Muscle-targeting complexes and their uses

Abstract

Aspects of the present disclosure relate to conjugates comprising a muscle targeting agent covalently linked to a molecular payload. In some embodiments, the muscle targeting agent specifically binds to an internalized cell surface receptor on muscle cells. In some embodiments, the molecular payload inhibits the activity of a disease allele associated with a muscle disease. In some embodiments, the molecular payload is an oligonucleotide, such as an antisense oligonucleotide or an RNAi oligonucleotide.

Description

[Technical Field]

[0001] Related Applications This application is a continuation of U.S. Provisional Patent Application No. 63 / 181450, entitled "MUSCLE-TARGETING COMPLEXES AND USES THEREOF," filed April 29, 2021; U.S. Provisional Patent Application No. 63 / 143831, entitled "MUSCLE-TARGETING COMPLEXES AND USES THEREOF," filed January 30, 2021; U.S. Provisional Patent Application No. 63 / 069078, entitled "MUSCLE-TARGETING COMPLEXES AND USES THEREOF," filed August 23, 2020; U.S. Provisional Patent Application No. 63 / 061842, entitled "MUSCLE-TARGETING COMPLEXES AND USES THEREOF," filed August 6, 2020; and U.S. Provisional Patent Application No. 63 / 061842, entitled "MUSCLE-TARGETING COMPLEXES AND USES THEREOF," filed July 23, 2020. This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 63 / 055,785, entitled "THEREOF," the contents of each of which are incorporated herein by reference in their entireties.

[0002] FIELD OF THE INVENTION This application relates to targeting complexes for delivering molecular payloads (eg, oligonucleotides) to cells and their uses, particularly for the treatment of disease.

[0003] Reference to a sequence listing submitted as a text file via EFS-WEB This application contains a Sequence Listing, which has been submitted via EFS-Web in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy created on July 8, 2021, is designated D082470041WO00-SEQ-DWY and is 152,275 bytes in size. [Background technology]

[0004] Muscle diseases are often associated with muscle weakness and / or (for example, and) muscle dysfunction, leading to life-threatening complications. Many examples of such diseases have been characterized, including muscular dystrophies (e.g., Duchenne, facioscapulohumeral, myotonic, and oculopharyngeal), Pompe disease, centronuclear myopathy, familial hypertrophic cardiomyopathy, Leyne distal myopathy, fibrodysplasia ossificans progressiva, Friedreich ataxia, myofibrillar myopathy, and various other forms. These diseases are generally inherited but can arise spontaneously. These diseases are often congenital but can also arise later. Many rare muscle diseases are single-gene disorders associated with gain-of-function or loss-of-function mutations that may have dominant or recessive phenotypes. For example, activating mutations have been identified in genes encoding ion channels, structural proteins, metabolic proteins, and signaling proteins that contribute to muscle diseases. Despite advances in understanding the genetic etiology of muscle diseases, effective treatment options remain limited. Summary of the Invention

[0005] According to some aspects, the present disclosure provides a complex that targets muscle cells for the purpose of delivering a molecular payload to those cells. In some embodiments, the complexes of the present disclosure facilitate muscle-specific delivery of a molecular payload that targets a muscle disease allele. For example, in some embodiments, the complexes provided herein are particularly useful for delivering a molecular payload that modulates the expression or activity of a gene in a subject having or suspected of having a muscle disease associated with that gene (e.g., a gene / disease in Table 1). In some embodiments, the complexes provided herein include a muscle targeting agent (e.g., a muscle-targeting antibody) that specifically binds to a receptor on the surface of muscle cells for the purpose of delivering the molecular payload to muscle cells. In some embodiments, the complex is internalized into the cell via receptor-mediated internalization (e.g., a transferrin receptor), whereupon the molecular payload can be released inside the cell to perform its function. For example, a complex modified to deliver an oligonucleotide can release the oligonucleotide so that the oligonucleotide can modulate the expression or activity of a muscle disease allele. In some embodiments, the oligonucleotide is released by endosomal cleavage of a covalent linker connecting the oligonucleotide and the muscle-targeting agent of the conjugate.

[0006] One aspect of the present disclosure relates to a conjugate comprising an anti-transferrin receptor (TfR) antibody covalently linked to a molecular payload configured to modulate the expression or activity of a muscle disease gene, wherein the antibody comprises: (i) 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; (ii) a heavy chain variable region (VH) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 69; and / or a light chain variable region (VL) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 70; (iii) a heavy chain variable region (VH) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 71; and / or a light chain variable region (VL) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 70; (iv) a heavy chain variable region (VH) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 72; and / or a light chain variable region (VL) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 70; (v) a heavy chain variable region (VH) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 73; and / or a light chain variable region (VL) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 74; (vi) a heavy chain variable region (VH) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 73; and / or a light chain variable region (VL) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 75; (vii) 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: 74; (viii) a heavy chain variable region (VH) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 77; and / or a light chain variable region (VL) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 78; (ix) a heavy chain variable region (VH) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 79; and / or a light chain variable region (VL) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 80; or (x) a heavy chain variable region (VH) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 77; a light chain variable region (VL) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 80.

[0007] In some embodiments, the antibody comprises: (i) 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; (ii) 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; (iii) 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; (iv) 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; (v) 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; (vi) 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; (vii) 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; (viii) 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; (ix) 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; or (x) VH comprising the amino acid sequence of SEQ ID NO: 77 and VL comprising the amino acid sequence of SEQ ID NO: 80.

[0008] In some embodiments, the 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 antibody is a Fab fragment.

[0009] In some embodiments, the antibody comprises: (i) 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; (ii) a heavy chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 97; and / or a light chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 85; (iii) a heavy chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 98; and / or a light chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 85; (iv) a heavy chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 99; and / or a light chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 85; (v) a heavy chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 100; and / or a light chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 89; (vi) a heavy chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 100; and / or a light chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 90; (vii) a heavy chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 101; and / or a light chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 89; (viii) a heavy chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 102; and / or a light chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 93; (ix) a heavy chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 103; and / or a light chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 95; or (x) a heavy chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 102; and / or a light chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 95.

[0010] In some embodiments, the antibody comprises: (i) 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; (ii) 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; (iii) 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; (iv) 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; (v) 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; (vi) 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; (vii) 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; (viii) 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; (ix) 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; or (x) 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.

[0011] In some embodiments, the antibody does not specifically bind to the transferrin binding site of the transferrin receptor and / or the antibody does not inhibit binding of transferrin to the transferrin receptor.

[0012] In some embodiments, the antibodies are cross-reactive with two or more extracellular epitopes of human, non-human primate, and rodent transferrin receptors.

[0013] In some aspects, the complex is configured to promote transferrin receptor-mediated internalization of the molecular payload into muscle cells.

[0014] In some embodiments, the molecular payload is an oligonucleotide. In some embodiments, the oligonucleotide comprises a region of complementarity to a muscle disease gene having a gain-of-function disease allele.

[0015] In some embodiments, the oligonucleotide comprises at least one modified internucleoside linkage. In some embodiments, at least one modified internucleoside linkage is a phosphorothioate linkage.

[0016] In some embodiments, the oligonucleotide comprises one or more modified nucleosides. In some embodiments, the one or more modified nucleosides are 2'-modified nucleosides.

[0017] In some embodiments, the oligonucleotide is a gapmer oligonucleotide that directs RNAse H-mediated cleavage of mRNA transcripts encoded by muscle disease genes in cells.

[0018] In some embodiments, the oligonucleotide is a mixmer oligonucleotide.

[0019] In some embodiments, the oligonucleotide is an RNAi oligonucleotide that promotes RNAi-mediated cleavage of an mRNA transcript encoded by a muscle disease gene.

[0020] In some embodiments, the 2'-modified nucleoside is selected from the group consisting of 2'-O-methyl, 2'-fluoro (2'-F), 2'-O-methoxyethyl (2'-MOE), and 2',4'-bridged nucleosides.

[0021] In some embodiments, one or more modified nucleosides is a 2',4'-bridged nucleoside.

[0022] In some embodiments, the oligonucleotide is a phosphorodiamidate morpholino oligomer.

[0023] In some embodiments, the antibody is covalently linked to the molecular payload via a cleavable linker. In some embodiments, the cleavable linker comprises a valine-citrulline sequence.

[0024] In some embodiments, the antibody is covalently linked to the molecular payload via conjugation to a lysine or cysteine ​​residue of the antibody.

[0025] In some embodiments, modulating the expression or activity of a muscle disease gene comprises decreasing RNA and / or protein expression.

[0026] Another aspect of the present disclosure relates to a method of modulating the expression or activity of a muscle disease gene in a cell, the method comprising contacting the cell with a complex disclosed herein in an amount effective to promote internalization of a molecular payload into the cell, optionally wherein the cell is a muscle cell.

[0027] In some embodiments, the muscle disease is a disease selected from the group consisting of adult Pompe disease, centronuclear myopathy (CNM), Duchenne muscular dystrophy, facioscapulohumeral muscular dystrophy (FSHD), familial hypertrophic cardiomyopathy, fibrodysplasia ossificans progressiva (FOP), Friedreich's ataxia (FRDA), type 2 inclusion body myopathy, distal Lehn myopathy, myofibrillar myopathy, myotonia congenita (autosomal dominant, Thomsen's disease), myotonic dystrophy type I, myotonic dystrophy type II, myotubular myopathy, oculopharyngeal muscular dystrophy, and congenital myotonia.

[0028] Another aspect of the present disclosure relates to a method of treating a subject having a muscle disease, the method comprising administering to the subject an effective amount of a conjugate disclosed herein, optionally wherein the muscle disease is selected from the group consisting of adult Pompe disease, centronuclear myopathy (CNM), Duchenne muscular dystrophy, facioscapulohumeral muscular dystrophy (FSHD), familial hypertrophic cardiomyopathy, fibrodysplasia ossificans progressiva (FOP), Friedreich's ataxia (FRDA), type 2 inclusion body myopathy, distal Lehn myopathy, myofibrillar myopathy, myotonia congenita (autosomal dominant, Thomsen's disease), myotonic dystrophy type I, myotonic dystrophy type II, myotubular myopathy, oculopharyngeal muscular dystrophy, and congenital myotonia. [Brief explanation of the drawings]

[0029] [Figure 1] FIG. 1 depicts a non-limiting schematic diagram showing the effect of transfecting Hepa1-6 cells with antisense oligonucleotides targeting DMPK (ASO300) on DMPK expression levels compared to vehicle transfection.

[0030] [Figure 2A] FIG. 2A depicts a non-limiting schematic diagram showing an HIL-HPLC trace obtained during purification of a muscle-targeting complex comprising an anti-transferrin receptor antibody covalently linked to a DMPK antisense oligonucleotide.

[0031] [Figure 2B] FIG. 2B depicts a non-limiting image of an SDS-PAGE analysis of the muscle-targeting complex.

[0032] [Figure 3] 1 depicts a non-limiting schematic diagram showing the ability of muscle-targeted RI7 217 Fab antibody-oligonucleotide (DTX-C-008) containing ASO300 to reduce DMPK expression levels.

[0033] [Figure 4A] Figures 4A-4E depict non-limiting schematic diagrams showing the ability of a muscle-targeted RI7 217 Fab antibody-oligonucleotide conjugate (DTX-C-008) containing ASO300 to reduce DMPK expression levels in mouse muscle tissue in vivo compared to vehicle treatment, treatment with naked ASO300, or treatment with a control non-targeted conjugate (DTX-C-007). (N=3 C57B1 / 6 WT mice) [Figure 4B] Figures 4A-4E depict non-limiting schematic diagrams showing the ability of a muscle-targeted RI7 217 Fab antibody-oligonucleotide conjugate (DTX-C-008) containing ASO300 to reduce DMPK expression levels in mouse muscle tissue in vivo compared to vehicle treatment, treatment with naked ASO300, or treatment with a control non-targeted conjugate (DTX-C-007). (N=3 C57B1 / 6 WT mice) [Figure 4C]Figures 4A-4E depict non-limiting schematic diagrams showing the ability of a muscle-targeted RI7 217 Fab antibody-oligonucleotide conjugate (DTX-C-008) containing ASO300 to reduce DMPK expression levels in mouse muscle tissue in vivo compared to vehicle treatment, treatment with naked ASO300, or treatment with a control non-targeted conjugate (DTX-C-007). (N=3 C57B1 / 6 WT mice) [Figure 4D] Figures 4A-4E depict non-limiting schematic diagrams showing the ability of a muscle-targeted RI7 217 Fab antibody-oligonucleotide conjugate (DTX-C-008) containing ASO300 to reduce DMPK expression levels in mouse muscle tissue in vivo compared to vehicle treatment, treatment with naked ASO300, or treatment with a control non-targeted conjugate (DTX-C-007). (N=3 C57B1 / 6 WT mice) [Figure 4E] Figures 4A-4E depict non-limiting schematic diagrams showing the ability of a muscle-targeted RI7 217 Fab antibody-oligonucleotide conjugate (DTX-C-008) containing ASO300 to reduce DMPK expression levels in mouse muscle tissue in vivo compared to vehicle treatment, treatment with naked ASO300, or treatment with a control non-targeted conjugate (DTX-C-007). (N=3 C57B1 / 6 WT mice)

[0034] [Figure 5A] Figures 5A-5B depict non-limiting schematic diagrams demonstrating the tissue selectivity of muscle-targeted RI7 217 Fab antibody-oligonucleotide (DTX-C-008) containing ASO300. The muscle-targeted complex containing ASO300 (DTX-C-008) does not reduce DMPK expression levels in mouse brain or spleen tissue in vivo compared to vehicle treatment, treatment with naked ASO300, or treatment with a control non-targeted complex (DTX-C-007). (N=3 C57B1 / 6 WT mice) [Figure 5B]Figures 5A-5B depict non-limiting schematic diagrams demonstrating the tissue selectivity of muscle-targeted RI7 217 Fab antibody-oligonucleotide (DTX-C-008) containing ASO300. The muscle-targeted complex containing ASO300 (DTX-C-008) does not reduce DMPK expression levels in mouse brain or spleen tissue in vivo compared to vehicle treatment, treatment with naked ASO300, or treatment with a control non-targeted complex (DTX-C-007). (N=3 C57B1 / 6 WT mice)

[0035] [Figure 6A] Figures 6A-6F depict non-limiting schematic diagrams showing the ability of muscle-targeted RI7 217 Fab antibody-oligonucleotide conjugates containing ASO300 (DTX-C-008) to reduce DMPK expression levels in mouse muscle tissue in vivo compared to vehicle treatment, treatment with naked ASO300, or treatment with a control non-targeted conjugate (DTX-C-007). (N=5 C57B1 / 6 WT mice) [Figure 6B] Figures 6A-6F depict non-limiting schematic diagrams showing the ability of muscle-targeted RI7 217 Fab antibody-oligonucleotide conjugates containing ASO300 (DTX-C-008) to reduce DMPK expression levels in mouse muscle tissue in vivo compared to vehicle treatment, treatment with naked ASO300, or treatment with a control non-targeted conjugate (DTX-C-007). (N=5 C57B1 / 6 WT mice) [Figure 6C] Figures 6A-6F depict non-limiting schematic diagrams showing the ability of muscle-targeted RI7 217 Fab antibody-oligonucleotide conjugates containing ASO300 (DTX-C-008) to reduce DMPK expression levels in mouse muscle tissue in vivo compared to vehicle treatment, treatment with naked ASO300, or treatment with a control non-targeted conjugate (DTX-C-007). (N=5 C57B1 / 6 WT mice) [Figure 6D]Figures 6A-6F depict non-limiting schematic diagrams showing the ability of muscle-targeted RI7 217 Fab antibody-oligonucleotide conjugates containing ASO300 (DTX-C-008) to reduce DMPK expression levels in mouse muscle tissue in vivo compared to vehicle treatment, treatment with naked ASO300, or treatment with a control non-targeted conjugate (DTX-C-007). (N=5 C57B1 / 6 WT mice) [Figure 6E] Figures 6A-6F depict non-limiting schematic diagrams showing the ability of muscle-targeted RI7 217 Fab antibody-oligonucleotide conjugates containing ASO300 (DTX-C-008) to reduce DMPK expression levels in mouse muscle tissue in vivo compared to vehicle treatment, treatment with naked ASO300, or treatment with a control non-targeted conjugate (DTX-C-007). (N=5 C57B1 / 6 WT mice) [Figure 6F] Figures 6A-6F depict non-limiting schematic diagrams showing the ability of muscle-targeted RI7 217 Fab antibody-oligonucleotide conjugates containing ASO300 (DTX-C-008) to reduce DMPK expression levels in mouse muscle tissue in vivo compared to vehicle treatment, treatment with naked ASO300, or treatment with a control non-targeting conjugate (DTX-C-007). (N=5 C57B1 / 6 WT mice)

[0036] [Figure 7A] 7A-7L depict non-limiting schematic diagrams showing the ability of a muscle-targeting antibody-oligonucleotide conjugate (DTX-C-012) comprising ASO300 covalently linked to an anti-hTfR antibody to reduce DMPK expression levels in cynomolgus monkey muscle tissue in vivo compared to vehicle treatment (saline) and compared to naked DMPK ASO (ASO300). (N=3 male cynomolgus monkeys) [Figure 7B]7A-7L depict non-limiting schematic diagrams showing the ability of a muscle-targeting antibody-oligonucleotide conjugate (DTX-C-012) comprising ASO300 covalently linked to an anti-hTfR antibody to reduce DMPK expression levels in cynomolgus monkey muscle tissue in vivo compared to vehicle treatment (saline) and compared to naked DMPK ASO (ASO300). (N=3 male cynomolgus monkeys) [Figure 7C] 7A-7L depict non-limiting schematic diagrams showing the ability of a muscle-targeting antibody-oligonucleotide conjugate (DTX-C-012) comprising ASO300 covalently linked to an anti-hTfR antibody to reduce DMPK expression levels in cynomolgus monkey muscle tissue in vivo compared to vehicle treatment (saline) and compared to naked DMPK ASO (ASO300). (N=3 male cynomolgus monkeys) [Figure 7D] 7A-7L depict non-limiting schematic diagrams showing the ability of a muscle-targeting antibody-oligonucleotide conjugate (DTX-C-012) comprising ASO300 covalently linked to an anti-hTfR antibody to reduce DMPK expression levels in cynomolgus monkey muscle tissue in vivo compared to vehicle treatment (saline) and compared to naked DMPK ASO (ASO300). (N=3 male cynomolgus monkeys) [Figure 7E] 7A-7L depict non-limiting schematic diagrams showing the ability of a muscle-targeting antibody-oligonucleotide conjugate (DTX-C-012) comprising ASO300 covalently linked to an anti-hTfR antibody to reduce DMPK expression levels in cynomolgus monkey muscle tissue in vivo compared to vehicle treatment (saline) and compared to naked DMPK ASO (ASO300). (N=3 male cynomolgus monkeys) [Figure 7F]7A-7L depict non-limiting schematic diagrams showing the ability of a muscle-targeting antibody-oligonucleotide conjugate (DTX-C-012) comprising ASO300 covalently linked to an anti-hTfR antibody to reduce DMPK expression levels in cynomolgus monkey muscle tissue in vivo compared to vehicle treatment (saline) and compared to naked DMPK ASO (ASO300). (N=3 male cynomolgus monkeys) [Figure 7G] 7A-7L depict non-limiting schematic diagrams showing the ability of a muscle-targeting antibody-oligonucleotide conjugate (DTX-C-012) comprising ASO300 covalently linked to an anti-hTfR antibody to reduce DMPK expression levels in cynomolgus monkey muscle tissue in vivo compared to vehicle treatment (saline) and compared to naked DMPK ASO (ASO300). (N=3 male cynomolgus monkeys) [Figure 7H] 7A-7L depict non-limiting schematic diagrams showing the ability of a muscle-targeting antibody-oligonucleotide conjugate (DTX-C-012) comprising ASO300 covalently linked to an anti-hTfR antibody to reduce DMPK expression levels in cynomolgus monkey muscle tissue in vivo compared to vehicle treatment (saline) and compared to naked DMPK ASO (ASO300). (N=3 male cynomolgus monkeys) [Figure 7I] 7A-7L depict non-limiting schematic diagrams showing the ability of a muscle-targeting antibody-oligonucleotide conjugate (DTX-C-012) comprising ASO300 covalently linked to an anti-hTfR antibody to reduce DMPK expression levels in cynomolgus monkey muscle tissue in vivo compared to vehicle treatment (saline) and compared to naked DMPK ASO (ASO300). (N=3 male cynomolgus monkeys) [Figure 7J]7A-7L depict non-limiting schematic diagrams showing the ability of a muscle-targeting antibody-oligonucleotide conjugate (DTX-C-012) comprising ASO300 covalently linked to an anti-hTfR antibody to reduce DMPK expression levels in cynomolgus monkey muscle tissue in vivo compared to vehicle treatment (saline) and compared to naked DMPK ASO (ASO300). (N=3 male cynomolgus monkeys) [Figure 7K] 7A-7L depict non-limiting schematic diagrams showing the ability of a muscle-targeting antibody-oligonucleotide conjugate (DTX-C-012) comprising ASO300 covalently linked to an anti-hTfR antibody to reduce DMPK expression levels in cynomolgus monkey muscle tissue in vivo compared to vehicle treatment (saline) and compared to naked DMPK ASO (ASO300). (N=3 male cynomolgus monkeys) [Figure 7L] 7A-7L depict non-limiting schematic diagrams showing the ability of a muscle-targeting antibody-oligonucleotide conjugate (DTX-C-012) comprising ASO300 covalently linked to an anti-hTfR antibody to reduce DMPK expression levels in cynomolgus monkey muscle tissue in vivo compared to vehicle treatment (saline) and compared to naked DMPK ASO (ASO300). (N=3 male cynomolgus monkeys)

[0037] [Figure 8A] 8A-8B depict non-limiting schematic diagrams showing the ability of a muscle-targeting antibody-oligonucleotide conjugate (DTX-C-012) comprising ASO300 covalently linked to an anti-hTfR antibody compared to a naked DMPK ASO (ASO300) to reduce DMPK expression levels in cynomolgus monkey smooth muscle tissue in vivo compared to vehicle treatment (saline). (N=3 male cynomolgus monkeys) [Figure 8B]8A-8B depict non-limiting schematic diagrams showing the ability of a muscle-targeting antibody-oligonucleotide conjugate (DTX-C-012) comprising ASO300 covalently linked to an anti-hTfR antibody compared to a naked DMPK ASO (ASO300) to reduce DMPK expression levels in cynomolgus monkey smooth muscle tissue in vivo compared to vehicle treatment (saline). (N=3 male cynomolgus monkeys)

[0038] [Figure 9A] Figures 9A-9D depict non-limiting schematic diagrams demonstrating the tissue selectivity of a muscle-targeting antibody-oligonucleotide (DTX-C-012) complex containing ASO300 covalently linked to an anti-hTfR antibody. The muscle-targeting complex containing DMPK-ASO does not reduce DMPK expression levels in kidney, brain, or spleen tissues of cynomolgus monkeys in vivo compared to vehicle treatment. (N = 3 male cynomolgus monkeys) [Figure 9B] Figures 9A-9D depict non-limiting schematic diagrams demonstrating the tissue selectivity of a muscle-targeting antibody-oligonucleotide (DTX-C-012) complex containing ASO300 covalently linked to an anti-hTfR antibody. The muscle-targeting complex containing DMPK-ASO does not reduce DMPK expression levels in kidney, brain, or spleen tissues of cynomolgus monkeys in vivo compared to vehicle treatment. (N = 3 male cynomolgus monkeys) [Figure 9C] Figures 9A-9D depict non-limiting schematic diagrams demonstrating the tissue selectivity of a muscle-targeting antibody-oligonucleotide (DTX-C-012) complex containing ASO300 covalently linked to an anti-hTfR antibody. The muscle-targeting complex containing DMPK-ASO does not reduce DMPK expression levels in kidney, brain, or spleen tissues of cynomolgus monkeys in vivo compared to vehicle treatment. (N = 3 male cynomolgus monkeys) [Figure 9D]Figures 9A-9D depict non-limiting schematic diagrams demonstrating the tissue selectivity of a muscle-targeting antibody-oligonucleotide (DTX-C-012) complex containing ASO300 covalently linked to an anti-hTfR antibody. The muscle-targeting complex containing DMPK-ASO does not reduce DMPK expression levels in kidney, brain, or spleen tissues of cynomolgus monkeys in vivo compared to vehicle treatment. (N = 3 male cynomolgus monkeys)

[0039] [Figure 10] Figure 10 shows normalized DMPK mRNA tissue expression levels across several tissue types in cynomolgus monkeys. (N=3 male cynomolgus monkeys)

[0040] [Figure 11A] 11A-11B depict non-limiting schematic diagrams showing the ability of a muscle-targeted RI7 217 Fab antibody-oligonucleotide conjugate (DTX-C-008) containing ASO300 compared to vehicle treatment (saline) and compared to a naked DMPK ASO (ASO300) to reduce DMPK expression levels in mouse muscle tissue in vivo for up to 28 days following DTX-C-008 dosing. [Figure 11B] 11A-11B depict non-limiting schematic diagrams showing the ability of a muscle-targeted RI7 217 Fab antibody-oligonucleotide conjugate (DTX-C-008) containing ASO300 compared to vehicle treatment (saline) and compared to a naked DMPK ASO (ASO300) to reduce DMPK expression levels in mouse muscle tissue in vivo for up to 28 days following DTX-C-008 dosing.

[0041] [Figure 12] Figure 12 shows that a single dose of a muscle-targeting conjugate (DTX-C-012) comprising ASO300 covalently linked to an anti-hTfR antibody is safe and tolerated in cynomolgus monkeys. (N=3 male cynomolgus monkeys)

[0042] [Figure 13A]Figures 13A-13B depict non-limiting schematic diagrams showing the ability of a muscle-targeted RI7 217 Fab antibody-oligonucleotide conjugate (DTX-C-008) containing ASO300 compared to a control IgG2a Fab antibody-oligonucleotide conjugate (DTX-C-007) and a naked DMPK ASO (ASO300) to reduce DMPK expression levels in mouse muscle tissue in vivo for up to 12 weeks after DTX-C-008 dosing, compared to vehicle treatment (PBS). (N=5 C57B1 / 6 WT mice) [Figure 13B] Figures 13A-13B depict non-limiting schematic diagrams showing the ability of a muscle-targeted RI7 217 Fab antibody-oligonucleotide conjugate (DTX-C-008) containing ASO300 compared to a control IgG2a Fab antibody-oligonucleotide conjugate (DTX-C-007) and a naked DMPK ASO (ASO300) to reduce DMPK expression levels in mouse muscle tissue in vivo for up to 12 weeks after DTX-C-008 dosing, compared to vehicle treatment (PBS). (N=5 C57B1 / 6 WT mice)

[0043] [Figure 14A] Figures 14A-14B depict non-limiting schematic diagrams showing the ability of muscle-targeted RI7 217 Fab antibody-oligonucleotide conjugates (DTX-C-008) containing ASO300 to target nuclear mutant DMPK RNA in a mouse model. (N=6 mice) [Figure 14B] Figures 14A-14B depict non-limiting schematic diagrams showing the ability of muscle-targeted RI7 217 Fab antibody-oligonucleotide conjugates (DTX-C-008) containing ASO300 to target nuclear mutant DMPK RNA in a mouse model. (N=6 mice)

[0044] [Figure 15A]Figures 15A-15B depict non-limiting schematic diagrams showing the ability of muscle-targeted RI7 217 Fab antibody-ASO conjugates containing actin-targeting oligonucleotides (DTX-actin) to dose-dependently reduce actin expression levels in muscle tissue and the functional grade of myotonia (N=2 HSALR mice). [Figure 15B] Figures 15A-15B depict non-limiting schematic diagrams showing the ability of muscle-targeted RI7 217 Fab antibody-ASO conjugates containing actin-targeting oligonucleotides (DTX-actin) to dose-dependently reduce actin expression levels in muscle tissue and the functional grade of myotonia (N=2 HSALR mice).

[0045] [Figure 16A] Figures 16A-16C show non-limiting schematic diagrams demonstrating that the muscle-targeted RI7 217 Fab antibody-oligonucleotide conjugate (DTX-C-008) containing ASO300 can significantly shorten the prolonged QTc interval in a mouse model to validate functional correction of arrhythmia in the DM1 cardiac model. (N=10 mice) [Figure 16B] Figures 16A-16C show non-limiting schematic diagrams demonstrating that the muscle-targeted RI7 217 Fab antibody-oligonucleotide conjugate (DTX-C-008) containing ASO300 can significantly shorten the prolonged QTc interval in a mouse model to validate functional correction of arrhythmia in the DM1 cardiac model. (N=10 mice) [Figure 16C] Figures 16A-16C show non-limiting schematic diagrams demonstrating that the muscle-targeted RI7 217 Fab antibody-oligonucleotide conjugate (DTX-C-008) containing ASO300 can significantly shorten the prolonged QTc interval in a mouse model to validate functional correction of arrhythmia in the DM1 cardiac model. (N=10 mice)

[0046] [Figure 17A]Figures 17A-17B depict non-limiting schematic diagrams showing that a muscle-targeting antibody-oligonucleotide conjugate (DTX-C-012) comprising an ASO300 antisense oligonucleotide covalently linked to an anti-hTfR antibody can reduce the expression level of DMPK and correct the splicing of a DMPK-specific target gene (Bin1) in human cells from DM1 patients. (N=3) [Figure 17B] Figures 17A-17B depict non-limiting schematic diagrams showing that a muscle-targeting antibody-oligonucleotide conjugate (DTX-C-012) comprising an ASO300 antisense oligonucleotide covalently linked to an anti-hTfR antibody can reduce the expression level of DMPK and correct the splicing of a DMPK-specific target gene (Bin1) in human cells from DM1 patients. (N=3)

[0047] [Figure 18] FIG. 18 depicts a non-limiting schematic diagram showing the ability of a muscle-targeting complex (anti-TfR antibody-FM10) comprising an anti-TfR1 Fab (RI7 217) conjugated to an FM10 antisense oligonucleotide compared to naked FM10 antisense oligonucleotide to reduce expression levels of downstream DUX4 genes (ZSCAN4, MBD3L2, TRIM43) in human U-2 OS cells.

[0048] [Figure 19] FIG. 19 depicts a non-limiting schematic diagram showing the ability of an anti-transferrin receptor muscle-targeting complex containing an exon 23 skipping phosphorodiamidate morpholino oligomer (PMO) to dose-dependently enhance exon skipping in muscle tissue of an mdx mouse model.

[0049] [Figure 20A] 20A-20B depict non-limiting schematic diagrams showing the ability of an anti-transferrin receptor muscle-targeting complex containing an exon 23 skipping PMO to dose-dependently increase dystrophin in skeletal muscle (quadriceps) of an mdx mouse model. [Figure 20B] 20A-20B depict non-limiting schematic diagrams showing the ability of an anti-transferrin receptor muscle-targeting complex containing an exon 23 skipping PMO to dose-dependently increase dystrophin in skeletal muscle (quadriceps) of an mdx mouse model.

[0050] [Figure 21A] Figures 21A-21E depict non-limiting schematic diagrams showing the ability of an anti-transferrin receptor muscle-targeting complex containing an exon 23 skipping PMO to improve functional performance (Figures 21A, 21B, 21C, and 21D) and reduce creatine kinase levels (Figure 21E) in an mdx mouse model. (**p<0.01; ***p<0.001; ****p<0.0001) [Figure 21B] Figures 21A-21E depict non-limiting schematic diagrams showing the ability of an anti-transferrin receptor muscle-targeting complex containing an exon 23 skipping PMO to improve functional performance (Figures 21A, 21B, 21C, and 21D) and reduce creatine kinase levels (Figure 21E) in an mdx mouse model. (**p<0.01; ***p<0.001; ****p<0.0001) [Figure 21C] Figures 21A-21E depict non-limiting schematic diagrams showing the ability of an anti-transferrin receptor muscle-targeting complex containing an exon 23 skipping PMO to improve functional performance (Figures 21A, 21B, 21C, and 21D) and reduce creatine kinase levels (Figure 21E) in an mdx mouse model. (**p<0.01; ***p<0.001; ****p<0.0001) [Figure 21D] Figures 21A-21E depict non-limiting schematic diagrams showing the ability of an anti-transferrin receptor muscle-targeting complex containing an exon 23 skipping PMO to improve functional performance (Figures 21A, 21B, 21C, and 21D) and reduce creatine kinase levels (Figure 21E) in an mdx mouse model. (**p<0.01; ***p<0.001; ****p<0.0001) [Figure 21E]Figures 21A-21E depict non-limiting schematic diagrams showing the ability of an anti-transferrin receptor muscle-targeting complex containing an exon 23 skipping PMO to improve functional performance (Figures 21A, 21B, 21C, and 21D) and reduce creatine kinase levels (Figure 21E) in an mdx mouse model. (**p<0.01; ***p<0.001; ****p<0.0001)

[0051] [Figure 22A] Figures 22A-22C depict non-limiting schematic diagrams showing the dose response of selected antisense oligonucleotides (DMPK-ASO-1, DMPK-ASO-2, and DMPK-ASO-3) in DMPK knockdown in human DM1 myotubes. ASO300 was used as a control. All tested oligonucleotides demonstrated DMPK knockdown activity. Statistical analysis: One-way ANOVA with Tukey's HSD post-hoc test vs. naked ASO300 treatment; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 22B] Figures 22A-22C depict non-limiting schematic diagrams showing the dose response of selected antisense oligonucleotides (DMPK-ASO-1, DMPK-ASO-2, and DMPK-ASO-3) in DMPK knockdown in human DM1 myotubes. ASO300 was used as a control. All tested oligonucleotides demonstrated DMPK knockdown activity. Statistical analysis: One-way ANOVA with Tukey's HSD post-hoc test vs. naked ASO300 treatment; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 22C]Figures 22A-22C depict non-limiting schematic diagrams showing the dose response of selected antisense oligonucleotides (DMPK-ASO-1, DMPK-ASO-2, and DMPK-ASO-3) in DMPK knockdown in human DM1 myotubes. ASO300 was used as a control. All tested oligonucleotides demonstrated DMPK knockdown activity. Statistical analysis: One-way ANOVA with Tukey's HSD post-hoc test vs. naked ASO300 treatment; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0052] [Figure 23A] Figures 23A-23B depict non-limiting schematic diagrams showing the dose response of selected antisense oligonucleotides (DMPK-ASO-1, DMPK-ASO-2, and DMPK-ASO-3) in DMPK knockdown in non-human primate (NHP) DM1 myotubes. ASO300 was used as a control. All tested oligonucleotides demonstrated DMPK knockdown activity. [Figure 23B] Figures 23A-23B depict non-limiting schematic diagrams showing the dose response of selected antisense oligonucleotides (DMPK-ASO-1, DMPK-ASO-2, and DMPK-ASO-3) in DMPK knockdown in non-human primate (NHP) DM1 myotubes. ASO300 was used as a control. All tested oligonucleotides demonstrated DMPK knockdown activity.

[0053] [Figure 24] FIG. 24 shows the serum stability over time of linkers used to connect anti-TfR antibodies and molecular payloads (e.g., oligonucleotides) in various species after intravenous administration.

[0054] [Figure 25A]Figures 25A-25F show the binding of humanized anti-TfR Fabs to human TfR1 (hTfR1) or cynomolgus monkey TfR1 (cTfR1) as measured by ELISA. Figure 25A shows the binding of humanized 3M12 variants to hTfR1. [Figure 25B] Figures 25A-25F show the binding of humanized anti-TfR Fabs to human TfR1 (hTfR1) or cynomolgus monkey TfR1 (cTfR1) as measured by ELISA. Figure 25B shows the binding of humanized 3M12 variants to cTfR1. [Figure 25C] Figures 25A-25F show the binding of humanized anti-TfR Fabs to human TfR1 (hTfR1) or cynomolgus monkey TfR1 (cTfR1) as measured by ELISA. Figure 25C shows the binding of humanized 3A4 variants to hTfR1. [Figure 25D] Figures 25A-25F show the binding of humanized anti-TfR Fabs to human TfR1 (hTfR1) or cynomolgus monkey TfR1 (cTfR1) as measured by ELISA. Figure 25D shows the binding of humanized 3A4 variants to cTfR1. [Figure 25E] Figures 25A-25F show the binding of humanized anti-TfR Fabs to human TfR1 (hTfR1) or cynomolgus monkey TfR1 (cTfR1) as measured by ELISA. Figure 25E shows the binding of humanized 5H12 variants to hTfR1. [Figure 25F] Figures 25A-25F show the binding of humanized anti-TfR Fabs to human TfR1 (hTfR1) or cynomolgus monkey TfR1 (cTfR1) as measured by ELISA. Figure 25F shows the binding of humanized 5H12 variants to hTfR1.

[0055] [Figure 26]Figure 26 shows the quantified cellular uptake of anti-TfR Fab conjugates into rhabdomyosarcoma (RD) cells. The molecular payload in the tested conjugates was a DMPK-targeting oligonucleotide, and conjugate uptake was promoted by the indicated anti-TfR Fab. Conjugates with a negative control Fab (anti-mouse TfR) or a positive control Fab (anti-human TfR1) were also included in the assay. Cells were incubated with the indicated conjugates at a concentration of 100 nM for 4 hours. Cell uptake was measured by mean Cypher5e fluorescence.

[0056] [Figure 27A] Figures 27A-27F show the binding of oligonucleotide-conjugated or unconjugated humanized anti-TfR Fabs to human TfR1 (hTfR1) and cynomolgus monkey TfR1 (cTfR1) as measured by ELISA. Figure 27A shows the binding of humanized 3M12 variants alone or conjugated with DMPK-targeting oligos to hTfR1. The respective EC50 values ​​are also shown. [Figure 27B] Figures 27A-27F show the binding of oligonucleotide-conjugated or unconjugated humanized anti-TfR Fabs to human TfR1 (hTfR1) and cynomolgus monkey TfR1 (cTfR1) as measured by ELISA. Figure 27B shows the binding of humanized 3M12 variants alone or conjugated with DMPK-targeting oligos to cTfR1. The respective EC50 values ​​are also shown. [Figure 27C] Figures 27A-27F show the binding of oligonucleotide-conjugated or unconjugated humanized anti-TfR Fabs to human TfR1 (hTfR1) and cynomolgus monkey TfR1 (cTfR1) as measured by ELISA. Figure 27C shows the binding of the humanized 3A4 variant alone or conjugated with a DMPK-targeting oligo to hTfR1. The respective EC50 values ​​are also shown. [Figure 27D]Figures 27A-27F show the binding of oligonucleotide-conjugated or unconjugated humanized anti-TfR Fabs to human TfR1 (hTfR1) and cynomolgus monkey TfR1 (cTfR1) as measured by ELISA. Figure 27D shows the binding of the humanized 3A4 variant alone or conjugated with a DMPK-targeting oligo to cTfR1. The respective EC50 values ​​are also shown. [Figure 27E] Figures 27A-27F show the binding of oligonucleotide-conjugated or unconjugated humanized anti-TfR Fabs to human TfR1 (hTfR1) and cynomolgus monkey TfR1 (cTfR1) as measured by ELISA. Figure 27E shows the binding of humanized 5H12 variants alone or conjugated with DMPK-targeting oligos to hTfR1. The respective EC50 values ​​are also shown. [Figure 27F] Figures 27A-27F show the binding of oligonucleotide-conjugated or unconjugated humanized anti-TfR Fabs to human TfR1 (hTfR1) and cynomolgus monkey TfR1 (cTfR1) as measured by ELISA. Figure 27F shows the binding of humanized 5H12 variants alone or conjugated with DMPK-targeting oligos to cTfR1. The respective EC50 values ​​are also shown.

[0057] [Figure 28] Figure 28 shows DMPK expression in RD cells treated with DMPK-targeting oligonucleotides compared to cells treated with PBS. The duration of treatment was 3 days. The DMPK-targeting oligonucleotides were delivered to cells as free oligonucleotides (gymnotic uptake, "free") or using a transfection reagent ("Trans").

[0058] [Figure 29]Figure 29 shows DMPK expression in RD cells treated with various concentrations of conjugates containing the indicated humanized anti-TfR antibodies conjugated to a DMPK-targeting oligonucleotide (ASO300). The duration of treatment was 3 days. ASO300 delivered using a transfection agent (labeled "Trans") was used as a control.

[0059] [Figure 30] Figure 30 shows the results of splicing correction of Atp2a1 by anti-TfR1 antibody-oligonucleotide conjugates (Ab-ASOs) in the HSA-LR mouse model of DM1, measured in the gastrocnemius muscle. The anti-TfR antibody used was RI7 217, and the oligonucleotide targets human skeletal actin.

[0060] [Figure 31-1] Figure 31 shows splicing corrections in over 30 different RNAs for DM1 measured in the gastrocnemius muscle of HSA-LR mice treated with anti-TfR1 antibody-oligonucleotide (Ab-ASO) conjugates or saline. The anti-TfR antibody used was RI7 217, and the oligonucleotide targets human skeletal actin. [Figure 31-2] Figure 31 shows splicing corrections in over 30 different RNAs for DM1 measured in the gastrocnemius muscle of HSA-LR mice treated with anti-TfR1 antibody-oligonucleotide (Ab-ASO) conjugates or saline. The anti-TfR antibody used was RI7 217, and the oligonucleotide targets human skeletal actin. [Figure 31-3] Figure 31 shows splicing corrections in over 30 different RNAs for DM1 measured in the gastrocnemius muscle of HSA-LR mice treated with anti-TfR1 antibody-oligonucleotide (Ab-ASO) conjugates or saline. The anti-TfR antibody used was RI7 217, and the oligonucleotide targets human skeletal actin.

[0061] [Figure 32] Figure 32 shows splicing abnormalities in the quadriceps, gastrocnemius, or tibialis anterior muscles of HSA-LR mice treated with anti-TfR1 antibody-oligonucleotide conjugates (Ab-ASOs) or saline. The data represent the combined splicing abnormalities measured in over 30 RNAs shown in Figure 31.

[0062] [Figure 33] Figure 33 shows the myotonia grade measured in the quadriceps, gastrocnemius, and tibialis anterior muscles of HSA-LR mice treated with saline, unconjugated oligonucleotide (ASO), or anti-TfR1 antibody-oligonucleotide conjugate (Ab-ASO). Myotonia was measured by electromyography (EMG) and graded as 0, 1, 2, or 3 based on the frequency of myotonic discharges.

[0063] [Figure 34] Figure 34 shows exon 51 skipping in human DMD myotubes promoted by DMD exon 51 skipping oligonucleotides (PMOs). Cells were treated with naked PMOs or PMOs conjugated to anti-TfR1 Fab (Ab-PMO).

[0064] [Figure 35] Figure 35 shows a dose-dependent increase in dystrophin expression measured by Western blotting in mdx mouse quadriceps muscle after treatment with anti-mouse TfR1 (RI7 217) conjugated to an oligonucleotide (PMO) targeted to exon 23, using alpha-actin as a loading control. Standards were generated using pooled wild-type protein and pooled mdx protein. Percentages indicate the amount of WT protein spiked into the sample.

[0065] [Figure 36]FIG. 36 shows quantification of dystrophin protein levels in the quadriceps muscle of mdx mice after treatment with various doses of anti-mouse TfR (RI7 217) conjugated to an oligonucleotide (PMO) targeting exon 23.

[0066] [Figure 37] Figure 37 shows immunofluorescence staining images of quadriceps muscles from wild-type (WT) mice treated with saline, or mdx mice treated with saline, naked oligonucleotide, or oligonucleotide conjugated to anti-mouse TfR1 (RI7 217).

[0067] [Figure 38A] Figures 38A-38B show the expression of a wide range of MBD3L2, TRIM43, and ZSCAN4 transcripts in myotubes from FSHD patients treated with naked FM10 (Figure 38A) or FM10 conjugated to anti-TfR1 (Figure 38B). [Figure 38B] Figures 38A-38B show the expression of a wide range of MBD3L2, TRIM43, and ZSCAN4 transcripts in myotubes from FSHD patients treated with naked FM10 (Figure 38A) or FM10 conjugated to anti-TfR1 (Figure 38B).

[0068] [Figure 39] Figure 39 shows data demonstrating that conjugates containing the indicated anti-TfR Fab's (3M12 VH3 / VK2, 3M12 VH4 / VK3, and 3A4 VH3 N54S / VK4) conjugated to DMD exon skipping oligonucleotides resulted in enhanced exon skipping in DMD patient myotubes compared to naked DMD exon skipping oligos.

[0069] [Figure 40A]Figures 40A-40E show the in vivo activity of conjugates containing the indicated anti-TfR Fabs (control, 3M12 VH3 / VK2, 3M12 VH4 / VK3, and 3A4 VH3 N54S / VK4) conjugated to DMPK-targeting oligonucleotides in reducing DMPK mRNA expression in mice expressing human TfR1 (hTfR1 knock-in mice). Figure 40A shows the experimental design (e.g., IV dose, dosing frequency). DMPK mRNA levels were measured in the tibialis anterior muscle (Figure 40B), gastrocnemius muscle (Figure 40C), heart (Figure 40D), and diaphragm (Figure 40E) of mice 14 days after the first dose. [Figure 40B] Figures 40A-40E show the in vivo activity of conjugates containing the indicated anti-TfR Fabs (control, 3M12 VH3 / VK2, 3M12 VH4 / VK3, and 3A4 VH3 N54S / VK4) conjugated to DMPK-targeting oligonucleotides in reducing DMPK mRNA expression in mice expressing human TfR1 (hTfR1 knock-in mice). Figure 40A shows the experimental design (e.g., IV dose, dosing frequency). DMPK mRNA levels were measured in the tibialis anterior muscle (Figure 40B), gastrocnemius muscle (Figure 40C), heart (Figure 40D), and diaphragm (Figure 40E) of mice 14 days after the first dose. [Figure 40C] Figures 40A-40E show the in vivo activity of conjugates containing the indicated anti-TfR Fabs (control, 3M12 VH3 / VK2, 3M12 VH4 / VK3, and 3A4 VH3 N54S / VK4) conjugated to DMPK-targeting oligonucleotides in reducing DMPK mRNA expression in mice expressing human TfR1 (hTfR1 knock-in mice). Figure 40A shows the experimental design (e.g., IV dose, dosing frequency). DMPK mRNA levels were measured in the tibialis anterior muscle (Figure 40B), gastrocnemius muscle (Figure 40C), heart (Figure 40D), and diaphragm (Figure 40E) of mice 14 days after the first dose. [Figure 40D]Figures 40A-40E show the in vivo activity of conjugates containing the indicated anti-TfR Fabs (control, 3M12 VH3 / VK2, 3M12 VH4 / VK3, and 3A4 VH3 N54S / VK4) conjugated to DMPK-targeting oligonucleotides in reducing DMPK mRNA expression in mice expressing human TfR1 (hTfR1 knock-in mice). Figure 40A shows the experimental design (e.g., IV dose, dosing frequency). DMPK mRNA levels were measured in the tibialis anterior muscle (Figure 40B), gastrocnemius muscle (Figure 40C), heart (Figure 40D), and diaphragm (Figure 40E) of mice 14 days after the first dose. [Figure 40E] Figures 40A-40E show the in vivo activity of conjugates containing the indicated anti-TfR Fabs (control, 3M12 VH3 / VK2, 3M12 VH4 / VK3, and 3A4 VH3 N54S / VK4) conjugated to DMPK-targeting oligonucleotides in reducing DMPK mRNA expression in mice expressing human TfR1 (hTfR1 knock-in mice). Figure 40A shows the experimental design (e.g., IV dose, dosing frequency). DMPK mRNA levels were measured in the tibialis anterior muscle (Figure 40B), gastrocnemius muscle (Figure 40C), heart (Figure 40D), and diaphragm (Figure 40E) of mice 14 days after the first dose.

[0070] [Figure 41A] Figures 41A-41C show that a conjugate containing an anti-TfR antibody conjugated to a DMPK-targeting oligonucleotide corrected splicing and reduced foci in CM-DM1-32F primary cells expressing a DMPK mutant mRNA containing 380 CUG repeats. Figure 41A shows that the conjugate reduced mutant DMPK mRNA expression. [Figure 41B] Figures 41A-41C show that a conjugate containing an anti-TfR antibody conjugated to a DMPK-targeting oligonucleotide corrected splicing and reduced foci in CM-DM1-32F primary cells expressing a DMPK mutant mRNA containing 380 CUG repeats. Figure 41B shows that the conjugate corrected BIN1 exon 11 splicing. [Figure 41C] Figures 41A-41C show that a conjugate containing an anti-TfR antibody conjugated to a DMPK-targeting oligonucleotide corrected splicing and reduced foci in CM-DM1-32F primary cells expressing a DMPK mutant mRNA containing 380 CUG repeats. Figure 41C shows images of fluorescence in situ hybridization (FISH) analysis and quantification of the images, demonstrating that the conjugate reduced nuclear foci formed by mutant DMPK mRNA. In the microscopy image shown in the top panel of Figure 41C, bright round shapes indicate cell nuclei, and bright spots within the nuclei of DM1 cells (right three microscopy panels) indicate CUG foci.

[0071] [Figure 42] Figure 42 shows ELISA measurements of the binding of anti-TfR Fab 3M12 VH4 / Vk3 to recombinant human (circles), cynomolgus monkey (squares), mouse (upward triangles), or rat (downward triangles) TfR1 protein over a Fab concentration range of 230 pM to 500 nM. The results show that the anti-TfR Fab is reactive with human and cynomolgus monkey TfR1. No binding was observed to mouse or rat recombinant TfR1. Data are presented as relative fluorescence units normalized to baseline.

[0072] [Figure 43] Figure 43 shows the results of an ELISA testing the affinity of anti-TfR Fab 3M12 VH4 / Vk3 for recombinant human TfR1 or TfR2 over a Fab concentration range of 230 pM to 500 nM. Data are presented as relative fluorescence units normalized to baseline. The results demonstrate that the Fab does not bind to recombinant human TfR2.

[0073] [Figure 44]Figure 44 shows the serum stability of the linker used to connect anti-TfR Fab 3M12 VH4 / Vk3 to the control antisense oligonucleotide over 72 hours of incubation in PBS or in rat, mouse, cynomolgus monkey or human serum.

[0074] [Figure 45] Figure 45 shows that a conjugate containing anti-TfR Fab 3M12 VH4 / Vk3 conjugated to a DUX4-targeting oligonucleotide (SEQ ID NO: 147) inhibited the DUX4 transcriptome in C6 (AB1080)-immortalized FSHD1 cells, as indicated by reduced mRNA expression of MDB3L2, TRIM43, and ZSCAN4. The conjugate showed superior activity in inhibiting the DUX4 transcriptome compared to unconjugated DUX4-targeting oligonucleotide.

[0075] [Figure 46A] Figures 46A-46B show dose-response curves for gene knockdown. Figure 46A shows MBD3L2 knockdown in C6 (AB1080)-immortalized FSHD1 cells treated with a conjugate containing anti-TfR Fab 3M12 VH4 / Vk3 conjugated to a DUX4-targeting oligonucleotide (SEQ ID NO: 147). [Figure 46B] Figures 46A-46B show dose-response curves for gene knockdown. Figure 46B shows MBD3L2, TRIM43, and ZSCAN4 knockdown in FSHD patient myotubes treated with a conjugate containing anti-TfR Fab 3M12 VH4 / Vk3 conjugated to a DUX4-targeting oligonucleotide (SEQ ID NO: 147). Figure 46B includes the MBD3L2 data shown in Figure 46A.

[0076] [Figure 47A]Figures 47A-47C show EMG myotonia grades in the quadriceps (Figure 47A), gastrocnemius (Figure 47B), and tibialis anterior (Figure 47C) muscles of HSA-LR mice treated with vehicle, a single dose of unconjugated ASO, or a single dose of anti-TfR1 antibody-ASO conjugate (Ab-ASO). The anti-TfR1 antibody used was RI7 217 Fab, and the oligonucleotide targets human skeletal actin (ACTA1). [Figure 47B] Figures 47A-47C show EMG myotonia grades in the quadriceps (Figure 47A), gastrocnemius (Figure 47B), and tibialis anterior (Figure 47C) muscles of HSA-LR mice treated with vehicle, a single dose of unconjugated ASO, or a single dose of anti-TfR1 antibody-ASO conjugate (Ab-ASO). The anti-TfR1 antibody used was RI7 217 Fab, and the oligonucleotide targets human skeletal actin (ACTA1). [Figure 47C] Figures 47A-47C show EMG myotonia grades in the quadriceps (Figure 47A), gastrocnemius (Figure 47B), and tibialis anterior (Figure 47C) muscles of HSA-LR mice treated with vehicle, a single dose of unconjugated ASO, or a single dose of anti-TfR1 antibody-ASO conjugate (Ab-ASO). The anti-TfR1 antibody used was RI7 217 Fab, and the oligonucleotide targets human skeletal actin (ACTA1).

[0077] [Figure 48] Figure 48 shows human ACTA1 expression measured by qPCR in HSALR DM1 mice after a single dose of naked ASO or a dose-equivalent anti-TFR1 antibody-ASO conjugate (Ab-ASO) compared to vehicle-treated mice. The anti-TFR1 antibody used was RI7 217 Fab, and the oligonucleotide targets human skeletal actin (ACTA1).

[0078] [Figure 49A]Figures 49A-49C show ACTA1 expression in the quadriceps (Figure 49A), gastrocnemius (Figure 49B), and tibialis anterior (Figure 49C) muscles of HSALR DM1 mice after a single dose of 10 mg / kg naked ASO, 20 mg / kg naked ASO, or the equivalent dose of an anti-TFR antibody-ASO conjugate (Ab-ASO) compared to vehicle-treated mice. The anti-TFR1 antibody used was RI7 217 Fab, and the oligonucleotide targets human skeletal actin (ACTA1). (*p<0.05; ***p<0.001) [Figure 49B] Figures 49A-49C show ACTA1 expression in the quadriceps (Figure 49A), gastrocnemius (Figure 49B), and tibialis anterior (Figure 49C) muscles of HSALR DM1 mice after a single dose of 10 mg / kg naked ASO, 20 mg / kg naked ASO, or the equivalent dose of an anti-TFR antibody-ASO conjugate (Ab-ASO) compared to vehicle-treated mice. The anti-TFR1 antibody used was RI7 217 Fab, and the oligonucleotide targets human skeletal actin (ACTA1). (*p<0.05; ***p<0.001) [Figure 49C] Figures 49A-49C show ACTA1 expression in the quadriceps (Figure 49A), gastrocnemius (Figure 49B), and tibialis anterior (Figure 49C) muscles of HSALR DM1 mice after a single dose of 10 mg / kg naked ASO, 20 mg / kg naked ASO, or the equivalent dose of an anti-TFR antibody-ASO conjugate (Ab-ASO) compared to vehicle-treated mice. The anti-TFR1 antibody used was RI7 217 Fab, and the oligonucleotide targets human skeletal actin (ACTA1). (*p<0.05; ***p<0.001)

[0079] [Figure 50A]Figures 50A-50C show quantification of exon 23 skipping in the quadriceps (Figure 50A), heart (Figure 50B), and diaphragm (Figure 50C) of wild-type (WT) and mdx mice 2 or 4 weeks after administration of a single dose of saline, an unconjugated oligonucleotide (ASO) that induces exon 23 skipping in DMD, or a conjugate containing anti-TfR1 RI7217 Fab conjugated to an ASO (Ab-ASO). Little or no exon 23 skipping was observed in tissues from WT or mdx mice administered saline or unconjugated ASO, whereas significant levels of exon 23 skipping were observed in tissues from mdx mice treated with Ab-ASO. (*p<0.05, **p<0.01, ****p<0.0001) [Figure 50B] Figures 50A-50C show quantification of exon 23 skipping in the quadriceps (Figure 50A), heart (Figure 50B), and diaphragm (Figure 50C) of wild-type (WT) and mdx mice 2 or 4 weeks after administration of a single dose of saline, an unconjugated oligonucleotide (ASO) that induces exon 23 skipping in DMD, or a conjugate containing anti-TfR1 RI7217 Fab conjugated to an ASO (Ab-ASO). Little or no exon 23 skipping was observed in tissues from WT or mdx mice administered saline or unconjugated ASO, whereas significant levels of exon 23 skipping were observed in tissues from mdx mice treated with Ab-ASO. (*p<0.05, **p<0.01, ****p<0.0001) [Figure 50C]Figures 50A-50C show quantification of exon 23 skipping in the quadriceps (Figure 50A), heart (Figure 50B), and diaphragm (Figure 50C) of wild-type (WT) and mdx mice 2 or 4 weeks after administration of a single dose of saline, an unconjugated oligonucleotide (ASO) that induces exon 23 skipping in DMD, or a conjugate containing anti-TfR1 RI7217 Fab conjugated to an ASO (Ab-ASO). Little or no exon 23 skipping was observed in tissues from WT or mdx mice administered saline or unconjugated ASO, whereas significant levels of exon 23 skipping were observed in tissues from mdx mice treated with Ab-ASO. (*p<0.05, **p<0.01, ****p<0.0001)

[0080] [Figure 51A] Figures 51A-51D show measurements of dystrophin protein in the quadriceps muscle of mdx mice after a single dose of unconjugated oligonucleotides (ASOs) or a conjugate containing anti-TfR1 RI7217 Fab conjugated to an ASO (Ab-ASO), which induces exon 23 skipping in DMD. Figure 51A shows Western blots of dystrophin and alpha-actinin protein in muscle tissue two weeks after injection of ASOs or Ab-ASOs. The standard curves in Figures 51A and 51C were generated by pooling tissue from wild-type (WT) and mdx mouse samples, and percent WT indicates the amount of WT protein spiked into each sample. (*p<0.05; ns, not significant). [Figure 51B] Figures 51A-51D show measurements of dystrophin protein in the quadriceps muscle of mdx mice after a single dose of unconjugated oligonucleotide (ASO) or a conjugate containing anti-TfR1 RI7217 Fab conjugated to an ASO (Ab-ASO) that induces exon 23 skipping in DMD. Figure 51B shows quantification of dystrophin in the Western blot of Figure 51A compared to dystrophin protein in wild-type muscle. [Figure 51C]Figures 51A-51D show measurements of dystrophin protein in the quadriceps muscle of mdx mice after a single dose of unconjugated oligonucleotides (ASOs) or a conjugate containing anti-TfR1 RI7217 Fab conjugated to an ASO (Ab-ASO), which induces exon 23 skipping in DMD. Figure 51C shows Western blots of dystrophin and alpha-actinin protein in muscle tissue 4 weeks after injection of ASOs or Ab-ASOs. The standard curves in Figures 51A and 51C were generated by pooling tissue from wild-type (WT) and mdx mouse samples, and percent WT indicates the amount of WT protein spiked into each sample. (*p<0.05; ns, not significant). [Figure 51D] Figures 51A-51D show measurements of dystrophin protein in the quadriceps muscle of mdx mice after a single dose of unconjugated oligonucleotide (ASO) or a conjugate containing anti-TfR1 RI7217 Fab conjugated to an ASO (Ab-ASO) that induces exon 23 skipping in DMD. Figure 51D shows quantification of dystrophin in the Western blot of Figure 51C compared to dystrophin protein in wild-type muscle.

[0081] [Figure 52A] Figures 52A-52D show measurements of dystrophin protein in cardiac muscle of mdx mice after a single dose of unconjugated oligonucleotides (ASOs) or a conjugate containing anti-TfR1 RI7217 Fab conjugated to an ASO (Ab-ASO), which induces exon 23 skipping in DMD. Figure 52A shows Western blots of dystrophin and alpha-actinin protein in muscle tissue two weeks after injection of ASOs or Ab-ASOs. The standard curves in Figures 52A and 52C were generated by pooling tissue from wild-type (WT) and mdx mouse samples, and percent WT indicates the amount of WT protein spiked into each sample. (*p<0.05; ****p<0.0001) [Figure 52B]Figures 52A-52D show measurements of dystrophin protein in cardiac muscle of mdx mice after a single dose of unconjugated oligonucleotide (ASO) or a conjugate containing anti-TfR1 RI7217 Fab conjugated to an ASO (Ab-ASO) that induces exon 23 skipping in DMD. Figure 52B shows quantification of dystrophin in the Western blot of Figure 52A compared to dystrophin protein in wild-type muscle. [Figure 52C] Figures 52A-52D show measurements of dystrophin protein in cardiac muscle of mdx mice after a single dose of unconjugated oligonucleotides (ASOs) or a conjugate containing anti-TfR1 RI7217 Fab conjugated to an ASO (Ab-ASO), which induces exon 23 skipping in DMD. Figure 52C shows Western blots of dystrophin and alpha-actinin protein in muscle tissue 4 weeks after injection of ASOs or Ab-ASOs. The standard curves in Figures 52A and 52C were generated by pooling tissue from wild-type (WT) and mdx mouse samples, and percent WT indicates the amount of WT protein spiked into each sample. (*p<0.05; ****p<0.0001) [Figure 52D] Figures 52A-52D show measurements of dystrophin protein in cardiac muscle of mdx mice after a single dose of unconjugated oligonucleotide (ASO) or a conjugate containing anti-TfR1 RI7217 Fab conjugated to an ASO (Ab-ASO) that induces exon 23 skipping in DMD. Figure 52D shows quantification of dystrophin in the Western blot of Figure 52C compared to dystrophin protein in wild-type muscle.

[0082] [Figure 53A]Figures 53A-53D show measurements of dystrophin protein in the diaphragm muscle of mdx mice after a single dose of unconjugated oligonucleotides (ASOs) or a conjugate containing anti-TfR1 RI7217 Fab conjugated to an ASO (Ab-ASO), which induces exon 23 skipping in DMD. Figure 53A shows Western blots of dystrophin and alpha-actinin protein in muscle tissue two weeks after injection of ASOs or Ab-ASOs. The standard curves in Figures 53A and 53C were generated by pooling tissue from wild-type (WT) and mdx mouse samples, and percent WT indicates the amount of WT protein spiked into each sample. (**p<0.01; ***p<0.001) [Figure 53B] Figures 53A-53D show measurements of dystrophin protein in the diaphragm muscle of mdx mice after a single dose of unconjugated oligonucleotide (ASO) or a conjugate containing anti-TfR1 RI7217 Fab conjugated to an ASO (Ab-ASO) that induces exon 23 skipping in DMD. Figure 53B shows quantification of dystrophin in the Western blot of Figure 53A compared to dystrophin protein in wild-type muscle. [Figure 53C] Figures 53A-53D show measurements of dystrophin protein in the diaphragm muscle of mdx mice after a single dose of unconjugated oligonucleotides (ASOs) or a conjugate containing anti-TfR1 RI7217 Fab conjugated to an ASO (Ab-ASO), which induces exon 23 skipping in DMD. Figure 53C shows Western blots of dystrophin and alpha-actinin protein in muscle tissue 4 weeks after injection of ASOs or Ab-ASOs. The standard curves in Figures 53A and 53C were generated by pooling tissue from wild-type (WT) and mdx mouse samples, and percent WT indicates the amount of WT protein spiked into each sample. (**p<0.01; ***p<0.001) [Figure 53D]Figures 53A-53D show measurements of dystrophin protein in the diaphragm muscle of mdx mice after a single dose of unconjugated oligonucleotide (ASO) or a conjugate containing anti-TfR1 RI7217 Fab conjugated to an ASO (Ab-ASO) that induces exon 23 skipping in DMD. Figure 53D shows quantification of dystrophin in the Western blot of Figure 53C compared to dystrophin protein in wild-type muscle.

[0083] [Figure 54A] Figures 54A-54C show quantification of the amount of oligonucleotide (ASO) administered to the quadriceps (Figure 54A), diaphragm (Figure 54B), and heart (Figure 54C) of wild-type (WT) or mdx mice 2 or 4 weeks after administration of a single dose of saline, unconjugated exon 23 skipping oligonucleotide (ASO), or a conjugate containing anti-TfR1 RI7217 Fab conjugated to an ASO (Ab-ASO). [Figure 54B] Figures 54A-54C show quantification of the amount of oligonucleotide (ASO) administered to the quadriceps (Figure 54A), diaphragm (Figure 54B), and heart (Figure 54C) of wild-type (WT) or mdx mice 2 or 4 weeks after administration of a single dose of saline, unconjugated exon 23 skipping oligonucleotide (ASO), or a conjugate containing anti-TfR1 RI7217 Fab conjugated to an ASO (Ab-ASO). [Figure 54C] Figures 54A-54C show quantification of the amount of oligonucleotide (ASO) administered to the quadriceps (Figure 54A), diaphragm (Figure 54B), and heart (Figure 54C) of wild-type (WT) or mdx mice 2 or 4 weeks after administration of a single dose of saline, unconjugated exon 23 skipping oligonucleotide (ASO), or a conjugate containing anti-TfR1 RI7217 Fab conjugated to an ASO (Ab-ASO).

[0084] [Figure 55]Figure 55 shows non-human primate plasma levels of DUX4-targeted oligonucleotide (SEQ ID NO: 147) over time following administration of 30 mg / kg unconjugated ("naked") oligonucleotide or 3, 10, or 30 mg / kg oligonucleotide equivalents of a conjugate comprising anti-TfR1 Fab 3M12 VH4 / Vk3 covalently linked to a DUX4-targeted oligonucleotide ("Fab-oligonucleotide conjugate").

[0085] [Figure 56] Figure 56 shows tissue levels of DUX4-targeted oligonucleotide (SEQ ID NO: 147) measured in non-human primate muscle tissue samples two weeks after administration of 30 mg / kg unconjugated ("naked") oligonucleotide or 3, 10, or 30 mg / kg oligonucleotide equivalents of a conjugate comprising anti-TfR1 Fab 3M12 VH4 / Vk3 covalently linked to a DUX4-targeted oligonucleotide ("Fab-oligonucleotide conjugate").

[0086] [Figure 57] Figure 57 shows tissue levels of DUX4-targeted oligonucleotide (SEQ ID NO: 147) measured in non-human primate muscle tissue samples collected by biopsy (left five bars) one week after administration of 30 mg / kg unconjugated oligonucleotide ("oligo") or 3, 10, or 30 mg / kg oligonucleotide equivalents of a conjugate comprising anti-TfR1 Fab 3M12 VH4 / Vk3 covalently linked to a DUX4-targeted oligonucleotide ("conjugate"), or by autopsy (right five bars) two weeks after administration.

[0087] [Figure 58A-1]Figures 58A-58B show splicing corrections in over 30 different RNAs known to be misspliced ​​in DM1 patients, measured in the tibialis anterior (Figure 58A) or quadriceps (Figure 58B) muscles of HSA-LR mice treated with a single dose of anti-TfR1 antibody-oligonucleotide (Ab-ASO) conjugate or saline. The anti-TfR1 antibody used was RI7 217 Fab, and the oligonucleotide targets skeletal actin (ACTA1). [Figure 58A-2] Figures 58A-58B show splicing corrections in over 30 different RNAs known to be misspliced ​​in DM1 patients, measured in the tibialis anterior (Figure 58A) or quadriceps (Figure 58B) muscles of HSA-LR mice treated with a single dose of anti-TfR1 antibody-oligonucleotide (Ab-ASO) conjugate or saline. The anti-TfR1 antibody used was RI7 217 Fab, and the oligonucleotide targets skeletal actin (ACTA1). [Figure 58A-3] Figures 58A-58B show splicing corrections in over 30 different RNAs known to be misspliced ​​in DM1 patients, measured in the tibialis anterior (Figure 58A) or quadriceps (Figure 58B) muscles of HSA-LR mice treated with a single dose of anti-TfR1 antibody-oligonucleotide (Ab-ASO) conjugate or saline. The anti-TfR1 antibody used was RI7 217 Fab, and the oligonucleotide targets skeletal actin (ACTA1).

[0088] [Figure 58B-1] Figures 58A-58B show splicing corrections in over 30 different RNAs known to be misspliced ​​in DM1 patients, measured in the tibialis anterior (Figure 58A) or quadriceps (Figure 58B) muscles of HSA-LR mice treated with a single dose of anti-TfR1 antibody-oligonucleotide (Ab-ASO) conjugate or saline. The anti-TfR1 antibody used was RI7 217 Fab, and the oligonucleotide targets skeletal actin (ACTA1). [Figure 58B-2] Figures 58A-58B show splicing corrections in over 30 different RNAs known to be misspliced ​​in DM1 patients, measured in the tibialis anterior (Figure 58A) or quadriceps (Figure 58B) muscles of HSA-LR mice treated with a single dose of anti-TfR1 antibody-oligonucleotide (Ab-ASO) conjugate or saline. The anti-TfR1 antibody used was RI7 217 Fab, and the oligonucleotide targets skeletal actin (ACTA1). [Figure 58B-3] Figures 58A-58B show splicing corrections in over 30 different RNAs known to be misspliced ​​in DM1 patients, measured in the tibialis anterior (Figure 58A) or quadriceps (Figure 58B) muscles of HSA-LR mice treated with a single dose of anti-TfR1 antibody-oligonucleotide (Ab-ASO) conjugate or saline. The anti-TfR1 antibody used was RI7 217 Fab, and the oligonucleotide targets skeletal actin (ACTA1).

[0089] [Figure 59] Figure 59 shows the % exon 53 skipping in DMD patient cells harboring a DMD exon 52 deletion after gymnotic incorporation of exon 53 skipping oligonucleotides over a range of concentrations.

[0090] [Figure 60] Figure 60 shows the % exon 53 skipping in DMD patient cells harboring a deletion of DMD exon 52 after treatment with various concentrations of exon 53 skipping PMOs either unbound to antibody ("naked ASO") or covalently linked to anti-TfR1 Fab ("anti-TfR1 Fab-ASO complex"). DETAILED DESCRIPTION OF THE INVENTION

[0091] Aspects of the present disclosure relate to the recognition that while certain molecular payloads (e.g., oligonucleotides, peptides, small molecules) can have beneficial effects on muscle cells, effectively targeting such cells can be challenging. As described herein, the present disclosure provides conjugates comprising a muscle targeting agent covalently linked to a molecular payload to overcome such challenges. In some embodiments, the conjugates are particularly useful for delivering molecular payloads that modulate target gene expression or activity in muscle cells, e.g., in subjects with or suspected of having a muscle disease. For example, in some embodiments, the conjugates are useful for treating subjects with rare muscle diseases, including Pompe disease, centronuclear myopathy, fibrodysplasia ossificans progressiva, Friedreich's ataxia, or Duchenne muscular dystrophy. In some embodiments, different molecular payloads may be used in such conjugates depending on the disease being treated. For example, if the underlying mutation confers a splicing defect, an oligonucleotide or other payload may be used to correct the splicing defect (e.g., an oligonucleotide that inhibits exon skipping or promotes alternative splicing). If the underlying mutation results in a gain-of-function allele, oligonucleotides (e.g., RNAi, PMO, ASO-gapmer) may be used to inhibit the expression or activity of the allele. In some embodiments, for example, if the mutation results in a loss-of-function allele, the payload may include, for example, an expression construct to express a wild-type version of the allele. In some embodiments, the payload may include machinery (e.g., a guide nucleic acid, an expression construct encoding a gene editing enzyme) to correct the underlying defect, for example, by gene editing.

[0092] Further aspects of the disclosure, including a description of defined terms, are provided below.

[0093] I. Definition Administering: As used herein, the term "administering" or "administration" means providing a conjugate to a subject in a physiologically and / or pharmacologically useful manner (e.g., treating a disease in a subject).

[0094] 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 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 a workable value).

[0095] 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., 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, 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, for example, 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).

[0096] 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.

[0097] 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 6. Table 6. CDR definition [Table 1]

[0098] 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.

[0099] 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.

[0100] 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.

[0101] 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.

[0102] 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.

[0103] 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.

[0104] Disease allele: As used herein, the term "disease allele" refers to any one of alternative forms (e.g., mutant forms) of a gene in which the allele correlates with and / or (e.g., and) directly or indirectly contributes to or causes a disease. A disease allele may contain genetic alterations compared to a wild-type (non-disease) allele, including, but not limited to, insertions (e.g., disease-associated repeats described below), deletions, missense mutations, nonsense mutations, and splice site mutations. In some embodiments, a disease allele has a loss-of-function mutation. In some embodiments, a disease allele has a gain-of-function mutation. In some embodiments, a disease allele encodes an activating mutation (e.g., encodes a constitutively active protein). In some embodiments, a disease allele is a recessive allele with a recessive phenotype. In some embodiments, a disease allele is a dominant allele with a dominant phenotype.

[0105] Disease-associated repeat: As used herein, the term "disease-associated repeat" refers to a repetitive nucleotide sequence at a genomic location where several units of the repetitive nucleotide sequence correlate with and / or (for example, and) directly or indirectly contribute to or cause a genetic disease. Each repeat unit of the disease-associated repeat may be 2, 3, 4, 5, or more nucleotides in length. For example, in some embodiments, the disease-associated repeat is a dinucleotide repeat. In some embodiments, the disease-associated repeat is a trinucleotide repeat. In some embodiments, the disease-associated repeat is a tetranucleotide repeat. In some embodiments, the disease-associated repeat is a pentanucleotide repeat. In some embodiments, the disease-associated repeat comprises a CAG repeat, a CTG repeat, a CUG repeat, a CGG repeat, a CCTG repeat, or the nucleotide complement of any thereof. In some embodiments, the disease-associated repeat is in a non-coding portion of a gene. However, in some embodiments, the disease-associated repeat is in a coding region of a gene. In some embodiments, the disease-associated repeat is expanded from the normal state to a length that directly or indirectly contributes to or causes a genetic disease. In some embodiments, the disease-associated repeats are in RNA (e.g., RNA transcripts). In some embodiments, the disease-associated repeats are in DNA (e.g., chromosomes, plasmids). In some embodiments, the disease-associated repeats are expanded in chromosomes of germline cells. In some embodiments, the disease-associated repeats are expanded in chromosomes of somatic cells. In some embodiments, the disease-associated repeats are expanded into multiple repeat units associated with congenital onset. In some embodiments, the disease-associated repeats are expanded into multiple repeat units associated with childhood onset of the disease. In some embodiments, the disease-associated repeats are expanded into multiple repeat units associated with adult onset of the disease.

[0106] 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.

[0107] 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.

[0108] 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).

[0109] 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.

[0110] 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.

[0111] 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.

[0112] 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 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.

[0113] Muscle disease gene: As used herein, the term "muscle disease gene" refers to a gene having at least one disease allele that correlates with and / or directly or indirectly contributes to or causes a muscle disease. In some embodiments, the muscle disease is a rare disease, e.g., as defined by the Genetic and Rare Diseases Information Center (GARD), a program of the National Center for Advancing Translational Sciences (NCATS). In some embodiments, the muscle disease is a rare disease characterized as affecting fewer than 200,000 people. In some embodiments, the muscle disease is a monogenic disease. In some embodiments, the muscle disease gene is a gene listed in Table 1.

[0114] 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.

[0115] 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.

[0116] 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, phosphorodiamidates, 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.

[0117] 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, e.g., an antibody expressed using a recombinant expression vector transfected into a host cell (as described in more detail in this disclosure), an antibody 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), or an antibody 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 generated using techniques well known in the art, such as, 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.).

[0118] 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.

[0119] 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 M or less K D In some embodiments, the antibody specifically binds to a transferrin receptor, e.g., an epitope in the apical domain of the transferrin receptor.

[0120] 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, e.g., a human patient, having or suspected of having a disease. In some embodiments, the subject is a human patient having or suspected of having a muscle disease (e.g., any of the diseases provided in Table 1).

[0121] 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).

[0122] 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 nucleotides, and oligonucleotides comprising the 2'-modified nucleotides have increased affinity for target sequences compared to unmodified oligonucleotides. Examples of structures of 2'-modified nucleosides are provided below: [ka]

[0123] II. Complexes Provided herein are conjugates comprising a targeting agent, e.g., 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 antigenic site, or an antibody that binds to at least two antigenic sites, which may be on the same antigen or different antigens. The conjugate may be used to modulate the activity or function of at least one gene, protein, and / or (e.g., and) nucleic acid. In some embodiments, the molecular payload present with the conjugate is responsible for modulating the gene, protein, and / or (e.g., 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 (e.g., and) nucleic acid in a cell. In some embodiments, the molecular payload is an oligonucleotide that targets a muscle disease allele in a muscle cell.

[0124] 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 that targets a muscle disease allele).

[0125] In some embodiments, the conjugates are useful for treating muscle diseases in which the molecular payload affects the activity of a corresponding gene provided in Table 1. For example, depending on the disease, the molecular payload may modulate (e.g., decrease, increase) the transcription or expression of the gene, modulate the expression of a protein encoded by the gene, or modulate the activity of the encoded protein. In some embodiments, the molecular payload is an oligonucleotide comprising a strand having a region of complementarity to a target gene provided in Table 1.

[0126] Table 1 - List of muscle diseases and corresponding genes. [Table 2-1] [Table 2-2] [Table 2-3]

[0127] 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.

[0128] 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.

[0129] 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.

[0130] 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.

[0131] 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.

[0132] Muscle cells encompassed by this disclosure include, but are not limited to, skeletal muscle cells, smooth muscle cells, cardiomyocytes, myoblasts, and myocytes.

[0133] 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 bind myosin IIb," Mol Immunol. 2003 Mar,39(13):78309, the entire contents of each of which are incorporated herein by reference.

[0134] 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 are internalized into muscle cells along with any attached molecular payload. As used herein, antibodies that bind to the transferrin receptor may be referred to interchangeably as transferrin receptor antibodies, anti-transferrin receptor antibodies, or anti-TfR 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.

[0135] 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 Dec. 4, 1979, entitled "Monoclonal antibody to a human early thymocyte antigen and methods for preparing same"; U.S. Pat. No. 8,409,573, filed Jun. 14, 2006, entitled "Anti-CD71 monoclonal antibodies and uses thereof for treating malignant tumor cells"; U.S. Pat. No. 9,708,406, filed May 20, 2014, entitled "Anti-transferrin receptor antibodies and methods of use"; U.S. Pat. No. 9,611,323, filed Dec. 19, 2014, entitled "Low affinity blood brain barrier receptor antibodies and uses therefor"; WO 2015 / 098989, filed Dec. 24, 2014, entitled "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 (See Pharmacol. Exp. Ther., 292:1048-1052).

[0136] Provided herein are, in some aspects, novel anti-TfR antibodies for use as muscle-targeting agents (e.g., in muscle-targeting complexes). In some embodiments, the anti-TfR antibodies described herein bind to the transferrin receptor with high specificity and affinity. In some embodiments, the anti-TfR 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-TfR antibodies provided herein specifically bind to the transferrin receptor from humans, non-human primates, mice, rats, etc. In some embodiments, the anti-TfR antibodies provided herein bind to the human transferrin receptor. In some embodiments, the anti-TfR 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 anti-TfR 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 NOS: 105, which is not in the apical domain of the transferrin receptor.

[0137] 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)

[0138] 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)

[0139] 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)

[0140] 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)

[0141] 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.

[0142] 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).

[0143] 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."

[0144] In some embodiments, an anti-TfR antibody of the present disclosure comprises a VL domain and / or (by way of example and) a VH domain of any one of the anti-TfR 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 (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or of any subclass (e.g., 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.

[0145] In some embodiments, agents that bind to the transferrin receptor, e.g., anti-TfR 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.

[0146] 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-TfR 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-TfR antibodies provided herein specifically bind to transferrin receptor from humans, non-human primates, mice, rats, etc. In some embodiments, the humanized anti-TfR antibodies provided herein bind to the human transferrin receptor. In some embodiments, the humanized anti-TfR 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-TfR 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-TfR antibodies described herein bind to TfR1 but not TfR2.

[0147] In some embodiments, the anti-TFR 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-TfR 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-TfR antibodies described herein bind to TfR1 with a Kd in the sub-nanomolar range. In some embodiments, the anti-TfR antibodies described herein selectively bind to transferrin receptor 1 (TfR1) but not to transferrin receptor 2 (TfR2). In some embodiments, the anti-TfR 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-TfR antibodies do not bind to mouse TfR1 (by virtue of a smaller Kd, M, or smaller). The affinity and binding kinetics of the anti-TfR 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-TfR antibodies described herein does not compete with or inhibit transferrin binding to TfR1. In some embodiments, the binding of any one of the anti-TfR antibodies described herein does not compete with or inhibit HFE-beta2-microglobulin binding to TfR1.

[0148] The anti-TfR antibodies described herein are humanized antibodies. The CDR and variable region amino acid sequences of the murine monoclonal anti-TfR antibodies from which the humanized anti-TfR antibodies described herein are derived are provided in Table 2. Table 2. Mouse monoclonal anti-TfR antibodies [Table 3-1] [Table 3-2] [Table 3-3]

[0149] In some embodiments, an anti-TfR antibody of the disclosure is a humanized variant of any one of the anti-TfR antibodies provided in Table 2. In some embodiments, an anti-TfR 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-TfR 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.

[0150] 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.

[0151] Humanized antibodies and methods for making them are known, see, e.g., 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); 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.

[0152] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises 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).

[0153] In some embodiments, a humanized anti-TfR 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), a humanized anti-TfR antibody of the present disclosure includes a humanized VL 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 VL of any one of the anti-TfR antibodies listed in Table 2 (e.g., any one of SEQ ID NOs: 18, 44, and 62).

[0154] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising an amino acid sequence that is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regions to the VH of any of the anti-TfR antibodies listed in Table 2 (e.g., any one of SEQ ID NOs: 17, 22, 26, 43, 61, 65, and 68). Alternatively or additionally (e.g., additionally), in some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VL comprising an amino acid sequence that is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regions to the VL of any of the anti-TfR antibodies listed in Table 2 (e.g., any one of SEQ ID NOs: 18, 44, and 62).

[0155] In some embodiments, a humanized anti-TfR antibody of the 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 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), the anti-TfR antibodies of the present disclosure include 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 humanized VL containing no more than 25 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: 18.

[0156] In some embodiments, a humanized anti-TfR antibody of the disclosure comprises a humanized VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 1 (according to the IMGT definition system), a CDR-H2 having the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 19, or SEQ ID NO: 23 (according to the IMGT definition system), and a CDR-H3 having the amino acid sequence of SEQ ID NO: 3 (according to the IMGT definition system), and which 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), a humanized anti-TfR antibody of the present disclosure comprises a humanized VL comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 4 (according to the IMGT definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 5 (according to the IMGT definition system), and a CDR-L3 having the amino acid sequence of SEQ ID NO: 6 (according to the IMGT definition system), and is at least 75% (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.

[0157] In some embodiments, a humanized anti-TfR 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 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), a humanized anti-TfR antibody of the present disclosure comprises 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 includes a humanized VL containing no more than 25 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: 18.

[0158] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO:7 (according to the Kabat definition system), a CDR-H2 having the amino acid sequence of SEQ ID NO:8, SEQ ID NO:20, or SEQ ID NO:24 (according to the Kabat definition system), and a CDR-H3 having the amino acid sequence of SEQ ID NO:9 (according to the Kabat definition system), and which 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 humanized anti-TfR antibody of the present disclosure comprises a humanized VL comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 10 (according to the Kabat definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 11 (according to the Kabat definition system), and a CDR-L3 having the amino acid sequence of SEQ ID NO: 6 (according to the Kabat definition system), and is at least 75% (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.

[0159] In some embodiments, a humanized anti-TfR 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 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), a humanized anti-TfR 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 includes a humanized VL containing no more than 25 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: 18.

[0160] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 12 (according to the Chothia definition system), a CDR-H2 having the amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 21, or SEQ ID NO: 25 (according to the Chothia definition system), and a CDR-H3 having the amino acid sequence of SEQ ID NO: 14 (according to the Chothia definition system), and which 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-TfR antibody of the present disclosure comprises a humanized VL comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 15 (according to the Chothia-defined system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 5 (according to the Chothia-defined system), and a CDR-L3 having the amino acid sequence of SEQ ID NO: 16 (according to the Chothia-defined system), 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.

[0161] In some embodiments, a humanized anti-TfR antibody of the disclosure comprises a humanized VH that 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 that contains no more than 25 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), a humanized anti-TfR 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 humanized VL containing no more than 25 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.

[0162] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 27 (according to the IMGT definition system), a CDR-H2 having the amino acid sequence of SEQ ID NO: 28 (according to the IMGT definition system), 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 humanized anti-TfR antibody of the present disclosure comprises a humanized VL comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 30 (according to the IMGT definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 31 (according to the IMGT definition system), and a CDR-L3 having the amino acid sequence of SEQ ID NO: 32 (according to the IMGT definition system), and is at least 75% (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.

[0163] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH that includes 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 that contains no more than 25 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), the humanized anti-TfR antibodies of the present disclosure include 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 humanized VL containing no more than 25 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.

[0164] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 33 (according to the 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 humanized anti-TfR antibody of the present disclosure comprises a humanized 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, which is at least 75% (for example, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regions to the VL as set forth in SEQ ID NO: 44.

[0165] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH that 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 that contains no more than 25 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), a humanized anti-TfR 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 includes a humanized VL containing no more than 25 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.

[0166] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 38 (according to the Chothia definition system), a CDR-H2 having the amino acid sequence of SEQ ID NO: 39 (according to the Chothia definition system), and a CDR-H3 having the amino acid sequence of SEQ ID NO: 40 (according to the Chothia definition system), which 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), a humanized anti-TfR antibody of the present disclosure comprises a humanized VL comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 41 (according to the Chothia-defined system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 31 (according to the Chothia-defined system), and a CDR-L3 having the amino acid sequence of SEQ ID NO: 42 (according to the Chothia-defined system), which 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.

[0167] In some embodiments, a humanized anti-TfR antibody of the 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 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:61, SEQ ID NO:65, or SEQ ID NO:68. Alternatively or additionally (for example, additionally), a humanized anti-TfR 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 includes a humanized VL containing no more than 25 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.

[0168] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO:45, SEQ ID NO:63, or SEQ ID NO:66 (according to the IMGT definition system), a CDR-H2 having the amino acid sequence of SEQ ID NO:46 (according to the IMGT definition system), and a CDR-H3 having the amino acid sequence of SEQ ID NO:47 (according to the IMGT definition system), and which 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), a humanized anti-TfR antibody of the present disclosure comprises a humanized VL comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 48 (according to the IMGT definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 49 (according to the IMGT definition system), and a CDR-L3 having the amino acid sequence of SEQ ID NO: 50 (according to the IMGT definition system), and which 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.

[0169] In some embodiments, a humanized anti-TfR 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 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:61, SEQ ID NO:65, or SEQ ID NO:68. Alternatively or additionally (for example, additionally), the humanized anti-TfR antibodies of the present disclosure include a humanized VL that comprises 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 that contains no more than 25 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.

[0170] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO:51, SEQ ID NO:64, or SEQ ID NO:67 (according to the 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 which 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 humanized anti-TfR antibody of the present disclosure comprises a humanized VL comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 54 (according to the 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.

[0171] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH that 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 that contains no more than 25 amino acid variations in the framework regions (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: 61, SEQ ID NO: 65, or SEQ ID NO: 68. Alternatively or additionally (for example, additionally), a humanized anti-TfR 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 includes a humanized VL containing no more than 25 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.

[0172] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 56 (according to the Chothia definition system), a CDR-H2 having the amino acid sequence of SEQ ID NO: 57 (according to the Chothia definition system), 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), a humanized anti-TfR antibody of the present disclosure comprises a humanized VL comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 59 (according to the Chothia definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 49 (according to the Chothia definition system), and a CDR-L3 having the amino acid sequence of SEQ ID NO: 60 (according to the Chothia definition system), which 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.

[0173] Examples of amino acid sequences of humanized anti-TfR antibodies described herein are provided in Table 3. Table 3. Variable regions of humanized anti-TfR antibodies [Table 4-1] [Table 4-2] [Table 4-3]

[0174] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising CDR-H1, CDR-H2, and CDR-H3 of any one of the anti-TfR antibodies provided in Table 2, and comprises one or more (e.g., other than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid variations compared to the respective humanized VH provided in Table 3. Alternatively or additionally (e.g., additionally), a humanized anti-TfR antibody of the present disclosure comprises a humanized VL comprising CDR-L1, CDR-L2, and CDR-L3 of any one of the anti-TfR antibodies provided in Table 2, and comprises one or more (e.g., other than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid variations compared to the respective humanized VL provided in Table 3.

[0175] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized 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 humanized VL comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 70. In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising the amino acid sequence of SEQ ID NO: 69 and a humanized VL comprising the amino acid sequence of SEQ ID NO: 70.

[0176] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized 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 humanized 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, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising the amino acid sequence of SEQ ID NO: 71 and a humanized VL comprising the amino acid sequence of SEQ ID NO: 70.

[0177] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized 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 humanized VL comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 70. In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising the amino acid sequence of SEQ ID NO: 72 and a humanized VL comprising the amino acid sequence of SEQ ID NO: 70.

[0178] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized 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 humanized 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, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising the amino acid sequence of SEQ ID NO: 73 and a humanized VL comprising the amino acid sequence of SEQ ID NO: 74.

[0179] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized 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 humanized VL comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 75. In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising the amino acid sequence of SEQ ID NO: 73 and a humanized VL comprising the amino acid sequence of SEQ ID NO: 75.

[0180] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized 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 humanized 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, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising the amino acid sequence of SEQ ID NO: 76 and a humanized VL comprising the amino acid sequence of SEQ ID NO: 74.

[0181] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized 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 humanized VL comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 75. In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising the amino acid sequence of SEQ ID NO: 76 and a humanized VL comprising the amino acid sequence of SEQ ID NO: 75.

[0182] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized 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 humanized 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, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising the amino acid sequence of SEQ ID NO: 77 and a humanized VL comprising the amino acid sequence of SEQ ID NO: 78.

[0183] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized 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 humanized 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, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising the amino acid sequence of SEQ ID NO: 79 and a humanized VL comprising the amino acid sequence of SEQ ID NO: 80.

[0184] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized 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 humanized 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, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising the amino acid sequence of SEQ ID NO: 77 and a humanized VL comprising the amino acid sequence of SEQ ID NO: 80.

[0185] In some embodiments, the humanized anti-TfR 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-TfR 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 a 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)

[0186] In some embodiments, the heavy chain of any of the anti-TfR 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]

[0187] In some embodiments, the light chain of any of the anti-TfR 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)

[0188] 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.

[0189] In some embodiments, the humanized anti-TfR 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 humanized anti-TfR 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 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 SEQ ID NO: 81 or SEQ ID NO: 82. In some embodiments, the humanized anti-TfR antibodies described herein comprise a heavy chain comprising any one of the VHs listed in Table 3 or any variant thereof, and a heavy chain constant region set forth in SEQ ID NO: 81. In some embodiments, the humanized anti-TfR antibodies described herein comprise a heavy chain comprising any one of the VHs listed in Table 3 or any variant thereof, and a heavy chain constant region set forth in SEQ ID NO: 82.

[0190] In some embodiments, the humanized anti-TfR antibodies described herein comprise a light chain comprising any one of the VLs listed in Table 3 or any variant thereof, and a light chain constant region that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 83. In some embodiments, the humanized anti-TfR antibodies described herein comprise a light chain comprising any one of the VLs listed in Table 3 or any variant thereof, 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 humanized anti-TfR antibodies described herein comprise a light chain comprising any one of the VLs listed in Table 3 or any variant thereof, and a light chain constant region set forth in SEQ ID NO: 83.

[0191] Examples of IgG heavy and light chain amino acid sequences of the described anti-TfR antibodies are provided in Table 4 below. Table 4. Heavy and light chain sequences of examples of humanized anti-TfR IgG [Table 5-1] [Table 5-2] [Table 5-3]

[0192] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a heavy chain containing no more than 25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) 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), a humanized anti-TfR antibody of the present disclosure comprises 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.

[0193] In some embodiments, the humanized anti-TfR 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 humanized anti-TfR 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-TfR 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-TfR 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.

[0194] In some embodiments, a humanized anti-TfR 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, a humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 84 and a light chain comprising the amino acid sequence of SEQ ID NO: 85.

[0195] In some embodiments, a humanized anti-TfR 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, a humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 86 and a light chain comprising the amino acid sequence of SEQ ID NO: 85.

[0196] In some embodiments, a humanized anti-TfR 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, a humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 87 and a light chain comprising the amino acid sequence of SEQ ID NO: 85.

[0197] In some embodiments, a humanized anti-TfR 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, a humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 88 and a light chain comprising the amino acid sequence of SEQ ID NO: 89.

[0198] In some embodiments, a humanized anti-TfR 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, a humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 88 and a light chain comprising the amino acid sequence of SEQ ID NO: 90.

[0199] In some embodiments, a humanized anti-TfR 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, a humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 91 and a light chain comprising the amino acid sequence of SEQ ID NO: 89.

[0200] In some embodiments, a humanized anti-TfR 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, a humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 91 and a light chain comprising the amino acid sequence of SEQ ID NO: 90.

[0201] In some embodiments, a humanized anti-TfR 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, a humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 92 and a light chain comprising the amino acid sequence of SEQ ID NO: 93.

[0202] In some embodiments, a humanized anti-TfR 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, a humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 94 and a light chain comprising the amino acid sequence of SEQ ID NO: 95.

[0203] In some embodiments, a humanized anti-TfR 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, a humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 92 and a light chain comprising the amino acid sequence of SEQ ID NO: 95.

[0204] In some embodiments, the anti-TfR 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 using 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).

[0205] In some embodiments, the humanized anti-TfR 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 humanized anti-TfR 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 humanized anti-TfR 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.

[0206] In some embodiments, the humanized anti-TfR antibodies described herein comprise a light chain comprising any one of the VLs listed in Table 3 or any variant thereof, and a light chain constant region that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 83. In some embodiments, the humanized anti-TfR antibodies described herein comprise a light chain comprising any one of the VLs listed in Table 3 or any variant thereof, 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 humanized anti-TfR antibodies described herein comprise a light chain comprising any one of the VLs listed in Table 3 or any variant thereof, and a light chain constant region set forth in SEQ ID NO: 83.

[0207] Examples of Fab heavy and light chain amino acid sequences of the described anti-TfR antibodies are provided in Table 5 below. Table 5. Heavy and light chain sequences of examples of humanized anti-TfR Fabs [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4]

[0208] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a heavy chain containing no more than 25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) compared to the heavy chain set forth in any one of SEQ ID NOs: 97-103. Alternatively or additionally (e.g., additionally), a humanized anti-TfR antibody of the present disclosure comprises 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.

[0209] In some embodiments, the humanized anti-TfR 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 humanized anti-TfR 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-TfR 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-TfR 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.

[0210] In some embodiments, a humanized anti-TfR 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, a humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 97 and a light chain comprising the amino acid sequence of SEQ ID NO: 85.

[0211] In some embodiments, a humanized anti-TfR 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, a humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 98 and a light chain comprising the amino acid sequence of SEQ ID NO: 85.

[0212] In some embodiments, a humanized anti-TfR 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, a humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 99 and a light chain comprising the amino acid sequence of SEQ ID NO: 85.

[0213] In some embodiments, a humanized anti-TfR 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, a humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 100 and a light chain comprising the amino acid sequence of SEQ ID NO: 89.

[0214] In some embodiments, a humanized anti-TfR 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, a humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 100 and a light chain comprising the amino acid sequence of SEQ ID NO: 90.

[0215] In some embodiments, a humanized anti-TfR 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, a humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 101 and a light chain comprising the amino acid sequence of SEQ ID NO: 89.

[0216] In some embodiments, a humanized anti-TfR 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, a humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 101 and a light chain comprising the amino acid sequence of SEQ ID NO: 90.

[0217] In some embodiments, a humanized anti-TfR 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, a humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 102 and a light chain comprising the amino acid sequence of SEQ ID NO:93.

[0218] In some embodiments, a humanized anti-TfR 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, a humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 103 and a light chain comprising the amino acid sequence of SEQ ID NO: 95.

[0219] In some embodiments, a humanized anti-TfR 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, a humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 102 and a light chain comprising the amino acid sequence of SEQ ID NO: 95.

[0220] In some embodiments, the humanized anti-TfR receptor antibodies described herein can be in any antibody form, including, but not limited to, intact (i.e., full-length) antibodies, antigen-binding fragments thereof (such as Fab, Fab', F(ab')2, and Fv), single-chain antibodies, bispecific antibodies, or nanobodies. In some embodiments, the humanized anti-TfR antibodies described herein are scFvs. In some embodiments, the humanized anti-TfR antibodies described herein are scFv-Fabs (e.g., scFvs fused to a portion of a constant region). In some embodiments, the anti-TfR 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).

[0221] 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-TfR 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.

[0222] 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.

[0223] 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.

[0224] In some embodiments, one or more amino acid mutations (i.e., substitutions, insertions, or deletions) are introduced into an IgG constant region or FcRn-binding fragment thereof (preferably an 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 an antibody in vivo.

[0225] 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 antibodies 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. Mutant IgGs of this type, termed "YTE mutants," have 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.

[0226] 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).

[0227] 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.

[0228] 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.

[0229] 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.

[0230] 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."

[0231] 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).

[0232] 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 8. 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., the anti-transferrin receptor antibodies listed in Table 8.

[0233] Table 8 - List of anti-transferrin receptor antibody clones, including relevant references and binding epitope information [Table 7-1] [Table 7-2] [Table 7-3]

[0234] In some embodiments, a transferrin receptor antibody of the present 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 8. 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 8. 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 8. 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 8. 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 8. 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 8.

[0235] 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 (e.g., and) CDR-L3 sequences from one of the anti-transferrin receptor antibodies selected from Table 8. 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.

[0236] 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 8), 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 8), 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 can 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 8), 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 8), 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 8), 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 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 8), 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.

[0237] 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 8. For example, an antibody can include one or more CDR sequence(s) from any of the anti-transferrin receptor antibodies selected from Table 8 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 8), 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 8. 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 8.

[0238] 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 8. 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 8.

[0239] 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 8. 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 8.

[0240] 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 8. 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 anti-transferrin receptor antibody, such as any one of the anti-transferrin receptor antibodies selected from Table 8. 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 anti-transferrin receptor antibody, such as any one of the anti-transferrin receptor antibodies selected from Table 8. 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 any anti-transferrin receptor antibody, such as any one of the anti-transferrin receptor antibodies selected from Table 8. In some embodiments, the light chain variable framework region derived from the amino acid sequence described above consists of the amino acid sequence described above 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 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.

[0241] 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 8. 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 8). 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 8. 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.

[0242] 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 8.

[0243] 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 8.

[0244] 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 8, 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.

[0245] 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).

[0246] The heavy and light chain CDRs of antibodies according to various definition systems are provided in Table 9. 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 9. Heavy and light chain CDRs of mouse transferrin receptor antibodies [Table 8]

[0247] Heavy chain variable domain (VH) and light chain variable domain sequences are also provided:

[0248] VH QVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGEINPTNGRTNYIEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARGTRAYHYWGQGTSVTVSS (SEQ ID NO: 124)

[0249] VL DIQMTQSPASLSVSVGETVTITCRASDNLYSNLAWYQQKQGKSPQLLVYDATNLADGVPSRFSGSGSGTQYSLKINSLQSEDFGTYYCQHFWGTPLTFGAGTKLELK (SEQ ID NO: 125)

[0250] 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 9. 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 9.

[0251] 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 9. "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 9.

[0252] 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 9. 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 9.

[0253] 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 9. 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 9. 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 a 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 9, 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.

[0254] 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 set forth in Table 9. 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 set forth in Table 9.

[0255] 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.

[0256] 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.

[0257] 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.

[0258] 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 9, and comprise a humanized heavy chain variable region and / or (by way of example and) a humanized light chain variable region.

[0259] 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.

[0260] In some embodiments, humanization is achieved by grafting CDRs (e.g., as shown in Table 9) 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.

[0261] 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.

[0262] 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:

[0263] Humanized VH EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQRLEWIGEINPTNGRTNYIEKFKSRATLTVDKSASTAYMELSSLRSEDTAVYYCARGTRAYHYWGQGTMVTVSS (SEQ ID NO: 128)

[0264] Humanized VL DIQMTQSPSSLSASVGDRVTITCRASDNLYSNLAWYQQKPGKSPKLLVYDATNLADGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQHFWGTPLTFGQGTKVEIK (SEQ ID NO: 129)

[0265] 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.

[0266] 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.

[0267] 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.

[0268] In some embodiments, the transferrin receptor antibody of the disclosure is a humanized variant comprising 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 humanized variant comprising 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: A67V, L69I, V71R, and K73T compared to the VH set forth in SEQ ID NO: 124.

[0269] In some embodiments, the transferrin receptor antibodies of the disclosure are humanized 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.

[0270] 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.

[0271] 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.

[0272] 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: 130)

[0273] 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)

[0274] 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.

[0275] Examples of heavy and light chain amino acid sequences of the described anti-transferrin receptor antibodies are provided below:

[0276] Heavy chain (VH + human IgG1 constant region) QVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGEINPTNGRTNYIEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARGTRAYHYWGQGTSVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 132)

[0277] Light chain (VL + kappa light chain) DIQMTQSPASLSVSVGETVTITCRASDNLYSNLAWYQQKQGKSPQLLVYDATNLADGVPSRFSGSGSGTQYSLKINSLQSEDFGTYYCQHFWGTPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 133)

[0278] Heavy chain (humanized VH + human IgG1 constant region) EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQRLEWIGEINPTNGRTNYIEKFKSRATLTVDKSASTAYMELSSLRSEDTAVYYCARGTRAYHYWGQGTMVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 134)

[0279] Light chain (humanized VL + kappa light chain) DIQMTQSPSSLSASVGDRVTITCRASDNLYSNLAWYQQKPGKSPKLLVYDATNLADGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQHFWGTPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 135)

[0280] 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.

[0281] 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.

[0282] 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.

[0283] 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 humanized 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.

[0284] 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:

[0285] Heavy chain Fab (VH + part of human IgG1 constant region) QVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGEINPTNGRTNYIEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARGTRAYHYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP (SEQ ID NO: 136)

[0286] Heavy chain Fab (humanized VH + part of human IgG1 constant region) EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQRLEWIGEINPTNGRTNYIEKFKSRATLTVDKSASTAYMELSSLRSEDTAVYYCARGTRAYHYWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP (SEQ ID NO: 137)

[0287] 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.

[0288] 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.

[0289] 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: 130).

[0290] In some embodiments, any one of the anti-TfR 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.

[0291] 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.

[0292] 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.

[0293] 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.

[0294] 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.

[0295] 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.

[0296] In some embodiments, one or more amino acid mutations (i.e., substitutions, insertions, or deletions) are introduced into an IgG constant region or FcRn-binding fragment thereof (preferably an 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 an antibody in vivo.

[0297] 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 antibodies 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. Mutant IgGs of this type, termed "YTE mutants," have 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.

[0298] 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).

[0299] 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.

[0300] 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.

[0301] 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.

[0302] 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 include 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 include VH and VL domains, or antigen-binding portions thereof, combined with any suitable constant region.

[0303] 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.

[0304] 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."

[0305] 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 DMD, 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.

[0306] 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).

[0307] 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).

[0308] 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.

[0309] 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.

[0310] 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.

[0311] 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.

[0312] 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.

[0313] 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).

[0314] 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.

[0315] 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, expression of a protein, or activity of a protein). In some embodiments, the molecular payload is linked or otherwise associated with a muscle-targeting agent. In some embodiments, such molecular payloads can target 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 complementary to a gene provided in Table 1. 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.

[0316] In some embodiments, at least one (e.g., at least two, at least three, at least four, at least five, at least ten) molecular payloads (e.g., oligonucleotides) are linked to the muscle-targeting agent. In some embodiments, all molecular payloads linked to the muscle-targeting agent are the same, e.g., target the same gene. In some embodiments, all molecular payloads linked to the muscle-targeting agent are different, e.g., molecular payloads may target different portions of the same target gene, or molecular payloads may target at least two different target genes. In some embodiments, a muscle-targeting agent may be linked to some molecular payloads that are the same and some that are different.

[0317] The present disclosure also provides compositions comprising a plurality of conjugates, wherein at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) of the conjugates comprise a muscle-targeting agent linked to the same number of molecular payloads (e.g., oligonucleotides).

[0318] i. Oligonucleotides Any suitable oligonucleotide may be used as a molecular payload as described herein. In some embodiments, the oligonucleotide may be designed to cause degradation of mRNA (for example, 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 mRNA (for example, the oligonucleotide may be a mixmer, siRNA, or aptamer that blocks translation). In some embodiments, the oligonucleotide may be designed to cause degradation of mRNA to block its translation. In some embodiments, the oligonucleotide may be a guide nucleic acid (for example, a guide RNA) to direct the activity of an enzyme (for example, a gene editing enzyme). Other examples of oligonucleotides are provided herein. It should be understood that in some embodiments, an oligonucleotide of one format (for example, an antisense oligonucleotide) may be suitably adapted to another format (for example, an siRNA oligonucleotide) by incorporating a functional sequence (for example, an antisense strand sequence) from one format into the other format.

[0319] In some embodiments, the oligonucleotides may comprise a region of complementarity to a target gene provided in Table 1. Further non-limiting examples are provided below for selected genes in Table 1.

[0320] DMPK / DM1 In some embodiments, examples of useful oligonucleotides for targeting DMPK, e.g., for the treatment of DM1, include those described in U.S. Patent Application Publication No. 20100016215A1, published January 1, 2010, entitled "Compound And Method For Treating Myotonic Dystrophy"; U.S. Patent Application Publication No. 20130237585A1, published July 19, 2010, entitled "Modulation Of Dystrophia Myotonica-Protein Kinase (DMPK) Expression"; U.S. Patent Application Publication No. 20150064181A1, published March 5, 2015, entitled "Antisense Conjugates For Decreasing Expression Of Dmpk"; and U.S. Patent Application Publication No. 20150064181A1, published March 5, 2015, entitled "Peptide-Linked Morpholino Antisense Oligonucleotides For Treatment Of Myotonic Dystrophy." U.S. Patent Application Publication No. 20150238627A1, published August 27, 2015, entitled "Myotonic Dystrophy"; Pandey, S.K. et al., "Identification and Characterization of Modified Antisense Oligonucleotides Targeting DMPK in Mice and Nonhuman Primates for the Treatment of Myotonic Dystrophy Type 1," J. of Pharmacol Exp Ther, 2015, 355:329-340; Langlois, M. et al., "Cytoplasmic and Nuclear Retained DMPK mRNAs Are Targets for RNA Interference in Myotonic Dystrophy Cells," J. Biological Chemistry, 2005, 280:17, 16949-16954; Jauvin, D. et al."Targeting DMPK with Antisense Oligonucleotide Improves Muscle Strength in Myotonic Dystrophy Type 1 Mice", Mol.Ther:Nucleic Acids,2017,7:465-474.;Mulders, SAet al. "Triplet-repeat oligonucleotide-mediated reversal of RNA toxicity in myotonic dystrophy” PNAS, 2009, 106:33, 13915-13920.; Wheeler, TMet al., “Targeting nuclear RNA for in vivo correction of myotonic dystrophy” Nature, 2012, 488(7409): 111-115.; and “Compounds And Methods For Modulation Of Dystrophia Myotonica-Protein and U.S. Patent Application Publication No. 20160304877A1, published October 20, 2016, entitled "Methods for Promoting the Expression of Glucocorticoids in a Cellular Cell Line," the entire contents of each of which are incorporated herein by reference.

[0321] Examples of oligonucleotides for facilitating DMPK gene editing include U.S. Patent Application Publication No. 20170088819A1, published March 3, 2017, entitled "Genetic Correction Of Myotonic Dystrophy Type 1"; and International Patent Application Publication No. WO18002812A1, published April 1, 2018, entitled "Materials And Methods For Treatment Of Myotonic Dystrophy Type 1 (DM1) And Other Related Disorders," the entire contents of each of which are incorporated herein by reference.

[0322] In some embodiments, the oligonucleotide may have a region of complementarity to a mutant form of DMPK, e.g., a mutant form reported in Botta A. et al., "The CTG repeat expansion size correlates with splicing defect in muscle from myotonic dystrophy type 1 patients," J Med Genet. 2008 Oct;45(10):639-46; and Machuca-Tzili L. et al., "Clinical and molecular aspects of the myotonic dystrophies: a review," Muscle Nerve. 2005 Jul;32(1):1-18, the contents of each of which are incorporated herein by reference in their entirety.

[0323] In some embodiments, the oligonucleotide provided herein is an antisense oligonucleotide targeting DMPK. In some embodiments, the oligonucleotide targeting is any one of the DMPK-targeting antisense oligonucleotides (e.g., Gapmers) described in U.S. Patent Application Publication US20160304877A1, entitled "Compounds and Methods for Modulation of Dystrophia Myotonica-Protein Kinase (DMPK) Expression," published on October 20, 2016, which is incorporated herein by reference. In some embodiments, the DMPK-targeting oligonucleotide targets the region of the DMPK gene sequence represented by Genbank Accession No. NM_001081560.2 or Genbank Accession No. NG_009784.1.

[0324] In some embodiments, the DMPK-targeting oligonucleotide comprises a nucleotide sequence comprising a region complementary to a target region in Genbank Accession No. NM_001081560.2 that is at least 10 contiguous nucleotides (e.g., at least 10, at least 12, at least 14, at least 16, or more contiguous nucleotides).

[0325] In some embodiments, the DMPK-targeting oligonucleotide comprises a gapmer motif. "Gapmer" refers to a chimeric antisense compound in which an internal region having multiple nucleotides that support RNase H cleavage is positioned between external regions having one or more nucleotides, where the nucleotides comprising the internal region are chemically distinct from the nucleotides comprising the external regions. The internal region may be referred to as a "gap segment," and the external regions may be referred to as a "wing segment." In some embodiments, the DMPK-targeting oligonucleotide comprises one or more modified nucleotides and / or (for example, and) one or more modified internucleotide linkages. In some embodiments, the internucleotide linkages are phosphorothioate linkages. In some embodiments, the oligonucleotide comprises a complete phosphorothioate backbone. In some embodiments, the oligonucleotide is a DNA gapmer with a cET terminus (e.g., 3-10-3; cET-DNA-cET). In some embodiments, the DMPK-targeting oligonucleotide comprises one or more 6'-(S)-CH3 bicyclic nucleotides, one or more β-D-2'-deoxyribonucleotides, and / or (by way of example and not limitation) one or more 5-methyl-cytosine nucleotides.

[0326] DUX4 / FSHD In some embodiments, examples of useful oligonucleotides for targeting DUX4, e.g., for the treatment of FSHD, are described in U.S. Patent No. 9,988,628, published February 2, 2017, entitled "AGENTS USEFUL IN TREATING FACIOSCAPULOHUMERAL MUSCULAR DYSTROPHY"; U.S. Patent No. 9,469,851, published October 30, 2014, entitled "RECOMBINANT VIRUS PRODUCTS AND METHODS FOR INHIBITING EXPRESSION OF DUX4"; U.S. Patent Application Publication No. 20120225034, published September 6, 2012, entitled "AGENTS USEFUL IN TREATING FACIOSCAPULOHUMERAL MUSCULAR DYSTROPHY ...MORPHOLINO TARGETING DUX4 FOR TREATING FACIOSCAPULOHUMERAL MUSCULAR DYSTROPHY"; and Ansseau et al., "Antisense Oligonucleotides Used to Target the DUX4 mRNA as Therapeutic Approaches in Facioscapulohumeral Muscular Dystrophy (FSHD)," Genes, 2017, 8, 93, the entire contents of each of which are incorporated herein. In some embodiments, the oligonucleotide is an antisense oligonucleotide, morpholino, siRNA, shRNA, or another nucleotide that hybridizes to the target DUX4 gene or mRNA.

[0327] In some embodiments, by way of example, for the treatment of FSHD, the oligonucleotide may have a region complementary to a hypomethylated, shortened D4Z4 repeat as in Daxinger, et al., "Genetic and Epigenetic Contributors to FSHD," published in Curr Opin Genet Dev in 2015; Lim JW, et al., DICER / AGO-dependent epigenetic silencing of D4Z4 repeats enhanced by exogenous siRNA suggests mechanisms and therapies for FSHD Hum Mol Genet. 2015 Sep 1;24(17):4817-4828 (the entire contents of each of which are incorporated herein).

[0328] DNM2 / CNM In some embodiments, examples of useful oligonucleotides for targeting DNM2, e.g., for the treatment of CNM, are provided in U.S. Patent Application Publication No. 20180142008, published May 24, 2018, entitled "DYNAMIN 2 INHIBITOR FOR THE TREATMENT OF DUCHENNE'S MUSCULAR DYSTROPHY," and PCT Application Publication No. WO 2018 / 100010A1, published June 7, 2018, entitled "ALLELE-SPECIFIC SILENCING THERAPY FOR DYNAMIN 2-RELATED DISEASES." For example, in some embodiments, the oligonucleotide is an RNAi, antisense nucleic acid, siRNA, or ribozyme that specifically interferes with DNM2 expression. Other examples of useful oligonucleotides for targeting DNM2 are provided in Tasfaout, et al., "Single Intramuscular Injection of AAV-shRNA Reduces DNM2 and Prevents Myotubular Myopathy in Mice," and Tasfaout, et al., "Antisense oligonucleotide-mediated Dnm2 knockdown prevents and reverts myotubular myopathy in mice," Nature Communications volume 8, Article number: 15661 (2017), published in Mol. Ther. on April 4, 2018. In some embodiments, the oligonucleotide is an shRNA or morpholino that efficiently targets DNM2 mRNA.In some embodiments, the oligonucleotide encodes wild-type DNM2 that is resistant to miR-133 activity, such as in Todaka, et al., "Overexpression of NF90-NF45 Represses Myogenic MicroRNA Biogenesis, Resulting in Development of Skeletal Muscle Atrophy and Centronuclear Muscle Fibers," published in Mol. Cell Biol. in July 2015. Further examples of oligonucleotides useful for targeting DNM2 are provided in Gibbs, et al., "Two Dynamin-2 Genes are Required for Normal Zebrafish Development," published in PLoS One in 2013, the contents of each of which are incorporated herein in their entirety.

[0329] In some embodiments, for example, for the treatment of CNM, the oligonucleotide may have a region complementary to a mutation in DNM2 associated with CNM, as in Boehm et al., "Mutation Spectrum in the Large GTPase Dynamin 2, and Genotype-Phenotype Correlation in Autosomal Dominant Centronuclear Myopathy," published in Hum. Mutat. in 2012 (the entire contents of which are incorporated herein).

[0330] Pompe disease In some embodiments, by way of example, for the treatment of Pompe disease, the oligonucleotide mediates exon 2 inclusion in the GAA disease allele as in van der Wal, et al., "GAA Deficiency in Pompe Disease is Alleviated by Exon Inclusion in iPSC-Derived Skeletal Muscle Cells," Mol Ther Nucleic Acids. 2017 Jun 16;7:101-115, the entire contents of which are incorporated herein. Consequently, in some embodiments, the oligonucleotide may have a region of complementarity to the GAA disease allele.

[0331] In some embodiments, for example, for the treatment of Pompe disease, oligonucleotides such as RNAi or antisense oligonucleotides are utilized to suppress expression of wild-type GYS1 in muscle cells, as reported, for example, in Clayton, et al., "Antisense Oligonucleotide-Mediated Suppression of Muscle Glycogen Synthase 1 Synthesis as an Approach for Substrate Reduction Therapy of Pompe Disease," published in Mol Ther Nucleic Acids in 2017, or in U.S. Patent Application Publication No. 2017182189, published June 29, 2017, entitled "INHIBITING OR DOWNREGULATING GLYCOGEN SYNTHASE BY CREATING PREMATURE STOP CODONS USING ANTISENSE OLIGONUCLEOTIDES," the contents of which are incorporated herein by reference. Consequently, in some embodiments, the oligonucleotide may have an antisense strand that has a region of complementarity to a sequence that is the human GYS1 sequence corresponding to RefSeq number NM_002103.4 and / or (by way of example) the mouse GYS1 sequence corresponding to RefSeq number NM_030678.3.

[0332] ACVR1 / FOP For example, examples of oligonucleotides useful for targeting ACVR1 for the treatment of FOP are described in U.S. Patent Application No. 2009 / 0253132, published 10 / 8 / 2009, entitled "Mutated ACVR1 for diagnosis and treatment of fibrodysplasia ossificans progressiva (FOP)"; WO 2015 / 152183, published 10 / 8 / 2015, entitled "Prophylactic agent and therapeutic agent for fibrodysplasia ossificans progressive"; Lowery, J. Wet et al., "Allele-specific RNA Interference in FOP-Silencing the FOP gene," GENE THERAPY, vol. 19, 2012, pages 701-702; Takahashi, M. et al., "Disease-causing allele-specific silencing against the ALK2 mutants, R206H and G356D, in fibrodysplasia ossificans progressiva," Gene Therapy(2012)19,781-785;Shi,S.et al. "Antisense-Oligonucleotide Mediated Exon Skipping in Activin-Receptor-Like Kinase 2:Inhibiting the Receptor That Is Overactive in Fibrodysplasia Ossificans Progressiva" Plos One,July 2013,Vol 8:7,e69096.U.S. Patent Application No. 2017 / 0159056, published June 8, 2017, entitled "Antisense oligonucleotides and methods of use thereof"; U.S. Patent No. 8,859,752, published October 4, 2014, entitled "SIRNA-based therapy of Fibrodyplasia Ossificans Progressiva (FOP)"; and WO2004 / 094636, published November 4, 2004, entitled "Effective siRNA knockdown constructs," the contents of each of which are incorporated herein in their entirety.

[0333] FXN / Friedreich's ataxia In some embodiments, examples of useful oligonucleotides for targeting FXN and / or otherwise compensating for frataxin deficiency, e.g., for the treatment of Friedreich's ataxia, include those described in Li, L. et al., "Activating frataxin expression by repeat-targeted nucleic acids," Nat. Comm. 2016, 7:10606; WO 2016 / 094374, published 6 / 16 / 2016, "Compositions and methods for treatment of Friedreich's ataxia."; WO 2015 / 020993, published 2 / 12 / 2015, "RNAi COMPOSITIONS AND METHODS FOR TREATMENT OF FRIEDREICH'S ATAXIA."; WO 2017 / 186815, published 11 / 2 / 2017, "Antisense oligonucleotides for enhanced expression of frataxin."; WO U.S. Patent Application No. 2008 / 018795, published 2 / 14 / 2008, "Methods and means for treating DNA repeat instability associated genetic disorders"; U.S. Patent Application No. 2018 / 0028557, published 2 / 1 / 2018, "Hybrid oligonucleotides and uses thereof"; WO 2015 / 023975, published 2 / 19 / 2015, "Compositions and methods for modulating RNA"; WO 2015 / 023939, published 2 / 19 / 2015, "Compositions and methods for modulating expression of frataxin"; U.S. Patent Application No. 2017 / 0281643, published 10 / 5 / 2017, "Compounds and methods for modulating frataxin expression"; Li L. et al., "Activating frataxin expression by repeat-targeted nucleic acids," Nature Communications, published February 4, 2016; and Li L. et al., "Activation of Frataxin Protein Expression by Antisense Oligonucleotides Targeting the Mutant Expanded Repeat," Nucleic Acid Ther. 2018 February;28(1):23-33, the entire contents of each of which are incorporated herein.

[0334] In some embodiments, the oligonucleotide payload is configured to inhibit expression of natural antisense transcripts that inhibit FXN expression (e.g., as gapmers or RNAi oligonucleotides), as disclosed, for example, in U.S. Patent No. 9,593,330, filed 6 / 9 / 2011, entitled "Treatment of frataxin (FXN)-related diseases by inhibition of natural antisense transcript to FXN," the entire contents of which are incorporated herein by reference.

[0335] Examples of oligonucleotides for facilitating FXN gene editing include WO 2016 / 094845, published June 16, 2016, entitled "Compositions and methods for editing nucleic acids in cells utilizing oligonucleotides"; WO 2015 / 089354, published June 18, 2015, entitled "Compositions and methods of use of CRISPR-Cas systems in nucleotide repeat disorders"; WO 2015 / 139139, published September 24, 2015, entitled "CRISPR-based methods and products for increasing frataxin levels and uses thereof"; and WO 2018 / 002783, published January 4, 2018, entitled "Materials and methods for treatment of Friedreich ataxia and other related disorders," the entire contents of each of which are incorporated herein.

[0336] Examples of oligonucleotides for promoting FXN gene expression through targeting non-FXN genes, such as epigenetic regulators of FXN, include WO 2015 / 023938, published 2 / 19 / 2015, entitled "Epigenetic regulators of frataxin," the entire contents of which are incorporated herein.

[0337] In some embodiments, the oligonucleotide may have a region of complementarity to a sequence defined as the FXN gene from humans (Gene ID 2395; NC_000009.12) and / or (by way of example) the FXN gene from mice (Gene ID 14297; NC_000085.6). In some embodiments, the oligonucleotide may have a region of complementarity to a mutant form of FXN, as reported in, for example, Montermini, L. et al., "The Friedreich ataxia GAA triplet repeat: premutation and normal alleles," Hum. Molec. Genet., 1997, 6:1261-1266; Filla, A. et al., "The relationship between trinucleotide (GAA) repeat length and clinical features in Friedreich ataxia," Am. J. Hum. Genet., 1996, 59:554-560; Pandolfo, M. Friedreich ataxia: the clinical picture. J. Neurol., 2009, 256, 3-8, the contents of each of which are incorporated herein by reference in their entirety.

[0338] DMD / Dystrophinopathy 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 Application Publication No. 8,361,979, issued January 29, 2013, entitled "MEANS AND METHOD FOR INDUCING EXON-SKIPPING"; U.S. Patent Application Publication No. 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 No. 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.

[0339] 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.

[0340] 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.

[0341] MYH7 / hypertrophic cardiomyopathy Examples of useful oligonucleotides for targeting, e.g., MYH7, as a payload include U.S. Patent Application Publication No. 20180094262, published April 5, 2018, entitled "Inhibitors of MYH7B and Uses Thereof"; U.S. Patent Application Publication No. 20160348103, published December 1, 2016, entitled "Oligonucleotides and Methods for Treatment of Cardiomyopathy Using RNA Interference"; U.S. Patent Application Publication No. 20160237430, published August 18, 2016, entitled "Allele-specific RNA Silencing for the Treatment of Hypertrophic Cardiomyopathy"; U.S. Patent Application Publication No. 20160032286, published February 4, 2016, entitled "Inhibitors of MYH7B and Uses Thereof"; and U.S. Patent Application Publication No. 20160032286, published February 4, 2016, entitled "MicroRNA Inhibitors Comprising Locked U.S. Patent Application Publication No. 20140187603, published July 3, 2014, entitled "Mutant Nucleotides"; U.S. Patent Application Publication No. 20140179764, published June 26, 2014, entitled "Dual Targeting of miR-208 and miR-499 in the Treatment of Cardiac Disorders"; and U.S. Patent Application Publication No. 20120114744, published May 10, 2012, entitled "Compositions and Methods to Treat Muscular and Cardiovascular Disorders," the entire contents of each of which are incorporated herein.

[0342] 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.

[0343] In some embodiments, any one of the oligonucleotides may be in a salt form, for example, as a sodium, potassium, or magnesium salt.

[0344] 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 unsubstituted heteroaliphatic, substituted or unsubstituted carbocyclylene, substituted or unsubstituted heterocyclylene, substituted or unsubstituted arylene, substituted or unsubstituted heteroarylene, -O-, -N(R A )-, -S-, -C(=O)-, -C(=O)O-, -C(=O)NR A -, -NR A C(=O)-, -NR A C(=O)R A -, -C(=O)R A -, -NR A C(=O)O-, -NR A C(=O)N(R A )-, -OC(=O)-, -OC(=O)O-, -OC(=O)N(R A )-, -S(O)NR A -, -NR A S(O)2-, or a combination thereof; each R A are independently hydrogen or substituted or unsubstituted alkyl. In certain embodiments, the spacer is a substituted or unsubstituted alkylene, a substituted or unsubstituted heterocyclylene, a substitute...

Claims

1. 1. A conjugate comprising an anti-transferrin receptor (TfR) antibody covalently linked to a molecular payload configured to modulate the expression or activity of a muscle disease gene, wherein the antibody comprises a heavy chain variable region (VH) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 76; and a light chain variable region (VL) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 75, wherein the VH and VL of the antibody are selected from the following: (i) heavy chain complementarity determining region 1 (CDR-H1) represented by SEQ ID NO: 27, heavy chain complementarity determining region 2 (CDR-H2) represented by SEQ ID NO: 28, heavy chain complementarity determining region 3 (CDR-H3) represented by SEQ ID NO: 29, light chain complementarity determining region 1 (CDR-L1) represented by SEQ ID NO: 30, light chain complementarity determining region 2 (CDR-L2) represented by SEQ ID NO: 31, and light chain complementarity determining region 3 (CDR-L3) represented by SEQ ID NO: 32; (ii) CDR-H1 represented by SEQ ID NO: 33, CDR-H2 represented by SEQ ID NO: 34, CDR-H3 represented by SEQ ID NO: 35, CDR-L1 represented by SEQ ID NO: 36, CDR-L2 represented by SEQ ID NO: 37, and CDR-L3 represented by SEQ ID NO: 32; or (iii) CDR-H1 represented by SEQ ID NO: 38, CDR-H2 represented by SEQ ID NO: 39, CDR-H3 represented by SEQ ID NO: 40, CDR-L1 represented by SEQ ID NO: 41, CDR-L2 represented by SEQ ID NO: 31, and CDR-L3 represented by SEQ ID NO: 42 Including, The complex.

2. The conjugate of claim 1, wherein the 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.

3. 3. The conjugate of claim 1 or 2, wherein the 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.

4. The conjugate of any one of claims 1 to 3, wherein the antibody is a Fab fragment.

5. The conjugate of any one of claims 1 to 4, wherein the antibody comprises a heavy chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 101; and a light chain comprising an amino acid sequence at least 85% identical to SEQ ID NO:

90.

6. the antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 101; and a light chain comprising the amino acid sequence of SEQ ID NO:

90. The complex of any one of claims 1 to 5, comprising:

7. The conjugate of any one of claims 1 to 6, wherein the heavy chain of the antibody comprises an N-terminal pyroglutamic acid.

8. The conjugate of any one of claims 1 to 7, wherein the molecular payload comprises an oligonucleotide.

9. 9. The conjugate of claim 8, wherein the oligonucleotide comprises a region of complementarity to a muscle disease gene having a gain-of-function disease allele.

10. 10. The conjugate of any one of claims 1 to 9, wherein modulating the expression or activity of a muscle disease gene comprises decreasing RNA and / or protein expression.

11. The conjugate of any one of claims 8 to 10, wherein the oligonucleotide comprises at least one modified internucleoside linkage.

12. 12. The conjugate of claim 11, wherein at least one modified internucleoside linkage is a phosphorothioate linkage.

13. The conjugate of any one of claims 8 to 12, wherein the oligonucleotide comprises one or more modified nucleosides.

14. 14. The conjugate of claim 13, wherein the one or more modified nucleosides comprise a 2'-modified nucleoside.

15. 15. The conjugate of claim 14, wherein the one or more 2'-modified nucleosides are each selected from the group consisting of 2'-O-methyl, 2'-fluoro (2'-F), 2'-O-methoxyethyl (2'-MOE), and 2',4'-bridged nucleosides.

16. The oligonucleotide is (i) gapmer oligonucleotides that direct RNAse H-mediated cleavage of mRNA transcripts encoded by muscle disease genes in cells; (ii) mixmer oligonucleotides; (iii) is a phosphorodiamidate morpholino oligomer; or (iv) an RNAi oligonucleotide that promotes RNAi-mediated cleavage of an mRNA transcript encoded by a muscle disease gene; Including, The complex according to any one of claims 8 to 15.

17. 17. The conjugate of any one of claims 1 to 16, wherein the antibody is covalently linked to the molecular payload via a cleavable linker.

18. 18. The conjugate of claim 17, wherein the cleavable linker comprises a valine-citrulline sequence.

19. 17. The conjugate of any one of claims 1 to 16, wherein the antibody is covalently linked to the molecular payload via a non-cleavable linker.

20. 20. The conjugate of claim 19, wherein the non-cleavable linker comprises an optionally substituted alkyl group.

21. 21. The conjugate of any one of claims 1 to 20, wherein the antibody is covalently linked to the molecular payload via a lysine residue on the antibody.

22. 21. The conjugate of any one of claims 1 to 20, wherein the antibody is covalently linked to the molecular payload via a cysteine ​​residue of the antibody.

23. 23. The complex of any one of claims 1 to 22 for use in a method of treating a disease or condition that can be ameliorated or prevented by modulating the expression or activity of a muscle disease gene in a cell, the method comprising contacting a cell with the complex.

24. 24. The complex of claim 23, wherein the cell is a muscle cell.

25. 25. The conjugate of claim 23 or 24, wherein the disease or condition is a muscle disorder selected from the group consisting of adult Pompe disease, centronuclear myopathy (CNM), Duchenne muscular dystrophy, facioscapulohumeral muscular dystrophy (FSHD), familial hypertrophic cardiomyopathy, fibrodysplasia ossificans progressiva (FOP), Friedreich's ataxia (FRDA), type 2 inclusion body myopathy, distal Lehn myopathy, myofibrillar myopathy, myotonia congenita (autosomal dominant, Thomsen's disease), myotonic dystrophy type I, myotonic dystrophy type II, myotubular myopathy, oculopharyngeal muscular dystrophy, and congenital myotonia.

26. 23. The conjugate of any one of claims 1 to 22 for use in a method of treating a subject having a muscular disease, the method comprising administering the conjugate to the subject.

27. 27. The conjugate of claim 26, wherein the muscle disease is selected from the group consisting of adult Pompe disease, centronuclear myopathy (CNM), Duchenne muscular dystrophy, facioscapulohumeral muscular dystrophy (FSHD), familial hypertrophic cardiomyopathy, fibrodysplasia ossificans progressiva (FOP), Friedreich's ataxia (FRDA), type 2 inclusion body myopathy, distal Lehn myopathy, myofibrillar myopathy, congenital myotonia (autosomal dominant, Thomsen's disease), myotonic dystrophy type I, myotonic dystrophy type II, myotubular myopathy, oculopharyngeal muscular dystrophy, and congenital myotonia.

28. 27. The conjugate of any one of claims 23, 24, and 26, wherein the disease or condition is adult Pompe disease.

29. 27. The conjugate of any one of claims 23, 24, and 26, wherein the disease or condition is Duchenne muscular dystrophy.

30. 27. The conjugate of any one of claims 23, 24, and 26, wherein the disease or condition is facioscapulohumeral muscular dystrophy (FSHD).

31. 27. The conjugate of any one of claims 23, 24, and 26, wherein the disease or condition is myotonic dystrophy type I.

32. The conjugate according to any one of claims 26 to 31, wherein the subject is a human.

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