Muscle targeting complexes and uses thereof

Abstract

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

Description

[0001] Related applications

[0002] This application claims priority under 35 U.S.C. §119(e) to: 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”; U.S. Provisional Application Serial No. 63 / 069079, filed August 23, 2020, entitled “MUSCLE-TARGETING COMPLEXES AND USES THEREOF”; and U.S. Provisional Application Serial No. 63 / 181450, filed April 29, 2021, entitled “MUSCLE-TARGETING COMPLEXES AND USES THEREOF”. THEREOF”; and U.S. Provisional Application Serial No. 63 / 055,785, filed on July 23, 2020, entitled “MUSCLE-TARGETING COMPLEXES AND USES THEREOF”; the contents of each of which are incorporated herein by reference in their entirety. Technical Field

[0003] The present application relates to targeting complexes for delivering molecular payloads (eg, oligonucleotides) to cells and their uses, particularly in connection with the treatment of diseases.

[0004] Reference sequence listing submitted as a text file via EFS-Web

[0005] This application contains a sequence listing that has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. The ASCII copy created on July 8, 2021 is named D082470041WO00-SEQ-DWY and is 152,275 bytes in size. Background Art

[0006] Muscle diseases are generally associated with muscle weakness and / or (for example, and) muscle dysfunction leading to life-threatening complications. Many examples of such diseases have been characterized, including various forms of muscular dystrophy (for example, Duchenne, facioscapulohumeral, myotonia, and oculopharyngeal), Pompe disease, centronuclear myopathy, familial hypertrophic cardiomyopathy, Laing distal myopathy, Fibrodysplasia Ossificans Progressiva, Friedreich's ataxia, myofibrillar myopathy, etc. These conditions are generally hereditary, but can occur spontaneously. These conditions are generally congenital, but can occur in later 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 that cause muscle diseases have been identified in genes encoding ion channels, structural proteins, metabolic proteins, and signaling 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, the present disclosure provides a compound targeting muscle cells for delivering molecular payloads to these cells. In some embodiments, the compound of the present disclosure promotes muscle-specific delivery of molecular payloads targeting muscle disease alleles. For example, in some embodiments, the compound provided herein is particularly useful for delivering a molecular payload that regulates the expression or activity of a gene in an object suffering from or suspected of suffering from a muscle disease associated with a gene (e.g., the genes / diseases of Table 1). In some embodiments, the compound provided herein comprises a muscle targeting agent (e.g., a muscle targeting antibody) that specifically binds to a receptor on the surface of a muscle cell for delivering a molecular payload to a muscle cell. In some embodiments, the compound is taken up into the cell by receptor (e.g., transferrin receptor)-mediated internalization, and then the molecular payload can be released to perform functions inside the cell. For example, a compound engineered to deliver an oligonucleotide can release the oligonucleotide so that the oligonucleotide can regulate the expression or activity of a muscle disease allele. In some embodiments, the oligonucleotide is released by endosome cleavage of the covalent linker of the oligonucleotide and muscle targeting agent connecting the compound.

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

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

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

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

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

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

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

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

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

[0018] (ix) a heavy chain variable region (VH) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 79; and / or a light chain variable region (VL) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 80; or

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

[0020] In some embodiments, the antibody comprises:

[0021] (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;

[0022] (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;

[0023] (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;

[0024] (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;

[0025] (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;

[0026] (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;

[0027] (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;

[0028] (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;

[0029] (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

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

[0031] In some embodiments, 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. In some embodiments, the antibody is a Fab fragment.

[0032] In some embodiments, the antibody comprises:

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

[0034] (ii) a heavy chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 97; and / or a light chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 85;

[0035] (iii) a heavy chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 98; and / or a light chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 85;

[0036] (iv) a heavy chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 99; and / or a light chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 85;

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

[0038] (vi) a heavy chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 100; and / or a light chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 90;

[0039] (vii) a heavy chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 101; and / or a light chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 89;

[0040] (viii) a heavy chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 102; and / or a light chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 93;

[0041] (ix) a heavy chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 103; and / or a light chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 95; or

[0042] (x) a heavy chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 102; and / or a light chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 95.

[0043] In some embodiments, the antibody comprises:

[0044] (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;

[0045] (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;

[0046] (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;

[0047] (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;

[0048] (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;

[0049] (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;

[0050] (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;

[0051] (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;

[0052] (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

[0053] (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.

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

[0055] In some embodiments, the antibody is cross-reactive with extracellular epitopes of two or more of the human, non-human primate, and rodent transferrin receptors.

[0056] In some embodiments, the complex is configured to promote transferrin receptor-mediated internalization of a molecular cargo into myocytes.

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

[0058] In some embodiments, the oligonucleotide comprises at least one modified internucleoside linkage. In some embodiments, the at least one modified internucleoside linkage is a phosphorothioate linkage.

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

[0060] In some embodiments, 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.

[0061] In some embodiments, the oligonucleotide is a mixed-mer oligonucleotide.

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

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

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

[0065] In some embodiments, the oligonucleotide is a phosphorodiamidate morpholino oligomer.

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

[0067] In some embodiments, the antibody is covalently linked to the molecular cargo via conjugation to a lysine residue or a cysteine ​​residue of the antibody.

[0068] In some embodiments, modulating the expression or activity of a muscle disease gene comprises decreasing the expression of RNA and / or protein.

[0069] Another aspect of the disclosure relates to a method of modulating the expression or activity of a muscle disease gene in a cell, the method comprising contacting the cell with an effective amount of a complex described herein for promoting internalization of a molecular cargo into the cell, optionally wherein the cell is a muscle cell.

[0070] In some embodiments, 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 Ryan myopathy, myofibrillar myopathy, myotonia congenita (autosomal dominant form, Thomson disease), myotonic dystrophy type I, myotonic dystrophy type II, myotubular myopathy, oculopharyngeal muscular dystrophy, and paramyotonia congenita.

[0071] Another aspect of the present disclosure relates to a method of treating a subject having a muscle disease, comprising administering to the subject an effective amount of a complex described herein, 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 Ryan myopathy, myofibrillar myopathy, myotonia congenita (autosomal dominant form, Thomson disease), myotonic dystrophy type I, myotonic dystrophy type II, myotubular myopathy, oculopharyngeal muscular dystrophy, and paramyotonia congenita. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figure 1 Depicted is a non-limiting schematic diagram showing the effect of transfection of Hepa 1-6 cells with an antisense oligonucleotide targeting DMPK (ASO300) on DMPK expression levels relative to vehicle transfection.

[0074] Figure 2A Depicted is a non-limiting schematic diagram showing a HIL-HPLC trace obtained during the purification of a muscle-targeted complex comprising an anti-transferrin receptor antibody covalently linked to a DMPK antisense oligonucleotide.

[0075] Figure 2B Depicted are non-limiting images of SDS-PAGE analysis of the muscle targeting complex.

[0076] Figure 3 Depicted is a non-limiting schematic diagram showing the ability of a muscle-targeted RI7 217 Fab antibody-oligonucleotide complex (DTX-C-008) comprising ASO300 to reduce DMPK expression levels.

[0077] Figures 4A to 4EDepicted is a non-limiting schematic diagram showing the ability of a muscle-targeted RI7 217 Fab antibody-oligonucleotide complex (DTX-C-008) comprising ASO300 to reduce DMPK expression levels in mouse muscle tissue in vivo relative to vehicle treatment, treatment with naked ASO300, or treatment with a control non-targeting complex (DTX-C-007). (N=3 C57Bl / 6WT mice).

[0078] Figures 5A to 5B Depicted is a non-limiting schematic diagram showing the tissue selectivity of a muscle-targeted RI7 217 Fab antibody-oligonucleotide complex (DTX-C-008) containing ASO300. The muscle-targeted complex (DTX-C-008) containing ASO300 did not reduce DMPK expression levels in mouse brain or spleen tissue in vivo relative to vehicle treatment, treatment with naked ASO300, or treatment with a control non-targeted complex (DTX-C-007). (N=3 C57Bl / 6 WT mice).

[0079] Figures 6A to 6F Depicted is a non-limiting schematic diagram showing the ability of a muscle-targeted RI7 217 Fab antibody-oligonucleotide complex (DTX-C-008) comprising ASO300 to reduce DMPK expression levels in mouse muscle tissue in vivo relative to vehicle treatment, treatment with naked ASO300, or treatment with a control non-targeting complex (DTX-C-007). (N=5 C57Bl / 6 WT mice).

[0080] Figures 7A to 7L Depicted is a non-limiting schematic diagram showing the ability of a muscle-targeted antibody-oligonucleotide complex (DTX-C-012) comprising ASO300 covalently linked to an anti-hTfR antibody to reduce DMPK expression levels in cynomolgus monkey muscle tissue in vivo relative to vehicle treatment (saline) and compared to naked DMPK ASO (ASO300). (N=3 male cynomolgus monkeys).

[0081] Figures 8A to 8B Depicted is a non-limiting schematic diagram showing the ability of a muscle-targeted antibody-oligonucleotide complex (DTX-C-012) comprising ASO300 covalently linked to an anti-hTfR antibody to reduce DMPK expression levels in cynomolgus monkey smooth muscle tissue in vivo relative to vehicle treatment (saline) and compared to naked DMPK ASO (ASO300). (N=3 male cynomolgus monkeys).

[0082] Figures 9A to 9DA non-limiting schematic diagram illustrates the tissue selectivity of a muscle-targeted antibody-oligonucleotide complex (DTX-C-012) comprising ASO300 covalently linked to an anti-hTfR antibody. The muscle-targeted complex comprising DMPK-ASO did not reduce DMPK expression levels in kidney, brain, or spleen tissue in cynomolgus monkeys in vivo relative to vehicle treatment. (N = 3 male cynomolgus monkeys).

[0083] Figure 10 Shown are normalized DMPK mRNA tissue expression levels between several tissue types in cynomolgus monkeys. (N=3 male cynomolgus monkeys).

[0084] Figures 11A to 11B Depicted are non-limiting schematic diagrams showing the ability of DTX-C-008 to reduce DMPK expression levels in mouse muscle tissue in vivo for up to 28 days following administration of a muscle-targeted RI7 217 Fab antibody-oligonucleotide complex (DTX-C-008) comprising ASO300 relative to vehicle treatment (saline) and compared to naked DMPKASO (ASO300).

[0085] Figure 12 A single dose of a muscle-targeted complex (DTX-C-012) comprising ASO300 covalently linked to an anti-hTfR antibody was shown to be safe and tolerable in cynomolgus monkeys (N=3 male cynomolgus monkeys).

[0086] Figures 13A to 13B Depicted is a non-limiting schematic diagram showing the ability of DTX-C-008 to reduce DMPK expression levels in mouse muscle tissue in vivo for up to 12 weeks following administration of muscle-targeted RI7 217 Fab antibody-oligonucleotide complex (DTX-C-008) containing ASO300 relative to vehicle treatment (PBS) and compared to a control IgG2a Fab antibody-oligonucleotide complex (DTX-C-007) and a naked DMPK ASO (ASO300). (N=5 C57Bl / 6 WT mice).

[0087] Figures 14A to 14B Depicted is a non-limiting schematic diagram showing the ability of the muscle-targeted RI7 217 Fab antibody-oligonucleotide complex (DTX-C-008) containing ASO300 to target nuclear mutant DMPK RNA in a mouse model. (N=6 mice).

[0088] Figures 15A to 15BDepicted is a non-limiting schematic diagram illustrating the ability of a muscle-targeted RI7 217 Fab antibody-ASO complex (DTX-actin) comprising an oligonucleotide targeting actin to dose-dependently reduce the expression level of actin and the functional grade of myotonia in muscle tissue. (N=2 HSA LR mice).

[0089] Figures 16A to 16C Depicted is a non-limiting schematic diagram showing that a muscle-targeted RI7 217Fab antibody-oligonucleotide complex containing ASO300 (DTX-C-008) was able to significantly reduce prolonged QTc intervals in a mouse model for validation of functional correction of arrhythmias in the DM1 heart model (N=10 mice).

[0090] Figures 17A to 17B Depicted is a non-limiting schematic diagram showing that a muscle-targeted antibody-oligonucleotide complex (DTX-C-012) comprising an ASO300 antisense oligonucleotide covalently linked to an anti-hTfR antibody is able to reduce DMPK expression levels and correct splicing of a DMPK-specific target gene (Bin1) in human cells from a DM1 patient. (N=3)

[0091] Figure 18 Depicted is a non-limiting schematic diagram showing the ability of a muscle targeting complex (anti-TfR antibody-FM10) comprising an anti-TfR1 Fab (RI7 217) conjugated to an FM10 antisense oligonucleotide to reduce the expression levels of downstream DUX4 genes (ZSCAN4, MBD3L2, TRIM43) in human U-2 OS cells relative to naked FM10 antisense oligonucleotide.

[0092] Figure 19 Depicted is a non-limiting schematic diagram showing the ability of an anti-transferrin receptor muscle-targeted complex comprising an exon-23 skipping phosphorodiamidate morpholino oligomer (PMO) to dose-dependently enhance exon skipping in muscle tissue of an mdx mouse model.

[0093] Figures 20A to 20B Depicted is a non-limiting schematic diagram illustrating the ability of an anti-transferrin receptor muscle targeting complex containing an exon-23 skipping PMO to dose-dependently elevate dystrophin in skeletal muscle (quadriceps) of the mdx mouse model.

[0094] Figures 21A to 21E Depicted is a non-limiting schematic diagram showing that an anti-transferrin receptor muscle-targeted complex comprising an exon-23 skipping PMO improves functional performance in the mdx mouse model ( Figure 21A 、 21B , 21C and 21D) and decreased creatine kinase levels ( Figure 21E ) ability. (**p<0.01; ***p<0.001; ****p<0.0001).

[0095] Figures 22A to 22C A non-limiting schematic diagram is depicted showing the dose response of selected antisense oligonucleotides (DMPK-ASO-1, DMPK-ASO-2, and DMPK-ASO-3) in DMPK knockdown in human DM1 myotubes. ASO300 was used as a control. All oligonucleotides tested showed activity in DMPK knockdown. Statistical analysis: One-way ANOVA with Tukey's HSD post hoc test vs. bare ASO300 treatment; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0096] Figures 23A to 23B Depicted is a non-limiting schematic diagram showing the dose response of selected antisense oligonucleotides (DMPK-ASO-1, DMPK-ASO-2, and DMPK-ASO-3) in DMPK knockdown in non-human primate (NHP) DM1 myotubes. ASO300 was used as a control. All oligonucleotides tested showed activity in DMPK knockdown.

[0097] Figure 24 The serum stability of linkers used to link anti-TfR antibodies to molecular cargo (e.g., oligonucleotides) over time following intravenous administration in multiple species is shown.

[0098] Figures 25A to 25F Shown are the binding of humanized anti-TfR Fabs to human TfR1 (hTfR1) or cynomolgus monkey TfR1 (cTfR1) measured by ELISA. Figure 25A Binding of humanized 3M12 variants to hTfR1 is shown. Figure 25B Binding of humanized 3M12 variants to cTfR1 is shown. Figure 25C Binding of humanized 3A4 variants to hTfR1 is shown. Figure 25D Binding of humanized 3A4 variants to cTfR1 is shown. Figure 25E Binding of humanized 5H12 variants to hTfR1 is shown. Figure 25F Binding of humanized 5H12 variants to hTfR1 is shown.

[0099] Figure 26The quantitative cellular uptake of anti-TfR Fab conjugates into rhabdomyosarcoma (RD) cells is shown. The molecular load in the tested conjugate is a DMPK targeting oligonucleotide, and the uptake of the conjugate is promoted by the specified anti-TfR Fab. The assay also includes a conjugate with a negative control Fab (anti-mouse TfR) or a positive control Fab (anti-human TfR1). Cells are incubated for 4 hours with the specified conjugates at a concentration of 100 nM. Cell uptake is measured by average Cypher5e fluorescence.

[0100] Figures 27A to 27F Shown are the binding of oligonucleotide-conjugated or unconjugated humanized anti-TfR Fab to human TfR1 (hTfR1) and cynomolgus monkey TfR1 (cTfR1) measured by ELISA. Figure 27A Binding of humanized 3M12 variants to hTfR1, alone or conjugated to DMPK targeting oligonucleotides, is shown. Figure 27B Binding of humanized 3M12 variants to cTfR1, alone or conjugated to DMPK targeting oligonucleotides, is shown. Figure 27C Binding of humanized 3A4 variants to hTfR1, alone or conjugated to DMPK targeting oligonucleotides, is shown. Figure 27D Binding of humanized 3A4 variants to cTfR1, alone or conjugated to DMPK targeting oligonucleotides, is shown. Figure 27E Binding of humanized 5H12 variants to hTfR1, alone or conjugated to DMPK targeting oligonucleotides, is shown. Figure 27F Binding of humanized 5H12 variants to cTfR1 alone or conjugated to DMPK targeting oligonucleotides is shown. Also shown are the respective EC 50 value.

[0101] Figure 28 DMPK expression in RD cells treated with DMPK targeting oligonucleotides relative to cells treated with PBS is shown. Treatment duration was 3 days. DMPK targeting oligonucleotides were delivered to cells as free oligonucleotides (gymnotic uptake, "free") or delivered to cells using a transfection reagent ("trans").

[0102] Figure 29 Figure 2 shows DMPK expression in RD cells treated with various concentrations of a conjugate comprising the indicated humanized anti-TfR antibodies conjugated to a DMPK targeting oligonucleotide (ASO300). Treatment duration was 3 days. ASO300 delivered using a transfection agent was used as a control (labeled "Trans").

[0103] Figure 30Shown are the results of splicing correction of Atp2a1 by anti-TfR1 antibody-oligonucleotide conjugate (Ab-ASO) measured in gastrocnemius muscle in the HSA-LR mouse model of DM1. The anti-TfR antibody used was RI7 217, and the oligonucleotide targeted human skeletal actin.

[0104] Figure 31 Shown are splicing corrections in more than 30 different RNAs associated with DM1 measured in gastrocnemius muscle of HSA-LR mice treated with anti-TfR1 antibody-oligonucleotide (Ab-ASO) conjugates or saline. The anti-TfR antibody used was RI7217, and the oligonucleotides targeted human skeletal actin.

[0105] Figure 32 Shown are splicing perturbations in the quadriceps, gastrocnemius, or tibialis anterior muscles of HSA-LR mice treated with anti-TfR1 antibody-oligonucleotide conjugates (Ab-ASO) or saline. Figure 31 Complex splicing perturbations were detected in more than 30 RNAs shown in .

[0106] Figure 33 Shown are the myotonic grades measured in the quadriceps, gastrocnemius, and tibialis anterior muscles of HSA-LR mice treated with saline, unconjugated oligonucleotide (ASO), or anti-TfR1 antibody-oligonucleotide conjugates (Ab-ASO). Myotonia was measured by electromyography (EMG) and graded as 0, 1, 2, or 3 based on the frequency of myotonic discharges.

[0107] Figure 34 Shown is exon 51 skipping in human DMD myotubes, which is promoted by DMD exon 51 skipping oligonucleotide (PMO). Cells were treated with naked PMO or with PMO conjugated to anti-TfR1 Fab (Ab-PMO).

[0108] Figure 35 Shown is a dose-dependent increase in dystrophin expression in the quadriceps of mdx mice following treatment with anti-mouse TfR1 (RI7217) conjugated to an oligonucleotide targeting exon 23 (PMO), with α-actin as a loading control, as measured by western blot for dystrophin. Standards were generated using pooled wild-type and pooled mdx proteins. Percentages represent the amount of WT protein incorporated into the sample.

[0109] Figure 36Shown is the quantification of dystrophin levels in the quadriceps muscles of mdx mice after treatment with various doses of anti-mouse TfR (RI7 217) conjugated to an oligonucleotide targeting exon 23 (PMO).

[0110] Figure 37 Shown are images of immunofluorescence staining of quadriceps muscles from wild-type (WT) mice treated with saline or mdx mice treated with saline, naked oligonucleotide, or oligonucleotide conjugated to anti-mouse TfR1 (RI7 217).

[0111] Figures 38A to 38B It shows that the concentration of bare FM10 ( Figure 38A ) or FM10 conjugated with anti-TfR1 ( Figure 38B )-treated FSHD patient-derived myotubes.

[0112] Figure 39 Data are shown demonstrating that conjugates comprising the indicated anti-TfRFabs (3M12 VH3 / VK2, 3M12 VH4 / VK3, and 3A4 VH3 N54S / VK4) conjugated to DMD exon-skipping oligonucleotides resulted in enhanced exon skipping in DMD patient myotubes compared to naked DMD exon-skipping oligonucleotides.

[0113] Figures 40A to 40E Shown are the in vivo activities of conjugates comprising the indicated anti-TfR Fabs (control, 3M12 VH3 / VK2, 3M12 VH4 / VK3, and 3A4 VH3N54S / VK4) conjugated to DMPK targeting oligonucleotides in reducing DMPK mRNA expression in mice expressing human TfR1 (hTfR1 knock-in mice). Figure 40A The experimental design (e.g., IV dose, dosing frequency) is shown. 14 days after the first dose, the tibialis anterior muscle of mice ( Figure 40B ), gastrocnemius( Figure 40C ),heart( Figure 40D ) and diaphragm ( Figure 40E ) in the DMPK mRNA levels.

[0114] Figures 41A to 41C It was shown that a conjugate comprising an anti-TfR antibody conjugated to a DMPK targeting oligonucleotide corrected splicing and reduced foci in CM-DM1-32F primary cells expressing a DMPK mutant mRNA comprising 380 CUG repeats. Figure 41A The conjugate was shown to reduce mutant DMPK mRNA expression. Figure 41B The conjugate is shown to correct BIN1 exon 11 splicing. Figure 41C Images of fluorescence in situ hybridization (FISH) analysis and quantification of the images are shown, demonstrating that the conjugation reduced nuclear foci formed by mutant DMPK mRNA. Figure 41C In the microscopy images shown in the upper panel of FIG, the light circles show the cell nuclei, and the bright spots within the nuclei of DM1 cells (three microscopy images on the right) show CUG foci.

[0115] Figure 42 ELISA measurements of the binding of anti-TfR Fab 3M12 VH4 / Vk3 to recombinant human (circles), cynomolgus monkey (squares), mouse (upward triangles), or rat (downward triangles) TfR1 proteins at Fab concentrations ranging from 230 pM to 500 nM are shown. The measurements show that the anti-TfR Fab is reactive with human and cynomolgus monkey TfR1. No binding was observed with mouse or rat recombinant TfR1. Data are shown as relative fluorescence units normalized to baseline.

[0116] Figure 43 Figure 2 shows ELISA results testing the affinity of anti-TfR Fab 3M12 VH4 / Vk3 to recombinant human TfR1 or TfR2 at concentrations ranging from 230 pM to 500 nM Fab. Data are given as relative fluorescence units normalized to baseline. The results indicate that the Fab does not bind to recombinant human TfR2.

[0117] Figure 44 Shown are the serum stabilities of linkers used to link anti-TfR Fab 3M12 VH4 / Vk3 to control antisense oligonucleotides during 72 hours of incubation in PBS or in rat, mouse, cynomolgus monkey, or human serum.

[0118] Figure 45 The conjugate containing the anti-TfR Fab 3M12VH4 / Vk3 conjugated to a DUX4 targeting oligonucleotide (SEQ ID NO: 147) inhibits the DUX4 transcriptome in C6 (AB1080) immortalized FSHD1 cells, as shown by reduced mRNA expression of MDB3L2, TRIM43, and ZSCAN4. The conjugate exhibits superior activity in inhibiting the DUX4 transcriptome relative to the unconjugated DUX4 targeting oligonucleotide.

[0119] Figures 46A to 46B Dose response curves for gene knockdown are shown. Figure 46AShown is MBD3L2 knockdown in C6 (AB1080) immortalized FSHD1 cells treated with a conjugate containing the anti-TfR Fab 3M12 VH4 / Vk3 conjugated to a DUX4 targeting oligonucleotide (SEQ ID NO: 147). Figure 46B Shown are knockdown of MBD3L2, TRIM43, and ZSCAN4 in FSHD patient myotubes treated with a conjugate containing the anti-TfR Fab 3M12 VH4 / Vk3 conjugated to a DUX4 targeting oligonucleotide (SEQ ID NO: 147). Figure 46B include Figure 46A MBD3L2 data shown in .

[0120] Figures 47A to 47C Shown are the quadriceps muscles ( Figure 47A ), gastrocnemius( Figure 47B ) and tibialis anterior ( Figure 47C ) The anti-TfR1 antibody used was RI7 217 Fab and the oligonucleotides targeted human skeletal actin (ACTA1).

[0121] Figure 48 Shown are HSA levels relative to vehicle-treated mice after a single dose of naked ASO or a comparable dose of anti-TFR1 antibody-ASO conjugate (Ab-ASO). LR Human ACTA1 expression measured by qPCR in DM1 mice. The anti-TfR1 antibody used was RI7 217 Fab and the oligonucleotides targeted human skeletal actin (ACTA1).

[0122] Figures 49A to 49C Shown are HSA levels relative to vehicle-treated mice after a single dose of 10 mg / kg naked ASO, 20 mg / kg naked ASO, or a dose-equivalent anti-TFR antibody-ASO conjugate (Ab-ASO). LR Quadriceps muscle of DM1 mice ( Figure 49A ), gastrocnemius( Figure 49B ) and tibialis anterior ( Figure 49C ) ACTA1 expression in . The anti-TfR1 antibody used was RI7 217 Fab and the oligonucleotide targeted human skeletal actin (ACTA1). (*p<0.05; ***p<0.001).

[0123] Figures 50A to 50CFigure 2 shows the effect of a single dose of saline, an unconjugated oligonucleotide (ASO) that induces exon 23 skipping in DMD, or a conjugate comprising anti-TfR1 RI7217 Fab conjugated to an ASO (Ab-ASO) on the quadriceps muscles of wild-type (WT) and mdx mice 2 or 4 weeks after administration of a single dose of saline, an unconjugated oligonucleotide (ASO) that induces exon 23 skipping in DMD, or an Ab-ASO conjugated to an anti-TfR1 RI7217 Fab Figure 50A ),heart( Figure 50B ) and diaphragm ( Figure 50C Quantification of exon 23 skipping in tissues from WT mice or mdx mice administered saline or unconjugated ASOs. Significant levels of exon 23 skipping were observed in tissues from mdx mice treated with Ab-ASOs (*p<0.05, **p<0.01, ****p<0.0001).

[0124] Figures 51A to 51D Shown are measurements of dystrophin in the quadriceps muscles of mdx mice following administration of a single dose of either an unconjugated oligonucleotide (ASO) or a conjugate comprising anti-TfR1 RI7217 Fab conjugated to an ASO (Ab-ASO) that induces exon 23 skipping in DMD. Figure 51A Shown are western blots of dystrophin and α-actinin in muscle tissue two weeks after injection of ASO or Ab-ASO. Figure 51B The dystrophin expression in wild-type muscle is shown. Figure 51A Quantification of dystrophin in western blots. Figure 51C Shown are western blots of dystrophin and α-actinin in muscle tissue four weeks after injection of ASO or Ab-ASO. Figure 51D The dystrophin expression in wild-type muscle is shown. Figure 51C Quantification of dystrophin in western blots. Figure 51A and 51C The standard curves in were generated by pooling tissues from wild-type (WT) and mdx mouse samples, and the WT percentage represents the amount of WT protein incorporated into each sample. (*p<0.05; ns, not significant)

[0125] Figures 52A to 52D Shown are measurements of dystrophin in the myocardium of mdx mice following administration of a single dose of either an unconjugated oligonucleotide (ASO) or a conjugate comprising anti-TfR1 RI7217 Fab conjugated to an ASO (Ab-ASO) that induces exon 23 skipping in DMD. Figure 52A Shown are western blots of dystrophin and α-actinin in muscle tissue two weeks after injection of ASO or Ab-ASO. Figure 52BShown is the expression of dystrophin relative to wild-type muscle. Figure 52A Quantification of dystrophin in western blots. Figure 52C Shown are western blots of dystrophin and α-actinin in muscle tissue four weeks after injection of ASO or Ab-ASO. Figure 52D Shown is the expression of dystrophin relative to wild-type muscle. Figure 52C Quantification of dystrophin in western blots. Figure 52A and 52C The standard curves in the Figures were generated by pooling tissues from wild-type (WT) and mdx mouse samples, and the WT percentage represents the amount of WT protein spiked into each sample. (*p<0.05, ****p<0.0001)

[0126] Figures 53A to 53D Shown are measurements of dystrophin in the diaphragm of mdx mice following administration of a single dose of either an unconjugated oligonucleotide (ASO) or a conjugate comprising anti-TfR1 RI7217 Fab conjugated to an ASO (Ab-ASO) that induces exon 23 skipping in DMD. Figure 53A Shown are western blots of dystrophin and α-actinin in muscle tissue two weeks after injection of ASO or Ab-ASO. Figure 53B Shown is the expression of dystrophin relative to wild-type muscle. Figure 53A Quantification of dystrophin in western blots. Figure 53C Shown are western blots of dystrophin and α-actinin in muscle tissue four weeks after injection of ASO or Ab-ASO. Figure 53D Shown is the expression of dystrophin relative to wild-type muscle. Figure 53C Quantification of dystrophin in western blots. Figure 53A and 53C The standard curves in the Figures were generated by pooling tissues from wild-type (WT) and mdx mouse samples, and the WT percentage represents the amount of WT protein spiked into each sample. (**p<0.01, ***p<0.001)

[0127] Figures 54A to 54C Figure 2 shows the effect of single doses of saline, unconjugated exon 23 skipping oligonucleotide (ASO), or a conjugate comprising anti-TfR1 RI7217 Fab conjugated to ASO (Ab-ASO) on the quadriceps muscles of wild-type (WT) or mdx mice ( Figure 54A ), diaphragm( Figure 54B ) and heart ( Figure 54C ) Quantification of the amount of oligonucleotide (ASO) administered.

[0128] Figure 55 Shown are non-human primate plasma levels of a DUX4-targeting oligonucleotide (SEQ ID NO: 147) over time following administration of 30 mg / kg unconjugated ('naked') oligonucleotide or 3, 10, or 30 mg / kg oligonucleotide equivalents of a conjugate comprising anti-TfR1 Fab3M12 VH4 / Vk3 covalently linked to a DUX4-targeting oligonucleotide ('Fab-oligonucleotide conjugate').

[0129] Figure 56 Shown are tissue levels of a DUX4 targeting oligonucleotide (SEQ ID NO: 147) measured in non-human primate muscle tissue samples two weeks after administration of 30 mg / kg unconjugated ('naked') oligonucleotide or 3, 10, or 30 mg / kg oligonucleotide equivalents of a conjugate comprising anti-TfR1 Fab 3M12 VH4 / Vk3 covalently linked to a DUX4 targeting oligonucleotide ('Fab-oligonucleotide conjugate').

[0130] Figure 57 Shown are tissue levels of a DUX4-targeting oligonucleotide (SEQ ID NO: 147) measured in non-human primate muscle tissue samples collected by biopsy (5 bars on the left) one week after administration of 30 mg / kg unconjugated oligonucleotide ('Oligo') or 3, 10, or 30 mg / kg oligonucleotide equivalents of a conjugate comprising anti-TfR1 Fab3M12 VH4 / Vk3 covalently linked to the DUX4-targeting oligonucleotide ('Conjugate').

[0131] Figures 58A to 58B Shown are the tibialis anterior muscles ( Figure 58A ) or quadriceps ( Figure 58B ) were measured in a 24-well assay for splicing correction of more than 30 different RNAs known to be mis-spliced ​​in DM1 patients. The anti-TfR1 antibody used was RI7 217 Fab and the oligonucleotides targeted skeletal actin (ACTA1).

[0132] Figure 59 Shown is the % exon 53 skipping in DMD patient cells carrying a DMD exon 52 deletion following gymnotic uptake of a range of concentrations of exon 53 skipping oligonucleotides.

[0133] Figure 60Shown is the % exon 53 skipping in DMD patient cells carrying DMD exon 52 deletions after treatment with various concentrations of exon 53 skipping PMOs that were not linked to an antibody ("naked ASO") or covalently linked to an anti-TfR1 Fab ("anti-TfR1 Fab-ASO complex"). Detailed Description of the Invention

[0135] Some aspects of the present disclosure relate to the recognition that although certain molecular payloads (e.g., oligonucleotides, peptides, small molecules) can have beneficial effects in muscle cells, it has been shown that effectively targeting such cells is challenging. As described herein, the present disclosure provides complexes comprising muscle targeting agents covalently linked to molecular payloads to overcome such challenges. In some embodiments, the complexes are particularly useful for delivering molecular payloads that modulate the expression or activity of target genes in muscle cells, such as in subjects suffering from or suspected of having muscle diseases. For example, in some embodiments, the complexes can be used to treat subjects with rare muscle diseases including Pompe disease, centronuclear myopathy, fibrous dysplasia ossificans progressiva, Friedreich's ataxia, or Duchenne muscular dystrophy. In some embodiments, different molecular payloads can be used in such complexes depending on the condition to be treated. For example, if the underlying mutation causes a splicing defect, an oligonucleotide or other payload can be used to correct the splicing defect (e.g., an oligonucleotide that inhibits exon skipping or promotes alternative splicing). If the underlying mutation results in a gain-of-function allele, oligonucleotides (e.g., RNAi, PMOs, ASO-spacers) can be used to inhibit the expression or activity of the allele. In some embodiments, for example, when the mutation results in a loss-of-function allele, the payload may comprise an expression construct, for example, for expressing the wild-type form of the allele. In some embodiments, the payload may comprise a mechanism for correcting the underlying defect, for example, by gene editing (e.g., a guide nucleic acid, an expression construct encoding a gene editing enzyme).

[0136] Additional aspects of the disclosure, including a description of defined terms, are provided below.

[0137] I. Definition

[0138] Administer: As used herein, the term "administer" means providing a complex to a subject (eg, to treat a condition in a subject) in a physiologically and / or (eg, and) pharmacologically usable manner.

[0139] Approximately: As used herein, the terms "approximately" or "about," as applied to one or more values ​​of interest, refer to a value similar to a stated reference value. In certain embodiments, the terms "approximately" or "about" refer to a range of values ​​that fall within 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of a stated reference value, unless otherwise specified or otherwise apparent from the context (unless such a number exceeds 100% of the possible values).

[0140] Antibody: The term "antibody" as used herein 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, a Fab' fragment, a F(ab')2 fragment, an Fv fragment, or a scFv fragment. In some embodiments, the antibody is a nanobody derived from a camelid antibody or a nanobody derived from a shark antibody. In some embodiments, the antibody is a diabody. 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 (abbreviated herein as VH) and / or (e.g., and) a light (L) chain variable region (abbreviated herein 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 of human IgG heavy and light chain constant domains and their functional variations are known. With respect to the heavy chain, in some embodiments, the heavy chain of the antibodies 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 antibodies described herein may comprise a human alpha (α), delta (Δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain. In a specific embodiment, the antibodies described herein comprise human γ1 CH1, 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 a 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 EA et al., (1991) supra. In some embodiments, the VH domain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or at least 99% identical to 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 by N-glycosylation, O-glycosylation, C-glycosylation, glycosylphosphatidylinositolization (GPI anchor 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-acetylgalactosamine units, galactose units, fucose units, or phospholipid units. In some embodiments, the antibody is a construct comprising a polypeptide comprising one or more antigen-binding fragments of the present disclosure connected to a linker polypeptide or an immunoglobulin constant domain. The linker polypeptide comprises two or more amino acid residues linked by a peptide bond and is used to connect one or more antigen-binding portions. Some examples of linker polypeptides have been reported (see, e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448; Poljak, RJ, et al. (1994) Structure 2: 1121-1123). In addition, an antibody can be part of a larger immunoadhesion molecule formed by covalent or non-covalent association of the antibody or antibody portion with one or more other proteins or peptides. Some examples of such immunoadhesion molecules include the use of a streptavidin core region to prepare tetrameric scFv molecules (Kipriyanov, SM, et al. (1995) Human Antibodies and Hybridomas 6:93-101), and the use of cysteine ​​residues, a labeling peptide, and a C-terminal polyhistidine tag to prepare bivalent and biotinylated scFv molecules (Kipriyanov, SM, et al. (1994) Mol. Immunol. 31:1047-1058).

[0141] CDR: The term "CDR" as used herein refers to the complementarity determining regions within an antibody variable sequence. A typical antibody molecule comprises a heavy chain variable region (VH) and a light chain variable region (VL), which are generally involved in antigen binding. The VH and VL regions can be further subdivided into hypervariable regions, also known as "complementarity determining regions" (CDRs), interspersed with more conserved regions called "framework regions" (FRs). Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The extent of the framework regions and CDRs can be precisely identified using methods known in the art, for example, by the Kabat definition, the IMGT definition, the Chothia definition, the AbM definition, and / or (e.g., and) contact definitions, 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); Lefranc, M.-P. et al. 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. As used herein, CDRs may refer to CDRs defined by any method known in the art. Two antibodies having the same CDRs means that the amino acid sequences of the CDRs of the two antibodies are identical, as determined by the same method (e.g., IMGT definition).

[0142] There are three CDRs in each variable region of the heavy and light chains, referred to as CDR1, CDR2, and CDR3 for each variable region. As used herein, the term "CDR set" refers to the group of three CDRs that appear within a single variable region and are capable of binding an antigen. The exact boundaries of these CDRs have been defined differently according to different systems. 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 an unambiguous residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries that define the three CDRs. These CDRs may be referred to as Kabat CDRs. Subportions of the CDRs may be designated L1, L2, and L3 or H1, H2, and H3, where "L" and "H" designate light and heavy chain regions, respectively. These regions may be referred to as Chothia CDRs, which have boundaries that overlap with the Kabat CDRs. Padlan (FASEB J. 9: 133-139 (1995)) and MacCallum (J Mol Biol 262(5): 732-45 (1996)) have described residue boundaries that overlap with the Kabat CDRs. Other boundaries of the CDR that CDR overlaps.Other CDR boundary definitions may not strictly follow one of the above-mentioned systems, but still overlap with Kabat CDRs, although they can be shortened or extended according to predictions or experimental findings that specific residues or groups of residues or even entire CDRs do not significantly affect antigen binding.The method used herein can utilize CDRs defined according to any one of these systems.Some examples of CDR definition systems are provided in Table 6.

[0143] Table 6. CDR definitions

[0144]

[0145] 1 the international IrmMunoGeneTics information imgt.org, Lefranc, M.-P. et al., Nucleic Acids Res., 27: 209-212 (1999)

[0146] 2Kabat et al. (1991) Sequences of Proteins of Immunological Interest. Fifth Edition, USDepartment of Health and Human Services, NIHPublication No.91-3242

[0147] 3 Chothia et al., J. Mol. Biol. 196: 901-917 (1987))

[0148] CDR-grafted antibody: The term "CDR-grafted antibody" refers to an antibody comprising heavy and light chain variable region sequences from one species but in which the sequences of 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 murine heavy and light chain variable regions in which one or more murine CDRs (e.g., CDR3) have been replaced by human CDR sequences.

[0149] Chimeric antibody: The term "chimeric antibody" refers to an antibody that comprises heavy and light chain variable region sequences from one species and constant region sequences from another species, for example, an antibody having murine heavy and light chain variable regions linked to human constant regions.

[0150] Complementary: the term "complementary" as used herein refers to the ability to accurately pair between two nucleotides or two groups of nucleotides. In particular, complementary is a term that characterizes the degree of combination between two nucleotides or two groups of nucleotides caused by hydrogen bond pairing. For example, if the base at one position of an oligonucleotide can hydrogen bond with the base at the corresponding position of a target nucleic acid (e.g., mRNA), it is considered that the bases at that position are complementary to each other. Base pairing can include both standard Watson-Crick base pairing and non-Watson-Crick base pairing (e.g., Wobble base pairing and Hoogsteen base pairing). For example, in some embodiments, for complementary base pairing, adenosine base (A) is complementary to thymidine base (T) or uracil base (U), cytosine base (C) is complementary to 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 to be complementary. Inosine (I) is also considered in the art to be a universal base and is considered complementary to any of A, C, U, or T.

[0151] Conservative amino acid substitutions: As used herein, "conservative amino acid substitutions" refer to amino acid substitutions that do not change the relative charge or size characteristics of the protein in which the amino acid substitution is made. Variants can be prepared according to methods known to those of ordinary skill in the art for altering polypeptide sequences, such as those found in references compiling such methods: for example, 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, FM Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Conservative amino acid substitutions include substitutions between amino acids within 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.

[0152] Covalently linked: As used herein, the term "covalently linked" refers to the characteristic of two or more molecules being linked together by at least one covalent bond. In some embodiments, two molecules can be covalently linked together by a single bond, such as a disulfide bond or a disulfide bridge, that acts as a linker between the molecules. However, in some embodiments, two or more molecules can be covalently linked together by a molecule that acts as a linker, which links the two or more molecules together through multiple covalent bonds. In some embodiments, the linker can be a cleavable linker. However, in some embodiments, the linker can be a non-cleavable linker.

[0153] Cross-reactivity: As used herein and in the case of targeting agents (e.g., antibodies), the term "cross-reactivity" refers to the property of a substance that can specifically bind to more than one antigen of a similar type or class (e.g., antigens of multiple homologs, paralogs, or orthologs) with similar affinity or avidity. 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) can bind to human antigens and non-human primate antigens with similar affinity or avidity. In some embodiments, antibodies are cross-reactive to similar types or classes of human antigens and rodent antigens. In some embodiments, antibodies are cross-reactive to similar types or classes of rodent antigens and non-human primate antigens. In some embodiments, antibodies are cross-reactive to similar types or classes of human antigens, non-human primate antigens, and rodent antigens.

[0154] Disease allele: The term "disease allele" as used herein refers to any alternative form (e.g., mutant form) of a gene in which the allele is associated with a disease and / or (e.g., and) directly or indirectly contributes to or causes a disease. Relative to the wild-type (non-disease) allele, the disease allele may comprise genetic alterations including, but not limited to, insertions (e.g., disease-associated duplications described below), deletions, missense mutations, nonsense mutations, and splice site mutations. In some embodiments, the disease allele has a loss-of-function mutation. In some embodiments, the disease allele has a gain-of-function mutation. In some embodiments, the disease allele encodes an activating mutation (e.g., encoding a protein with constitutive activity). In some embodiments, the disease allele is a recessive allele with a recessive phenotype. In some embodiments, the disease allele is a dominant allele with a dominant phenotype.

[0155] Disease-associated repeats: The term "disease-associated repeats" as used herein refers to a repetitive nucleotide sequence at a genomic location, wherein the number of units of the repetitive nucleotide sequence is associated with a genetic disease and / or (e.g., and) directly or indirectly contributes to or causes a genetic disease. The length of each repetitive unit of a disease-associated repeat can be 2, 3, 4, 5 or more nucleotides. For example, in some embodiments, the disease-associated repeat is a dinucleotide repeat. In some embodiments, the disease-associated repeat is a trinucleotide repeat. In some embodiments, the disease-associated repeat is a tetranucleotide repeat. In some embodiments, the disease-associated repeat is a pentanucleotide repeat. In some embodiments, some embodiments, the disease-associated repeat comprises CAG repeats, CTG repeats, CUG repeats, CGG repeats, CCTG repeats, or any nucleotide complementary sequence thereof. In some embodiments, the disease-associated repeat is in the non-coding portion of a gene. However, in some embodiments, the disease-associated repeat is in the coding region of a gene. In some embodiments, the disease-associated repeat is amplified from a normal state to a length that directly or indirectly contributes to or causes a genetic disease. In some embodiments, the disease-associated repeat is in RNA (e.g., RNA transcript). In some embodiments, the disease-associated repeats are in DNA (e.g., chromosomes, plasmids). In some embodiments, the disease-associated repeats are amplified in the chromosomes of germ cells. In some embodiments, the disease-associated repeats are amplified in the chromosomes of somatic cells. In some embodiments, the disease-associated repeats are amplified to the number of repeat units associated with the congenital onset of the disease. In some embodiments, the disease-associated repeats are amplified to the number of repeat units associated with the onset of the disease in childhood. In some embodiments, the disease-associated repeats are amplified to the number of repeat units associated with the onset of the disease in adulthood.

[0156] Framework: The term "framework" or "framework sequence" as used herein refers to the remaining sequence of the variable region minus the CDRs. Since the exact definition of the CDR sequence can be determined by different systems, the meaning of the framework sequence has different interpretations 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 the framework on the light chain and heavy chain into four subregions (FR1, FR2, FR3 and FR4) on each chain, wherein CDR1 is located between FR1 and FR2, CDR2 is located between FR2 and FR3, and CDR3 is located between FR3 and FR4. Where a specific subregion is not designated as FR1, FR2, FR3 or FR4, the framework region referred to by others represents the combined FR within the variable region of a single naturally occurring immunoglobulin chain. As used herein, FR represents one of the four subregions, and FRs represents two or more of the four subregions that make up the framework region. Human heavy and light chain linker sequences are known in the art. In one embodiment, acceptor sequences known in the art can be used in the antibodies disclosed herein.

[0157] Human antibody: The term "human antibody" as used herein is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the present disclosure may contain amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example, in the CDRs, particularly in CDR3. However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species (e.g., a mouse) have been grafted onto human framework sequences.

[0158] Humanized antibody: The term "humanized antibody" refers to an antibody comprising heavy and light chain variable region sequences from a non-human species (e.g., mouse) but wherein at least a portion of the VH and / or (e.g., and) VL sequences have been altered to be more "human-like" (i.e., more similar to human germline variable sequences). One type of humanized antibody is a CDR-grafted antibody in which human CDR sequences are introduced into non-human VH and VL sequences to replace the corresponding non-human CDR sequences. In one embodiment, humanized anti-transferrin receptor antibodies and antigen-binding portions are provided. Such antibodies can be produced by obtaining mouse anti-transferrin receptor monoclonal antibodies using traditional hybridoma technology and then humanizing them using in vitro genetic engineering, such as those disclosed in PCT Publication No. WO 2005 / 123126 A2 by Kasaian et al.

[0159] Internalized cell surface receptor: The term "internalized cell surface receptor" as used herein refers to a cell surface receptor that is internalized by a cell, for example, under external stimulation (e.g., ligand binding to a receptor). In some embodiments, the internalized cell surface receptor is internalized by endocytosis. In some embodiments, the internalized cell surface receptor is internalized by clathrin-mediated endocytosis. However, in some embodiments, the internalized cell surface receptor is internalized by a clathrin-independent pathway, 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 further comprise a ligand binding domain. In some embodiments, the cell surface receptor is internalized by the cell after 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.

[0160] Isolated antibody: As used herein, "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds to transferrin receptor is substantially free of antibodies that specifically bind to antigens other than transferrin receptor). However, an isolated antibody that specifically binds to the transferrin receptor complex may have cross-reactivity with other antigens (e.g., transferrin receptor molecules from other species). Furthermore, an isolated antibody may be substantially free of other cellular material and / or (e.g., and) chemical substances.

[0161] Kabat numbering: The terms "Kabat numbering," "Kabat definition," and "Kabat notation" are used interchangeably herein. These terms, recognized in the art, refer to a system for numbering amino acid residues that are more variable (i.e., hypervariable) than other amino acid residues in the variable regions of the heavy and light chains of an antibody, or antigen-binding portion thereof (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, US Department of Health and Human Services, NIH Publication No. 5501068. No. 91-3242). For the heavy chain variable region, the hypervariable region of CDR1 is from amino acids 31 to 35, that of CDR2 is from amino acids 50 to 65, and that of CDR3 is from amino acids 95 to 102. For the light chain variable region, the hypervariable region of CDR1 is from amino acids 24 to 34, that of CDR2 is from amino acids 50 to 56, and that of CDR3 is from amino acids 89 to 97.

[0162] Molecular payload: As used herein, the term "molecular payload" refers to a molecule or substance that acts to modulate a biological outcome. In some embodiments, the molecular payload is linked to or otherwise associated with a muscle targeting agent. In some embodiments, the molecular payload is a small molecule, protein, peptide, nucleic acid, or oligonucleotide. In some embodiments, the molecular payload acts to modulate the transcription of a DNA sequence, modulate the expression of a protein, or modulate the activity of a protein. In some embodiments, the molecular payload is an oligonucleotide comprising a strand having a complementary region to a target gene.

[0163] Muscle disease gene: As used herein, the term "muscle disease gene" refers to a gene having at least one disease allele that is associated with a muscle disease and / or (e.g., and) directly or indirectly contributes to or causes a muscle disease. In some embodiments, the muscle disease is a rare disease, such as defined by the Genetic and Rare Diseases Information Center (GARD), which is a program of the National Center for Advancing Translational Sciences (NCATS). In some embodiments, the muscle disease is a rare disease characterized by affecting fewer than 200,000 people. In some embodiments, the muscle disease is a monogenic disease. In some embodiments, the muscle disease gene is a gene listed in Table 1.

[0164] Muscle targeting agent: As used herein, the term "muscle targeting agent" refers to a molecule that specifically binds to an antigen expressed on a muscle cell. The antigen within or on the muscle cell can be a membrane protein, such as an integral membrane protein or a peripheral membrane protein. Generally speaking, the muscle targeting agent specifically binds to the antigen on the muscle cell, which helps internalize the muscle targeting agent (and any associated molecular cargo) into the muscle cell. In some embodiments, the muscle targeting agent specifically binds to an internalizing cell surface receptor on the muscle and is capable of internalization into the muscle cell via receptor-mediated internalization. In some embodiments, the muscle targeting agent is a small molecule, a protein, a peptide, a nucleic acid (e.g., an aptamer), or an antibody. In some embodiments, the muscle targeting agent is linked to a molecular cargo.

[0165] Muscle-targeting antibody: As used herein, the term "muscle-targeting antibody" refers to a muscle-targeting agent that is an antibody that specifically binds to an antigen present in or on a muscle cell. In some embodiments, the muscle-targeting antibody specifically binds to an antigen on a muscle cell, which facilitates internalization of the muscle-targeting antibody (and any associated molecular cargo) into the muscle cell. In some embodiments, the muscle-targeting antibody specifically binds to an internalizing cell surface receptor present on a muscle cell. In some embodiments, the muscle-targeting antibody is an antibody that specifically binds to the transferrin receptor.

[0166] Oligonucleotide: The term "oligonucleotide" as used herein refers to an oligomeric nucleic acid compound up to 200 nucleotides in length. Some examples of oligonucleotides include, but are not limited to, RNAi oligonucleotides (e.g., siRNA, shRNA), microRNA, spacer polymers, mixed polymers, phosphorodiamidate morpholinos, peptide nucleic acids, aptamers, guide nucleic acids (e.g., Cas9 guide RNA), etc. Oligonucleotides can be single-stranded or double-stranded. In some embodiments, the oligonucleotide may comprise one or more modified nucleotides (e.g., 2'-O-methyl sugar modifications, purine or pyrimidine modifications). In some embodiments, the oligonucleotide may comprise one or more modified internucleotide linkages. In some embodiments, the oligonucleotide may comprise one or more phosphorothioate linkages, which may be Rp or Sp stereochemical conformations.

[0167] Recombinant antibody: As used herein, the term "recombinant human antibody" is intended to include all human antibodies prepared, expressed, generated or isolated by recombinant means, e.g., antibodies expressed using recombinant expression vectors 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), antibodies isolated from animals (e.g., mice) transgenic for human immunoglobulin genes (see, e.g., 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, produced, or isolated by any other means involving splicing human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or in vivo somatic mutagenesis when an animal transgenic for human Ig sequences is used), and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, although derived from and related to human germline VH and VL sequences, may not naturally occur in the human antibody germline repertoire in vivo. One embodiment of the present disclosure provides fully human antibodies capable of binding to human transferrin receptor, which can be generated using techniques well known in the art, such as, but not limited to, using human Ig phage libraries, such as those disclosed in PCT Publication No. WO 2005 / 007699 A2 to Jermutus et al.

[0168] Complementary region: As used herein, the term "complementary region" refers to a nucleotide sequence, such as an oligonucleotide, that is sufficiently complementary to a homologous nucleotide sequence, such as 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 fully 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 comprises 1, 2, 3, or 4 mismatches compared to the homologous nucleotide sequence of the target nucleic acid.

[0169] 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 avidity that enables the molecule to be used to distinguish the binding partner from a suitable control in a binding assay or other binding context. With respect to antibodies, the term "specific binding" refers to the ability of an antibody to bind to a specific antigen with a degree of affinity or avidity that enables the antibody to be used to distinguish the specific antigen from other antigens, e.g., to a degree that allows for preferential targeting of certain cells (e.g., muscle cells) by binding to an antigen as described herein. In some embodiments, an antibody may bind to a target with a KD of at least about 10 -4 M, 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 - 12 M, 10 -13 M or less, the antibody specifically binds to the target. In some embodiments, the antibody specifically binds to the transferrin receptor (e.g., an epitope of the apical domain of the transferrin receptor).

[0170] Subject: As used herein, the term "subject" refers to a mammal. In some embodiments, the subject is a non-human primate or rodent. In some embodiments, the subject is a human. In some embodiments, the subject is a patient, such as a human patient having or suspected of having a disease. In some embodiments, the subject is a human patient having or suspected of having a muscle disease (e.g., any of the diseases provided in Table 1).

[0171] Transferrin receptor: The term "transferrin receptor" as used herein (also referred to as TFRC, CD71, p90, TFR, or TFR1) refers to a cell surface receptor that binds transferrin to promote the internalization of iron uptake by endocytosis. In some embodiments, the transferrin receptor can 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). In addition, a variety of human transcript variants encoding different isoforms of the receptor have been characterized (e.g., as annotated with the following GenBank RefSeq accession numbers: NP_001121620.1, NP_003225.2, NP_001300894.1, and NP_001300895.1).

[0172] 2'-modified nucleosides: As used herein, the terms "2'-modified nucleosides" and "2'-modified ribonucleosides" are used interchangeably 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., through a methylene, ethylene, or (S)-constrained ethyl bridge). In some embodiments, the 2'-modified nucleoside is a non-bicyclic 2'-modified nucleoside, e.g., 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'-O-N-methylacetamido (2'-O-NMA), locked nucleic acid (LNA, methylene bridged nucleic acid), ethylene bridged nucleic acid (ENA), and (S)-constrained 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 having increased affinity for a target sequence relative to an unmodified oligonucleotide. Some examples of the structures of 2'-modified nucleosides are provided below:

[0173]

[0174] II. Complex

[0175] Provided herein are complexes comprising targeting agents (e.g., antibodies) 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 regulate 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 genes, proteins, and / or (e.g., and) nucleic acids. The molecular payload can be a small molecule, protein, nucleic acid, oligonucleotide, or any molecular entity that is capable of regulating the activity or function of genes, proteins, and / or (e.g., and) nucleic acids in a cell. In some embodiments, the molecular payload is an oligonucleotide that targets a muscle disease allele in a muscle cell.

[0176] In some embodiments, the complex comprises a muscle targeting agent, such as an anti-transferrin receptor antibody, covalently linked to a molecular cargo, such as an antisense oligonucleotide that targets a muscle disease allele.

[0177] In some embodiments, the complex can be used to treat muscle diseases, wherein the molecular payload affects the activity of the corresponding gene provided in Table 1. For example, depending on the condition, the molecular payload can modulate (e.g., reduce, increase) the transcription or expression of the gene, modulate the expression of the protein encoded by the gene, or modulate the activity of the encoded protein. In some embodiments, the molecular payload is an oligonucleotide comprising a strand having a complementary region to the target gene provided in Table 1.

[0178] Table 1 - List of muscle diseases and corresponding genes.

[0179]

[0180]

[0181]

[0182] A. Muscle Targeting Agents

[0183] Some aspects of the present disclosure provide muscle targeting agents, for example, for delivering molecular payloads to muscle cells. In some embodiments, such muscle targeting agents can bind to muscle cells, for example, by specifically binding to an antigen on the muscle cell, and deliver the associated molecular payload to the muscle cell. In some embodiments, the molecular payload is bound to the muscle targeting agent (e.g., covalently bound), and after the muscle targeting agent binds to the antigen on the muscle cell, it is internalized into the muscle cell, for example, by endocytosis. It should be understood that various types of muscle targeting agents can be used in accordance with the present disclosure. For example, a muscle targeting agent can comprise a nucleic acid (e.g., DNA or RNA), a peptide (e.g., an antibody), a lipid (e.g., a microvesicle), or a sugar moiety (e.g., a polysaccharide), or consist of the same. 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 meant to be limiting.

[0184] Some aspects of the present 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.

[0185] 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 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 allows even macromolecules (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 can then be endocytosed, for example, by clathrin-mediated endocytosis.

[0186] 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 in the subject. In some embodiments, the muscle targeting agent concentrates the bound molecular payload in muscle cells (e.g., skeletal muscle, smooth muscle, or cardiac muscle cells) in an amount that is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times greater than the amount in non-muscle cells (e.g., liver, neurons, blood, or adipocytes). In some embodiments, the toxicity of a molecular payload in a 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% when delivered to the subject in conjunction with a muscle targeting agent.

[0187] In some embodiments, a muscle recognition element (e.g., a myocyte antigen) may be required to achieve muscle selectivity. As an example, a muscle targeting agent can be a small molecule that is a substrate for a muscle-specific uptake transporter. As another example, a muscle targeting agent can be an antibody that enters muscle cells via transporter-mediated endocytosis. As another example, a muscle targeting agent can be a ligand that binds to a cell surface receptor on a muscle cell. It should be understood that while transporter-based approaches provide a direct route for cell entry, receptor-based targeting may involve stimulated endocytosis to reach the desired site of action.

[0188] Myocytes encompassed by the present disclosure include, but are not limited to, skeletal muscle cells, smooth muscle cells, cardiomyocytes, myoblasts, and myocytes.

[0189] i. Muscle-targeted antibodies

[0190] In some embodiments, the muscle targeting agent is an antibody. Generally speaking, the high specificity of antibodies to their target antigens provides the potential for selective targeting of muscle cells (e.g., skeletal muscle, smooth muscle and / or (e.g., and) cardiac muscle cells). This specificity can also limit off-target toxicity. Some examples of antibodies capable of targeting myocyte surface antigens have been reported and are within the scope of the present disclosure. For example, antibodies targeting the surface of muscle cells are described in: Arahata K., et al. "Immunostaining of skeletal and cardiac muscle surface membrane with antibody against Duchenne muscular dystrophy peptide" Nature 1988; 333: 861-3; Song KS, et al. "Expression of caveolin-3 in skeletal, cardiac, and smooth muscle cells. Caveolin-3 is a component of the sarcolemma and co-fractionates with dystrophin and dystrophin-associated glycoproteins" J Biol Chem 1996; 271: 15160-5; and Weisbart RH et al., "Cell type specific targeted intracellular delivery into muscle of a monoclonal antibody that binds myosin IIb" Mol Immunol. 2003 Mar, 39(13):78309; the entire contents of each of which are incorporated herein by reference.

[0191] a. Anti-transferrin receptor antibodies

[0192] Some aspects of the present disclosure are based on such understanding: the substance (for example, anti-transferrin receptor antibody) that is bound to the transferrin receptor can target myocytes. The transferrin receptor is an internalized cell surface receptor that transfers transferrin across the cell membrane and participates in the regulation and homeostasis of intracellular iron levels. Some aspects of the present disclosure provide transferrin receptor-bound proteins that can be bound to the transferrin receptor. Therefore, some aspects of the present disclosure provide binding proteins (for example, antibodies) that are bound to the transferrin receptor. In some embodiments, the binding proteins bound to the transferrin receptor are internalized into myocytes together with any combined molecular payload. The antibodies bound to the transferrin receptor used herein can be interchangeably referred to as transferrin receptor antibodies, anti-transferrin receptor antibodies or anti-TfR antibodies. The antibodies bound to the transferrin receptor (for example, specifically bound) can be internalized into cells after being bound to the transferrin receptor, for example, by receptor-mediated endocytosis.

[0193] It should be understood that several known methods (e.g., library design using phage display) can be used to produce, synthesize, and / or (e.g., and) derive anti-transferrin receptor antibodies. Exemplary methods have been characterized in the art and incorporated by reference (Díez, P. et al. "High-throughput phage-display screening in array format", Enzyme and microbial technology, 2015, 79, 34-41.; Christoph MH and Stanley, JR "Antibody Phage Display: Technique and Applications" J Invest Dermatol. 2014, 134: 2.; Engleman, Edgar (Ed.) "Human Hybridomas and Monoclonal Antibodies." 1985, Springer). In other embodiments, anti-transferrin antibodies have been previously characterized or disclosed.Antibodies that specifically bind to transferrin receptor are known in the art (see, e.g., U.S. Patent No. 4,364,934, filed December 4, 1979, “Monoclonal antibody to a human early thymocyte antigen 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).

[0194] In some aspects, provided herein are new anti-TfR antibodies for use as muscle targeting agents (e.g., in muscle targeting complexes). In some embodiments, the anti-TfR antibodies described herein bind to the transferrin receptor with high specificity and affinity. In some embodiments, the anti-TfR antibodies described herein specifically bind to any extracellular epitope of the transferrin receptor or an epitope exposed to the antibody. In some embodiments, the anti-TfR antibodies provided herein specifically bind to transferrin receptors 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 an amino acid segment of a human or non-human primate transferrin receptor (such as provided in SEQ ID NOs: 105 to 108). In some embodiments, the anti-TfR antibodies described herein bind to an amino acid segment corresponding to amino acids 90 to 96 of the human transferrin receptor (as shown in SEQ ID NO: 105), which is not in the apical domain of the transferrin receptor.

[0195] An exemplary human transferrin receptor amino acid sequence corresponding to NCBI sequence NP_003225.2 (transferrin receptor protein 1 isoform 1, homosapiens) is as follows:

[0196]

[0197] An exemplary non-human primate transferrin receptor amino acid sequence corresponding to NCBI sequence NP_001244232.1 (Transferrin receptor protein 1, Rhesus monkey (Macaca mulatta)) is as follows:

[0198]

[0199] An exemplary non-human primate transferrin receptor amino acid sequence corresponding to NCBI sequence XP_005545315.1 (transferrin receptor protein 1, Macaca fascicularis) is as follows:

[0200]

[0201] An exemplary mouse transferrin receptor amino acid sequence corresponding to NCBI sequence NP_001344227.1 (transferrin receptor protein 1, mus musculus) is as follows:

[0202]

[0203] In some embodiments, the anti-transferrin receptor antibody binds to the following receptor amino acid segment:

[0204]

[0205] TCRMVTSESKNVKLTVSNVLKE (SEQ ID NO: 109) and does not inhibit the binding interaction between transferrin receptor and transferrin and / or (e.g., and) human hemochromatosis protein (also known as HFE). In some embodiments, the anti-transferrin receptor antibodies described herein do not bind to the epitope in SEQ ID NO: 109.

[0206] Appropriate methods can be used to obtain and / or (e.g., and) produce antibodies, antibody fragments, or antigen-binding agents, for example, by using recombinant DNA protocols. In some embodiments, antibodies can also be produced by the production of hybridomas (see, e.g., Kohler, G and Milstein, C. "Continuous cultures of fused cells secreting antibody of predefined specificity" Nature, 1975, 256: 495-497). The antigen of interest can be used as an immunogen in any form or entity (e.g., recombinant or naturally occurring form or entity). Hybridomas are screened using standard methods (e.g., ELISA screening) to find at least one hybridoma that produces an antibody targeting a specific antigen. Antibodies can also be produced by screening protein expression libraries (e.g., phage display libraries) that express antibodies. In some embodiments, phage display library design can also be used (see, e.g., U.S. Patent No. 5,223,409, “Directed evolution of novel binding proteins,” filed March 1, 1991; WO 1992 / 18619, “Heterodimeric receptor libraries using phagemids,” filed April 10, 1992; WO 1991 / 17271, “Recombinant library screening methods,” filed May 1, 1991; WO 1992 / 20791, “Methods for producing members of specific binding pairs,” filed May 15, 1992; WO 1992 / 15679, “Improved epitope displaying phage,” filed February 28, 1992). In some embodiments, the antigen of interest can be used to immunize non-human animals, such as rodents or goats. In some embodiments, 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, e.g., Harlow et al. "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory, 1988.).

[0207] 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 sugars or carbohydrate molecules. In some embodiments, one or more sugars or carbohydrate molecules are conjugated to the antibody by N-glycosylation, O-glycosylation, C-glycosylation, glycosylphosphatidylinositolization (GPI anchor attachment) and / or (e.g., and) phosphoglycosylation. In some embodiments, one or more sugars or carbohydrate molecules are monosaccharides, disaccharides, oligosaccharides or polysaccharides. In some embodiments, one or more sugars or carbohydrate molecules are branched oligosaccharides or branched polysaccharides. In some embodiments, one or more sugars or carbohydrate molecules comprise mannose units, glucose units, N-acetylglucosamine units, N-acetylgalactosamine units, galactose units, fucose units or phospholipid units. In some embodiments, there are 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. In some embodiments, the glycosylated antibody is fully or partially glycosylated. In some embodiments, the antibody is glycosylated by a chemical reaction or by enzymatic means. In some embodiments, the antibody is glycosylated in vitro or in a cell, which may optionally lack 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 No. WO2014065661, entitled “Modified antibody, antibody-conjugate and process for the preparation thereof,” published on May 1, 2014.

[0208] In some embodiments, an anti-TfR antibody of the present disclosure comprises a VL domain and / or (e.g., and) a VH domain selected from any one of the anti-TfR antibodies of Table 2, and comprises a constant region comprising the amino acid sequence of a constant region of an IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or any subclass (e.g., IgG2a and IgG2b) of immunoglobulin molecules. Some non-limiting examples of human constant regions are described in the art, e.g., see Kabat EA et al., (1991), supra.

[0209] In some embodiments, the substance bound to transferrin receptor, such as anti-TfR antibodies, can target myocytes and / or (for example, and) mediate the transport of substances across the blood-brain barrier. Transferrin receptor is a cell surface receptor that transports transferrin across the cell membrane and participates in the regulation of intracellular iron levels and the internalization of homeostasis. Some aspects of the present disclosure provide transferrin receptor-binding proteins that can be bound to transferrin receptor. Antibodies bound to transferrin receptor (for example, specifically bound) can be internalized into cells after being bound to transferrin receptor, for example, by receptor-mediated endocytosis and be internalized into cells.

[0210] In some aspects, provided herein are humanized antibodies that bind to transferrin receptor with high specificity and affinity. In some embodiments, the humanized anti-TfR antibodies described herein specifically bind to any extracellular epitope of transferrin receptor or an epitope exposed to the antibody. In some embodiments, the humanized anti-TfR antibodies provided herein specifically bind to transferrin receptors 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 an amino acid segment of a human or non-human primate transferrin receptor as provided in SEQ ID NOs: 105 to 108. In some embodiments, the humanized anti-TfR antibodies described herein bind to an amino acid segment corresponding to amino acids 90 to 96 of the human transferrin receptor as shown in SEQ ID NO: 105, which is not in the apical domain of the transferrin receptor. In some embodiments, the humanized anti-TfR antibodies described herein bind to TfR1 but not TfR2.

[0211] In some embodiments, the anti-TFR antibody is expressed in an amount of at least about 10 -4 M, 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, 10 -13In some embodiments, the anti-TfR antibodies described herein bind to TfR1 (e.g., human or non-human primate TfR1) with a binding affinity of 10 M or less (e.g., as shown by Kd). In some embodiments, the anti-TfR antibodies described herein bind to TfR1 with a KD in the sub-nanomolar range. In some embodiments, the anti-TfR antibodies described herein selectively bind to transferrin receptor 1 (TfR1) but not to transferrin receptor 2 (TfR2). In some embodiments, the anti-TfR antibodies described herein bind to human TfR1 and cynomolgus monkey TfR1 (e.g., with a Kd of 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, 10 -13 M or less), but does not bind to mouse TfR1. The affinity and binding kinetics of the anti-TfR antibodies can be tested using any suitable method, including but not limited to biosensor technology (e.g., OCTET or BIACORE). In some embodiments, the binding of any 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.

[0212] The anti-TfR antibodies described herein are humanized antibodies. Table 2 provides the CDR and variable region amino acid sequences of the mouse monoclonal anti-TfR antibodies from which the humanized anti-TfR antibodies described herein are derived.

[0213] Table 2. Mouse monoclonal anti-TfR antibodies

[0214]

[0215]

[0216]

[0217] *The mutation positions are according to the Kabat numbering of the corresponding VH sequence containing the mutation

[0218] In some embodiments, an anti-TfR antibody of the present disclosure is a humanized variant of any one of the anti-TfR antibodies provided in Table 2. In some embodiments, an anti-TfR antibody of the present disclosure comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 that are identical to CDR-H1, CDR-H2, and CDR-H3 in any one 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.

[0219] Humanized antibody is human immunoglobulin (acceptor antibody), wherein the residue from the complementary determining region (complementary determining region, CDR) of acceptor is replaced by the residue of the CDR of non-human species (donor antibody) such as mouse, rat or rabbit with desired specificity, affinity and capacity (capacity).In some embodiments, the Fv framework region (framework region, FR) residue of human immunoglobulin is replaced by corresponding non-human residue.In addition, humanized antibody can be included in acceptor antibody or the CDR or framework sequence of importing and all do not find but be included in to further improve and optimize the residue of antibody performance.In general, humanized antibody will comprise at least one and typically two variable domains substantially all, wherein all or substantially all of CDR districts correspond to those of non-human immunoglobulins, and all or substantially all of FR districts are those of human immunoglobulin consensus sequences.Humanized antibody will also comprise at least a portion of immunoglobulin constant region or domain (Fc) (typically those of human immunoglobulins) optimally. 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, six) that are altered relative to the original antibody, also referred to as one or more CDRs derived from one or more CDRs from the original antibody. Humanized antibodies may also involve affinity maturation.

[0220] Humanized antibodies and methods for their preparation are known, for example, as described in: 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); Osboum et al., Methods 36:61-68 (2005); and Klimka et al., Br. J. Cancer, 83:252-260 (2000), the entire contents of which are incorporated herein by reference. Human framework regions that can be used for humanization are described, for example, in Sims et al. J. Immunol. 151: 2296 (1993); Carter et al. Proc. Natl. Acad. Sci. USA, 89: 4285 (1992); Presta et al. J. Immunol., 151: 2623 (1993); Almagro et al., Front. Biosci. 13: 1619-1633 (2008); Baca et al., J. Biol. Chem. 272: 10678-10684 (1997); and Rosok et al., J Biol. Chem. 271: 22611-22618 (1996), the entire contents of which are incorporated herein by reference.

[0221] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises the following: a humanized VH comprising one or more amino acid variations compared to any one of the VHs listed in Table 2 (e.g., in the VH framework region), and / or (e.g., and) a humanized VL comprising one or more amino acid variations compared to any one of the VLs listed in Table 2 (e.g., in the VL framework region).

[0222] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH 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 the VH of any anti-TfR antibody listed in Table 2 (e.g., any one of SEQ ID NOs: 17, 22, 26, 43, 61, 65, and 68). Alternatively or in addition (e.g., supplementally), a humanized anti-TfR antibody of the present disclosure comprises a humanized VL 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 the VL of any anti-TfR antibody listed in Table 2 (e.g., any one of SEQ ID NOs: 18, 44, and 62).

[0223] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising an amino acid sequence in the framework regions that is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to a VH of any of the anti-TfR antibodies listed in Table 2 (e.g., any one of SEQ ID NOs: 17, 22, 26, 43, 61, 65, and 68). Alternatively or additionally (e.g., supplementally), in some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VL comprising an amino acid sequence in the framework regions that is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to a VL of any of the anti-TfR antibodies listed in Table 2 (e.g., any one of SEQ ID NOs: 18, 44, and 62).

[0224] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising a CDR-H1 having an amino acid sequence of SEQ ID NO: 1 (according to the IMGT definition system), a CDR-H2 having an amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 19, or SEQ ID NO: 23 (according to the IMGT definition system), a CDR-H3 having an amino acid sequence of SEQ ID NO: 3 (according to the IMGT definition system), and comprising no more than 25 amino acid variations in the framework region (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: 17, SEQ ID NO: 22, or SEQ ID NO: 26. As an alternative or supplement (e.g., supplement), the anti-TfR antibody of the present disclosure comprises a humanized VL, wherein the humanized VL comprises a CDR-L1 having an amino acid sequence of SEQ ID NO: 4 (according to the IMGT definition system), a CDR-L2 having an amino acid sequence of SEQ ID NO: 5 (according to the IMGT definition system), and a CDR-L3 having an amino acid sequence of SEQ ID NO: 6 (according to the IMGT definition system), and comprises no more than 25 amino acid variations in the framework region (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 shown in SEQ ID NO: 18.

[0225] In some embodiments, the humanized anti-TfR antibody of the present disclosure comprises a humanized VH, which comprises a CDR-H1 having an amino acid sequence of SEQ ID NO: 1 (according to the IMGT definition system), a CDR-H2 having an amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 19 or SEQ ID NO: 23 (according to the IMGT definition system), a CDR-H3 having an amino acid sequence of SEQ ID NO: 3 (according to the IMGT definition system), and has at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98% or 99%) identity in the framework region with the VH shown in SEQ ID NO: 17, SEQ ID NO: 22 or SEQ ID NO: 26. Alternatively or in addition (e.g., supplementally), the humanized anti-TfR antibody of the present disclosure comprises a humanized VL comprising a CDR-L1 having an amino acid sequence of SEQ ID NO: 4 (according to the IMGT definition system), a CDR-L2 having an amino acid sequence of SEQ ID NO: 5 (according to the IMGT definition system), and a CDR-L3 having an amino acid sequence of SEQ ID NO: 6 (according to the IMGT definition system), and having at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98% or 99%) identity in the framework region with the VL shown in any one of SEQ ID NO: 18.

[0226] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising a CDR-H1 having an amino acid sequence of SEQ ID NO: 7 (according to the Kabat definition system), a CDR-H2 having an amino acid sequence of SEQ ID NO: 8, SEQ ID NO: 20, or SEQ ID NO: 24 (according to the Kabat definition system), a CDR-H3 having an amino acid sequence of SEQ ID NO: 9 (according to the Kabat definition system), and comprising no more than 25 amino acid variations in the framework region (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: 17, SEQ ID NO: 22, or SEQ ID NO: 26. As an alternative or supplement (e.g., supplement), the humanized anti-TfR antibody of the present disclosure comprises a humanized VL, wherein the humanized VL comprises a CDR-L1 having an amino acid sequence of SEQ ID NO: 10 (according to the Kabat definition system), a CDR-L2 having an amino acid sequence of SEQ ID NO: 11 (according to the Kabat definition system), and a CDR-L3 having an amino acid sequence of SEQ ID NO: 6 (according to the Kabat definition system), and comprises no more than 25 amino acid variations in the framework region (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 shown in SEQ ID NO: 18.

[0227] In some embodiments, the humanized anti-TfR antibody of the present disclosure comprises a humanized VH, which comprises a CDR-H1 having an amino acid sequence of SEQ ID NO: 7 (according to the Kabat definition system), a CDR-H2 having an amino acid sequence of SEQ ID NO: 8, SEQ ID NO: 20 or SEQ ID NO: 24 (according to the Kabat definition system), a CDR-H3 having an amino acid sequence of SEQ ID NO: 9 (according to the Kabat definition system), and has at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98% or 99%) identity in the framework region with the VH shown in SEQ ID NO: 17, SEQ ID NO: 22 or SEQ ID NO: 26. As an alternative or supplement (e.g., supplement), the humanized anti-TfR antibody of the present disclosure comprises a humanized VL, wherein the humanized VL comprises a CDR-L1 having an amino acid sequence of SEQ ID NO: 10 (according to the Kabat definition system), a CDR-L2 having an amino acid sequence of SEQ ID NO: 11 (according to the Kabat definition system) and a CDR-L3 having an amino acid sequence of SEQ ID NO: 6 (according to the Kabat definition system), and has at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98% or 99%) identity in the framework region with the VL shown in any one of SEQ ID NO: 18.

[0228] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising a CDR-H1 having an amino acid sequence of SEQ ID NO: 12 (according to the Chothia definition system), a CDR-H2 having an amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 21 or SEQ ID NO: 25 (according to the Chothia definition system), a CDR-H3 having an amino acid sequence of SEQ ID NO: 14 (according to the Chothia definition system), and comprising no more than 25 amino acid variations in the framework region (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: 17, SEQ ID NO: 22 or SEQ ID NO: 26. As an alternative or supplement (e.g., supplement), the humanized anti-TfR antibody of the present disclosure comprises a humanized VL, wherein the humanized VL comprises a CDR-L1 having an amino acid sequence of SEQ ID NO: 15 (according to the Chothia definition system), a CDR-L2 having an amino acid sequence of SEQ ID NO: 5 (according to the Chothia definition system), and a CDR-L3 having an amino acid sequence of SEQ ID NO: 16 (according to the Chothia definition system), and comprises no more than 25 amino acid variations in the framework 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).

[0229] In some embodiments, the humanized anti-TfR antibody of the present disclosure comprises a humanized VH, which comprises a CDR-H1 having an amino acid sequence of SEQ ID NO: 12 (according to the Chothia definition system), a CDR-H2 having an amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 21 or SEQ ID NO: 25 (according to the Chothia definition system), a CDR-H3 having an amino acid sequence of SEQ ID NO: 14 (according to the Chothia definition system), and has at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98% or 99%) identity in the framework region with the VH shown in SEQ ID NO: 17, SEQ ID NO: 22 or SEQ ID NO: 26. Alternatively or in addition (e.g., supplementally), the anti-TfR antibody of the present disclosure comprises a humanized VL, wherein the humanized VL comprises a CDR-L1 having an amino acid sequence of SEQ ID NO: 15 (according to the Chothia definition system), a CDR-L2 having an amino acid sequence of SEQ ID NO: 5 (according to the Chothia definition system), and a CDR-L3 having an amino acid sequence of SEQ ID NO: 16 (according to the Chothia definition system), and has at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98% or 99%) identity in the framework region with the VL shown in any one of SEQ ID NO: 18.

[0230] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising a CDR-H1 having an amino acid sequence of SEQ ID NO: 27 (according to the IMGT definition system), a CDR-H2 having an amino acid sequence of SEQ ID NO: 28 (according to the IMGT definition system), a CDR-H3 having an amino acid sequence of SEQ ID NO: 29 (according to the IMGT definition system), and comprising no more than 25 amino acid variations in the framework region (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: 43. As an alternative or supplement (e.g., supplement), the humanized anti-TfR antibody of the present disclosure comprises a humanized VL, wherein the humanized VL comprises a CDR-L1 having an amino acid sequence of SEQ ID NO: 30 (according to the IMGT definition system), a CDR-L2 having an amino acid sequence of SEQ ID NO: 31 (according to the IMGT definition system), and a CDR-L3 having an amino acid sequence of SEQ ID NO: 32 (according to the IMGT definition system), and comprises no more than 25 amino acid variations in the framework region (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 shown in SEQ ID NO: 44.

[0231] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising a CDR-H1 having an amino acid sequence of SEQ ID NO: 27 (according to the IMGT definition system), a CDR-H2 having an amino acid sequence of SEQ ID NO: 28 (according to the IMGT definition system), a CDR-H3 having an amino acid sequence of SEQ ID NO: 29 (according to the IMGT definition system), and having at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98% or 99%) identity in the framework region with the VH shown in SEQ ID NO: 43. Alternatively or additionally (e.g., supplementally), the humanized anti-TfR antibody of the present disclosure comprises a humanized VL comprising a CDR-L1 having an amino acid sequence of SEQ ID NO: 30 (according to the IMGT definition system), a CDR-L2 having an amino acid sequence of SEQ ID NO: 31 (according to the IMGT definition system), and a CDR-L3 having an amino acid sequence of SEQ ID NO: 32 (according to the IMGT definition system), and having at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98% or 99%) identity in the framework regions with the VL shown in SEQ ID NO: 44.

[0232] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising a CDR-H1 having an amino acid sequence of SEQ ID NO: 33 (according to the Kabat definition system), a CDR-H2 having an amino acid sequence of SEQ ID NO: 34 (according to the Kabat definition system), a CDR-H3 having an amino acid sequence of SEQ ID NO: 35 (according to the Kabat definition system), and comprising no more than 25 amino acid variations in the framework region (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid variations) compared to the VH shown in SEQ ID NO: 43. As an alternative or supplement (e.g., supplement), the humanized anti-TfR antibody of the present disclosure comprises a humanized VL, wherein the humanized VL comprises a CDR-L1 having an amino acid sequence of SEQ ID NO: 36 (according to the Kabat definition system), a CDR-L2 having an amino acid sequence of SEQ ID NO: 37 (according to the Kabat definition system), and a CDR-L3 having an amino acid sequence of SEQ ID NO: 32 (according to the Kabat definition system), and comprises no more than 25 amino acid variations in the framework region (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 shown in SEQ ID NO: 44.

[0233] In some embodiments, the humanized anti-TfR antibody of the present disclosure comprises a humanized VH, which comprises a CDR-H1 having an amino acid sequence of SEQ ID NO: 33 (according to the Kabat definition system), a CDR-H2 having an amino acid sequence of SEQ ID NO: 34 (according to the Kabat definition system), a CDR-H3 having an amino acid sequence of SEQ ID NO: 35 (according to the Kabat definition system), and has at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98% or 99%) identity in the framework region with the VH shown in SEQ ID NO: 43. As an alternative or supplement (e.g., supplement), the humanized anti-TfR antibody of the present disclosure comprises a humanized VL, wherein the humanized VL comprises a CDR-L1 having an amino acid sequence of SEQ ID NO: 36 (according to the Kabat definition system), a CDR-L2 having an amino acid sequence of SEQ ID NO: 37 (according to the Kabat definition system) and a CDR-L3 having an amino acid sequence of SEQ ID NO: 32 (according to the Kabat definition system), and has at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98% or 99%) identity in the framework region with the VL shown in SEQ ID NO: 44.

[0234] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising a CDR-H1 having an amino acid sequence of SEQ ID NO: 38 (according to the Chothia definition system), a CDR-H2 having an amino acid sequence of SEQ ID NO: 39 (according to the Chothia definition system), a CDR-H3 having an amino acid sequence of SEQ ID NO: 40 (according to the Chothia definition system), and comprising no more than 25 amino acid variations in the framework region (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: 43. As an alternative or supplement (e.g., supplement), the humanized anti-TfR antibody of the present disclosure comprises a humanized VL, wherein the humanized VL comprises a CDR-L1 having an amino acid sequence of SEQ ID NO: 41 (according to the Chothia definition system), a CDR-L2 having an amino acid sequence of SEQ ID NO: 31 (according to the Chothia definition system), and a CDR-L3 having an amino acid sequence of SEQ ID NO: 42 (according to the Chothia definition system), and comprises no more than 25 amino acid variations in the framework region (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 shown in SEQ ID NO: 44.

[0235] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising a CDR-H1 having an amino acid sequence of SEQ ID NO: 38 (according to the Chothia definition system), a CDR-H2 having an amino acid sequence of SEQ ID NO: 39 (according to the Chothia definition system), a CDR-H3 having an amino acid sequence of SEQ ID NO: 40 (according to the Chothia definition system), and having at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98% or 99%) identity in the framework region with the VH shown in SEQ ID NO: 43. As an alternative or supplement (e.g., supplement), the humanized anti-TfR antibody of the present disclosure comprises a humanized VL, wherein the humanized VL comprises a CDR-L1 having an amino acid sequence of SEQ ID NO: 41 (according to the Chothia definition system), a CDR-L2 having an amino acid sequence of SEQ ID NO: 31 (according to the Chothia definition system), and a CDR-L3 having an amino acid sequence of SEQ ID NO: 42 (according to the Chothia definition system), and has at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98% or 99%) identity in the framework region with the VL shown in SEQ ID NO: 44.

[0236] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising a CDR-H1 having an amino acid sequence of SEQ ID NO: 45, SEQ ID NO: 63, or SEQ ID NO: 66 (according to the IMGT definition system), a CDR-H2 having an amino acid sequence of SEQ ID NO: 46 (according to the IMGT definition system), a CDR-H3 having an amino acid sequence of SEQ ID NO: 47 (according to the IMGT definition system), and comprising no more than 25 amino acid variations in the framework region (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: 61, SEQ ID NO: 65, or SEQ ID NO: 68. As an alternative or supplement (e.g., supplement), the humanized anti-TfR antibody of the present disclosure comprises a humanized VL, wherein the humanized VL comprises a CDR-L1 having an amino acid sequence of SEQ ID NO: 48 (according to the IMGT definition system), a CDR-L2 having an amino acid sequence of SEQ ID NO: 49 (according to the IMGT definition system), and a CDR-L3 having an amino acid sequence of SEQ ID NO: 50 (according to the IMGT definition system), and comprises no more than 25 amino acid variations in the framework region (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 shown in SEQ ID NO: 62.

[0237] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising a CDR-H1 having an amino acid sequence of SEQ ID NO: 45, SEQ ID NO: 63, or SEQ ID NO: 66 (according to the IMGT definition system), a CDR-H2 having an amino acid sequence of SEQ ID NO: 46 (according to the IMGT definition system), a CDR-H3 having an amino acid sequence of SEQ ID NO: 47 (according to the IMGT definition system), and having at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identity in the framework region with the VH shown in SEQ ID NO: 61, SEQ ID NO: 65, SEQ ID NO: 68. Alternatively or in addition (e.g., supplementally), the humanized anti-TfR antibody of the present disclosure comprises a humanized VL comprising a CDR-L1 having an amino acid sequence of SEQ ID NO: 48 (according to the IMGT definition system), a CDR-L2 having an amino acid sequence of SEQ ID NO: 49 (according to the IMGT definition system), and a CDR-L3 having an amino acid sequence of SEQ ID NO: 50 (according to the IMGT definition system), and having at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98% or 99%) identity in the framework region with the VL shown in SEQ ID NO: 62.

[0238] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH, comprising a CDR-H1 having an amino acid sequence of SEQ ID NO: 51, SEQ ID NO: 64, or SEQ ID NO: 67 (according to the Kabat definition system), a CDR-H2 having an amino acid sequence of SEQ ID NO: 52 (according to the Kabat definition system), a CDR-H3 having an amino acid sequence of SEQ ID NO: 53 (according to the Kabat definition system), and comprising no more than 25 amino acid variations in the framework region (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: 61, SEQ ID NO: 65, or SEQ ID NO: 68. As an alternative or supplement (e.g., supplement), the humanized anti-TfR antibody of the present disclosure comprises a humanized VL, wherein the humanized VL comprises a CDR-L1 having an amino acid sequence of SEQ ID NO: 54 (according to the Kabat definition system), a CDR-L2 having an amino acid sequence of SEQ ID NO: 55 (according to the Kabat definition system), and a CDR-L3 having an amino acid sequence of SEQ ID NO: 50 (according to the Kabat definition system), and comprises no more than 25 amino acid variations in the framework region (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 shown in SEQ ID NO: 62.

[0239] In some embodiments, the humanized anti-TfR antibody of the present disclosure comprises a humanized VH, which comprises a CDR-H1 having an amino acid sequence of SEQ ID NO: 51, SEQ ID NO: 64 or SEQ ID NO: 67 (according to the Kabat definition system), a CDR-H2 having an amino acid sequence of SEQ ID NO: 52 (according to the Kabat definition system), a CDR-H3 having an amino acid sequence of SEQ ID NO: 53 (according to the Kabat definition system), and has at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98% or 99%) identity in the framework region with the VH shown in SEQ ID NO: 61, SEQ ID NO: 65, SEQ ID NO: 68. As an alternative or supplement (e.g., supplement), the humanized anti-TfR antibody of the present disclosure comprises a humanized VL, wherein the humanized VL comprises a CDR-L1 having an amino acid sequence of SEQ ID NO: 54 (according to the Kabat definition system), a CDR-L2 having an amino acid sequence of SEQ ID NO: 55 (according to the Kabat definition system) and a CDR-L3 having an amino acid sequence of SEQ ID NO: 50 (according to the Kabat definition system), and has at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98% or 99%) identity in the framework region with the VL shown in SEQ ID NO: 62.

[0240] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising a CDR-H1 having an amino acid sequence of SEQ ID NO: 56 (according to the Chothia definition system), a CDR-H2 having an amino acid sequence of SEQ ID NO: 57 (according to the Chothia definition system), a CDR-H3 having an amino acid sequence of SEQ ID NO: 58 (according to the Chothia definition system), and comprising no more than 25 amino acid variations in the framework region (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: 61, SEQ ID NO: 65, or SEQ ID NO: 68. As an alternative or supplement (e.g., supplement), the humanized anti-TfR antibody of the present disclosure comprises a humanized VL, wherein the humanized VL comprises a CDR-L1 having an amino acid sequence of SEQ ID NO: 59 (according to the Chothia definition system), a CDR-L2 having an amino acid sequence of SEQ ID NO: 49 (according to the Chothia definition system), and a CDR-L3 having an amino acid sequence of SEQ ID NO: 60 (according to the Chothia definition system), and comprises no more than 25 amino acid variations in the framework region (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 shown in SEQ ID NO: 62.

[0241] In some embodiments, the humanized anti-TfR antibody of the present disclosure comprises a humanized VH, which comprises a CDR-H1 having an amino acid sequence of SEQ ID NO: 56 (according to the Chothia definition system), a CDR-H2 having an amino acid sequence of SEQ ID NO: 57 (according to the Chothia definition system), a CDR-H3 having an amino acid sequence of SEQ ID NO: 58 (according to the Chothia definition system), and has at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98% or 99%) identity in the framework region with the VH shown in SEQ ID NO: 61, SEQ ID NO: 65, SEQ ID NO: 68. Alternatively or in addition (e.g., supplementally), the humanized anti-TfR antibody of the present disclosure comprises a humanized VL comprising a CDR-L1 having an amino acid sequence of SEQ ID NO: 59 (according to the Chothia definition system), a CDR-L2 having an amino acid sequence of SEQ ID NO: 49 (according to the Chothia definition system), and a CDR-L3 having an amino acid sequence of SEQ ID NO: 60 (according to the Chothia definition system), and having at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98% or 99%) identity in the framework region with the VL shown in SEQ ID NO: 62.

[0242] Some examples of amino acid sequences of humanized anti-TfR antibodies described herein are provided in Table 3.

[0243] Table 3. Variable regions of humanized anti-TfR antibodies

[0244]

[0245]

[0246]

[0247] *The mutation positions are according to the Kabat numbering of the corresponding VH sequence containing the mutation

[0248] **CDRs according to the Kabat numbering system are in bold

[0249] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising CDR-H1, CDR-H2, and CDR-H3 of any one of the anti-TfR antibodies provided in Table 2, and comprising one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid variations in the framework regions compared to the corresponding humanized VH provided in Table 3. Alternatively or additionally (e.g., supplementally), a humanized anti-TfR antibody of the present disclosure comprises a humanized VL comprising CDR-L1, CDR-L2, and CDR-L3 of any one of the anti-TfR antibodies provided in Table 2, and comprising one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid variations in the framework regions compared to the corresponding humanized VL provided in Table 3.

[0250] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 69, and / or (e.g., and) a humanized VL comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 70. In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising the amino acid sequence of SEQ ID NO: 69 and a humanized VL comprising the amino acid sequence of SEQ ID NO: 70.

[0251] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 71, and / or (e.g., and) a humanized VL comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 70. In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising the amino acid sequence of SEQ ID NO: 71 and a humanized VL comprising the amino acid sequence of SEQ ID NO: 70.

[0252] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 72, and / or (e.g., and) a humanized VL comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 70. In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising the amino acid sequence of SEQ ID NO: 72 and a humanized VL comprising the amino acid sequence of SEQ ID NO: 70.

[0253] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 73, and / or (e.g., and) a humanized VL comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 74. In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising the amino acid sequence of SEQ ID NO: 73 and a humanized VL comprising the amino acid sequence of SEQ ID NO: 74.

[0254] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 73, and / or (e.g., and) a humanized VL comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 75. In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising the amino acid sequence of SEQ ID NO: 73 and a humanized VL comprising the amino acid sequence of SEQ ID NO: 75.

[0255] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 76, and / or (e.g., and) a humanized VL comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 74. In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising the amino acid sequence of SEQ ID NO: 76 and a humanized VL comprising the amino acid sequence of SEQ ID NO: 74.

[0256] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 76, and / or (e.g., and) a humanized VL comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 75. In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising the amino acid sequence of SEQ ID NO: 76 and a humanized VL comprising the amino acid sequence of SEQ ID NO: 75.

[0257] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 77, and / or (e.g., and) a humanized VL comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 78. In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising the amino acid sequence of SEQ ID NO: 77 and a humanized VL comprising the amino acid sequence of SEQ ID NO: 78.

[0258] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 79, and / or (e.g., and) a humanized VL comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 80. In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising the amino acid sequence of SEQ ID NO: 79 and a humanized VL comprising the amino acid sequence of SEQ ID NO: 80.

[0259] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 77, and / or (e.g., and) a humanized VL comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 80. In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising the amino acid sequence of SEQ ID NO: 77 and a humanized VL comprising the amino acid sequence of SEQ ID NO: 80.

[0260] In some embodiments, the humanized anti-TfR antibodies described herein are full-length IgGs, which may include a heavy constant region and a light constant region from a human antibody. In some embodiments, the heavy chain of any anti-TfR antibody described herein may include a heavy chain constant region (CH) or a portion thereof (e.g., CH1, CH2, CH3, or a combination thereof). The heavy chain constant region may be of any suitable origin, such as human, mouse, rat, or rabbit. In a specific example, the heavy chain constant region is from human IgG, such as IgG1, IgG2, or IgG4 (γ heavy chain). An example of a human IgG1 constant region is given below:

[0261]

[0262] In some embodiments, the heavy chain of any anti-TfR antibody described herein comprises a mutant human IgG1 constant region. For example, it is known that the introduction of a LALA mutation in the CH2 domain of human IgG1 (derived from a mutant of mAb b12, which has mutated to replace the lower hinge residues Leu234Leu235 with Ala234 and Ala235) reduces Fcg receptor binding (Bruhns, P., et al. (2009) and Xu, D. et al. (2000)). The mutant human IgG1 constant region is provided below (the mutation is bold and underlined):

[0263]

[0264] 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 kappa light chain. In other instances, the CL is a lambda light chain. In some embodiments, the CL is a kappa light chain, the sequence of which is provided below:

[0265]

[0266] Additional antibody heavy and light chain constant regions are known in the art, such as those provided in the IMGT database (www.imgt.org) or www.vbase2.org / vbstat.php., both of which are incorporated herein by reference.

[0267] In some embodiments, the humanized anti-TfR antibodies described herein comprise a heavy chain comprising any one of the VHs listed in Table 3, or any variant thereof, and a heavy chain constant region that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 81 or SEQ ID NO: 82. In some embodiments, the humanized anti-TfR antibodies described herein comprise a heavy chain comprising any one of the VHs listed in Table 3, or any variant thereof, and a heavy chain constant region that comprises 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 antibodies described herein comprise a heavy chain comprising any one of the VHs listed in Table 3 or any variant thereof and a heavy chain constant region set forth in SEQ ID NO: 81. In some embodiments, the humanized anti-TfR antibodies described herein comprise a heavy chain comprising any one of the VHs listed in Table 3 or any variant thereof and a heavy chain constant region set forth in SEQ ID NO: 82.

[0268] In some embodiments, the humanized anti-TfR antibodies described herein comprise a light chain comprising any one of the VLs listed in Table 3, or any variant thereof, and a light chain constant region that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 83. In some embodiments, the humanized anti-TfR antibodies described herein comprise a light chain comprising any one of the VLs listed in Table 3, or any variant thereof, and a light chain constant region that comprises 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, a humanized anti-TfR antibody described herein comprises a light chain comprising any one of the VLs listed in Table 3 or any variant thereof and a light chain constant region set forth in SEQ ID NO: 83.

[0269] Some examples of IgG heavy chain amino acid sequences and light chain amino acid sequences of the anti-TfR antibodies are provided in Table 4 below.

[0270] Table 4. Heavy and light chain sequences of humanized anti-TfR IgG examples

[0271]

[0272]

[0273]

[0274] *The mutation positions are according to the Kabat numbering of the corresponding VH sequence containing the mutation

[0275] **CDRs according to the Kabat numbering system are bold; VH / VL sequences are underlined

[0276] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a heavy chain 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 variations) compared to the heavy chain set forth in any one of SEQ ID NOs: 84, 86, 87, 88, 91, 92, and 94. Alternatively or in addition (e.g., supplementally), the humanized anti-TfR antibodies of the present disclosure comprise a light chain 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 the light chain set forth in any one of SEQ ID NOs: 85, 89, 90, 93, and 95.

[0277] In some embodiments, the humanized anti-TfR antibodies described herein comprise a heavy chain comprising an amino acid sequence that is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to any one of SEQ ID NOs: 84, 86, 87, 88, 91, 92, and 94. Alternatively or additionally (e.g., supplementally), the humanized anti-TfR antibodies described herein comprise a light chain comprising an amino acid sequence that is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to any one of SEQ ID NOs: 85, 89, 90, 93, and 95. In some embodiments, the anti-TfR antibodies described herein comprise a heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 84, 86, 87, 88, 91, 92, and 94. Alternatively or additionally (eg, supplementally), the anti-TfR antibodies described herein comprise a light chain comprising the amino acid sequence of any one of SEQ ID NOs: 85, 89, 90, 93, and 95.

[0278] In some embodiments, the humanized anti-TfR antibodies of the present disclosure comprise a heavy chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 84, and / or (e.g., and) a light chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 85. In some embodiments, the humanized anti-TfR antibodies of the present disclosure comprise a heavy chain comprising the amino acid sequence of SEQ ID NO: 84 and a light chain comprising the amino acid sequence of SEQ ID NO: 85.

[0279] In some embodiments, the humanized anti-TfR antibodies of the present disclosure comprise a heavy chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 86, and / or (e.g., and) a light chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 85. In some embodiments, the humanized anti-TfR antibodies of the present disclosure comprise a heavy chain comprising the amino acid sequence of SEQ ID NO: 86 and a light chain comprising the amino acid sequence of SEQ ID NO: 85.

[0280] In some embodiments, the humanized anti-TfR antibodies of the present disclosure comprise a heavy chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 87, and / or (e.g., and) a light chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 85. In some embodiments, the humanized anti-TfR antibodies of the present disclosure comprise a heavy chain comprising the amino acid sequence of SEQ ID NO: 87 and a light chain comprising the amino acid sequence of SEQ ID NO: 85.

[0281] In some embodiments, the humanized anti-TfR antibodies of the present disclosure comprise a heavy chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 88, and / or (e.g., and) a light chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 89. In some embodiments, the humanized anti-TfR antibodies of the present disclosure comprise a heavy chain comprising the amino acid sequence of SEQ ID NO: 88 and a light chain comprising the amino acid sequence of SEQ ID NO: 89.

[0282] In some embodiments, the humanized anti-TfR antibodies of the present disclosure comprise a heavy chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 88, and / or (e.g., and) a light chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 90. In some embodiments, the humanized anti-TfR antibodies of the present disclosure comprise a heavy chain comprising the amino acid sequence of SEQ ID NO: 88 and a light chain comprising the amino acid sequence of SEQ ID NO: 90.

[0283] In some embodiments, the humanized anti-TfR antibodies of the present disclosure comprise a heavy chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 91, and / or (e.g., and) a light chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 89. In some embodiments, the humanized anti-TfR antibodies of the present disclosure comprise a heavy chain comprising the amino acid sequence of SEQ ID NO: 91 and a light chain comprising the amino acid sequence of SEQ ID NO: 89.

[0284] In some embodiments, the humanized anti-TfR antibodies of the present disclosure comprise a heavy chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 91, and / or (e.g., and) a light chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 90. In some embodiments, the humanized anti-TfR antibodies of the present disclosure comprise a heavy chain comprising the amino acid sequence of SEQ ID NO: 91 and a light chain comprising the amino acid sequence of SEQ ID NO: 90.

[0285] In some embodiments, the humanized anti-TfR antibodies of the present disclosure comprise a heavy chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 92, and / or (e.g., and) a light chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 93. In some embodiments, the humanized anti-TfR antibodies of the present disclosure comprise a heavy chain comprising the amino acid sequence of SEQ ID NO: 92 and a light chain comprising the amino acid sequence of SEQ ID NO: 93.

[0286] In some embodiments, the humanized anti-TfR antibodies of the present disclosure comprise a heavy chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 94, and / or (e.g., and) a light chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 95. In some embodiments, the humanized anti-TfR antibodies of the present disclosure comprise a heavy chain comprising the amino acid sequence of SEQ ID NO: 94 and a light chain comprising the amino acid sequence of SEQ ID NO: 95.

[0287] In some embodiments, the humanized anti-TfR antibodies of the present disclosure comprise a heavy chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 92, and / or (e.g., and) a light chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 95. In some embodiments, the humanized anti-TfR antibodies of the present disclosure comprise a heavy chain comprising the amino acid sequence of SEQ ID NO: 92 and a light chain comprising the amino acid sequence of SEQ ID NO: 95.

[0288] In some embodiments, the anti-TfR antibody is a Fab fragment, a Fab' fragment or a F(ab')2 fragment of a complete antibody (full-length antibody). The antigen-binding fragment of a complete antibody (full-length antibody) can be prepared by conventional methods (e.g., recombinantly prepared or by digesting the heavy chain constant region of a full-length IgG with an enzyme such as papain). For example, a F(ab')2 fragment can be produced by digesting the antibody molecule with pepsin or papain, and a Fab' fragment can be produced by reducing the disulfide bridges of the F(ab')2 fragment. In some embodiments, the heavy chain constant region in the Fab fragment of the anti-TfR1 antibody described herein comprises the following amino acid sequence:

[0289]

[0290] In some embodiments, the humanized anti-TfR antibodies described herein comprise a heavy chain comprising any one of the VHs listed in Table 3, or any variant thereof, and a heavy chain constant region that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 96. In some embodiments, the humanized anti-TfR antibodies described herein comprise a heavy chain comprising any one of the VHs listed in Table 3, or any variant thereof, and a heavy chain constant region that comprises 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, a humanized anti-TfR antibody described herein comprises a heavy chain comprising any one of the VHs listed in Table 3 or any variant thereof and a heavy chain constant region set forth in SEQ ID NO: 96.

[0291] In some embodiments, the humanized anti-TfR antibodies described herein comprise a light chain comprising any one of the VLs listed in Table 3, or any variant thereof, and a light chain constant region that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 83. In some embodiments, the humanized anti-TfR antibodies described herein comprise a light chain comprising any one of the VLs listed in Table 3, or any variant thereof, and a light chain constant region that comprises 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, a humanized anti-TfR antibody described herein comprises a light chain comprising any one of the VLs listed in Table 3 or any variant thereof and a light chain constant region set forth in SEQ ID NO: 83.

[0292] Some examples of Fab heavy and light chain amino acid sequences of the anti-TfR antibodies are provided in Table 5 below.

[0293] Table 5. Heavy and light chain sequences of humanized anti-TfR Fab examples

[0294]

[0295]

[0296]

[0297]

[0298] *The mutation positions are according to the Kabat numbering of the corresponding VH sequence containing the mutation

[0299] **CDRs according to the Kabat numbering system are bold; VH / VL sequences are underlined

[0300] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a heavy chain 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 variations) compared to the heavy chain shown in any one of SEQ ID NOs: 97 to 103. Alternatively or in addition (e.g., supplementally), the humanized anti-TfR antibodies of the present disclosure comprise a light chain having no more than 25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) compared to the light chain shown in any one of SEQ ID NOs: 85, 89, 90, 93, and 95.

[0301] In some embodiments, the humanized anti-TfR antibodies described herein comprise a heavy chain comprising an amino acid sequence that is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to any one of SEQ ID NOs: 97 to 103. Alternatively or additionally (e.g., supplementally), the humanized anti-TfR antibodies described herein comprise a light chain comprising an amino acid sequence that is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to any one of SEQ ID NOs: 85, 89, 90, 93, and 95. In some embodiments, the anti-TfR antibodies described herein comprise a heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 97 to 103. Alternatively or additionally (eg, supplementally), the anti-TfR antibodies described herein comprise a light chain comprising the amino acid sequence of any one of SEQ ID NOs: 85, 89, 90, 93, and 95.

[0302] In some embodiments, the humanized anti-TfR antibodies of the present disclosure comprise a heavy chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 97, and / or (e.g., and) a light chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 85. In some embodiments, the humanized anti-TfR antibodies of the present disclosure comprise 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.

[0303] In some embodiments, the humanized anti-TfR antibodies of the present disclosure comprise a heavy chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 98, and / or (e.g., and) a light chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 85. In some embodiments, the humanized anti-TfR antibodies of the present disclosure comprise 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.

[0304] In some embodiments, the humanized anti-TfR antibodies of the present disclosure comprise a heavy chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 99, and / or (e.g., and) a light chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 85. In some embodiments, the humanized anti-TfR antibodies of the present disclosure comprise 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.

[0305] In some embodiments, the humanized anti-TfR antibodies of the present disclosure comprise a heavy chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 100, and / or (e.g., and) a light chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 89. In some embodiments, the humanized anti-TfR antibodies of the present disclosure comprise 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.

[0306] In some embodiments, the humanized anti-TfR antibodies of the present disclosure comprise a heavy chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 100, and / or (e.g., and) a light chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 90. In some embodiments, the humanized anti-TfR antibodies of the present disclosure comprise 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.

[0307] In some embodiments, the humanized anti-TfR antibodies of the present disclosure comprise a heavy chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 101, and / or (e.g., and) a light chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 89. In some embodiments, the humanized anti-TfR antibodies of the present disclosure comprise 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.

[0308] In some embodiments, the humanized anti-TfR antibodies of the present disclosure comprise a heavy chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 101, and / or (e.g., and) a light chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 90. In some embodiments, the humanized anti-TfR antibodies of the present disclosure comprise 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.

[0309] In some embodiments, the humanized anti-TfR antibodies of the present disclosure comprise a heavy chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 102, and / or (e.g., and) a light chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 93. In some embodiments, the humanized anti-TfR antibodies of the present disclosure comprise 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.

[0310] In some embodiments, the humanized anti-TfR antibodies of the present disclosure comprise a heavy chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 103, and / or (e.g., and) a light chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 95. In some embodiments, the humanized anti-TfR antibodies of the present disclosure comprise 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.

[0311] In some embodiments, the humanized anti-TfR antibodies of the present disclosure comprise a heavy chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 102, and / or (e.g., and) a light chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 95. In some embodiments, the humanized anti-TfR antibodies of the present disclosure comprise 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.

[0312] In some embodiments, the humanized anti-TfR receptor antibodies described herein can be any antibody form, including but not limited to complete (ie, full-length) antibodies, antigen-binding fragments thereof (eg, Fab, Fab', F(ab')2, Fv), single-chain antibodies, bispecific antibodies, or nanobodies. In some embodiments, the humanized anti-TfR antibodies described herein are scFv. In some embodiments, the humanized anti-TfR antibodies described herein are scFv-Fab (eg, scFv fused to a portion of a constant region). In some embodiments, the anti-TfR receptor antibodies described herein are scFv fused to a constant region (eg, a human IgG1 constant region shown in SEQ ID NO: 81 or SEQ ID NO: 82, or a portion thereof, such as an Fc portion) at the C-terminus or N-terminus.

[0313] In some embodiments, conservative mutations can be introduced into the antibody sequence (e.g., CDR or framework sequence) at positions where the residues are unlikely to participate in interactions with the target antigen (e.g., transferrin receptor) (e.g., as determined based on the crystal structure). In some embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced into the Fc region of an 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., the 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 cellular cytotoxicity.

[0314] 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) such that the number of cysteine ​​residues in the hinge region is altered (e.g., increased or decreased), as described, for example, in U.S. Patent No. 5,677,425. The number of cysteine ​​residues in the hinge region of the CH1 domain can be altered, for example, to facilitate assembly of the light and heavy chains, or to alter (e.g., increase or decrease) antibody stability, or to facilitate linker conjugation.

[0315] In some embodiments, one, two or more mutations (e.g., amino acid substitutions) are introduced into the Fc region of the muscle-targeted antibodies 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., the EU index in Kabat)) to increase or decrease the affinity of the antibody for an Fc receptor on the surface of an effector cell (e.g., an activated Fc receptor). Mutations in the Fc region of an antibody that decrease or increase the affinity of an antibody for an Fc receptor and techniques for introducing such mutations into an Fc receptor or fragment thereof are known to those of skill in the art. Some examples of mutations in antibody Fc receptors that can be made to alter the affinity of the antibody for the Fc receptor are described, for example, in Smith P et al., (2012) PNAS 109:6181-6186, U.S. Patent No. 6,737,056, and International Publication Nos. WO 02 / 060919, WO 98 / 23289, and WO 97 / 34631, which are incorporated herein by reference.

[0316] In some embodiments, one, two or more amino acid mutations (i.e., substitutions, insertions or deletions) are introduced into the IgG constant domain or FcRn binding fragment thereof (preferably, Fc or hinge-Fc domain fragment) to alter (e.g., reduce or increase) the half-life of the antibody in vivo. See, e.g., International Publication Nos. WO 02 / 060919, WO 98 / 23289, and WO 97 / 34631, and U.S. Patent Nos. 5,869,046, 6,121,022, 6,277,375, and 6,165,745, for example, mutations that alter (e.g., reduce or increase) the half-life of the antibody in vivo.

[0317] In some embodiments, one, two or more amino acid mutations (i.e., substitutions, insertions or deletions) are introduced into the IgG constant domain or its FcRn binding fragment (preferably, 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 IgG constant domain or its FcRn binding fragment (preferably, 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 EA et al., (1991) supra)). In some embodiments, the constant region of the IgG1 of the antibodies described herein comprises a methionine (M) to tyrosine (Y) substitution at position 252, a serine (S) to threonine (T) substitution at position 254, and a threonine (T) to glutamic acid (E) substitution at position 256, as numbered according to the EU index as in Kabat. See U.S. Patent No. 7,658,921, which is incorporated herein by reference. This type of mutant IgG (referred to as a "YTE mutant") has been shown to have a 4-fold increase in half-life compared to the wild-type form of the same antibody (see Dall'Acqua WF et al., (2006) J Biol Chem 281: 23514-24). In some embodiments, the antibody comprises an IgG constant domain comprising one, two, three, or more amino acid substitutions at amino acid residues at positions 251 to 257, 285 to 290, 308 to 314, 385 to 389, and 428 to 436, as numbered according to the EU index as in Kabat.

[0318] 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 to which affinity is altered can be, for example, an Fc receptor or the C1 component of complement. This approach 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 region domain (by point mutation or other means) can reduce Fc receptor binding of circulating antibodies, thereby improving tumor localization. For descriptions of mutations that delete or inactivate 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 can be introduced into the Fc region of the antibodies described herein to remove potential glycosylation sites on the Fc region, which can reduce Fc receptor binding (see, for example, Shields RL et al., (2001) J Biol Chem 276: 6591-604).

[0319] In some embodiments, one or more amino groups in the constant region of the anti-TfR antibodies described herein can be replaced with different amino acid residues so that the antibody has 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 changed to change the ability of the antibody 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 improve the antibody's ability to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or (e.g., and) improve the antibody's affinity for Fcγ receptors. This method is further described in International Publication No. WO 00 / 42072.

[0320] 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 produce, for example, CDR-grafted, chimeric, humanized or composite human antibodies or antigen-binding fragments, as described elsewhere herein. As understood by those of ordinary skill in the art, any variant (CDR-grafted, chimeric, humanized or composite antibody) derived from any antibody provided herein can be used in the compositions and methods described herein, and will maintain the ability to specifically bind to transferrin receptor, such that relative to the original antibody from which it is derived, the variant (CDR-grafted, chimeric, humanized or composite antibody) has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or more binding to transferrin receptor.

[0321] In some embodiments, the antibodies provided herein include mutations that confer desired properties on the antibodies. For example, to avoid potential complications due to known Fab arm exchange with natural IgG4 mAbs, the antibodies provided herein may include a stability '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 the serine at position 228 (EU numbering, residue 241 according to Kabat numbering) is converted to a proline, thereby generating an IgG1-like hinge sequence. Thus, any antibody may include a stability 'Adair' mutation.

[0322] 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 sugars or carbohydrate molecules. In some embodiments, one or more sugars or carbohydrate molecules are conjugated to the antibody by N-glycosylation, O-glycosylation, C-glycosylation, glycosylphosphatidylinositolization (GPI anchor attachment) and / or (e.g., and) phosphoglycosylation. In some embodiments, one or more sugars or carbohydrate molecules are monosaccharides, disaccharides, oligosaccharides or polysaccharides. In some embodiments, one or more sugars or carbohydrate molecules are branched oligosaccharides or branched polysaccharides. In some embodiments, one or more sugars or carbohydrate molecules comprise mannose units, glucose units, N-acetylglucosamine units, N-acetylgalactosamine units, galactose units, fucose units or phospholipid units. In some embodiments, there are 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. In some embodiments, the glycosylated antibody is fully or partially glycosylated. In some embodiments, the antibody is glycosylated by a chemical reaction or by enzymatic means. In some embodiments, the antibody is glycosylated in vitro or in a cell, which may optionally lack 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 No. WO2014065661, entitled “Modified antibody, antibody-conjugate and process for the preparation thereof,” published on May 1, 2014.

[0323] In some embodiments, any of the anti-TfR1 antibodies described herein may comprise a signal peptide (e.g., an N-terminal signal peptide) in the heavy chain sequence and / or (e.g., and) light chain sequence. In some embodiments, the anti-TfR1 antibodies described herein comprise 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 comprise 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).

[0324] Other known anti-transferrin receptor antibodies

[0325] Any other suitable anti-transferrin receptor antibody known in the art can be used as a muscle targeting agent in the complex 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 comprises the complementary 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).

[0326] Table 8. List of anti-transferrin receptor antibody clones, including relevant references and binding epitope information.

[0327]

[0328]

[0329]

[0330]

[0331] In some embodiments, the transferrin receptor antibodies of the present disclosure comprise one or more CDR-H (e.g., CDR-H1, CDR-H2, and CDR-H3) amino acid sequences from any one of the anti-transferrin receptor antibodies selected from Table 8. In some embodiments, the transferrin receptor antibodies comprise CDR-H1, CDR-H2, and CDR-H3 as provided for any one of the anti-transferrin receptor antibodies selected from Table 8. In some embodiments, the anti-transferrin receptor antibodies comprise CDR-L1, CDR-L2, and CDR-L3 as provided for any one of the anti-transferrin receptor antibodies selected from Table 8. In some embodiments, the anti-transferrin antibodies comprise CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 as provided for any one of the anti-transferrin receptor antibodies selected from Table 8. The present 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 anti-transferrin receptor antibody selected from Table 8. In some embodiments, the antibody heavy chain and light chain CDR3 domains can play a particularly important role in the binding specificity / affinity of the antibody to the antigen. Therefore, the anti-transferrin receptor antibody of the present disclosure can at least comprise a heavy chain and / or (e.g., and) light chain CDR3 selected from any anti-transferrin receptor antibody of Table 8.

[0332] In some examples, any anti-transferrin receptor antibodies of the present disclosure have 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 antibodies 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 can be altered by one, two, three, four, five, or six amino acid positions, so long as immunospecific binding to transferrin receptor (e.g., human transferrin receptor) is maintained (e.g., substantially maintaining, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the binding of the original antibody from which it was derived). For example, in some embodiments, the positions defining the CDRs of any of the antibodies described herein can be altered by shifting the N-terminal and / or (e.g., and) the C-terminal boundaries of the CDRs by one, two, three, four, five, or six amino acids relative to the positions of the CDRs of any of the antibodies described herein, so long as immunospecific binding to transferrin receptor (e.g., human transferrin receptor) is maintained (e.g., substantially maintaining, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the binding of the original antibody from which it was derived). In another embodiment, the length of one or more CDRs of the antibodies described herein can be altered (e.g., made shorter or longer) by one, two, three, four, five, or more amino acids 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, so long as immunospecific binding to transferrin receptor (e.g., human transferrin receptor) is maintained (e.g., substantially maintains, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the binding of the original antibody from which it is derived).

[0333] Thus, 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 shorter than one or more CDRs described herein (e.g., a CDR from any anti-transferrin receptor antibody selected from Table 8) by one, two, three, four, five, or more amino acids, so long as immunospecific binding to transferrin receptor (e.g., human transferrin receptor) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% 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., a CDR from any anti-transferrin receptor antibody selected from Table 8), so long as immunospecific binding to transferrin receptor (e.g., human transferrin receptor) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% relative to the binding of the original antibody from which it is derived). In some embodiments, the amino portion of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and / or (e.g., and) CDR-H3 described herein can be extended by one, two, three, four, five, or more amino acids compared to one or more CDRs described herein (e.g., a CDR from any anti-transferrin receptor antibody selected from Table 8), so long as immunospecific binding to transferrin receptor (e.g., human transferrin receptor) is maintained (e.g., substantially maintaining, e.g., 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 portion of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and / or (e.g., and) CDR-H3 described herein can be extended by one, two, three, four, five, or more amino acids compared to one or more CDRs described herein (e.g., a CDR from any anti-transferrin receptor antibody selected from Table 8), so long as immunospecific binding to transferrin receptor (e.g., human transferrin receptor) is maintained (e.g., substantially maintaining, e.g., 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 portion of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and / or (e.g., and) CDR-H3 described herein can be shortened by one, two, three, four, five, or more amino acids compared to one or more CDRs described herein (e.g., a CDR from any anti-transferrin receptor antibody selected from Table 8), so long as immunospecific binding to transferrin receptor (e.g., human transferrin receptor) is maintained (e.g., substantially maintaining, e.g., 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 portion of the CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and / or (e.g., and) CDR-H3 described herein can be shortened by one, two, three, four, five, or more amino acids compared to one or more CDRs described herein (e.g., a CDR from any anti-transferrin receptor antibody selected from Table 8), as long as immunospecific binding to transferrin receptor (e.g., human transferrin receptor) is maintained (e.g., substantially maintaining, 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 can be used to determine whether immunospecific binding to transferrin receptor (e.g., human transferrin receptor) is maintained, for example, using binding assays and conditions described in the art.

[0334] In some examples, any anti-transferrin receptor antibody of the present disclosure has one or more CDR (e.g., CDR-H or CDR-L) sequences that are substantially similar to any anti-transferrin receptor antibody selected from Table 8. For example, the antibody may comprise one or more CDR sequences from any anti-transferrin receptor antibody selected from Table 8, which comprise 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., a CDR from any anti-transferrin receptor antibody selected from Table 8), as long as immunospecific binding to transferrin receptor (e.g., human transferrin receptor) is maintained (e.g., substantially maintaining, 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, any amino acid variation in any CDR provided herein may be a conservative variation. Conservative variation can be introduced into CDR at the position (such as determined based on crystal structure) where residue is unlikely to participate in interacting with transferrin receptor protein (such as, human transferrin receptor protein). Some aspects of the present disclosure provide transferrin receptor antibodies, which include one or more heavy chain variable (VH) and / or (such as, and) light chain variable (VL) domains provided herein. In some embodiments, any VH domain provided herein includes one or more CDR-H sequences (such as, CDR-H1, CDR-H2 and CDR-H3) provided herein, such as any CDR-H sequence provided in any anti-transferrin receptor antibody selected from Table 8. In some embodiments, any VL domain provided herein includes one or more CDR-L sequences (such as, CDR-L1, CDR-L2 and CDR-L3) provided herein, such as any CDR-L sequence provided in any anti-transferrin receptor antibody selected from Table 8.

[0335] In some embodiments, the anti-transferrin receptor antibodies of the present disclosure include any antibodies comprising a heavy chain variable domain and / or (e.g., and) a light chain variable domain of any anti-transferrin receptor antibody (e.g., any anti-transferrin receptor antibody selected from Table 8). In some embodiments, the anti-transferrin receptor antibodies of the present disclosure include any antibodies comprising a heavy chain variable and light chain variable pair of any anti-transferrin receptor antibody (e.g., any anti-transferrin receptor antibody selected from Table 8).

[0336] Some aspects of the present disclosure provide anti-transferrin receptor antibodies having heavy chain variable (VH) and / or (e.g., and) light chain variable (VL) domain amino acid sequences homologous to those described herein. In some embodiments, anti-transferrin receptor antibodies include a heavy chain variable sequence and / or any light chain variable sequence with at least 75% (e.g., 80%, 85%, 90%, 95%, 98% or 99%) homology to any anti-transferrin receptor antibody (e.g., any anti-transferrin receptor antibody selected from Table 8). In some embodiments, homologous heavy chain variable and / or (e.g., and) light chain variable amino acid sequences do not vary within any CDR sequence 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 a heavy chain variable and / or (e.g., and) light chain variable sequence that does not include any CDR sequence provided herein. In some embodiments, any of the anti-transferrin receptor antibodies provided herein comprise a heavy chain variable sequence and a light chain variable sequence comprising a framework sequence that is at least 75%, 80%, 85%, 90%, 95%, 98% or 99% identical to the framework sequence of any anti-transferrin receptor antibody (e.g., any one of the anti-transferrin receptor antibodies selected from Table 8).

[0337] In some embodiments, an anti-transferrin receptor antibody that specifically binds to a transferrin receptor (e.g., a human transferrin receptor) comprises a light chain variable VL domain comprising any CDR-L domain (CDR-L1, CDR-L2, and CDR-L3) of any anti-transferrin receptor antibody selected from Table 8, or a CDR-L domain variant provided herein. In some embodiments, an anti-transferrin receptor antibody that specifically binds to a transferrin receptor (e.g., a human transferrin receptor) comprises a light chain variable VL domain comprising CDR-L1, CDR-L2, and CDR-L3 of any anti-transferrin receptor antibody (e.g., any one 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 framework regions of a light chain variable region sequence 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 one, two, three, or four framework regions of a light chain variable region sequence that are at least 75%, 80%, 85%, 90%, 95%, or 100% identical to one, two, three, or four framework regions of a light chain variable region sequence of any anti-transferrin receptor antibody (e.g., any anti-transferrin receptor antibody selected from Table 8). In some embodiments, the light chain variable framework 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 framework region derived from the amino acid sequence consists of the amino acid sequence, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues are substituted for amino acids at analogous positions in the corresponding non-human primate or human light chain variable framework region.

[0338] In some embodiments, the anti-transferrin receptor antibody that specifically binds to transferrin receptor comprises 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 antibody further comprises one, two, three or all four VL framework regions of the VL derived from a human antibody or primate antibody. The primate or human antibody light chain framework region selected for use with the light chain CDR sequences described herein can have, for example, at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98% or at least 99%) identity with the light chain framework region of the non-human parent antibody. The amino acid numbering of the selected primate or human antibody in its light chain complementary determining region can be identical or substantially identical to the amino acid numbering in the light chain complementary determining region of any antibody provided herein (e.g., any anti-transferrin receptor antibody selected from Table 8). In some embodiments, the primate or human light chain framework region amino acid residues are from a natural primate or human antibody light chain framework region, which has at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, at least 99% (or more) identity to the light chain framework region of any anti-transferrin receptor antibody (e.g., any one of the anti-transferrin receptor antibodies selected from Table 8). In some embodiments, the anti-transferrin receptor antibody further comprises one, two, three, or all four VL framework regions derived from the human light chain variable 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.

[0339] In some embodiments, any anti-transferrin receptor antibody provided herein comprises a light chain variable domain, which further comprises a light chain constant region. In some embodiments, the light chain constant region is a kappa or lambda light chain constant region. In some embodiments, the kappa or lambda light chain constant region is from a mammal, for example, from a human, monkey, rat or mouse. In some embodiments, the light chain constant region is a human kappa light chain constant region. In some embodiments, the light chain constant region is a human lambda light chain constant region. It should be understood that any light chain constant region provided herein can 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).

[0340] In some embodiments, the anti-transferrin receptor antibody is any anti-transferrin receptor antibody, such as any one of the anti-transferrin receptor antibodies selected from Table 8.

[0341] In some embodiments, the anti-transferrin receptor antibody comprises a VL domain comprising the amino acid sequence of any anti-transferrin receptor antibody (e.g., any one of the anti-transferrin receptor antibodies selected from Table 8), and wherein the constant region comprises the amino acid sequence of a 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 VL domain variant, and any VH domain or VH domain variant, wherein the VL and VH domains or variants thereof are from the same antibody clone, and wherein the constant region comprises the amino acid sequence of a constant region of an IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, or any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or any subclass (e.g., IgG2a and IgG2b) of an immunoglobulin molecule. Some non-limiting examples of human constant regions are described in the art, see, eg, Kabat EA et al., (1991), supra.

[0342] In some embodiments, the muscle targeting agent is a transferrin receptor antibody (e.g., an antibody and variants thereof as described in International Application Publication No. WO 2016 / 081643, which is incorporated herein by reference).

[0343] Heavy and light chain CDRs of antibodies according to different definition systems are provided in Table 9. Different definition systems have been described, such as the Kabat definition, the Chothia definition and / or (e.g., and) the contact definition. See, e.g., (e.g., Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed., US Department of Health and Human Services, NIH Publication No. 91-3242, Chothia et al., (1989) Nature 342:877; Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917, Al-lazikani et al (1997) J. 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).

[0344] Table 9 Heavy and light chain CDRs of mouse transferrin receptor antibodies

[0345]

[0346] Also provided are the heavy chain variable domain (VH) and light chain variable domain sequences:

[0347] VH

[0348]

[0349] VL

[0350]

[0351] In some embodiments, the transferrin receptor antibodies of the present disclosure comprise CDR-H1, CDR-H2, and CDR-H3 that are identical to the CDR-H1, CDR-H2, and CDR-H3 shown in Table 9. Alternatively or additionally (e.g., supplementally), the transferrin receptor antibodies of the present disclosure comprise CDR-L1, CDR-L2, and CDR-L3 that are identical to the CDR-L1, CDR-L2, and CDR-L3 shown in Table 9.

[0352] In some embodiments, the transferrin receptor antibodies of the present disclosure comprise CDR-H1, CDR-H2, and CDR-H3, which together comprise no more than 5 amino acid variations (e.g., no more than 5, 4, 3, 2, or 1 amino acid variations) compared to the CDR-H1, CDR-H2, and CDR-H3 shown in Table 9. "Common" means that the total number of amino acid variations in all three heavy chain CDRs is within a defined range. Alternatively or in addition (e.g., supplementing), the transferrin receptor antibodies of the present disclosure may comprise CDR-L1, CDR-L2, and CDR-L3, which together comprise no more than 5 amino acid variations (e.g., no more than 5, 4, 3, 2, or 1 amino acid variations) compared to the CDR-L1, CDR-L2, and CDR-L3 shown in Table 9.

[0353] In some embodiments, the transferrin receptor antibodies of the present disclosure comprise CDR-H1, CDR-H2, and CDR-H3, at least one of which comprises no more than 3 amino acid variations (e.g., no more than 3, 2, or 1 amino acid variations) compared to the corresponding heavy chain CDR shown in Table 9. Alternatively or additionally (e.g., supplementally), the transferrin receptor antibodies of the present disclosure may comprise CDR-L1, CDR-L2, and CDR-L3, at least one of which comprises no more than 3 amino acid variations (e.g., no more than 3, 2, or 1 amino acid variations) compared to the corresponding light chain CDR shown in Table 9.

[0354] In some embodiments, the transferrin receptor antibodies of the present disclosure comprise a CDR-L3 comprising 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 antibodies of the present disclosure comprise a CDR-L3 comprising 1 amino acid variation compared to the CDR-L3 shown in Table 9. In some embodiments, the transferrin receptor antibodies of the present disclosure comprise a CDR-L3 of QHFAGTPLT (SEQ ID NO: 126) (according to the Kabat and Chothia definition system) or a CDR-L3 of QHFAGTPL (SEQ ID NO: 127) (according to the Contact definition system). In some embodiments, a transferrin receptor antibody of the present disclosure comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, and CDR-L2 that are identical to the CDR-H1, CDR-H2, and CDR-H3 shown in Table 9, and comprises CDR-L3 of QHFAGTPLT (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).

[0355] In some embodiments, the transferrin receptor antibodies of the present disclosure comprise heavy chain CDRs that collectively are at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identical to the heavy chain CDRs shown in Table 9. Alternatively or additionally (e.g., supplementally), the transferrin receptor antibodies of the present disclosure comprise light chain CDRs that collectively are at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identical to the light chain CDRs shown in Table 9.

[0356] In some embodiments, the transferrin receptor antibodies of the present disclosure comprise a VH comprising the amino acid sequence of SEQ ID NO: 124. Alternatively or additionally (e.g., supplementally), the transferrin receptor antibodies of the present disclosure comprise a VL comprising the amino acid sequence of SEQ ID NO: 125.

[0357] In some embodiments, the transferrin receptor antibodies of the present disclosure comprise a VH 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 variations) compared to the VH set forth in SEQ ID NO: 124. Alternatively or additionally (e.g., supplementally), the transferrin receptor antibodies of the present disclosure comprise a VL comprising 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 variations) compared to the VL set forth in SEQ ID NO: 125.

[0358] In some embodiments, the transferrin receptor antibodies of the present disclosure comprise a VH comprising an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identical to the VH shown in SEQ ID NO: 124. Alternatively or additionally (e.g., supplementally), the transferrin receptor antibodies of the present disclosure comprise a VL comprising an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identical to the VL shown in SEQ ID NO: 125.

[0359] In some embodiments, the transferrin receptor antibodies of the present disclosure are humanized antibodies (e.g., humanized variants of antibodies). In some embodiments, the transferrin receptor antibodies of the present disclosure comprise CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 identical to those shown in Table 9, and comprise a humanized heavy chain variable region and / or (e.g., and) a humanized light chain variable region.

[0360] Humanized antibody is human immunoglobulin (acceptor antibody), wherein the residue from the complementary determining region (CDR) of acceptor is replaced by the residue from the CDR of non-human species (donor antibody) such as mouse, rat or rabbit with desired specificity, affinity and capacity.In some embodiments, the Fv framework region (FR) residue of human immunoglobulin is replaced by corresponding non-human residue.In addition, humanized antibody can be included in acceptor antibody or the CDR or framework sequence of importing and all do not find but be included in to further improve and optimize the residue of antibody performance.In general, humanized antibody will comprise at least one and generally all of two variable domains, wherein all or substantially all of CDR districts correspond to those of non-human immunoglobulin, and all or substantially all of FR districts are those of human immunoglobulin consensus sequence.Humanized antibody will also comprise at least a portion of immunoglobulin constant region or domain (Fc) (generally those of human immunoglobulin) optimally.Antibody can have the Fc district modified as described in WO 99 / 58572. Other forms of humanized antibodies have one or more CDRs (one, two, three, four, five, six) that are changed relative to the original antibody, which is also referred to as one or more CDRs derived from one or more CDRs from the original antibody.Humanized antibodies may also involve affinity maturation.

[0361] In some embodiments, humanization is achieved by grafting CDRs (e.g., as shown in Table 9) into the IGKV1-NL1*01 and IGHV1-3*01 human variable domains. In some embodiments, the transferrin receptor antibodies of the present disclosure are humanized variants comprising 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) comprising 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 antibodies of the present disclosure are humanized variants comprising amino acid substitutions at all of positions 9, 13, 17, 18, 40, 45, and 70 compared to the VL set forth in SEQ ID NO: 125, and / or (e.g., and) comprising amino acid substitutions at all of positions 1, 5, 7, 11, 12, 20, 38, 40, 44, 66, 75, 81, 83, 87, and 108 compared to the VH set forth in SEQ ID NO: 124.

[0362] In some embodiments, the transferrin receptor antibodies of the present disclosure are humanized antibodies and comprise residues at positions 43 and 48 of the VL set forth in SEQ ID NO: 125. Alternatively or additionally (e.g., supplementally), the transferrin receptor antibodies of the present disclosure are humanized antibodies and comprise residues at positions 48, 67, 69, 71, and 73 of the VH set forth in SEQ ID NO: 124.

[0363] The VH and VL amino acid sequences of exemplary humanized antibodies that can be used according to the present disclosure are provided:

[0364] Humanized VH

[0365]

[0366] Humanized VL

[0367]

[0368] In some embodiments, the transferrin receptor antibodies of the present disclosure comprise a VH comprising the amino acid sequence of SEQ ID NO: 128. Alternatively or additionally (e.g., supplementally), the transferrin receptor antibodies of the present disclosure comprise a VL comprising the amino acid sequence of SEQ ID NO: 129.

[0369] In some embodiments, the transferrin receptor antibodies of the present disclosure comprise a VH 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 variations) compared to the VH set forth in SEQ ID NO: 128. Alternatively or additionally (e.g., supplementally), the transferrin receptor antibodies of the present disclosure comprise a VL comprising 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 variations) compared to the VL set forth in SEQ ID NO: 129.

[0370] In some embodiments, the transferrin receptor antibodies of the present disclosure comprise a VH comprising an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identical to the VH shown in SEQ ID NO: 128. Alternatively or additionally (e.g., supplementally), the transferrin receptor antibodies of the present disclosure comprise a VL comprising an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identical to the VL shown in SEQ ID NO: 129.

[0371] In some embodiments, the transferrin receptor antibodies of the present disclosure are humanized variants comprising an amino acid substitution at one or more of positions 43 and 48 compared to the VL set forth in SEQ ID NO: 125, and / or (e.g., and) an amino acid substitution at one or more of positions 48, 67, 69, 71, and 73 compared to the VH set forth in SEQ ID NO: 124. In some embodiments, the transferrin receptor antibodies of the present disclosure are humanized variants comprising an S43A and / or (e.g., and) a V48L mutation compared to the VL set forth in SEQ ID NO: 125, and / or (e.g., and) one or more of an A67V, L69I, V71R, and K73T mutation compared to the VH set forth in SEQ ID NO: 124.

[0372] In some embodiments, the transferrin receptor antibodies of the present disclosure are humanized variants comprising an amino acid substitution at one or more of positions 9, 13, 17, 18, 40, 43, 48, 45, and 70 compared to the VL set forth in SEQ ID NO: 125, and / or (e.g., and) an amino acid substitution at one or more of positions 1, 5, 7, 11, 12, 20, 38, 40, 44, 48, 66, 67, 69, 71, 73, 75, 81, 83, 87, and 108 compared to the VH set forth in SEQ ID NO: 124.

[0373] In some embodiments, the transferrin receptor antibodies of the present disclosure are chimeric antibodies, which may include a heavy constant region and a light constant region from a human antibody. A chimeric antibody refers to an antibody having a variable region from a first species or a part of a variable region and a constant region from a second species. Generally speaking, in these chimeric antibodies, the variable region simulations of the light chain and heavy chain are derived from the variable region of an antibody of a mammal (e.g., non-human mammals, such as mice, rabbits, and rats), while the constant portion is homologous to the sequence in the antibody derived from another mammal (e.g., people). In some embodiments, amino acid modifications may be performed in the variable region and / or (e.g., and) constant region.

[0374] In some embodiments, the transferrin receptor antibodies described herein are chimeric antibodies, which may include a heavy constant region and a light constant region from a human antibody. A chimeric antibody refers to an antibody having a variable region from a first species or a portion of a variable region and a constant region from a second species. Generally speaking, in these chimeric antibodies, the variable region simulations of the light and heavy chains are derived from the variable region of an antibody of a mammal (e.g., non-human mammals, such as mice, rabbits, and rats), while the constant portion is homologous to the sequence in the antibody derived from another mammal (e.g., people). In some embodiments, amino acid modifications may be made in the variable region and / or (e.g., and) constant region.

[0375] In some embodiments, the heavy chain of any transferrin receptor antibody as described herein may comprise a heavy chain constant region (CH) or a portion thereof (e.g., CH1, CH2, CH3, or a combination thereof). The heavy chain constant region may be of any suitable origin, such as human, mouse, rat, or rabbit. In a specific example, the heavy chain constant region is from human IgG, such as IgG1, IgG2, or IgG4 (γ heavy chain). An example of a human IgG1 constant region is given below:

[0376]

[0377] In some embodiments, the light chain of any transferrin receptor 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 kappa light chain. In other instances, the CL is a lambda light chain. In some embodiments, the CL is a kappa light chain, the sequence of which is provided below:

[0378]

[0379] Additional antibody heavy and light chain constant regions are known in the art, such as those provided in the IMGT database (www.imgt.org) or www.vbase2.org / vbstat.php., both of which are incorporated herein by reference.

[0380] Some examples of heavy and light chain amino acid sequences of transferrin receptor antibodies are provided below:

[0381] Heavy chain (VH + human IgG1 constant region)

[0382]

[0383] Light chain (VL + kappa light chain)

[0384]

[0385] Heavy chain (humanized VH + human IgG1 constant region)

[0386]

[0387] Light chain (humanized VL + kappa light chain)

[0388]

[0389] In some embodiments, the transferrin receptor antibodies described herein comprise a heavy chain comprising an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identical to SEQ ID NO: 132. Alternatively or additionally (e.g., supplementally), the transferrin receptor antibodies described herein comprise a light chain comprising an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identical to SEQ ID NO: 133. In some embodiments, the transferrin receptor antibodies described herein comprise a heavy chain comprising the amino acid sequence of SEQ ID NO: 132. Alternatively or additionally (e.g., supplementally), the transferrin receptor antibodies described herein comprise a light chain comprising the amino acid sequence of SEQ ID NO: 133.

[0390] In some embodiments, the transferrin receptor antibodies of the present disclosure comprise a heavy chain 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 the heavy chain set forth in SEQ ID NO: 132. Alternatively or additionally (e.g., supplementally), the transferrin receptor antibodies of the present disclosure comprise a light chain comprising 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 set forth in SEQ ID NO: 133.

[0391] In some embodiments, the transferrin receptor antibodies described herein comprise a heavy chain comprising an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identical to SEQ ID NO: 134. Alternatively or additionally (e.g., supplementally), the transferrin receptor antibodies described herein comprise a light chain comprising an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identical to SEQ ID NO: 135. In some embodiments, the transferrin receptor antibodies described herein comprise a heavy chain comprising the amino acid sequence of SEQ ID NO: 134. Alternatively or additionally (e.g., supplementally), the transferrin receptor antibodies described herein comprise a light chain comprising the amino acid sequence of SEQ ID NO: 135.

[0392] In some embodiments, the transferrin receptor antibodies of the present disclosure comprise a heavy chain 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 variations) compared to the heavy chain of the humanized antibody set forth in SEQ ID NO: 134. Alternatively or additionally (e.g., supplementally), the transferrin receptor antibodies of the present disclosure comprise a light chain comprising 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 variations) compared to the light chain of the humanized antibody set forth in SEQ ID NO: 135.

[0393] In some embodiments, the transferrin receptor antibody is an antigen-binding fragment (Fab) of a complete antibody (full-length antibody). Antigen-binding fragments of a complete antibody (full-length antibody) can be prepared by conventional methods. For example, F(ab')2 fragments can be produced by pepsin digestion of antibody molecules, and Fab' fragments can be produced by reducing the disulfide bridges of F(ab')2 fragments. Some examples of Fab amino acid sequences of transferrin receptor antibodies described herein are provided below:

[0394] Heavy chain Fab (VH + part of the human IgG1 constant region)

[0395]

[0396] Heavy chain Fab (humanized VH + part of human IgG1 constant region)

[0397]

[0398] In some embodiments, the transferrin receptor antibodies described herein comprise a heavy chain comprising the amino acid sequence of SEQ ID NO: 136. Alternatively or additionally (e.g., supplementally), the transferrin receptor antibodies described herein comprise a light chain comprising the amino acid sequence of SEQ ID NO: 133.

[0399] In some embodiments, the transferrin receptor antibodies described herein comprise a heavy chain comprising the amino acid sequence of SEQ ID NO: 137. Alternatively or additionally (e.g., supplementally), the transferrin receptor antibodies described herein comprise a light chain comprising the amino acid sequence of SEQ ID NO: 135.

[0400] The transferrin receptor antibodies described herein can be in any antibody form, including but not limited to complete (i.e., full-length) antibodies, antigen-binding fragments thereof (e.g., Fab, Fab', F(ab')2, Fv), single-chain antibodies, bispecific antibodies, or nanobodies. In some embodiments, the transferrin receptor antibodies described herein are scFv. In some embodiments, the transferrin receptor antibodies described herein are scFv-Fab (e.g., scFv fused to a portion of a constant region). In some embodiments, the transferrin receptor antibodies described herein are scFv fused to a constant region (e.g., the human IgG1 constant region shown in SEQ ID NO: 130).

[0401] In some embodiments, any of the anti-TfR antibodies described herein are produced by recombinant DNA technology in Chinese hamster ovary (CHO) cell suspension culture, optionally in CHO-K1 cell suspension culture (e.g., CHO-K1 cells from the European Collection of Animal Cell Culture, catalog number 85051005).

[0402] In some embodiments, the antibodies provided herein may have one or more post-translational modifications. In some embodiments, the N-terminal cyclization also referred to as pyroglutamate formation (pyro-Glu) may occur at the N-terminal glutamate (Glu) and / or glutamine (Gln) residues of the antibody during production. Therefore, it should be understood that antibodies designated as having a sequence comprising an N-terminal glutamate or glutamine residue encompass antibodies that have undergone pyroglutamate formation caused by 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.

[0403] b. Other muscle-targeted antibodies

[0404] In some embodiments, the muscle targeting antibody is an antibody that specifically binds 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, myogenin, 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, beta-Sarcoglycan, calpain inhibitor, creatine kinase MM / CKMM, eIF5A, enolase 2 / neuron-specific enolase, epsilon-Sarcoglycan, FABP3 / H-FABP, GDF-8 / myostatin, GDF-11 / GDF-8, integrin alpha 7, integrin alpha 7 beta 1, integrin beta 1 / CD29, MCAM / CD146, MyoD, myogenin, myosin light chain kinase inhibitor, NCAM-1 / CD56, and troponin I. In some embodiments, the muscle-targeting antibody is an antibody that specifically binds to a smooth muscle protein. 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 H2R, motilin R / GPR38, transglutamin / TAGLN, and vimentin. However, it will be understood that antibodies to other targets are within the scope of the present disclosure, and the exemplary list of targets provided herein is not meant to be limiting.

[0405] c. Antibody characteristics / changes

[0406] In some embodiments, conservative mutations can be introduced into the antibody sequence (e.g., CDR or framework sequence) at positions where the residues are unlikely to be involved in interacting with the target antigen (e.g., transferrin receptor) (e.g., as determined based on the crystal structure). In some embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced into the Fc region of the muscle-targeted antibodies 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., the 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 cellular cytotoxicity.

[0407] 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) such that the number of cysteine ​​residues in the hinge region is altered (e.g., increased or decreased), as described, for example, in U.S. Patent No. 5,677,425. The number of cysteine ​​residues in the hinge region of the CH1 domain can be altered, for example, to facilitate assembly of the light and heavy chains, or to alter (e.g., increase or decrease) antibody stability, or to facilitate linker conjugation.

[0408] In some embodiments, one, two or more mutations (e.g., amino acid substitutions) are introduced into the Fc region of the muscle-targeted antibodies 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., the EU index in Kabat)) to increase or decrease the affinity of the antibody for an Fc receptor on the surface of an effector cell (e.g., an activated Fc receptor). Mutations in the Fc region of an antibody that decrease or increase the affinity of an antibody for an Fc receptor and techniques for introducing such mutations into an Fc receptor or fragment thereof are known to those of skill in the art. Some examples of mutations in the Fc receptor of an antibody that can be made to alter the affinity of the antibody for the Fc receptor are described in, e.g., Smith P et al., (2012) PNAS 109:6181-6186, U.S. Patent No. 6,737,056, and International Publication Nos. WO 02 / 060919, WO 98 / 23289, and WO 97 / 34631, which are incorporated herein by reference.

[0409] In some embodiments, one, two or more amino acid mutations (i.e., substitutions, insertions or deletions) are introduced into the IgG constant domain or FcRn binding fragment thereof (preferably, Fc or hinge-Fc domain fragment) to alter (e.g., reduce or increase) the half-life of the antibody in vivo. See, e.g., International Publication Nos. WO 02 / 060919, WO 98 / 23289, and WO 97 / 34631, and U.S. Patent Nos. 5,869,046, 6,121,022, 6,277,375, and 6,165,745, for example, mutations that alter (e.g., reduce or increase) the half-life of the antibody in vivo.

[0410] In some embodiments, one, two or more amino acid mutations (i.e., substitutions, insertions or deletions) are introduced into the IgG constant domain or its FcRn binding fragment (preferably, 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 IgG constant domain or its FcRn binding fragment (preferably, 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 EA et al., (1991) supra)). In some embodiments, the constant region of the IgG1 of the antibodies described herein comprises a methionine (M) to tyrosine (Y) substitution at position 252, a serine (S) to threonine (T) substitution at position 254, and a threonine (T) to glutamic acid (E) substitution at position 256, as numbered according to the EU index as in Kabat. See U.S. Patent No. 7,658,921, which is incorporated herein by reference. This type of mutant IgG (referred to as a "YTE mutant") has been shown to have a 4-fold increase in half-life compared to the wild-type form of the same antibody (see Dall'Acqua WF et al., (2006) J Biol Chem 281: 23514-24). In some embodiments, the antibody comprises an IgG constant domain comprising one, two, three, or more amino acid substitutions at amino acid residues at positions 251 to 257, 285 to 290, 308 to 314, 385 to 389, and 428 to 436, as numbered according to the EU index as in Kabat.

[0411] In some embodiments, one, two, or more amino acid substitutions are introduced into the Fc region of an IgG constant domain to alter the effector function of an anti-transferrin receptor antibody. The effector ligand to which affinity is altered can be, for example, an Fc receptor or the C1 component of complement. This approach is described in more detail in U.S. Patent Nos. 5,624,821 and 5,648,260. In some embodiments, deletion or inactivation of a constant region domain (by point mutation or otherwise) can reduce Fc receptor binding of circulating antibodies, thereby improving tumor localization. For descriptions of mutations that delete or inactivate constant domains 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 can be introduced into the Fc region of an antibody described herein to remove potential glycosylation sites on the Fc region, which can reduce Fc receptor binding (see, for example, Shields RL et al., (2001) J Biol Chem 276: 6591-604).

[0412] In some embodiments, one or more amino groups in the constant region of the muscle-targeting antibodies described herein can be replaced with different amino acid residues so that the antibody has 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 antibodies described herein are altered to alter 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 antibodies described herein is modified to improve the antibody's ability to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or (e.g., and) to improve the antibody's affinity for Fcγ receptors. This approach is further described in International Publication No. WO 00 / 42072.

[0413] 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 produce, for example, CDR-grafted, chimeric, humanized or composite human antibodies or antigen-binding fragments, as described elsewhere herein. As understood by those of ordinary skill in the art, any variant (CDR-grafted, chimeric, humanized or composite antibody) derived from any antibody provided herein can be used in the compositions and methods described herein, and will maintain the ability to specifically bind to transferrin receptor, such that relative to the original antibody from which it is derived, the variant (CDR-grafted, chimeric, humanized or composite antibody) has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or more binding to transferrin receptor.

[0414] In some embodiments, the antibodies provided herein include mutations that confer desired properties on the antibodies. For example, to avoid potential complications due to known Fab arm exchange with natural IgG4 mAb, the antibodies provided herein may include a stability '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), wherein the 228th position (EU numbering, according to Kabat numbering, the 241st residue) serine is converted to a proline, thereby generating an IgG1-like hinge sequence. Thus, any antibody may include a stability 'Adair' mutation.

[0415] As provided herein, the antibodies of the present disclosure may optionally comprise a constant region or a portion thereof. For example, the VL domain may be connected to a light chain constant domain, such as Cκ or Cλ, at its C-terminus. Similarly, the VH domain or a portion thereof may be connected to all or a portion of a heavy chain such as IgA, IgD, IgE, IgG, and IgM (and any isotype subclass). The antibody may include a suitable constant region (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, No. 91-3242, National Institutes of Health Publications, Bethesda, Md. (1991)). Thus, antibodies within the scope of the present disclosure may include VH and VL domains or their antigen-binding portion(s) in combination with any suitable constant region.

[0416] ii. Muscle-targeted peptides

[0417] Some aspects of the present disclosure provide muscle-targeting peptides as muscle-targeting agents.Short peptide sequences (eg, peptide sequences 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: 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, issued 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 each of which are incorporated herein by reference. By designing peptides to interact with specific cell surface antigens (e.g., receptors), selectivity for desired tissues, such as muscle, can be achieved. Skeletal muscle targeting has been studied and can deliver a range of molecular payloads. These approaches can be highly selective for muscle tissue without many of the practical disadvantages of large antibodies or viral particles. Thus, in some embodiments, the muscle targeting agent is a muscle targeting peptide having a length of 4 to 50 amino acids. In some embodiments, the muscle targeting peptide is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length.Muscle-targeting peptides can be produced using any of several methods, such as phage display.

[0418] In some embodiments, the muscle-targeting peptide can bind to an internalized cell surface receptor that is overexpressed or relatively highly expressed in muscle cells compared to certain other cells, such as the transferrin receptor. In some embodiments, the muscle-targeting peptide can target the transferrin receptor (e.g., bind thereto). In some embodiments, the peptide targeting the transferrin receptor can 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 on November 30, 2000, “RECEPTOR-MEDIATED UPTAKE OF PEPTIDES THAT 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 hybrid peptide 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, “TRANSFERRIN / TRANSFERRIN RECEPTOR-MEDIATED SIRNA DELIVERY.”

[0419] As discussed above, some examples of muscle-targeting peptides have been reported. For example, muscle-specific peptides were 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 murine myotubes in vitro and to mouse muscle tissue in vivo. Thus, in some embodiments, a muscle-targeting agent comprises the amino acid sequence ASSLNIA (SEQ ID NO: 138). This peptide exhibits increased specificity for binding to cardiac and skeletal muscle tissue, and reduced binding to liver, kidney, and brain following intravenous injection in mice. Additional muscle-specific peptides have been identified using phage display. For example, a 12-amino acid peptide was identified using a phage display library for muscle targeting in the context of DMD treatment. 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 having the sequence SKTFNTHPQSTP (SEQ ID NO: 139) was identified and this muscle-targeting peptide showed improved binding to C2C12 cells relative to the ASSLNIA (SEQ ID NO: 138) peptide.

[0420] Another method for identifying peptides that are selective for muscle (e.g., skeletal muscle) relative to other cell types involves in vitro selection, which is 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 a random 12-mer peptide phage display library 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. Thus, in some embodiments, the muscle targeting agent comprises the amino acid sequence TARGEHKEEELI (SEQ ID NO: 140).

[0421] Muscle targeting agents can be amino acid-containing molecules or peptides. Muscle targeting peptides can correspond to protein sequences that preferentially bind to protein receptors found in muscle cells. In some embodiments, muscle targeting peptides contain highly propensity hydrophobic amino acids, such as valine, such that the peptide preferentially targets muscle cells. In some embodiments, muscle targeting peptides are previously uncharacterized 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-scanning synthetic peptide combinatorial libraries). Exemplary methods have been characterized in the art and incorporated by reference (Gray, BP and Brown, KC “Combinatorial Peptide Libraries: Mining for Cell-Binding Peptides” Chem Rev. 2014, 114: 2, 1020-1081.; Samoylova, TI and Smith, BF “Elucidation of muscle-binding peptides byphage display screening.” Muscle Nerve, 1999, 22: 4.460-6.).In some embodiments, muscle-targeting peptides have been previously disclosed (see, e.g., Writer MJ et al. “Targeted gene delivery to human airway epithelial cells with synthetic vectors incorporating novel targeting peptides selected by phage display.” J. Drug Targeting. 2004; 12: 185; Cai, D. “BDNF-mediated enhancement of inflammation and injury in the aging heart.” Physiol Genomics. 2006, 24: 3, 191-7.; Zhang, L. “Molecular profiling of heart endothelial cells.” Circulation, 2005, 112: 11, 1601-11.; McGuire, MJ et al. “In vitro selection of a peptide with high selectivity for cardiomyocytes in vivo.” J Mol Biol. 2004, 342: 1, 171-82.). Exemplary muscle targeting peptides include amino acid sequences from the group consisting of 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).

[0422] 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, 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, e.g., Silvana, MG et al. Mol. Therapy, 2018, 26: 1, 132-147.).

[0423] iii. Muscle-targeted receptor ligands

[0424] The muscle targeting agent can be a ligand, such as a ligand that binds to a receptor protein. The muscle targeting ligand can be a protein, such as transferrin, that binds to an internalized cell surface receptor expressed by muscle cells. Thus, in some embodiments, the muscle targeting agent is transferrin or a transferrin derivative that binds to a transferrin receptor. The muscle targeting ligand can alternatively be a small molecule, such as a lipophilic small molecule that preferentially targets 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, linolene, linoleic acid, myristic acid, sterols, dihydrotestosterone, testosterone derivatives, glycerol, alkyl chains, trityl groups, and alkoxy acids.

[0425] iv. Muscle-targeting aptamers

[0426] Muscle targeting agents can be aptamers, such as RNA aptamers, which preferentially target muscle cells relative to other cell types. In some embodiments, muscle targeting aptamers are previously uncharacterized or disclosed. Any of several methods (e.g., systematic evolution of ligands by exponential enrichment) can be used to conceive, produce, synthesize, and / or (e.g., and) derive these aptamers. Exemplary methods have been characterized in the art and incorporated by reference into (Yan, AC and Levy, M. "Aptamers and aptamer targeted delivery" RNA biology, 2009, 6: 3, 316-20.; Germer, K. et al. "RNA aptamers and their therapeutic and diagnostic applications." Int. J. Biochem. Mol. Biol. 2013; 4: 27-40.). In some embodiments, muscle-targeting aptamers have been previously disclosed (see, e.g., Phillippou, S. et al. "Selection and Identification of Skeletal-Muscle-Targeted RNA Aptamers." Mol Ther Nucleic Acids. 2018, 10: 199-214.; Thiel, W. et al. "Smooth Muscle Cell-targeted RNA Aptamer Inhibits Neointimal Formation." Mol Ther. 2016, 24: 4, 779-87.). Exemplary muscle-targeting aptamers include A01B RNA aptamer and RNA Apt 14. In some embodiments, the aptamer is a nucleic acid-based aptamer, an oligonucleotide aptamer, or a peptide aptamer. In some embodiments, the aptamer can 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.

[0427] v. Other muscle-targeting agents

[0428] 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 targeting agent is a substrate of an influx transporter that is specific for muscle tissue. In some embodiments, the influx transporter is specific for skeletal muscle tissue. Two major classes of transporters are expressed on the skeletal muscle sarcolemma: (1) the adenosine triphosphate (ATP) binding cassette (ABC) superfamily, which facilitates efflux from skeletal muscle tissue and (2) the solute carrier (SLC) superfamily, which facilitates substrate influx into skeletal muscle. In some embodiments, the muscle targeting agent is a substrate that binds to the ABC superfamily or the SLC superfamily of transporters. In some embodiments, the substrate that binds to the ABC or SLC superfamily of transporters is a naturally occurring substrate. In some embodiments, the substrate that binds to the ABC or SLC superfamily of transporters is a non-naturally occurring substrate, for example, a synthetic derivative thereof that binds to the ABC or SLC superfamily of transporters.

[0429] In some embodiments, the muscle targeting agent is a substrate of the SLC superfamily of transporters. SLC transporters are equilibrative, or use a proton or sodium ion gradient across the membrane to drive transport of substrates. Exemplary SLC transporters with high skeletal muscle expression include, but are not limited to, SATT transporter (ASCT1; SLC1A4), GLUT4 transporter (SLC2A4), GLUT7 transporter (GLUT7; SLC2A7), ATRC2 transporter (CAT-2SLC7A2), LAT3 transporter (KIAA0245; SLC7A6), PHT1 transporter (PTR4; SLC15A4), OATP-J transporter (OATP5A1; SLC21A15), OCT3 transporter (EMT; SLC22A3), OCTN2 transporter (FLJ46769; SLC22A5), ENT transporter (ENT1; SLC29A1 and ENT2; SLC29A2), PAT2 transporter (SLC36A2), and SAT2 transporter (KIAA1382; SLC38A2). These transporters can facilitate substrate influx into skeletal muscle, providing opportunities for muscle targeting.

[0430] In some embodiments, the muscle targeting agent is a substrate for the equilibrative nucleoside transporter 2 (ENT2) transporter. ENT2 has one of the highest mRNA expressions in skeletal muscle relative to other transporters. Although 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 absorption of its substrate, which is dependent on the concentration gradient of the substrate. ENT2 plays a role in maintaining nucleoside homeostasis by transporting a wide range of purine and pyrimidine nucleobases. The hENT2 transporter has low affinity for all nucleosides (adenosine, guanosine, uridine, thymidine, and cytidine) except inosine. Therefore, in some embodiments, the muscle targeting agent is an ENT2 substrate. Exemplary ENT2 substrates include, but are not limited to, inosine, 2',3'-dideoxyinosine, and calofarabine. In some embodiments, any muscle targeting agent provided herein is associated with a molecular payload (e.g., an oligonucleotide payload). In some embodiments, the muscle targeting agent is covalently linked to the molecular payload. In some embodiments, the muscle targeting agent is non-covalently linked to the molecular payload.

[0431] In some embodiments, the muscle targeting agent is a substrate of the organic cation / carnitine transporter (OCTN2), which is a sodium-dependent, high-affinity carnitine transporter. In some embodiments, the muscle targeting agent is carnitine, meldonium, acetylcarnitine, or any derivative thereof that binds to OCTN2. In some embodiments, carnitine, meldonium, acetylcarnitine, or a derivative thereof is covalently linked to a molecular payload (e.g., an oligonucleotide payload).

[0432] The muscle targeting agent can be a protein that is present in at least one soluble form that targets muscle cells. In some embodiments, the muscle targeting protein can be hemojuvelin (also known as repulsive guidance molecule C or hemochromatosis type 2 protein), a protein involved in iron overload and homeostasis. In some embodiments, hemojuvelin 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 to a functional hemojuvelin protein. In some embodiments, the hemojuvelin mutant can be a soluble fragment, can lack an N-terminal signaling domain, and / or (for example, and) lack a C-terminal anchoring domain. In some embodiments, hemojuvelin can be annotated with GenBank RefSeq accession numbers NM_001316767.1, NM_145277.4, NM_202004.3, NM_213652.3, or NM_213653.3. It will be understood that hemojuvelin can be of human, non-human primate, or rodent origin.

[0433] B. Molecular Loading

[0434] Some aspects of the present disclosure provide molecular payloads, for example, for regulating biological outcomes, such as transcription of a DNA sequence, expression of a protein, or activity of a protein. In some embodiments, the molecular payload is connected to or otherwise associated with a muscle targeting agent. In some embodiments, such a molecular payload is capable of targeting muscle cells, for example, by specifically binding to nucleic acids or proteins in muscle cells after being delivered to muscle cells by an associated muscle targeting agent. It should be understood that various types of muscle targeting agents can be used according to the present disclosure. For example, a molecular payload may comprise or consist of an oligonucleotide (e.g., an antisense oligonucleotide), a peptide (e.g., a peptide that binds to a disease-associated nucleic acid or protein in a muscle cell), a protein (e.g., a protein that binds to a disease-associated nucleic acid or protein in a muscle cell), or a small molecule (e.g., a small molecule that regulates the function of a disease-associated nucleic acid or protein in a muscle cell). In some embodiments, the molecular payload is an oligonucleotide comprising a chain having a complementary region to the 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.

[0435] In some embodiments, at least one (e.g., at least 2, at least 3, at least 4, at least 5, at least 10) molecular payloads (e.g., oligonucleotides) are attached to a muscle targeting agent. In some embodiments, all molecular payloads attached to a muscle targeting agent are identical, e.g., targeting the same gene. In some embodiments, all molecular payloads attached to a muscle targeting agent are different, e.g., the molecular payloads can target different portions of the same target gene, or the molecular payloads can target at least two different target genes. In some embodiments, a muscle targeting agent can be attached to some identical molecular payloads and others different molecular payloads.

[0436] The present disclosure also provides compositions comprising a plurality of 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 comprise a molecular targeting agent linked to the same number of molecular payloads (e.g., oligonucleotides).

[0437] i. Oligonucleotides

[0438] As described herein, any suitable oligonucleotide can be used as molecular payload.In some embodiments, oligonucleotide can be designed to cause the degradation of mRNA (for example, oligonucleotide can be the interval polymer, siRNA, ribozyme or aptamer that causes degradation).In some embodiments, oligonucleotide can be designed to block the translation of mRNA (for example, oligonucleotide can be the mixed polymer, siRNA or aptamer that blocks translation).In some embodiments, oligonucleotide can be designed to cause the degradation of mRNA and block the translation of mRNA.In some embodiments, oligonucleotide can be the guide nucleic acid (for example, guide RNA) for guiding enzyme (for example, gene editing enzyme) activity.Other examples of oligonucleotide are provided herein.It should be understood that in some embodiments, it is possible to make the oligonucleotide (for example, antisense oligonucleotide) of a format suitably adapted to another format (for example, siRNA oligonucleotide) by incorporating functional sequence (for example, antisense strand sequence) from a format into another format.

[0439] In some embodiments, the oligonucleotide may comprise a complementary region to a target gene provided in Table 1. Additional non-limiting examples are provided below for selected genes of Table 1.

[0440] DMPK / DM1

[0441] In some embodiments, some 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 on January 1, 2010, entitled Compound And Method For Treating Myotonic Dystrophy; U.S. Patent Application Publication 20130237585A1, published on July 19, 2010, entitled Modulation Of Dystrophia Myotonica-Protein Kinase (DMPK) Expression; U.S. Patent Application Publication 20150064181A1, published on March 5, 2015, entitled “Antisense Conjugates For Decreasing Expression Of Dmpk”; U.S. Patent Application Publication 20150238627A1, published on August 27, 2015, entitled “Peptide-Linked Morpholino Antisense 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 Type1 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 No. 20160304877A1, published on October 20, 2016, entitled "Compounds And Methods For Modulation Of Dystrophia Myotonica-Protein Kinase (Dmpk) Expression", the contents of each of which are incorporated herein by reference in their entirety.

[0442] Some examples of oligonucleotides for promoting 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 each of which are incorporated herein by reference in their entirety.

[0443] In some embodiments, the oligonucleotide may have a complementary region to a mutant form of DMPK, such as those reported in: Botta A. et al. "The CTG repeat expansion size correlates 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 each of which are incorporated herein by reference in their entirety.

[0444] In some embodiments, the oligonucleotide provided herein is the antisense oligonucleotide of targeting DMPK. In some embodiments, the oligonucleotide of targeting is any antisense oligonucleotide (for example, spaced polymer) of targeting DMPK, as described in U.S. Patent Application Publication US20160304877A1, which is disclosed on 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 oligonucleotide targeting of targeting DMPK is as shown in Genbank Login No.NM_001081560.2 or as shown in Genbank Login No.NG_009784.1. The region of the DMPK gene sequence.

[0445] In some embodiments, an oligonucleotide targeting DMPK comprises a nucleotide sequence comprising a complementary region to a target region of 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.

[0446] In some embodiments, the oligonucleotide targeting DMPK comprises a spacer motif. "Spacer" means a chimeric antisense compound wherein the inner region with a plurality of nucleotides supporting RNAse H cleavage is located between the outer regions with one or more nucleotides, wherein the nucleotides comprising the inner region are chemically different from the one or more nucleotides comprising the outer region. The inner region can be referred to as a "spacer segment" and the outer region can be referred to as a "wing segment". In some embodiments, the oligonucleotide targeting DMPK comprises one or more modified nucleotides and / or (for example, and) one or more modified internucleotide linkages. In some embodiments, the internucleotide linkage is a thiophosphate linkage. In some embodiments, the oligonucleotide comprises a backbone (backbone) of a complete thiophosphate. In some embodiments, the oligonucleotide is a DNA spacer with a cET end (for example, 3-10-3; cET-DNA-cET). In some embodiments, an oligonucleotide targeting DMPK comprises one or more 6'-(S)-CH3 biocyclic nucleotides, one or more β-D-2'-deoxyribonucleotides and / or (eg, and) one or more 5-methylcytosine nucleotides.

[0447] DUX4 / FSHD

[0448] In some embodiments, some examples of oligonucleotides that can be used to target DUX4 (e.g., for the treatment of FSHD) are provided below: U.S. Patent No. 9,988,628, which published on February 2, 2017, and is entitled “AGENTS USEFUL INTREATING FACIOSCAPULOHUMERAL MUSCULAR DYSTROPHY”; U.S. Patent No. 9,469,851, which published on October 30, 2014, and is entitled “RECOMBINANT VIRUS PRODUCTS AND METHODS FOR INHIBITING EXPRESSION OF DUX4”; U.S. Patent Application Publication No. 20120225034, which published on September 6, 2012, and is 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 each of which are incorporated herein in their entirety. In some embodiments, the oligonucleotide is an antisense oligonucleotide, a morpholino, an siRNA, an shRNA, or other nucleotide that hybridizes to the target DUX4 gene or mRNA.

[0449] In some embodiments, for example, for the treatment of FSHD, the oligonucleotide may have a region complementary to the hypomethylated compact D4Z4 repeats, as described in Daxinger, et al., "Genetic and Epigenetic Contributors to FSHD," published in Curr Opin Genet Dev in 2015, Lim JW, et al., DICER / AGO-dependent epigenetic silencing of D4ZA repeats enhanced by exogenous siRNA suggests mechanisms and therapies for FSHD Hum Mol Genet. 2015 Sep 1; 24(17): 4817-4828, the contents of each of which are incorporated herein in their entirety.

[0450] DNM2 / CNM

[0451] In some embodiments, some examples of oligonucleotides that can be used to target DNM2 (e.g., for treating CNMs) are provided in U.S. Patent Application Publication No. 20180142008, published on May 24, 2018, entitled “DYNAMIN 2 INHIBITOR FOR THE TREATMENT OF DUCHENNE'S MUSCULAR DYSTROPHY,” and PCT Application Publication No. WO 2018 / 100010A1, published on June 7, 2018, entitled “ALLELE-SPECIFIC SILENCING THERAPY FOR DYNAMIN 2-RELATED DISEASES.” For example, in some embodiments, the oligonucleotide is an RNAi, antisense nucleic acid, siRNA, or ribozyme that specifically interferes with DNM2 expression. Other examples of 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 in Mol. Ther. on April 4, 2018, 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 a wild-type DNM2 that is 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.Additional 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 PLoS One in 2013, the contents of each of which are incorporated herein in their entirety.

[0452] In some embodiments, for example, for the treatment of CNM, the oligonucleotide may have a region complementary to a mutant in DNM2 associated with CNM, as described in B6hm et al, "Mutation Spectrum in the Large GTPase Dynamin 2, and Genotype-Phenotype Correlation in Autosomal Dominant Centronuclear Myopathy," as published in Hum. Mutat., 2012, the contents of which are incorporated herein in their entirety.

[0453] Pompe disease

[0454] In some embodiments, for example, 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., "GAA Deficiency in Pompe Disease is Alleviated by Exon Inclusion in iPSC-Derived Skeletal Muscle Cells," Mol Ther Nucleic Acids. 2017 Jun 16; 7: 101-115, the contents of which are incorporated herein by reference. Thus, in some embodiments, the oligonucleotide may have a complementary region to the GAA disease allele.

[0455] In some embodiments, for example, for the treatment of Pompe disease, oligonucleotides (e.g., RNAi or antisense oligonucleotides) are utilized to inhibit the expression of wild-type GYS1 in muscle cells, as reported, for example, in Clayton, et al., “Antisense Oligonucleotide-mediated Suppression of Muscle Glycogen Synthase 1 Synthesis as an Approach for Substrate Reduction Therapy of Pompe Disease,” published in Mol Ther Nucleic Acids in 2017, or 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. Thus, in some embodiments, an oligonucleotide can have an antisense strand having a complementary region to a sequence corresponding to the human GYS1 sequence of RefSeq No. NM_002103.4 and / or (eg, and) a sequence corresponding to the mouse GYS1 sequence of RefSeq No. NM_030678.3.

[0456] ACVR1 / FOP

[0457] In some embodiments, some examples of oligonucleotides that can be used to target ACVR1 (e.g., for the treatment of FOP) are provided below: U.S. Patent Application 2009 / 0253132, published on October 8, 2009, “Mutated ACVR1 for diagnosis and treatment of fibrodyplasia ossificans progressiva (FOP)”; WO 2015 / 152183, published on October 8, 2015, “Prophylactic agent and therapeutic agent for fibrodysplasia ossificans progressive”; Lowery, J. Wet 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” Gene Therapy (2012) 19, 781-785; Shi, S. et al. “Antisense-Oligonucleotide Mediated Exon Skipping in Activin-Receptor-Like Kinase 2: Inhibiting the Receptor That Is Overactive in Fibrodysplasia Ossificans Progressiva” Plos One, July 2013, Vol. 8:7, e69096.; U.S. Patent Application 2017 / 0159056, published on June 8, 2017, “Antisense oligonucleotides and methods of use thereof”; U.S. Patent No.8,859,752, granted on October 4, 2014, “SIRNA-based therapy of Fibrodyplasia Ossificans Progressiva (FOP)”; WO2004 / 094636, published on November 4, 2004, “Effective siRNA knock-down constructs,” the contents of each of which are incorporated herein in their entirety.

[0458] FXN / Friedreich's Ataxia

[0459] In some embodiments, some examples of oligonucleotides that can be used to target FXN and / or (e.g., and) otherwise compensate for frataxin deficiency (e.g., for treating 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"; WO 2017 / 186815, published November 2, 2017, "Antisense oligonucleotides for enhanced expression of frataxin"; WO 2008 / 018795, published on February 14, 2008, “Methods and means for treating DNA repeat instability associated genetic disorders”; U.S. patent application 2018 / 0028557, published on February 1, 2018, “Hybrid oligonucleotides and uses thereof”; WO 2015 / 023975, published on February 19, 2015, “Compositions and methods for modulating RNA”; WO 2015 / 023939, published on February 19, 2015, “Compositions and methods for modulating expression of frataxin”; U.S. patent application 2017 / 0281643, published on October 5, 2017, “Compounds and methods for modulating frataxin expression”; Li L. et al."Activating frataxin expression by repeat-targeted nucleic acids" Nature Communications, published on February 4, 2016; and Li L. et al. "Activation of Frataxin Protein Expression by Antisense Oligonucleotides Targeting the Mutant Expanded Repeat" Nucleic Acid Ther. 2018 Feb; 28(1): 23-33., the contents of each of which are incorporated herein in their entirety.

[0460] In some embodiments, the oligonucleotide payload is configured (e.g., as a spacer or RNAi oligonucleotide) to inhibit expression of a natural antisense transcript that inhibits FXN expression, e.g., as disclosed 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.

[0461] Some examples of oligonucleotides used to facilitate FXN gene editing include WO 2016 / 094845, published on June 16, 2016, “Compositions and methods for editing nucleic acids in cells utilizing oligonucleotides”; WO 2015 / 089354, published on June 18, 2015, “Compositions and methods of use of CRISPR-Cas systems in nucleotide repeat disorders”; WO 2015 / 139139, published on September 24, 2015, “CRISPR-based methods and products for increasing frataxin levels and uses thereof”; and WO 2018 / 002783, published on January 4, 2018, “Materials and methods for treatment of Friedreich ataxia and other related disorders,” the contents of each of which are incorporated herein in their entirety.

[0462] Some examples of oligonucleotides for promoting FXN gene expression by targeting non-FXN genes (e.g., epigenetic regulators of FXN) include WO 2015 / 023938, published February 19, 2015, "Epigenetic regulators of frataxin," the contents of which are incorporated herein in their entirety.

[0463] In some embodiments, the oligonucleotide can have a complementary region to the FXN gene from human (Gene ID 2395; NC_000009.12) and / or (eg, and) the FXN gene from mouse (Gene ID 14297; NC_000085.6). In some embodiments, the oligonucleotide may have a complementary region to a mutant form of FXN, e.g., as reported in: e.g., 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 each of which are incorporated herein by reference in their entirety.

[0464] DMD / Dystrophinopathy

[0465] Some examples of oligonucleotides that can be used to target DMD are provided in the following: U.S. Patent Application Publication US20100130591A1, which was published on May 27, 2010, and is entitled “MULTIPLE EXON SKIPPING COMPOSITIONS FOR DMD”; U.S. Patent No. 8,361,979, which was issued on January 29, 2013, and is entitled “MEANS AND METHOD FOR INDUCING EXON-SKIPPING”; U.S. Patent Application Publication 20120059042, which was published on March 8, 2012, and is entitled “METHOD FOR EFFICIENT EXON (44) SKIPPING IN DUCHENNE MUSCULARDYSTROPHY AND ASSOCIATED MEANS”; U.S. Patent Application Publication 20140329881, which was published on November 6, 2014, and is entitled “EXON SKIPPING COMPOSITIONS FOR TREATING U.S. Patent No. 8,232,384, issued on July 31, 2012, entitled “ANTISENSE OLIGONUCLEOTIDES FOR INDUCING EXON SKIPPING AND METHODS OF USE THEREOF”; U.S. Patent Application Publication 20120022134A1, published on January 26, 2012, entitled “METHODS AND MEANS FOR EFFICIENTSKIPPING OF EXON 45 IN DUCHENNE MUSCULAR DYSTROPHY PRE-MRNA”; U.S. Patent Application Publication 20120077860, published on March 29, 2012, entitled “ADENO-ASSOCIATED VIRAL VECTOR FOR EXON SKIPPING IN A GENE ENCODING A DISPENSABLE DOMAN PROTEIN”; U.S. Patent No. 8,324,371, issued on December 4, 2012, entitled “OLIGOMERS”; U.S. Patent No. 9,078,911, issued on July 14, 2015, entitled “ANTISENSE OLIGONUCLEOTIDES”; U.S. Patent No. 9,079,934, issued on July 14, 2015, entitled “ANTISENSE NUCLEIC ACIDS”; U.S. Patent No.9,034,838, issued May 19, 2015, entitled “MIR-31 IN DUCHENNE MUSCULAR DYSTROPHY THERAPY”; and International Patent Publication WO2017062862A3, published April 13, 2017, entitled “OLIGONUCLEOTIDE COMPOSITIONS AND METHODS THEREOF”; the contents of each of which are incorporated herein in their entirety.

[0466] Some examples of oligonucleotides used to facilitate DMD gene editing include International Patent Publication WO2018053632A1, published on March 29, 2018, entitled “METHODS OF MODIFYING THE DYSTROPHIN GENE AND RESTORING DYSTROPHIN EXPRESSION AND USES THEREOF”; International Patent Publication WO2017049407A1, published on March 30, 2017, entitled “MODIFICATION OF THE DYSTROPHIN GENE AND USES THEREOF”; International Patent Publication WO2016161380A1, published on October 6, 2016, entitled “CRISPR / CAS-RELATED METHODS AND COMPOSITIONS FOR TREATING DUCHENNE MUSCULAR DYSTROPHY AND BECKER MUSCULAR DYSTROPHY”; 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 METHODS FOR TREATMENT OF DUCHENNE MUSCULAR DYSTROPHY”; International Patent Publication WO2018098480A1, published on May 31, 2018, entitled “PREVENTION OF MUSCULAR DYSTROPHY BY CRISPR / CPF 1-MEDIATED GENE EDITING”; U.S. 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 No. 2016 / 0201089, published on July 14, 2016, entitled “RNA-GUIDED GENE EDITING AND GENE REGULATION”; and U.S. Patent Application Publication No. 2013 / 0145487, published on June 6, 2013, entitled “MEGANUCLEASE VARIANTSCLEAVING A DNA TARGET SEQUENCE FROM THE DYSTROPHN GENE AND USES THEREOF,” the contents of each of which are incorporated herein in their entirety. In some embodiments, the oligonucleotides may have complementary regions to DMD gene sequences from multiple species (e.g., selected from human, mouse, and non-human species).

[0467] In some embodiments, the oligonucleotide can have a complementary region to a mutant DMD allele, such as a DMD allele having at least one mutation in any one of exons 1 to 79 of human DMD that results in a frameshift and improper RNA splicing / processing.

[0468] MYH7 / Hypertrophic Cardiomyopathy

[0469] Some examples of oligonucleotides that can be used as payloads (e.g., for targeting MYH7) are provided in: U.S. Patent Application Publication 20180094262, published on April 5, 2018, entitled Inhibitors of MYH7B and Uses Thereof; U.S. Patent Application Publication 20160348103, published on December 1, 2016, entitled Oligonucleotides and Methods for Treatment of Cardiomyopathy Using RNA Interference; U.S. Patent Application Publication 20160237430, published on August 18, 2016, entitled “Allele-specific RNA Silencing for the Treatment of Hypertrophic Cardiomyopathy”; U.S. Patent Application Publication 20160032286, published on February 4, 2016, entitled “Inhibitors of MYH7B and Uses Thereof”. Thereof”; U.S. Patent Application Publication 20140187603, published on July 3, 2014, entitled “MicroRNA Inhibitors Comprising Locked Nucleotides”; U.S. Patent Application Publication 20140179764, published on 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 on May 10, 2012, entitled “Compositions and Methods to Treat Muscular and Cardiovascular Disorders”; the contents of each of which are incorporated herein in their entirety.

[0470] In some embodiments, oligonucleotides can target lncRNA or mRNA, for example, for degradation. In some embodiments, oligonucleotides can target (for example, 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 (wherein the mRNA encoding such proteins can be targeted by the oligonucleotides described herein) are described in: 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" DNA Repair (Amst). 2016 Feb; 38: 117-26.

[0471] In some embodiments, any of the oligonucleotides can be in the form of a salt, for example, as a sodium salt, potassium salt, or magnesium salt.

[0472] In some embodiments, the 5' or 3' nucleoside (e.g., terminal nucleoside) of any oligonucleotide described herein is optionally conjugated to an amine 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 linkage is present between the spacer and the 5' or 3' nucleoside of the oligonucleotide. In some embodiments, the 5' or 3' nucleoside (e.g., terminal nucleoside) of any oligonucleotide described herein is conjugated to a spacer, wherein the spacer is a substituted or unsubstituted aliphatic, a substituted or unsubstituted heteroaliphatic, a substituted or unsubstituted carbocyclylene, a substituted or unsubstituted heterocyclylene, a substituted or unsubstituted arylene, a substituted or unsubstituted heteroarylene, -O-, -N(R A )-, -S-, -C(=O)-, -C(=O)O-, -C(=O)NR A -,-NR A C(=O)-,-NR A C(=O)R A -,-C(=O)R A -,-NR A C(=O)O-,-NR A C(=O)N(R A )-, -OC(=O)-, -OC(=O)O-, -OC(=O)N(R A)-,-S(O)2NR A -,-NR A S(O)2-, or a combination thereof; each R A are independently hydrogen or substituted or unsubstituted alkyl. In certain embodiments, the spacer is substituted or unsubstituted alkylene, substituted or unsubstituted heterocyclylene, substituted or unsubstituted heteroarylene, -O-, -N(R A )-or-C(=O)N(R A )2, or a combination thereof.

[0473] In some embodiments, the 5' or 3' nucleoside of any of the oligonucleotides described herein is in the form of -NH2-(CH2) n -, wherein 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 linkage is present in the formula NH2-(CH2) n In some embodiments, the compound of formula NH2-(CH2)6- is conjugated to the oligonucleotide by reaction between 6-amino-1-hexanol (NH2-(CH2)6-OH) and the 5' phosphate of the oligonucleotide.

[0474] In some embodiments, the oligonucleotide is conjugated to a targeting agent, eg, a muscle targeting agent such as an anti-TfR antibody, eg, via an amine group.

[0475] a. Oligonucleotide size / sequence

[0476] In some embodiments, the oligonucleotide has a length of 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 oligonucleotide has a length of 8 to 50 nucleotides, a length of 8 to 40 nucleotides, a length of 8 to 30 nucleotides, a length of 10 to 15 nucleotides, a length of 10 to 20 nucleotides, a length of 15 to 25 nucleotides, a length of 21 to 23 nucleotides, etc.

[0477] In some embodiments, when the binding of the complementary nucleic acid sequence of the oligonucleotide to the target molecule (e.g., mRNA) interferes with the normal function of the target (e.g., mRNA) resulting in loss of activity (e.g., inhibition of translation) or expression (e.g., degradation of the target mRNA), and has a sufficient degree of complementarity to avoid non-specific binding of the sequence to non-target sequences under the following conditions, for the purposes of the present disclosure, the complementary nucleic acid sequence of the oligonucleotide can specifically hybridize with the target nucleic acid or has specificity for the target nucleic acid: under conditions where it is desired to avoid non-specific binding, such as under physiological conditions in the case of in vivo assays or therapeutic treatments, and in the case of in vitro assays, under conditions where the assay is performed under appropriate stringent conditions. Thus, in some embodiments, the oligonucleotide can 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 consecutive nucleotides of the target nucleic acid. In some embodiments, a complementary nucleotide sequence need not be 100% complementary to its targeted sequence to be specifically hybridizable to or specific for a target nucleic acid.

[0478] In some embodiments, the oligonucleotide comprises a complementary region to the target nucleic acid, the length of the 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 some embodiments, the length of the complementary region 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 some embodiments, the complementary region is complementary to at least 8 consecutive nucleotides of the target nucleic acid. In some embodiments, the oligonucleotide may comprise 1, 2, or 3 base mismatches compared to the continuous nucleotide portion of the target nucleic acid. In some embodiments, an oligonucleotide can have up to 3 mismatches over 15 bases, or up to 2 mismatches over 10 bases.

[0479] In some embodiments, the oligonucleotide is complementary (e.g., at least 85%, at least 90%, at least 95%, or 100% complementary) to the target sequence of any of the oligonucleotides provided herein. In some embodiments, such a target sequence is 100% complementary to an oligonucleotide described herein.

[0480] In some embodiments, any one or more thymine bases (T) in any of the oligonucleotides provided herein can optionally be uracil bases (U), and / or any one or more U can optionally be T.

[0481] b. Oligonucleotide modification:

[0482] The oligonucleotides described herein may be modified, for example, to comprise a modified sugar moiety, a modified internucleoside linkage, a modified nucleotide, and / or (e.g., and) a combination thereof. Additionally, in some embodiments, the oligonucleotide may exhibit one or more of the following properties: not mediating alternative splicing; not immunostimulatory; having nuclease resistance; having increased cellular uptake compared to unmodified oligonucleotides; being non-toxic to cells or mammals; increasing internal excretion of endosomes in cells; minimizing TLR stimulation; or avoiding pattern recognition receptors. Any modified chemical composition (chemistry) or form of the oligonucleotides described herein may be combined with one another. For example, one, two, three, four, five or more different types of modifications may be included in the same oligonucleotide.

[0483] In some embodiments, certain nucleotide modifications can be used that render the oligonucleotides into which they are incorporated more resistant to nuclease digestion than native oligodeoxynucleotide or oligoribonucleotide molecules; these modified oligonucleotides survive intact for longer periods of time than unmodified oligonucleotides. Some specific examples of modified oligonucleotides include those containing modified backbones, such as modified internucleoside linkages, such as phosphorothioate linkages, phosphotriester linkages, methylphosphonate linkages, short-chain alkyl linkages, or cycloalkyl sugar inter-linkages, or short-chain heteroatom linkages, or heterocyclic sugar inter-linkages. Thus, the oligonucleotides of the present disclosure can be stabilized against nucleolytic degradation, for example, by incorporating modifications, such as nucleotide modifications.

[0484] In some embodiments, the length of the oligonucleotide can be as many as 50 or as many as 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 can have every nucleotide except 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 modified nucleotides.Oligonucleotide modifications are further described herein.

[0485] c. Modified nucleosides

[0486] In some embodiments, the oligonucleotides described herein comprise at least one nucleoside modified at the 2' position of the sugar. In some embodiments, the oligonucleotides comprise at least one 2'-modified nucleoside. In some embodiments, all nucleosides in the oligonucleotides are 2'-modified nucleosides.

[0487] 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'-O-N-methylacetamido (2'-O-NMA) modified nucleosides.

[0488] In some embodiments, the oligonucleotides described herein comprise one or more 2'-4' bicyclic nucleosides in which the ribose ring comprises a bridging moiety connecting the two atoms in the ring, for example, a methylene (LNA) bridge, an ethylene (ENA) bridge, or an (S)-constrained ethyl (cEt) bridge connecting the 2'-O atom to the 4'-C atom. Some examples of LNAs are described in International Patent Application Publication No. WO / 2008 / 043753, published April 17, 2008, and entitled "RNA Antagonist Compounds For The Modulation Of PCSK9," the contents of which are incorporated herein by reference in their entirety. Some examples of ENAs are provided in International Patent Publication No. WO 2005 / 042777, published May 12, 2005, and entitled "APP / ENA Antisense"; Morita et al., Nucleic Acid Res., Suppl 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 entireties. Some examples of cEt are provided in: US Patents 7,101,993, 7,399,845, and 7,569,686, each of which is incorporated herein by reference in its entirety.

[0489] In some embodiments, the oligonucleotide comprises a modified nucleoside disclosed in one of the following U.S. patents or patent application publications: U.S. Patent 7,399,845, which issued on July 15, 2008, and is entitled “6-Modified Bicyclic Nucleic Acid Analogs”; U.S. Patent 7,741,457, which issued on June 22, 2010, and is entitled “6-Modified Bicyclic Nucleic Acid Analogs”; U.S. Patent 8,022,193, which issued on September 20, 2011, and is entitled “6-Modified Bicyclic Nucleic A cid Analogs”; U.S. Patent 7,569,686, which issued on August 4, 2009, and is entitled “Compounds And Methods For Synthesis Of Bicyclic Nucleic Acid Analogs”; U.S. Patent 7,335,765, which issued on February 26, 2008, and is entitled “Novel Nucleoside And Oligonucleotide Analogues”; U.S. Patent 7,314,923, issued on January 1, 2008, and entitled “Novel Nucleoside And Oligonucleotide Analogues”; U.S. Patent 7,816,333, issued on October 19, 2010, and entitled “Oligonucleotide Analogues And Methods Utilizing The Same”; and U.S. Publication No. 2011 / 0009471, now U.S. Patent 8,957,201, issued on February 17, 2015, and entitled “Oligonucleotide Analogues And Methods Utilizing The Same,” the entire contents of each of which are incorporated herein by reference for all purposes.

[0490] In some embodiments, an oligonucleotide comprises at least one modified nucleoside that results in an increase in the Tm of the oligonucleotide by 1° C., 2° C., 3° ​​C., 4° C., or 5° C. compared to an oligonucleotide that does not have at least one modified nucleoside. An oligonucleotide may have a plurality of modified nucleosides that result in an overall increase in the Tm of the oligonucleotide 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 more compared to an oligonucleotide that does not have modified nucleosides.

[0491] Oligonucleotides may comprise a mixture of different types of nucleosides. For example, an oligonucleotide may comprise a mixture of 2'-deoxyribonucleosides or ribonucleosides and 2'-fluoro modified nucleosides. An oligonucleotide may comprise a mixture of deoxyribonucleosides or ribonucleosides and 2'-O-Me modified nucleosides. An oligonucleotide may comprise a mixture of 2'-fluoro modified nucleosides and 2'-O-Me modified nucleosides. An oligonucleotide may comprise a mixture of 2'-4' bicyclic nucleosides 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).

[0492] Oligonucleotides may comprise different types of alternative nucleosides. For example, an oligonucleotide may comprise alternative 2'-deoxyribonucleosides or ribonucleosides and 2'-fluoro modified nucleosides. An oligonucleotide may comprise alternative deoxyribonucleosides or ribonucleosides and 2'-O-Me modified nucleosides. An oligonucleotide may comprise alternative 2'-fluoro modified nucleosides and 2'-O-Me modified nucleosides. An oligonucleotide may comprise alternative 2'-4' bicyclic nucleosides and 2'-MOE, 2'-fluoro or 2'-O-Me modified nucleosides. An oligonucleotide may comprise alternative 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).

[0493] In some embodiments, an oligonucleotide described herein comprises a 5'-vinylphosphonate modification, one or more abasic residues, and / or one or more inverted abasic residues.

[0494] d. Internucleoside linkages / backbone

[0495] In some embodiments, the oligonucleotide may comprise a phosphorothioate linkage or other modified internucleoside linkage. In some embodiments, the oligonucleotide comprises a phosphorothioate internucleoside linkage. In some embodiments, the oligonucleotide comprises a phosphorothioate internucleoside linkage between at least two nucleotides. In some embodiments, the oligonucleotide comprises a phosphorothioate internucleoside linkage between all nucleotides. For example, in some embodiments, the oligonucleotide comprises a modified internucleoside linkage at the first, second, and / or (e.g., and) third internucleoside linkage at the 5' or 3' end of the nucleotide sequence.

[0496] Phosphorus-containing linkages that may be used include, but are not limited to, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methylphosphonates and other alkylphosphonates including 3'alkylenephosphonates, as well as chiral phosphonates, phosphinates, phosphoramidates including 3'-aminophosphoramidates and aminoalkylphosphoramidates, thionylphosphoramidates, thionylalkylphosphonates, thionylalkylphosphotriesters and boranophosphates with normal 3'-5' linkages, 2'-5' linked analogs of these, and those with opposite polarity where adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'; see U.S. Patent No.

[0497] 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.

[0498] 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, with the nucleotides being bound directly or indirectly to aza nitrogen atoms of the polyamide backbone; see Nielsen et al., Science 1991, 254, 1497).

[0499] e. Stereospecific oligonucleotides

[0500] In some embodiments, the internucleoside phosphorus atom of the oligonucleotide is chiral, and the properties of the oligonucleotide are adjusted based on the configuration of the chiral phosphorus atom. In some embodiments, an appropriate method 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 phosphorusatoms. Chem Soc Rev. 2011 Dec; 40 (12): 5829-43). In some embodiments, a thiophosphate-containing oligonucleotide is provided, which comprises nucleoside units linked together by substantially all Sp or substantially all Rp thiophosphate sugar interlinkages. In some embodiments, such thiophosphate oligonucleotides having substantially chiral pure sugar interlinkages are prepared by enzymatic or chemical synthesis, as described, for example, in U.S. Pat. No. 5,587,261, issued on 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 of the target nucleic acid. For example, in some embodiments, chirality-controlled oligonucleotides provide for single-site cleavage within the complementary sequence of a nucleic acid, as described, for example, in U.S. Patent Application Publication No. 20170037399A1, published on February 2, 2017, entitled “CHIRAL DESIGN,” the contents of which are incorporated herein by reference in their entirety.

[0501] f. Morpholino

[0502] In some embodiments, the oligonucleotide can be a morpholino-based compound. Morpholino-based oligomeric compounds 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; Nasevicius et al., Nat. Genet., 2000, 26, 216-220; Lacerra 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 morpholino-based oligomeric compound 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 herein incorporated by reference in their entireties).

[0503] g. Peptide nucleic acid (PNA)

[0504] In some embodiments, the sugar and internucleoside linkages (main chain) of the nucleotide units of the oligonucleotides are both replaced by new groups. In some embodiments, the base unit is maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound (an oligonucleotide mimic having excellent hybridization properties) is referred to as peptide nucleic acid (PNA). In PNA compounds, the sugar-main chain of the oligonucleotide is replaced by an amide-containing main chain (e.g., an aminoethylglycine main chain). The core base is retained and directly or indirectly bound to the nitrogen-nitrogen atom of the main chain amide portion. Representative publications reporting the preparation of PNA compounds include, but are not limited to, U.S. Patents no. 5,539,082; 5,714,331; and 5,719,262, each of which is incorporated herein by reference. Further teachings of PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.

[0505] h. Gap polymers

[0506] In some embodiments, the oligonucleotides described herein are spacer polymers. Spacer polymer oligonucleotides generally have the formula 5'-XYZ-3', wherein X and Z serve as flanking regions around spacer Y. In some embodiments, the flanking region X of formula 5'-XYZ-3' is also referred to as X district, 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 Z district, flanking sequence Z, 3' wing region Z, or 3' wing segment. In some embodiments, the spacer Y of formula 5'-XYZ-3' is also referred to as Y district, Y segment, or spacer segment Y. In some embodiments, each nucleoside in spacer Y is a 2'-deoxyribonucleoside, and 5' wing region X or 3' wing region Z do not contain any 2'-deoxyribonucleoside.

[0507] In some embodiments, the Y region is a continuous extension of nucleotides, for example, a region of 6 or more DNA nucleotides, which can recruit RNA enzymes (e.g., RNAse H). In some embodiments, the spacer binds to the target nucleic acid, at which point the RNAse is recruited and can subsequently cut the target nucleic acid. In some embodiments, the flanks of the Y region 5' and 3' are X and Z regions comprising high-affinity modified nucleosides, for example, 1 to 6 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 length 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 lengths or different lengths. In some embodiments, the spacer segment Y can be a nucleotide sequence of 5 to 20 nucleotides, 5 to 15 dodecanucleotides, or 6 to 10 nucleotides in length.

[0508] In some embodiments, in addition to DNA nucleotides, the spacer of the spacer oligonucleotide may include modified nucleotides known to be acceptable for efficient RNase H action, such as C4'-substituted nucleotides, acyclic nucleotides, and nucleotides of arabino configuration. In some embodiments, the spacer comprises one or more unmodified inner cores. In some embodiments, one or two flanking regions each independently comprise one or more thiophosphate nucleoside linkages (e.g., thiophosphate nucleoside linkages or other linkages) between at least two, at least three, at least four, at least five or more nucleotides. In some embodiments, the spacer and two flanking regions each independently comprise modified nucleoside linkages (e.g., thiophosphate nucleoside linkages or other linkages) between at least two, at least three, at least four, at least five or more nucleotides.

[0509] Suitable methods can be used to generate gapmers. Representative U.S. patents, U.S. patent publications, and PCT publications that teach the preparation of gapmers include, but are not limited to: U.S. Patent Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775;

[0510] 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;

[0511] US Patent Publication Nos. US20050074801, US20090221685, US20090286969, US20100197762, and US20110112170; PCT Publication Nos. WO2004069991, WO2005023825, WO2008049085, and WO2009090182; and EP Patent No. EP2,149,605, each of which is herein incorporated by reference in its entirety.

[0512] In some embodiments, the length of the gapmer is 10 to 40 nucleosides. For example, the length of the gapmer 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 embodiments, the gapmer 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 in length.

[0513] In some embodiments, the length of the spacer Y in the spacer polymer is 5 to 20 nucleosides. For example, the length of spacer Y can be 5 to 20, 5 to 15, 5 to 10, 10 to 20, 10 to 15 or 15 to 20 nucleosides. In some embodiments, the length of spacer Y is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleosides. In some embodiments, each nucleoside in spacer Y is a 2'-deoxyribonucleoside. In some embodiments, all nucleosides in spacer Y are 2'-deoxyribonucleosides. In some embodiments, one or more nucleosides in spacer Y are modified nucleosides (for example, 2' modified nucleosides, such as those described herein). In some embodiments, one or more cytosines in spacer Y are optionally 5-methyl-cytosines. In some embodiments, each cytosine in spacer Y is 5-methyl-cytosines.

[0514] In some embodiments, the 5'-wing of the gapmer (X in the 5'-XYZ-3' formula) and the 3'-wing of the gapmer (Z in the 5'-XYZ-3' formula) are independently 1 to 20 nucleosides in length. For example, the 5'-wing of the gapmer (X in the 5'-XYZ-3' formula) and the 3'-wing of the gapmer (Z in the 5'-XYZ-3' formula) can independently be 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 nucleosides in length. In some embodiments, the 5' wing of the gapmer (X in the 5'-XYZ-3' formula) and the 3' wing of the gapmer (Z in the 5'-XYZ-3' formula) are independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleosides in length. In some embodiments, the 5' wing of the gapmer (X in the 5'-XYZ-3' formula) and the 3' wing of the gapmer (Z in the 5'-XYZ-3' formula) are the same length. In some embodiments, the 5' wing of the gapmer (X in the 5'-XYZ-3' formula) and the 3' wing of the gapmer (Z in the 5'-XYZ-3' formula) are different lengths. In some embodiments, the 5' wing of the gapmer (X in the 5'-XYZ-3' formula) is longer than the 3' wing of the gapmer (Z in the 5'-XYZ-3' formula). In some embodiments, the 5' wing of the gapmer (X in the 5'-XYZ-3' formula) is shorter than the 3' wing of the gapmer (Z in the 5'-XYZ-3' formula).

[0515] In some embodiments, the gapmer 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, 1-14-4, 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, 3-1 1-6, 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. ,

[0516] The numbers indicate the number of nucleosides in the X, Y, and Z regions of the 5'-XYZ-3' gapmer.

[0517] In some embodiments, one or more nucleosides in the 5' wing of the gapmer (X in the 5'-XYZ-3' formula) or the 3' wing of the gapmer (Z in the 5'-XYZ-3' formula) are modified nucleotides (e.g., high affinity modified nucleosides). In some embodiments, the modified nucleosides (e.g., high affinity modified nucleosides) are 2'-modified nucleosides. In some embodiments, the 2'-modified nucleosides are 2'-4' bicyclic nucleosides or non-bicyclic 2'-modified nucleosides. 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)).

[0518] In some embodiments, one or more nucleosides in the 5' wing (X in the 5'-XYZ-3' formula) of a gapmer are high-affinity modified nucleosides. In some embodiments, each nucleoside in the 5' wing (X in the 5'-XYZ-3' formula) of a gapmer is a high-affinity modified nucleoside. In some embodiments, one or more nucleosides in the 3' wing (Z in the 5'-XYZ-3' formula) of a gapmer are high-affinity modified nucleosides. In some embodiments, each nucleoside in the 3' wing (Z in the 5'-XYZ-3' formula) of a gapmer is a high-affinity modified nucleoside. In some embodiments, one or more nucleosides in the 5' wing (X in the 5'-XYZ-3' formula) of a gapmer are high-affinity modified nucleosides, and one or more nucleosides in the 3' wing (Z in the 5'-XYZ-3' formula) of a gapmer are high-affinity modified nucleosides. In some embodiments, each nucleoside in the 5'-wing (X in the 5'-XYZ-3' formula) of the gapmer is a high affinity modified nucleoside, and each nucleoside in the 3'-wing (Z in the 5'-XYZ-3' formula) of the gapmer is a high affinity modified nucleoside.

[0519] In some embodiments, the 5' wing of the gapmer (X in the 5'-XYZ-3' formula) comprises the same high-affinity nucleoside as the 3' wing of the gapmer (Z in the 5'-XYZ-3' formula). For example, the 5' wing of the gapmer (X in the 5'-XYZ-3' formula) and the 3' wing of the gapmer (Z in the 5'-XYZ-3' formula) can comprise one or more non-bicyclic 2'-modified nucleosides (e.g., 2'-MOE or 2'-O-Me). In another example, the 5' wing of the gapmer (X in the 5'-XYZ-3' formula) and the 3' wing of the gapmer (Z in the 5'-XYZ-3' formula) can comprise one or more 2'-4' bicyclic nucleosides (e.g., LNA or cEt). In some embodiments, each nucleoside in the 5'-wing (X in the 5'-XYZ-3' formula) of the gapmer and the 3'-wing (Z in the 5'-XYZ-3' formula) of the gapmer is a non-bicyclic 2'-modified nucleoside (e.g., 2'-MOE or 2'-O-Me). In some embodiments, each nucleoside in the 5'-wing (X in the 5'-XYZ-3' formula) of the gapmer and the 3'-wing (Z in the 5'-XYZ-3' formula) of the gapmer is a 2'-4' bicyclic nucleoside (e.g., LNA or cEt).

[0520] In some embodiments, the gapmer comprises a 5'-XYZ-3' configuration, wherein X and Z are independently 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleosides in length, and Y is 6 to 10 (e.g., 6, 7, 8, 9, or 10) nucleosides in length, wherein each nucleoside of X and Z is a non-bicyclic 2'-modified nucleoside (e.g., 2'-MOE or 2'-O-Me) and each nucleoside of Y is a 2'-deoxyribonucleoside. In some embodiments, the gapmer comprises a 5'-XYZ-3' configuration, wherein X and Z are independently 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleosides in length, and Y is 6 to 10 (e.g., 6, 7, 8, 9, or 10) nucleosides in length, wherein each nucleoside of X and Z is a 2'-4' bicyclic nucleoside (e.g., LNA or cEt), and each nucleoside of Y is a 2'-deoxyribonucleoside. In some embodiments, the 5'-wing of the gapmer (X in the 5'-XYZ-3' formula) comprises a different high-affinity nucleoside than the 3'-wing of the gapmer (Z in the 5'-XYZ-3' formula). For example, the 5'-wing of the gapmer (X in the 5'-XYZ-3' formula) can comprise one or more non-bicyclic 2'-modified nucleosides (e.g., 2'-MOE or 2'-O-Me), and the 3'-wing of the gapmer (Z in the 5'-XYZ-3' formula) can comprise one or more 2'-4' bicyclic nucleosides (e.g., LNA or cEt). In another example, the 3'-wing of the gapmer (Z in the 5'-XYZ-3' formula) can comprise one or more non-bicyclic 2'-modified nucleosides (e.g., 2'-MOE or 2'-O-Me), and the 5'-wing of the gapmer (X in the 5'-XYZ-3' formula) can comprise one or more 2'-4' bicyclic nucleosides (e.g., LNA or cEt).

[0521] In some embodiments, the gapmer comprises a 5'-XYZ-3' configuration, wherein X and Z are independently 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleosides in length, and Y is 6 to 10 (e.g., 6, 7, 8, 9, or 10) nucleosides in length, 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 gapmer comprises a 5'-XYZ-3' configuration, wherein X and Z are independently 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleosides in length, and Y is 6 to 10 (e.g., 6, 7, 8, 9, or 10) nucleosides in length, 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.

[0522] In some embodiments, the 5'-wing of the gapmer (X in the 5'-XYZ-3' formula) 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 of the gapmer (Z in the 5'-XYZ-3' formula) 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 of the gapmer (X in the 5'-XYZ-3' formula) and the 3'-wing of the gapmer (Z in the 5'-XYZ-3' formula) comprise 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).

[0523] In some embodiments, the gapmer comprises a 5'-XYZ-3' configuration, wherein X and Z are independently 2 to 7 (e.g., 2, 3, 4, 5, 6, or 7) nucleosides in length and Y is 6 to 10 (e.g., 6, 7, 8, 9, or 10) nucleosides in length, 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 (the 5'-most position being position 1) of X is a non-bicyclic 2'-modified nucleoside (e.g., 2'-MOE or 2'-O-Me), wherein the remaining nucleosides in both X and Z are 2'-4' bicyclic nucleosides (e.g., LNA or cEt), and wherein each nucleoside in Y is a 2' deoxyribonucleoside. In some embodiments, the gapmer comprises a 5'-XYZ-3' configuration, wherein X and Z are independently 2 to 7 (e.g., 2, 3, 4, 5, 6, or 7) nucleosides in length and Y is 6 to 10 (e.g., 6, 7, 8, 9, or 10) nucleosides in length, 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 (the 5'-most position being position 1) of Z is a non-bicyclic 2'-modified nucleoside (e.g., 2'-MOE or 2'-O-Me), wherein the remaining nucleosides in both X and Z are 2'-4' bicyclic nucleosides (e.g., LNA or cEt), and wherein each nucleoside in Y is a 2' deoxyribonucleoside. In some embodiments, the gapmer comprises a 5'-XYZ-3' configuration, wherein X and Z are independently 2 to 7 (e.g., 2, 3, 4, 5, 6, or 7) nucleosides in length and Y is 6 to 10 (e.g., 6, 7, 8, 9, or 10) nucleosides in length, 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...

Claims

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

42.

2. The complex of claim 1, wherein the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 76 and a VL comprising the amino acid sequence of SEQ ID NO:

75.

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

5. The complex of claim 3, wherein the antibody is a Fab fragment and comprises a heavy chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 101; and / or a light chain comprising an amino acid sequence at least 85% identical to SEQ ID NO:

90.

6. The complex of claim 3, wherein the antibody is a Fab fragment and comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 101; and a light chain comprising the amino acid sequence of SEQ ID NO:

90.

7. The complex of claim 1 or claim 2, wherein the heavy chain of the antibody comprises an N-terminal pyroglutamate.

8. The complex of claim 1 or claim 2, wherein the molecular cargo comprises an oligonucleotide.

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

10. The complex of claim 1 or claim 2, wherein modulating the expression or activity of a muscle disease gene comprises reducing RNA and / or protein expression.

11. The complex of claim 8, wherein the oligonucleotide comprises at least one modified internucleoside linkage.

12. The complex of claim 11, wherein the modified internucleoside linkage is a phosphorothioate linkage.

13. The complex of claim 8, wherein the oligonucleotide comprises one or more modified nucleosides. The complex of claim 13 , wherein the one or more modified nucleosides comprises one or more 2′-modified nucleosides.

15. The complex of claim 14, wherein each 2'-modified nucleoside is independently selected from the group consisting of: 2'-O-methyl, 2'-fluoro (2'-F), 2'-O-methoxyethyl (2'-MOE), and 2',4'-bridged nucleosides.

16. The complex of claim 8, wherein the oligonucleotide comprises: (i) a gapmer oligonucleotide that directs RNase H-mediated cleavage of an mRNA transcript encoded by the muscle disease gene in a cell; (ii) mixed-mer oligonucleotides; (iii) phosphorodiamidate morpholino oligomers; or (iv) an RNAi oligonucleotide that promotes RNAi-mediated cleavage of an mRNA transcript encoded by said muscle disease gene.

17. The complex of claim 1 or claim 2, wherein the antibody is covalently linked to the molecular cargo via: (i) a cleavable linker; or (ii) Non-cleavable linker.

18. The complex of claim 17, wherein the antibody is covalently attached to the molecular cargo via a cleavable linker comprising a valine-citrulline sequence.

19. The complex of claim 17, wherein the antibody is covalently attached to the molecular cargo via a non-cleavable linker comprising an optionally substituted alkyl group.

20. The complex of claim 17, wherein the molecular cargo is covalently linked to the antibody via conjugation to a lysine residue of the antibody.

21. The complex of claim 17, wherein the molecular cargo is covalently linked to the antibody via conjugation to a cysteine ​​residue of the antibody.

22. Use of the complex according to any one of claims 1 to 21 in the preparation of a medicament for treating a disease or condition that can be improved or prevented by modulating the expression or activity of a muscle disease gene in a cell, wherein the medicament is formulated to promote internalization of the molecular cargo into the cell; wherein the disease or disorder is a muscle 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 Ryan myopathy, myofibrillar myopathy; an autosomal dominant form of myotonia congenita, i.e., Thomson's disease; myotonic dystrophy type I, myotonic dystrophy type II, myotubular myopathy, oculopharyngeal muscular dystrophy, and paramyotonia congenita.

23. The use of claim 22, wherein the cell is a muscle cell.

24. Use of the complex of any one of claims 1 to 21 in the preparation of a medicament for treating a subject suffering from a muscle disease, wherein the medicament is formulated for treating the muscle disease, wherein the muscle disease is 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 Ryan myopathy, myofibrillar myopathy; autosomal dominant form of myotonia congenita, i.e., Thomson disease; myotonic dystrophy type I, myotonic dystrophy type II, myotubular myopathy, oculopharyngeal muscular dystrophy, and paramyotonia congenita.

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