Transferrin receptor binding molecule conjugates for delivery of oligonucleotides to cells

Abstract

Provided herein are conjugates comprising: an anti-TfR antibody antigen binding domain that binds to a transferrin receptor, and an oligonucleotide. The conjugates can be used to deliver oligonucleotides to cells expressing the transferrin receptor. Delivery of the oligonucleotides to a cell can be used to modulate expression of a target gene or sequence in the cell.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 357,959, filed on July 1, 2022, which is incorporated herein by reference.

[0003] Sequence Listing

[0004] The sequence listing written in the file DNL-041-01-WO_SeqListing.txt is 177 kilobytes in size, created on June 30, 2023, and is hereby incorporated by reference. Technical Field

[0005] The subject matter disclosed herein relates to TfR binder-oligonucleotide conjugates that bind to transferrin receptors on target cells and modulate expression of a target gene or sequence in the cell, and methods of using the same. Background Art

[0006] In vivo delivery of nucleic acid-based molecules such as antisense oligonucleotides or RNAi agents generally requires specific targeting to reach certain tissues or cell types. In particular, delivery to non-liver tissues remains an obstacle and limits the use of such therapies. Delivery of oligonucleotides to the central nervous system (CNS) causes significant problems due to the blood-brain barrier (BBB). A means of delivering oligonucleotides to CNS is intrathecal delivery. However, intrathecal delivery is invasive, has a higher risk of side effects, and often results in uneven distribution.

[0007] Transferrin receptor can be targeted for delivery of cancer diagnostics and therapeutics. 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.

[0008] What is needed is a therapeutic modality that can target the transferrin receptor to deliver cargo to cells via the transferrin receptor. Summary of the invention

[0009] TfR binder-oligonucleotide conjugates for delivering oligonucleotides to CNS or cells expressing transferrin receptor (TfR) are described, the TfR binder-oligonucleotide conjugates comprising: an oligonucleotide connected 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, Fab, F(ab′)2, a single-chain Fab, (scFab), an Fv fragment, a single-chain variable fragment (scFv), a bivalent scFv, a heavy chain antibody variable domain only (nanobody, such as VHH or vNAR) or a nanobody. In some embodiments, the anti-TfR antibody antigen binding domain comprises or consists of scFv. In some embodiments, the anti-TfR antibody antigen binding domain comprises or consists of Fab. In some embodiments, the anti-TfR antibody antigen binding domain comprises or consists of scFab. The anti-TfR antibody antigen binding domain may be from any known antibody that specifically binds to TfR. In some embodiments, the anti-TfR antibody antigen binding domain comprises a VH domain and a VL domain, the VH domain comprising a CDR having a sequence of SEQ ID NO: 12-14, and the VL domain comprising a CDR having a sequence of SEQ ID NO: 15-17. In some embodiments, the anti-TfR antibody antigen binding domain comprises a VH domain and a VL domain, the VH domain comprising a CDR having a sequence of SEQ ID NO: 21-23, and the VL domain comprising a CDR having a sequence of SEQ ID NO: 24-26. In some embodiments, the anti-TfR antibody antigen binding domain comprises a VH domain and a VL domain, the VH domain comprising a CDR having a sequence of SEQ ID NO: 114-116, and the VL domain comprising a CDR having a sequence of SEQ ID NO: 117-119. In some embodiments, the anti-TfR antibody antigen binding domain comprises a VH domain and a VL domain, the VH domain comprising a CDR having a sequence of SEQ ID NO: 126-128, and the VL domain comprising a CDR having a sequence of SEQ ID NO: 129-131. In some embodiments, the anti-TfR antibody antigen binding domain comprises a VH domain and a VL domain, the VH domain comprising a CDR having a sequence of SEQ ID NO: 134-136, and the VL domain comprising a CDR having a sequence of SEQ ID NO: 137-139. In some embodiments, the anti-TfR antibody antigen binding domain comprises a VH domain and a VL domain, the VH domain comprising a CDR having a sequence of SEQ ID NO: 154-156, and the VL domain comprising a CDR having a sequence of SEQ ID NO: 157-159.In some embodiments, the anti-TfR antibody antigen binding domain comprises a VH domain and a VL domain, the VH domain comprising a CDR having a sequence of SEQ ID NO: 161-163, and the VL domain comprising a CDR having a sequence of SEQ ID NO: 164-166. In some embodiments, the anti-TfR antibody antigen binding domain specifically binds to human TfR. In some embodiments, the TfR binding region binds to the apical domain of TfR. The oligonucleotide may be directly or indirectly connected to the anti-TfR antibody antigen binding domain. For indirect attachment, the oligonucleotide may be connected to the anti-TfR antibody antigen binding domain via a chemical linker and / or a peptide. The peptide may be, but is not limited to, an Fc polypeptide and an Fc dimer or albumin. The oligonucleotide may be, but is not limited to, an antisense oligonucleotide (ASO) or an RNA interference oligonucleotide. The anti-TfR antibody antigen binding domain may have a substitution or modification that promotes oligonucleotide conjugation. The peptide (eg, Fc polypeptide, Fc dimer or albumin), if present, may have substitutions or modifications that facilitate conjugation to the oligonucleotide.

[0010] In some embodiments, the TfR binder-oligonucleotide conjugate comprises an oligonucleotide connected to an anti-TfR Fab or scFab. In some embodiments, the TfR binder-oligonucleotide conjugate comprises an oligonucleotide connected to a monomeric anti-TfR Fab or scFab (single Fab). Single Fab indicates that the TfR binder-oligonucleotide conjugate comprises a single Fab or scFab (i.e., the TfR binder-oligonucleotide conjugate does not contain a second antibody antigen binding domain). In some embodiments, the single Fab is connected to an Fc polypeptide or an Fc dimer. In some embodiments, the single Fab is connected to an Fc polypeptide or an Fc dimer, wherein the oligonucleotide is connected to an Fc polypeptide, an Fc dimer. Fc polypeptide, Fc dimer, Fab or scFab may have a substitution or modification that promotes oligonucleotide conjugation.

[0011] In some embodiments, the TfR binder-oligonucleotide conjugate comprises or consists of:

[0012]

[0013] in,

[0014] P contains the anti-TfR antibody antigen binding domain,

[0015] F is optionally present or absent, and if present, comprises a peptide, an Fc polypeptide, an Fc dimer, or albumin;

[0016] L is optionally present or absent, and if present, is a linking group;

[0017] 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 to transferrin;

[0018] O is an oligonucleotide;

[0019] y is an integer greater than or equal to 1 (e.g., 1, 2, 3, or 4); and

[0020] n is an integer greater than or equal to 1 (eg, 1, 2, 3, 4, 5, 6, 7, or 8).

[0021] PFP' may be referred to as a TfR binder. In some embodiments, PFP' comprises an anti-TfR antibody. In some embodiments, PF comprises a monovalent anti-TfR antibody. If P' is present and F comprises an Fc dimer, then P or the heavy chain component of P may form a single polypeptide chain with one Fc polypeptide of the Fc dimer, and P' or the heavy chain component of P' may form a single polypeptide chain with the other Fc polypeptide of the Fc dimer. If n is greater than or equal to 2, then for each (L-(O) y ), y is independently greater than or equal to 1 (eg, 1, 2, 3, or 4). In some embodiments, the oligonucleotide comprises an ASO.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] In some embodiments, the TfR binder-oligonucleotide conjugate comprises:

[0028] Protein, which contains:

[0029] Antibody Fc constant domain dimer,

[0030] a first Fab that specifically binds to transferrin receptor (TfR), and

[0031] Covalent conjugation modifications; and,

[0032] Oligonucleotides conjugated at the modification site.

[0033] 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 and a VL domain, the VH domain comprises a CDR having a sequence of SEQ ID NO: 12-14, and the VL domain comprises a CDR having a sequence of SEQ ID NO: 15-17. In some embodiments, the first Fab comprises a VH domain and a VL domain, the VH domain comprises a CDR having a sequence of SEQ ID NO: 21-23, and the VL domain comprises a CDR having a sequence of SEQID NO: 24-26. In some embodiments, the first Fab comprises a VH domain and a VL domain, the VH domain comprises a CDR having a sequence of SEQ ID NO: 114-116, and the VL domain comprises a CDR having a sequence of SEQ ID NO: 117-119. In some embodiments, the first Fab comprises a VH domain and a VL domain, the VH domain comprising a CDR having a sequence of SEQ ID NOs: 126-128, and the VL domain comprising a CDR having a sequence of SEQ ID NOs: 129-131. In some embodiments, the first Fab comprises a VH domain and a VL domain, the VH domain comprising a CDR having a sequence of SEQ ID NOs: 134-139, and the VL domain comprising a CDR having a sequence of SEQ ID NOs: 137-139. In some embodiments, the first Fab comprises a VH domain and a VL domain, the VH domain comprising a CDR having a sequence of SEQ ID NOs: 154-156, and the VL domain comprising a CDR having a sequence of SEQ ID NOs: 157-159. In some embodiments, the first Fab comprises a VH domain and a VL domain, the VH domain comprising a CDR having a sequence of SEQ ID NO: 161-163, and the VL domain comprising a CDR having a sequence of SEQ ID NO: 164-166. The first Fab may be connected to a first Fc polypeptide or a second Fc polypeptide to form a Fab-Fc fusion. In some embodiments, the TfR binder-oligonucleotide conjugate further comprises a second Fab. The second Fab may be, but is not limited to, a Fab, a non-binding Fab, or a non-binding variable region (NBVR) that specifically binds to TfR. The second Fab may be connected to a first Fc polypeptide or a second Fc polypeptide to form a Fab-Fc. In some embodiments, the TfR binder-oligonucleotide conjugate comprises a second Fab, wherein the first Fab is connected to a first Fc polypeptide and the second Fab is connected to a second Fc polypeptide. In some embodiments, the TfR binder-oligonucleotide conjugate comprises a second Fab, wherein the first Fab is connected to a second Fc polypeptide and the second Fab is connected to a first Fc polypeptide.In some embodiments, the oligonucleotide is conjugated to the antibody via a linker "L".

[0034] In certain embodiments, the TfR binder-oligonucleotide conjugate comprises an antibody-oligonucleotide conjugate comprising:

[0035] An antibody that binds to transferrin receptor (TfR), wherein the antibody comprises a heavy chain CDR of SEQ ID NO: 12-14, 21-23, 114-116, 126-128, 134-136, 154-156, or 161-163 and a light chain CDR of SEQ ID NO: 15-17, 24-26, 117-119, 129-131, 137-139, 157-159, or 164-166; and

[0036] Oligonucleotides conjugated to cysteine ​​modifications on the constant domains of this antibody.

[0037] In some embodiments, the oligonucleotide is conjugated to the antibody via a linker "L".

[0038] In certain embodiments, the TfR binder-oligonucleotide conjugate comprises an antibody-oligonucleotide conjugate comprising:

[0039] An antibody that binds to transferrin receptor (TfR), wherein the antibody comprises a heavy chain CDR of SEQ ID NO: 12-14, 21-23, 114-116, 126-128, 134-136, 154-156, or 161-163 and a light chain CDR of SEQ ID NO: 15-17, 24-26, 117-119, 129-131, 137-139, 157-159, or 164-166; and

[0040] Oligonucleotides conjugated to cysteine ​​modifications on the constant domains of this antibody.

[0041] In certain embodiments, the TfR binder-oligonucleotide conjugate described herein has the following structure:

[0042]

[0043] In certain embodiments, the subject matter described herein relates to a method of modulating expression of a target gene in a myocyte or CNS cell of a patient, the method comprising administering to the patient a conjugate as described herein or a pharmaceutical composition comprising the conjugate.

[0044] These and other embodiments are fully described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 Depicted are huIgG, intact drug, % intact drug, and total ASO in the CNS, i.e., cortex, spinal cord, 24 hours after a single dose for TfR-MonoFab conjugate.

[0047] Figure 2A Demonstrated are huIgG and intact drug in the CNS, i.e., cortex, spinal cord, 72 hours after the last dose for TfR-MonoFab conjugate in the multiple dose study.

[0048] Figure 2B Total ASO and Malat1 knockdown in the CNS, ie, cortex, spinal cord, 72 hours after the last dose in the multiple dose study for the TfR mono-Fab conjugate is demonstrated.

[0049] Figure 3 Depicted are huIgG, intact drug, % intact drug, and total ASO in the periphery 24 hours after a single dose for the TfR mono-Fab conjugate.

[0050] Figure 4 Depicted are huIgG, intact drug, and total ASO 72 hours after the last dose in the multiple dose study for the TfR mono-Fab conjugate.

[0051] Figure 5 Malat1 72 hours after the last dose in the multiple dose study for the TfR mono-Fab conjugate is illustrated.

[0052] Figure 6 Malat1 knockdown in the CNS and periphery is demonstrated for anti-TfR bivalent antibody conjugates.

[0053] Figure 7 Plasma clearance of TfR-monoFab:ASO conjugates is illustrated.

[0054] Figure 8 The huIgG concentrations of TfR-monoFab:ASO conjugates in brain, spinal cord and peripheral tissues are demonstrated.

[0055] Fig. 9 ASO concentrations in brain, spinal cord, and peripheral tissues are illustrated for TfR-Single Fab:ASO conjugates. Unconjugated ASO is the first bar for each tissue (not visible for brain and SC). TfR-Single Fab is the middle bar for each tissue. TfR-Single Fab 2 is the third bar for each tissue.

[0056] Fig.10 ASO concentrations in the brain at 72 hours are illustrated for Tfr albumin:ASO conjugates.

[0057] Fig.11ASO concentrations in kidney and liver at 72 hours are illustrated for Tfr albumin:ASO conjugates.

[0058] Fig.12 Plasma clearance of Tfr albumin:ASO conjugates is illustrated.

[0059] Fig.13 Exemplary TfR binder-oligonucleotide conjugates with: (a) anti-TfR Fab / non-binding Fab antibody (top left), (b) anti-TfR single Fab antibody (top right), and (c) anti-TfR scFv-albumin (bottom) are illustrated. (a), (b), and (c) are shown with ASO attached. (c) is shown with an optional 6×His tag. DETAILED DESCRIPTION

[0060] I. Introduction

[0061] Oligonucleotide therapy for disorders caused by genetic abnormalities or increased protein accumulation is becoming an increasingly popular approach to modulate gene expression to treat the disorder. Delivery of oligonucleotides to cells remains a challenge. Disclosed herein are TfR binder-oligonucleotide conjugates that utilize transferrin receptor to deliver oligonucleotides to target cells.

[0062] In certain embodiments, the TfR binding agent-oligonucleotide conjugate can cross the blood-brain barrier (BBB). Generally, the BBB represents the challenge of delivering systemically administered oligonucleotides to relevant action sites in the CNS. Intrathecal (IT) delivery (wherein the drug is directly administered into the cerebrospinal fluid (CSF) space) can bypass the BBB. However, a limitation of the method is that these oligonucleotide therapies are directly delivered to the CSF via the IT method and cannot achieve effective distribution in the CNS.

[0063] In certain embodiments, the TfR binder-oligonucleotide conjugate is capable of delivering the conjugated oligonucleotide to the CNS or to a cell expressing the transferrin receptor. The cell may be, but is not limited to, a muscle cell or a cancer cell. The muscle cell may be, but is not limited to, a skeletal muscle cell or a cardiomyocyte.

[0064] TfR binders-oligonucleotide conjugates and methods of using them are described herein. In some embodiments, the TfR binder comprises a monovalent antibody (single Fab; that is, an antibody with a single anti-TfR antibody antigen binding domain, for example, a single Fab arm or scFv). In some embodiments, the TfR binder comprises a bispecific bivalent antibody (i.e., an antibody with a single anti-TfR antibody antigen binding domain and a single non-binding Fab or NBVR). In some embodiments, the TfR binder comprises an anti-TfR scFab, svFc, VHH, vNAR or nanobody connected to albumin. In some embodiments, the TfR binder comprises a bivalent anti-TfR antibody (e.g., anti-TfR (Fab) 2). In some embodiments, the TfR binder-oligonucleotide conjugate comprises an oligonucleotide covalently linked to an anti-TfR antibody.

[0065] In certain embodiments, the oligonucleotide is conjugated at a cysteine ​​modification on the constant domain of an antibody, Fc polypeptide, or Fc dimer.

[0066] Anti-TfR antibody antigen binding domains used to form TfR binders can be derived from antibodies known to have affinity for transferrin receptor. Derived from an antigen binding region indicating that the anti-TfR antibody antigen binding domain comprises an antibody and an antigen binding fragment of an antibody, or a CDR sequence 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.

[0067] The oligonucleotide may also be referred to as a cargo delivered to the target cell by the TfR binder-oligonucleotide conjugate. The oligonucleotide may be, but is not limited to, an antisense oligonucleotide ("ASO") or a RNAi agent (eg, siRNA or shRNA).

[0068] In further embodiments, provided herein are methods of treatment and methods of targeting an oligonucleotide (eg, an ASO or RNAi agent) to a transferrin receptor expressing cell, eg, delivering the oligonucleotide to the cell, using the conjugates as described herein.

[0069] II. Definitions

[0070] As used herein, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise.

[0071] As used herein, the terms "about" and "approximately," when used to modify an amount specified in a value or range, represent the value and reasonable deviations from the value known to those skilled in the art, e.g., ±20%, ±10%, or ±5%, within the intended meaning of the value.

[0072] The term "non-targeting Fab fragment" refers to a Fab fragment that does not specifically bind to an antigen via its heavy or light chain variable domain, or does not specifically bind to an antigen expressed in a given mammal, such as a primate, e.g., a human and non-human primate, or a rodent, e.g., a mouse, via its heavy or light chain variable domain.

[0073] "Transferrin receptor" or "TfR" refers to transferrin receptor protein 1. The human transferrin receptor 1 polypeptide sequence is shown 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; cattle, 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. The 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 contains residues 189-383 of human TfR. The sequence of the apical domain of human transferrin receptor 1 is shown in SEQ ID NO:3.

[0074] The term "constant region" refers to the light chain constant region domain polypeptide (CL) and the CH1, CH2 and CH3 domain polypeptides from the heavy chain.

[0075] The terms "CH1 domain", "CH3 domain" and "CH2 domain" refer to immunoglobulin constant region domain polypeptides. In the context of IgG antibodies, a CH3 domain polypeptide refers to an amino acid fragment from about position 341 to about position 447 numbered according to the EU numbering scheme, a CH2 domain polypeptide refers to an amino acid fragment from about position 231 to about position 340 numbered according to the EU numbering scheme, and a CH1 domain polypeptide refers to an amino acid fragment from about position 118 to about position 215 numbered according to the EU numbering scheme. CH1, CH2 and CH3 domain polypeptides may also be numbered by the IMGT (ImMunoGeneTics) numbering scheme, wherein 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, an Fc polypeptide refers to the amino acid stretch from about position 231 to about position 447 as numbered according to the EU numbering scheme.

[0076] The term "variable region" refers to the light chain variable region domain polypeptide (VL) and the heavy chain variable region domain polypeptide (VH). VL contains three complementary determining regions (CDR) regions, namely CDR-L1, CDR-L2, CDR-L3, and VH contains three complementary determining regions, namely CDR-H1, CDR-H2 and CDR-H3. The CDR regions together form the antibody binding site that binds to the antigen.

[0077] 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 domain. An Fc polypeptide typically contains a constant region sequence that includes at least a CH2 domain and / or a CH3 domain, and may contain at least a portion of a hinge region. Illustrative hinge region sequences or portions thereof are shown in SEQ ID NOs: 4-6.

[0078] "Fc polypeptide dimer" refers to a dimer of two Fc polypeptides. In some embodiments, the Fc polypeptide dimer is capable of binding to an Fc receptor (e.g., FcγR). In the Fc polypeptide dimer, the two Fc polypeptides dimerize through the interaction between the two CH3 antibody constant domains. In some embodiments, the two Fc polypeptides may also dimerize via one or more disulfide bonds formed between the hinge domains of the two dimerized Fc domain monomers. The Fc polypeptide dimer may be a heterodimer or a homodimer. The Fc polypeptide dimer may comprise two wild-type Fc polypeptides, a wild-type Fc polypeptide and a modified Fc polypeptide, or two modified Fc polypeptides. For an Fc polypeptide dimer comprising two modified Fc polypeptides, the two modified Fc polypeptides may be the same or different.

[0079] The antibody antigen binding domain comprises an antigen binding domain of an immunoglobulin or a peptide having a structure similar to an antigen binding domain of an immunoglobulin. An immunoglobulin may be, but is not limited to, IgG, IgM, IgE, IgA, IgD or a heavy chain antibody. The antibody antigen binding domain may be, but is not limited to, a Fab, a scFab, a Fv fragment, a scFv or only a heavy chain antibody variable domain (nanoantibody, such as VHH or vNAR).

[0080] The term "CL domain" refers to the immunoglobulin constant domain of the light chain. In the context of an IgG antibody, a kappa CL domain polypeptide refers to the amino acid segment from about position 108 to about position 214 as numbered according to the EU numbering scheme. Alternatively, the kappa and lambda CL domains may be numbered by the IMGT (ImMunoGeneTics) numbering scheme, wherein the kappa CL domain is numbered 1-107 and the lambda CL domain is numbered 1-106 according to the IMGT scientific chart numbering (IMGT website).

[0081] The term "Fab" or "Fab fragment" refers to a monovalent fragment consisting of the VL, VH, CL and CH1 domains. L ) and the light chain constant region (CL) (together the antibody light chain) and the heavy chain variable region (V H ) and the heavy chain CH1 constant region (together the antibody Fd fragment). Fab or Fab fragments may or may not contain all or part of the antibody hinge region.

[0082] The term "single-chain Fab" or "scFab" refers to an antigen-binding fragment consisting of Fab in which the Fd fragment and the light chain are linked together via a peptide linker. The linker can connect the N-terminus of the Fd fragment to the C-terminus of the light chain, or connect the N-terminus of the light chain to the C-terminus of the Fd fragment.

[0083] The term "Fv fragment" refers to a fragment of V H and V L The antigen-binding fragments.

[0084] 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 connected together via a peptide linker. H The N-terminus of L C-terminus of the L The N-terminus of H The C-terminus of the scFv is connected. The scFv lacks a constant region. Modified scFvs and methods of modifying scFvs to bind to target proteins are described in WO2022 / 258841 (which is incorporated herein by reference).

[0085] The term "nanobody" refers to an antibody fragment consisting of a single monomeric variable antibody domain. Nanobodies derived from camel 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 of modifying VHH fragments to bind to target proteins (including TfR) are described in WO 2020 / 056327, WO 2022 / 103769, and WO 2023 / 023166 (each of which is incorporated herein by reference).

[0086] The term "non-targeted Fab fragment" or "NTF" refers to a Fab fragment that does not specifically bind to a naturally occurring human antigen via its heavy or light chain variable domain or does not specifically bind to a naturally occurring antigen expressed in a given mammal or in a specific tissue within such a mammal via its heavy or light chain variable domain, such as a primate, such as a human and a non-human primate, or a rodent, such as a mouse. In certain embodiments, the Fab used in the Fab-Fc fusion or Fab-Fc dimer fusion described herein does not specifically bind to transferrin via its heavy or light chain variable domain. Non-limiting examples of non-targeted Fab fragments include (a) RSV (palivizumab) Fab fragments, which are non-targeted in mice and non-human primates, and (b) Fab fragments for dinitrophenyl hapten (DNP) (see Leahy, PNAS 3661-3665, 1988).

[0087] The terms "wild type," "native," and "naturally occurring" with respect to a CH3 or CH2 domain refer to the domain having a sequence that occurs in nature.

[0088] As used herein, the term "mutant" with respect to a mutant polypeptide or mutant polynucleotide can be used interchangeably with "variant". Variants with respect to a given wild-type CH3 or CH2 domain reference sequence may include naturally occurring allelic variants. A "non-natural" CH3 or CH2 domain refers to a variant or mutant domain that does not exist in a natural cell, which is produced by genetic modification of a natural CH3 domain or CH2 domain polynucleotide or polypeptide, such as using genetic engineering techniques or mutagenesis techniques. "Variant" includes any domain comprising at least one amino acid mutation relative to the wild type. Mutations may include substitutions, insertions, and deletions.

[0089] 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. The modification site may include, for example, an insertion or substitution. The term "substitution" refers to a change in which one amino acid is replaced with another amino acid. For example, "cysteine ​​substitution" or "cysteine ​​modification" refers to the replacement of an amino acid with cysteine. The modification can be represented by the symbol XnumberY, where X represents the amino acid at the position represented by the number in the parent polypeptide, and Y represents the substituted amino acid that replaces the amino acid X. For example, S239C means that the serine at position 239 is replaced by cysteine.

[0090]

[0063] The term "amino acid" refers to naturally occurring and 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.

[0091] Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. "Amino acid analogs" refers to compounds that have the same basic chemical structure as naturally occurring amino acids, i.e., an alpha carbon bound to a hydrogen, a carboxyl group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. "Amino acid mimetics" refers to chemical compounds that have a general chemical structure that is different from that of an amino acid, but function in a manner similar to the naturally occurring amino acids.

[0092] 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.

[0093] 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.

[0094] The terms "polypeptide" and "peptide" are used interchangeably to refer to a polymer of amino acid residues in a single chain. These terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of the corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. Amino acid polymers may comprise entirely L-amino acids, entirely D-amino acids, or a mixture of L and D amino acids.

[0095] The term "protein" refers to a single-chain polypeptide or a dimer (ie, two) or multimer (ie, three or more) of a single-chain polypeptide. The dimer or multimer single-chain polypeptides may be linked by covalent bonds such as disulfide bonds or non-covalent interactions.

[0096] The term "conservative substitution", "conservative mutation" or "conservatively modified variant" refers to a change that results in the substitution of an amino acid by another amino acid that can be classified as having similar characteristics. Examples of categories of conservative amino acid groups defined in this manner may include: "charged / polar groups" 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); "aromatic groups" including Phe (phenylalanine or F), Tyr (tyrosine or Y), Trp (tryptophan or W) and (histidine or H); and "aliphatic groups" 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 may be subdivided into subgroups including: a "positively charged subgroup" comprising Lys, Arg, and His; a "negatively charged subgroup" comprising Glu and Asp; and a "polar subgroup" comprising Asn and Gln. In another example, aromatic or cyclic groups may be subdivided into subgroups including: a "nitrogen ring subgroup" comprising Pro, His, and Trp; and a "phenyl subgroup" comprising Phe and Tyr. In another further example, aliphatic groups may be subdivided into subgroups, for example, an "aliphatic nonpolar subgroup" comprising Val, Leu, Gly, and Ala; and an "aliphatic weakly polar subgroup" comprising Met, Ser, Thr, and Cys. Examples of conservative mutation categories include amino acid substitutions of amino acids in the above subgroups, such as, but not limited to: Lys for Arg or vice versa, so that a positive charge can be maintained; Glu for Asp or vice versa, so that a negative charge can be maintained; Ser for Thr or vice versa, so that a free -OH can be maintained; and Gln for Asn or vice versa, so that a free -NH2 can be maintained. In some embodiments, a hydrophobic amino acid replaces a naturally occurring hydrophobic amino acid, such as in an active site, to maintain hydrophobicity.

[0097] In the context of two or more polypeptide sequences, the terms "identical" or "percent identity" means that two or more sequences or subsequences are identical or have a specified percentage of amino acid residues, for example, 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 greater, when compared and aligned for maximum correspondence over a comparison window or designated region when measured using a sequence comparison algorithm or by manual alignment and visual inspection.

[0098] In order to compare the sequence of polypeptide, usually an amino acid sequence serves as a reference sequence and is compared with the candidate sequence. The various methods available to those skilled in the art can be used, for example, visual comparison or use publicly available software to perform comparison using a known algorithm for realizing maximum comparison. Such programs include BLAST programs, ALIGN, ALIGN-2 (Genentech, South San Francisco, Calif.) or Megalign (DNASTAR). The parameters used for comparison to realize maximum comparison can be determined by those skilled in the art. For the purpose of this application, in order to compare the sequence of polypeptide sequences, the BLASTP algorithm standard protein BLAST is used to compare two protein sequences with default parameters.

[0099] The terms "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 residue position in a specified reference sequence when the given amino acid sequence is maximally aligned and compared to the reference sequence. Thus, for example, when optimally aligned with SEQ ID NO: 1, amino acid residues in a polypeptide "correspond to" amino acids in the region from amino acids 114-220 in SEQ ID NO: 1 when the amino acid residues in SEQ ID NO: 1 are aligned. A polypeptide aligned to a reference sequence need not be the same length as the reference sequence.

[0100] "Binding affinity" refers to the strength of a non-covalent interaction between two molecules, e.g., a single binding site on a polypeptide / protein and its bound target, e.g., transferrin receptor. Thus, for example, the term may refer to a 1:1 interaction between a polypeptide / protein and its target, unless otherwise stated or clear from the context. Binding affinity can be measured by measuring the equilibrium dissociation constant (K D ) is used to quantify the equilibrium dissociation constant, which refers to the dissociation rate constant (k d ,time -1 ) divided by the association rate constant (k a ,time -1 M -1 ). D This can be determined by measuring the kinetics of complex formation and dissociation, for example using surface plasmon resonance (SPR) methods such as Biacore TM system; kinetic exclusion assays such as and BioLayer interferometry (e.g., using As used herein, "binding affinity" includes not only formal binding affinities, such as those reflecting a 1:1 interaction between a polypeptide / protein and its target, but also calculated K DThe apparent affinity of the binding may be reflected.

[0101] The phrase "specific binding" or "selective binding" target, such as transferrin receptor, refers to such a binding reaction in which a protein binds to a target with greater affinity, greater avidity and / or longer duration than a target that is structurally different from its binding structure (e.g., a target that is not in the transferrin receptor family). In typical embodiments, when measured under the same affinity assay conditions, the protein has at least 5 times, 10 times, 100 times, 1000 times, 10,000 times or higher affinity to transferrin receptor compared to an unrelated target. In certain embodiments, the protein can specifically bind to human transferrin receptor.

[0102] The terms "nucleic acid" and "polynucleotide" refer to deoxyribonucleotides or ribonucleotides and polymers thereof in single-stranded or double-stranded form, which are composed of monomers (nucleotides) containing a sugar portion, a phosphate ester and a nucleobase. Unless otherwise specified, the term encompasses both modified and unmodified nucleic acids.

[0103] The term "nucleobase" refers to a nitrogenous compound that can be linked to a sugar moiety to form a nucleoside, which in turn is a component of a nucleotide. The ability of a nucleobase to form base pairs and to 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.

[0104] The term "nucleoside" refers to a compound comprising a nucleobase and a sugar moiety (eg, deoxyribose or ribose, or modified versions thereof). The term nucleoside includes modified and unmodified nucleosides.

[0105] The term "nucleotide" refers to a compound comprising a nucleobase, a sugar moiety and one or more phosphate groups. The term nucleotide includes modified and unmodified nucleotides.

[0106] The term "internucleoside linkage" refers to a covalent bond between two nucleosides in an oligonucleotide. Nucleosides can be linked via a natural (ie, phosphodiester (PO) bond) or a modified bond.

[0107] The terms "chemical modification", "modification" or "modified" may refer to a chemical change in a compound compared to its naturally occurring counterpart. For example, a nucleobase, a sugar moiety or an internucleoside bond may be chemically modified. An amino acid in a protein or polypeptide may be modified. The modification may be a modification of an existing amino acid or a substitution of one amino acid by another. Examples of modifications of one amino acid to another include, but are not limited to, cysteine ​​modifications, where a naturally occurring amino acid at one position is substituted with cysteine ​​(i.e., cysteine ​​modifications).

[0108] The terms "nucleotide sequence" and "nucleic acid sequence" and "nucleic acid chain" refer to a sequence of bases (purines and / or pyrimidines or synthetic derivatives thereof) in a polymer of DNA or RNA, which may be single-stranded or double-stranded, optionally containing synthetic, non-natural or altered nucleotides capable of incorporation into a DNA or RNA polymer, and / or backbone modifications (e.g., modified oligomers). The terms "oligomer," "oligonucleotide," and "oligomer" are used interchangeably and refer to such sequences of purines and / or pyrimidines. For example, an oligonucleotide may comprise a chemically modified or unmodified nucleic acid molecule (RNA or DNA) less than about, for example, about 200 nucleotides (e.g., less than about 100 or 50 nucleotides) in length. An oligonucleotide may, for example, be a single-stranded DNA or RNA (e.g., ASO); a double-stranded DNA or RNA (e.g., a small interfering RNA (siRNA)), including a double-stranded DNA or RNA with a hairpin loop; or a DNA / RNA hybrid. In one embodiment, an oligonucleotide has a length in the range of about 5 to about 60 nucleotides, or about 10 to about 50 nucleotides. In another embodiment, the oligonucleotide has a length in the range of about 5 to about 30 nucleotides or about 15 to about 30 nucleotides. In another embodiment, the oligonucleotide has a length in the range of about 18 to about 24 nucleotides.

[0109] The terms "modified oligomer," "modified oligonucleotide," or "modified oligomer" are similarly used interchangeably and refer to such sequences containing synthetic, non-natural or altered bases, sugars and / or backbone modifications.

[0110] Oligonucleotides as described herein can be synthesized using standard solid phase or liquid phase synthesis techniques known in the art. In certain embodiments, oligonucleotides are synthesized using solid phase phosphoramidite chemistry (U.S. Patent number 6,773,885) using an automatic synthesizer. Chemical synthesis of nucleic acids allows the production of various forms of nucleic acids with modified bonds, chimeric compositions, and non-standard bases or modified groups attached at selected positions across the full length of nucleic acids.

[0111] As used herein, the term "complementary" refers to the broad concept of complementary base pairing between two nucleic acids, which are arranged in antisense positions relative to each other. When the nucleotide positions in the two molecules are occupied by nucleotides that are usually capable of base pairing with each other, it is considered that the nucleic acids are complementary to each other at this position. Therefore, when at least about 50%, at least about 60% or at least about 80% of the corresponding positions in each molecule are occupied by nucleotides that are usually base paired with each other (for example, A:T (A:U of RNA) and G:C nucleotide pairs), the two nucleic acids are substantially complementary to each other.

[0112] In the context of two or more nucleotide sequences, the terms "identical" or percent "identity" refer to two or more sequences or subsequences that are identical or have a specified percentage of nucleotides, for example, 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 greater, when compared and aligned for maximum correspondence over a comparison window or specified region when measured using a sequence comparison algorithm or by manual alignment and visual inspection.

[0113] For sequence comparison of oligonucleotides (e.g., to determine identity or complementarity), typically a nucleotide sequence serves as a reference sequence to be compared with the candidate sequence. Various methods available to those skilled in the art can be used, for example, visual comparison or using publicly available software using known algorithms for achieving maximum alignment to perform the comparison. Such programs include BLAST programs, ALIGN, ALIGN-2 (Genentech, South San Francisco, Calif.) or Megalign (DNASTAR). Parameters for comparison to achieve maximum alignment can be determined by those skilled in the art.

[0114] As used herein, "hybridize" or "hybridization" means the pairing of complementary nucleotide sequences (e.g., an antisense compound and its target nucleic acid; or between an antisense strand and a sense strand). As used herein, "specific hybridization" refers to the ability of a reference nucleic acid to hybridize to one nucleic acid molecule with an affinity greater than the affinity for hybridization to another nucleic acid molecule.

[0115] "Expression" refers to the transcription and / or translation of an endogenous gene, a heterologous gene, or a nucleic acid fragment 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.

[0116] The term "gene" refers to a nucleic acid (eg, DNA or RNA) sequence that contains coding sequences necessary for the production of a polypeptide or precursor.

[0117] The phrase "modulates the expression of a target gene or sequence" means a change (e.g., an increase or decrease) in the expression of a target gene or sequence (e.g., via degradation or translational inhibition of the target). For example, it includes inhibiting, reducing or decreasing the expression of a target gene or sequence. This also includes changes in alternative splicing, which can result in changes in the absolute or relative amount of a particular splicing variant.

[0118] The term "halo" is fluoro, chloro, bromo or iodo. Alkyl, alkoxy, etc. represent both straight and branched chain groups; however, reference to individual groups such as propyl includes only the straight chain group, and branched chain isomers such as isopropyl are specifically mentioned.

[0119] Unless otherwise indicated, the term "alkyl" by itself or as part of another substituent means a straight or branched chain hydrocarbon radical (i.e., C 1-6 means one to six carbons). Examples include (C1-C6)alkyl, (C2-C6)alkyl, and (C3-C6)alkyl. Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, and higher homologs and isomers.

[0120] The term "alkoxy" refers to an alkyl group attached to the remainder of the molecule through an oxygen atom (the "oxy" group).

[0121] The term "alkylthio" refers to an alkyl group attached to the rest of the molecule via a sulfur group.

[0122] The term "alkoxycarbonyl" refers to the group (alkyl)-OC(=O)-, wherein the term alkyl has the meaning defined herein.

[0123] The term "alkanoyloxy" refers to the group (alkyl)-C(=O)-O-, wherein the term alkyl has the meaning defined herein.

[0124] The term "aryloxy" refers to an aryl group attached to the remainder of the molecule via an oxygen atom (aryl-O-).

[0125] The term "heteroaryloxy" refers to a heteroaryl group attached to the remainder of the molecule via an oxygen atom (heteroaryl-O-).

[0126] As used herein, the term "heteroatom" includes oxygen (O), nitrogen (N), sulfur (S) and silicon (Si).

[0127] The term "cycloalkyl" refers to a saturated or partially unsaturated (non-aromatic) all-carbon ring (ie, (C3-C6) carbocycle) having 3 to 6 carbon atoms. Non-limiting examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0128] The term "aryl" refers to a single all-carbon aromatic ring or a plurality of fused all-carbon ring systems, wherein at least one ring is aromatic. For example, in certain embodiments, the aryl group has 6 to 20 carbon atoms, 6 to 14 carbon atoms, 6 to 12 carbon atoms or 6 to 10 carbon atoms. Aryl includes phenyl. Aryl also includes a plurality of fused carbocyclic ring systems (for example, a ring system comprising 2, 3 or 4 rings) with about 9 to 20 carbon atoms, wherein at least one ring is aromatic, and wherein other rings can be aromatic or non-aromatic (i.e., cycloalkyl). When valence requirements allow, the rings of the polycondensed ring systems can be connected to each other via condensation, spiral and bridge bonds. It should be understood that the attachment point of the polycondensed ring system as defined above can be at any position of the ring system, including the aromatic or carbocyclic portion 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, etc.

[0129] The term "heterocycle" refers to a single saturated or partially unsaturated ring having at least one atom other than carbon in the ring, wherein the atom is selected from the group consisting of oxygen, nitrogen and sulfur; The term also includes a polycondensed ring system having at least one such saturated or partially unsaturated ring, which will be further described below. Therefore, the term includes a single saturated or partially unsaturated ring (e.g., 3, 4, 5, 6 or 7-membered ring) having 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. Sulfur and nitrogen atoms can also exist in their oxidized forms. Exemplary heterocycles include, but are not limited to, azetidinyl, tetrahydrofuranyl and piperidinyl. The term "heterocycle" also includes polycondensed ring systems (e.g., ring systems comprising 2, 3 or 4 rings), wherein a single heterocycle (as defined above) can be fused with one or more groups selected from cycloalkyl, aryl and heterocycle to form the polycondensed ring system. When valence requirements allow, the rings of the polycondensed ring system can be connected to each other via fusion, spiral and bridge bonds. It should be understood that the various rings of the polycondensed ring system can be connected in any order relative to each other. It should also be understood that the attachment point of the polycondensed ring system (as defined above for heterocycle) can be at any position of the polycondensed ring system, including the heterocycle, aryl and carbocyclic ring parts of the ring. In one embodiment, the term heterocycle includes 3-12 heterocycles. In one embodiment, the term heterocycle includes 3-7 heterocycles. In one embodiment, the term heterocycle includes 3-6 heterocycles. In one embodiment, the term heterocycle includes 4-6 heterocycles. In one embodiment, the term heterocycle includes 3-12 monocyclic or bicyclic heterocycles containing 1 to 3 heteroatoms. In one embodiment, the term heterocycle includes 3-6 monocyclic heterocycles containing 1 to 2 heteroatoms. In one embodiment, the term heterocycle includes 4-6 monocyclic heterocycles containing 1 to 2 heteroatoms. Exemplary heterocycles include, but are not limited to, aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, homopiperidinyl, morpholinyl, thiomorpholinyl, piperazinyl, tetrahydrofuranyl, dihydrooxazolyl, tetrahydropyranyl, tetrahydrothiopyranyl, 1,2,3,4-tetrahydroquinolinyl, benzoxazinyl, dihydrooxazolyl, chromanyl, 1,2-dihydropyridinyl, 2,3-dihydrobenzofuranyl, 1,3-benzodioxazolyl, 1,4-benzodioxanyl, spiro[cyclopropane-1,1′-isoindolyl]-3′-one, isoindolinyl-1-one, 2-oxa-6-azaspiro[3.3]heptyl, imidazolidin-2-one, imidazolidine, pyrazolidine, butyrolactam, valerolactam, imidazolinone, hydantoin, dioxolane, phthalimide, 1,4-dioxane, and

[0130] In one embodiment, the heterocycle can be divalent, i.e., attached to the rest of the molecule or a linker at two positions of 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: (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), and carboxyl.

[0131] As used herein, a wavy line intersecting a bond in a chemical structure Represents the point of attachment of a bond where a wavy bond meets the rest of the molecule in a chemical structure.

[0132] The terms "subject," "individual," and "patient," as 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.

[0133] The terms "treatment", "treating", etc. are used herein to generally refer to obtaining the desired pharmacological and / or physiological effects. "Treatment" or "treatment" may refer to any indication of success in the treatment or improvement of an injury, disease or condition, including any objective or subjective parameter, such as mitigation, alleviation, improvement of patient survival, increase of survival time or survival rate, reduction of symptoms, or making the injury, disease or condition more tolerable to the patient, slowing down the rate of degeneration or decline, or improving the patient's physical or mental health. In addition, "treating" or "treatment" may refer to the regulation of target gene expression, such as gene knockdown or gene knockout. For example, compared with the expression in the control, the expression of the target gene or sequence is inhibited or reduced, for example, by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100%. The treatment or improvement of symptoms can be based on objective or subjective parameters. The effect of treatment can be compared to an individual or pool of individuals who have not received treatment, or to the same patient before or at different times during treatment.

[0134] The term "pharmaceutically acceptable excipient" refers to inactive drug ingredients that are biologically or pharmacologically compatible for use in humans or animals, such as, but not limited to, buffers, carriers, or preservatives.

[0135] A "therapeutic amount" or "therapeutically effective amount" of an agent is an amount of the agent that treats, alleviates, lessens, or reduces the severity of a subject's disease symptoms. A "therapeutic amount" or "therapeutically effective amount" of an agent can improve patient survival, increase survival time or survival rate, alleviate symptoms, make an injury, disease, or condition more tolerable, slow the rate of degeneration or decline, or improve the patient's physical or mental health.

[0136] 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 delivery, parenteral delivery, intravenous delivery, intradermal delivery, intramuscular delivery, intrathecal delivery, colon delivery, rectal delivery, or intraperitoneal delivery. In one embodiment, the protein described herein is administered intravenously.

[0137] III. TFR Binder-Oligonucleotide Conjugates

[0138] In some embodiments, the TfR binder-oligonucleotide conjugate comprises or consists of:

[0139]

[0140] in,

[0141] P contains the anti-TfR antibody antigen binding domain,

[0142] F is optionally present or absent, and if present, comprises a peptide, an Fc polypeptide, an Fc dimer, or albumin;

[0143] L is optionally present or absent, and if present, is a linking group;

[0144] 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);

[0145] O is an oligonucleotide;

[0146] y is an integer greater than or equal to 1 (e.g., 1, 2, 3, or 4); and

[0147] n is an integer greater than or equal to 1 (eg, 1, 2, 3, 4, 5, 6, 7, or 8).

[0148] PFP′ (optionally, PF or P if P′ or F and P′ are absent) may be referred to as a TfR binder. In some embodiments, PFP′ comprises an anti-TfR antibody. The antibody may be a bivalent antibody (having two Fab arms that each specifically bind to TfR) or a bispecific antibody (having a first Fab arm that specifically binds to TfR and a second Fab arm that does not specifically bind to TfR). If P′ is present and F comprises an Fc dimer, the heavy chain component of P or P may form a single polypeptide chain with one Fc polypeptide of the Fc dimer, and the heavy chain component of P′ or P′ may form a single polypeptide chain with the other Fc polypeptide of the Fc dimer. If n is greater than or equal to 2, then for each (L-(O) y ), y is independently greater than or equal to 1 (e.g., 1, 2, 3, or 4). In some embodiments, the oligonucleotide comprises an ASO. In some embodiments, the TfR binder (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.

[0149] A. Monovalent anti-TfR (single Fab) antibody-oligonucleotide conjugate

[0150] In some embodiments, the TfR binder-oligonucleotide conjugate comprises or consists of:

[0151] PF-(L-(O) y ) n

[0152] in,

[0153] P contains the anti-TfR antibody antigen binding domain,

[0154] F comprises an Fc polypeptide or an Fc dimer;

[0155] L is a linking group;

[0156] O is an oligonucleotide;

[0157] y is an integer greater than or equal to 1 (e.g., 1, 2, 3, or 4); and

[0158] n is an integer greater than or equal to 1 (e.g., 1, 2, 3, 4, 5, 6, 7, or 8);

[0159] Wherein the TfR binder-oligonucleotide conjugate comprises a single anti-TfR antibody antigen binding domain and does not comprise any additional antibody antigen binding domains, non-binding Fab or NBVR (e.g., Fig.13As shown). 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 may be 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 binder (P and / or F) comprises at least one substitution or modification that promotes covalent conjugation of the oligonucleotide O, optionally via a linker L. The oligonucleotide may be connected to P or F. If the oligonucleotide is connected to F and F is an Fc dimer, it may be connected to an Fc polypeptide connected to P or an Fc polypeptide that is not connected to P.

[0160] B. Anti-TfR / Non-binding Fab Antibody-Oligonucleotide Conjugate

[0161] In some embodiments, the TfR binder-oligonucleotide conjugate comprises or consists of:

[0162]

[0163] in,

[0164] P contains the anti-TfR antibody antigen binding domain,

[0165] F contains Fc dimer;

[0166] L is a linking group;

[0167] P′ contains the non-binding Fab or NBVR;

[0168] O is an oligonucleotide;

[0169] y is an integer greater than or equal to 1 (e.g., 1, 2, 3, or 4); and

[0170] n is an integer greater than or equal to 1 (eg, 1, 2, 3, 4, 5, 6, 7, or 8).

[0171] In some embodiments, P comprises an anti-TfR Fab (e.g., Fig.13As shown). 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 may be from any anti-TfR antibody known to specifically bind to TfR. In some embodiments, the non-binding Fab or NVBR may be any of the non-binding Fab or NVBR described herein. In some embodiments, the oligonucleotide comprises an ASO. In some embodiments, the TfR binder (P, P' and / or F) comprises at least one substitution or modification that promotes covalent conjugation of the oligonucleotide O, optionally via a linker L. The oligonucleotide may be connected to P, F or P'. If the oligonucleotide is connected to F (Fc dimer), it may be connected to an Fc polypeptide connected to P or to an Fc polypeptide connected to P'.

[0172] C. Anti-TfR scFv-albumin-oligonucleotide conjugate

[0173] In some embodiments, the TfR binder-oligonucleotide conjugate comprises or consists of:

[0174] PF-(L-(O) y ) n

[0175] in,

[0176] P contains the anti-TfR antibody antigen binding domain,

[0177] F contains albumin;

[0178] L is a linking group;

[0179] O is an oligonucleotide;

[0180] y is an integer greater than or equal to 1 (e.g., 1, 2, 3, or 4); and

[0181] n is an integer greater than or equal to 1 (e.g., 1, 2, 3, 4, 5, 6, 7, or 8);

[0182] In some embodiments, P comprises an anti-TfR scFv (e.g., 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 may be from any anti-TfR antibody known to specifically bind to TfR. In some embodiments, albumin is white albumin. In some embodiments, the oligonucleotide comprises an ASO. In some embodiments, the TfR binder (P and / or F) comprises at least one substitution or modification that promotes covalent conjugation of the oligonucleotide O, optionally via a linker L.

[0183] D. Bivalent anti-TfR antibody (anti-TfR (Fab) 2)-oligonucleotide conjugate

[0184] In some embodiments, the TfR binder-oligonucleotide conjugate comprises or consists of:

[0185]

[0186] in,

[0187] P comprises a first anti-TfR antibody antigen binding domain,

[0188] F contains Fc dimer;

[0189] L is a linking group;

[0190] P′ comprises a second anti-TfR antibody antigen binding domain;

[0191] O is an oligonucleotide;

[0192] y is an integer greater than or equal to 1 (e.g., 1, 2, 3, or 4); and

[0193] n is an integer greater than or equal to 1 (eg, 1, 2, 3, 4, 5, 6, 7, or 8).

[0194] In some embodiments, P and P' comprise anti-TfR Fab. In some embodiments, P and P' comprise anti-TfR scFv. In some embodiments, P comprises anti-TfR Fab and P' comprises anti-TfR scFv. In some embodiments, P comprises anti-TfR scFv and P' comprises anti-TfR Fab. In some embodiments, the first anti-TfR antibody antigen binding domain and / or the second anti-TfR antibody antigen binding domain comprises anti-TfR VHH, vNAR or nanobody. The anti-TfR antibody antigen binding domain may be from any anti-TfR antibody known to specifically bind TfR. In some embodiments, the oligonucleotide comprises an ASO. In some embodiments, the oligonucleotide comprises an ASO. In some embodiments, the TfR binder (P, P' and / or F) comprises at least one substitution or modification that promotes covalent conjugation of oligonucleotide O, optionally via a linker L.

[0195] IV. Oligonucleotides

[0196] As described herein, one or more oligonucleotides (eg, ASOs or RNAi agents) can be optionally linked to a TfR binder described herein via a linker "L" to form a TfR binder-oligonucleotide conjugate.

[0197] Although the length of the oligonucleotide can vary, in certain embodiments, the length of the oligonucleotide is about 10 to about 60 nucleotides, or about 10 to about 30 nucleotides, or about 18 to about 30 nucleotides, or about 15 to about 25 nucleotides, or about 16 to about 20 nucleotides. In addition, as described below, the oligonucleotide may include certain chemical modifications, such as modified internucleoside bonds, modified core bases, modified sugars or combinations thereof. In certain embodiments, one or more oligonucleotides are connected (i.e., by a linking group "L") to a TfR binding agent. In certain embodiments, two or more oligonucleotides are connected to a TfR binding agent (e.g., 1, 2, 3, 4, 5, 6, 7 or 8 or more). In certain embodiments, an oligonucleotide is connected to a TfR binding agent. In certain embodiments, two oligonucleotides are connected to a TfR binding agent. In certain embodiments, four oligonucleotides are connected to a TfR binding agent.

[0198] In certain embodiments, 1 oligonucleotide is attached to a single linking group (L). In certain embodiments, 2 oligonucleotides are attached to a single linking group (L). For example, oligonucleotides can be connected in series with each other. In certain embodiments, L is attached to the 5' end of the first oligonucleotide and the second oligonucleotide is connected to the 3' end of the first oligonucleotide. In certain embodiments, oligonucleotides can be connected via nucleic acid linkers or non-oligonucleotide cleavable linkers.

[0199] In other embodiments, the linker is a branched linker and two or more oligonucleotides are each attached to a single linker (L) (ie, y is 2 or greater).

[0200] When two or more oligonucleotides are attached to a TfR binder, the oligonucleotides may be the same or different. In certain embodiments, the oligonucleotides are the same.

[0201] ASO

[0202] In one embodiment, each oligonucleotide is independently an ASO. The term "antisense oligonucleotide (ASO)" refers to a single strand (e.g., a modified nucleotide such as those described herein) of a DNA-like or RNA-like molecule that is complementary or partially complementary to a selected target polynucleotide sequence (e.g., mRNA). By binding to a complementary target sequence, ASOs can change or regulate gene expression by a variety of mechanisms, including, for example, by altering splicing (exon exclusion or exon inclusion); by recruiting RNase H to cause target degradation; by translation inhibition; and by small RNA inhibition.

[0203] Typically, the length of an ASO ranges from about 10 to 30 base pairs (bp), but may be longer or shorter. For example, in certain embodiments, the length of an ASO is from about 10 to about 60 nucleotides, or from about 10 to about 50 nucleotides, or from about 10 to about 40 nucleotides. In certain embodiments, the length of an ASO is from about 10 to 30 nucleotides, or from about 12 to 30 nucleotides, or from about 14 to about 30 nucleotides, or from about 15 to about 30 nucleotides, or from about 16 to about 30 nucleotides, or from about 17 to about 30 nucleotides, or from about 18 to about 30 nucleotides, or from about 18 to about 28 nucleotides, or from about 18 to 26 nucleotides, or from about 18 to about 24 nucleotides, or from about 15 to about 25 nucleotides, or from about 16 to about 20 nucleotides. In certain embodiments, the ASO is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

[0204] The selection of an antisense oligonucleotide sequence specific for a given target sequence is based on analysis of the chosen target sequence and determination of a number of factors, including secondary structure, T m , binding energy and relative stability. In addition, antisense oligonucleotides can be selected based on their relative inability to form dimers, hairpins or other secondary structures that will reduce or prevent the specific binding to the target mRNA in the host cell. The target region of mRNA includes those regions at or near the AUG translation initiation codon and those sequences that are substantially complementary to the 5' region of mRNA. Secondary structure analysis and target site selection considerations can be performed using software and algorithms known in the art, such as using the 4th edition of OLIGO primer analysis software (Molecular Biology Insights) and / or BLASTN 2.0.5 algorithm software (Altschul et al., Nucleic Acids Res. 1997, 25 (17): 3389-402).

[0205] RNAi Agents

[0206] In certain other embodiments, each oligonucleotide is independently an RNAi agent (e.g., siRNA or shRNA). The term "RNA interference (RNAi) agent" refers to 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) or a molecule that can be cut into an RNA agent. The RNAi agent can be single-stranded or double-stranded. If the RNAi agent is single-stranded, it may include 5' modifications, such as one or more phosphate groups or one or more analogs of a phosphate group. In one embodiment, the RNAi agent is double-stranded and comprises a sense strand and an antisense strand (e.g., short interfering RNA (siRNA)).

[0207] RNAi agents generally include a region with sufficient homology to the target gene, and have sufficient length so that the RNAi agent can mediate the downregulation of the target gene. The complementarity between the RNAi agent and the target sequence should be sufficient to enable the RNAi agent or its cleavage product to guide sequence-specific silencing. In certain embodiments, the RNAi agent is or includes a region that is at least partially complementary to the target RNA. In certain other embodiments, the RNAi agent is or includes a region that is fully complementary to the target RNA.

[0208] In some embodiments, the RNAi agent comprises an unpaired region at one or both ends of the molecule. For example, a double-stranded RNAi agent can pair its strands with overhangs, such as 5' and / or 3' overhangs, such as overhangs of 1-3 nucleotides. In certain embodiments, the RNAi agent will comprise an unpaired overhang of 1, 2, 3, or 4 nucleotides in length at each end. 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.

[0209] The length of the duplex region in the RNAi agent can vary, but generally the length ranges from about 5 to about 30 nucleotides. In certain embodiments, the duplex region length is between 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 certain embodiments, the duplex region length is between about 20-24 or about 21-23 nucleotides. In certain embodiments, the length of the duplex region is about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more nucleotides.

[0210] As used herein, a "single-stranded RNAi agent" or "ssRNAi agent" consists of a single molecule. It may include a duplex region formed by intrachain pairing, for example, it may be or include a hairpin or a coiled handle structure. The single-stranded RNAi agent may be antisense with respect to the target molecule. The single-stranded RNAi agent may be long enough so that it can enter RISC and participate in the cleavage of the target mRNA mediated by RISC. In certain embodiments, the length of the single-stranded RNAi agent is at least 10, 15, 20, 25, 30, 35, 40 or 50 nucleotides. In certain embodiments, its length is less than 200, 100, 80 or 60 nucleotides.

[0211] Small hairpin RNA (shRNA) agents typically have a duplex region less than 200, 100 or 50 in length. In certain embodiments, the length of the duplex region ranges from 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 certain embodiments, the length of the duplex region is between 17-23, or about 19-23, or about 20-23, or about 21-23, or about 19-21 nucleotides. In certain embodiments, the length of the duplex region is at least about 17, 18, 19, 29, 21, 22, 23, 24 or 25 nucleotide pairs. A hairpin may have a single-stranded overhang or an unpaired terminal region. In certain embodiments, the overhang is 2-3 nucleotides in length. In some embodiments, the overhang is on the sense side of a hairpin, and in some embodiments on the antisense side of a hairpin.

[0212] As used herein, a "double-stranded RNAi agent" or "dsRNAi agent" includes more than one strand, wherein interstrand hybridization can form a duplex region within the molecule (e.g., hybridization between a sense strand and an antisense strand). In certain embodiments, the RNAi agent is large enough so that it can be cleaved by an endogenous molecule (such as Dicer) to produce a smaller molecule.

[0213] In certain embodiments, the RNAi agent is a siRNA molecule comprising a sense strand and an antisense strand.

[0214] As used herein, the term "antisense strand" refers to a strand of an RNAi agent that is fully complementary to a target polynucleotide (e.g., target mRNA). In certain embodiments, the length of 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 certain embodiments, the antisense strand length of a double-stranded RNAi agent is less than about 200, 100, or 50 nucleotides. In certain embodiments, the length range of the antisense strand is about 17 to 25 nucleotides, or about 19 to 23 nucleotides, or about 19 to 21 nucleotides.

[0215] As used herein, the term "sense strand" refers to a strand of a RNAi agent that is fully complementary to the antisense strand. In certain embodiments, the length of 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 certain embodiments, the length of the sense strand of a double-stranded RNAi agent is less than about 200, 100, or 50 nucleotides. In certain embodiments, the length of the sense strand ranges from about 17 to 25 nucleotides, or about 19 to 23 nucleotides, or about 19 to 21 nucleotides.

[0216] In certain embodiments, the double-stranded portion 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 sense strand ranges in length from about 17 to 25 nucleotides, or from about 19 to 23 nucleotides, or from about 19 to 21 nucleotides.

[0217] In certain embodiments, sense strand and antisense strand can be selected so that dsRNAi agent comprises unpaired region at one end or both ends of molecule.Therefore, dsRNAi agent can contain sense and antisense strand, and pairing is to contain overhang, for example, 5' and / or 3' overhang of length between 1,2,3 or 4 nucleotides.Overhang can be the result that one chain is longer than another chain, or the result that two chains of the same length are staggered.In certain embodiments, dsRNAi agent comprises at least one 3' overhang.In certain embodiments, the two ends of dsRNAi agent comprise 3' overhang (for example, length is 2 nucleotides).

[0218] The length of the duplex region in a dsRNAi agent can vary, but generally the length ranges from about 5 to about 30 nucleotides. In certain embodiments, the length of the duplex region is about 5-60, or 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 certain embodiments, the length of the duplex region is between 17-23, or about 19-23, or about 20-23, or about 21-23, or about 19-21 nucleotides. In certain embodiments, the length of the duplex region is about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more nucleotides.

[0219] Methods for producing RNAi agents (such as siRNA and shRNA) are known in the art and can be easily adapted to produce RNAi agents targeting any polynucleotide sequence. In certain embodiments, RNAi agents are chemically synthesized. For example, oligonucleotides can be synthesized using a variety of techniques, such as 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: those described in 59 (1997).

[0220] Illustrative Oligonucleotide Modifications

[0221] In certain embodiments, the oligonucleotides described herein may include at least one nucleic acid modification, such as those selected from the group consisting of modified internucleoside bonds, modified nucleobases, modified sugars, and combinations thereof. Such modifications can be used to change pharmacokinetics (improved nuclease resistance leads to longer half-life), pharmacodynamics (excellent affinity to target RNA), or endocytic uptake. However, many modifications exclude RNase H cleavage, which is the desired mechanism of action of many ASOs. Therefore, some RNase H ASOs can be designed as chimeras, in which different bases are mixtures of different chemicals, or are designed as gapmers, in which some modifications are placed on the "wings" instead of the central bases. On the contrary, for RNAi agents and ASOs intended to change mRNA splicing or translation, considerations about RNase H are unnecessary.

[0222] Thus, the oligonucleotides described herein may comprise one or more nucleic acid modifications. In certain embodiments, the oligonucleotides comprise 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.

[0223] In certain embodiments, oligonucleotides as described herein comprise one or more nucleotide modifications (e.g., modifications to a 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 in the oligonucleotide are modified.

[0224] In certain embodiments, the oligonucleotide comprises one or more core base modifications. In certain embodiments, the oligonucleotide comprises one or more modifications to the sugar moiety (e.g., furanose group comprises a substituent at the 2'-position, 3'-position, 4'-position and / or 5'-position). In certain embodiments, the substituted sugar moiety comprises a bicyclic sugar moiety.

[0225] In certain embodiments, the nucleic acid modification is included in a pattern with the oligonucleotide. In certain embodiments, the oligonucleotide is a gapmer. The modification pattern of the gapmer oligonucleotide generally has the formula 5′-X a -Y a -Z a -3′, where X a and Z a As the gap region Y a In certain embodiments, Y a A region is a stretch of contiguous nucleotides, such as a region of at least 6 DNA nucleotides, that is capable of recruiting RNAse, such as RNAse H. In certain embodiments, Y a In certain embodiments, Y a Region is about 9 to about 15 DNA nucleotides. In certain embodiments, Y a The region is about 11 to about 13 DNA nucleotides. In certain embodiments, Y a The region is 10, 11, 12 or 13 DNA nucleotides. In certain embodiments, the gap body binds to the target nucleic acid, and RNAse is recruited, which can then cut the target nucleic acid. In certain embodiments, Y a The area on both sides of 5' and 3' is area X a and Z a , which contain high affinity modified nucleotides, such as in X a and Z a In certain embodiments, Y a The area on both sides of 5' and 3' is area X a and Z a , where X a and Z a In certain embodiments, X a and Z aEach 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, the modified nucleotide is present in the 5' and 3' regions of the oligonucleotide, and certain modified nucleotides and / or modified bonds may or may not be present in the central portion of the molecule. In certain embodiments, the modified nucleotide is present in the 5' and 3' regions of the oligonucleotide, and certain modified nucleotides are 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 bond, such as a PS bond. In certain embodiments, X a and Z a Each independently is about 3 to about 6 nucleotides long. In certain embodiments, X a and Z a Each independently is 3, 4 or 5 nucleotides long. In certain embodiments, X a and Z a Each comprises 3 modified nucleotides. In certain embodiments, the 3 modified nucleotides are in X a and Z a are arranged in series in each of .

[0226] Modified nucleosides / nucleotides are known in the art and include, but are not limited to, 2'-O methyl (2'OMe) residues, 2'O-methoxyethyl (MOE) residues, restricted 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 2019 April, 5(2)e323). In certain embodiments, the oligonucleotide comprises one or more MOE residues. In certain embodiments, the oligonucleotide comprises one or more OMe residues or F residues (e.g., 2'-F or 2'OMe). In certain embodiments, the oligonucleotide comprises one or more restricted residues (e.g., S-cEt, R-cEt, S-cMOE, and R-cMOE) and / or LNA residues. When nucleic acids have a methylene bridge formed between the 2'-oxygen and 4'-carbon of the ribose sugar molecule, they are considered to be "locked". In certain embodiments, the oligonucleotide is a morpholino (ie, comprises certain modifications to the sugar moiety). In certain embodiments, the oligonucleotides described herein comprise one or more LNA residues and one or more 5-methylcytidine residues.

[0227] In certain embodiments, the oligonucleotide comprises one or more modifications to the internucleoside backbone (i.e., the natural phosphodiester (PO) bond is modified). In certain embodiments, such modifications are performed, for example, to reduce nuclease activity. Therefore, 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 bonds. In certain embodiments, 25% or more of the internucleoside bonds are modified. In certain embodiments, 50% or more of the internucleoside bonds are modified. In certain embodiments, 75% or more of the internucleoside bonds are modified. In certain embodiments, 100% of the internucleoside bonds in the oligonucleotide are modified.

[0228] Backbone modifications are known in the art and include, but are not limited to, phosphorothioate (PS) bonds, chiral phosphorothioate bonds, phosphoramidate bonds, methylsulfonylphosphoramidate bonds and phosphorodiamidate bonds, phosphorodithioate bonds, aminoalkylphosphotriester bonds, phosphotriester bonds, phosphorothioate bonds, phosphonate bonds, methylphosphonate bonds, alkylphosphonate bonds, 3′-alkylenephosphonate bonds, chiral phosphonate bonds, 3′-aminophosphoramidate bonds, aminoalkylphosphoramidate bonds, phosphinate bonds, thionoalkylphosphonate bonds, thionophosphoramidate bonds, thionoalkylphosphotriester bonds, boranophosphate bonds, morpholino bonds, and peptide nucleic acid (PNA) bonds. 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) internucleoside bonds in an oligonucleotide are replaced by thiophosphate (PS) bonds. In certain embodiments, an oligonucleotide comprises a mixture of modified and unmodified bonds. The modification at an internucleoside bond can be independent of the modification at another internucleoside bond. In certain embodiments, each internucleoside bond in a MAPT ASO is a modified bond. In certain embodiments, each internucleoside bond in a MAPT ASO is a PS bond. In some embodiments, each internucleoside bond in a LPA ASO is a thiophosphate or a methylsulfonyl phosphoramidate. In certain other 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 or 25 or more) nucleoside internucleoside bonds in the oligonucleotide are replaced by diaminophosphoroside bonds. In certain embodiments, the oligonucleotide is a diaminophosphoroside morpholino (PMO).

[0229] In certain embodiments, the internucleoside linkage is stereo-random about the chiral center (Rp and Sp). In certain other embodiments, the Rp and Sp configurations in the oligonucleotide are optimized in a particular configuration.

[0230] In certain embodiments, the oligonucleotide is a gapmer comprising LNA and PS modifications. For example, in certain embodiments, the oligonucleotide is a gapmer having the formula 5′-X a -Y a -Z a -3′ modification pattern of the gap body, where X a and Z a As the gap region Y a The surrounding flanking regions, where X a and Z a Each comprises 3 LNA modified nucleotides (e.g., 3 consecutive LNA modified nucleotides), and wherein the gap region Y a In some embodiments, each internucleotide bond in the antisense oligonucleotide comprises a PS bond. In certain embodiments, the oligonucleotide further comprises one or more 5′-methylcytidine residues. In certain embodiments, the gap region Y a No LNA residues are included.

[0231] V. Linking Group

[0232] In some embodiments, the oligonucleotide is conjugated to a TfR binder (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 joins each oligonucleotide to a TfR binder. The linking group can be any group suitable for joining an oligonucleotide to a protein or polypeptide such as an antibody.

[0233] The linking group may be attached to any region of the TfR binder (e.g., attached to the N-terminal region, attached to the C-terminal region, or attached to an amino acid within a protein, such as a cysteine ​​residue or a glutamine residue), as long as the oligonucleotide does not prevent the binding of the TfR binder to the TfR. Similarly, the linking group may be attached to any region of the oligonucleotide (e.g., the 5′ end, the 3′ end, or attached to a nucleic acid residue within the molecule), as long as the TfR binder does not interfere with the functionality of the oligonucleotide (e.g., complementary binding to the target nucleic acid). For example, the linker may be attached to the oligonucleotide at any number of synthetically feasible points located throughout the oligonucleotide, such as at the 3′ or 5′ terminal residue of the oligonucleotide; at the sugar portion; at the base portion; or at a residue located within the backbone.

[0234] 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 the residue within the oligonucleotide. In certain embodiments, the oligonucleotide is a double-stranded RNAi molecule, wherein 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, wherein the linker is attached to the antisense strand (e.g., at the 5' or 3' terminal residue). In certain embodiments, the oligonucleotide is a siRNA, wherein 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 C6 amine.

[0235] 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).

[0236] The linking group can be a homobifunctional linker or a heterobifunctional linker.

[0237] 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, the 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 the linking group are unmodified. In certain embodiments, the linking group comprises one or more nucleotides with unmodified bases, unmodified sugar groups, and / or unmodified phosphate groups. In certain embodiments, the linking group comprises one or more nucleosides with unmodified bases and / or unmodified sugar groups. In certain embodiments, the linking group comprises TCA (thymine-cytosine-adenine) trinucleotides. In certain embodiments, TCA is modified with C6 amine at the T position. In certain embodiments, the linking group does not comprise TCA.

[0238] In certain embodiments, the linking group is enzyme cleavable. In certain embodiments, the linking group can be cleaved by an enzyme present in the central nervous system (CNS) or muscle. In certain embodiments, the cleavable linking group is applicable to a conjugate comprising an ASO (e.g., enabling the ASO to be dissociated from the remainder of the conjugate to be transported to 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 linker or a valine-alanine cleavable linker.

[0239] In certain embodiments, the cleavable linking group is an acid cleavable linker. In certain embodiments, the acid cleavable linker is a carbonate linker or a hydrazone linker.

[0240] In certain embodiments, the cleavable linking group comprises one or more PEG spacers.

[0241] In certain embodiments, the cleavable linking group is a disulfide, such as SPDP (succinimidyl 3-(2-pyridyldithio)propionate) or a lys-conjugated acid-cleavable hydrazide.

[0242] 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 is derived from 3-aryl propionitrile (APN) or acrylamide. In certain embodiments, the covalent linking group includes the group -CH2CH2C(=O)-. In certain embodiments, the covalent linking group includes the following groups:

[0243]

[0244] In certain embodiments, the covalent linking group can be derived from a haloacetamide, such as bromoacetamide, chloroacetamide, iodoacetamide.

[0245] In certain embodiments, the linking group includes a C6 amine group having the formula -(CH2)6-NH-.

[0246] In certain embodiments, the linking group may be derived from maleimide. For example, in certain embodiments, the linking group includes the following groups:

[0247]

[0248] In certain embodiments, the linking group can be attached to P at the valency labeled * (eg, to the sulfur atom of the modification site within P).

[0249] In certain embodiments, the maleimide is a modified maleimide. In certain embodiments, the modified maleimide is an alkyl-, aryl-, cycloalkyl-, or exocyclic-maleimide.

[0250] In certain embodiments, the linking group is a self-hydrolyzable linking group.

[0251] Certain specific non-limiting embodiments of exemplary linking groups (abbreviated as Linker Embodiments LE1-LE42) are described below.

[0252] 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.

[0253] In linker embodiment LE4, the linker group has a length of about 5 angstroms to about 60 angstroms. In linker embodiment LE5, the linker group separates the oligonucleotide from the TfR binder of formula (I) by a length of about 5 angstroms to about 40 angstroms, inclusive.

[0254] In linker embodiment LE6, 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—), (—S—), amino acids, hydrazones (—C(R′)═N═N(R′)—), nucleotides, or 3-12 membered divalent heterocycles, wherein the chain and any 3-12 membered divalent heterocycles are optionally replaced by one or more (e.g., 1, 2, 3, or 4) carbon atoms. The invention further comprises a substituent selected from the group consisting of (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), hydrazone (=N=N(R′)-), carboxyl, aryl, aryloxy, heteroaryl and heteroaryloxy; wherein each R′ is independently H or (C1-C6)alkyl.

[0255] In linker embodiment LE7, the linking group is a divalent, branched or unbranched, saturated or unsaturated hydrocarbon chain having 2-25 carbon atoms, wherein one or more (e.g., 1, 2, 3 or 4) carbon atoms are optionally replaced by (-O-), (-NH-) or a 3-12 membered divalent heterocycle, wherein the chain and any 3-12 membered divalent heterocycle are optionally substituted by one or more (e.g., 1, 2, 3 or 4) substituents independently selected from the group consisting of: (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), carboxyl, aryl, aryloxy, heteroaryl and heteroaryloxy.

[0256] In linker embodiment LE8, the linking group is a divalent, branched or unbranched, saturated or unsaturated hydrocarbon chain having 2-10 carbon atoms, wherein one or more (e.g., 1, 2, 3, or 4) carbon atoms are optionally replaced by (—O—), (—NH—), (—S—), amino acids, hydrazones (—C(R′)═N═N(R′)—), nucleotides, or 3-12 membered divalent heterocycles, wherein the chain and any 3-12 membered divalent heterocycles are optionally replaced by one or more (e.g., 1, 2, 3, or 4) carbon atoms. The invention further comprises a substituent selected from the group consisting of (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), hydrazone (=N=N(R′)-), carboxyl, aryl, aryloxy, heteroaryl and heteroaryloxy; wherein each R′ is independently H or (C1-C6)alkyl.

[0257] In linker embodiment LE9, the linking group is a divalent, branched or unbranched, saturated or unsaturated hydrocarbon chain having 2-10 carbon atoms, wherein one or more (e.g., 1, 2, 3 or 4) carbon atoms are optionally replaced by (-O-), (-NH-) or a 3-12 membered divalent heterocycle, wherein the chain and any 3-12 membered divalent heterocycle are optionally substituted by one or more (e.g., 1, 2, 3 or 4) substituents independently selected from the group consisting of: (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), carboxyl, aryl, aryloxy, heteroaryl and heteroaryloxy.

[0258] In linker embodiment LE10, the linking group is a divalent, branched or unbranched, saturated or unsaturated hydrocarbon chain having 2-25 carbon atoms, wherein the chain is optionally substituted on carbon with one or more (e.g., 1, 2, 3 or 4) substituents selected from the group consisting of: (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), carboxyl, aryl, aryloxy, heteroaryl and heteroaryloxy.

[0259] In linker embodiment LE11, the linking group is a divalent, branched or unbranched, saturated or unsaturated hydrocarbon chain having 2-10 carbon atoms, wherein the chain is optionally substituted on carbon with one or more (e.g., 1, 2, 3 or 4) substituents selected from the group consisting of: (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), carboxyl, aryl, aryloxy, heteroaryl and heteroaryloxy.

[0260] 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.

[0261] In linker embodiment LE13, the linking group is a divalent, branched or unbranched, saturated hydrocarbon chain having from 2 to 10 carbon atoms.

[0262] In linker embodiment LE14, the linking group is a divalent, unbranched, saturated hydrocarbon chain having from 2 to 10 carbon atoms.

[0263] In linker embodiment LE15, the linking group is a divalent, branched or unbranched, saturated or unsaturated chain having from 2 to 25 atoms selected from carbon, oxygen, nitrogen and sulfur, wherein one or more disulfide bonds are contained in the chain.

[0264] In linker embodiment LE16, the linking group is a divalent, branched or unbranched, saturated or unsaturated chain having from 2 to 25 atoms selected from carbon, oxygen, nitrogen and sulfur, wherein the chain includes one or more hydrazone groups in the chain or attached to a carbon atom of the chain.

[0265] In linker embodiment LE17, the linking group is a divalent, branched or unbranched, saturated or unsaturated chain having from 2 to 35 atoms selected from carbon, oxygen, nitrogen and sulfur, wherein one or more amino acids in the chain are included in the chain.

[0266] In linker embodiment LE18, the linking group is a divalent, branched or unbranched, saturated or unsaturated chain having from 2 to 35 atoms selected from carbon, oxygen, nitrogen and sulfur, wherein the chain comprises a dipeptide in the chain.

[0267] In linker embodiment LE19, the linking group is a divalent, branched or unbranched, saturated or unsaturated chain having from 2 to 35 atoms selected from carbon, oxygen, nitrogen and sulfur, wherein the dipeptide valine-citrulline is included in the chain.

[0268] In linker embodiment LE20, the linking group comprises one or more nucleotides in the chain.

[0269] In linker embodiment LE21, the linking group comprises two or more nucleotides in the chain.

[0270] In linker embodiment LE22, the linking group comprises trinucleotide groups in the chain.

[0271] In linker embodiment LE23, at least one linking group is attached to two or more oligonucleotides (e.g., for compounds of formula (I), for (L-(O) y ), y is greater than or equal to 2).

[0272] In linker embodiment LE24, only one linking group is attached to two or more oligonucleotides (e.g., for a compound of formula (I), for a single (L-(O) y ), a y greater than or equal to 2).

[0273] In linker embodiment LE24b, the TfR binder-oligonucleotide conjugate contains a single linking group attached to two or more oligonucleotides (eg, for compounds of Formula (I), n=1 and y is greater than or equal to 2).

[0274] 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 greater than or equal to 2, and for at least two (L-(O) y ), y is greater than or equal to 2).

[0275] In linker embodiment LE26, at least two linking groups are attached to two oligonucleotides (e.g., for compounds of formula (I), n is greater than or equal to 2, and for at least two (L-(O) y ), y=2).

[0276] 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).

[0277] In linker embodiment LE28, the linking group is attached to the oligonucleotide via the phosphorothioate of the oligonucleotide (eg, associated with the 5' terminal residue).

[0278] 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.

[0279] In linker embodiment LE30, the linking group comprises a 5-membered divalent heterocyclic ring.

[0280] In linker embodiment LE31, the linking group has the structure:

[0281]

[0282] wherein L′ 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—), (—S—), an amino acid, a hydrazone (—C(R′)═N═N(R′)—), a nucleotide or a 3-12 membered divalent heterocycle, wherein the chain and any 3-12 membered divalent heterocycle are optionally substituted by one or more (e.g., 1, 2, 3 or 4) substituents independently selected from the group consisting of: (C1- (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), hydrazone (-NH-N=C(R')-), carboxyl, aryl, aryloxy, heteroaryl and heteroaryloxy; wherein each R' is independently H or (C1-C6)alkyl; and wherein the valences marked * in formula (I) are attached to P and the valences marked ** 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 one or more disulfide bonds are contained in the chain. 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 one or more hydrazone groups in the chain or attached to the carbon atoms of the chain are included in 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 one or more amino acids in the chain are included in 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 a dipeptide in the chain is included in 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 a dipeptide in the chain is included in 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.

[0283] In linker embodiment LE32, L' has the following structure:

[0284]

[0285] 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 , R2 and R 3 Each is independently a nucleotide.

[0286] In linker embodiment LE33, L' has the following structure:

[0287]

[0288] In linker embodiment LE34, the linking group has the structure:

[0289]

[0290] 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.

[0291] In linker embodiment LE35, the linking group has the structure:

[0292]

[0293] 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, wherein in formula (I) the valencies marked * are attached to P and the valencies marked ** are attached to O. In certain embodiments, the valencies marked ** are attached to O via a phosphate of the oligonucleotide (e.g., associated with the 5′ terminal residue).

[0294] In linker embodiment LE36, the linking group has the structure:

[0295]

[0296] In linker embodiment LE37, the linking group has the following structure:

[0297]

[0298] wherein in formula (I) the valences marked * are attached to P and the valences marked ** are attached to O. In certain embodiments, the valences marked ** are attached to O via a phosphate of the oligonucleotide (e.g., associated with the 5′ terminal residue). Thus, in embodiments, the A group in the linker structure may be It is covalently bound to -O-PO3, which itself is covalently bound to the oligonucleotide.

[0299] In linker embodiment LE38, the linking group has the structure:

[0300]

[0301] In linker embodiment LE39, the linker is a peptide linker or is formed by a protein, a peptide, or an amino acid. For example, in certain embodiments, the linking group is a divalent group formed by a protein. In another embodiment, the linking group is a divalent group formed by a peptide. In another embodiment, the linking group is a divalent group formed by an amino acid.

[0302] In linker embodiment LE40, the linker group can be configured such that it allows the oligonucleotide and the TfR binder to rotate relative to each other; and / or is resistant to digestion by proteases. In some embodiments, the linker group can be a flexible linker, for example containing amino acids such as Gly, Asn, Ser, Thr, Ala, etc. Such linkers are designed using known parameters. For example, the linker group can have repeats, such as Gly-Ser repeats.

[0303] In linker embodiment LE41, the linking group has or comprises a formula selected from the group consisting of:

[0304]

[0305]

[0306] in,

[0307] Each A is independently (C1-C 15 )alkyl;

[0308] Each D is –(CH2-CH2-O) m -;and

[0309] 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.

[0310] In linker embodiment LE42, the linking group has or comprises a formula selected from the group consisting of:

[0311]

[0312]

[0313] In various embodiments, the conjugates can be generated using well-known chemical cross-linking reagents and protocols. For example, there are a large number of chemical cross-linking agents known to those skilled in the art and that can be used to cross-link proteins to the agent of interest. For example, the cross-linking agent is a heterobifunctional cross-linking agent, which can be used to connect molecules in a step-wise manner. Heterobifunctional cross-linking agents provide the ability to design more specific coupling methods for conjugating proteins, thereby reducing the occurrence of unwanted side reactions such as homotypic protein aggregates. A variety of heterobifunctional cross-linking agents are 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), succinimidyl 4 -(p-maleimidophenyl)butyrate (SMPB), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC); 4-succinimidyloxycarbonyl-a-methyl-a-(2-pyridyldithio)-toluene (SMPT), N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP), and succinimidyl 6-[3-(2-pyridyldithio) propionate] hexanoate (LC-SPDP). Those cross-linkers with N-hydroxysuccinimide moieties are available as N-hydroxysulfosuccinimide analogs, which generally have greater water solubility. Additionally, those cross-linkers with disulfide bonds in the linker can be synthesized as alkyl derivatives to reduce the amount of linker cleavage in vivo. In addition to heterobifunctional cross-linkers, many other cross-linkers exist, including homobifunctional and photoreactive cross-linkers. Disuccinimidyl suberate (DSS), bismaleimidohexane (BMH), and dimethyl pimelimidate.2HCl (DMP) are examples of useful homobifunctional cross-linkers, and bis-[B-(4-azidosalicylamido)ethyl]disulfide (BASED) and N-succinimidyl-6(4′-azido-2′-nitrophenylamino)hexanoate (SANPAH) are examples of useful photoreactive cross-linkers.

[0314] VI. Anti-TFR Antibody Antigen Binding Domain

[0315] The anti-TfR antibody antigen binding domain suitable for the TfR binder-oligonucleotide conjugate can be an anti-TfR antibody antigen binding domain derived from an antibody known to specifically bind to TfR. The anti-TfR antibody antigen binding domain can be derived from, but is not limited to, any of the antibodies or protein molecules described in the following documents: US20130028891, US2018282408, US20190092870, US2020071413, US20210138083, WO2014 / 033074, WO2015 / 101588, WO2016 / 081640, W WO2016 / 208695, WO2018 / 124121, WO2018 / 210898, WO2020 / 132584, WO2021 / 076546, WO2021 / 205358, WO2022 / 101633, WO2022 / 103769, WO2022 / 221505, Candelaria et al. (Front. Immunol. 12 March 17, 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.

[0316] In some embodiments, the TfR binder comprises an antibody having at least one variable domain or antigen binding site that specifically binds to TfR. In some embodiments, the TfR binder comprises an antibody having a single variable domain or antigen binding site that specifically binds to TfR. In some embodiments, the TfR binder comprises an antibody having a single variable domain or antigen binding site that specifically binds to TfR (monovalent, i.e., wherein the TfR-binding agent does not include any additional antibody antigen binding domains, non-binding Fabs or NBVRs (anti-TfR single Fabs)). In some embodiments, the TfR binder 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 that comprises non-binding Fabs or NBVRs. In some embodiments, the TfR binder comprises an anti-TfR antibody binding domain (e.g., anti-TfR Fab, scFv, VHH, vNAR or nanobody) connected to albumin (e.g., human albumin). In some embodiments, the TfR binder comprises an anti-TfR antibody having a first anti-TfR antibody antigen binding domain (e.g., Fab or scFv) and a second anti-TfR antibody antigen binding domain (e.g., Fab or scFv).

[0317] Illustrative proteins comprising Fabs that specifically bind to TfR

[0318] Exemplary Fabs that specifically bind to TfR include a heavy chain variable region of SEQ ID NO: 10, 19, 102, 104, 110, 122, 132, or 143 and a light chain variable region of SEQ ID NO: 9, 18, 103, 105, 111, 123, 133, or 144. Unless the context indicates otherwise, reference to a Fab that specifically binds to TfR should be understood to refer to any of the mouse, chimeric, veneered, humanized, and modified forms.

[0319] In some embodiments, the 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, the 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, the 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, the 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, the 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, the 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, the 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, the 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.

[0320] In some embodiments, the 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, the 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, the 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, the 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, the 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, the 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, the 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, the 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.

[0321] In some embodiments, the Fab that specifically binds to TfR comprises a heavy chain CH1 and a variable region of SEQ ID NO: 10 and a light chain containing SEQ ID NO: 9. In some embodiments, the Fab that specifically binds to TfR comprises a heavy chain CH1 and a variable region of SEQ ID NO: 19 and a light chain containing SEQ ID NO: 18. In some embodiments, the Fab that specifically binds to TfR comprises a heavy chain containing SEQ ID NO: 102 and a light chain containing SEQ ID NO: 103. In some embodiments, the Fab that specifically binds to TfR comprises a heavy chain containing SEQ ID NO: 104 and a light chain containing SEQ ID NO: 105. In some embodiments, the Fab that specifically binds to TfR comprises a heavy chain containing SEQ ID NO: 110 and a light chain containing SEQ ID NO: 111. In some embodiments, the Fab that specifically binds to TfR comprises a heavy chain containing SEQ ID NO: 122 and a light chain containing SEQ ID NO: 123. In some embodiments, the 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, the Fab that specifically binds to TfR comprises a heavy chain comprising SEQ ID NO: 143 and a light chain comprising SEQ ID NO: 144.

[0322] In some embodiments, the Fab that specifically binds to TfR consists of the heavy chain CH1 and variable region of SEQ ID NO: 10 and the light chain of SEQ ID NO: 9. In some embodiments, the Fab that specifically binds to TfR consists of the heavy chain CH1 and variable region of SEQ ID NO: 19 and the light chain of SEQ ID NO: 18. In some embodiments, the Fab that specifically binds to TfR consists of SEQ ID NO: 102 and SEQ ID NO: 103. In some embodiments, the Fab that specifically binds to TfR consists of SEQ ID NO: 104 and SEQ ID NO: 105. In some embodiments, the Fab that specifically binds to TfR consists of SEQ ID NO: 110 and SEQ ID NO: 111. In some embodiments, the Fab that specifically binds to TfR consists of SEQ ID NO: 122 and SEQ ID NO: 123. In some embodiments, the Fab that specifically binds to TfR consists of SEQ ID NO: 132 and SEQ ID NO: 133. In some embodiments, the Fab that specifically binds to TfR consists of SEQ ID NO:143 and SEQ ID NO:144.

[0323] In some embodiments, the 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.

[0324] In some embodiments, the Fab that specifically binds to TfR comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 having sequences of SEQ ID NOs: 12, 13, 14, 15, 16, and 17, respectively. In some embodiments, the Fab that specifically binds to TfR comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 having sequences of SEQ ID NOs: 21, 22, 23, 24, 25, and 26, respectively. In some embodiments, the Fab that specifically binds to TfR comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 having sequences of SEQ ID NOs: 114, 115, 116, 117, 118, and 119, respectively. In some embodiments, the Fab that specifically binds to TfR comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 having sequences of SEQ ID NOs: 126, 127, 128, 129, 130, and 131, respectively. In some embodiments, the Fab that specifically binds to TfR comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 having sequences of SEQ ID NOs: 134, 135, 136, 137, 138, and 139, respectively. In some embodiments, the Fab that specifically binds to TfR comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 having sequences of SEQ ID NOs: 154, 155, 156, 157, 158, and 159, respectively. In some embodiments, the 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.

[0325] In some embodiments, the Fab that specifically binds to TfR comprises a light chain comprising 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 heavy chain comprising 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 the VH and CH1 regions of SEQ ID NO: 10, 19, 102, 104, 110, 122, 132 or 143, and a heavy chain comprising 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 the VH and CH1 regions of SEQ ID NO: CDR sequences of 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.

[0326] In some embodiments, the Fab that specifically binds to TfR comprises light chain 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.

[0327] 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.

[0328] 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 NO: 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.

[0329] 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 NO: 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.

[0330] 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.

[0331] 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 sequences 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.

[0332] 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: 110 and SEQ ID NO: 111, and contains CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 having the sequences of SEQ ID NO: 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.

[0333] 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: 122 and SEQ ID NO: 123, and contains CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 having the sequences of SEQ ID NO: 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.

[0334] 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: 132 and SEQ ID NO: 133, and contains CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 having the sequences of SEQ ID NO: 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.

[0335] 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: 143 and SEQ ID NO: 144, and contains CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 having the sequences of SEQ ID NO: 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.

[0336] ScFab can be prepared by forming a fusion protein comprising the heavy and light chains of any of the Fabs using methods known in the art.

[0337] Illustrative proteins comprising scFvs that specifically bind to TfR

[0338] Exemplary scFvs that specifically bind to TfR include heavy chain variable regions of SEQ ID NOs: 10, 19, 102, 104, 110, 122, 132, or 143 and light chain variable regions of SEQ ID NOs: 9, 18, 103, 105, 111, 123, 133, or 144. Unless the context indicates otherwise, reference to scFvs that specifically bind to TfR should be understood to refer to any of mouse, chimeric, veneered, humanized, and modified forms.

[0339] In some embodiments, the scFv 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, the scFv 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, the scFv 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, the scFv 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, the scFv 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, the scFv 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, the scFv 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, the scFv 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.

[0340] In some embodiments, the scFv that specifically binds to TfR comprises SEQ ID NO: 106. In some embodiments, the scFv that specifically binds to TfR comprises SEQ ID NO: 107. In some embodiments, the scFv that specifically binds to TfR comprises SEQ ID NO: 171. In some embodiments, the scFv that specifically binds to TfR comprises SEQ ID NO: 153. In some embodiments, the scFv that specifically binds to TfR comprises SEQ ID NO: 160. In some embodiments, the scFv that specifically binds to TfR comprises SEQ ID NOs: 112 and 113. In some embodiments, the scFv that specifically binds to TfR comprises SEQ ID NOs: 124 and 125. In some embodiments, the scFv that specifically binds to TfR comprises SEQ ID NOs: 145 and 146.

[0341] In some embodiments, the scFv that specifically binds to TfR consists of SEQ ID NO: 106. In some embodiments, the scFv that specifically binds to TfR consists of SEQ ID NO: 107. In some embodiments, the scFv that specifically binds to TfR consists of SEQ ID NO: 171. In some embodiments, the scFv that specifically binds to TfR consists of SEQ ID NO: 153. In some embodiments, the scFv that specifically binds to TfR consists of SEQ ID NO: 160.

[0342] In some embodiments, a 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.

[0343] 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 sequences of SEQ ID NOs: 12, 13, 14, 15, 16, and 17, 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 sequences of SEQ ID NOs: 21, 22, 23, 24, 25, and 26, 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 sequences of SEQ ID NOs: 114, 115, 116, 117, 118, and 119, 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 sequences of SEQ ID NOs: 126, 127, 128, 129, 130, and 131, 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 sequences of SEQ ID NOs: 134, 135, 136, 137, 138, and 139, 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 sequences of SEQ ID NOs: 154, 155, 156, 157, 158, and 159, respectively. In some embodiments, a scFv that specifically binds to TfR comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 having sequences of SEQ ID NOs: 161, 162, 163, 164, 165, and 166, respectively.

[0344] In some embodiments, the 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 NO: 102 and 103. In some embodiments, the 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 NO: 154, 155, 156, 157, 158, and 159, respectively. In some embodiments, the 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 NO: 161, 162, 163, 164, 165 and 166, respectively.

[0345] In some embodiments, the 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).

[0346] In some embodiments, the 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).

[0347] In some embodiments, the 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).

[0348] In some embodiments, the scFv that specifically binds to TfR comprises light chain 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.

[0349] Illustrative proteins comprising antibodies that specifically bind to TfR

[0350] Exemplary antibodies that specifically bind to TfR include a heavy chain variable region of SEQ ID NO: 10, 19, 102, 104, 110, 122, 132, or 143 and a light chain variable region of SEQ ID NO: 9, 18, 103, 105, 111, 123, 133, or 144. Unless the context indicates otherwise, references to antibodies that specifically bind to TfR should be understood to refer to any of the mouse, chimeric, veneered, humanized, and modified forms.

[0351] 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, the 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, the 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.

[0352] In some embodiments, an antibody that specifically binds to TfR comprises a heavy chain CH1 and a 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 a 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.

[0353] In some embodiments, the antibody that specifically binds to TfR comprises SEQ ID NO: 108 and SEQ ID NO: 109. In some embodiments, the antibody that specifically binds to TfR comprises SEQ ID NO: 120 and SEQ ID NO: 121. In some embodiments, the antibody that specifically binds to TfR comprises SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11. In some embodiments, the antibody that specifically binds to TfR comprises SEQ ID NO: 18 and SEQ ID NO: 19.

[0354] 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.

[0355] 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 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 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 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 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 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 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.

[0356] In some embodiments, an antibody that specifically binds to TfR comprises: a heavy chain comprising 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 sequences of SEQ ID NO: 10, 19, 102, 104, 110, 122, 132 or 143; a light chain comprising 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 comprising 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: CDR sequences of IDNO: 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.

[0357] In some embodiments, an antibody that specifically binds to TfR comprises: a first heavy chain comprising 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 comprising 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:11; a light chain comprising 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 comprising the CDR sequences of SEQ ID NOs:10 and 9.

[0358] In some embodiments, the antibody that specifically binds to TfR comprises: a heavy chain comprising 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; a light chain comprising 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 comprising the CDR sequences of SEQ ID NOs:19 and 18.

[0359] In some embodiments, an antibody that specifically binds to TfR comprises: a heavy chain comprising 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: 108; a light chain comprising 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 comprising the CDR sequences of SEQ ID NOs: 108 and 109 (i.e., SEQ ID NOs 114-119).

[0360] In some embodiments, an antibody that specifically binds to TfR comprises: a heavy chain comprising 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: 120; a light chain comprising 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 comprising the CDR sequences of SEQ ID NOs: 120 and 121 (i.e., SEQ ID NOs 126-131).

[0361] Any of the antibodies that specifically bind to TfR may 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.

[0362] Any of the antibodies that specifically bind to TfR may 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 may be modified to increase serum stability, modulate effector function, affect glycosylation, reduce immunogenicity in humans, promote heterodimerization, and / or promote conjugation of oligonucleotides.

[0363] In some embodiments, an antibody that specifically binds to TfR comprises light chain 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.

[0364] Additional exemplary anti-TfR antibody antigen binding domains include, but are not limited to: 17H10 anti-TfR Fab or scFv; 17H10.1 anti-TfRFab or scFv; JC-141 anti-TfR antibody; JC-141 anti-TfRFab; JC-141 anti-TfR scFv; anti-TfR antibody, Fab, scFab, Fv fragment or scFv having the heavy and light chain CDR1, CDR2 and CDR3 sequences of JC-141 antibody (WO2016208695); JC-171 anti-TfR antibody; JC-171 anti-TfR Fab; JC-171 anti-TfR scFv; anti-TfR antibody, Fab, scFab, Fv fragment or scFv having the heavy chain and light chain CDR1, CDR2 and CDR3 sequences of JR-171 antibody (WO2018124121); "brain shuttle" (BS) anti-TfR Fab; anti-TfR antibody, Fab, scFab, Fv fragment or scFv having the heavy chain and light chain CDR1, CDR2 and CDR3 sequences of BS anti-TfR Fab (WO2018210898, WO2015101588 and WO2014033074); 13E4v2ii anti-TfR antibody; 13E4v2ii anti-TfR Fab; 13E4v2ii anti-TfR scFv; anti-TfR antibody, Fab, scFab, Fv fragment or scFv having the heavy chain and light chain CDR1, CDR2 and CDR3 sequences of 13E4v2ii antibody (WO2020132584); TfR12 anti-TfR scFv; anti-TfR antibody, Fab, scFab, Fv fragment or scFv having the heavy chain and light chain CDR1, CDR2 and CDR3 sequences of TfR12 anti-TfR scFv (WO2021 / 205358); TfR13 anti-TfR scFv; anti-TfR antibody, Fab, scFab, Fv fragment or scFv having the heavy chain and light chain CDR1, CDR2 and CDR3 sequences of Tfr13 anti-TfR scFv (WO2021 / 205358) (sequences are shown in Table 1).

[0365] Additional anti-TfR antibodies are described in WO2021 / 205358, and the anti-TfR antibody antigen binding domains of the present invention may include TfR1, TfR2, TfR3, TfR4, TfR5, TfR6, TfR7, TfR8, TfR9, TfR10, TfR11, TfR12, TfR13, TfR14, TfR15, TfR16, TfR17, Any antibody antigen binding domain of the CDRs or variable regions of any of TfR18, TfR19, TfR20, TfR21, TfR22, TfR23, TfR24, TfR25, TfR26, TfR27, TfR28, TfR29, TfR30, TfR31, TfR32, TfR33, TfR34, TfR35, TfR36, TfR37 and TfR38.

[0366] Table 1. Exemplary TfR-binding regions comprising antibody antigen binding domains.

[0367]

[0368]

[0369]

[0370]

[0371]

[0372]

[0373]

[0374] Additional anti-TfR antibodies are known in the art and / or available from various commercial sources. In some embodiments, the TfR binding fragment of an anti-TfR antibody or an anti-TfR antibody binds to the apical domain of TfR. In some embodiments, the binding of an anti-TfR antibody or an anti-TfR antibody TfR binding fragment to TfR does not inhibit the binding of transferrin 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 (March 17, 2021), 2021), Ri7, 8D3 (Weber et al. Cell Reports 22: 149-162, 2018). Exemplary anti-TfR antibodies are also described in the following U.S. Patent Publications: US2018282408A1, US2020071413A1, US20210138083A1, US20190092870A1, and US20130028891 (each of which is incorporated herein by reference).

[0375] Exemplary anti-TfR vNARs are described in WO 2022 / 103769.

[0376] Brain shuttles containing anti-TfR antibody antigen binding domains are described in WO 2014 / 033074 and WO 2015 / 101588 (each of which is incorporated herein by reference).

[0377] In some embodiments, the anti-TfR antibody antigen binding domain binds to 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 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 of TfR with an affinity of less than 1 nM.

[0378] Illustrative proteins comprising non-targeted Fab fragments

[0379] In some embodiments, the TfR binder comprises a non-binding Fab or a NBVR.

[0380] 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 an epitope naturally present 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 may be a mammalian antigen or an antigen found in a mammal, such as an antigen from an infectious organism such as a virus, bacteria, fungus, or parasite. The mammal may be, but is not limited to, a non-human primate, a human, or a rodent (e.g., a mouse). The NBVR may be, but is not limited to, a scFv.

[0381] Specific binding of an antibody to an antigen means at least 10 6 M -1 Specific binding is detectably higher in magnitude and distinguishable from nonspecific binding occurring to at least one unrelated target. Nonspecific binding is typically the result of van der Waals forces. Non-binding does not mean that the NBVR does not bind any antigen with any affinity. Rather, in some embodiments, the NBVR does not exhibit specific binding to (a) any protein or epitope in a mammalian cell, mammalian tissue, or 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.

[0382] The NBVR may be part of a scFv or a Fab. The Fab may or may not contain all or part of the hinge region of the antibody. The NBVR may be produced by recombinant DNA technology, by enzymatic or chemical separation of intact immunoglobulins, or by chemical peptide synthesis. In some embodiments, the NBVR is part of a non-binding Fab that comprises a light chain and a heavy chain, wherein the light chain comprises a V L The heavy chain comprises a V H region and heavy chain CH1 constant region.

[0383] Exemplary NBVRs include NBVR1 or NBVR2. Unless the context indicates otherwise, references to NBVR1 or NBVR2 should be understood to refer to any of the mouse, chimeric, veneered, humanized, and modified forms of NBVR1 or NBVR2.

[0384] Exemplary NTFs include NBVR1 or NBVR2. Unless the context indicates otherwise, references to NBVR1 or NBVR2 should be understood to refer to any of the mouse, chimeric, veneered, humanized, and modified forms of NBVR1 or NBVR2.

[0385] The sequences of the light chain and heavy chain variable regions of NBVR1 are designated as SEQ ID NOs: 35 and 36, respectively. The sequences of the light chain and heavy chain of NBVR1 are designated as SEQ ID NOs: 37 and 38, respectively.

[0386] In some embodiments, the NBVR comprises the CDR sequences of NBVR1. The CDRs of the light chain of NBVR1 (L1, L2, and L3) are designated as SEQ ID NOs: 39, 41, and 43, respectively. The CDRs of the heavy chain of NBVR1 (H1, H2, and H3) are designated 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.

[0387] 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.

[0388] In some embodiments, the NBVR comprises a light chain comprising 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 comprising 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 retains the non-binding properties of NBVR1.

[0389] In some embodiments, the NBVR comprises 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 NBVRs 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 (but preferably Kabat).

[0390] The sequences of the light chain and heavy chain variable regions of NBVR2 are designated as SEQ ID NOs: 53 and 60, respectively. The sequences of the light chain and heavy chain of NBVR2 are designated as SEQ ID NOs: 53 and 61, respectively.

[0391] In some embodiments, the NBVR comprises the CDR sequences of NBVR2. The CDRs of the light chain of NBVR2 (L1, L2, and L3) are designated as SEQ ID NOs: 40, 42, and 44, respectively. The CDRs of the heavy chain of NBVR2 (H1, H2, and H3) are designated 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.

[0392] In some embodiments, the NBVR comprises a light chain comprising 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 comprising 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 retains the non-binding properties of NBVR2.

[0393] In some embodiments, the NBVR comprises 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 NBVRs 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 (but preferably Kabat).

[0394] In some embodiments, the NBVR comprises light and heavy chain variable regions having some or all (e.g., 3, 4, 5, and 6) CDRs that are completely or substantially derived from NBVR1 or NBVR2. Such NBVRs may include a heavy chain variable region having at least two, and typically all three, CDRs that are completely or substantially derived from the heavy chain variable region of NBVR1 or NBVR2 and / or a light chain variable region having at least two, and typically all three, CDRs that are completely or substantially derived from the light chain variable region of NBVR1 or NBVR2. When a CDR contains no more than 4, 3, 2, or 1 substitutions, insertions, or deletions, it is substantially derived from the corresponding NBVR1 or NBVR2 CDR, except that CDR-H2 (when defined by Kabat) may have no more than 6, 5, 4, 3, 2, or 1 substitutions, insertions, or deletions. Such antibodies may be 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 any of the NBVR1 or NBVR2 light and heavy chain amino acid sequences and retain their functional properties, and / or be different from NBVR1 or NBVR2. In some embodiments, the NBVR does not exhibit specific binding to (a) any protein or epitope naturally occurring in a mammalian cell, mammalian tissue, or mammal; (b) any surface accessible protein or epitope on a naturally occurring mammalian cell or mammalian tissue; or (c) any serum accessible protein or epitope in a naturally occurring mammalian tissue or mammal.

[0395] 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.

[0396] In some embodiments, the nucleic acid encoding the NBVR light chain comprises a nucleotide sequence that is 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% identical to the nucleotide sequence of SEQ ID NO: 72 or 73. In some embodiments, the nucleic acid encoding the NBVR heavy chain comprises a nucleotide sequence that is 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% identical to the nucleotide sequence of SEQ ID NO: 74 or 75.

[0397] Cells containing nucleic acids encoding the heavy and light chains of any of the NBVRs are described. In some embodiments, the cell contains nucleic acids encoding a NBVR light chain comprising the amino acid sequence of SEQ ID NO: 35, 37, or 52, and nucleic acids encoding a 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 cell contains nucleic acids encoding a NBVR light chain comprising the amino acid sequence of SEQ ID NO: 62, 63, or 64, and nucleic acids encoding a NBVR heavy chain comprising the amino acid sequence of SEQ ID NO: 60, 61, 65, 66, 67, 68, 69, 70, or 71. The cell can be a bacterial cell, a yeast cell, an insect cell, or a mammalian cell.

[0398] In some embodiments, the non-binding Fab is a RSV (palivizumab) Fab fragment (light chain comprising SEQ ID NO: 101), which is non-targeted in mice and non-human primates.

[0399] Anti-TfR antibodies in which one of the Fab arms was removed or replaced with a non-binding Fab or NBVR.

[0400] Humanized Antibody Antigen Binding Domain

[0401] Any of the anti-TfR antibody antigen binding domains, non-binding Fab, NBVR or antibodies described herein can be humanized. Humanized antibody antigen binding domains can be humanized in one or more of the following: light chain variable domains, heavy chain variable domains, light chain constant domains and heavy chain constant (CH1) domains. Humanized antibody antigen binding domains are genetically engineered antibody antigen binding domains in which CDRs from non-human "donor" antibodies are transplanted into human "acceptor" antibody heavy and / or light chain variable regions, light chain constant regions and / or heavy chain CH1 region sequences (see, e.g., Queen, US 5,530,101 and 5,585,089; Winter, US 5,225,539; Carter, US 6,407,213; Adair, US 5,859,205; and Foote, US 6,881,557). The receptor antibody sequence can be, for example, a mature human antibody sequence (e.g., a sequence from one or more of the following: CH1 district, CH2 district, CH3 district, heavy chain variable region, light chain constant region or light chain variable region), a complex of such sequence, a consensus sequence of a human antibody sequence or a germline region sequence. Therefore, a humanized antibody antigen-binding domain is an antibody antigen-binding domain with at least three, four, five or all completely or substantially from a donor antibody and completely or substantially from a human antibody variable region framework sequence and / or a constant region sequence of a CDR. Similarly, a humanized heavy chain has at least one, two and usually all three completely or substantially from a donor antibody heavy chain CDR, and if present, a heavy chain variable region framework sequence and a heavy chain constant region sequence substantially from a human heavy chain variable region framework and a constant region sequence. Similarly, a humanized light chain has at least one, two and usually all three completely or substantially from a donor antibody light chain CDR, and if present, a light chain variable region framework sequence and a light chain constant region sequence substantially from a human light chain variable region framework and a constant region. The CDRs in a humanized antibody are substantially derived from the corresponding CDRs in a non-human antibody when at least 85%, 90%, 95% or 100% of the corresponding residues (as defined by any conventional definition, but preferably by the Kabat definition) are identical between the corresponding CDRs. The variable region framework sequence of the antibody chain or the constant region of the antibody chain are substantially derived from human variable region framework sequences or human constant regions, respectively, when at least 85%, 90%, 95% or 100% of the corresponding residues as defined by Kabat are identical.

[0402] In some embodiments, the Fab is a chimeric Fab. The 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.

[0403] In some embodiments, the Fab is a veneered Fab. The veneered Fab comprises partially humanized light chain and / or heavy chain variable regions and human heavy chain (CH1) and / or light chain constant regions.

[0404] VII. Fc polypeptide or Fc dimer

[0405] In some embodiments, the TfR binder comprises an Fc polypeptide or an Fc dimer. The Fc polypeptide or Fc dimer may comprise 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 conjugation of oligonucleotides.

[0406] In some embodiments, an Fc polypeptide as described herein has 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).

[0407] One or both of the Fc polypeptides may each comprise an independently selected modification (e.g., a mutation), or one or both of the Fc polypeptides may be a wild-type Fc polypeptide, such as a human IgG1 Fc polypeptide. In some embodiments, an Fc polypeptide as described herein has 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 with a corresponding wild-type Fc polypeptide (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc polypeptide). Non-limiting examples of mutations that may be introduced into one or both Fc polypeptides include mutations, for example, to provide for heterodimerization of the knob and hole of the polypeptides, modulate effector function, increase serum half-life, affect glycosylation, and / or reduce immunogenicity in humans.

[0408] Fc peptide modification for heterodimerization

[0409] In some embodiments, the Fc polypeptide of the Fc dimer includes mutations that promote heterodimer formation and hinder homodimer formation. These modifications are useful, for example, when it is desired that only one Fc polypeptide in the dimer has a TfR binding site (i.e., a monovalent TfR binder).

[0410] In some embodiments, the polypeptide present in the Fc dimer may include knob and hole mutations to promote heterodimer formation. Generally, the method includes introducing a protrusion ("knob") at the interface of one polypeptide and introducing a corresponding cavity ("hole") in the interface of another 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). Compensatory cavities of the same or similar size as the protrusion are generated in the interface of the second polypeptide by replacing a large amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine).

[0411] The knob-and-hole approach generally involves introducing a protrusion ("knob") at the interface of one Fc polypeptide and a corresponding cavity ("hole") in the interface of another Fc polypeptide, such that the protrusion can be positioned in the cavity to promote heterodimer formation and thereby hinder homodimer formation. The protrusion is constructed by replacing small amino acid side chains from the interface of one Fc polypeptide with larger side chains (e.g., Tyr or Trp). Compensatory cavities of the same or similar size to the protrusion are generated in the interface of another Fc polypeptide by replacing large amino acid side chains with smaller amino acid side chains (e.g., Ala or Thr). In some embodiments, such additional mutations are located at positions in the Fc polypeptide that do not have a negative impact on the binding of the polypeptide to TfR.

[0412] In an illustrative embodiment of the knob and hole method for dimerization, position 366 of one of the Fc polypeptides comprises Trp in place of a native Thr. The other Fc polypeptide in the dimer has Val in place of a native Tyr at position 407. The other Fc polypeptide may also comprise a substitution in which the native Thr at position 366 is substituted with Ser and the native Leu at position 368 is substituted with Ala. Thus, one of the Fc polypeptides has a T366W knob mutation and the other Fc polypeptide has a Y407V hole mutation, which is typically accompanied by T366S and L368A hole mutations. As described above, all positions are numbered according to EU numbering. In certain embodiments, a first Fc polypeptide comprises T366S, L368A, and Y407V substitutions according to EU numbering, and a second Fc polypeptide further comprises a T366W substitution according to EU numbering.

[0413] In some embodiments, one or both Fc polypeptides present in the Fc polypeptide dimer may also be engineered to contain other modifications for heterodimerization, such as electrostatic engineering of naturally charged contact residues or hydrophobic patch modifications within the CH3-CH3 interface.

[0414] Fc peptide modifications for modulation of effector function

[0415] In some embodiments, one or both Fc polypeptides in the Fc polypeptide dimer may comprise a modification that reduces effector function, i.e., the ability to induce certain biological functions when binding to Fc receptors expressed on effector cells that mediate effector function is reduced. 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), downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0416] In some embodiments, one or both Fc polypeptides in the Fc polypeptide dimer may comprise a modification that reduces or eliminates effector function. Illustrative Fc polypeptide mutations that reduce effector function include, but are not limited to, substitutions in the CH2 domain, for example, at positions 234 and 235 and / or at position 329 according to the EU numbering scheme. For example, in some embodiments, the two Fc polypeptides comprise an Ala residue at positions 234 and 235 (also referred to herein as "LALA"). In some embodiments, the two 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, the two Fc polypeptides comprise an Ala residue at positions 234 and 235 and a Gly residue at position 329 (also referred to herein as "LALA PG"). In some embodiments, the two Fc polypeptides comprise an Ala residue at positions 234 and 235 and a Ser residue at position 329 (also referred to herein as "LALAPS").

[0417] Additional Fc polypeptide mutations that modulate effector function include, but are not limited to, the following: Position 329 may have a mutation in which Pro is substituted with Gly, Ala, Ser, or Arg or an amino acid residue that is large enough to disrupt the Fc / Fcγ receptor interface formed between Proline 329 of Fc and Trp residues Trp87 and Trp110 of FcγRIII. Additional illustrative substitutions include S228P, E233P, L235E, N297A, N297D, and P331S according to the EU numbering scheme. Multiple substitutions may also be present, e.g., according to the EU numbering scheme, L234A, L235A, and P329G of human IgG1; S228P and L235E of human IgG4; L234A and G237A of human IgG1; L234A, L235A, and G237A of human IgG1; V234A and G237A of human IgG2; L235A, G237A, and E318A of human IgG4; and S228P and L236E of human IgG4.

[0418] Fc peptide modification for extended serum half-life

[0419] In some embodiments, modifications that enhance serum half-life can be introduced into any Fc polypeptide described herein. For example, in some embodiments, two Fc polypeptides in an Fc polypeptide dimer may include M428L and N434S substitutions (also referred to as LS substitutions) numbered according to the EU numbering scheme. Alternatively, two Fc polypeptides in an Fc polypeptide dimer may have N434S or N434A substitutions. Alternatively, two Fc polypeptides in an Fc polypeptide dimer may have M428L substitutions. In other embodiments, two Fc polypeptides in an Fc polypeptide dimer may include M252Y, S254T, and T256E substitutions.

[0420] Fc peptide with C-terminal lysine residue removed

[0421] In some embodiments, one or both of the Fc polypeptides may have its C-terminal lysine removed (e.g., the Lys residue at position 447 of the Fc polypeptide according to EU numbering). The C-terminal lysine residue is highly conserved in immunoglobulins across many species and may be completely or partially removed by cellular mechanisms during protein production. In some embodiments, removal of the C-terminal lysine in the Fc polypeptide may improve the stability of the protein.

[0422] Exemplary Fc polypeptides are provided in SEQ ID NOs:76-100.

[0423] Engineered anti-TfR antibody variants for conjugation via linker groups

[0424] 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—as described herein) can be connected to an oligonucleotide via a linker "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 attach 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 attached to L via a cysteine ​​residue of the antibody (e.g., via a sulfur atom of a cysteine ​​residue). In some embodiments, cysteine ​​is a cysteine ​​modification, wherein 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 attached to L via a glutamine residue (e.g., via an amide bond in the side chain of a glutamine residue).

[0425] In other aspects, it may be desirable to produce engineered antibodies with one or more modification sites. These modification sites can be used to promote the attachment of TfR binders to each L. For example, TfR binders can be attached to each L at the modification site. In other embodiments, the modification site may enable L to be attached 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, such as within 2 or 3 amino acids of the modification site). In a specific embodiment, such modification sites are replacement residues occurring at accessible sites of 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) include one or more modification sites (e.g., one or more amino acid substitutions, such as cysteine, alanine or glycine substitutions). In certain embodiments, the antibody includes 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.

[0426] In certain embodiments, the modification site within the antibody is an amino acid substitution or insertion. In certain embodiments, the antibody comprises an Fc dimer (or a protein that is an Fc dimer). In certain embodiments, the Fc polypeptide may be part of a Fab-Fc fusion or a Fab-Fc dimer fusion, and the modification site may be in a Fab-Fc polypeptide that binds to TfR and / or in a Fab-Fc polypeptide that has formed a dimer with a Fab-Fc polypeptide that binds to TfR.

[0427] 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.

[0428] In certain embodiments, the modification site is an amino acid substitution. In certain embodiments, the modification site is a cysteine, glycine or alanine substitution.

[0429] In certain embodiments, the modification site is a cysteine ​​substitution. By replacing those residues with cysteine, reactive thiol groups are thus positioned at accessible sites of the antibody, and can be used to conjugate the antibody to an oligonucleotide via a linking group (L), to produce a conjugate as described herein. In certain embodiments, the antibody contains an Fc polypeptide or an Fc polypeptide dimer and includes a cysteine ​​substitution selected from the group consisting of S239C, S442C, A330C and T289C, wherein the position and substitution are numbered according to EU. In other embodiments, the Fc polypeptide is attached to the CH1 domain and includes an A114C substitution. In other embodiments, the antibody includes a Fab-Fc fusion, and the light chain includes a K149C substitution.

[0430] In other aspects, the modification site is an alanine or glycine substitution. Such modified amino acids can promote enzymatic conjugation of L to antibodies at nearby amino acids (such as glutamine residues) (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 numbered according to EU. These substitutions eliminate the glycosylation at position 297, which will hinder the enzymatic conjugation of the linker to the antibody at position Q295 (i.e., the linker is attached to the antibody via an amide bond in the glutamine side chain). Therefore, in certain embodiments, the modification site is N297A or N297G, and the antibody is attached to L at Q295 (e.g., by enzymatic conjugation).

[0431] 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)).

[0432] 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., 1 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 the following items: S239C, S442C, A330C, T289C, N297A and N297G in the heavy chain (according to EU numbering), K149C in the light chain (according to EU numbering), and A114C in the heavy chain (according to Kabat numbering). 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 the first Fc polypeptide comprises A330C. In certain embodiments, the Fc polypeptide or the first Fc polypeptide comprises T289C. In certain embodiments, the Fc polypeptide or the first Fc polypeptide comprises N297A. In certain embodiments, the Fc polypeptide or the first Fc polypeptide comprises N297G. In certain embodiments, the Fc polypeptide or the first Fc polypeptide comprises S239C and A330C.

[0433] In certain embodiments, the second Fc polypeptide (of the Fc dimer) comprises one or more amino substitutions (e.g., 1 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 in the heavy chain (according to EU numbering), K149C in the light chain (according to EU numbering), and A114C in the heavy chain (according to Kabat numbering). 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. In certain embodiments, the second Fc polypeptide comprises A114C.

[0434] In certain embodiments, the Fc polypeptide comprises a sequence that is at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO:1.

[0435] In certain embodiments, the first Fc polypeptide and the second Fc polypeptide of the Fc dimer each comprise one or more amino acid substitutions (e.g., 1 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 and the second Fc polypeptide each comprise an amino acid substitution (e.g., 1 cysteine ​​substitution) that promotes conjugation of an oligonucleotide. In certain embodiments, the first Fc polypeptide and the second Fc polypeptide each comprise a cysteine ​​substitution at S239C. In certain embodiments, the first Fc polypeptide and the second Fc polypeptide each comprise two amino acid substitutions (e.g., 2 cysteine ​​substitutions). In certain embodiments, the first Fc polypeptide and the second Fc polypeptide each comprise a cysteine ​​substitution at S239C and A330C.

[0436] Fc polypeptides or dimers thereof comprising one or more modification sites (eg, cysteine ​​substitutions) may be used in the conjugates as described herein.

[0437] In some embodiments, the anti-TfR antibody antigen binding domain comprises a Fab or scFab, wherein the Fab or scFab comprises a K149C substitution on the light chain (according to EU numbering) or an A114C substitution on the heavy chain (according to Kabat numbering).

[0438] In certain embodiments, 1 or more oligonucleotides are attached to a linking group (L). In certain embodiments, 2 or more oligonucleotides are attached to a linking group (L). In certain embodiments, 1 oligonucleotide is attached to a linking group (L). In certain embodiments, 2 oligonucleotides are attached to a linking group (L).

[0439] VIII. Albumin

[0440] For TfR binder-oligonucleotide conjugates comprising 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 number P0276, GenBank: AAA98797.1, NCBANP_000468.1, gene ID: 213, mRNA NM_000477.7). The oligonucleotide can be linked to the albumin, optionally to a surface accessible free cysteine ​​in the albumin (e.g., C58 of mouse prealbumin (position 34 (box) of SEQ ID NO: 167 and 168)) via a linking group. The albumin can be modified to contain one or more amino acid substitutions, such as cysteine ​​substitutions, to facilitate conjugation with the oligonucleotide.

[0441] In some embodiments, the TfR binder-oligonucleotide conjugate comprises an anti-TfR scFv fused to albumin. The anti-TfR scFv fused to albumin can be provided as a single polypeptide chain fusion protein. The anti-TfR scFv can be fused to the amino or carboxyl terminus of albumin. In some embodiments, the anti-TfR scFv is fused to the amino terminus of albumin. The fusion portion may contain a connecting peptide between scFc and albumin. The connecting peptide may 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 contain 17H10 anti-TfR scFv fused to mouse and human albumin, respectively. The anti-TfR scFv-albumin fusion protein may also contain peptides that promote purification, such as epitope tags or polyhistidine tags (e.g., His6). The epitope tag or polyhistidine may be located at the amino or carboxyl terminus of the fusion protein.

[0442] Table 2.

[0443]

[0444]

[0445] IX. Nucleic Acids, Vectors and Host Cells

[0446] The TfR binding agents described herein can be prepared using recombinant methods. Therefore, the isolated nucleic acid containing the sequence encoding any one or part of the TfR binding agents described herein can be easily produced using methods available in the art. The host cell into which the nucleic acid is introduced and can be used to replicate the nucleic acid encoding the polypeptide and / or express the polypeptide is also available in the art. The host cell can be, but is not limited to, a prokaryotic cell or a eukaryotic cell. Eukaryotic cells are, but are not limited to, yeast cells, insect cells, or mammalian cells (e.g., human cells).

[0447] The nucleic acid encoding a TfR binder or portion thereof can be DNA, RNA, cDNA, mRNA, single-stranded, double-stranded, linear or circular.

[0448] The TfR binder may comprise two or more (e.g., three) polypeptides, each of which may be encoded by a separate nucleic acid sequence. The isolated 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 isolated nucleic acid sequences may be expressed from a single promoter or different promoters. Methods for expressing nucleic acids encoding separate polypeptides from a single promoter are known in the art, including but not limited to the use of 2A elements and internal ribosome entry sites.

[0449] The nucleic acid encoding the TfR binder or a portion thereof may be provided in a plasmid or vector. The plasmid or vector may be used to replicate the nucleic acid or promote expression of the nucleic acid. The plasmid or vector may be, but is not limited to, a viral vector, a phagemid, a yeast chromosomal vector, or a non-additive mammalian vector.

[0450] In some embodiments, a nucleic acid encoding a TfR binder or a portion thereof is operably linked to one or more regulatory sequences in an expression construct. The expression construct may be suitable for expressing the polypeptide in a system that produces a dual transporter. Such a system may 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.

[0451] Expression vectors for producing recombinant polypeptides include plasmids and other vectors. For example, suitable vectors include the following types of plasmids: pBR322 derived plasmids, pEMBL derived plasmids, pEX derived plasmids, pBTac derived plasmids and pETC derived plasmids for expression in prokaryotic cells such as Escherichia coli. 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 transfecting eukaryotic cells. Alternatively, viral derivatives such as bovine papilloma virus (BPV-1) or Epstein-Barr virus (pREP derived pHEBo and p205) can be used for transient expression of polypeptides in eukaryotic cells. In some embodiments, it may be desirable to express recombinant polypeptides by 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.

[0452] An expression vector for expressing a TfR binder 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 baby hamster kidney (BHK) cell, a NSO cell, a YO cell, a HEK293 cell, a COS cell, a Vero cell or a HeLa cell. The host cell containing the expression vector can be cultured under suitable conditions to allow expression of the TfR binder or a portion thereof.

[0453] TfR-binding agents can be prepared by culturing host cells comprising one or more nucleic acids encoding the TfR-binding agents, expressing the TfR-binding agents, and isolating the expressed TfR-binding agents from the culture.

[0454] X. How to use

[0455] The conjugates described herein can be used for a variety of purposes, including therapeutic indications.

[0456] In some embodiments, the conjugate is used to deliver an oligonucleotide (e.g., an ASO or RNAi agent) to a target cell type expressing a transferrin receptor. In some embodiments, the conjugate can be used to transport an oligonucleotide (e.g., an ASO or RNAi agent) across the endothelium (e.g., the blood-brain barrier) to be absorbed by the brain.

[0457] For example, certain embodiments provide methods for oligonucleotide (e.g., ASO or RNAi agent) transendothelial transcytosis, the method comprising contacting the endothelium (e.g., blood-brain barrier (BBB)) with a conjugate as described herein. Therefore, certain embodiments provide methods for transporting oligonucleotides through the BBB of a subject in need, the method comprising administering a conjugate as described herein to the subject. In certain embodiments, a conjugate as described herein used in transporting oligonucleotides through the BBB of a subject in need is provided. In certain embodiments, a conjugate as described herein used in transporting oligonucleotides to a muscle cell of a subject in need is provided.

[0458] Certain embodiments also provide a method of regulating 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 as described herein. In some embodiments, a conjugate as described herein for use in regulating expression of a target gene is provided.

[0459] In certain embodiments, the target gene or sequence is expressed in cells in the subject's brain. In certain embodiments, the target gene or sequence is expressed in cells expressing TfR. In certain embodiments, the target gene or sequence is expressed in muscle cells such as skeletal muscle cells or cardiomyocytes.

[0460] In certain embodiments, the regulation of target gene expression is gene knockdown or gene knockout. Thus, in certain embodiments, the expression of the target gene or sequence is inhibited or reduced, for example, by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100%, compared to expression in a control (e.g., a subject to which the conjugate is not administered).

[0461] The conjugates as described herein are administered to a subject in a therapeutically effective amount or dosage. However, the dosage may vary according to several factors, including the chosen route of administration, the formulation of the composition, the patient's response, the severity of the condition, the subject's weight, and the judgment of the prescribing physician. The dosage may be increased or decreased over time according to the needs of the individual patient.

[0462] In various embodiments, the conjugate as described herein is administered parenterally. In some embodiments, the conjugate is administered intravenously. Intravenous administration can be performed by infusion, for example, over a period of about 10 to about 30 minutes, or over a period of at least 1 hour, 2 hours, or 3 hours. In some embodiments, the conjugate is administered as an intravenous push. A combination of infusion and push administration can also be used.

[0463] 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.

[0464] In other embodiments, the conjugates as described herein can be administered orally, by pulmonary administration, intranasally, intraocularly, or by topical administration. Pulmonary administration can also be employed, for example, by using an inhaler or nebulizer, and formulations with aerosolizing agents.

[0465] XI. Pharmaceutical Compositions and Kits

[0466] In another aspect, pharmaceutical compositions and kits comprising the conjugates as described herein are provided.

[0467] Pharmaceutical composition

[0468] Guidance for the preparation of formulations for use as described herein can be found in numerous pharmaceutical preparation and formulation manuals known to those skilled in the art.

[0469] In some embodiments, the pharmaceutical composition comprises a conjugate as described herein and further comprises one or more pharmaceutically acceptable carriers and / or excipients. In certain embodiments, the composition comprises a variety of conjugates as described herein, which may be identical or different (e.g., mixtures of different conjugates). In certain embodiments, the ratio of oligonucleotides to proteins in the composition is from about 1:1 to about 4:1. In certain embodiments, the ratio of oligonucleotides to proteins in the composition is from about 1:1 to about 2:1. In certain embodiments, the ratio of oligonucleotides to proteins in the composition is about 1.23. In certain embodiments, the ratio of oligonucleotides to proteins in the composition is from about 2:1 to about 3:1. In certain embodiments, the ratio of oligonucleotides to proteins in the composition is about 2.5.

[0470] As used herein, the term 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 agent. Various 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. A pharmaceutically acceptable carrier may contain one or more physiologically acceptable compounds, which function, 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 agent; or excipients or other stabilizers and / or buffers. Other pharmaceutically acceptable carriers and formulations thereof are also available in the art.

[0471] The pharmaceutical compositions described herein can be manufactured in a manner known to those skilled in the art, for example, by conventional mixing, dissolving, granulating, dragee-making, emulsifying, encapsulating, embedding or lyophilizing processes. The following methods and excipients are exemplary only and are by no means limiting.

[0472] For oral administration, the conjugates as described herein can be formulated by combining them with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compound to be formulated into tablets, pills, dragees, capsules, emulsions, lipophilic and hydrophilic suspensions, liquids, gels, syrups, slurries, suspensions, etc., for oral ingestion by patients to be treated. Pharmaceutical preparations for oral use can be obtained by mixing the conjugate with a solid excipient, optionally grinding the resulting mixture, and, if necessary, processing the mixture of particles after adding a suitable adjuvant to obtain a tablet or dragee core. 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, hydroxypropyl methylcellulose, sodium carboxymethylcellulose and / or polyvinyl pyrrolidone. If necessary, disintegrants such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate can be added.

[0473] As disclosed above, the conjugates as described herein can be formulated for parenteral administration by injection (e.g., by 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 oils or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids, or propylene glycol); and, if necessary, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers and preservatives. In some embodiments, the conjugates can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution or physiological saline buffer. Preparations for injection can be present in unit dosage form, for example, in ampoules or in multi-dose containers, and preservatives are added. The composition can take the form of a suspension, solution or emulsion in an oily or aqueous vehicle, and can contain formulation agents such as suspending agents, stabilizers and / or dispersants.

[0474] Typically, pharmaceutical compositions for in vivo administration are sterile. Sterilization can be accomplished according to methods known in the art, such as heat sterilization, steam sterilization, sterile filtration or irradiation.

[0475] The dosage and desired drug concentration of the pharmaceutical composition as described herein may vary depending on the specific use envisioned. The determination of an appropriate dosage or route of administration is entirely within the capabilities of those skilled in the art. Suitable dosages are also described above.

[0476] Reagent test kit

[0477] In some embodiments, a kit comprising a conjugate as described herein is provided. In some embodiments, the kit is used to modulate 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 used to modulate the expression of a target gene.

[0478] In some embodiments, the kit also includes one or more additional therapeutic agents. For example, in some embodiments, the kit includes a conjugate as described herein and also includes one or more additional therapeutic agents. In some embodiments, the kit also includes explanatory materials (e.g., instructions for administering the composition across the blood-brain barrier using a kit) containing instructions (i.e., schemes) for implementing the methods described herein. Although explanatory materials typically include written or printed materials, they are not limited thereto. 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., disks, tapes, cassettes (cartridges), chips), optical media (e.g., CD-ROMs), etc. Such media may include the website address of an Internet site that provides such explanatory materials.

[0479] Table 3. List of informal sequences

[0480]

[0481]

[0482]

[0483]

[0484]

[0485]

[0486]

[0487]

[0488]

[0489]

[0490]

[0491]

[0492]

[0493]

[0494] Example

[0495] Subject matter will be described in further 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 that various non-critical parameters that can be changed or modified to produce substantially the same results. Efforts have been made to ensure the accuracy of the numbers used (e.g., amount, temperature, etc.), but there may be some experimental errors and deviations. Unless otherwise indicated, the implementation of the present invention will adopt conventional methods of protein chemistry, biochemistry, recombinant DNA technology and pharmacology within the technical scope of the art. These technologies are fully explained in the literature.

[0496] Example 1: Mono-Fab and bivalent antibody conjugates

[0497] For single Fab and bivalent antibodies, the heavy chain vector and the corresponding light chain vector were cotransfected into Expi293 cells with a knob: mortar: light chain ratio of 1: 1: 2. The protein expressed was 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). The protein was eluted with 50mM sodium citrate (pH 3.0, containing 150mM NaCl) and immediately neutralized with 200mM arginine, 137mM succinic acid (pH 5.0). Using 200mM arginine, 137mM succinic acid (pH 5.0) as running buffer, the protein was further purified by size exclusion chromatography (SEC) (GE Superdex200). The purified protein was confirmed by intact mass LC / MS, and purity>95% was confirmed by SDS-PAGE and analytical HPLC-SEC. The combination with the human and cynomolgus monkey TfR apical domain was tested via biacore.

[0498] Table 4.

[0499] molecular HC1 HC2 LC1 LC2 TfR-Single Fab SEQ ID NO:19 SEQ ID NO:20 SEQ ID NO:18 SEQ ID NO:101 TfR-Single Fab 2 SEQ ID NO:173 SEQ ID NO:174 SEQ ID NO:18 N / A Bivalent anti-TfR antibody SEQ ID NO:10 SEQ ID NO:11 SEQ ID NO:9 SEQ ID NO:9

[0500] The mono-Fab and bivalent antibodies generated above contain cysteine ​​modifications for conjugation and are first reduced using a reducing agent (e.g., TCEP). After reduction, the remaining reducing agent is removed (purified by, for example, dialysis) and the antibody is reoxidized with an oxidant (e.g., dHAA). ASOs containing linking groups are also generated, followed by reduction and oxidation steps. The reduced and oxidized linker-ASOs are then conjugated to free cysteine ​​on the mono-Fab and bivalent antibodies. The resulting conjugates are purified to remove unwanted and unconjugated products, and the purity is determined by LC / MS and SEC.

[0501] An exemplary ASO sequence targeting MALAT1 used herein is:

[0502] 5′-G ks m C ks A ks T ds T ds m C ds 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). The abbreviations refer to the following components: d: DNA; k: LNA; m C: 5-methylcytidine (methylated cytosine); s: phosphorothioate backbone (PS). The ASO is modified with a 5′C6 amine. Another exemplary ASO sequence targeting MALAT1 is SEQ ID NO: 172 (cynomolgus monkey MALAT1)

[0503] An exemplary linker group for use herein is shown below, where the linker group is attached to the sulfur atom of a cysteine ​​residue within a mono-Fab or bivalent antibody and is attached to the ASO via a phosphate associated with the 5' terminal residue of the ASO:

[0504]

[0505] Example 2: In vivo pharmacokinetics and Malat1 knockout using TfR mono-Fab conjugate.

[0506] The monovalent TfR Fab conjugate prepared above ("TfR monoFab") was diluted in sterile saline before administration. As controls, saline, unconjugated ASO, and RSV-ASO groups were included.

[0507] In the single-dose study, the TfR was tested at 2 months of age according to the groups (n=4) in Table 4 below. ms / hu Female mice were administered intravenous doses. Tissues were collected 24 hours after a single dose. In particular, brain, spinal cord and peripheral organs (kidney, lung, liver and quadriceps) were harvested. Peripheral blood was also collected 24 hours after a single dose.

[0508] In the multiple-dose study, 2-month-old TfR ms / hu Female mice were administered intravenous doses. Plasma was collected at 30 minutes, 4 hours, 24 hours, 48 ​​hours, 72 hours and 1 week. Tissues were collected 72 hours after the last administration. In particular, brain, spinal cord and peripheral organs (kidney, lung, liver and quadriceps) were harvested. Peripheral blood was also collected 72 hours after the last administration.

[0509] Table 5

[0510] Group Details Dosage (mg / kg) 1 Negative control: saline 2 Negative control: naked ASO (unconjugated ASO) 1 3 Negative control: RSV-ASO 25 8 Single Fab 25

[0511] Intact drug and total ASO were measured according to the following methods.

[0512] huIgG assay

[0513] The quantification of humanized antibodies in mouse plasma and tissue lysates is measured using a general electrochemiluminescent immunoassay (ECLIA). In short, in the wells of the MSD GOLD 96-well streptavidin-coated microtiter plates (Meso Scale Discovery, Rockville, MD), the biotinylated goat anti-human IgG polyclonal primary antibody (Southern Biotech, Birmingham, AL) of the working concentration prepared in the assay diluent is incubated for about 1 hour. After the incubation and plate washing steps, the prepared test sample (pre-dilution of the sample, when appropriate) and the relevant standard are added to the assay plate and allowed to be incubated for about 1 hour. After the test sample incubation and plate washing steps, the ruthenium (SULFO-TAG) goat anti-human IgG secondary antibody (Meso Scale Discovery, Rockville, MD) of the working concentration in the assay diluent is added to the assay plate and incubated for about 1 hour. After the plate is washed, 1x MSD is then added to read buffer T (Meso Scale Discovery, Rockville, MD) to generate electrochemiluminescence (ECL) assay signals, which are then expressed in ECL units (ECLU). All assay reaction steps are performed at ambient temperature on a plate oscillator (when appropriate); and all test samples are pre-diluted with a 1:20 assay MRD before analysis in the assay plate. The sample ECLU signal generated in the assay is then processed into a concentration by reverse calculation of the assay calibration (CS) curve. The CS curve is fitted with a weighted four-parameter nonlinear logistic regression to calculate the concentration of unknown / test samples.

[0514] Complete drug assay

[0515] The quantification of intact drug (anti-TfR antibody conjugated with antisense oligonucleotide (ASO)) in mouse plasma and tissue lysates was measured using hybridization-based electrochemiluminescent immunoassay (ECLIA). Briefly, custom biotinylated antisense probes (synthesized by Integrated DNA Technologies, Coralville, IA) were incubated at working concentrations with prepared test samples (pre-dilution of samples, as appropriate) and relevant standards in TE buffer (10 mM Tris-HCL containing 1 mM EDTA) and hybridized at an appropriate temperature for 45 minutes. After incubation, the hybridization products were added to the wells of MSD GOLD 96-well streptavidin-coated microtiter plates (Meso Scale Discovery, Rockville, MD) and incubated for approximately 30 minutes. After the hybridization product incubation and plate washing steps, the ruthenium-labeled (SULFO-TAG) goat anti-human IgG secondary antibody (Meso Scale Discovery, Rockville, MD) at a working concentration in the assay diluent is added to the assay plate and incubated for about 1 hour. After the plate is washed, 1x MSD reading buffer T (Meso Scale Discovery, Rockville, MD) is then added to generate an electrochemiluminescence (ECL) assay signal, which is then expressed in ECL units (ECLU). All assay reaction steps are performed at ambient temperature on a plate oscillator (when appropriate); and all test samples are pre-diluted with a 1:20 assay MRD before analysis in the assay plate. The sample ECLU signal generated in the assay is then processed into a concentration by reverse calculation of the assay calibration (CS) curve. The CS curve is fitted with a weighted four-parameter nonlinear logistic regression to calculate the concentration of unknown / test samples.

[0516] Total ASO assay

[0517] The total ASO (in conjugated and free form) in mouse plasma and tissue homogenate was measured using hybridization-based electrochemiluminescent immunoassay (ECLIA). In short, the custom biotinylated and digoxigenin-conjugated antisense probe (synthesized by Integrated DNA Technologies, Coralville, IA) was combined with the prepared test sample (pre-dilution of the sample when appropriate) and the relevant standard in TE buffer (10mM Tris-HCL containing 1mM EDTA) at a working concentration. The samples prepared in TE buffer were added to 1x SSC buffer (Sigma-Aldrich, St.Louis, MO) containing a working concentration of recombinant proteinase K enzyme (ThermoFisher, Waltham, MA) with a 1:1 mixture. The hybridization / enzyme mixture was then digested, denatured, annealed and cooled in a thermal cycler. After the hybridization product incubation, the sample was added to the well of the MSDGOLD 96-well streptavidin-coated microtiter plate (Meso Scale Discovery, Rockville, MD) and incubated for about 30 minutes. After the incubation and plate washing steps, the ruthenium-labeled (SULFO-TAG) sheep anti-digoxigenin antibody (Novus Biologicals, Littleton, CO) at the working concentration in the assay dilution night was added to the plate and incubated for about 30 minutes. After the plate was washed, 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 shaken on a plate oscillator (when appropriate) at ambient temperature; and all test samples were pre-diluted with a 1:20 assay MRD before analysis in the assay plate. The sample ECLU signal generated in the assay was then processed into a concentration by reverse calculation of the assay calibration (CS) curve. The assay CS curves were fitted with a weighted four-parameter nonlinear logistic regression and used to calculate the concentrations of unknown / test samples.

[0518] Malat 1 expression analysis

[0519] Malat1 expression in brain, spinal cord, liver, heart, quadriceps, diaphragm and sciatic nerve was measured as follows. Tissue blocks <50 mg were homogenized in Trizol with a bead homogenizer for large RNA isolation. The homogenized tissue was incubated with chloroform for 3-5 minutes to allow phase separation after centrifugation. The aqueous phase was then incubated with isopropanol for 10 minutes to allow RNA precipitation, followed by washing with 75% ethanol and resuspending in nuclease-free water. The expression of Malat1 was then measured by qPCR using the Express One-Step Superscript kit and normalized to the expression of the housekeeping gene Gapdh.

[0520] The results are shown in Figures 1 to 5 Increased delivery of intact drug and total ASO to the CNS was observed with the TfR mono-Fab conjugate compared to naked ASO and RSV-ASO controls ( Figure 1 Increased Malat1 knockdown in the CNS was also observed in both single-dose and multiple-dose studies compared to controls (Figure 2). Malat1 knockdown was also observed in peripheral tissues ( Figure 5 In single-dose studies, the liver was the site of ASO accumulation 24 hours after dosing ( Figure 3 In multiple-dose studies, accumulation of ASO in the liver and kidneys was observed 72 hours after the last dose ( Figure 4 ).

[0521] Example 3: In vivo Malat1 knockdown using anti-TfR bivalent antibody conjugate.

[0522] The bivalent anti-TfR antibody conjugated to Malat1 ASO prepared as in Example 1 was diluted in sterile saline and administered intravenously to the TfR at a dose of 50 mg / kg per week. ms / hu knock-in mice for 4 weeks. ms / hu Two control groups of mice were intravenously administered sterile saline or unconjugated ASO. Three days after the fourth dose, tissues were collected and frozen for molecular and biochemical analysis. Tissues included brain, spinal cord, liver, heart, quadriceps, diaphragm, and sciatic nerve.

[0523] Malatl expression in brain, spinal cord, liver, heart, quadriceps muscle, diaphragm and sciatic nerve was measured as described above.

[0524] The results are shown in Figure 6 Some Malat1 knockdown was observed in the CNS, and higher Malat1 knockdown was observed in the periphery.

[0525] Example 4: In vivo pharmacokinetics and biodistribution using TfR mono-Fab conjugates

[0526] Two TfR-monoFab conjugates (TfR monoFab and TfR monoFab 2) were generated in Example 1 and diluted in sterile saline before application.TfR monoFab conjugate has TfR binding arm and non-binding RSV arm and is conjugated to mouse MALAT1 (SEQ ID NO:8) (anti-TfR / non-binding Fab antibody-oligonucleotide), and TfR-monoFab 2 conjugate has TfR binding arm (second arm does not exist; monoFab) and is conjugated to cyno MALAT1 (SEQ ID NO:172).Unconjugated ASO is used as a control.The dosage of naked ASO (0.9mg / kg (mpk)), TfR-monoFab conjugate (25mpk) or TfR-monoFab 2 conjugate (17.2mpk) was intravenously administered to two-month-old TfR ms / hu female mice.The following tissues were collected 24 hours after a single dose: brain, spinal cord, kidney, diaphragm, liver and quadriceps. Plasma was also collected 15 minutes, 4 hours, and 24 hours after a single dose.

[0527] Total ASO and total huIgG were measured according to the method described in Example 2 above. The results are shown in Figures 7 to 9 The two TfR-monoFab conjugate molecules showed similar pharmacokinetic characteristics in plasma ( Figure 7 ) and similar biodistribution patterns throughout the body ( Figure 8 Molar equivalent amounts of two TfR-monoFab molecules achieved robust CNS ASO uptake compared to unconjugated ASO, which was rapidly cleared from the circulation after a 0.9 mpk dose and was not detected in the brain or spinal cord ( Fig. 9 In addition, both TfR-monoFab molecules resulted in more ASO in the diaphragm, quadriceps, and liver, but significantly less ASO in the kidney ( Fig. 9 ).

[0528] Example 5: TfR-albumin-ASO construction

[0529] TfR-albumin-ASO molecules are generated by fusing the scFv that binds TfR to mouse serum albumin via a linker. Linker-ASOs were 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 purposes. For bioconjugation of linker-ASO to TfR-albumin, TCEP (30 molar equivalents) was first used to reduce TfR-albumin. The linker-ASO was then conjugated to the free cysteine ​​(1.2 molar equivalents) on TfR-albumin. The resulting conjugate was purified by cation exchange chromatography (mobile phase A: 20mM sodium acetate, pH 5; mobile phase B1: 20mm sodium acetate, 1M NaCl, pH 5) to remove unwanted and unconjugated products, and the purity was determined by LC / MS and analytical SEC.

[0530] Example 6: In vivo pharmacokinetics and biodistribution using TfR-albumin-ASO conjugates

[0531] The TfR-albumin-ASO molecules prepared above were diluted in sterile saline before administration. As a control, unconjugated ASO was also administered.

[0532] TfR for 4-8 months ms / hu Female mice were dosed intravenously. TfR-albumin-ASO was dosed at 9.5 mg / kg (n=2). Unconjugated ASO ("naked ASO") was dosed at 1.37 mg / kg (n=3). Plasma was collected 15 minutes (unconjugated ASO only), 4 hours, and 24 hours after dosing. Tissues including brain, liver, and kidney, as well as terminal plasma were collected 72 hours after dosing. ASO concentrations were measured as described in Example 2.

[0533] The results are shown in Figures 10 to 12 Compared with naked ASO, increased ASO delivery to the brain was observed with TfR-albumin-ASO conjugates ( Fig.10 The liver and kidney are the sinks of naked ASO, and TfR-albumin-ASO conjugates reduced ASO concentrations in these organs ( Fig.11 In plasma, clearance was similar between the two molecules, but clearance was slightly faster for the TfR-albumin-ASO conjugate ( Fig.12 ).

[0534] Many modifications and other embodiments of the subject matter set forth herein will occur to those skilled in the art having the benefit of the foregoing description and the teachings presented in the associated drawings. Therefore, it should be understood that the invention is not limited to the specific embodiments disclosed, and 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. A TfR binder-oligonucleotide conjugate, comprising: in, P contains the 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 greater than or equal to 1 (e.g., 1, 2, 3, or 4); and n is an integer greater than or equal to 1 (eg, 1, 2, 3, 4, 5, 6, 7, or 8).

2. The TfR binder-oligonucleotide conjugate of claim 1, wherein F is an Fc dimer; and P′ does not exist.

3. The TfR binder-oligonucleotide conjugate of claim 2, wherein P is anti-TfR Fab, single-chain Fab (scFab), or single-chain variable fragment (scFv); 4. The TfR binder-oligonucleotide conjugate of claim 3, wherein P is anti-TfR Fab or scFab.

5. The TfR binder-oligonucleotide conjugate of claim 1, wherein F is an Fc dimer; and P' is non-binding Fab or NBVR.

6. The TfR binder-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. The TfR binder-oligonucleotide conjugate of claim 6, wherein P is anti-TfR Fab or scFab.

8. The TfR binder-oligonucleotide conjugate of claim 1, wherein F is albumin; and P′ does not exist.

9. The TfR binder-oligonucleotide conjugate of claim 8, wherein P is a scFv or a nanobody.

10. The TfR binder-oligonucleotide conjugate of claim 9, wherein P is a scFv.

11. The TfR binder-oligonucleotide conjugate of claim 1, wherein F is an Fc dimer; and P′ is the anti-TfR antibody antigen binding domain.

12. The TfR binder-oligonucleotide conjugate of claim 11, wherein PFP' comprises an antibody.

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

14. The TfR binder-oligonucleotide conjugate of claim 11, wherein P is anti-TfR Fab, single-chain Fab (scFab), single-chain variable fragment (scFv) or nanobody.

15. The TfR binder-oligonucleotide conjugate of claim 14, wherein P is anti-TfR Fab or scFab.

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

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

18. The TfR binder-oligonucleotide conjugate of any one of claims 1-17, wherein P comprises 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, and 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, and CDR-L3 comprises SEQ ID NO:26; (c) CDR-H1 comprises SEQ ID NO: 114, and 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, and CDR-L3 comprises SEQ ID NO: 119; (d) CDR-H1 comprises SEQ ID NO: 126, and 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, and 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, and 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, and 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, and CDR-L3 comprises SEQ ID NO:

166.

19. The TfR binder-oligonucleotide conjugate of any one of claims 1-8 and 11-17, wherein P is an anti-TfR Fab comprising: (a) SEQ ID NO: 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; Optionally wherein P comprises one or more modifications for covalent conjugation of said oligonucleotide.

20. The TfR binder-oligonucleotide conjugate of any one of claims 1-3, 5-6, 8-10, 11, 14, and 16-17, wherein P is a scFv comprising: (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 NO: 112 and 113 (g) SEQ ID NO: 124 and 125 (h) SEQ ID NOs: 145 and 146.

21. The TfR binder-oligonucleotide conjugate of any one of claims 1-3, 5-6, 8-10, 11, 14, and 16-17, wherein P is an antibody comprising: (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 NO: 18, 19; Optionally wherein the antibody 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. The TfR binder-oligonucleotide of any one of claims 1-21, wherein P, F or P' comprises a modification for conjugation of the oligonucleotide.

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

24. The TfR binder-oligonucleotide conjugate of claim 23, wherein F comprises the Fc polypeptide or the Fc dimer, and wherein the cysteine ​​modification is a S239C, S442C, A330C or T289C substitution, wherein positions are numbered according to EU.

25. The TfR binder-oligonucleotide conjugate of claim 24, wherein the cysteine ​​modification is the S239C substitution.

26. A TfR binder-oligonucleotide conjugate as described in claim 23, wherein P comprises Fab or scFab, and wherein the cysteine ​​modification comprises a K149C substitution, wherein the position is numbered according to EU; or an A114C substitution, wherein the position is numbered according to Kabat.

27. The TfR binder-oligonucleotide conjugate of claim 22, wherein F comprises an Fc polypeptide or an Fc dimer, and wherein the modification comprises an N297A substitution or an N297G substitution, wherein positions are numbered according to EU.

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

29. The TfR binder-oligonucleotide conjugate of claim 28, wherein the at least one modification comprises: (a) L234A substitution and L235A substitution; (b) P329G substitution; or (c) L234A substitution, L235A substitution, and P329G substitution; The positions are numbered according to EU.

30. The TfR binder-oligonucleotide conjugate of claim 28 or 29, wherein the at least one modification comprises: (a) M428L substitution and N434S substitution; (b) M428L substitution; (b) N434S substitution; (c) N434A substitution; or (d) M252Y substitution, S254T substitution, and T256E substitution; The positions are numbered according to EU.

31. The TfR binder-oligonucleotide conjugate of any one of claims 28-30, wherein the at least one modification comprises: Deletion of the carboxyl-terminal lysine.

32. The TfR binder-oligonucleotide conjugate of any one of claims 28-31, wherein the at least one modification comprises: (a) M428L substitution and N434S substitution; (b) M428L substitution; (b) N434S substitution; (c) N434A substitution; or (d) M252Y substitution, S254T substitution, and T256E substitution; The positions are numbered according to EU.

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

34. A TfR binder-oligonucleotide conjugate as described in claim 33, wherein the knob mutation comprises a T366W substitution according to the EU numbering scheme; and the hole mutation comprises a Y407V substitution according to the EU numbering scheme and optionally a T366S substitution and a L368A substitution.

35. The TfR binder-oligonucleotide conjugate of any one of claims 1, 5-7, 11-34, wherein P′ is present and comprises a non-binding Fab or NBVR, wherein the non-binding Fab or NBVR comprises three heavy chain CDRs and three light chain CDRs, wherein: (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, and 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, and 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, and CDR-L3 comprises SEQ ID NO:44; or 36. The TfR binder-oligonucleotide conjugate of claim 35, wherein P′ comprises: (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; (1) 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) SEQ ID NO:62 or 64 and SEQ ID NO:

71.

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

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

39. The conjugate of any one of claims 22-36, wherein the oligonucleotide is conjugated to the modified site via the linker.

40. The TfR binder-oligonucleotide conjugate of claim 1, comprising: A protein comprising: Fc dimer, a first Fab known to specifically bind TfR, and S239C substitution, where positions are numbered according to EU; and An oligonucleotide conjugated to the protein at the S239C substitution.

41. A method for regulating target gene expression in a muscle cell of a subject, the method comprising administering to the subject a TfR binder-oligonucleotide conjugate as described in any one of claims 1-40.

42. The method of claim 41, wherein the muscle cell is a cardiac muscle cell or a skeletal muscle cell.

43. A method of delivering an oligonucleotide to the central nervous system (CNS) of a subject, the method comprising administering to the subject a TfR binder-oligonucleotide conjugate as described in any one of claims 1-40.

44. A method of delivering an oligonucleotide to a cancer in a subject, the method comprising administering to the subject a TfR binder-oligonucleotide conjugate as described in any one of claims 1-40.

45. A TfR binder-oligonucleotide conjugate, comprising: An antibody that specifically binds to TfR, wherein the antibody comprises 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, and 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, and 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, and CDR-L3 comprises SEQ ID NO: 119; (d) CDR-H1 comprises SEQ ID NO: 126, and 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, and 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, and 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, and 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, and CDR-L3 comprises SEQ ID NO: 166; and Oligonucleotides conjugated to cysteine ​​modifications on the constant domains of said antibodies.

46. ​​A TfR binder-oligonucleotide conjugate as described in claim 45, wherein the cysteine ​​modification is a S239C, S442C, A330C, K149C or T289C substitution, wherein the position is numbered according to EU; or an A114C substitution, wherein the position is numbered according to Kabat.

47. The TfR binder-oligonucleotide conjugate of claim 46, wherein the cysteine ​​modification is the S239C substitution.

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

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

50. A TfR binder-oligonucleotide conjugate, comprising: An antibody that specifically binds to TfR, wherein the antibody comprises 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, and 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, and CDR-L3 comprises SEQ ID NO:26; (c) CDR-H1 comprises SEQ ID NO: 114, and 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, and CDR-L3 comprises SEQ ID NO: 119; (d) CDR-H1 comprises SEQ ID NO: 126, and 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, and 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, and 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, and 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, and CDR-L3 comprises SEQ ID NO: 166; and The oligonucleotide was conjugated via a linker to the S239C substitution on the constant domain of the antibody, where positions are numbered according to EU.

51. A method for regulating target gene expression in a muscle cell of a subject, the method comprising administering to the subject a TfR binder-oligonucleotide conjugate as described in any one of claims 45-50.

52. The method of claim 51, wherein the muscle cell is a cardiac muscle cell or a skeletal muscle cell.

53. A method of delivering an oligonucleotide to the CNS of a subject, the method comprising administering to the subject a TfR binder-oligonucleotide conjugate as described in any one of claims 45-50.

54. A method of delivering an oligonucleotide to a cancer cell in a subject, the method comprising administering to the subject a TfR binder-oligonucleotide conjugate as described in any one of claims 45-50.

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