Methods and compositions for inhibiting the progression of intramuscular fibrosis

Abstract

The present application relates to conjugates comprising a muscle targeting agent covalently linked to a molecular payload for delivery to cells (e.g., muscle cells) and uses thereof, particularly in connection with the treatment and / or prevention of disease, amelioration, delay in onset, inhibition of progression, or prevention of fibrosis (e.g., intramuscular fibrosis such as endomysial fibrosis, perimysial fibrosis, and epimysial fibrosis).

Description

[Technical Field]

[0001] Related Applications This application claims the benefit under 35 U.S.C. §119(e) of Provisional Patent Application No. 63 / 487,035, entitled "METHODS AND COMPOSITIONS FOR INHIBITING PROGRESSION OF INTRAMUSCULAR FIBROSIS," filed February 27, 2023, the entire contents of which are incorporated herein by reference.

[0002] FIELD OF THE INVENTION The present application relates to targeted complexes for delivering molecular payloads to muscle cells, formulations containing such complexes, and uses thereof, particularly for inhibiting the progression of and / or reducing intramuscular fibrosis.

[0003] Reference to Electronic Sequence Listing The contents of the Electronic Sequence Listing (D082470084WO00-SEQ-COB.xml; size: 1,892,295 bytes; and creation date: February 22, 2024) are incorporated herein by reference in their entirety. [Background technology]

[0004] background Dystrophinopathies are a group of distinct neuromuscular diseases caused by mutations in the dystrophin gene. Dystrophinopathies include Duchenne muscular dystrophy, Becker muscular dystrophy, and X-linked dilated cardiomyopathy. Dystrophin (DMD) is a large gene containing 79 exons and approximately 2.6 million total base pairs. Many DMD mutations, including exonic frameshifts, deletions, substitutions, and duplications, can reduce functional dystrophin expression, resulting in dystrophinopathy. Many patients with dystrophinopathies exhibit degeneration of muscle tissue, including myofibrosis. Summary of the Invention

[0005] overview According to some aspects, the present disclosure provides a conjugate that targets muscle cells for delivering a molecular payload to these cells. In some embodiments, the conjugates provided herein are particularly useful for delivering a molecular payload that increases or restores the expression or activity of a functional dystrophin protein. Thus, in some embodiments, the conjugates provided herein include a muscle-targeting agent (e.g., a muscle-targeting antibody) that specifically binds to a receptor on the muscle cell surface for the purpose of delivering the molecular payload to the muscle cell. In some embodiments, the conjugate is taken up into the cell via receptor-mediated internalization, after which the molecular payload may be released to perform its function inside the cell. For example, a conjugate modified to deliver an oligonucleotide may release the oligonucleotide such that the oligonucleotide can promote the expression of DMD and / or functional dystrophin protein in the muscle cell (e.g., through an exon skipping mechanism).

[0006] It has been surprisingly demonstrated herein that timely administration of a complex described herein to a subject having or at risk of having Duchenne muscular dystrophy inhibits the progression of intramuscular fibrosis in the subject, resulting in benefits such as prolonged muscle integrity and function. In some embodiments, timely administration of a complex described herein to a subject having or at risk of having Duchenne muscular dystrophy results in a reduction in intramuscular fibrosis in the subject. In some embodiments, timely administration of a complex described herein to a subject having or at risk of having Duchenne muscular dystrophy results in a reduction in fibrosis (e.g., muscle fibrosis) in the subject. In some embodiments, the fibrosis is endomysial fibrosis. In some embodiments, the fibrosis is perimysial fibrosis. In some embodiments, the fibrosis is epimysial fibrosis. For example, in some embodiments, timely administration of a complex described herein results in a prolonged period of time during which the subject's skeletal muscle remains in a pre-fibrotic state. In some embodiments, timely administration of a complex described herein results in a longer period of time before the onset (e.g., substantial onset) of fibrosis (e.g., endomysial fibrosis) in skeletal muscles (e.g., appendicular muscles, quadriceps muscles) that control a subject's ability to walk, compared to the absence of treatment with a complex described herein. In some embodiments, timely administration of a complex described herein results in a longer period of time before loss (e.g., decline) of motor function in a subject. In some embodiments, timely administration of a complex described herein results in a longer period of time before loss of locomotion (e.g., independent walking) in a subject.

[0007] In some embodiments, timely administration includes initiating treatment with a complex described herein in a subject having or at risk of having Duchenne muscular dystrophy, which is shown herein to be particularly beneficial to the subject in inhibiting the progression of intramuscular fibrosis, at an early stage of the disease (e.g., when the subject's skeletal muscle is in a pre-fibrotic state). In some embodiments, timely administration includes initiating treatment with a complex described herein in a subject having or at risk of having Duchenne muscular dystrophy, which is shown herein to be particularly beneficial to the subject in inhibiting the progression of fibrosis (e.g., myofibrosis), at an early stage of the disease (e.g., when the subject's skeletal muscle is in a pre-fibrotic state). In some embodiments, timely administration is beneficial to the subject in reducing fibrosis (e.g., myofibrosis, such as intramuscular fibrosis). In some embodiments, the fibrosis is endomysial fibrosis. In some embodiments, the fibrosis is perimysial fibrosis. In some embodiments, the fibrosis is epimysial fibrosis.In some embodiments, timely administration includes at least one or more of the following: (i) initiating administration of a complex described herein as soon as a subject is diagnosed with or at risk of having Duchenne muscular dystrophy; (ii) initiating administration of a complex described herein prior to the progression of muscle degeneration that leads to muscle fibrosis; (ii) initiating administration of a complex described herein prior to the progression of extracellular matrix protein (e.g., collagen and fibronectin) accumulation in muscle tissue that leads to the replacement of muscle connective tissue with fibrotic tissue; (iii) initiating administration of a complex described herein prior to the progression of muscle degeneration that leads to the replacement of muscle connective tissue with fibrotic tissue in a subject's skeletal muscle (e.g., quadriceps, etc.); (iv) initiating administration of a complex described herein before the subject loses motor function; (v) initiating administration of a complex described herein before the subject loses walking; and (vi) once administration has begun in a subject (e.g., at any time during (i)-(v)), continuing administration of a complex described herein for a period during which the subject's skeletal muscle is in a pre-fibrotic state (e.g., with delivery of the complex administered multiple times during that period, e.g., monthly, bimonthly).

[0008] According to some aspects, methods for inhibiting the progression of intramuscular fibrosis in a subject are provided. According to some aspects, methods for inhibiting the progression of fibrosis (e.g., myofibrosis) in a subject are provided. In some embodiments, the method for inhibiting the progression of intramuscular fibrosis in a subject having a loss-of-function mutation in the dystrophin (DMD) gene that abolishes dystrophin production comprises timely administering to the subject an effective amount of a conjugate comprising an anti-TfR1 antibody covalently linked to a molecular payload configured to promote dystrophin expression or activity, resulting in the inhibition of the progression of intramuscular fibrosis in the subject. In some embodiments, the method for inhibiting the progression of fibrosis (e.g., myofibrosis) in a subject having a loss-of-function mutation in the dystrophin (DMD) gene that abolishes dystrophin production comprises timely administering to the subject an effective amount of a conjugate comprising an anti-TfR1 antibody covalently linked to a molecular payload configured to promote dystrophin expression or activity, resulting in the inhibition of the progression of fibrosis (e.g., myofibrosis) in the subject. In some embodiments, the fibrosis is endomysial fibrosis. In some embodiments, the fibrosis is perimysial fibrosis. In some embodiments, the fibrosis is epimysial fibrosis.

[0009] In some embodiments, administration of the complex is initiated when the subject's skeletal muscle tissue is in a pre-degenerative state. In some embodiments, administration of the complex is initiated when the subject's skeletal muscle tissue is in a pre-fibrotic state.

[0010] In some embodiments, the complex is administered to the subject multiple times over a period of time before substantial onset of endomysial fibrosis in the subject's quadriceps muscle. In some embodiments, the complex is administered to the subject multiple times over a period of time before substantial onset of fibrosis (e.g., endomysial fibrosis, perimysial fibrosis, and / or epimysial fibrosis) in the subject's quadriceps muscle. In some embodiments, the complex is administered to the subject multiple times over a period of time before the subject becomes unable to walk.

[0011] According to some aspects, methods for treating a subject diagnosed with or at risk of having Duchenne muscular dystrophy are provided. In some embodiments, the methods for treating a subject diagnosed with or at risk of having Duchenne muscular dystrophy include administering to the subject a conjugate comprising an anti-TfR1 antibody covalently linked to a molecular payload configured to promote dystrophin expression or activity, wherein the administration is for a period during which the subject's skeletal muscle is in a pre-fibrotic state.

[0012] In some embodiments, administration of the conjugate increases the period of time that a subject's skeletal muscle remains in a pre-fibrotic state compared to when the conjugate is not administered.

[0013] In some embodiments, the prefibrotic state precedes the substantial onset of fibrosis (e.g., endomysial fibrosis, perimysial fibrosis, and / or epimysial fibrosis) in skeletal muscles that control the subject's ability to walk. In some embodiments, the prefibrotic state precedes the substantial onset of endomysial fibrosis in skeletal muscles that control the subject's ability to walk. In some embodiments, the prefibrotic state precedes the substantial onset of fibrosis (e.g., endomysial fibrosis, perimysial fibrosis, and / or epimysial fibrosis) in limb muscles of the subject. In some embodiments, the prefibrotic state precedes the substantial onset of endomysial fibrosis in limb muscles of the subject. In some embodiments, the prefibrotic state precedes the substantial onset of fibrosis (e.g., endomysial fibrosis, perimysial fibrosis, and / or epimysial fibrosis) in the quadriceps muscles of the subject. In some embodiments, the prefibrotic state precedes the substantial onset of endomysial fibrosis in the quadriceps muscles of the subject. In some embodiments, the pre-fibrotic state is before there is a substantial decline in motor function in the limb muscles of the subject.

[0014] According to some aspects, methods of treating a subject diagnosed with or at risk of having Duchenne muscular dystrophy are provided. In some embodiments, the method comprises administering to the subject a conjugate comprising an anti-TfR1 antibody covalently linked to a molecular payload configured to promote dystrophin expression or activity, wherein the subject has a DMD gene modifier that promotes LTBP4-dependent, TGF-β1-mediated fibrosis.

[0015] In some embodiments, the subject has a hyperfibrotic polymorphism in LTBP4.

[0016] In some embodiments, the subject is undergoing or has undergone treatment with a corticosteroid. In some embodiments, the corticosteroid is a glucocorticoid or a dissociative steroid. In some embodiments, the corticosteroid is prednisone, prednisolone, dexamethasone, deflazacort, or vamorolone.

[0017] In some embodiments, the method further comprises administering a corticosteroid to the subject. In some embodiments, the corticosteroid is a glucocorticoid or a dissociative steroid. In some embodiments, the corticosteroid is prednisone, prednisolone, dexamethasone, deflazacort, or vamorolone.

[0018] In some embodiments, administration of the complex to a subject inhibits the progression of fibrosis (e.g., myofibrosis) in the subject. In some embodiments, fibrosis (e.g., myofibrosis) is measured by histological analysis of skeletal muscle tissue in a muscle biopsy sample from the subject or by evaluation of magnetic resonance imaging (MRI) of skeletal muscle tissue in the subject. In some embodiments, administration of the complex to a subject inhibits the progression of intramuscular fibrosis in the subject. In some embodiments, intramuscular fibrosis is measured by histological analysis of skeletal muscle tissue in a muscle biopsy sample from the subject or by evaluation of magnetic resonance imaging (MRI) of skeletal muscle tissue in the subject. In some embodiments, the fibrosis is endomysial fibrosis. In some embodiments, the fibrosis is perimysial fibrosis. In some embodiments, the fibrosis is epimysial fibrosis.

[0019] In some embodiments, the histological analysis comprises staining for one or more extracellular matrix components in a muscle biopsy sample from the subject and determining the percentage of tissue in the muscle biopsy sample that stains positive for one or more extracellular matrix components. In some embodiments, the stain is picrosirius red staining.

[0020] In some embodiments, the subject is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 years of age or younger.

[0021] In some embodiments, the molecular payload comprises an oligonucleotide, wherein the oligonucleotide promotes exon skipping in the DMD RNA and / or the oligonucleotide comprises a region of complementarity to the DMD RNA.

[0022] In some embodiments, the subject has a DMD gene that is prone to exon skipping. In some embodiments, the exon ranges from exon 8 to exon 55. In some embodiments, the exon is exon 8, exon 23, exon 43, exon 44, exon 45, exon 46, exon 50, exon 51, exon 52, exon 53, or exon 55. In particular embodiments, the exon is exon 51.

[0023] In some embodiments, the oligonucleotides promote skipping of exons ranging from exon 8 to exon 55, and / or the oligonucleotides comprise a region of complementarity to exons ranging from exon 8 to exon 55. In some embodiments, the oligonucleotides promote skipping of exon 8, exon 23, exon 43, exon 44, exon 45, exon 46, exon 50, exon 51, exon 52, exon 53, and / or exon 55, and / or the oligonucleotides comprise a region of complementarity to exon 8, exon 23, exon 43, exon 44, exon 45, exon 46, exon 50, exon 51, exon 52, exon 53, and / or exon 55. In a particular embodiment, the oligonucleotides promote skipping of exon 51.

[0024] In some embodiments, the oligonucleotide comprises a region of complementarity to one or more complete or partial exonic splicing enhancers (ESEs) of the DMD transcript. In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence comprising one or more complete or partial ESEs as set forth in SEQ ID NOs: 402-436 and 2043-2238.

[0025] In some embodiments, the oligonucleotide promotes skipping of exon 51 and / or the oligonucleotide comprises a region of complementarity to exon 51.

[0026] In some embodiments, the oligonucleotide is 20 to 30 nucleotides in length and comprises a region of complementarity to the target sequence comprising at least four consecutive nucleotides of an ESE as set forth in any one of SEQ ID NOs: 402 to 436.

[0027] In some embodiments, the oligonucleotide comprises any one of SEQ ID NOs: 437-1241, or comprises a region of complementarity to any one of SEQ ID NOs: 1242-2046.

[0028] In some embodiments, the oligonucleotide comprises one or more phosphorodiamidate morpholinos. In some embodiments, the oligonucleotide is a phosphorodiamidate morpholino oligomer (PMO).

[0029] In some embodiments, the anti-TfR1 antibody comprises heavy chain complementarity determining region 1 (CDR-H1), heavy chain complementarity determining region 2 (CDR-H2), heavy chain complementarity determining region 3 (CDR-H3), light chain complementarity determining region 1 (CDR-L1), light chain complementarity determining region 2 (CDR-L2), and light chain complementarity determining region 3 (CDR-L3) of an antibody provided in any one of Tables 2-6.

[0030] In some embodiments, the anti-TfR1 antibody comprises a heavy chain variable region (VH) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 76; and / or a light chain variable region (VL) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 75. In some embodiments, the anti-TfR1 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 76, and a VL comprising the amino acid sequence of SEQ ID NO: 75.

[0031] In some embodiments, the anti-TfR1 antibody is selected from the group consisting of a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an scFv, an Fv, and a full-length IgG. In some embodiments, the anti-TfR1 antibody is a Fab fragment.

[0032] In some embodiments, the anti-TfR1 antibody comprises a heavy chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 101; and / or a light chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 90. In some embodiments, the anti-TfR1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 101; and a light chain comprising the amino acid sequence of SEQ ID NO: 90.

[0033] In some embodiments, the anti-TfR1 antibody is covalently linked to the molecular payload via a cleavable linker. In some embodiments, the cleavable linker comprises a valine-citrulline sequence.

[0034] In some embodiments, the conjugate has a structure represented by formula (E): [ka] or a pharmaceutically acceptable salt thereof, wherein n is 0-15 and m is 0-15, optionally where n is 3 and / or m is 4.

[0035] In some embodiments, L1 is [ka] or a pharmaceutically acceptable salt thereof, wherein L2 is [ka] where a labels the site that is directly linked to the carbamate moiety of formula (E); and b labels the site that is covalently linked to the molecular payload.

[0036] In some embodiments, the molecular payload comprises an oligonucleotide and L1 is linked to the 5' phosphate of the oligonucleotide.

[0037] In some embodiments, the conjugate is administered to the subject via intravenous infusion.

[0038] In some embodiments, the subject is a human. In some embodiments, the subject is a cynomolgus monkey. In some embodiments, the subject is a rodent. [Brief explanation of the drawings]

[0039] [Figure 1] FIG. 1 shows microscopic images of hematoxylin and eosin (H&E) stained tissue sections of muscle tissue taken from D2-mdx mice treated with a vehicle control ("vehicle"), D2-mdx mice treated early with an anti-TfR1 Fab-oligonucleotide conjugate ("early dose of conjugate"), and D2-mdx mice treated late with an anti-TfR1 Fab-oligonucleotide conjugate ("late dose of conjugate").

[0040] [Figure 2] 2 shows fluorescence microscopy images of histological sections of muscle tissue taken from wild-type mice ("WT"), D2-mdx mice treated with a vehicle control ("vehicle"), D2-mdx mice treated early with an anti-TfR1 Fab-oligonucleotide conjugate ("early dosing of conjugate"), and D2-mdx mice treated late with an anti-TfR1 Fab-oligonucleotide conjugate ("late dosing of conjugate"). The sections were stained for dystrophin and laminin proteins.

[0041] [Figure 3]Figure 3 shows microscopic images of histological sections of muscle tissue taken from D2-mdx mice treated with a vehicle control ("vehicle"), D2-mdx mice treated early with an anti-TfR1 Fab-oligonucleotide conjugate ("early dosing of conjugate"), and D2-mdx mice treated late with an anti-TfR1 Fab-oligonucleotide conjugate ("late dosing of conjugate"). Sections were stained with picrosirius red to visualize collagen deposition, which increases during the progression of fibrosis.

[0042] [Figure 4] Figures 4A-4B show the effect of early or late treatment with anti-TfR1 Fab-oligonucleotide conjugates on fibrosis progression in the quadriceps muscles of D2-mdx mice. Figure 4A shows microscopic images of picrosirius red-stained tissue sections from the quadriceps muscles of D2-mdx mice treated with vehicle control (top row, showing samples taken at 5 and 22 weeks of age), D2-mdx mice treated early with anti-TfR1 Fab-oligonucleotide conjugates ("early dosing of conjugate"; middle row), and D2-mdx mice treated late with anti-TfR1 Fab-oligonucleotide conjugates ("late dosing of conjugate"; bottom row). Figure 4B shows quantification of fibrosis area in picrosirius red-stained tissue sections of quadriceps muscle taken from D2-mdx mice treated with vehicle control ("vehicle"), D2-mdx mice treated early with anti-TfR1 Fab-oligonucleotide conjugate ("early dose of conjugate"), and D2-mdx mice treated late with anti-TfR1 Fab-oligonucleotide conjugate ("late dose of conjugate"). Baseline and treatment values ​​for early- and late-dosed mice are shown. Baseline measurements correspond to 5 weeks of age for early-dosed mice and 12 weeks of age for late-dosed mice. Tissues from vehicle, early-dosed, and late-dosed mice were harvested at 22 weeks of age.

[0043] [Figure 5]Figure 5 shows the muscle mass of the quadriceps muscles of D2-mdx mice treated with a vehicle control ("Vehicle") or early treated with an anti-TfR1 Fab-oligonucleotide conjugate ("Early Dose"). (**, P<0.01)

[0044] [Figure 6] Figure 6 shows exon 23 skipping measured in quadriceps, diaphragm, and myocardial tissues from D2-mdx mice treated with a vehicle control ("Vehicle"), early treatment with an anti-TfR1 Fab-oligonucleotide conjugate ("Early Dosing"), or late treatment with an anti-TfR1 Fab-oligonucleotide conjugate ("Late Dosing"). The oligonucleotide in the conjugate used in this experiment is an exon 23 skipping oligonucleotide. DETAILED DESCRIPTION OF THE INVENTION

[0045] DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS Aspects of the present disclosure relate to the recognition that certain disorders (e.g., dystrophinopathies such as Duchenne muscular dystrophy) involve muscle degeneration, such as muscle fibrosis. As described herein, the present disclosure provides complexes comprising a muscle-targeting agent covalently linked to a molecular payload (e.g., a molecular payload configured to promote dystrophin expression or activity) to inhibit or reduce the progression of muscle degeneration. For example, in some embodiments, complexes are provided to target the DMD gene, e.g., a mutated DMD allele, to inhibit or reduce the progression of muscle degeneration (e.g., degeneration associated with fibrosis) in a subject. In some embodiments, the complexes provided herein may comprise an oligonucleotide that promotes normal dystrophin expression and activity. As another example, the complex may comprise an oligonucleotide that induces exon skipping in DMD mRNA. In some embodiments, synthetic nucleic acid payloads (e.g., DNA or RNA payloads) expressing one or more proteins that promote normal dystrophin expression and activity may be used.

[0046] Aspects of the present disclosure relate to methods of administering a conjugate to a subject having or at risk of having Duchenne muscular dystrophy to inhibit the progression of fibrosis (e.g., muscle fibrosis) in the subject, resulting in benefits such as prolonged muscle integrity and function. Aspects of the present disclosure relate to methods of administering a conjugate to a subject having or at risk of having Duchenne muscular dystrophy to inhibit the progression of intramuscular fibrosis in the subject, resulting in benefits such as prolonged muscle integrity and function. In some embodiments, administering a conjugate to a subject having or at risk of having Duchenne muscular dystrophy reduces fibrosis (e.g., muscle fibrosis) in the subject, resulting in benefits such as prolonged muscle integrity and function. In some embodiments, administering a conjugate to a subject having or at risk of having Duchenne muscular dystrophy reduces intramuscular fibrosis in the subject, resulting in benefits such as prolonged muscle integrity and function. In some embodiments, administering a conjugate to a subject having or at risk of having Duchenne muscular dystrophy reduces intramuscular fibrosis in the subject, resulting in benefits such as prolonged muscle integrity and function. In some embodiments, the fibrosis is endomysial fibrosis. In some embodiments, the fibrosis is perimysial fibrosis. In some embodiments, the fibrosis is epimysial fibrosis. For example, in some embodiments, the methods of administering a complex described herein result in a prolonged period of time during which a subject's skeletal muscle remains in a pre-fibrotic state. In some embodiments, the methods of administering a complex described herein result in a prolonged period of time before the onset (e.g., substantial onset) of fibrosis (e.g., endomysial fibrosis) in a skeletal muscle (e.g., appendicular muscle, quadriceps muscle) that controls the subject's ability to walk, compared to the absence of treatment with a complex described herein. In some embodiments, the methods of administering a complex described herein result in a prolonged period of time before a subject loses motor function. In some embodiments, the methods of administering a complex described herein result in a prolonged period of time before a subject loses ambulation (e.g., independent ambulation).

[0047] In some embodiments, the method of administration comprises initiating treatment with a complex described herein in a subject having or at risk of having Duchenne muscular dystrophy at an early stage of the disease (e.g., when the subject's skeletal muscle is in a pre-fibrotic state), which in some embodiments results in inhibition of the progression of fibrosis (e.g., myofibrosis) in the subject. In some embodiments, the method of administration comprises initiating treatment with a complex described herein in a subject having or at risk of having Duchenne muscular dystrophy at an early stage of the disease (e.g., when the subject's skeletal muscle is in a pre-fibrotic state), which in some embodiments results in inhibition of the progression of intramuscular fibrosis in the subject. In some embodiments, treatment with a complex described herein results in a reduction of fibrosis (e.g., myofibrosis) in the subject. In some embodiments, treatment with a complex described herein results in a reduction of intramuscular fibrosis in the subject. In some embodiments, the fibrosis is endomysial fibrosis. In some embodiments, the fibrosis is perimysial fibrosis. In some embodiments, the fibrosis is epimysial fibrosis.In some embodiments, the method of administration includes at least one or more of the following: (i) initiating administration of a complex described herein as soon as a subject is diagnosed with or at risk of having Duchenne muscular dystrophy; (ii) initiating administration of a complex described herein prior to the progression of muscle degeneration that leads to muscle fibrosis; (iii) initiating administration of a complex described herein prior to the progression of extracellular matrix protein (e.g., collagen and fibronectin) accumulation in muscle tissue that leads to the replacement of muscle connective tissue with fibrotic tissue; (iv) initiating administration of a complex described herein prior to the progression of extracellular matrix protein (e.g., collagen and fibronectin) accumulation in muscle tissue that leads to the replacement of muscle connective tissue with fibrotic tissue; (v) initiating administration of a complex described herein prior to the progression of extracellular matrix protein (e.g., collagen and fibronectin) accumulation in muscle tissue that leads to the replacement of muscle connective tissue with fibrotic tissue; (v) initiating administration of a complex described herein before the subject loses motor function; (vi) initiating administration of a complex described herein before the subject loses walking; and (vii) once administration has commenced in a subject (e.g., at any time during (i)-(vi)), continuing administration of a complex described herein for a period during which the subject's skeletal muscle is in a pre-fibrotic state.

[0048] Further aspects of the disclosure, including a description of defined terms, are provided below.

[0049] I. Definition Administering: As used herein, the terms "administering" or "administration" mean providing a conjugate to a subject in a physiologically and / or pharmacologically useful manner (e.g., treating a disease in a subject).

[0050] Approximately: As used herein, the term "approximately" or "about," when applied to one or more values ​​of interest, refers to a value similar to a stated reference value. In certain embodiments, the term "approximately" or "about" refers to a broad range of values ​​that fall within plus or minus (more than or less than) 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the stated reference value, unless otherwise stated or clear from the context (except when such number exceeds 100% of the possible values).

[0051] Antibody: As used herein, the term "antibody" refers to a polypeptide that includes at least one immunoglobulin variable domain or at least one antigenic determinant, e.g., a paratope, that specifically binds to an antigen. In some embodiments, the antibody is a full-length antibody. In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a humanized antibody. However, in some embodiments, the antibody is a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an Fv fragment, or an scFv fragment. In some embodiments, the antibody is a nanobody derived from a camelid antibody or a nanobody derived from a shark antibody. In some embodiments, the antibody is a bispecific antibody. In some embodiments, the antibody comprises a framework with human germline sequences. In another embodiment, the antibody comprises a heavy chain constant region selected from the group consisting of the constant regions of IgG, IgG1, IgG2, IgG2A, IgG2B, IgG2C, IgG3, IgG4, IgA1, IgA2, IgD, IgM, and IgE. In some embodiments, an antibody comprises a heavy (H) chain variable region (abbreviated herein as VH) and / or (for example, and), a light (L) chain variable region (abbreviated herein as VL). In some embodiments, an antibody comprises a constant region, for example, an Fc region. An immunoglobulin constant region refers to a heavy or light chain constant region. Human IgG heavy and light chain constant region amino acid sequences and their functional variations are known. With respect to the heavy chain, in some embodiments, the heavy chain of an antibody described herein can be an alpha (α), delta (Δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain. In some embodiments, the heavy chain of an antibody described herein can comprise a human alpha (α), delta (Δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain. In specific embodiments, the antibody described herein comprises a human gamma 1 CH1 domain, CH2 domain, and / or (for example, and), CH3 domain. In some embodiments, the amino acid sequence of the VH domain comprises the amino acid sequence of a human gamma (γ) heavy chain constant region, such as any sequence known in the art.Non-limiting examples of human constant region sequences are described in the art; see, e.g., U.S. Patent No. 5,693,780 and Kabat EA et al. (1991), supra. In some embodiments, the VH domain comprises an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or at least 99% identical to any of the variable chain constant regions provided herein. In some embodiments, the antibody is modified, e.g., via glycosylation, phosphorylation, sumoylation, and / or (e.g., and) methylation. In some embodiments, the antibody is a glycosylated antibody conjugated to one or more sugar or carbohydrate molecules. In some embodiments, one or more sugar or carbohydrate molecules are conjugated to the antibody via N-glycosylation, O-glycosylation, C-glycosylation, glypiation (GPI anchor attachment), and / or (e.g., and) phosphoglycosylation. In some embodiments, the one or more sugar or carbohydrate molecules are monosaccharides, disaccharides, oligosaccharides, or glycans. In some embodiments, the one or more sugar or carbohydrate molecules are branched oligosaccharides or branched glycans. In some embodiments, the one or more sugar or carbohydrate molecules comprise a mannose unit, a glucose unit, an N-acetylglucosamine unit, an N-acetylgalactosamine unit, a galactose unit, a fucose unit, or a phospholipid unit. In some embodiments, the antibody is a construct comprising a polypeptide comprising one or more antigen-binding fragments of the present disclosure linked to a linker polypeptide or an immunoglobulin constant region. The linker polypeptide comprises two or more amino acid residues linked by a peptide bond and is used to link one or more antigen-binding moieties. Examples of linker polypeptides have been reported (see, e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, RJ, et al. (1994) Structure 2:1121-1123).Furthermore, an antibody may be part of a larger immunoadhesion molecule formed by covalent or noncovalent association of one or more other proteins or peptides with the antibody or antibody portion. Examples of such immunoadhesion molecules include the use of streptavidin core regions to generate tetrameric scFv molecules (Kipriyanov, SM, et al. (1995) Human Antibodies and Hybridomas 6:93-101), and the use of cysteine ​​residues, marker peptides, and C-terminal polyhistidine tags to generate bivalent and biotinylated scFv molecules (Kipriyanov, SM, et al. (1994) Mol. Immunol. 31:1047-1058).

[0052] CDR: As used herein, the term "CDR" refers to a complementarity-determining region within an antibody variable sequence. A typical antibody molecule comprises a heavy chain variable region (VH) and a light chain variable region (VL), which are typically involved in antigen binding. The VH and VL regions can be further subdivided into hypervariable regions, also known as "complementarity-determining regions" ("CDRs"), interspersed with more conserved regions known as "framework regions" ("FRs"). Each VH and VL typically consists of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The extent of the framework regions and CDRs can be precisely identified using methodologies known in the art, such as the Kabat definition, the IMGT definition, the Chothia definition, the AbM definition, and / or (for example, and) the contact definition, all of which are well known in the art.For example, Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242; IMGT (registered trademark), the international ImMunoGeneTics information system (registered trademark) http: / / www.imgt.org, Lefranc, M.-P. et al., Nucleic Acids. Res.,27:209-212(1999);Ruiz,M.et al.,Nucleic Acids Res.,28:219-221(2000);Lefranc,M.-P.,Nucleic Acids Res.,29:207-209(2001);Lefranc,M.-P.,Nucleic Acids Res.,31:307-310(2003);Lefranc,M.-P.et al.,In Silico Biol.,5,0006(2004)[Epub],5:45-60(2005);Lefranc,M.-P.et al.,Nucleic Acids Res.,33:D593-597(2005);Lefranc,M.-P.et al.,Nucleic Acids Res.,37:D1006-1012(2009);Lefranc,M.-P.et al.,Nucleic Acids Res.,43:D413-422(2015);Chothia et al.,(1989)Nature 342:877;Chothia,C.et al. (1987) J. Mol. Biol. 196:901-917, Al-lazikani et al. al (1997) J. Molec. Biol. 273:927-948; and Almagro, J. Mol. Recognit. 17:132-143 (2004). See also hgmp.mrc.ac.uk and bioinf.org.uk / abs. As used herein, CDRs may refer to CDRs defined by any method known in the art.Two antibodies having the same CDRs means that the two antibodies have the same amino acid sequence of their CDRs when determined by the same method, for example, the IMGT definition.

[0053] There are three CDRs in each of the heavy and light chain variable regions, designated CDR1, CDR2, and CDR3 for each variable region. The term "CDR set" as used herein refers to a group of three CDRs occurring in a single variable region capable of binding to an antigen. The exact boundaries of these CDRs have been defined differently according to various systems. The system described by Kabat (Kabat et al., Sequence of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides an unambiguous residue numbering system applicable to any antibody variable region, but also provides precise residue boundaries defining the three CDRs. These CDRs are sometimes referred to as Kabat CDRs. Sub-portions of the CDRs are sometimes designated L1, L2, and L3, or H1, H2, and H3, where "L" and "H" designate the light chain and heavy chain regions, respectively. These regions are sometimes referred to as Chothia CDRs, whose boundaries overlap with the Kabat CDRs. Other boundaries defining CDRs that overlap with the Kabat CDRs are described by Padlan (FASEB J. 9:133-139 (1995)) and MacCallum (J Mol Biol 262(5):732-45(1996)). Still other CDR boundary definitions may not strictly adhere to one of the above systems, but may still overlap with the Kabat CDRs, and may be shortened or extended in light of predictions or experimental findings that a particular residue or group of residues, or even an entire CDR, does not significantly affect antigen binding. The methods used herein may utilize CDRs defined according to any of these systems. Examples of CDR definition systems are shown in Table 1. Table 1. CDR definition [Table 1]

[0054] CDR-grafted antibody: The term "CDR-grafted antibody" refers to an antibody that contains heavy and light chain variable region sequences from one species but in which the sequence of one or more of the CDR regions of its VH and / or VL has been replaced with CDR sequences from another species, such as an antibody having murine heavy and light chain variable regions but in which one or more of the murine CDRs (e.g., CDR3) have been replaced with human CDR sequences.

[0055] Chimeric antibody: The term "chimeric antibody" refers to an antibody that contains heavy and light chain variable region sequences from one species and constant region sequences from another species, such as an antibody having murine heavy and light chain variable regions linked to human constant regions.

[0056] Complementary: As used herein, the term "complementary" refers to the ability for precise pairing between two nucleotides or two pairs of nucleotides. In particular, complementary is a term that characterizes the degree of hydrogen bond pairing that results in binding between two nucleotides or two pairs of nucleotides. For example, if the base of an oligonucleotide at a certain position can hydrogen bond with the base of a target nucleic acid (e.g., mRNA) at the corresponding position, then the bases are considered to be complementary to each other at that position. Base pairing may include both standard Watson-Crick base pairing and non-Watson-Crick base pairing (e.g., Wobble base pairing and Hoogsteen base pairing). For example, in some embodiments, for complementary base pairing, an adenosine-type base (A) is complementary to a thymidine-type base (T) or a uracil-type base (U), a cytosine-type base (C) is complementary to a guanosine-type base (G), and a universal base such as 3-nitropyrrole or 5-nitroindole can hybridize with any A, C, U, or T. Inosine (I) is also considered a universal base in the art and is considered complementary to any A, C, U, or T.

[0057] Conservative amino acid substitution: As used herein, "conservative amino acid substitution" refers to an amino acid substitution that does not change the relative charge or size characteristics of the protein in which the amino acid substitution is made.Variants can be prepared according to methods for modifying polypeptide sequences known to those skilled in the art, and can be found, for example, in references that summarize such methods, such as Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Fourth Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2012, or Current Protocols in Molecular Biology, FMA Usubel, et al., eds., John Wiley & Sons, Inc., New York.Conservative amino acid substitutions include those made to amino acids in the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

[0058] Covalently linked (or linked): As used herein, the term "covalently linked (or linked)" refers to the characteristic of two or more molecules being linked together via at least one covalent bond. In some embodiments, two molecules may be covalently linked together by a single bond (e.g., a disulfide bond or disulfide bridge) that acts as an intermolecular linker. However, in some embodiments, two or more molecules may be covalently linked together via a molecule that acts as a linker that connects two or more molecules together through multiple covalent bonds. In some embodiments, the linker may be a cleavable linker. However, in some embodiments, the linker may be a non-cleavable linker.

[0059] Cross-reacting: As used herein, and in the context of targeting agents (e.g., antibodies), the term "cross-reacting" refers to the property of an agent that is capable of specifically binding to more than one antigen of the same type or class (e.g., multiple homologous, paralogous, or orthologous antigens) with similar affinity or avidity. For example, in some embodiments, an antibody that cross-reacts with a similar type or class of human and non-human primate antigens (e.g., human transferrin receptor and non-human primate transferrin receptor) is capable of binding to a human antigen and a non-human primate antigen with similar affinity or avidity. In some embodiments, an antibody cross-reacts with a similar type or class of human antigen and rodent antigen. In some embodiments, an antibody cross-reacts with a similar type or class of rodent antigen and non-human primate antigen. In some embodiments, an antibody cross-reacts with a similar type or class of human antigen, non-human primate antigen, and rodent antigen.

[0060] DMD: As used herein, the term "DMD" refers to the gene encoding the dystrophin protein, which is an important component of the internal cytoskeleton and extracellular matrix in muscle cells, specifically the dystrophin-glycoprotein complex that bridges muscle fibers. Deletions, duplications, and point mutations in DMD can cause dystrophinopathies, such as Duchenne muscular dystrophy, Becker muscular dystrophy, or cardiomyopathy (e.g., Duchenne muscular dystrophy-associated dilated cardiomyopathy). Alternative promoter usage and alternative splicing result in multiple distinct transcript variants and protein isoforms for this gene. In some embodiments, the dystrophin gene may be a human (gene ID: 1756), non-human primate (e.g., gene ID: 465559), or rodent gene (e.g., gene ID: 13405; gene ID: 24907). Additionally, multiple human transcript variants (e.g., shown in GenBank RefSeq Accession Numbers: NM_000109.3, NM_004006.2 (SEQ ID NO: 2239), NM_004009.3, NM_004010.3, and NM_004011.3) encoding different protein isoforms have been characterized.

[0061] DMD allele: As used herein, the term "DMD allele" refers to any one of the alternative forms (e.g., wild-type or mutant) of the DMD gene. In some embodiments, a DMD allele may encode dystrophin that retains its normal and typical function. In some embodiments, a DMD allele may contain one or more mutations that result in muscular dystrophy. Common mutations that lead to Duchenne muscular dystrophy include frameshift, deletion, substitution, and duplication mutations in one or more of the 79 exons present in dystrophin alleles, such as exon 8, exon 23, exon 41, exon 44, exon 50, exon 51, exon 52, exon 53, or exon 55. Further examples of DMD mutations are disclosed, e.g., in Flanigan KM, et al., Mutational spectrum of DMD mutations in dystrophinopathy patients: application of modern diagnostic techniques to a large cohort. Hum Mutat. 2009 Dec; 30 (12):1657-66, the entire contents of which are incorporated herein by reference.

[0062] DMD gene modifier: As used herein, the term "DMD gene modifier" refers to a genomic variant that alleviates (e.g., suppresses) or exacerbates (e.g., enhances) the severity of Duchenne muscular dystrophy. DMD gene modifiers can modify the phenotypic outcome of the primary disease-causing mutation. DMD gene modifiers result in phenotypic variability in different Duchenne muscular dystrophy patients, e.g., between patients with different DMD gene modifiers and between patients with and without DMD gene modifiers. Even when patients have the same Duchenne muscular dystrophy-causing mutation (e.g., siblings with identical mutations in the DMD gene), their disease phenotypic expression can differ based on, e.g., the presence or absence of a particular DMD gene modifier. DMD gene modifiers can either increase the severity of Duchenne muscular dystrophy (an "enhancer" DMD gene modifier) ​​or decrease the severity of Duchenne muscular dystrophy (a "suppressor" DMD gene modifier). DMD genetic modifiers can alter disease phenotypes by genetically, biochemically, or functionally interacting with one or more target genes or gene products associated with Duchenne muscular dystrophy, the DMD gene, or the dystrophin protein. The extent of the effect of a given DMD genetic modifier can vary between subjects, for example, based on other genetic variants in the subject, potentially resulting in wide phenotypic variation and varying penetrance. DMD genetic modifiers are discussed in Rahit and Tarailo-Graovac, "Genetic Modifiers and Rare Mendelian Disease," Genes 11(3):239 (2020), the entire contents of which are incorporated herein by reference for this purpose. In some embodiments, the DMD genetic modifier is a mutation or polymorphism (e.g., a single nucleotide polymorphism) in a latent TGF-β binding protein (LTBP), such as LTBP4.In some embodiments, the genetic modifier is a hyperfibrotic polymorphism in a chromosomal locus or gene, such as LTBP4. In some embodiments, the DMD genetic modifier is a mutation or polymorphism that results in reduced or lost expression or function of alpha-7 integrin (ITGA7), as discussed in Hightower RM and Alexander MS, Genetic Modifiers of Duchenne and Facioscapulohumeral Muscular Dystrophies, Muscle Nerve. 2018 Jan; 57(1): 6-15, the entire contents of which are incorporated herein by reference. Further examples of DMD genetic modifiers are reported in Pascual-Morena, C, et al., Genetic Modifiers and Phenotype of Duchenne Muscular Dystrophy: A Systematic Review and Meta-Analysis, Pharmaceuticals 2021, 14(8), 798, the entire contents of which are incorporated herein by reference.

[0063] Dystrophinopathy: As used herein, the term "dystrophinopathy" refers to a muscle disease resulting from one or more mutated DMD alleles. Dystrophinopathy encompasses a spectrum of conditions (varying from mild to severe), including Duchenne muscular dystrophy, Becker muscular dystrophy, and Duchenne muscular dystrophy-associated dilated cardiomyopathy (DCM). In some embodiments, at one end of the spectrum, dystrophinopathy is phenotypically associated with asymptomatic increases in plasma concentrations of creatine phosphokinase (CK) and / or muscle spasms with (for example, and) myoglobinuria. In some embodiments, at the other end of the spectrum, dystrophinopathy is phenotypically associated with a progressive muscle disease, generally classified as Duchenne muscular dystrophy or Becker muscular dystrophy when it primarily affects skeletal muscles, and as Duchenne muscular dystrophy-associated dilated cardiomyopathy (DCM) when it primarily affects the heart. Symptoms of Duchenne muscular dystrophy include muscle loss or degeneration, reduced muscle function, pseudohypertrophy of tongue and calf muscles, high-risk neurological abnormalities, and shortened lifespan. Duchenne muscular dystrophy is associated with Online Mendelian Inheritance in Man (OMIM) Entry # 310200. Becker muscular dystrophy is associated with OMIM Entry # 300376. Dilated cardiomyopathy is associated with OMIM Entry X# 302045.

[0064] Exonic splicing enhancer (ESE): As used herein, the term "exonic splicing enhancer" or "ESE" refers to a nucleic acid sequence motif within an exon of a gene, pre-mRNA, or mRNA that directs or enhances splicing of pre-mRNA into mRNA, as described, for example, in Blencowe et al., Trends Biochem Sci 25, 106-10 (2000), incorporated herein by reference. An ESE may direct or enhance splicing, for example, to remove one or more introns and / or one or more exons from a gene transcript. ESE motifs are typically 6-8 bases in length. SR proteins (e.g., proteins encoded by the genes SRSF1, SRSF2, SRSF3, SRSF4, SRSF5, SRSF6, SRSF7, SRSF8, SRSF9, SRSF10, SRSF11, SRSF12, TRA2A, or TRA2B) bind to ESEs through their RNA recognition motif regions to promote splicing. ESE motifs can be identified by a number of methods, including those described in Cartegni et al., Nucleic Acids Research, 2003, Vol. 31, No. 13, 3568-3571, which is incorporated herein by reference.

[0065] Framework: As used herein, the term "framework" or "framework sequence" refers to the remaining sequence of a variable region minus the CDRs. Because the precise definition of a CDR sequence can be determined by various systems, the meaning of a framework sequence is subject to correspondingly different interpretations. The six CDRs (CDR-L1, CDR-L2, and CDR-L3 of the light chain and CDR-H1, CDR-H2, and CDR-H3 of the heavy chain) also divide the framework regions on the light and heavy chains into four subregions (FR1, FR2, FR3, and FR4) on each chain, where CDR1 is located between FR1 and FR2, CDR2 is located between FR2 and FR3, and CDR3 is located between FR3 and FR4. When framework regions that do not specify a specific subregion as FR1, FR2, FR3, or FR4 are referred to by others, they represent the combined FRs in the variable region of a single naturally occurring immunoglobulin chain. As used herein, FR refers to one of the four subregions, and FR(s) refers to two or more of the four subregions containing framework regions. Human heavy and light chain acceptor sequences are known in the art. In one embodiment, acceptor sequences known in the art may be used in the antibodies disclosed herein.

[0066] Human antibody: The term "human antibody," as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the present disclosure may include, for example, in the CDRs, particularly CDR3, amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody," as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0067] Humanized antibody: The term "humanized antibody" refers to an antibody that contains heavy and light chain variable region sequences from a non-human species (e.g., mouse), but in which at least a portion of the VH and / or (e.g., and) VL sequences have been altered to be more "human-like," i.e., more similar to human germline variable sequences. One type of humanized antibody is a CDR-grafted antibody in which human CDR sequences are introduced onto non-human VH and VL sequences to replace the corresponding non-human CDR sequences. In one embodiment, humanized anti-transferrin receptor antibodies and antigen-binding portions are provided. Such antibodies may be produced by obtaining a murine anti-transferrin receptor monoclonal antibody using conventional hybridoma technology, followed by humanization using in vitro genetic engineering (such as that disclosed in Kasaian et al., PCT Publication No. WO 2005 / 123126 A2).

[0068] Internalizing cell surface receptor: As used herein, the term "internalizing cell surface receptor" refers to a cell surface receptor that is internalized by a cell upon an external stimulus (e.g., a ligand binding to the receptor). In some embodiments, the internalizing cell surface receptor is internalized by endocytosis. In some embodiments, the internalizing cell surface receptor is internalized by clathrin-mediated endocytosis. However, in some embodiments, the internalizing cell surface receptor is internalized by a clathrin-independent pathway, such as phagocytosis, macropinocytosis, caveolae- and raft-mediated uptake, or clathrin-independent constitutive endocytosis. In some embodiments, the internalizing cell surface receptor comprises an intracellular domain, a transmembrane domain, and / or (e.g., and) an extracellular domain, which optionally further comprise a ligand-binding domain. In some embodiments, the cell surface receptor becomes internalized by a cell after ligand binding. In some embodiments, the ligand may be a muscle-targeting agent or a muscle-targeting antibody. In some embodiments, the internalizing cell surface receptor is a transferrin receptor.

[0069] Isolated antibody: As used herein, an "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds to the transferrin receptor is substantially free of antibodies that specifically bind to antigens other than the transferrin receptor). However, an isolated antibody that specifically binds to the transferrin receptor complex may have cross-reactivity to other antigens, such as transferrin receptor molecules from other species. Moreover, an isolated antibody may be substantially free of other cellular material and / or (by way of example and) chemicals.

[0070] Kabat numbering: The terms "Kabat numbering," "Kabat definition," and "Kabat labeling" are used interchangeably herein. These terms, recognized in the art, refer to a system for numbering amino acid residues that are more variable (i.e., hypervariable) than other amino acid residues in the heavy and light chain variable regions of an antibody or its antigen-binding portion (Kabat et al. (1971) Ann. NY Acad. Sci. 190:382-391 and Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242). In the heavy chain variable region, the hypervariable region spans amino acid positions 31-35 for CDR1, positions 50-65 for CDR2, and positions 95-102 for CDR3. In the light chain variable region, the hypervariable region spans amino acid positions 24-34 for CDR1, amino acid positions 50-56 for CDR2, and amino acid positions 89-97 for CDR3.

[0071] Molecular payload: As used herein, the term "molecular payload" refers to a molecule or species that functions to modulate a biological outcome. In some embodiments, the molecular payload is linked to or otherwise associated with a muscle-targeting agent. In some embodiments, the molecular payload is a small molecule, protein, peptide, nucleic acid, or oligonucleotide. In some embodiments, the molecular payload functions to modulate the transcription of a DNA sequence, to modulate the expression of a protein, or to modulate the activity of a protein. In some embodiments, the molecular payload is an oligonucleotide comprising a strand having a region complementary to a target gene.

[0072] Muscle targeting agent: As used herein, the term "muscle targeting agent" refers to a molecule that specifically binds to an antigen expressed on a muscle cell. The antigen in or on a muscle cell may be a membrane protein, e.g., an integral membrane protein or a peripheral membrane protein. Typically, a muscle targeting agent specifically binds to an antigen on a muscle cell, facilitating internalization of the muscle targeting agent (and any associated molecular payload) into the muscle cell. In some embodiments, the muscle targeting agent specifically binds to an internalizing cell surface receptor on muscle and can be internalized into the muscle cell through receptor-mediated internalization. In some embodiments, the muscle targeting agent is a small molecule, protein, peptide, nucleic acid (e.g., an aptamer), or antibody. In some embodiments, the muscle targeting agent is linked to a molecular payload.

[0073] Muscle-targeting antibody: As used herein, the term "muscle-targeting antibody" refers to a muscle-targeting agent that is an antibody that specifically binds to an antigen found in or on muscle cells. In some embodiments, the muscle-targeting antibody specifically binds to an antigen on muscle cells that facilitates internalization of the muscle-targeting antibody (and any attached molecular payload) into the muscle cells. In some embodiments, the muscle-targeting antibody specifically binds to an internalizing cell surface receptor present on muscle cells. In some embodiments, the muscle-targeting antibody is an antibody that specifically binds to the transferrin receptor.

[0074] Oligonucleotide: As used herein, the term "oligonucleotide" refers to an oligomeric nucleic acid compound up to 200 nucleotides in length. Examples of oligonucleotides include, but are not limited to, RNAi oligonucleotides (e.g., siRNA, shRNA), microRNA, gapmers, mixmers, phosphorodiamidate morpholinos, peptide nucleic acids, aptamers, guide nucleic acids (e.g., Cas9 guide RNA), and the like. Oligonucleotides may be single-stranded or double-stranded. In some embodiments, oligonucleotides may contain one or more modified nucleotides (e.g., 2'-O-methyl sugar modifications, purine or pyrimidine modifications). In some embodiments, oligonucleotides may contain one or more modified internucleotide linkages. In some embodiments, oligonucleotides may contain one or more phosphorothioate linkages, which may be in an Rp or Sp stereochemical configuration.

[0075] Pharmaceutically acceptable salts: As used herein, the term "pharmaceutically acceptable salts" refers to salts that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like, and that are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, which is incorporated herein by reference. Pharmaceutically acceptable salts of the compounds of the present disclosure include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts of amino groups formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, perchloric acid, and the like, or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, malonic acid, and the like, or by using other methods known in the art, such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, dicruconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium, and N+(C) salts. 1-4 alkyl) 4-Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc. Further pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium, and amine cations, optionally formed using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkylsulfonates, and arylsulfonates.

[0076] Prefibrotic state: The "prefibrotic state" of a tissue (e.g., muscle tissue) precedes substantial fibrosis in the tissue (e.g., endomysial, perimysial, and / or epimysial fibrosis in muscle tissue). In some embodiments, the prefibrotic state precedes substantial replacement of normal tissue (e.g., muscle tissue) with fat and / or extracellular matrix components (e.g., extracellular matrix proteins such as collagen and fibronectin). In some embodiments, the prefibrotic state precedes adipocyte replacement of normal cells, e.g., following fibrotic remodeling (e.g., late fibrotic remodeling). In some embodiments, the prefibrotic state precedes progression of muscle fibrosis (e.g., intramuscular fibrosis such as endomysial, perimysial, and / or epimysial fibrosis) to the extent that a subject loses significant muscle strength and / or function. Cholok et al. "Traumatic muscle fibrosis: From pathway to prevention" J Trauma Acute Care Surg. 82(1):174-184 (2017); and Mann et al. "Aberrant repair and fibrosis development in skeletal muscle" Skeletal Muscle 1:21 (2011), the entire contents of each of which are incorporated herein by reference, discuss muscle fibrosis and its development and progression from a prefibrotic state to fulminant fibrosis, including that resulting from dystrophinopathies such as Duchenne muscular dystrophy.

[0077] Pre-degeneration state: The "pre-degeneration state" of tissue (e.g., muscle tissue) is prior to substantial degeneration in the tissue. In some embodiments, the pre-degeneration state refers to prior to substantial loss of normal tissue (e.g., muscle tissue) in the tissue prior to degeneration. In some embodiments, the pre-degeneration state is prior to the loss of a substantial portion of muscle fibers in muscle tissue. In some embodiments, the pre-degeneration state is prior to substantial replacement of normal tissue (e.g., muscle tissue) with adipose tissue. In some embodiments, the pre-degeneration state is prior to the replacement of normal cells with adipocytes. In some embodiments, the pre-degeneration state is prior to significant loss of muscle strength and / or function in the muscle tissue prior to degeneration. For example, the pre-degeneration state may be prior to the inability of a subject to sit upright or the inability of a subject to walk. Abdel-Salam et al. "Markers of degeneration and regeneration in Duchenne muscular dystrophy" Acta Myol. 28(3):94-100 (2009); Chemello et al. "Degenerative and regenerative pathways underlying Duchenne muscular dystrophy revealed by single-nucleus RNA sequencing" PNAS 117(47): 29691-29701 (2020); and Duan et al. "Duchenne muscular dystrophy" Nature Reviews Disease Primers 7:13 (2021), the entire contents of each of which are incorporated herein by reference, discuss muscle degeneration and its progression associated with Duchenne muscular dystrophy.

[0078] Recombinant antibody: The term "recombinant human antibody," as used herein, refers to any human antibody that is prepared, expressed, created, or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell (as described in more detail in this disclosure), antibodies isolated from a recombinant combinatorial human antibody library (Hoogenboom HR, (1997) TIB Tech. 15:62-70; Azzazy H., and Highsmith WE, (2002) Clin. Biochem. 35:425-445; Gavilondo JV, and Larrick JW (2002) BioTechniques 29:128-145; Hoogenboom H., and Chames P. (2000) Immunology Today 21:371-378), antibodies isolated from a human immunoglobulin gene transgenic animal (e.g., a mouse) (see, e.g., Taylor, LD, et al. (See, e.g., Kellermann SA, and Green LL (2002) Current Opinion in Biotechnology 13:593-597; Little M. et al (2000) Immunology Today 21:364-370), or any other means involving splicing of human immunoglobulin gene sequences with other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when human Ig sequence transgenic animals are used, in vivo somatic mutagenesis), such that the amino acid sequences of the VH and VL regions of the recombinant antibodies, while derived from and related to human germline VH and VL sequences, are sequences that may not naturally occur within the germline repertoire of human antibodies in vivo.One aspect of the present disclosure provides fully human antibodies capable of binding to the human transferrin receptor that can be produced using techniques well known in the art, including, but not limited to, techniques using human Ig phage libraries (e.g., those disclosed in PCT Publication No. WO 2005 / 007699 A2 to Jermutus et al.).

[0079] Region of Complementarity: As used herein, the term "region of complementarity" refers to a nucleotide sequence (e.g., a nucleotide sequence of an oligonucleotide) that is sufficiently complementary to a cognate nucleotide sequence (e.g., a nucleotide sequence of a target nucleic acid) such that the two nucleotide sequences are capable of annealing to each other under physiological conditions (e.g., in a cell). In some embodiments, the region of complementarity is fully complementary to the cognate nucleotide sequence of the target nucleic acid. However, in some embodiments, the region of complementarity is only partially complementary (e.g., at least 80%, 90%, 95%, or 99% complementary) to the cognate nucleotide sequence of the target nucleic acid. In some embodiments, the region of complementarity contains one, two, three, or four mismatches compared to the cognate nucleotide sequence of the target nucleic acid.

[0080] Specific binding: As used herein, the term "specific binding" refers to the ability of a molecule to bind to a binding partner in a binding assay or other binding context, with a degree of affinity or avidity that can be used to distinguish the binding partner from an appropriate control. With respect to an antibody, the term "specific binding" refers to the ability of an antibody to bind to a specific antigen, relative to an appropriate reference antigen, or an antigen that can be used to distinguish the specific antigen from other antigens, with a degree of affinity or avidity (e.g., that allows preferential targeting to certain cells (e.g., muscle cells) through binding to the antigen, as described herein). In some embodiments, the antibody binds to the target with at least about 10 -4 M, 10 -5 M, 10-6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, 10 -13 An antibody specifically binds to a target if it has a KD at or below M. In some embodiments, the antibody specifically binds to a transferrin receptor, e.g., an epitope in the apical domain of the transferrin receptor.

[0081] Subject: As used herein, the term "subject" refers to a mammal. In some embodiments, the subject is a non-human primate animal or a rodent. In some embodiments, the subject is a human. In some embodiments, the subject is a patient having or suspected of having a disease, e.g., a human patient. In some embodiments, the subject is a human patient having or suspected of having a disease resulting from a mutated DMD gene sequence, e.g., a mutation in an exon of the DMD gene sequence. In some embodiments, the subject has a dystrophinopathy, e.g., Duchenne muscular dystrophy.

[0082] Transferrin receptor: As used herein, the term "transferrin receptor" (also known as TFRC, CD71, p90, TFR, or TFR1) refers to an internalized cell surface receptor that binds to transferrin to facilitate iron uptake by endocytosis. In some embodiments, the transferrin receptor may originate from humans (NCBI Gene ID 7037), non-human primates (e.g., NCBI Gene ID 711568 or NCBI Gene ID 102136007), or rodents (e.g., NCBI Gene ID 22042). In addition, multiple human transcript variants encoding different isoforms of the receptor have been characterized (e.g., those annotated with GenBank RefSeq accession numbers: NP_001121620.1, NP_003225.2, NP_001300894.1, and NP_001300895.1).

[0083] Treat: As used herein, the terms "treat" and "treatment" refer to both therapeutic procedures and measures that can alleviate symptoms or provide some benefit to a subject, the purpose of which is to prevent or delay (lessen) undesirable physiological changes or disorders, such as the progression, onset, or spread of a disease (e.g., Duchenne muscular dystrophy) or condition (e.g., fibrosis). Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, whether detectable or undetectable, reduction in the extent of the disease, stabilization of the disease state (i.e., not worsening), slowing or slowing disease progression, improvement or palliation of the disease state, and remission (whether partial or total). In some embodiments, "treating" can also mean prolonging survival compared to expected survival in the absence of treatment. In some embodiments, "treating" can also mean slowing the progression of a disease compared to the progression of the disease in the absence of treatment. Patients in need of treatment include those who already suffer from a condition, disease, or disorder, or those who are suspected of having or are susceptible to a condition, disease, or disorder.Therefore, the term "treat" or "treatment" also encompasses the prophylactic use of therapeutic agents.As described herein, the term "treat fibrosis" or "treating fibrosis" (and similar terms) encompasses reducing, preventing, and / or increasing resistance to any type of fibrosis (e.g., muscle fibrosis, such as intramuscular fibrosis) as known or described in the art in subjects with or susceptible to Duchenne muscular dystrophy.In some embodiments, the fibrosis to be treated includes endomysial fibrosis, perimysial fibrosis, and / or epimysial fibrosis.

[0084] 2'-modified nucleoside: As used herein, the terms "2'-modified nucleoside" and "2'-modified ribonucleoside" are used interchangeably and refer to a nucleoside having a sugar moiety modified at the 2'-position. In some embodiments, the 2'-modified nucleoside is a 2'-4' bicyclic nucleoside, in which the 2' and 4' positions of the sugar are bridged (e.g., by a methylene, ethylene, or (S)-constrained ethyl bridge). In some embodiments, the 2'-modified nucleoside is a non-bicyclic 2'-modified nucleoside, e.g., in which the 2' position of the sugar moiety is substituted. Non-limiting examples of 2'-modified nucleosides include the following: 2'-deoxy, 2'-fluoro (2'-F), 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), 2'-ON-methylacetamide (2'-O-NMA), locked nucleic acids (LNA, methylene-bridged nucleic acids), ethylene-bridged nucleic acids (ENA), and (S)-constrained ethyl-bridged nucleic acids (cEt). In some embodiments, the 2'-modified nucleosides described herein are high-affinity modified nucleosides, and oligonucleotides comprising the 2'-modified nucleosides have increased affinity for target sequences compared to unmodified oligonucleotides. Examples of structures of 2'-modified nucleosides are provided below: [ka] Although these examples are shown with a phosphate group, any internucleoside linkage is contemplated between 2'-modified nucleosides.

[0085] Ranges: All ranges provided in this disclosure are inclusive of the endpoints.

[0086] II. Complex Provided herein are conjugates comprising a targeting agent, for example, an antibody covalently linked to a molecular payload. In some embodiments, the conjugate comprises a muscle-targeting antibody covalently linked to an oligonucleotide. The conjugate may comprise an antibody that specifically binds to a single antigen site, or an antibody that binds to at least two antigen sites, which may be present on the same antigen or different antigens.

[0087] The complex may be used to modulate the activity or function of at least one gene, protein, and / or (for example, and) nucleic acid. In some embodiments, the molecular payload present with the complex is responsible for modulating the gene, protein, and / or (for example, and) nucleic acid. The molecular payload may be a small molecule, protein, nucleic acid, oligonucleotide, or any molecular entity capable of modulating the activity or function of a gene, protein, and / or (for example, and) nucleic acid in a cell. In some embodiments, the molecular payload is an oligonucleotide that targets a disease-associated repeat in muscle cells.

[0088] The complexes disclosed herein can be used to regulate fibrotic pathways, e.g., the initiation and / or progression of fibrosis in muscle tissue. In some embodiments, the complexes inhibit the initiation and / or progression of fibrosis in muscle tissue. In some embodiments, the complexes inhibit the initiation and / or progression of intramuscular fibrosis (e.g., endomysial fibrosis, perimysial fibrosis, and / or epimysial fibrosis). In some embodiments, the complexes reduce fibrosis in muscle tissue. In some embodiments, the complexes reduce intramuscular fibrosis (e.g., endomysial fibrosis, perimysial fibrosis, and / or epimysial fibrosis).

[0089] In some embodiments, the conjugate comprises a muscle targeting agent (e.g., an anti-transferrin receptor antibody) covalently linked to a molecular payload (e.g., an antisense oligonucleotide targeting a mutated DMD allele) to facilitate exon skipping.

[0090] A. Muscle-targeting agents Some aspects of the present disclosure provide muscle-targeting agents, e.g., muscle-targeting agents for delivering molecular payloads to muscle cells. In some embodiments, such muscle-targeting agents are capable of binding to muscle cells and delivering the associated molecular payload to muscle cells, e.g., via specific binding to an antigen on the muscle cells. In some embodiments, the molecular payload is attached (e.g., covalently attached) to the muscle-targeting agent, and is internalized into the muscle cells upon binding to the antigen on the muscle cells, e.g., via endocytosis. It should be understood that various types of muscle-targeting agents may be used in accordance with the present disclosure. For example, the muscle-targeting agent may comprise or consist of a nucleic acid (e.g., DNA or RNA), a peptide (e.g., an antibody), a lipid (e.g., a microvesicle), or a sugar moiety (e.g., a polysaccharide). Exemplary muscle-targeting agents are described in further detail herein; however, it should be understood that the exemplary muscle-targeting agents provided herein are not intended to be limiting.

[0091] Some aspects of the present disclosure provide muscle targeting agents that specifically bind to antigens on muscles, such as skeletal muscle, smooth muscle, or cardiac muscle. In some embodiments, any of the muscle targeting agents provided herein bind (e.g., specifically bind) to antigens on skeletal muscle cells, smooth muscle cells, and / or (e.g., and) cardiac muscle cells.

[0092] Interaction with muscle-specific cell surface recognition elements (e.g., cell membrane proteins) can achieve both tissue localization and selective uptake into muscle cells. In some embodiments, molecules that are substrates for muscle uptake transporters are useful for delivering molecular payloads into muscle tissue. Binding to muscle surface recognition elements, followed by endocytosis, can allow even macromolecules, such as antibodies, to enter muscle cells. As another example, molecular payloads conjugated to transferrin or anti-transferrin receptor antibodies can be taken up by muscle cells via binding to the transferrin receptor and then endocytosed, e.g., via clathrin-mediated endocytosis.

[0093] The use of muscle-targeting agents can be useful for concentrating molecular payloads (e.g., oligonucleotides) in muscle while reducing toxicity associated with effects in other tissues. In some embodiments, muscle-targeting agents concentrate the bound molecular payload in muscle cells compared to other cell types within a subject. In some embodiments, muscle-targeting agents concentrate the bound molecular payload in muscle cells (e.g., skeletal muscle cells, smooth muscle cells, or cardiomyocytes) at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold greater than the amount in non-muscle cells (e.g., liver cells, nerve cells, blood cells, or adipocytes). In some embodiments, the toxicity in a subject of the molecular payload when conjugated to a muscle-targeting agent is reduced by at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, or 95% when delivered to a subject.

[0094] In some embodiments, a muscle recognition element (e.g., a muscle cell antigen) may be required to achieve muscle selectivity. As one example, the muscle targeting agent may be a small molecule that is a substrate for a muscle-specific uptake transporter. As another example, the muscle targeting agent may be an antibody that enters muscle cells via transporter-mediated endocytosis. As another example, the muscle targeting agent may be a ligand that binds to a cell surface receptor on muscle cells. It should be understood that transporter-based approaches provide a direct pathway for cell entry, whereas receptor-based targeting may involve stimulated endocytosis to reach the desired site of action.

[0095] i. Muscle targeting antibody In some embodiments, the muscle targeting agent is an antibody. Generally, the high specificity of antibodies for their target antigens offers the potential to selectively target muscle cells (e.g., skeletal muscle cells, smooth muscle cells, and / or (e.g., and) cardiac muscle cells). This specificity may also limit off-target toxicity. Examples of antibodies capable of targeting surface antigens of muscle cells have been reported and are within the scope of the present disclosure. For example, antibodies that target the surface of muscle cells are described in Arahata K., et al., "Immunostaining of skeletal and cardiac muscle surface membrane with antibody against Duchenne muscular dystrophy peptide," Nature 1988;333:861-3; Song KS, et al., "Expression of caveolin-3 in skeletal, cardiac, and smooth muscle cells. Caveolin-3 is a component of the sarcolemma and co-fractionates with dystrophin and dystrophin-associated glycoproteins," J Biol Chem 1996;271:15160-5; and Weisbart RH et al., "Cell type-specific targeted intracellular delivery into muscle of a monoclonal antibody that binds myosin IIb," Mol Immunol. 2003 Mar,39(13):78309; the entire contents of each of which are incorporated herein by reference.

[0096] anti-transferrin receptor antibody Some aspects of the present disclosure are based on the recognition that agents that bind to the transferrin receptor, e.g., anti-transferrin receptor antibodies, can target muscle cells. The transferrin receptor is an internalized cell surface receptor that transports transferrin across the cell membrane and participates in the regulation and homeostasis of intracellular iron levels. Some aspects of the present disclosure provide transferrin receptor binding proteins capable of binding to the transferrin receptor. Consequently, aspects of the present disclosure provide binding proteins (e.g., antibodies) that bind to the transferrin receptor. In some embodiments, the binding proteins that bind to the transferrin receptor, along with any attached molecular payload, are internalized into muscle cells. As used herein, antibodies that bind to the transferrin receptor may be referred to interchangeably as transferrin receptor antibodies, anti-transferrin receptor antibodies, anti-TfR antibodies, or anti-TfR1 antibodies. Antibodies that bind, e.g., specifically bind, to the transferrin receptor may be internalized into cells upon binding to the transferrin receptor, e.g., through receptor-mediated endocytosis.

[0097] It should be understood that anti-transferrin receptor antibodies may be produced, synthesized, and / or (for example, and) derivatized using several known methodologies, for example, library design using phage display. Exemplary methodologies have been characterized in the art and are incorporated by reference (Diez, P. et al. "High-throughput phage-display screening in array format," Enzyme and microbial technology, 2015, 79, 34-41; Christoph MH and Stanley, JR "Antibody Phage Display: Technique and Applications," J Invest Dermatol. 2014, 134:2; Engleman, Edgar (Ed.) "Human Hybridomas and Monoclonal Antibodies," 1985, Springer). In other embodiments, the anti-transferrin antibody has been previously characterized or disclosed.Antibodies that specifically bind to the transferrin receptor are known in the art (see, e.g., U.S. Pat. No. 4,364,934, filed December 4, 1979, "Monoclonal antibody to a human early thymocyte antigen and methods for preparing the same"; U.S. Pat. No. 8,409,573, filed June 14, 2006, "Anti-CD71 monoclonal antibodies and uses thereof for treating malignant tumor cells"; U.S. Pat. No. 9,708,406, filed May 20, 2014, "Anti-transferrin receptor antibodies and methods of use"; U.S. Pat. No. 9,611,323, filed December 19, 2014, "Low affinity blood-brain barrier receptor antibodies and uses therefor"; WO 2015 / 098989, filed December 24, 2014, "Novel anti-Transferrin receptor antibody that passes through the blood-brain barrier"; Schneider C. et al., "Structural features of the cell surface receptor for transferrin that is recognized by the monoclonal antibody OKT9.”J Biol Chem.1982,257:14,8516-8522.;Lee et al.“Targeting Rat Anti-Mouse Transferrin Receptor Monoclonal Antibodies through Blood-Brain Barrier in Mouse”2000,J Pharmacol. Exp. Ther., 292:1048-1052).

[0098] Provided herein are, in some aspects, novel anti-TfR1 antibodies for use as muscle-targeting agents (e.g., in muscle-targeting complexes). In some embodiments, the anti-TfR1 antibodies described herein bind to the transferrin receptor with high specificity and affinity. In some embodiments, the anti-TfR1 antibodies described herein specifically bind to any extracellular epitope of the transferrin receptor or an epitope that becomes exposed to the antibody. In some embodiments, the anti-TfR1 antibodies provided herein specifically bind to the transferrin receptor from humans, non-human primates, mice, rats, etc. In some embodiments, the anti-TfR1 antibodies provided herein bind to the human transferrin receptor. In some embodiments, the anti-TfR1 antibodies described herein bind to the amino acid segment of the human or non-human primate transferrin receptor provided in SEQ ID NOS: 105-108. In some embodiments, the anti-TfR1 antibodies described herein bind to the amino acid segment corresponding to amino acids 90-96 of the human transferrin receptor as set forth in SEQ ID NOS: 105, which is not in the apical domain of the transferrin receptor.

[0099] An example of a human transferrin receptor amino acid sequence, corresponding to the NCBI sequence NP_003225.2 (transferrin receptor protein 1 isoform 1, Homo sapiens), is as follows: (SEQ ID NO: 105)

[0100] An example of a primate non-human transferrin receptor amino acid sequence, corresponding to the NCBI sequence NP_001244232.1 (Transferrin receptor protein 1, Macaca mulatta), is as follows: MMDQARSAFSNLFGGEPLSYTRFSLARQVDGDNSHVEMKLGVDEEENTDNNTKPNGTKPKRCGGNICYGTIAVIIFFLIGFMIGYLGYCKGVEPKTECERLAGTESPAREEPEEDFPAAPRLYWDDLKRKLSEKLDTTDFTSTIKLLNENLYVPREAGSQKDENLALYIENQFREFKLSKVWRDQHFVKI QVKDSAQNSVIIVDKNGGLVYLVENPGGYVAYSKAATVTGKLVHANFGTKKDFEDLDSPVNGSIVIVRAGKITFAEKVANAESLNAIGVLIYMDQTKFPIVKADLSFFGHAHLGTGDPYTPGFPSFNHTQFPPSQSSGLPNIPVQTISRAAAEKLFGNMEGDCPSDWKTDSTCKMVTSENKSVKLTVSNV LKETKILNIFGVIKGFVEPDHYVVVGAQRDAWGPGAAKSSVGTALLLKLAQMFSDMVLKDGFQPSRSIIFASWSAGDFGSVGATEWLEGYLSSLHLKAFTYINLDKAVLGTSNFKVSASPLLYTLIEKTMQDVKHPVTGRSLYQDSNWASKVEKLTLDNAAFPFLAYSGIPAVSFCFCEDTDYPYLGTTM DTYKELVERIPELNKVARAAAEVAGQFVIKLTHDTELNLDYERYNSQLLLFLRDLNQYRADVKEMGLSLQWLYSARGDFFRATSRLTTDFRNAEKRDKFVMKKLNDRVMRVEYYFLSPYVSPKESPFRHVFWGSGSHTLSALLESLKLRRQNNSAFNETLFRNQLALATWTIQGAANALSGDVWDIDNEF (SEQ ID NO: 106)

[0101] An example of a primate non-human transferrin receptor amino acid sequence corresponding to the NCBI sequence XP_005545315.1 (transferrin receptor protein 1, Macaca fascicularis) is as follows: (SEQ ID NO: 107)

[0102] An example of a mouse transferrin receptor amino acid sequence, corresponding to the NCBI sequence NP_001344227.1 (transferrin receptor protein 1, Mus musculus), is as follows: MMDQARSAFSNLFGGEPLSYTRFSLARQVDGDNSHVEMCLAADEEENADNNMKASVRKPKRFNGRLCFAAIALVIFFLIGFMSGYLGYCKRVEQKEECVKLAETEETDKSETMETEDVPTSSRLYWADLKTLLSEKLNSIEFADTIKQLSQNTYTPREAGSQKDESLAYYIENQFHEFKFSKVWRDEHYV KIQVKSsIGQNMVTIVQSNGNLDPVESPEGYVAFSKPTEVSGKLVHANFGTKKDFEELSYSVNGSLVIVRAGEITFAEKVANAQSFNAIGVLIYMDKNKFPVVEADLALFGHAHHLGTGDPYTPGPFPSFNHTQFPPSQSSGLPNIPVQTISRAAAEKLFGKMEGSCPARWNIDSSCKLELSQNQNVKLIVKN VLKERRILNIFGVIKGYEEPDRYVVVGAQRDALGAGVAAKSSVGTGLLLKLAQVFSDMISKDGFRPSRSIIFASWTAGDFGAVGATEWLEGYLSSLHLKAFTYINLDKVVLGTSNFKVSASPLLYTLMGKIMQDVKHPVDGKSLYRDSNWISKVEKLSFDNAAYPFLAYSGIPAVSFCCEDADYPYLGTR LDTYEALTQKVPQLNQMVRTAAEVAGQLIIKLTHDVELNLDYEMYNSKLLSFMKDLNQFKTDIRDMGLSLQWLYSARGDYFRATSRLTTDFHNAEKTNRFVMREINDRIMKVEYHFLSPYVSPRESPFRHIFWGSGSHTLSALVENLKLRQKNITAFNETLFRNQLALATEWTIQGVANALSGDIWNIDNEF (query number 108)

[0103] In some embodiments, an anti-transferrin receptor antibody binds to an amino acid segment of the receptor as follows:FVKIQVKDSAQNSVIIVDKNGRLVYLVENPGGYVAYSKAATVTGKLVHANFGTKKDFEDLYTPVNGSIVIVRAGKITFAEKVANAESLNAIGVLIYMDQTKFPIVNAELSFFGHAHLGTGDPYTPGFPSFNHTQFPPSRSSGLPNIPVQTISRAAAEKLFGNMEGDCPSDWKTDSTCRMVTSESKNVKLTVSNVLKE (SEQ ID NO: 109) and does not inhibit the binding interaction between the transferrin receptor and transferrin and / or (by way of example and not limitation) human hemochromatosis protein (also known as HFE). In some embodiments, an anti-transferrin receptor antibody described herein does not bind to the epitope of SEQ ID NO: 109.

[0104] Suitable methodologies may be used to obtain and / or (for example, and) produce antibodies, antibody fragments, or antigen-binding agents, for example, through the use of recombinant DNA protocols. In some embodiments, antibodies may also be produced through the generation of hybridomas (see, for example, Kohler, G. and Milstein, C. "Continuous cultures of fused cells secreting antibody of predefined specificity," Nature, 1975, 256:495-497). The antigen of interest may be used as an immunogen in any form or entity, for example, in a recombinant or naturally occurring form or entity. Hybridomas are screened using standard methods, for example, ELISA screening, to find at least one hybridoma producing an antibody that targets a specific antigen. Antibodies may also be produced through screening of protein expression libraries (for example, phage display libraries) that express antibodies. Phage display library design may also be used in some embodiments (see, e.g., U.S. Pat. No. 5,223,409, filed March 1, 1991, entitled "Directed evolution of novel binding proteins"; WO 1992 / 18619, filed April 10, 1992, entitled "Heterodimeric receptor libraries using phagemids"; WO 1991 / 17271, filed May 1, 1991, entitled "Recombinant library screening methods"; WO 1992 / 20791, filed May 15, 1992, entitled "Methods for producing members of specific binding pairs"; and WO 1992 / 15679, filed February 28, 1992, entitled "Improved epitope displaying phage"). In some embodiments, the antigen of interest may be used to immunize a non-human animal, e.g., a rodent or goat.In some embodiments, once the antibody is obtained from the non-human animal, it may then optionally be modified using a number of methodologies, for example, using recombinant DNA techniques. Additional examples of antibody production and methodologies are also known in the art (see, for example, Harlow et al., "Antibodies: A Laboratory Manual," Cold Spring Harbor Laboratory, 1988).

[0105] In some embodiments, the antibody is modified (e.g., modified via glycosylation, phosphorylation, sumoylation, and / or (e.g., and) methylation). In some embodiments, the antibody is a glycosylated antibody conjugated to one or more sugar or carbohydrate molecules. In some embodiments, the one or more sugar or carbohydrate molecules are conjugated to the antibody via N-glycosylation, O-glycosylation, C-glycosylation, glypiation (GPI anchor attachment), and / or (e.g., and) phosphoglycosylation. In some embodiments, the one or more sugar or carbohydrate molecules are monosaccharides, disaccharides, oligosaccharides, or glycans. In some embodiments, the one or more sugar or carbohydrate molecules are branched oligosaccharides or branched glycans. In some embodiments, the one or more sugar or carbohydrate molecules include a mannose unit, a glucose unit, an N-acetylglucosamine unit, an N-acetylgalactosamine unit, a galactose unit, a fucose unit, or a phospholipid unit. In some embodiments, there are about 1-10, about 1-5, about 5-10, about 1-4, about 1-3, or about 2 sugar molecules. In some embodiments, the glycosylated antibody is fully or partially glycosylated. In some embodiments, the antibody is glycosylated by chemical reaction or by enzymatic means. In some embodiments, the antibody is glycosylated in vitro or inside a cell (optionally deficient in an enzyme (e.g., glycosyltransferase) in the N- or O-glycosylation pathway). In some embodiments, the antibody is functionalized with a sugar or carbohydrate molecule as described in International Patent Application Publication WO2014065661, published May 1, 2014, entitled "Modified antibody, antibody-conjugate and process for the preparation thereof."

[0106] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a VL domain and / or (for example, and) a VH domain of any one of the anti-TfR1 antibodies selected from Table 2, and comprises a constant region comprising the amino acid sequence of the constant region of an IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, of any class (for example, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or of any subclass (for example, IgG2a and IgG2b) of immunoglobulin molecules. Non-limiting examples of human constant regions are described in the art; see, e.g., Kabat EA et al. (1991), supra.

[0107] In some embodiments, agents that bind to the transferrin receptor, e.g., anti-TfR1 antibodies, can target muscle cells and / or mediate transport of agents across the blood-brain barrier (e.g., and). The transferrin receptor is an internalized cell surface receptor that transports transferrin across the cell membrane and participates in the regulation and homeostasis of intracellular iron levels. Some aspects of the present disclosure provide transferrin receptor-binding proteins capable of binding to the transferrin receptor. An antibody that binds, e.g., specifically binds, to the transferrin receptor may be internalized into the cell upon binding to the transferrin receptor, e.g., through receptor-mediated endocytosis.

[0108] In some embodiments, provided herein are humanized antibodies that bind to the transferrin receptor with high specificity and affinity. In some embodiments, the humanized anti-TfR1 antibodies described herein specifically bind to any extracellular epitope of the transferrin receptor, or to an epitope that becomes exposed to the antibody. In some embodiments, the humanized anti-TfR1 antibodies provided herein specifically bind to transferrin receptor from humans, non-human primates, mice, rats, etc. In some embodiments, the humanized anti-TfR1 antibodies provided herein bind to the human transferrin receptor. In some embodiments, the humanized anti-TfR1 antibodies described herein bind to an amino acid segment of the human or non-human primate transferrin receptor, such as those provided in SEQ ID NOS: 105-108. In some embodiments, the humanized anti-TfR1 antibodies described herein bind to an amino acid segment corresponding to amino acids 90-96 of the human transferrin receptor set forth in SEQ ID NO: 105, which is not in the apical domain of the transferrin receptor. In some embodiments, the humanized anti-TfR1 antibodies described herein bind to TfR1 but not to TfR2.

[0109] In some embodiments, the anti-TfR1 antibody is at least about 10 -4 M, 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, 10 -13In some embodiments, the anti-TfR1 antibodies described herein bind to TfR1 (e.g., human or non-human primate TfR1) with a binding affinity (e.g., indicated by Kd) of 1 M or less. In some embodiments, the anti-TfR1 antibodies described herein bind to TfR1 with a Kd in the sub-nanomolar range. In some embodiments, the anti-TfR1 antibodies described herein selectively bind to transferrin receptor 1 (TfR1) but not transferrin receptor 2 (TfR2). In some embodiments, the anti-TfR1 antibodies described herein bind to human TfR1 and cynomolgus TfR1 (e.g., 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, 10 -13 The anti-TfR1 antibodies do not bind to mouse TfR1 (by virtue of a smaller Kd, or M). The affinity and binding kinetics of the anti-TfR1 antibodies can be tested using any suitable method, including, but not limited to, biosensor technology (e.g., OCTET or BIACORE). In some embodiments, the binding of any one of the anti-TfR1 antibodies described herein does not compete with or inhibit transferrin binding to TfR1. In some embodiments, the binding of any one of the anti-TfR1 antibodies described herein does not compete with or inhibit HFE-beta2-microglobulin binding to TfR1.

[0110] In some embodiments, the anti-TfR1 antibodies described herein are humanized antibodies. The CDR and variable region amino acid sequences of the murine monoclonal anti-TfR1 antibodies from which the humanized anti-TfR1 antibodies described herein are derived are provided in Table 2. Table 2. Mouse monoclonal anti-TfR1 antibodies [Table 2-1] [Table 2-2] [Table 2-3]

[0111] In some embodiments, an anti-TfR1 antibody of the disclosure is a humanized variant of any one of the anti-TfR1 antibodies provided in Table 2. In some embodiments, an anti-TfR1 antibody of the disclosure comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 that are the same as the CDR-H1, CDR-H2, and CDR-H3 of any one of the shown anti-TfR1 antibodies provided in Table 2, and comprises a humanized heavy chain variable region and / or (by way of example and) a humanized light chain variable region.

[0112] Humanized antibodies are human immunoglobulins (recipient antibody) in which residues from the recipient's complementarity-determining regions (CDRs) are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. In some embodiments, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the imported CDR or framework sequences, but may also include residues incorporated to further refine and optimize antibody performance. In general, humanized antibodies will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. A humanized antibody will optimally also comprise at least a portion of an immunoglobulin (typically a human immunoglobulin) constant region or domain (Fc). The antibody may have an Fc region modified as described in WO 99 / 58572. Other forms of humanized antibodies have one or more CDRs (1, 2, 3, 4, 5, 6) that are altered with respect to the original antibody, also referred to as one or more CDRs derived from one or more CDRs from the original antibody. Humanized antibodies may also be affinity matured.

[0113] Humanized antibodies and methods for making them are known, see, for example, Almagro et al., Front. Biosci. 13:1619-1633 (2008); Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005); Padlan et al., Mol. Immunol. 28:489-498 (1991); Dall'Acqua et al., Methods 36:43-60 (2005); As described in Osbourn et al., Methods 36:61-68 (2005); and Klimka et al., Br. J. Cancer, 83:252-260 (2000), the contents of all of which are incorporated herein by reference. Human framework regions that can be used for humanization are described, for example, in Sims et al. J. Immunol. 151:2296(1993); Carter et al. Proc. Natl. Acad. Sci. USA. 89:4285(1992); Presta et al. J. Immunol. 151:2623(1993); Almagro et al., Front. Biosci. 13:1619-1633(2008)); Baca et al., J. Biol. Chem. 272:10678-10684(1997); and Rosok et al., J. Biol. Chem. 271:22611-22618(1996), the contents of all of which are incorporated herein by reference.

[0114] In some embodiments, the anti-TfR1 antibodies of the present disclosure comprise a humanized VH (e.g., in the VH framework region) that comprises one or more amino acid variations compared to any one of the VHs listed in Table 2, and / or a humanized VL (e.g., in the VL framework region) that comprises one or more amino acid variations compared to any one of the VLs listed in Table 2 (e.g., and).

[0115] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a humanized VH that contains no more than 25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) compared to the VH of any of the anti-TfR antibodies listed in Table 2 (e.g., any one of SEQ ID NOs: 17, 22, 26, 43, 61, 65, and 68). Alternatively or additionally (e.g., additionally), the anti-TfR1 antibodies of the present disclosure include a humanized VL that contains 25 or fewer amino acid variations (e.g., 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid variations) compared to the VL of any one of the anti-TfR1 antibodies listed in Table 2 (e.g., any one of SEQ ID NOs: 18, 44, and 62).

[0116] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a VH comprising an amino acid sequence that is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regions to the VH of any of the anti-TfR1 antibodies listed in Table 2 (e.g., any one of SEQ ID NOs: 17, 22, 26, 43, 61, 65, and 68). Alternatively or additionally (e.g., additionally), in some embodiments, an anti-TfR1 antibody of the present disclosure comprises a humanized VL comprising an amino acid sequence that is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regions to the VL of any of the anti-TfR1 antibodies listed in Table 2 (e.g., any one of SEQ ID NOs: 18, 44, and 62).

[0117] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a CDR-H1 having the amino acid sequence of SEQ ID NO: 1 (according to the IMGT definition system), a CDR-H2 having the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 19, or SEQ ID NO: 23 (according to the IMGT definition system), and a CDR-H3 having the amino acid sequence of SEQ ID NO: 3 (according to the IMGT definition system), and comprises a humanized VH containing no more than 25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) in the framework regions compared to the VH set forth in SEQ ID NO: 17, SEQ ID NO: 22, or SEQ ID NO: 26. Alternatively or additionally (for example, additionally), an anti-TfR1 antibody of the present disclosure comprises a CDR-L1 having the amino acid sequence of SEQ ID NO: 4 (according to the IMGT definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 5 (according to the IMGT definition system), a CDR-L3 having the amino acid sequence of SEQ ID NO: 6 (according to the IMGT definition system), and a VL containing 25 or fewer amino acid variations (for example, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid variations) in the framework regions compared to the VL set forth in SEQ ID NO: 18.

[0118] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 1 (according to the IMGT definition system), a CDR-H2 having the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 19, or SEQ ID NO: 23 (according to the IMGT definition system), and a CDR-H3 having the amino acid sequence of SEQ ID NO: 3 (according to the IMGT definition system), and is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regions to a VH set forth in SEQ ID NO: 17, SEQ ID NO: 22, or SEQ ID NO: 26. Alternatively or additionally (for example, additionally), the anti-TfR1 antibodies of the present disclosure comprise a VL comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 4 (according to the IMGT definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 5 (according to the IMGT definition system), and a CDR-L3 having the amino acid sequence of SEQ ID NO: 6 (according to the IMGT definition system), and are at least 75% (for example, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regions to a VL set forth in any one of SEQ ID NOs: 18.

[0119] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a CDR-H1 having the amino acid sequence of SEQ ID NO:7 (according to the Kabat definition system), a CDR-H2 having the amino acid sequence of SEQ ID NO:8, SEQ ID NO:20, or SEQ ID NO:24 (according to the Kabat definition system), and a CDR-H3 having the amino acid sequence of SEQ ID NO:9 (according to the Kabat definition system), and a modified VH containing 25 or fewer amino acid variations (e.g., 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid variations) in the framework regions compared to the VH set forth in SEQ ID NO:17, SEQ ID NO:22, or SEQ ID NO:26. Alternatively or additionally (for example, additionally), the anti-TfR1 antibodies of the present disclosure include a CDR-L1 having the amino acid sequence of SEQ ID NO: 10 (according to the Kabat definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 11 (according to the Kabat definition system), and a CDR-L3 having the amino acid sequence of SEQ ID NO: 6 (according to the Kabat definition system), and a humanized VL containing 25 or fewer amino acid variations (for example, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid variations) in the framework regions compared to the VL set forth in SEQ ID NO: 18.

[0120] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO:7 (according to the Kabat definition system), a CDR-H2 having the amino acid sequence of SEQ ID NO:8, SEQ ID NO:20, or SEQ ID NO:24 (according to the Kabat definition system), and a CDR-H3 having the amino acid sequence of SEQ ID NO:9 (according to the Kabat definition system), and is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regions to the VH set forth in SEQ ID NO:17, SEQ ID NO:22, or SEQ ID NO:26. Alternatively or additionally (for example, additionally), the anti-TfR1 antibodies of the present disclosure comprise a VL comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 10 (according to the Kabat definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 11 (according to the Kabat definition system), and a CDR-L3 having the amino acid sequence of SEQ ID NO: 6 (according to the Kabat definition system), and which is at least 75% (for example, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regions to a VL set forth in any one of SEQ ID NOs: 18.

[0121] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a CDR-H1 having the amino acid sequence of SEQ ID NO: 12 (according to the Chothia definition system), a CDR-H2 having the amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 21, or SEQ ID NO: 25 (according to the Chothia definition system), a CDR-H3 having the amino acid sequence of SEQ ID NO: 14 (according to the Chothia definition system), and a VH containing 25 or fewer amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) in the framework regions compared to the VH set forth in SEQ ID NO: 17, SEQ ID NO: 22, or SEQ ID NO: 26. Alternatively or additionally (for example, additionally), an anti-TfR1 antibody of the present disclosure comprises a CDR-L1 having the amino acid sequence of SEQ ID NO: 15 (according to the Chothia definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 5 (according to the Chothia definition system), and a CDR-L3 having the amino acid sequence of SEQ ID NO: 16 (according to the Chothia definition system), and a VL containing 25 or fewer amino acid variations (for example, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid variations) in the framework regions compared to the VL set forth in SEQ ID NO: 18.

[0122] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 12 (according to the Chothia definition system), a CDR-H2 having the amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 21, or SEQ ID NO: 25 (according to the Chothia definition system), and a CDR-H3 having the amino acid sequence of SEQ ID NO: 14 (according to the Chothia definition system), and is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regions to the VH set forth in SEQ ID NO: 17, SEQ ID NO: 22, or SEQ ID NO: 26. Alternatively or additionally (for example, additionally), an anti-TfR1 antibody of the present disclosure comprises a VL comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 15 (according to the Chothia definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 5 (according to the Chothia definition system), and a CDR-L3 having the amino acid sequence of SEQ ID NO: 16 (according to the Chothia definition system), and is at least 75% (for example, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regions to a VL set forth in any one of SEQ ID NOs: 18.

[0123] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a CDR-H1 having the amino acid sequence of SEQ ID NO: 27 (according to the IMGT definition system), a CDR-H2 having the amino acid sequence of SEQ ID NO: 28 (according to the IMGT definition system), and a CDR-H3 having the amino acid sequence of SEQ ID NO: 29 (according to the IMGT definition system), and a VH that contains 25 or fewer amino acid variations in the framework regions compared to the VH set forth in SEQ ID NO: 43 (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations). Alternatively or additionally (for example, additionally), an anti-TfR1 antibody of the present disclosure comprises a CDR-L1 having the amino acid sequence of SEQ ID NO: 30 (according to the IMGT definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 31 (according to the IMGT definition system), a CDR-L3 having the amino acid sequence of SEQ ID NO: 32 (according to the IMGT definition system), and a VL containing 25 or fewer amino acid variations (for example, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid variations) in the framework regions compared to the VL set forth in SEQ ID NO: 44.

[0124] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 27 (according to the IMGT definition system), a CDR-H2 having the amino acid sequence of SEQ ID NO: 28 (according to the IMGT definition system), and a CDR-H3 having the amino acid sequence of SEQ ID NO: 29 (according to the IMGT definition system), and is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regions to the VH set forth in SEQ ID NO: 43. Alternatively or additionally (for example, additionally), the anti-TfR1 antibody of the present disclosure comprises a VL comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 30 (according to the IMGT definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 31 (according to the IMGT definition system), and a CDR-L3 having the amino acid sequence of SEQ ID NO: 32 (according to the IMGT definition system), and is at least 75% (for example, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regions to the VL set forth in SEQ ID NO: 44.

[0125] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a CDR-H1 having the amino acid sequence of SEQ ID NO: 33 (according to the Kabat definition system), a CDR-H2 having the amino acid sequence of SEQ ID NO: 34 (according to the Kabat definition system), and a CDR-H3 having the amino acid sequence of SEQ ID NO: 35 (according to the Kabat definition system), and a VH containing 25 or fewer amino acid variations in the framework regions compared to the VH set forth in SEQ ID NO: 43 (e.g., 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid variations). Alternatively or additionally (for example, additionally), the anti-TfR1 antibodies of the present disclosure comprise a CDR-L1 having the amino acid sequence of SEQ ID NO: 36 (according to the Kabat definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 37 (according to the Kabat definition system), and a CDR-L3 having the amino acid sequence of SEQ ID NO: 32 (according to the Kabat definition system), and a VL containing 25 or fewer amino acid variations (for example, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid variations) in the framework regions compared to the VL set forth in SEQ ID NO: 44.

[0126] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 33 (according to the Kabat definition system), a CDR-H2 having the amino acid sequence of SEQ ID NO: 34 (according to the Kabat definition system), and a CDR-H3 having the amino acid sequence of SEQ ID NO: 35 (according to the Kabat definition system), and is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regions to the VH set forth in SEQ ID NO: 43. Alternatively or in addition (for example, in addition), the anti-TfR1 antibodies of the present disclosure comprise a VL comprising a CDR-L1 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO: 36, a CDR-L2 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO: 37, and a CDR-L3 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO: 32, and which is at least 75% (for example, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regions to a VL as set forth in SEQ ID NO: 44.

[0127] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a CDR-H1 having the amino acid sequence of SEQ ID NO: 38 (according to the Chothia definition system), a CDR-H2 having the amino acid sequence of SEQ ID NO: 39 (according to the Chothia definition system), and a CDR-H3 having the amino acid sequence of SEQ ID NO: 40 (according to the Chothia definition system), and a VH containing 25 or fewer amino acid variations in the framework regions compared to the VH set forth in SEQ ID NO: 43 (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations). Alternatively or additionally (for example, additionally), an anti-TfR1 antibody of the present disclosure comprises a CDR-L1 having the amino acid sequence of SEQ ID NO: 41 (according to the Chothia definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 31 (according to the Chothia definition system), and a CDR-L3 having the amino acid sequence of SEQ ID NO: 42 (according to the Chothia definition system), and a VL containing 25 or fewer amino acid variations (for example, no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) in the framework regions compared to the VL set forth in SEQ ID NO: 44.

[0128] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 38 (according to the Chothia definition system), a CDR-H2 having the amino acid sequence of SEQ ID NO: 39 (according to the Chothia definition system), and a CDR-H3 having the amino acid sequence of SEQ ID NO: 40 (according to the Chothia definition system), and is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regions to the VH set forth in SEQ ID NO: 43. Alternatively or additionally (for example, additionally), an anti-TfR1 antibody of the present disclosure comprises a VL comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 41 (according to the Chothia definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 31 (according to the Chothia definition system), and a CDR-L3 having the amino acid sequence of SEQ ID NO: 42 (according to the Chothia definition system), and is at least 75% (for example, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regions to the VL set forth in SEQ ID NO: 44.

[0129] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a CDR-H1 having the amino acid sequence of SEQ ID NO: 45, SEQ ID NO: 63, or SEQ ID NO: 66 (according to the IMGT definition system), a CDR-H2 having the amino acid sequence of SEQ ID NO: 46 (according to the IMGT definition system), and a CDR-H3 having the amino acid sequence of SEQ ID NO: 47 (according to the IMGT definition system), and a VH containing 25 or fewer amino acid variations (e.g., 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid variations) in the framework regions compared to the VH set forth in SEQ ID NO: 61, SEQ ID NO: 65, or SEQ ID NO: 68. Alternatively or additionally (for example, additionally), an anti-TfR1 antibody of the present disclosure comprises a CDR-L1 having the amino acid sequence of SEQ ID NO: 48 (according to the IMGT definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 49 (according to the IMGT definition system), and a CDR-L3 having the amino acid sequence of SEQ ID NO: 50 (according to the IMGT definition system), and a VL containing 25 or fewer amino acid variations (for example, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid variations) in the framework regions compared to the VL set forth in SEQ ID NO: 62.

[0130] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 45, SEQ ID NO: 63, or SEQ ID NO: 66 (according to the IMGT definition system), a CDR-H2 having the amino acid sequence of SEQ ID NO: 46 (according to the IMGT definition system), and a CDR-H3 having the amino acid sequence of SEQ ID NO: 47 (according to the IMGT definition system), and is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regions to the VH set forth in SEQ ID NO: 61, SEQ ID NO: 65, or SEQ ID NO: 68. Alternatively or additionally (for example, additionally), an anti-TfR1 antibody of the present disclosure comprises a VL comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 48 (according to the IMGT definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 49 (according to the IMGT definition system), and a CDR-L3 having the amino acid sequence of SEQ ID NO: 50 (according to the IMGT definition system), and is at least 75% (for example, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regions to the VL set forth in SEQ ID NO: 62.

[0131] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a CDR-H1 having the amino acid sequence of SEQ ID NO: 51, SEQ ID NO: 64, or SEQ ID NO: 67 (according to the Kabat definition system), a CDR-H2 having the amino acid sequence of SEQ ID NO: 52 (according to the Kabat definition system), and a CDR-H3 having the amino acid sequence of SEQ ID NO: 53 (according to the Kabat definition system), and a VH containing 25 or fewer amino acid variations (e.g., 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid variations) in the framework regions compared to the VH set forth in SEQ ID NO: 61, SEQ ID NO: 65, or SEQ ID NO: 68. Alternatively or additionally (for example, additionally), the anti-TfR1 antibodies of the present disclosure comprise a CDR-L1 having the amino acid sequence of SEQ ID NO: 54 (according to the Kabat definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 55 (according to the Kabat definition system), and a CDR-L3 having the amino acid sequence of SEQ ID NO: 50 (according to the Kabat definition system), and a VL containing 25 or fewer amino acid variations (for example, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid variations) in the framework regions compared to the VL set forth in SEQ ID NO: 62.

[0132] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 51, SEQ ID NO: 64, or SEQ ID NO: 67 (according to the Kabat definition system), a CDR-H2 having the amino acid sequence of SEQ ID NO: 52 (according to the Kabat definition system), and a CDR-H3 having the amino acid sequence of SEQ ID NO: 53 (according to the Kabat definition system), and is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regions to the VH set forth in SEQ ID NO: 61, SEQ ID NO: 65, or SEQ ID NO: 68. Alternatively or additionally (for example, additionally), the anti-TfR1 antibody of the present disclosure comprises a VL comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 54 (according to the Kabat definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 55 (according to the Kabat definition system), and a CDR-L3 having the amino acid sequence of SEQ ID NO: 50 (according to the Kabat definition system), and is at least 75% (for example, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regions to the VL set forth in SEQ ID NO: 62.

[0133] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a CDR-H1 having the amino acid sequence of SEQ ID NO: 56 (according to the Chothia definition system), a CDR-H2 having the amino acid sequence of SEQ ID NO: 57 (according to the Chothia definition system), and a CDR-H3 having the amino acid sequence of SEQ ID NO: 58 (according to the Chothia definition system), and a VH that contains 25 or fewer amino acid variations in the framework regions compared to the VHs set forth in SEQ ID NO: 61, SEQ ID NO: 65, and SEQ ID NO: 68 (e.g., 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid variations). Alternatively or additionally (for example, additionally), an anti-TfR1 antibody of the present disclosure comprises a CDR-L1 having the amino acid sequence of SEQ ID NO: 59 (according to the Chothia definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 49 (according to the Chothia definition system), and a CDR-L3 having the amino acid sequence of SEQ ID NO: 60 (according to the Chothia definition system), and a VL containing 25 or fewer amino acid variations (for example, no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) in the framework regions compared to the VL set forth in SEQ ID NO: 62.

[0134] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 56 (according to the Chothia definition system), a CDR-H2 having the amino acid sequence of SEQ ID NO: 57 (according to the Chothia definition system), and a CDR-H3 having the amino acid sequence of SEQ ID NO: 58 (according to the Chothia definition system), and is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regions to the VH set forth in SEQ ID NO: 61, SEQ ID NO: 65, or SEQ ID NO: 68. Alternatively or additionally (for example, additionally), an anti-TfR1 antibody of the present disclosure comprises a VL comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 59 (according to the Chothia definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 49 (according to the Chothia definition system), and a CDR-L3 having the amino acid sequence of SEQ ID NO: 60 (according to the Chothia definition system), and is at least 75% (for example, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regions to the VL set forth in SEQ ID NO: 62.

[0135] Exemplary amino acid sequences of anti-TfR1 antibodies described herein are provided in Table 3. Table 3. Variable regions of anti-TfR1 antibodies [Table 3-1] [Table 3-2] [Table 3-3]

[0136] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a VH comprising CDR-H1, CDR-H2, and CDR-H3 of any one of the anti-TfR1 antibodies provided in Table 2, and comprises one or more (e.g., other than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid variations compared to the respective VHs provided in Table 3. Alternatively or additionally (e.g., additionally), an anti-TfR1 antibody of the present disclosure comprises a VL comprising CDR-L1, CDR-L2, and CDR-L3 of any one of the anti-TfR1 antibodies provided in Table 2, and comprises one or more (e.g., other than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid variations compared to the respective VLs provided in Table 3.

[0137] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a VH comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 69, and / or a VL comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to (e.g., and) SEQ ID NO: 70. In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO: 69 and a VL comprising the amino acid sequence of SEQ ID NO: 70.

[0138] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a VH comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 71, and / or a VL comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to (e.g., and) SEQ ID NO: 70. In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO: 71 and a VL comprising the amino acid sequence of SEQ ID NO: 70.

[0139] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a VH comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 72, and / or a VL comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to (e.g., and) SEQ ID NO: 70. In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO: 72 and a VL comprising the amino acid sequence of SEQ ID NO: 70.

[0140] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a VH comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 73, and / or a VL comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to (e.g., and) SEQ ID NO: 74. In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO: 73 and a VL comprising the amino acid sequence of SEQ ID NO: 74.

[0141] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a VH comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 73, and / or a VL comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to (e.g., and) SEQ ID NO: 75. In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO: 73 and a VL comprising the amino acid sequence of SEQ ID NO: 75.

[0142] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a VH comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 76, and / or a VL comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to (e.g., and) SEQ ID NO: 74. In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO: 76 and a VL comprising the amino acid sequence of SEQ ID NO: 74.

[0143] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a VH comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 76, and / or a VL comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to (e.g., and) SEQ ID NO: 75. In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO: 76 and a VL comprising the amino acid sequence of SEQ ID NO: 75.

[0144] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a VH comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 77, and / or a VL comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to (e.g., and) SEQ ID NO: 78. In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO: 77 and a VL comprising the amino acid sequence of SEQ ID NO: 78.

[0145] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a VH comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 79, and / or a VL comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to (e.g., and) SEQ ID NO: 80. In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO: 79 and a VL comprising the amino acid sequence of SEQ ID NO: 80.

[0146] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a VH comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 77, and / or a VL comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to (e.g., and) SEQ ID NO: 80. In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO: 77 and a VL comprising the amino acid sequence of SEQ ID NO: 80.

[0147] In some embodiments, the anti-TfR1 antibodies described herein are full-length IgGs, which may include heavy and light chain constant regions from a human antibody. In some embodiments, the heavy chain of any of the anti-TfR1 antibodies described herein may include a heavy chain constant region (CH) or a portion thereof (e.g., CH1, CH2, CH3, or a combination thereof). The heavy chain constant region may belong to any suitable source, e.g., human, mouse, rat, or rabbit. In a specific example, the heavy chain constant region is from human IgG (gamma heavy chain), e.g., IgG1, IgG2, or IgG4. An example of a human IgG1 constant region is provided below: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 81)

[0148] In some embodiments, the heavy chain of any of the anti-TfR1 antibodies described herein comprises a mutant human IgG1 constant region. For example, the introduction of a LALA mutation on the CH2 domain of human IgG1 (a mutant derived from mAb b12 mutated to replace the lower hinge residues Leu234 and Leu235 with Ala234 and Ala235) is known to reduce Fcg receptor binding (Bruhns, P., et al. (2009) and Xu, D. et al. (2000)). The mutant human IgG1 constant region is given below (mutations are bolded and underlined): [ka]

[0149] In some embodiments, the light chain of any of the anti-TfR1 antibodies described herein may further comprise a light chain constant region (CL), which may be any CL known in the art. In some examples, the CL is a kappa light chain. In other examples, the CL is a lambda light chain. In some embodiments, the CL is a kappa light chain, the sequence of which is given below: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 83)

[0150] Heavy and light chain constant regions of other antibodies are well known in the art and are provided, for example, in the IMGT database (imgt.org) or at vbase2.org / vbstat.php., both of which are incorporated herein by reference.

[0151] In some embodiments, the anti-TfR1 antibodies described herein comprise a heavy chain comprising any one of a VH or any variant thereof listed in Table 3, and a heavy chain constant region that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 81 or SEQ ID NO: 82. In some embodiments, the anti-TfR1 antibodies described herein comprise a heavy chain comprising any one of a VH or any variant thereof listed in Table 3, and a heavy chain constant region that contains 25 or fewer amino acid variations (e.g., 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid variations) compared to SEQ ID NO: 81 or SEQ ID NO: 82. In some embodiments, the anti-TfR1 antibodies described herein comprise a heavy chain comprising any one of a VH or any variant thereof listed in Table 3, and a heavy chain constant region set forth in SEQ ID NO: 81. In some embodiments, the anti-TfR1 antibodies described herein comprise any one of the VHs listed in Table 3 or any variant thereof, and a heavy chain comprising the heavy chain constant region set forth in SEQ ID NO:82.

[0152] In some embodiments, the anti-TfR1 antibodies described herein comprise a light chain comprising any one of a VL or any variant thereof listed in Table 3, and a light chain constant region that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 83. In some embodiments, the anti-TfR1 antibodies described herein comprise a light chain comprising any one of a VL or any variant thereof listed in Table 3, and a light chain constant region that contains 25 or fewer amino acid variations (e.g., 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid variations) compared to SEQ ID NO: 83. In some embodiments, the anti-TfR1 antibodies described herein comprise a light chain comprising any one of a VL or any variant thereof listed in Table 3, and a light chain constant region set forth in SEQ ID NO: 83.

[0153] Examples of IgG heavy and light chain amino acid sequences of the described anti-TfR1 antibodies are provided in Table 4 below. Table 4. Heavy and light chain sequences of examples of humanized anti-TfR1 IgG [Table 4-1] [Table 4-2] [Table 4-3]

[0154] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a heavy chain containing no more than 25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) compared to the heavy chain set forth in any one of SEQ ID NOs: 84, 86, 87, 88, 91, 92, and 94. Alternatively or additionally (e.g., additionally), an anti-TfR1 antibody of the present disclosure includes a light chain that contains no more than 25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) compared to the light chain set forth in any one of SEQ ID NOs: 85, 89, 90, 93, and 95.

[0155] In some embodiments, the anti-TfR1 antibodies described herein comprise a heavy chain comprising an amino acid sequence at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to any one of SEQ ID NOs: 84, 86, 87, 88, 91, 92, and 94. Alternatively or additionally (e.g., additionally), the anti-TfR1 antibodies described herein comprise a light chain comprising an amino acid sequence at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to any one of SEQ ID NOs: 85, 89, 90, 93, and 95. In some embodiments, the anti-TfR1 antibodies described herein comprise a heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 84, 86, 87, 88, 91, 92, and 94. Alternatively or additionally (for example, additionally), the anti-TfR1 antibodies described herein comprise a light chain comprising the amino acid sequence of any one of SEQ ID NOs: 85, 89, 90, 93 and 95.

[0156] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 84, and / or a light chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to (e.g., and) SEQ ID NO: 85. In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 84 and a light chain comprising the amino acid sequence of SEQ ID NO: 85.

[0157] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 86, and / or a light chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to (e.g., and) SEQ ID NO: 85. In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 86 and a light chain comprising the amino acid sequence of SEQ ID NO: 85.

[0158] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 87, and / or a light chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to (e.g., and) SEQ ID NO: 85. In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 87 and a light chain comprising the amino acid sequence of SEQ ID NO: 85.

[0159] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 88, and / or a light chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to (e.g., and) SEQ ID NO: 89. In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 88 and a light chain comprising the amino acid sequence of SEQ ID NO: 89.

[0160] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 88, and / or a light chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to (e.g., and) SEQ ID NO: 90. In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 88 and a light chain comprising the amino acid sequence of SEQ ID NO: 90.

[0161] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 91, and / or a light chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to (e.g., and) SEQ ID NO: 89. In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 91 and a light chain comprising the amino acid sequence of SEQ ID NO: 89.

[0162] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 91, and / or a light chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to (e.g., and) SEQ ID NO: 90. In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 91 and a light chain comprising the amino acid sequence of SEQ ID NO: 90.

[0163] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 92, and / or a light chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to (e.g., and) SEQ ID NO: 93. In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 92 and a light chain comprising the amino acid sequence of SEQ ID NO: 93.

[0164] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 94, and / or a light chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to (e.g., and) SEQ ID NO: 95. In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 94 and a light chain comprising the amino acid sequence of SEQ ID NO: 95.

[0165] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 92, and / or a light chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to (e.g., and) SEQ ID NO: 95. In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 92 and a light chain comprising the amino acid sequence of SEQ ID NO: 95.

[0166] In some embodiments, the anti-TfR1 antibody is a Fab fragment, Fab' fragment, or F(ab')2 fragment of an intact antibody (full-length antibody). Antigen-binding fragments of intact antibodies (full-length antibodies) can be prepared by conventional methods (e.g., recombinantly or by digesting the heavy chain constant region of a full-length IgG with an enzyme such as papain). For example, F(ab')2 fragments can be produced by pepsin or papain digestion of antibody molecules, and Fab' fragments can be generated by reducing the disulfide bridges of F(ab')2 fragments. In some embodiments, the heavy chain region of the Fab fragment of an anti-TfR1 antibody described herein comprises the amino acid sequence ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT (SEQ ID NO: 96).

[0167] In some embodiments, the anti-TfR1 antibodies described herein comprise a heavy chain comprising any one of a VH listed in Table 3 or any variant thereof, and a heavy chain constant region that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 96. In some embodiments, the anti-TfR1 antibodies described herein comprise a heavy chain comprising any one of a VH listed in Table 3 or any variant thereof, and a heavy chain constant region that contains 25 or fewer amino acid variations (e.g., 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid variations) compared to SEQ ID NO: 96. In some embodiments, the anti-TfR1 antibodies described herein comprise a heavy chain comprising any one of a VH listed in Table 3 or any variant thereof, and a heavy chain constant region set forth in SEQ ID NO:96.

[0168] In some embodiments, the anti-TfR1 antibodies described herein comprise a light chain comprising any one of a VL or any variant thereof listed in Table 3, and a light chain constant region that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 83. In some embodiments, the anti-TfR1 antibodies described herein comprise a light chain comprising any one of a VL or any variant thereof listed in Table 3, and a light chain constant region that contains 25 or fewer amino acid variations (e.g., 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid variations) compared to SEQ ID NO: 83. In some embodiments, the anti-TfR1 antibodies described herein comprise a light chain comprising any one of a VL or any variant thereof listed in Table 3, and a light chain constant region set forth in SEQ ID NO: 83.

[0169] Examples of Fab heavy and light chain amino acid sequences of the described anti-TfR1 antibodies are provided in Table 5 below. Table 5. Heavy and light chain sequences of example anti-TfR1 Fabs [Table 5-1] [Table 5-2]

[0170] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a heavy chain containing no more than 25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) compared to a heavy chain set forth in any one of SEQ ID NOs: 97-103. Alternatively or additionally (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) compared to a light chain set forth in any one of SEQ ID NOs: 85, 89, 90, 93, and 95.

[0171] In some embodiments, the anti-TfR1 antibodies described herein comprise a heavy chain comprising an amino acid sequence at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to any one of SEQ ID NOs: 97-103. Alternatively or additionally (e.g., additionally), the anti-TfR1 antibodies described herein comprise a light chain comprising an amino acid sequence at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to any one of SEQ ID NOs: 85, 89, 90, 93, and 95. In some embodiments, the anti-TfR1 antibodies described herein comprise a heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 97-103. Alternatively or additionally (e.g., additionally), the anti-TfR1 antibodies described herein comprise a light chain comprising the amino acid sequence of any one of SEQ ID NOs: 85, 89, 90, 93, and 95.

[0172] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 97, and / or a light chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to (e.g., and) SEQ ID NO: 85. In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 97 and a light chain comprising the amino acid sequence of SEQ ID NO: 85.

[0173] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 98, and / or a light chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to (e.g., and) SEQ ID NO: 85. In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 98 and a light chain comprising the amino acid sequence of SEQ ID NO: 85.

[0174] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 99, and / or a light chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to (e.g., and) SEQ ID NO: 85. In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 99 and a light chain comprising the amino acid sequence of SEQ ID NO: 85.

[0175] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 100, and / or a light chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to (e.g., and) SEQ ID NO: 89. In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 100 and a light chain comprising the amino acid sequence of SEQ ID NO:89.

[0176] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 100, and / or a light chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to (e.g., and) SEQ ID NO: 90. In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 100 and a light chain comprising the amino acid sequence of SEQ ID NO: 90.

[0177] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 101, and / or a light chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to (e.g., and) SEQ ID NO: 89. In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 101 and a light chain comprising the amino acid sequence of SEQ ID NO: 89.

[0178] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 101, and / or a light chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to (e.g., and) SEQ ID NO: 90. In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 101 and a light chain comprising the amino acid sequence of SEQ ID NO: 90.

[0179] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 102, and / or a light chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to (e.g., and) SEQ ID NO: 93. In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 102 and a light chain comprising the amino acid sequence of SEQ ID NO:93.

[0180] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 103, and / or a light chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to (e.g., and) SEQ ID NO: 95. In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 103 and a light chain comprising the amino acid sequence of SEQ ID NO: 95.

[0181] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 102, and / or a light chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to (e.g., and) SEQ ID NO: 95. In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 102 and a light chain comprising the amino acid sequence of SEQ ID NO: 95.

[0182] In some embodiments, the anti-TfR1 receptor antibodies described herein can be in any antibody form, including, but not limited to, intact (i.e., full-length) antibodies, antigen-binding fragments thereof (such as Fab, Fab', F(ab')2, and Fv), single-chain antibodies, bispecific antibodies, or nanobodies. In some embodiments, the anti-TfR1 antibodies described herein are scFvs. In some embodiments, the anti-TfR1 antibodies described herein are scFv-Fabs (e.g., scFvs fused to a portion of a constant region). In some embodiments, the anti-TfR1 receptor antibodies described herein are scFvs fused at either the N-terminus or the C-terminus to a constant region (e.g., the human IgG1 constant region set forth in SEQ ID NO: 81 or SEQ ID NO: 82, or a portion thereof, such as an Fc portion).

[0183] In some embodiments, conservative mutations may be introduced into an antibody sequence (e.g., a CDR or framework sequence) at a position where the residue is unlikely to be involved in interactions with the target antigen (e.g., transferrin receptor), as determined, for example, based on a crystal structure. In some embodiments, one or more mutations (e.g., amino acid substitutions) are introduced into the Fc region (e.g., in the CH2 domain (residues 231-340 of human IgG1), and / or (e.g., in the CH3 domain (residues 341-447 of human IgG1), and / or (e.g., in the hinge region), numbered according to the Kabat numbering system (e.g., EU index of Kabat)) of an anti-TfR1 antibody described herein to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or (e.g., and) antigen-dependent cellular cytotoxicity.

[0184] In some embodiments, one or more mutations (e.g., amino acid substitutions) are introduced into the hinge region of the Fc region (CH1 domain) such that the number of cysteine ​​residues in the hinge region can be varied (e.g., increased or decreased), e.g., as described in U.S. Patent No. 5,677,425. The number of cysteine ​​residues in the hinge region of the CH1 domain can be altered, e.g., to facilitate assembly of the light and heavy chains, or to alter (e.g., increase or decrease) the stability of the antibody, or to facilitate linker conjugation.

[0185] In some embodiments, one or more mutations (e.g., amino acid substitutions) are introduced into the Fc region of a muscle-targeting antibody described herein (e.g., in the CH2 domain (residues 231-340 of human IgG1), and / or (e.g., and) in the CH3 domain (residues 341-447 of human IgG1), and / or (e.g., and) in the hinge region, numbered according to the Kabat numbering system (e.g., EU index of Kabat)) to increase or decrease the affinity of the antibody for an Fc receptor (e.g., an activated Fc receptor) on the surface of an effector cell. Mutations in the Fc region of an antibody that increase or decrease the affinity of the antibody for an Fc receptor, and techniques for introducing such mutations into an Fc receptor or fragment thereof, are known to those of skill in the art. Examples of mutations in the Fc receptor of an antibody that can be made to alter the affinity of the antibody to the Fc receptor are described, for example, in Smith P et al., (2012) PNAS 109:6181-6186, U.S. Patent No. 6,737,056, and International Publication Nos. WO 02 / 060919; WO 98 / 23289; and WO 97 / 34631, which are incorporated herein by reference.

[0186] In some embodiments, one or more amino acid mutations (i.e., substitutions, insertions, or deletions) are introduced into the IgG constant region or FcRn-binding fragment thereof (preferably, the Fc or hinge-Fc domain fragment) to alter (e.g., increase or decrease) the half-life of the antibody in vivo. See, e.g., International Publication Nos. WO 02 / 060919; WO 98 / 23289; and WO 97 / 34631; and U.S. Patent Nos. 5,869,046, 6,121,022, 6,277,375, and 6,165,745, for mutations that may alter (e.g., increase or decrease) the half-life of the antibody in vivo.

[0187] In some embodiments, one or more amino acid mutations (i.e., substitutions, insertions, or deletions) are introduced into the IgG constant region or FcRn-binding fragment thereof (preferably, the Fc or hinge-Fc domain fragment) to decrease the half-life of the anti-TfR antibody in vivo. In some embodiments, one or more amino acid mutations (i.e., substitutions, insertions, or deletions) are introduced into the IgG constant region or FcRn-binding fragment thereof (preferably, the Fc or hinge-Fc domain fragment) to increase the half-life of the antibody in vivo. In some embodiments, the antibody may have one or more amino acid mutations (e.g., substitutions) in the second constant (CH2) domain (residues 231-340 of human IgG1) and / or (for example, and) in the third constant (CH3) domain (residues 341-447 of human IgG1) numbered according to the EU index of Kabat (Kabat EA et al. (1991) supra). In some embodiments, the IgG1 constant region of the antibody described herein comprises a methionine (M) to tyrosine (Y) substitution at position 252, a serine (S) to threonine (T) substitution at position 254, and a threonine (T) to glutamic acid (E) substitution at position 256, numbered according to the EU index as in Kabat. See U.S. Patent No. 7,658,921, which is incorporated herein by reference. This type of mutant IgG, termed a "YTE mutant," has been shown to exhibit a four-fold increased half-life compared to the wild-type version of the same antibody (see Dall'Acqua WF et al., (2006) J Biol Chem 281:23514-24). In some embodiments, the antibody comprises an IgG constant region comprising one, two, three or more amino acid substitutions of amino acid residues at positions 251-257, 285-290, 308-314, 385-389, and 428-436, numbered according to the EU index as in Kabat.

[0188] In some embodiments, one, two, or more amino acid substitutions are introduced into the IgG constant region Fc region to alter the effector function(s) of the anti-transferrin receptor antibody. The effector ligand with altered affinity can be, for example, an Fc receptor or the C1 component of complement. This approach is described in further detail in U.S. Patent Nos. 5,624,821 and 5,648,260. In some embodiments, deletion or inactivation of the constant region domain (through point mutation or other means) can reduce binding of circulating antibodies to Fc receptors, thereby increasing tumor localization. See, e.g., U.S. Patent Nos. 5,585,097 and 8,591,886 for a description of mutations that delete or inactivate constant regions, thereby increasing tumor localization. In some embodiments, one or more amino acid substitutions may be introduced into the Fc region of an antibody described herein to remove potential glycosylation sites in the Fc region, which may reduce binding to Fc receptors (see, e.g., Shields RL et al., (2001) J Biol Chem 276:6591-604).

[0189] In some embodiments, one or more amino acid residues in the constant region of an anti-TfR antibody described herein can be replaced with a different amino acid residue so that the antibody can have altered Clq binding and / or (for example, and) reduced or eliminated complement-dependent cytotoxicity (CDC). This approach is described in further detail in U.S. Pat. No. 6,194,551 (Idusogie et al.). In some embodiments, one or more amino acid residues in the N-terminal region of the CH2 domain of an antibody described herein are altered to thereby alter the antibody's ability to fix complement. This approach is further described in International Publication No. WO 94 / 29351. In some embodiments, the Fc region of an antibody described herein is modified to increase the antibody's ability to mediate antibody-dependent cellular cytotoxicity (ADCC) to cells and / or (for example, and) increase the antibody's affinity for Fcγ receptors. This approach is further described in International Publication No. WO 00 / 42072.

[0190] In some embodiments, the heavy and / or (by way of example) light chain variable domain(s) sequence(s) of the antibodies provided herein can be used to generate, for example, CDR-grafted, chimeric, humanized, or composite human antibodies, or antigen-binding fragments, as described elsewhere herein. As will be understood by one of skill in the art, any variant, CDR-grafted, chimeric, humanized, or composite antibody derived from any of the antibodies provided herein may be useful in the compositions and methods described herein and will retain the ability to specifically bind to the transferrin receptor, such that the variant, CDR-grafted, chimeric, humanized, or composite antibody may have at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or more binding to the transferrin receptor compared to the original antibody from which it was derived.

[0191] In some embodiments, the antibodies provided herein contain mutations that confer desired properties to the antibody. For example, to avoid potential complications due to Fab-arm exchange, which is known to occur in native IgG4 mAbs, the antibodies provided herein may contain the stabilizing "Adair" mutation (Angal S., et al., "A single amino acid substitution abolishes the heterogeneity of chimeric mouse / human (IgG4) antibody," Mol Immunol 30, 105-108; 1993), in which serine 228 (EU numbering; residue 241 Kabat numbering) is converted to proline, resulting in an IgG1-like hinge sequence. Consequently, any of the antibodies may include the stabilizing "Adair" mutation.

[0192] In some embodiments, the antibody is modified (e.g., modified via glycosylation, phosphorylation, sumoylation, and / or (e.g., and) methylation). In some embodiments, the antibody is a glycosylated antibody conjugated to one or more sugar or carbohydrate molecules. In some embodiments, the one or more sugar or carbohydrate molecules are conjugated to the antibody via N-glycosylation, O-glycosylation, C-glycosylation, glypiation (GPI anchor attachment), and / or (e.g., and) phosphoglycosylation. In some embodiments, the one or more sugar or carbohydrate molecules are monosaccharides, disaccharides, oligosaccharides, or glycans. In some embodiments, the one or more sugar or carbohydrate molecules are branched oligosaccharides or branched glycans. In some embodiments, the one or more sugar or carbohydrate molecules include a mannose unit, a glucose unit, an N-acetylglucosamine unit, an N-acetylgalactosamine unit, a galactose unit, a fucose unit, or a phospholipid unit. In some embodiments, there are about 1-10, about 1-5, about 5-10, about 1-4, about 1-3, or about 2 sugar molecules. In some embodiments, the glycosylated antibody is fully or partially glycosylated. In some embodiments, the antibody is glycosylated by chemical reaction or by enzymatic means. In some embodiments, the antibody is glycosylated in vitro or inside a cell (optionally deficient in an enzyme (e.g., glycosyltransferase) in the N- or O-glycosylation pathway). In some embodiments, the antibody is functionalized with a sugar or carbohydrate molecule as described in International Patent Application Publication WO2014065661, published May 1, 2014, entitled "Modified antibody, antibody-conjugate and process for the preparation thereof."

[0193] In some embodiments, any one of the anti-TfR1 antibodies described herein can include a signal peptide (e.g., an N-terminal signal peptide) on the heavy and / or (e.g., and) light chain sequence. In some embodiments, the anti-TfR1 antibodies described herein include any one of the VH and VL sequences, any one of the IgG heavy and light chain sequences, or any one of the Fab heavy and light chain sequences described herein, and further include a signal peptide (e.g., an N-terminal signal peptide). In some embodiments, the signal peptide includes the amino acid sequence MGWSCIILFLVATATGVHS (SEQ ID NO: 104).

[0194] Other known anti-transferrin receptor antibodies Any other suitable anti-transferrin receptor antibody known in the art can be used as a muscle-targeting agent in the conjugates disclosed herein. Examples of known anti-transferrin receptor antibodies (including associated references and binding epitopes) are listed in Table 6. In some embodiments, the anti-transferrin receptor antibody comprises the complementarity-determining regions (CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3) of any of the anti-transferrin receptor antibodies provided herein, e.g., those listed in Table 6. Table 6 - List of anti-TfR1 antibody clones, including relevant references and binding epitope information [Table 6-1] [Table 6-2] The entire contents of the publications listed in Table 6 are incorporated herein by reference.

[0195] In some embodiments, a transferrin receptor antibody of the disclosure includes one or more CDR-H (e.g., CDR-H1, CDR-H2, and CDR-H3) amino acid sequences from any one of the anti-transferrin receptor antibodies selected from Table 6. In some embodiments, a transferrin receptor antibody includes CDR-H1, CDR-H2, and CDR-H3 provided for any one of the anti-transferrin receptor antibodies selected from Table 6. In some embodiments, an anti-transferrin receptor antibody includes CDR-L1, CDR-L2, and CDR-L3 provided for any one of the anti-transferrin receptor antibodies selected from Table 6. In some embodiments, an anti-transferrin antibody includes CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 provided for any one of the anti-transferrin receptor antibodies selected from Table 6. The present disclosure also encompasses any nucleic acid sequence encoding a molecule comprising CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, or CDR-L3 as provided for any one of the anti-transferrin receptor antibodies selected from Table 6. In some embodiments, the heavy and light chain CDR3 domains of an antibody can play a particularly important role in the binding specificity / affinity of the antibody for a given antigen. Thus, an anti-transferrin receptor antibody of the present disclosure can comprise at least the heavy and / or (by way of example) light chain CDR3 of any one of the anti-transferrin receptor antibodies selected from Table 6.

[0196] In some examples, any of the anti-transferrin receptor antibodies of the present disclosure have one or more CDR (e.g., CDR-H or CDR-L) sequences substantially similar to any of the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and / or (by way of example) CDR-L3 sequences from one of the anti-transferrin receptor antibodies selected from Table 6. In some embodiments, the position of one or more CDRs on the VH (e.g., CDR-H1, CDR-H2, or CDR-H3) and / or (e.g., and) VL (e.g., CDR-L1, CDR-L2, or CDR-L3) regions of an antibody described herein may be varied by 1, 2, 3, 4, 5, or 6 amino acid positions, so long as immunospecific binding to a transferrin receptor (e.g., a human transferrin receptor) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%) of the binding of the original antibody from which it is derived. For example, in some embodiments, the positions defining the CDRs of any of the antibodies described herein can be varied by shifting the N-terminal and / or (e.g., and) C-terminal boundaries of the CDR by 1, 2, 3, 4, 5, or 6 amino acids compared to the CDR positions of any one of the antibodies described herein, so long as immunospecific binding to a transferrin receptor (e.g., human transferrin receptor) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%) of the binding of the original antibody from which it is derived. In another embodiment, the length of one or more CDRs on the VH (e.g., CDR-H1, CDR-H2, or CDR-H3) and / or (e.g., and) VL (e.g., CDR-L1, CDR-L2, or CDR-L3) regions of an antibody described herein is such that immunospecific binding to a transferrin receptor (e.g., human transferrin receptor) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%) of the binding of the original antibody from which it is derived.For example, it may vary by 1, 2, 3, 4, 5 amino acids or more (e.g., shorter or longer) as long as it has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the binding of the original antibody from which it is derived.

[0197] Thus, in some embodiments, CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and / or (for example, and) CDR-H3 described herein can be 1, 2, 3, 4, 5, or more amino acids shorter than one or more of the CDRs described herein (for example, a CDR from any of the anti-transferrin receptor antibodies selected from Table 6), so long as immunospecific binding to a transferrin receptor (for example, a human transferrin receptor) is maintained (for example, substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% compared to the binding of the original antibody from which it is derived). In some embodiments, CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and / or (for example, and) CDR-H3 described herein can be 1, 2, 3, 4, 5, or more amino acids longer than one or more of the CDRs described herein (for example, a CDR from any of the anti-transferrin receptor antibodies selected from Table 6), so long as immunospecific binding to a transferrin receptor (for example, a human transferrin receptor) is maintained (for example, substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% compared to the binding of the original antibody from which it is derived). In some embodiments, the amino portion of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and / or (by way of example and) CDR-H3 described herein may be extended by 1, 2, 3, 4, 5, or more amino acids compared to one or more of the CDRs described herein (e.g., a CDR from any of the anti-transferrin receptor antibodies selected from Table 6), so long as immunospecific binding to a transferrin receptor (e.g., a human transferrin receptor) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% compared to the binding of the original antibody from which it is derived).In some embodiments, the carboxy portion of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and / or (by way of example) CDR-H3 described herein may be extended by 1, 2, 3, 4, 5, or more amino acids compared to one or more of the CDRs described herein (e.g., a CDR from any of the anti-transferrin receptor antibodies selected from Table 6), so long as immunospecific binding to a transferrin receptor (e.g., a human transferrin receptor) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% compared to the binding of the original antibody from which it is derived). In some embodiments, the amino portion of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and / or (by way of example and) CDR-H3 described herein can be shortened by 1, 2, 3, 4, 5, or more amino acids compared to one or more of the CDRs described herein (e.g., a CDR from any of the anti-transferrin receptor antibodies selected from Table 6), so long as immunospecific binding to a transferrin receptor (e.g., a human transferrin receptor) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% compared to the binding of the original antibody from which it is derived). In some embodiments, the carboxy portion of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and / or (by way of example) CDR-H3 described herein can be shortened by 1, 2, 3, 4, 5, or more amino acids compared to one or more of the CDRs described herein (e.g., a CDR from any of the anti-transferrin receptor antibodies selected from Table 6), so long as immunospecific binding to a transferrin receptor (e.g., a human transferrin receptor) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% compared to the binding of the original antibody from which it is derived).Any method can be used to determine whether immunospecific binding to the transferrin receptor (e.g., the human transferrin receptor) is maintained, for example, using binding assays and conditions described in the art.

[0198] In some examples, any of the anti-transferrin receptor antibodies of the present disclosure have one or more CDR (e.g., CDR-H or CDR-L) sequences substantially similar to any one of the anti-transferrin receptor antibodies selected from Table 6. For example, an antibody can include one or more CDR sequence(s) from any of the anti-transferrin receptor antibodies selected from Table 6 containing up to 5, 4, 3, 2, or 1 amino acid residue variations compared to the corresponding CDR region of any one of the CDRs provided herein (e.g., a CDR from any of the anti-transferrin receptor antibodies selected from Table 6), so long as immunospecific binding to transferrin receptor (e.g., human transferrin receptor) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% compared to the binding of the original antibody from which it is derived). In some embodiments, any of the amino acid variations in any of the CDRs provided herein can be conservative variations. Conservative variations can be introduced into CDRs at positions where the residues are unlikely to be involved in interactions with the transferrin receptor protein (e.g., human transferrin receptor protein), e.g., as determined based on a crystal structure. Some aspects of the present disclosure provide transferrin receptor antibodies comprising one or more of the heavy chain variable (VH) and / or (e.g., and) light chain variable (VL) domains provided herein. In some embodiments, any of the VH domains provided herein include one or more of the CDR-H sequences (e.g., CDR-H1, CDR-H2, and CDR-H3) provided herein, e.g., any of the CDR-H sequences provided in any one of the anti-transferrin receptor antibodies selected from Table 6. In some embodiments, any of the VL domains provided herein include one or more of the CDR-L sequences (e.g., CDR-L1, CDR-L2, and CDR-L3) provided herein, e.g., any of the CDR-L sequences provided in any one of the anti-transferrin receptor antibodies selected from Table 6.

[0199] In some embodiments, anti-transferrin receptor antibodies of the disclosure include any antibody that includes the heavy chain variable domain and / or (by way of example and not limitation) the light chain variable domain of any anti-transferrin receptor antibody, such as any one of the anti-transferrin receptor antibodies selected from Table 6. In some embodiments, anti-transferrin receptor antibodies of the disclosure include any antibody that includes the heavy chain variable and light chain variable pair of any anti-transferrin receptor antibody, such as any one of the anti-transferrin receptor antibodies selected from Table 6.

[0200] Aspects of the present disclosure provide anti-transferrin receptor antibodies having heavy chain variable (VH) and / or (by way of example, and) light chain variable (VL) domain amino acid sequences homologous to any of those described herein. In some embodiments, the anti-transferrin receptor antibody comprises a heavy chain variable sequence or a light chain variable sequence that is at least 75% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to the heavy chain variable sequence and / or any light chain variable sequence of any anti-transferrin receptor antibody, such as any one of the anti-transferrin receptor antibodies selected from Table 6. In some embodiments, the homologous heavy chain variable and / or (by way of example, and) light chain variable amino acid sequence does not vary within any of the CDR sequences provided herein. For example, in some embodiments, a degree of sequence variation (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) can occur within the heavy chain variable and / or (by way of example, and) light chain variable sequence that excludes any of the CDR sequences provided herein. In some embodiments, any of the anti-transferrin receptor antibodies provided herein comprise heavy chain and light chain variable sequences comprising framework sequences that are at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to the framework sequences of any anti-transferrin receptor antibody, such as any one of the anti-transferrin receptor antibodies selected from Table 6.

[0201] In some embodiments, an anti-transferrin receptor antibody that specifically binds to a transferrin receptor (e.g., a human transferrin receptor) comprises a light chain variable VL domain that includes any of the CDR-L domains (CDR-L1, CDR-L2, and CDR-L3) provided herein or a CDR-L domain variant of any of the anti-transferrin receptor antibodies selected from Table 6. In some embodiments, an anti-transferrin receptor antibody that specifically binds to a transferrin receptor (e.g., a human transferrin receptor) comprises a light chain variable VL domain that includes CDR-L1, CDR-L2, and CDR-L3 of any of the anti-transferrin receptor antibodies selected from Table 6. In some embodiments, an anti-transferrin receptor antibody comprises a light chain variable (VL) region sequence that includes one, two, three, or four of the framework regions of the light chain variable region sequence of any of the anti-transferrin receptor antibodies selected from Table 6. In some embodiments, the anti-transferrin receptor antibody comprises one, two, three, or four framework regions of a light chain variable region sequence that are at least 75%, 80%, 85%, 90%, 95%, or 100% identical to one, two, three, or four framework regions of the light chain variable region sequence of an anti-transferrin receptor antibody, such as any one of the anti-transferrin receptor antibodies selected from Table 6. In some embodiments, the light chain variable framework region derived from the above amino acid sequence consists of the above amino acid sequence except for the presence of up to 10 amino acid substitutions, deletions, and / or (by way of example and) insertions, preferably up to 10 amino acid substitutions. In some embodiments, the light chain variable framework region derived from the above amino acid sequence consists of the amino acid sequence with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues substituted for amino acids found in the analogous positions in the light chain variable framework region of the corresponding non-human primate animal or human.

[0202] In some embodiments, an anti-transferrin receptor antibody that specifically binds to transferrin receptor comprises CDR-L1, CDR-L2, and CDR-L3 of any anti-transferrin receptor antibody, such as any one of the anti-transferrin receptor antibodies selected from Table 6. In some embodiments, the antibody further comprises one, two, three, or all four VL framework regions derived from the VL of a human or primate antibody. The primate or human light chain framework regions of the selected antibodies for use with the light chain CDR sequences described herein can have, for example, at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, or at least 99%) identity to the light chain framework regions of the non-human parent antibody. The selected primate or human antibody can have the same or substantially the same number of amino acids in its light chain complementarity determining regions as the amino acids in the light chain complementarity determining regions of any of the antibodies provided herein (e.g., any of the anti-transferrin receptor antibodies selected from Table 6). In some embodiments, the amino acid residues of the primate or human light chain framework region are from a naturally occurring primate or human antibody light chain framework region that has at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, at least 99% (or more) identity to the light chain framework region of any anti-transferrin receptor antibody, such as any one of the anti-transferrin receptor antibodies selected from Table 6. In some embodiments, the anti-transferrin receptor antibody further comprises one, two, three, or all four VL framework regions derived from the human light chain variable kappa subfamily. In some embodiments, the anti-transferrin receptor antibody further comprises one, two, three, or all four VL framework regions derived from the human light chain variable lambda subfamily.

[0203] In some embodiments, any of the anti-transferrin receptor antibodies provided herein comprises a light chain variable domain that further comprises a light chain constant region. In some embodiments, the light chain constant region is a kappa or lambda light chain constant region. In some embodiments, the kappa or lambda light chain constant region is from a mammal, e.g., a human, a monkey, a rat, or a mouse. In some embodiments, the light chain constant region is a human kappa light chain constant region. In some embodiments, the light chain constant region is a human lambda light chain constant region. It should be understood that any of the light chain constant regions provided herein may be a variant of any of the light chain constant regions provided herein. In some embodiments, the light chain constant region comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to any of the light chain constant regions of any anti-transferrin receptor antibody, such as any one of the anti-transferrin receptor antibodies selected from Table 6.

[0204] In some embodiments, the anti-transferrin receptor antibody is any anti-transferrin receptor antibody, such as any one of the anti-transferrin receptor antibodies selected from Table 6.

[0205] In some embodiments, the anti-transferrin receptor antibody comprises a VL domain comprising the amino acid sequence of any anti-transferrin receptor antibody, such as any one of the anti-transferrin receptor antibodies selected from Table 6, wherein the constant region comprises the amino acid sequence of an IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, or the constant region of a human IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule. In some embodiments, the anti-transferrin receptor antibody comprises a VL domain, or any variant of the VL domain, and a VH domain, or any variant of the VH domain, wherein the VL and VH domains, or variants thereof, are from the same antibody clone, and wherein the constant region comprises the amino acid sequence of the constant region of an IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, of any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or any subclass (e.g., IgG2a and IgG2b) of an immunoglobulin molecule. Non-limiting examples of human constant regions have been described in the art; see, e.g., Kabat EA et al. (1991), supra.

[0206] In some embodiments, the muscle-targeting agent is an anti-transferrin receptor antibody (e.g., an antibody and variants thereof as described in International Application Publication WO 2016 / 081643, which is incorporated herein by reference).

[0207] The heavy and light chain CDRs of antibodies according to various definition systems are provided in Table 7. Various definition systems, such as the Kabat definition, the Chothia definition, and / or the Contact definition, are described. See, for example, (see, e.g., Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242, Chothia et al., (1989) Nature 342:877; Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917, Al-lazikani et al. (1997) J. Mol. Biol. 273:927-948; and Almagro, J. Mol. Recognit. 17:132-143 (2004). See also hgmp.mrc.ac.uk and bioinf.org.uk / abs). Table 7. Heavy and light chain CDRs of mouse transferrin receptor antibodies [Table 7]

[0208] Heavy chain variable domain (VH) and light chain variable domain sequences are also provided:

[0209] VH QVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGEINPTNGRTNYIEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARGTRAYHYWGQGTSVTVSS (SEQ ID NO: 124)

[0210] VL DIQMTQSPASLSVSVGETVTITCRASDNLYSNLAWYQQKQGKSPQLLVYDATNLADGVPSRFSGSGSGTQYSLKINSLQSEDFGTYYCQHFWGTPLTFGAGTKLELK (SEQ ID NO: 125)

[0211] In some embodiments, a transferrin receptor antibody of the disclosure comprises a CDR-H1, CDR-H2, and CDR-H3 that are the same as the CDR-H1, CDR-H2, and CDR-H3 shown in Table 7. Alternatively or additionally (for example, in addition), a transferrin receptor antibody of the disclosure comprises a CDR-L1, CDR-L2, and CDR-L3 that are the same as the CDR-L1, CDR-L2, and CDR-L3 shown in Table 7.

[0212] In some embodiments, a transferrin receptor antibody of the disclosure comprises CDR-H1, CDR-H2, and CDR-H3, which collectively contain no more than five amino acid variations (e.g., no more than five, four, three, two, or one amino acid variations) compared to CDR-H1, CDR-H2, and CDR-H3 shown in Table 7. "Combined" means that the total number of amino acid variations in all three heavy chain CDRs is within a defined range. Alternatively or in addition (e.g., in addition), a transferrin receptor antibody of the disclosure may comprise CDR-L1, CDR-L2, and CDR-L3, which collectively contain no more than five amino acid variations (e.g., no more than five, four, three, two, or one amino acid variations) compared to CDR-L1, CDR-L2, and CDR-L3 shown in Table 7.

[0213] In some embodiments, transferrin receptor antibodies of the disclosure comprise CDR-H1, CDR-H2, and CDR-H3, at least one of which contains no more than three amino acid variations (e.g., no more than three, two, or one amino acid variations) compared to the counterpart heavy chain CDR shown in Table 7. Alternatively or additionally (e.g., in addition), transferrin receptor antibodies of the disclosure may comprise CDR-L1, CDR-L2, and CDR-L3, at least one of which contains no more than three amino acid variations (e.g., no more than three, two, or one amino acid variations) compared to the counterpart light chain CDR shown in Table 7.

[0214] In some embodiments, a transferrin receptor antibody of the disclosure comprises a CDR-L3 that contains no more than three amino acid variations (e.g., no more than three, two, or one amino acid variations) compared to the CDR-L3 shown in Table 7. In some embodiments, a transferrin receptor antibody of the disclosure comprises a CDR-L3 that contains one amino acid variation compared to the CDR-L3 shown in Table 7. In some embodiments, a transferrin receptor antibody of the disclosure comprises a CDR-L3 of QHFAGTPLT (SEQ ID NO: 126) according to the Kabat and Chothia definition system or QHFAGTPL (SEQ ID NO: 127) according to the Contact definition system. In some embodiments, a transferrin receptor antibody of the disclosure comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, and CDR-L2 that are the same as the CDR-H1, CDR-H2, and CDR-H3 shown in Table 7, and comprises a CDR-L3 of QHFAGTPLT (SEQ ID NO: 126) according to the Kabat and Chothia definition system) or QHFAGTPL (SEQ ID NO: 127) according to the Contact definition system).

[0215] In some embodiments, transferrin receptor antibodies of the disclosure comprise heavy chain CDRs that, collectively, are at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identical to the heavy chain CDRs shown in Table 7. Alternatively or additionally (e.g., in addition), transferrin receptor antibodies of the disclosure comprise light chain CDRs that, collectively, are at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identical to the light chain CDRs shown in Table 7.

[0216] In some embodiments, a transferrin receptor antibody of the disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO: 124. Alternatively or additionally (for example, in addition), a transferrin receptor antibody of the disclosure comprises a VL comprising the amino acid sequence of SEQ ID NO: 125.

[0217] In some embodiments, transferrin receptor antibodies of the disclosure include a VH that contains 25 or fewer amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) compared to the VH set forth in SEQ ID NO: 124. Alternatively or in addition (e.g., in addition), transferrin receptor antibodies of the disclosure include a VL that contains 15 or fewer amino acid variations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) compared to the VL set forth in SEQ ID NO: 125.

[0218] In some embodiments, a transferrin receptor antibody of the disclosure comprises a VH comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identical to the VH set forth in SEQ ID NO: 124. Alternatively or additionally (e.g., in addition), a transferrin receptor antibody of the disclosure comprises a VL comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identical to the VL set forth in SEQ ID NO: 125.

[0219] In some embodiments, the transferrin receptor antibodies of the disclosure are humanized antibodies (e.g., humanized variants of antibodies). In some embodiments, the transferrin receptor antibodies of the disclosure comprise CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 that are the same as the CDR-H1, CDR-H2, and CDR-H3 shown in Table 7, and comprise a humanized heavy chain variable region and / or (by way of example) a humanized light chain variable region.

[0220] Humanized antibodies are human immunoglobulins (recipient antibody) in which residues from the recipient's complementarity-determining regions (CDRs) are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. In some embodiments, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the imported CDR or framework sequences, but may also include residues incorporated to further refine and optimize antibody performance. In general, humanized antibodies will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. A humanized antibody will optimally also comprise at least a portion of an immunoglobulin (typically a human immunoglobulin) constant region or domain (Fc). The antibody may have an Fc region modified as described in WO 99 / 58572. Other forms of humanized antibodies have one or more CDRs (1, 2, 3, 4, 5, 6) that are altered with respect to the original antibody, also referred to as one or more CDRs derived from one or more CDRs from the original antibody. Humanized antibodies may also be affinity matured.

[0221] In some embodiments, humanization is achieved by grafting CDRs (e.g., as shown in Table 7) into IGKV1-NL1*01 and IGHV1-3*01 human variable domains. In some embodiments, the transferrin receptor antibodies of the disclosure are humanized variants that include one or more amino acid substitutions at positions 9, 13, 17, 18, 40, 45, and 70 compared to the VL set forth in SEQ ID NO: 125, and / or one or more amino acid substitutions at positions 1, 5, 7, 11, 12, 20, 38, 40, 44, 66, 75, 81, 83, 87, and 108 compared to the VH set forth in SEQ ID NO: 124 (by way of example and not limitation). In some embodiments, the transferrin receptor antibodies of the disclosure are humanized variants that include amino acid substitutions at all of positions 9, 13, 17, 18, 40, 45, and 70 compared to the VL set forth in SEQ ID NO: 125, and / or (by way of example and not limitation) amino acid substitutions at all of positions 1, 5, 7, 11, 12, 20, 38, 40, 44, 66, 75, 81, 83, 87, and 108 compared to the VH set forth in SEQ ID NO: 124.

[0222] In some embodiments, a transferrin receptor antibody of the disclosure is a humanized antibody and contains residues at positions 43 and 48 of the VL set forth in SEQ ID NO: 125. Alternatively or in addition (for example, in addition), a transferrin receptor antibody of the disclosure is a humanized antibody and contains residues at positions 48, 67, 69, 71, and 73 of the VH set forth in SEQ ID NO: 124.

[0223] The VH and VL amino acid sequences of examples of humanized antibodies that may be used in accordance with the present disclosure are provided below:

[0224] VH EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQRLEWIGEINPTNGRTNYIEKFKSRATLTVDKSASTAYMELSSLRSEDTAVYYCARGTRAYHYWGQGTMVTVSS (SEQ ID NO: 128)

[0225] VL DIQMTQSPSSLSASVGDRVTITCRASDNLYSNLAWYQQKPGKSPKLLVYDATNLADGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQHFWGTPLTFGQGTKVEIK (SEQ ID NO: 129)

[0226] Alternatively or additionally (for example, in addition), a transferrin receptor antibody of the disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO: 128. Alternatively or additionally (for example, in addition), a transferrin receptor antibody of the disclosure comprises a VL comprising the amino acid sequence of SEQ ID NO: 129.

[0227] In some embodiments, transferrin receptor antibodies of the disclosure include a VH that contains 25 or fewer amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) compared to the VH set forth in SEQ ID NO: 128. Alternatively or in addition (e.g., in addition), transferrin receptor antibodies of the disclosure include a VL that contains 15 or fewer amino acid variations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) compared to the VL set forth in SEQ ID NO: 129.

[0228] In some embodiments, a transferrin receptor antibody of the disclosure comprises a VH comprising an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identical to a VH as set forth in SEQ ID NO: 128. Alternatively or additionally (e.g., in addition), a transferrin receptor antibody of the disclosure comprises a VL comprising an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identical to a VL as set forth in SEQ ID NO: 129.

[0229] In some embodiments, the transferrin receptor antibody of the disclosure is a variant that includes an amino acid substitution at one or more of positions 43 and 48 compared to the VL set forth in SEQ ID NO: 125, and / or (by way of example and not limitation) an amino acid substitution at one or more of positions 48, 67, 69, 71, and 73 compared to the VH set forth in SEQ ID NO: 124. In some embodiments, the transferrin receptor antibody of the disclosure is a variant that includes an S43A and / or (by way of example and not limitation) a V48L mutation compared to the VL set forth in SEQ ID NO: 125, and / or (by way of example and not limitation) one or more of the following mutations compared to the VH set forth in SEQ ID NO: 124: A67V, L69I, V71R, and K73T.

[0230] In some embodiments, the transferrin receptor antibodies of the disclosure are variants that include amino acid substitutions at one or more of positions 9, 13, 17, 18, 40, 43, 48, 45, and 70 compared to the VL set forth in SEQ ID NO: 125, and / or (by way of example and not limitation) amino acid substitutions at one or more of positions 1, 5, 7, 11, 12, 20, 38, 40, 44, 48, 66, 67, 69, 71, 73, 75, 81, 83, 87, and 108 compared to the VH set forth in SEQ ID NO: 124.

[0231] In some embodiments, the anti-transferrin receptor antibody of the present disclosure is a chimeric antibody that can include heavy and light chain constant regions from a human antibody. A chimeric antibody refers to an antibody having a variable region or a portion of a variable region from a first species and a constant region from a second species. Typically, in these chimeric antibodies, the variable regions of both the light and heavy chains mimic the variable regions of antibodies from a certain mammal (e.g., a non-human mammal such as a mouse, rabbit, or rat), while the constant regions are homologous to sequences in antibodies from another mammal, such as a human. In some embodiments, amino acid modifications can be made in the variable region and / or (e.g., and) the constant region.

[0232] In some embodiments, the anti-transferrin receptor antibodies described herein are chimeric antibodies that can include heavy and light chain constant regions from a human antibody. A chimeric antibody refers to an antibody having a variable region or a portion of a variable region from a first species and a constant region from a second species. Typically, in these chimeric antibodies, the variable regions of both the light and heavy chains mimic the variable regions of antibodies from a certain mammal (e.g., a non-human mammal such as a mouse, rabbit, or rat), while the constant regions are homologous to sequences in antibodies from another mammal, such as a human. In some embodiments, amino acid modifications can be made in the variable region and / or (e.g., and) the constant region.

[0233] In some embodiments, any heavy chain of an anti-transferrin receptor antibody as described herein may comprise a heavy chain constant region (CH) or a portion thereof (e.g., CH1, CH2, CH3, or a combination thereof). The heavy chain constant region may belong to any suitable origin, e.g., human, mouse, rat, or rabbit. In one particular example, the heavy chain constant region is from human IgG (gamma heavy chain), e.g., IgG1, IgG2, or IgG4. An example of a human IgG1 constant region is given below: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 81)

[0234] In some embodiments, the light chain of any of the anti-transferrin receptor antibodies described herein may further comprise a light chain constant region (CL), which may be any CL known in the art. In some examples, the CL is a kappa light chain. In other examples, the CL is a lambda light chain. In some embodiments, the CL is a kappa light chain, the sequence of which is given below: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 83)

[0235] Heavy and light chain constant regions of other antibodies are well known in the art and are provided, for example, in the IMGT database (www.imgt.org) or at www.vbase2.org / vbstat.php, both of which are incorporated herein by reference.

[0236] Examples of heavy and light chain amino acid sequences of the described anti-transferrin receptor antibodies are provided below:

[0237] Heavy chain (VH + human IgG1 constant region) QVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGEINPTNGRTNYIEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARGTRAYHYWGQGTSVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 132)

[0238] Light chain (VL + kappa light chain) DIQMTQSPASLSVSVGETVTITCRASDNLYSNLAWYQQKQGKSPQLLVYDATNLADGVPSRFSGSGSGTQYSLKINSLQSEDFGTYYCQHFWGTPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 133)

[0239] Heavy chain (VH + human IgG1 constant region) EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQRLEWIGEINPTNGRTNYIEKFKSRATLTVDKSASTAYMELSSLRSEDTAVYYCARGTRAYHYWGQGTMVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 134)

[0240] Light chain (VL + kappa light chain) DIQMTQSPSSLSASVGDRVTITCRASDNLYSNLAWYQQKPGKSPKLLVYDATNLADGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQHFWGTPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 135)

[0241] In some embodiments, the transferrin receptor antibodies described herein comprise a heavy chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identical to SEQ ID NO: 132. Alternatively or additionally (e.g., in addition), the transferrin receptor antibodies described herein comprise a light chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identical to SEQ ID NO: 133. In some embodiments, the transferrin receptor antibodies described herein comprise a heavy chain comprising the amino acid sequence of SEQ ID NO: 132. Alternatively or additionally (e.g., in addition), the transferrin receptor antibodies described herein comprise a light chain comprising the amino acid sequence of SEQ ID NO: 133.

[0242] In some embodiments, transferrin receptor antibodies of the disclosure include a heavy chain that contains no more than 25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) compared to the heavy chain set forth in SEQ ID NO: 132. Alternatively or additionally (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) compared to the light chain set forth in SEQ ID NO: 133.

[0243] In some embodiments, the transferrin receptor antibodies described herein comprise a heavy chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identical to SEQ ID NO: 134. Alternatively or additionally (e.g., in addition), the transferrin receptor antibodies described herein comprise a light chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identical to SEQ ID NO: 135. In some embodiments, the transferrin receptor antibodies described herein comprise a heavy chain comprising the amino acid sequence of SEQ ID NO: 134. Alternatively or additionally (e.g., in addition), the transferrin receptor antibodies described herein comprise a light chain comprising the amino acid sequence of SEQ ID NO: 135.

[0244] In some embodiments, an anti-transferrin receptor antibody of the disclosure comprises a heavy chain that contains no more than 25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) compared to the heavy chain of the antibody set forth in SEQ ID NO: 134. Alternatively or additionally (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) compared to the light chain of the humanized antibody set forth in SEQ ID NO: 135.

[0245] In some embodiments, the transferrin receptor antibody is an antigen-binding fragment (Fab) of an intact antibody (full-length antibody). Antigen-binding fragments of intact antibodies (full-length antibodies) can be prepared via routine methods. For example, F(ab')2 fragments can be produced by pepsin digestion of the antibody molecule, and Fab' fragments can be generated by reducing the disulfide bridges of the F(ab')2 fragment. Examples of Fab amino acid sequences of the transferrin receptor antibodies described herein are provided below:

[0246] Heavy chain Fab (VH + part of human IgG1 constant region) QVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGEINPTNGRTNYIEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARGTRAYHYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP (SEQ ID NO: 136)

[0247] Heavy chain Fab (VH + part of human IgG1 constant region) EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQRLEWIGEINPTNGRTNYIEKFKSRATLTVDKSASTAYMELSSLRSEDTAVYYCARGTRAYHYWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP (SEQ ID NO: 137)

[0248] In some embodiments, the transferrin receptor antibodies described herein comprise a heavy chain comprising the amino acid sequence of SEQ ID NO: 136. Alternatively or additionally (for example, in addition), the transferrin receptor antibodies described herein comprise a light chain comprising the amino acid sequence of SEQ ID NO: 133.

[0249] In some embodiments, the transferrin receptor antibodies described herein comprise a heavy chain comprising the amino acid sequence of SEQ ID NO: 137. Alternatively or additionally (for example, in addition), the transferrin receptor antibodies described herein comprise a light chain comprising the amino acid sequence of SEQ ID NO: 135.

[0250] The anti-transferrin receptor antibodies described herein can be in any antibody form, including, but not limited to, intact (i.e., full-length) antibodies, antigen-binding fragments thereof (such as Fab, Fab', F(ab')2, Fv, etc.), single-chain antibodies, bispecific antibodies, or nanobodies. In some embodiments, the anti-transferrin receptor antibodies described herein are scFvs. In some embodiments, the anti-transferrin receptor antibodies described herein are scFv-Fabs (e.g., scFvs fused with a portion of a constant region). In some embodiments, the transferrin receptor antibodies described herein are scFvs fused with a constant region (e.g., the human IgG1 constant region set forth in SEQ ID NO: 81).

[0251] In some embodiments, any one of the anti-TfR1 antibodies described herein is produced by recombinant DNA technology from a Chinese hamster ovary (CHO) cell suspension culture, optionally from a CHO-K1 cell (e.g., CHO-K1 cells from the European Collection of Animal Cell Culture, Cat. No. 85051005) suspension culture.

[0252] Additional anti-TfR1 antibodies are described in International Patent Application Publications WO2021 / 1544761A1, WO2021 / 154477A1, and WO2021 / 142307A1, the entire contents of each of which are incorporated herein by reference for this purpose.

[0253] In some embodiments, the antibodies provided herein may have one or more post-translational modifications. In some embodiments, N-terminal cyclization, also referred to as pyroglutamic acid formation (pyroGlu), may occur in antibodies at N-terminal glutamic acid (Glu) and / or glutamine (Gln) residues during production. Thus, it should be understood that an antibody identified as having a sequence containing an N-terminal glutamate or glutamine residue encompasses antibodies that have undergone pyroglutamate formation due to post-translational modification. In some embodiments, pyroglutamic acid formation occurs in the heavy chain sequence. In some embodiments, pyroglutamic acid formation occurs in the light chain sequence.

[0254] b. Other muscle-targeting antibodies In some embodiments, the muscle-targeting antibody specifically binds to hemojuvelin, caveolin-3, Duchenne muscular dystrophy peptide, myosin Iib, or CD63. In some embodiments, the muscle-targeting antibody specifically binds to a myogenic precursor protein. Exemplary myogenic precursor proteins include, but are not limited to, ABCG2, M-cadherin / cadherin-15, caveolin-1, CD34, FoxK1, integrin alpha7, integrin alpha7beta1, MYF-5, MyoD, myogenin, NCAM-1 / CD56, Pax3, Pax7, and Pax9. In some embodiments, the muscle-targeting antibody specifically binds to a skeletal muscle protein. Exemplary skeletal muscle proteins include, without limitation, alpha-sarcoglycan, beta-sarcoglycan, calpain inhibitor, creatine kinase MM / CKMM, eIF5A, enolase 2 / neuron-specific enolase, epsilon-sarcoglycan, FABP3 / H-FABP, GDF-8 / myostatin, GDF-11 / GDF-8, integrin alpha7, integrin alpha7beta1, integrin beta1 / CD29, MCAM / CD146, MyoD, myogenin, myosin light chain kinase inhibitor, NCAM-1 / CD56, and troponin I. In some embodiments, the muscle-targeting antibody is an antibody that specifically binds to a smooth muscle protein. Exemplary smooth muscle proteins include, but are not limited to, alpha-smooth muscle actin, VE-cadherin, caldesmon / CALD1, calponin 1, desmin, histamine H2R, motilin R / GPR38, transgelin / TAGLN, and vimentin. However, it should be understood that antibodies to additional targets are within the scope of this disclosure and that the exemplary list of targets provided herein is not intended to be limiting.

[0255] c. Antibody Features / Modifications In some embodiments, conservative mutations may be introduced into an antibody sequence (e.g., a CDR or framework sequence) at a position where the residue is unlikely to be involved in interactions with the target antigen (e.g., a transferrin receptor), as determined, for example, based on a crystal structure. In some embodiments, one or more mutations (e.g., amino acid substitutions) are introduced into the Fc region (e.g., in the CH2 domain (residues 231-340 of human IgG1), and / or (e.g., in the CH3 domain (residues 341-447 of human IgG1), and / or (e.g., in the hinge region), numbered according to the Kabat numbering system (e.g., EU index of Kabat)) of a muscle-targeting antibody described herein to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or (e.g., and) antigen-dependent cellular cytotoxicity.

[0256] In some embodiments, one or more mutations (e.g., amino acid substitutions) are introduced into the hinge region of the Fc region (CH1 domain) such that the number of cysteine ​​residues in the hinge region can be varied (e.g., increased or decreased), e.g., as described in U.S. Patent No. 5,677,425. The number of cysteine ​​residues in the hinge region of the CH1 domain can be altered, e.g., to facilitate assembly of the light and heavy chains, or to alter (e.g., increase or decrease) the stability of the antibody, or to facilitate linker conjugation.

[0257] In some embodiments, one or more mutations (e.g., amino acid substitutions) are introduced into the Fc region of a muscle-targeting antibody described herein (e.g., in the CH2 domain (residues 231-340 of human IgG1), and / or (e.g., and) in the CH3 domain (residues 341-447 of human IgG1), and / or (e.g., and) in the hinge region, numbered according to the Kabat numbering system (e.g., EU index of Kabat)) to increase or decrease the affinity of the antibody for an Fc receptor (e.g., an activated Fc receptor) on the surface of an effector cell. Mutations in the Fc region of an antibody that increase or decrease the affinity of the antibody for an Fc receptor, and techniques for introducing such mutations into an Fc receptor or fragment thereof, are known to those of skill in the art. Examples of mutations in the Fc receptor of an antibody that can be made to alter the affinity of the antibody to the Fc receptor are described, for example, in Smith P et al., (2012) PNAS 109:6181-6186, U.S. Patent No. 6,737,056, and International Publication Nos. WO 02 / 060919; WO 98 / 23289; and WO 97 / 34631, which are incorporated herein by reference.

[0258] In some embodiments, one or more amino acid mutations (i.e., substitutions, insertions, or deletions) are introduced into the IgG constant region or FcRn-binding fragment thereof (preferably, the Fc or hinge-Fc domain fragment) to alter (e.g., increase or decrease) the half-life of the antibody in vivo. See, e.g., International Publication Nos. WO 02 / 060919; WO 98 / 23289; and WO 97 / 34631; and U.S. Patent Nos. 5,869,046, 6,121,022, 6,277,375, and 6,165,745, for mutations that may alter (e.g., increase or decrease) the half-life of the antibody in vivo.

[0259] In some embodiments, one or more amino acid mutations (i.e., substitutions, insertions, or deletions) are introduced into the IgG constant region or FcRn-binding fragment thereof (preferably, the Fc or hinge-Fc domain fragment) to decrease the half-life of the anti-transferrin receptor antibody in vivo. In some embodiments, one or more amino acid mutations (i.e., substitutions, insertions, or deletions) are introduced into the IgG constant region or FcRn-binding fragment thereof (preferably, the Fc or hinge-Fc domain fragment) to increase the half-life of the antibody in vivo. In some embodiments, the antibody may have one or more amino acid mutations (e.g., substitutions) in the second constant (CH2) domain (residues 231-340 of human IgG1) and / or (for example, and) in the third constant (CH3) domain (residues 341-447 of human IgG1) numbered according to the EU index of Kabat (Kabat EA et al. (1991) supra). In some embodiments, the IgG1 constant region of the antibody described herein comprises a methionine (M) to tyrosine (Y) substitution at position 252, a serine (S) to threonine (T) substitution at position 254, and a threonine (T) to glutamic acid (E) substitution at position 256, numbered according to the EU index as in Kabat. See U.S. Patent No. 7,658,921, which is incorporated herein by reference. This type of mutant IgG, termed a "YTE mutant," has been shown to exhibit a four-fold increased half-life compared to the wild-type version of the same antibody (see Dall'Acqua WF et al., (2006) J Biol Chem 281:23514-24). In some embodiments, the antibody comprises an IgG constant region comprising one, two, three or more amino acid substitutions of amino acid residues at positions 251-257, 285-290, 308-314, 385-389, and 428-436, numbered according to the EU index as in Kabat.

[0260] In some embodiments, one or more amino acid substitutions are introduced into the IgG constant region Fc region to alter the effector function(s) of the anti-transferrin receptor antibody. The effector ligand with altered affinity can be, for example, an Fc receptor or the C1 component of complement. This approach is described in further detail in U.S. Patent Nos. 5,624,821 and 5,648,260. In some embodiments, deletion or inactivation of the constant region domain (through point mutation or other means) can reduce binding of circulating antibodies to Fc receptors, thereby increasing tumor localization. See, e.g., U.S. Patent Nos. 5,585,097 and 8,591,886 for a description of mutations that delete or inactivate constant regions, thereby increasing tumor localization. In some embodiments, one or more amino acid substitutions may be introduced into the Fc region of an antibody described herein to remove potential glycosylation sites in the Fc region, which may reduce binding to Fc receptors (see, e.g., Shields RL et al., (2001) J Biol Chem 276:6591-604).

[0261] In some embodiments, one or more amino acid residues in the constant region of the muscle-targeting antibody described herein can be replaced with a different amino acid residue so that the antibody can have altered Clq binding and / or (for example, and) reduced or eliminated complement-dependent cytotoxicity (CDC). This approach is described in further detail in U.S. Patent No. 6,194,551 (Idusogie et al.). In some embodiments, one or more amino acid residues in the N-terminal region of the CH2 domain of the antibody described herein are altered to thereby alter the antibody's ability to fix complement. This approach is further described in International Publication No. WO 94 / 29351. In some embodiments, the Fc region of the antibody described herein is modified to increase the antibody's ability to mediate antibody-dependent cellular cytotoxicity (ADCC) to cells and / or (for example, and) increase the antibody's affinity for Fcγ receptors. This approach is further described in International Publication No. WO 00 / 42072.

[0262] In some embodiments, the heavy and / or (by way of example) light chain variable domain(s) sequence(s) of the antibodies provided herein can be used to generate, for example, CDR-grafted, chimeric, humanized, or composite human antibodies, or antigen-binding fragments, as described elsewhere herein. As will be understood by one of skill in the art, any variant, CDR-grafted, chimeric, humanized, or composite antibody derived from any of the antibodies provided herein may be useful in the compositions and methods described herein and will retain the ability to specifically bind to the transferrin receptor, such that the variant, CDR-grafted, chimeric, humanized, or composite antibody may have at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or more binding to the transferrin receptor compared to the original antibody from which it was derived.

[0263] In some embodiments, the antibodies provided herein contain mutations that confer desired properties to the antibody. For example, to avoid potential complications due to Fab-arm exchange, which is known to occur in native IgG4 mAbs, the antibodies provided herein may contain the stabilizing "Adair" mutation (Angal S., et al., "A single amino acid substitution abolishes the heterogeneity of chimeric mouse / human (IgG4) antibody," Mol Immunol 30, 105-108; 1993), in which serine 228 (EU numbering; residue 241 Kabat numbering) is converted to proline, resulting in an IgG1-like hinge sequence. Consequently, any of the antibodies may include the stabilizing "Adair" mutation.

[0264] As provided herein, the antibodies of the present disclosure may optionally comprise a constant region or a portion thereof. For example, a VL domain may be attached at its C-terminus to a light chain constant region like Cκ or Cλ. Similarly, a VH domain or a portion thereof may be attached to all or a portion of a heavy chain like IgA, IgD, IgE, IgG, and IgM, and any isotype subclass. The antibody may comprise any suitable constant region (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, No. 91-3242, National Institutes of Health Publications, Bethesda, Md. (1991)). Thus, antibodies within the scope of the present disclosure may comprise VH and VL domains, or antigen-binding portions thereof, combined with any suitable constant region.

[0265] ii. Muscle-targeting peptides Some aspects of the present disclosure provide muscle-targeting peptides as muscle-targeting agents. Short peptide sequences (e.g., peptide sequences 5-20 amino acids in length) that bind to specific cell types have been described. For example, cell-targeting peptides are described in Vines e., et al., A., "Cell-penetrating and cell-targeting peptides in drug delivery," Biochim Biophys Acta 2008, 1786:126-38; Jarver P., et al., "In vivo biodistribution and efficacy of peptide mediated delivery," Trends Pharmacol Sci 2010;31:528-35; Samoylova TI, et al., "Elucidation of muscle-binding peptides by phage display screening," Muscle Nerve 1999;22:460-6; ​​U.S. Patent No. 6,329,501, issued December 11, 2001, entitled "METHODS AND COMPOSITIONS FOR TARGETING COMPOUNDS TO MUSCLE"; and Samoylov AM, et al., "Recognition of cell-specific binding of phage display derived peptides using an acoustic wave Sensor," Biomol Eng 2002;18:269-72, the entire contents of each of which are incorporated herein by reference. By designing peptides to interact with specific cell surface antigens (e.g., receptors), selectivity for a desired tissue, e.g., muscle, can be achieved. Skeletal muscle targeting has been explored, and a wide range of molecular payloads can be delivered. These approaches, which lack many of the practical disadvantages of large antibodies or viral particles, may have high selectivity for muscle tissue. Consequently, in some embodiments, the muscle-targeting agent is a muscle-targeting peptide from 4 to 50 amino acids in length.In some embodiments, the muscle-targeting peptide is 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length. Muscle-targeting peptides can be generated using any of several methods, such as phage display.

[0266] In some embodiments, the muscle-targeting peptide may bind to an internalized cell surface receptor (e.g., transferrin receptor) that is overexpressed or relatively highly expressed in muscle cells compared to certain other cells. In some embodiments, the muscle-targeting peptide may target (e.g., bind to) the transferrin receptor. In some embodiments, the transferrin receptor-targeting peptide may comprise a segment of a naturally occurring ligand, e.g., transferrin. In some embodiments, the transferrin receptor-targeting peptide is as described in U.S. Patent No. 6,743,893, filed 11 / 30 / 2000, entitled "RECEPTOR-MEDIATED UPTAKE OF PEPTIDES THAT BIND THE HUMAN TRANSFERRIN RECEPTOR." In some embodiments, the transferrin receptor-targeting peptide is as described in Kawamoto, M. et al, "A novel transferrin receptor-targeted hybrid peptide disintegrates cancer cell membrane to induce rapid killing of cancer cells," BMC Cancer. 2011 Aug 18;11:359. In some embodiments, the transferrin receptor-targeting peptide is as described in U.S. Patent No. 8,399,653, filed 5 / 20 / 2011, entitled "TRANSFERRIN / TRANSFERRIN RECEPTOR-MEDIATED SIRNA DELIVERY."

[0267] As mentioned above, examples of muscle-targeting peptides have been reported. For example, muscle-specific peptides have been identified using phage display libraries that display surface heptapeptides. As an example, a peptide having the amino acid sequence ASSLNIA (SEQ ID NO: 138) bound to C2C12 mouse myotubes in vitro and to mouse muscle tissue in vivo. Consequently, in some embodiments, a muscle-targeting agent comprises the amino acid sequence ASSLNIA (SEQ ID NO: 138). This peptide exhibited improved specificity for binding to cardiac and skeletal muscle tissue after intravenous injection into mice, with reduced binding to the liver, kidney, and brain. Additional muscle-specific peptides have been identified using phage display. For example, in the context of treating Duchenne muscular dystrophy, a 12-amino acid peptide was identified by a phage display library for muscle targeting. See Yoshida D., et al., "Targeting of salicylate to skin and muscle following topical injections in rats," Int J Pharm 2002;231:177-84, the entire contents of which are incorporated herein by reference. Herein, a 12-amino acid peptide having the sequence SKTFNTHPQSTP (SEQ ID NO: 139) was identified, and this muscle-targeting peptide showed improved binding to C2C12 cells compared to the ASSLNIA (SEQ ID NO: 138) peptide.

[0268] Additional methods for identifying peptides selective for muscle (e.g., skeletal muscle) over other cell types include in vitro selection, as described in Ghosh D., et al., "Selection of muscle-binding peptides from context-specific peptide-presenting phage libraries for adenoviral vector targeting," J Virol 2005;79:13667-72, the entire contents of which are incorporated herein by reference. Nonspecific cell binders were selected by preincubating a random 12-mer peptide phage display library with a mixture of non-muscle cell types. After repeated selection, the 12-amino acid peptide TARGEHKEEELI (SEQ ID NO: 140) emerged most frequently. Consequently, in some embodiments, the muscle-targeting agent comprises the amino acid sequence TARGEHKEEELI (SEQ ID NO: 140).

[0269] The muscle-targeting agent may be an amino acid-containing molecule or peptide. The muscle-targeting peptide may correspond to the sequence of a protein that preferentially binds to a protein receptor found on muscle cells. In some embodiments, the muscle-targeting peptide contains a high propensity of hydrophobic amino acids (e.g., valine) so that the peptide can preferentially target muscle cells. In some embodiments, the muscle-targeting peptide has not been previously characterized or disclosed. These peptides may be conceived, produced, synthesized, and / or derivatized (e.g., and) using any of several methodologies, e.g., phage-displayed peptide libraries, one-bead-one-compound peptide libraries, or positional scanning synthetic peptide combinatorial libraries. Exemplary methodologies have been characterized in the art and are incorporated by reference (Gray, BP and Brown, KC, "Combinatorial Peptide Libraries: Mining for Cell-Binding Peptides," Chem Rev. 2014, 114:2, 1020-1081; Samoylova, TI and Smith, BF, "Elucidation of muscle-binding peptides by phage display screening," Muscle Nerve, 1999, 22:4, 460-6).In some embodiments, muscle-targeting peptides have been previously disclosed (see, e.g., Writer MJ et al., "Targeted gene delivery to human airway epithelial cells with synthetic vectors incorporating novel targeting peptides selected by phage display," J. Drug Targeting. 2004;12:185; Cai, D., "BDNF-mediated enhancement of inflammation and injury in the aging heart," Physiol Genomics. 2006,24:3,191-7; Zhang, L., "Molecular profiling of heart endothelial cells," Circulation, 2005,112:11,1601-11; McGuire, MJ et al., "In vitro selection of a peptide with high selectivity for cardiomyocytes in vivo," J. Mol. Biol. 2004,342:1,171-82). Exemplary muscle-targeting peptides include the following group of amino acid sequences: CQAQGQLVC (SEQ ID NO: 141), CSERSMNFC (SEQ ID NO: 142), CPKTRRVPC (SEQ ID NO: 143), WLSEAGPVVTVRALRGTGSW (SEQ ID NO: 144), ASSLNIA (SEQ ID NO: 138), CMQHSMRVC (SEQ ID NO: 145), and DDTRHWG (SEQ ID NO: 146). In some embodiments, muscle-targeting peptides may contain about 2-25 amino acids, about 2-20 amino acids, about 2-15 amino acids, about 2-10 amino acids, or about 2-5 amino acids. Muscle-targeting peptides may contain naturally occurring amino acids, such as cysteine, alanine, or non-naturally occurring or modified amino acids. Non-naturally occurring amino acids include β-amino acids, homo-amino acids, proline derivatives, 3-substituted alanine derivatives, linear core amino acids, N-methyl amino acids, and other amino acids known in the art.In some embodiments, the muscle-targeting peptide may be linear; in other embodiments, the muscle-targeting peptide may be cyclic (e.g., bicyclic) (see, e.g., Silvana, MGet al. Mol. Therapy, 2018, 26:1, 132-147).

[0270] iii. Muscle-targeted receptor ligands The muscle-targeting agent may be a ligand, e.g., a ligand that binds to a receptor protein. The muscle-targeting ligand may be a protein, e.g., transferrin, which binds to an internalized cell surface receptor expressed by muscle cells. Consequently, in some embodiments, the muscle-targeting agent is transferrin or a derivative thereof that binds to the transferrin receptor. Alternatively, the muscle-targeting ligand may be a small molecule, e.g., a lipophilic small molecule that preferentially targets muscle cells over other cell types. Exemplary lipophilic small molecules that may target muscle cells include compounds containing cholesterol, cholesteryl, stearic acid, palmitic acid, oleic acid, oleyl, linolenic acid, linoleic acid, myristic acid, sterol, dihydrotestosterone, testosterone derivatives, glycerin, alkyl chains, trityl groups, and alkoxy acids.

[0271] iv. Muscle-targeting aptamers The muscle-targeting agent may be an aptamer, e.g., an RNA aptamer, that preferentially targets muscle cells over other cell types. In some embodiments, the muscle-targeting aptamer has not previously been characterized or disclosed. These aptamers may be conceived, produced, synthesized, and / or (e.g., and) derivatized using any of several methodologies, e.g., Systematic Evolution of Ligands by Exponential Enrichment. Exemplary methodologies are described in the art and are incorporated by reference (Yan, A.C. and Levy, M., "Aptamers and aptamer targeted delivery," RNA biology, 2009, 6:3, 316-20; Germer, K. et al., "RNA aptamers and their therapeutic and diagnostic applications," Int. J. Biochem. Mol. Biol. 2013; 4:27-40). In some embodiments, muscle-targeting aptamers have been previously disclosed (see, e.g., Phillippou, S. et al., "Selection and Identification of Skeletal-Muscle-Targeted RNA Aptamers," Mol Ther Nucleic Acids. 2018, 10:199-214; Thiel, W. H. et al., "Smooth Muscle Cell-Targeted RNA Aptamer Inhibits Neointimal Formation," Mol Ther. 2016, 24:4, 779-87). Exemplary muscle-targeting aptamers include the A01B RNA aptamer and RNA Apt 14. In some embodiments, the aptamer is a nucleic acid-based aptamer, an oligonucleotide aptamer, or a peptide aptamer. In some embodiments, the aptamer may be about 5-15 kDa, about 5-10 kDa, about 10-15 kDa, about 1-5 Da, about 1-3 kDa, or smaller.

[0272] v. Other muscle-targeting agents One strategy for targeting muscle cells (e.g., skeletal muscle cells) is to use a substrate of a muscle transporter protein, such as a transporter protein expressed on the sarcolemma. In some embodiments, the muscle targeting agent is a substrate of an influx transporter specific to muscle tissue. In some embodiments, the influx transporter is specific to skeletal muscle tissue. Two major classes of transporters expressed on the sarcolemma of skeletal muscle are (1) the adenosine triphosphate (ATP)-binding cassette (ABC) superfamily, which facilitates efflux from skeletal muscle tissue, and (2) the solute carrier (SLC) superfamily, which can facilitate the influx of substrates into skeletal muscle. In some embodiments, the muscle targeting agent is a substrate that binds to the ABC or SLC superfamily of transporters. In some embodiments, the substrate that binds to the ABC or SLC superfamily of transporters is a naturally occurring substrate. In some embodiments, the substrate that binds to the ABC or SLC superfamily of transporters is a non-naturally occurring substrate, for example, a synthetic derivative thereof that binds to the ABC or SLC superfamily of transporters.

[0273] In some embodiments, the muscle-targeting agent is a substrate for the SLC superfamily of transporters. SLC transporters are either equilibrium or use proton or sodium ion gradients created across the membrane to drive transport of the substrate. Exemplary SLC transporters with high expression in skeletal muscle include, without limitation, the SATT transporter (ASCT1; SLC1A4), the GLUT4 transporter (SLC2A4), the GLUT7 transporter (GLUT7; SLC2A7), the ATRC2 transporter (CAT-2; SLC7A2), the LAT3 transporter (KIAA0245; SLC7A6), the PHT1 transporter (PTR4; SLC15A4), the OATP-J transporter (OATP5A1; SLC21A15), the OCT3 transporter (EMT; SLC22A3), the OCTN2 transporter (FLJ46769; SLC22A5), the ENT transporters (ENT1; SLC29A1 and ENT2; SLC29A2), the PAT2 transporter (SLC36A2), and the SAT2 transporter (KIAA1382; SLC38A2). These transporters may facilitate the entry of substrates into skeletal muscle, thereby providing opportunities for muscle targeting.

[0274] In some embodiments, the muscle-targeting agent is a substrate for the equilibrative nucleoside transporter 2 (ENT2) transporter. Compared with other transporters, ENT2 has one of the highest mRNA expression levels in skeletal muscle. Human ENT2 (hENT2) is expressed in most body organs, such as the brain, heart, placenta, thymus, pancreas, prostate, and kidney, but is particularly abundant in skeletal muscle. Human ENT2 facilitates the uptake of its substrates according to their concentration gradient. ENT2 plays a role in maintaining nucleoside homeostasis by transporting a wide range of purine and pyrimidine nucleobases. The hENT2 transporter has low affinity for all nucleosides (adenosine, guanosine, uridine, thymidine, and cytidine) except for inosine. Consequently, in some embodiments, the muscle-targeting agent is an ENT2 substrate. Exemplary ENT2 substrates include, but are not limited to, inosine, 2',3'-dideoxyinosine, and clofarabine. In some embodiments, any of the muscle-targeting agents provided herein is associated with a molecular payload (e.g., an oligonucleotide payload). In some embodiments, the muscle-targeting agent is covalently linked to the molecular payload. In some embodiments, the muscle-targeting agent is non-covalently linked to the molecular payload.

[0275] In some embodiments, the muscle-targeting agent is a substrate of the organic cation / carnitine transporter (OCTN2), a sodium ion-dependent, high-affinity carnitine transporter. In some embodiments, the muscle-targeting agent is carnitine, mildronate, acetylcarnitine, or a derivative thereof that binds to OCTN2. In some embodiments, carnitine, mildronate, acetylcarnitine, or a derivative thereof is covalently linked to a molecular payload (e.g., an oligonucleotide payload).

[0276] A muscle-targeting agent may be a protein that exists in at least one soluble form and targets muscle cells. In some embodiments, the muscle-targeting protein may be hemojuvelin (also known as repulsive guidance molecule C or hemochromatosis type 2 protein), a protein involved in iron overload and homeostasis. In some embodiments, hemojuvelin may be full-length, a fragment, or a mutant having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to a functional hemojuvelin protein. In some embodiments, the hemojuvelin mutant may be a soluble fragment, may lack the N-terminal signaling domain, and / or (for example, and) may lack the C-terminal anchoring domain. In some embodiments, the hemojuvelin may be annotated with GenBank RefSeq accession numbers NM_001316767.1, NM_145277.4, NM_202004.3, NM_213652.3, or NM_213653.3. It is to be understood that the hemojuvelin may be of human, non-human primate, or rodent origin.

[0277] B. Molecular Payload Some aspects of the present disclosure provide molecular payloads, e.g., molecular payloads for modulating a biological outcome (e.g., transcription of a DNA sequence, splicing and processing of an RNA sequence, protein expression, or protein activity). In some embodiments, the molecular payload is linked or otherwise associated with a muscle-targeting agent. In some embodiments, such molecular payloads are capable of targeting muscle cells, e.g., via specific binding to a nucleic acid or protein in muscle cells upon delivery to the muscle cells by the associated muscle-targeting agent. It should be understood that various types of muscle-targeting agents may be used in accordance with the present disclosure. For example, the molecular payload may comprise or consist of an oligonucleotide (e.g., an antisense oligonucleotide), a peptide (e.g., a peptide that binds to a nucleic acid or protein in a muscle cell associated with a disease), a protein (e.g., a protein that binds to a nucleic acid or protein in a muscle cell associated with a disease), or a small molecule (e.g., a small molecule that modulates the function of a nucleic acid or protein in a muscle cell associated with a disease). In some embodiments, the molecular payload is an oligonucleotide comprising a strand having a region of complementarity to a mutated DMD allele. Exemplary molecular payloads are described in further detail herein; however, it should be understood that the exemplary molecular payloads provided herein are not intended to be limiting.

[0278] In some embodiments, the molecular payloads disclosed herein are configured to promote dystrophin expression or activity. In some embodiments, the molecular payloads configured to promote dystrophin expression or activity promote transcription of the DMD gene, e.g., to produce a DMD pre-mRNA. In some embodiments, the molecular payloads configured to promote dystrophin expression or activity regulate pre-mRNA processing, e.g., by promoting exon skipping in the pre-mRNA to produce a mature mRNA lacking one or more exons (e.g., an exon containing a mutation). In some embodiments, the molecular payloads configured to promote dystrophin expression or activity comprise an oligonucleotide comprising a region complementary to an exon of the DMD gene. In some embodiments, the molecular payloads configured to promote dystrophin expression or activity comprise an oligonucleotide comprising a region complementary to an ESE of the DMD gene. In some embodiments, a molecular payload configured to enhance dystrophin expression or activity facilitates increased levels of mRNA encoding a truncated dystrophin protein, wherein the truncated dystrophin protein is at least partially functional (e.g., the truncated dystrophin protein is partially functional compared to a full-length, wild-type dystrophin protein).

[0279] i. Oligonucleotides Any suitable oligonucleotide may be used as a molecular payload as described herein. In some embodiments, oligonucleotides may be designed to induce exon skipping (e.g., EXONDYS 51 oligonucleotide (Sarepta Therapeutics, Inc.), which comprises SEQ ID NO: 343 (CUCCAACAUCAAGGAAGAUGGCAUUUCUAG); WVE-210201 (Wave Life Sciences), which comprises SEQ ID NO: 334 (UCAAGGAAGAUGGCAUUUCU); Casimersen (Sarepta Therapeutics, Inc.), which comprises SEQ ID NO: 302, or (CAAUGCCAUCCUGGAGUUCCUG); or Golodirsen (Sarepta Therapeutics, Inc.), which comprises SEQ ID NO: 380 (GUUGCCUCCGGUUCUGAAGGUGUUC). In some embodiments, oligonucleotides may be designed to induce exon skipping (e.g., viltolarsen (NS Pharma, Inc.), which comprises SEQ ID NO: 2257 (CCTCCGGTTCTGAAGGTGTTC), or renadirsen (Daiichi Sankyo Company), which comprises SEQ ID NO: 2252 (CGCUGCCCAAUGCCAUCC). In some embodiments, the oligonucleotide comprises a sequence, or a portion thereof (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more consecutive nucleosides), of a sequence provided in Table 8, and / or the oligonucleotide comprises a region of complementarity to a target sequence provided in Table 8. Any one or more of the thymine bases (T) in any one of the oligonucleotides provided herein (e.g., the oligonucleotides listed in Table 8) can optionally be a uracil base (U), and / or any one of the U's in the oligonucleotides provided herein can optionally be a T. Table 8. Examples of oligonucleotide molecular payloads [Table 8]

[0280] In some embodiments, the oligonucleotide may be designed to cause degradation of the mRNA (e.g., the oligonucleotide may be a gapmer, siRNA, ribozyme, or aptamer that causes degradation). In some embodiments, the oligonucleotide may be designed to block translation of the mRNA (e.g., the oligonucleotide may be a mixmer, siRNA, or aptamer that blocks translation). In some embodiments, the oligonucleotide may be designed to cause degradation of the mRNA to block its translation. In some embodiments, the oligonucleotide may be designed to promote mRNA stability. In some embodiments, the oligonucleotide may be designed to promote mRNA translation. In some embodiments, the oligonucleotide may be designed to promote mRNA stability to promote its translation. In some embodiments, the oligonucleotide may be a guide nucleic acid (e.g., a guide RNA) to direct the activity of an enzyme (e.g., a gene editing enzyme). In some embodiments, the guide nucleic acid may direct the enzyme to delete all or part of the mutated DMD allele (e.g., to facilitate in-frame exon skipping). In some embodiments, the oligonucleotide may be designed to target a repressive regulator of DMD expression (e.g., miR-31). Other examples of oligonucleotides are provided herein. It should be understood that in some embodiments, an oligonucleotide of one format (e.g., an antisense oligonucleotide) may be suitably adapted to another format (e.g., an siRNA oligonucleotide) by incorporating a functional sequence (e.g., an antisense strand sequence) from one format into the other.

[0281] Examples of useful oligonucleotides for targeting DMD are described in U.S. Patent Application Publication US20100130591A1, published May 27, 2010, entitled "MULTIPLE EXON SKIPPING COMPOSITIONS FOR DMD"; U.S. Patent No. 8,361,979, issued January 29, 2013, entitled "MEANS AND METHOD FOR INDUCING EXON-SKIPPING"; U.S. Patent Application Publication 20120059042, published March 8, 2012, entitled "METHOD FOR EFFICIENT EXON (44) SKIPPING IN DUCHENNE MUSCULAR DYSTROPHY AND ASSOCIATED MEANS"; and U.S. Patent Application Publication 20120059042, published March 8, 2012, entitled "EXON SKIPPING COMPOSITIONS FOR TREATING MUSCULAR DYSTROPHY"; U.S. Patent Application Publication No. 20140329881, published November 6, 2014, entitled "ANTISENSE OLIGONUCLEOTIDES FOR INDUCING EXON SKIPPING AND METHODS OF USE THEREOF"; U.S. Patent Application Publication No. 8,232,384, issued July 31, 2012, entitled "METHODS AND MEANS FOR EFFICIENT SKIPPING OF EXON 45 IN DUCHENNE MUSCULAR DYSTROPHY PRE-MRNA"; U.S. Patent Application Publication No. 20120022134A1, published January 26, 2012, entitled "ADENO-ASSOCIATED VIRAL VECTOR FOR EXON SKIPPING IN A GENE ENCODING A DISPENSABLE DOG U.S. Patent Application Publication No. 20120077860, published March 29, 2012, entitled "Oligomers," U.S. Patent No. 8,324,371, issued December 4, 2012, entitled "Oligomers," U.S. Patent No. 9,078,911, issued July 14, 2015, entitled "Antisense Oligonucleotides," U.S. Patent No. 9,079,934, issued July 14, 2015, entitled "Antisense Nucleic Acid ...No. 9,034,838, issued May 19, 2015, entitled "MIR-31 IN DUCHENNE MUSCULAR DYSTROPHY THERAPY"; and International Patent Publication WO2017062862A3, published April 13, 2017, entitled "OLIGONUCLEOTIDE COMPOSITIONS AND METHODS THEREOF," the entire contents of each of which are incorporated herein.

[0282] Table 9 provides non-limiting examples of oligonucleotide sequences that are useful for targeting DMD (e.g., for exon skipping). In some embodiments, the oligonucleotide may comprise any of the sequences provided in Table 9. Table 9 - Oligonucleotide sequences for targeting DMD. [Table 9-1] [Table 9-2] [Table 9-3] [Table 9-4] [Table 9-5] [Table 9-6] [Table 9-7] †Each uracil base (U) in any one of the oligonucleotide sequences provided in Table 9 may independently and optionally be replaced with a thymine base (T).

[0283] In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) target a region of DMD RNA (e.g., the Dp427m transcript of SEQ ID NO: 2239). In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) comprise a region complementary to a DMD RNA (e.g., the Dp427m transcript of SEQ ID NO: 2239). In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) comprise a region complementary to an exon of DMD RNA (e.g., any one of SEQ ID NOs: 2240-2250). Examples of DMD RNA sequences and exon sequences are provided below.

[0284] Homo sapiens dystrophin (DMD), transcript variant Dp427m, mRNA (NCBI Reference Sequence: NM_004006.2)

[0285] Homo sapiens dystrophin (DMD), transcript variant Dp427m, exon 8 (nucleotides 894-1075 of NCBI reference sequence: NM_004006.2) ATGTTGATACCACCTATCCAGATAAGAAGTCCATCTTAATGTACATCACATCACTCTTCCAAGTTTTGCCTCAACAAGTGAGCATTGAAGCCATCCAGGAAGTGGAAATGTTGCCAAGGCCACCTAAAGTGACTAAAGAAGAACATTTTCAGTTACATCATCAAATGCACTATTCTCAACAG (SEQ ID NO: 2240)

[0286] Homo sapiens dystrophin (DMD), transcript variant Dp427m, exon 23 (nucleotides 3194-3406 of the NCBI reference sequence: NM_004006.2) GCTTTACAAAGTTCTCTGCAAGAGCAACAAAGTGGCCTATACTATCTCAGCACCACTGTGAAAGAGATGTCGAAGAAAGCGCCCTCTGAAATTAGCCGGAAATATCAATCAGAATTTGAAGAAATTGAGGGACGCTGGAAGAAGCTCTCCTCCCAGCTGGTTGAGCATTGTCAAAGCTAGAGGAGCAAATGAATAAACTCCGAAAAATTCAG (SEQ ID NO: 2241)

[0287] Homo sapiens dystrophin (DMD), transcript variant Dp427m, exon 43 (nucleotides 6362-6534 of the NCBI reference sequence: NM_004006.2) AATATAAAGATAGTCTACAACAAAGCTCAGGTCGGATTGACATTATTCATAGCAAGAAGACAGCAGCATTGCAAAGTGCAACGCCTGTGGAAAGGGTGAAGCTACAGGAAGCTCTCCCAGCTTGATTTCCAATGGGAAAAGTTAACAAAATGTACAAGGACCGACAAGG (SEQ ID NO: 2242)

[0288] Homo sapiens dystrophin (DMD), transcript variant Dp427m, exon 44 (nucleotides 6535-6682 of the NCBI reference sequence: NM_004006.2) GCGATTTGACAGATCTGTTGAGAAATGGCGGCGTTTTCATTATGATATAAAGATATTTAATCAGTGGCTAACAGAAGCTGAACAGTTTCTCAGAAAGACACAAATTCCTGAGAATTGGGAACATGCTAAATACAAATGGTATCTTAAG (SEQ ID NO: 2243)

[0289] Homo sapiens dystrophin (DMD), transcript variant Dp427m, exon 45 (nucleotides 6683-6858 of the NCBI reference sequence: NM_004006.2) GAACTCCAGGATGGCATTGGGCAGCGCAAACTGTTGTCAGAACATTGAATGCAACTGGGGAAGAAATAATTCAGCAATCCTCAAAAACAGATGCCAGTATTCTACAGGAAAAATTGGGAAGCCTGAATCTGCGGTGGCAGGAGGTCTGCAAACAGCTGTCAGACAGAAAAAAGAG (SEQ ID NO: 2244)

[0290] Homo sapiens dystrophin (DMD), transcript variant Dp427m, exon 46 (nucleotides 6859-7006 of the NCBI reference sequence: NM_004006.2) GCTAGAAGAACAAAAGAATATCTTGTCAGAATTTCAAAGAGATTTAAATGAATTTGTTTTATGGTTGGAGGAAGCAGATAACATTGCTAGTATCCCACTTGAACCTGGAAAAGAGCAGCAACTAAAAGAAAAGCTTGAGCAAGTCAAG (SEQ ID NO: 2245)

[0291] Homo sapiens dystrophin (DMD), transcript variant Dp427m, exon 50 (nucleotides 7445-7553 of the NCBI reference sequence: NM_004006.2) AGGAAGTTAGAAGATCTGAGCTCTGAGTGGAAGGCGGTAACCGTTTACTTCAAGAGCTGAGGGCAAAGCAGCCTGACCTAGCTCCTGGACTGACCACTATTGGAGCCT (SEQ ID NO: 2246)

[0292] Homo sapiens dystrophin (DMD), transcript variant Dp427m, exon 51 (nucleotides 7554-7786 of the NCBI reference sequence: NM_004006.2) CTCCTACTCAGACTGTTACTCTGGTGACACAACCTGTGGTTACTAAGGAAACTGCCATCTCCAAACTAGAAATGCCATCTTCCTTGATGTTGGAGGTACCTGCTCTGGCAGATTTCAACCG GGCTTGGACAGAACTTACCGACTGGCTTTCTCTGCTTGATCAAGTTATAAAATCACAGAGGGTGATGGTGGGTGACCTTGAGGATATCAACGAGATGATCATCAAGCAGAAG (SEQ ID NO: 2247)

[0293] Homo sapiens dystrophin (DMD), transcript variant Dp427m, exon 52 (nucleotides 7787-7904 of the NCBI reference sequence: NM_004006.2) GCAACAATGCAGGATTTGGAACAGAGGCGTCCCCAGTTGGAAGAACTCATTACCGCTGCCCAAAATTTGAAAAACAAGACCAGCAATCAAGAGGCTAGAACAATCATTACGGATCGAA (SEQ ID NO: 2248)

[0294] Homo sapiens dystrophin (DMD), transcript variant Dp427m, exon 53 (nucleotides 7905-8116 of the NCBI reference sequence: NM_004006.2) TTGAAAGAATTCAGAATCAGTGGGATGAAGTACAAGAACACCTTCAGAACCGGAGGCAACAGTTGAATGAAATGTTAAAGGATTCAACACAATGGCTGGAAGCTAAGGAAGAAGCTGAGCAGGTCTTAGGACAGGCCAGAGCCAAGCTTGAGTCATGGAAGGAGGGTCCCTATACAGTAGATGCAATCCAAAAGAAAATCACAGAAACCAAG (SEQ ID NO: 2249)

[0295] Homo sapiens dystrophin (DMD), transcript variant Dp427m, exon 55 (nucleotides 8272-8461 of the NCBI reference sequence: NM_004006.2) GGTGAGTGAGCGAGAGGCTGCTTTGGAAGAAACTCATAGATTACTGCAACAGTTCCCCTGGACCTGGAAAAGTTTCTTGCCTGGCTTACAGAAGCTGAAACAACTGCCAATGTCCTACAGGATGCTACCCGTAAGGAAAGGCTCCTAGAAGACTCCAAGGGAGTAAAAGAGCTGATGAAACAATGGCAA (SEQ ID NO: 2250)

[0296] In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) target exonic splicing enhancer (ESE) sequences in DMD (e.g., the ESE sequences of exons 23, 44, 45, 46, 50, 51, 52, 53, or 55). In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) target exonic splicing enhancer (ESE) sequences in DMD (e.g., the ESE sequences of exons 8, 23, 43, 44, 45, 46, 50, 51, 52, 53, or 55). In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) target the ESE sequences of DMD exon 51 (e.g., the ESEs listed in Table 10). In some embodiments, oligonucleotides useful for targeting DMD target ESE sequences in DMD exons 8, 23, 42, 44, 45, 46, 50, 52, 53, or 55 (eg, the ESEs listed in Table 11).

[0297] In some embodiments, oligonucleotides useful for targeting DMD (e.g., for skipping one or more of exons 8, 23, 42, 44, 45, 46, 50, 52, 53, and 55) comprise a region of complementarity to a target sequence comprising one or more complete or partial ESEs of the DMD transcript (e.g., one or more complete or partial ESEs listed in Table 10 or Table 11). In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence comprising one or more complete or partial ESEs set forth in SEQ ID NOs: 402-436 and 2043-2238. In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence comprising at least four (e.g., four, five, six, seven, or eight) consecutive nucleotides of an ESE set forth in any one of SEQ ID NOs: 402-436 and 2043-2238. Table 10. Exonic splicing enhancers within exons of DMD [Table 10] Table 11. Exonic splicing enhancers within exons 8, 23, 43, 44, 45, 46, 50, 52, 53, and 55 of DMD [Table 11-1] [Table 11-2] [Table 11-3] [Table 11-4] [Table 11-5] [Table 11-6]

[0298] In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence comprising one or more complete or partial ESEs of DMD exon 8. In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence comprising at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE of DMD exon 8. In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence comprising one or more complete or partial ESEs as set forth in SEQ ID NOs: 2047-2062. In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence comprising at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as set forth in any one of SEQ ID NOs: 2047-2062.

[0299] In some embodiments, the oligonucleotide comprises a region complementary to a target sequence comprising at least 6 (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) nucleotides of one or more ESEs (e.g., 2, 3, 4, or more contiguous ESEs) of DMD exon 8. In some embodiments, the oligonucleotide comprises a region complementary to a target sequence comprising at least 6 (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) nucleotides (e.g., 2, 3, 4, or more contiguous ESEs) of one or more ESEs as set forth in SEQ ID NOs: 2047-2062.

[0300] In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) are 18-35 nucleotides in length and contain a region of complementarity to a target sequence comprising at least 4 (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as set forth in any one of SEQ ID NOs: 2047-2062. In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) are 20-30 (e.g., 20, 25, 30) nucleotides in length and contain a region of complementarity to a target sequence comprising at least 4 (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as set forth in any one of SEQ ID NOs: 2047-2062. In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) are 20 nucleotides in length and comprise a region of complementarity to a target sequence comprising at least 4 (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as set forth in any one of SEQ ID NOs: 2047-2062. In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) are 30 nucleotides in length and comprise a region of complementarity to a target sequence comprising at least 4 (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as set forth in any one of SEQ ID NOs: 2047-2062.

[0301] In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence comprising one or more complete or partial ESEs of DMD exon 23. In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence comprising at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE of DMD exon 23. In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence comprising one or more complete or partial ESEs as set forth in SEQ ID NOs: 429 and 2063-2086. In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence comprising at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as set forth in any one of SEQ ID NOs: 429 and 2063-2086.

[0302] In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence comprising at least 6 (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) nucleotides of one or more ESEs (e.g., 2, 3, 4, or more contiguous ESEs) of DMD exon 23. In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence comprising at least 6 (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) nucleotides (e.g., 2, 3, 4, or more contiguous ESEs) of one or more ESEs as set forth in SEQ ID NOS: 429 and 2063-2086.

[0303] In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) are 18-35 nucleotides in length and include a region of complementarity to a target sequence comprising at least 4 (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as set forth in any one of SEQ ID NOs: 429 and 2063-2086. In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) are 20-30 (e.g., 20, 25, 30) nucleotides in length and include a region of complementarity to a target sequence comprising at least 4 (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE set forth in any one of SEQ ID NOs: 429 and 2063-2086. In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) are 20 nucleotides in length and comprise a region of complementarity to a target sequence comprising at least 4 (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as set forth in any one of SEQ ID NOs: 429 and 2063-2086. In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) are 30 nucleotides in length and comprise a region of complementarity to a target sequence comprising at least 4 (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as set forth in any one of SEQ ID NOs: 429 and 2063-2086.

[0304] In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence comprising one or more complete or partial ESEs of DMD exon 43. In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence comprising at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE of DMD exon 43. In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence comprising one or more complete or partial ESEs as set forth in SEQ ID NOs: 412, 2078-2080, and 2087-2111. In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence comprising at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as set forth in any one of SEQ ID NOs: 412, 2078-2080, and 2087-2111.

[0305] In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence comprising at least six (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) nucleotides of one or more ESEs (e.g., 2, 3, 4, or more adjacent ESEs) of DMD exon 43. In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence comprising at least six (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) nucleotides of one or more ESEs (e.g., 2, 3, 4, or more adjacent ESEs) as set forth in SEQ ID NOs: 412, 2078-2080, and 2087-2111.

[0306] In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) are 18-35 nucleotides in length and include a region of complementarity to a target sequence comprising at least 4 (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as set forth in any one of SEQ ID NOs: 412, 2078-2080, and 2087-2111. In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) are 20-30 (e.g., 20, 25, 30) nucleotides in length and include a region of complementarity to a target sequence comprising at least 4 (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as set forth in any one of SEQ ID NOs: 412, 2078-2080, and 2087-2111. In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) are 20 nucleotides in length and comprise a region of complementarity to a target sequence comprising at least 4 (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as set forth in any one of SEQ ID NOs: 412, 2078-2080, and 2087-2111. In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) are 30 nucleotides in length and comprise a region of complementarity to a target sequence comprising at least 4 (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as set forth in any one of SEQ ID NOs: 412, 2078-2080, and 2087-2111.

[0307] In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence comprising one or more complete or partial ESEs of DMD exon 44. In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence comprising at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE of DMD exon 44. In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence comprising one or more complete or partial ESEs as set forth in SEQ ID NOs: 409 and 2112-2121. In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence comprising at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as set forth in any one of SEQ ID NOs: 409 and 2112-2121.

[0308] In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence comprising at least 6 (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) nucleotides of one or more ESEs (e.g., 2, 3, 4, or more contiguous ESEs) of DMD exon 44. In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence comprising at least 6 (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) nucleotides of one or more ESEs (e.g., 2, 3, 4, or more contiguous ESEs) as set forth in SEQ ID NOS: 409 and 2112-2121.

[0309] In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) are 18-35 nucleotides in length and include a region of complementarity to a target sequence comprising at least 4 (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as set forth in any one of SEQ ID NOs: 409 and 2112-2121. In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) are 20-30 (e.g., 20, 25, 30) nucleotides in length and include a region of complementarity to a target sequence comprising at least 4 (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as set forth in any one of SEQ ID NOs: 409 and 2112-2121. In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) are 20 nucleotides in length and comprise an ESE as set forth in any one of SEQ ID NOs: 409 and 2112-2121 that comprises at least 4 (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides. In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) are 30 nucleotides in length and comprise a region of complementarity to a target sequence that comprises at least 4 (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as set forth in any one of SEQ ID NOs: 409 and 2112-2121.

[0310] In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence comprising one or more complete or partial ESEs of DMD exon 45. In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence comprising at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE of DMD exon 45. In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence comprising one or more complete or partial ESEs as set forth in SEQ ID NOs: 2097, 2102, 2103, and 2122-2146. In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence comprising at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as set forth in any one of SEQ ID NOs: 2097, 2102, 2103, and 2122-2146.

[0311] In some embodiments, the oligonucleotide comprises a region complementary to a target sequence comprising at least 6 (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) nucleotides of one or more ESEs (e.g., 2, 3, 4, or more contiguous ESEs) of DMD exon 45. In some embodiments, the oligonucleotide comprises a region complementary to a target sequence comprising at least 6 (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) nucleotides of one or more ESEs (e.g., 2, 3, 4, or more contiguous ESEs) set forth in SEQ ID NOs: 2097, 2102, 2103, and 2122-2146.

[0312] In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) are 18-35 nucleotides in length and include a region of complementarity to a target sequence comprising at least 4 (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as set forth in any one of SEQ ID NOs: 2097, 2102, 2103, and 2122-2146. In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) are 20-30 (e.g., 20, 25, 30) nucleotides in length and include a region of complementarity to a target sequence comprising at least 4 (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as set forth in any one of SEQ ID NOs: 2097, 2102, 2103, and 2122-2146. In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) are 20 nucleotides in length and comprise a region of complementarity to a target sequence comprising at least 4 (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as set forth in any one of SEQ ID NOs: 2097, 2102, 2103, and 2122-2146. In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) are 30 nucleotides in length and comprise a region of complementarity to a target sequence comprising at least 4 (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as set forth in any one of SEQ ID NOs: 2097, 2102, 2103, and 2122-2146.

[0313] In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence comprising one or more complete or partial ESEs of DMD exon 46. In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence comprising at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE of DMD exon 46. In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence comprising one or more complete or partial ESEs as set forth in SEQ ID NOs: 2096 and 2147-2158. In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence comprising at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as set forth in any one of SEQ ID NOs: 2096 and 2147-2158.

[0314] In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence comprising at least 6 (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) nucleotides of one or more ESEs (e.g., 2, 3, 4, or more contiguous ESEs) of DMD exon 46. In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence comprising at least 6 (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) nucleotides of one or more ESEs (e.g., 2, 3, 4, or more contiguous ESEs) as set forth in SEQ ID NOs: 2096 and 2147-2158.

[0315] In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) are 18-35 nucleotides in length and include a region of complementarity to a target sequence comprising at least 4 (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as set forth in any one of SEQ ID NOs: 2096 and 2147-2158. In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) are 20-30 (e.g., 20, 25, 30) nucleotides in length and include a region of complementarity to a target sequence comprising at least 4 (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as set forth in any one of SEQ ID NOs: 2096 and 2147-2158. In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) are 20 nucleotides in length and comprise a region of complementarity to a target sequence comprising at least 4 (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as set forth in any one of SEQ ID NOs: 2096 and 2147-2158. In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) are 30 nucleotides in length and comprise a region of complementarity to a target sequence comprising at least 4 (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as set forth in any one of SEQ ID NOs: 2096 and 2147-2158.

[0316] In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence comprising one or more complete or partial ESEs of DMD exon 50. In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence comprising at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE of DMD exon 50. In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence comprising one or more complete or partial ESEs as set forth in SEQ ID NOs: 2096 and 2160-2177. In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence comprising at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as set forth in any one of SEQ ID NOs: 2096 and 2160-2177.

[0317] In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence comprising at least 6 (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) nucleotides of one or more ESEs (e.g., 2, 3, 4, or more contiguous ESEs) of DMD exon 50. In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence comprising at least 6 (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) nucleotides of one or more ESEs (e.g., 2, 3, 4, or more contiguous ESEs) as set forth in SEQ ID NOs: 2096 and 2160-2177.

[0318] In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) are 18 to 35 nucleotides in length and include a region of complementarity to a target sequence comprising at least 4 (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as set forth in any one of SEQ ID NOs: 2096 and 2160-2177. In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) are 20 to 30 (e.g., 20, 25, 30) nucleotides in length and include a region of complementarity to a target sequence comprising at least 4 (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as set forth in any one of SEQ ID NOs: 2096 and 2160-2177. In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) are 20 nucleotides in length and comprise a region of complementarity to a target sequence comprising at least 4 (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as set forth in any one of SEQ ID NOs: 2096 and 2160-2177. In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) are 30 nucleotides in length and comprise a region of complementarity to a target sequence comprising at least 4 (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as set forth in any one of SEQ ID NOs: 2096 and 2160-2177.

[0319] In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence comprising one or more complete or partial ESEs of DMD exon 51. In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence comprising at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE of DMD exon 51. In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence comprising one or more complete or partial ESEs set forth in SEQ ID NOs: 402-436. In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence comprising at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as set forth in any one of SEQ ID NOs: 402-436. In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence comprising at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as set forth in SEQ ID NO: 419.

[0320] In some embodiments, the oligonucleotide comprises a region complementary to a target sequence comprising at least 6 (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) nucleotides of one or more ESEs (e.g., 2, 3, 4, or more contiguous ESEs) of DMD exon 51. In some embodiments, the oligonucleotide comprises a region complementary to a target sequence comprising at least 6 (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) nucleotides of one or more ESEs (e.g., 2, 3, 4, or more contiguous ESEs) as set forth in SEQ ID NOs: 402-436. In some embodiments, the oligonucleotide comprises a region of complementarity to the target sequence comprising at least 6 (e.g., 6, 7, 8, 9, 10, 11, 12, 13, or 14) nucleotides of the ESE as set forth in SEQ ID NO:418 and SEQ ID NO:419.

[0321] In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) are 18-35 nucleotides in length and contain a region of complementarity to a target sequence comprising at least 4 (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as set forth in any one of SEQ ID NOs: 402-436. In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) are 20-30 (e.g., 20, 25, 30) nucleotides in length and contain a region of complementarity to a target sequence comprising at least 4 (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as set forth in any one of SEQ ID NOs: 402-436. In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) are 20 nucleotides in length and comprise a region of complementarity to a target sequence comprising at least 4 (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as set forth in any one of SEQ ID NOs: 402-436. In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) are 30 nucleotides in length and comprise a region of complementarity to a target sequence comprising at least 4 (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as set forth in any one of SEQ ID NOs: 402-436.

[0322] In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) are 20-30 (e.g., 20, 25, 30) nucleotides in length and contain a region of complementarity to a target sequence comprising at least 4 (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as set forth in SEQ ID NO: 419. In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) are 30 nucleotides in length and contain a region of complementarity to a target sequence comprising at least 4 (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as set forth in SEQ ID NO: 419.

[0323] In some embodiments, the oligonucleotide is 20 to 30 (e.g., 20, 25, 30) nucleotides in length and comprises a region of complementarity to the target sequence comprising at least 6 (e.g., 6, 7, 8, 9, 10, 11, 12, 13, or 14) nucleotides of the ESEs as set forth in SEQ ID NO: 418 and SEQ ID NO: 419. In some embodiments, the oligonucleotide is 30 nucleotides in length and comprises a region of complementarity to the target sequence comprising at least 6 (e.g., 6, 7, 8, 9, 10, 11, 12, 13, or 14) nucleotides of the ESEs as set forth in SEQ ID NO: 418 and SEQ ID NO: 419.

[0324] Non-limiting examples of oligonucleotides useful for DMD exon 51 skipping and their target sequences are provided in SEQ ID NOs: 437-1241 and 1242-2046, respectively. In some embodiments, the oligonucleotide is 20-30 nucleotides in length and comprises a region of complementarity to a target sequence comprising at least 20 contiguous nucleotides of any one of SEQ ID NOs: 1242-2046. In some embodiments, the oligonucleotide is 20-30 nucleotides in length and comprises at least 20 contiguous nucleotides of any one of SEQ ID NOs: 437-1241. In some embodiments, the oligonucleotide comprises the nucleotide sequence of any one of SEQ ID NOs: 437-1241. In some embodiments, the oligonucleotide is at least 30 nucleotides (e.g., 30, 31, 32, 33, 34, or 35) in length and comprises the nucleotide sequence of any one of SEQ ID NOs: 437-1241.

[0325] In some embodiments, the oligonucleotide is 20-30 nucleotides in length and comprises a region of complementarity to a target sequence comprising at least 20 contiguous nucleotides of any one of SEQ ID NOs: 1548, 1550, 1551, 1552, 1555, 1558, 1559, 1562, 1565, 1569, 1577, 1583, 1589, 1595, 1600, 1606, 1610, 1614, 1621, 1626, 1629, 1632, 1637, 1640, 1643, 1646, 1650, 1655, 1658, and 1662. In some embodiments, the oligonucleotide is 20-30 nucleotides in length and comprises 20 consecutive nucleotides of any one of SEQ ID NOs: 743, 745, 746, 747, 750, 753, 754, 757, 760, 764, 772, 778, 784, 790, 795, 801, 805, 809, 816, 821, 824, 827, 832, 835, 838, 841, 845, 850, 853, and 857. In some embodiments, the oligonucleotide comprises the nucleobase sequence of any one of SEQ ID NOs: 743, 745, 746, 747, 750, 753, 754, 757, 760, 764, 772, 778, 784, 790, 795, 801, 805, 809, 816, 821, 824, 827, 832, 835, 838, 841, 845, 850, 853, and 857. In some embodiments, the oligonucleotide is 30 nucleotides in length and comprises the nucleic acid sequence of any one of SEQ ID NOs: 743, 745, 746, 747, 750, 753, 754, 757, 760, 764, 772, 778, 784, 790, 795, 801, 805, 809, 816, 821, 824, 827, 832, 835, 838, 841, 845, 850, 853 and 857.

[0326] In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence comprising one or more complete or partial ESEs of DMD exon 52. In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence comprising at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE of DMD exon 52. In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence comprising one or more complete or partial ESEs as set forth in SEQ ID NOs: 432 and 2178-2192. In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence comprising at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as set forth in any one of SEQ ID NOs: 432 and 2178-2192.

[0327] In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence comprising at least 6 (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) nucleotides of one or more ESEs (e.g., 2, 3, 4, or more contiguous ESEs) of DMD exon 52. In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence comprising at least 6 (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) nucleotides of one or more ESEs (e.g., 2, 3, 4, or more contiguous ESEs) as set forth in SEQ ID NOs: 432 and 2178-2192.

[0328] In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) are 18-35 nucleotides in length and include a region of complementarity to a target sequence comprising at least 4 (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as set forth in any one of SEQ ID NOs: 432 and 2178-2192. In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) are 20-30 (e.g., 20, 25, 30) nucleotides in length and include a region of complementarity to a target sequence comprising at least 4 (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as set forth in any one of SEQ ID NOs: 432 and 2178-2192. In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) are 20 nucleotides in length and comprise a region of complementarity to a target sequence comprising at least 4 (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as set forth in any one of SEQ ID NOs: 432 and 2178-2192. In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) are 30 nucleotides in length and comprise a region of complementarity to a target sequence comprising at least 4 (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as set forth in any one of SEQ ID NOs: 432 and 2178-2192.

[0329] In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence comprising one or more complete or partial ESEs of DMD exon 53. In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence comprising at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE of DMD exon 53. In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence comprising one or more complete or partial ESEs as set forth in SEQ ID NOs: 416, 430, 431, 2108, 2114, 2127, and 2193-2213. In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence comprising at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as set forth in any one of SEQ ID NOs: 416, 430, 431, 2108, 2114, 2127, and 2193-2213.

[0330] In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence comprising at least 6 (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) nucleotides of one or more ESEs (e.g., 2, 3, 4, or more contiguous ESEs) of DMD exon 53. In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence comprising at least 6 (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) nucleotides of one or more ESEs (e.g., 2, 3, 4, or more contiguous ESEs) set forth in SEQ ID NOs: 416, 430, 431, 2108, 2114, 2127, and 2193-2213.

[0331] In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) are 18-35 nucleotides in length and include a region of complementarity to a target sequence comprising at least 4 (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as set forth in any one of SEQ ID NOs: 416, 430, 431, 2108, 2114, 2127, and 2193-2213. In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) are 20-30 (e.g., 20, 25, 30) nucleotides in length and include a region of complementarity to a target sequence comprising at least 4 (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as set forth in any one of SEQ ID NOs: 416, 430, 431, 2108, 2114, 2127, and 2193-2213. In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) are 20 nucleotides in length and comprise a region of complementarity to a target sequence comprising at least 4 (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as set forth in any one of SEQ ID NOs: 416, 430, 431, 2108, 2114, 2127, and 2193-2213. In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) are 30 nucleotides in length and comprise a region of complementarity to a target sequence comprising at least 4 (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as set forth in any one of SEQ ID NOs: 416, 430, 431, 2108, 2114, 2127, and 2193-2213.

[0332] In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence comprising one or more complete or partial ESEs of DMD exon 55. In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence comprising at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE of DMD exon 55. In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence comprising one or more complete or partial ESEs as set forth in SEQ ID NOs: 2097, 2102, 2103, 2116, 2147, 2199, and 2214-2238. In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence comprising at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as set forth in any one of SEQ ID NOs: 2097, 2102, 2103, 2116, 2147, 2199, and 2214-2238.

[0333] In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence comprising at least six (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) nucleotides of one or more ESEs (e.g., 2, 3, 4, or more contiguous ESEs) of DMD exon 55. In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence comprising at least six (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) nucleotides of one or more ESEs (e.g., 2, 3, 4, or more contiguous ESEs) as set forth in SEQ ID NOs: 2097, 2102, 2103, 2116, 2147, 2199, and 2214-2238).

[0334] In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) are 18-35 nucleotides in length and include a region of complementarity to the target sequence comprising at least 4 consecutive nucleotides (e.g., 4, 5, 6, 7, or 8 of an ESE as set forth in any one of SEQ ID NOS: 2097, 2102, 2103, 2116, 2147, 2199, and 2214-2238). In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) are 20-30 (e.g., 20, 25, 30) nucleotides in length and include a region of complementarity to the target sequence comprising at least 4 consecutive nucleotides (e.g., 4, 5, 6, 7, or 8 of an ESE as set forth in any one of SEQ ID NOS: 2097, 2102, 2103, 2116, 2147, 2199, and 2214-2238). In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) are 20 nucleotides in length and comprise a region of complementarity to a target sequence comprising at least 4 (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as set forth in any one of SEQ ID NOs: 2097, 2102, 2103, 2116, 2147, 2199, and 2214-2238. In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) are 30 nucleotides in length and comprise a region of complementarity to a target sequence comprising at least 4 (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as set forth in any one of SEQ ID NOs: 2097, 2102, 2103, 2116, 2147, 2199, and 2214-2238.

[0335] In some embodiments, any one of the oligonucleotides useful for targeting DMD (eg, for exon skipping) is a phosphorodiamidate morpholino oligomer (PMO).

[0336] Additional examples of oligonucleotides targeting DMD (e.g., for exon skipping) are described in U.S. Patent Application Publication No. 2013-072541, published March 21, 2013, entitled "ADENO-ASSOCIATED VIRAL VECTOR FOR EXON SKIPPING IN A GENE ENCODING A DISPENSIBLE-DOMAIN PROTEIN"; U.S. Patent Application Publication No. 2015-191725, published July 9, 2015, entitled "OLIGONUCLEOTIDE FOR THE TREATMENT OF MUSCULAR DYSTROPHY PATIENTS"; U.S. Patent Application Publication No. 2015-196670, published July 16, 2015, entitled "COMPOSITIONS AND METHODS FOR DUCHENNE MUSCULAR DYSTROPHY GENE THERAPY"; and U.S. Patent Application Publication No. 2015-196670, published July 16, 2015, entitled "GENOME EDITING WITH SPLIT CAS9 EXPRESSED FROM U.S. Patent Application Publication No. 2017-349905, published December 7, 2017, entitled "OLIGOMERS HAVING BICYCLIC SCAFFOLD MOLECULES," U.S. Patent Application Publication No. 2018-028554, published February 1, 2018, entitled "ANTISENSE MOLECULES AND METHODS FOR TREATING PATHOLOGIES," U.S. Patent Application Publication No. 2018-171333, published June 21, 2018, entitled "ANTISENSE NUCLEIC ACIDS," U.S. Patent Application Publication No. 2018-179538, published June 28, 2018, entitled "MODIFICATION OF THE DYSTROPHIN GENE AND USES U.S. Patent Application Publication No. 2018-265859, published September 20, 2018, entitled "THEREOF"; U.S. Patent Application Publication No. 2018-369400, published December 27, 2018, entitled "NUCLEIC ACID-POLYPEPTIDE COMPOSITIONS AND METHODS OF INDUCING EXON SKIPPING";U.S. Patent Application Publication No. 2019-000986, published January 3, 2019, entitled "NUCLEIC ACID-POLYPEPTIDE COMPOSITIONS AND METHODS OF INDUCING EXON SKIPPING"; U.S. Patent Application Publication No. 2019-00898, published January 10, 2019, entitled "OLIGONUCLEOTIDE COMPOSITIONS AND METHODS THEREOF"; U.S. Patent Application Publication No. 2019-112604, published April 18, 2019, entitled "METHODS AND MEANS FOR EFFICIENT SKIPPING OF EXON 45 IN DUCHENNE MUSCULAR DYSTROPHY PRE-MRNA"; and U.S. Patent Application Publication No. 2019-112604, published April 18, 2019, entitled "METHODS AND MEANS FOR EFFICIENT SKIPPING OF EXON 45 IN DUCHENNE MUSCULAR DYSTROPHY U.S. Patent Application Publication No. 2019-119679, published April 25, 2019, entitled "OLIGONUCLEOTIDE COMPOSITIONS AND METHODS THEREOF," U.S. Patent Application Publication No. 2019-127733, published May 2, 2019, entitled "THERAPEUTIC APPLICATIONS OF CPF1-BASED GENOME EDITING," U.S. Patent Application Publication No. 2019-151476, published May 23, 2019, entitled "COMPOSITIONS AND METHODS FOR TREATING DUCHENNE MUSCULAR DYSTROPHY AND RELATED DISORDERS," U.S. Patent Application Publication No. 2019-177723, published June 13, 2019, entitled "METHODS AND MEANS FOR EFFICIENT SKIPPING OF EXONS" U.S. Patent Application Publication No. 2019-177725, published June 13, 2019, entitled "45 IN DUCHENNE MUSCULAR DYSTROPHY PRE-MRNA";U.S. Patent Application Publication No. 2019-209604, published July 11, 2019, entitled "OLIGONUCLEOTIDES, COMPOSITIONS AND METHODS THEREOF"; U.S. Patent Application Publication No. 2019-249173, published August 15, 2019, entitled "METHODS AND COMPOSITIONS OF BIOLOGICALLY ACTIVE AGENTS"; U.S. Patent Application Publication No. 2019-270994, published September 5, 2019, entitled "ANTISENSE MOLECULES AND METHODS FOR TREATING PATHOLOGIES"; U.S. Patent Application Publication No. 2019-284556, published September 19, 2019, entitled "MULTIPLE EXON SKIPPING COMPOSITIONS FOR DMD"; and U.S. Patent Application Publication No. 2019-284556, published September 19, 2019, entitled "ANTISENSE OLIGONUCLEOTIDES FOR INDUCING EXON SKIPPING U.S. Patent Application Publication No. 2019-323010, published October 24, 2019, entitled "COMPOUNDS AND METHODS FOR USE THEREOF"; U.S. Patent Application Publication No. 2019-330626, published January 31, 2019, entitled "COMPOUNDS AND METHODS FOR USE IN DYSTROPHIN TRANSCRIPT"; U.S. Patent Application Publication No. 2019-338311, published November 7, 2019, entitled "OPTIMIZED STRATEGY FOR EXON SKIPPING MODIFICATIONS USING CRISPR / CAS9 WITH TRIPLE GUIDE SEQUENCES"; 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[0337] Examples of oligonucleotides for facilitating DMD gene editing are described in International Patent Publication WO2018053632A1, published March 29, 2018, entitled "METHODS OF MODIFYING THE DYSTROPHIN GENE AND RESTORING DYSTROPHIN EXPRESSION AND USES THEREOF"; International Patent Publication WO2017049407A1, published March 30, 2017, entitled "MODIFICATION OF THE DYSTROPHIN GENE AND USES THEREOF"; International Patent Publication WO2016161380A1, published October 6, 2016, entitled "CRISPR / CAS-RELATED METHODS AND COMPOSITIONS FOR TREATING DUCHENNE MUSCULAR DYSTROPHY AND BECKER MUSCULAR DYSTROPHY"; and International Patent Publication WO2016161380A1, published October 6, 2016, entitled "THERAPEUTIC TARGETS FOR THE CORRECTION OF International Patent Publication WO2017095967, published June 8, 2017, entitled "THE HUMAN DYSTROPHIN GENE BY GENE EDITING AND METHODS OF USE"; International Patent Publication WO2017072590A1, published May 4, 2017, entitled "MATERIALS AND METHODS FOR TREATMENT OF DUCHENNE MUSCULAR DYSTROPHY"; International Patent Publication WO2018098480A1, published May 31, 2018, entitled "PREVENTION OF MUSCULAR DYSTROPHY BY CRISPR / CPF1-MEDIATED GENE EDITING"; and "RNA-Guided Systems for In Vivo Gene Editing." U.S. Patent Application Publication US20170266320A1, published September 21, 2017, entitled "PREVENTION OF MUSCULAR DYSTROPHY BY CRISPR / CAS9-MEDIATED GENE EDITING"; International Patent Publication WO2016025469A1, published February 18, 2016, entitled "PREVENTION OF MUSCULAR DYSTROPHY BY CRISPR / CAS9-MEDIATED GENE EDITING";and U.S. Patent Application Publication No. 2013 / 0145487, published June 6, 2013, entitled "MEGANUCLEASE VARIANTS CLEAVING A DNA TARGET SEQUENCE FROM THE DYSTROPHN GENE AND USES THEREOF," the entire contents of each of which are incorporated herein. In some embodiments, the oligonucleotide may have regions complementary to DMD gene sequences of multiple species, e.g., selected from human, mouse, and non-human species.

[0338] In some embodiments, the oligonucleotide may have a region of complementarity to a mutant DMD allele, e.g., a DMD allele with at least one mutation in any of exons 1-79 of DMD in humans that leads to a frameshift and improper RNA splicing / processing.

[0339] In some embodiments, the oligonucleotide may target lncRNA or mRNA, for example, for degradation. In some embodiments, the oligonucleotide may target nucleic acids encoding proteins involved in the mismatch repair pathway, for example, MSH2, MutL alpha, MutS beta, MutL alpha, for example, for degradation. Non-limiting examples of proteins involved in the mismatch repair pathway (the mRNA encoding such proteins may be targeted by the oligonucleotides described herein) are described in Iyer, RR et al., "DNA triplet repeat expansion and mismatch repair," Annu Rev Biochem. 2015;84:199-226; and Schmidt MH and Pearson CE, "Disease-associated repeat instability and mismatch repair," DNA Repair (Amst). 2016 Feb;38:117-26.

[0340] In some embodiments, any one of the oligonucleotides may be in a salt form, for example, as a sodium, potassium, or magnesium salt.

[0341] In some embodiments, the 5' or 3' nucleoside (e.g., the terminal nucleoside) of any one of the oligonucleotides described herein is conjugated to an amine group, optionally via a spacer. In some embodiments, the spacer comprises an aliphatic moiety. In some embodiments, the spacer comprises a polyethylene glycol moiety. In some embodiments, a phosphodiester linkage is present between the spacer and the 5' or 3' nucleoside of the oligonucleotide. In some embodiments, the 5' or 3' nucleoside (e.g., the terminal nucleoside) of any one of the oligonucleotides described herein is conjugated to a spacer, which is a substituted or unsubstituted aliphatic, substituted or u...

Claims

1. A method for inhibiting the progression of intramuscular fibrosis in a subject having a loss-of-function mutation in the dystrophin (DMD) gene that prevents dystrophin production, the method comprising timely administering to the subject an effective amount of a conjugate comprising an anti-TfR1 antibody covalently linked to a molecular payload configured to promote dystrophin expression or activity, the administration resulting in inhibition of the progression of intramuscular fibrosis in the subject.

2. 10. The method of claim 1, wherein administration of the complex is initiated when the subject's skeletal muscle tissue is in a pre-degenerative state or when the subject's skeletal muscle tissue is in a pre-fibrotic state.

3. 3. The method of claim 1 or claim 2, wherein the complex is administered to the subject multiple times over a period of time prior to substantial development of endomysial fibrosis in the subject's quadriceps muscle, or the complex is administered to the subject multiple times over a period of time prior to the subject becoming unable to walk.

4. A method for treating a subject diagnosed with or at risk of having Duchenne muscular dystrophy, the method comprising administering to the subject a conjugate comprising an anti-TfR1 antibody covalently linked to a molecular payload configured to promote dystrophin expression or activity, wherein the administration is for a period during which the subject's skeletal muscle is in a pre-fibrotic state.

5. The method of claim 4, wherein administration of the complex prolongs the period during which the subject's skeletal muscle remains in a pre-fibrotic state compared to when the complex is not administered.

6. The method according to any one of claims 2 to 5, wherein the pre-fibrotic state is: (a) Prior to the substantial onset of endomysial fibrosis in the skeletal muscles controlling the subject's walking ability; (b) prior to the substantial onset of endomysial fibrosis in the limb muscles of interest; (c) prior to the substantial onset of endomysial fibrosis in the subject's quadriceps muscle; and / or (d) Before there is a substantial decline in motor function in the limb muscles of the subject.

7. A method for treating a subject diagnosed with or at risk of having Duchenne muscular dystrophy, the method comprising administering to the subject a conjugate comprising an anti-TfR1 antibody covalently linked to a molecular payload configured to promote dystrophin expression or activity, the subject having a DMD gene modification factor that promotes LTBP4-dependent TGF-β1-mediated fibrosis, and optionally wherein the subject has a hyperfibrotic polymorphism in LTBP4.

8. 8. The method of any one of claims 1-7, wherein the subject is undergoing or has undergone treatment with a corticosteroid, optionally wherein the corticosteroid is a glucocorticoid or a dissociative steroid, further optionally wherein the corticosteroid is prednisone, prednisolone, dexamethasone, deflazacort, or vamorolone.

9. 9. The method of any one of claims 1-8, further comprising administering to the subject a corticosteroid, optionally wherein the corticosteroid is a glucocorticoid or a dissociative steroid, and further optionally wherein the corticosteroid is prednisone, prednisolone, dexamethasone, deflazacort, or vamorolone.

10. 10. The method of any one of claims 1-9, wherein administration of the complex to the subject inhibits the progression of intramuscular fibrosis in the subject, optionally wherein intramuscular fibrosis is measured by histological analysis of skeletal muscle tissue in a muscle biopsy sample from the subject or by magnetic resonance imaging (MRI) assessment of skeletal muscle tissue in the subject.

11. 11. The method of claim 10, wherein the histological analysis comprises staining for one or more extracellular matrix components in a muscle biopsy sample from the subject and measuring the percentage of tissue in the muscle biopsy sample that stains positive for the one or more extracellular matrix components, optionally wherein the staining is picrosirius red staining.

12. 12. The method of any one of claims 1 to 11, wherein the subject is aged 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 years or less.

13. 13. The method of any one of claims 1 to 12, wherein the molecular payload comprises an oligonucleotide, wherein the oligonucleotide promotes exon skipping in the DMD RNA and / or the oligonucleotide comprises a region of complementarity to the DMD RNA.

14. 14. The method of any one of claims 1 to 13, wherein the subject has a DMD gene prone to exon skipping, optionally wherein the exon is in the range of exon 8 to exon 55, and further optionally wherein the exon is exon 8, exon 23, exon 43, exon 44, exon 45, exon 46, exon 50, exon 51, exon 52, exon 53, or exon 55.

15. 15. The method of claim 13 or claim 14, wherein the oligonucleotide promotes skipping of an exon in the range of exon 8 to exon 55 and / or the oligonucleotide comprises a region of complementarity to an exon in the range of exon 8 to exon 55, optionally wherein the oligonucleotide promotes skipping of exon 8, exon 23, exon 43, exon 44, exon 45, exon 46, exon 50, exon 51, exon 52, exon 53 and / or exon 55 and / or the oligonucleotide comprises a region of complementarity to exon 8, exon 23, exon 43, exon 44, exon 45, exon 46, exon 50, exon 51, exon 52, exon 53 and / or exon 55.

16. 16. The method of any one of claims 13 to 15, wherein the oligonucleotide comprises a region of complementarity to one or more complete or partial exonic splicing enhancers (ESEs) of the DMD transcript, and optionally wherein the oligonucleotide comprises a region of complementarity to a target sequence comprising one or more complete or partial ESEs as set forth in SEQ ID NOs: 402-436 and 2043-2238.

17. 17. The method of any one of claims 13 to 16, wherein the oligonucleotide promotes skipping of exon 51 and / or the oligonucleotide comprises a region complementary to exon 51.

18. The method of any one of claims 13 to 17, wherein the oligonucleotide is 20 to 30 nucleotides in length and comprises a region of complementarity to the target sequence comprising at least four consecutive nucleotides of an ESE as set forth in any one of SEQ ID NOs: 402 to 436.

19. 18. The method of any one of claims 13 to 17, wherein the oligonucleotide comprises any one of SEQ ID NOs: 437 to 1241 or comprises a region complementary to any one of SEQ ID NOs: 1242 to 2046.

20. 20. The method of any one of claims 13 to 19, wherein the oligonucleotide comprises one or more phosphorodiamidate morpholinos, optionally wherein the oligonucleotide is a phosphorodiamidate morpholino oligomer (PMO).

21. 21. The method of any one of claims 1 to 20, wherein the anti-TfR1 antibody comprises: Heavy chain complementarity determining region 1 (CDR-H1), heavy chain complementarity determining region 2 (CDR-H2), heavy chain complementarity determining region 3 (CDR-H3), light chain complementarity determining region 1 (CDR-L1), light chain complementarity determining region 2 (CDR-L2), and light chain complementarity determining region 3 (CDR-L3) of an antibody provided in any one of Tables 2-6.

22. The method of any one of claims 1 to 21, wherein the anti-TfR1 antibody comprises a heavy chain variable region (VH) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 76; and / or a light chain variable region (VL) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 75, optionally wherein the anti-TfR1 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 76 and a VL comprising the amino acid sequence of SEQ ID NO:

75.

23. The method of any one of claims 1 to 22, wherein the anti-TfR1 antibody is selected from the group consisting of a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a scFv, an Fv, and a full-length IgG.

24. The method of claim 23, wherein the anti-TfR1 antibody is a Fab fragment.

25. The method of claim 24, wherein the anti-TfR1 antibody comprises a heavy chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 101; and / or a light chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 90, optionally wherein the anti-TfR1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 101 and a light chain comprising the amino acid sequence of SEQ ID NO:

90.

26. 26. The method of any one of claims 1 to 25, wherein the anti-TfR1 antibody is covalently linked to the molecular payload via a cleavable linker, optionally wherein the cleavable linker comprises a valine-citrulline sequence.

27. 27. The method of any one of claims 1 to 26, wherein the conjugate has a structure represented by formula (E): 【Chemistry 1】 or a pharmaceutically acceptable salt thereof, wherein n is 0 to 15 and m is 0 to 15, optionally where n is 3 and / or m is 4.

28. 28. The method of claim 27, wherein L1 is 【Chemistry 2】 or a pharmaceutically acceptable salt thereof, wherein L2 is 【Transformation 3】 wherein a labels the moiety that is directly linked to the carbamate moiety of formula (E); and b labels the moiety that is covalently linked to the molecular payload.

29. 29. The method of claim 27 or claim 28, wherein the molecular payload comprises an oligonucleotide and L1 is linked to the 5' phosphate of the oligonucleotide.

30. 30. The method of any one of claims 1 to 29, wherein the conjugate is administered to the subject via intravenous infusion.

31. 31. The method of any one of claims 1 to 30, wherein the subject is a human, a cynomolgus monkey, or a rodent.

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