Muscle targeting complexes and uses thereof

Abstract

Muscle targeting complexes and uses thereof. Some aspects of the present disclosure relate to complexes comprising a muscle targeting agent covalently linked to a molecular load. In some embodiments, the muscle targeting agent specifically binds to an internalized cell surface receptor on a muscle cell. In some embodiments, the molecular load inhibits the activity of disease alleles associated with muscle disease. In some embodiments, the molecular load is an oligonucleotide, such as an antisense oligonucleotide or an RNAi oligonucleotide.

Description

[0001] This application is a divisional application of Chinese patent application No. 202180064691.X, entitled "Muscle Targeting Complex and Its Use Therein". The parent application is PCT international patent application PCT / US2021 / 041003 filed on July 9, 2021, which entered the Chinese national phase.

[0002] Related applications

[0003] This application claims priority under 35 U.S. SC § 119(e): U.S. Provisional Application Serial No. 63 / 181450, filed April 29, 2021, entitled “MUSCLE-TARGETING COMPLEXES AND USES THEREOF”; U.S. Provisional Application Serial No. 63 / 143831, filed January 30, 2021, entitled “MUSCLE-TARGETING COMPLEXES AND USES THEREOF”; U.S. Provisional Application Serial No. 63 / 069078, filed August 23, 2020, entitled “MUSCLE-TARGETING COMPLEXES AND USES THEREOF”; and U.S. Provisional Application Serial No. 63 / 069078, filed August 6, 2020, entitled “MUSCLE-TARGETING COMPLEXES AND USES THEREOF”. The contents of U.S. Provisional Application No. 63 / 061842 entitled “THREOF”, filed on July 23, 2020, and U.S. Provisional Application No. 63 / 055785 entitled “MUSCLE-TARGETING COMPLEXES AND USES THEREOF”, are incorporated herein by reference in their entirety. Technical Field

[0004] This application relates to targeted complexes for delivering molecular payloads (e.g., oligonucleotides) to cells and their uses, particularly in relation to disease treatment.

[0005] The reference is the sequence list submitted as a text file via EFS-Web.

[0006] This application contains a sequence list that 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 named D082470041WO00-SEQ-DWY and has a size of 152,275 bytes. Background Technology

[0007] Muscle diseases are commonly associated with muscle weakness and / or (e.g., muscle dysfunction) that can lead to life-threatening complications. Numerous examples of such diseases have been characterized, including various forms of muscular dystrophy (e.g., Duchenne, facioscapulohumeral, myotonia, and oculopharyngeal), Pompe disease, centronuclear myopathy, familial hypertrophic cardiomyopathy, Laing distal myopathy, Fibrodysplasia Ossificans Progressiva, Friedreich's ataxia, myofibrillar myopathy, and others. These conditions are often hereditary but can occur spontaneously. Other conditions are often congenital but can develop later in life. Many rare muscle diseases are monogenic disorders associated with gain-of-function or loss-of-function mutations, which can have dominant or recessive phenotypes. For example, activating mutations leading to muscle diseases have been identified in genes encoding ion channels, structural proteins, metabolic proteins, and signal transduction proteins. Despite advances in understanding the genetic etiology of muscle diseases, effective treatment options remain limited. Summary of the Invention

[0008] According to some aspects, this disclosure provides complexes that target muscle cells for delivering molecular payloads to these cells. In some embodiments, the complexes of this disclosure facilitate muscle-specific delivery of molecular payloads targeting muscle disease alleles. For example, in some embodiments, the complexes provided herein are particularly useful for delivering molecular payloads that regulate gene expression or activity in subjects who have or are suspected of having a gene-related muscle disease (e.g., the genes / diseases in Table 1). In some embodiments, the complexes provided herein comprise a muscle-targeting agent (e.g., a muscle-targeting antibody) that specifically binds to a receptor on the surface of muscle cells for delivering the molecular payload to the muscle cells. In some embodiments, the complex is taken up into the cell via receptor-mediated internalization (e.g., transferrin receptor), and the molecular payload can then be released to perform its function within the cell. For example, a complex engineered to deliver an oligonucleotide can release the oligonucleotide such that the oligonucleotide can regulate the expression or activity of a muscle disease allele. In some embodiments, the oligonucleotide is released via endosomal cleavage of a covalent linker connecting the oligonucleotide of the complex and the muscle-targeting agent.

[0009] One aspect of this disclosure relates to a complex comprising an anti-transferrin receptor (TfR) antibody covalently linked to a molecular payload configured to regulate the expression or activity of a muscle disease gene, wherein the antibody comprises:

[0010] (i) Contains a heavy chain variable region (VH) having an amino acid sequence that is at least 95% identical to that of SEQ ID NO:76; and / or contains a light chain variable region (VL) having an amino acid sequence that is at least 95% identical to that of SEQ ID NO:75;

[0011] (ii) Containing a heavy chain variable region (VH) having an amino acid sequence that is at least 95% identical to that of SEQ ID NO:69; and / or containing a light chain variable region (VL) having an amino acid sequence that is at least 95% identical to that of SEQ ID NO:70;

[0012] (iii) Containing a heavy chain variable region (VH) having an amino acid sequence that is at least 95% identical to that of SEQ ID NO:71; and / or containing a light chain variable region (VL) having an amino acid sequence that is at least 95% identical to that of SEQ ID NO:70;

[0013] (iv) A heavy chain variable region (VH) containing an amino acid sequence having at least 95% identity with SEQ ID NO:72; and / or a light chain variable region (VL) containing an amino acid sequence having at least 95% identity with SEQ ID NO:70;

[0014] (v) Containing a heavy chain variable region (VH) having an amino acid sequence that is at least 95% identical to that of SEQ ID NO:73; and / or containing a light chain variable region (VL) having an amino acid sequence that is at least 95% identical to that of SEQ ID NO:74;

[0015] (vi) Containing a heavy chain variable region (VH) having an amino acid sequence that is at least 95% identical to that of SEQ ID NO:73; and / or containing a light chain variable region (VL) having an amino acid sequence that is at least 95% identical to that of SEQ ID NO:75;

[0016] (vii) A heavy chain variable region (VH) containing an amino acid sequence having at least 95% identity with SEQ ID NO:76; and / or a light chain variable region (VL) containing an amino acid sequence having at least 95% identity with SEQ ID NO:74;

[0017] (viii) Contains a heavy chain variable region (VH) having an amino acid sequence that is at least 95% identical to that of SEQ ID NO:77; and / or contains a light chain variable region (VL) having an amino acid sequence that is at least 95% identical to that of SEQ ID NO:78;

[0018] (ix) Containing a heavy chain variable region (VH) having at least 95% identity with an amino acid sequence of SEQ ID NO:79; and / or containing a light chain variable region (VL) having at least 95% identity with an amino acid sequence of SEQ ID NO:80; or

[0019] (x) contains a heavy chain variable region (VH) having an amino acid sequence that is at least 95% identical to that of SEQ ID NO:77; and / or contains a light chain variable region (VL) having an amino acid sequence that is at least 95% identical to that of SEQ ID NO:80.

[0020] In some embodiments, the antibody comprises:

[0021] (i) VH containing the amino acid sequence of SEQ ID NO:76 and VL containing the amino acid sequence of SEQ ID NO:75;

[0022] (ii) VH containing the amino acid sequence of SEQ ID NO:69 and VL containing the amino acid sequence of SEQ ID NO:70;

[0023] (iii) VH containing the amino acid sequence of SEQ ID NO:71 and VL containing the amino acid sequence of SEQ ID NO:70;

[0024] (iv) VH containing the amino acid sequence of SEQ ID NO:72 and VL containing the amino acid sequence of SEQ ID NO:70;

[0025] (v) VH containing the amino acid sequence of SEQ ID NO:73 and VL containing the amino acid sequence of SEQ ID NO:74;

[0026] (vi) VH containing the amino acid sequence of SEQ ID NO:73 and VL containing the amino acid sequence of SEQ ID NO:75;

[0027] (vii) VH containing the amino acid sequence of SEQ ID NO:76 and VL containing the amino acid sequence of SEQ ID NO:74;

[0028] (viii) VH containing the amino acid sequence of SEQ ID NO:77 and VL containing the amino acid sequence of SEQ ID NO:78;

[0029] (ix) VH containing the amino acid sequence of SEQ ID NO:79 and VL containing the amino acid sequence of SEQ ID NO:80; or

[0030] (x) VH containing the amino acid sequence of SEQ ID NO:77 and VL containing the amino acid sequence of SEQ ID NO:80.

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

[0032] In some embodiments, the antibody comprises:

[0033] (i) a heavy chain containing an amino acid sequence having at least 85% identity with SEQ ID NO:101; and / or a light chain containing an amino acid sequence having at least 85% identity with SEQ ID NO:90;

[0034] (ii) a heavy chain containing an amino acid sequence having at least 85% identity with SEQ ID NO:97; and / or a light chain containing an amino acid sequence having at least 85% identity with SEQ ID NO:85;

[0035] (iii) A heavy chain containing an amino acid sequence having at least 85% identity with SEQ ID NO:98; and / or a light chain containing an amino acid sequence having at least 85% identity with SEQ ID NO:85;

[0036] (iv) A heavy chain containing an amino acid sequence having at least 85% identity with SEQ ID NO:99; and / or a light chain containing an amino acid sequence having at least 85% identity with SEQ ID NO:85;

[0037] (v) a heavy chain containing an amino acid sequence having at least 85% identity with SEQ ID NO:100; and / or a light chain containing an amino acid sequence having at least 85% identity with SEQ ID NO:89;

[0038] (vi) A heavy chain containing an amino acid sequence having at least 85% identity with SEQ ID NO:100; and / or a light chain containing an amino acid sequence having at least 85% identity with SEQ ID NO:90;

[0039] (vii) A heavy chain containing an amino acid sequence having at least 85% identity with SEQ ID NO:101; and / or a light chain containing an amino acid sequence having at least 85% identity with SEQ ID NO:89;

[0040] (viii) A heavy chain containing an amino acid sequence having at least 85% identity with SEQ ID NO:102; and / or a light chain containing an amino acid sequence having at least 85% identity with SEQ ID NO:93;

[0041] (ix) A heavy chain containing an amino acid sequence having at least 85% identity with SEQ ID NO:103; and / or a light chain containing an amino acid sequence having at least 85% identity with SEQ ID NO:95; or

[0042] (x) A heavy chain containing an amino acid sequence having at least 85% identity with SEQ ID NO:102; and / or a light chain containing an amino acid sequence having at least 85% identity with SEQ ID NO:95.

[0043] In some embodiments, the antibody comprises:

[0044] (i) a heavy chain containing the amino acid sequence of SEQ ID NO:101; and a light chain containing the amino acid sequence of SEQ ID NO:90;

[0045] (ii) a heavy chain containing the amino acid sequence of SEQ ID NO:97; and a light chain containing the amino acid sequence of SEQ ID NO:85;

[0046] (iii) a heavy chain containing the amino acid sequence of SEQ ID NO:98; and a light chain containing the amino acid sequence of SEQ ID NO:85;

[0047] (iv) a heavy chain containing the amino acid sequence of SEQ ID NO:99; and a light chain containing the amino acid sequence of SEQ ID NO:85;

[0048] (v) a heavy chain containing the amino acid sequence of SEQ ID NO:100; and a light chain containing the amino acid sequence of SEQ ID NO:89;

[0049] (vi) A heavy chain containing the amino acid sequence of SEQ ID NO:100; and a light chain containing the amino acid sequence of SEQ ID NO:90;

[0050] (vii) A heavy chain containing the amino acid sequence of SEQ ID NO:101; and a light chain containing the amino acid sequence of SEQ ID NO:89;

[0051] (viii) A heavy chain containing the amino acid sequence of SEQ ID NO:102; and a light chain containing the amino acid sequence of SEQ ID NO:93;

[0052] (ix) a heavy chain containing the amino acid sequence of SEQ ID NO: 103; and a light chain containing the amino acid sequence of SEQ ID NO: 95; or

[0053] (x) A heavy chain containing the amino acid sequence of SEQ ID NO:102; and a light chain containing the amino acid sequence of SEQ ID NO:95.

[0054] In some implementations, the antibody does not specifically bind to the transferrin binding site of the transferrin receptor and / or the antibody does not inhibit the binding of transferrin to the transferrin receptor.

[0055] In some implementations, the antibody is cross-reactive with two or more extracellular sites of transferrin receptors in humans, non-human primates, and rodents.

[0056] In some embodiments, the complex is configured to facilitate transferrin receptor-mediated internalization of molecular loads into muscle cells.

[0057] In some embodiments, the molecular payload is an oligonucleotide. In some embodiments, the oligonucleotide contains the complementary region of a muscle disease gene having an allele of gain-of-function disease.

[0058] In some embodiments, the oligonucleotide comprises at least one modified nucleoside link. In some embodiments, the at least one modified nucleoside link is a phosphate thioester link.

[0059] In some embodiments, the oligonucleotide comprises one or more modified nucleosides. In some embodiments, the one or more modified nucleosides are 2'-modified nucleosides.

[0060] In some implementations, the oligonucleotide is a spacer polymer oligonucleotide that directs RNase H-mediated cleavage of the mRNA transcript encoded by the muscle disease gene in the cell.

[0061] In some implementations, the oligonucleotide is a mixed polymeric oligonucleotide.

[0062] In some implementations, the oligonucleotide is an RNAi oligonucleotide that promotes RNAi-mediated cleavage of the mRNA transcript encoded by the muscle disease gene.

[0063] In some embodiments, the 2'-modified nucleoside is selected from: 2'-O-methyl, 2'-fluoro(2'-F), 2'-O-methoxyethyl(2'-MOE), and 2',4'-bridged nucleoside.

[0064] In some embodiments, the one or more modified nucleosides are 2',4'-bridged nucleosides.

[0065] In some implementations, the oligonucleotide is a phosphodiamidomorpholine oligomer.

[0066] In some embodiments, the antibody is covalently linked to the molecular payload via a cleavable adapter. In some embodiments, the cleavable adapter comprises a valine-citrulline sequence.

[0067] In some embodiments, the antibody is covalently linked to the molecular payload, the covalent linking being achieved through conjugation with lysine or cysteine ​​residues of the antibody.

[0068] In some implementations, regulating the expression or activity of muscle disease genes includes reducing the expression of RNA and / or proteins.

[0069] Another aspect of this disclosure relates to a method for regulating the expression or activity of muscle disease genes in cells, the method comprising contacting the cells with a complex described herein for promoting the internalization of molecular loads into the cells in an effective amount, optionally wherein the cells are muscle cells.

[0070] In some embodiments, the muscle disease is selected from the following: adult Pompey's disease, central nucleus myopathy (CNM), Dechené muscular dystrophy, facioscapulohumeral muscular dystrophy (FSHD), familial hypertrophic cardiomyopathy, progressive ossifying fibrous dysplasia (FOP), Friedreich ataxia (FRDA), inclusion body myopathy 2, Lane distal myopathy, myofibril myopathy, congenital myotonia (autosomal dominant form, Thomson's disease), type I myotonia, type II myotonia, myotubular myopathy, oculopharyngeal muscular dystrophy, and congenital paramyotonia.

[0071] Another aspect of this disclosure relates to a method of treating a subject suffering from a muscle disease, the method comprising administering to the subject an effective amount of the complex described herein, wherein the muscle disease is optionally selected from the following: adult Pompey's disease, central nucleus myopathy (CNM), Dechené muscular dystrophy, facioscapulohumeral muscular dystrophy (FSHD), familial hypertrophic cardiomyopathy, progressive ossifying fibrous dysplasia (FOP), Friedreich ataxia (FRDA), inclusion body myopathy 2, Lane distal myopathy, myofibril myopathy, congenital myotonia (autosomal dominant form, Thomson's disease), type I myotonia, type II myotonia, myotubular myopathy, oculopharyngeal muscular dystrophy, and congenital paramyotonia. Attached Figure Description

[0072] Figure 1 A non-restrictive schematic diagram is depicted, illustrating the effect of transfection of Hepa 1-6 cells with an antisense oligonucleotide targeting DMPK (ASO300) on DMPK expression levels relative to carrier transfection.

[0073] Figure 2A A non-limiting schematic diagram is depicted, illustrating the HIL-HPLC trace obtained during the purification of a muscle-targeting complex containing an anti-transferrin receptor antibody covalently linked to a DMPK antisense oligonucleotide.

[0074] Figure 2B Unrestricted images depicting the SDS-PAGE analysis of the muscle-targeting complex.

[0075] Figure 3 A non-limiting schematic diagram is depicted, illustrating the ability of the muscle-targeting RI7217Fab antibody-oligonucleotide complex (DTX-C-008) containing ASO300 to reduce DMPK expression levels.

[0076] Figures 4A to 4EA non-limiting schematic diagram is depicted, illustrating the ability of the muscle-targeting RI7 217Fab antibody-oligonucleotide complex (DTX-C-008) containing ASO300 to reduce DMPK expression levels in mouse muscle tissue in vivo, relative to load-based treatment, treatment with naked ASO300, or treatment with the control non-targeted complex (DTX-C-007). (N = 3 C57Bl / 6WT mice).

[0077] Figures 5A to 5B A non-limiting schematic diagram is provided, illustrating the tissue selectivity of the muscle-targeting RI7 217Fab antibody-oligonucleotide complex (DTX-C-008) containing ASO300. The muscle-targeting complex (DTX-C-008) containing ASO300 did not reduce DMPK expression levels in mouse brain or spleen tissue in vivo, relative to load-bearing treatment, treatment with naked ASO300, or treatment with the control non-targeting complex (DTX-C-007). (N = 3 C57Bl / 6WT mice).

[0078] Figures 6A to 6F A non-limiting schematic diagram is depicted, illustrating the ability of the muscle-targeting RI7 217Fab antibody-oligonucleotide complex (DTX-C-008) containing ASO300 to reduce DMPK expression levels in mouse muscle tissue in vivo, relative to load-based treatment, treatment with naked ASO300, or treatment with the control non-targeted complex (DTX-C-007). (N = 5 C57Bl / 6WT mice).

[0079] Figures 7A to 7L A non-limiting schematic diagram is depicted, illustrating the ability of a muscle-targeting antibody-oligonucleotide complex (DTX-C-012) containing ASO300 covalently linked to an anti-hTfR antibody in vivo to reduce DMPK expression levels in cynomolgus monkey muscle tissue, relative to load treatment (saline) and compared to naked DMPKASO (ASO300). (N = 3 male cynomolgus monkeys).

[0080] Figures 8A to 8B A non-limiting schematic diagram is depicted, illustrating the ability of a muscle-targeting antibody-oligonucleotide complex (DTX-C-012) containing ASO300 covalently linked to an anti-hTfR antibody to reduce DMPK expression levels in cynomolgus smooth muscle tissue in vivo, relative to load treatment (saline) and compared to naked DMPKASO (ASO300). (N = 3 male cynomolgus monkeys).

[0081] Figures 9A to 9DA non-limiting schematic diagram is provided, illustrating the tissue selectivity of a muscle-targeting antibody-oligonucleotide complex (DTX-C-012) comprising ASO300 covalently linked to an anti-hTfR antibody. The muscle-targeting complex comprising DMPK-ASO did not reduce DMPK expression levels in the kidney, brain, or spleen tissues of cynomolgus monkeys in vivo, relative to load treatment (N = 3 male cynomolgus monkeys).

[0082] Figure 10 Normalized DMPK mRNA tissue expression levels were shown across several tissue types in cynomolgus monkeys (N = 3 male cynomolgus monkeys).

[0083] Figures 11A to 11B A non-limiting schematic diagram is depicted, illustrating the ability of DTX-C-008 to reduce DMPK expression levels in mouse muscle tissue in vivo for up to 28 days after administration of a muscle-targeting RI7 217Fab antibody-oligonucleotide complex (DTX-C-008) containing ASO300, relative to carrier treatment (saline) and naked DMPKASO (ASO300).

[0084] Figure 12 A single dose of the muscle-targeting complex (DTX-C-012) containing ASO300 covalently linked to an anti-hTfR antibody was demonstrated to be safe and well-tolerated in cynomolgus monkeys (N = 3 male cynomolgus monkeys).

[0085] Figures 13A to 13B A non-limiting schematic diagram is depicted, illustrating the ability of DTX-C-008 to reduce DMPK expression levels in mouse muscle tissue in vivo for up to 12 weeks after administration of a muscle-targeting RI7 217Fab antibody-oligonucleotide complex (DTX-C-008) containing ASO300, compared to load treatment (PBS) and control IgG2a Fab antibody-oligonucleotide complex (DTX-C-007) and naked DMPK ASO (ASO300). (N = 5 C57Bl / 6WT mice).

[0086] Figures 14A to 14B A non-limiting schematic diagram is depicted, illustrating the ability of the muscle-targeting RI7 217Fab antibody-oligonucleotide complex (DTX-C-008) containing ASO300 to target nuclear mutant DMPK RNA in a mouse model (N = 6 mice).

[0087] Figures 15A to 15BA non-limiting schematic diagram is depicted, illustrating the ability of a muscle-targeting RI7 217Fab antibody-ASO complex (DTX-actin) containing oligonucleotides targeting actin to dose-dependently reduce actin expression levels and functional grades of myotonia in muscle tissue. (N = 2 HSA) LR Mice).

[0088] Figures 16A to 16C A non-limiting schematic diagram is depicted, showing that the muscle-targeting RI7 217Fab antibody-oligonucleotide complex (DTX-C-008) containing ASO300 significantly reduces the prolonged QTc interval in a mouse model to validate the functional correction of arrhythmias in a DM1 cardiac model (N = 10 mice).

[0089] Figures 17A to 17B A non-limiting schematic diagram is provided, illustrating that a muscle-targeting antibody-oligonucleotide complex (DTX-C-012) containing an ASO300 antisense oligonucleotide covalently linked to an anti-hTfR antibody can reduce DMPK expression levels in human cells from DM1 patients and correct splicing of the DMPK-specific target gene (Bin1). (N=3)

[0090] Figure 18 A non-limiting schematic diagram is depicted, illustrating the ability of a muscle-targeting complex (anti-TfR antibody-FM10) containing an anti-TfR1 Fab (RI7 217) conjugated with an anti-FM10 antisense oligonucleotide to reduce the expression levels of downstream DUX4 genes (ZSCAN4, MBD3L2, TRIM43) in human U-2OS cells relative to the naked FM10 antisense oligonucleotide.

[0091] Figure 19 A non-limiting schematic diagram is depicted, showing that the anti-transferrin receptor muscle-targeting complex containing exon-23 skipping phosphodiamide morpholino oligomer (PMO) dose-dependently enhances the ability of exon skipping in muscle tissue of an MDX mouse model.

[0092] Figures 20A to 20B A non-limiting schematic diagram is depicted, illustrating the ability of an anti-transferrin receptor muscle-targeting complex containing exon-23 skip-read PMO to dose-dependently enhance myotrophin in skeletal muscle (quadriceps) of an MDX mouse model.

[0093] Figures 21A to 21E A non-limiting schematic diagram is depicted, illustrating the improved functional performance of an anti-transferrin receptor muscle-targeting complex containing exon-23 skip-read PMO in an MDX mouse model. Figure 21A , 21B (21C and 21D) and reduced creatine kinase levels ( Figure 21E The ability to (**p<0.01; ***p<0.001; ****p<0.0001).

[0094] Figures 22A to 22C A non-restrictive schematic diagram is provided, illustrating the dose-response of selected antisense oligonucleotides (DMPK-ASO-1, DMPK-ASO-2, and DMPK-ASO-3) in human DM1 myotubes in response to DMPK knockdown. ASO300 was used as a control. All tested oligonucleotides showed activity in response to DMPK knockdown. Statistical analysis: One-way ANOVA versus Tukey's HSD post-hoc test vs. naked ASO300 treatment; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0095] Figures 23A to 23B A non-restrictive schematic diagram is provided, illustrating 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 showed activity in DMPK knockdown.

[0096] Figure 24 The serum stability of the linker for connecting anti-TfR antibodies to molecular payloads (e.g., oligonucleotides) over time after intravenous administration in multiple species is shown.

[0097] Figures 25A to 25F The binding of humanized anti-TfR Fab to human TfR1 (hTfR1) or cynomolgus monkey TfR1 (cTfR1) as measured by ELISA is shown. Figure 25A The binding of the humanized 3M12 variant with hTfR1 is shown. Figure 25B The binding of the humanized 3M12 variant to cTfR1 is shown. Figure 25C The binding of the humanized 3A4 variant with hTfR1 is shown. Figure 25D The binding of the humanized 3A4 variant with cTfR1 is shown. Figure 25E The binding of the humanized 5H12 variant to hTfR1 is shown. Figure 25F The binding of the humanized 5H12 variant to hTfR1 is shown.

[0098] Figure 26This study demonstrates quantitative cellular uptake of anti-TfR Fab conjugates into rhabdomyosarcoma (RD) cells. The molecular load in the test conjugate is a DMPK-targeting oligonucleotide, and uptake is facilitated by the specified anti-TfR Fab. The assay also included conjugates with either a negative control Fab (anti-mouse TfR) or a positive control Fab (anti-human TfR1). Cells were incubated with the specified conjugate at a concentration of 100 nM for 4 hours. Cellular uptake was measured by mean Cypher5e fluorescence.

[0099] Figures 27A to 27F The binding of oligonucleotide-conjugated or unconjugated humanized anti-TfR Fab to human TfR1 (hTfR1) and cynomolgus monkey TfR1 (cTfR1) was shown by ELISA. Figure 27A The binding of humanized 3M12 variants, alone or conjugated with DMPK-targeting oligonucleotides, to hTfR1 is shown. Figure 27B The binding of humanized 3M12 variants, alone or conjugated with DMPK-targeting oligonucleotides, to cTfR1 is shown. Figure 27C The binding of humanized 3A4 variants, alone or conjugated with DMPK-targeting oligonucleotides, to hTfR1 is shown. Figure 27D The binding of humanized 3A4 variants, alone or conjugated with DMPK-targeting oligonucleotides, to cTfR1 is shown. Figure 27E The binding of humanized 5H12 variants, alone or conjugated with DMPK-targeting oligonucleotides, to hTfR1 is shown. Figure 27F The binding of humanized 5H12 variants, alone or conjugated with DMPK-targeting oligonucleotides, to cTfR1 is shown. The respective EC50 values ​​are also shown. 50 value.

[0100] Figure 28 The image shows DMPK expression in RD cells treated with DMPK-targeting oligonucleotides, relative to cells treated with PBS. The treatment lasted for 3 days. The DMPK-targeting oligonucleotides were delivered to the cells either as free oligonucleotides (gymnotic uptake, “free”) or using a transfection reagent (“trans”).

[0101] Figure 29 DMPK expression in RD cells treated with various concentrations of conjugates containing a specified humanized anti-TfR antibody conjugated to a DMPK-targeting oligonucleotide (ASO300). Treatment lasted for 3 days. ASO300 delivered using a transfection agent was used as a control (labeled "Trans").

[0102] Figure 30The results of Atp2a1 splicing correction by anti-TfR1 antibody-oligonucleotide conjugate (Ab-ASO) measured in the gastrocnemius muscle in a DM1 HSA-LR mouse model are shown. The anti-TfR antibody used was RI7 217, and the oligonucleotide targeted human skeletal muscle actin.

[0103] Figure 31 Splicing corrections in more than 30 different RNAs associated with DM1, measured in the gastrocnemius muscle of HSA-LR mice treated with an anti-TfR1 antibody-oligonucleotide (Ab-ASO) conjugate or saline, are shown. The anti-TfR antibody used was RI7217, and the oligonucleotide targeted human skeletal muscle actin.

[0104] Figure 32 This study illustrates splicing disorder in the quadriceps, gastrocnemius, or tibialis anterior muscles of HSA-LR mice treated with anti-TfR1 antibody-oligonucleotide conjugates (Ab-ASO) or saline. Data represent [data missing]. Figure 31 The diagram shows multiple splicing disorder detected in more than 30 RNAs.

[0105] Figure 33 The grades of myotonia, gastrocnemius, and tibialis anterior muscles in HSA-LR mice treated with saline, unconjugated oligonucleotides (ASO), or anti-TfR1 antibody-oligonucleotide conjugates (Ab-ASO) are shown. Myotonia was measured by electromyography (EMG) and graded as 0, 1, 2, or 3 based on the frequency of myotonic discharges.

[0106] Figure 34 The skipping of exon 51 in human DMD myotubes is shown, which is promoted by DMD exon 51 skipping oligonucleotides (PMOs). Cells were treated with naked PMOs or with PMOs conjugated with anti-TfR1 Fab (Ab-PMOs).

[0107] Figure 35 This study demonstrates a dose-dependent increase in myoglobin expression in the quadriceps muscle of MDX mice after treatment with an anti-mouse TfR1 (RI7 217) conjugated to an oligonucleotide (PMO) targeting exon 23, as measured by Western blot analysis for myoglobin, with α-actin as a loading control. Standards were generated using both pooled wild-type and pooled MDX proteins. Percentages represent the amount of WT protein incorporated into the sample.

[0108] Figure 36The study demonstrates the quantification of intramuscular myoglobin levels in mdx mice after treatment with multiple doses of anti-mouse TfR (RI7 217) conjugated with oligonucleotides (PMO) targeting exon 23.

[0109] Figure 37 Immunofluorescence staining images of quadriceps muscles from wild-type (WT) mice treated with saline or from mdx mice treated with saline, naked oligonucleotides, or oligonucleotides conjugated with anti-mouse TfR1 (RI7 217) are shown.

[0110] Figures 38A to 38B This demonstrates the use of bare FM10 ( ) within a certain concentration range. Figure 38A ) or FM10 conjugated with anti-TfR1 ( Figure 38B Expression of MBD3L2, TRIM43 and ZSCAN4 transcripts in myotubes from FSHD patients treated with [the treatment].

[0111] Figure 39 Data show that conjugates containing specified anti-TfRFabs (3M12 VH3 / VK2, 3M12 VH4 / VK3, and 3A4 VH3N54S / VK4) conjugated to DMD exon skipping oligonucleotides result in enhanced exon skipping in myotubes of DMD patients compared to naked DMD exon skipping oligonucleotides.

[0112] Figures 40A to 40E The in vivo activity of conjugates containing specific anti-TfR Fabs (control, 3M12VH3 / VK2, 3M12 VH4 / VK3, and 3A4 VH3 N54S / VK4) conjugated to DMPK-targeting oligonucleotides in reducing DMPK mRNA expression was demonstrated in mice expressing human TfR1 (hTfR1 knock-in mice). Figure 40A The experimental design (e.g., IV dose, dosing frequency) is shown. Fourteen days after the first dose, the tibialis anterior muscle of mice was measured. Figure 40B ), gastrocnemius muscle ( Figure 40C ),heart( Figure 40D ) and diaphragm ( Figure 40E DMPK mRNA levels in ).

[0113] Figures 41A to 41C The study demonstrated that conjugates containing anti-TfR antibodies conjugated to DMPK-targeting oligonucleotides corrected splicing and reduced lesions in CM-DM1-32F primary cells expressing DMPK mutant mRNA containing 380 CUG repeats. Figure 41A The conjugate was shown to reduce DMPK mRNA expression in mutants. Figure 41B The conjugate correction of exon 11 splicing of BIN1 is shown. Figure 41C Images from fluorescence in situ hybridization (FISH) analysis and quantification of these images are shown, indicating that the conjugation reduced nuclear lesions formed by mutant DMPK mRNA. Figure 41C In the microscopic images shown above, light circles indicate cell nuclei, and bright spots within the nuclei of DM1 cells (three microscopic images on the right) indicate CUG lesions.

[0114] Figure 42 ELISA measurements of the binding of anti-TfR Fab 3M12 VH4 / Vk3 to recombinant human (circle), cynomolgus monkey (square), mouse (upward triangle), or rat (downward triangle) TfR1 protein at Fab concentrations ranging from 230 pM to 500 nM are shown. The results demonstrate the reactivity of anti-TfR Fab with human and cynomolgus monkey TfR1. No binding with recombinant TfR1 in mice or rats was observed. Data are shown as relative fluorescence units normalized to baseline.

[0115] Figure 43 ELISA results testing the affinity of anti-TfR Fab 3M12 VH4 / Vk3 for recombinant human TfR1 or TfR2 at concentrations ranging from 230 pM to 500 nM Fab are presented. Data are given as relative fluorescence units normalized relative to baseline. The results indicate that Fab does not bind to recombinant human TfR2.

[0116] Figure 44 The linker used to connect anti-TfR Fab 3M12 VH4 / Vk3 to the control antisense oligonucleotide is shown to exhibit serum stability during incubation in PBS or in the serum of rats, mice, cynomolgus monkeys or humans for 72 hours.

[0117] Figure 45 The study demonstrated that a conjugate containing an anti-TfR Fab 3M12VH4 / 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. This conjugate exhibited superior activity in inhibiting the DUX4 transcriptome compared to the unconjugated DUX4-targeting oligonucleotide.

[0118] Figures 46A to 46B The dose-response curve for gene knockdown is shown. Figure 46AMBD3L2 knockdown was demonstrated in C6(AB1080) immortalized FSHD1 cells treated with a conjugate containing an anti-TfR Fab 3M12VH4 / Vk3 conjugated with a DUX4-targeting oligonucleotide (SEQ ID NO:147). Figure 46B The knockdown of MBD3L2, TRIM43, and ZSCAN4 in myotubes of FSHD patients treated with a conjugate containing an anti-TfR Fab 3M12 VH4 / Vk3 conjugated with a DUX4-targeting oligonucleotide (SEQ ID NO:147) was demonstrated. Figure 46B include Figure 46A The MBD3L2 data shown is shown.

[0119] Figures 47A to 47C The image shows the quadriceps muscle of HSA-LR mice treated with a loader, a single dose of unconjugated ASO, or a single dose of anti-TfR1 antibody-ASO conjugate (Ab-ASO). Figure 47A ), gastrocnemius muscle ( Figure 47B ) and tibialis anterior muscle ( Figure 47C The EMG myotonia level was determined using the anti-TfR1 antibody RI7 217Fab and the oligonucleotide targeting human skeletal actin (ACTA1).

[0120] Figure 48 The results show that, following a single dose of naked ASO or a comparable dose of anti-TFR1 antibody-ASO conjugate (Ab-ASO), HSA levels in mice compared to those treated with the load cell were significantly reduced. LR Human ACTA1 expression in DM1 mice was measured by qPCR. The anti-TfR1 antibody used was RI7 217Fab, and the oligonucleotide targeted human skeletal actin (ACTA1).

[0121] Figures 49A to 49C The results show that, following a single dose of 10 mg / kg naked ASO, 20 mg / kg naked ASO, or an equivalent dose of anti-TFR antibody-ASO conjugate (Ab-ASO), HSA levels were lower in mice compared to those treated with the load cell. LR Quadriceps muscle of DM1 mice Figure 49A ), gastrocnemius muscle ( Figure 49B ) and tibialis anterior muscle ( Figure 49C ACTA1 expression in ) . The anti-TfR1 antibody used was RI7 217Fab and the oligonucleotide targeted human skeletal actin (ACTA1). (*p<0.05; ***p<0.001).

[0122] Figures 50A to 50CThe results showed that, 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 RI7217Fab conjugated to ASO (Ab-ASO), the effects on quadriceps muscle in wild-type (WT) and mdx mice were significant. Figure 50A ),heart( Figure 50B ) and diaphragm ( Figure 50C Quantification of exon 23 skipping. Few or no exon 23 skipping was observed in tissues from WT mice or mdx mice treated with saline or unconjugated ASO, while 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).

[0123] Figures 51A to 51D The measurement of myoglobin in the quadriceps muscle of mdx mice is shown after administration of a single dose of an unconjugated oligonucleotide (ASO) with exon 23 skipping in induced DMD or a conjugate containing anti-TfR1 RI7217 Fab conjugated to ASO (Ab-ASO). Figure 51A Western blots of gamma-gamma and α-actin in muscle tissue were shown two weeks after injection of ASO or Ab-ASO. Figure 51B It shows the effect of gamma glycoprotein relative to wild-type muscle. Figure 51A Quantification of gamma proteins in Western blot. Figure 51C Western blots of glycoprotein and α-actin in muscle tissue four weeks after injection of ASO or Ab-ASO are shown. Figure 51D It shows the effect of gamma glycoprotein relative to wild-type muscle. Figure 51C Quantification of gamma proteins in Western blot. Figure 51A and 51C The standard curve was generated by combining tissue samples from wild-type (WT) and mdx mice, and the WT percentage represents the amount of WT protein incorporated into each sample. (*p<0.05; ns, not significant)

[0124] Figures 52A to 52D The measurement of myoglobin in the myocardium of mdx mice is shown after administration of a single dose of an unconjugated oligonucleotide (ASO) with exon 23 skipping in induced DMD or a conjugate containing anti-TfR1 RI7217 Fab conjugated to ASO (Ab-ASO). Figure 52A Western blots of gamma-gamma and α-actin in muscle tissue were shown two weeks after injection of ASO or Ab-ASO. Figure 52BIt shows the effect of gamma proteins relative to wild-type muscle on Figure 52A Quantification of gamma proteins in Western blot. Figure 52C Western blots of glycoprotein and α-actin in muscle tissue four weeks after injection of ASO or Ab-ASO are shown. Figure 52D It shows the effect of gamma proteins relative to wild-type muscle on Figure 52C Quantification of gamma proteins in Western blot. Figure 52A and 52C The standard curve was generated by combining tissue samples from wild-type (WT) and mdx mice, and the WT percentage represents the amount of WT protein incorporated into each sample. (*p<0.05, ****p<0.0001)

[0125] Figures 53A to 53D The measurement of myoglobin in the diaphragm of mdx mice is shown after administration of a single dose of an unconjugated oligonucleotide (ASO) with exon 23 skipping in induced DMD or a conjugate containing anti-TfR1 RI7217 Fab conjugated to ASO (Ab-ASO). Figure 53A Western blots of gamma-gamma and α-actin in muscle tissue were shown two weeks after injection of ASO or Ab-ASO. Figure 53B It shows the effect of gamma proteins relative to wild-type muscle on Figure 53A Quantification of glycoprotein in Western blot. Figure 53C Western blots of glycoprotein and α-actin in muscle tissue four weeks after injection of ASO or Ab-ASO are shown. Figure 53D It shows the effect of gamma proteins relative to wild-type muscle on Figure 53C Quantification of gamma proteins in Western blot. Figure 53A and 53C The standard curves in the data were generated by combining tissue samples from wild-type (WT) and mdx mice, and the WT percentage represents the amount of WT protein incorporated into each sample. (**p<0.01,***p<0.001)

[0126] Figures 54A to 54C The study demonstrated the effects on quadriceps muscle development in wild-type (WT) or mdx mice two or four weeks after administration of a single dose of saline, unconjugated exon 23 skipped-read oligonucleotide (ASO), or a conjugate containing anti-TfR1 RI7217 Fab conjugated to ASO (Ab-ASO). Figure 54A ), diaphragm ( Figure 54B ) and heart ( Figure 54C Quantification of the amount of oligonucleotides (ASO) applied in the process.

[0127] Figure 55 The nonhuman primate plasma levels of DUX4-targeting oligonucleotide (SEQ ID NO: 147) over time following administration of 30 mg / kg of unconjugated ('naked') oligonucleotide or conjugates at doses of 3, 10, or 30 mg / kg of oligonucleotide equivalents, are shown. The conjugates ('Fab-oligonucleotide conjugates') contain anti-TfR1 Fab3M12 VH4 / Vk3 covalently linked to the DUX4-targeting oligonucleotide.

[0128] Figure 56 The tissue levels of DUX4-targeting oligonucleotide (SEQ ID NO: 147) measured in non-human primate muscle tissue samples two weeks after administration of 30 mg / kg of unconjugated ('naked') oligonucleotide or conjugates at doses of 3, 10, or 30 mg / kg of oligonucleotide equivalents, are shown. The conjugates ('Fab-oligonucleotide conjugates') contain anti-TfR1Fab 3M12 VH4 / Vk3 covalently linked to the DUX4-targeting oligonucleotide.

[0129] Figure 57 The tissue levels of DUX4-targeting oligonucleotide (SEQ ID NO: 147) measured one week after administration of 30 mg / kg of unconjugated oligonucleotide ('oligonucleotide') or conjugates at doses of 3, 10, or 30 mg / kg of oligonucleotide equivalents, collected by biopsy (5 bars on the left) or two weeks after administration by autopsy (5 bars on the right), said conjugate ('conjugate') containing anti-TfR1 Fab3M12VH4 / Vk3 covalently linked to the DUX4-targeting oligonucleotide are shown.

[0130] Figures 58A to 58B The anterior tibialis muscle of HSA-LR mice treated with a single dose of anti-TfR1 antibody-oligonucleotide (Ab-ASO) conjugate or saline is shown. Figure 58A ) or quadriceps ( Figure 58B The splicing correction of more than 30 different RNAs known to be misspliced ​​in DM1 patients was measured in the study. The anti-TfR1 antibody used was RI7 217Fab and the oligonucleotide targeted skeletal actin (ACTA1).

[0131] Figure 59 This illustrates exon 53 skipping in DMD patient cells carrying exon 52 deletions after gymnotic uptake of exon 53 skipping oligonucleotides within a certain concentration range.

[0132] Figure 60The exon 53 skipping percentage in DMD patient cells carrying DMD exon 52 deletion is shown after treatment with multiple concentrations of exon 53 skipping PMO, which is neither linked to an antibody (“naked ASO”) nor covalently linked to anti-TfR1 Fab (“anti-TfR1 Fab-ASO complex”). Detailed Implementation

[0133] Some aspects of this disclosure relate to the understanding that while certain molecular payloads (e.g., oligonucleotides, peptides, small molecules) may have beneficial effects in muscle cells, effectively targeting such cells has proven challenging. As described herein, this disclosure provides complexes comprising a muscle target covalently linked to a molecular payload to overcome this challenge. In some embodiments, the complexes are particularly useful for delivering molecular payloads that regulate the expression or activity of target genes in muscle cells, for example, in subjects who have or are suspected of having a muscle disease. For example, in some embodiments, the complexes can be used to treat subjects with rare muscle diseases, including Pompey's disease, central nucleus myopathy, progressive ossifying fibrous dysplasia, Friedreich's ataxia, or Dichené muscular dystrophy. In some embodiments, depending on the condition to be treated, different molecular payloads may be used in such complexes. For example, if an underlying mutation causes a splicing defect, an oligonucleotide or other payload may be used to correct that splicing defect (e.g., an oligonucleotide that inhibits exon skipping or promotes alternative splicing). If the potential mutation results in a gain-of-function allele, oligonucleotides (e.g., RNAi, PMO, ASO-spacer polymers) can be used to suppress the expression or activity of the allele. In some embodiments, such as when the mutation results in a loss-of-function allele, the payload may contain an expression construct, such as a wild-type form for expressing the allele. In some embodiments, the payload may contain a mechanism for correcting the potential defect, for example, through gene editing (e.g., an expression construct that directs nucleic acid or encodes a gene-editing enzyme).

[0134] Other aspects of this disclosure are provided below, including a description of the qualified terminology.

[0135] I. Definition

[0136] Application: As used herein, the term “application” means providing a compound to a subject in a physiologically and / or (e.g., and) pharmacologically available manner (e.g., to treat a condition in the subject).

[0137] Approximately: As used herein, the term “approximately” or “about”, when applied to one or more intended values, refers to a value similar to the stated reference value. In some embodiments, the term “approximately” or “about” refers to a range of values ​​falling within (greater than or less than) 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in any direction of the stated reference value, unless otherwise stated or otherwise apparent from the context (unless such a figure exceeds 100% of the possible value).

[0138] Antibody: As used herein, the term "antibody" refers to a polypeptide comprising 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, Fab' fragment, F(ab')2 fragment, Fv fragment, or scFv fragment. In some embodiments, the antibody is a nanobody derived from a camel antibody or a nanobody derived from a shark antibody. In some embodiments, the antibody is a biantibody. In some embodiments, the antibody comprises a framework having a human germline sequence. In another embodiment, the antibody comprises a heavy chain constant domain selected from IgG, IgG1, IgG2, IgG2A, IgG2B, IgG2C, IgG3, IgG4, IgA1, IgA2, IgD, IgM, and IgE constant domains. In some embodiments, the antibody comprises a heavy (H) chain variable region (hereinafter referred to as VH) and / or (e.g., and) a light (L) chain variable region (hereinafter referred to as VL). In some embodiments, the antibody comprises a constant domain, such as an Fc region. An immunoglobulin constant domain refers to a heavy chain or light chain constant domain. The amino acid sequences and functional variations of the human IgG heavy and light chain constant domains are known. Regarding the heavy chain, in some embodiments, the heavy chain of the antibody described herein may be an alpha (α), delta (Δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain. In some embodiments, the heavy chain of the antibody described herein may comprise a human alpha (α), delta (Δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain. In one specific embodiment, the antibody described herein comprises human γ1CH1, CH2, and / or (e.g., and) CH3 domains. In some embodiments, the amino acid sequence of the VH domain comprises the amino acid sequence of the human gamma (γ) heavy chain constant region, such as any known in the art. Non-limiting examples of human constant region sequences have been described in the art, for example, see U.S. Patent No. 5,693,780 and Kabat E. Aetal., (1991), ibid. In some embodiments, the VH domain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or at least 99% identity with any variable chain constant region provided herein. In some embodiments, the antibody is modified, for example, by 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, glycosylphosphatidylinositylation (GPI anchoring attachment), and / or (e.g., and) phosphoglycosylation. In some embodiments, one or more sugar or carbohydrate molecules are monosaccharides, disaccharides, oligosaccharides, or polysaccharides. In some embodiments, one or more sugar or carbohydrate molecules are branched oligosaccharides or branched polysaccharides. In some embodiments, one or more sugar or carbohydrate molecules comprise mannose units, glucose units, N-acetylglucosamine units, N-acetylglucosamine units, galactose units, fucose units, or phospholipid units. In some embodiments, the antibody is a construct comprising a polypeptide containing one or more antigen-binding fragments of this disclosure linked to a linker polypeptide or an immunoglobulin constant domain. The linker polypeptide comprises two or more amino acid residues linked by peptide bonds and is used to link one or more antigen-binding moieties. Examples of linker peptides 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). Additionally, antibodies can be part of larger immunoadhesion molecules, which are formed by the covalent or non-covalent association of antibodies or antibody portions with one or more other proteins or peptides. Some examples of such immunoadhesion molecules include the use of streptavidin core regions to prepare tetrameric scFv molecules (Kipriyanov, SM, et al. (1995) Human Antibodies and Hybridomas 6:93-101), and the use of cysteine ​​residues, labeled peptides, and C-terminal multihistidine tags to prepare divalent and biotinylated scFv molecules (Kipriyanov, SM, et al. (1994) Mol. Immunol. 31:1047-1058).

[0139] CDR: As used herein, the term "CDR" refers to the complementarity determining region within the variable sequence of an antibody. A typical antibody molecule contains 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), within which are interspersed more conserved regions called "framework regions" (FRs). Each VH and VL typically consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The extent of the frame region and CDRs can be precisely identified using methods known in the art, such as the Kabat definition, IMGT definition, Chothia definition, AbM definition, and / or (e.g., and) contact definition (all of which are well known in the art). See, for example, Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIHPublication No. 91-3242; the international ImMunoGeneTics information 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); 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 (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. The CDR used herein can refer to a CDR defined by any method known in the art. Two antibodies having the same CDR means that the amino acid sequence of that CDR is identical for both antibodies, as determined by the same method (e.g., IMGT definition).

[0140] Each variable region of the heavy and light chains contains three CDRs, designated CDR1, CDR2, and CDR3 for each variable region. The term "CDR group" used herein refers to a group of three CDRs capable of binding the antigen that appear within a single variable region. The exact boundaries of these CDRs have been defined differently depending on the system. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides a definitive residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries defining three CDRs. These CDRs may be referred to as Kabat CDRs. Sub-regions of a CDR may be designated as L1, L2, and L3 or H1, H2, and H3, where “L” and “H” designate the light chain and heavy chain regions, respectively. These regions may be referred to as Chothia CDRs, which have boundaries that overlap with Kabat CDRs. Padlan (FASEB J.9:133-139 (1995)) and MacCallum (J MolBiol 262 (5):732-45 (1996)) have described the definition of Kabat CDRs. Other CDR boundaries that overlap with CDRs. Some other CDR boundary definitions may not strictly follow one of the systems described above, but still overlap with Kabat CDRs, although they can be shortened or lengthened based on predictions or experimental findings that specific residues or groups of residues, or even the entire CDR, do not significantly affect antigen binding. The methods used in this paper can utilize CDRs defined according to any of these systems. Some examples of CDR definition systems are provided in Table 6.

[0141] Table 6. CDR Definition

[0142]

[0143] 1 the international ImMunoGeneTics information imgt.org,Lefranc,M.-P.et al.,Nucleic Acids Res.,27:209-212(1999)

[0144] 2Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242

[0145] 3 Chothia et al., J.Mol.Biol.196:901-917(1987))

[0146] 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 one or more CDR regions of VH and / or (e.g., and) VL are replaced by CDR sequences from another species, such as an antibody having mouse heavy and light chain variable regions and in which one or more mouse CDRs (e.g., CDR3) have been replaced by human CDR sequences.

[0147] 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 a mouse heavy and light chain variable region linked to a human constant region.

[0148] Complementarity: As used herein, the term “complementarity” refers to the ability to precisely pair between two nucleotides or groups of nucleotides. Specifically, complementarity is a term characterizing the degree to which hydrogen-bonded pairing results in binding between two nucleotides or groups of nucleotides. For example, if a base at a position of an oligonucleotide is able to hydrogen bond with a base at a corresponding position of a target nucleic acid (e.g., mRNA), the bases at that position are considered complementary to each other. Base pairing can include both canonical 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 base (A) is complementary to a thymidine base (T) or a uracil base (U), a cytosine base (C) is complementary to a guanosine base (G), and universal bases such as 3-nitropyrrole or 5-nitroindole can hybridize with any A, C, U, or T and are considered complementary. Inosine (I) is also considered a universal base in the art and is believed to be complementary to any A, C, U or T.

[0149] Conservative amino acid substitution: As used herein, “conservative amino acid substitution” refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein to which the substitution is made. Variants can be prepared according to methods known to those skilled in the art for altering polypeptide sequences, methods which can be found, for example, in compilations of such methods: 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, FMAusubel, et al., eds., John Wiley & Sons, Inc., New York. Conservative substitution of amino acids includes substitutions between 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.

[0150] Covalent Linkage: As used herein, the term "covalent linkage" refers to the feature of two or more molecules being linked together by at least one covalent bond. In some embodiments, two molecules may be covalently linked together by a single bond, such as a disulfide bond or disulfide bridge, acting as a joint between the molecules. However, in some embodiments, two or more molecules may be covalently linked together by a molecule acting as a joint, which links the two or more molecules together by multiple covalent bonds. In some embodiments, the joint may be a cutterable joint. However, in some embodiments, the joint may be an incutterable joint.

[0151] Cross-reactivity: As used herein and in the case of a target agent (e.g., an antibody), the term "cross-reactivity" refers to the property of a substance to specifically bind with more than one antigen of similar type or class (e.g., multiple homologs, paralogs, or orthologs) with similar affinity or coercivity. For example, in some embodiments, antibodies that are cross-reactive to similar types or classes of human and non-human primate antigens (e.g., human transferrin receptor and non-human primate transferrin receptor) are capable of binding to human and non-human primate antigens with similar affinity or coercivity. In some embodiments, antibodies are cross-reactive to similar types or classes of human and rodent antigens. In some embodiments, antibodies are cross-reactive to similar types or classes of rodent and non-human primate antigens. In some embodiments, antibodies are cross-reactive to similar types or classes of human, non-human primate, and rodent antigens.

[0152] Disease Alleles: As used herein, the term "disease allele" refers to any alternative form (e.g., a mutant form) of a gene whose allele is associated with and / or (e.g., directly or indirectly contributes to or causes disease). Disease alleles may contain gene alterations relative to wild-type (non-disease) alleles, including but not limited to insertions (e.g., disease-associated duplications as described below), deletions, missense mutations, nonsense mutations, and splice site mutations. In some embodiments, disease alleles have loss-of-function mutations. In some embodiments, disease alleles have gain-of-function mutations. In some embodiments, disease alleles encode activating mutations (e.g., proteins with constitutive activity). In some embodiments, disease alleles are recessive alleles with a recessive phenotype. In some embodiments, disease alleles are dominant alleles with a dominant phenotype.

[0153] Disease-Related Repeats: As used herein, the term "disease-related repeat" refers to a repetitive nucleotide sequence at a genomic location, wherein the number of units of the repetitive nucleotide sequence is associated with and / or (e.g., directly or indirectly contributes to or causes a genetic disease). Each repeat unit of a disease-related repeat can be 2, 3, 4, 5, or more nucleotides in length. For example, in some embodiments, the disease-related repeat is a dinucleotide repeat. In some embodiments, the disease-related repeat is a trinucleotide repeat. In some embodiments, the disease-related repeat is a tetranucleotide repeat. In some embodiments, the disease-related repeat is a pentanucleotide repeat. In some embodiments, the disease-related repeat comprises a CAG repeat, a CTG repeat, a CUG repeat, a CGG repeat, a CCTG repeat, or any nucleotide complement thereof. In some embodiments, the disease-related repeat is located in a non-coding region of a gene. However, in some embodiments, the disease-related repeat is located in a coding region of a gene. In some embodiments, the disease-related repeat is amplified from its normal state to a length that directly or indirectly contributes to or causes a genetic disease. In some embodiments, the disease-related repeat is located in RNA (e.g., RNA transcripts). In some embodiments, the disease-related repeats are located in DNA (e.g., chromosomes, plasmids). In some embodiments, the disease-related repeats are amplified in the chromosomes of germ cells. In some embodiments, the disease-related repeats are amplified in the chromosomes of somatic cells. In some embodiments, the disease-related repeats are amplified to the number of repeat units associated with the congenital onset of the disease. In some embodiments, the disease-related repeats are amplified to the number of repeat units associated with the onset of the disease in childhood. In some embodiments, the disease-related repeats are amplified to the number of repeat units associated with the onset of the disease in adulthood.

[0154] Frame: As used herein, the term “frame” or “frame sequence” refers to the sequence remaining after subtracting the CDR from the variable region. Because the exact definition of a CDR sequence can be determined by different systems, the meaning of a frame sequence can be interpreted accordingly. 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 distinguish frames on both the light and heavy chains into four sub-regions (FR1, FR2, FR3, and FR4) on each chain, where CDR1 lies between FR1 and FR2, CDR2 lies between FR2 and FR3, and CDR3 lies between FR3 and FR4. Where no specific sub-region is designated as FR1, FR2, FR3, or FR4, other references to frame regions refer to combinations of FRs within the variable region of a single naturally occurring immunoglobulin chain. As used herein, FR represents one of the four sub-regions, and FRs represent two or more of the four sub-regions constituting a frame region. Human heavy and light chain receptor sequences are known in the art. In one embodiment, an acceptor sequence known in the art can be used in the antibody disclosed herein.

[0155] Human Antibodies: As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies of this disclosure may contain amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced through random or site-specific mutagenesis in vitro or through somatic mutations in vivo), such as in CDRs, particularly CDR3. However, the term "human antibody" as used herein is not intended to include antibodies in which a CDR sequence derived from another mammalian species (e.g., mouse) has been grafted onto a human frame sequence.

[0156] Humanized Antibody: The term "humanized antibody" refers to an antibody containing 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 has 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 a human CDR sequence is introduced into non-human VH and VL sequences to replace the corresponding non-human CDR sequences. In one embodiment, a humanized anti-transferrin receptor antibody and an antigen-binding moiety are provided. Such antibodies can be produced by obtaining mouse anti-transferrin receptor monoclonal antibodies using conventional hybridoma techniques and then humanizing them using in vitro genetic engineering, such as those disclosed in Kasaian et al., PCT Publication No. WO 2005 / 123126A2.

[0157] Internalized cell surface receptors: As used herein, the term "internalized cell surface receptor" refers to a cell surface receptor that is internalized by a cell in response to, for example, an external stimulus (e.g., ligand binding to a receptor). In some embodiments, the internalized cell surface receptor is internalized via endocytosis. In some embodiments, the internalized cell surface receptor is internalized via clathrin-mediated endocytosis. However, in some embodiments, the internalized cell surface receptor is internalized via clathrin-independent pathways, such as phagocytosis, macropinocytosis, pit and raft-mediated uptake, or constitutive clathrin-independent endocytosis. In some embodiments, the internalized cell surface receptor comprises an intracellular domain, a transmembrane domain, and / or (e.g., and) an extracellular domain, which may optionally also include a ligand-binding domain. In some embodiments, the cell surface receptor is internalized by a cell upon ligand binding. In some embodiments, the ligand may be a muscle-targeting agent or a muscle-targeting antibody. In some embodiments, the internalized cell surface receptor is a transferrin receptor.

[0158] Isolated Antibodies: As used herein, “isolated antibodies” are intended to refer to antibodies that are substantially free of other antibodies with different antigen specificities (e.g., isolated antibodies that specifically bind to the transferrin receptor are substantially free of antibodies that specifically bind to antigens other than the transferrin receptor). However, isolated antibodies that specifically bind to the transferrin receptor complex may be cross-reactive with other antigens (e.g., transferrin receptor molecules from other species). Furthermore, isolated antibodies may be substantially free of other cellular material and / or (e.g., and) chemicals.

[0159] Kabat Numbering: The terms “Kabat numbering,” “Kabat definition,” and “Kabat labeling” are used interchangeably in this document. These terms, as accepted in the art, refer to a system for numbering amino acid residues in the variable regions of the heavy and light chains of antibodies or their antigen-binding moieties that are more variable (i.e., hypervariable) than other amino acid residues (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, Department of Health and Human Services, NIH Publication). (No. 91-3242). For the heavy chain variable region, the hypervariable region of CDR1 is amino acids 31 to 35, that of CDR2 is amino acids 50 to 65, and that of CDR3 is amino acids 95 to 102. For the light chain variable region, the hypervariable region of CDR1 is amino acids 24 to 34, that of CDR2 is amino acids 50 to 56, and that of CDR3 is amino acids 89 to 97.

[0160] Molecular payload: As used herein, the term "molecular payload" refers to a molecule or substance that plays a role in regulating biological outcomes. In some embodiments, the molecular payload is linked to or otherwise associated with a muscle target. In some embodiments, the molecular payload is a small molecule, protein, peptide, nucleic acid, or oligonucleotide. In some embodiments, the molecular payload plays a role in regulating the transcription of a DNA sequence, regulating protein expression, or regulating protein activity. In some embodiments, the molecular payload is an oligonucleotide containing a strand with a complementary region of a target gene.

[0161] Muscle disease gene: As used herein, the term "muscle disease gene" refers to a gene that has at least one disease allele associated with and / or (e.g., and) directly or indirectly contributes to or causes muscle disease. In some embodiments, muscle disease is a rare disease, such as 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, muscle disease is a rare disease characterized by affecting fewer than 200,000 people. In some embodiments, muscle disease is a single-gene disease. In some embodiments, muscle disease genes are the genes listed in Table 1.

[0162] Muscle Target: As used herein, the term "muscle target" refers to a molecule that specifically binds to an antigen expressed on a muscle cell. The antigen within or on a muscle cell can be a membrane protein, such as an integrated membrane protein or a peripheral membrane protein. Typically, the muscle target binds specifically to the antigen on the muscle cell, which facilitates the internalization of the muscle target (and any associated molecular payload) into the muscle cell. In some embodiments, the muscle target specifically binds to an internalized cell surface receptor on the muscle and is internalized into the muscle cell via receptor-mediated internalization. In some embodiments, the muscle target is a small molecule, protein, peptide, nucleic acid (e.g., an aptamer), or antibody. In some embodiments, the muscle target is linked to a molecular payload.

[0163] Muscle-targeting antibody: As used herein, the term "muscle-targeting antibody" refers to a muscle-targeting agent that specifically binds to an antigen present within or on muscle cells. In some embodiments, the muscle-targeting antibody specifically binds to an antigen on muscle cells, which facilitates the internalization of the muscle-targeting antibody (and any associated molecular payload) into the muscle cells. In some embodiments, the muscle-targeting antibody specifically binds to an internalized cell surface receptor present on muscle cells. In some embodiments, the muscle-targeting antibody is an antibody that specifically binds to the transferrin receptor.

[0164] Oligonucleotide: As used herein, the term "oligonucleotide" refers to an oligonucleotide compound up to 200 nucleotides in length. Examples of oligonucleotides include, but are not limited to, RNAi oligonucleotides (e.g., siRNA, shRNA), microRNAs, spacer polymers, hybrid polymers, phosphodiesteramide morpholino, peptide nucleic acids, aptamers, and guide nucleic acids (e.g., Cas9 guide RNA). Oligonucleotides can be single-stranded or double-stranded. In some embodiments, an oligonucleotide may contain one or more modified nucleotides (e.g., 2'-O-methyl sugar modification, purine or pyrimidine modification). In some embodiments, an oligonucleotide may contain one or more modified nucleotide links. In some embodiments, an oligonucleotide may contain one or more phosphate thioester links, which may be in the Rp or Sp stereochemical conformation.

[0165] Recombinant Antibody: The term “recombinant human antibody” as used herein is intended to include all human antibodies prepared, expressed, generated, or isolated in a recombinant manner, such as antibodies expressed using a recombinant expression vector transfected into host cells (described in more detail in this disclosure), antibodies isolated from recombinant, combinatorial human antibody libraries (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), and antibodies isolated from animals transgenic with human immunoglobulin genes (e.g., mice) (see, for example, Taylor, LD, et al. (1992) Nucl. Acids Res. 20:6287-6295; Kellermann SA., and Green LL (2002) Current Opinion in Biotechnology 13:593-597; Little M. et al (2000) Immunology Today 21:364-370), or antibodies prepared, expressed, generated, or isolated by any other means involving splicing human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in some embodiments, such recombinant human antibodies are mutagenized in vitro (or in vivo somatic cell mutagenized when using animals transgenic with human Ig sequences), and therefore the amino acid sequences of the VH and VL regions of the recombinant antibody are sequences that, although derived from and associated with human germline VH and VL sequences, may not be naturally present in an in vivo human antibody germline library. One embodiment of this disclosure provides a fully human antibody capable of binding to the human transferrin receptor, which can be generated using techniques known in the art, such as, but not limited to, using human Ig phage libraries, such as those disclosed in Jermutus et al. PCT Publication No. WO 2005 / 007699 A2.

[0166] Complementary region: As used herein, the term "complementary region" refers to a nucleotide sequence, such as an oligonucleotide, that is fully complementary to a homologous nucleotide sequence of, for example, a target nucleic acid, such that the two nucleotide sequences can anneal to each other under physiological conditions (e.g., in a cell). In some embodiments, the complementary region is completely complementary to the homologous nucleotide sequence of the target nucleic acid. However, in some embodiments, the complementary region is partially complementary to the homologous nucleotide sequence of the target nucleic acid (e.g., at least 80%, 90%, 95%, or 99% complementary). In some embodiments, the complementary region contains 1, 2, 3, or 4 mismatches compared to the homologous nucleotide sequence of the target nucleic acid.

[0167] Specific binding: As used herein, the term "specific binding" refers to the ability of a molecule to bind to a binding partner with a degree of affinity or affinity that allows the molecule to be used to distinguish the binding partner from a suitable control in a binding assay or other binding setting. Regarding antibodies, the term "specific binding" refers to the ability of an antibody to bind to a specific antigen with a degree of affinity or affinity compared to one or more suitable reference antigens that allows the antibody to be used to distinguish the specific antigen from other antigens, for example, to the extent that it allows preferential targeting of certain cells (e.g., muscle cells) by binding to antigens as described herein. In some embodiments, if the antibody binds to the target K... D For 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 If M is smaller, the antibody binds specifically to the target. In some embodiments, the antibody binds specifically to the transferrin receptor (e.g., an epitope of the apical domain of the transferrin receptor).

[0168] Object: As used herein, the term "object" refers to a mammal. In some embodiments, the object is a non-human primate or rodent. In some embodiments, the object is a human. In some embodiments, the object is a patient, such as a person who has or is suspected of having a disease. In some embodiments, the object is a person who has or is suspected of having a muscle disease (e.g., any of the diseases provided in Table 1).

[0169] 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 via endocytosis. In some embodiments, the transferrin receptor may be of human origin (NCBI gene ID 7037), non-human primate origin (e.g., NCBI gene ID 711568 or NCBI gene ID 102136007), or rodent origin (e.g., NCBI gene ID 22042). Additionally, several human transcript variants encoding different isotypes of the receptor have been characterized (e.g., as annotated with the following GenBankRefSeq accession numbers: NP_001121620.1, NP_003225.2, NP_001300894.1, and NP_001300895.1).

[0170] 2'-Modified Nucleosides: The terms "2'-modified nucleosides" and "2'-modified ribonucleosides" are used interchangeably herein and refer to nucleosides having a modified sugar moiety at the 2' position. In some embodiments, the 2'-modified nucleoside is a 2'-4' bicyclic nucleoside, wherein the 2' and 4' positions of the sugar are bridged (e.g., by methylene, ethylene, or (S)-restricted ethyl bridging). In some embodiments, the 2'-modified nucleoside is a non-bicyclic 2'-modified nucleoside, for example, wherein the 2' position of the sugar moiety is substituted. Some non-limiting examples of 2'-modified nucleosides include: 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-methylacetamido(2'-O-NMA), locked nucleic acid (LNA, methylene-bridged nucleic acid), ethyl-bridged nucleic acid (ENA), and (S)-bound ethyl-bridged nucleic acid (cEt). In some embodiments, the 2'-modified nucleosides described herein are high-affinity modified nucleotides and oligonucleotides comprising 2'-modified nucleotides that have increased affinity for target sequences relative to unmodified oligonucleotides. The following are some examples of the structures of 2'-modified nucleosides:

[0171]

[0172] II. complex

[0173] This document provides for complexes comprising a target agent (e.g., an antibody) covalently linked to a molecular payload. In some embodiments, the complex comprises a muscle-targeting antibody covalently linked to an oligonucleotide. The complex may comprise an antibody that specifically binds to a single antigenic site or binds to at least two antigenic sites that may be present on the same or different antigens. The complex can 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 complex, is responsible for the regulation of 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 targeting a muscle disease allele in muscle cells.

[0174] In some embodiments, the complex comprises a muscle target, such as an anti-transferrin receptor antibody, covalently linked to a molecular payload (e.g., an antisense oligonucleotide targeting a muscle disease allele).

[0175] In some embodiments, the complex can be used to treat muscle diseases, wherein the molecular payload affects the activity of the corresponding genes provided in Table 1. For example, depending on the condition, the molecular payload can regulate (e.g., decrease, increase) the transcription or expression of a gene, regulate the expression of a protein encoded by the gene, or regulate the activity of the encoded protein. In some embodiments, the molecular payload is an oligonucleotide comprising a strand having a complementary region of the target gene provided in Table 1.

[0176] Table 1 - List of muscle diseases and corresponding genes.

[0177]

[0178]

[0179]

[0180] A. Muscle-targeting agents

[0181] Some aspects of this disclosure provide muscle targeting agents, for example, for delivering molecular payloads to muscle cells. In some embodiments, such muscle targeting agents are capable of binding to muscle cells, for example, by specifically binding to an antigen on the muscle cell, and delivering the associated molecular payload to the muscle cell. In some embodiments, the molecular payload binds to the muscle targeting agent (e.g., covalently) and is internalized into the muscle cell after the muscle targeting agent binds to the antigen on the muscle cell, for example, through endocytosis. It should be understood that various types of muscle targeting agents can be used according to this disclosure. For example, muscle targeting agents may comprise, or be composed of, nucleic acids (e.g., DNA or RNA), peptides (e.g., antibodies), lipids (e.g., microvesicles), or glycosides (e.g., polysaccharides). 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.

[0182] Some aspects of this disclosure provide muscle-targeting agents that specifically bind to antigens on muscles (e.g., skeletal muscle, smooth muscle, or cardiac muscle). In some embodiments, any muscle-targeting agent provided herein binds (e.g., specifically binds) to antigens on skeletal muscle cells, smooth muscle cells, and / or (e.g., and) cardiac muscle cells.

[0183] By interacting with muscle-specific cell surface recognition elements (e.g., cell membrane proteins), both tissue localization and selective uptake into muscle cells can be achieved. In some embodiments, molecules that serve as substrates for muscle uptake transporters can be used to deliver molecular payloads into muscle tissue. Binding to muscle surface recognition elements is followed by endocytosis, which can allow even large molecules (e.g., 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 by binding to transferrin receptors and then endocytosed, for example, via clathrin-mediated endocytosis.

[0184] The use of muscle-targeting agents can be used to concentrate molecular payloads (e.g., oligonucleotides) in muscle while reducing toxicity associated with effects in other tissues. In some embodiments, the muscle-targeting agent concentrates the bound molecular payload in muscle cells compared to another cell type within the subject. In some embodiments, the muscle-targeting agent concentrates the bound molecular payload in muscle cells (e.g., skeletal muscle, smooth muscle, or cardiomyocytes) in an amount at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times higher than the amount found in non-muscle cells (e.g., liver, neurons, blood, or fat cells). In some embodiments, when the molecular payload is delivered to the subject while bound to a muscle-targeting agent, its toxicity in the subject 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%.

[0185] In some implementations, muscle recognition elements (e.g., myocyte antigens) may be required to achieve muscle selectivity. As an example, a muscle target may be a small molecule that serves as a substrate for a transporter that specifically takes over muscle. As another example, a muscle target may be an antibody that enters muscle cells via transporter-mediated endocytosis. As yet another example, a muscle target may be a ligand that binds to cell surface receptors on muscle cells. It should be understood that while transporter-based approaches provide a direct pathway for cell entry, receptor-based targeting may involve stimulating endocytosis to reach the desired site of action.

[0186] The muscle cells covered by this disclosure include, but are not limited to, skeletal muscle cells, smooth muscle cells, cardiomyocytes, myoblasts, and myocytes.

[0187] i. Muscle-targeting antibodies

[0188] In some embodiments, the muscle target is an antibody. Generally, the high specificity of antibodies to their target antigens offers the potential for selectively targeting muscle cells (e.g., skeletal muscle, smooth muscle, and / or (e.g., cardiomyocytes)). This specificity can also limit off-target toxicity. Examples of antibodies capable of targeting muscle cell surface antigens have been reported and are within the scope of this disclosure. For example, antibodies targeting the surface of muscle cells are described in the following: 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., "Celltype specific targeted intracellular delivery into muscle of a monoclonal antibody that binds myosinIIb”MolImmunol.2003Mar,39(13):78309; all of its contents are incorporated herein by reference.

[0189] a. Anti-transferrin receptor antibody

[0190] Some aspects of this disclosure are based on the understanding that substances 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 translocates transferrin across the cell membrane and participates in the regulation and homeostasis of intracellular iron levels. Some aspects of this disclosure provide transferrin receptor-binding proteins capable of binding to the transferrin receptor. Therefore, some aspects of this disclosure provide binding proteins (e.g., antibodies) that bind to the transferrin receptor. In some embodiments, the binding protein that binds to the transferrin receptor is internalized into muscle cells along with any bound molecular payload. Antibodies that bind to the transferrin receptor as used herein may be interchangeably referred to as transferrin receptor antibodies, anti-transferrin receptor antibodies, or anti-TfR antibodies. Antibodies that bind to the transferrin receptor (e.g., specifically) can be internalized into cells after binding to the transferrin receptor, for example, through receptor-mediated endocytosis.

[0191] It should be understood that several known methods (e.g., using phage display library design) can be used to generate, synthesize, and / or (e.g., and) derive anti-transferrin receptor antibodies. Exemplary methods have been characterized in the art and are incorporated by reference (Díez, P. et al. "High-throughput phage-display screening in arrayformat", 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, anti-transferrin antibodies have been previously characterized or disclosed.Antibodies that specifically bind to the transferrin receptor are known in the art (see, for example, U.S. Patent No. 4,364,934, filed December 4, 1979, “Monoclonal antibody to a human early thymocyte antibody and methods for preparing same”; U.S. Patent No. 8,409,573, filed June 14, 2006, “Anti-CD71 monoclonal antibodies and uses thereof for treating malignant tumor cells”; U.S. Patent No. 9,708,406, filed May 20, 2014, “Anti-transferrin receptor antibodies and methods of use”; U.S. Patent No. 9,611,323, filed December 19, 2014, “Lowaffinity blood brain barrier receptor antibodies and uses therefor”; WO 2015 / 098989, filed December 24, 2014, “Novel anti-Transferrin receptor antibody that passes through blood-brain barrier”; Schneider C. et al. "Structural features of the cell surface receptor for transferrin that is recognized by the monoclonal antibody OKT9." J Biol Chem.1982,257:14,8516-8522.; Lee et al. "Targeting Rat Anti-Mouse Transferrin Receptor Monoclonal Antibodies through Blood-Brain Barrier in Mouse" 2000, J Pharmacol. Exp. Ther., 292:1048-1052).

[0192] In some aspects, novel anti-TfR antibodies are provided herein for use as muscle targeting agents (e.g., in muscle-targeting complexes). In some embodiments, the anti-TfR antibodies described herein bind to transferrin receptors with high specificity and affinity. In some embodiments, the anti-TfR antibodies described herein specifically bind to any extracellular site of the transferrin receptor or an epitope exposed to the antibody. In some embodiments, the anti-TfR antibodies provided herein specifically bind to transferrin receptors derived from humans, non-human primates, mice, rats, etc. In some embodiments, the anti-TfR antibodies provided herein bind to human transferrin receptors. In some embodiments, the anti-TfR antibodies described herein bind to amino acid segments of human or non-human primate transferrin receptors (as provided in SEQ ID NO: 105 to 108). In some embodiments, the anti-TfR antibodies described herein bind to amino acid segments corresponding to amino acids 90 to 96 of the human transferrin receptor (as shown in SEQ ID NO: 105) that are not located in the apical domain of the transferrin receptor.

[0193] The exemplary human transferrin receptor amino acid sequence corresponding to NCBI sequence NP_003225.2 (transferrin receptor protein 1 isotype 1, Homo sapiens) is as follows:

[0194]

[0195] The exemplary non-human primate transferrin receptor amino acid sequence corresponding to the NCBI sequence NP_001244232.1 (transferrin receptor protein 1, rhesus monkey (Macacamulatta)) is as follows:

[0196] The exemplary non-human primate transferrin receptor amino acid sequence corresponding to the NCBI sequence XP_005545315.1 (transferrin receptor protein 1, cynomolgus monkey) is as follows:

[0197]

[0198] The exemplary mouse transferrin receptor amino acid sequence corresponding to the NCBI sequence NP_001344227.1 (transferrin receptor protein 1, mus musculus) is as follows:

[0199]

[0200] In some implementations, the anti-transferrin receptor antibody binds to the following receptor amino acid segment:

[0201]

[0202] Furthermore, it does not inhibit the binding interaction between the transferrin receptor and transferrin and / or (e.g., and) human hemochromatosis protein (also known as HFE). In some embodiments, the anti-transferrin receptor antibody described herein does not bind to the epitope in SEQ ID NO:109.

[0203] Suitable methods can be used to obtain and / or (e.g., and) generate antibodies, antibody fragments, or antigen-binding agents, for example, by using recombinant DNA protocols. In some embodiments, antibodies can also be generated by the production 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 target antigen can be used as an immunogen in any form or entity (e.g., recombinant or naturally occurring forms or entities). Hybridomas are screened using standard methods (e.g., ELISA screening) to identify at least one hybridoma that produces an antibody targeting a specific antigen. Antibodies can also be generated by screening protein expression libraries (e.g., phage display libraries) that express antibodies. In some embodiments, phage display library designs may also be used (see, for example, U.S. Patent No. 5,223,409, filed March 1, 1991, “Directed evolution of novel binding proteins”; WO 1992 / 18619, filed April 10, 1992, “Heterodimeric receptor libraries using phagemids”; WO 1991 / 17271, filed May 1, 1991, “Recombinant library screening methods”; WO 1992 / 20791, filed May 15, 1992, “Methods for producing members of specific binding pairs”; WO 1992 / 15679, filed February 28, 1992, “Improved epitope displaying phage”). In some embodiments, the target antigen may be used for immunization of non-human animals, such as rodents or goats. In some implementations, antibodies are then obtained from non-human animals and optionally modified using various methods (e.g., using recombinant DNA technology). Other examples and methods of antibody production are known in the art (see, for example, Harlow et al., “Antibodies: A Laboratory Manual”, Cold Spring Harbor Laboratory, 1988).

[0204] In some embodiments, the antibody is modified, for example, by 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 by N-glycosylation, O-glycosylation, C-glycosylation, glycosylphosphatidylinositol (GPI-anchored attachment), and / or (e.g., and) phosphorylated glycosylation. In some embodiments, one or more sugar or carbohydrate molecules are monosaccharides, disaccharides, oligosaccharides, or glycans. In some embodiments, one or more sugar or carbohydrate molecules are branched oligosaccharides or branched glycans. In some embodiments, one or more sugar or carbohydrate molecules comprise mannose units, glucose units, N-acetylglucosamine units, N-acetylglucosamine units, galactose units, fucose units, or phospholipid units. In some embodiments, about 1 to 10, about 1 to 5, about 5 to 10, about 1 to 4, about 1 to 3, or about 2 sugar molecules are present. In some embodiments, the glycosylated antibody is fully or partially glycosylated. In some embodiments, the antibody is glycosylated by a chemical reaction or by an enzymatic means. In some embodiments, the antibody is glycosylated in vitro or intracellularly, optionally lacking enzymes in the N- or O-glycosylation pathway, such as glycosyltransferases. In some embodiments, the antibody is functionalized with sugar or carbohydrate molecules, as described in International Patent Application Publication WO2014065661, published May 1, 2014, entitled "Modified antibody, antibody-conjugate and process for the preparation thereof".

[0205] In some embodiments, the anti-TfR antibody of this disclosure comprises a VL domain and / or (e.g., and) a VH domain selected from any of the anti-TfR antibodies in 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, any class of immunoglobulin molecules (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or any subclass (e.g., IgG2a and IgG2b). Some non-limiting examples of human constant regions are described in the art, for example, see above, Kabat E A et al., (1991).

[0206] In some embodiments, substances that bind to transferrin receptors, such as anti-TfR antibodies, are capable of targeting muscle cells and / or (e.g., and) mediating the transport of substances across the blood-brain barrier. Transferrin receptors are internalized cell surface receptors that transport transferrin across the cell membrane and participate in the regulation and homeostasis of intracellular iron levels. Some aspects of this disclosure provide transferrin receptor-binding proteins capable of binding to transferrin receptors. Antibodies that bind to transferrin receptors (e.g., specifically) can be internalized into cells after binding to the transferrin receptor, for example, through receptor-mediated endocytosis.

[0207] In some aspects, this document provides 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 site of the transferrin receptor or an epitope exposed to the antibody. In some embodiments, the humanized anti-TfR antibodies provided herein specifically bind to transferrin receptors derived from humans, non-human primates, mice, rats, etc. In some embodiments, the humanized anti-TfR antibodies provided herein bind to human transferrin receptors. In some embodiments, the humanized anti-TfR antibodies described herein bind to amino acid segments of human or non-human primate transferrin receptors as provided in SEQ ID NO:105 to 108. In some embodiments, the humanized anti-TfR antibodies described herein bind to amino acid segments corresponding to amino acids 90 to 96 of the human transferrin receptor shown in SEQ ID NO:105, which are not located in the apical domain of the transferrin receptor. In some implementations, the humanized anti-TfR antibody described herein binds to TfR1 but not to TfR2.

[0208] In some implementations, 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 -13M or smaller binding affinity (e.g., as indicated by Kd) specifically binds to TfR1 (e.g., human or non-human primate TfR1). In some embodiments, the anti-TfR antibodies described herein bind to TfR1 with a KD in the range of 1000 N / A. 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 monkey TfR1 (e.g., Kd is 10). -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, 10 -13 (M or smaller), but does not bind to mouse TfR1. The affinity and binding kinetics of anti-TfR antibodies can be tested using any suitable method, including but not limited to biosensor technologies (e.g., OCTET or BIACORE). In some embodiments, the binding of any of the anti-TfR antibodies described herein does not compete with or inhibit the binding of transferrin to TfR1. In some embodiments, the binding of any of the anti-TfR antibodies described herein does not compete with or inhibit the binding of HFE-β-2-microglobulin to TfR1.

[0209] The anti-TfR antibody described in this article is a humanized antibody. Table 2 provides the CDR and variable region amino acid sequences of the mouse monoclonal anti-TfR antibody from which the humanized anti-TfR antibody described in this article is derived.

[0210] Table 2. Mouse monoclonal anti-TfR antibodies

[0211]

[0212]

[0213]

[0214] *The mutation location is determined by the Kabat number of the corresponding VH sequence containing the mutation.

[0215] In some embodiments, the anti-TfR antibody of this disclosure is a humanized variant of any of the anti-TfR antibodies provided in Table 2. In some embodiments, the anti-TfR antibody of this disclosure comprises the same CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 as CDR-H1, CDR-H2, and CDR-H3 of any of the anti-TfR antibodies provided in Table 2, and comprises a humanized heavy chain variable region and / or (e.g., and) a humanized light chain variable region.

[0216] Humanized antibodies are human immunoglobulins (receptor antibodies) in which residues from the complementary determining region (CDR) of the receptor are replaced by residues from the CDR of a non-human species (donor antibody) with the desired specificity, affinity, and capacity, such as mouse, rat, or rabbit. In some embodiments, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may include residues not found in either the receptor antibody or the introduced CDR or framework sequence but included to further refine and optimize antibody performance. Generally, humanized antibodies will contain substantially all of at least one, and typically two, variable domains, wherein all or substantially all of the CDR regions correspond to those of non-human immunoglobulins, and all or substantially all of the FR regions are those of human immunoglobulin common sequences. Humanized antibodies will also preferably also contain at least a portion of the immunoglobulin constant region or Fc domain (typically those of human immunoglobulins). Antibodies may have an Fc region modified as described in WO 99 / 58572. Other forms of humanized antibodies have one or more CDRs (one, two, three, four, five, or six) altered relative to the original antibody, also referred to as one or more CDRs derived from one or more CDRs of the original antibody. Humanized antibodies may also involve affinity maturation.

[0217] Humanized antibodies and their preparation methods are known, for example, as described in the following: 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), all of which are incorporated herein by reference. Human frames that can be used for humanization are described, for example, in the following: 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), all of which are incorporated herein by reference.

[0218] In some embodiments, the humanized anti-TfR antibody of this disclosure comprises: a humanized VH containing one or more amino acid variations compared to any of the VHs listed in Table 2 (e.g., in the VH frame region), and / or (e.g., and) a humanized VL containing one or more amino acid variations compared to any of the VLs listed in Table 2 (e.g., in the VL frame region).

[0219] In some embodiments, the humanized anti-TfR antibody of this 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 variation) compared to the VH of any anti-TfR antibody listed in Table 2 (e.g., any one of SEQ ID NO: 17, 22, 26, 43, 61, 65 and 68). As an alternative or supplement (e.g., supplement), the humanized anti-TfR antibody of this disclosure comprises 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 variation) compared to the VL of any anti-TfR antibody listed in Table 2 (e.g., any one of SEQ ID NO: 18, 44 and 62).

[0220] In some embodiments, the humanized anti-TfR antibody of this disclosure comprises a humanized VH, which contains an amino acid sequence in the frame region having at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identity with the VH of any anti-TfR antibody listed in Table 2 (e.g., any one of SEQ ID NO: 17, 22, 26, 43, 61, 65, and 68). Alternatively or supplementally (e.g., as an adjunct), in some embodiments, the humanized anti-TfR antibody of this disclosure comprises a humanized VL, which contains an amino acid sequence in the frame region having at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identity with the VL of any anti-TfR antibody listed in Table 2 (e.g., any one of SEQ ID NO: 18, 44, and 62).

[0221] In some embodiments, the humanized anti-TfR antibody of this disclosure comprises a humanized VH, said humanized VH comprising CDR-H1 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO:1, CDR-H2 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO:2, SEQ ID NO:19 or SEQ ID NO:23, and CDR-H3 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO:3, and comprising no more than 25 amino acid variations in the frame region compared to the VH shown in SEQ ID NO:17, SEQ ID NO:22 or SEQ ID NO:26 (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid variation). As an alternative or supplement (e.g., supplement), the anti-TfR antibody of this disclosure comprises a humanized VL comprising CDR-L1 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO:4, CDR-L2 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO:5, and CDR-L3 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO:6, and comprising no more than 25 amino acid variations in the frame region compared to the VL shown in SEQ ID NO:18 (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).

[0222] In some embodiments, the humanized anti-TfR antibody of this disclosure comprises a humanized VH, said humanized VH comprising CDR-H1 having the amino acid sequence SEQ ID NO:1 (according to the IMGT definition system), CDR-H2 having the amino acid sequence SEQ ID NO:2, SEQ ID NO:19 or SEQ ID NO:23 (according to the IMGT definition system), CDR-H3 having the amino acid sequence SEQ ID NO:3 (according to the IMGT definition system), and having at least 75% (e.g. 75%, 80%, 85%, 90%, 95%, 98% or 99%) identity with the VH shown in SEQ ID NO:17, SEQ ID NO:22 or SEQ ID NO:26 in the frame region. As an alternative or supplement (e.g., supplement), the humanized anti-TfR antibody of this disclosure comprises a humanized VL comprising CDR-L1 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO:4, CDR-L2 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO:5, and CDR-L3 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO:6, and having at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identity with any of the VLs shown in SEQ ID NO:18 in the frame region.

[0223] In some embodiments, the humanized anti-TfR antibody of this disclosure comprises a humanized VH, said humanized VH comprising CDR-H1 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO:7, CDR-H2 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO:8, SEQ ID NO:20 or SEQ ID NO:24, and CDR-H3 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO:9, and comprising no more than 25 amino acid variations in the frame region compared to the VH shown in SEQ ID NO:17, SEQ ID NO:22 or SEQ ID NO:26 (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid variation). As an alternative or supplement (e.g., supplement), the humanized anti-TfR antibody of this disclosure comprises a humanized VL comprising CDR-L1 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO:10, CDR-L2 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO:11, and CDR-L3 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO:6, and comprising no more than 25 amino acid variations in the frame region compared to the VL shown in SEQ ID NO:18 (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).

[0224] In some embodiments, the humanized anti-TfR antibody of this disclosure comprises a humanized VH, said humanized VH comprising CDR-H1 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO:7, CDR-H2 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO:8, SEQ ID NO:20 or SEQ ID NO:24, and CDR-H3 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO:9, and having at least 75% (e.g. 75%, 80%, 85%, 90%, 95%, 98% or 99%) identity with the VH shown in SEQ ID NO:17, SEQ ID NO:22 or SEQ ID NO:26 in the frame region. As an alternative or supplement (e.g., supplement), the humanized anti-TfR antibody of this disclosure comprises a humanized VL comprising CDR-L1 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO:10, CDR-L2 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO:11, and CDR-L3 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO:6, and having at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identity with any of the VLs shown in SEQ ID NO:18 in the frame region.

[0225] In some embodiments, the humanized anti-TfR antibody of this disclosure comprises a humanized VH, said humanized VH comprising CDR-H1 (according to the Chothia definition system) having the amino acid sequence of SEQ ID NO:12, CDR-H2 (according to the Chothia definition system) having the amino acid sequence of SEQ ID NO:13, SEQ ID NO:21 or SEQ ID NO:25, and CDR-H3 (according to the Chothia definition system) having the amino acid sequence of SEQ ID NO:14, and comprising no more than 25 amino acid variations in the frame region compared to the VH shown in SEQ ID NO:17, SEQ ID NO:22 or SEQ ID NO:26 (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid variation). As an alternative or supplement (e.g., supplement), the humanized anti-TfR antibody of this disclosure comprises a humanized VL comprising CDR-L1 (according to the Chothia definition system) having the amino acid sequence of SEQ ID NO:15, CDR-L2 (according to the Chothia definition system) having the amino acid sequence of SEQ ID NO:5, and CDR-L3 (according to the Chothia definition system) having the amino acid sequence of SEQ ID NO:16, and comprising no more than 25 amino acid variations in the frame region compared to the VL shown in SEQ ID NO:18 (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).

[0226] In some embodiments, the humanized anti-TfR antibody of this disclosure comprises a humanized VH, said humanized VH comprising CDR-H1 (according to the Chothia definition system) having the amino acid sequence of SEQ ID NO:12, CDR-H2 (according to the Chothia definition system) having the amino acid sequence of SEQ ID NO:13, SEQ ID NO:21 or SEQ ID NO:25, and CDR-H3 (according to the Chothia definition system) having the amino acid sequence of SEQ ID NO:14, and having at least 75% (e.g. 75%, 80%, 85%, 90%, 95%, 98% or 99%) identity with the VH shown in SEQ ID NO:17, SEQ ID NO:22 or SEQ ID NO:26 in the frame region. As an alternative or supplement (e.g., supplement), the anti-TfR antibody of this disclosure comprises a humanized VL comprising CDR-L1 (according to the Chothia definition system) having the amino acid sequence of SEQ ID NO:15, CDR-L2 (according to the Chothia definition system) having the amino acid sequence of SEQ ID NO:5, and CDR-L3 (according to the Chothia definition system) having the amino acid sequence of SEQ ID NO:16, and having at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identity with any of the VLs shown in SEQ ID NO:18 in the frame region.

[0227] In some embodiments, the humanized anti-TfR antibody of this disclosure comprises a humanized VH, said humanized VH comprising CDR-H1 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO:27, CDR-H2 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO:28, CDR-H3 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO:29, and containing no more than 25 amino acid variations in the frame region compared to the VH shown 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 variation). As an alternative or supplement (e.g., supplement), the humanized anti-TfR antibody of this disclosure comprises a humanized VL comprising CDR-L1 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO:30, CDR-L2 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO:31, and CDR-L3 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO:32, and containing no more than 25 amino acid variations in the frame region compared to the VL shown in SEQ ID NO:44 (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).

[0228] In some embodiments, the humanized anti-TfR antibody of this disclosure comprises a humanized VH, said humanized VH comprising CDR-H1 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO:27, CDR-H2 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO:28, and CDR-H3 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO:29, and having at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identity with the VH shown in SEQ ID NO:43 in the frame region. As an alternative or supplement (e.g., an addition), the humanized anti-TfR antibody of this disclosure comprises a humanized VL comprising CDR-L1 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO:30, CDR-L2 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO:31, and CDR-L3 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO:32, and having at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identity with the VL shown in SEQ ID NO:44 in the frame region.

[0229] In some embodiments, the humanized anti-TfR antibody of this disclosure comprises a humanized VH, said humanized VH comprising CDR-H1 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO:33, CDR-H2 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO:34, CDR-H3 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO:35, and containing no more than 25 amino acid variations in the frame region compared to the VH shown 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 variation). As an alternative or supplement (e.g., supplement), the humanized anti-TfR antibody of this disclosure comprises a humanized VL comprising CDR-L1 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO:36, CDR-L2 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO:37, and CDR-L3 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO:32, and containing no more than 25 amino acid variations in the frame region compared to the VL shown in SEQ ID NO:44 (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).

[0230] In some embodiments, the humanized anti-TfR antibody of this disclosure comprises a humanized VH comprising CDR-H1 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO:33, CDR-H2 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO:34, and CDR-H3 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO:35, and having at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identity with the VH shown in SEQ ID NO:43 in the frame region. As an alternative or supplement (e.g., supplement), the humanized anti-TfR antibody of this disclosure comprises a humanized VL comprising CDR-L1 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO:36, CDR-L2 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO:37, and CDR-L3 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO:32, and having at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identity with the VL shown in SEQ ID NO:44 in the frame region.

[0231] In some embodiments, the humanized anti-TfR antibody of this disclosure comprises a humanized VH, said humanized VH comprising CDR-H1 (according to the Chothia definition system) having the amino acid sequence of SEQ ID NO:38, CDR-H2 (according to the Chothia definition system) having the amino acid sequence of SEQ ID NO:39, CDR-H3 (according to the Chothia definition system) having the amino acid sequence of SEQ ID NO:40, and comprising no more than 25 amino acid variations in the frame region compared to the VH shown 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 variation). As an alternative or supplement (e.g., supplement), the humanized anti-TfR antibody of this disclosure comprises a humanized VL comprising CDR-L1 (according to the Chothia definition system) having the amino acid sequence of SEQ ID NO:41, CDR-L2 (according to the Chothia definition system) having the amino acid sequence of SEQ ID NO:31, and CDR-L3 (according to the Chothia definition system) having the amino acid sequence of SEQ ID NO:42, and comprising no more than 25 amino acid variations in the frame region compared to the VL shown in SEQ ID NO:44 (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).

[0232] In some embodiments, the humanized anti-TfR antibody of this disclosure comprises a humanized VH comprising CDR-H1 (according to the Chothia definition system) having the amino acid sequence of SEQ ID NO:38, CDR-H2 (according to the Chothia definition system) having the amino acid sequence of SEQ ID NO:39, and CDR-H3 (according to the Chothia definition system) having the amino acid sequence of SEQ ID NO:40, and having at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identity with the VH shown in SEQ ID NO:43 in the frame region. As an alternative or supplement (e.g., supplement), the humanized anti-TfR antibody of this disclosure comprises a humanized VL comprising CDR-L1 (according to the Chothia definition system) having the amino acid sequence of SEQ ID NO:41, CDR-L2 (according to the Chothia definition system) having the amino acid sequence of SEQ ID NO:31, and CDR-L3 (according to the Chothia definition system) having the amino acid sequence of SEQ ID NO:42, and having at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identity with the VL shown in SEQ ID NO:44 in the frame region.

[0233] In some embodiments, the humanized anti-TfR antibody of this disclosure comprises a humanized VH, said humanized VH comprising CDR-H1 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO:45, SEQ ID NO:63 or SEQ ID NO:66, CDR-H2 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO:46, CDR-H3 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO:47, and containing no more than 25 amino acid variations in the frame region compared to the VH shown in SEQ ID NO:61, SEQ ID NO:65 or SEQ ID NO:68 (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid variation). As an alternative or supplement (e.g., supplement), the humanized anti-TfR antibody of this disclosure comprises a humanized VL comprising CDR-L1 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO:48, CDR-L2 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO:49, and CDR-L3 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO:50, and comprising no more than 25 amino acid variations in the frame region compared to the VL shown in SEQ ID NO:62 (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).

[0234] In some embodiments, the humanized anti-TfR antibody of this disclosure comprises a humanized VH, said humanized VH comprising CDR-H1 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO:45, SEQ ID NO:63 or SEQ ID NO:66, CDR-H2 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO:46, and CDR-H3 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO:47, and having at least 75% (e.g. 75%, 80%, 85%, 90%, 95%, 98% or 99%) identity with the VH shown in SEQ ID NO:61, SEQ ID NO:65, SEQ ID NO:68 in the frame region. As an alternative or supplement (e.g., an addition), the humanized anti-TfR antibody of this disclosure comprises a humanized VL comprising CDR-L1 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO:48, CDR-L2 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO:49, and CDR-L3 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO:50, and having at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identity with the VL shown in SEQ ID NO:62 in the frame region.

[0235] In some embodiments, the humanized anti-TfR antibody of this disclosure comprises a humanized VH, said humanized VH comprising CDR-H1 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO:51, SEQ ID NO:64 or SEQ ID NO:67, CDR-H2 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO:52, CDR-H3 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO:53, and containing no more than 25 amino acid variations in the frame region compared to the VH shown in SEQ ID NO:61, SEQ ID NO:65, SEQ ID NO:68 (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid variation). As an alternative or supplement (e.g., supplement), the humanized anti-TfR antibody of this disclosure comprises a humanized VL containing CDR-L1 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO:54, CDR-L2 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO:55, and CDR-L3 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO:50, and containing no more than 25 amino acid variations in the frame region compared to the VL shown in SEQ ID NO:62 (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).

[0236] In some embodiments, the humanized anti-TfR antibody of this disclosure comprises a humanized VH, said humanized VH comprising CDR-H1 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO:51, SEQ ID NO:64 or SEQ ID NO:67, CDR-H2 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO:52, and CDR-H3 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO:53, and having at least 75% (e.g. 75%, 80%, 85%, 90%, 95%, 98% or 99%) identity with the VH shown in SEQ ID NO:61, SEQ ID NO:65, SEQ ID NO:68 in the frame region. As an alternative or supplement (e.g., supplement), the humanized anti-TfR antibody of this disclosure comprises a humanized VL comprising CDR-L1 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO:54, CDR-L2 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO:55, and CDR-L3 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO:50, and having at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identity with the VL shown in SEQ ID NO:62 in the frame region.

[0237] In some embodiments, the humanized anti-TfR antibody of this disclosure comprises a humanized VH, said humanized VH comprising CDR-H1 (according to the Chothia definition system) having the amino acid sequence of SEQ ID NO:56, CDR-H2 (according to the Chothia definition system) having the amino acid sequence of SEQ ID NO:57, and CDR-H3 (according to the Chothia definition system) having the amino acid sequence of SEQ ID NO:58, and containing no more than 25 amino acid variations in the frame region compared to the VH shown in SEQ ID NO:61, SEQ ID NO:65, and SEQ ID NO:68 (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation). As an alternative or supplement (e.g., supplement), the humanized anti-TfR antibody of this disclosure comprises a humanized VL comprising CDR-L1 (according to the Chothia definition system) having the amino acid sequence of SEQ ID NO:59, CDR-L2 (according to the Chothia definition system) having the amino acid sequence of SEQ ID NO:49, and CDR-L3 (according to the Chothia definition system) having the amino acid sequence of SEQ ID NO:60, and comprising no more than 25 amino acid variations in the frame region compared to the VL shown in SEQ ID NO:62 (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).

[0238] In some embodiments, the humanized anti-TfR antibody of this disclosure comprises a humanized VH comprising CDR-H1 (according to the Chothia definition system) having the amino acid sequence of SEQ ID NO:56, CDR-H2 (according to the Chothia definition system) having the amino acid sequence of SEQ ID NO:57, and CDR-H3 (according to the Chothia definition system) having the amino acid sequence of SEQ ID NO:58, and having at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identity with the VH shown in SEQ ID NO:61, SEQ ID NO:65, and SEQ ID NO:68 in the frame region. As an alternative or supplement (e.g., supplement), the humanized anti-TfR antibody of this disclosure comprises a humanized VL comprising CDR-L1 (according to the Chothia definition system) having the amino acid sequence of SEQ ID NO:59, CDR-L2 (according to the Chothia definition system) having the amino acid sequence of SEQ ID NO:49, and CDR-L3 (according to the Chothia definition system) having the amino acid sequence of SEQ ID NO:60, and having at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identity with the VL shown in SEQ ID NO:62 in the frame region.

[0239] Table 3 provides some examples of the amino acid sequences of the humanized anti-TfR antibodies described in this article.

[0240] Table 3. Variable regions of humanized anti-TfR antibodies

[0241]

[0242]

[0243]

[0244] *The mutation location is determined by the Kabat number of the corresponding VH sequence containing the mutation.

[0245] **The CDRs based on the Kabat numbering system are in bold.

[0246] In some embodiments, the humanized anti-TfR antibody of this disclosure comprises a humanized VH, which comprises CDR-H1, CDR-H2, and CDR-H3 of any of the anti-TfR antibodies provided in Table 2, and contains one or more amino acid variations (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) in the frame region compared to the corresponding humanized VH provided in Table 3. Alternatively or supplementally (e.g., as an adjunct), the humanized anti-TfR antibody of this disclosure comprises a humanized VL, which comprises CDR-L1, CDR-L2, and CDR-L3 of any of the anti-TfR antibodies provided in Table 2, and contains one or more amino acid variations (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) in the frame region compared to the corresponding humanized VL provided in Table 3.

[0247] In some embodiments, the humanized anti-TfR antibody of this disclosure comprises: a humanized VH containing an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identity with SEQ ID NO:69, and / or (e.g., and) a humanized VL containing an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identity with SEQ ID NO:70. In some embodiments, the humanized anti-TfR antibody of this disclosure comprises a humanized VH containing the amino acid sequence of SEQ ID NO:69 and a humanized VL containing the amino acid sequence of SEQ ID NO:70.

[0248] In some embodiments, the humanized anti-TfR antibody of this disclosure comprises: a humanized VH containing an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identity with SEQ ID NO:71, and / or (e.g., and) a humanized VL containing an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identity with SEQ ID NO:70. In some embodiments, the humanized anti-TfR antibody of this disclosure comprises a humanized VH containing the amino acid sequence of SEQ ID NO:71 and a humanized VL containing the amino acid sequence of SEQ ID NO:70.

[0249] In some embodiments, the humanized anti-TfR antibody of this disclosure comprises: a humanized VH containing an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identity with SEQ ID NO:72, and / or (e.g., and) a humanized VL containing an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identity with SEQ ID NO:70. In some embodiments, the humanized anti-TfR antibody of this disclosure comprises a humanized VH containing the amino acid sequence of SEQ ID NO:72 and a humanized VL containing the amino acid sequence of SEQ ID NO:70.

[0250] In some embodiments, the humanized anti-TfR antibody of this disclosure comprises: a humanized VH containing an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identity with SEQ ID NO:73, and / or (e.g., and) a humanized VL containing an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identity with SEQ ID NO:74. In some embodiments, the humanized anti-TfR antibody of this disclosure comprises a humanized VH containing the amino acid sequence of SEQ ID NO:73 and a humanized VL containing the amino acid sequence of SEQ ID NO:74.

[0251] In some embodiments, the humanized anti-TfR antibody of this disclosure comprises: a humanized VH containing an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identity with SEQ ID NO:73, and / or (e.g., and) a humanized VL containing an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identity with SEQ ID NO:75. In some embodiments, the humanized anti-TfR antibody of this disclosure comprises a humanized VH containing the amino acid sequence of SEQ ID NO:73 and a humanized VL containing the amino acid sequence of SEQ ID NO:75.

[0252] In some embodiments, the humanized anti-TfR antibody of this disclosure comprises: a humanized VH containing an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identity with SEQ ID NO:76, and / or (e.g., and) a humanized VL containing an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identity with SEQ ID NO:74. In some embodiments, the humanized anti-TfR antibody of this disclosure comprises a humanized VH containing the amino acid sequence of SEQ ID NO:76 and a humanized VL containing the amino acid sequence of SEQ ID NO:74.

[0253] In some embodiments, the humanized anti-TfR antibody of this disclosure comprises: a humanized VH containing an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identity with SEQ ID NO:76, and / or (e.g., and) a humanized VL containing an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identity with SEQ ID NO:75. In some embodiments, the humanized anti-TfR antibody of this disclosure comprises a humanized VH containing the amino acid sequence of SEQ ID NO:76 and a humanized VL containing the amino acid sequence of SEQ ID NO:75.

[0254] In some embodiments, the humanized anti-TfR antibody of this disclosure comprises: a humanized VH containing an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identity with SEQ ID NO:77, and / or (e.g., and) a humanized VL containing an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identity with SEQ ID NO:78. In some embodiments, the humanized anti-TfR antibody of this disclosure comprises a humanized VH containing the amino acid sequence of SEQ ID NO:77 and a humanized VL containing the amino acid sequence of SEQ ID NO:78.

[0255] In some embodiments, the humanized anti-TfR antibody of this disclosure comprises: a humanized VH containing an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identity with SEQ ID NO:79, and / or (e.g., and) a humanized VL containing an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identity with SEQ ID NO:80. In some embodiments, the humanized anti-TfR antibody of this disclosure comprises a humanized VH containing the amino acid sequence of SEQ ID NO:79 and a humanized VL containing the amino acid sequence of SEQ ID NO:80.

[0256] In some embodiments, the humanized anti-TfR antibody of this disclosure comprises: a humanized VH containing an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identity with SEQ ID NO:77, and / or (e.g., and) a humanized VL containing an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identity with SEQ ID NO:80. In some embodiments, the humanized anti-TfR antibody of this disclosure comprises a humanized VH containing the amino acid sequence of SEQ ID NO:77 and a humanized VL containing the amino acid sequence of SEQ ID NO:80.

[0257] In some embodiments, the humanized anti-TfR antibody described herein is a full-length IgG that may contain a heavy chain constant region and a light chain constant region derived from a human antibody. In some embodiments, the heavy chain of any anti-TfR antibody described herein may contain a heavy chain constant region (CH) or a portion thereof (e.g., CH1, CH2, CH3, or combinations thereof). The heavy chain constant region may have any suitable source, such as human, mouse, rat, or rabbit. In a specific example, the heavy chain constant region is derived from human IgG, such as IgG1, IgG2, or IgG4 (γ heavy chain). An example of the human IgG1 constant region is given below:

[0258]

[0259] In some embodiments, the heavy chain of any anti-TfR antibody described herein contains a mutant human IgG1 constant region. For example, it is known that introducing the LALA mutation (a mutant derived from mAb b12, which has been mutated to replace the lower hinge residues Leu234 and Leu235 with Ala234 and Ala235) into the CH2 domain of human IgG1 reduces Fcg receptor binding (Bruhns, P., et al. (2009) and Xu, D. et al. (2000)). The mutant human IgG1 constant region is shown below (mutations are bolded and underlined):

[0260]

[0261] In some embodiments, the light chain of any anti-TfR antibody described herein may further comprise a light chain constant region (CL), which may be any CL known in the art. In some instances, the CL is a κ light chain. In other instances, the CL is a λ light chain. In some embodiments, the CL is a κ light chain, the sequence of which is provided below:

[0262]

[0263] Other antibody heavy and light chain constant regions are well known in the art, such as those available in the IMGT database (www.imgt.org) or www.vbase2.org / vbstat.php, both of which are incorporated herein by reference.

[0264] In some embodiments, the humanized anti-TfR antibody described herein comprises a heavy chain containing any VH or any variant thereof listed in Table 3, and a heavy chain constant region having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with SEQ ID NO:81 or SEQ ID NO:82. In some embodiments, the humanized anti-TfR antibody described herein comprises a heavy chain containing any VH or any variant thereof listed in Table 3, and a heavy chain constant region 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 SEQ ID NO:81 or SEQ ID NO:82. In some embodiments, the humanized anti-TfR antibody described herein comprises a heavy chain, which includes any VH or any variant thereof listed in Table 3 and the heavy chain constant region shown in SEQ ID NO:81. In some embodiments, the humanized anti-TfR antibody described herein comprises a heavy chain, which includes any VH or any variant thereof listed in Table 3 and the heavy chain constant region shown in SEQ ID NO:82.

[0265] In some embodiments, the humanized anti-TfR antibody described herein comprises a light chain comprising any VL or any variant thereof listed in Table 3 and a light chain constant region having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with SEQ ID NO:83. In some embodiments, the humanized anti-TfR antibody described herein comprises a light chain comprising any VL or any variant thereof listed in Table 3 and a light chain constant region comprising 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 SEQ ID NO:83. In some embodiments, the humanized anti-TfR antibody described herein comprises a light chain comprising any of the VLs listed in Table 3 or any variant thereof, and the light chain constant region shown in SEQ ID NO:83.

[0266] Table 4 below provides some examples of the IgG heavy chain amino acid sequences and light chain amino acid sequences of the anti-TfR antibody.

[0267] Table 4. Heavy and light chain sequences of humanized anti-TfR IgG samples

[0268]

[0269]

[0270]

[0271] *The mutation location is determined by the Kabat number of the corresponding VH sequence containing the mutation.

[0272] **The CDRs according to the Kabat numbering system are bolded; VH / VL sequences are underlined.**

[0273] In some embodiments, the humanized anti-TfR antibody of this 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 variation) compared to the heavy chain shown in any of SEQ ID NO: 84, 86, 87, 88, 91, 92 and 94. As an alternative or supplement (e.g., supplement), the humanized anti-TfR antibody of this disclosure comprises a light chain containing no more than 25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid variation) compared to the light chain shown in any one of SEQ ID NO: 85, 89, 90, 93 and 95.

[0274] In some embodiments, the humanized anti-TfR antibody described herein comprises a heavy chain containing an amino acid sequence having at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identity with any one of SEQ ID NO: 84, 86, 87, 88, 91, 92, and 94. Alternatively or supplementally (e.g., additionally), the humanized anti-TfR antibody described herein comprises a light chain containing an amino acid sequence having at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identity with any one of SEQ ID NO: 85, 89, 90, 93, and 95. In some embodiments, the anti-TfR antibody described herein comprises a heavy chain containing an amino acid sequence of any one of SEQ ID NO: 84, 86, 87, 88, 91, 92, and 94. As an alternative or supplement (e.g., supplement), the anti-TfR antibodies described herein comprise a light chain containing an amino acid sequence of any one of SEQ ID NO: 85, 89, 90, 93 and 95.

[0275] In some embodiments, the humanized anti-TfR antibody of this disclosure comprises: a heavy chain containing an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identity with SEQ ID NO:84, and / or (e.g., and) a light chain containing an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identity with SEQ ID NO:85. In some embodiments, the humanized anti-TfR antibody of this disclosure comprises a heavy chain containing the amino acid sequence of SEQ ID NO:84 and a light chain containing the amino acid sequence of SEQ ID NO:85.

[0276] In some embodiments, the humanized anti-TfR antibody of this disclosure comprises: a heavy chain containing an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identity with SEQ ID NO:86, and / or (e.g., and) a light chain containing an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identity with SEQ ID NO:85. In some embodiments, the humanized anti-TfR antibody of this disclosure comprises a heavy chain containing the amino acid sequence of SEQ ID NO:86 and a light chain containing the amino acid sequence of SEQ ID NO:85.

[0277] In some embodiments, the humanized anti-TfR antibody of this disclosure comprises: a heavy chain containing an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identity with SEQ ID NO:87, and / or (e.g., and) a light chain containing an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identity with SEQ ID NO:85. In some embodiments, the humanized anti-TfR antibody of this disclosure comprises a heavy chain containing the amino acid sequence of SEQ ID NO:87 and a light chain containing the amino acid sequence of SEQ ID NO:85.

[0278] In some embodiments, the humanized anti-TfR antibody of this disclosure comprises: a heavy chain containing an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identity with SEQ ID NO:88, and / or (e.g., and) a light chain containing an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identity with SEQ ID NO:89. In some embodiments, the humanized anti-TfR antibody of this disclosure comprises a heavy chain containing the amino acid sequence of SEQ ID NO:88 and a light chain containing the amino acid sequence of SEQ ID NO:89.

[0279] In some embodiments, the humanized anti-TfR antibody of this disclosure comprises: a heavy chain containing an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identity with SEQ ID NO:88, and / or (e.g., and) a light chain containing an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identity with SEQ ID NO:90. In some embodiments, the humanized anti-TfR antibody of this disclosure comprises a heavy chain containing the amino acid sequence of SEQ ID NO:88 and a light chain containing the amino acid sequence of SEQ ID NO:90.

[0280] In some embodiments, the humanized anti-TfR antibody of this disclosure comprises: a heavy chain containing an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identity with SEQ ID NO:91, and / or (e.g., and) a light chain containing an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identity with SEQ ID NO:89. In some embodiments, the humanized anti-TfR antibody of this disclosure comprises a heavy chain containing the amino acid sequence of SEQ ID NO:91 and a light chain containing the amino acid sequence of SEQ ID NO:89.

[0281] In some embodiments, the humanized anti-TfR antibody of this disclosure comprises: a heavy chain containing an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identity with SEQ ID NO:91, and / or (e.g., and) a light chain containing an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identity with SEQ ID NO:90. In some embodiments, the humanized anti-TfR antibody of this disclosure comprises a heavy chain containing the amino acid sequence of SEQ ID NO:91 and a light chain containing the amino acid sequence of SEQ ID NO:90.

[0282] In some embodiments, the humanized anti-TfR antibody of this disclosure comprises: a heavy chain containing an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identity with SEQ ID NO:92, and / or (e.g., and) a light chain containing an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identity with SEQ ID NO:93. In some embodiments, the humanized anti-TfR antibody of this disclosure comprises a heavy chain containing the amino acid sequence of SEQ ID NO:92 and a light chain containing the amino acid sequence of SEQ ID NO:93.

[0283] In some embodiments, the humanized anti-TfR antibody of this disclosure comprises: a heavy chain containing an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identity with SEQ ID NO:94, and / or (e.g., and) a light chain containing an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identity with SEQ ID NO:95. In some embodiments, the humanized anti-TfR antibody of this disclosure comprises a heavy chain containing the amino acid sequence of SEQ ID NO:94 and a light chain containing the amino acid sequence of SEQ ID NO:95.

[0284] In some embodiments, the humanized anti-TfR antibody of this disclosure comprises: a heavy chain containing an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identity with SEQ ID NO:92, and / or (e.g., and) a light chain containing an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identity with SEQ ID NO:95. In some embodiments, the humanized anti-TfR antibody of this disclosure comprises a heavy chain containing the amino acid sequence of SEQ ID NO:92 and a light chain containing the amino acid sequence of SEQ ID NO:95.

[0285] In some embodiments, the anti-TfR antibody is a Fab fragment, Fab' fragment, or F(ab')2 fragment of a complete antibody (full-length antibody). The antigen-binding fragment of the complete antibody (full-length antibody) can be prepared by conventional methods (e.g., recombinant preparation or preparation by digesting the heavy chain constant region of full-length IgG with an enzyme such as papain). For example, the F(ab')2 fragment can be produced by digesting the antibody molecule with pepsin or papain, and the Fab' fragment can be produced by reducing the disulfide bridge of the F(ab')2 fragment. In some embodiments, the heavy chain constant region of the Fab fragment of the anti-TfR1 antibody described herein contains the following amino acid sequence:

[0286]

[0287] In some embodiments, the humanized anti-TfR antibody described herein comprises a heavy chain containing any VH or any variant thereof listed in Table 3, and a heavy chain constant region having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with SEQ ID NO:96. In some embodiments, the humanized anti-TfR antibody described herein comprises a heavy chain containing any VH or any variant thereof listed in Table 3, and a heavy chain constant region 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 SEQ ID NO:96. In some embodiments, the humanized anti-TfR antibody described herein comprises a heavy chain comprising any VH or any variant thereof listed in Table 3 and the heavy chain constant region shown in SEQ ID NO:96.

[0288] In some embodiments, the humanized anti-TfR antibody described herein comprises a light chain comprising any VL or any variant thereof listed in Table 3 and a light chain constant region having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with SEQ ID NO:83. In some embodiments, the humanized anti-TfR antibody described herein comprises a light chain comprising any VL or any variant thereof listed in Table 3 and a light chain constant region comprising 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 SEQ ID NO:83. In some embodiments, the humanized anti-TfR antibody described herein comprises a light chain comprising any of the VLs listed in Table 3 or any variant thereof, and the light chain constant region shown in SEQ ID NO:83.

[0289] Table 5 below provides some examples of the Fab heavy and light chain amino acid sequences of the anti-TfR antibody.

[0290] Table 5. Heavy and light chain sequences of humanized anti-TfR Fab instances

[0291]

[0292]

[0293]

[0294]

[0295] *The mutation location is determined by the Kabat number of the corresponding VH sequence containing the mutation.

[0296] **The CDRs according to the Kabat numbering system are bolded; VH / VL sequences are underlined.**

[0297] In some embodiments, the humanized anti-TfR antibody of this 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 variation) compared to the heavy chain shown in any of SEQ ID NO: 97 to 103. As an alternative or supplement (e.g., supplement), the humanized anti-TfR antibody of this disclosure comprises a light chain with no more than 25 amino acid variations compared to the light chain shown in any one of SEQ ID NO: 85, 89, 90, 93 and 95 (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).

[0298] In some embodiments, the humanized anti-TfR antibody described herein comprises a heavy chain containing an amino acid sequence having at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identity with any one of SEQ ID NO: 97 to 103. Alternatively or supplementally (e.g., additionally), the humanized anti-TfR antibody described herein comprises a light chain containing an amino acid sequence having at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identity with any one of SEQ ID NO: 85, 89, 90, 93, and 95. In some embodiments, the anti-TfR antibody described herein comprises a heavy chain containing an amino acid sequence of any one of SEQ ID NO: 97 to 103. As an alternative or supplement (e.g., as an adjunct), the anti-TfR antibody described herein comprises a light chain containing the amino acid sequence of any one of SEQ ID NO: 85, 89, 90, 93 and 95.

[0299] In some embodiments, the humanized anti-TfR antibody of this disclosure comprises: a heavy chain containing an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identity with SEQ ID NO:97, and / or (e.g., and) a light chain containing an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identity with SEQ ID NO:85. In some embodiments, the humanized anti-TfR antibody of this disclosure comprises a heavy chain containing the amino acid sequence of SEQ ID NO:97 and a light chain containing the amino acid sequence of SEQ ID NO:85.

[0300] In some embodiments, the humanized anti-TfR antibody of this disclosure comprises: a heavy chain containing an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identity with SEQ ID NO:98, and / or (e.g., and) a light chain containing an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identity with SEQ ID NO:85. In some embodiments, the humanized anti-TfR antibody of this disclosure comprises a heavy chain containing the amino acid sequence of SEQ ID NO:98 and a light chain containing the amino acid sequence of SEQ ID NO:85.

[0301] In some embodiments, the humanized anti-TfR antibody of this disclosure comprises: a heavy chain containing an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identity with SEQ ID NO:99, and / or (e.g., and) a light chain containing an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identity with SEQ ID NO:85. In some embodiments, the humanized anti-TfR antibody of this disclosure comprises a heavy chain containing the amino acid sequence of SEQ ID NO:99 and a light chain containing the amino acid sequence of SEQ ID NO:85.

[0302] In some embodiments, the humanized anti-TfR antibody of this disclosure comprises: a heavy chain containing an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identity with the amino acid sequence of SEQ ID NO:100, and / or (e.g., and) a light chain containing an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identity with the amino acid sequence of SEQ ID NO:89. In some embodiments, the humanized anti-TfR antibody of this disclosure comprises a heavy chain containing the amino acid sequence of SEQ ID NO:100 and a light chain containing the amino acid sequence of SEQ ID NO:89.

[0303] In some embodiments, the humanized anti-TfR antibody of this disclosure comprises: a heavy chain containing an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identity with the amino acid sequence of SEQ ID NO:100, and / or (e.g., and) a light chain containing an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identity with the amino acid sequence of SEQ ID NO:90. In some embodiments, the humanized anti-TfR antibody of this disclosure comprises a heavy chain containing the amino acid sequence of SEQ ID NO:100 and a light chain containing the amino acid sequence of SEQ ID NO:90.

[0304] In some embodiments, the humanized anti-TfR antibody of this disclosure comprises: a heavy chain containing an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identity with SEQ ID NO:101, and / or (e.g., and) a light chain containing an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identity with SEQ ID NO:89. In some embodiments, the humanized anti-TfR antibody of this disclosure comprises a heavy chain containing the amino acid sequence of SEQ ID NO:101 and a light chain containing the amino acid sequence of SEQ ID NO:89.

[0305] In some embodiments, the humanized anti-TfR antibody of this disclosure comprises: a heavy chain containing an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identity with SEQ ID NO:101, and / or (e.g., and) a light chain containing an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identity with SEQ ID NO:90. In some embodiments, the humanized anti-TfR antibody of this disclosure comprises a heavy chain containing the amino acid sequence of SEQ ID NO:101 and a light chain containing the amino acid sequence of SEQ ID NO:90.

[0306] In some embodiments, the humanized anti-TfR antibody of this disclosure comprises: a heavy chain containing an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identity with SEQ ID NO:102, and / or (e.g., and) a light chain containing an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identity with SEQ ID NO:93. In some embodiments, the humanized anti-TfR antibody of this disclosure comprises a heavy chain containing the amino acid sequence of SEQ ID NO:102 and a light chain containing the amino acid sequence of SEQ ID NO:93.

[0307] In some embodiments, the humanized anti-TfR antibody of this disclosure comprises: a heavy chain containing an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identity with SEQ ID NO:103, and / or (e.g., and) a light chain containing an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identity with SEQ ID NO:95. In some embodiments, the humanized anti-TfR antibody of this disclosure comprises a heavy chain containing the amino acid sequence of SEQ ID NO:103 and a light chain containing the amino acid sequence of SEQ ID NO:95.

[0308] In some embodiments, the humanized anti-TfR antibody of this disclosure comprises: a heavy chain containing an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identity with SEQ ID NO:102, and / or (e.g., and) a light chain containing an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identity with SEQ ID NO:95. In some embodiments, the humanized anti-TfR antibody of this disclosure comprises a heavy chain containing the amino acid sequence of SEQ ID NO:102 and a light chain containing the amino acid sequence of SEQ ID NO:95.

[0309] In some embodiments, the humanized anti-TfR receptor antibody described herein can be any antibody form, including but not limited to full-length antibodies, their antigen-binding fragments (e.g., Fab, Fab', F(ab')2, Fv), single-chain antibodies, bispecific antibodies, or nanobodies. In some embodiments, the humanized anti-TfR antibody described herein is an scFv. In some embodiments, the humanized anti-TfR antibody described herein is an scFv-Fab (e.g., an scFv fused to a portion of a constant region). In some embodiments, the anti-TfR receptor antibody described herein is an scFv fused to a constant region (e.g., the human IgG1 constant region shown in SEQ ID NO:81 or SEQ ID NO:82, or a portion thereof, such as the Fc portion) at the C-terminus or N-terminus.

[0310] In some embodiments, conserved mutations may be introduced into the antibody sequence (e.g., CDR or framework sequence) at locations where residues are unlikely to interact with the target antigen (e.g., transferrin receptor) (e.g., as determined based on crystal structure). In some embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced into the Fc region of the anti-TfR antibody described herein (e.g., in the CH2 domain (residues 231 to 340 of human IgG1) and / or (e.g., and) the CH3 domain (residues 341 to 447 of human IgG1) and / or (e.g., and) the hinge region, numbered according to the Kabat numbering system (e.g., EU index in Kabat) to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or (e.g., and) antigen-dependent cytotoxicity.

[0311] In some embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced into the hinge region of the Fc region (CH1 domain) to alter (e.g., increase or decrease) the number of cysteine ​​residues in the hinge region, as described, for example, in U.S. Patent No. 5,677,425. Altering the number of cysteine ​​residues in the hinge region of the CH1 domain can, for example, promote the assembly of light and heavy chains, or alter (e.g., increase or decrease) antibody stability or promote linker conjugation.

[0312] In some embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced into the Fc region of the muscle-targeting antibody described herein (e.g., in the CH2 domain (residues 231 to 340 of human IgG1) and / or (e.g., and) the CH3 domain (residues 341 to 447 of human IgG1) and / or (e.g., and) the hinge region, numbered according to the Kabat numbering system (e.g., EU index in Kabat)) to increase or decrease the antibody's affinity for an Fc receptor (e.g., an activated Fc receptor) on the surface of effector cells. Mutations in the Fc region of antibodies that decrease or increase the antibody's affinity for Fc receptors, and techniques for introducing such mutations into the Fc receptor or fragments thereof, are known to those skilled in the art. Some examples of mutations in the Fc receptor that can be used to alter the affinity of an antibody for the Fc receptor are described below: for example, Smith P et al., (2012) PNAS109:6181-6186, U.S. Patent No. 6,737,056, and International Publications Nos. WO 02 / 060919, WO 98 / 23289, and WO 97 / 34631, which are incorporated herein by reference.

[0313] In some embodiments, one, two, or more amino acid mutations (i.e., substitutions, insertions, or deletions) are introduced into the constant domain of IgG or its FcRn binding fragment (preferably, an Fc or hinge-Fc domain fragment) to alter (e.g., decrease or increase) the half-life of the antibody in vivo. See, for example, International Publications Nos. WO 02 / 060919, WO 98 / 23289, and WO97 / 34631, and U.S. Patent Nos. 5,869,046, 6,121,022, 6,277,375, and 6,165,745, for example, mutations that alter (e.g., decrease or increase) the half-life of the antibody in vivo.

[0314] In some embodiments, one, two, or more amino acid mutations (i.e., substitutions, insertions, or deletions) are introduced into the constant domain of IgG or its FcRn binding fragment (preferably, an Fc or hinge-Fc domain fragment) to reduce the half-life of the anti-TfR antibody in vivo. In some embodiments, one, two, or more amino acid mutations (i.e., substitutions, insertions, or deletions) are introduced into the constant domain of IgG or its FcRn binding fragment (preferably, an 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 to 340 of human IgG1) and / or (e.g., and) the third constant (CH3) domain (residues 341 to 447 of human IgG1) (numbered according to the EU index in Kabat (Kabat E A et al., (1991) ibid.)). In some embodiments, the constant region of the IgG1 of the antibody described herein includes 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, the positions of which are based on the EU index number in Kabat. See U.S. Patent No. 7,658,921, which is incorporated herein by reference. This type of mutant IgG (referred to as the “YTE mutant”) has been shown to have a 4-fold increased half-life compared to the wild-type form of the same antibody (see Dall'Acqua WF et al., (2006) JBiolChem 281:23514-24). In some embodiments, the antibody comprises an IgG constant domain containing one, two, three or more amino acid substitutions at positions 251 to 257, 285 to 290, 308 to 314, 385 to 389 and 428 to 436, according to the EU index number in Kabat.

[0315] In some embodiments, one, two, or more amino acid substitutions are introduced into the Fc region of the IgG constant domain to alter the effector function of the anti-TfR antibody. The effector ligand whose affinity is altered may be, for example, an Fc receptor or a C1 component of complement. This method is described in more detail in U.S. Patent Nos. 5,624,821 and 5,648,260. In some embodiments, deletion or inactivation of the constant domain (through point mutations or other means) can reduce Fc receptor binding of the circulating antibody, thereby improving tumor localization. For a description of mutations that result in deletion or inactivation of the constant domain to improve tumor localization, see, for example, U.S. Patent Nos. 5,585,097 and 8,591,886. In some embodiments, one or more amino acid substitutions may be introduced into the Fc region of the antibody described herein to remove potential glycosylation sites on the Fc region, which can reduce Fc receptor binding (see, for example, Shields R Let al., (2001) J BiolChem 276:6591-604).

[0316] In some embodiments, one or more amino groups in the constant region of the anti-TfR antibody described herein may be replaced with different amino acid residues, such that the antibody exhibits altered Clq binding and / or (e.g., and) reduced or eliminated complement-dependent cytotoxicity (CDC). This method is described in more 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 modified, thereby altering the antibody's ability to fix complement. This method is further described in International Publication No. WO 94 / 29351. In some embodiments, the Fc region of the antibody described herein is modified to enhance the antibody's ability to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or (e.g., and) increase the antibody's affinity for the Fcγ receptor. This method is further described in International Publication No. WO 00 / 42072.

[0317] In some embodiments, the heavy chain and / or (e.g., and) light chain variable domain sequences of the antibodies provided herein can be used to generate, for example, CDR-grafted, chimeric, humanized, or complexed human antibodies or antigen-binding fragments, as described elsewhere herein. As will be understood by those skilled in the art, any variant (CDR-grafted, chimeric, humanized, or complexed antibody) derived from any antibody provided herein can be used 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 complexed antibody) has 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 relative to the original antibody from which it is derived.

[0318] In some embodiments, the antibodies provided herein contain mutations that confer the desired properties to the antibody. For example, to avoid potential complications attributable to Fab arm exchanges known to occur with native IgG4 mAb, the antibodies provided herein may contain a stable 'Adair' mutation (Angal S., et al., "Asingle amino acid substitution abolishes the heterogeneity of chimeric mouse / human (IgG4) antibody," Mol Immunol 30, 105-108; 1993), in which serine at position 228 (EU number, residue 241 according to Kabat number) is converted to proline, thereby producing an IgG1-like hinge sequence. Therefore, any antibody may contain a stable 'Adair' mutation.

[0319] In some embodiments, the antibody is modified, for example, by 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 by N-glycosylation, O-glycosylation, C-glycosylation, glycosylphosphatidylinositol (GPI-anchored attachment), and / or (e.g., and) phosphorylated glycosylation. In some embodiments, one or more sugar or carbohydrate molecules are monosaccharides, disaccharides, oligosaccharides, or glycans. In some embodiments, one or more sugar or carbohydrate molecules are branched oligosaccharides or branched glycans. In some embodiments, one or more sugar or carbohydrate molecules comprise mannose units, glucose units, N-acetylglucosamine units, N-acetylglucosamine units, galactose units, fucose units, or phospholipid units. In some embodiments, about 1 to 10, about 1 to 5, about 5 to 10, about 1 to 4, about 1 to 3, or about 2 sugar molecules are present. In some embodiments, the glycosylated antibody is fully or partially glycosylated. In some embodiments, the antibody is glycosylated by a chemical reaction or by an enzymatic means. In some embodiments, the antibody is glycosylated in vitro or intracellularly, optionally lacking enzymes in the N- or O-glycosylation pathway, such as glycosyltransferases. In some embodiments, the antibody is functionalized with sugar or carbohydrate molecules, as described in International Patent Application Publication WO2014065661, published May 1, 2014, entitled "Modified antibody, antibody-conjugate and process for the preparation thereof".

[0320] In some embodiments, any of the anti-TfR1 antibodies described herein may include a signal peptide (e.g., an N-terminal signal peptide) in the heavy chain sequence and / or (e.g., and) the light chain sequence. In some embodiments, the anti-TfR1 antibody described herein comprises any of the VH and VL sequences, any of the IgG heavy chain and light chain sequences, or any of the Fab' heavy chain and light chain sequences described herein, and further comprises a signal peptide (e.g., an N-terminal signal peptide). In some embodiments, the signal peptide comprises the amino acid sequence MGWSCIILFLVATATGVHS (SEQ ID NO: 104).

[0321] Other known anti-transferrin receptor antibodies

[0322] Any other suitable anti-transferrin receptor antibody known in the art may be used as a muscle target in the complexes disclosed herein. Table 8 lists some examples of known anti-transferrin receptor antibodies, including relevant references and binding epitopes. In some embodiments, the anti-transferrin receptor antibody contains the complementarity-determining regions (CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3) of any anti-transferrin receptor antibody provided herein (e.g., the anti-transferrin receptor antibodies listed in Table 8).

[0323] Table 8. List of anti-transferrin receptor antibody clones, including relevant references and binding epitope information.

[0324]

[0325]

[0326]

[0327]

[0328] In some embodiments, the transferrin receptor antibody of this disclosure comprises one or more CDR-H (e.g., CDR-H1, CDR-H2, and CDR-H3) amino acid sequences selected from any of the anti-transferrin receptor antibodies listed in Table 8. In some embodiments, the transferrin receptor antibody comprises CDR-H1, CDR-H2, and CDR-H3 as provided for any of the anti-transferrin receptor antibodies selected in Table 8. In some embodiments, the anti-transferrin receptor antibody comprises CDR-L1, CDR-L2, and CDR-L3 as provided for any of the anti-transferrin receptor antibodies selected in Table 8. In some embodiments, the anti-transferrin antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 as provided for any of the anti-transferrin receptor antibodies selected in Table 8. This disclosure also includes 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 of the anti-transferrin receptor antibodies selected from Table 8. In some embodiments, the CDR3 domains of both the antibody heavy and light chains may play a particularly important role in the antibody's binding specificity / affinity to the antigen. Therefore, the anti-transferrin receptor antibody of this disclosure may comprise at least the heavy chain and / or (e.g., and) light chain CDR3 of any of the anti-transferrin receptor antibodies selected from Table 8.

[0329] In some instances, any anti-transferrin receptor antibody of this disclosure has one or more CDR (e.g., CDR-H or CDR-L) sequences that are substantially similar to any CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and / or (e.g., and) CDR-L3 sequences from an anti-transferrin receptor antibody selected from Table 8. In some embodiments, the position of one or more CDRs of the antibody described herein along 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 may vary by one, two, three, four, five, or six amino acid positions, as long as immune-specific binding to the transferrin receptor (e.g., human transferrin receptor) is maintained (e.g., substantially maintaining binding to the original antibody from which it is derived, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). For example, in some embodiments, the location of the CDR of any antibody described herein can be altered by shifting the N-terminal and / or (e.g., and) C-terminal boundary of the CDR relative to the CDR location of any antibody described herein by one, two, three, four, five, or six amino acids, as long as immune-specific binding to the transferrin receptor (e.g., human transferrin receptor) is maintained (e.g., substantially maintaining binding to the original antibody from which it is derived, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). In another embodiment, the length of one or more CDRs of the antibody described herein along 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 may be altered (e.g., become shorter or longer) by one, two, three, four, five, or more amino acids, as long as immune-specific binding to the transferrin receptor (e.g., human transferrin receptor) is maintained (e.g., substantially maintaining binding to the original antibody from which it is derived, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%).

[0330] Therefore, in some embodiments, the CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and / or (e.g., and) CDR-H3 described herein may be one, two, three, four, five or more amino acids shorter than one or more CDRs described herein (e.g., CDRs from any anti-transferrin receptor antibody selected from Table 8), as long as the immune-specific binding to the transferrin receptor (e.g., human transferrin receptor) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% relative to the binding of the original antibody from which it is derived). In some embodiments, the CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and / or (e.g., and) CDR-H3 described herein may be one, two, three, four, five or more amino acids longer than one or more CDRs described herein (e.g., CDRs from any anti-transferrin receptor antibody selected from Table 8), provided that an immune-specific binding to the transferrin receptor (e.g., human transferrin receptor) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% relative to the binding of the original antibody from which it is derived). In some embodiments, the amino moiety of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and / or (e.g., and) CDR-H3 described herein may be extended by one, two, three, four, five or more amino acids compared to one or more CDRs described herein (e.g., CDRs from any anti-transferrin receptor antibody selected from Table 8), as long as the immune-specific binding to the transferrin receptor (e.g., human transferrin receptor) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%) relative to the binding of the original antibody from which it originates. In some embodiments, the carboxyl moiety of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and / or (e.g., and) CDR-H3 described herein may be extended by one, two, three, four, five or more amino acids compared to one or more CDRs described herein (e.g., CDRs from any anti-transferrin receptor antibody selected from Table 8), as long as the immune-specific binding to the transferrin receptor (e.g., human transferrin receptor) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%) relative to the binding of the original antibody from which it originates.In some embodiments, the amino moiety of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and / or (e.g., and) CDR-H3 described herein may be shortened by one, two, three, four, five or more amino acids compared to one or more CDRs described herein (e.g., CDRs from any anti-transferrin receptor antibody selected from Table 8), as long as the immune-specific binding to the transferrin receptor (e.g., human transferrin receptor) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%) relative to the binding of the original antibody from which it is derived. In some embodiments, the carboxyl moiety of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and / or (e.g., and) CDR-H3 described herein may be shortened by one, two, three, four, five or more amino acids compared to one or more CDRs described herein (e.g., CDRs from any anti-transferrin receptor antibody selected from Table 8), as long as immune-specific binding to the transferrin receptor (e.g., human transferrin receptor) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, relative to the binding of the original antibody from which it is derived). Any method may be used to determine whether immune-specific binding to the transferrin receptor (e.g., human transferrin receptor) is maintained, for example using binding assays and conditions described in the art.

[0331] In some instances, any anti-transferrin receptor antibody of this disclosure has one or more CDR (e.g., CDR-H or CDR-L) sequences substantially similar to any anti-transferrin receptor antibody selected from Table 8. For example, an antibody may comprise one or more CDR sequences from any anti-transferrin receptor antibody 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 CDR provided herein (e.g., CDRs from any anti-transferrin receptor antibody selected from Table 8), provided that immune-specific binding to the transferrin receptor (e.g., human transferrin receptor) is maintained (e.g., substantially maintained, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% relative to the binding of the original antibody from which it originates). In some embodiments, any amino acid variation in any CDR provided herein may be a conserved variation. Conserved variations can be introduced into the CDR at locations where residues are unlikely to interact with transferrin receptor proteins (e.g., human transferrin receptor protein) (e.g., as determined based on crystal structure). Some aspects of this disclosure provide transferrin receptor antibodies comprising one or more heavy chain variable (VH) and / or (e.g., and) light chain variable (VL) domains provided herein. In some embodiments, any VH domain provided herein comprises one or more CDR-H sequences (e.g., CDR-H1, CDR-H2, and CDR-H3) provided herein, such as any CDR-H sequence provided in any of the anti-transferrin receptor antibodies listed in Table 8. In some embodiments, any VL domain provided herein comprises one or more CDR-L sequences (e.g., CDR-L1, CDR-L2, and CDR-L3) provided herein, such as any CDR-L sequence provided in any of the anti-transferrin receptor antibodies listed in Table 8.

[0332] In some embodiments, the anti-transferrin receptor antibody of this disclosure comprises any antibody containing a heavy chain variable domain and / or (e.g., and) a light chain variable domain of any anti-transferrin receptor antibody (e.g., selected from any anti-transferrin receptor antibody in Table 8). In some embodiments, the anti-transferrin receptor antibody of this disclosure comprises any antibody containing a heavy chain variable and a light chain variable pair of any anti-transferrin receptor antibody (e.g., selected from any anti-transferrin receptor antibody in Table 8).

[0333] Some aspects of this disclosure provide anti-transferrin receptor antibodies having a heavy chain variable (VH) and / or (e.g., and) light chain variable (VL) domain amino acid sequence homologous to any of those described herein. In some embodiments, the anti-transferrin receptor antibody comprises a heavy chain variable sequence or light chain variable sequence having at least 75% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identity with any heavy chain variable sequence and / or any light chain variable sequence of any anti-transferrin receptor antibody (e.g., selected from any of the anti-transferrin receptor antibodies in Table 8). In some embodiments, the homologous heavy chain variable and / or (e.g., and) light chain variable amino acid sequences are not varied within any CDR sequences provided herein. For example, in some embodiments, the degree of sequence variation (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) may occur in the heavy chain variable and / or (e.g., and) light chain variable sequences excluding any CDR sequences provided herein. In some embodiments, any anti-transferrin receptor antibody provided herein comprises a heavy chain variable sequence and a light chain variable sequence, which comprises a frame sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity with the frame sequence of any anti-transferrin receptor antibody (e.g., any anti-transferrin receptor antibody selected from Table 8).

[0334] In some embodiments, anti-transferrin receptor antibodies that specifically bind to a transferrin receptor (e.g., human transferrin receptor) include a light chain variable VL domain comprising any CDR-L domain (CDR-L1, CDR-L2, and CDR-L3) selected from any anti-transferrin receptor antibody in Table 8, or CDR-L domain variants provided herein. In some embodiments, anti-transferrin receptor antibodies that specifically bind to a transferrin receptor (e.g., human transferrin receptor) include a light chain variable VL domain comprising CDR-L1, CDR-L2, and CDR-L3 of any anti-transferrin receptor antibody (e.g., any anti-transferrin receptor antibody selected from Table 8). In some embodiments, the anti-transferrin receptor antibody comprises a light chain variable (VL) region sequence comprising one, two, three, or four frame regions of the light chain variable region sequence of any anti-transferrin receptor antibody (e.g., selected from any of the anti-transferrin receptor antibodies in Table 8). In some embodiments, the anti-transferrin receptor antibody comprises one, two, three, or four frame regions of the light chain variable region sequence, which have at least 75%, 80%, 85%, 90%, 95%, or 100% identity with one, two, three, or four frame regions of the light chain variable region sequence of any anti-transferrin receptor antibody (e.g., selected from any of the anti-transferrin receptor antibodies in Table 8). In some embodiments, the light chain variable frame region derived from the amino acid sequence consists of the amino acid sequence, but with up to 10 amino acid substitutions, deletions, and / or (e.g., and) insertions, preferably up to 10 amino acid substitutions. In some embodiments, the light chain variable frame region derived from the amino acid sequence is composed of the amino acid sequence, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues replace amino acids present at similar positions in the corresponding non-human primate or human light chain variable frame region.

[0335] In some embodiments, the anti-transferrin receptor antibody that specifically binds to the transferrin receptor comprises CDR-L1, CDR-L2, and CDR-L3 of any anti-transferrin receptor antibody (e.g., selected from any of the anti-transferrin receptor antibodies in Table 8). In some embodiments, the antibody also comprises one, two, three, or all four VL framework regions derived from human or primate antibodies. The primate or human antibody light chain framework region selected for use with the light chain CDR sequence described herein may have, for example, at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, or at least 99%) identity with the light chain framework region of the non-human parent antibody. The amino acid numbering in the light chain complementarity-determining region of the selected primate or human antibody may be the same as or substantially the same as the amino acid numbering in the light chain complementarity-determining region of any antibody provided herein (e.g., selected from any of the anti-transferrin receptor antibodies in Table 8). In some embodiments, the amino acid residues of the primate or human light chain framework region are derived from the light chain framework region of a natural primate or human antibody, and have at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% (or more) identity with the light chain framework region of any anti-transferrin receptor antibody (e.g., selected from any of the anti-transferrin receptor antibodies in 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 κ 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 λ subfamily.

[0336] In some embodiments, any anti-transferrin receptor antibody provided herein comprises a light chain variable domain, which also comprises a light chain constant region. In some embodiments, the light chain constant region is a κ or λ light chain constant region. In some embodiments, the κ or λ light chain constant region is derived from mammals, such as from humans, monkeys, rats, or mice. In some embodiments, the light chain constant region is a human κ light chain constant region. In some embodiments, the light chain constant region is a human λ light chain constant region. It should be understood that any light chain constant region provided herein may be a variant of any light chain constant region provided herein. In some embodiments, the light chain constant region comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity with any light chain constant region of any anti-transferrin receptor antibody (e.g., any anti-transferrin receptor antibody selected from Table 8).

[0337] In some implementations, the anti-transferrin receptor antibody is any anti-transferrin receptor antibody, such as any of the anti-transferrin receptor antibodies selected from Table 8.

[0338] In some embodiments, the anti-transferrin receptor antibody comprises a VL domain containing the amino acid sequence of any anti-transferrin receptor antibody (e.g., selected from any of the anti-transferrin receptor antibodies in Table 8), and wherein the constant region contains the amino acid sequence of the constant region of an IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule or a human IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule. In some embodiments, the anti-transferrin receptor antibody comprises any VL domain or a variant of the VL domain, and any VH domain or a variant of the VH domain, wherein the VL and VH domains or variants thereof originate from the same antibody clone, and wherein the constant region contains the amino acid sequence of the constant region of an IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule or any class of immunoglobulin molecules (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or any subclass (e.g., IgG2a and IgG2b). Some non-limiting examples of human constant regions are described in this art, for example, see above Kabat E A et al., (1991).

[0339] In some embodiments, the muscle target is a transferrin receptor antibody (e.g., an antibody and variants thereof as described in International Application Publication WO2016 / 081643, which is incorporated herein by reference).

[0340] Table 9 provides the heavy and light chain CDRs of antibodies according to different definition systems. Different definition systems have been described, such as the Kabat definition, the Chothia definition, and / or (e.g., and) the contact definition. See, for example (e.g., Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, 5th 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-lazikanie et 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).

[0341] Table 9. Heavy and light chain CDRs of mouse transferrin receptor antibodies.

[0342]

[0343] It also provides sequences of heavy-chain variable domains (VH) and light-chain variable domains:

[0344] VH

[0345]

[0346] VL

[0347]

[0348] In some embodiments, the transferrin receptor antibody of this disclosure comprises the same CDR-H1, CDR-H2, and CDR-H3 as CDR-H1, CDR-H2, and CDR-H3 shown in Table 9. Alternatively or supplementally (e.g., additionally), the transferrin receptor antibody of this disclosure comprises the same CDR-L1, CDR-L2, and CDR-L3 as CDR-L1, CDR-L2, and CDR-L3 shown in Table 9.

[0349] In some embodiments, the transferrin receptor antibody of this disclosure comprises CDR-H1, CDR-H2, and CDR-H3, which, compared to CDR-H1, CDR-H2, and CDR-H3 shown in Table 9, collectively contain no more than 5 amino acid variations (e.g., no more than 5, 4, 3, 2, or 1 amino acid variation). "Common" means that the total number of amino acid variations in all three heavy chain CDRs is within the defined range. Alternatively or supplementally (e.g., as an adjunct), the transferrin receptor antibody of this disclosure may comprise CDR-L1, CDR-L2, and CDR-L3, which, compared to CDR-L1, CDR-L2, and CDR-L3 shown in Table 9, collectively contain no more than 5 amino acid variations (e.g., no more than 5, 4, 3, 2, or 1 amino acid variation).

[0350] In some embodiments, the transferrin receptor antibody of this disclosure comprises CDR-H1, CDR-H2, and CDR-H3, wherein at least one contains no more than 3 amino acid variations (e.g., no more than 3, 2, or 1 amino acid variation) compared to the corresponding heavy chain CDR shown in Table 9. Alternatively or supplementally (e.g., additionally), the transferrin receptor antibody of this disclosure may comprise CDR-L1, CDR-L2, and CDR-L3, wherein at least one contains no more than 3 amino acid variations (e.g., no more than 3, 2, or 1 amino acid variation) compared to the corresponding light chain CDR shown in Table 9.

[0351] In some embodiments, the transferrin receptor antibody of this disclosure comprises CDR-L3, which contains no more than 3 amino acid variations (e.g., no more than 3, 2, or 1 amino acid variations) compared to the CDR-L3 shown in Table 9. In some embodiments, the transferrin receptor antibody of this disclosure comprises CDR-L3, which contains 1 amino acid variation compared to the CDR-L3 shown in Table 9. In some embodiments, the transferrin receptor antibody of this disclosure comprises CDR-L3 of QHFAGPTLT (SEQ ID NO: 126) (according to the Kabat and Chothia definition system) or CDR-L3 of QHFAGPTLT (SEQ ID NO: 127) (according to the Contact definition system). In some embodiments, the transferrin receptor antibody of this disclosure comprises the same CDR-H1, CDR-H2, CDR-H3, CDR-L1, and CDR-L2 as CDR-H1, CDR-H2, and CDR-H3 shown in Table 9, and comprises CDR-L3 of QHFAGPTLT (SEQ ID NO: 126) (according to the Kabat and Chothia definition system) or CDR-L3 of QHFAGTPL (SEQ ID NO: 127) (according to the Contact definition system).

[0352] In some embodiments, the transferrin receptor antibody of this disclosure comprises a heavy chain CDR that collectively has at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identity with the heavy chain CDRs shown in Table 9. Alternatively or supplementally (e.g., additionally), the transferrin receptor antibody of this disclosure comprises a light chain CDR that collectively has at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identity with the light chain CDRs shown in Table 9.

[0353] In some embodiments, the transferrin receptor antibody of this disclosure comprises VH containing the amino acid sequence of SEQ ID NO:124. Alternatively or as an alternative (e.g., supplement), the transferrin receptor antibody of this disclosure comprises VL containing the amino acid sequence of SEQ ID NO:125.

[0354] In some embodiments, the transferrin receptor antibody of this disclosure comprises a VH containing no more than 25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) compared to the VH shown in SEQ ID NO: 125. Alternatively or supplementally (e.g., as an adjunct), the transferrin receptor antibody of this disclosure comprises a VL containing no more than 15 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 variation) compared to the VL shown in SEQ ID NO: 125.

[0355] In some embodiments, the transferrin receptor antibody of this disclosure comprises VH, said VH containing an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identity with the VH shown in SEQ ID NO:124. Alternatively or supplementally (e.g., as an adjunct), the transferrin receptor antibody of this disclosure comprises VL, said VL containing an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identity with the VL shown in SEQ ID NO:125.

[0356] In some embodiments, the transferrin receptor antibody of this disclosure is a humanized antibody (e.g., a humanized variant of the antibody). In some embodiments, the transferrin receptor antibody of this disclosure comprises the same CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 as CDR-H1, CDR-H2, and CDR-H3 shown in Table 9, and comprises a humanized heavy chain variable region and / or (e.g., and) a humanized light chain variable region.

[0357] Humanized antibodies are human immunoglobulins (receptor antibodies) in which residues of the complementarity-determining region (CDR) from the receptor are replaced by residues of the CDR from a non-human species (donor antibody) having the desired specificity, affinity, and capacity, such as mouse, rat, or rabbit. In some embodiments, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may include residues not found in either the receptor antibody or the introduced CDR or framework sequence but included to further refine and optimize antibody performance. Generally, humanized antibodies will contain substantially all of at least one, and typically two, variable domains, wherein all or substantially all of the CDR regions correspond to those of non-human immunoglobulins, and all or substantially all of the FR regions are those of human immunoglobulin common sequences. Humanized antibodies will preferably also contain at least a portion of the immunoglobulin constant region or domain (Fc) (typically those of human immunoglobulins). The antibody may have an Fc region modified as described in WO 99 / 58572. Other forms of humanized antibodies have one or more CDRs (one, two, three, four, five, or six) that are altered relative to the original antibody; these are also referred to as one or more CDRs derived from one or more CDRs of the original antibody. Humanized antibodies may also involve affinity maturation.

[0358] In some embodiments, humanization is achieved by grafting CDRs (e.g., as shown in Table 9) into the human variable domains of IGKV1-NL1*01 and IGHV1-3*01. In some embodiments, the transferrin receptor antibody of this disclosure is a humanized variant containing one or more amino acid substitutions at positions 9, 13, 17, 18, 40, 45, and 70 compared to the VL shown in SEQ ID NO:125, and / or (e.g., and) containing 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 shown in SEQ ID NO:124. In some embodiments, the transferrin receptor antibody of this disclosure is a humanized variant containing amino acid substitutions at all positions 9, 13, 17, 18, 40, 45, and 70 compared to VL shown in SEQ ID NO:125, and / or (e.g., and) containing amino acid substitutions at all positions 1, 5, 7, 11, 12, 20, 38, 40, 44, 66, 75, 81, 83, 87, and 108 compared to VH shown in SEQ ID NO:124.

[0359] In some embodiments, the transferrin receptor antibody of this disclosure is a humanized antibody and contains residues at positions 43 and 48 of VL as shown in SEQ ID NO:125. Alternatively or supplementally (e.g., additionally), the transferrin receptor antibody of this disclosure is a humanized antibody and contains residues at positions 48, 67, 69, 71, and 73 of VH as shown in SEQ ID NO:124.

[0360] The following are provided: VH and VL amino acid sequences of exemplary humanized antibodies that can be used in accordance with this disclosure:

[0361] Humanized VH

[0362]

[0363] Humanized VL

[0364]

[0365] In some embodiments, the transferrin receptor antibody of this disclosure comprises VH containing the amino acid sequence of SEQ ID NO:128. Alternatively or supplementally (e.g., as an alternative), the transferrin receptor antibody of this disclosure comprises VL containing the amino acid sequence of SEQ ID NO:129.

[0366] In some embodiments, the transferrin receptor antibody of this disclosure comprises a VH containing no more than 25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) compared to the VH shown in SEQ ID NO: 129. Alternatively or supplementally (e.g., as an adjunct), the transferrin receptor antibody of this disclosure comprises a VL containing no more than 15 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 variation) compared to the VL shown in SEQ ID NO: 129.

[0367] In some embodiments, the transferrin receptor antibody of this disclosure comprises VH, said VH containing an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identity with the VH shown in SEQ ID NO:128. Alternatively or supplementally (e.g., as an adjunct), the transferrin receptor antibody of this disclosure comprises VL, said VL containing an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identity with the VL shown in SEQ ID NO:129.

[0368] In some embodiments, the transferrin receptor antibody of this disclosure is a humanized variant containing amino acid substitutions at one or more of positions 43 and 48 compared to VL shown in SEQ ID NO:125, and / or (e.g., and) amino acid substitutions at one or more of positions 48, 67, 69, 71, and 73 compared to VH shown in SEQ ID NO:124. In some embodiments, the transferrin receptor antibody of this disclosure is a humanized variant containing S43A and / or (e.g., and) V48L mutations compared to VL shown in SEQ ID NO:125, and / or (e.g., and) A67V, L69I, V71R, and K73T mutations compared to VH shown in SEQ ID NO:124.

[0369] In some embodiments, the transferrin receptor antibody of this disclosure is a humanized variant that contains amino acid substitutions at one or more of positions 9, 13, 17, 18, 40, 43, 48, 45, and 70 compared to the VL shown in SEQ ID NO: 125, and / or (e.g., and) contains 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 shown in SEQ ID NO: 124.

[0370] In some embodiments, the transferrin receptor antibody of this disclosure is a chimeric antibody, which may comprise a heavy constant region and a light constant region derived from a human antibody. A chimeric antibody is an antibody having a variable region or a portion thereof 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 derived from an antibody of one mammal (e.g., a non-human mammal, such as a mouse, rabbit, or rat), while the constant portion is sequence homologous to an antibody derived from another mammal (e.g., a human). In some embodiments, amino acid modifications may be made in the variable region and / or (e.g., and) the constant region.

[0371] In some embodiments, the transferrin receptor antibody described herein is a chimeric antibody, which may comprise a heavy constant region and a light constant region derived from a human antibody. A chimeric antibody is an antibody having a variable region or a portion thereof derived from a first species and a constant region derived from a second species. Typically, in these chimeric antibodies, the variable regions of both the light and heavy chains mimic the variable regions derived from an antibody of one mammal (e.g., a non-human mammal, such as a mouse, rabbit, or rat), while the constant portion is sequence homologous to that of an antibody derived from another mammal (e.g., a human). In some embodiments, amino acid modifications may be made in the variable region and / or (e.g., and) the constant region.

[0372] In some implementations, the heavy chain of any transferrin receptor antibody as described herein may include a heavy chain constant region (CH) or a portion thereof (e.g., CH1, CH2, CH3, or combinations thereof). The heavy chain constant region may have any suitable source, such as human, mouse, rat, or rabbit. In a specific example, the heavy chain constant region is derived from human IgG, such as IgG1, IgG2, or IgG4 (γ heavy chain). An example of the human IgG1 constant region is given below:

[0373]

[0374] In some embodiments, the light chain of any transferrin receptor antibody described herein may also include a light chain constant region (CL), which may be any CL known in the art. In some instances, the CL is a κ light chain. In other instances, the CL is a λ light chain. In some embodiments, the CL is a κ light chain, the sequence of which is provided below:

[0375]

[0376] Other antibody heavy and light chain constant regions are well known in the art, such as those available in the IMGT database (www.imgt.org) or www.vbase2.org / vbstat.php, both of which are incorporated herein by reference.

[0377] The following are some examples of the heavy and light chain amino acid sequences of the aforementioned transferrin receptor antibody:

[0378] Heavy chain (VH+ human IgG1 constant region)

[0379]

[0380] Light chain (VL+κ light chain)

[0381]

[0382] Heavy chain (humanized VH+ human IgG1 constant region)

[0383]

[0384] Light chain (humanized VL+κ light chain)

[0385]

[0386] In some embodiments, the transferrin receptor antibody described herein comprises a heavy chain containing an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identity with SEQ ID NO:132. Alternatively or supplementally (e.g., as an adjunct), the transferrin receptor antibody described herein comprises a light chain containing an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identity with SEQ ID NO:133. In some embodiments, the transferrin receptor antibody described herein comprises a heavy chain containing the amino acid sequence of SEQ ID NO:132. Alternatively or supplementally (e.g., as an adjunct), the transferrin receptor antibody described herein comprises a light chain containing the amino acid sequence of SEQ ID NO:133.

[0387] In some embodiments, the transferrin receptor antibody of this 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 shown in SEQ ID NO: 132. Alternatively or supplementally (e.g., as an adjunct), the transferrin receptor antibody of this disclosure comprises a light chain containing no more than 15 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 variation) compared to the light chain shown in SEQ ID NO: 133.

[0388] In some embodiments, the transferrin receptor antibody described herein comprises a heavy chain containing an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identity with SEQ ID NO:134. Alternatively or supplementally (e.g., as an adjunct), the transferrin receptor antibody described herein comprises a light chain containing an amino acid sequence having at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identity with SEQ ID NO:135. In some embodiments, the transferrin receptor antibody described herein comprises a heavy chain containing the amino acid sequence of SEQ ID NO:134. Alternatively or supplementally (e.g., as an adjunct), the transferrin receptor antibody described herein comprises a light chain containing the amino acid sequence of SEQ ID NO:135.

[0389] In some embodiments, the transferrin receptor antibody of this 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 of the humanized antibody shown in SEQ ID NO: 135. Alternatively or supplementally (e.g., additionally), the transferrin receptor antibody of this disclosure comprises a light chain containing no more than 15 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 variation) compared to the light chain of the humanized antibody shown in SEQ ID NO: 135.

[0390] In some embodiments, the transferrin receptor antibody is an antigen-binding fragment (Fab) of a full-length antibody (full-length antibody). The antigen-binding fragment of a full-length antibody (full-length antibody) can be prepared using conventional methods. For example, the F(ab')2 fragment can be produced by pepsin digestion of the antibody molecule, and the Fab' fragment can be produced by reducing the disulfide bridge of the F(ab')2 fragment. Some examples of the Fab amino acid sequences of the transferrin receptor antibodies described herein are provided below:

[0391] Heavy chain Fab (part of the constant region of VH+ human IgG1)

[0392]

[0393] Heavy chain Fab (part of the humanized VH+ human IgG1 constant region)

[0394]

[0395] In some embodiments, the transferrin receptor antibody described herein comprises a heavy chain containing the amino acid sequence of SEQ ID NO:136. Alternatively or supplementally (e.g., as an adjunct), the transferrin receptor antibody described herein comprises a light chain containing the amino acid sequence of SEQ ID NO:133.

[0396] In some embodiments, the transferrin receptor antibody described herein comprises a heavy chain containing the amino acid sequence of SEQ ID NO:137. Alternatively or supplementally (e.g., as an adjunct), the transferrin receptor antibody described herein comprises a light chain containing the amino acid sequence of SEQ ID NO:135.

[0397] The transferrin receptor antibody described herein can be in any antibody form, including but not limited to full-length antibodies, their antigen-binding fragments (e.g., Fab, Fab', F(ab')2, Fv), single-chain antibodies, bispecific antibodies, or nanobodies. In some embodiments, the transferrin receptor antibody described herein is an scFv. In some embodiments, the transferrin receptor antibody described herein is an scFv-Fab (e.g., an scFv fused to a portion of the constant region). In some embodiments, the transferrin receptor antibody described herein is an scFv fused to a constant region (e.g., the human IgG1 constant region shown in SEQ ID NO:130).

[0398] In some embodiments, any of the anti-TfR antibodies described herein are generated using recombinant DNA technology in Chinese hamster ovary (CHO) cell suspension cultures, optionally in CHO-K1 cell suspension cultures (e.g., CHO-K1 cells from the European Collection of Animal Cell Culture, catalog number 85051005).

[0399] In some embodiments, the antibodies provided herein may have one or more post-translational modifications. In some embodiments, N-terminal cyclization, also known as pyroglutamate formation (pyro-Glu), may occur during production at the N-terminal glutamate (Glu) and / or glutamine (Gln) residues of the antibody. Therefore, it should be understood that antibodies specified as having sequences containing N-terminal glutamate or glutamine residues encompass antibodies that have undergone pyroglutamate formation due to post-translational modifications. In some embodiments, pyroglutamate formation occurs in the heavy chain sequence. In some embodiments, pyroglutamate formation occurs in the light chain sequence.

[0400] b. Other muscle-targeting antibodies

[0401] In some embodiments, the muscle-targeting antibody is an antibody that specifically binds to hemojuvelin, caveolin-3, Duchenne muscular dystrophy peptide, myosin Iib, or CD63. In some embodiments, the muscle-targeting antibody is an antibody that specifically binds to myogenic precursor proteins. Some exemplary myogenic precursor proteins include, but are not limited to, ABCG2, M-cadherin / cadherin-15, caveolin-1, CD34, FoxK1, integrin α7, integrin α7β1, MYF-5, MyoD, myocyte cytogenes, NCAM-1 / CD56, Pax3, Pax7, and Pax9. In some embodiments, the muscle-targeting antibody is an antibody that specifically binds to skeletal muscle proteins. Some exemplary skeletal muscle proteins include, but are not limited to, alpha-sarcoglycan, β-sarcoglycan, calpain inhibitor, creatine kinase MM / CKMM, eIF5A, enolase 2 / neuron-specific enolase, ε-sarcoglycan, FABP3 / H-FABP, GDF-8 / myogenic inhibitor, GDF-11 / GDF-8, integrin α7, integrin α7β1, integrin β1 / CD29, MCAM / CD146, MyoD, myocyte cytokinase, 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 smooth muscle proteins. Some exemplary smooth muscle proteins include, but are not limited to, α-smooth muscle actin, VE-cadherin, calmodulin-binding protein / CALD1, calmodulin 1, desmin, histamine H2 R, motilin R / GPR38, TAGLN, and vimentin. However, it should be understood that antibodies against other targets are within the scope of this disclosure, and the exemplary list of targets provided herein is not intended to be limiting.

[0402] c. Antibody characteristics / changes

[0403] In some embodiments, conserved mutations may be introduced into the antibody sequence (e.g., CDR or framework sequence) at locations where residues are unlikely to interact with the target antigen (e.g., transferrin receptor) (e.g., as determined based on crystal structure). In some embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced into the Fc region of the muscle-targeting antibody described herein (e.g., in the CH2 domain (residues 231 to 340 of human IgG1) and / or (e.g., and) the CH3 domain (residues 341 to 447 of human IgG1) and / or (e.g., and) the hinge region, numbered according to the Kabat numbering system (e.g., EU index in Kabat) to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or (e.g., and) antigen-dependent cytotoxicity.

[0404] In some embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced into the hinge region of the Fc region (CH1 domain) to alter (e.g., increase or decrease) the number of cysteine ​​residues in the hinge region, as described, for example, in U.S. Patent No. 5,677,425. Altering the number of cysteine ​​residues in the hinge region of the CH1 domain can, for example, promote the assembly of light and heavy chains, or alter (e.g., increase or decrease) antibody stability or promote linker conjugation.

[0405] In some embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced into the Fc region of the muscle-targeting antibody described herein (e.g., in the CH2 domain (residues 231 to 340 of human IgG1) and / or (e.g., and) the CH3 domain (residues 341 to 447 of human IgG1) and / or (e.g., and) the hinge region, numbered according to the Kabat numbering system (e.g., EU index in Kabat)) to increase or decrease the antibody's affinity for an Fc receptor (e.g., an activated Fc receptor) on the surface of effector cells. Mutations in the Fc region of antibodies that decrease or increase the antibody's affinity for Fc receptors, and techniques for introducing such mutations into the Fc receptor or fragments thereof, are known to those skilled in the art. Some examples of mutations in the Fc receptor of antibodies that can be altered to change the affinity of the antibody for the Fc receptor are described below: for example, Smith P et al., (2012) PNAS 109:6181-6186, U.S. Patent No. 6,737,056, and International Publications Nos. WO 02 / 060919, WO98 / 23289, and WO 97 / 34631, which are incorporated herein by reference.

[0406] In some embodiments, one, two, or more amino acid mutations (i.e., substitutions, insertions, or deletions) are introduced into the constant domain of IgG or its FcRn binding fragment (preferably, an Fc or hinge-Fc domain fragment) to alter (e.g., decrease or increase) the half-life of the antibody in vivo. See, for example, International Publications Nos. WO 02 / 060919, WO 98 / 23289, and WO97 / 34631, and U.S. Patent Nos. 5,869,046, 6,121,022, 6,277,375, and 6,165,745, for example, mutations that alter (e.g., decrease or increase) the half-life of the antibody in vivo.

[0407] In some embodiments, one, two, or more amino acid mutations (i.e., substitutions, insertions, or deletions) are introduced into the constant domain of IgG or its FcRn-binding fragment (preferably, an Fc or hinge-Fc domain fragment) to reduce the half-life of the anti-transferrin receptor antibody in vivo. In some embodiments, one, two, or more amino acid mutations (i.e., substitutions, insertions, or deletions) are introduced into the constant domain of IgG or its FcRn-binding fragment (preferably, an 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 to 340 of human IgG1) and / or (e.g., and) the third constant (CH3) domain (residues 341 to 447 of human IgG1) (numbered according to the EU index in Kabat (Kabat E Aet al., (1991) ibid.)). In some embodiments, the constant region of the IgG1 of the antibody described herein includes 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 glutamate (E) substitution at position 256, the positions of which are based on the EU index number in Kabat. See U.S. Patent No. 7,658,921, which is incorporated herein by reference. This type of mutant IgG (referred to as the “YTE mutant”) has been shown to have a 4-fold increased half-life compared to the wild-type form of the same antibody (see Dall'Acqua WF et al., (2006) J BiolChem 281:23514-24). In some embodiments, the antibody comprises an IgG constant domain containing one, two, three or more amino acid substitutions at positions 251 to 257, 285 to 290, 308 to 314, 385 to 389 and 428 to 436, according to the EU index number in Kabat.

[0408] In some embodiments, one, two, or more amino acid substitutions are introduced into the Fc region of the IgG constant domain to alter the effector function of the anti-transferrin receptor antibody. The effector ligand whose affinity is altered may be, for example, an Fc receptor or a C1 component of complement. This method is described in more detail in U.S. Patent Nos. 5,624,821 and 5,648,260. In some embodiments, deletion or inactivation of the constant domain (through point mutations or other means) can reduce Fc receptor binding of the circulating antibody, thereby improving tumor localization. For a description of mutations that result in deletion or inactivation of the constant domain to improve tumor localization, see, for example, U.S. Patent Nos. 5,585,097 and 8,591,886. In some embodiments, one or more amino acid substitutions may be introduced into the Fc region of the antibody described herein to remove potential glycosylation sites on the Fc region, which can reduce Fc receptor binding (see, for example, Shields R Let al., (2001) J BiolChem 276:6591-604).

[0409] In some embodiments, one or more amino groups in the constant region of the muscle-targeting antibody described herein may be replaced with different amino acid residues, such that the antibody exhibits altered Clq binding and / or (e.g., and) reduced or eliminated complement-dependent cytotoxicity (CDC). This approach is described in more 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, thereby changing the antibody's ability to fix complement. This approach is further described in International Publication No. WO94 / 29351. In some embodiments, the Fc region of the antibody described herein is modified to enhance the antibody's ability to mediate antibody-dependent cytotoxicity (ADCC) and / or (e.g., and) to enhance the antibody's affinity for the Fcγ receptor. This approach is further described in International Publication No. WO 00 / 42072.

[0410] In some embodiments, the heavy chain and / or (e.g., and) light chain variable domain sequences of the antibodies provided herein can be used to generate, for example, CDR-grafted, chimeric, humanized, or complexed human antibodies or antigen-binding fragments, as described elsewhere herein. As will be understood by those skilled in the art, any variant (CDR-grafted, chimeric, humanized, or complexed antibody) derived from any antibody provided herein can be used 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 complexed antibody) has 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 relative to the original antibody from which it is derived.

[0411] In some embodiments, the antibodies provided herein contain mutations that confer the desired properties. For example, to avoid potential complications attributable to Fab arm exchanges known to occur with native IgG4 mAb, the antibodies provided herein may contain a stable '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 at position 228 (EU number, or residue 241 according to Kabat number) is converted to proline, resulting in an IgG1-like hinge sequence. Therefore, any antibody may contain a stable 'Adair' mutation.

[0412] As provided herein, antibodies of this disclosure may optionally include a constant region or a portion thereof. For example, the VL domain may be linked at its C-terminus to a light chain constant domain, such as Cκ or Cλ. Similarly, the VH domain or a portion thereof may be linked to all or a portion of a heavy chain such as IgA, IgD, IgE, IgG, and IgM (and any isotype subclass). Antibodies may include suitable constant regions (see, for example, 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 this disclosure may include VH and VL domains or their antigen-binding portions in combination with any suitable constant region.

[0413] ii. Muscle-targeting peptides

[0414] Some aspects of this disclosure provide muscle-targeting peptides as muscle-targeting agents. Short peptide sequences (e.g., peptide sequences of 5 to 20 amino acids in length) that bind to specific cell types have been described. For example, cell-targeting peptides have been described in the following: 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., “Invivo 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, granted on December 11, 2001, entitled “METHODS AND COMPOSITIONS FORTARGETING COMPOUNDS TO MUSCLE”; and Samoylov AM, et al., “Recognition of cell-specific binding of Phage display derived peptides using an acoustic wavesensor.” Biomol Eng 2002; 18:269-72; the entire contents of which are incorporated herein by reference. Selectivity for desired tissues, such as muscle, can be achieved by designing peptides to interact with specific cell surface antigens (e.g., receptors). Skeletal muscle targeting has been studied and a range of molecular payloads can be delivered. These methods offer high selectivity for muscle tissue without many of the practical drawbacks of large antibody or viral particles. Therefore, in some embodiments, the muscle target is a muscle-targeting peptide of 4 to 50 amino acids in length. In some implementations, the muscle-targeting peptide has a length of 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.Muscle-targeting peptides can be generated using any of several methods (such as phage display).

[0415] In some embodiments, the muscle-targeting peptide may bind to an internalized cell surface receptor, such as the 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 peptide targeting the transferrin receptor may comprise a segment of a naturally occurring ligand (e.g., transferrin). In some embodiments, the peptide targeting the transferrin receptor is as described in U.S. Patent No. 6,743,893, filed November 30, 2000, “RECEPTOR-MEDIATED UPTAKE OF PEPTIDESTHAT BIND THE HUMAN TRANSFERRIN RECEPTOR”. In some embodiments, the peptide targeting the transferrin receptor is as described in Kawamoto, M. et al, “A novel transferrin receptor-targeted hybridpeptide disintegrates cancer cell membrane to induce rapid killing of cancer cells.” BMC Cancer. 2011 Aug 18; 11:359. In some embodiments, the peptide targeting the transferrin receptor is as described in U.S. Patent No. 8,399,653, filed May 20, 2011, entitled “TRANSFERRIN / TRANSFERRINRECEPTOR-MEDIATED SIRNADELIVERY”.

[0416] As discussed above, several examples of muscle-targeting peptides have been reported. For example, muscle-specific peptides have been identified using phage display libraries presenting surface heptapeptides. As an example, a peptide having the amino acid sequence ASSLNIA (SEQ ID NO: 138) binds to C2C12 mouse myotubes in vitro and to mouse muscle tissue in vivo. Therefore, in some embodiments, the muscle-targeting agent comprises the amino acid sequence ASSLNIA (SEQ ID NO: 138). This peptide, after intravenous injection in mice, exhibits increased specificity for binding to cardiac and skeletal muscle tissue, and decreased binding to the liver, kidney, and brain. Other muscle-specific peptides have been identified using phage display. For example, a 12-amino acid peptide for muscle targeting in the context of DMD treatment has been identified using a phage display library. 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. Here, a 12-amino acid peptide with the sequence SKTFNTHPQSTP (SEQ ID NO:139) was identified, and this muscle-targeting peptide showed enhanced binding to C2C12 cells compared to the ASSLNIA (SEQ ID NO:138) peptide.

[0417] Another method for identifying peptides that are selective for muscle (e.g., skeletal muscle) relative to other cell types includes in vitro selection, which has been 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. Non-specific cell binders were selected by pre-incubating random 12-mer peptide phage display libraries with a mixture of non-muscle cell types. After several rounds of selection, the 12-amino acid peptide TARGEHKEEELI (SEQ ID NO:140) appeared most frequently. Therefore, in some embodiments, the muscle target comprises the amino acid sequence TARGEHKEEELI (SEQ ID NO:140).

[0418] Muscle-targeting agents can be amino acid-containing molecules or peptides. Muscle-targeting peptides may correspond to protein sequences that preferentially bind to protein receptors found in muscle cells. In some embodiments, muscle-targeting peptides contain highly favored hydrophobic amino acids, such as valine, such that the peptide preferentially targets muscle cells. In some embodiments, muscle-targeting peptides are not previously characterized or disclosed. These peptides can be conceived, generated, synthesized, and / or (e.g., and) derived using any of several methods (e.g., phage display peptide libraries, single-bead single-compound peptide libraries, or position-scan synthetic peptide combinatorial libraries). Exemplary methods have been characterized in the art and incorporated by reference (Gray, B.P. and Brown, K.C. "Combinatorial Peptide Libraries: Mining for Cell-Binding Peptides" Chem Rev. 2014, 114:2, 1020–1081.; Samoylova, T.I. and Smith, B.F. "Elucidation of muscle-binding peptides byphage display screening." Muscle Nerve, 1999, 22:4, 460-6.).In some implementations, muscle-targeting peptides have been previously disclosed (see, for example, 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. “Invitro selection of a peptide with high selectivity for cardiomyocytes invivo.” J MolBiol. 2004, 342:1, 171-82.). Exemplary muscle-targeting peptides comprise the amino acid sequences of the following groups: 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).

[0419] In some embodiments, the muscle-targeting peptide may comprise about 2 to 25 amino acids, about 2 to 20 amino acids, about 2 to 15 amino acids, about 2 to 10 amino acids, or about 2 to 5 amino acids. The muscle-targeting peptide may comprise naturally occurring amino acids such as cysteine ​​and 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, such as bicyclic (see, for example, Silvana, M. Get al. Mol. Therapy, 2018, 26:1, 132–147.).

[0420] iii. Muscle-targeting receptor ligand

[0421] Muscle-targeting agents can be ligands, such as ligands that bind to receptor proteins. Muscle-targeting ligands can be proteins, such as transferrin, which binds to internalized cell surface receptors expressed by muscle cells. Therefore, in some embodiments, the muscle-targeting agent is transferrin or a transferrin derivative that binds to a transferrin receptor. Alternatively, muscle-targeting ligands can be small molecules, such as lipophilic small molecules that preferentially target muscle cells relative to other cell types. Some exemplary lipophilic small molecules that can target muscle cells include compounds comprising: cholesterol, cholesterol groups, stearic acid, palmitic acid, oleic acid, oleyl alcohol, linolene, linoleic acid, myristic acid, sterols, dihydrotestosterone, testosterone derivatives, glycerol, alkyl chains, triphenylmethyl groups, and alkoxy acids.

[0422] iv. Muscle-targeting aptamers

[0423] Muscle-targeting agents can be aptamers, such as RNA aptamers, that preferentially target muscle cells relative to other cell types. In some embodiments, the muscle-targeting aptamers are not previously characterized or disclosed. These aptamers can be conceived, generated, synthesized, and / or (e.g., and) derived using any of several methods (e.g., through phylogenetic evolution of exponentially enriched ligands). Exemplary methods have been characterized in the art and are incorporated herein 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, for example, Phillippou, S. et al. “Selection and Identification of Skeletal-Muscle-Targeted RNA Aptamers.” Mol Ther Nucleic Acids. 2018, 10:199-214.; Thiel, W. He et al. “Smooth Muscle Cell-targeted RNA Aptamer Inhibits NeointimalFormation.” Mol Ther. 2016, 24:4,779-87.). Exemplary muscle-targeting aptamers include A01B RNA aptamer and RNAApt 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 to 15 kDa, about 5 to 10 kDa, about 10 to 15 kDa, about 1 to 5 Da, about 1 to 3 kDa, or less.

[0424] v. Other muscle-targeting agents

[0425] One strategy for targeting muscle cells (e.g., skeletal muscle cells) is to use substrates of muscle transporter proteins (e.g., transporter proteins expressed on the sarcolemma). In some embodiments, the muscle target is a substrate of an inflow transporter that is specific to muscle tissue. In some embodiments, the inflow transporter is specific to skeletal muscle tissue. Two main classes of transporters are expressed on the sarcolemma of skeletal muscle: (1) the adenosine triphosphate (ATP) binding cassette (ABC) superfamily, which promotes outflow from skeletal muscle tissue and (2) the solute transporter (SLC) superfamily, which promotes inflow of substrates into skeletal muscle. In some embodiments, the muscle target is a substrate that binds to the ABC or SLC superfamily of the transporter. In some embodiments, the substrate that binds to the ABC or SLC superfamily of the transporter is a naturally occurring substrate. In some embodiments, the substrate that binds to the ABC or SLC superfamily of the transporter is a non-natural substrate, such as a synthetic derivative thereof that binds to the ABC or SLC superfamily of the transporter.

[0426] In some implementations, the muscle-targeting agent is a substrate of the SLC superfamily of transporters. The SLC transporters are either balanced or use a transmembrane proton or sodium ion gradient to drive substrate transport. Exemplary SLC transporters with high skeletal muscle expression include, but are not limited to, 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 facilitate substrate inflow into skeletal muscle, providing opportunities for muscle targeting.

[0427] In some embodiments, the muscle target is a substrate of the balanced nucleoside transporter 2 (ENT2) transporter. ENT2 has one of the highest mRNA expression rates in skeletal muscle compared to other transporters. While human ENT2 (hENT2) is expressed in most body organs such as the brain, heart, placenta, thymus, pancreas, prostate, and kidney, it is particularly abundant in skeletal muscle. Human ENT2 promotes the uptake of its substrate, depending on the concentration gradient of that substrate. ENT2 plays a role in maintaining nucleoside homeostasis by transporting a broad range of purine and pyrimidine nucleoside bases. The hENT2 transporter has low affinity for all nucleosides except inosine (adenosine, guanosine, uridine, thymidine, and cytidine). Therefore, in some embodiments, the muscle target is an ENT2 substrate. Exemplary ENT2 substrates include, but are not limited to, inosine, 2',3'-dideoxyinosine, and calofarabine. In some embodiments, any muscle target provided herein is associated with a molecular payload (e.g., an oligonucleotide payload). In some embodiments, the muscle target is covalently linked to the molecular payload. In some embodiments, the muscle target is non-covalently linked to the molecular payload.

[0428] In some embodiments, the muscle target is a substrate of an organic cation / carnitine transporter (OCTN2), which is a sodium-dependent, high-affinity carnitine transporter. In some embodiments, the muscle target is carnitine, mildronate, acetylcarnitine, or any derivative thereof bound to OCTN2. In some embodiments, carnitine, mildronate, acetylcarnitine, or a derivative thereof is covalently linked to a molecular load (e.g., an oligonucleotide load).

[0429] Muscle-targeting agents can be proteins that exist in at least one soluble form targeting muscle cells. In some embodiments, the muscle-targeting protein can be hemoblastin (also known as rejection-directing molecule C or hemochromatosis type 2 protein), which is a protein involved in iron overload and homeostasis. In some embodiments, hemoblastin can be full-length or 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 with a functional hemoblastin protein. In some embodiments, hemoblastin mutants can be soluble fragments, may lack N-terminal signaling, and / or (e.g., and) lack a C-terminal anchoring domain. In some implementations, hemojuglin 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 should be understood that hemojuglin may be of human, non-human primate, or rodent origin.

[0430] B. Molecular Loading

[0431] Some aspects of this disclosure provide molecular payloads, for example, for regulating biological outcomes, such as transcription of DNA sequences, expression of proteins, or activity of proteins. In some embodiments, the molecular payload is linked to or otherwise associated with a muscle target. In some embodiments, such a molecular payload is capable of targeting muscle cells, for example, by specifically binding to nucleic acids or proteins in the muscle cells after delivery to the muscle cells via an associated muscle target. It should be understood that various types of muscle targets can be used according to this disclosure. For example, the molecular payload may comprise or consist of: oligonucleotides (e.g., antisense oligonucleotides), peptides (e.g., peptides that bind to disease-related nucleic acids or proteins in muscle cells), proteins (e.g., proteins that bind to disease-related nucleic acids or proteins in muscle cells), or small molecules (e.g., small molecules that regulate the function of disease-related nucleic acids or proteins in muscle cells). In some embodiments, the molecular payload is an oligonucleotide comprising a strand having a complementary region of 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.

[0432] In some embodiments, at least one (e.g., at least two, three, four, five, or ten) molecular payloads (e.g., oligonucleotides) are linked to a muscle target. In some embodiments, all molecular payloads linked to the muscle target are identical, for example, targeting the same gene. In some embodiments, all molecular payloads linked to the muscle target are different, for example, the molecular payloads may target different parts of the same target gene, or the molecular payloads may target at least two different target genes. In some embodiments, the muscle target may be linked to some identical molecular payloads and other different molecular payloads.

[0433] This disclosure also provides compositions comprising multiple complexes 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 complexes contain a molecular target linked to the same number of molecular loads (e.g., oligonucleotides).

[0434] i. Oligonucleotides

[0435] As described herein, any suitable oligonucleotide can be used as a molecular payload. In some embodiments, the oligonucleotide may be programmed to cause mRNA degradation (e.g., the oligonucleotide may be a spacer polymer, siRNA, ribozyme, or aptamer that causes degradation). In some embodiments, the oligonucleotide may be programmed to block mRNA translation (e.g., the oligonucleotide may be a hybrid polymer, siRNA, or aptamer that blocks translation). In some embodiments, the oligonucleotide may be programmed to both cause mRNA degradation and block mRNA translation. In some embodiments, the oligonucleotide may be a guide nucleic acid (e.g., guide RNA) for guiding the activity of an enzyme (e.g., gene editing enzyme). Other examples of oligonucleotides are provided herein. It should be understood that in some embodiments, an oligonucleotide of one format (e.g., an antisense oligonucleotide) can be appropriately adapted to another format (e.g., siRNA oligonucleotide) by incorporating a functional sequence (e.g., an antisense sequence) from one format to another format.

[0436] In some implementations, the oligonucleotide may contain the complementary region of the target gene provided in Table 1. Further non-limiting examples of the genes selected in Table 1 are provided below.

[0437] DMPK / DM1

[0438] In some implementations, examples of oligonucleotides that can be used to target DMPK (e.g., for the treatment of DM1) are provided in the following: U.S. Patent Application Publication 20100016215A1, published January 1, 2010, entitled "Compound And Method For Treating Myotonic Dystrophy"; U.S. Patent Application Publication 20130237585A1, published July 19, 2010, entitled "Modulation Of Dystrophia Myotonica-Protein Kinase (DMPK) Expression"; U.S. Patent Application Publication 20150064181A1, published March 5, 2015, entitled "Antisense Conjugates For Decreasing Expression Of Dmpk"; and U.S. Patent Application Publication 20150238627A1, published August 27, 2015, entitled "Peptide-Linked MorpholinoAntisense Oligonucleotides For Treatment Of Myotonic Dystrophy"; Pandey, S Ketal. "Identification and Characterization of Modified Antisense Oligonucleotides Targeting DMPK in Mice and Nonhuman Primates for theTreatment of Myotonic Dystrophy Type 1" J. of Pharmacol Exp Ther, 2015, 355:329-340.; Langlois, M. et al. "Cytoplasmic and Nuclear Retained DMPK mRNAs AreTargets 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, SA et al. “Triplet-repeat oligonucleotide-mediated reversal of RNA toxicity in myotonic dystrophy” PNAS, 2009, 106:33, 13915-13920.; Wheeler, TM et al., “Targeting nuclear RNA for in vivo correction of myotonic dystrophy” Nature, 2012, 488(7409):111-115.; and U.S. Patent Application Publication 20160304877A1, published on October 20, 2016, entitled “Compounds and Methods For Modulation Of Dystrophia Myotonica-Protein Kinase (Dmpk) Expression”, the contents of which are incorporated herein by reference in their entirety.

[0439] Some examples of oligonucleotides used to facilitate DMPK gene editing include U.S. Patent Application Publication 20170088819A1, published on March 3, 2017, entitled “Genetic Correction Of Myotonic Dystrophy Type 1”; and International Patent Application Publication WO18002812A1, published on April 1, 2018, entitled “Materials And Methods For Treatment Of Myotonic Dystrophy Type 1 (DM1) And Other Related Disorders”, the contents of which are incorporated herein by reference in their entirety.

[0440] In some embodiments, the oligonucleotide may have a complementary region of a mutant form of DMPK, such mutant form as those reported in: Botta A. et al. “The CTG repeat expansion sizecorrelates with the splicing defects observed in muscles 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 which are incorporated herein by reference in their entirety.

[0441] In some embodiments, the oligonucleotides provided herein are antisense oligonucleotides targeting DMPK. In some embodiments, the targeted oligonucleotide is any antisense oligonucleotide targeting DMPK (e.g., spacer polymers), as described in U.S. Patent Application Publication US20160304877A1, published October 20, 2016, entitled “Compounds and Methods For Modulation Of Dystrophia Myotonica-Protein Kinase (DMPK) Expression,” which is incorporated herein by reference. In some embodiments, the DMPK-targeting oligonucleotide targets a region of the DMPK gene sequence as shown in Genbank Accession No. NM_001081560.2 or as shown in Genbank Accession No. NG_009784.1.

[0442] In some embodiments, the oligonucleotide targeting DMPK comprises a nucleotide sequence containing a complementary region to the target region, which is at least 10 consecutive nucleotides (e.g., at least 10, at least 12, at least 14, at least 16 or more consecutive nucleotides) in Genbank accession No. NM_001081560.2.

[0443] In some embodiments, the DMPK-targeting oligonucleotide comprises a spacer polymer motif. "Spacer polymer" refers to a chimeric antisense compound in which an inner region containing multiple nucleotides supporting RNase H cleavage is located between an outer region containing one or more nucleotides, wherein the nucleotides containing the inner region are chemically different from the one or more nucleotides containing the outer region. The inner region may be referred to as a "spacer segment" and the outer region may be referred to as a "wing segment." In some embodiments, the DMPK-targeting oligonucleotide comprises one or more modified nucleotides and / or (e.g., and) one or more modified nucleotides linked together. In some embodiments, the nucleotide linking is a phosphate thioester linking. In some embodiments, the oligonucleotide comprises a complete phosphate thioester backbone. In some embodiments, the oligonucleotide is a DNA spacer polymer with cET ends (e.g., 3-10-3; cET-DNA-cET). In some implementations, the oligonucleotide targeting DMPK comprises one or more 6'-(S)-CH3 biocyclic nucleotides, one or more β-D-2'-deoxyribonucleotides, and / or (e.g., and) one or more 5-methylcytosine nucleotides.

[0444] DUX4 / FSHD

[0445] In some embodiments, examples of oligonucleotides that can be used to target DUX4 (e.g., for the treatment of FSHD) are provided in the following: U.S. Patent No. 9,988,628, published February 2, 2017, entitled “AGENTS USEFUL INTREATING 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 20120225034, published September 6, 2012, entitled “AGENTS USEFUL IN TREATING FACIOSCAPULOHUMERAL MUSCULAR DYSTROPHY”; PCT Patent Application Publication No. WO 2013 / 120038, published on August 15, 2013, entitled “MORPHOLINO TARGETING DUX4 FOR TREATING FSHD”; Chen et al., “Morpholino-mediated Knockdown of DUX4 Toward Facioscapulohumeral Muscular Dystrophy Therapeutics,” Molecular Therapy, 2016, 24:8, 1405-1411.; 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 contents of which are incorporated herein by reference in their entirety. In some embodiments, the oligonucleotide is an antisense oligonucleotide, morpholino, siRNA, shRNA, or other nucleotide that hybridizes to the target DUX4 gene or mRNA.

[0446] In some implementations, such as for the treatment of FSHD, the oligonucleotide may have a region complementary to a compact, hypomethylated D4Z4 repeat, as described in: Daxinger, et al., “Genetic and Epigenetic Contributors to FSHD,” published in Curr Opin Genet Dev 2015; Lim JW, et al., DICER / AGO-dependent epigenetic silencing of D4Z4 repeats enhanced by exogenous siRNAs suggests mechanisms and therapies for FSHD Hum MolGenet. 2015 Sep 1; 24(17):4817–4828, the contents of which are incorporated herein by reference in their entirety.

[0447] DNM2 / CNM

[0448] In some embodiments, examples of oligonucleotides that can be used to target 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-SPECIFICSILENCING THERAPY FOR DYNAMIN 2-RELATEDDISEASES”. 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 oligonucleotides that can be used to target DNM2 are provided in: Tasfaout, et al., “Single Intramuscular Injection of AAV-shRNA Reduces DNM2 and Prevents Myotubular Myopathy in Mice,” published April 4, 2018 in Mol. Ther., and Tasfaout, et al., “Antisense oligonucleotide-mediated Dnm2 knockdown prevents and reverts myotubular myopathy in mice,” Nature Communications volume 8, Article number: 15661 (2017). In some embodiments, the oligonucleotide is an shRNA or morpholino that effectively targets DNM2 mRNA. In some embodiments, the oligonucleotide encodes wild-type DNM2 resistant to miR-133 activity, as described 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., July 2015.Other examples of oligonucleotides that can be used to target DNM2 are provided in Gibbs, et al., “Two Dynamin-2 Genes are Required for Normal Zebrafish Development”, published in PLoSOne in 2013, the contents of which are incorporated herein by reference in their entirety.

[0449] In some implementations, such as for treating CNM, the oligonucleotide may have a region complementary to a mutant in CNM-associated DNM2, as described below: et al, “Mutation Spectrumin the Large GTPase Dynamin 2, and Genotype-Phenotype Correlation in Autosomal Dominant Centronuclear Myopathy,” published in Hum. Mutat. in 2012, the contents of which are incorporated herein by reference in their entirety.

[0450] Pompeo disease

[0451] In some embodiments, such as for the treatment of Pompe disease, oligonucleotides mediate the inclusion of exon 2 in the GAA disease allele, as described in van der Wal, et al., “GAADeficiency in Pompe Disease is Alleviated by Exon Inclusion in iPSC-Derived Skeletal Muscle Cells,” Mol Ther NucleicAcids. 2017 Jun 16; 7:101–115, the contents of which are incorporated herein by reference. Therefore, in some embodiments, the oligonucleotide may have a complementary region to the GAA disease allele.

[0452] In some implementations, such as for treating Pompe disease, oligonucleotides (e.g., RNAi or antisense oligonucleotides) are used to suppress the expression of wild-type GYS1 in muscle cells, as reported, for example, in Clayton, et al., “Antisense Oligonucleotide-mediated Suppression of Muscle Glycogen Synthase1 Synthesis as an Approach for Substrate Reduction Therapy of Pompe Disease,” published in MolTher Nucleic Acids in 2017, or U.S. Patent Application Publication No. 2017182189, published on 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. Therefore, in some embodiments, the oligonucleotide may have an antisense strand having complementary regions of a human GYS1 sequence corresponding to RefSeq number NM_002103.4 and / or (e.g., and) a mouse GYS1 sequence corresponding to RefSeq number NM_030678.3.

[0453] ACVR1 / FOP

[0454] In some implementations, examples of oligonucleotides that can be used to target ACVR1 (e.g., for the treatment of FOP) are provided in the following: U.S. Patent Application 2009 / 0253132, published October 8, 2009, “Mutated ACVR1 for diagnosis and treatment of fibrodyplasia ossificans progressiva (FOP)”; WO2015 / 152183, published October 8, 2015, “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, pp. 701-702; Takahashi, M. et al., “Disease-causing allele-specific silencing against the ALK2 mutants, R206H and G356D, in fibrodysplasia ossificans progressiva”. Therapy (2012) 19, 781–785; Shi, S. et al. “Antisense-Oligonucleotide Mediated Exon Skipping in Activin-Receptor-Like Kinase 2: Inhibiting the Receptor ThatIs Overactive in Fibrodysplasia Ossificans Progressiva” Plos One, July 2013, Vol. 8:7, e69096; U.S. Patent Application 2017 / 0159056, published June 8, 2017, “Antisenseoligonucleotides and methods of use thereof”; U.S. Patent No.Authorized on October 4, 2014, 8,859,752, “SIRNA-based therapy of Fibrodyplasia Ossificans Progressiva (FOP)”; and published on November 4, 2004, WO2004 / 094636, “Effective sirna knock-down constructs”, the contents of which are incorporated herein by reference in their entirety.

[0455] FXN / Friedreich Ataxia

[0456] In some implementations, examples of oligonucleotides that can be used to target FXN and / or (e.g., and) otherwise compensate for frataxin deficiency (e.g., for the treatment of Friedreich's ataxia) are provided in: Li, L. et al., “Activating frataxin expression by repeat-targeted nucleic acids”, Nat. Comm. 2016, 7:10606.; WO 2016 / 094374, published June 16, 2016, “Compositions and methods for treatment of Friedreich's ataxia”; WO 2015 / 020993, published February 12, 2015, “RNAi COMPOSITIONS AND METHODS FOR TREATMENT OF FRIEDREICH'S ATAXIA”; WO2017 / 186815, published November 2, 2017, “Antisense oligonucleotides for enhanced expression of frataxin”; WO US Patent Application No. 2008 / 018795, published February 14, 2008, "Methods and means for treating DNA repeat instability associated genetic disorders"; US Patent Application No. 2018 / 0028557, published February 1, 2018, "Hybrid oligonucleotides and uses thereof"; WO2015 / 023975, published February 19, 2015, "Compositions and methods for modulating RNA"; WO 2015 / 023939, published February 19, 2015, "Compositions and methods for modulating expression of frataxin"; US Patent Application No. 2017 / 0281643, published October 5, 2017, "Compounds and methods for modulating frataxin expression"; Li L. et al.The articles “Activating frataxin expression by repeat-targeted nucleic acids” (Nature Communications, February 4, 2016) and “Activation of Frataxin Protein Expression by Antisense Oligonucleotides Targeting the Mutant Expanded Repeat” (Nucleic Acid Ther., 2018 Feb; 28(1):23-33) are incorporated herein by reference in their entirety.

[0457] In some embodiments, the oligonucleotide payload is configured (e.g., as a spacer polymer or RNAi oligonucleotide) to inhibit the expression of a natural antisense transcript that inhibits FXN expression, as disclosed, for example, in U.S. Patent No. 9,593,330, filed June 9, 2011, “Treatment of frataxin (FXN) related diseases by inhibition of natural antisense transcript to FXN,” the contents of which are incorporated herein by reference in their entirety.

[0458] Examples of oligonucleotides used to facilitate FXN gene editing include WO 2016 / 094845, published June 16, 2016, “Compositions and methods for editing nucleic acids in cells utilizing oligonucleotides”; WO 2015 / 089354, published June 18, 2015, “Compositions and methods of use of CRISPR-Cas systems in nucleotide repeat disorders”; WO 2015 / 139139, published September 24, 2015, “CRISPR-based methods and products for increasing frataxin levels and uses thereof”; and WO 2018 / 002783, published January 4, 2018, “Materials and methods for treatment of Friedreich ataxia and other related disorders”, the contents of which are incorporated herein by reference in their entirety.

[0459] Some examples of oligonucleotides used to promote FXN gene expression by targeting non-FXN genes (such as epigenetic regulators of FXN) include WO 2015 / 023938, published on February 19, 2015, “Epigenetic regulators of frataxin”, the contents of which are incorporated herein by reference in their entirety.

[0460] In some embodiments, the oligonucleotide may have complementary regions of sequences as shown below: from the human FXN gene (gene ID 2395; NC_000009.12) and / or (e.g., and) from the mouse FXN gene (gene ID 14297; NC_000085.6). In some implementations, the oligonucleotide may have a complementary region of a mutant form of FXN, as reported in, for example, the following: Montemini, 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 which are incorporated herein by reference in their entirety.

[0461] Dystrophinopathy (DMD)

[0462] Some examples of oligonucleotides that can be used to target DMD are provided below: U.S. Patent Application Publication US20100130591A1, published May 27, 2010, entitled “MULTIPLE EXON SKIPPING COMPOSITIONS FOR DMD”; U.S. Patent No. 8,361,979, granted January 29, 2013, entitled “MEANS AND METHOD FOR INDUCING EXON-SKIPPING”; U.S. Patent Application Publication 20120059042, published March 8, 2012, entitled “METHOD FOR EFFICIENT EXON(44)SKIPPING IN DUCHENNE MUSCULARDYSTROPHY AND ASSOCIATED MEANS”; U.S. Patent Application Publication 20140329881, published November 6, 2014, entitled “EXON SKIPPING COMPOSITIONS FOR TREATING”. "MUSCULAR DYSTROPHY"; U.S. Patent No. 8,232,384, granted July 31, 2012, entitled "ANTISENSE OLIGONUCLEOTIDES FOR INDUCING EXON SKIPPING AND METHODS OF USE THEREOF"; U.S. Patent Application Publication 20120022134A1, published January 26, 2012, entitled "METHODS AND MEANS FOR EFFICIENTSKIPPING OF EXON 45IN DUCHENNE MUSCULAR DYSTROPHY PRE-MRNA"; U.S. Patent Application Publication 20120077860, published March 29, 2012, entitled "ADENO-ASSOCIATED VIRAL VECTOR FOR EXON SKIPPING IN A GENE ENCODING ADISPENSABLE DOMAN "PROTEIN"; 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 ACIDS"; U.S. Patent No.Patent No. 9,034,838, granted on May 19, 2015, entitled “MIR-31IN DUCHENNE MUSCULAR DYSTROPHY THERAPY”; and International Patent Publication WO2017062862A3, published on April 13, 2017, entitled “OLIGONUCLEOTIDE COMPOSITIONS AND METHODS THEREOF”; the contents of each are incorporated herein by reference in their entirety.

[0463] Some examples of oligonucleotides used to facilitate DMD gene editing include International Patent Publication WO2018053632A1, published on March 29, 2018, entitled "METHODS OF MODIFYING THEDYSTROPHIN GENE AND RESTORING DYSTROPHIN EXPRESSION AND USES THEREOF"; International Patent Publication WO2017049407A1, published on March 30, 2017, entitled "MODIFICATION OF THEDYSTROPHIN GENE AND USES THEREOF"; and International Patent Publication WO2016161380A1, published on October 6, 2016, entitled "CRISPR / CAS-RELATED METHODS AND COMPOSITIONS FOR TREATINGDUCHENNE MUSCULAR DYSTROPHY AND BECKER MUSCULAR". International patent publication WO2017095967, published on June 8, 2017, entitled "THERAPEUTIC TARGETS FOR THE CORRECTION OF THE HUMAN DYSTROPHIN GENE BY GENE EDITING AND METHODS OF USE"; International patent publication WO2017072590A1, published on May 4, 2017, entitled "MATERIALS AND METHODSFOR TREATMENT OF DUCHENNE MUSCULAR DYSTROPHY"; International patent publication WO2018098480A1, published on May 31, 2018, entitled "PREVENTION OF MUSCULAR DYSTROPHY BY CRISPR / CPF1-MEDIATED GENE". "EDITING"; US Patent Application Publication US20170266320A1, published on September 21, 2017, entitled "RNA-Guided Systems for In Vivo Gene Editing"; International Patent Publication WO2016025469A1, published on February 18, 2016, entitled "PREVENTION OF MUSCULAR DYSTROPHYBY CRISPR / CAS9-MEDIATED GENE EDITING";U.S. Patent Application Publication 2016 / 0201089, published July 14, 2016, entitled "RNA-GUIDED GENE EDITING AND GENE REGULATION"; and U.S. Patent Application Publication 2013 / 0145487, published June 6, 2013, entitled "MEGANUCLEASE VARIANTSCLEAVING A DNA TARGET SEQUENCE FROM THE DYSTROPHN GENE AND USES THEREOF", the contents of which are incorporated herein by reference in their entirety. In some embodiments, the oligonucleotide may have complementary regions to DMD gene sequences from multiple species (e.g., selected from human, mouse, and non-human species).

[0464] In some embodiments, the oligonucleotide may have a complementary region to a mutant DMD allele, such as having at least one mutated DMD allele in any one of exons 1 to 79 of human DMD, which results in frameshift and incorrect RNA splicing / processing.

[0465] MYH7 / Hypertrophic Cardiomyopathy

[0466] Some examples of oligonucleotides that can be used as payloads (e.g., for targeting MYH7) are provided below: U.S. Patent Application Publication 20180094262, published April 5, 2018, entitled "Inhibitors of MYH7B and Uses Thereof"; U.S. Patent Application Publication 20160348103, published December 1, 2016, entitled "Oligonucleotides and Methods for Treatment of Cardiomyopathy Using RNA Interference"; U.S. Patent Application Publication 20160237430, published August 18, 2016, entitled "Allele-specific RNA Silencing for the Treatment of Hypertrophic Cardiomyopathy"; and U.S. Patent Application Publication 20160032286, published February 4, 2016, entitled "Inhibitors of MYH7B and Uses" Thereof; U.S. Patent Application Publication 20140187603, published July 3, 2014, entitled “MicroRNA Inhibitors Comprising Locked Nucleotides”; U.S. Patent Application Publication 20140179764, published June 26, 2014, entitled “Dual Targeting of miR-208 and miR-499 in the Treatment of Cardiac Disorders”; U.S. Patent Application Publication 20120114744, published May 10, 2012, entitled “Compositions and Methods to Treat Muscular and Cardiovascular Disorders”; the contents of each of these patents are incorporated herein by reference in their entirety.

[0467] In some embodiments, the oligonucleotide may target lncRNA or mRNA, for example, for degradation. In some embodiments, the oligonucleotide may target (e.g., for degradation) nucleic acids encoding proteins involved in mismatch repair pathways (e.g., MSH2, MutLα, MutSβ, MutLα). Some non-limiting examples of proteins involved in mismatch repair pathways (where the mRNA encoding such proteins can be targeted by the oligonucleotides described herein) are described in the following: Iyer, RR et al., “DNAtriplet 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” DNARepair (Amst). 2016 Feb; 38:117-26.

[0468] In some implementations, any of the oligonucleotides may be in salt form, for example, as a sodium salt, potassium salt, or magnesium salt.

[0469] In some embodiments, the 5' or 3' nucleotide (e.g., terminal nucleotide) of any oligonucleotide described herein is optionally conjugated to an amino group 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 bond is present between the spacer and the 5' or 3' nucleotide of the oligonucleotide. In some embodiments, the 5' or 3' nucleotide (e.g., terminal nucleotide) of any oligonucleotide described herein is conjugated to a spacer that is a substituted or unsubstituted aliphatic, substituted or unsubstituted heteroaliphatic, substituted or unsubstituted carbocyclic, substituted or unsubstituted heterocyclic, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, -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)2NR A -,-NR A S(O)2-, or combinations thereof; each R A Independently, it is hydrogen or a substituted or unsubstituted alkyl group. In some embodiments, the spacer group is a substituted or unsubstituted alkylene group, a substituted or unsubstituted heterocyclic group, a substituted or unsubstituted heteroaryl group, -O-, -N(R) A - or -C(=O)N(R) A )2, or a combination thereof.

[0470] In some embodiments, the 5' or 3' nucleotide of any of the oligonucleotides described herein is combined with the formula -NH2-(CH2). n - The compound conjugation, where n is an integer from 1 to 12. In some embodiments, n is 6, 7, 8, 9, 10, 11, or 12. In some embodiments, the phosphodiester bond exists in the formula NH2-(CH2). n - The compound is associated with the 5' or 3' nucleoside of the oligonucleotide. In some embodiments, the compound of formula NH2-(CH2)6- is conjugated to the oligonucleotide via a reaction between 6-amino-1-hexanol (NH2-(CH2)6-OH) and the 5' phosphate of the oligonucleotide.

[0471] In some implementations, oligonucleotides are conjugated to a targeting agent, such as a muscle targeting agent or an anti-TfR antibody, for example, via an amino group.

[0472] a. Oligonucleotide size / sequence

[0473] Oligonucleotides can have a variety of different lengths, for example, depending on the format. In some embodiments, the length of the oligonucleotide is 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75 or more nucleotides. In some embodiments, the length of the oligonucleotide is 8 to 50 nucleotides, 8 to 40 nucleotides, 8 to 30 nucleotides, 10 to 15 nucleotides, 10 to 20 nucleotides, 15 to 25 nucleotides, 21 to 23 nucleotides, etc.

[0474] In some embodiments, when the binding of the complementary nucleic acid sequence of the oligonucleotide to a target molecule (e.g., mRNA) interferes with the normal function of the target (e.g., mRNA) leading to loss of activity (e.g., inhibition of translation) or expression (e.g., degradation of target mRNA), and has sufficient complementarity to avoid nonspecific binding of the sequence to a non-target sequence, for the purposes of this disclosure, the complementary nucleic acid sequence of the oligonucleotide may specifically hybridize with or be specific to the target nucleic acid under conditions in which nonspecific binding is desired to be avoided, such as in vivo assays or therapeutic treatments under physiological conditions, and in in vitro assays, under suitable stringent conditions. Therefore, in some embodiments, the oligonucleotide may be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to the consecutive nucleotides of the target nucleic acid. In some implementations, the complementary nucleotide sequence does not need to be 100% complementary to the target nucleic acid in order to specifically hybridize with or be specific to the target nucleic acid.

[0475] In some embodiments, the oligonucleotide comprises a complementary region of the target nucleic acid, said complementary region being 8 to 15, 8 to 30, 8 to 40, or 10 to 50, or 5 to 50, or 5 to 40 nucleotides in length. In some embodiments, the complementary region of the oligonucleotide to the target nucleic acid is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length. In some embodiments, the complementary region is complementary to at least 8 consecutive nucleotides of the target nucleic acid. In some embodiments, the oligonucleotide may contain 1, 2, or 3 base mismatches compared to the consecutive nucleotide portions of the target nucleic acid. In some implementations, the oligonucleotide may have up to 3 mismatches on 15 bases or up to 2 mismatches on 10 bases.

[0476] In some embodiments, the oligonucleotide is complementary to the target sequence of any of the oligonucleotides provided herein (e.g., at least 85%, at least 90%, at least 95%, or 100% complementary). In some embodiments, such a target sequence is 100% complementary to the oligonucleotides described herein.

[0477] In some embodiments, any one or more thymine bases (T) in any oligonucleotide provided herein may optionally be uracil bases (U), and / or any one or more Us may optionally be T.

[0478] b. Oligonucleotide modification:

[0479] The oligonucleotides described herein may be modified, for example, to include modified sugar moieties, modified nucleoside-to-nucleotide linkages, modified nucleotides, and / or (e.g., and) combinations thereof. Additionally, in some embodiments, the oligonucleotides may exhibit one or more of the following properties: not mediating alternative splicing; not immunostimulatory; nuclease resistant; having increased cellular uptake compared to unmodified oligonucleotides; non-toxic to cells or mammals; increased intracellular endosome expulsion; minimizing TLR stimulation; or avoiding pattern recognition receptors. Any modified chemical composition or form of the oligonucleotides described herein may be combined with each other. For example, the same oligonucleotide may contain one, two, three, four, five, or more different types of modifications.

[0480] In some embodiments, certain nucleotide modifications may be used that make the incorporated oligonucleotides more resistant to nuclease digestion than native oligodeoxynucleotides or oligoribonucleotide molecules; these modified oligonucleotides survive intact for a longer time than unmodified oligonucleotides. Specific examples of modified oligonucleotides include those containing a modified backbone, such as modified internucleotide bonds, such as phosphate thioester bonds, phosphate triester bonds, methylphosphonate bonds, short-chain alkyl bonds, or cycloalkyl sugar bonds, or short-chain heteroatom bonds, or heterocyclic sugar bonds. Therefore, the oligonucleotides of this disclosure can be stabilized against nucleolytic degradation, for example, through incorporation modifications such as nucleotide modifications.

[0481] In some embodiments, the length of the oligonucleotide can be up to 50 or up to 100 nucleotides, wherein 2 to 10, 2 to 15, 2 to 16, 2 to 17, 2 to 18, 2 to 19, 2 to 20, 2 to 25, 2 to 30, 2 to 40, 2 to 45 or more nucleotides of the oligonucleotide are modified nucleotides. The length of the oligonucleotide can be 8 to 30 nucleotides, wherein 2 to 10, 2 to 15, 2 to 16, 2 to 17, 2 to 18, 2 to 19, 2 to 20, 2 to 25, 2 to 30 nucleotides of the oligonucleotide are modified nucleotides. The length of the oligonucleotide can be 8 to 15 nucleotides, wherein 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, 2 to 10, 2 to 11, 2 to 12, 2 to 13, 2 to 14 nucleotides of the oligonucleotide are modified nucleotides. Optionally, the oligonucleotide may have each nucleotide other than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 modified nucleotides. Oligonucleotide modifications are further described herein.

[0482] c. Modified nucleosides

[0483] In some embodiments, the oligonucleotide described herein comprises at least one nucleoside modified at the 2' position of the sugar. In some embodiments, the oligonucleotide comprises at least one 2'-modified nucleoside. In some embodiments, all nucleosides in the oligonucleotide are 2'-modified nucleosides.

[0484] In some embodiments, the oligonucleotides described herein comprise one or more non-bicyclic 2'-modified nucleosides, such as 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), or 2'-ON-methylacetamido (2'-O-NMA) modified nucleosides.

[0485] In some embodiments, the oligonucleotides described herein comprise one or more 2'-4' bicyclic nucleotides, wherein the ribose ring includes a bridging portion connecting two atoms in the linking ring, for example, by linking the 2'-O atom to the 4'-C atom via methylene (LNA) bridging, ethylene (ENA) bridging, or (S)-restricted ethyl (cEt) bridging. Some examples of LNA are described in International Patent Application Publication WO / 2008 / 043753, published on April 17, 2008, entitled "RNA Antagonist Compounds For The Modulation Of PCSK9", the contents of which are incorporated herein by reference in their entirety. Examples of ENAs are provided in the following: International Patent Publication No. WO 2005 / 042777, published on May 12, 2005, entitled “APP / ENA Antisense”; Morita et al., Nucleic Acid Res., Supplement 1:241-242, 2001; Surono et al., Hum. Gene Ther., 15:749-757, 2004; Koizumi, Curr. Opin. Mol. Ther., 8:144-149, 2006; and Horie et al., Nucleic Acids Symp. Ser(Oxf), 49:171-172, 2005; the disclosures of which are incorporated herein by reference in their entirety. Some examples of cEt are provided in: U.S. Patents 7,101,993, 7,399,845 and 7,569,686, each of which is incorporated herein by reference in its entirety.

[0486] In some embodiments, the oligonucleotide comprises a modified nucleoside disclosed in one of the following U.S. patent or patent application publications: U.S. Patent 7,399,845, issued July 15, 2008, entitled "6-Modified Bicyclic Nucleic Acid Analogs"; U.S. Patent 7,741,457, issued June 22, 2010, entitled "6-Modified Bicyclic Nucleic Acid Analogs"; U.S. Patent 8,022,193, issued September 20, 2011, entitled "6-Modified Bicyclic Nucleic Acid Analogs"; U.S. Patent 7,569,686, issued August 4, 2009, entitled "Compounds and Methods For Synthesis Of Bicyclic Nucleic Acid Analogs"; U.S. Patent 7,335,765, issued February 26, 2008, entitled "Novel Nucleoside and “Oligonucleotide Analogues”; U.S. Patent 7,314,923, issued January 1, 2008, entitled “Novel Nucleoside And Oligonucleotide Analogues”; U.S. Patent 7,816,333, issued October 19, 2010, entitled “Oligonucleotide Analogues And Methods Utilizing The Same”; and U.S. Publication No. 2011 / 0009471, now U.S. Patent 8,957,201, issued February 17, 2015, entitled “Oligonucleotide Analogues And Methods Utilizing The Same”, the entire contents of which are incorporated herein by reference for all purposes.

[0487] In some embodiments, the oligonucleotide comprises at least one modified nucleoside, which causes the Tm of the oligonucleotide to increase by 1°C, 2°C, 3°C, 4°C, or 5°C compared to an oligonucleotide without at least one modified nucleoside. The oligonucleotide may have multiple modified nucleosides, which cause the overall Tm of the oligonucleotide to increase by 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, or higher compared to an oligonucleotide without modified nucleosides.

[0488] Oligonucleotides may comprise mixtures of different types of nucleosides. For example, an oligonucleotide may comprise a mixture of 2'-deoxyribonucleoside or ribonucleoside and 2'-fluoro-modified nucleoside. An oligonucleotide may comprise a mixture of deoxyribonucleoside or ribonucleoside and 2'-O-Me-modified nucleoside. An oligonucleotide may comprise a mixture of 2'-fluoro-modified nucleoside and 2'-O-Me-modified nucleoside. An oligonucleotide may comprise a mixture of 2'-4' bicyclic nucleoside and 2'-MOE, 2'-fluoro, or 2'-O-Me-modified nucleosides. An oligonucleotide may comprise a mixture of non-bicyclic 2'-modified nucleosides (e.g., 2'-MOE, 2'-fluoro, or 2'-O-Me) and 2'-4' bicyclic nucleosides (e.g., LNA, ENA, cEt).

[0489] Oligonucleotides may contain various types of substituted nucleosides. For example, oligonucleotides may contain substituted 2'-deoxyribonucleosides or ribonucleosides and 2'-fluoro-modified nucleosides. Oligonucleotides may contain substituted deoxyribonucleosides or ribonucleosides and 2'-O-Me-modified nucleosides. Oligonucleotides may contain substituted 2'-fluoro-modified nucleosides and 2'-O-Me-modified nucleosides. Oligonucleotides may contain substituted 2'-4' bicyclic nucleosides and 2'-MOE, 2'-fluoro, or 2'-O-Me-modified nucleosides. Oligonucleotides may contain substituted non-bicyclic 2'-modified nucleosides (e.g., 2'-MOE, 2'-fluoro, or 2'-O-Me) and 2'-4' bicyclic nucleosides (e.g., LNA, ENA, cEt).

[0490] In some embodiments, the oligonucleotides described herein comprise 5'-vinylphosphonate modification, one or more baseless residues, and / or one or more inverted baseless residues.

[0491] d. Nucleoside linkages / backbone

[0492] In some embodiments, the oligonucleotide may contain phosphate-thioester links or other modified nucleoside links. In some embodiments, the oligonucleotide contains phosphate-thioester nucleoside links. In some embodiments, the oligonucleotide contains phosphate-thioester nucleoside links between at least two nucleotides. In some embodiments, the oligonucleotide contains phosphate-thioester nucleoside links between all nucleotides. For example, in some embodiments, the oligonucleotide contains modified nucleoside links at the first, second, and / or (e.g., and) third nucleoside links at the 5' or 3' ends of the nucleotide sequence.

[0493] Permissible phosphorus-containing linkages include, but are not limited to: thiophosphates, chiral thiophosphates, dithiophosphates, phosphate triesters, aminoalkyl phosphate triesters, methylphosphonates, and other alkylphosphonates containing 3'-alkylene phosphonates, as well as chiral phosphonates, hypophosphonates, phosphatidates containing 3'-aminophosphatidates and aminoalkylphosphatidates, thiocarbonylphosphatidates, thiocarbonylalkylphosphonates, thiocarbonylalkyl phosphate triesters, and borane phosphates having normal 3'-5' linkages, their 2'-5' linkage analogues, and those wherein adjacent nucleoside units are linked at 3'-5' to 5'-3' or 2'-5' to 5'-2' with opposite polarities; see U.S. Patent No. 3,687,808 ;4,469,863;4,476,301;5,023,243;5,177,196;5,188,897;5,264,423;5,276,019;5,278,302;5,286,717;5,321,131;5,399,676;5,405,939;5,453,496;5,455,233;5,466,677;5,476,925;5,519,126;5,536,821;5,541,306;5,550,111;5,563,253;5,571,799;5,587,361;and 5,625,050.

[0494] In some embodiments, the oligonucleotide may have a heteroatom backbone, such as a methylene (methylimino) or MMI backbone; an amide backbone (see De Mesmaeker et al. Ace. Chem. Res. 1995, 28:366-374); a morpholino backbone (see Summerton and Weller, U.S. Patent No. 5,034,506); or a peptide nucleic acid (PNA) backbone (in which the phosphodiester backbone of the oligonucleotide is replaced by a polyamide backbone, and the nucleotide is directly or indirectly bound to the aza-nitrogen atom of the polyamide backbone, see Nielsen et al., Science 1991, 254, 1497).

[0495] e. Stereospecific oligonucleotides

[0496] In some embodiments, the internucleotide phosphorus atom of the oligonucleotide is chiral, and the properties of the oligonucleotide are modulated based on the configuration of the chiral phosphorus atom. In some embodiments, suitable methods can be used to synthesize P-chiral oligonucleotide analogs in a stereocontrolled manner (e.g., as described in Oka N, Wada T, Stereocontrolled synthesis of oligonucleotide analogs containing chiral internucleotidic phosphorus atoms. Chem Soc Rev. 2011 Dec; 40(12):5829-43). In some embodiments, phosphate-thioester-containing oligonucleotides are provided, comprising nucleoside units linked together by substantially all Sp or substantially all Rp phosphate-thioester sugar interlinkings. In some embodiments, such phosphate-thioester oligonucleotides having substantially chiral pure sugar interlinkings are prepared by enzymatic or chemical synthesis, as described, for example, in U.S. Patent 5,587,261, issued December 12, 1996, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the chiral-controlled oligonucleotide provides a selective cleavage pattern for the target nucleic acid. For example, in some embodiments, chiral-controlled oligonucleotides provide single-site cleavage within the complementary sequence of a nucleic acid, as described, for example, in U.S. Patent Application Publication 20170037399A1, published February 2, 2017, entitled "CHIRAL DESIGN," the contents of which are incorporated herein by reference in their entirety.

[0497] f. Morpholin

[0498] In some embodiments, the oligonucleotide may be a morpholino-based compound. Morpholino-based oligomers are described in Dwaine A. Braasch and David R. Corey, Biochemistry, 2002, 41(14), 4503-4510; Genesis, volume 30, issue 3, 2001; Heasman, J., Dev. Biol., 2002, 243, 209-214; Nasevichius et al., Nat. Genet., 2000, 26, 216-220; Lacera et al., Proc. Natl. Acad. Sci., 2000, 97, 9591-9596; and U.S. Patent No. 5,034,506, issued July 23, 1991. In some embodiments, the morpholine-based oligomer is a phosphorodiamidate morpholino oligomer (PMO) (e.g., as described in Iverson, Curr. Opin. Mol. Ther., 3:235-238, 2001; and Wang et al., J. Gene Med., 12:354-364, 2010; the disclosures of which are incorporated herein by reference in their entirety).

[0499] g. Peptide Nucleic Acid (PNA)

[0500] In some embodiments, both the sugar and nucleoside linkages (backbone) of the nucleotide units of the oligonucleotide are replaced with new groups. In some embodiments, the base units are maintained for hybridization with a suitable nucleic acid target compound. One such oligomeric compound (an oligonucleotide mimic that has shown excellent hybridization properties) is called a peptide nucleic acid (PNA). In a PNA compound, the sugar-backbone of the oligonucleotide is replaced with an amide-containing backbone (e.g., an aminoethylglycine backbone). The nucleobases are retained and are directly or indirectly bound to the aza-nitrogen atoms of the amide moiety of the backbone. Representative publications reporting the preparation of PNA compounds include, but are not limited to, U.S. Patent Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is incorporated herein by reference. Further teachings on PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.

[0501] h. Spacer

[0502] In some embodiments, the oligonucleotides described herein are spacer polymers. Spacer polymer oligonucleotides typically have the formula 5'-XYZ-3', where X and Z serve as flanking regions surrounding the spacer region Y. In some embodiments, the flanking region X of formula 5'-XYZ-3' is also referred to as the X region, flanking sequence X, 5' wing region X, or 5' wing segment. In some embodiments, the flanking region Z of formula 5'-XYZ-3' is also referred to as the Z region, flanking sequence Z, 3' wing region Z, or 3' wing segment. In some embodiments, the spacer region Y of formula 5'-XYZ-3' is also referred to as the Y region, Y segment, or spacer segment Y. In some embodiments, each nucleotide in the spacer region Y is a 2'-deoxyribonucleoside, and neither the 5' wing region X nor the 3' wing region Z contains any 2'-deoxyribonucleoside.

[0503] In some embodiments, the Y region is a continuous extension of nucleotides, for example, a region of six or more DNA nucleotides, capable of recruiting RNases (e.g., RNase H). In some embodiments, the spacer aggregate binds to the target nucleic acid at a point where the RNase is recruited and subsequently cleaves the target nucleic acid. In some embodiments, the 5' and 3' flanks of the Y region are X and Z regions containing high-affinity modified nucleosides, such as one to six high-affinity modified nucleosides. Some examples of high-affinity modified nucleosides include, but are not limited to, 2'-modified nucleosides (e.g., 2'-MOE, 2'O-Me, 2'-F) or 2'-4' bicyclic nucleosides (e.g., LNA, cEt, ENA). In some embodiments, the lengths of the flanking sequences X and Z can be 1 to 20 nucleotides, 1 to 8 nucleotides, or 1 to 5 nucleotides. The flanking sequences X and Z can have similar or different lengths. In some implementations, the spacer segment Y can be a nucleotide sequence of 5 to 20 nucleotides, 5 to 15 twelve nucleotides, or 6 to 10 nucleotides in length.

[0504] In some embodiments, in addition to DNA nucleotides, the spacer region of the spacer oligonucleotide may contain modified nucleotides known to be acceptable for efficient RNase H action, such as C4'-substituted nucleotides, acyclic nucleotides, and arabinonucleotides. In some embodiments, the spacer region contains one or more unmodified nucleotides. In some embodiments, one or two flanking regions each independently contain one or more phosphate-thioester nucleoside links (e.g., phosphate-thioester nucleoside links or other links) between at least two, at least three, at least four, at least five, or more nucleotides. In some embodiments, the spacer region and the two flanking regions each independently contain modified nucleoside links (e.g., phosphate-thioester nucleoside links or other links) between at least two, at least three, at least four, at least five, or more nucleotides.

[0505] Spacer polymers can be generated using appropriate methods. Representative U.S. patents, U.S. patent publications, and PCT publications that teach the preparation of spacer polymers include, but are not limited to: U.S. Patent Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; 5,700,922; 5,898,031; 7,015,315; 7,101,993; 7,399,845; 7,432,250; 7,569,686; 7,683,036; 7,750,131; 8,580 ,756; 9,045,754; 9,428,534; 9.695,418; 10,017,764; 10,260.069; 9,428,534; 8,580,756; US Patent Publications Nos. US20050074801, US20090221685, US20090286969, US20100197762 and US20110112170; PCT Publications Nos. WO2004069991, WO2005023825, WO2008049085 and WO2009090182; and EP Patent No. EP2,149,605, each of which is incorporated herein by reference in its entirety.

[0506] In some embodiments, the length of the spacer polymer is 10 to 40 nucleosides. For example, the length of the spacer polymer can be 10 to 40, 10 to 35, 10 to 30, 10 to 25, 10 to 20, 10 to 15, 15 to 40, 15 to 35, 15 to 30, 15 to 25, 15 to 20, 20 to 40, 20 to 35, 20 to 30, 20 to 25, 25 to 40, 25 to 35, 25 to 30, 30 to 40, 30 to 35, or 35 to 40 nucleosides. In some implementations, the length of the spacer polymer is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 nucleosides.

[0507] In some embodiments, the length of spacer region Y in the spacer polymer is 5 to 20 nucleotides. For example, the length of spacer region Y can be 5 to 20, 5 to 15, 5 to 10, 10 to 20, 10 to 15, or 15 to 20 nucleotides. In some embodiments, the length of spacer region Y is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides. In some embodiments, each nucleotide in spacer region Y is a 2'-deoxyribonucleoside. In some embodiments, all nucleotides in spacer region Y are 2'-deoxyribonucleoside. In some embodiments, one or more nucleotides in spacer region Y are modified nucleotides (e.g., 2'-modified nucleotides, such as those described herein). In some embodiments, one or more cytosines in spacer region Y are optionally 5-methylcytosine. In some embodiments, each cytosine in spacer region Y is 5-methylcytosine.

[0508] In some embodiments, the 5' wing region (X in the formula 5'-XYZ-3') and the 3' wing region (Z in the formula 5'-XYZ-3') of the spacer polymer are independently 1 to 20 nucleotide lengths. For example, the 5' wing region (X in the formula 5'-XYZ-3') and the 3' wing region (Z in the formula 5'-XYZ-3') of the spacer polymer may be independently 1 to 20, 1 to 15, 1 to 10, 1 to 7, 1 to 5, 1 to 3, 1 to 2, 2 to 5, 2 to 7, 3 to 5, 3 to 7, 5 to 20, 5 to 15, 5 to 10, 10 to 20, 10 to 15, or 15 to 20 nucleotide lengths. In some embodiments, the 5' wing region (X in 5'-XYZ-3' formula) and the 3' wing region (Z in 5'-XYZ-3' formula) of the spacer polymer are independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotide lengths. In some embodiments, the 5' wing region (X in 5'-XYZ-3' formula) and the 3' wing region (Z in 5'-XYZ-3' formula) of the spacer polymer are of the same length. In some embodiments, the 5' wing region (X in 5'-XYZ-3' formula) and the 3' wing region (Z in 5'-XYZ-3' formula) of the spacer polymer are of different lengths. In some embodiments, the 5' wing region of the spacer polymer (X in the formula 5'-XYZ-3') is longer than the 3' wing region of the spacer polymer (Z in the formula 5'-XYZ-3'). In some embodiments, the 5' wing region of the spacer polymer (X in the formula 5'-XYZ-3') is shorter than the 3' wing region of the spacer polymer (Z in the formula 5'-XYZ-3').

[0509] In some embodiments, the spacer polymer comprises the following 5'-XYZ-3': 5-10-5, 4-12-4, 3-14-3, 2-16-2, 1-18-1, 3-10-3, 2-10-2, 1-10-1, 2-8-2, 4-6-4, 3-6-3, 2-6-2, 4-7-4, 3-7-3, 2-7-2, 4-8-4, 3-8-3, 2-8-2, 1-8-1, 2-9-2, 1-9-1, 2-10-2, 1-10-1, 1-12-1, 1-16 -1, 2-15-1, 1-15-2, 1-14-3, 3-14-1, 2-14-2, 1-13-4, 4-13-1, 2-13-3, 3-13-2, 1-12-5, 5-12-1, 2-12-4, 4-12-2, 3-12-3, 1-11-6, 6-11-1, 2-11-5, 5-11-2, 3-11-4, 4-11-3, 1-17-1, 2-16-1, 1-16-2, 1-15-3, 3-15-1, 2-15-2, 1-1 4-4, 4-14-1, 2-14-3, 3-14-2, 1-13-5, 5-13-1, 2-13-4, 4-13-2, 3-13-3, 1-12-6, 6-12-1, 2-12-5, 5-12-2, 3-12-4, 4-12-3, 1-11-7, 7-11-1, 2-11-6, 6-11-2, 3-11-5, 5-11-3, 4-11-4, 1-18-1, 1-17-2, 2-17-1, 1-16-3, 1-16-3, 2 -16-2, 1-15-4, 4-15-1, 2-15-3. 3-15-2, 1-14-5, 5-14-1, 2-14-4, 4-14-2, 3-14-3, 1-13-6, 6-13-1, 2-13-5, 5-13-2, 3-13-4, 4-13-3, 1-12-7, 7-12-1, 2-12-6. 6-12-2, 3-12-5, 5-12-3, 1-11-8, 8-11-1, 2-11-7, 7-11-2, 3-11-6.6-11-3，4-11-5，5-11-4，1-18-1，1-17-2，2-17-1，1-16-3，3-16-1，2-16-2，1-15-4，4-15-1，2-15-3，3-15-2，1-14-5，2-14-4，4-14-2，3-14-3，1-13-6，6-13-1，2-13-5，5-13-2，3-13-4，4-13-3，1-12-7，7-12-1，2-12-6，6-12-2，3-12-5，5-12-3，1-11-8，8-11-1，2-11-7，7-11-2，3-11-6，6-11-3，4-11-5，5-11-4.1-19-1，1-18-2，2-18-1.1-17-3，3-17-1，2-17-2，1-16-4，4-16-1，2-16-3.3-16-2，1-15-5，2-15-4，4-15-2，3-15-3，1-14-6，6-14-1，2-14-5.5-14-2，3-14-4，4-14-3，1-13-7，7-13-1，2-13-6，6-13-2，3-13-5，5-13-3.4-13-4，1-12-8.8-12-1，2-12-7，7-12-2，3-12-6，6-12-3，4-12-5，5-12-4，2-11-8，8-11-2，3-11-7，7-11-3，4-11-6，6-11-4，5-11-5，1-20-1，1-19-2，2-19-1，1-18-3.3-18-1，2-18-2.1-17-4，4-17-1，2-17-3，3-17-2，1-16-5，2-16-4，4-16-2，3-16-3，1-15-6，6-15-1，2-15-5，5-15-2，3-15-4，4-15-3，1-14-7，7-14-1，2-14-6，6-14-2，3-14-5，5-14-3，4-14-4，1-13-8，8-13-1，2-13-7，7-13-2，3-13-6.6-13-3，4-13-5，5-13-4，2-12-8，8-12-2，3-12-7，7-12-3.4-12-6，6-12-4，5-12-5，3-11-8.8-11-3, 4-11-7, 7-11-4, 5-11-6, 6-11-5, 1-21-1, 1-20-2, 2-20-1, 1-20-3, 3-19-1, 2-19-2, 1-18-4, 4-18-1, 2-18-3, 3-18-2, 1-17-5, 2-17-4, 4-17-2, 3-17-3, 1-16-6, 6-16-1, 2-16-5, 5-16-2, 3-16-4, 4-16-3, 1-15-7, 7-15-1, 2-15-6, 6-15-2, 3-15- 5, 5-15-3, 4-15-4.1-14-8.8-14-1, 2-14-7, 7-14-2, 3-14-6, 6-14-3, 4-14-5.5-14-4, 2-13-8.8-13-2, 3-13-7, 7-13-3.4-13-6, 6-13-4, 5-13-5, 1-12-10, 10-12-1, 2-12-9, 9-12-2, 3-12-8.8-12-3.4-12-7, 7-12-4, 5-12-6.6-12-5, 4-11-8, 8-11-4, 5- 11-7, 7-11-5, 6-11-6.1-22-1, 1-21-2, 2-21-1.1-21-3, 3-20-1, 2-20-2, 1-19-4, 4-19-1, 2-19-3, 3-19-2, 1-18-5, 2-18-4, 4-18-2, 3-18-3, 1-17-6, 6-17-1, 2-17-5, 5-17-2, 3-17-4, 4-17-3, 1-16-7, 7-16-1, 2-16-6, 6-16-2, 3-16-5, 5-16-3, 4-16-4, 1 -15-8, 8-15-1, 2-15-7, 7-15-2, 3-15-6, 6-15-3, 4-15-5, 5-15-4, 2-14-8, 8-14-2, 3-14-7, 7-14-3, 4-14-6, 6-14-4, 5-14-5, 3-13-8, 8-13-3, 4-13-7, 7-13-4, 5-13-6, 6-13-5, 4-12-8, 8-12-4, 5-12-7, 7-12-5, 6-12-6, 5-11-8, 8-11-5, 6-11-7 or 7-11-6.

[0510] The numbers represent the number of nucleosides in the X, Y, and Z regions of the 5'-XYZ-3' spacer polymer.

[0511] In some embodiments, one or more nucleotides in the 5' wing region (X in the formula 5'-XYZ-3') or the 3' wing region (Z in the formula 5'-XYZ-3') of the spacer polymer are modified nucleotides (e.g., high-affinity modified nucleotides). In some embodiments, the modified nucleotide (e.g., high-affinity modified nucleotide) is a 2'-modified nucleotide. In some embodiments, the 2'-modified nucleotide is a 2'-4' bicyclic nucleotide or a non-bicyclic 2'-modified nucleotide. In some embodiments, the high-affinity modified nucleoside is a 2'-4' bicyclic nucleoside (e.g., LNA, cEt, or ENA) or a non-bicyclic 2'-modified nucleoside (e.g., 2'-fluoro(2'-F), 2'-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), or 2'-ON-methylacetamido(2'-O-NMA)).

[0512] In some embodiments, one or more nucleotides in the 5' wing region (X in the formula 5'-XYZ-3') of the spacer polymer are high-affinity modified nucleotides. In some embodiments, each nucleotide in the 5' wing region (X in the formula 5'-XYZ-3') of the spacer polymer is a high-affinity modified nucleotide. In some embodiments, one or more nucleotides in the 3' wing region (Z in the formula 5'-XYZ-3') of the spacer polymer are high-affinity modified nucleotides. In some embodiments, each nucleotide in the 3' wing region (Z in the formula 5'-XYZ-3') of the spacer polymer is a high-affinity modified nucleotide. In some embodiments, one or more nucleotides in the 5' wing region (X in the formula 5'-XYZ-3') of the spacer polymer are high-affinity modified nucleotides, and one or more nucleotides in the 3' wing region (Z in the formula 5'-XYZ-3') of the spacer polymer are high-affinity modified nucleotides. In some embodiments, each nucleotide in the 5' wing region (X in the formula 5'-XYZ-3') of the spacer polymer is a high-affinity modified nucleotide, and each nucleotide in the 3' wing region (Z in the formula 5'-XYZ-3') of the spacer polymer is a high-affinity modified nucleotide.

[0513] In some embodiments, the 5' wing region (X in 5'-XYZ-3' formula) of the spacer polymer contains the same high-affinity nucleotide as the 3' wing region (Z in 5'-XYZ-3' formula). For example, the 5' wing region (X in 5'-XYZ-3' formula) and the 3' wing region (Z in 5'-XYZ-3' formula) of the spacer polymer may contain one or more non-bicyclic 2'-modified nucleotides (e.g., 2'-MOE or 2'-O-Me). In another example, the 5' wing region (X in 5'-XYZ-3' formula) and the 3' wing region (Z in 5'-XYZ-3' formula) of the spacer polymer may contain one or more 2'-4' bicyclic nucleotides (e.g., LNA or cEt). In some embodiments, each nucleotide in the 5' wing region (X in the formula 5'-XYZ-3') and the 3' wing region (Z in the formula 5'-XYZ-3') of the spacer polymer is a non-bicyclic 2'-modified nucleotide (e.g., 2'-MOE or 2'-O-Me). In some embodiments, each nucleotide in the 5' wing region (X in the formula 5'-XYZ-3') and the 3' wing region (Z in the formula 5'-XYZ-3') of the spacer polymer is a 2'-4' bicyclic nucleotide (e.g., LNA or cEt).

[0514] In some embodiments, the spacer polymer comprises a 5'-XYZ-3' configuration, wherein the lengths of X and Z are independently 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotides, and the length of Y is 6 to 10 (e.g., 6, 7, 8, 9, or 10) nucleotides, wherein each nucleotide in X and Z is a non-bicyclic 2'-modified nucleotide (e.g., 2'-MOE or 2'-O-Me), and each nucleotide in Y is a 2'-deoxyribonucleoside. In some embodiments, the spacer polymer comprises a 5'-XYZ-3' configuration, wherein the lengths of X and Z are independently 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleotides, and the length of Y is 6 to 10 (e.g., 6, 7, 8, 9, or 10) nucleotides, wherein each nucleotide in X and Z is a 2'-4' bicyclic nucleotide (e.g., LNA or cEt), and each nucleotide in Y is a 2'-deoxyribonucleoside. In some embodiments, the 5' wing region of the spacer polymer (X in the 5'-XYZ-3' formula) comprises a high-affinity nucleotide that is different from the 3' wing region of the spacer polymer (Z in the 5'-XYZ-3' formula). For example, the 5' wing region of the spacer polymer (X in the formula 5'-XYZ-3') may contain one or more non-bicyclic 2'-modified nucleosides (e.g., 2'-MOE or 2'-O-Me), and the 3' wing region of the spacer polymer (Z in the formula 5'-XYZ-3') may contain one or more 2'-4' bicyclic nucleosides (e.g., LNA or cEt). In another example, the 3' wing region of the spacer polymer (Z in the formula 5'-XYZ-3') may contain one or more non-bicyclic 2'-modified nucleosides (e.g., 2'-MOE or 2'-O-Me), and the 5' wing region of the spacer polymer (X in the formula 5'-XYZ-3') may contain one or more 2'-4' bicyclic nucleosides (e.g., LNA or cEt).

[0515] In some embodiments, the spacer polymer comprises a 5'-XYZ-3' configuration, wherein the lengths of X and Z are independently 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleosides, and the length of Y is 6 to 10 (e.g., 6, 7, 8, 9, or 10) nucleosides, wherein each nucleoside in X is a non-bicyclic 2'-modified nucleoside (e.g., 2'-MOE or 2'-O-Me), each nucleoside in Z is a 2'-4' bicyclic nucleoside (e.g., LNA or cEt), and each nucleoside in Y is a 2'-deoxyribonucleoside. In some embodiments, the spacer polymer comprises a 5'-XYZ-3' configuration, wherein the lengths of X and Z are independently 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleosides, and the length of Y is 6 to 10 (e.g., 6, 7, 8, 9, or 10) nucleosides, wherein each nucleoside in X is a 2'-4' bicyclic nucleoside (e.g., LNA or cEt), each nucleoside in Z is a non-bicyclic 2'-modified nucleoside (e.g., 2'-MOE or 2'-O-Me), and each nucleoside in Y is a 2'-deoxyribonucleoside.

[0516] In some embodiments, the 5' wing region of the spacer polymer (X in the formula 5'-XYZ-3') comprises one or more non-bicyclic 2'-modified nucleosides (e.g., 2'-MOE or 2'-O-Me) and one or more 2'-4' bicyclic nucleosides (e.g., LNA or cEt). In some embodiments, the 3' wing region of the spacer polymer (Z in the formula 5'-XYZ-3') comprises one or more non-bicyclic 2'-modified nucleosides (e.g., 2'-MOE or 2'-O-Me) and one or more 2'-4' bicyclic nucleosides (e.g., LNA or cEt). In some embodiments, both the 5' wing region of the spacer polymer (X in the formula 5'-XYZ-3') and the 3' wing region of the spacer polymer (Z in the formula 5'-XYZ-3') contain one or more non-bicyclic 2'-modified nucleosides (e.g., 2'-MOE or 2'-O-Me) and one or more 2'-4' bicyclic nucleosides (e.g., LNA or cEt).

[0517] In some embodiments, the spacer polymer comprises a 5'-XYZ-3' configuration, wherein the lengths of X and Z are independently 2 to 7 (e.g., 2, 3, 4, 5, 6, or 7) nucleotides, and the length of Y is 6 to 10 (e.g., 6, 7, 8, 9, or 10) nucleotides, wherein at least one, but not all (e.g., 1, 2, 3, 4, 5, or 6) of the 1st, 2nd, 3rd, 4th, 5th, 6th, or 7th position (the 5th position is the 1st position) of X are non-bicyclic 2'-modified nucleotides (e.g., 2'-MOE or 2'-O-Me), wherein the remaining nucleotides in both X and Z are 2'-4' bicyclic nucleotides (e.g., LNA or cEt), and wherein each nucleotide in Y is a 2'-deoxyribonucleoside. In some embodiments, the spacer polymer comprises a 5'-XYZ-3' configuration, wherein the lengths of X and Z are independently 2 to 7 (e.g., 2, 3, 4, 5, 6, or 7) nucleotides, and the length of Y is 6 to 10 (e.g., 6, 7, 8, 9, or 10) nucleotides, wherein at least one, but not all (e.g., 1, 2, 3, 4, 5, or 6) of the 1st, 2nd, 3rd, 4th, 5th, 6th, or 7th position (the 5th position is the 1st position) of Z are non-bicyclic 2'-modified nucleotides (e.g., 2'-MOE or 2'-O-Me), wherein the remaining nucleotides in both X and Z are 2'-4' bicyclic nucleotides (e.g., LNA or cEt), and wherein each nucleotide in Y is a 2'-deoxyribonucleoside. In some embodiments, the spacer polymer comprises a 5'-XYZ-3' configuration, wherein the lengths of X and Z are independently 2 to 7 (e.g., 2, 3, 4, 5, 6, or 7) nucleotides, and the length of Y is 6 to 10 (e.g., 6, 7, 8, 9, or 10) nucleotides, wherein at least one but not all (e.g., 1, 2, 3, 4, 5, or 6) of positions 1, 2, 3, 4, 5, 6, or 7 of X and at least one but not all (e.g., 1, 2, 3, 4, 5, or 6) of positions 1, 2, 3, 4, 5, 6, or 7 of Z (the 5' position is position 1) are non-bicyclic 2'-modified nucleotides (e.g., 2'-MOE or 2'-O-Me), wherein the remaining nucleotides in both X and Z are 2'-4' bicyclic nucleotides (e.g., LNA or cEt), and wherein each nucleotide in Y is a 2'-deoxyribonucleoside.

[0518] Some non-limiting examples of spacer polymer configurations having a mixture of non-bicyclic 2'-modified nucleotides (e.g., 2'-MOE or 2'-O-Me) and 2'-4' bicyclic nucleotides (e.g., LNA or cEt) in the 5' wing region (X in the formula 5'-XYZ-3') and / or the 3' wing region (Z in the formula 5'-XYZ-3') of the spacer polymer include: BBB-(D)n-BBBAA; KKK-(D)n-KKKAA; LLL-(D)n-LLLAA; BBB-(D)n-BBBEE; KKK-(D)n-KKKEE; LLL-(D)n-LLLEE; BBB-(D)n-BBBAA; KKK-(D)n-KKKAA ; LLL-(D)n-LLLAA; BBB-(D)n-BBBEE; KKK-(D)n-KKKEE; LLL-(D)n-LLLEE; BBB-(D)n-BBBAAA; KKK-(D)n-KKKAAA; LLL-(D)n-LLLAAA; BBB-(D)n-BBBEEE ; KKK-(D)n-KKKEEE; LLL-(D)n-LLLEEE; BBB-(D)n-BBBAAA; KKK-(...

Claims

1. A complex comprising an anti-transferrin receptor (TfR) antibody covalently linked to a molecular payload configured to modulate expression or activity of a muscle disease gene, wherein the antibody comprises: (i) a heavy chain variable region (VH) comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 76; and / or a light chain variable region (VL) comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 75; (ii) a heavy chain variable region (VH) comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 69; and / or a light chain variable region (VL) comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 70; (iii) a heavy chain variable region (VH) comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 71; and / or a light chain variable region (VL) comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 70; (iv) a heavy chain variable region (VH) comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 72; and / or a light chain variable region (VL) comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 70; (v) a heavy chain variable region (VH) comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 73; and / or a light chain variable region (VL) comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 74; (vi) a heavy chain variable region (VH) comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 73; and / or a light chain variable region (VL) comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 75; (vii) a heavy chain variable region (VH) comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 76; and / or a light chain variable region (VL) comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 74; (viii) a heavy chain variable region (VH) comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 77; and / or a light chain variable region (VL) comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 78; (ix) a heavy chain variable region (VH) comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 79; and / or a light chain variable region (VL) comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 80; or (x) a heavy chain variable region (VH) comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 77; and / or a light chain variable region (VL) comprising an amino acid sequence that is at least 95% identical to SEQ ID NO:

80.

2. The complex of claim 1, wherein 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) a VH comprising the amino acid sequence of SEQ ID NO: 77 and a VL comprising the amino acid sequence of SEQ ID NO:

80.

3. The complex of claim 1 or claim 2, wherein the antibody is selected from the group consisting of a Fab fragment, a Fab’ fragment, a F(ab’)2 fragment, a scFv, a Fv, and a full-length IgG.

4. The complex of claim 3, wherein the antibody is a Fab fragment.

5. The complex of claim 4, wherein the antibody comprises: (i) a heavy chain comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 101; and / or a light chain comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 90; (ii) a heavy chain comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 97; and / or a light chain comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 85; (iii) a heavy chain comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 98; and / or a light chain comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 85; (iv) a heavy chain comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 99; and / or a light chain comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 85; (v) a heavy chain comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 100; and / or a light chain comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 89; (vi) a heavy chain comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 100; and / or a light chain comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 90; (vii) a heavy chain comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 101; and / or a light chain comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 89; (viii) a heavy chain comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 102; and / or a light chain comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 93; (ix) a heavy chain comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 103; and / or a light chain comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 95; or (x) a heavy chain comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 102; and / or a light chain comprising an amino acid sequence that is at least 85% identical to SEQ ID NO:

95.

6. The complex of claim 4 or claim 5, wherein 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. (x) a heavy chain that comprises the amino acid sequence of SEQ ID NO: 102; and a light chain that comprises the amino acid sequence of SEQ ID NO:

95.

7. The complex of any one of claims 1 to 6, wherein the antibody does not specifically bind to the transferrin binding site of the transferrin receptor and / or wherein the antibody does not inhibit the binding of transferrin to the transferrin receptor.

8. The complex of any one of claims 1 to 7, wherein the antibody is cross-reactive with an extracellular epitope of the transferrin receptor in two or more of human, non-human primates, and rodents.

9. The complex of any one of claims 1 to 8, wherein the complex is configured to facilitate transferrin receptor-mediated internalization of the molecular payload into a muscle cell.

10. The complex of any one of claims 1 to 9, wherein the molecular payload is an oligonucleotide.

11. The complex of claim 10, wherein the oligonucleotide comprises a complement of a region of a muscle disease gene having a gain-of-function disease allele.

12. The complex of claim 10 or 11, wherein the oligonucleotide comprises at least one modified internucleoside linkage.

13. The complex of claim 12, wherein the at least one modified internucleoside linkage is a phosphorothioate linkage.

14. The complex of any one of claims 10 to 13, wherein the oligonucleotide comprises one or more modified nucleosides.

15. The complex of claim 14, wherein the one or more modified nucleosides are 2’-modified nucleosides.

16. The complex of any one of claims 10 to 15, wherein the oligonucleotide is a gapmer oligonucleotide that directs RNAse H-mediated cleavage of an mRNA transcript encoded by the muscle disease gene in a cell.

17. The complex of any one of claims 10 to 15, wherein the oligonucleotide is a mixed-mer oligonucleotide.

18. The complex of any one of claims 10 to 15, wherein the oligonucleotide is an RNAi oligonucleotide that facilitates RNAi-mediated cleavage of an mRNA transcript encoded by the muscle disease gene.

19. The complex of claim 15, wherein each 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.

20. The complex of claim 14, wherein the one or more modified nucleosides are 2’,4’- bridged nucleosides.

21. The complex of any one of claims 10 to 15, wherein the oligonucleotide is a phosphorodiamidate morpholino oligomer.

22. The complex of any one of claims 1 to 21, wherein the antibody is covalently linked to the molecular payload by a cleavable linker.

23. The complex of claim 22, wherein the cleavable linker comprises a valine-citrulline sequence.

24. The complex of any one of claims 1 to 23, wherein the antibody is covalently linked to the molecular payload, the covalent linkage being by conjugation to a lysine residue or a cysteine residue of the antibody.

25. The complex of any one of claims 1 to 24, wherein modulating expression or activity of a muscle disease gene comprises reducing expression of RNA and / or protein.

26. A method of modulating expression or activity of a muscle disease gene in a cell, the method comprising contacting the cell with an effective amount of the complex of any one of claims 1 to 25 for promoting internalization of the molecular payload into the cell, optionally wherein the cell is a muscle cell.

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

28. A method of treating a subject having a muscle disease, the method comprising administering to the subject an effective amount of the complex of any one of claims 1 to 25, optionally wherein the muscle disease is a disease selected from the group consisting of adult-onset Pompe disease, centronuclear myopathy (CNM), Duchenne muscular dystrophy, facioscapulohumeral muscular dystrophy (FSHD), familial hypertrophic cardiomyopathy, fibrodysplasia ossificans progressiva (FOP), Friedreich’s ataxia (FRDA), inclusion body myopathy 2, distal myopathy of Laing, myofibrillar myopathy, myotonia congenita (autosomal dominant form, Thomsen disease), myotonic dystrophy type I, myotonic dystrophy type II, myotubular myopathy, oculopharyngeal muscular dystrophy, and paramyotonia congenita.

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