR, are described in WO2020 / 056327, WO2022 / 103769, and WO2023 / 023166 (each of which is incorporated herein by reference).

[0047] The term "non-targeting Fab fragment" or "NTF" refers to a Fab fragment that does not specifically bind to a naturally occurring antigen through the heavy or light chain variable domain of the antigen, or that does not specifically bind to a naturally occurring antigen expressed in a given mammal, e.g., a primate (e.g., human or non-human primate) or rodent (e.g., mouse), or in a specific tissue within such a mammal, through the heavy or light chain variable domain of the antigen. In certain embodiments, a Fab for use in the Fab-Fc fusions or Fab-Fc dimer fusions described herein does not specifically bind to transferrin through its heavy or light chain variable domain. Non-limiting examples of non-targeting Fab fragments include (a) RSV (palivizumab) Fab fragments, which are non-targeting in mice and non-human primates, and (b) Fab fragments against dinitrophenyl hapten (DNP) (see Leahy, PNAS 3661-3665, 1988).

[0048] The terms "wild-type," "native," and "naturally occurring" with respect to a CH3 or CH2 domain refer to a domain having a sequence found in nature.

[0049] As used herein, the term "mutant" with respect to a mutant polypeptide or mutant polynucleotide is used interchangeably with "variant." Variants with respect to a given wild-type CH3 or CH2 domain reference sequence can include naturally occurring allelic variants. A "non-naturally" occurring CH3 or CH2 domain refers to a variant or mutant domain that does not occur in natural cells, produced by genetic modification of a native CH3 domain or CH2 domain polynucleotide or polypeptide, for example, using genetic engineering or mutagenesis techniques. A "variant" includes any domain that contains at least one amino acid mutation with respect to the wild-type. Mutations can include substitutions, insertions, and deletions.

[0050] The term "modification site" refers to a specific position within a polypeptide that contains a mutation or variant relative to a corresponding wild-type polypeptide (e.g., a wild-type CL, CH1, CH2, or CH3 domain). In certain embodiments, the mutation or variant is non-naturally occurring. A modified site can include, for example, an insertion or substitution. The term "substitution" refers to a modification that replaces one amino acid with another. For example, a "cysteine substitution" or "cysteine modification" refers to the replacement of an amino acid with a cysteine. A modification can be indicated using the notation X number Y, where X represents the amino acid in the parent polypeptide at the position indicated by the number, and Y represents the replacement amino acid that replaces amino acid X. For example, S239C indicates that the serine at position 239 is replaced with a cysteine.

[0051] The term "amino acid" refers to naturally occurring amino acids, synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.

[0052] Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that have been later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. An "amino acid analog" refers to a compound that has the same basic chemical structure as a naturally occurring amino acid, i.e., an α-carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, or methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. An "amino acid mimetic" refers to a chemical compound that has a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

[0053] Naturally occurring α-amino acids include, but are not limited to, alanine (Ala), cysteine (Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile), arginine (Arg), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gln), serine (Ser), threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), and combinations thereof. Naturally occurring stereoisomers of α-amino acids include, but are not limited to, D-alanine (D-Ala), D-cysteine (D-Cys), D-aspartic acid (D-Asp), D-glutamic acid (D-Glu), D-phenylalanine (D-Phe), D-histidine (D-His), D-isoleucine (D-Ile), D-arginine (D-Arg), D-lysine (D-Lys), D-leucine (D-Leu), D-methionine (D-Met), D-asparagine (D-Asn), D-proline (D-Pro), D-glutamine (D-Gln), D-serine (D-Ser), D-threonine (D-Thr), D-valine (D-Val), D-tryptophan (D-Trp), D-tyrosine (D-Tyr), and combinations thereof.

[0054] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0055] The terms "polypeptide" and "peptide" are used interchangeably and refer to a polymer of amino acid residues in a single chain. The terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of corresponding naturally occurring amino acids, as well as to naturally occurring and non-naturally occurring amino acid polymers. An amino acid polymer can contain entirely L-amino acids, entirely D-amino acids, or a mixture of L- and D-amino acids.

[0056] The term "protein" refers to either a single-chain polypeptide, or a dimer (i.e., two) or multimer (i.e., three or more) of single-chain polypeptides. The dimeric or multimeric single-chain polypeptides can be linked by covalent bonds, e.g., disulfide bonds, or by non-covalent interactions.

[0057] The terms "conservative substitution," "conservative mutation," or "conservatively modified variant" refer to alterations that result in the substitution of an amino acid with another amino acid that can be classified as having similar characteristics. Examples of such defined conservative amino acid group classifications include the "charged / polar group" including Glu (glutamic acid or E), Asp (aspartic acid or D), Asn (asparagine or N), Gln (glutamine or Q), Lys (lysine or K), Arg (arginine or R), and His (histidine or H); the "aromatic group" including Phe (phenylalanine or F), Tyr (tyrosine or Y), Trp (tryptophan or W), and (histidine or H); and the "aliphatic group" including Gly (glycine or G), Ala (alanine or A), Val (valine or V), Leu (leucine or L), Ile (isoleucine or I), Met (methionine or M), Ser (serine or S), Thr (threonine or T), and Cys (cysteine or C). Within each group, subgroups may also be identified. For example, the group of charged or polar amino acids can be subdivided into subgroups including a "positively charged subgroup" consisting of Lys, Arg, and His, a "negatively charged subgroup" consisting of Glu and Asp, and a "polar subgroup" consisting of Asn and Gln. In another example, the aromatic or cyclic group can be subdivided into subgroups including a "nitrogen ring subgroup" consisting of Pro, His, and Trp, and a "phenyl subgroup" consisting of Phe and Tyr. In yet another example, the aliphatic group can be subdivided into subgroups, for example, an "aliphatic nonpolar subgroup" consisting of Val, Leu, Gly, and Ala, and an "aliphatic slightly polar subgroup" consisting of Met, Ser, Thr, and Cys.Examples of conservative mutation categories include amino acid substitutions of amino acids within the above subgroups, such as, but not limited to, Arg for Lys, or vice versa, to maintain a positive charge; Glu for Asp, or vice versa, to maintain a negative charge; Ser for Thr, or vice versa, to maintain a free -OH; and Gln for Asn, or vice versa, to maintain a free -NH. In some embodiments, a hydrophobic amino acid is substituted with a naturally occurring hydrophobic amino acid, e.g., in the active site, to preserve hydrophobicity.

[0058] In the context of two or more polypeptide sequences, the terms "identical" or percent "identity" refer to two or more sequences or subsequences that are identical, are the same, or have a specified percentage of amino acid residues, e.g., at least 60% identity, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% or more, over a particular region when compared and aligned for maximum correspondence over a comparison window or designated region, as measured using a sequence comparison algorithm or by manual alignment and visual inspection.

[0059] For polypeptide sequence comparison, typically, one amino acid sequence serves as a reference sequence to which candidate sequences are compared.Alignment can be performed by various methods available to those skilled in the art, such as visual alignment, or by using publicly available software with known algorithms to achieve maximum alignment.Such programs include the BLAST program, ALIGN, ALIGN-2 (Genentech, South San Francisco, Calif.) or Megalign (DNASTAR).The parameters used for alignment to achieve maximum alignment can be determined by those skilled in the art.For the purpose of this application, for the sequence comparison of polypeptide sequences, the BLASTP algorithm, standard protein BLAST, is used to align two protein sequences with default parameters.

[0060] The phrases "corresponding to," "determined with reference to," or "numbered with reference to," when used in the context of identifying a given amino acid residue in a polypeptide or protein sequence, refer to the position of the residue in a particular reference sequence when the given amino acid sequence is maximally aligned and compared to the reference sequence. Thus, for example, an amino acid residue in a polypeptide "corresponds to" an amino acid in SEQ ID NO: 1 if that residue matches an amino acid in the region of SEQ ID NO: 1 from amino acids 114 to 220 when optimally aligned to SEQ ID NO: 1. A polypeptide aligned to a reference sequence need not be the same length as the reference sequence.

[0061] "Binding affinity" refers to the strength of the non-covalent interaction between two molecules, e.g., between a single binding site on a polypeptide / protein and the target to which it binds, e.g., the transferrin receptor. Thus, for example, unless otherwise indicated or clear from the context, the term may refer to a 1:1 interaction between a polypeptide / protein and its target. Binding affinity is measured by the equilibrium dissociation constant (K D ) and the equilibrium dissociation constant (K D) is the dissociation rate constant (k d ,time -1 ) to the association rate constant (k a ,time -1 M-1) K D can be determined by measuring the kinetics of complex formation and dissociation, for example, using surface plasmon resonance (SPR) methods, such as the Biacore™ system; kinetic exclusion assays such as KinExA®; and BioLayer interferometry (e.g., using the ForteBio® Octet® platform). As used herein, "binding affinity" refers not only to formal binding affinities, such as those reflecting a 1:1 interaction between a polypeptide / protein and its target, but also to K values that may reflect strong binding. D It also includes the apparent affinity, from which the

[0062] The phrases "specifically bind" or "selectively bind" to a target, e.g., transferrin receptor, refer to a binding reaction in which a protein binds to a target with higher affinity, higher avidity, and / or longer duration than it binds to a structurally different target, e.g., a target that is not in the transferrin receptor family. In typical embodiments, the protein has at least 5-fold, 10-fold, 100-fold, 1000-fold, or 10,000-fold or more greater affinity for transferrin receptor compared to an unrelated target when assayed under the same affinity assay conditions. In some embodiments, the protein may bind exclusively to the human transferrin receptor.

[0063] The terms "nucleic acid" and "polynucleotide" refer to deoxyribonucleotides or ribonucleotides and polymers thereof, in either single- or double-stranded form, composed of monomers (nucleotides) containing sugar moieties, phosphates, and nucleobases. Unless otherwise limited, the terms encompass both modified and unmodified nucleic acids.

[0064] The term "nucleobase" refers to a nitrogen-containing compound that can be linked to a sugar moiety to form a nucleoside, which is the building block of a nucleotide. The ability of nucleobases to base pair and stack with each other directly leads to long-chain helical structures such as ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). Nucleobases can be naturally occurring (i.e., adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U)) or modified.

[0065] The term "nucleoside" refers to a compound comprising a nucleobase and a sugar moiety (e.g., deoxyribose or ribose, or modified variants thereof). The term nucleoside includes both modified and unmodified nucleosides.

[0066] The term "nucleotide" refers to a compound comprising a nucleobase, a sugar moiety, and one or more phosphate groups. The term nucleotide includes both modified and unmodified nucleotides.

[0067] The term "internucleoside linkage" means the covalent bond between two nucleosides in an oligonucleotide. The nucleosides can be linked via a natural bond (i.e., a phosphodiester (PO) bond) or a modified bond.

[0068] The terms "chemical modification," "modification," or "modified" can refer to a chemical change in a compound compared to its naturally occurring counterpart. For example, a nucleic acid base, a sugar moiety, or an internucleoside bond can be chemically modified. Amino acids in proteins or polypeptides can be modified. The modification can be a modification of an existing amino acid or a substitution of one amino acid for another. An example of another modification of one amino acid includes, but is not limited to, a cysteine modification, in which a naturally occurring amino acid at a position is replaced with a cysteine (i.e., a cysteine modification).

[0069] The terms "nucleotide sequence," "nucleic acid sequence," and "nucleic acid strand" refer to a sequence of bases (purines and / or pyrimidines, or synthetic derivatives thereof) in a DNA or RNA polymer, which may be single-stranded or double-stranded, and optionally contain synthetic, non-natural, or modified nucleotides and / or backbone modifications (e.g., modified oligomers) that can be incorporated into the DNA or RNA polymer. The terms "oligo," "oligonucleotide," and "oligomer" may be used interchangeably and refer to such a sequence of purines and / or pyrimidines. For example, an oligonucleotide may comprise a chemically modified or unmodified nucleic acid molecule (RNA or DNA) having a length of about, e.g., less than about 200 nucleotides (e.g., less than about 100 or 50 nucleotides). An oligonucleotide may be, for example, a single-stranded DNA or RNA (e.g., an ASO), a double-stranded DNA or RNA containing a hairpin loop (e.g., a small interfering RNA (siRNA)), or a DNA / RNA hybrid. In one embodiment, the oligonucleotide has a length ranging from about 5 to about 60 nucleotides, or from about 10 to about 50 nucleotides. In another embodiment, the oligonucleotide has a length ranging from about 5 to about 30 nucleotides, or from about 15 to about 30 nucleotides, and in yet another embodiment, the oligonucleotide has a length ranging from about 18 to about 24 nucleotides.

[0070] The terms "modified oligo," "modified oligonucleotide," or "modified oligomer" may also be used interchangeably and refer to such sequences containing synthetic, non-natural, or altered base, sugar, and / or backbone modifications.

[0071] The oligonucleotides described herein can be synthesized using standard solid-phase or solution-phase synthesis techniques well known in the art. In certain embodiments, oligonucleotides are synthesized using solid-phase phosphoramidite chemistry (U.S. Patent No. 6,773,885) on an automated synthesizer. Chemical synthesis of nucleic acids allows the production of various forms of nucleic acids with modified linkages, chimeric compositions, and non-standard bases or modified groups attached at selected locations throughout the entire length of the nucleic acid.

[0072] The term " complementary " used herein refers to the broad concept of complementary base pairing between two nucleic acids aligned with each other at antisense position.When the nucleotide position of both molecules is normally occupied by the nucleotide that can base pair with each other, the nucleic acids are considered to be complementary to each other at this position.Therefore, two nucleic acids are substantially complementary to each other when at least about 50%, at least about 60%, or at least about 80% of the corresponding position in each molecule is normally occupied by the nucleotide that base pair with each other (for example, A:T (A:U in the case of RNA) and G:C nucleotide pair).

[0073] The terms "identical" or percent "identity" in the context of two or more nucleotide sequences refer to two or more sequences or subsequences that are identical, are the same, or have a specified percentage of nucleotides, e.g., at least 60% identity, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% or more, over a particular region when compared and aligned for maximum correspondence over a comparison window or designated region as measured using a sequence comparison algorithm or by manual alignment and visual inspection.

[0074] For the sequence comparison of oligonucleotides (for example, to identify identity or complementarity), typically, one nucleotide sequence serves as the reference sequence to which candidate sequences are compared.Alignment can be performed by various methods available to those skilled in the art, such as visual alignment, or by using publicly available software with known algorithms to achieve maximum alignment.Such programs include BLAST program, ALIGN, ALIGN-2 (Genentech, South San Francisco, Calif.) or Megalign (DNASTAR).The parameters used for alignment to achieve maximum alignment can be determined by those skilled in the art.

[0075] As used herein, "hybridize" or "hybridization" refers to the pairing of complementary nucleotide sequences (e.g., between an antisense compound and its target nucleic acid, or between an antisense strand and a sense strand). As used herein, "specifically hybridize" refers to the ability of a reference nucleic acid to hybridize to one nucleic acid molecule with greater affinity than it hybridizes to another nucleic acid molecule.

[0076] "Expression" refers to the transcription and / or translation of an endogenous gene, a heterologous gene or nucleic acid segment, or a transgene in a cell. For example, expression can refer to the transcription and stable accumulation of sense (mRNA) or functional RNA. Expression can also refer to the production of a protein.

[0077] The term "gene" refers to a nucleic acid (eg, DNA or RNA) sequence that comprises coding sequences necessary for the production of a polypeptide or precursor.

[0078] The phrase "modulating the expression of a target gene or sequence" refers to a change (e.g., an increase or decrease) in the expression of the target gene or sequence (e.g., via target degradation or translational inhibition). For example, this includes inhibiting, reducing, or decreasing the expression of the target gene or sequence. This also includes changes in alternative splicing, which can result in changes in the absolute or relative amounts of particular splice variants.

[0079] The term "halo" refers to fluoro, chloro, bromo, or iodo. Alkyl, alkoxy, etc. refer to both straight-chain and branched groups; however, reference to individual radicals such as propyl encompasses only the straight-chain radical, as well as only the branched-chain isomer specifically mentioned, such as isopropyl.

[0080] The term "alkyl," by itself or as part of another substituent, means, unless otherwise stated, a straight or branched chain hydrocarbon having the stated number of carbon atoms (i.e., C1-6 means 1 to 6 carbons). Examples include (C1-C6) alkyl, (C2-C6) alkyl, and (C3-C6) alkyl. Examples of alkyl groups include methyl, ethyl, n-propyl, iso-propyl, n-butyl, t-butyl, iso-butyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, and higher homologs and isomers.

[0081] The term "alkoxy" refers to an alkyl group attached to the remainder of the molecule through an oxygen atom ("oxy").

[0082] The term "alkylthio" refers to an alkyl group attached to the remainder of the molecule via a thio group.

[0083] The term "alkoxycarbonyl" refers to the group (alkyl)-OC(=O)-, where the term alkyl has the meaning defined herein.

[0084] The term "alkanoyloxy" refers to the group (alkyl)-C(=O)-O-, where the term alkyl has the meaning defined herein.

[0085] The term "aryloxy" refers to an aryl group attached to the remainder of the molecule through an oxygen atom (aryl-O-).

[0086] The term "heteroaryloxy" refers to a heteroaryl group attached to the remainder of the molecule through an oxygen atom (heteroaryl-O-).

[0087] As used herein, the term "heteroatom" includes oxygen (O), nitrogen (N), sulfur (S) and silicon (Si).

[0088] The term "cycloalkyl" refers to a saturated or partially unsaturated (non-aromatic) all-carbon ring having 3 to 6 carbon atoms (i.e., (C3-C6) carbocycle). Non-limiting examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0089] The term "aryl" refers to a single all-carbon aromatic ring or a multiple condensed all-carbon ring system, where at least one of the rings is aromatic. For example, in certain embodiments, an aryl group has 6 to 20 carbon atoms, 6 to 14 carbon atoms, or 6 to 12 carbon atoms, or 6 to 10 carbon atoms. Aryl includes the phenyl radical. Aryl also includes multiple condensed ring systems (e.g., ring systems containing 2, 3, or 4 rings) having about 9 to 20 carbon atoms, where at least one ring is aromatic and the other rings may or may not be aromatic (i.e., cycloalkyl). Rings in multiple condensed ring systems can be connected to each other via fused, spiro, and bridged bonds, where valence requirements allow. As defined above, it should be understood that the point of attachment of multiple condensed ring systems may be at any position on the ring system, including the aromatic or carbocyclic portions of the ring. Non-limiting examples of aryl groups include, but are not limited to, phenyl, indenyl, indanyl, naphthyl, 1,2,3,4-tetrahydronaphthyl, anthracenyl, and the like.

[0090] The term "heterocycle" refers to a single saturated or partially unsaturated ring having at least one atom other than carbon in the ring, where the atom is selected from the group consisting of oxygen, nitrogen, and sulfur; the term also includes multiple condensed ring systems having at least one such saturated or partially unsaturated ring, which are further described below. Thus, the term includes a single saturated or partially unsaturated ring (e.g., a 3-, 4-, 5-, 6-, or 7-membered ring) of about 1 to 6 carbon atoms and about 1 to 3 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur in the ring. The sulfur and nitrogen atoms can also be present in their oxidized forms. Exemplary heterocycles include, but are not limited to, azetidinyl, tetrahydrofuranyl, and piperidinyl. The term "heterocycle" also includes multiple condensed ring systems (e.g., ring systems containing two, three, or four rings), where a single heterocycle (as defined above) can be fused with one or more groups selected from cycloalkyl, aryl, and heterocycle to form the multiple condensed ring system. The rings of a multiple condensed ring system can be connected to each other via fused, spiro, and bridged bonds, where valence requirements allow. It should be understood that the individual rings of a multiple condensed ring system can be connected to each other in any order. It should also be understood that the point of attachment of a multiple condensed ring system (as defined above for heterocycle) can be at any position in the multiple condensed ring system, including the heterocyclic, aryl, and carbocyclic portions of the ring. In one embodiment, the term heterocycle includes a 3- to 12-membered heterocycle. In one embodiment, the term heterocycle includes a 3- to 7-membered heterocycle. In one embodiment, the term heterocycle includes a 3- to 6-membered heterocycle. In one embodiment, the term heterocycle includes a 4- to 6-membered heterocycle. In one embodiment, the term heterocycle includes 3- to 12-membered monocyclic or bicyclic heterocycles containing 1-3 heteroatoms. In one embodiment, the term heterocycle includes 3- to 6-membered monocyclic heterocycles containing 1-2 heteroatoms. In one embodiment, the term heterocycle includes 4- to 6-membered monocyclic heterocycles containing 1-2 heteroatoms.Exemplary heterocycles include aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, homopiperidinyl, morpholinyl, thiomorpholinyl, piperazinyl, tetrahydrofuranyl, dihydrooxazolyl, tetrahydropyranyl, tetrahydrothiopyranyl, 1,2,3,4-tetrahydroquinolyl, benzoxazinyl, dihydrooxazolyl, chromanyl, 1,2-dihydropyridinyl, 2,3-dihydrobenzoyl, and the like. Furanyl, 1,3-benzodioxolyl, 1,4-benzodioxanyl, spiro[cyclopropane-1,1'-isoindolinyl]-3'-one, isoindolinyl-1-one, 2-oxa-6-azaspiro[3.3]heptanyl, imidazolidin-2-one, imidazolidine, pyrazolidine, butyrolactam, valerolactam, imidazolidinone, hydantoin, dioxolane, phthalimide, 1,4-dioxane, and. [ka] These include, but are not limited to:

[0091] In one embodiment, the heterocycle can be divalent, i.e., attached to the remainder of the molecule or to a linking group at two positions on the heterocycle (-heterocycle-). In one embodiment, the heterocycle is substituted with one or more (e.g., 1, 2, 3, or 4) substituents independently selected from the group consisting of (C-C)alkoxy, (C-C)cycloalkyl, (C-C)alkanoyl, (C-C)alkanoyloxy, (C-C)alkoxycarbonyl, (C-C)alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (=O), and carboxy.

[0092] As used herein, a wavy line crossing a bond in a chemical structure [ka] indicates the point of attachment of the bond that the wavy bond meets in the chemical structure to the rest of the molecule.

[0093] The terms "subject," "individual," and "patient," used interchangeably, refer to mammals, including, but not limited to, humans, non-human primates, rodents (e.g., rats, mice, and guinea pigs), rabbits, cows, pigs, horses, and other mammalian species. In one embodiment, the patient is a human.

[0094] The terms "treatment," "treating," and the like are used herein generally to mean obtaining a desired pharmacological and / or physiological effect. "Treating" or "treatment" can refer to any indication of successful treatment or amelioration of an injury, disease, or condition, including any objective or subjective parameter, such as alleviation, remission, improved patient survival, increased survival time or survival rate, alleviation of symptoms, or increasing the patient's tolerance of the injury, disease, or condition, slowing the rate of degeneration or decline, or improving the patient's physical or mental health. Furthermore, "treating" or "treatment" can refer to modulation of target gene expression, such as gene knockdown or gene knockout. For example, expression of a target gene or sequence is inhibited or reduced, e.g., by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%, compared to expression in a control. Treatment or amelioration of symptoms can be based on objective or subjective parameters. The effect of treatment can be compared to an individual or pool of individuals not receiving the treatment, or to the same patient at different time points before or during treatment.

[0095] The term "pharmaceutically acceptable excipient" refers to non-active pharmaceutical ingredients, such as, but not limited to, buffers, carriers, or preservatives, that are biologically or pharmacologically compatible for use in humans or animals.

[0096] A "therapeutic amount" or "therapeutically effective amount" of a drug is an amount of drug that treats, alleviates, relieves, or reduces the severity of symptoms of a disease in a subject. A "therapeutic amount" or "therapeutically effective amount" of a drug can improve survival, increase the length or rate of survival of a patient, reduce symptoms, make an injury, disease, or condition more bearable, slow the rate of degeneration or decline, or improve the physical or mental well-being of a patient.

[0097] The term "administering" refers to a method of delivering an agent, compound, or composition to a desired biological site of action. These methods include, but are not limited to, topical, parenteral, intravenous, intradermal, intramuscular, intrathecal, colonic, rectal, or intraperitoneal delivery. In one embodiment, the proteins described herein are administered intravenously.

[0098] III. TFR-binding agent-oligonucleotide conjugates In some embodiments, the TfR-binding agent-oligonucleotide conjugate comprises: PF-P' | Formula (I) (L-(O) y ) n comprising or consisting of: P comprises an anti-TfR antibody antigen-binding domain; F is optionally present or absent, and if present comprises a peptide, an Fc polypeptide, an Fc dimer, or albumin; L is optionally present or absent and, if present, is a linking group; P' is optionally present or absent, and if present, comprises an anti-TfR antibody antigen-binding domain, or a non-binding Fab, or a non-binding variable region (NBVR); O is an oligonucleotide, y is an integer greater than or equal to 1 (e.g., 1, 2, 3, or 4); n is an integer greater than or equal to 1 (e.g., 1, 2, 3, 4, 5, 6, 7, or 8).

[0099] PF-P' (or, optionally, PF or P if P' or F and P' are absent) may be referred to as a TfR-binding agent. In some embodiments, PF-P' comprises an anti-TfR antibody. The antibody may be bivalent (having two Fab arms that each specifically bind to TfR) or bispecific (having a first Fab arm that specifically binds to TfR and a second Fab arm that does not specifically bind to TfR). When P' is present and F comprises an Fc dimer, the heavy chain component of P or P can form a single polypeptide chain with one Fc polypeptide of the Fc dimer and P', or the heavy chain component of P' can form a single polypeptide chain with the other Fc polypeptide of the Fc dimer. When n is 2 or more, y is independently 1 or more (e.g., 1, 2, 3, or 4) for each (L-(O)y). In some embodiments, the oligonucleotide comprises an ASO. In some embodiments, the TfR binding agent (P, P' (if present), and / or F (if present)) comprises at least one substitution or modification that facilitates covalent conjugation of the oligonucleotide O, optionally via a linker L.

[0100] A. Monovalent anti-TfR (mono-Fab) antibody-oligonucleotide conjugates In some embodiments, the TfR-binding agent-oligonucleotide conjugate comprises: PF-(L-(O) y ) n comprising or consisting of: P comprises an anti-TfR antibody antigen-binding domain; F comprises an Fc polypeptide or an Fc dimer, L is a linking group, O is an oligonucleotide, y is an integer greater than or equal to 1 (e.g., 1, 2, 3, or 4); n is an integer equal to or greater than 1 (e.g., 1, 2, 3, 4, 5, 6, 7, or 8); wherein the TfR-binding agent-oligonucleotide conjugate comprises a single anti-TfR antibody antigen-binding domain and does not comprise any additional antibody antigen-binding domains, non-binding Fabs, or NBVRs (e.g., as shown in FIG. 13). In some embodiments, P comprises an anti-TfR Fab. In some embodiments, P comprises an anti-TfR scFv. In some embodiments, the anti-TfR antibody antigen-binding domain comprises an anti-TfR VHH, vNAR, or nanobody. The anti-TfR antibody antigen-binding domain can be derived from any anti-TfR antibody known to specifically bind to TfR. In some embodiments, F comprises an Fc dimer. In some embodiments, the oligonucleotide comprises an ASO. In some embodiments, the TfR-binding agent (P and / or F) comprises at least one substitution or modification that facilitates covalent conjugation of an oligonucleotide O, optionally via a linker L. An oligonucleotide can be linked to P or F. When an oligonucleotide is linked to F and F is an Fc dimer, it can be linked to an Fc polypeptide that is linked to P or an Fc polypeptide that is not linked to P.

[0101] B. Anti-TfR / non-binding Fab antibody-oligonucleotide conjugate In some embodiments, the TfR-binding agent-oligonucleotide conjugate comprises: PF-P' | (L-(O) y ) n comprising or consisting of: P comprises an anti-TfR antibody antigen-binding domain; F comprises an Fc dimer, L is a linking group, P' comprises a non-binding Fab or NBVR; O is an oligonucleotide, y is an integer greater than or equal to 1 (e.g., 1, 2, 3, or 4); n is an integer greater than or equal to 1 (e.g., 1, 2, 3, 4, 5, 6, 7, or 8).

[0102] In some embodiments, P comprises an anti-TfR Fab (e.g., as shown in FIG. 13). In some embodiments, P comprises an anti-TfR scFv. In some embodiments, the anti-TfR antibody antigen-binding domain comprises an anti-TfR VHH, vNAR, or nanobody. The anti-TfR antibody antigen-binding domain can be derived from any anti-TfR antibody known to specifically bind to TfR. In some embodiments, the non-binding Fab or NVBR can be any of the non-binding Fabs or NVBRs described herein. In some embodiments, the oligonucleotide comprises an ASO. In some embodiments, the TfR binding agent (P, P', and / or F) comprises at least one substitution or modification that facilitates covalent conjugation of the oligonucleotide O, optionally via a linker L. The oligonucleotide can be linked to P, F, or P'. When the oligonucleotide is linked to F (Fc dimer), it can be linked to an Fc polypeptide linked to P or to an Fc polypeptide linked to P'.

[0103] C. Anti-TfR scFv-albumin-oligonucleotide conjugate In some embodiments, the TfR-binding agent-oligonucleotide conjugate comprises: PF-(L-(O) y ) n comprising or consisting of: P comprises an anti-TfR antibody antigen-binding domain; F includes albumin, L is a linking group, O is an oligonucleotide, y is an integer greater than or equal to 1 (e.g., 1, 2, 3, or 4); n is an integer greater than or equal to 1 (e.g., 1, 2, 3, 4, 5, 6, 7, or 8).

[0104] In some embodiments, P comprises an anti-TfR scFv (e.g., as shown in FIG. 13). In some embodiments, P comprises an anti-TfR Fab. In some embodiments, the anti-TfR antibody antigen-binding domain comprises an anti-TfR VHH, vNAR, or nanobody. The anti-TfR antibody antigen-binding domain can be derived from any anti-TfR antibody known to specifically bind to TfR. In some embodiments, the albumin is human albumin. In some embodiments, the oligonucleotide comprises an ASO. In some embodiments, the TfR binding agent (P and / or F) comprises at least one substitution or modification that facilitates covalent conjugation of the oligonucleotide O, optionally via a linker L.

[0105] D. Bivalent anti-TfR antibody (anti-TfR(Fab)2)-oligonucleotide conjugate In some embodiments, the TfR-binding agent-oligonucleotide conjugate comprises: PF-P' | (L-(O) y ) n comprising or consisting of: P comprises a first anti-TfR antibody antigen-binding domain; F comprises an Fc dimer, L is a linking group, P' comprises a second anti-TfR antibody antigen-binding domain; O is an oligonucleotide, y is an integer greater than or equal to 1 (e.g., 1, 2, 3, or 4); n is an integer greater than or equal to 1 (e.g., 1, 2, 3, 4, 5, 6, 7, or 8).

[0106] In some embodiments, P and P' comprise an anti-TfR Fab. In some embodiments, P and P' comprise an anti-TfR scFv. In some embodiments, P comprises an anti-TfR Fab and P' comprises an anti-TfR scFv. In some embodiments, P comprises an anti-TfR scFv and P' comprises an anti-TfR Fab. In some embodiments, the first and / or second anti-TfR antibody antigen-binding domain comprises an anti-TfR VHH, vNAR, or nanobody. The anti-TfR antibody antigen-binding domain can be derived from any anti-TfR antibody known to specifically bind to TfR. In some embodiments, the oligonucleotide comprises an ASO. In some embodiments, the oligonucleotide comprises an ASO. In some embodiments, the TfR binding agent (P, P', and / or F) comprises at least one substitution or modification that facilitates covalent conjugation of the oligonucleotide O, optionally via a linker L.

[0107] IV. Oligonucleotides As described herein, one or more oligonucleotides (e.g., ASOs or RNAi agents) can be linked, optionally via a linker "L," to a TfR-binding agent described herein to form a TfR-binding agent-oligonucleotide conjugate.

[0108] While the length of the oligonucleotides can vary, in certain embodiments, the oligonucleotides are about 10 to about 60 nucleotides in length, or about 10 to about 30 nucleotides in length, or about 18 to about 30 nucleotides in length, or about 15 to about 25 nucleotides in length, or about 16 to about 20 nucleotides in length. Furthermore, as described below, the oligonucleotides may contain certain chemical modifications, such as modified internucleoside linkages, modified nucleobases, modified sugars, or combinations thereof. In certain embodiments, one or more oligonucleotides are linked to the TfR binding agent (i.e., via a linking group "L"). In certain embodiments, two or more oligonucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 or more) are linked to the TfR binding agent. In certain embodiments, one oligonucleotide is linked to the TfR binding agent. In certain embodiments, two oligonucleotides are linked to the TfR binding agent. In certain embodiments, four oligonucleotides are linked to the TfR binding agent.

[0109] In certain embodiments, one oligonucleotide is linked to a single linking group (L). In certain embodiments, two oligonucleotides are linked to a single linking group (L). For example, the oligonucleotides may be linked to each other in tandem. In certain embodiments, L is linked at the 5' end of the first oligonucleotide, and the second oligonucleotide is linked to the 3' end of the first oligonucleotide. In certain embodiments, the oligonucleotides may be linked via a nucleic acid linker or a non-oligonucleotide cleavable linker.

[0110] In other embodiments, the linking group is a branched linking group, and two or more oligonucleotides are separately attached to a single linking group (L) (ie, y is 2 or greater).

[0111] When two or more oligonucleotides are conjugated to a TfR-binding agent, the oligonucleotides can be the same or different. In certain embodiments, the oligonucleotides are the same.

[0112] ASO In one embodiment, each oligonucleotide is independently an ASO. The term "antisense oligonucleotide (ASO)" refers to a single strand of DNA-like or RNA-like molecule (e.g., modified nucleotides such as those described herein) that is complementary or partially complementary to a selected target polynucleotide sequence, such as mRNA. ASO can modify or regulate gene expression by binding to a complementary target sequence through several mechanisms, including, for example, by modifying splicing (exon exclusion or exon inclusion); by employing RNase H, which leads to target degradation; by translation inhibition; and by small RNA inhibition.

[0113] Typically, the length of an ASO is in the range of about 10 to 30 base pairs (bp), but it can be longer or shorter. For example, in certain embodiments, the ASO is about 10 to about 60 nucleotides in length, or about 10 to about 50 nucleotides in length, or about 10 to about 40 nucleotides in length. In certain embodiments, the ASO is about 10 to 30 nucleotides in length, or about 12 to 30 nucleotides in length, or about 14 to about 30 nucleotides in length, or about 15 to about 30 nucleotides in length, or about 16 to about 30 nucleotides in length, or about 17 to about 30 nucleotides in length, or about 18 to about 30 nucleotides in length, or about 18 to about 28 nucleotides in length, or about 18 to about 26 nucleotides in length, or about 18 to about 24 nucleotides in length, or about 15 to about 25 nucleotides in length, or about 16 to about 20 nucleotides in length. In certain embodiments, the ASO is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

[0114] The selection of antisense oligonucleotide sequences specific for a given target sequence is determined by analysis of the selected target sequence, as well as secondary structure, T mAntisense oligonucleotides are selected based on the determination of several factors, including their binding affinity, binding energy, and relative stability. Additionally, antisense oligonucleotides can be selected based on their relative inability to form dimers, hairpins, or other secondary structures that reduce or prohibit specific binding to the target mRNA in the host cell. Target regions of mRNA include the region at or near the AUG translation initiation codon and sequences that are substantially complementary to the 5' region of the mRNA. Secondary structure analysis and target site selection considerations can be performed using software and algorithms well known in the art, such as OLIGO Primer Analysis Software (Molecular Biology Insights) version 4 and / or BLASTN 2.0.5 algorithm software (Altschul et al., Nucleic Acids Res. 1997, 25(17):3389-402).

[0115] RNAi agents In certain other embodiments, each oligonucleotide is independently an RNAi agent (e.g., an siRNA or shRNA). The term "RNA interference (RNAi) agent" refers to an RNA agent, or a molecule that can be cleaved into an RNA agent, that can inhibit the expression of a target gene or sequence (e.g., mRNA, tRNA, or viral RNA) in a sequence-specific manner (e.g., via Dicer / RISC). An RNAi agent can be single-stranded or double-stranded. If the RNAi agent is single-stranded, it can include 5' modifications, such as one or more phosphate groups or phosphate group analogs. In one embodiment, the RNAi agent is double-stranded and includes a sense and an antisense strand (e.g., a small interfering RNA (siRNA)).

[0116] RNAi agent usually comprises a region that is sufficiently homologous to target gene, and is long enough so that RNAi agent can mediate the down-regulation of target gene.The complementarity between RNAi agent and target sequence should be sufficient so that RNAi agent or its cleavage product can lead to sequence-specific silencing.In certain embodiments, RNAi agent is or comprises the region that is at least partially complementary to target RNA.In certain other embodiments, RNAi agent is or comprises the region that is completely complementary to target RNA.

[0117] In some embodiments, an RNAi agent includes an unpaired region at one or both ends of the molecule. For example, a double-stranded RNAi agent may have its strands paired with overhangs, e.g., 5' and / or 3' overhangs, e.g., 1-3 nucleotide overhangs. In certain embodiments, the RNAi agent will include unpaired overhangs of 1, 2, 3, or 4 nucleotides in length at each end. The overhangs can be the result of one strand being longer than the other, or the result of two strands of the same length being staggered.

[0118] The duplex region within an RNAi agent can vary in length, but typically ranges from about 5 to about 30 nucleotides in length. In certain embodiments, the duplex region is about 15-60, or about 15-50, or about 15-40, or about 15-30, or about 15-25, or about 19-25 nucleotides in length. In certain embodiments, the duplex region is about 20-24, or about 21-23 nucleotides in length. In certain embodiments, the duplex region can be about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or more nucleotides in length.

[0119] As used herein, a "single-stranded RNAi agent" or "ssRNAi agent" is composed of a single molecule. It may include a double-stranded region formed by intrastrand pairing, for example, it may be or include a hairpin or panhandle structure. A single-stranded RNAi agent may be antisense with respect to a target molecule. A single-stranded RNAi agent may be long enough to enter RISC and participate in RISC-mediated cleavage of target mRNA. In certain embodiments, a single-stranded RNAi agent is at least 10, 15, 20, 25, 30, 35, 40, or 50 nucleotides in length. In certain embodiments, it is less than 200, 100, 80, or 60 nucleotides in length.

[0120] Small hairpin RNA (shRNA) agents typically have a duplex region less than 200, 100, or 50 nucleotides in length. In certain embodiments, the length of the duplex region ranges from about 15 to 60, about 15 to 50, about 15 to 40, about 15 to 30, about 15 to 25, or about 19 to 25 nucleotides in length. In certain embodiments, the duplex region is about 17 to 23, about 19 to 23, about 20 to 23, about 21 to 23, or about 19 to 21 nucleotides in length. In certain embodiments, the duplex region is at least about 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs in length. The hairpin may have single-stranded overhangs or terminal unpaired regions. In certain embodiments, the overhangs are 2 to 3 nucleotides in length. In some embodiments, the overhang is on the sense side of the hairpin, and in some embodiments, the overhang is on the antisense side of the hairpin.

[0121] As used herein, "double-stranded RNAi agent" or "dsRNAi agent" comprises multiple strands, where interstrand hybridization can form a duplex region within the molecule (e.g., hybridization between sense and antisense strands).In certain embodiments, the RNAi agent is large enough that it can be cleaved by endogenous molecules such as Dicer to generate smaller molecules.

[0122] In certain embodiments, the RNAi agent is an siRNA molecule comprising a sense and an antisense strand.

[0123] As used herein, the term "antisense strand" refers to the strand of an RNAi agent that is sufficiently complementary to a target polynucleotide, e.g., a target mRNA. In certain embodiments, the antisense strand of a double-stranded RNAi agent is at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, or 60 nucleotides in length. In certain embodiments, the antisense strand of a double-stranded RNAi agent is less than about 200, 100, or 50 nucleotides in length. In certain embodiments, the length of the antisense strand is in the range of about 17-25, about 19-23, or about 19-21 nucleotides in length.

[0124] As used herein, the term "sense strand" refers to the strand of an RNAi agent that is sufficiently complementary to the antisense strand. In certain embodiments, the sense strand of a double-stranded RNAi agent is at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, or 60 nucleotides in length. In certain embodiments, the sense strand of a double-stranded RNAi agent is less than about 200, 100, or 50 nucleotides in length. In certain embodiments, the length of the sense strand is in the range of about 17-25, about 19-23, or about 19-21 nucleotides in length.

[0125] In certain embodiments, the double-stranded portion of the double-stranded RNAi agent is at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, or 60 nucleotides in length. In certain embodiments, the sense strand of the double-stranded RNAi agent is less than about 200, 100, or 50 nucleotides in length. In certain embodiments, the length of the sense strand is in the range of about 17-25, or about 19-23, or about 19-21 nucleotides in length.

[0126] In certain embodiments, sense strand and antisense strand can be selected so that dsRNAi agent comprises unpaired region at one or both ends of molecule.Therefore, dsRNAi agent can comprise paired sense strand and antisense strand to contain overhang, for example, 5' and / or 3' overhang of 1, 2, 3 or 4 nucleotides in length.Overhang can be the result of one strand being longer than the other strand, or the result of two strands of the same length being staggered.In certain embodiments, dsRNAi agent comprises at least one 3' overhang.In certain embodiments, both ends of dsRNAi agent comprise 3' overhang (for example, 2 nucleotides in length).

[0127] The duplex region within a dsRNAi agent can vary in length, but typically ranges from about 5 to about 30 nucleotides in length. In certain embodiments, the length of the duplex region ranges from about 5 to 60, or about 15 to 60, or about 15 to 50, or about 15 to 40, or about 15 to 30, or about 15 to 25, or about 19 to 25 nucleotides in length. In certain embodiments, the duplex region is about 17 to 23, or about 19 to 23, or about 20 to 23, or about 21 to 23, or about 19 to 21 nucleotides in length. In certain embodiments, the duplex region can be about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or more nucleotides in length.

[0128] The method of generating RNAi agents such as siRNA and shRNA is well known in the art and can be easily adapted to generate RNAi agents that target any polynucleotide sequence.In certain embodiments, RNAi agents are chemically synthesized.For example, oligonucleotides can be synthesized using a variety of techniques, such as those described in Usman et al., J.Am.Chem.Soc., 109:7845(1987); Scaringe et al., Nucl.Acids Res., 18:5433(1990); Wincott et al., Nucl.Acids Res., 23:2677-2684(1995); and Wincott et al., Methods Mol.Bio., 74:59(1997).

[0129] Exemplary Oligonucleotide Modifications In certain embodiments, the oligonucleotides described herein may contain at least one nucleic acid modification, such as a modified internucleoside linkage, a modified nucleic acid base, a modified sugar, or a combination thereof. Such modifications can be used to modify pharmacokinetics (improved nuclease resistance, prolonged half-life), pharmacodynamics (superior affinity for target RNA), or endocytic uptake. However, many modifications prevent RNase H cleavage, the desired mechanism of action of many ASOs. Therefore, certain RNase HASSOs may be designed as chimeras, where different bases are mixtures of different chemicals, or as gapmers, where some modifications are located in the "wings" rather than the central base. In contrast, for RNAi agents and ASOs intended to modify mRNA splicing or translation, RNase H considerations are not necessary.

[0130] Therefore, the oligonucleotides described herein may contain one or more nucleic acid modifications. In certain embodiments, the oligonucleotides contain 1, 2, 3, 4, 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, or 40 or more modifications.

[0131] In certain embodiments, the oligonucleotides described herein contain one or more nucleotide modifications (e.g., to the nucleobase or sugar moiety). In certain embodiments, 25% or more of the nucleotides present in the oligonucleotide are modified. In certain embodiments, 50% or more of the nucleotides present in the oligonucleotide are modified. In certain embodiments, 75% or more of the nucleotides present in the oligonucleotide are modified. In certain embodiments, 100% of the nucleotides present in the oligonucleotide are modified.

[0132] In certain embodiments, the oligonucleotide comprises one or more nucleobase modifications. In certain embodiments, the oligonucleotide comprises one or more modifications to the sugar moiety (e.g., a furanosyl containing a substituent at the 2', 3', 4', and / or 5' position). In certain embodiments, the substituted sugar moiety comprises a bicyclic sugar moiety.

[0133] In certain embodiments, the nucleic acid modifications of the oligonucleotide are included in a pattern. In certain embodiments, the oligonucleotide is a gapmer. The modification pattern of a gapmer oligonucleotide generally has the formula 5'-X a -Y a -Z a -3', and X a and Z a is the gap region Y a In certain embodiments, Y a The region is a contiguous stretch of nucleotides, e.g., a region of at least 6 DNA nucleotides, that can recruit an RNAse, such as RNase H. In certain embodiments, Ya The region is at least 8 DNA nucleotides. In certain embodiments, Y a The region is about 9 to about 15 DNA nucleotides. a The region is about 11 to about 13 DNA nucleotides. a The region is 10, 11, 12, or 13 DNA nucleotides. In certain embodiments, the gapmer binds to the target nucleic acid, at which point an RNAse can be recruited and then cleave the target nucleic acid. In certain embodiments, the Y a The region is located at both the 5' and 3' ends of region X a and Z a and these are flanked by high affinity modified nucleotides, e.g., X a and Z a In certain embodiments, Y a The region is located at both the 5' and 3' ends of region X a and Z a and adjacent, where X a and Z a comprises a modified nucleotide having a modified sugar. a and Z a Each nucleotide in comprises a modified nucleotide having a sugar modification. The modified nucleotide may be, but is not limited to, a 2-MOE modified nucleotide, a bicyclic nucleotide, an LNA nucleotide, or a cET modified nucleotide. In certain embodiments, modified nucleotides are present in the 5' and 3' regions of the oligonucleotide, although the particular modified nucleotide and / or modified linkage may or may not be present in the central portion of the molecule. In certain embodiments, modified nucleotides are present in the 5' and 3' regions of the oligonucleotide, but the particular modified nucleotide is not present in the central portion of the molecule (e.g., LNA residues are not present in the central portion). However, the central region may contain a modified linkage, such as a PS linkage. In certain embodiments, X a and Z a are each independently about 3 to about 6 nucleotides in length. aand Z a are each independently 3, 4, or 5 nucleotides in length. a and Z a each comprises three modified nucleotides. In certain embodiments, the three modified nucleotides are a and Z a are arranged in series in each of the

[0134] Modified nucleosides / nucleotides are well known in the art and include, but are not limited to, 2'-O-methyl (2'OMe) residues, 2'O-methoxyethyl (MOE) residues, constrained nucleic acid residues (e.g., S-cEt, R-cEt, S-cMOE, and R-cMOE), peptide nucleic acid (PNA) residues, locked nucleic acid (LNA) residues, and 5-methylcytidine residues (methylated cytosine residues) (see also Scoles, et al., Neurol Genet Apr 2019, 5(2)e323). In certain embodiments, an oligonucleotide comprises one or more MOE residues. In certain embodiments, an oligonucleotide comprises one or more OMe or F residues (e.g., 2'-F or 2'OMe). In certain embodiments, an oligonucleotide comprises one or more constrained (e.g., S-cEt, R-cEt, S-cMOE, and R-cMOE) and / or LNA residues. A nucleic acid is considered "locked" if it has a methylene bridge connection made between the 2'-oxygen and the 4'-carbon of the ribose sugar molecule. In certain embodiments, the oligonucleotide is morpholino (i.e., contains certain modifications in the sugar moiety). In certain embodiments, the oligonucleotides described herein contain one or more LNA residues and one or more 5-methylcytidine residues.

[0135] In certain embodiments, the oligonucleotide comprises one or more modifications to the internucleoside backbone (i.e., the native phosphodiester (PO) linkage is modified). In certain embodiments, such modifications are made, for example, to reduce nuclease activity. Thus, in certain embodiments, the oligonucleotide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 or more modified internucleoside linkages. In certain embodiments, 25% or more of the modified internucleoside linkages are modified. In certain embodiments, 50% or more of the modified internucleoside linkages are modified. In certain embodiments, 75% or more of the modified internucleoside linkages are modified. In certain embodiments, 100% of the modified internucleoside linkages present in the oligonucleotide are modified.

[0136] Backbone modifications are well known in the art and include, but are not limited to, phosphorothioate (PS) linkages, chiral phosphorothioate linkages, phosphoramidate linkages, mesylphosphoramidate linkages, and phosphorodiamidate linkages, phosphorodithioate linkages, aminoalkylphosphotriester linkages, phosphotriester linkages, thiophosphate linkages, phosphonate linkages, methylphosphonate linkages, alkylphosphonate linkages, 3' alkylenephosphonate linkages, chiral phosphonate linkages, 3'-aminophosphoramidate linkages, aminoalkylphosphoramidate linkages, phosphinic acid linkages, thionoalkylphosphonate linkages, thionophosphoramidate linkages, thionoalkyl-phosphotriester linkages, boranophosphate linkages, morpholino linkages, and peptide nucleic acid (PNA) linkages. For example, in certain embodiments, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more) nucleoside linkages in an oligonucleotide are replaced with phosphorothioate (PS) linkages. In certain embodiments, an oligonucleotide contains a mixture of modified and unmodified linkages. Modifications at one internucleoside linkage can be independent of modifications at another internucleoside linkage. In certain embodiments, all internucleoside linkages in a MAPT ASO are modified linkages. In certain embodiments, all internucleoside linkages in a MAPT ASO are PS linkages. In some embodiments, all internucleoside linkages in an LPA ASO are phosphorothioate or mesyl phosphoramidate. In certain other embodiments, one or more of the nucleoside bonds in the oligonucleotide (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 or more) are replaced with phosphorodiamidate bonds.In certain embodiments, the oligonucleotide is phosphorodiamidate morpholino (PMO).

[0137] In certain embodiments, the internucleoside linkages are sterically random with respect to the chiral centers (Rp and Sp). In certain other embodiments, the Rp and Sp configurations of the oligonucleotide are optimized to specific configurations.

[0138] In certain embodiments, the oligonucleotide is a gapmer comprising LNA and PS modifications. For example, in certain embodiments, the oligonucleotide has the formula 5'-X a -Y a -Z a - a gapmer with a 3' modification pattern, wherein X a and Z a is the gap region Y a Let X be the neighboring region around a and Z a each contain three LNA modified nucleotides (e.g., three consecutive LNA modified nucleotides), and the gap region Y a contains a PS bond. In some embodiments, all internucleotide linkages in the antisense oligonucleotide contain a PS bond. In certain embodiments, the oligonucleotide further comprises one or more 5'-methylcytidine residues. In certain embodiments, the gap region Y a does not contain LNA residues.

[0139] V. Linking group In some embodiments, the oligonucleotides are conjugated to the TfR binding agent (e.g., an anti-TfR antibody antigen-binding domain or an anti-TfR antibody) via a linker "L." In certain embodiments, L is a linking group that links each oligonucleotide to the TfR binding agent. The linking group can be any group suitable for linking an oligonucleotide to a protein or polypeptide, such as an antibody.

[0140] The linking group can be attached to any region of the TfR-binding agent (e.g., to the N-terminal region, the C-terminal region, or to an amino acid within a protein, such as a cysteine or glutamine residue), as long as the oligonucleotide does not interfere with the binding of the TfR-binding agent to the TfR. Similarly, the linking group can be attached to any region of the oligonucleotide (e.g., the 5'-end, the 3'-end, or a nucleic acid residue within the molecule), as long as the TfR-binding agent does not interfere with the functionality of the oligonucleotide (e.g., complementary binding to a target nucleic acid). For example, the linker can be attached to the oligonucleotide through any number of synthetically feasible points located throughout the oligo, such as the 3'- or 5'-terminal residue of the oligo; the sugar moiety; the base moiety; or a residue located within the backbone.

[0141] In certain embodiments, the linker is attached to the oligonucleotide at the 5'-terminal residue of the oligonucleotide. In certain embodiments, the linker is attached to the oligonucleotide at the 3'-terminal residue of the oligonucleotide. In certain embodiments, the linker is attached to the oligonucleotide at a residue within the oligonucleotide. In certain embodiments, the oligonucleotide is a double-stranded RNAi molecule, and the linker is attached to the sense strand (e.g., at the 5'- or 3'-terminal residue). In certain embodiments, the oligonucleotide is a double-stranded RNAi molecule, and the linker is attached to the antisense strand (e.g., at the 5'- or 3'-terminal residue). In certain embodiments, the oligonucleotide is an siRNA, and the linker is attached to the 3'-end of the sense strand. In certain embodiments, the 3'-end of the sense strand of the siRNA is modified with a C6 amine.

[0142] In certain embodiments, the linking group comprises at least one spacer. In certain embodiments, the spacer is a hydrophilic spacer. In certain embodiments, the hydrophilic spacer is polyethylene glycol (PEG).

[0143] The linking group can be a homobifunctional linker or a heterobifunctional linker.

[0144] In some embodiments, the linking group is cleavable (e.g., a nuclease-cleavable linker, an acid-labile linker, a peptidase-sensitive linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker (Chari et al., Cancer Res. 52:127-131 (1992), U.S. Pat. No. 5,208,020). In certain embodiments, a linking group comprises one or more nucleotides (e.g., 1, 2, 3, or more) or one or more nucleosides (e.g., 1, 2, 3, or more). In certain embodiments, one or more nucleotides or one or more nucleosides of a linking group are unmodified. In certain embodiments, a linking group comprises one or more nucleotides having an unmodified base, an unmodified sugar group, and / or an unmodified phosphate group. In certain embodiments, a linking group comprises one or more nucleotides having an unmodified base and / or an unmodified sugar group. In certain embodiments, a linking group comprises a TCA (thymine-cytosine-adenine) trinucleotide. In certain embodiments, the TCA is modified with a C6 amine at the T position. In certain embodiments, a linking group does not comprise a TCA.

[0145] In certain embodiments, the linking group is enzymatically cleavable. In certain embodiments, the linking group is cleavable by enzymes present in the central nervous system (CNS) or muscle. In certain embodiments, a cleavable linking group is suitable for conjugates containing ASOs (e.g., allowing the ASO to dissociate from the remainder of the conjugate for transport into the nucleus). In certain embodiments, the cleavable linking group is a cleavable dipeptide linker. In certain embodiments, the cleavable dipeptide linker is a valine-citrulline cleavable linking group or a valine-alanine cleavable linker.

[0146] In certain embodiments, the cleavable linker is an acid-cleavable linker. In certain embodiments, the acid-cleavable linker is a carbonate linker or a hydrazone linker.

[0147] In certain embodiments, the cleavable linker comprises one or more PEG spacers.

[0148] In certain embodiments, the cleavable linking group is an amide such as SPDP (succinimidyl 3-(2-pyridyldithio)propionate) or a lys-conjugated acid-cleavable hydrazide.

[0149] In certain embodiments, the linking group is a non-cleavable linking group. In certain embodiments, the linking group is a covalent linking group. In certain embodiments, the covalent linking group may be derived from 3-arylpropionitrile (APN) or acrylamide. In certain embodiments, the covalent linking group comprises the group -CHCHC(=O)-. In certain embodiments, the covalent linking group comprises the group: [ka] Includes.

[0150] In certain embodiments, the covalent linking group can be derived from a haloacetamide, for example, bromoacetamide, chloroacetamide, iodoacetamide.

[0151] In certain embodiments, the linking group comprises a C6 amine group having the formula -(CH2)6-NH-.

[0152] In certain embodiments, the linking group may be derived from a maleimide. For example, in certain embodiments, the linking group may be the group: [ka] Includes.

[0153] In certain embodiments, the linking group may be attached to P at the valence marked with * (eg, to the sulfur atom of the modification site within P).

[0154] In certain embodiments, the maleimide is a modified maleimide. In certain embodiments, the modified maleimide is an alkyl-, aryl-, cycloalkyl-, or exocyclic-maleimide.

[0155] In certain embodiments, the linking group is a self-hydrolyzable linking group.

[0156] Certain specific non-limiting embodiments of exemplary linking groups (abbreviated as linker embodiments LE1-LE42) are described below.

[0157] In linker embodiment LE1, the linking group has a molecular weight of about 20 daltons to about 5,000 daltons. In linker embodiment LE2, the linking group has a molecular weight of about 20 daltons to about 1,000 daltons. In linker embodiment LE3, the linking group has a molecular weight of about 20 daltons to about 200 daltons.

[0158] In linker embodiment LE4, the linking group has a length of about 5 angstroms to about 60 angstroms. In linker embodiment LE5, the linking group separates the oligonucleotide from the TfR binding agent of Formula (I) by a length of about 5 angstroms to about 40 angstroms, inclusive.

[0159] In linker embodiment LE6, the linking group is a divalent branched or unbranched, saturated or unsaturated hydrocarbon chain having 2 to 25 carbon atoms, in which one or more (e.g., 1, 2, 3, or 4) carbon atoms are optionally replaced by (-O-), (-NH-), (-S-), an amino acid, a hydrazone (-C(R')=N=N(R')-), a nucleotide, or a 3- to 12-membered divalent heterocycle, and the chain and any 3- to 12-membered divalent heterocycle are optionally selected from the group consisting of (C1-C6)alkoxy, (C3-C6)cyclo ... and substituted with one or more (e.g., 1, 2, 3, or 4) substituents independently selected from the group consisting of alkyl, (C1-C6)alkanoyl, (C1-C6)alkanoyloxy, (C1-C6)alkoxycarbonyl, (C1-C6)alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (=O), hydrazone (=N=N(R')-)carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy, wherein each R' is independently H or (C1-C6)alkyl.

[0160] In linker embodiment LE7, the linking group is a divalent branched or unbranched, saturated or unsaturated hydrocarbon chain having 2 to 25 carbon atoms, wherein one or more (e.g., 1, 2, 3, or 4) carbon atoms are optionally replaced by (-O-), (-NH-), or a 3- to 12-membered divalent heterocycle, and the chain and any 3- to 12-membered divalent heterocycle are optionally substituted with one or more (e.g., 1, 2, 3, or 4) substituents independently selected from the group consisting of (C-C)alkoxy, (C-C)cycloalkyl, (C-C)alkanoyl, (C-C)alkanoyloxy, (C-C)alkoxycarbonyl, (C-C)alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

[0161] In linker embodiment LE8, the linking group is a divalent branched or unbranched, saturated or unsaturated hydrocarbon chain having 2 to 10 carbon atoms, in which one or more (e.g., 1, 2, 3, or 4) carbon atoms are optionally replaced by (-O-), (-NH-), (-S-), an amino acid, a hydrazone (-C(R')=N=N(R')-), a nucleotide, or a 3- to 12-membered divalent heterocycle, and the chain and any 3- to 12-membered divalent heterocycle are optionally selected from the group consisting of (C1-C6)alkoxy, (C3-C6)cyclo ... and substituted with one or more (e.g., 1, 2, 3, or 4) substituents independently selected from the group consisting of alkyl, (C1-C6)alkanoyl, (C1-C6)alkanoyloxy, (C1-C6)alkoxycarbonyl, (C1-C6)alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (=O), hydrazone (=N=N(R')-)carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy, wherein each R' is independently H or (C1-C6)alkyl.

[0162] In linker embodiment LE9, the linking group is a divalent branched or unbranched, saturated or unsaturated hydrocarbon chain having 2 to 10 carbon atoms, wherein one or more (e.g., 1, 2, 3, or 4) carbon atoms are optionally replaced by (-O-), (-NH-), or a 3- to 12-membered divalent heterocycle, and the chain and any 3- to 12-membered divalent heterocycle are optionally substituted with one or more (e.g., 1, 2, 3, or 4) substituents independently selected from the group consisting of (C-C)alkoxy, (C-C)cycloalkyl, (C-C)alkanoyl, (C-C)alkanoyloxy, (C-C)alkoxycarbonyl, (C-C)alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

[0163] In linker embodiment LE10, the linking group is a divalent branched or unbranched, saturated or unsaturated hydrocarbon chain having from 2 to 25 carbon atoms, which chain is optionally substituted on carbon with one or more (e.g., 1, 2, 3, or 4) substituents selected from (C1-C6)alkoxy, (C3-C6)cycloalkyl, (C1-C6)alkanoyl, (C1-C6)alkanoyloxy, (C1-C6)alkoxycarbonyl, (C1-C6)alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

[0164] In linker embodiment LE11, the linking group is a divalent branched or unbranched, saturated or unsaturated hydrocarbon chain having from 2 to 10 carbon atoms, optionally substituted on carbon with one or more (e.g., 1, 2, 3, or 4) substituents selected from (C1-C6)alkoxy, (C3-C6)cycloalkyl, (C1-C6)alkanoyl, (C1-C6)alkanoyloxy, (C1-C6)alkoxycarbonyl, (C1-C6)alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

[0165] In linker embodiment LE12, the linking group is a divalent branched or unbranched, saturated or unsaturated hydrocarbon chain having from 2 to 10 carbon atoms.

[0166] In linker embodiment LE13, the linking group is a divalent branched or unbranched saturated hydrocarbon chain having from 2 to 10 carbon atoms.

[0167] In linker embodiment LE14, the linking group is a divalent unbranched saturated hydrocarbon chain having from 2 to 10 carbon atoms.

[0168] In linker embodiment LE15, the linking group is a bivalent branched or unbranched, saturated or unsaturated chain having 2 to 25 atoms selected from carbon, oxygen, nitrogen, and sulfur, the chain containing one or more disulfide bonds.

[0169] In linker embodiment LE16, the linking group is a divalent branched or unbranched, saturated or unsaturated chain having 2 to 25 atoms selected from carbon, oxygen, nitrogen, and sulfur, which chain includes one or more hydrazone groups within the chain or attached to a carbon atom of the chain.

[0170] In linker embodiment LE17, the linking group is a bivalent branched or unbranched, saturated or unsaturated chain having 2 to 35 atoms selected from carbon, oxygen, nitrogen, and sulfur, the chain including one or more amino acids within the chain.

[0171] In linker embodiment LE18, the linking group is a divalent branched or unbranched, saturated or unsaturated chain having 2 to 35 atoms selected from carbon, oxygen, nitrogen, and sulfur, which chain comprises a dipeptide within the chain.

[0172] In linker embodiment LE19, the linking group is a bivalent branched or unbranched, saturated or unsaturated chain having 2 to 35 atoms selected from carbon, oxygen, nitrogen, and sulfur, the chain including the dipeptide valine-citrulline within the chain.

[0173] In linker embodiment LE20, the linking group comprises one or more nucleotides in the chain.

[0174] In linker embodiment LE21, the linking group comprises two or more nucleotides in the chain.

[0175] In linker embodiment LE22, the linking group comprises a trinucleotide group within the chain.

[0176] In linker embodiment LE23, at least one linking group is attached to two or more oligonucleotides (e.g., for compounds of formula (I), y is at least one (L-(O) y ) is 2 or more).

[0177] In linker embodiment LE24, only one linking group is attached to two or more oligonucleotides (e.g., for compounds of formula (I), y is a single (L-(O) y ) is 2 or more).

[0178] In linker embodiment LE24b, the TfR-binding agent-oligonucleotide conjugate contains a single linking group attached to two or more oligonucleotides (e.g., for compounds of formula (I), n=1 and y is 2 or greater).

[0179] In linker embodiment LE25, at least two linking groups are attached to two or more oligonucleotides (e.g., for compounds of formula (I), n is 2 or more and at least two (L-(O) y ), where y is greater than or equal to 2).

[0180] In linker embodiment LE26, at least two linking groups are attached to two or more oligonucleotides (e.g., for compounds of formula (I), n is 2 or more and at least two (L-(O) y ) for which y=2).

[0181] In linker embodiment LE27, the linking group is attached to the oligonucleotide via a phosphate of the oligonucleotide (eg, associated with the 5'-terminal residue).

[0182] In linker embodiment LE28, the linking group is attached to the oligonucleotide via a phosphorothioate group of the oligonucleotide (eg, associated with the 5' terminal residue).

[0183] In linker embodiment LE29, the linking group comprises a polyethyleneoxy chain. In another embodiment of the invention, the polyethyleneoxy chain comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeating ethyleneoxy units.

[0184] In linker embodiment LE30, the linking group comprises a 5-membered divalent heterocycle.

[0185] In linker embodiment LE31, the linking group has the following structure: [ka] wherein L' is a divalent branched or unbranched, saturated or unsaturated hydrocarbon chain having 2 to 25 carbon atoms, in which one or more (e.g., 1, 2, 3, or 4) carbon atoms are optionally replaced by (-O-), (-NH-), (-S-), an amino acid, a hydrazone (-C(R')=N=N(R')-), a nucleotide, or a 3- to 12-membered divalent heterocycle, and the chain and any 3- to 12-membered divalent heterocycle are optionally selected from the group consisting of (C1-C6)alkoxy, (C3-C6)cycloalkyl, (C1-C6)alkanoyl, (C1-C6)alkanoyl, and the like. and L' is substituted with one or more (e.g., 1, 2, 3, or 4) substituents independently selected from the group consisting of aryloxy, (C1-C6)alkoxycarbonyl, (C1-C6)alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (=O), hydrazone (-NH-N=C(R')-), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy, wherein each R' is independently H or (C1-C6)alkyl, and in formula (I), valences marked with * are attached to P and valences marked with ** are attached to O. In another embodiment, L' is a divalent branched or unbranched, saturated or unsaturated chain having 2 to 25 atoms selected from carbon, oxygen, nitrogen, and sulfur, wherein the chain contains one or more disulfide bonds. In another embodiment, L' is a divalent branched or unbranched, saturated or unsaturated chain having 2 to 25 atoms selected from carbon, oxygen, nitrogen, and sulfur, wherein the chain contains one or more hydrazone groups within the chain or attached to a carbon atom of the chain. In another embodiment, L' is a divalent branched or unbranched, saturated or unsaturated chain having 2 to 35 atoms selected from carbon, oxygen, nitrogen, and sulfur, wherein the chain contains one or more amino acids within the chain. In another embodiment, L' is a divalent branched or unbranched, saturated or unsaturated chain having 2 to 35 atoms selected from carbon, oxygen, nitrogen, and sulfur, wherein the chain contains a dipeptide within the chain. In another embodiment, L' is a divalent branched or unbranched, saturated or unsaturated chain having 2 to 35 atoms selected from carbon, oxygen, nitrogen, and sulfur, wherein the chain contains the dipeptide valine-citrulline within the chain.In another embodiment, L' comprises one or more nucleotides. In another embodiment, L' comprises two or more nucleotides. In another embodiment, L' comprises a trinucleotide group. In another embodiment, L' comprises one or more nucleotides having an unmodified base, an unmodified sugar group, and / or an unmodified phosphate group.

[0186] In linker embodiment LE32, L' has the following structure: [ka] wherein t is 1, 2, 3, 4, 5, 6, 7, or 8; z is 0, 1, 2, 3, 4, 5, 6, 7, or 8; and R 1 , R 2 , and R 3 Each of is independently a nucleotide.

[0187] In linker embodiment LE33, L' has the following structure: [ka] Includes.

[0188] In linker embodiment LE34, the linking group has the following structure: [ka] wherein t is 1, 2, 3, 4, 5, 6, 7, or 8; and z is 0, 1, 2, 3, 4, 5, 6, 7, or 8.

[0189] In linker embodiment LE35, the linking group has the following structure: [ka] wherein t is 1, 2, 3, 4, 5, 6, 7, or 8, and z is 0, 1, 2, 3, 4, 5, 6, 7, or 8, and in formula (I), the valence marked with * is attached to P and the valence marked with ** is attached to O. In certain embodiments, the valence marked with ** is attached to O via a phosphate of the oligonucleotide (e.g., associated with the 5'-terminal residue).

[0190] In linker embodiment LE36, the linking group has the following structure: [ka] Includes.

[0191] In linker embodiment LE37, the linking group has the following structure: [ka] In formula (I), the valence marked with * is attached to P and the valence marked with ** is attached to O. In certain embodiments, the valence marked with ** is attached to O via a phosphate of the oligonucleotide (e.g., associated with the 5'-terminal residue). Thus, the A group in the linker structure is, in embodiments, itself covalently attached to the oligonucleotide. [ka] It may be covalently bonded to -O-PO3 via

[0192] In linker embodiment LE38, the linking group has the following structure: [ka] Includes.

[0193] In linker embodiment LE39, the linker is a peptide linker or formed from a protein, peptide, or amino acid. For example, in certain embodiments, the linking group is a divalent radical formed from a protein. In another embodiment, the linking group is a divalent radical formed from a peptide. In another embodiment, the linking group is a divalent radical formed from an amino acid.

[0194] In linker embodiment LE40, the linking group can be configured to allow relative rotation of the oligonucleotide and the TfR-binding agent and / or be resistant to digestion by proteases. In some embodiments, the linking group can be a flexible linker containing amino acids such as, for example, Gly, Asn, Ser, Thr, Ala, etc. Such linking groups are designed using known parameters. For example, the linking group can have repeats such as Gly-Ser repeats.

[0195] In linker embodiment LE41, the linking group is [ka] [ka] having or comprising a formula selected from the group consisting of: Each A is independently (C1 to C 15 ) alkyl, Each D is -(CH2-CH2-O) m - and Each m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24.

[0196] In linker embodiment LE42, the linking group is [ka] [ka] having or comprising a formula selected from the group consisting of:

[0197] In various embodiments, conjugates can be produced using well-known chemical cross-linking reagents and protocols. For example, there are numerous chemical cross-linking agents known to those skilled in the art that are useful for cross-linking proteins with target agents. For example, the cross-linking agent is a heterobifunctional cross-linker that can be used to link molecules in a stepwise manner. Heterobifunctional cross-linkers allow for the design of more specific coupling methods for protein conjugation, thereby reducing the occurrence of undesired side reactions such as homoprotein polymers. A wide variety of heterobifunctional cross-linkers are well known in the art, including N-hydroxysuccinimide (NHS) or its water-soluble analog, N-hydroxysulfosuccinimide (sulfo-NHS), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS); N-succinimidyl(4-iodoacetyl)aminobenzoate (SIAB), Examples include succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC); 4-succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)-toluene (SMPT), N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), and succinimidyl 6-[3-(2-pyridyldithio)propionate]hexanoate (LC-SPDP). These crosslinkers containing an N-hydroxysuccinimide moiety can be obtained as N-hydroxysulfosuccinimide analogs, which generally have higher water solubility. Furthermore, these crosslinkers containing disulfide bridges within the linking chain can be synthesized as alkyl derivatives instead, to reduce the amount of linker cleavage in vivo. In addition to heterobifunctional crosslinkers, numerous other crosslinkers exist, including homobifunctional and photoreactive crosslinkers.Disuccinimidyl suberate (DSS), bismaleimidohexane (BMH), and dimethylpimelimidate·2HCl (DMP) are examples of useful homobifunctional crosslinkers, and bis-[B-(4-azidosalicylamido)ethyl]disulfide (BASED) and N-succinimidyl-6(4'-azido-2'-nitrophenylamino)hexanoate (SANPAH) are examples of useful photoreactive crosslinkers.

[0198] VI. Anti-TFR Antibody Antigen-Binding Domain Anti-TfR antibody antigen-binding domains suitable for use in the described TfR-binding agent-oligonucleotide conjugates can be anti-TfR antibody antigen-binding domains derived from antibodies known to specifically bind to TfR. Anti-TfR antibody antigen-binding domains are disclosed in US20130028891, US2018282408, US20190092870, US2020071413, US20210138083, WO2014 / 033074, WO2015 / 101588, WO2016 / 081640, WO2016 / 208695, WO2018 / 124121, WO2018 / 210898, WO2020 / 132584, WO2021 / 076546, WO2021 / 205358, WO2022 / 101633, WO2022 / 103769, WO2022 / 221505, Candelaria et al. (Front. Immunol. 12 17 March 2021, 2021) and Weber et al. (Cell Reports 22:149-162, 2018), each of which is incorporated herein by reference in its entirety. The anti-TfR antibody antigen-binding domain may comprise an antibody, Fab (including F(ab')2), scFab, Fv fragment, scFv, VHH, vNAR, or nanobody.

[0199] In some embodiments, the TfR-binding agent comprises an antibody having at least one variable domain or antigen-binding site that specifically binds to TfR. In some embodiments, the TfR-binding agent comprises an antibody having a single variable domain or antigen-binding site that specifically binds to TfR. In some embodiments, the TfR-binding agent comprises an antibody having a single variable domain or antigen-binding site that specifically binds to TfR (monovalent, i.e., the TfR-binding agent does not comprise any additional antibody antigen-binding domain, non-binding Fab, or NBVR (anti-TfR monoFab)). In some embodiments, the TfR-binding agent comprises a bispecific, bivalent antibody having a first variable domain or antigen-binding site that specifically binds to TfR and a second variable domain or antigen-binding site comprising a non-binding Fab or NBVR. In some embodiments, the TfR-binding agent comprises an anti-TfR antibody binding domain (e.g., an anti-TfR Fab, scFv, VHH, vNAR, or nanobody) linked to albumin (e.g., human albumin). In some embodiments, the TfR binding agent comprises an anti-TfR antibody having a first anti-TfR antibody antigen-binding domain (e.g., a Fab or scFv) and a second anti-TfR antibody antigen-binding domain (e.g., a Fab or scFv).

[0200] Exemplary Proteins Comprising Fabs That Specifically Bind TfR Exemplary Fabs that specifically bind to TfR include the heavy chain variable region of SEQ ID NO: 10, 19, 102, 104, 110, 122, 132, or 143, and the light chain variable region of SEQ ID NO: 9, 18, 103, 105, 111, 123, 133, or 144. Unless otherwise clear from the context, reference to a Fab that specifically binds to TfR should be understood to refer to any of the murine, chimeric, veneered, humanized, and modified forms.

[0201] In some embodiments, a Fab that specifically binds to TfR comprises a heavy chain variable region of SEQ ID NO: 10 and a light chain variable region of SEQ ID NO: 9. In some embodiments, a Fab that specifically binds to TfR comprises a heavy chain variable region of SEQ ID NO: 19 and a light chain variable region of SEQ ID NO: 18. In some embodiments, a Fab that specifically binds to TfR comprises a heavy chain variable region of SEQ ID NO: 102 and a light chain variable region of SEQ ID NO: 103. In some embodiments, a Fab that specifically binds to TfR comprises a heavy chain variable region of SEQ ID NO: 104 and a light chain variable region of SEQ ID NO: 105. In some embodiments, a Fab that specifically binds to TfR comprises a heavy chain variable region of SEQ ID NO: 110 and a light chain variable region of SEQ ID NO: 111. In some embodiments, a Fab that specifically binds to TfR comprises a heavy chain variable region of SEQ ID NO: 122 and a light chain variable region of SEQ ID NO: 123. In some embodiments, a Fab that specifically binds to TfR comprises a heavy chain variable region of SEQ ID NO: 132 and a light chain variable region of SEQ ID NO: 133. In some embodiments, a Fab that specifically binds to TfR comprises a heavy chain variable region of SEQ ID NO:143 and a light chain variable region of SEQ ID NO:144.

[0202] In some embodiments, a Fab that specifically binds to TfR consists of a heavy chain variable region of SEQ ID NO: 10 and a light chain variable region of SEQ ID NO: 9. In some embodiments, a Fab that specifically binds to TfR consists of a heavy chain variable region of SEQ ID NO: 19 and a light chain variable region of SEQ ID NO: 18. In some embodiments, a Fab that specifically binds to TfR consists of a heavy chain variable region of SEQ ID NO: 102 and a light chain variable region of SEQ ID NO: 103. In some embodiments, a Fab that specifically binds to TfR consists of a heavy chain variable region of SEQ ID NO: 104 and a light chain variable region of SEQ ID NO: 105. In some embodiments, a Fab that specifically binds to TfR consists of a heavy chain variable region of SEQ ID NO: 110 and a light chain variable region of SEQ ID NO: 111. In some embodiments, a Fab that specifically binds to TfR consists of a heavy chain variable region of SEQ ID NO: 122 and a light chain variable region of SEQ ID NO: 123. In some embodiments, a Fab that specifically binds to TfR consists of a heavy chain variable region of SEQ ID NO: 132 and a light chain variable region of SEQ ID NO: 133. In some embodiments, a Fab that specifically binds to TfR consists of a heavy chain variable region of SEQ ID NO:143 and a light chain variable region of SEQ ID NO:144.

[0203] In some embodiments, a Fab that specifically binds to TfR comprises a heavy chain CH1 and variable region of SEQ ID NO: 10, and a light chain comprising SEQ ID NO: 9. In some embodiments, a Fab that specifically binds to TfR comprises a heavy chain CH1 and variable region of SEQ ID NO: 19, and a light chain comprising SEQ ID NO: 18. In some embodiments, a Fab that specifically binds to TfR comprises a heavy chain comprising SEQ ID NO: 102, and a light chain comprising SEQ ID NO: 103. In some embodiments, a Fab that specifically binds to TfR comprises a heavy chain comprising SEQ ID NO: 104, and a light chain comprising SEQ ID NO: 105. In some embodiments, a Fab that specifically binds to TfR comprises a heavy chain comprising SEQ ID NO: 110, and a light chain comprising SEQ ID NO: 111. In some embodiments, a Fab that specifically binds to TfR comprises a heavy chain comprising SEQ ID NO: 122, and a light chain comprising SEQ ID NO: 123. In some embodiments, a Fab that specifically binds to TfR comprises a heavy chain comprising SEQ ID NO: 132, and a light chain comprising SEQ ID NO: 133. In some embodiments, a Fab that specifically binds to TfR comprises a heavy chain comprising SEQ ID NO: 143, and a light chain comprising SEQ ID NO: 144.

[0204] In some embodiments, a Fab that specifically binds to TfR consists of a heavy chain CH1 and variable region of SEQ ID NO: 10 and a light chain of SEQ ID NO: 9. In some embodiments, a Fab that specifically binds to TfR consists of a heavy chain CH1 and variable region of SEQ ID NO: 19 and a light chain of SEQ ID NO: 18. In some embodiments, a Fab that specifically binds to TfR consists of SEQ ID NO: 102 and SEQ ID NO: 103. In some embodiments, a Fab that specifically binds to TfR consists of SEQ ID NO: 104 and SEQ ID NO: 105. In some embodiments, a Fab that specifically binds to TfR consists of SEQ ID NO: 110 and SEQ ID NO: 111. In some embodiments, a Fab that specifically binds to TfR consists of SEQ ID NO: 122 and SEQ ID NO: 123. In some embodiments, a Fab that specifically binds to TfR consists of SEQ ID NO: 132 and SEQ ID NO: 133. In some embodiments, a Fab that specifically binds to TfR consists of SEQ ID NO: 143 and SEQ ID NO: 144.

[0205] In some embodiments, a Fab that specifically binds to TfR comprises the CDR sequences of SEQ ID NOs: 10 and 9, 19 and 18, 102 and 103, 104 and 105, 110 and 111, 122 and 123, 132 and 133, or 143 and 144.

[0206] In some embodiments, a Fab that specifically binds to TfR comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 having the sequences of SEQ ID NOs: 12, 13, 14, 15, 16, and 17, respectively. In some embodiments, a Fab that specifically binds to TfR comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 having the sequences of SEQ ID NOs: 21, 22, 23, 24, 25, and 26, respectively. In some embodiments, a Fab that specifically binds to TfR comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 having the sequences of SEQ ID NOs: 114, 115, 116, 117, 118, and 119, respectively. In some embodiments, a Fab that specifically binds to TfR comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 having the sequences of SEQ ID NOs: 126, 127, 128, 129, 130, and 131, respectively. In some embodiments, a Fab that specifically binds to TfR comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 having the sequences of SEQ ID NOs: 134, 135, 136, 137, 138, and 139, respectively. In some embodiments, a Fab that specifically binds to TfR comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 having the sequences of SEQ ID NOs: 154, 155, 156, 157, 158, and 159, respectively. In some embodiments, a Fab that specifically binds to TfR comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 having the sequences of SEQ ID NOs: 161, 162, 163, 164, 165, and 166, respectively.

[0207] In some embodiments, a Fab that specifically binds to TfR comprises a light chain containing an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 9, 18, 103, 105, 111, 123, 133, or 144, and a light chain containing an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 10, 19, 102, 104, 110, 122, 132, or 14 and a heavy chain containing an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the VH and CH1 region amino acid sequence of SEQ ID NO:3, and contains the CDR sequences of SEQ ID NO:9 and 10, 18 and 19, 102 and 103, 104 and 105, 110 and 111, 122 and 123, 132 and 133, or 143 and 144.

[0208] In some embodiments, a Fab that specifically binds to TfR comprises light and heavy chain variable regions that differ from the variable regions of SEQ ID NOs: 9 and 10, 18 and 19, 102 and 103, 104 and 105, 110 and 111, 122 and 123, 132 and 133, or 143 and 144 by a small number of functionally insignificant amino acid substitutions (e.g., conservative substitutions), deletions, or insertions.

[0209] In some embodiments, the Fab-Fc fusion comprises SEQ ID NO: 10 and SEQ ID NO: 9. In some embodiments, the Fab-Fc fusion comprises SEQ ID NO: 19 and SEQ ID NO: 18. In some embodiments, the Fab-Fc fusion comprises SEQ ID NO: 102 and SEQ ID NO: 103. In some embodiments, the Fab-Fc fusion comprises SEQ ID NO: 104 and SEQ ID NO: 105. In some embodiments, the Fab-Fc fusion comprises SEQ ID NO: 110 and SEQ ID NO: 111. In some embodiments, the Fab-Fc fusion comprises SEQ ID NO: 122 and SEQ ID NO: 123. In some embodiments, the Fab-Fc fusion comprises SEQ ID NO: 132 and SEQ ID NO: 133. In some embodiments, the Fab-Fc fusion comprises SEQ ID NO: 143 and SEQ ID NO: 144.

[0210] In some embodiments, the Fab-Fc fusion comprises an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 10 and SEQ ID NO: 9, and contains CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 having the sequences of SEQ ID NOs: 12, 13, 14, 15, 16, and 17, respectively. In some embodiments, the Fab-Fc fusion consists of SEQ ID NO: 10 and SEQ ID NO: 9.

[0211] In some embodiments, the Fab-Fc fusion comprises an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 19 and SEQ ID NO: 18, and contains CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 having the sequences of SEQ ID NOs: 21, 22, 23, 24, 25, and 16, respectively. In some embodiments, the Fab-Fc fusion consists of SEQ ID NO: 19 and SEQ ID NO: 18.

[0212] In some embodiments, the Fab-Fc fusion comprises an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 102 and SEQ ID NO: 103. In some embodiments, the Fab-Fc fusion consists of SEQ ID NO: 102 and SEQ ID NO: 103.

[0213] In some embodiments, the Fab-Fc fusion comprises an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 104 and SEQ ID NO: 105, and contains the CDR sequences of SEQ ID NOs: 102 and 103. In some embodiments, the Fab-Fc fusion consists of SEQ ID NO: 104 and SEQ ID NO: 105.

[0214] In some embodiments, the Fab-Fc fusion contains CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 that comprise an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 110 and SEQ ID NO: 111, and have the sequences of SEQ ID NOs: 114, 115, 116, 117, 118, and 119, respectively. In some embodiments, the Fab-Fc fusion consists of SEQ ID NO: 110 and SEQ ID NO: 111.

[0215] In some embodiments, the Fab-Fc fusion contains CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 that comprise an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 122 and SEQ ID NO: 123, and have the sequences of SEQ ID NOs: 126, 127, 128, 129, 130, and 131, respectively. In some embodiments, the Fab-Fc fusion consists of SEQ ID NO: 122 and SEQ ID NO: 123.

[0216] In some embodiments, the Fab-Fc fusion contains CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 that comprise an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 132 and SEQ ID NO: 133, and have the sequences of SEQ ID NOs: 134, 135, 136, 137, 138, and 139, respectively. In some embodiments, the Fab-Fc fusion consists of SEQ ID NO: 132 and SEQ ID NO: 133.

[0217] In some embodiments, the Fab-Fc fusion contains CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 that comprise an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 143 and SEQ ID NO: 144, and have the sequences of SEQ ID NOs: 147, 148, 149, 150, 151, and 152, respectively. In some embodiments, the Fab-Fc fusion consists of SEQ ID NO: 143 and SEQ ID NO: 144.

[0218] An ScFab can be made by forming a fusion protein containing the heavy and light chains of any of the described Fabs using methods well known in the art.

[0219] Exemplary Proteins Comprising scFvs That Specifically Bind TfR Exemplary scFvs that specifically bind to TfR include the heavy chain variable region of SEQ ID NO: 10, 19, 102, 104, 110, 122, 132, or 143, and the light chain variable region of SEQ ID NO: 9, 18, 103, 105, 111, 123, 133, or 144. Unless otherwise clear from the context, reference to scFvs that specifically bind to TfR should be understood to refer to any of the murine, chimeric, veneered, humanized, and modified forms.

[0220] In some embodiments, scFvs that specifically bind to TfR comprise a heavy chain variable region of SEQ ID NO: 10 and a light chain variable region of SEQ ID NO: 9. In some embodiments, scFvs that specifically bind to TfR comprise a heavy chain variable region of SEQ ID NO: 19 and a light chain variable region of SEQ ID NO: 18. In some embodiments, scFvs that specifically bind to TfR comprise a heavy chain variable region of SEQ ID NO: 102 and a light chain variable region of SEQ ID NO: 103. In some embodiments, scFvs that specifically bind to TfR comprise a heavy chain variable region of SEQ ID NO: 104 and a light chain variable region of SEQ ID NO: 105. In some embodiments, scFvs that specifically bind to TfR comprise a heavy chain variable region of SEQ ID NO: 110 and a light chain variable region of SEQ ID NO: 111. In some embodiments, scFvs that specifically bind to TfR comprise a heavy chain variable region of SEQ ID NO: 122 and a light chain variable region of SEQ ID NO: 123. In some embodiments, scFvs that specifically bind to TfR comprise a heavy chain variable region of SEQ ID NO: 132 and a light chain variable region of SEQ ID NO: 133. In some embodiments, scFvs that specifically bind to TfR comprise a heavy chain variable region of SEQ ID NO: 143 and a light chain variable region of SEQ ID NO: 144.

[0221] In some embodiments, an scFv that specifically binds to TfR comprises SEQ ID NO: 106. In some embodiments, an scFv that specifically binds to TfR comprises SEQ ID NO: 107. In some embodiments, an scFv that specifically binds to TfR comprises SEQ ID NO: 171. In some embodiments, an scFv that specifically binds to TfR comprises SEQ ID NO: 153. In some embodiments, an scFv that specifically binds to TfR comprises SEQ ID NO: 160. In some embodiments, an scFv that specifically binds to TfR comprises SEQ ID NOs: 112 and 113. In some embodiments, an scFv that specifically binds to TfR comprises SEQ ID NOs: 124 and 125. In some embodiments, an scFv that specifically binds to TfR comprises SEQ ID NOs: 145 and 146.

[0222] In some embodiments, an scFv that specifically binds to TfR consists of SEQ ID NO: 106. In some embodiments, an scFv that specifically binds to TfR consists of SEQ ID NO: 107. In some embodiments, an scFv that specifically binds to TfR consists of SEQ ID NO: 171. In some embodiments, an scFv that specifically binds to TfR consists of SEQ ID NO: 153. In some embodiments, an scFv that specifically binds to TfR consists of SEQ ID NO: 160.

[0223] In some embodiments, an scFv that specifically binds to TfR comprises the CDR sequences of SEQ ID NOs: 10 and 9, 19 and 18, 102 and 103, 104 and 105, 110 and 111, 122 and 123, 132 and 133, or 143 and 144.

[0224] In some embodiments, an scFv that specifically binds to TfR comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 having the sequences of SEQ ID NOs: 12, 13, 14, 15, 16, and 17, respectively. In some embodiments, an scFv that specifically binds to TfR comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 having the sequences of SEQ ID NOs: 21, 22, 23, 24, 25, and 26, respectively. In some embodiments, an scFv that specifically binds to TfR comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 having the sequences of SEQ ID NOs: 114, 115, 116, 117, 118, and 119, respectively. In some embodiments, an scFv that specifically binds to TfR comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 having the sequences of SEQ ID NOs: 126, 127, 128, 129, 130, and 131, respectively. In some embodiments, an scFv that specifically binds to TfR comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 having the sequences of SEQ ID NOs: 134, 135, 136, 137, 138, and 139, respectively. In some embodiments, an scFv that specifically binds to TfR comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 having the sequences of SEQ ID NOs: 154, 155, 156, 157, 158, and 159, respectively. In some embodiments, the scFv that specifically binds to TfR comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 having the sequences of SEQ ID NOs: 161, 162, 163, 164, 165, and 166, respectively.

[0225] In some embodiments, an scFv that specifically binds to TfR comprises an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 106, 107, or 171, and contains the CDR sequences of SEQ ID NOs: 102 and 103. In some embodiments, an scFv that specifically binds to TfR comprises an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 153, and contains CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 having the sequences of SEQ ID NOs: 154, 155, 156, 157, 158, and 159, respectively. In some embodiments, an scFv that specifically binds to TfR comprises an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 160, and contains CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 having the sequences of SEQ ID NOs: 161, 162, 163, 164, 165, and 166, respectively.

[0226] In some embodiments, an scFv that specifically binds to TfR comprises an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 112, and an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 113, and contains CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 having the sequences of SEQ ID NOs: 114, 115, 116, 117, 118, and 119, respectively (i.e., the CDR sequences of SEQ ID NOs: 110 and 111).

[0227] In some embodiments, an scFv that specifically binds to TfR comprises an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 124, and an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 125, and contains CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 having the sequences of SEQ ID NOs: 126, 127, 128, 129, 130, and 131, respectively (i.e., the CDR sequences of SEQ ID NOs: 120 and 121).

[0228] In some embodiments, an scFv that specifically binds to TfR comprises an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 145, and an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 146, and contains CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 having the sequences of SEQ ID NOs: 147, 148, 149, 150, 151, and 152, respectively (i.e., the CDR sequences of SEQ ID NOs: 140 and 142).

[0229] In some embodiments, an scFv that specifically binds to TfR comprises light and heavy chain variable regions that differ from the variable regions of SEQ ID NOs: 9 and 10, 18 and 19, 102 and 103, 104 and 105, 110 and 111, 122 and 123, 132 and 133, or 143 and 144 by a small number of functionally insignificant amino acid substitutions (e.g., conservative substitutions), deletions, or insertions.

[0230] Exemplary Proteins, Including Antibodies That Specifically Bind TfR Exemplary antibodies that specifically bind to TfR include the heavy chain variable region of SEQ ID NO: 10, 19, 102, 104, 110, 122, 132, or 143, and the light chain variable region of SEQ ID NO: 9, 18, 103, 105, 111, 123, 133, or 144. Unless otherwise clear from the context, reference to an antibody that specifically binds to TfR should be understood to refer to any of the murine, chimeric, veneered, humanized, and modified forms.

[0231] In some embodiments, an antibody that specifically binds to TfR comprises a heavy chain variable region of SEQ ID NO: 10 and a light chain variable region of SEQ ID NO: 9. In some embodiments, an antibody that specifically binds to TfR comprises a heavy chain variable region of SEQ ID NO: 19 and a light chain variable region of SEQ ID NO: 18. In some embodiments, an antibody that specifically binds to TfR comprises a heavy chain variable region of SEQ ID NO: 102 and a light chain variable region of SEQ ID NO: 103. In some embodiments, an antibody that specifically binds to TfR comprises a heavy chain variable region of SEQ ID NO: 104 and a light chain variable region of SEQ ID NO: 105. In some embodiments, an antibody that specifically binds to TfR comprises a heavy chain variable region of SEQ ID NO: 110 and a light chain variable region of SEQ ID NO: 111. In some embodiments, an antibody that specifically binds to TfR comprises a heavy chain variable region of SEQ ID NO: 122 and a light chain variable region of SEQ ID NO: 123. In some embodiments, an antibody that specifically binds to TfR comprises a heavy chain variable region of SEQ ID NO: 132 and a light chain variable region of SEQ ID NO: 133. In some embodiments, an antibody that specifically binds to TfR comprises a heavy chain variable region of SEQ ID NO: 143 and a light chain variable region of SEQ ID NO: 144.

[0232] In some embodiments, an antibody that specifically binds to TfR comprises a heavy chain CH1 and variable region of SEQ ID NO: 10 and a light chain comprising SEQ ID NO: 9. In some embodiments, an antibody that specifically binds to TfR comprises a heavy chain CH1 and variable region of SEQ ID NO: 19 and a light chain comprising SEQ ID NO: 18. In some embodiments, an antibody that specifically binds to TfR comprises a heavy chain comprising SEQ ID NO: 102 and a light chain comprising SEQ ID NO: 103. In some embodiments, an antibody that specifically binds to TfR comprises a heavy chain comprising SEQ ID NO: 104 and a light chain comprising SEQ ID NO: 105. In some embodiments, an antibody that specifically binds to TfR comprises a heavy chain comprising SEQ ID NO: 110 and a light chain comprising SEQ ID NO: 111. In some embodiments, an antibody that specifically binds to TfR comprises a heavy chain comprising SEQ ID NO: 122 and a light chain comprising SEQ ID NO: 123. In some embodiments, an antibody that specifically binds to TfR comprises a heavy chain comprising SEQ ID NO: 132 and a light chain comprising SEQ ID NO: 133. In some embodiments, an antibody that specifically binds to TfR comprises a heavy chain comprising SEQ ID NO: 143 and a light chain comprising SEQ ID NO: 144.

[0233] In some embodiments, an antibody that specifically binds to TfR comprises SEQ ID NO: 108 and SEQ ID NO: 109. In some embodiments, an antibody that specifically binds to TfR comprises SEQ ID NO: 120 and SEQ ID NO: 121. In some embodiments, an antibody that specifically binds to TfR comprises SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11. In some embodiments, an antibody that specifically binds to TfR comprises SEQ ID NO: 18 and SEQ ID NO: 19.

[0234] In some embodiments, an antibody that specifically binds to TfR comprises the CDR sequences of SEQ ID NOs: 10 and 9, 19 and 18, 102 and 103, 104 and 105, 110 and 111, 122 and 123, 132 and 133, or 143 and 144.

[0235] In some embodiments, an antibody that specifically binds to TfR comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 having the sequences of SEQ ID NOs: 12, 13, 14, 15, 16, and 17, respectively. In some embodiments, an antibody that specifically binds to TfR comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 having the sequences of SEQ ID NOs: 21, 22, 23, 24, 25, and 26, respectively. In some embodiments, an antibody that specifically binds to TfR comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 having the sequences of SEQ ID NOs: 114, 115, 116, 117, 118, and 119, respectively. In some embodiments, an antibody that specifically binds to TfR comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 having the sequences of SEQ ID NOs: 126, 127, 128, 129, 130, and 131, respectively. In some embodiments, an antibody that specifically binds to TfR comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 having the sequences of SEQ ID NOs: 134, 135, 136, 137, 138, and 139, respectively. In some embodiments, an antibody that specifically binds to TfR comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 having the sequences of SEQ ID NOs: 154, 155, 156, 157, 158, and 159, respectively. In some embodiments, an antibody that specifically binds to TfR comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 having the sequences of SEQ ID NOs: 161, 162, 163, 164, 165, and 166, respectively.

[0236] In some embodiments, an antibody that specifically binds to TfR comprises a heavy chain containing an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the VH and CH1 region amino acid sequence of SEQ ID NO: 10, 19, 102, 104, 110, 122, 132, or 143, and a heavy chain containing an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the VH and CH1 region amino acid sequence of SEQ ID NO: 9, 18, 103, 105, 111, 123, 133 and a light chain containing an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 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, 40, 41, 42, 43, 44, and 55, and contains the CDR sequences of SEQ ID NO: 10 and 9, 19 and 18, 102 and 103, 104 and 105, 110 and 111, 122 and 123, 132 and 133, or 143 and 144, respectively.

[0237] In some embodiments, an antibody that specifically binds to TfR comprises a first heavy chain containing an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 10; a second heavy chain containing an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 11; and a light chain containing an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 9, and contains the CDR sequences of SEQ ID NOs: 10 and 9.

[0238] In some embodiments, an antibody that specifically binds to TfR comprises a heavy chain containing an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 19, and a light chain containing an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 18, and contains the CDR sequences of SEQ ID NOs: 19 and 18.

[0239] In some embodiments, an antibody that specifically binds to TfR comprises a heavy chain containing an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 108, and a light chain containing an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 109, and contains the CDR sequences of SEQ ID NOs: 108 and 109 (i.e., SEQ ID NOs: 114-119).

[0240] In some embodiments, an antibody that specifically binds to TfR comprises a heavy chain containing an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 120, and a light chain containing an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 121, and contains the CDR sequences of SEQ ID NOs: 120 and 121 (i.e., SEQ ID NOs: 126-131).

[0241] Any of the described antibodies that specifically bind to TfR can have one or more modifications to increase serum stability, modulate effector function, affect glycosylation, reduce immunogenicity in humans, promote heterodimerization, and / or promote conjugation of oligonucleotides.

[0242] Any of the described antibodies that specifically bind to TfR can have an Fc polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 27-34, 79-90, and 92-98. The Fc polypeptide can be modified to increase serum stability, modulate effector function, affect glycosylation, reduce immunogenicity in humans, promote heterodimerization, and / or promote conjugation of oligonucleotides.

[0243] In some embodiments, an antibody that specifically binds to TfR comprises light and heavy chain variable regions that differ from the variable regions of SEQ ID NOs: 9 and 10, 18 and 19, 102 and 103, 104 and 105, 110 and 111, 122 and 123, 132 and 133, or 143 and 144 by a small number of functionally insignificant amino acid substitutions (e.g., conservative substitutions), deletions, or insertions.

[0244] Further exemplary anti-TfR antibody antigen-binding domains include 17H10 anti-TfR Fab or scFv; 17H10.1 anti-TfR Fab or scFv; JC-141 anti-TfR antibody; JC-141 anti-TfR Fab; JC-141 anti-TfR scFv; an anti-TfR antibody, Fab, scFab, Fv fragment, or scFv having the heavy and light chain CDR1, CDR2, and CDR3 sequences of the JR-141 antibody (WO2016208695); JC-171 anti-TfR antibody; JC-171 anti-TfR Fab; JC-171 anti-TfR scFv; an anti-TfR antibody, Fab, scFab, Fv fragment, or scFv having the heavy and light chain CDR1, CDR2, and CDR3 sequences of the JR-171 antibody (WO2018124121); "Brain Shuttle" (BS) anti-TfR Fab; an anti-TfR antibody, Fab, scFab, Fv fragment, or scFv having the heavy and light chain CDR1, CDR2, and CDR3 sequences of the BS anti-TfR Fab (WO2018210898, WO2015101588, and WO2014033074); 13E4v2ii anti-TfR antibody; 13E4v2ii anti-TfR Fab; 13E4v2ii anti-TfR scFv; an anti-TfR antibody, Fab, scFab, Fv fragment, or scFv having the heavy and light chain CDR1, CDR2, and CDR3 sequences of the 13E4v2ii antibody (WO2020132584); a TfR12 anti-TfR scFv; an anti-TfR antibody, Fab, scFab, Fv fragment, or scFv having the heavy and light chain CDR1, CDR2, and CDR3 sequences of the TfR12 anti-TfR scFv (WO2021 / 205358); a TfR13 anti-TfR scFv; or an anti-TfR antibody, Fab, scFab, Fv fragment, or scFv having the heavy and light chain CDR1, CDR2, and CDR3 sequences of the Tfr13 anti-TfR scFv (WO2021 / 205358) (sequences are shown in Table 1).

[0245] Further anti-TfR antibodies are described in WO2021 / 205358, and the anti-TfR antibody antigen-binding domains of the invention may be any of the TfR1, TfR2, TfR3, TfR4, TfR5, TfR6, TfR7, TfR8, TfR9, TfR10, TfR11, TfR12, TfR13, TfR14, TfR15, TfR16, TfR17, TfR18, TfR19, TfR20, TfR21, TfR22, TfR23, TfR24, TfR25, TfR26, TfR27, TfR28, TfR29, TfR30, TfR31, TfR32, TfR33, TfR34, TfR35, TfR36, TfR37, TfR38, TfR39, TfR40, TfR41, TfR42, TfR43, TfR44, TfR45, TfR46, TfR47, TfR48, TfR49, TfR50, TfR51, TfR52, TfR53, TfR54, TfR55, TfR56, TfR57, TfR58, TfR59, TfR60, TfR61, TfR62, TfR63, TfR64, TfR65, TfR66, TfR67, TfR68, TfR69, TfR70, TfR71, TfR72, TfR73, TfR74, TfR75, TfR76, TfR77, TfR78, TfR79, T The antigen-binding domain may comprise any antibody having the CDRs or variable regions of any one of TfR19, TfR20, TfR21, TfR22, TfR23, TfR24, TfR25, TfR26, TfR27, TfR28, TfR29, TfR30, TfR31, TfR32, TfR33, TfR34, TfR35, TfR36, TfR37, and TfR38. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9]

[0246] Additional anti-TfR antibodies are known in the art and / or available from various commercial sources. In some embodiments, the anti-TfR antibody or TfR-binding fragment of the anti-TfR antibody binds to the apical domain of TfR. In some embodiments, binding of the anti-TfR antibody or TfR-binding fragment of the anti-TfR antibody to TfR does not inhibit transferrin binding to TfR. Exemplary anti-TfR antibodies include, but are not limited to, B3 / 25, RBC4, 7579, E2.3, A27.15, D65.30, D2C, ch128.1Av, ch128.1 / IgG3, ch128.1 / IgG1, hu128.1 (Candelaria et al. Front. Immunol. 12 (17 March 2021), 2021), Ri7,8D3 (Weber et al. Cell Reports 22:149-162, 2018). Exemplary anti-TfR antibodies are also described in U.S. Patent Publication Nos. US2018282408A1, US2020071413A1, US20210138083A1, US20190092870A1, and US20130028891, each of which is incorporated herein by reference.

[0247] Exemplary anti-TfR vNARs are described in WO2022 / 103769.

[0248] Brain shuttles containing anti-TfR antibody antigen-binding domains are described in WO2014 / 033074 and WO2015 / 101588, each of which is incorporated herein by reference.

[0249] In some embodiments, the anti-TfR antibody antigen-binding domain binds to human TfR with an affinity of about 1 nM to about 1000 nM (e.g., about 1 nM, about 2 nM, about 5 nM, about 10 nM, about 20 nM, about 30 nM, about 40 nM, about 50 nM, about 75 nM, about 100 nM, about 150 nM, about 200 nM, about 250 nM, about 300 nM, about 400 nM, about 500 nM, about 750 nM, or about 1000 nM). In some embodiments, the anti-TfR antibody antigen-binding domain binds to human TfR with an affinity of about 1 nM to about 500 nM. In some embodiments, the anti-TfR antibody antigen-binding domain binds to human TfR with an affinity of about 1 nM to about 100 nM (e.g., about 1 nM, about 2 nM, about 5 nM, about 10 nM, about 20 nM, about 30 nM, about 40 nM, about 50 nM, about 60 nM, about 70 nM, about 80 nM, about 90 nM, or about 100 nM). In some embodiments, the anti-TfR antibody antigen-binding domain binds to the apical domain of human TfR with an affinity of about 1 nM to about 1000 nM (e.g., about 1 nM, about 2 nM, about 5 nM, about 10 nM, about 20 nM, about 30 nM, about 40 nM, about 50 nM, about 75 nM, about 100 nM, about 150 nM, about 200 nM, about 250 nM, about 300 nM, about 400 nM, about 500 nM, about 750 nM, or about 1000 nM). In some embodiments, the anti-TfR antibody antigen-binding domain binds to the apical domain of human TfR with an affinity of about 1 nM to about 500 nM. In some embodiments, the anti-TfR antibody antigen-binding domain binds to the apical domain of human TfR with an affinity of about 1 nM to about 100 nM (e.g., about 1 nM, about 2 nM, about 5 nM, about 10 nM, about 20 nM, about 30 nM, about 40 nM, about 50 nM, about 60 nM, about 70 nM, about 80 nM, about 90 nM, or about 100 nM). In some embodiments, the anti-TfR antibody antigen-binding domain binds to TfR or the apical domain or TfR with an affinity of less than 1 nM.

[0250] Exemplary Proteins Comprising Non-Targeting Fab Fragments In some embodiments, the TfR-binding agent comprises a non-binding Fab or NBVR.

[0251] In some embodiments, the non-binding Fab or portion thereof comprises a non-binding variable region (NBVR). The NBVR comprises a light chain variable region and a heavy chain variable region and does not specifically bind to a naturally occurring epitope in a subject. In some embodiments, the NBVR does not specifically bind to an antigen expressed in a given mammal, mammalian tissue, or mammalian cell type. The antigen can be an antigen found in a mammal, such as one derived from a mammalian source or an infectious organism such as a virus, bacterium, fungus, or parasite. The mammal can be, but is not limited to, a non-human primate, a human, or a rodent (e.g., a mouse). The NBVR can be, but is not limited to, an scFv.

[0252] Specific binding of an antibody to an antigen is defined as a binding activity of at least 10 6 M -1 Specific binding refers to an affinity of at least one antigen. Specific binding is detectably greater in magnitude and can be distinguished from nonspecific binding that occurs to at least one unrelated target. Nonspecific binding is often the result of van der Waals forces. Non-binding does not mean that the NBVR does not bind to any antigen with any affinity. Rather, in some embodiments, the NBVR does not exhibit specific binding to (a) any protein or epitope on a mammalian cell, mammalian tissue, or within a mammal, (b) any surface-accessible protein or epitope on a mammalian cell or mammalian tissue, or (c) any serum-accessible protein or epitope in a mammalian tissue or mammal.

[0253] An NBVR can be part of an scFv or Fab. A Fab may or may not include all or part of an antibody hinge region. An NBVR can be produced by recombinant DNA techniques, by enzymatic or chemical separation of intact immunoglobulins, or by chemical peptide synthesis. In some embodiments, an NBVR is part of an unlinked Fab comprising a light chain and a heavy chain, wherein the light chain is a V L The heavy chain comprises a V region and a light chain constant region (CL). H The heavy chain CH1 constant region and the heavy chain CH2 constant region.

[0254] Exemplary NBVRs include NBVR1 or NBVR2. Unless otherwise clear from the context, reference to NBVR1 or NBVR2 should be understood to refer to any of the murine, chimeric, veneered, humanized, and modified forms of NBVR1 or NBVR2.

[0255] Exemplary NTFs include NBVR1 or NBVR2. Unless otherwise clear from the context, reference to NBVR1 or NBVR2 should be understood to refer to any of the murine, chimeric, veneered, humanized, and modified forms of NBVR1 or NBVR2.

[0256] The sequences of the light chain variable region and heavy chain variable region of NBVR1 are shown as SEQ ID NOs: 35 and 36, respectively. The sequences of the light chain and heavy chain of NBVR1 are shown as SEQ ID NOs: 37 and 38, respectively.

[0257] In some embodiments, the NBVR comprises the CDR sequences of NBVR1. The light chain CDRs (L1, L2, and L3) of NBVR1 are set forth as SEQ ID NOs: 39, 41, and 43, respectively. The heavy chain CDRs (H1, H2, and H3) of NBVR1 are set forth as SEQ ID NOs: 45, 47, and 49, respectively. In some embodiments, the NBVR comprises the CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 sequences of NBVR1, and a CDR-H2 sequence comprising SEQ ID NO: 50.

[0258] In some embodiments, the NBVR comprises a light chain comprising the amino acid sequence of SEQ ID NO: 37 or 52, and a heavy chain comprising the amino acid sequence of SEQ ID NO: 38, 53, 54, 55, 56, 57, 58, or 59.

[0259] In some embodiments, the NBVR comprises a light chain containing an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 35, 37, or 51, and a heavy chain containing an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 36, 38, 53, 54, 55, 56, 57, 58, or 59, and contains the CDR sequences of NBVR1 and maintains the non-binding properties of NBVR1.

[0260] In some embodiments, NBVRs comprise light and heavy chain variable regions that differ from the NBVR1 light and heavy chain variable regions by a small number of functionally insignificant amino acid substitutions (e.g., conservative substitutions), deletions, or insertions. Also included are NBVR(s) having 1, 2, 3, 4, 5, or 6 CDRs that are 90%, 95%, 99%, or 100% identical to the corresponding CDRs of NBVR1 or NBVR2, as defined by any conventional definition, preferably Kabat.

[0261] The sequences of the light chain variable region and heavy chain variable region of NBVR2 are shown as SEQ ID NOs: 53 and 60, respectively. The light chain and heavy chain of NBVR2 are shown as SEQ ID NOs: 53 and 61, respectively.

[0262] In some embodiments, the NBVR comprises the CDR sequences of NBVR2. The light chain CDRs (L1, L2, and L3) of NBVR2 are set forth as SEQ ID NOs: 40, 42, and 44, respectively. The heavy chain CDRs (H1, H2, and H3) of NBVR2 are set forth as SEQ ID NOs: 46, 48, and 49, respectively. In some embodiments, the NBVR comprises the CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 sequences of NBVR2, and a CDR-H2 sequence comprising SEQ ID NO: 50.

[0263] In some embodiments, the NBVR comprises a light chain containing an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 62, 63, or 64, and a heavy chain containing an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 60, 61, 65, 66, 67, 68, 69, 70, or 71, and contains the CDR sequences of NBVR2 and maintains the non-binding properties of NBVR2.

[0264] In some embodiments, NBVRs comprise light and heavy chain variable regions that differ from the NBVR2 light and heavy chain variable regions by a small number of functionally insignificant amino acid substitutions (e.g., conservative substitutions), deletions, or insertions. Also included are NBVR(s) having 1, 2, 3, 4, 5, or 6 CDRs that are 90%, 95%, 99%, or 100% identical to the corresponding CDRs of NBVR1 or NBVR2, as defined by any conventional definition, preferably Kabat.

[0265] In some embodiments, an NBVR comprises a light chain variable region and a heavy chain variable region having some or all (e.g., 3, 4, 5, and 6) CDRs that are derived entirely or substantially from NBVR1 or NBVR2. Such an NBVR may comprise a heavy chain variable region having at least two, and usually all three, CDRs that are derived entirely or substantially from the heavy chain variable region of NBVR1 or NBVR2, and / or a light chain variable region having at least two, and usually all three CDRs that are derived entirely or substantially from the light chain variable region of NBVR1 or NBVR2. A CDR is substantially derived from the corresponding NBVR1 or NBVR2 CDR if it contains no more than 4, 3, 2, or 1 substitution, insertion, or deletion, with the proviso that CDR-H2 (as defined by Kabat) can have no more than 6, 5, 4, 3, 2, or 1 substitution, insertion, or deletion. Such antibodies may have at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any of the described NBVR1 or NBVR2 light and heavy chain amino acid sequences, maintain their functional properties, and / or differ from NBVR1 or NBVR2. In some embodiments, the NBVR does not exhibit specific binding to (a) any naturally occurring protein or epitope in a mammalian cell, mammalian tissue, or mammal, (b) any naturally occurring surface-accessible protein or epitope on a mammalian cell or mammalian tissue, or (c) any naturally occurring serum-accessible protein or epitope in a mammalian tissue or mammal.

[0266] In some embodiments, the nucleic acid encoding the NBVR light chain comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 35, 37, 52, 62, 63, or 64. In some embodiments, the nucleic acid encoding the NBVR heavy chain comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 36, 38, 53, 54, 55, 56, 57, 58, 59, 60, 61, 65, 66, 67, 68, 69, 70, or 71.

[0267] In some embodiments, a nucleic acid encoding an NBVR light chain comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% identity to the nucleotide sequence of SEQ ID NO: 72 or 73. In some embodiments, a nucleic acid encoding an NBVR heavy chain comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% identity to the nucleotide sequence of SEQ ID NO: 74 or 75.

[0268] Cells containing nucleic acids encoding the heavy and light chains of any of the described NBVRs are described. In some embodiments, the cells contain a nucleic acid encoding an NBVR light chain comprising the amino acid sequence of SEQ ID NO: 35, 37, or 52, and a nucleic acid encoding an NBVR heavy chain comprising the amino acid sequence of SEQ ID NO: 36, 38, 53, 54, 55, 56, 57, 58, or 59. In some embodiments, the cells contain a nucleic acid encoding an NBVR light chain comprising the amino acid sequence of SEQ ID NO: 62, 63, or 64, and a nucleic acid encoding an NBVR heavy chain comprising the amino acid sequence of SEQ ID NO: 60, 61, 65, 66, 67, 68, 69, 70, or 71. The cells can be bacterial cells, yeast cells, insect cells, or mammalian cells.

[0269] In some embodiments, the non-binding Fab is a RSV (palivizumab) Fab fragment (light chain comprising SEQ ID NO: 101), which is non-targeting in mice and non-human primates.

[0270] Anti-TfR antibodies in which one Fab arm has been removed or replaced with a non-binding Fab or NBVR.

[0271] Humanized antibody antigen-binding domain Any of the anti-TfR antibody antigen-binding domains, non-binding Fabs, NBVRs, or antibodies described herein can be humanized. Humanized antibody antigen-binding domains can be humanized in one or more of the light chain variable domain, heavy chain variable domain, light chain constant domain, and heavy chain constant (CH1) domain. Humanized antibody antigen-binding domains are engineered antibody antigen-binding domains in which CDRs from a non-human "donor" antibody are grafted onto the heavy and / or light chain variable region, light chain constant region, and / or heavy chain CH1 region sequences of a human "acceptor" antibody (see, e.g., Queen, US Pat. Nos. 5,530,101 and 5,585,089; Winter, US Pat. No. 5,225,539; Carter, US Pat. No. 6,407,213; Adair, US Pat. No. 5,859,205; and Foote, US Pat. No. 6,881,557). The acceptor antibody sequences can be, for example, mature human antibody sequences (e.g., sequences derived from one or more of the CH1 region, CH2 region, CH3 region, heavy chain variable region, light chain constant region, or light chain variable region), a composite of such sequences, a consensus sequence of human antibody sequences, or germline region sequences. Thus, a humanized antibody antigen-binding domain is an antibody antigen-binding domain having at least three, four, five, or all CDRs derived entirely or substantially from a donor antibody and entirely or substantially from human antibody variable region framework and / or constant region sequences. Similarly, a humanized heavy chain is derived entirely or substantially from a donor antibody heavy chain and heavy chain variable region framework and heavy chain constant region sequences, with at least one, two, and usually all three CDRs, if present, derived from substantially human heavy chain variable region framework and constant region sequences. Similarly, a humanized light chain is derived entirely or substantially from a donor antibody light chain and light chain variable region framework and light chain constant region sequences, and has at least one, two, and usually all three CDRs, if present, derived substantially from human light chain variable region framework and constant regions. CDRs in a humanized antibody are derived substantially from corresponding residues in a non-human antibody when at least 85%, 90%, 95%, or 100% of the corresponding residues (defined by any conventional definition, but preferably by Kabat) are identical between the respective CDRs.An antibody chain variable region framework sequence or antibody chain constant region is substantially derived from a human variable region framework sequence or human constant region, respectively, if at least 85%, 90%, 95%, or 100% of the corresponding residues as defined by Kabat are identical.

[0272] In some embodiments, the Fab is a chimeric Fab. A chimeric Fab comprises a non-human light chain and / or heavy chain variable region and a human heavy chain (CH1) and / or light chain constant region.

[0273] In some embodiments, the Fab is a veneered Fab, which comprises partially humanized light and / or heavy chain variable regions and human heavy chain (CH1) and / or light chain constant regions.

[0274] VII. Fc Polypeptides or Fc Dimers In some embodiments, the TfR-binding agent comprises an Fc polypeptide or Fc dimer that may contain one or more mutations or substitutions to increase serum stability, modulate effector function, affect glycosylation, reduce immunogenicity in humans, promote heterodimerization (e.g., knob and hole mutations), and / or promote oligonucleotide conjugation.

[0275] In some embodiments, the Fc polypeptides described herein have at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to a corresponding wild-type Fc polypeptide (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc polypeptide).

[0276] One or both of the Fc polypeptides may each comprise an independently selected modification (e.g., mutation), or one or both of the Fc polypeptides may be a wild-type Fc polypeptide, e.g., a human IgG1 Fc polypeptide. In some embodiments, the Fc polypeptides described herein have at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to a corresponding wild-type Fc polypeptide (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc polypeptide). Non-limiting examples of mutations that can be introduced into one or both Fc polypeptides include, for example, specific mutations to provide peptide knob and hole heterodimerization, modulate effector function, extend serum half-life, affect glycosylation, and / or reduce immunogenicity in humans.

[0277] Fc polypeptide modification for heterodimerization In some embodiments, the Fc polypeptides of the Fc dimer contain mutations that promote heterodimer formation and prevent homodimer formation. These modifications are useful, for example, when it is desired that only one of the Fc polypeptides of the dimer has a TfR binding site (i.e., a monovalent TfR binder).

[0278] In some embodiments, the polypeptides present in the Fc dimer may contain knob and hole mutations that promote heterodimer formation. Generally, the method involves introducing a protrusion ("knob") into the interface of one polypeptide and a corresponding cavity ("hole") into the interface of the other polypeptide. The protrusion is constructed by replacing a small amino acid side chain from the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). A complementary cavity of identical or similar size to the protrusion is created in the interface of the second polypeptide by replacing the large amino acid side chain with a smaller one (e.g., alanine or threonine).

[0279] The knobs-into-holes approach involves introducing protrusions ("knobs") into the interface of one Fc polypeptide and corresponding cavities ("holes") into the interface of the other Fc polypeptide, such that the protrusions can be positioned in the cavities to promote heterodimer formation and thereby prevent homodimer formation. The protrusions are constructed by replacing small amino acid side chains from the interface of one Fc polypeptide with larger side chains (e.g., Tyr or Trp). A complementary cavity of identical or similar size to the protrusion is created in the interface of the other Fc polypeptide by replacing the large amino acid side chains with smaller ones (e.g., Ala or Thr). In some embodiments, such additional mutations are at positions in the Fc polypeptide that do not adversely affect binding of the polypeptide to TfR.

[0280] In one exemplary embodiment of the knob-and-hole technique for dimerization, one of the Fc polypeptides contains a Trp at position 366 instead of the native Thr. The other Fc polypeptide in the dimer has a Val at position 407 instead of the native Tyr. The other Fc polypeptide may further contain a substitution of Ser for the native Thr at position 366 and Ala for the native Leu at position 368. Thus, one Fc polypeptide has a T366W knob mutation and the other Fc polypeptide has a Y407V hole mutation, typically accompanied by T366S and L368A hole mutations. As noted above, all positions are numbered according to EU numbering. In a specific embodiment, a first Fc polypeptide contains the T366S, L368A, and Y407V substitutions according to EU numbering, and a second Fc polypeptide further contains the T366W substitution according to EU numbering.

[0281] In some embodiments, one or both Fc polypeptides present in an Fc polypeptide dimer can also be engineered to contain other modifications for heterodimerization, such as electrostatic manipulation of contact residues within the CH3-CH3 interface that are naturally charged or hydrophobic patch modifications.

[0282] Fc polypeptide modifications to modulate effector function In some embodiments, one or both Fc polypeptides in an Fc polypeptide dimer can contain a modification that reduces effector function, i.e., a modification that reduces the ability to induce a particular biological function upon binding to an Fc receptor expressed on an effector cell that mediates effector function. Effector cells include, but are not limited to, monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, B cells, large granular lymphocytes, Langerhans cells, natural killer (NK) cells, and cytotoxic T cells. Examples of antibody effector functions include, but are not limited to, C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0283] In some embodiments, one or both Fc polypeptides in an Fc polypeptide dimer may comprise a modification that reduces or eliminates effector function. Exemplary Fc polypeptide mutations that reduce effector function include, but are not limited to, substitutions in the CH2 domain, e.g., at positions 234 and 235 and / or 329, according to the EU numbering scheme. For example, in some embodiments, both Fc polypeptides comprise Ala residues at positions 234 and 235 (also referred to herein as "LALA"). In some embodiments, both Fc polypeptides comprise a Gly residue at position 329 (also referred to herein as "P329G" or "PG") or a Ser residue at position 329 (also referred to herein as "P329S" or "PS"). In some embodiments, both Fc polypeptides comprise Ala residues at positions 234 and 235 and a Gly residue at position 329 (also referred to herein as "LALA PG"). In some embodiments, both Fc polypeptides comprise Ala residues at positions 234 and 235 and a Ser residue at position 329 (also referred to herein as "LALA PS").

[0284] Additional Fc polypeptide mutations that modulate effector function include, but are not limited to, the following: position 329 can be mutated to substitute Pro with Gly, Ala, Ser, or Arg, or an amino acid residue large enough to disrupt the Fc / Fcγ receptor interface formed between proline 329 of Fc and Trp residues Trp87 and Trp110 of FcγRIII. Further exemplary substitutions include S228P, E233P, L235E, N297A, N297D, and P331S according to the EU numbering scheme. Multiple substitutions according to the EU numbering scheme may also be present, for example, L234A, L235A, and P329G in human IgG1, S228P and L235E in human IgG4, L234A and G237A in human IgG1, L234A, L235A and G237A in human IgG1, V234A and G237A in human IgG2, L235A, G237A and E318A in human IgG4, and S228P and L236E in human IgG4.

[0285] Fc polypeptide modifications to extend serum half-life In some embodiments, modifications to enhance serum half-life can be introduced into any Fc polypeptide described herein. For example, in some embodiments, both Fc polypeptides in an Fc polypeptide dimer can contain M428L and N434S substitutions (also referred to as LS substitutions) numbered according to the EU numbering scheme. Alternatively, both Fc polypeptides in an Fc polypeptide dimer can have an N434S or N434A substitution. Alternatively, both Fc polypeptides in an Fc polypeptide dimer can have an M428L substitution. In other embodiments, both Fc polypeptides in an Fc polypeptide dimer can contain M252Y, S254T, and T256E substitutions.

[0286] Fc polypeptide with C-terminal lysine residue removed In some embodiments, one or both of the Fc polypeptides can have their C-terminal lysine removed (e.g., Lys residue at position 447 of the Fc polypeptide according to EU numbering). C-terminal lysine residues are highly conserved in immunoglobulins across many species and may be completely or partially removed by cellular machinery during protein production. In some embodiments, removal of the C-terminal lysine in the Fc polypeptide can improve protein stability.

[0287] Exemplary Fc polypeptides are provided in SEQ ID NOs: 76-100.

[0288] Anti-TfR antibody variants engineered for conjugation via linking groups As described herein, a TfR-binding antibody (or other TfR-binding agent, e.g., a monovalent anti-TfR antibody, an anti-TfR / non-binding Fab bispecific antibody, or an anti-TfR / NBVR bispecific antibody described herein) can be linked to an oligonucleotide(s) via a linking group "L." In one aspect, the antibody comprises one or more amino acid residues (e.g., amino acid residues present at accessible sites in the antibody) that can be used to link the antibody to L. For example, in one aspect, the antibody comprises one or more cysteine residues (e.g., cysteine residues present at accessible sites in the antibody). In certain embodiments, the antibody is linked to L through a cysteine residue of the antibody (e.g., through the sulfur atom of the cysteine residue). In some embodiments, the cysteine is cysteine modified, and an amino acid residue other than cysteine present at an accessible site in the antibody is modified to cysteine. In other embodiments, the antibody comprises one or more glutamine residues. In certain embodiments, the antibody is linked to L through a glutamine residue (e.g., through an amide bond in the side chain of the glutamine residue).

[0289] In other aspects, it may be desirable to create engineered antibodies with one or more modification sites. These modification sites can be used to facilitate attachment of a TfR-binding agent to each L. For example, a TfR-binding agent can be attached to each L at the modification site. In other embodiments, the modification site can allow attachment of an L to an amino acid residue located near the modification site (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids of the modification site, e.g., within 2 or 3 amino acids of the modification site). In certain embodiments, such modification sites are substituted residues that occur at accessible sites on the antibody. In certain embodiments, the anti-TfR antibodies described herein (e.g., monovalent anti-TfR antibodies, anti-TfR / non-binding Fab bispecific antibodies, or anti-TfR / NBVR bispecific antibodies) comprise one or more modification sites (e.g., one or more amino acid substitutions, e.g., cysteine, alanine, or glycine substitutions). In certain embodiments, the antibody comprises at least or exactly 1, 2, 3, 4, 5, 6, 7, or 8 modification sites. In certain embodiments, the antibody comprises 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 modification sites. In certain embodiments, the antibody comprises 2 to 4 modification sites.

[0290] In certain embodiments, the site of modification within the antibody is an amino acid substitution or insertion. In certain embodiments, the antibody comprises an Fc dimer (or the antibody is an Fc dimer that is a protein). In certain embodiments, the Fc polypeptide(s) can be part of a Fab-Fc fusion or a Fab-Fc dimer fusion, and the site of modification is in the Fab-Fc polypeptide that binds to TfR and / or in the Fab-Fc polypeptide that dimerizes with the Fab-Fc polypeptide that binds TfR.

[0291] In certain embodiments, the modification site is present in the CL domain. In certain embodiments, the modification site is present in the CH1 domain. In certain embodiments, the modification site is present in the CH2 domain. In certain embodiments, the modification site is present in the CH3 domain.

[0292] In certain embodiments, the modification site is an amino acid substitution, hi certain embodiments, the modification site is a cysteine, glycine, or alanine substitution.

[0293] In certain embodiments, the modification site is a cysteine substitution. By substituting these residues with cysteine, a reactive thiol group is thereby positioned at an accessible site on the antibody, which can be used to conjugate the antibody to an oligonucleotide via a linking group (L) to generate a conjugate as described herein. In certain embodiments, the antibody contains an Fc polypeptide or an Fc polypeptide dimer and contains a cysteine substitution selected from the group consisting of S239C, S442C, A330C, and T289C, where the positions and substitutions are according to EU numbering. In other embodiments, the Fc polypeptide is attached to the CH1 domain and contains an A114C substitution. In other embodiments, it comprises a Fab-Fc fusion, and the light chain contains a K149C substitution.

[0294] In other aspects, the modification site is an alanine or glycine substitution. Such modified amino acids may facilitate enzymatic conjugation of L to the antibody at a nearby amino acid, such as a glutamine residue (e.g., using bacterial transglutaminase (BTG)). For example, in certain embodiments, the alanine / glycine substitution is N297A or N297G, where the position and substitution are according to EU numbering. These substitutions eliminate glycosylation at position 297, thereby preventing enzymatic conjugation of a linker to the antibody at position Q295 (i.e., the linker is attached to the antibody via an amide bond in the side chain of glutamine). Thus, in certain embodiments, the modification site is N297A or N297G, and the antibody is attached to L at Q295 (e.g., by enzymatic conjugation).

[0295] In certain embodiments, the N-terminus of the Fc polypeptide includes a portion of the hinge region (eg, DKTHTCP (SEQ ID NO: 4) or DKTHTCPPCP (SEQ ID NO: 5)).

[0296] In some embodiments, the TfR-binding agent-oligonucleotide conjugate comprises an Fc polypeptide or an Fc dimer. The Fc dimer comprises a first Fc polypeptide and a second Fc polypeptide. In certain embodiments, the Fc polypeptide or the first Fc polypeptide comprises one or more amino acid substitutions (e.g., one or more cysteine substitutions). In certain embodiments, the Fc polypeptide or the first Fc polypeptide comprises one or more substitutions selected from the group consisting of S239C, S442C, A330C, T289C, N297A, and N297G (according to EU numbering) in the heavy chain, K149C (according to EU numbering) in the light chain, and A114C (according to Kabat numbering) in the heavy chain. In certain embodiments, the Fc polypeptide or the first Fc polypeptide comprises S239C. In certain embodiments, the Fc polypeptide or the first Fc polypeptide comprises S442C. In certain embodiments, the Fc polypeptide or first Fc polypeptide comprises A330C. In certain embodiments, the Fc polypeptide or first Fc polypeptide comprises T289C. In certain embodiments, the Fc polypeptide or first Fc polypeptide comprises N297A. In certain embodiments, the Fc polypeptide or first Fc polypeptide comprises N297G. In certain embodiments, the Fc polypeptide or first Fc polypeptide comprises S239C and A330C.

[0297] In certain embodiments, the second Fc polypeptide (of an Fc dimer) comprises one or more amino substitutions (e.g., one or more cysteine substitutions). In certain embodiments, the second Fc polypeptide comprises one or more substitutions selected from the group consisting of S239C, S442C, A330C, T289C, N297A, and N297G (according to EU numbering) in the heavy chain, K149C (according to EU numbering) in the light chain, and A114C (according to Kabat numbering) in the heavy chain. In certain embodiments, the second Fc polypeptide comprises S239C. In certain embodiments, the second Fc polypeptide comprises S442C. In certain embodiments, the second Fc polypeptide comprises A330C. In certain embodiments, the second Fc polypeptide comprises T289C. In certain embodiments, the second Fc polypeptide comprises N297A. In certain embodiments, the second Fc polypeptide comprises N297G. In certain embodiments, the second Fc polypeptide comprises S239C and A330C, hi certain embodiments, the second Fc polypeptide comprises A114C.

[0298] In certain embodiments, the Fc polypeptide comprises a sequence having at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to SEQ ID NO:1.

[0299] In certain embodiments, the first Fc polypeptide or the second Fc polypeptide of the Fc dimer each comprises one or more amino acid substitutions (e.g., one or more cysteine substitutions). In certain embodiments, the one or more substitutions are S239C, S442C, A330C, T289C, N297A, and / or N297G according to EU numbering, and / or A114C according to Kabat numbering. In certain embodiments, the one or more substitutions are S239C, S442C, A330C, A114C, and / or T289C. In certain embodiments, the one or more substitutions are S239C, S442C, A114C, and / or T289C. In certain embodiments, the one or more substitutions are N297A and / or N297G. In certain embodiments, the first Fc polypeptide or the second Fc polypeptide of the Fc dimer each comprises one amino acid substitution (e.g., one cysteine substitution) to facilitate conjugation of an oligonucleotide. In certain embodiments, the first and second Fc polypeptides each comprise a cysteine substitution at S239C. In certain embodiments, the first and second Fc polypeptides each comprise two amino acid substitutions (e.g., two cysteine substitutions). In certain embodiments, the first and second Fc polypeptides each comprise cysteine substitutions at S239C and A330C.

[0300] Fc polypeptides or dimers thereof containing one or more modification sites (eg, cysteine substitutions) may be used in the conjugates described herein.

[0301] In some embodiments, the anti-TfR antibody antigen-binding domain comprises a Fab or scFab, wherein the Fab or scFab comprises a K149C substitution (according to EU numbering) on the light chain or an A114C substitution (according to Kabat numbering) on the heavy chain.

[0302] In certain embodiments, one or more oligonucleotides are bound to the linking group (L). In certain embodiments, two or more oligonucleotides are bound to the linking group (L). In certain embodiments, one oligonucleotide is bound to the linking group (L). In certain embodiments, two oligonucleotides are bound to the linking group (L).

[0303] VIII. Albumin In the case of TfR-binding agent-oligonucleotide conjugates containing albumin, the albumin can be human albumin or albumin from another mammalian species, such as, but not limited to, mouse albumin or non-human primate albumin. In some embodiments, the albumin is human albumin (SEQ ID NO: 167; UNIPROT accession P0276, GenBank: AAA98797.1, NCBANP_000468.1, GeneID: 213, mRNA NM_000477.7). The oligonucleotide can be linked to the albumin, optionally via a linking group, to a surface-accessible free cysteine in the albumin (e.g., C58 of mouse preproalbumin, position 34 (boxed) in SEQ ID NOs: 167 and 168). The albumin can be modified to include one or more amino acid substitutions, such as cysteine substitutions, to facilitate conjugation to the oligonucleotide.

[0304] In some embodiments, the TfR binding agent-oligonucleotide conjugate comprises an anti-TfR scFv fused to an albumin protein. The anti-TfR scFv fused to an albumin protein can be provided as a single polypeptide chain fusion protein. The anti-TfR scFv can be fused to the amino or carboxy terminus of albumin. In some embodiments, the anti-TfR scFv is fused to the amino terminus of albumin. The fusion moiety can contain a linking peptide between the scFc and albumin. The linking peptide can be, but is not limited to, a GGGS (glycine)3-serine) peptide. Exemplary anti-TfR scFv-albumin fusion proteins are provided in SEQ ID NOs: 169 and 170, which comprise the 17H10 anti-TfR scFv fused to mouse and human albumin, respectively. The anti-TfR scFv-albumin fusion protein can further comprise a peptide to facilitate purification, such as an epitope tag or a polyhistidine tag (e.g., His6). The epitope tag or polyhistidine can be placed at the amino- or carboxy-terminus of the fusion protein. [Table 2-1] [Table 2-2]

[0305] IX. Nucleic Acids, Vectors, and Host Cells The TfR-binding agents described herein can be prepared using recombinant methods. Thus, isolated nucleic acids comprising sequences encoding any of the TfR-binding agents described herein, or portions thereof, can be readily produced using methods available in the art. Host cells into which the nucleic acids can be introduced and used to replicate the nucleic acid encoding the polypeptide and / or to express the polypeptide are also available in the art. Host cells can be, but are not limited to, prokaryotic or eukaryotic cells. Eukaryotic cells can be, but are not limited to, yeast cells, insect cells, or mammalian cells (e.g., human cells).

[0306] The nucleic acid encoding the TfR-binding agent or a portion thereof can be DNA, RNA, cDNA, mRNA, single-stranded, double-stranded, linear or circular.

[0307] A TfR-binding agent may comprise two or more (e.g., three) polypeptides, each of which may be encoded by a separate nucleic acid sequence. The separate nucleic acid sequences may be present on the same plasmid or vector, or on different plasmids or vectors. If present on the same plasmid or vector, the separate nucleic acid sequences may be expressed from a single promoter or from different promoters. Methods for expressing nucleic acids encoding separate polypeptides from a single promoter are well known in the art, including, but not limited to, the use of 2A elements and internal ribosome entry sites.

[0308] The nucleic acid encoding the TfR-binding agent or a portion thereof may be provided in a plasmid or vector. The plasmid or vector can be used to replicate the nucleic acid or to facilitate expression of the nucleic acid. The plasmid or vector can be, but is not limited to, a viral vector, a phagemid, a yeast chromosomal vector, and a non-episomal mammalian vector.

[0309] In some embodiments, the nucleic acid encoding the TfR-binding agent or a portion thereof is operably linked to one or more regulatory sequences in an expression construct. The expression construct can be adapted for expression of the polypeptide in a system that produces the dual transporter. Such a system can be, but is not limited to, a mammalian cell expression system, an insect cell expression system, a yeast cell expression system, or a bacterial cell expression system.

[0310] Expression vehicles for producing recombinant polypeptides include plasmids and other vectors. For example, suitable vectors include the following types of plasmids for expression in prokaryotic cells such as E. coli: pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids, and pETC-derived plasmids. pcDNAI / amp, pcDNAEneo, pRc / CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo, and pHyg-derived vectors are examples of mammalian expression vectors suitable for transfection of eukaryotic cells. Alternatively, derivatives of viruses such as bovine papillomavirus (BPV-1) or Epstein-Barr virus (pHEBo, pREP-derived, and p205) can be used for transient expression of polypeptides in eukaryotic cells. In some embodiments, it may be desirable to express recombinant polypeptides using a baculovirus expression system. Examples of such baculovirus expression systems include pVL-derived vectors (such as pVL1392, pVL1393, and pVL941), pAcUW-derived vectors (such as pAcUW1), and pBlueBac-derived vectors. Additional expression systems include adenovirus, adeno-associated virus, and other viral expression systems.

[0311] An expression vector for expressing a TfR-binding agent or a portion thereof, or a plasmid or vector containing a nucleic acid, can be transformed, transfected, or transduced into a host cell. The host cell can be, but is not limited to, a mammalian cell, a yeast cell, an insect cell, a prokaryotic cell, a Chinese hamster ovary (CHO) cell, a dwarf hamster kidney (BHK) cell, an NSO cell, a YO cell, an HEK293 cell, a COS cell, a Vero cell, or a HeLa cell. The host cell containing the expression vector can be cultured under appropriate conditions that allow expression of the TfR-binding agent or a portion thereof.

[0312] A TfR-binding agent can be produced by culturing host cells containing one or more nucleic acids encoding the TfR-binding agent, expressing the TfR-binding agent, and isolating the expressed TfR-binding agent from the culture.

[0313] X.How to use The conjugates described herein can be used for a variety of purposes, including therapeutic indications.

[0314] In some embodiments, the conjugates are used to deliver oligonucleotides (e.g., ASOs or RNAi agents) to target cell types that express the transferrin receptor. In some embodiments, the conjugates are used to transport the oligonucleotides (e.g., ASOs or RNAi agents) across the endothelium, e.g., the blood-brain barrier, for uptake by the brain.

[0315] For example, certain embodiments provide methods for transcytosis of an oligonucleotide (e.g., an ASO or an RNAi agent) across the endothelium, the method comprising contacting the endothelium (e.g., the blood-brain barrier (BBB)) with a conjugate described herein. Accordingly, certain embodiments provide methods for transporting an oligonucleotide across the BBB in a subject in need thereof, the method comprising administering to the subject a conjugate described herein. In certain embodiments, a conjugate described herein is provided for use in transporting an oligonucleotide across the BBB in a subject in need thereof. In certain embodiments, a conjugate described herein is provided for use in transporting an oligonucleotide to a muscle cell in a subject in need thereof.

[0316] Certain embodiments also provide methods of modulating expression of a target gene or sequence in a subject in need thereof, the method comprising administering to the subject an effective amount of a conjugate described herein. In some embodiments, there is provided a conjugate described herein for use in modulating expression of a target gene.

[0317] In certain embodiments, the target gene or sequence is expressed in cells in the brain of a subject.In certain embodiments, the target gene or sequence is expressed in cells that express TfR.In certain embodiments, the target gene or sequence is expressed in muscle cells, such as skeletal muscle cells or cardiac muscle cells.

[0318] In certain embodiments, the modulation of target gene expression is gene knockdown or gene knockout.Therefore, in certain embodiments, the expression of target gene or sequence is inhibited or reduced by, for example, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% compared with the expression in control (for example, the subject that is not administered with the conjugate).

[0319] The conjugate described herein is administered to the subject in a therapeutically effective amount or therapeutically effective dose.However, the dosage may vary depending on several factors, including the selected administration route, the formulation of the composition, the patient's response, the severity of the condition, the subject's weight, and the prescribing physician's judgment.Dosage may be increased or decreased over time according to the needs of each individual patient.

[0320] In various embodiments, the conjugates described herein are administered parenterally. In some embodiments, the conjugates are administered intravenously. Intravenous administration can be, for example, by infusion over about 10 minutes to about 30 minutes, or over at least 1 hour, 2 hours, or 3 hours. In some embodiments, the conjugates are administered as an intravenous bolus. A combination of infusion and bolus administration can also be used.

[0321] In some parenteral embodiments, the conjugate is administered intraperitoneally, subcutaneously, intradermally, or intramuscularly. In some embodiments, the conjugate is administered intradermally or intramuscularly. In some embodiments, the conjugate is administered intrathecally, such as by epidural administration, or intracerebroventricularly.

[0322] In other embodiments, the conjugates described herein can be administered orally, pulmonary, intranasally, intraocularly, or topically. Pulmonary administration can be employed, for example, by use of an inhaler or nebulizer and a formulation including an aerosolizing agent.

[0323] XI. Pharmaceutical Compositions and Kits In another aspect, pharmaceutical compositions and kits are provided that include the conjugates described herein.

[0324] Pharmaceutical Composition For the uses described herein In some embodiments, a pharmaceutical composition comprises a conjugate described herein and further comprises one or more pharmaceutically acceptable carriers and / or excipients. In certain embodiments, a composition comprises multiple conjugates described herein, which may be the same or different (e.g., a mixture of different conjugates). In certain embodiments, the ratio of oligonucleotide to protein in the composition is about 1:1 to about 4:1. In certain embodiments, the ratio of oligonucleotide to protein in the composition is about 1:1 to about 2:1. In certain embodiments, the ratio of oligonucleotide to protein in the composition is about 1.23. In certain embodiments, the ratio of oligonucleotide to protein in the composition is about 2:1 to about 3:1. In certain embodiments, the ratio of oligonucleotide to protein in the composition is about 2.5.

[0325] As used herein, a pharmaceutically acceptable carrier includes any solvent, dispersion medium, or coating that is physiologically compatible and preferably does not interfere with or otherwise inhibit the activity of the active substance. A variety of pharmaceutically acceptable excipients are well known. In some embodiments, the carrier is suitable for intravenous, intrathecal, intracerebroventricular, intramuscular, oral, intraperitoneal, transdermal, topical, or subcutaneous administration. The pharmaceutically acceptable carrier may contain one or more physiologically acceptable compounds that act, for example, to stabilize the composition or increase or decrease the absorption of the conjugate. Physiologically acceptable compounds may include, for example, carbohydrates such as glucose, sucrose, or dextran, antioxidants such as ascorbic acid or glutathione, chelating agents, low-molecular-weight proteins, compositions that reduce the clearance or hydrolysis of the active substance, or excipients or other stabilizers and / or buffers. Other pharmaceutically acceptable carriers and their formulations are also available in the art.

[0326] The pharmaceutical compositions described herein can be manufactured in a manner well known to those skilled in the art, for example, by conventional mixing, dissolving, granulating, dragee-making, emulsifying, encapsulating, entrapping, or lyophilizing processes. The following methods and excipients are merely illustrative and in no way limiting.

[0327] For oral administration, the conjugates described herein can be formulated by combining them with pharmaceutically acceptable carriers well known in the art. Such carriers allow the compounds to be formulated as tablets, pills, dragees, capsules, emulsions, lipophilic and hydrophilic suspensions, liquids, gels, syrups, slurries, suspensions, etc., to be orally ingested by the patient to be treated. Pharmaceutical preparations for oral use can be obtained by mixing the conjugates with solid excipients, optionally grinding the resulting mixture, and processing the granular mixture to obtain tablets or dragee cores, optionally after adding suitable excipients. Suitable excipients include, for example, fillers such as sugars including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidone. If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate.

[0328] As disclosed above, the conjugates described herein can be formulated for parenteral administration, for example, by injection via bolus injection or continuous infusion. For injection, the conjugates can be formulated into preparations by dissolving, suspending, or emulsifying them in aqueous or non-aqueous solvents, such as vegetable or other similar oils, synthetic fatty acid glycerides, esters of higher fatty acids, or propylene glycol, along with conventional additives, such as solubilizers, isotonicity agents, suspending agents, emulsifiers, stabilizers, and preservatives, as needed. In some embodiments, the conjugates can be formulated in aqueous solutions, preferably physiologically compatible buffers such as Hank's solution, Ringer's solution, and physiological saline buffer. Injectable preparations can be provided in unit dosage form, e.g., ampoules or multi-dose containers, with added preservatives. The compositions can take the form of suspensions, solutions, or emulsions in oily or aqueous vehicles, and can contain formulating agents, such as suspending agents, stabilizers, and / or dispersing agents.

[0329] Typically, pharmaceutical compositions for use in in vivo administration are sterile. Sterilization can be achieved by methods well known in the art, such as heat sterilization, steam sterilization, sterile filtration, or irradiation.

[0330] The dosage and desired drug concentration of the pharmaceutical compositions described herein may vary depending on the specific intended use. The determination of the appropriate dosage or route of administration is well within the skill of a person skilled in the art. Suitable dosages have been described above.

[0331] kit In some embodiments, a kit is provided that includes the conjugate described herein. In some embodiments, the kit is for use in regulating the expression of a target gene or sequence (e.g., a target gene expressed in the brain or central nervous system (CNS)). In some embodiments, the kit is for use in regulating the expression of a target gene.

[0332] In some embodiments, the kit further comprises one or more additional therapeutic agents. For example, in some embodiments, the kit comprises a conjugate described herein and further comprises one or more additional therapeutic agents. In some embodiments, the kit further comprises instructional materials containing instructions (i.e., protocols) for practicing the methods described herein (e.g., instructions for using the kit to administer a composition across the blood-brain barrier). The instructional materials typically comprise written or printed material, but are not limited to such. Any medium capable of storing such instructions and transmitting them to an end user is contemplated herein. Such media include, but are not limited to, electronic storage media (e.g., magnetic disks, magnetic tapes, magnetic cartridges, magnetic chips), optical media (e.g., CD-ROMs), and the like. Such media may include addresses to internet sites providing such instructional materials. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 3-7] [Table 3-8] [Table 3-9] [Table 3-10] [Table 3-11] [Table 3-12] [Table 3-13] [Table 3-14] [Table 3-15] [Table 3-16] [Table 3-17] [Table 3-18] [Table 3-19] [Example]

[0333] The present subject matter will be described in more detail by specific examples. The following examples are provided for illustrative purposes only and are not intended to limit the present invention in any way. Those skilled in the art will readily recognize a variety of non-critical parameters that can be changed or modified to yield essentially the same results. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.), but some experimental error and deviation may exist. The practice of the present disclosure employs, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA technology, and pharmacology, within the skill of the art. Such techniques are fully explained in the literature.

[0334] Example 1: Mono-Fab and bivalent antibody conjugates Heavy chain vectors were cotransfected with the corresponding light chain vectors into Expi293 cells at a knob:hole:light chain ratio of 1:1:2 for mono-Fab and bivalent antibodies. Expressed proteins were purified from the conditioned medium by loading the supernatant onto a Protein A column. The column was washed with 10 column volumes of PBS, pH 7.4. Proteins were eluted with 50 mM sodium citrate, pH 3.0, containing 150 mM NaCl and immediately neutralized with 200 mM arginine, 137 mM succinic acid, pH 5.0. Proteins were further purified by size-exclusion chromatography (SEC) (GE Superdex200) using 200 mM arginine, 137 mM succinic acid, pH 5.0 as the running buffer. The purified proteins were confirmed by intact mass LC / MS, and greater than 95% purity was confirmed by SDS-PAGE and analytical HPLC-SEC. Binding to the apical domain of human and cynomolgus TfR was tested via biacore. [Table 4]

[0335] The mono-Fab and bivalent antibodies produced above contained cysteine modifications for conjugation and were first reduced using a reducing agent (e.g., TCEP). After reduction, the remaining reducing agent was removed (e.g., purified by dialysis), and the antibodies were reoxidized with an oxidizing agent (e.g., dHAA). ASOs containing linking groups were also produced, followed by reduction and oxidation steps. The reduced and oxidized linker ASOs were then conjugated to free cysteines on the mono-Fab and bivalent antibodies. The resulting conjugates were purified to remove unwanted and unconjugated products, and purity was determined by LC / MS and SEC.

[0336] Exemplary ASO sequences used herein targeting MALAT1: 5'-G ks m C ks A ks T ds T ds m Cds T ds A ds A ds T ds A ds G ds m C ds A ks G ks m C k -3' (SEQ ID NO: 8; mouse MALAT1). Abbreviations refer to the following components: d: DNA, k: LNA, m C: 5-methylcytidine (methylated cytosine), s: phosphorothioate backbone (PS). The ASO is modified at the 5'C6 amine. Another exemplary ASO sequence targeting MALAT1 is SEQ ID NO: 172 (cynomolgus monkey MALAT1).

[0337] Exemplary linking groups used herein are shown below, where the linking group is attached to the sulfur atom of a cysteine residue in the monoFab or bivalent antibody and is attached to the ASO via the phosphate associated with the 5'-terminal residue of the ASO: [ka]

[0338] Example 2: In vivo pharmacokinetics and Malat1 knockdown using TfR mono-Fab conjugates. The monovalent TfR Fab conjugates ("TfR monoFabs") prepared above were diluted with sterile saline prior to administration. Saline, unconjugated ASO, and RSV-ASO groups served as controls.

[0339] In a single-dose study, 2-month-old TfR ms / hu Female mice were intravenously administered doses according to the groups (n=4) in Table 4 below. Tissues were collected 24 hours after the single dose. Specifically, brain, spinal cord, and peripheral organs (kidney, lung, liver, and quadriceps) were collected. Peripheral blood was also collected 24 hours after the single dose.

[0340] In multiple-dose studies, 2-month-old TfR ms / huFemale mice were intravenously dosed on days 1, 7, and 14 according to the groups (n=6) in Table 5. Plasma was collected at 30 minutes, 4 hours, 24 hours, 48 hours, 72 hours, and 1 week. Tissues were collected 72 hours after the final dose. Specifically, brain, spinal cord, and peripheral organs (kidney, lung, liver, and quadriceps) were collected. Peripheral blood was also collected 72 hours after the final dose. [Table 5]

[0341] Intact drug and total ASO were measured according to the methods described below.

[0342] huIgG assay Quantitation of humanized antibodies in mouse plasma and tissue lysates was measured using a universal electrochemiluminescence immunoassay (ECLIA). Briefly, wells of an MSD GOLD 96-well streptavidin-coated microtiter plate (Meso Scale Discovery, Rockville, MD) were incubated with a working concentration of biotinylated goat anti-human IgG polyclonal antibody (Southern Biotech, Birmingham, AL) prepared in assay diluent for approximately 1 hour. After this incubation and a plate wash step, prepared test samples (including sample predilutions, if necessary) and relevant standards were added to the assay plate and incubated for approximately 1 hour. After test sample incubation and a plate wash step, a secondary ruthenylated (SULFO-TAG) goat anti-human IgG antibody (Meso Scale Discovery, Rockville, MD) in assay diluent was added to the assay plate at the working concentration and incubated for approximately 1 hour. After plate washing, 1x MSD Read Buffer T (Meso Scale Discovery, Rockville, MD) was then added to generate electrochemiluminescence (ECL) assay signals, which were then expressed in ECL units (ECLU). All assay reaction steps were performed at ambient temperature with shaking on a plate shaker (where appropriate), and all test samples were prediluted 1:20 with the assay MRD before analysis in the assay plate. Sample ECLU signals generated in the assay were then processed into concentrations by back-calculation from the assay calibration (CS) curve. The assay CS curve was fitted with a weighted four-parameter nonlinear logistic regression for use in calculating unknown / test sample concentrations.

[0343] Intact Drug Assay Quantification of intact drug (anti-TfR antibody conjugated to antisense oligonucleotide (ASO)) in mouse plasma and tissue lysates was measured using a hybridization-based electrochemiluminescence immunoassay (ECLIA). Briefly, working concentrations of custom biotinylated antisense probes (synthesized by Integrated DNA Technologies, Coralvia, IA) were incubated with prepared test samples (including sample predilutions, if necessary) and relevant standards in TE buffer (10 mM Tris-HCl containing 1 mM EDTA) and hybridized for 45 minutes at the appropriate temperature. After incubation, the hybridized products were added to wells of an MSD GOLD 96-well streptavidin-coated microtiter plate (Meso Scale Discovery, Rockville, MD) and incubated for approximately 30 minutes. After hybrid product incubation and plate washing steps, a secondary ruthenylated (SULFO-TAG) goat anti-human IgG antibody (Meso Scale Discovery, Rockville, MD) in assay diluent was added to the assay plate at the working concentration and incubated for approximately 1 hour. After plate washing, 1x MSD Read Buffer T (Meso Scale Discovery, Rockville, MD) was then added to generate an electrochemiluminescence (ECL) assay signal, which was then expressed in ECL units (ECLU). All assay reaction steps were performed at ambient temperature with shaking on a plate shaker (where appropriate), and all test samples were prediluted 1:20 with the assay MRD before analysis in the assay plate. Sample ECLU signals generated in the assay were then processed into concentrations by back-calculation from the assay calibration (CS) curve. The assay CS curve was fitted with a weighted four-parameter nonlinear logistic regression for use in calculating unknown / test sample concentrations.

[0344] Total ASO assay Quantification of total ASOs (conjugated and free forms) in mouse plasma and tissue homogenates was measured using a hybridization-based electrochemiluminescence immunoassay (ECLIA). Briefly, working concentrations of custom biotinylated and digoxigenin-conjugated antisense probes (synthesized by Integrated DNA Technologies, Coralvia, IA) were mixed with prepared test samples (including sample predilutions, if necessary) and relevant standards in TE buffer (10 mM Tris-HCl containing 1 mM EDTA). The prepared samples in TE buffer were added 1:1 to 1x SSC buffer (Sigma-Aldrich, St. Louis, MO) containing a working concentration of recombinant proteinase K enzyme (ThermoFisher, Waltham, MA). The hybridization / enzyme mixture was then digested, detanned, annealed, and cooled in a thermal cycler instrument. Following hybridization product incubation, samples were added to wells of an MSD GOLD 96-well streptavidin-coated microtiter plate (Meso Scale Discovery, Rockville, MD) and incubated for approximately 30 minutes. After incubation and a plate wash step, a working concentration of secondary luteinized (SULFO-TAG) sheep anti-digoxigenin antibody (Novus Biologicals, Littleton, CO) in assay diluent was added to the plate and incubated for approximately 30 minutes. After plate washing, 1x MSD Read Buffer T (Meso Scale Discovery, Rockville, MD) was then added to generate an electrochemiluminescence (ECL) assay signal, which was then expressed in ECL units (ECLU). All assay reaction steps were performed at ambient temperature with shaking (where appropriate) on a plate shaker, and all test samples were prediluted 1:20 in the assay MRD before analysis in the assay plate. The sample ECLU signals generated in the assay were then processed into concentrations by back-calculation from the assay calibration (CS) curve.The assay CS curves were fitted with a weighted four-parameter nonlinear logistic regression for use in calculating unknown / test sample concentrations.

[0345] Malat1 expression assay Malat1 expression was measured in the brain, spinal cord, liver, heart, quadriceps, diaphragm, and sciatic nerve as follows. For bulk RNA isolation, tissue fragments of less than 50 mg were homogenized using a bead homogenizer in Trizol. The homogenized tissue was incubated with chloroform for 3–5 minutes, followed by phase separation after centrifugation. The aqueous phase was then incubated with isopropanol for 10 minutes to precipitate the RNA, followed by washing with 75% ethanol and resuspension in nuclease-free water. Malat1 expression was then measured by qPCR using the Express One-Step Superscript Kit and normalized to the expression of the housekeeping gene Gapdh.

[0346] The results are shown in Figures 1-5. Increased delivery of intact drug and total ASO to the CNS was observed with the TfR mono-Fab conjugate compared to the naked ASO and RSV-ASO controls (Figures 1 and 2). Increased Malat1 knockdown in the CNS compared to controls was also observed in both single- and multiple-dose studies (Figure 2). Malat1 knockdown was also observed in peripheral tissues (Figure 5). The liver was the sink for ASO 24 hours after dosing in the single-dose study (Figure 3). Accumulation of ASO in the liver and kidney was observed 72 hours after the final dose in the multiple-dose study (Figure 4).

[0347] Example 3: In vivo Malat1 knockdown using anti-TfR bivalent antibody conjugates. The bivalent anti-TfR antibody conjugated to Malat1 ASO prepared in Example 1 was diluted in sterile saline and administered to TfR at a weekly dose of 50 mg / kg for 4 weeks. ms / hu The knock-in mice were intravenously administered TfR. ms / huMice were intravenously administered either sterile saline or unconjugated ASO. Three days after the fourth dose, tissues were harvested and frozen for molecular and biochemical analysis. Tissues included brain, spinal cord, liver, heart, quadriceps, diaphragm, and sciatic nerve.

[0348] Malat1 expression was measured in the brain, spinal cord, liver, heart, quadriceps, diaphragm, and sciatic nerve as described above.

[0349] The results are shown in Figure 6. Some Malat1 knockdown was observed in the CNS, with higher Malat1 knockdown observed in the periphery.

[0350] Example 4: In vivo pharmacokinetics and biodistribution using TfR mono-Fab conjugates Two TfR-mono Fab conjugates (TfR mono Fab and TfR mono Fab2) were prepared as described in Example 1 and diluted in sterile saline prior to administration. The TfR mono Fab conjugate had a TfR-binding arm and a non-binding RSV arm and was conjugated to mouse MALAT1 (SEQ ID NO: 8) (anti-TfR / non-binding Fab antibody-oligonucleotide), while the TfR-mono Fab2 conjugate had a TfR-binding arm (no second arm; mono Fab) and was conjugated to cynoMALAT1 (SEQ ID NO: 172). Unconjugated ASO was administered as a control. Two-month-old TfRms / hu female mice received intravenous doses of either naked ASO (0.9 mg per kg (mpk)), TfR-mono Fab conjugate (25 mpk), or TfR-mono Fab2 conjugate (17.2 mpk). The following tissues were collected 24 hours after the single dose: brain, spinal cord, kidney, diaphragm, liver, and quadriceps. Plasma was also collected 15 minutes, 4 hours, and 24 hours after the single dose.

[0351] Total ASO and total huIgG were measured according to the method described in Example 2 above. The results are shown in Figures 7-9. These two TfR-mono Fab conjugate molecules exhibited similar pharmacokinetic profiles in plasma (Figure 7) and similar biodistribution patterns throughout the body (Figure 8). Compared to unconjugated ASO, which was rapidly cleared from the circulation and not detected in the brain or spinal cord after a dose of 0.9 mpk, molar equivalent amounts of both TfR-mono Fab molecules achieved robust CNS ASO uptake (Figure 9). Furthermore, both TfR mono Fab molecules delivered more ASO to the diaphragm, quadriceps, and liver, but significantly less ASO to the kidney (Figure 9).

[0352] Example 5: TfR-Albumin-ASO Construction A TfR-albumin-ASO molecule was generated by fusing a TfR-binding scFv to mouse serum albumin via a linker. A linker-ASO was also generated using the linker shown in Example 1 and mouse MALAT1 (SEQ ID NO: 8). The cysteine at position 34 (of SEQ ID NO: 168) was used for conjugation. For bioconjugation of the linker-ASO to the TfR-albumin protein, the TfR-albumin protein was first reduced using TCEP (30 molar equivalents). The linker-ASO was then conjugated to the free cysteine (1.2 molar equivalents) on the TfR-albumin protein. The resulting conjugate was purified by cation exchange chromatography (mobile phase A: 20 mM sodium acetate, pH 5; mobile phase B1: 20 mM sodium acetate, 1 M NaCl, pH 5) to remove unwanted unconjugated products, and purity was determined by LC / MS and analytical SEC.

[0353] Example 6: In vivo pharmacokinetics and biodistribution using TfR-albumin-ASO conjugates The TfR-albumin-ASO molecules prepared above were diluted with sterile saline before administration. As a control, unconjugated ASOs were also administered.

[0354] TfR at 4-8 months of age ms / huFemale mice were administered doses intravenously. TfR-albumin-ASO was administered at 9.5 mg / kg (n=2). Unconjugated ASO ("naked ASO") was administered at 1.37 mg / kg (n=3). Plasma was collected 15 minutes (unconjugated ASO only), 4 hours, and 24 hours after administration. Tissues including brain, liver, and kidney, as well as terminal plasma, were collected 72 hours after administration. ASO concentrations were measured as described in Example 2.

[0355] The results are shown in Figures 10-12. Compared to naked ASO, increased ASO delivery to the brain was observed with the TfR-albumin-ASO conjugate (Figure 10). The liver and kidney are sinks for naked ASO, and ASO concentrations in these organs were reduced with the TfR-albumin-ASO conjugate (Figure 11). In plasma, clearance was similar between the two molecules, with a slight acceleration of clearance with the TfR-albumin-ASO conjugate (Figure 12).

[0356] Many modifications and other embodiments of the subject matter described herein will come to mind to one skilled in the art to which this invention pertains having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. It is to be understood, therefore, that the invention is not to be limited to the particular embodiments disclosed, and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

Claims

1. 1. A TfR-binding agent-oligonucleotide conjugate comprising: P-F-P' | Formula (I) (L-(O) ｙ ) ｎ wherein P comprises an anti-TfR antibody antigen-binding domain; F is present or absent, and if present, comprises a peptide, an Fc polypeptide, an Fc dimer, or albumin; L is present or absent and, if present, comprises a linking group; P' is present or absent, and if present, comprises an anti-TfR antibody antigen-binding domain, or a non-binding Fab, or a non-binding variable region (NBVR); O comprises an oligonucleotide; y is an integer equal to or greater than 1 (e.g., 1, 2, 3, or 4); The TfR-binding agent-oligonucleotide conjugate, wherein n is an integer of 1 or greater (eg, 1, 2, 3, 4, 5, 6, 7, or 8).

2. During the ceremony, F is an Fc dimer, 2. The TfR-binding agent-oligonucleotide conjugate of claim 1, wherein P' is absent.

3. During the ceremony, 3. The TfR-binding agent-oligonucleotide conjugate of claim 2, wherein P is an anti-TfR Fab, a single-chain Fab (scFab), or a single-chain variable fragment (scFv).

4. During the ceremony, 4. The TfR binding agent-oligonucleotide conjugate of claim 3, wherein P is an anti-TfR Fab or scFab.

5. During the ceremony, F is an Fc dimer, 2. The TfR-binding agent-oligonucleotide conjugate of claim 1, wherein P' is a non-binding Fab or NBVR.

6. During the ceremony, 6. The TfR-binding agent-oligonucleotide conjugate of claim 5, wherein P is an anti-TfR Fab, a single-chain Fab (scFab), or a single-chain variable fragment (scFv).

7. During the ceremony, 7. The TfR binding agent-oligonucleotide conjugate of claim 6, wherein P is an anti-TfR Fab or scFab.

8. During the ceremony, F is albumin, 2. The TfR-binding agent-oligonucleotide conjugate of claim 1, wherein P' is absent.

9. During the ceremony, 9. The TfR-binding agent-oligonucleotide conjugate of claim 8, wherein P is an scFv or a nanobody.

10. During the ceremony, 10. The TfR-binding agent-oligonucleotide conjugate of claim 9, wherein P is an scFv.

11. During the ceremony, F is an Fc dimer, 2. The TfR binding agent-oligonucleotide conjugate of claim 1, wherein P' is an anti-TfR antibody antigen-binding domain.

12. During the ceremony, 12. The TfR-binding agent-oligonucleotide conjugate of claim 11, wherein P-FP' comprises an antibody.

13. 13. The TfR-binding agent-oligonucleotide conjugate of claim 12, wherein the antibody comprises a bivalent anti-TfR antibody or a bispecific antibody.

14. During the ceremony, 12. The TfR-binding agent-oligonucleotide conjugate of claim 11, wherein P is an anti-TfR Fab, a single-chain Fab (scFab), a single-chain variable fragment (scFv), or a nanobody.

15. During the ceremony, 15. The TfR binding agent-oligonucleotide conjugate of claim 14, wherein P is an anti-TfR Fab or scFab.

16. During the ceremony, P is a first Fab known to specifically bind to TfR; F is an Fc dimer, 2. The TfR-binding agent-oligonucleotide conjugate of claim 1, wherein PF comprises a modification for covalent conjugation, and O is conjugated to PF at the site of said modification.

17. 17. The conjugate of claim 16, further comprising a second Fab that is a non-targeting Fab or that does not bind to TfR.

18. wherein P comprises three heavy chain CDRs and three light chain CDRs; (a) CDR-H1 comprises SEQ ID NO: 12; CDR-H2 comprises SEQ ID NO: 13; CDR-H3 comprises SEQ ID NO: 14; CDR-L1 comprises SEQ ID NO: 15; CDR-L2 comprises SEQ ID NO: 16; CDR-L3 comprises SEQ ID NO: 17; (b) CDR-H1 comprises SEQ ID NO: 21; CDR-H2 comprises SEQ ID NO: 22; CDR-H3 comprises SEQ ID NO: 23; CDR-L1 comprises SEQ ID NO: 24; CDR-L2 comprises SEQ ID NO: 25; CDR-L3 comprises SEQ ID NO: 26; (c) CDR-H1 comprises SEQ ID NO: 114; CDR-H2 comprises SEQ ID NO: 115; CDR-H3 comprises SEQ ID NO: 116; CDR-L1 comprises SEQ ID NO: 117; CDR-L2 comprises SEQ ID NO: 118; CDR-L3 comprises SEQ ID NO: 119; (d) CDR-H1 comprises SEQ ID NO: 126; CDR-H2 comprises SEQ ID NO: 127; CDR-H3 comprises SEQ ID NO: 128; CDR-L1 comprises SEQ ID NO: 129; CDR-L2 comprises SEQ ID NO: 130; CDR-L3 comprises SEQ ID NO: 131; (e) CDR-H1 comprises SEQ ID NO: 134; CDR-H2 comprises SEQ ID NO: 135; CDR-H3 comprises SEQ ID NO: 136; CDR-L1 comprises SEQ ID NO: 137; CDR-L2 comprises SEQ ID NO: 138; CDR-L3 comprises SEQ ID NO: 139; (f) CDR-H1 comprises SEQ ID NO: 154; CDR-H2 comprises SEQ ID NO: 155; CDR-H3 comprises SEQ ID NO: 156; CDR-L1 comprises SEQ ID NO: 157; CDR-L2 comprises SEQ ID NO: 158; CDR-L3 comprises SEQ ID NO: 159; or (g) CDR-H1 comprises SEQ ID NO: 161; CDR-H2 comprises SEQ ID NO: 162; CDR-H3 comprises SEQ ID NO: 163; CDR-L1 comprises SEQ ID NO: 164; CDR-L2 comprises SEQ ID NO: 165; The TfR-binding agent-oligonucleotide conjugate of any one of claims 1 to 17, wherein CDR-L3 comprises SEQ ID NO:

166.

19. wherein P is an anti-TfR Fab; (a) SEQ ID NOs: 102 and 103; (b) SEQ ID NOs: 104 and 105; (c) SEQ ID NOs: 110 and 111; (d) SEQ ID NOs: 122 and 123; (e) SEQ ID NOs: 132 and 133, or (f) SEQ ID NOs: 143 and 144; 18. The TfR-binding agent-oligonucleotide conjugate of any one of claims 1 to 8 and 11 to 17, wherein optionally, P comprises one or more modifications for covalent conjugation of said oligonucleotide.

20. wherein P is an scFv; (a) SEQ ID NO: 106; (b) SEQ ID NO: 107; (c) SEQ ID NO: 171; (d) SEQ ID NO: 153; (e) SEQ ID NO: 160; (f) SEQ ID NOs: 112 and 113; (g) SEQ ID NOs: 124 and 125; (h) the TfR-binding agent-oligonucleotide conjugate of any one of claims 1 to 3, 5 to 6, 8 to 10, 11, 14, and 16 to 17, comprising SEQ ID NOs: 145 and 146.

21. wherein P is an antibody; (a) SEQ ID NOs: 108 and 109; (b) SEQ ID NOs: 120 and 121; (c) SEQ ID NOs: 9, 10, and 11, or (d) SEQ ID NOs: 18 and 19; 18. The TfR-binding agent-oligonucleotide conjugate of any one of claims 1 to 3, 5 to 6, 8 to 10, 11, 14, and 16 to 17, wherein the antibody optionally contains one or more modifications to increase serum stability, modulate effector function, affect glycosylation, reduce immunogenicity in humans, promote heterodimerization, and / or promote conjugation of the oligonucleotide.

22. 22. The TfR-binding agent-oligonucleotide of any one of claims 1 to 21, wherein P, F, or P' comprises a modification for conjugation of said oligonucleotide.

23. 23. The TfR binding agent-oligonucleotide conjugate of claim 22, wherein the modification for conjugation is a cysteine modification.

24. 24. The TfR-binding agent-oligonucleotide conjugate of claim 23, wherein F comprises the Fc polypeptide or the Fc dimer, and the cysteine modification is a S239C, S442C, A330C, or T289C substitution (positions according to EU numbering).

25. 25. The TfR-binding agent-oligonucleotide conjugate of claim 24, wherein the cysteine modification is a S239C substitution.

26. 24. The TfR-binding agent-oligonucleotide conjugate of claim 23, wherein P comprises a Fab or scFab, and the cysteine modification comprises a K149C substitution (position according to EU numbering) or an A114C substitution (position according to Kabat numbering).

27. 23. The TfR-binding agent-oligonucleotide conjugate of claim 22, wherein F comprises an Fc polypeptide or an Fc dimer, and the modification comprises an N297A or N297G substitution (positions according to EU numbering).

28. 28. The TfR-binding agent-oligonucleotide conjugate of any one of claims 1 to 7 and 11 to 27, wherein F comprises an Fc polypeptide or an Fc dimer, and wherein said Fc polypeptide or one or both Fc polypeptides of said Fc dimer contain at least one modification to increase serum stability, modulate effector function, affect glycosylation, reduce immunogenicity in humans, and / or promote heterodimerization.

29. The at least one modification is (a) L234A and L235A substitutions; (b) a P329G substitution, or (c) comprising an L234A substitution, an L235A substitution, and a P329G substitution; 29. The TfR-binding agent-oligonucleotide conjugate of claim 28, wherein the positions are according to EU numbering.

30. The at least one modification is (a) M428L and N434S substitutions; (b) M428L substitution, (b) N434S substitution, (c) an N434A substitution, or (d) comprising an M252Y substitution, an S254T substitution, and a T256E substitution; 30. The TfR-binding agent-oligonucleotide conjugate of claim 28 or 29, wherein said positions are according to EU numbering.

31. 31. The TfR-binding agent-oligonucleotide conjugate of any one of claims 28 to 30, wherein said at least one modification comprises a deletion of a carboxy-terminal lysine.

32. The at least one modification is (a) M428L and N434S substitutions; (b) M428L substitution, (b) N434S substitution, (c) an N434A substitution, or (d) comprising an M252Y substitution, an S254T substitution, and a T256E substitution; The TfR-binding agent-oligonucleotide conjugate of any one of claims 28 to 31, wherein said positions are according to EU numbering.

33. 33. The TfR-binding agent-oligonucleotide conjugate of any one of claims 28 to 32, wherein F comprises the Fc dimer, a first Fc polypeptide of the Fc dimer comprises a knob mutation, and a second Fc polypeptide of the Fc dimer comprises a hole mutation.

34. 34. The TfR-binding agent-oligonucleotide conjugate of claim 33, wherein the knob mutation comprises a T366W substitution according to the EU numbering scheme and the hole mutation comprises a Y407V substitution, and optionally a T366S substitution and a L368A substitution, according to the EU numbering scheme.

35. wherein P' is present and comprises a non-binding Fab or NBVR, said non-binding Fab or NBVR comprising three heavy chain CDRs and three light chain CDRs, (a) CDR-H1 comprises SEQ ID NO: 45; CDR-H2 comprises SEQ ID NO: 47; CDR-H3 comprises SEQ ID NO: 49; CDR-L1 comprises SEQ ID NO: 39; CDR-L2 comprises SEQ ID NO: 41; CDR-L3 comprises SEQ ID NO: 43; (b) CDR-H1 comprises SEQ ID NO: 46; CDR-H2 comprises SEQ ID NO: 48; CDR-H3 comprises SEQ ID NO: 49; CDR-L1 comprises SEQ ID NO: 40; CDR-L2 comprises SEQ ID NO: 42; CDR-L3 comprises SEQ ID NO: 44; or (c) CDR-H1 comprises SEQ ID NO: 46; CDR-H2 comprises SEQ ID NO: 50; CDR-H3 comprises SEQ ID NO: 49; CDR-L1 comprises SEQ ID NO: 40; CDR-L2 comprises SEQ ID NO: 42; The TfR-binding agent-oligonucleotide conjugate of any one of claims 1, 5-7, 11-34, wherein CDR-L3 comprises SEQ ID NO:

44.

36. In the formula, P′ is (a) SEQ ID NO: 35 and SEQ ID NO: 36; (b) SEQ ID NO: 37 or 52 and SEQ ID NO: 38; (c) SEQ ID NO: 37 or 52 and SEQ ID NO: 53; (d) SEQ ID NO: 37 or 52 and SEQ ID NO: 54; (e) SEQ ID NO: 37 or 52 and SEQ ID NO: 55; (f) SEQ ID NO: 37 or 52 and SEQ ID NO: 56; (g) SEQ ID NO: 37 or 52 and SEQ ID NO: 57; (h) SEQ ID NO: 37 or 52 and SEQ ID NO: 58; (i) SEQ ID NO: 37 or 52 and SEQ ID NO: 59; (j) SEQ ID NO: 51 and SEQ ID NO: 60; (k) SEQ ID NO: 62 or 64 and SEQ ID NO: 61; (l) SEQ ID NO: 62 or 64 and SEQ ID NO: 65; (m) SEQ ID NO: 62 or 64 and SEQ ID NO: 66; (n) SEQ ID NO: 62 or 64 and SEQ ID NO: 67; (o) SEQ ID NO: 62 or 64 and SEQ ID NO: 68; (p) SEQ ID NO: 62 or 64 and SEQ ID NO: 69; (q) SEQ ID NO: 62 or 64 and SEQ ID NO: 70, or (r) the TfR-binding agent-oligonucleotide conjugate of claim 35, comprising SEQ ID NO: 62 or 64 and SEQ ID NO:

71.

37. The conjugate of any one of claims 1 to 36, wherein the oligonucleotide is a single-stranded oligonucleotide.

38. 38. The conjugate of claim 37, wherein the oligonucleotide is an antisense oligonucleotide.

39. The conjugate of any one of claims 22 to 36, wherein the oligonucleotide is conjugated to the site of modification via the linking group.

40. A protein, Fc dimer, a first Fab known to specifically bind to TfR, and the protein comprising a S239C substitution (position according to EU numbering); and an oligonucleotide conjugated to said protein with a S239C substitution.

41. 41. A method of regulating expression of a target gene in muscle cells of a subject, the method comprising administering to the subject a TfR-binding agent-oligonucleotide conjugate of any one of claims 1 to 40.

42. 42. The method of claim 41, wherein the muscle cells are cardiac muscle cells or skeletal muscle cells.

43. 41. A method of delivering an oligonucleotide to the central nervous system (CNS) of a subject, comprising administering to the subject a TfR-binding agent-oligonucleotide conjugate according to any one of claims 1 to 40.

44. 41. A method of delivering an oligonucleotide to cancer in a subject, the method comprising administering to the subject a TfR-binding agent-oligonucleotide conjugate according to any one of claims 1 to 40.

45. 1. A TfR-binding agent-oligonucleotide conjugate comprising:

1. An antibody that specifically binds to TfR, comprising three heavy chain CDRs and three light chain CDRs, wherein: (a) CDR-H1 comprises SEQ ID NO: 12; CDR-H2 comprises SEQ ID NO: 13; CDR-H3 comprises SEQ ID NO: 14; CDR-L1 comprises SEQ ID NO: 15; CDR-L2 comprises SEQ ID NO: 16; CDR-L3 comprises SEQ ID NO: 17; (b) CDR-H1 comprises SEQ ID NO: 21; CDR-H2 comprises SEQ ID NO: 22; CDR-H3 comprises SEQ ID NO: 23; CDR-L1 comprises SEQ ID NO: 24; CDR-L2 comprises SEQ ID NO: 25; CDR-L3 comprises SEQ ID NO: 26; (c) CDR-H1 comprises SEQ ID NO: 114; CDR-H2 comprises SEQ ID NO: 115; CDR-H3 comprises SEQ ID NO: 116; CDR-L1 comprises SEQ ID NO: 117; CDR-L2 comprises SEQ ID NO: 118; CDR-L3 comprises SEQ ID NO: 119; (d) CDR-H1 comprises SEQ ID NO: 126; CDR-H2 comprises SEQ ID NO: 127; CDR-H3 comprises SEQ ID NO: 128; CDR-L1 comprises SEQ ID NO: 129; CDR-L2 comprises SEQ ID NO: 130; CDR-L3 comprises SEQ ID NO: 131; (e) CDR-H1 comprises SEQ ID NO: 134; CDR-H2 comprises SEQ ID NO: 135; CDR-H3 comprises SEQ ID NO: 136; CDR-L1 comprises SEQ ID NO: 137; CDR-L2 comprises SEQ ID NO: 138; CDR-L3 comprises SEQ ID NO: 139; (f) CDR-H1 comprises SEQ ID NO: 154; CDR-H2 comprises SEQ ID NO: 155; CDR-H3 comprises SEQ ID NO: 156; CDR-L1 comprises SEQ ID NO: 157; CDR-L2 comprises SEQ ID NO: 158; CDR-L3 comprises SEQ ID NO: 159; or (g) CDR-H1 comprises SEQ ID NO: 161; CDR-H2 comprises SEQ ID NO: 162; CDR-H3 comprises SEQ ID NO: 163; CDR-L1 comprises SEQ ID NO: 164; CDR-L2 comprises SEQ ID NO: 165; the antibody, wherein CDR-L3 comprises SEQ ID NO: 166; an oligonucleotide conjugated to a cysteine modification on the constant domain of the antibody.

46. 46. The TfR-binding agent-oligonucleotide conjugate of claim 45, wherein the cysteine modification is a S239C, S442C, A330C, K149C, or T289C substitution (positions according to EU numbering), or an A114C substitution (positions according to Kabat numbering).

47. 47. The TfR-binding agent-oligonucleotide conjugate of claim 46, wherein the cysteine modification is a S239C substitution.

48. The TfR-binding agent-oligonucleotide conjugate of any one of claims 45 to 47, wherein the oligonucleotide is a single-stranded oligonucleotide.

49. 49. The TfR-binding agent-oligonucleotide conjugate of claim 48, wherein the oligonucleotide is an antisense oligonucleotide.

50. 1. A TfR-binding agent-oligonucleotide conjugate comprising:

1. An antibody that specifically binds to TfR, comprising three heavy chain CDRs and three light chain CDRs, wherein: (a) CDR-H1 comprises SEQ ID NO: 12; CDR-H2 comprises SEQ ID NO: 13; CDR-H3 comprises SEQ ID NO: 14; CDR-L1 comprises SEQ ID NO: 15; CDR-L2 comprises SEQ ID NO: 16; CDR-L3 comprises SEQ ID NO: 17; (b) CDR-H1 comprises SEQ ID NO: 21; CDR-H2 comprises SEQ ID NO: 22; CDR-H3 comprises SEQ ID NO: 23; CDR-L1 comprises SEQ ID NO: 24; CDR-L2 comprises SEQ ID NO: 25; CDR-L3 comprises SEQ ID NO: 26; (c) CDR-H1 comprises SEQ ID NO: 114; CDR-H2 comprises SEQ ID NO: 115; CDR-H3 comprises SEQ ID NO: 116; CDR-L1 comprises SEQ ID NO: 117; CDR-L2 comprises SEQ ID NO: 118; CDR-L3 comprises SEQ ID NO: 119; (d) CDR-H1 comprises SEQ ID NO: 126; CDR-H2 comprises SEQ ID NO: 127; CDR-H3 comprises SEQ ID NO: 128; CDR-L1 comprises SEQ ID NO: 129; CDR-L2 comprises SEQ ID NO: 130; CDR-L3 comprises SEQ ID NO: 131; (e) CDR-H1 comprises SEQ ID NO: 134; CDR-H2 comprises SEQ ID NO: 135; CDR-H3 comprises SEQ ID NO: 136; CDR-L1 comprises SEQ ID NO: 137; CDR-L2 comprises SEQ ID NO: 138; CDR-L3 comprises SEQ ID NO: 139; (f) CDR-H1 comprises SEQ ID NO: 154; CDR-H2 comprises SEQ ID NO: 155; CDR-H3 comprises SEQ ID NO: 156; CDR-L1 comprises SEQ ID NO: 157; CDR-L2 comprises SEQ ID NO: 158; CDR-L3 comprises SEQ ID NO: 159; or (g) CDR-H1 comprises SEQ ID NO: 161; CDR-H2 comprises SEQ ID NO: 162; CDR-H3 comprises SEQ ID NO: 163; CDR-L1 comprises SEQ ID NO: 164; CDR-L2 comprises SEQ ID NO: 165; the antibody, wherein CDR-L3 comprises SEQ ID NO: 166; an oligonucleotide conjugated via a linker to the S239C substitution (position according to EU numbering) on the constant domain of the antibody.

51. 51. A method of modulating expression of a target gene in muscle cells of a subject, the method comprising administering to the subject a TfR binding agent-oligonucleotide conjugate according to any one of claims 45 to 50.

52. 52. The method of claim 51, wherein the muscle cells are cardiac muscle cells or skeletal muscle cells.

53. 51. A method of delivering an oligonucleotide to the CNS of a subject, comprising administering to said subject a TfR binding agent-oligonucleotide conjugate according to any one of claims 45 to 50.

54. 51. A method of delivering an oligonucleotide to cancer cells in a subject, the method comprising administering to the subject a TfR-binding agent-oligonucleotide conjugate according to any one of claims 45 to 50.

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