Muscle-targeting complexes and their use for treating dystrophin disorders

Abstract

The present invention provides a complex containing a muscle targeting agent covalently linked to a molecular payload. [Solution] In some embodiments, the muscle targeting agent specifically binds to internalized cell surface receptors on muscle cells. In some embodiments, the molecular payload promotes the expression or activity of a functional dystrophin protein. In some embodiments, the molecular payload is an antisense oligonucleotide, such as an oligonucleotide that causes exon skipping in mRNA expressed from a mutant DMD allele.

Description

[Technical Field]

[0001] Related applications This application claims priority under U.S. Provisional Application No. 63 / 143829, filed January 30, 2021, entitled "MUSCLE TARGETING COMPLEXES AND USES THEREOF FOR TREATING DYSTROPHINOPATHIES"; U.S. Provisional Application No. 63 / 069077, filed August 23, 2021, entitled "MUSCLE TARGETING COMPLEXES AND USES THEREOF FOR TREATING DYSTROPHINOPATHIES"; and U.S. Provisional Application No. 63 / 055777, filed July 23, 2020; the contents of each of these are incorporated herein by reference in their entirety.

[0002] Field of the present invention This application relates to targeted complexes for delivering molecular payloads (e.g., oligonucleotides) to cells, and their uses, particularly in relation to the treatment of diseases.

[0003] References to sequence listings submitted as text files via EFS-WEB This application includes a sequence listing. This was filed from EFS Web in ASCII format, and the entire ASCII copy, created on 8 January 2021, is incorporated herein by reference and is named D082470040WO00-SEQ-DWGDWY, with a size of 575,156 bytes. [Background technology]

[0004] Background of the Invention Dystrophin disorders are a distinct group of neuromuscular diseases resulting from mutations in the dystrophin gene. Dystrophin disorders include Duchenne muscular dystrophy, Becker muscular dystrophy, and X-linked dilated cardiomyopathy. Dystrophin (DMD) is a large gene containing 79 exons and approximately 26 million total base pairs. Numerous mutations in DMD, including exon frameshifts, deletions, substitutions, and duplications, can reduce the expression of functional dystrophin, leading to dystrophin disorders. One drug targeting exon 51 of human DMD has been provisionally approved by the U.S. Food and Drug Administration (FDA), but its efficacy is still under evaluation. [Overview of the project]

[0005] Summary of the Invention According to some aspects, the present disclosure provides a complex that targets muscle cells for the purpose of delivering a molecular payload to those cells. In some embodiments, the complexes provided herein are particularly useful for delivering molecular payloads that increase or restore the expression or activity of functional DMD. In some embodiments, the complex comprises an oligonucleotide-based molecular payload that promotes the normal expression of functional DMD via an in-frame exon skipping mechanism or suppression of a stop codon. In some embodiments, the complex is configured to deliver a mini-dystrophin gene or synthetic mRNA that increases or restores functional dystrophin activity. Thus, in some embodiments, the complexes provided herein comprise a muscle targeting agent (e.g., a muscle targeting antibody) that specifically binds to a receptor on the surface of a muscle cell for the purpose of delivering a molecular payload to the muscle cell. In some embodiments, the complex is taken up into the cell via receptor-mediated endocytosis, and in response, the molecular payload may be released inside the cell to perform its function. For example, a complex modified to deliver an oligonucleotide may release the oligonucleotide such that the oligonucleotide can promote the expression of functional DMD in muscle cells (e.g., via an exon skipping mechanism). In some embodiments, the oligonucleotide is released by endosomal cleavage of a covalent linker that connects the oligonucleotide of the complex to the muscle targeting agent.

[0006] One aspect of the present disclosure relates to a complex comprising an anti-transferrin receptor (TfR) antibody covalently linked to a molecular payload configured to promote the expression or activity of the DMD gene, the antibody comprising: (i) a heavy chain variable region (VH) comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 76; and / or a light chain variable region (VL) comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 75; (ii) a heavy chain variable region (VH) comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 69; and / or a light chain variable region (VL) comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 70; (iii) a heavy chain variable region (VH) comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 71; and / or a light chain variable region (VL) comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 70; (iv) a heavy chain variable region (VH) comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 72; and / or a light chain variable region (VL) comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 70; (v) a heavy chain variable region (VH) comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 73; and / or a light chain variable region (VL) comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 74; (vi) a heavy chain variable region (VH) comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 73; and / or a light chain variable region (VL) comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 75; (vii) a heavy chain variable region (VH) comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 76; and / or a light chain variable region (VL) comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 74; (viii) a heavy chain variable region (VH) comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 77; and / or a light chain variable region (VL) comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 78; (ix) a heavy chain variable region (VH) comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 79; and / or a light chain variable region (VL) comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 80; or (x) a heavy chain variable region (VH) comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 77; and / or a light chain variable region (VL) comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 80.

[0007] In some embodiments, the antibody comprises: (i) VH containing the amino acid sequence of SEQ ID NO: 76 and VL containing the amino acid sequence of SEQ ID NO: 75; (ii) VH containing the amino acid sequence of SEQ ID NO: 69 and VL containing the amino acid sequence of SEQ ID NO: 70; (iii) VH containing the amino acid sequence of SEQ ID NO: 71 and VL containing the amino acid sequence of SEQ ID NO: 70; (iv) VH containing the amino acid sequence of SEQ ID NO: 72 and VL containing the amino acid sequence of SEQ ID NO: 70; (v) VH containing the amino acid sequence of SEQ ID NO: 73 and VL containing the amino acid sequence of SEQ ID NO: 74; (vi) VH containing the amino acid sequence of SEQ ID NO: 73 and VL containing the amino acid sequence of SEQ ID NO: 75; (vii) VH containing the amino acid sequence of SEQ ID NO: 76 and VL containing the amino acid sequence of SEQ ID NO: 74; (viii) VH containing the amino acid sequence of SEQ ID NO: 77 and VL containing the amino acid sequence of SEQ ID NO: 78; (ix) VH containing the amino acid sequence of SEQ ID NO: 79 and VL containing the amino acid sequence of SEQ ID NO: 80; or (x) VH containing the amino acid sequence of SEQ ID NO: 77 and VL containing the amino acid sequence of SEQ ID NO: 80.

[0008] In some embodiments, the antibody is selected from the group consisting of Fab fragment, Fab' fragment, F(ab')2 fragment, scFv, Fv, and full-length IgG. In some embodiments, the antibody is a Fab fragment.

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

[0010] In some embodiments, the antibody includes: (i) A heavy chain containing the amino acid sequence of SEQ ID NO: 101; and a light chain containing the amino acid sequence of SEQ ID NO: 90; (ii) A heavy chain containing the amino acid sequence of SEQ ID NO: 97; and a light chain containing the amino acid sequence of SEQ ID NO: 85; (iii) A heavy chain containing the amino acid sequence of SEQ ID NO: 98; and a light chain containing the amino acid sequence of SEQ ID NO: 85; (iv) A heavy chain containing the amino acid sequence of SEQ ID NO: 99; and a light chain containing the amino acid sequence of SEQ ID NO: 85; (v) Heavy chain containing the amino acid sequence of SEQ ID NO: 100; and light chain containing the amino acid sequence of SEQ ID NO: 89; (vi) A heavy chain containing the amino acid sequence of SEQ ID NO: 100; and a light chain containing the amino acid sequence of SEQ ID NO: 90; (vii) A heavy chain containing the amino acid sequence of SEQ ID NO: 101; and a light chain containing the amino acid sequence of SEQ ID NO: 89; (viii) A heavy chain containing the amino acid sequence of SEQ ID NO: 102; and a light chain containing the amino acid sequence of SEQ ID NO: 93; (ix) A heavy chain containing the amino acid sequence of SEQ ID NO: 103; and a light chain containing the amino acid sequence of SEQ ID NO: 95; or (x) A heavy chain containing the amino acid sequence of SEQ ID NO: 102; and a light chain containing the amino acid sequence of SEQ ID NO: 95.

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

[0012] In some embodiments, the molecular payload is an oligonucleotide. In some embodiments, the oligonucleotide facilitates exon skipping in DMD RNA. In some embodiments, the oligonucleotide facilitates exon skipping of DMD in the range of exon 8 to exon 55. In some embodiments, the oligonucleotide facilitates the skipping of exon 8, exon 23, exon 43, exon 44, exon 45, exon 46, exon 50, exon 51, exon 52, exon 53, and / or exon 55.

[0013] In some embodiments, the oligonucleotide comprises a region complementary to one or more complete or partial exonic splicing enhancers (ESEs) of the DMD transcript. In some embodiments, the oligonucleotide comprises a region complementary to a target sequence comprising one or more complete or partial ESEs as described in SEQ ID NOs. 402-436 and 2043-2238.

[0014] In some embodiments, oligonucleotides facilitate the skipping of exon 51.

[0015] In some embodiments, the oligonucleotide is 20 to 30 nucleotides in length and includes a region complementary to a target sequence containing at least four consecutive nucleotides of the ESE described in any one of SEQ ID NOs. 402 to 436.

[0016] In some embodiments, the oligonucleotide contains one of sequence numbers 437-1241 or a region complementary to one of sequence numbers 1242-2046.

[0017] In some embodiments, the oligonucleotide includes a region complementary to the target sequence of the oligonucleotide listed in Table 14. In some embodiments, the oligonucleotide includes the sequences listed in Table 14, and one or more of the uracil bases (U) in the oligonucleotide may optionally be thymine bases (T).

[0018] In some embodiments, the oligonucleotide comprises at least one modified nucleoside linkage. In some embodiments, the at least one modified nucleoside linkage is a phosphorothioate linkage.

[0019] In some embodiments, the oligonucleotide comprises one or more modified nucleosides. In some embodiments, one or more modified nucleosides are 2'-modified nucleosides.

[0020] In some embodiments, the oligonucleotide comprises one or more phosphorodiamidate morpholino oligomers (PMOs), wherein the oligonucleotide is optionally a phosphorodiamidate morpholino oligomer (PMO).

[0021] 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.

[0022] In some embodiments, the antibody is covalently linked to the molecular payload via conjugation to a lysine or cysteine ​​residue of the antibody.

[0023] Another aspect of the present disclosure relates to a method for promoting the expression or activity of a DMD gene in a cell, the method comprising contacting the cell with an amount of the complex disclosed herein that is effective in promoting the internalization of a molecular payload into the cell, optionally the cell being a muscle cell.

[0024] Another aspect of this disclosure relates to a method for treating a subject having a mutated DMD allele associated with dystrophinosis, the method comprising administering an effective amount of the complex disclosed herein to the subject. [Brief explanation of the drawing]

[0025] [Figure 1] Figure 1 shows a non-limiting schematic diagram illustrating the effect of transfecting cells with siRNA.

[0026] [Figure 2] Figure 2 shows a non-restrictive schematic diagram illustrating the activity of a muscle targeting complex containing siRNA.

[0027] [Figure 3A-B] Figures 3A-3B show non-restrictive schematic diagrams illustrating the in vivo activity of a muscle-targeting complex containing siRNA in mouse muscle tissue (gastrocnemius and heart) compared to a control of a non-targeting complex containing the same siRNA. (N=4 C57BL / 6 WT mice)

[0028] [Figure 4A-B] Figures 4A-4E illustrate non-limiting schematic diagrams showing the tissue selectivity of muscle-targeting complexes containing siRNA. [Figure 4C-D] Figures 4A-4E illustrate non-limiting schematic diagrams showing the tissue selectivity of muscle-targeting complexes containing siRNA. [Figure 4E] Figures 4A-4E illustrate non-limiting schematic diagrams showing the tissue selectivity of muscle-targeting complexes containing siRNA.

[0029] [Figure 5] Figure 5 shows a non-limiting schematic diagram illustrating the dose-dependent enhancement of exon skipping in mdx mouse model muscle tissue by an anti-transferrin receptor muscle targeting complex containing a phosphorodiamidate morpholino oligomer (PMO) for exon 23 skipping.

[0030] [Figure 6A] Figures 6A-6B illustrate a non-limiting schematic diagram showing the dose-dependent increase in dystrophin in mdx mouse model skeletal muscle (quadriceps femoris) by the anti-transferrin receptor muscle targeting complex containing exon 23 skipping PMO. [Figure 6B] Figures 6A-6B illustrate a non-limiting schematic diagram showing the dose-dependent increase in dystrophin in mdx mouse model skeletal muscle (quadriceps femoris) by the anti-transferrin receptor muscle targeting complex containing exon 23 skipping PMO.

[0031] [Figure 7A] Figures 7A–7E provide a non-limiting schematic diagram showing the ability of an anti-transferrin receptor muscle targeting complex containing exon 23 skipping PMO to improve functional performance (Figures 7A, 7B, 7C, and 7D) and reduce creatine kinase levels (Figure 7E) in the mdx mouse model (** p<0.01; *** p<0.001; ****; p<0.0001; not NS significant). [Figure 7B]Figures 7A–7C illustrate a non-limiting schematic diagram showing the ability of an anti-transferrin receptor muscle targeting complex containing exon 23 skipping PMO to improve functional performance (Figures 7A, 7B, 7C, and 7D) and reduce creatine kinase levels (Figure 7E) in the mdx mouse model (** p<0.01; *** p<0.001; ****; p<0.0001; not NS significant). [Figure 7C] Figures 7A–7E provide a non-limiting schematic diagram showing the ability of an anti-transferrin receptor muscle targeting complex containing exon 23 skipping PMO to improve functional performance (Figures 7A, 7B, 7C, and 7D) and reduce creatine kinase levels (Figure 7E) in the mdx mouse model (** p<0.01; *** p<0.001; ****; p<0.0001; not NS significant). [Figure 7D] Figures 7A–7E provide a non-limiting schematic diagram showing the ability of an anti-transferrin receptor muscle targeting complex containing exon 23 skipping PMO to improve functional performance (Figures 7A, 7B, 7C, and 7D) and reduce creatine kinase levels (Figure 7E) in the mdx mouse model (** p<0.01; *** p<0.001; ****; p<0.0001; not NS significant). [Figure 7E] Figures 7A–7E provide a non-limiting schematic diagram showing the ability of an anti-transferrin receptor muscle targeting complex containing exon 23 skipping PMO to improve functional performance (Figures 7A, 7B, 7C, and 7D) and reduce creatine kinase levels (Figure 7E) in the mdx mouse model (** p<0.01; *** p<0.001; ****; p<0.0001; not NS significant).

[0032] [Figure 8]Figure 8 shows the time-dependent serum stability of the linker used to ligate anti-TfR antibodies and molecular payloads (e.g., oligonucleotides) in various organisms after intravenous administration.

[0033] [Figure 9A] Figures 9A–9F show the binding of humanized anti-TfR Fab to human TfR1 (hTfR1) or cynomolgus monkey TfR1 (cTfR1) as measured by ELISA. Figure 9A shows the binding of the humanized 3M12 variant to hTfR1. [Figure 9B] Figures 9A–9F show the binding of humanized anti-TfR Fab to human TfR1 (hTfR1) or cynomolgus monkey TfR1 (cTfR1) as measured by ELISA. Figure 9B shows the binding of the humanized 3M12 variant to cTfR1. [Figure 9C] Figures 9A–9F show the binding of humanized anti-TfR Fab to human TfR1 (hTfR1) or cynomolgus monkey TfR1 (cTfR1) as measured by ELISA. Figure 9C shows the binding of the humanized 3A4 variant to hTfR1. [Figure 9D] Figures 9A–9F show the binding of humanized anti-TfR Fab to human TfR1 (hTfR1) or cynomolgus monkey TfR1 (cTfR1) as measured by ELISA. Figure 9D shows the binding of the humanized 3A4 variant to cTfR1. [Figure 9E] Figures 9A–9F show the binding of humanized anti-TfR Fab to human TfR1 (hTfR1) or cynomolgus monkey TfR1 (cTfR1) as measured by ELISA. Figure 9E shows the binding of the humanized 5H12 variant to hTfR1. [Figure 9F] Figures 9A–9F show the binding of humanized anti-TfR Fab to human TfR1 (hTfR1) or cynomolgus monkey TfR1 (cTfR1) as measured by ELISA. Figure 9F shows the binding of the humanized 5H12 variant to hTfR1.

[0034] [Figure 10]Figure 10 shows the quantified cellular uptake of anti-TfR Fab conjugates into rhabdomyosarcoma (RD) cells. The molecular payload in the tested conjugates was a DMPK-targeted oligonucleotide, and conjugate uptake was facilitated by the indicated anti-TfR Fab. Conjugates with negative control Fab (anti-mouse TfR) or positive control Fab (anti-human TfR1) are also included in this assay. Cells were incubated with the indicated conjugates at a concentration of 100 nM for 4 hours. Cell uptake was measured by mean Cypher5e fluorescence.

[0035] [Figure 11A] Figures 11A–11F show the binding of oligonucleotide-conjugated or unconjugated humanized anti-TfR Fab to human TfR1 (hTfR1) and cynomolgus monkey TfR1 (cTfR1), as measured by ELISA. Figure 11A shows the binding of the humanized 3M12 variant to hTfR1, either alone or conjugated with a DMPK-targeted oligonucleotide. The respective EC50 values ​​are also shown. [Figure 11B] Figures 11A–11F show the binding of oligonucleotide-conjugated or unconjugated humanized anti-TfR Fab to human TfR1 (hTfR1) and cynomolgus monkey TfR1 (cTfR1), as measured by ELISA. Figure 11B shows the binding of the humanized 3M12 variant to cTfR1, either alone or conjugated with a DMPK-targeted oligonucleotide. The respective EC50 values ​​are also shown. [Figure 11C] Figures 11A–11F show the binding of oligonucleotide-conjugated or unconjugated humanized anti-TfR Fab to human TfR1 (hTfR1) and cynomolgus monkey TfR1 (cTfR1), as measured by ELISA. Figure 11C shows the binding of the humanized 3A4 variant to hTfR1, either alone or conjugated with a DMPK-targeted oligonucleotide. The respective EC50 values ​​are also shown. [Figure 11D]Figures 11A–11F show the binding of oligonucleotide-conjugated or unconjugated humanized anti-TfR Fab to human TfR1 (hTfR1) and cynomolgus monkey TfR1 (cTfR1), as measured by ELISA. Figure 11D shows the binding of the humanized 3A4 variant to cTfR1, either alone or conjugated with a DMPK-targeted oligonucleotide. The respective EC50 values ​​are also shown. [Figure 11E] Figures 11A–11F show the binding of oligonucleotide-conjugated or unconjugated humanized anti-TfR Fab to human TfR1 (hTfR1) and cynomolgus monkey TfR1 (cTfR1), as measured by ELISA. Figure 11E shows the binding of the humanized 5H12 variant to hTfR1, either alone or conjugated with a DMPK-targeted oligonucleotide. The respective EC50 values ​​are also shown. [Figure 11F] Figures 11A–11F show the binding of oligonucleotide-conjugated or unconjugated humanized anti-TfR Fab to human TfR1 (hTfR1) and cynomolgus monkey TfR1 (cTfR1), as measured by ELISA. Figure 11F shows the binding of the humanized 5H12 variant to cTfR1, either alone or conjugated with a DMPK-targeted oligonucleotide. The respective EC50 values ​​are also shown.

[0036] [Figure 12] Figure 12 shows DMPK expression in RD cells treated with conjugates containing the indicated humanized anti-TfR antibody, conjugated to the DMPK-targeted oligonucleotide ASO300, at various concentrations. The treatment duration was 3 days. ASO300 delivered using a transfection agent was used as a control.

[0037] [Figure 13] Figure 13 shows exon 51 skipping in human DMD myotubes, facilitated by DMD exon 51 skipping oligonucleotides (PMOs). Cells were treated with naked PMO or PMO conjugated to anti-TfR1 Fab (Ab-PMO).

[0038] [Figure 14] Figure 14 shows the dose-dependent increase in dystrophin expression, measured by Western blotting of dystrophin using alpha-actin as a loading control, in the quadriceps muscle of mdx mice after treatment with anti-mouse TfR1 (RI7 217) conjugated to an exon 23-targeted oligonucleotide (PMO). Standards were generated using pooled wild-type and pooled mdx proteins. Percentages indicate the amount of WT protein spiked into the sample.

[0039] [Figure 15] Figure 15 shows the quantification of dystrophin protein levels in the quadriceps muscle of mdx mice after treatment with various doses of anti-mouse TfR (RI7 217) conjugated to an oligonucleotide (PMO) targeting exon 23.

[0040] [Figure 16] Figure 16 shows immunofluorescence staining images of quadriceps muscles from wild-type (WT) mice treated with saline, or from mdx mice treated with saline, naked oligonucleotides, or oligonucleotides conjugated to anti-mouse TfR1 (RI7 217).

[0041] [Figure 17] Figure 17 shows data illustrating that conjugates containing specified anti-TfR Fab' (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.

[0042] [Figure 18]Figure 18 shows ELISA measurements of the binding of anti-TfR Fab 3M12 VH4 / Vk3 to recombinant human (circle), cynomolgus monkey (square), mouse (upward triangle), or rat (downward triangle) TfR1 proteins at Fab concentrations ranging from 230 pM to 500 nM. The results indicate that anti-TfR Fab is reactive with human and cynomolgus monkey TfR1. Binding to recombinant mouse or rat TfR1 was not observed. Data are presented as relative fluorescence units normalized to baseline.

[0043] [Figure 19] Figure 19 shows the results of ELISA tests testing the affinity of anti-TfR Fab 3M12 VH4 / Vk3 for recombinant human TfR1 or TfR2 across a Fab concentration range of 230 pM to 500 nM. Data are presented as relative fluorescence units normalized to baseline. The results demonstrate that Fab does not bind to recombinant human TfR2.

[0044] [Figure 20] Figure 20 shows the serum stability of the linker used to ligate anti-TfR Fab 3M12 VH4 / Vk3 to a control antisense oligonucleotide in PBS or in rat, mouse, cynomolgus monkey, or human serum over a 72-hour incubation period.

[0045] [Figure 21A]Figures 21A–21C show the quantification of exon 23 skipping in the quadriceps (Figure 21A), heart (Figure 21B), and diaphragm (Figure 21C) of wild-type (WT) and mdx mice two or four weeks after administration of a single dose of saline, an unconjugated oligonucleotide (ASO) that induces exon 23 skipping in DMD, or a conjugate containing anti-TfR1 RI7217 Fab conjugated to ASO (Ab-ASO). In tissues from WT or mdx mice administered with saline or unconjugated ASO, little to no exon 23 skipping was observed, while significant levels of exon 23 skipping were observed in tissues from mdx mice treated with Ab-ASO. (*p<0.05, **p<0.01, ****p<0.0001) [Figure 21B] Figures 21A–21C show the quantification of exon 23 skipping in the quadriceps (Figure 21A), heart (Figure 21B), and diaphragm (Figure 21C) of wild-type (WT) and mdx mice two or four weeks after administration of a single dose of saline, an unconjugated oligonucleotide (ASO) that induces exon 23 skipping in DMD, or a conjugate containing anti-TfR1 RI7217 Fab conjugated to ASO (Ab-ASO). In tissues from WT or mdx mice administered with saline or unconjugated ASO, little to no exon 23 skipping was observed, while significant levels of exon 23 skipping were observed in tissues from mdx mice treated with Ab-ASO. (*p<0.05, **p<0.01, ****p<0.0001) [Figure 21C]Figures 21A–21C show the quantification of exon 23 skipping in the quadriceps (Figure 21A), heart (Figure 21B), and diaphragm (Figure 21C) of wild-type (WT) and mdx mice two or four weeks after administration of a single dose of saline, an unconjugated oligonucleotide (ASO) that induces exon 23 skipping in DMD, or a conjugate containing anti-TfR1 RI7217 Fab conjugated to ASO (Ab-ASO). In tissues from WT or mdx mice administered with saline or unconjugated ASO, little to no exon 23 skipping was observed, while significant levels of exon 23 skipping were observed in tissues from mdx mice treated with Ab-ASO. (*p<0.05, **p<0.01, ****p<0.0001)

[0046] [Figure 22A] Figures 22A–22D show the measurement of dystrophin protein in the quadriceps muscle of mdx mice after a single dose of either an unconjugated oligonucleotide (ASO) that induces exon 23 skipping in DMD, or a conjugate containing anti-TfR1 RI7217 Fab conjugated to ASO (Ab-ASO). Figure 22A shows Western blots of dystrophin and alpha-actinin proteins in muscle tissue two weeks after injection of ASO or Ab-ASO. The standard curves in Figures 22A and 22C were generated by pooling tissue from wild-type (WT) and mdx mouse samples, and percent WT indicates the amount of WT protein spiked in each sample. (*p<0.05; ns, not significant) [Figure 22B] Figures 22A-22D show the measurement of dystrophin protein in the quadriceps muscle of mdx mice after a single dose of either an unconjugated oligonucleotide (ASO) that induces exon 23 skipping in DMD, or a conjugate (Ab-ASO) containing anti-TfR1 RI7217 Fab conjugated to an ASO. Figure 22B shows the quantification of dystrophin in the Western blot of Figure 22A compared to dystrophin protein in wild-type muscle. [Figure 22C]Figures 22A–22D show the measurement of dystrophin protein in the quadriceps muscle of mdx mice after a single dose of either an unconjugated oligonucleotide (ASO) that induces exon 23 skipping in DMD, or a conjugate containing anti-TfR1 RI7217 Fab conjugated to ASO (Ab-ASO). Figure 22C shows Western blots of dystrophin and alpha-actinin proteins in muscle tissue 4 weeks after injection of ASO or Ab-ASO. The standard curves in Figures 22A and 22C were generated by pooling tissue from wild-type (WT) and mdx mouse samples, and percent WT indicates the amount of WT protein spiked in each sample. (*p<0.05; ns, not significant) [Figure 22D] Figures 22A–22D show the measurement of dystrophin protein in the quadriceps muscle of mdx mice after a single dose of either an unconjugated oligonucleotide (ASO) that induces exon 23 skipping in DMD, or a conjugate (Ab-ASO) containing anti-TfR1 RI7217 Fab conjugated to an ASO. Figure 22D shows the quantification of dystrophin in the Western blot of Figure 22C compared to dystrophin protein in wild-type muscle.

[0047] [Figure 23A] Figures 23A–23D show the measurement of dystrophin protein in the cardiac muscle of mdx mice after a single dose of either an unconjugated oligonucleotide (ASO) that induces exon 23 skipping in DMD, or a conjugate containing anti-TfR1 RI7217 Fab conjugated to an ASO (Ab-ASO). Figure 23A shows Western blots of dystrophin and alpha-actinin proteins in muscle tissue two weeks after injection of ASO or Ab-ASO. The standard curves in Figures 23A and 23C were generated by pooling tissue from wild-type (WT) and mdx mouse samples, and percentage WT indicates the amount of WT protein spiked in each sample. (*p<0.05;****p<0.0001) [Figure 23B]Figures 23A–23D show the measurement of dystrophin protein in the cardiac muscle of mdx mice after a single dose of an unconjugated oligonucleotide (ASO) that induces exon 23 skipping in DMD, or a conjugate containing anti-TfR1 RI7217 Fab conjugated to an ASO (Ab-ASO). Figure 23B shows the quantification of dystrophin in the Western blot of Figure 23A compared to dystrophin protein in wild-type muscle. [Figure 23C] Figures 23A–23D show the measurement of dystrophin protein in the cardiac muscle of mdx mice after a single dose of either an unconjugated oligonucleotide (ASO) that induces exon 23 skipping in DMD, or a conjugate containing anti-TfR1 RI7217 Fab conjugated to an ASO (Ab-ASO). Figure 23C shows Western blots of dystrophin and alpha-actinin proteins in muscle tissue 4 weeks after injection of ASO or Ab-ASO. The standard curves in Figures 23A and 23C were generated by pooling tissue from wild-type (WT) and mdx mouse samples, and the percentage WT indicates the amount of WT protein spiked in each sample. (*p<0.05;****p<0.0001) [Figure 23D] Figures 23A–23D show the measurement of dystrophin protein in the cardiac muscle of mdx mice after a single dose of an unconjugated oligonucleotide (ASO) that induces exon 23 skipping in DMD, or a conjugate containing anti-TfR1 RI7217 Fab conjugated to an ASO (Ab-ASO). Figure 23D shows the quantification of dystrophin in the Western blot of Figure 23C compared to dystrophin protein in wild-type muscle.

[0048] [Figure 24A]Figures 24A–24D show the measurement of dystrophin protein in the diaphragmatic muscle of mdx mice after a single dose of either an unconjugated oligonucleotide (ASO) that induces exon 23 skipping in DMD, or a conjugate containing anti-TfR1 RI7217 Fab conjugated to ASO (Ab-ASO). Figure 24A shows Western blots of dystrophin and alpha-actinin proteins in muscle tissue two weeks after injection of ASO or Ab-ASO. The standard curves in Figures 24A and 24C were generated by pooling tissue from wild-type (WT) and mdx mouse samples, and percentage WT indicates the amount of WT protein spiked in each sample. (**p<0.01; ***p<0.001) [Figure 24B] Figures 24A-24D show the measurement of dystrophin protein in the diaphragmatic muscle of mdx mice after a single dose of either an unconjugated oligonucleotide (ASO) that induces exon 23 skipping in DMD, or a conjugate (Ab-ASO) containing anti-TfR1 RI7217 Fab conjugated to an ASO. Figure 24B shows the quantification of dystrophin in the Western blot of Figure 24A compared to dystrophin protein in wild-type muscle. [Figure 24C] Figures 24A–24D show the measurement of dystrophin protein in the diaphragmatic muscle of mdx mice after a single dose of either an unconjugated oligonucleotide (ASO) that induces exon 23 skipping in DMD, or a conjugate containing anti-TfR1 RI7217 Fab conjugated to ASO (Ab-ASO). Figure 24C shows Western blots of dystrophin and alpha-actinin proteins in muscle tissue 4 weeks after injection of ASO or Ab-ASO. The standard curves in Figures 24A and 24C were generated by pooling tissue from wild-type (WT) and mdx mouse samples, and percentage WT indicates the amount of WT protein spiked in each sample. (**p<0.01; ***p<0.001) [Figure 24D]Figures 24A–24D show the measurement of dystrophin protein in the diaphragmatic muscle of mdx mice after a single dose of either an unconjugated oligonucleotide (ASO) that induces exon 23 skipping in DMD, or a conjugate (Ab-ASO) containing anti-TfR1 RI7217 Fab conjugated to an ASO. Figure 24D shows the quantification of dystrophin in the Western blot of Figure 24C compared to dystrophin protein in wild-type muscle.

[0049] [Figure 25A] Figures 25A-25C show the quantification of oligonucleotide (ASO) amounts administered to the quadriceps (Figure 25A), diaphragm (Figure 25B), and heart (Figure 25C) of wild-type (WT) or mdx mice two or four weeks after administration of a single dose of physiological saline, unconjugated exon 23-skipping oligonucleotide (ASO), or a conjugate containing anti-TfR1 RI7217 Fab conjugated to ASO (Ab-ASO). [Figure 25B] Figures 25A-25C show the quantification of oligonucleotide (ASO) amounts administered to the quadriceps (Figure 25A), diaphragm (Figure 25B), and heart (Figure 25C) of wild-type (WT) or mdx mice two or four weeks after administration of a single dose of physiological saline, unconjugated exon 23-skipping oligonucleotide (ASO), or a conjugate containing anti-TfR1 RI7217 Fab conjugated to ASO (Ab-ASO). [Figure 25C] Figures 25A-25C show the quantification of oligonucleotide (ASO) amounts administered to the quadriceps (Figure 25A), diaphragm (Figure 25B), and heart (Figure 25C) of wild-type (WT) or mdx mice two or four weeks after administration of a single dose of physiological saline, unconjugated exon 23-skipping oligonucleotide (ASO), or a conjugate containing anti-TfR1 RI7217 Fab conjugated to ASO (Ab-ASO).

[0050] [Figure 26]Figure 26 shows the percentage of exon 53 skipping in DMD patient cells with DMD exon 52 deletion after gymnotic uptake of exon 53 skipping oligonucleotides above a certain concentration range.

[0051] [Figure 27] Figure 27 shows the exon 53 skipping percentage in DMD patient cells with DMD exon 52 deletion after treatment with various concentrations of exon 53 skipping PMO, either unbound to the antibody ("naked ASO") or covalently linked to anti-TfR1 Fab ("anti-TfR1 Fab-ASO complex"). [Modes for carrying out the invention]

[0052] Detailed description of the invention Aspect of this disclosure concerns the recognition that while certain molecular payloads (e.g., oligonucleotides, peptides, small molecules) may have beneficial effects on muscle cells, effectively targeting such cells is extremely difficult. As described herein, this disclosure provides a complex comprising a muscle targeting agent covalently linked to a molecular payload to overcome this challenge. In some embodiments, the complex is particularly useful for delivering a molecular payload that modulates (e.g., promotes) the expression or activity of a target gene in muscle cells, for example, in subjects with or suspected of having a rare muscle disease. For example, in some embodiments, the complex is provided to target DMD, for example, a mutated DMD allele. In some embodiments, the complex provided herein may include an oligonucleotide that promotes the normal expression and activity of DMD. As another example, the complex may include an oligonucleotide that induces exon skipping of DMD mRNA. In some embodiments, a synthetic nucleic acid payload (e.g., a DNA or RNA payload) expressing one or more proteins that promote the normal expression and activity of DMD may be used.

[0053] In some embodiments, the complex may include a molecular payload (e.g., a dystrophin minigene) of synthetic cDNA and / or (e.g., and) synthetic mRNA expressing dystrophin or a fragment thereof. In some embodiments, the complex may include a molecular payload such as a guide molecule (e.g., guide RNA) that can target a nucleic acid programmable nuclease (e.g., Cas9) to disease-related DMD mutations, e.g., in or near sequences in mutated DMD exons. In some embodiments, such a nucleic acid programmable nuclease may be used to cleave some or all of the disease-related DMD mutations, e.g., mutated DMD exons, in order to promote functional DMD expression. In some embodiments, the complex may include a molecular payload that upregulates the expression and / or (e.g., and) the activity of genes that can replace the function of dystrophin, such as eutrophin.

[0054] Further aspects of this disclosure, including the definition of terms, are provided below.

[0055] I. Definition Administering: As used herein, the term “administering” or “dosing” means providing a complex to a subject in a physiologically and / or pharmacologically useful manner (for example, treating a disease in the subject).

[0056] Approximately: As used herein, the terms “approximately” or “about” refer to a value similar to the given reference value when applied to one or more values ​​of interest. In some embodiments, unless otherwise stated or evident from the context (except where such a figure exceeds 100% of a feasible value), the terms “approximately” or “about” refer to a broad range of values ​​that fall within plus or minus (greater than or less than) 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less than these values.

[0057] Antibody: As used herein, the term “antibody” refers to a polypeptide comprising at least one immunoglobulin variable domain or at least one antigenic determinant, for example, 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, an F(ab')2 fragment, an Fv fragment, or an scFv fragment. In some embodiments, the antibody is a nanobody derived from an antibody of a camelid animal or a nanobody derived from a shark antibody. In some embodiments, the antibody is a bispecific antibody. In some embodiments, the antibody comprises a framework having a human germline sequence. In another embodiment, the antibody comprises a heavy chain constant region selected from the group consisting of constant regions of IgG, IgG1, IgG2, IgG2A, IgG2B, IgG2C, IgG3, IgG4, IgA1, IgA2, IgD, IgM, and IgE. In some embodiments, the antibody comprises a heavy (H) chain variable region (abbreviated herein as VH) and / or (for example, and), a light (L) chain variable region (abbreviated herein as VL). In some embodiments, the antibody comprises a constant region, for example, an Fc region. The immunoglobulin constant region refers to the heavy chain or light chain constant region. The amino acid sequences of the human IgG heavy chain and light chain constant region and their functional variations are known. With respect to the heavy chain, in some embodiments, the heavy chain of the antibody described herein may be an alpha (α), delta (Δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain. In some embodiments, the heavy chain of the antibody described herein may comprise a human alpha (α), delta (Δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain. In specific embodiments, the antibody described herein comprises a human gamma-1 CH1 domain, a CH2 domain, and / or (for example, and), a CH3 domain. In some embodiments, the amino acid sequence of the VH domain includes the amino acid sequence of the human gamma (γ) heavy chain constant region, for example, any sequence known in the art.Non-limiting examples of human constant region sequences are described in the art; see, for example, U.S. Patent No. 5,693,780 and Kabat EA et al. (1991) above. 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 of the variable chain constant regions provided herein. In some embodiments, the antibody is modified, for example, via glycosylation, phosphorylation, SUMOylation, and / or (for example, and), methylation. In some embodiments, the antibody is a glycosylated antibody conjugated to one or more sugar or carbohydrate molecules. In some embodiments, one or more sugar or carbohydrate molecules are conjugated to the antibody via N-glycosylation, O-glycosylation, C-glycosylation, glypiation (GPI anchor attachment), and / or (for example, and), phosphoglycosylation. In some embodiments, one or more sugar or carbohydrate molecules are monosaccharides, disaccharides, oligosaccharides, or glycans. In some embodiments, one or more sugar or carbohydrate molecules are branched oligosaccharides or branched glycans. In some embodiments, one or more sugar or carbohydrate molecules comprise mannose units, glucose units, N-acetylglucosamine units, N-acetylgalactosamine units, galactose units, fucose units, or phospholipid units. In some embodiments, the antibody is a construct comprising a polypeptide containing one or more antigen-binding fragments of the present disclosure linked to a linker polypeptide or an immunoglobulin constant region. The linker polypeptide comprises two or more amino acid residues linked by a peptide bond and is used to link one or more antigen-binding sites. Examples of linker polypeptides have been reported (see, for example, Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, RJ, et al. (1994) Structure 2:1121-1123).Furthermore, antibodies may also be part of larger immunoadhesion molecules formed by covalent or non-covalent bonds between the antibody or antibody moiety and one or more other proteins or peptides. Examples of such immunoadhesion molecules include the use of streptavidin core regions to construct tetrameric scFv molecules (Kipriyanov, SM, et al. (1995) Human Antibodies and Hybridomas 6:93-101), and the use of cysteine ​​residues, marker peptides, and C-terminal polyhistidine tags to construct divalent and biotinylated scFv molecules (Kipriyanov, SM, et al. (1994) Mol.Immunol. 31:1047-1058).

[0058] CDR: As used herein, the term “CDR” refers to the complementarity-determining region within an antibody variable sequence. A typical antibody molecule contains a heavy chain variable region (VH) and a light chain variable region (VL), which are typically involved in antigen binding. The VH and VL regions can be further subdivided into hypervariable regions, also known as “complementarity-determining regions” (“CDR”), which are interspersed with more conserved regions known as “framework regions” (“FR”). Each VH and VL typically consists of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The extent of the framework regions and CDRs can be precisely identified using methodologies known in the art, for example, by Kabat definitions, IMGT definitions, Chothia definitions, AbM definitions, and / or (as an example, and) contact definitions, all of which are well known in the art.For example, Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242; IMGT (registered trademark), the international ImMunoGeneTics information system (registered trademark) http: / / www.imgt.org, Lefranc, M.-P. et al., Nucleic Acids. Res.,27:209-212(1999);Ruiz,M.et al.,Nucleic Acids Res.,28:219-221(2000);Lefranc,M.-P.,Nucleic Acids Res.,29:207-209(2001);Lefranc,M.-P.,Nucleic Acids Res.,31:307-310(2003);Lefranc,M.-P.et al.,In Silico Biol.,5,0006(2004)[Epub],5:45-60(2005);Lefranc,M.-P.et al.,Nucleic Acids Res.,33:D593-597(2005);Lefranc,M.-P.et al.,Nucleic Acids Res.,37:D1006-1012(2009);Lefranc,M.-P.et al.,Nucleic Acids Res.,43:D413-422(2015);Chothia et al.,(1989)Nature 342:877;Chothia,C.et al. (1987) J. Mol. Biol. 196:901-917, Al-lazikani et al. See al(1997)J.Molec.Biol.273:927-948; and Almagro,J.Mol.Recognit.17:132-143(2004). Also see hgmp.mrc.ac.uk and bioinf.org.uk / abs. As used herein, CDR may mean CDR as defined by any method known in the art.Two antibodies having the same CDR mean that, if determined in the same way, for example by the IMGT definition, the two antibodies have the same amino acid sequence of their CDR.

[0059] There are three CDRs in each of the variable regions of the heavy and light chains, designated as CDR1, CDR2, and CDR3 for each variable region. The term “CDR set,” as used herein, refers to a group of three CDRs occurring in a single variable region capable of binding to an antigen. The precise boundaries of these CDRs are defined differently depending on the system. The system described by Kabat et al., Sequence of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and (1991)) not only provides a unique residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries that define three CDRs. These CDRs are sometimes referred to as Kabat CDRs. Subportions of the CDRs may be designated as L1, L2, and L3, or H1, H2, and H3, where "L" and "H" designate the light chain and heavy chain regions, respectively. These regions are sometimes referred to as Chothia CDRs, which have boundaries that overlap with Kabat CDRs. Other boundaries that define CDRs that overlap with Kabat CDRs are described by Padlan (FASEB J.9:133-139 (1995)) and MacCallum (J Mol Biol This is described in 262(5):732-45(1996). Further CDR boundary definitions do not have to strictly adhere to one of the systems above, but may still overlap with Kabat CDRs, and may be shortened or lengthened based on predictions or experimental findings that specific residues, groups of residues, or even the entire CDR do not significantly affect antigen binding. The methods used herein may utilize CDRs defined according to any of these systems. Examples of CDR definition systems are shown in Table 1. Table 1. CDR definition [Table 1]

[0060] CDR-grafted antibody: The term "CDR-grafted antibody" refers to an antibody that contains heavy chain and light chain variable region sequences from one species, but in which one or more sequences of the VH and / or VL CDR regions are replaced with CDR sequences from another species. This includes antibodies that have mouse heavy chain and light chain variable regions, but in which one or more of the mouse CDR (e.g., CDR3) are replaced with human CDR sequences.

[0061] Chimeric antibody: The term "chimeric antibody" refers to an antibody that contains heavy chain and light chain variable region sequences from one species and a constant region sequence from another species, such as an antibody that has mouse heavy chain and light chain variable regions linked to a human constant region.

[0062] Complementary: As used herein, the term “complementary” refers to the capacity for accurate pairing between two nucleotides or between two sets of nucleotides. In particular, complementarity is a term that characterizes the degree of hydrogen bond pairing that results in a bond between two nucleotides or between two sets of nucleotides. For example, if a base of an oligonucleotide at a certain position can hydrogen bond with a base of a target nucleic acid (e.g., mRNA) at a corresponding position, then the bases are considered complementary to each other at that position. Base pairing may include both standard Watson-Crick base pairings and non-Watson-Crick base pairings (e.g., Wobble base pairings and Hoogsteen base pairings). For example, in some embodiments, as complementary base pairings, an adenosine-type base (A) is complementary to a thymidine-type base (T) or a uracil-type base (U), a cytosine-type base (C) is complementary to a guanosine-type base (G), and a universal base such as 3-nitropyrrole or 5-nitroindole can hybridize with any of A, C, U, or T. Inosine (I) is also considered a universal base in the art and is considered complementary to any of A, C, U, or T.

[0063] Conservative amino acid substitutions: As used herein, “conservative amino acid substitutions” refer to amino acid substitutions that do not alter the relative charge or size characteristics of the protein to which the amino acid substitution is made. Variants may be prepared according to methods for modifying polypeptide sequences known to those skilled in the art, for example, references summarizing such methods, such as Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Fourth Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2012, or Current Protocols in Molecular Biology, FMAusubel, et al., eds., John Wiley & Sons, Inc., New York. Conservative amino acid substitutions include substitutions made to amino acids in the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

[0064] Covalently linked (or connected): As used herein, the term “covalently linked (or connected)” refers to the characteristic of two or more molecules that are linked together by at least one covalent bond. In some embodiments, two molecules may be covalently linked together by a single bond (e.g., a disulfide bond or disulfide bridge) acting as an intermolecular linker. However, in some embodiments, two or more molecules may be covalently linked together by a molecule that acts as a linker, connecting two or more molecules together through multiple covalent bonds. In some embodiments, the linker may be a cleavable linker. However, in some embodiments, the linker may be an incleavable linker.

[0065] Cross-reactivity: As used herein, and in the context of targeting agents (e.g., antibodies), the term “cross-reactivity” refers to the property of an agent to be able to specifically bind to one or more antigens of a similar type or class (e.g., multiple homologous, paralogous, or orthologous antigens) with similar affinity or binding activity. For example, in some embodiments, an antibody that cross-reacts to antigens of a similar type or class between humans and non-human primates (e.g., human transferrin receptors and non-human primate transferrin receptors) is capable of binding to human antigens and non-human primate antigens with similar affinity or binding activity. In some embodiments, an antibody cross-reacts to human antigens and rodent antigens of a similar type or class. In some embodiments, an antibody cross-reacts to rodent antigens of a similar type or class and non-human primate antigens. In some embodiments, an antibody cross-reacts to human antigens, non-human primate antigens, and rodent antigens of a similar type or class.

[0066] DMD: As used herein, the term “DMD” refers to the gene encoding the dystrophin protein, a key component of the dystrophin-glycoprotein complex that cross-links muscle fibers in the internal cytoskeleton and extracellular matrix of muscle cells. Deletions, duplications, and point mutations in DMDs can cause dystrophin disorders such as Duchenne muscular dystrophy, Becker muscular dystrophy, or cardiomyopathy (e.g., DMD-associated dilated cardiomyopathy). The use of alternative promoters and alternative splicings results in a number of distinct transcript variants and protein isoforms for this gene. In some embodiments, the dystrophin gene may be human (gene ID: 1756), non-human primate (e.g., gene ID: 465559), or rodent gene (e.g., gene ID: 13405; gene ID: 24907). Furthermore, several human transcript variants encoding different protein isoforms have been characterized (for example, indicated by GenBank RefSeq Accession Numbers: NM_000109.3, NM_004006.2 (SEQ ID NO: 2239), NM_004009.3, NM_004010.3, and NM_004011.3).

[0067] DMD allele: As used herein, the term “DMD allele” refers to any one of the alternative forms of the DMD gene (e.g., wild-type or mutant). In some embodiments, the DMD allele may encode dystrophin, which retains its normal and typical function. In some embodiments, the DMD allele may include one or more mutations that result in muscular dystrophy. Common mutations leading to Duchenne muscular dystrophy include one or more frameshift, deletion, substitution, and duplication mutations in the 79 exons present in the dystrophin allele (e.g., exon 8, exon 23, exon 41, exon 44, exon 50, exon 51, exon 52, exon 53, or exon 55). Further examples of DMD mutations are disclosed, for example, in Flanigan KM, et al., Mutational spectrum of DMD mutations in dystrophinopathy patients: application of modern diagnostic techniques to a large cohort. Hum Mutat. 2009 Dec; 30 (12):1657-66, which is incorporated herein by reference in its entirety.

[0068] Dystrophinic disorder: As used herein, the term “dystrophinic disorder” refers to a muscle disorder resulting from one or more mutated DMD alleles. Dystrophinic disorder encompasses a spectrum of conditions (ranging from mild to severe) that include Duchenne muscular dystrophy, Becker muscular dystrophy, and DMD-associated dilated cardiomyopathy (DCM). In some embodiments, at one end of the spectrum, dystrophinic disorder is phenotypically associated with asymptomatic increases in plasma concentrations of creatine phosphokinase (CK) and / or muscle spasms with myoglobinuria (as an example). In some embodiments, at the other end of the spectrum, dystrophinic disorder is phenotypically associated with progressive muscle disorders that are generally classified as Duchenne muscular dystrophy or Becker muscular dystrophy when skeletal muscle is primarily affected, and as DMD-associated dilated cardiomyopathy (DCM) when the heart is primarily affected. Symptoms of Duchenne muscular dystrophy include muscle loss or degeneration, reduced muscle function, pseudohypertrophy of the tongue and calf muscles, high-risk neurological abnormalities, and shortened lifespan. Duchenne muscular dystrophy is associated with Online Mendelian Inheritance in Man (OMIM) Entry # 310200. Becker muscular dystrophy is associated with OMIM Entry # 300376. Dilated cardiomyopathy is associated with OMIM Entry X# 302045.

[0069] Exonic splicing enhancer (ESE): As used herein, the term “exonic splicing enhancer” or “ESE” refers to a gene, premRNA, or nucleic acid sequence motif within an exon of mRNA that directs or enhances the splicing of premRNA to mRNA, as described, for example, in Blencowe et al., Trends Biochem Sci 25, 106-10 (2000), incorporated herein by reference. An ESE may direct or promote splicing, for example, to remove one or more introns and / or one or more exons from a gene transcript. ESE motifs are typically 6-8 nucleotides long. SR proteins (e.g., proteins encoded by genes SRSF1, SRSF2, SRSF3, SRSF4, SRSF5, SRSF6, SRSF7, SRSF8, SRSF9, SRSF10, SRSF11, SRSF12, TRA2A, or TRA2B) bind to ESEs through their RNA recognition motif regions to facilitate splicing. ESE motifs can be identified by many methods, including those described in Cartegni et al., Nucleic Acids Research, 2003, Vol. 31, No. 13, 3568-3571, which are incorporated herein by reference.

[0070] Framework: As used herein, the term “framework” or “framework sequence” refers to the sequence remaining in the variable region after subtracting the CDRs. Since the precise definition of a CDR sequence can be determined by various systems, the meaning of a framework sequence depends on correspondingly different interpretations. The six CDRs (CDR-L1, CDR-L2, and CDR-L3 on the light chain, and CDR-H1, CDR-H2, and CDR-H3 on the heavy chain) also divide the framework regions on the light and heavy chains into four sub-regions (FR1, FR2, FR3, and FR4) on each chain, where CDR1 is located between FR1 and FR2, CDR2 between FR2 and FR3, and CDR3 between FR3 and FR4. When referred to elsewhere, framework regions that do not specify a particular sub-region as FR1, FR2, FR3, or FR4 represent the combined FRs within the variable region of a naturally occurring single immunoglobulin chain. When used herein, FR represents one of four subregions, and FR(plural) represents two or more of the four subregions containing the framework region. Human heavy and light chain acceptor sequences are known in the art. In one embodiment, acceptor sequences known in the art may be used in the antibodies disclosed herein.

[0071] Human Antibodies: When used herein, the term “human antibodies” is intended to encompass antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies in this disclosure may include, for example, amino acid residues not encoded by human germline immunoglobulin sequences (mutations introduced, e.g., by random mutagenesis or site-directed mutagenesis in vitro, or by somatic mutation in vivo), particularly in CDRs, especially CDR3. However, when used herein, the term “human antibodies” is not intended to encompass antibodies in which CDR sequences derived from the germline of another mammalian species, such as mouse, are conjugated onto a human framework sequence.

[0072] Humanized Antibodies: The term “humanized antibody” refers to an antibody that contains heavy and light chain variable region sequences from a non-human species (e.g., mouse), but in which at least a portion of the VH sequence and / or (e.g., and) VL sequence has been modified to be more “human-like,” i.e., more similar to human germline variable sequences. One type of humanized antibody is a CDR-conjugated antibody in which a human CDR sequence is introduced onto non-human VH and VL sequences and replaced with the corresponding non-human CDR sequence. In one embodiment, a humanized anti-transferrin receptor antibody and antigen-binding moiety are provided. Such an antibody may be produced by obtaining a mouse anti-transferrin receptor monoclonal antibody using existing hybridoma technology followed by in vitro genetic engineering for humanization (e.g., disclosed in PCT publication WO 2005 / 123126 A2 by Kasaian et al.).

[0073] Internalizing cell surface receptors: As used herein, the term “internalizing cell surface receptors” refers, for example, to cell surface receptors that are internalized by a cell in response to an external stimulus (for example, a ligand that binds to the receptor). In some embodiments, internalizing cell surface receptors are internalized by endocytosis. In some embodiments, internalizing cell surface receptors are internalized by clathrin-mediated endocytosis. However, in some embodiments, internalizing cell surface receptors are internalized by clathrin-independent pathways, such as phagocytosis, macropinocytosis, caveolae- and raft-mediated uptake, or clathrin-independent constitutive endocytosis. In some embodiments, internalizing cell surface receptors include an intracellular domain, a transmembrane domain, and / or (for example, and), an extracellular domain, which optionally further include a ligand-binding domain. In some embodiments, cell surface receptors become 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 aspects, the internalized cell surface receptor is the transferrin receptor.

[0074] Isolated Antibodies: When used herein, “isolated antibodies” are intended to refer to antibodies for which there are substantially no other antibodies with different antigen specificities (for example, an isolated antibody that specifically binds to the transferrin receptor has substantially no other antibodies that specifically bind to antigens other than the transferrin receptor). However, an isolated antibody that specifically binds to the transferrin receptor complex may have cross-reactivity to other antigens, such as transferrin receptor molecules from other species. Furthermore, isolated antibodies may be substantially free of other cellular material and / or (for example, and) chemicals.

[0075] Kabat Numbering: The terms “Kabat numbering,” “Kabat definition,” and “Kabat labeling” are used interchangeably herein. These terms refer to a system of numbering amino acid residues that are recognized in the art but are more variable (i.e., highly variable) than other amino acid residues in the heavy and light chain variable regions of an antibody or its antigen-binding moiety (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, USD Department of Health and Human Services, NIH Publication No. 91-3242). In the heavy chain variable region, the hypervariable region extends from amino acid positions 31-35 for CDR1, amino acid positions 50-65 for CDR2, and amino acid positions 95-102 for CDR3. In the light chain variable region, the hypervariable region extends to amino acid positions 24-34 for CDR1, amino acid positions 50-56 for CDR2, and amino acid positions 89-97 for CDR3.

[0076] Molecular Payload: As used herein, the term “molecular payload” refers to a molecule or species that functions to modulate a biological outcome. In some embodiments, the molecular payload is ligated to or otherwise linked to a muscle targeting agent. In some embodiments, the molecular payload is a small molecule, protein, peptide, nucleic acid, or oligonucleotide. In some embodiments, the molecular payload functions to modulate the transcription of a DNA sequence, to modulate the expression of a protein, or to modulate the activity of a protein. In some embodiments, the molecular payload is an oligonucleotide containing a chain having a region complementary to a target gene.

[0077] Muscle Targeting Agent: As used herein, the term “muscle targeting agent” refers to a molecule that specifically binds to an antigen expressed on a muscle cell. The antigen in or on the muscle cell may be a membrane protein, e.g., an endogenous membrane protein or a superficial membrane protein. Typically, a muscle targeting agent specifically binds to an antigen on the muscle cell that facilitates the internalization of the muscle targeting agent (and any associated molecular payload) into the muscle cell. In some embodiments, the muscle targeting agent can specifically bind to an internalizing cell surface receptor on the muscle and be internalized into the muscle cell through receptor-mediated internalization. In some embodiments, the muscle targeting agent is a small molecule, protein, peptide, nucleic acid (e.g., an aptamer), or antibody. In some embodiments, the muscle targeting agent is linked to a molecular payload.

[0078] Muscle-Targeted Antibodies: As used herein, the term “muscle-targeted antibody” refers to a muscle-targeting agent that is an antibody that specifically binds to an antigen found in or on muscle cells. In some embodiments, a muscle-targeted antibody specifically binds to an antigen on a muscle cell that facilitates the internalization of the muscle-targeted antibody (and any associated molecular payload) into the muscle cell. In some embodiments, a muscle-targeted antibody specifically binds to an internalizing cell surface receptor present on the muscle cell. In some embodiments, a muscle-targeted antibody is an antibody that specifically binds to a transferrin receptor.

[0079] Oligonucleotides: As used herein, the term “oligonucleotide” refers to an oligomeric nucleic acid compound up to 200 nucleotides in length. Examples of oligonucleotides include, but are not limited to, RNAi oligonucleotides (e.g., siRNA, shRNA), microRNAs, gapmers, mixmers, phosphorodiamidate morpholino, peptide nucleic acids, aptamers, guide nucleic acids (e.g., Cas9 guide RNA), etc. Oligonucleotides may be single-stranded or double-stranded. In some embodiments, oligonucleotides may contain one or more modified nucleotides (e.g., 2'-O-methylglycosulfate, purine, or pyrimidine modification). In some embodiments, oligonucleotides may contain one or more modified nucleotide linkages. In some embodiments, oligonucleotides may contain one or more phosphorothioate linkages, which may be in the stereochemical configuration of Rp or Sp.

[0080] Recombinant Antibodies: When used herein, the term “recombinant human antibodies” refers to all human antibodies prepared, expressed, created, or isolated by recombinant means, such as antibodies expressed using recombinant expression vectors transfected into host cells (as described in detail herein), 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 human immunoglobulin gene transgenic animals (e.g., mice) (e.g., Taylor, LD, et al.) It is intended to include antibodies prepared, expressed, created, or isolated by any other means involving splicing of human immunoglobulin gene sequences with other DNA sequences, as described by 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). Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in some embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when human Ig sequence transgenic animals are used, in vivo somatic mutagenesis), and therefore, although the amino acid sequences of the VH and VL regions of the recombinant antibody are derived from and related to the VH and VL sequences of the human germline, they may not be naturally present in the germline repertoire of human antibodies in vivo.One aspect of the present disclosure provides fully human antibodies capable of binding to human transferrin receptors, which may be produced using techniques well known in the art, for example, but not limited to, techniques using a human Ig phage library (e.g., disclosed in PCT publication WO 2005 / 007699 A2 by Jermutus et al.).

[0081] Complementary Region: As used herein, the term “complementary region” refers to a nucleotide sequence (e.g., a nucleotide sequence of an oligonucleotide) that is sufficiently complementary to a cognate nucleotide sequence (e.g., a nucleotide sequence of a target nucleic acid) such that the two nucleotide sequences can anneal to each other under physiological conditions (e.g., in a cell). In some embodiments, the complementary region is fully complementary to the cognate nucleotide sequence of the target nucleic acid. However, in some embodiments, the complementary region is partially complementary to the cognate nucleotide sequence of the target nucleic acid (e.g., at least 80%, 90%, 95%, or 99% complementary). In some embodiments, the complementary region contains one, two, three, or four mismatches compared to the cognate nucleotide sequence of the target nucleic acid.

[0082] Specific binding: As used herein, the term “specific binding” refers to the ability of a molecule to bind to a binding partner to a degree of affinity or binding activity that can be used to distinguish the binding partner from a suitable control in a binding assay or other binding context. With respect to an antibody, the term “specific binding” refers to the ability of an antibody to bind to a particular antigen to a suitable reference antigen, or to an antigen that can be used to distinguish a particular antigen from other antigens, to a degree of affinity or binding activity that allows for preferential targeting to a cell (e.g., muscle cells) through binding to the antigen, as described herein, for example. In some embodiments, an antibody specifically binds to a target if it has a KD of at least about 10⁻⁴M, 10⁻⁵M, 10⁻⁶M, 10⁻⁷M, 10⁸M, 10⁹M, 10⁻¹⁰M, 10⁻¹¹M, 10⁻¹²M, 10⁻¹³M, or less, when the antibody binds to the target. In some embodiments, the antibody specifically binds to a transferrin receptor, for example, to an epitope in the tip domain of the transferrin receptor.

[0083] Subject: As used herein, the term “subject” refers to mammals. In some embodiments, subject is a non-human animal or rodent of the order Primates. In some embodiments, subject is a human. In some embodiments, subject is a patient having or suspected of having the disease, e.g., a human patient. In some embodiments, subject is a human patient having or suspected of having the disease resulting from a mutated DMD gene sequence, e.g., a mutation in an exon of the DMD gene sequence. In some embodiments, subject has a dystrophin disorder, e.g., Duchenne muscular dystrophy.

[0084] Transferrin receptor: As used herein, the term “transferrin receptor” (also known as TFRC, CD71, p90, TFR, or TFR1) refers to an internalized cell surface receptor that binds to transferrin to facilitate iron uptake by endocytosis. In some embodiments, the transferrin receptor may originate from humans (NCBI Gene ID 7037), non-human primates (e.g., NCBI Gene ID 711568 or NCBI Gene ID 102136007), or rodents (e.g., NCBI Gene ID 22042). In addition, several human transcript variants encoding various isoforms of the receptor have been characterized (e.g., as annotated with GenBank RefSeq accessions: NP_001121620.1, NP_003225.2, NP_001300894.1, and NP_001300895.1).

[0085] 2'-Modified Nucleoside: As used herein, the terms “2'-Modified Nucleoside” and “2'-Modified Ribonucleoside” are interchangeable and refer to nucleosides having a sugar moiety modified at the 2' position. In some embodiments, a 2'-modified nucleoside is a 2'-4' bicyclic nucleoside, where the 2' and 4' positions of the sugar are cross-linked (e.g., by methylene, ethylene, or (S)-restricted ethyl cross-linking). In some embodiments, a 2'-modified nucleoside is a non-bicyclic 2'-modified nucleoside, where, for example, the 2' position of the sugar moiety is substituted. Non-limiting examples of 2'-modified nucleosides include: 2'-deoxy, 2'-fluoro (2'-F), 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), 2'-ON-methylacetamide (2'-O-NMA), locked nucleic acids (LNA, methylene-bridged nucleic acids), ethylene-bridged nucleic acids (ENA), and (S)-restricted ethyl-bridged nucleic acids (cEt). In some embodiments, the 2'-modified nucleosides described herein are high-affinity modified nucleotides, and oligonucleotides containing 2'-modified nucleotides have increased affinity for target sequences compared to unmodified oligonucleotides. An example of the structure of a 2'-modified nucleoside is provided below: [ka]

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

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

[0088] In some embodiments, the complex comprises a muscle targeting agent (e.g., an anti-transferrin receptor antibody) covalently linked to a molecular payload (e.g., a mixmer antisense oligonucleotide targeting a mutated DMD allele) to facilitate exon skipping.

[0089] A. Muscle targeting agents Several aspects of this disclosure provide muscle targeting agents, for example, muscle targeting agents for delivering molecular payloads to muscle cells. In some embodiments, such muscle targeting agents can bind to muscle cells, for example, via specific binding to antigens on muscle cells, and deliver the bound molecular payload to the muscle cells. In some embodiments, the molecular payload is bound to the muscle targeting agent (for example, covalently), and when the muscle targeting agent binds to an antigen on a muscle cell, it is internalized into the muscle cell, for example, via endocytosis. It should be understood that various types of muscle targeting agents may be used in accordance with this disclosure. For example, muscle targeting agents may contain, or consist of, nucleic acids (e.g., DNA or RNA), peptides (e.g., antibodies), lipids (e.g., microvesicles), or sugar moieties (e.g., polysaccharides). Exemplary muscle targeting agents are described in more detail herein, but it should be understood that the exemplary muscle targeting agents provided herein are not intended to be limiting.

[0090] Several aspects of this disclosure provide muscle targeting agents that specifically bind to antigens on muscles, such as skeletal muscle, smooth muscle, or cardiac muscle. In some embodiments, any of the muscle targeting agents provided herein bind (specifically, for example) to antigens on skeletal muscle cells, smooth muscle cells, and / or (for example, and) cardiac muscle cells.

[0091] Interaction with muscle-specific cell surface recognition elements (e.g., cell membrane proteins) can achieve both tissue localization and selective uptake into muscle cells. In some embodiments, molecules that are substrates of muscle uptake transporters are useful for delivering molecular payloads into muscle tissue. Binding to muscle surface recognition elements, followed by endocytosis, can allow even macromolecules such as antibodies to enter muscle cells. As another example, a molecular payload conjugated to transferrin or an anti-transferrin receptor antibody may be taken up by muscle cells via binding to the transferrin receptor and then endocytosed, for example, via clathrin-mediated endocytosis.

[0092] The use of muscle-targeting agents can also be useful for enriching molecular payloads (e.g., oligonucleotides) in muscle while reducing toxicity associated with the effect in other tissues. In some embodiments, muscle-targeting agents enrich bound molecular payloads in muscle cells compared to other cell types within the subject. In some embodiments, muscle-targeting agents enrich bound molecular payloads in muscle cells (e.g., skeletal muscle cells, smooth muscle cells, or cardiomyocytes) to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times more than in non-muscle cells (e.g., liver cells, nerve cells, blood cells, or adipocytes). In some embodiments, the toxicity of the molecular payload in a target when conjugated to a muscle targeting agent is reduced by at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, or 95% when it is delivered to the target.

[0093] In some embodiments, muscle recognition elements (e.g., muscle cell antigens) may be required to achieve muscle selectivity. For example, the muscle targeter may be a small molecule that is a substrate of a muscle-specific uptake transporter. Another example is that the muscle targeter may be an antibody that enters muscle cells via transporter-mediated endocytosis. Yet another example is that the muscle targeter may be a ligand that binds to cell surface receptors on muscle cells. It should be understood that while transporter-based approaches provide a direct pathway to cell entry, receptor-based targeting may involve stimulated endocytosis to reach the desired site of action.

[0094] i. Muscle targeting antibody In some embodiments, muscle targeting agents are antibodies. Generally, the high specificity of antibodies against their target antigens provides the potential to selectively target muscle cells (e.g., skeletal muscle cells, smooth muscle cells, and / or cardiomyocytes). This specificity may also limit off-target toxicity. Examples of antibodies capable of targeting surface antigens of muscle cells have been reported and are within the scope of this disclosure. For example, antibodies that target the surface of muscle cells are described in Arahata K., et al. “Immunostaining of skeletal and cardiac muscle surface membrane with antibody against Duchenne muscular dystrophy peptide” Nature 1988;333:861-3; Song KS, et al. “Expression of caveolin-3 in skeletal, cardiac, and smooth muscle cells. Caveolin-3 is a component of the sarcolemma and co-fractionates with dystrophin and dystrophin-associated glycoproteins” J Biol Chem 1996;271:15160-5; and Weisbart RH et al., “Cell type specific targeted intracellular delivery into muscle of a monoclonal antibody that binds myosin IIb” Mol Immunol. 2003 Mar,39(13):78309; the entire contents of each of these are incorporated herein by reference.

[0095] a. Anti-transferrin receptor antibody Several aspects of this disclosure are based on the recognition that agents that bind to transferrin receptors, such as anti-transferrin receptor antibodies, can target muscle cells. Transferrin receptors are internalized cell surface receptors that transport transferrin across the cell membrane and contribute to the regulation of intracellular iron levels and homeostasis. Several aspects of this disclosure provide transferrin receptor-binding proteins capable of binding to transferrin receptors. Consequently, aspects of this disclosure provide binding proteins (e.g., antibodies) that bind to transferrin receptors. In some embodiments, binding proteins that bind to transferrin receptors are internalized into muscle cells along with any bound molecular payload. As used herein, antibodies that bind to transferrin receptors may also be interchangeably referred to as transferrin receptor antibodies, anti-transferrin receptor antibodies, or anti-TfR antibodies. Antibodies that bind to transferrin receptors, such as specifically binding antibodies, may be internalized into cells upon binding to the transferrin receptor, for example, through receptor-mediated endocytosis.

[0096] It should be understood that anti-transferrin receptor antibodies may be produced, synthesized, and / or (for example, and) derivatized using several known methodologies, e.g., library design using phage display. Exemplary methodologies are characterized in the art and incorporated by reference (Diez, P. et al. “High-throughput phage-display screening in array format”, Enzyme and microbial technology, 2015, 79, 34-41.; Christoph MH and Stanley, JR “Antibody Phage Display: Technique and Applications”, J Invest Dermatol. 2014, 134:2.; Engleman, Edgar (Ed.) “Human Hybridomas and Monoclonal Antibodies.” 1985, Springer). In other embodiments, anti-transferrin antibodies have been characterized or disclosed to date.Antibodies that specifically bind to transferrin receptors are known in the art (for example, U.S. Patent No. 4,364,934, filed 12 / 4 / 1979, “Monoclonal antibody to a human early thymocyte antigen and methods for preparing same”; U.S. Patent No. 8,409,573, filed 6 / 14 / 2006, “Anti-CD71 monoclonal antibodies and uses thereof for treating malignant tumor cells”; U.S. Patent No. 9,708,406, filed 5 / 20 / 2014, “Anti-transferrin receptor antibodies and methods of use”; U.S. Patent No. 9,611,323, filed 12 / 19 / 2014, “Low affinity blood brain barrier receptor antibodies and uses therefor”; WO 2015 / 098989, filed 12 / 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 (See Pharmacol. Exp. Ther., 292:1048-1052).

[0097] Provided herein are novel anti-TfR antibodies for use as muscle targeting agents (e.g., on muscle targeting complexes) in several aspects. In some aspects, the anti-TfR antibodies described herein bind to transferrin receptors with high specificity and affinity. In some aspects, the anti-TfR antibodies described herein bind specifically to any extracellular epitope of the transferrin receptor or to an epitope that will be exposed to the antibody. In some aspects, the anti-TfR antibodies provided herein bind specifically to transferrin receptors from humans, non-human primate animals, mice, rats, etc. In some aspects, the anti-TfR antibodies provided herein bind to human transferrin receptors. In some aspects, the anti-TfR antibodies described herein bind to amino acid segments of human or non-human primate animal transferrin receptors provided in SEQ ID NOs. 105-108. In some aspects, the anti-TfR antibodies described herein bind to amino acid segments corresponding to amino acids 90-96 of the human transferrin receptor as represented by SEQ ID NO. 105, which are not in the apex domain of the transferrin receptor.

[0098] An example of a human transferrin receptor amino acid sequence corresponding to the NCBI sequence NP_003225.2 (transferrin receptor protein 1 isoform 1, homo sapiens) is as follows: (Sequence ID 105)

[0099] Examples of non-human primate transferrin receptor amino acid sequences corresponding to the NCBI sequence NP_001244232.1 (transferrin receptor protein 1, Macaca mulatta) are as follows: MMDQARSAFSNLFGGEPLSYTRFSLARQVDGDNSHVEMKLGVDEEENTDNNTKPNGTKPKRCGGNICYGTIAVIIFFLIGFMIGYLGYCKGVEPKTECERLAGTESPAREEPEEDFPAAPRLYWDDLKRKLSEKLDTTDFTSTIKLLNENLYVPREAGSQKDENLALYIENQFREFKLSKVWRDQHFVKI QVKDSAQNSVIIVDKNGGLVYLVENPGGYVAYSKAATVTGKLVHANFGTKKDFEDLDSPVNGSIVIVRAGKITFAEKVANAESLNAIGVLIYMDQTKFPIVKADLSFFGHAHLGTGDPYTPGFPSFNHTQFPPSQSSGLPNIPVQTISRAAAEKLFGNMEGDCPSDWKTDSTCKMVTSENKSVKLTVSNV LKETKILNIFGVIKGFVEPDHYVVVGAQRDAWGPGAAKSSVGTALLLKLAQMFSDMVLKDGFQPSRSIIFASWSAGDFGSVGATEWLEGYLSSLHLKAFTYINLDKAVLGTSNFKVSASPLLYTLIEKTMQDVKHPVTGRSLYQDSNWASKVEKLTLDNAAFPFLAYSGIPAVSFCFCEDTDYPYLGTTM DTYKELVERIPELNKVARAAAEVAGQFVIKLTHDTELNLDYERYNSQLLLFLRDLNQYRADVKEMGLSLQWLYSARGDFFRATSRLTTDFRNAEKRDKFVMKKLNDRVMRVEYYFLSPYVSPKESPFRHVFWGSGSHTLSALLESLKLRRQNNSAFNETLFRNQLALATWTIQGAANALSGDVWDIDNEF (Sequence ID 106)

[0100] Examples of non-human primate transferrin receptor amino acid sequences corresponding to the NCBI sequence XP_005545315.1 (transferrin receptor protein 1, Macaca fascicularis) are as follows: (Sequence ID 107)

[0101] An example of a mouse transferrin receptor amino acid sequence corresponding to the NCBI sequence NP_001344227.1 (transferrin receptor protein 1, mus musculus) is as follows: MMDQARSAFSNLFGGEPLSYTRFSLARQVDGDNSHVEMCLAADEEENADNNMKASVRKPKRFNGRLCFAAIALVIFFLIGFMSGYLGYCKRVEQKEECVKLAETEETDKSETMETEDVPTSSRLYWADLKTLLSEKLNSIEFADTIKQLSQNTYTPREAGSQKDESLAYYIENQFHEFKFSKVWRDEHYV KIQVKSsIGQNMVTIVQSNGNLDPVESPEGYVAFSKPTEVSGKLVHANFGTKKDFEELSYSVNGSLVIVRAGEITFAEKVANAQSFNAIGVLIYMDKNKFPVVEADLALFGHAHHLGTGDPYTPGPFPSFNHTQFPPSQSSGLPNIPVQTISRAAAEKLFGKMEGSCPARWNIDSSCKLELSQNQNVKLIVKN VLKERRILNIFGVIKGYEEPDRYVVVGAQRDALGAGVAAKSSVGTGLLLKLAQVFSDMISKDGFRPSRSIIFASWTAGDFGAVGATEWLEGYLSSLHLKAFTYINLDKVVLGTSNFKVSASPLLYTLMGKIMQDVKHPVDGKSLYRDSNWISKVEKLSFDNAAYPFLAYSGIPAVSFCCEDADYPYLGTR LDTYEALTQKVPQLNQMVRTAAEVAGQLIIKLTHDVELNLDYEMYNSKLLSFMKDLNQFKTDIRDMGLSLQWLYSARGDYFRATSRLTTDFHNAEKTNRFVMREINDRIMKVEYHFLSPYVSPRESPFRHIFWGSGSHTLSALVENLKLRQKNITAFNETLFRNQLALATEWTIQGVANALSGDIWNIDNEF (query number 108)

[0102] In some embodiments, the anti-transferrin receptor antibody binds to the amino acid segment of the receptor as follows:FVKIQVKDSAQNSVIIVDKNGRLVYLVENPGGYVAYSKAATVTGKLVHANFGTKKDFEDLYTPVNGSIVIVRAGKITFAEKVANAESLNAIGVLIYMDQTKFPIVNAELSFFGHAHLGTGDPYTPGFPSFNHTQFPPSRSSGLPNIPVQTISRAAAEKLFGNMEGDCPSDWKTDSTCRMVTSESKNVKLTVSNVLKE (SEQ ID NO: 109) and does not inhibit the binding interaction between the transferrin receptor and transferrin and / or (for example, and) human hemoglobin proteins (also known as HFE). In some embodiments, the anti-transferrin receptor antibody described herein does not bind to the epitope of SEQ ID NO: 109.

[0103] Appropriate methodologies may be used, for example, through the use of recombinant DNA protocols to obtain and / or (for example, and) produce antibodies, antibody fragments, or antigen conjugates. In some embodiments, antibodies may also be produced through the generation of hybridomas (see, for example, Kohler, G and Milstein, C. "Continuous cultures of fused cells secreting antibody of predefined specificity," Nature, 1975, 256:495-497). The antigen of interest may be used as an immunogen of any type or entity, for example, recombinant or naturally occurring type or entity. Hybridomas are screened using standard methods, for example, ELISA screening, to find at least one hybridoma that produces an antibody targeting a specific antigen. Antibodies may also be produced through screening of protein expression libraries expressing the antibody (for example, phage display libraries). Phage display library designs may also be used in several embodiments (see, for example, U.S. Patent No. 5,223,409, 3 / 1 / 1991, "Directed evolution of novel binding proteins"; WO 1992 / 18619, 4 / 10 / 1992, "Heterodimeric receptor libraries using phagemids"; WO 1991 / 17271, 5 / 1 / 1991, "Recombinant library screening methods"; WO 1992 / 20791, 5 / 15 / 1992, "Methods for producing members of specific binding pairs"; and WO 1992 / 15679, 2 / 28 / 1992, "Improved epitope displaying phage"). In some embodiments, antigens of interest may be used to immunize non-human animals, for example, rodents or goats.In some embodiments, once antibodies have been obtained from non-human animals, they may be modified using a number of methodologies, including recombinant DNA techniques, as an option. Examples of antibody production and methodology additions are also known in the art (see, for example, Harlow et al., "Antibodies: A Laboratory Manual," Cold Spring Harbor Laboratory, 1988).

[0104] In some embodiments, the antibody is modified (e.g., modified via glycosylation, phosphorylation, SUMOylation, and / or (e.g., and) methylation). In some embodiments, the antibody is a glycosylated antibody conjugated to one or more sugar or carbohydrate molecules. In some embodiments, one or more sugar or carbohydrate molecules are conjugated to the antibody via N-glycosylation, O-glycosylation, C-glycosylation, glyciation (GPI anchor attachment), and / or (e.g., and) phosphoglycosylation. In some embodiments, one or more sugar or carbohydrate molecules are monosaccharides, disaccharides, oligosaccharides, or glycans. In some embodiments, one or more sugar or carbohydrate molecules are branched oligosaccharides or branched glycans. In some embodiments, one or more sugar or carbohydrate molecules comprise mannose units, glucose units, N-acetylglucosamine units, N-acetylgalactosamine units, galactose units, fucose units, or phospholipid units. In some embodiments, the sugar molecules are present in quantities of approximately 1–10, 1–5, 5–10, 1–4, 1–3, or 2. In some embodiments, the glycosylated antibody is glycosylated whole or partially. In some embodiments, the antibody is glycosylated by chemical reaction or by enzymatic means. In some embodiments, the antibody is glycosylated in vitro or in cells (which may optionally be deficient in enzymes in the N- or O-glycosylation pathway, e.g., glycosyltransferase). In some embodiments, the antibody is functionalized with sugar or carbohydrate molecules as described in International Patent Application Publication WO2014065661, published on May 1, 2014, entitled "Modified antibody, antibody-conjugate and process for the preparation thereof".

[0105] In some embodiments, the anti-TfR antibodies of this disclosure comprise the VL domain and / or (for example, and) the VH domain of any one of the anti-TfR antibodies selected from Table 2, and comprise a constant region comprising the amino acid sequence of the constant region of an IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, any class (for example, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or any subclass (for example, IgG2a and IgG2b). Non-limiting examples of human constant regions have been described in the art; for example, see Kabat EA et al. (1991) above.

[0106] In some embodiments, drugs that bind to transferrin receptors, such as anti-TfR antibodies, can target muscle cells and / or mediate the transport of drugs across the blood-brain barrier (for example, and). Transferrin receptors are internalized cell surface receptors that transport transferrin across the cell membrane and contribute to the regulation of intracellular iron levels and homeostasis. Some aspects of this disclosure provide transferrin receptor-binding proteins capable of binding to transferrin receptors. Antibodies that bind to transferrin receptors, such as specifically binding antibodies, may be internalized into cells upon binding to the transferrin receptor, for example, through receptor-mediated endocytosis.

[0107] In some embodiments, humanized antibodies that bind to the transferrin receptor with high specificity and affinity are provided herein. In some embodiments, the humanized anti-TfR antibodies described herein specifically bind to any extracellular epitope of the transferrin receptor, or an epitope that becomes exposed to the antibody. In some embodiments, the 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 the human transferrin receptor. In some embodiments, the humanized anti-TfR antibodies described herein bind to amino acid segments of human or non-human primate transferrin receptors, such as those provided in SEQ ID NOs: 105-108. In some embodiments, the humanized anti-TfR antibodies described herein bind to an amino acid segment corresponding to amino acids 90-96 of the human transferrin receptor 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 to TfR2.

[0108] In some embodiments, the anti-TfR antibody is at least about 10 -4 M, 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, 10 -13The antibodies specifically bind to TfR1 (e.g., human or non-human primate TfR1) by a binding affinity of M, or a smaller binding affinity (e.g., indicated by Kd). In some embodiments, the anti-TfR antibodies described herein bind to TfR1 by a KD in the sub-nanomole 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 cynoporin TfR1 (e.g., 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, 10 -13 The antibodies do not bind to mouse TfR1 (by M or a smaller Kd). The affinity and binding kinetics of anti-TfR antibodies can be tested using any preferred method, including but not limited to biosensor technologies (e.g., OCTET or BIACORE). In some embodiments, the binding of any one of the anti-TfR antibodies described herein does not compete with or inhibit transferrin binding to TfR1. In some embodiments, the binding of any one of the anti-TfR antibodies described herein does not compete with or inhibit HFE-beta-2-microglobulin binding to TfR1.

[0109] The anti-TfR antibodies described herein are humanized antibodies. Table 2 provides the CDR and variable region amino acid sequences of mouse monoclonal anti-TfR antibodies derived from the humanized anti-TfR antibodies described herein. Table 2. Mouse monoclonal anti-TfR antibodies [Table 2-1] [Table 2-2] [Table 2-3]

[0110] In some embodiments, the anti-TfR antibody of this disclosure is a humanized variant of any one of the anti-TfR antibodies provided in Table 2. In some embodiments, the anti-TfR antibody of this disclosure comprises the same CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 as any one of the indicated anti-TfR antibodies provided in Table 2, and comprises a humanized heavy chain variable region and / or (for example, and) a humanized light chain variable region.

[0111] A humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the recipient's complementarity-determining region (CDR) are replaced by residues from the CDR of a non-human species (donor antibody), such as mouse, rat, or rabbit, having the desired specificity, affinity, and capabilities. In some embodiments, the Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, the humanized antibody may also include residues not found in the recipient antibody or in the imported CDR or framework sequence, but may also include residues that are included to further refine and optimize the performance of the antibody. Generally, a humanized antibody will substantially include all of at least one, typically two, variable domains, in which all or substantially all of the CDR region corresponds to the CDR region of the non-human immunoglobulin, and all or substantially all of the FR region is the FR region of the human immunoglobulin consensus sequence. Humanized antibodies will also, optimally, contain at least a portion of the constant region or domain (Fc) of an immunoglobulin (typically human immunoglobulin). The antibody may have a modified Fc region as described in WO 99 / 58572. Other forms of humanized antibodies have one or more CDRs (1, 2, 3, 4, 5, 6) that are modified with respect to the original antibody, and these are also called one or more CDRs derived from one or more CDRs from the original antibody. Humanized antibodies may also undergo affinity maturation.

[0112] Humanized antibodies and methods for producing them are known, for example, as seen 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. Patents Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005); Padlan et al., Mol. Immunol. 28:489-498 (1991); Dall'Acqua et al., Methods 36:43-60 (2005); As described in Osbourn et al., Methods 36:61-68 (2005); and Klimka et al., Br. J. Cancer, 83:252-260 (2000), all of which are incorporated herein by reference. Human framework regions that can be used for humanization are described, for example, in Sims et al. J. Immunol. 151:2296 (1993); Carter et al. Proc. Natl. Acad. Sci. USA. 89:4285 (1992); Presta et al. J. Immunol. 151:2623 (1993); Almagro et al., Front. Biosci. 13:1619-1633 (2008)); Baca et al., J. Biol. Chem. 272:10678-10684 (1997); and Rosok et al., J Biol. Chem. 271:22611-22618 (1996), all of which are incorporated herein by reference.

[0113] In some embodiments, the humanized anti-TfR antibodies of this disclosure include humanized VH, which comprises 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 humanized VL, which comprises one or more amino acid variations compared to any one of the VLs listed in Table 2 (e.g., in the VL framework region).

[0114] In some embodiments, the humanized anti-TfR antibody of this disclosure includes a humanized VH containing 25 or fewer amino acid variations (for example, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid variations) compared to the VH of any of the anti-TfR antibodies listed in Table 2 (for example, any one of SEQ ID NOs. 17, 22, 26, 43, 61, 65, and 68). Alternatively or additionally (for example, additionally), the humanized anti-TfR antibodies of this disclosure include humanized VLs containing 25 or fewer amino acid variations (for example, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid variations) compared to the VL of any one of the anti-TfR antibodies listed in Table 2 (for example, any one of SEQ ID NOs. 18, 44, and 62).

[0115] In some embodiments, the humanized anti-TfR antibody of this disclosure comprises a humanized VH having an amino acid sequence identical to at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) of the framework region of any VH of any of the anti-TfR antibodies listed in Table 2 (e.g., one of SEQ ID NOs. 17, 22, 26, 43, 61, 65, and 68). Alternatively or additionally (e.g., additionally), in some embodiments, the humanized anti-TfR antibody of this disclosure comprises a humanized VL having an amino acid sequence identical to at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) of the framework region of any VL of any of the anti-TfR antibodies listed in Table 2 (e.g., one of SEQ ID NOs. 18, 44, and 62).

[0116] In some embodiments, the humanized anti-TfR antibody of the present disclosure comprises CDR-H1 having the amino acid sequence of SEQ ID NO: 1 (according to the IMGT definition system), CDR-H2 having the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 19, or SEQ ID NO: 23 (according to the IMGT definition system), and CDR-H3 having the amino acid sequence of SEQ ID NO: 3 (according to the IMGT definition system), and includes a humanized VH having 25 or fewer amino acid variations (for example, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid variations) in the framework region compared to the VH shown in SEQ ID NO: 17, SEQ ID NO: 22, or SEQ ID NO: 26. Alternatively or additionally (for example, additionally), the anti-TfR antibody of the present disclosure comprises CDR-L1 having the amino acid sequence of SEQ ID NO: 4 (according to the IMGT definition system), CDR-L2 having the amino acid sequence of SEQ ID NO: 5 (according to the IMGT definition system), and CDR-L3 having the amino acid sequence of SEQ ID NO: 6 (according to the IMGT definition system), and includes a humanized VL containing 25 or fewer amino acid variations (for example, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid variations) in the framework region compared to the VL shown in SEQ ID NO: 18.

[0117] In some embodiments, the humanized anti-TfR antibody of the present disclosure comprises a humanized VH having CDR-H1 having the amino acid sequence of SEQ ID NO: 1 (according to the IMGT definition system), CDR-H2 having the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 19, or SEQ ID NO: 23 (according to the IMGT definition system), and CDR-H3 having the amino acid sequence of SEQ ID NO: 3 (according to the IMGT definition system), which is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework region to the VH represented by SEQ ID NO: 17, SEQ ID NO: 22, or SEQ ID NO: 26. Alternatively or additionally (for example, additionally), the humanized anti-TfR antibody of the present disclosure comprises a humanized VL having CDR-L1 having the amino acid sequence of SEQ ID NO: 4 (according to the IMGT-defined system), CDR-L2 having the amino acid sequence of SEQ ID NO: 5 (according to the IMGT-defined system), and CDR-L3 having the amino acid sequence of SEQ ID NO: 6 (according to the IMGT-defined system), which is at least 75% (for example, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework region to the VL shown in any one of SEQ ID NOs: 18.

[0118] In some embodiments, the humanized anti-TfR antibody of the present disclosure comprises CDR-H1 having the amino acid sequence of SEQ ID NO: 7 (according to the Kabat definition system), CDR-H2 having the amino acid sequence of SEQ ID NO: 8, SEQ ID NO: 20, or SEQ ID NO: 24 (according to the Kabat definition system), and CDR-H3 having the amino acid sequence of SEQ ID NO: 9 (according to the Kabat definition system), and includes a humanized VH having 25 or fewer amino acid variations (for example, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid variations) in the framework region compared to the VH shown in SEQ ID NO: 17, SEQ ID NO: 22, or SEQ ID NO: 26. Alternatively or additionally (for example, additionally), the humanized anti-TfR antibody of the present disclosure comprises a CDR-L1 having the amino acid sequence of SEQ ID NO: 10 (according to the Kabat definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 11 (according to the Kabat definition system), and a CDR-L3 having the amino acid sequence of SEQ ID NO: 6 (according to the Kabat definition system), and includes a humanized VL containing 25 or fewer amino acid variations (for example, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid variations) in the framework region compared to the VL shown in SEQ ID NO: 18.

[0119] In some embodiments, the humanized anti-TfR antibody of the present disclosure comprises a humanized VH having CDR-H1 having the amino acid sequence of SEQ ID NO: 7 (according to the Kabat definition system), CDR-H2 having the amino acid sequence of SEQ ID NO: 8, SEQ ID NO: 20, or SEQ ID NO: 24 (according to the Kabat definition system), and CDR-H3 having the amino acid sequence of SEQ ID NO: 9 (according to the Kabat definition system), which is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework region to the VH represented by SEQ ID NO: 17, SEQ ID NO: 22, or SEQ ID NO: 26. Alternatively or additionally (for example, additionally), the humanized anti-TfR antibody of the present disclosure comprises a humanized VL having CDR-L1 having the amino acid sequence of SEQ ID NO: 10 (according to the Kabat definition system), CDR-L2 having the amino acid sequence of SEQ ID NO: 11 (according to the Kabat definition system), and CDR-L3 having the amino acid sequence of SEQ ID NO: 6 (according to the Kabat definition system), which is at least 75% (for example, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework region to the VL shown in any one of SEQ ID NO: 18.

[0120] In some embodiments, the humanized anti-TfR antibody of the present disclosure comprises CDR-H1 having the amino acid sequence of SEQ ID NO: 12 (according to the Chothia definition system), CDR-H2 having the amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 21, or SEQ ID NO: 25 (according to the Chothia definition system), and CDR-H3 having the amino acid sequence of SEQ ID NO: 14 (according to the Chothia definition system), and includes a humanized VH having 25 or fewer amino acid variations (for example, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid variations) in the framework region compared to the VH shown in SEQ ID NO: 17, SEQ ID NO: 22, or SEQ ID NO: 26. Alternatively or additionally (for example, additionally), the humanized anti-TfR antibody of the present disclosure comprises a CDR-L1 having the amino acid sequence of SEQ ID NO: 15 (according to the Chothia definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 5 (according to the Chothia definition system), and a CDR-L3 having the amino acid sequence of SEQ ID NO: 16 (according to the Chothia definition system), and includes a humanized VL containing 25 or fewer amino acid variations (for example, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid variations) in its framework region compared to the VL shown in SEQ ID NO: 18.

[0121] In some embodiments, the humanized anti-TfR antibody of the present disclosure comprises a humanized VH having CDR-H1 having the amino acid sequence of SEQ ID NO: 12 (according to the Chothia definition system), CDR-H2 having the amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 21, or SEQ ID NO: 25 (according to the Chothia definition system), and CDR-H3 having the amino acid sequence of SEQ ID NO: 14 (according to the Chothia definition system), which is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework region to the VH represented by SEQ ID NO: 17, SEQ ID NO: 22, or SEQ ID NO: 26. Alternatively or additionally (for example, additionally), the anti-TfR antibody of the present disclosure comprises a humanized VL having CDR-L1 having the amino acid sequence of SEQ ID NO: 15 (according to the Chothia definition system), CDR-L2 having the amino acid sequence of SEQ ID NO: 5 (according to the Chothia definition system), and CDR-L3 having the amino acid sequence of SEQ ID NO: 16 (according to the Chothia definition system), which is at least 75% (for example, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework region to the VL shown in any one of SEQ ID NO: 18.

[0122] In some embodiments, the humanized anti-TfR antibody of the present disclosure comprises CDR-H1 having the amino acid sequence of SEQ ID NO: 27 (according to the IMGT definition system), CDR-H2 having the amino acid sequence of SEQ ID NO: 28 (according to the IMGT definition system), and CDR-H3 having the amino acid sequence of SEQ ID NO: 29 (according to the IMGT definition system), and includes a humanized VH having 25 or fewer amino acid variations in the framework region compared to the VH shown in SEQ ID NO: 43 (e.g., 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid variations). Alternatively or additionally (for example, additionally), the humanized anti-TfR antibody of the present disclosure comprises a humanized VL having 25 or fewer amino acid variations (for example, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid variations) in the framework region compared to the VL shown in SEQ ID NO: 44.

[0123] In some embodiments, the humanized anti-TfR antibody of the present disclosure comprises a humanized VH having CDR-H1 having the amino acid sequence of SEQ ID NO: 27 (according to the IMGT definition system), CDR-H2 having the amino acid sequence of SEQ ID NO: 28 (according to the IMGT definition system), and CDR-H3 having the amino acid sequence of SEQ ID NO: 29 (according to the IMGT definition system), which is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to the VH shown in SEQ ID NO: 43 in the framework region. Alternatively or additionally (for example, additionally), the humanized anti-TfR antibody of the present disclosure comprises a humanized VL having CDR-L1 having the amino acid sequence of SEQ ID NO: 30 (according to the IMGT definition system), CDR-L2 having the amino acid sequence of SEQ ID NO: 31 (according to the IMGT definition system), and CDR-L3 having the amino acid sequence of SEQ ID NO: 32 (according to the IMGT definition system), which is at least 75% (for example, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework region to the VL shown in SEQ ID NO: 44.

[0124] In some embodiments, the humanized anti-TfR antibody of the present disclosure comprises CDR-H1 having the amino acid sequence of SEQ ID NO: 33 (according to the Kabat definition system), CDR-H2 having the amino acid sequence of SEQ ID NO: 34 (according to the Kabat definition system), and CDR-H3 having the amino acid sequence of SEQ ID NO: 35 (according to the Kabat definition system), and includes a humanized VH having 25 or fewer amino acid variations in the framework region compared to the VH shown in SEQ ID NO: 43 (e.g., 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid variations). Alternatively or additionally (for example, additionally), the humanized anti-TfR antibody of the present disclosure comprises CDR-L1 having the amino acid sequence of SEQ ID NO: 36 (according to the Kabat definition system), CDR-L2 having the amino acid sequence of SEQ ID NO: 37 (according to the Kabat definition system), and CDR-L3 having the amino acid sequence of SEQ ID NO: 32 (according to the Kabat definition system), and includes a humanized VL containing 25 or fewer amino acid variations (for example, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid variations) in the framework region compared to the VL shown in SEQ ID NO: 44.

[0125] In some embodiments, the humanized anti-TfR antibody of the present disclosure comprises a humanized VH having CDR-H1 having the amino acid sequence of SEQ ID NO: 33 (according to the Kabat definition system), CDR-H2 having the amino acid sequence of SEQ ID NO: 34 (according to the Kabat definition system), and CDR-H3 having the amino acid sequence of SEQ ID NO: 35 (according to the Kabat definition system), which is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to the VH shown in SEQ ID NO: 43 in the framework region. Alternatively or in addition (for example, in addition), the humanized anti-TfR antibody of the present disclosure comprises a humanized VL having the amino acid sequence of SEQ ID NO: 36 (according to the Kabat definition system), CDR-L2 having the amino acid sequence of SEQ ID NO: 37 (according to the Kabat definition system), and CDR-L3 having the amino acid sequence of SEQ ID NO: 32 (according to the Kabat definition system), which in the framework region is at least 75% (for example, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to the VL as represented by SEQ ID NO: 44.

[0126] In some embodiments, the humanized anti-TfR antibody of the present disclosure comprises CDR-H1 having the amino acid sequence of SEQ ID NO: 38 (according to the Chothia definition system), CDR-H2 having the amino acid sequence of SEQ ID NO: 39 (according to the Chothia definition system), and CDR-H3 having the amino acid sequence of SEQ ID NO: 40 (according to the Chothia definition system), and includes a humanized VH having 25 or fewer amino acid variations in the framework region compared to the VH shown in SEQ ID NO: 43 (e.g., 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid variations). Alternatively or additionally (for example, additionally), the humanized anti-TfR antibody of the present disclosure comprises CDR-L1 having the amino acid sequence of SEQ ID NO: 41 (according to the Chothia definition system), CDR-L2 having the amino acid sequence of SEQ ID NO: 31 (according to the Chothia definition system), and CDR-L3 having the amino acid sequence of SEQ ID NO: 42 (according to the Chothia definition system), and includes a humanized VL containing 25 or fewer amino acid variations (for example, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid variations) in the framework region compared to the VL shown in SEQ ID NO: 44.

[0127] In some embodiments, the humanized anti-TfR antibody of the present disclosure comprises a humanized VH having CDR-H1 having the amino acid sequence of SEQ ID NO: 38 (according to the Chothia definition system), CDR-H2 having the amino acid sequence of SEQ ID NO: 39 (according to the Chothia definition system), and CDR-H3 having the amino acid sequence of SEQ ID NO: 40 (according to the Chothia definition system), which is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to the VH shown in SEQ ID NO: 43 in the framework region. Alternatively or additionally (for example, additionally), the humanized anti-TfR antibody of the present disclosure comprises a humanized VL having CDR-L1 having the amino acid sequence of SEQ ID NO: 41 (according to the Chothia definition system), CDR-L2 having the amino acid sequence of SEQ ID NO: 31 (according to the Chothia definition system), and CDR-L3 having the amino acid sequence of SEQ ID NO: 42 (according to the Chothia definition system), which is at least 75% (for example, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework region to the VL shown in SEQ ID NO: 44.

[0128] In some embodiments, the humanized anti-TfR antibody of the present disclosure comprises CDR-H1 having the amino acid sequence of SEQ ID NO: 45, SEQ ID NO: 63, or SEQ ID NO: 66 (according to the IMGT definition system), CDR-H2 having the amino acid sequence of SEQ ID NO: 46 (according to the IMGT definition system), and CDR-H3 having the amino acid sequence of SEQ ID NO: 47 (according to the IMGT definition system), and includes a humanized VH having 25 or fewer amino acid variations (for example, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid variations) in the framework region compared to the VH shown in SEQ ID NO: 61, SEQ ID NO: 65, or SEQ ID NO: 68. Alternatively or additionally (for example, additionally), the humanized anti-TfR antibody of the present disclosure comprises a humanized VL having 25 or fewer amino acid variations (for example, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid variations) in the framework region compared to the VL shown in SEQ ID NO. 62.

[0129] In some embodiments, the humanized anti-TfR antibody of the present disclosure comprises a humanized VH having CDR-H1 having the amino acid sequence of SEQ ID NO: 45, SEQ ID NO: 63, or SEQ ID NO: 66 (according to the IMGT definition system), CDR-H2 having the amino acid sequence of SEQ ID NO: 46 (according to the IMGT definition system), and CDR-H3 having the amino acid sequence of SEQ ID NO: 47 (according to the IMGT definition system), which is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework region to the VH represented by SEQ ID NO: 61, SEQ ID NO: 65, or SEQ ID NO: 68. Alternatively or additionally (for example, additionally), the humanized anti-TfR antibody of the present disclosure comprises a humanized VL having CDR-L1 having the amino acid sequence of SEQ ID NO: 48 (according to the IMGT definition system), CDR-L2 having the amino acid sequence of SEQ ID NO: 49 (according to the IMGT definition system), and CDR-L3 having the amino acid sequence of SEQ ID NO: 50 (according to the IMGT definition system), which is at least 75% (for example, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework region to the VL shown in SEQ ID NO: 62.

[0130] In some embodiments, the humanized anti-TfR antibody of the present disclosure comprises CDR-H1 having the amino acid sequence of SEQ ID NO: 51, SEQ ID NO: 64, or SEQ ID NO: 67 (according to the Kabat definition system), CDR-H2 having the amino acid sequence of SEQ ID NO: 52 (according to the Kabat definition system), and CDR-H3 having the amino acid sequence of SEQ ID NO: 53 (according to the Kabat definition system), and includes a humanized VH having 25 or fewer amino acid variations (for example, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid variations) in the framework region compared to the VH shown in SEQ ID NO: 61, SEQ ID NO: 65, and SEQ ID NO: 68. Alternatively or additionally (for example, additionally), the humanized anti-TfR antibody of the present disclosure comprises CDR-L1 having the amino acid sequence of SEQ ID NO: 54 (according to the Kabat definition system), CDR-L2 having the amino acid sequence of SEQ ID NO: 55 (according to the Kabat definition system), and CDR-L3 having the amino acid sequence of SEQ ID NO: 50 (according to the Kabat definition system), and includes a humanized VL containing 25 or fewer amino acid variations (for example, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid variations) in the framework region compared to the VL shown in SEQ ID NO: 62.

[0131] In some embodiments, the humanized anti-TfR antibody of the present disclosure comprises a humanized VH having CDR-H1 having the amino acid sequence of SEQ ID NO: 51, SEQ ID NO: 64, or SEQ ID NO: 67 (according to the Kabat definition system), CDR-H2 having the amino acid sequence of SEQ ID NO: 52 (according to the Kabat definition system), and CDR-H3 having the amino acid sequence of SEQ ID NO: 53 (according to the Kabat definition system), which is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework region to the VH represented by SEQ ID NO: 61, SEQ ID NO: 65, or SEQ ID NO: 68. Alternatively or additionally (for example, additionally), the humanized anti-TfR antibody of the present disclosure comprises a humanized VL having CDR-L1 having the amino acid sequence of SEQ ID NO: 54 (according to the Kabat definition system), CDR-L2 having the amino acid sequence of SEQ ID NO: 55 (according to the Kabat definition system), and CDR-L3 having the amino acid sequence of SEQ ID NO: 50 (according to the Kabat definition system), which is at least 75% (for example, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework region to the VL shown in SEQ ID NO: 62.

[0132] In some embodiments, the humanized anti-TfR antibody of the present disclosure comprises CDR-H1 having the amino acid sequence of SEQ ID NO: 56 (according to the Chothia definition system), CDR-H2 having the amino acid sequence of SEQ ID NO: 57 (according to the Chothia definition system), and CDR-H3 having the amino acid sequence of SEQ ID NO: 58 (according to the Chothia definition system), and includes a humanized VH having 25 or fewer amino acid variations in the framework region compared to the VH shown in SEQ ID NO: 61, SEQ ID NO: 65, and SEQ ID NO: 68 (for example, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid variations). Alternatively or additionally (for example, additionally), the humanized anti-TfR antibody of the present disclosure comprises CDR-L1 having the amino acid sequence of SEQ ID NO: 59 (according to the Chothia definition system), CDR-L2 having the amino acid sequence of SEQ ID NO: 49 (according to the Chothia definition system), and CDR-L3 having the amino acid sequence of SEQ ID NO: 60 (according to the Chothia definition system), and includes a humanized VL containing 25 or fewer amino acid variations (for example, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid variations) in the framework region compared to the VL shown in SEQ ID NO: 62.

[0133] In some embodiments, the humanized anti-TfR antibody of the present disclosure comprises a humanized VH having CDR-H1 having the amino acid sequence of SEQ ID NO: 56 (according to the Chothia definition system), CDR-H2 having the amino acid sequence of SEQ ID NO: 57 (according to the Chothia definition system), and CDR-H3 having the amino acid sequence of SEQ ID NO: 58 (according to the Chothia definition system), and is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework region to the VH shown in SEQ ID NO: 61, SEQ ID NO: 65, and SEQ ID NO: 68. Alternatively or additionally (for example, additionally), the humanized anti-TfR antibody of the present disclosure comprises a humanized VL having CDR-L1 having the amino acid sequence of SEQ ID NO: 59 (according to the Chothia definition system), CDR-L2 having the amino acid sequence of SEQ ID NO: 49 (according to the Chothia definition system), and CDR-L3 having the amino acid sequence of SEQ ID NO: 60 (according to the Chothia definition system), which is at least 75% (for example, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework region to the VL shown in SEQ ID NO: 62.

[0134] Examples of amino acid sequences of humanized anti-TfR antibodies described herein are provided in Table 3. Table 3. Variable regions of humanized anti-TfR antibodies [Table 3-1] [Table 3-2] [Table 3-3]

[0135] In some embodiments, the humanized anti-TfR antibody of this disclosure comprises a humanized VH containing any one of the anti-TfR antibodies CDR-H1, CDR-H2, and CDR-H3 provided in Table 2, and includes one or more amino acid variations (e.g., other than 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10) compared to each humanized VH provided in Table 3. Alternatively or additionally (e.g., additionally), the humanized anti-TfR antibody of this disclosure comprises a humanized VL containing any one of the anti-TfR antibodies CDR-L1, CDR-L2, and CDR-L3 provided in Table 2, and includes one or more amino acid variations (e.g., other than 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10) compared to each humanized VL provided in Table 3.

[0136] In some embodiments, the humanized anti-TfR antibody of the Disclosure comprises a humanized VH having an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 69, and / or a humanized VL having an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 70. In some embodiments, the humanized anti-TfR antibody of the Disclosure comprises a humanized VH having the amino acid sequence of SEQ ID NO: 69 and a humanized VL having the amino acid sequence of SEQ ID NO: 70.

[0137] In some embodiments, the humanized anti-TfR antibody of the Disclosure comprises a humanized VH having an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 71, and / or a humanized VL having an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 70. In some embodiments, the humanized anti-TfR antibody of the Disclosure comprises a humanized VH having the amino acid sequence of SEQ ID NO: 71 and a humanized VL having the amino acid sequence of SEQ ID NO: 70.

[0138] In some embodiments, the humanized anti-TfR antibody of the Disclosure comprises a humanized VH having an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 72, and / or a humanized VL having an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 70. In some embodiments, the humanized anti-TfR antibody of the Disclosure comprises a humanized VH having the amino acid sequence of SEQ ID NO: 72 and a humanized VL having the amino acid sequence of SEQ ID NO: 70.

[0139] In some embodiments, the humanized anti-TfR antibody of the Disclosure comprises a humanized VH having an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 73, and / or a humanized VL having an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 74. In some embodiments, the humanized anti-TfR antibody of the Disclosure comprises a humanized VH having the amino acid sequence of SEQ ID NO: 73 and a humanized VL having the amino acid sequence of SEQ ID NO: 74.

[0140] In some embodiments, the humanized anti-TfR antibody of the Disclosure comprises a humanized VH having an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 73, and / or a humanized VL having an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 75. In some embodiments, the humanized anti-TfR antibody of the Disclosure comprises a humanized VH having the amino acid sequence of SEQ ID NO: 73 and a humanized VL having the amino acid sequence of SEQ ID NO: 75.

[0141] In some embodiments, the humanized anti-TfR antibody of the Disclosure comprises a humanized VH having an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 76, and / or a humanized VL having an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 74. In some embodiments, the humanized anti-TfR antibody of the Disclosure comprises a humanized VH having the amino acid sequence of SEQ ID NO: 76 and a humanized VL having the amino acid sequence of SEQ ID NO: 74.

[0142] In some embodiments, the humanized anti-TfR antibody of the Disclosure comprises a humanized VH having an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 76, and / or a humanized VL having an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 75. In some embodiments, the humanized anti-TfR antibody of the Disclosure comprises a humanized VH having the amino acid sequence of SEQ ID NO: 76 and a humanized VL having the amino acid sequence of SEQ ID NO: 75.

[0143] In some embodiments, the humanized anti-TfR antibody of the Disclosure comprises a humanized VH having an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 77, and / or a humanized VL having an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 78. In some embodiments, the humanized anti-TfR antibody of the Disclosure comprises a humanized VH having the amino acid sequence of SEQ ID NO: 77 and a humanized VL having the amino acid sequence of SEQ ID NO: 78.

[0144] In some embodiments, the humanized anti-TfR antibody of the Disclosure comprises a humanized VH having an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 79, and / or a humanized VL having an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 80. In some embodiments, the humanized anti-TfR antibody of the Disclosure comprises a humanized VH having the amino acid sequence of SEQ ID NO: 79 and a humanized VL having the amino acid sequence of SEQ ID NO: 80.

[0145] In some embodiments, the humanized anti-TfR antibody of the Disclosure comprises a humanized VH having an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 77, and / or a humanized VL having an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 80. In some embodiments, the humanized anti-TfR antibody of the Disclosure comprises a humanized VH having the amino acid sequence of SEQ ID NO: 77 and a humanized VL having the amino acid sequence of SEQ ID NO: 80.

[0146] In some embodiments, the humanized anti-TfR antibody described herein is full-length IgG, which may encompass the heavy chain constant region and light chain constant region from a human antibody. In some embodiments, the heavy chain of any of the anti-TfR antibodies described herein may comprise the heavy chain constant region (CH) or a portion thereof (e.g., CH1, CH2, CH3, or a combination thereof). The heavy chain constant region may belong to any preferred origin, e.g., human, mouse, rat, or rabbit. In a particular example, the heavy chain constant region is from human IgG (gamma heavy chain), e.g., IgG1, IgG2, or IgG4. An example of the human IgG1 constant region is given below: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(Sequence ID 81)

[0147] In some embodiments, the heavy chain of any of the anti-TfR antibodies described herein includes the constant region of mutant human IgG1. For example, the introduction of an LALA mutation on the CH2 domain of human IgG1 (a mutant derived from mAb b12 mutated to replace the lower hinge residues Leu234 Leu235 with Ala234 and Ala235) is known to reduce Fcg receptor binding (Bruhns, P., et al. (2009) and Xu, D. et al. (2000)). The constant region of mutant human IgG1 is given below (mutations are in bold and underlined): [ka] (Sequence No. 82)

[0148] In some embodiments, any light chain of any of the anti-TfR antibodies described herein may further comprise a light chain constant region (CL), which may be any CL known in the art. In some examples, the CL is a kappa light chain. In other examples, the CL is a lambda light chain. In some embodiments, the CL is a kappa light chain, the sequence of which is given below: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(Sequence ID 83)

[0149] The heavy and light chain constant regions of other antibodies are well known in the art and are provided, for example, in the IMGT database (www.imgt.org) or at www.vbase2.org / vbstat.php, both of which are incorporated herein by reference.

[0150] In some embodiments, the humanized anti-TfR antibody described herein comprises a heavy chain comprising a VH or any of its variants listed in Table 3, and a heavy chain comprising 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 antibody described herein comprises a heavy chain comprising a VH or any of its variants listed in Table 3, and a heavy chain comprising a heavy chain constant region containing 25 or fewer amino acid variations compared to SEQ ID NO: 81 or SEQ ID NO: 82 (for example, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid variations). In some embodiments, the humanized anti-TfR antibody described herein comprises a heavy chain including one of the VH variants listed in Table 3 and the heavy chain constant region indicated by SEQ ID NO: 81. In some embodiments, the humanized anti-TfR antibody described herein comprises a heavy chain including one of the VH variants listed in Table 3 and the heavy chain constant region indicated by SEQ ID NO: 82.

[0151] In some embodiments, the humanized anti-TfR antibody described herein comprises a light chain comprising one of the VLs listed in Table 3 or any of their variants, and a light chain comprising 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 antibody described herein comprises a light chain comprising one of the VLs listed in Table 3 or any of their variants, and a light chain comprising a light chain constant region containing 25 or fewer amino acid variations compared to SEQ ID NO: 83 (e.g., 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid variations). In some embodiments, the humanized anti-TfR antibody described herein comprises a light chain comprising one of the VLs listed in Table 3 or any of their variants, and a light chain comprising a light chain constant region shown in SEQ ID NO: 83.

[0152] Examples of IgG heavy and light chain amino acid sequences of the described anti-TfR antibodies are provided in Table 4 below. Table 4. Heavy and light chain sequences of examples of humanized anti-TfR IgG. [Table 4-1] [Table 4-2] [Table 4-3]

[0153] In some embodiments, the humanized anti-TfR antibody of the present disclosure comprises a heavy chain containing 25 or fewer amino acid variations (for example, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid variations) compared to the heavy chain shown in any one of SEQ ID NOs. Alternatively or additionally (for example, additionally), the humanized anti-TfR antibody of the present disclosure comprises a light chain containing 25 or fewer amino acid variations (for example, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid variations) compared to the light chain shown in any one of SEQ ID NOs.

[0154] In some embodiments, the humanized anti-TfR antibody described herein comprises a heavy chain containing 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. Alternatively or additionally (e.g., additionally), the humanized anti-TfR antibody described herein comprises a light chain containing 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. In some embodiments, the anti-TfR antibody described herein comprises a heavy chain containing 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 (for example, additionally), the anti-TfR antibodies described herein include a light chain comprising any one of the amino acid sequences of SEQ ID NOs. 85, 89, 90, 93, and 95.

[0155] In some embodiments, the humanized anti-TfR antibody of the Disclosure comprises a heavy chain containing an amino acid sequence that is 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 containing an amino acid sequence that is 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 antibody of the Disclosure comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 84 and a light chain containing the amino acid sequence of SEQ ID NO: 85.

[0156] In some embodiments, the humanized anti-TfR antibody of the Disclosure comprises a heavy chain containing an amino acid sequence that is 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 containing an amino acid sequence that is 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 antibody of the Disclosure comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 86 and a light chain containing the amino acid sequence of SEQ ID NO: 85.

[0157] In some embodiments, the humanized anti-TfR antibody of the Disclosure comprises a heavy chain containing an amino acid sequence that is 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 containing an amino acid sequence that is 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 antibody of the Disclosure comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 87 and a light chain containing the amino acid sequence of SEQ ID NO: 85.

[0158] In some embodiments, the humanized anti-TfR antibody of the Disclosure comprises a heavy chain having an amino acid sequence that is 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 having an amino acid sequence that is 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 antibody of the Disclosure comprises a heavy chain having the amino acid sequence of SEQ ID NO: 88 and a light chain having the amino acid sequence of SEQ ID NO: 89.

[0159] In some embodiments, the humanized anti-TfR antibody of the Disclosure comprises a heavy chain having an amino acid sequence that is 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 having an amino acid sequence that is 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 antibody of the Disclosure comprises a heavy chain having the amino acid sequence of SEQ ID NO: 88 and a light chain having the amino acid sequence of SEQ ID NO: 90.

[0160] In some embodiments, the humanized anti-TfR antibody of the Disclosure comprises a heavy chain containing an amino acid sequence that is 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 containing an amino acid sequence that is 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 antibody of the Disclosure comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 91 and a light chain containing the amino acid sequence of SEQ ID NO: 89.

[0161] In some embodiments, the humanized anti-TfR antibody of the Disclosure comprises a heavy chain containing an amino acid sequence that is 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 containing an amino acid sequence that is 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 antibody of the Disclosure comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 91 and a light chain containing the amino acid sequence of SEQ ID NO: 90.

[0162] In some embodiments, the humanized anti-TfR antibody of the Disclosure comprises a heavy chain having an amino acid sequence that is 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 having an amino acid sequence that is 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 antibody of the Disclosure comprises a heavy chain having the amino acid sequence of SEQ ID NO: 92 and a light chain having the amino acid sequence of SEQ ID NO: 93.

[0163] In some embodiments, the humanized anti-TfR antibody of the Disclosure comprises a heavy chain containing an amino acid sequence that is 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 containing an amino acid sequence that is 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 antibody of the Disclosure comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 94 and a light chain containing the amino acid sequence of SEQ ID NO: 95.

[0164] In some embodiments, the humanized anti-TfR antibody of the Disclosure comprises a heavy chain containing an amino acid sequence that is 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 containing an amino acid sequence that is 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 antibody of the Disclosure comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 92 and a light chain containing the amino acid sequence of SEQ ID NO: 95.

[0165] In some embodiments, the anti-TfR antibody is a Fab fragment, Fab' fragment, or F(ab')2 fragment of an intact antibody (full-length antibody). Antigen-binding fragments of intact antibodies (full-length antibodies) can be prepared by standard methods (e.g., recombinantly, or by digesting the heavy chain constant region of full-length IgG using an enzyme such as papain). For example, the F(ab')2 fragment can be produced by pepsin or papain digestion of the antibody molecule, and the Fab' fragment can be produced by reducing the disulfide crosslinks of the F(ab')2 fragment. In some embodiments, the heavy chain region in the Fab fragment of the anti-TfR1 antibody described herein comprises the amino acid sequence ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT (SEQ ID NO: 96).

[0166] In some embodiments, the humanized anti-TfR antibody described herein comprises a heavy chain comprising one of the VHs listed in Table 3 or any of their variants, and a heavy chain comprising 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 antibody described herein comprises a heavy chain comprising one of the VHs listed in Table 3 or any of their variants, and a heavy chain comprising a heavy chain constant region containing 25 or fewer amino acid variations compared to SEQ ID NO: 96 (e.g., 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid variations). In some embodiments, the humanized anti-TfR antibody described herein comprises a heavy chain comprising one of the VHs listed in Table 3 or any of their variants, and a heavy chain comprising the heavy chain constant region shown in SEQ ID NO: 96.

[0167] In some embodiments, the humanized anti-TfR antibody described herein comprises a light chain comprising one of the VLs listed in Table 3 or any of their variants, and a light chain comprising 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 antibody described herein comprises a light chain comprising one of the VLs listed in Table 3 or any of their variants, and a light chain comprising a light chain constant region containing 25 or fewer amino acid variations compared to SEQ ID NO: 83 (e.g., 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid variations). In some embodiments, the humanized anti-TfR antibody described herein comprises a light chain comprising one of the VLs listed in Table 3 or any of their variants, and a light chain comprising a light chain constant region shown in SEQ ID NO: 83.

[0168] Examples of Fab heavy and light chain amino acid sequences of the described anti-TfR antibodies are provided in Table 5 below. Table 5. Heavy and light chain sequences of examples of humanized anti-TfR Fabs [Table 5-1] [Table 5-2]

[0169] In some embodiments, the humanized anti-TfR antibody of the present disclosure comprises a heavy chain containing 25 or fewer amino acid variations (for example, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid variations) compared to the heavy chain shown in any one of SEQ ID NOs. Alternatively or additionally (for example, additionally), the humanized anti-TfR antibody of the present disclosure comprises a light chain containing 25 or fewer amino acid variations (for example, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid variations) compared to the light chain shown in any one of SEQ ID NOs.

[0170] In some embodiments, the humanized anti-TfR antibody described herein comprises a heavy chain containing 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. Alternatively or additionally (e.g., additionally), the humanized anti-TfR antibody described herein comprises a light chain containing 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. In some embodiments, the anti-TfR antibody described herein comprises a heavy chain containing an amino acid sequence of any one of SEQ ID NOs. 97–103. Alternatively or additionally (e.g., additionally), the anti-TfR antibody described herein comprises a light chain containing an amino acid sequence of any one of SEQ ID NOs. 85, 89, 90, 93, and 95.

[0171] In some embodiments, the humanized anti-TfR antibody of the Disclosure comprises a heavy chain having an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 97, and / or a light chain having an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 85.

[0172] In some embodiments, the humanized anti-TfR antibody of the Disclosure comprises a heavy chain having an amino acid sequence that is 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 having an amino acid sequence that is 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 antibody of the Disclosure comprises a heavy chain having the amino acid sequence of SEQ ID NO: 98 and a light chain having the amino acid sequence of SEQ ID NO: 85.

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

[0174] In some embodiments, the humanized anti-TfR antibody of the Disclosure comprises a heavy chain containing an amino acid sequence that is 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 containing an amino acid sequence that is 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 antibody of the Disclosure comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 100 and a light chain containing the amino acid sequence of SEQ ID NO: 89.

[0175] In some embodiments, the humanized anti-TfR antibody of the Disclosure comprises a heavy chain containing an amino acid sequence that is 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 containing an amino acid sequence that is 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 antibody of the Disclosure comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 100 and a light chain containing the amino acid sequence of SEQ ID NO: 90.

[0176] In some embodiments, the humanized anti-TfR antibody of the Disclosure comprises a heavy chain having an amino acid sequence that is 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 having an amino acid sequence that is 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 antibody of the Disclosure comprises a heavy chain having the amino acid sequence of SEQ ID NO: 101 and a light chain having the amino acid sequence of SEQ ID NO: 89.

[0177] In some embodiments, the humanized anti-TfR antibody of the Disclosure comprises a heavy chain containing an amino acid sequence that is 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 containing an amino acid sequence that is 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 antibody of the Disclosure comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 101 and a light chain containing the amino acid sequence of SEQ ID NO: 90.

[0178] In some embodiments, the humanized anti-TfR antibody of the Disclosure comprises a heavy chain containing an amino acid sequence that is 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 containing an amino acid sequence that is 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 antibody of the Disclosure comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 102 and a light chain containing the amino acid sequence of SEQ ID NO: 93.

[0179] In some embodiments, the humanized anti-TfR antibody of the Disclosure comprises a heavy chain containing an amino acid sequence that is 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 containing an amino acid sequence that is 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 antibody of the Disclosure comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 103 and a light chain containing the amino acid sequence of SEQ ID NO: 95.

[0180] In some embodiments, the humanized anti-TfR antibody of the Disclosure comprises a heavy chain containing an amino acid sequence that is 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 containing an amino acid sequence that is 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 antibody of the Disclosure comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 102 and a light chain containing the amino acid sequence of SEQ ID NO: 95.

[0181] In some embodiments, the humanized anti-TfR receptor antibodies described herein may be in any antibody form, including, but not limited to, intact (i.e., full-length) antibodies, their antigen-binding fragments (Fab, Fab', F(ab')2, Fv, etc.), single-chain antibodies, bispecific antibodies, or nanobodies. In some embodiments, the humanized anti-TfR antibody described herein is scFv. In some embodiments, the humanized anti-TfR antibody described herein is scFv-Fab (e.g., scFv fused to a portion of the constant region). In some embodiments, the anti-TfR receptor antibody described herein is scFv fused to a constant region (e.g., the human IgG1 constant region shown in SEQ ID NO: 81 or SEQ ID NO: 82, or a portion of that region such as the Fc moiety) at either the C-terminus or N-terminus.

[0182] In some embodiments, conservative mutations can be introduced into an antibody sequence (e.g., a CDR or framework sequence) at a position where, when determined, for example based on the crystal structure, the residue is unlikely to be involved in interaction with the target antigen (e.g., a transferrin receptor). In some embodiments, one or more mutations (e.g., amino acid substitutions) are introduced into the Fc region of the anti-TfR antibody described herein (e.g., in the CH2 domain (residues 231-340 of human IgG1) and / or (e.g., and) in the CH3 domain (residues 341-447 of human IgG1) and / or (e.g., and) in the hinge region) to alter one or more functional properties of the antibody, such as serum half-life, complement binding, Fc receptor binding, and / or (e.g., and).

[0183] In some embodiments, one or more mutations (e.g., amino acid substitutions) are introduced into the hinge region of the Fc region (CH1 domain) such that the number of cysteine ​​residues in the hinge region can be varied (e.g., increased or decreased) 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 may be altered, for example, to facilitate the assembly of the light and heavy chains, or to change the stability of the antibody (e.g., increased or decreased), or to facilitate linker conjugation.

[0184] In some embodiments, one 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 231-340 of human IgG1) and / or (e.g., and) in the CH3 domain (residues 341-447 of human IgG1) and / or (e.g., and) in the hinge region) to increase or decrease the antibody's affinity for Fc receptors on the surface of effector cells. Mutations in the Fc region of antibodies that increase or decrease the antibody's affinity for Fc receptors, and techniques for introducing such mutations into Fc receptors or fragments thereof are known to those skilled in the art. Examples of mutations in the Fc receptor of an antibody that may be made to alter the antibody's affinity 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 Publications WO 02 / 060919; WO 98 / 23289; and WO 97 / 34631 (these are incorporated herein by reference).

[0185] In some embodiments, one or more amino acid mutations (i.e., substitutions, insertions, or deletions) are introduced into the IgG constant region or its FcRn-binding fragment (preferably an Fc or hinge-Fc domain fragment) to alter (e.g., increase or decrease) the half-life of the antibody in vivo. For example, see International Publications WO 02 / 060919; WO 98 / 23289; and WO 97 / 34631; and U.S. Patents 5,869,046, 6,121,022, 6,277,375, and 6,165,745 for mutations that would alter (e.g., increase or decrease) the half-life of the antibody in vivo.

[0186] In some embodiments, one or more amino acid mutations (i.e., substitutions, insertions, or deletions) are introduced into the IgG constant region or its FcRn-binding fragment (preferably an Fc or hinge-Fc domain fragment) to reduce the half-life of the anti-TfR antibody in vivo. In some embodiments, one or more amino acid mutations (i.e., substitutions, insertions, or deletions) are introduced into the IgG constant region or its FcRn-binding fragment (preferably an Fc or hinge-Fc domain fragment) to increase the half-life of the antibody in vivo. In some embodiments, the antibody may have one or more amino acid mutations (e.g., substitutions) in the second constant (CH2) domain (residues 231-340 of human IgG1) and / or (e.g., and) the third constant (CH3) domain (residues 341-447 of human IgG1), numbered according to the Kabat EU index (Kabat EA et al. (1991) above). In some embodiments, the IgG1 constant region of the antibodies described herein includes a methionine (M) to tyrosine (Y) substitution at position 252, numbered according to the EU index as found in Kabat, a serine (S) to threonine (T) substitution at position 254, and a threonine (T) to glutamic acid (E) substitution at position 256. See U.S. Patent No. 7,658,921 (which is incorporated herein by reference). This type of mutant IgG, referred to as the “YTE mutant,” has been shown to exhibit a four-fold increased half-life compared to the wild-type version of the same antibody (see Dall'Acqua WF et al., (2006) J Biol Chem 281:23514-24). In some embodiments, the antibody includes an IgG constant region containing one, two, or three or more amino acid substitutions of amino acid residues at positions 251-257, 285-290, 308-314, 385-389, and 428-436, numbered according to the EU index as found in Kabat.

[0187] In some embodiments, one, two, or more amino acid substitutions are introduced into the IgG constant region Fc region to alter the effector function(s) of an anti-transferrin receptor antibody. The effector ligand with altered affinity to itself may be, for example, the Fc receptor or the C1 component of complement. This approach is described in more detail in U.S. Patents 5,624,821 and 5,648,260. In some embodiments, deletion or inactivation of the constant region domain (through point mutation or other means) may reduce the binding of the circulating antibody to the Fc receptor, thereby increasing tumor localization. For descriptions of mutations that delete or inactivate the constant region and thereby increase tumor localization, see, for example, U.S. Patents 5,585,097 and 8,591,886. In some embodiments, one or more amino acid substitutions may be introduced into the Fc region of the antibodies described herein to remove a potential glycosylation site on the Fc region (which may reduce binding to the Fc receptor) (see, for example, Shields RL et al., (2001) J Biol Chem 276:6591-604).

[0188] In some embodiments, one or more amino acid residues in the constant region of the anti-TfR antibody described herein may be replaced with different amino acid residues so that the antibody may have modified Clq binding and / or (for example, and) reduced or absent complement-dependent cytotoxicity (CDC). This approach is described in further detail in U.S. Patent No. 6,194,551 (Idusogie et al). In some embodiments, one or more amino acid residues in the N-terminal region of the CH2 domain of the antibody described herein are modified to alter the complement-binding ability of the antibody. This approach is described in further international publication WO 94 / 29351. In some embodiments, the Fc region of the antibody described herein is modified to increase the antibody's ability to mediate antibody-dependent cytotoxicity (ADCC) to cells and / or (for example, and) to increase the antibody's affinity for the Fcγ receptor. This approach is described in further international publication WO 00 / 42072.

[0189] In some embodiments, the heavy chain and / or (for example, and) light chain variable domain(s) sequences(s) of the antibodies provided herein may be used, as described elsewhere herein, to generate, for example, CDR-conjugated antibodies, chimeric antibodies, humanized antibodies, or compound human antibodies, or antigen-binding fragments. As will be understood by those skilled in the art, any variant, CDR-conjugated antibody, chimeric antibody, humanized antibody, or compound antibody derived from any of the antibodies provided herein may be useful in the compositions and methods described herein, and the variant, CDR-conjugated antibody, chimeric antibody, humanized antibody, or compound antibody will maintain its specific binding ability to the transferrin receptor such that it may have at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% binding to the transferrin receptor compared to the original antibody from which it is derived.

[0190] In some embodiments, the antibodies provided herein include mutations that confer desired properties to the antibody. For example, to avoid potential complications resulting from Fab-arm exchange, which is known to occur in native IgG4 mAbs, the antibodies provided herein may include a stabilizing "Adair" mutation (Angal S., et al., "A single amino acid substitution abolishes the heterogeneity of chimeric mouse / human (IgG4) antibody", Mol Immunol 30, 105-108; 1993), where serine 228 (EU numbering; residue 241 Kabat numbering) is converted to proline, resulting in an IgG1-like hinge sequence. Consequently, any of the antibodies may contain a stabilizing "Adair" mutation.

[0191] In some embodiments, the antibody is modified (e.g., modified via glycosylation, phosphorylation, SUMOylation, and / or (e.g., and) methylation). In some embodiments, the antibody is a glycosylated antibody conjugated to one or more sugar or carbohydrate molecules. In some embodiments, one or more sugar or carbohydrate molecules are conjugated to the antibody via N-glycosylation, O-glycosylation, C-glycosylation, glyciation (GPI anchor attachment), and / or (e.g., and) phosphoglycosylation. In some embodiments, one or more sugar or carbohydrate molecules are monosaccharides, disaccharides, oligosaccharides, or glycans. In some embodiments, one or more sugar or carbohydrate molecules are branched oligosaccharides or branched glycans. In some embodiments, one or more sugar or carbohydrate molecules comprise mannose units, glucose units, N-acetylglucosamine units, N-acetylgalactosamine units, galactose units, fucose units, or phospholipid units. In some embodiments, the sugar molecules are present in quantities of approximately 1–10, 1–5, 5–10, 1–4, 1–3, or 2. In some embodiments, the glycosylated antibody is glycosylated whole or partially. In some embodiments, the antibody is glycosylated by chemical reaction or by enzymatic means. In some embodiments, the antibody is glycosylated in vitro or in cells (which may optionally be deficient in enzymes in the N- or O-glycosylation pathway, e.g., glycosyltransferase). In some embodiments, the antibody is functionalized with sugar or carbohydrate molecules as described in International Patent Application Publication WO2014065661, published on May 1, 2014, entitled "Modified antibody, antibody-conjugate and process for the preparation thereof".

[0192] In some embodiments, any one of the anti-TfR1 antibodies described herein may include a signal peptide (e.g., an N-terminal signal peptide) on the heavy chain and / or (by way of example, and) light chain sequence. In some embodiments, the anti-TfR1 antibodies described herein include any one of the VH and VL sequences, any one of the IgG heavy and light chain sequences, or any one of the Fab heavy and light chain sequences described herein, and further include a signal peptide (e.g., an N-terminal signal peptide). In some embodiments, the signal peptide includes the amino acid sequence of MGWSCIILFLVATATGVHS (SEQ ID NO: 104).

[0193] Other known anti-transferrin receptor antibodies Any other suitable anti-transferrin receptor antibodies known in the art may be used as muscle targeting agents in the complexes disclosed herein. Examples of known anti-transferrin receptor antibodies (including relevant references and binding epitopes) are listed in Table 8. In some embodiments, the anti-transferrin receptor antibody includes any one of the complementarity determining regions (CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3) of the anti-transferrin receptor antibodies provided herein, e.g., the anti-transferrin receptor antibodies listed in Table 8.

[0194] Table 8 - List of anti-transferrin receptor antibody clones including relevant reference and binding epitope information

Table 6-1

Table 6-2

Table 6-3

[0195] In some embodiments, the transferrin receptor antibody of this disclosure comprises one or more CDR-H (e.g., CDR-H1, CDR-H2, and CDR-H3) amino acid sequences from any one of the anti-transferrin receptor antibodies selected from Table 8. In some embodiments, the transferrin receptor antibody comprises CDR-H1, CDR-H2, and CDR-H3 provided for any one of the anti-transferrin receptor antibodies selected from Table 8. In some embodiments, the anti-transferrin receptor antibody comprises CDR-L1, CDR-L2, and CDR-L3 provided for any one of the anti-transferrin receptor antibodies selected from Table 8. In some embodiments, the anti-transferrin antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 provided for any one of the anti-transferrin receptor antibodies selected from Table 8. This disclosure also encompasses any nucleic acid sequences encoding molecules containing CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, or CDR-L3, provided for any one of the anti-transferrin receptor antibodies selected from Table 8. In some embodiments, the heavy and light chain CDR3 domains of an antibody may play a particularly important role in the antibody's binding specificity / affinity for a given antigen. Thus, the anti-transferrin receptor antibodies of this disclosure may encompass at least the heavy chain and / or (for example, and) the light chain CDR3 of any one of the anti-transferrin receptor antibodies selected from Table 8.

[0196] In some examples, any of the anti-transferrin receptor antibodies of this disclosure has one or more CDR (e.g., CDR-H or CDR-L) sequences substantially similar to any of the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and / or (e.g., and) CDR-L3 sequences from one of the anti-transferrin receptor antibodies selected from Table 8. In some embodiments, the positions of one or more CDRs on the VH (e.g., CDR-H1, CDR-H2, or CDR-H3) and / or (e.g., and) VL (e.g., CDR-L1, CDR-L2, or CDR-L3) regions of the antibodies described herein may vary by 1, 2, 3, 4, 5, or 6 amino acid positions, insofar as immunospecific binding to the transferrin receptor (e.g., human transferrin receptor) is maintained (e.g., substantially maintained; for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the binding of the original antibody from which it originates). For example, in some embodiments, the position defining the CDR of any antibody described herein may be varied by shifting the N-terminal and / or (for example, and) C-terminal boundary of the CDR by 1, 2, 3, 4, 5, or 6 amino acids compared to the CDR position of any one of the antibodies described herein, as long as immunospecific binding to the transferrin receptor (e.g., human transferrin receptor) is maintained (for example, substantially maintained; 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 originates). In another embodiment, the length of one or more CDRs on the VH (e.g., CDR-H1, CDR-H2, or CDR-H3) and / or (for example, and) VL (e.g., CDR-L1, CDR-L2, or CDR-L3) regions of the antibodies described herein may be varied as long as immunospecific binding to the transferrin receptor (e.g., human transferrin receptor) is maintained (for example, substantially maintained).For example, it can vary by only 1, 2, 3, 4, 5 amino acids, or more (for example, shorter or longer), as long as it is 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 originates.

[0197] Accordingly, in some embodiments, the CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and / or (for example, and) CDR-H3 described herein may be 1, 2, 3, 4, 5 amino acids, or more shorter than one or more of the CDRs described herein (for example, CDRs from any of the anti-transferrin receptor antibodies selected from Table 8), insofar as immunospecific binding to the transferrin receptor (for example, human transferrin receptor) is maintained (for example, substantially maintained; for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% compared to the binding of the original antibody from which it is derived). In some embodiments, the CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and / or (for example, and) CDR-H3 described herein may be 1, 2, 3, 4, 5 amino acids, or more longer than one or more of the CDRs described herein (for example, CDRs from any of the anti-transferrin receptor antibodies selected from Table 8), insofar as immunospecific binding to the transferrin receptor (for example, human transferrin receptor) is maintained (for example, substantially maintained; for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% compared to the binding of the original antibody from which it is derived). In some embodiments, the amino portions of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and / or (for example, and) CDR-H3 described herein may be extended by only 1, 2, 3, 4, 5 amino acids, or more, compared to one or more of the CDRs described herein (for example, CDRs from any of the anti-transferrin receptor antibodies selected from Table 8), as long as immunospecific binding to the transferrin receptor (for example, human transferrin receptor) is maintained (for example, substantially maintained; for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% compared to the binding of the original antibody from which it is derived).In some embodiments, the carboxyl moieties of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and / or (for example, and) CDR-H3 described herein may be stretched by only 1, 2, 3, 4, 5 amino acids, or more, compared to one or more of the CDRs described herein (for example, CDRs from any of the anti-transferrin receptor antibodies selected from Table 8), as long as immunospecific binding to the transferrin receptor (for example, human transferrin receptor) is maintained (for example, substantially maintained; for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% compared to the binding of the original antibody from which it is derived). In some embodiments, the amino portion of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and / or (for example, and) CDR-H3 described herein may be shortened by only 1, 2, 3, 4, 5 amino acids, or more, compared to one or more of the CDRs described herein (for example, CDRs from any of the anti-transferrin receptor antibodies selected from Table 8), as long as immunospecific binding to the transferrin receptor (for example, human transferrin receptor) is maintained (for example, substantially maintained; for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% compared to the binding of the original antibody from which it is derived). In some embodiments, the carboxyl portions of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and / or (for example, and) CDR-H3 described herein may be shortened by only 1, 2, 3, 4, 5 amino acids, or more, compared to one or more of the CDRs described herein (for example, CDRs from any of the anti-transferrin receptor antibodies selected from Table 8), insofar as immunospecific binding to the transferrin receptor (for example, human transferrin receptor) is maintained (for example, substantially maintained; for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% compared to the binding of the original antibody from which it is derived).For example, any of the binding assays and conditions described in the art may be used to determine whether immunospecific binding to a transferrin receptor (e.g., human transferrin receptor) is maintained.

[0198] In some examples, any of the anti-transferrin receptor antibodies of this disclosure have one or more CDR (e.g., CDR-H or CDR-L) sequences substantially similar to any one of the anti-transferrin receptor antibodies selected from Table 8. For example, an antibody may contain one or more CDR sequences from any of the anti-transferrin receptor antibodies selected from Table 8, containing up to 5, 4, 3, 2, or 1 amino acid residue variations compared to the corresponding CDR region of any one of the CDRs provided herein (e.g., CDRs from any of the anti-transferrin receptor antibodies selected from Table 8). In some embodiments, any of the amino acid variations in any of the CDRs provided herein may be conservative variations. Conservative variations can be introduced into the CDR at positions where residues are unlikely to be involved in interaction with the transferrin receptor protein (e.g., human transferrin receptor protein), as determined, for example, based on the crystal structure. Several aspects of this disclosure provide transferrin receptor antibodies comprising one or more heavy-chain variable (VH) and / or (e.g., and) light-chain variable (VL) domains provided herein. In some embodiments, any of the VH domains provided herein encompasses one or more CDR-H sequences provided herein (e.g., CDR-H1, CDR-H2, and CDR-H3), for example, any of the CDR-H sequences provided in any of the anti-transferrin receptor antibodies selected from Table 8. In some embodiments, any of the VL domains provided herein encompass one or more CDR-L sequences provided herein (e.g., CDR-L1, CDR-L2, and CDR-L3), for example, any of the CDR-L sequences provided in any of the anti-transferrin receptor antibodies selected from Table 8.

[0199] In some embodiments, the anti-transferrin receptor antibody of this disclosure comprises any antibody that includes the heavy chain variable domain and / or (for example, and) the light chain variable domain of any anti-transferrin receptor antibody, such as any one of the anti-transferrin receptor antibodies selected from Table 8. In some embodiments, the anti-transferrin receptor antibody of this disclosure comprises any antibody that includes the heavy chain variable and light chain variable pair of any anti-transferrin receptor antibody, such as any one of the anti-transferrin receptor antibodies selected from Table 8.

[0200] Aspects of this 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 any of those described herein. In some embodiments, the anti-transferrin receptor antibody includes a heavy-chain variable sequence or a light-chain variable sequence that is at least 75% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to the heavy-chain variable sequence and / or any light-chain variable sequence of any anti-transferrin receptor antibody, such as any one of the anti-transferrin receptor antibodies selected from Table 8. In some embodiments, the homologous heavy-chain variable and / or (e.g., and) light-chain variable amino acid sequences are not varied in any of the CDR sequences provided herein. For example, in some embodiments, the degree of sequence variation (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) may occur within the heavy-chain variable and / or (e.g., and) light-chain variable sequences that exclude any of the CDR sequences provided herein. In some embodiments, any of the anti-transferrin receptor antibodies provided herein include a heavy-chain variable sequence and a light-chain variable sequence that include a framework sequence which is at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to the framework sequence of any anti-transferrin receptor antibody, such as any one of the anti-transferrin receptor antibodies selected from Table 8.

[0201] In some embodiments, an anti-transferrin receptor antibody that specifically binds to a transferrin receptor (e.g., the human transferrin receptor) comprises a light chain variable VL domain of any of the anti-transferrin receptor antibodies selected from Table 8, comprising either the CDR-L domain (CDR-L1, CDR-L2, and CDR-L3) 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., the human transferrin receptor) comprises a light chain variable VL domain comprising CDR-L1, CDR-L2, and CDR-L3 of any of the anti-transferrin receptor antibodies, such as any one of the anti-transferrin receptor antibodies selected from Table 8. In some embodiments, an anti-transferrin receptor antibody comprises a light chain variable (VL) region sequence comprising framework regions 1, 2, 3, or 4 of the light chain variable region sequence of any of the anti-transferrin receptor antibodies, such as any one of the anti-transferrin receptor antibodies selected from Table 8. In some embodiments, the anti-transferrin receptor antibody comprises one, two, three, or four framework regions of the light chain variable region sequence that are at least 75%, 80%, 85%, 90%, 95%, or 100% identical to one, two, three, or four framework regions of the light chain variable region sequence of any anti-transferrin receptor antibody, such as one of the anti-transferrin receptor antibodies selected from Table 8. In some embodiments, the light chain variable framework region derived from the amino acid sequence consists of the amino acid sequence except for the presence of substitutions, deletions, and / or (for example, and) insertions, preferably up to 10 amino acids. In some embodiments, the light chain variable framework region derived from the amino acid sequence consists of the amino acid sequence in which one, two, three, four, five, six, seven, eight, nine, or ten amino acid residues are substituted with amino acids found at similar positions in the corresponding primate non-human or human light chain variable framework region.

[0202] In some embodiments, an anti-transferrin receptor antibody that specifically binds to the transferrin receptor comprises CDR-L1, CDR-L2, and CDR-L3 of any anti-transferrin receptor antibody, such as any one of the anti-transferrin receptor antibodies selected from Table 8. In some embodiments, the antibody further comprises all one, two, three, or four VL framework regions derived from the VL of a human or primate antibody. The primate or human light chain framework region of the selected antibody for use with the light chain CDR sequences described herein may have at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, or at least 99%) identity with, for example, the light chain framework region of a non-human parent antibody. The selected primate or human antibody may have the same or substantially the same number of amino acids in its light chain complementarity-determining region as any of the amino acids in the light chain complementarity-determining region of any of the antibodies provided herein (e.g., any of the anti-transferrin receptor antibodies selected from Table 8). In some embodiments, the amino acid residues of the light chain framework region of a primate or human are from a natural primate or human antibody light chain framework region having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, and at least 99% (or more) identity with the light chain framework region of any anti-transferrin receptor antibody, such as any one of the anti-transferrin receptor antibodies selected from Table 8. In some embodiments, the anti-transferrin receptor antibody further comprises one, two, three, or all four VL framework regions derived from the human light chain variable kappa subfamily. In some embodiments, the anti-transferrin receptor antibody further comprises one, two, three, or all four VL framework regions derived from the human light chain variable lambda subfamily.

[0203] In some embodiments, any of the anti-transferrin receptor antibodies provided herein includes a light chain variable domain further comprising 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 of the light chain constant regions provided herein may be any variant of any of the light chain constant regions provided herein. In some embodiments, the light chain constant region contains an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to any of the light chain constant regions of any anti-transferrin receptor antibody, such as any one of the anti-transferrin receptor antibodies selected from Table 8.

[0204] In some embodiments, the anti-transferrin receptor antibody is any of the anti-transferrin receptor antibodies, such as any one of the anti-transferrin receptor antibodies selected from Table 8.

[0205] In some embodiments, the anti-transferrin receptor antibody comprises a VL domain containing the amino acid sequence of any anti-transferrin receptor antibody, such as one of the anti-transferrin receptor antibodies selected from Table 8, where the constant region contains the amino acid sequence of an IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, or the constant region of a human IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule. In some embodiments, an anti-transferrin receptor antibody comprises either a VL domain or a variant of the VL domain, and either a VH domain or a VH domain variant, where the VL and VH domains, or their variants, are from the same antibody clone, and where the constant region comprises the amino acid sequence of the constant region of an IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, of any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or any subclass (e.g., IgG2a and IgG2b) immunoglobulin molecule. Non-limiting examples of human constant regions have been described in the art; see, for example, Kabat EA et al. (1991) above.

[0206] In some embodiments, the muscle targeting agent is an anti-transferrin receptor antibody (for example, an antibody and its variant as described in the international patent publication WO 2016 / 081643 (incorporated herein by reference)).

[0207] The heavy and light chain CDRs of antibodies according to various definition systems are provided in Table 9. Various definition systems, such as the Kabat definition, Chothia definition, and / or Contact definition, are described. For example, see Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USD 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). Also see hgmp.mrc.ac.uk and bioinf.org.uk / abs. Table 9. Heavy and light chain CDRs of mouse transferrin receptor antibodies [Table 7]

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

[0209] VH QVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGEINPTNGRTNYIEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARGTRAYHYWGQGTSVTVSS(Sequence ID 124)

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

[0211] In some embodiments, the transferrin receptor antibodies of the present disclosure include CDR-H1, CDR-H2, and CDR-H3 that are the same as CDR-H1, CDR-H2, and CDR-H3 shown in Table 9. Alternatively or additionally (by way of example, additionally), the transferrin receptor antibodies of the present disclosure include CDR-L1, CDR-L2, and CDR-L3 that are the same as CDR-L1, CDR-L2, and CDR-L3 shown in Table 9.

[0212] In some embodiments, the transferrin receptor antibodies of the present disclosure include CDR-H1, CDR-H2, and CDR-H3, which together contain a total of only 5 amino acid variations (by way of example, only 5, 4, 3, 2, or 1 amino acid variations) compared to CDR-H1, CDR-H2, and CDR-H3 shown in Table 9. "Together" means that the total number of amino acid variations in all three heavy chain CDRs is within the defined range. Alternatively or additionally (by way of example, additionally), the transferrin receptor antibodies of the present disclosure may include CDR-L1, CDR-L2, and CDR-L3, which together contain a total of only 5 amino acid variations (by way of example, only 5, 4, 3, 2, or 1 amino acid variations) compared to CDR-L1, CDR-L2, and CDR-L3 shown in Table 9.

[0213] In some embodiments, the transferrin receptor antibodies of the present disclosure comprise CDR-H1, CDR-H2, and CDR-H3, at least one of which contains only three amino acid variations (e.g., only three, two, or one amino acid variation) compared to the counterpart heavy chain CDR shown in Table 9. Alternatively or in addition (e.g., in addition), the transferrin receptor antibodies of the present disclosure may comprise CDR-L1, CDR-L2, and CDR-L3, at least one of which contains only three amino acid variations (e.g., only three, two, or one amino acid variation) compared to the counterpart light chain CDR shown in Table 9.

[0214] In some embodiments, the transferrin receptor antibody of the present disclosure comprises CDR-L3 containing only three amino acid variations (e.g., only three, two, or one amino acid variation) compared to the CDR-L3 shown in Table 9. In some embodiments, the transferrin receptor antibody of the present disclosure comprises CDR-L3 containing one amino acid variation compared to the CDR-L3 shown in Table 9. In some embodiments, the transferrin receptor antibody of the present disclosure comprises CDR-L3 of QHFAGTPLT (SEQ ID NO: 126) (according to the Kabat and Chothia definition system) or QHFAGTPL (SEQ ID NO: 127) (according to the Contact definition system). In some embodiments, the transferrin receptor antibodies of this disclosure include CDR-H1, CDR-H2, CDR-H3, CDR-L1, and CDR-L2, which are the same as CDR-H1, CDR-H2, and CDR-H3 shown in Table 9, and CDR-L3, which is QHFAGTPLT (SEQ ID NO: 126) (according to the Kabat and Chothia definition system) or QHFAGTPL (SEQ ID NO: 127) (according to the Contact definition system).

[0215] In some embodiments, the transferrin receptor antibodies of the present disclosure include a heavy chain CDR that is identical to the heavy chain CDR shown in Table 9 by at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) in combination. Alternatively or in addition (e.g., in addition), the transferrin receptor antibodies of the present disclosure include a light chain CDR that is identical to the light chain CDR shown in Table 9 by at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) in combination.

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

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

[0218] In some embodiments, the transferrin receptor antibody of the present disclosure comprises a VH having 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 in addition (e.g., in addition), the transferrin receptor antibody of the present disclosure comprises a VL having 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.

[0219] In some embodiments, the transferrin receptor antibodies of the Disclosure are humanized antibodies (e.g., humanized variants of antibodies). In some embodiments, the transferrin receptor antibodies of the Disclosure include CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, which are the same as CDR-H1, CDR-H2, and CDR-H3 shown in Table 9, and include a humanized heavy chain variable region and / or (e.g., and) a humanized light chain variable region.

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

[0221] In some embodiments, humanization is achieved by conjugating a CDR (as shown, for example, in Table 9) into the IGKV1-NL1*01 and IGHV1-3*01 human variable domains. In some embodiments, the transferrin receptor antibodies of this 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 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 (as shown, for example). In some embodiments, the transferrin receptor antibody of the present disclosure is a humanized variant comprising amino acid substitutions at all positions 9, 13, 17, 18, 40, 45, and 70 compared to VL shown in SEQ ID NO: 125, and / or (as an example, and) amino acid substitutions at all positions 1, 5, 7, 11, 12, 20, 38, 40, 44, 66, 75, 81, 83, 87, and 108 compared to VH shown in SEQ ID NO: 124.

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

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

[0224] Humanized VH EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQRLEWIGEINPTNGRTNYIEKFKSRATLTVDKSASTAYMELSSLRSEDTAVYYCARGTRAYHYWGQGTMVTVSS(Sequence ID 128)

[0225] Humanized VL DIQMTQSPSSLSASVGDRVTITCRASDNLYSNLAWYQQKPGKSPKLLVYDATNLADGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQHFWGTPLTFGQGTKVEIK(Sequence ID 129)

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

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

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

[0229] In some embodiments, the transferrin receptor antibody of the present disclosure is a humanized variant comprising one or more amino acid substitutions at positions 43 and 48 compared to VL shown in SEQ ID NO: 125, and / or one or more amino acid substitutions at positions 48, 67, 69, 71 and 73 compared to VH shown in SEQ ID NO: 124 (for example). In some embodiments, the transferrin receptor antibody of the present disclosure is a humanized variant comprising S43A and / or (for example, and) V48L mutations compared to VL shown in SEQ ID NO: 125, and / or one or more mutations in A67V, L69I, V71R and K73T compared to VH shown in SEQ ID NO: 124 (for example).

[0230] In some embodiments, the transferrin receptor antibody of the present disclosure is a humanized variant comprising one or more amino acid substitutions at positions 9, 13, 17, 18, 40, 43, 48, 45, and 70 compared to VL shown in SEQ ID NO: 125, and / or (as an example, and) one or more amino acid substitutions at positions 1, 5, 7, 11, 12, 20, 38, 40, 44, 48, 66, 67, 69, 71, 73, 75, 81, 83, 87, and 108 compared to VH shown in SEQ ID NO: 124.

[0231] In some embodiments, the anti-transferrin receptor antibodies of this disclosure are chimeric antibodies that may incorporate heavy chain constant regions and light chain constant regions from a human antibody. A chimeric antibody refers to an antibody having a variable region or part of a variable region from a first species and a constant region from a second species. Typically, in these chimeric antibodies, both the light chain and heavy chain variable regions mimic the variable regions of antibodies derived from certain mammals (e.g., non-human mammals such as mice, rabbits, and rats), while the constant region is homologous to a sequence in an antibody derived from another mammal, such as a human. In some embodiments, amino acid modifications may be made in the variable region and / or (e.g., and) the constant region.

[0232] In some embodiments, the anti-transferrin receptor antibodies described herein are chimeric antibodies that may incorporate the heavy chain constant region and light chain constant region from a human antibody. A chimeric antibody refers to an antibody having a variable region or part of a variable region from a first species and a constant region from a second species. Typically, in these chimeric antibodies, both the light chain and heavy chain variable regions mimic the variable regions of antibodies derived from certain mammals (e.g., non-human mammals such as mice, rabbits, and rats), while the constant region is homologous to the sequence in an antibody derived from another mammal, such as a human. In some embodiments, amino acid modifications may be made in the variable region and / or (e.g., and) the constant region.

[0233] In some embodiments, any heavy chain of an anti-transferrin receptor antibody as described herein may include a heavy chain constant region (CH) or a portion thereof (e.g., CH1, CH2, CH3, or a combination thereof). The heavy chain constant region may belong to any preferred source, e.g., human, mouse, rat, or rabbit. In a particular example, the heavy chain constant region is from human IgG (gamma heavy chain), e.g., IgG1, IgG2, or IgG4. An example of a human IgG1 constant region is given below: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(Sequence ID 130)

[0234] In some embodiments, any light chain of the anti-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 examples, the CL is a kappa light chain. In other examples, the CL is a lambda light chain. In some embodiments, the CL is a kappa light chain, the sequence of which is given below: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(Sequence ID 83)

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

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

[0237] Heavy chain (VH + human IgG1 constant region) QVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGEINPTNGRTNYIEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARGTRAYHYWGQGTSVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(Sequence ID 132)

[0238] Lightweight chain (VL + Kappa lightweight chain) DIQMTQSPASLSVSVGETVTITCRASDNLYSNLAWYQQKQGKSPQLLVYDATNLADGVPSRFSGSGSGTQYSLKINSLQSEDFGTYYCQHFWGTPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence ID 133)

[0239] Heavy chain (humanized VH + human IgG1 constant region) EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQRLEWIGEINPTNGRTNYIEKFKSRATLTVDKSASTAYMELSSLRSEDTAVYYCARGTRAYHYWGQGTMVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(Sequence ID 134)

[0240] Light chain (humanized VL + kappa light chain) DIQMTQSPSSLSASVGDRVTITCRASDNLYSNLAWYQQKPGKSPKLLVYDATNLADGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQHFWGTPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence ID 135)

[0241] In some embodiments, the transferrin receptor antibody described herein comprises a heavy chain having at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) the same amino acid sequence as SEQ ID NO: 132. Alternatively or in addition (e.g., in addition), the transferrin receptor antibody described herein comprises a light chain having at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) the same amino acid sequence as SEQ ID NO: 133. In some embodiments, the transferrin receptor antibody described herein comprises a heavy chain having the amino acid sequence of SEQ ID NO: 132. Alternatively or in addition (e.g., in addition), the transferrin receptor antibody described herein comprises a light chain having the amino acid sequence of SEQ ID NO: 133.

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

[0243] In some embodiments, the transferrin receptor antibody described herein comprises a heavy chain having at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) the same amino acid sequence as SEQ ID NO: 134. Alternatively or in addition (e.g., in addition), the transferrin receptor antibody described herein comprises a light chain having at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) the same amino acid sequence as SEQ ID NO: 135. In some embodiments, the transferrin receptor antibody described herein comprises a heavy chain having the amino acid sequence of SEQ ID NO: 134. Alternatively or in addition (e.g., in addition), the transferrin receptor antibody described herein comprises a light chain having the amino acid sequence of SEQ ID NO: 135.

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

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

[0246] Heavy chain Fab (VH + part of the human IgG1 constant region) QVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGEINPTNGRTNYIEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARGTRAYHYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP(Sequence ID 136)

[0247] Heavy chain Fab (humanized VH + part of the human IgG1 constant region) EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQRLEWIGEINPTNGRTNYIEKFKSRATLTVDKSASTAYMELSSLRSEDTAVYYCARGTRAYHYWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP(Sequence ID 137)

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

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

[0250] The anti-transferrin receptor antibodies described herein may be in any form, but are not limited to, intact (i.e., full-length) antibodies, their antigen-binding fragments (such as Fab, Fab', F(ab')2, Fv, etc.), single-chain antibodies, bispecific antibodies, or antibodies comprising nanobodies. In some embodiments, the anti-transferrin receptor antibodies described herein are scFv. In some embodiments, the anti-transferrin receptor antibodies described herein are scFv-Fab (e.g., scFv condensed with a portion of the constant region). In some embodiments, the transferrin receptor antibodies described herein are scFv condensed with a constant region (e.g., the human IgG1 constant region shown in SEQ ID NO: 130).

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

[0252] In some embodiments, antibodies provided herein may have one or more post-translational modifications. In some embodiments, N-terminal cyclization, also called pyroglutamate formation (pyroGlu), may occur in the antibody at N-terminal glutamate (Glu) and / or glutamine (Gln) residues during production. Therefore, it should be understood that antibodies identified as having a sequence containing an N-terminal glutamate or glutamine residue include antibodies that have undergone pyroglutamate formation due to post-translational modifications. In some embodiments, pyroglutamate formation occurs in the heavy chain sequence. In some embodiments, pyroglutamate formation occurs in the light chain sequence.

[0253] b. Other muscle-targeting antibodies In some embodiments, muscle-targeted antibodies are antibodies that specifically bind to hemojuvelin, caveolin-3, Duchenne muscular dystrophy peptide, myosin Iib, or CD63. In some embodiments, muscle-targeted antibodies are antibodies that specifically bind to myogenic precursor proteins. Exemplary myogenic precursor proteins include, but are not limited to, ABCG2, M-cadherin / cadherin-15, caveolin-1, CD34, FoxK1, integrin alpha-7, integrin alpha-7 beta-1, MYF-5, MyoD, myogenin, NCAM-1 / CD56, Pax3, Pax7, and Pax9. In some embodiments, muscle-targeted antibodies are antibodies that specifically bind to skeletal muscle proteins. The exemplified skeletal muscle proteins include, but are not limited to, alpha-sarcoglycans, beta-sarcoglycans, calpain inhibitors, creatine kinase MM / CKMM, eIF5A, enolase 2 / neuron-specific enolase, epsilon-sarcoglycans, 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, muscle-targeted antibodies are antibodies that specifically bind to smooth muscle proteins. The exemplary smooth muscle proteins include, but are not limited to, alpha-smooth muscle actin, VE-cadherin, cardesmon / CALD1, carponin 1, desmin, histamine H2 R, motilin R / GPR38, transgelin / TAGLN, and vimentin. However, it should be understood that antibodies against additional targets are within the scope of this disclosure, and that the list of exemplary targets provided herein is not intended to be limiting.

[0254] c. Antibody characteristics / changes In some embodiments, conservative mutations can be introduced into an antibody sequence (e.g., a CDR or framework sequence) at a position where, when determined based on, for example, the crystal structure, the residue is unlikely to be involved in interaction with the target antigen (e.g., a transferrin receptor). In some embodiments, one or more mutations (e.g., amino acid substitutions) are introduced into the Fc region of the muscle-targeted antibody described herein (e.g., in the CH2 domain (residues 231-340 of human IgG1) and / or (e.g., and) in the CH3 domain (residues 341-447 of human IgG1) and / or (e.g., and) in the hinge region) to alter one or more functional properties of the antibody, such as serum half-life, complement binding, Fc receptor binding, and / or (e.g., and).

[0255] In some embodiments, one or more mutations (e.g., amino acid substitutions) are introduced into the hinge region of the Fc region (CH1 domain) such that the number of cysteine ​​residues in the hinge region can be varied (e.g., increased or decreased) 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 may be altered, for example, to facilitate the assembly of the light and heavy chains, or to change the stability of the antibody (e.g., increased or decreased), or to facilitate linker conjugation.

[0256] In some embodiments, one 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 231-340 of human IgG1) and / or (e.g., and) in the CH3 domain (residues 341-447 of human IgG1) and / or (e.g., and) in the hinge region) to increase or decrease the antibody's affinity for Fc receptors on the surface of effector cells. Mutations in the Fc region of antibodies that increase or decrease the antibody's affinity for Fc receptors, and techniques for introducing such mutations into Fc receptors or fragments thereof are known to those skilled in the art. Examples of mutations in the Fc receptor of an antibody that may be made to alter the antibody's affinity 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 Publications WO 02 / 060919; WO 98 / 23289; and WO 97 / 34631 (these are incorporated herein by reference).

[0257] In some embodiments, one or more amino acid mutations (i.e., substitutions, insertions, or deletions) are introduced into the IgG constant region or its FcRn-binding fragment (preferably an Fc or hinge-Fc domain fragment) to alter (e.g., increase or decrease) the half-life of the antibody in vivo. For example, see International Publications WO 02 / 060919; WO 98 / 23289; and WO 97 / 34631; and U.S. Patents 5,869,046, 6,121,022, 6,277,375, and 6,165,745 for mutations that would alter (e.g., increase or decrease) the half-life of the antibody in vivo.

[0258] In some embodiments, one or more amino acid mutations (i.e., substitutions, insertions, or deletions) are introduced into the IgG constant region or its FcRn-binding fragment (preferably Fc or hinge-Fc domain fragment) to decrease the half-life of the anti-transferrin receptor antibody in vivo. In some embodiments, one or more amino acid mutations (i.e., substitutions, insertions, or deletions) are introduced into the IgG constant region or 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 231-340 of human IgG1) and / or (e.g., and) the third constant (CH3) domain (residues 341-447 of human IgG1), numbered according to the Kabat EU index (Kabat EA et al. (1991) above). In some embodiments, the IgG1 constant region of the antibodies described herein includes a methionine (M) to tyrosine (Y) substitution at position 252, numbered according to the EU index as found in Kabat, a serine (S) to threonine (T) substitution at position 254, and a threonine (T) to glutamic acid (E) substitution at position 256. See U.S. Patent No. 7,658,921 (which is incorporated herein by reference). This type of mutant IgG, referred to as the “YTE mutant,” has been shown to exhibit a four-fold increased half-life compared to the wild-type version of the same antibody (see Dall'Acqua WF et al., (2006) J Biol Chem 281:23514-24). In some embodiments, the antibody includes an IgG constant region containing one, two, or three or more amino acid substitutions of amino acid residues at positions 251-257, 285-290, 308-314, 385-389, and 428-436, numbered according to the EU index as found in Kabat.

[0259] In some embodiments, one or more amino acid substitutions are introduced into the IgG constant region Fc region to alter the effector function (one or more) of an anti-transferrin receptor antibody. The effector ligand with altered affinity to itself may be, for example, the Fc receptor or the C1 component of complement. This approach is described in more detail in U.S. Patents 5,624,821 and 5,648,260. In some embodiments, deletion or inactivation of the constant region domain (through point mutation or other means) may reduce the binding of the circulating antibody to the Fc receptor, thereby increasing tumor localization. For descriptions of mutations that delete or inactivate the constant region and thereby increase tumor localization, see, for example, U.S. Patents 5,585,097 and 8,591,886. In some embodiments, one or more amino acid substitutions may be introduced into the Fc region of the antibodies described herein to remove potential glycosylation sites on the Fc region (which may reduce binding to the Fc receptor) (see, for example, Shields RL et al., (2001) J Biol Chem 276:6591-604).

[0260] In some embodiments, one or more amino acid residues in the constant region of the muscle-targeting antibody described herein may be replaced with different amino acid residues so that the antibody may have modified Clq binding and / or (for example, and) reduced or absent complement-dependent cytotoxicity (CDC). This approach is described in further detail in U.S. Patent No. 6,194,551 (Idusogie et al). In some embodiments, one or more amino acid residues in the N-terminal region of the CH2 domain of the antibody described herein are modified to alter the complement-binding ability of the antibody. This approach is described in further international publication WO 94 / 29351. In some embodiments, the Fc region of the antibody described herein is modified to increase the antibody's ability to mediate antibody-dependent cytotoxicity (ADCC) to cells and / or (for example, and) to increase the antibody's affinity for the Fcγ receptor. This approach is described in further international publication WO 00 / 42072.

[0261] In some embodiments, the heavy chain and / or (for example, and) light chain variable domain(s) sequences(s) of the antibodies provided herein may be used, as described elsewhere herein, to generate, for example, CDR-conjugated antibodies, chimeric antibodies, humanized antibodies, or compound human antibodies, or antigen-binding fragments. As will be understood by those skilled in the art, any variant, CDR-conjugated antibody, chimeric antibody, humanized antibody, or compound antibody derived from any of the antibodies provided herein may be useful in the compositions and methods described herein, and the variant, CDR-conjugated antibody, chimeric antibody, humanized antibody, or compound antibody will maintain its specific binding ability to the transferrin receptor such that it may have at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% binding to the transferrin receptor compared to the original antibody from which it is derived.

[0262] In some embodiments, the antibodies provided herein include mutations that confer desired properties to the antibody. For example, to avoid potential complications resulting from Fab-arm exchange, which is known to occur in native IgG4 mAbs, the antibodies provided herein may include a stabilizing "Adair" mutation (Angal S., et al., "A single amino acid substitution abolishes the heterogeneity of chimeric mouse / human (IgG4) antibody", Mol Immunol 30, 105-108; 1993), where serine 228 (EU numbering; residue 241 Kabat numbering) is converted to proline, resulting in an IgG1-like hinge sequence. Consequently, any of the antibodies may contain a stabilizing "Adair" mutation.

[0263] As provided herein, the antibodies of this disclosure may optionally include a constant region or a portion thereof. For example, the VL domain may be attached at its C-terminus to a light chain constant region-like Cκ or Cλ. Similarly, the VH domain or a portion thereof may be attached to all or some heavy chain-like IgA, IgD, IgE, IgG, and IgM and any isotype subclass. The antibody may also encompass a preferred constant region (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, No. 91-3242, National Institutes of Health Publications, Bethesda, Md. (1991)). Thus, antibodies within the scope of this disclosure may be combined with any preferred constant region to encompass the VH and VL domains, or their antigen-binding regions.

[0264] ii. Muscle-targeting peptides Several aspects of this disclosure provide muscle-targeting peptides as muscle-targeting agents. Short peptide sequences that bind to specific cell types (for example, peptide sequences with a length of 5 to 20 amino acids) are described. For example, cell-targeting peptides are described in Vines e., et al., A., "Cell-penetrating and cell-targeting peptides in drug delivery," Biochim Biophys Acta 2008, 1786:126-38; Jarver P., et al., "In vivo biodistribution and efficacy of peptide mediated delivery," Trends Pharmacol Sci 2010; 31:528-35; Samoylova TI, et al., "Elucidation of muscle-binding peptides by phage display screening," Muscle Nerve 1999; 22:460-6; ​​U.S. Patent No. 6,329,501, issued December 11, 2001, titled "METHODS AND COMPOSITIONS FOR TARGETING COMPOUNDS TO MUSCLE"; and Samoylov AM, et al., "Recognition of cell-specific binding of phage display derived peptides using an acoustic wave The term "sensor" is described in Biomol Eng 2002;18:269-72, and the entire contents of each of these are incorporated herein by reference. Selectivity to desired tissues, e.g., muscle, can be achieved by designing peptides to interact with specific cell surface antigens (e.g., receptors). Skeletal muscle targeting has been investigated, and a wide range of molecular payloads can be delivered. These approaches, which do not have many of the practical disadvantages of large antibodies or viral particles, may have high selectivity to muscle tissue. Consequently, in some embodiments, muscle targeting agents are muscle-targeting peptides ranging in length from 4 to 50 amino acids.In some embodiments, muscle-targeting peptides have a length of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids. Muscle-targeting peptides can be generated using one of several methods, such as phage display.

[0265] In some embodiments, muscle-targeting peptides may bind to internalized cell surface receptors (e.g., transferrin receptors) that are overexpressed or relatively highly expressed in muscle cells compared to other cells. In some embodiments, muscle-targeting peptides may target transferrin receptors (e.g., bind to transferrin receptors). In some embodiments, transferrin receptor-targeting peptides may contain naturally occurring ligands, e.g., segments of transferrin. In some embodiments, transferrin receptor-targeting peptides are described in U.S. Patent No. 6,743,893, 11 / 30 / 2000, “RECEPTOR-MEDIATED UPTAKE OF PEPTIDES THAT BIND THE HUMAN TRANSFERRIN RECEPTOR”. In some embodiments, the transferrin receptor-targeting peptide is as described in Kawamoto, M. et al, "A novel transferrin receptor-targeted hybrid peptide disintegrates cancer cell membrane to induce rapid killing of cancer cells," BMC Cancer. 2011 Aug 18;11:359. In some embodiments, the transferrin receptor-targeting peptide is as described in U.S. Patent No. 8,399,653, 5 / 20 / 2011, "TRANSFERRIN / TRANSFERRIN RECEPTOR-MEDIATED SIRNA DELIVERY."

[0266] As mentioned above, examples of muscle-targeting peptides have been reported. For example, muscle-specific peptides have been identified using phage display libraries that present ptapeptides on their surface. As an example, a peptide with the amino acid sequence ASSLNIA (SEQ ID NO: 138) bound to C2C12 mouse myotubes in vitro and to mouse muscle tissue in vivo. Consequently, in several embodiments, muscle-targeting agents contain the amino acid sequence ASSLNIA (SEQ ID NO: 138). This peptide exhibited improved specificity for binding to myocardial and skeletal muscle tissue after intravenous injection into mice, with reduced binding to the liver, kidneys, and brain. Additional muscle-specific peptides have been identified using phage display. For example, in the context of treatment for DMD, a 12-amino acid peptide was identified by a phage display library for muscle targeting. See Yoshida D., et al., "Targeting of salicylate to skin and muscle following topical injections in rats," Int J Pharm 2002;231:177-84; its entire content is 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 compared to the ASSLNIA (SEQ ID NO: 138) peptide.

[0267] Any additional method for identifying peptides selective to muscle (e.g., skeletal muscle) more than 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 content of this is incorporated herein by reference. Nonspecific cell binders were selected by pre-incubating a random 12-mer peptide phage display library with a mixture of non-muscle cell types. After repeated selection, the 12-amino acid peptide TARGEHKEEELI (SEQ ID NO: 140) appeared most frequently. Consequently, in some embodiments, muscle targeting agents include the amino acid sequence TARGEHKEEELI (SEQ ID NO: 140).

[0268] Muscle targeting agents may be amino acid-containing molecules or peptides. Muscle targeting peptides may correspond to sequences of proteins that preferentially bind to protein receptors found in muscle cells. In some embodiments, muscle targeting peptides contain high propensity of hydrophobic amino acids (e.g., valine) so that the peptides can preferentially target muscle cells. In some embodiments, muscle targeting peptides have not been characterized or disclosed to date. These peptides may be recalled, produced, synthesized, and / or (e.g., and) derivatized using one of several methodologies, e.g., a phage-dispenseed peptide library, a one-bead-one-compound peptide library, or a positional scanning synthetic peptide combinatorial library. The example methodologies have become characteristic in the relevant technical field and are incorporated by reference (Gray, BP and Brown, KC, "Combinatorial Peptide Libraries: Mining for Cell-Binding Peptides," Chem Rev. 2014, 114:2, 1020-1081; Samoylova, TI and Smith, BF, "Elucidation of muscle-binding peptides by phage display screening," Muscle Nerve, 1999, 22:4, 460-6).In some aspects, muscle-targeting peptides have been disclosed in the past (see, for example, Writer, MJet 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, MJet al. "In vitro selection of a peptide with high selectivity for cardiomyocytes in vivo" J Mol Biol. 2004, 342:1, 171-82). The exemplary muscle-targeting peptides include the following amino acid sequences: CQAQGQLVC (SEQ ID NO: 141), CSERSMNFC (SEQ ID NO: 142), CPKTRRVPC (SEQ ID NO: 143), WLSEAGPVVTVRALRGTGSW (SEQ ID NO: 144), ASSLNIA (SEQ ID NO: 138), CMQHSMRVC (SEQ ID NO: 145), and DDTRHWG (SEQ ID NO: 146). In some embodiments, the muscle-targeting peptides may contain about 2–25 amino acids, about 2–20 amino acids, about 2–15 amino acids, about 2–10 amino acids, or about 2–5 amino acids. The muscle-targeting peptides may contain naturally occurring amino acids, such as cysteine, alanine, or amino acids that are not naturally occurring or are modified amino acids. Amino acids that are not naturally occurring include β-amino acids, homo-amino acids, proline derivatives, 3-substituted alanine derivatives, linear core amino acids, N-methyl amino acids, and other amino acids known in the art.In some embodiments, the muscle-targeting peptide may be linear; in other embodiments, the muscle-targeting peptide may be cyclic (e.g., bicyclic) (see, for example, Silvana, MGet al. Mol. Therapy, 2018, 26:1, 132-147).

[0269] iii. Muscle-targeting receptor ligands The muscle targeting agent may be a ligand, for example, a ligand that binds to a receptor protein. The muscle targeting ligand may be a protein, for example, transferrin, which binds to an internalized cell surface receptor expressed by muscle cells. Consequently, in some embodiments, the muscle targeting agent is transferrin, or a derivative thereof that binds to a transferrin receptor. Alternatively, the muscle targeting ligand may be a small molecule, for example, a lipophilic small molecule that preferentially targets muscle cells compared to other cell types. Exemplary lipophilic small molecules that may target muscle cells include compounds containing cholesterol, cholesteryl, stearic acid, palmitic acid, oleic acid, oleyl, linolenic acid, linoleic acid, myristic acid, sterols, dihydrotestosterone, testosterone derivatives, glycerin, alkyl chains, trityl groups, and alkoxy acids.

[0270] iv. Muscle-targeting aptamers Muscle-targeting agents may be aptamers that preferentially target muscle cells compared to other cell types, such as RNA aptamers. In some embodiments, muscle-targeting aptamers have not been characterized or disclosed to date. These aptamers may be recalled, produced, synthesized, and / or (for example, and) derivatized using one of several methodologies, e.g., systematic evolution of ligands by exponential enrichment. The exemplary methodologies are characterized in the art and incorporated by reference (Yan, A. 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-targeted aptamers have been previously disclosed (see, for example, Phillippou, S. et al. "Selection and Identification of Skeletal-muscle-Targeted RNA Aptamers," Mol Ther Nucleic Acids. 2018, 10:199-214; Thiel, WH et al. "Smooth Muscle Cell-targeted RNA Aptamer Inhibits Neointimal Formation," Mol Ther. 2016, 24:4, 779-87). Exemplary muscle-targeted aptamers include the A01B RNA aptamer and RNA Apt 14. In some embodiments, the aptamer is a nucleic acid-based aptamer, an oligonucleotide aptamer, or a peptide aptamer. In some embodiments, the aptamer may be approximately 5–15 kDa, approximately 5–10 kDa, approximately 10–15 kDa, approximately 1–5 Daa, approximately 1–3 kDa, or smaller.

[0271] v. Other muscle targeting agents One strategy for targeting muscle cells (e.g., skeletal muscle cells) is to use substrates of muscle transporter proteins, such as transporter proteins expressed on the muscle fiber sheath. In some embodiments, muscle targeting agents are substrates of influx transporters specific to muscle tissue. In some embodiments, the influx transporters are specific to skeletal muscle tissue. The two major classes of transporters expressed on the muscle fiber sheath of skeletal muscle are (1) the adenosine triphosphate (ATP)-binding cassette (ABC) superfamily, which facilitates efflux from skeletal muscle tissue, and (2) the solute carrier (SLC) superfamily, which can facilitate the influx of substrates into skeletal muscle. In some embodiments, muscle targeting agents are substrates that bind to the ABC or SLC superfamily of transporters. In some embodiments, the substrates that bind to the ABC or SLC superfamily of transporters are naturally occurring substrates. In some embodiments, the substrates that bind to the ABC or SLC superfamily of transporters are naturally occurring substrates, such as synthetic derivatives that bind to the ABC or SLC superfamily of transporters.

[0272] In some embodiments, muscle targeting agents are substrates of the SLC superfamily of transporters. SLC transporters are either equilibrium-type or utilize a proton or sodium ion gradient created across the membrane to propel substrate transport. Exemplary SLC transporters with high expression in skeletal muscle include, but are not limited to, the SATT transporter (ASCT1; SLC1A4), GLUT4 transporter (SLC2A4), GLUT7 transporter (GLUT7; SLC2A7), ATRC2 transporter (CAT-2; SLC7A2), 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 provide an opportunity for muscle targeting by facilitating the influx of substrates into skeletal muscle.

[0273] In some embodiments, muscle targeting agents are substrates of the equilibrium nucleoside transporter 2 (ENT2) transporter. Compared to other transporters, ENT2 has one of the highest expression mRNAs in skeletal muscle. Human ENT2 (hENT2) is expressed in most body organs, including the brain, heart, placenta, thymus, pancreas, prostate, and kidneys, but is particularly abundant in skeletal muscle. Human ENT2 facilitates the uptake of its substrates according to their concentration gradients. ENT2 plays a role in maintaining nucleoside homeostasis by transporting a wide range of purine and pyrimidine nucleic acid bases. The hENT2 transporter has low affinity for all nucleosides except inosine (adenosine, guanosine, uridine, thymidine, and cytidine). Consequently, in some embodiments, muscle targeting agents are ENT2 substrates. Exemplary ENT2 substrates include, but are not limited to, inosine, 2',3'-dideoxyinosine, and clopharabine. In some embodiments, any of the muscle targeting agents provided herein are related to 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 noncovalently linked to the molecular payload.

[0274] In some embodiments, the muscle targeting agent is a substrate of an organic cation / carnitine transporter (OCTN2), which is a sodium ion-dependent high-affinity carnitine transporter. In some embodiments, the muscle targeting agent is carnitine, mildronate, acetylcarnitine, or any derivative thereof that binds to OCTN2. In some embodiments, carnitine, mildronate, acetylcarnitine, or their derivatives are covalently linked to a molecular payload (e.g., an oligonucleotide payload).

[0275] The muscle targeting agent may be a protein that exists in at least one soluble form that targets muscle cells. In some embodiments, the muscle targeting protein may be hemoduvelin (also known as repulsive guidance molecule C or hemochromatosis type 2 protein), a protein involved in iron overload and homeostasis. In some embodiments, hemoduvelin may be full length or fragmentary, or a mutant having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with a functional hemoduvelin protein. In some embodiments, the hemoduvelin mutant may be a soluble fragment, may lack N-terminal signaling, and / or (for example, and), may lack a C-terminal anchoring domain. In some embodiments, hemoduberin may be annotated with GenBank RefSeq accession numbers NM_001316767.1, NM_145277.4, NM_202004.3, NM_213652.3, or NM_213653.3. It should be understood that hemoduberin may originate from humans, non-human primates, or rodents.

[0276] B. Molecular payload Several aspects of this disclosure provide molecular payloads for modulating biological outcomes, such as transcription of DNA sequences, splicing and processing of RNA sequences, protein expression, or protein activity. In some embodiments, the molecular payload is ligated to or otherwise linked to a muscle targeting agent. In some embodiments, such a molecular payload can target muscle cells, for example, by specific binding to nucleic acids or proteins in muscle cells that have been delivered to muscle cells by the linked muscle targeting agent. It should be understood that various types of muscle targeting agents may be used in accordance with this disclosure. For example, the molecular payload may include, or consist of, oligonucleotides (e.g., antisense oligonucleotides), peptides (e.g., peptides that bind to nucleic acids or proteins in disease-associated muscle cells), proteins (e.g., proteins that bind to nucleic acids or proteins in disease-associated muscle cells), or small molecules (e.g., small molecules that modulate the function of nucleic acids or proteins in disease-associated muscle cells). In some embodiments, the molecular payload is an oligonucleotide containing a chain having a region complementary to a mutated DMD allele. While exemplary molecular payloads are described in more detail herein, it should be understood that the exemplary molecular payloads provided herein are not intended to be limiting.

[0277] i. oligonucleotides Any suitable oligonucleotide may be used as the molecular payload as described herein. In some embodiments, oligonucleotides may be designed to induce exon skipping (for example, EXONDYS 51 oligonucleotide (Sarepta Therapeutics, Inc.), which includes SEQ ID NO: 343 (CUCCAACAUCAAGGAAGAUGGCAUUUCUAG); WVE-210201 (Wave Life Sciences), which includes SEQ ID NO: 334 (UCAAGGAAGAUGGCAUUUCU); Casimersen (Sarepta Therapeutics, Inc.), which includes SEQ ID NO: 302 (CAAUGCCAUCCUGGAGUUCCUG); or Golodirsen (Sarepta Therapeutics, Inc.), which includes SEQ ID NO: 380 (GUUGCCUCCGGUUCUGAAGGUGUUC). In some embodiments, oligonucleotides may be designed to induce exon skipping (for example, viltolarsen (NS Pharma, Inc.), which includes SEQ ID NO: 2257 (CCTCCGGTTCTGAAGGTGTTC); or renadirsen (Daiichi Sankyo Company), this includes sequence number 2252 (CGCUGCCCAAUGCCAUCC). In some embodiments, the oligonucleotide comprises a sequence or a portion thereof of the sequences provided in Table 10 (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more consecutive nucleosides), and / or the oligonucleotide comprises a region complementary to the target sequence provided in Table 10. One or more thymine bases (T's) in any of the oligonucleotides provided herein (e.g., the oligonucleotides listed in Table 10) may optionally be uracil bases (U's), and / or one of the U's in the oligonucleotides provided herein may optionally be T's. Table 10. Examples of oligonucleotide molecular payloads [Table 8]

[0278] In some embodiments, oligonucleotides may be designed to induce the degradation of mRNA (for example, oligonucleotides may be gapmers, siRNAs, ribozymes, or aptamers that induce degradation). In some embodiments, oligonucleotides may be designed to block the translation of mRNA (for example, oligonucleotides may be mixmers, siRNAs, or aptamers that block translation). In some embodiments, oligonucleotides may be designed to induce the degradation of mRNA and thereby block its translation. In some embodiments, oligonucleotides may be designed to promote the stabilization of mRNA. In some embodiments, oligonucleotides may be designed to promote the translation of mRNA. In some embodiments, oligonucleotides may be designed to promote the stabilization of mRNA and thereby promote its translation. In some embodiments, oligonucleotides may be guide nucleic acids (for example, guide RNA) for directing the activity of an enzyme (for example, a gene editing enzyme). In some embodiments, the guide nucleic acid may direct the enzyme to delete all or part of a mutated DMD allele (for example, to facilitate in-frame exon skipping). In some embodiments, oligonucleotides may be designed to target repressive regulators of DMD expression (e.g., miR-31). Other examples of oligonucleotides are provided herein. In some embodiments, it should be understood that by incorporating a functional sequence (e.g., an antisense chain sequence) from one format into the other, an oligonucleotide of one format (e.g., an antisense oligonucleotide) may be suitably adapted to another format (e.g., an siRNA oligonucleotide).

[0279] Examples of useful oligonucleotides for targeting DMD include U.S. Patent Application Publication US20100130591A1, published May 27, 2010, entitled "MULTIPLE EXON SKIPPING COMPOSITIONS FOR DMD"; U.S. Patent No. 8,361,979, registered January 29, 2013, entitled "MEANS AND METHOD FOR INDUCING EXON-SKIPPING"; U.S. Patent Application Publication 20120059042, published March 8, 2012, entitled "METHOD FOR EFFICIENT EXON (44) SKIPPING IN DUCHENNE MUSCULAR DYSTROPHY AND ASSOCIATED MEANS"; and "EXON SKIPPING COMPOSITIONS FOR TREATING MUSCULAR U.S. Patent Application Publication 20140329881, published November 6, 2014, titled "DYSTROPHY"; U.S. Patent No. 8,232,384, registered July 31, 2012, titled "ANTISENSE OLIGONUCLEOTIDES FOR INDUCING EXON SKIPPING AND METHODS OF USE THEREOF"; U.S. Patent Application Publication 20120022134A1, published January 26, 2012, titled "METHODS AND MEANS FOR EFFICIENT SKIPPING OF EXON 45 IN DUCHENNE MUSCULAR DYSTROPHY PRE-MRNA"; "ADENO-ASSOCIATED VIRAL VECTOR FOR EXON SKIPPING IN A GENE ENCODING A DISPENSABLE DOMAN U.S. Patent Application Publication 20120077860, published on March 29, 2012, titled "PROTEIN"; U.S. Patent No. 8,324,371, registered on December 4, 2012, titled "OLIGOMERS"; U.S. Patent No. 9,078,911, registered on July 14, 2015, titled "ANTISENSE OLIGONUCLEOTIDES"; U.S. Patent No. 9,079,934, registered on July 14, 2015, titled "ANTISENSE NUCLEIC ACIDS";Provided in U.S. Patent No. 9,034,838, registered on May 19, 2015, entitled "MIR-31 IN DUCHENNE MUSCULAR DYSTROPHY THERAPY"; and in the international patent publication WO2017062862A3, published on April 13, 2017, entitled "OLIGONUCLEOTIDE COMPOSITIONS AND METHODS THEREOF," the entire contents of each of these are incorporated herein by reference.

[0280] Table 14 provides non-restrictive examples of oligonucleotide sequences useful for targeting DMDs (e.g., for exon skipping). In some embodiments, the oligonucleotides may include any sequence provided in Table 14. Table 14 - Oligonucleotide sequences for targeting DMD. [Table 9-1] [Table 9-2] [Table 9-3] [Table 9-4] [Table 9-5] [Table 9-6] [Table 9-7]

[0281] In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) target a region of DMD RNA (e.g., the Dp427m transcript of SEQ ID NO: 2239). In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) contain a region complementary to the RNA of DMD (e.g., the Dp427m transcript of SEQ ID NO: 2239). In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) contain a region complementary to an exon of DMD RNA (e.g., any one of SEQ ID NOs. 2240-2250). Examples of DMD RNA sequences and exon sequences are provided below.

[0282] Homo sapiens dystrophin (DMD), transcription variant Dp427m, mRNA (NCBI reference sequence: NM_004006.2)

[0283] Homo sapiens dystrophin (DMD), transcription variant Dp427m, exon 8 (nucleotides at positions 894-1075 of NCBI reference sequence: NM_004006.2) ATGTTGATACCACCTATCCAGATAAGAAGTCCATCTTAATGTACATCACATCACTCTTCCAAGTTTTGCCTCAACAAGTGAGCATTGAAGCCATCCAGGAAGTGGAAATGTTGCCAAGGCCACCTAAAGTGACTAAAGAAGAACATTTTCAGTTACATCATCAAATGCACTATTCTCAACAG (SEQ ID NO: 2240)

[0284] Homo sapiens dystrophin (DMD), transcription variant Dp427m, exon 23 (nucleotides 3194-3406 of NCBI reference sequence: NM_004006.2) GCTTTACAAAGTTCTCTGCAAGAGCAACAAAGTGGCCTATACTATCTCAGCACCACTGTGAAAGAGATGTCGAAGAAAGCGCCCTCTGAAATTAGCCGGAAATATCAATCAGAATTTGAAGAAATTGAGGGACGCTGGAAGAAGCTCTCCTCCCAGCTGGTTGAGCATTGTCAAAGCTAGAGGAGCAAATGAATAAACTCCGAAAAATTCAG (SEQ ID NO: 2241)

[0285] Homo sapiens dystrophin (DMD), transcription variant Dp427m, exon 43 (nucleotides at positions 6362-6534 of NCBI reference sequence: NM_004006.2) AATATAAAGATAGTCTACAACAAAGCTCAGGTCGGATTGACATTATTCATAGCAAGAAGACAGCAGCATTGCAAAGTGCAACGCCTGTGGAAAGGGTGAAGCTACAGGAAGCTCTCCCAGCTTGATTTCCAATGGGAAAAGTTAACAAAATGTACAAGGACCGACAAGG (SEQ ID NO: 2242)

[0286] Homo sapiens dystrophin (DMD), transcription variant Dp427m, exon 44 (nucleotides at positions 6535-6682 of NCBI reference sequence: NM_004006.2) GCGATTTGACAGATCTGTTGAGAAATGGCGGCGTTTTCATTATGATATAAAGATATTTAATCAGTGGCTAACAGAAGCTGAACAGTTTCTCAGAAAGACACAAATTCCTGAGAATTGGGAACATGCTAAATACAAATGGTATCTTAAG (SEQ ID NO: 2243)

[0287] Homo sapiens dystrophin (DMD), transcription variant Dp427m, exon 45 (nucleotides at positions 6683-6858 of NCBI reference sequence: NM_004006.2) GAACTCCAGGATGGCATTGGGCAGCGCAAACTGTTGTCAGAACATTGAATGCAACTGGGGAAGAAATAATTCAGCAATCCTCAAAAACAGATGCCAGTATTCTACAGGAAAAATTGGGAAGCCTGAATCTGCGGTGGCAGGAGGTCTGCAAACAGCTGTCAGACAGAAAAAAGAG (SEQ ID NO: 2244)

[0288] Homo sapiens dystrophin (DMD), transcription variant Dp427m, exon 46 (nucleotides at positions 6859-7006 of NCBI reference sequence: NM_004006.2) GCTAGAAGAACAAAAGAATATCTTGTCAGAATTTCAAAGAGATTTAAATGAATTTGTTTTATGGTTGGAGGAAGCAGATAACATTGCTAGTATCCCACTTGAACCTGGAAAAGAGCAGCAACTAAAAGAAAAGCTTGAGCAAGTCAAG (SEQ ID NO: 2245)

[0289] Homo sapiens dystrophin (DMD), transcription variant Dp427m, exon 50 (nucleotides at positions 7445-7553 of NCBI reference sequence: NM_004006.2) AGGAAGTTAGAAGATCTGAGCTCTGAGTGGAAGGCGGTAAACCGTTTACTTCAAGAGCTGAGGGCAAAGCAGCCTGACCTAGCTCCTGGACTGACCACTATTGGAGCCT (SEQ ID NO: 2246)

[0290] Homo sapiens dystrophin (DMD), transcription variant Dp427m, exon 51 (nucleotides at positions 7554-7786 of NCBI reference sequence: NM_004006.2) CTCCTACTCAGACTGTTACTCTGGTGACACAACCTGTGGTTACTAAGGAAACTGCCATCTCCAAACTAGAAATGCCATCTTCCTTGATGTTGGAGGTACCTGCTCTGGCAGATTTCAACCGGGCTTGGACAGAACTTACCGACTGGCTTTCTCTGCTTGATCAAGTTATAAAATCACAGAGGGTGATGGTGGTGACCTTGAGGATATCAACGAGATGATCATCAAGCAGAAG (Sequence number 2247)

[0291] Homo sapiens dystrophin (DMD), transcription variant Dp427m, exon 52 (nucleotides at positions 7787-7904 of NCBI reference sequence: NM_004006.2) GCAACAATGCAGGATTTGGAACAGAGGCGTCCCCAGTTGGAAGAACTCATTACCGCTGCCCAAAATTTGAAAAACAAGACCAGCAATCAAGAGGCTAGAACAATCATTACGGATCGAA (SEQ ID NO: 2248)

[0292] Homo sapiens dystrophin (DMD), transcription variant Dp427m, exon 53 (nucleotides at positions 7905-8116 of NCBI reference sequence: NM_004006.2) TTGAAAGAATTCAGAATCAGTGGGATGAAGTACAAGAACACCTTCAGAACCGGAGGCAACAGTTGAATGAAATGTTAAAGGATTCAACACAATGGCTGGAAGCTAAGGAAGAAGCTGAGCAGGTCTTAGGACAGGCCAGAGCCAAGCTTGAGTCATGGAAGGAGGGTCCCTATACAGTAGATGCAATCCAAAAGAAAATCACAGAAACCAAG (SEQ ID NO: 2249)

[0293] Homo sapiens dystrophin (DMD), transcription variant Dp427m, exon 55 (nucleotides at positions 8272-8461 of NCBI reference sequence: NM_004006.2) GGTGAGTGAGCGAGAGGCTGCTTTGGAAGAAACTCATAGATTACTGCAACAGTTCCCCTGGACCTGGAAAAGTTTCTTGCCTGGCTTACAGAAGCTGAAACAACTGCCAATGTCCTACAGGATGCTACCCGTAAGGAAAGGCTCCTAGAAGACTCCAAGGGAGTAAAAGAGCTGATGAAACAATGGCAA (SEQ ID NO: 2250)

[0294] In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) target exonic splicing enhancer (ESE) sequences in DMD (e.g., ESE sequences of exons 23, 44, 45, 46, 50, 51, 52, 53, or 55). In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) target exonic splicing enhancer (ESE) sequences in DMD (e.g., ESE sequences of exons 8, 23, 43, 44, 45, 46, 50, 51, 52, 53, or 55). In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) target the ESE sequence of DMD exon 51 (e.g., ESEs listed in Table 15). In some embodiments, oligonucleotides useful for targeting DMD target ESE sequences in DMD exons 8, 23, 42, 44, 45, 46, 50, 52, 53, or 55 (for example, the ESEs listed in Table 11).

[0295] In some embodiments, oligonucleotides useful for targeting DMD (e.g., for skipping one or more exons 8, 23, 42, 44, 45, 46, 50, 52, 53, and 55) include a region complementary to a target sequence containing one or more complete or partial ESEs of the DMD transcript (e.g., one or more complete or partial ESEs listed in Table 15 or Table 11). In some embodiments, the oligonucleotide includes a region complementary to a target sequence containing one or more complete or partial ESEs as described in SEQ ID NOs. 402-436 and 2043-2238. In some embodiments, the oligonucleotide includes a region complementary to a target sequence containing at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE described in any one of SEQ ID NOs. 402-436 and 2043-2238. Table 15. Exonics Splicing Enhancers within Exons of DMD [Table 10] Table 11. Exonic splicing enhancers in exons 8, 23, 43, 44, 45, 46, 50, 52, 53, and 55 of DMD [Table 11-1] [Table 11-2] [Table 11-3] [Table 11-4] [Table 11-5] [Table 11-6]

[0296] In some embodiments, the oligonucleotide comprises a region complementary to a target sequence containing one or more complete or partial ESEs of DMD exon 8. In some embodiments, the oligonucleotide comprises a region complementary to a target sequence containing at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE of DMD exon 8. In some embodiments, the oligonucleotide comprises a region complementary to a target sequence containing one or more complete or partial ESEs as represented by SEQ ID NOs. 2047-2062. In some embodiments, the oligonucleotide comprises a region complementary to a target sequence containing at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as represented by any one of SEQ ID NOs. 2047-2062.

[0297] In some embodiments, the oligonucleotide comprises a region complementary to a target sequence containing at least six nucleotides (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) of one or more ESEs (e.g., two, three, four, or more adjacent ESEs) of DMD exon 8. In some embodiments, the oligonucleotide comprises a region complementary to a target sequence containing at least six nucleotides (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) of one or more ESEs (e.g., two, three, four, or more adjacent ESEs) as represented by SEQ ID NOs. 2047-2062.

[0298] In some embodiments, oligonucleotides useful for targeting DMDs (e.g., for exon skipping) are 18 to 35 nucleotides in length and include a region complementary to a target sequence containing at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as represented by any one of SEQ ID NOs. 2047 to 2062. In some embodiments, oligonucleotides useful for targeting DMDs (e.g., for exon skipping) are 20 to 30 (e.g., 20, 25, 30) nucleotides in length and include a region complementary to a target sequence containing at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as represented by any one of SEQ ID NOs. 2047 to 2062. In some embodiments, an oligonucleotide useful for targeting DMD (e.g., for exon skipping) is 20 nucleotides long and includes a region complementary to a target sequence containing at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as represented by any one of SEQ ID NOs. 2047-2062. In some embodiments, an oligonucleotide useful for targeting DMD (e.g., for exon skipping) is 30 nucleotides long and includes a region complementary to a target sequence containing at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as represented by any one of SEQ ID NOs. 2047-2062.

[0299] In some embodiments, the oligonucleotide comprises a region complementary to a target sequence containing one or more complete or partial ESEs of DMD exon 23. In some embodiments, the oligonucleotide comprises a region complementary to a target sequence containing at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE of DMD exon 23. In some embodiments, the oligonucleotide comprises a region complementary to a target sequence containing one or more complete or partial ESEs as represented by SEQ ID NOs. 429 and 2063-2086. In some embodiments, the oligonucleotide comprises a region complementary to a target sequence containing at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as represented by any one of SEQ ID NOs. 429 and 2063-2086.

[0300] In some embodiments, the oligonucleotide comprises a region complementary to a target sequence containing at least six nucleotides (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) of one or more ESEs (e.g., 2, 3, 4, or more adjacent ESEs) of DMD exon 23. In some embodiments, the oligonucleotide comprises a region complementary to a target sequence containing at least six nucleotides (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) of one or more ESEs (e.g., 2, 3, 4, or more adjacent ESEs) as represented by SEQ ID NOs. 429 and 2063-2086.

[0301] In some embodiments, oligonucleotides useful for targeting DMDs (e.g., for exon skipping) are 18 to 35 nucleotides in length and include a region complementary to a target sequence containing at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as represented by any one of SEQ ID NOs. 429 and 2063-2086. In some embodiments, oligonucleotides useful for targeting DMDs (e.g., for exon skipping) are 20 to 30 (e.g., 20, 25, 30) nucleotides in length and include a region complementary to a target sequence containing at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as described in any one of SEQ ID NOs. 429 and 2063-2086. In some embodiments, an oligonucleotide useful for targeting DMD (e.g., for exon skipping) is 20 nucleotides long and includes a region complementary to a target sequence containing at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as represented by any one of SEQ ID NOs. 429 and 2063-2086. In some embodiments, an oligonucleotide useful for targeting DMD (e.g., for exon skipping) is 30 nucleotides long and includes a region complementary to a target sequence containing at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as represented by any one of SEQ ID NOs. 429 and 2063-2086.

[0302] In some embodiments, the oligonucleotide comprises a region complementary to a target sequence containing one or more complete or partial ESEs of DMD exon 43. In some embodiments, the oligonucleotide comprises a region complementary to a target sequence containing at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE of DMD exon 43. In some embodiments, the oligonucleotide comprises a region complementary to a target sequence containing one or more complete or partial ESEs as represented by SEQ ID NOs. 412, 2078-2080, and 2087-2111. In some embodiments, the oligonucleotide comprises a region complementary to a target sequence containing at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as represented by any one of SEQ ID NOs. 412, 2078-2080, and 2087-2111.

[0303] In some embodiments, the oligonucleotide includes a region complementary to a target sequence containing at least six nucleotides (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) of one or more ESEs (e.g., two, three, four, or more adjacent ESEs) of DMD exon 43. In some embodiments, the oligonucleotide includes a region complementary to a target sequence containing at least six nucleotides (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) of one or more ESEs (e.g., two, three, four, or more adjacent ESEs) as represented by SEQ ID NOs. 412, 2078-2080, and 2087-2111.

[0304] In some embodiments, oligonucleotides useful for targeting DMDs (e.g., for exon skipping) are 18–35 nucleotides in length and include a region complementary to a target sequence containing at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as represented by one of sequence numbers 412, 2078–2080, and 2087–2111. In some embodiments, oligonucleotides useful for targeting DMDs (e.g., for exon skipping) are 20–30 (e.g., 20, 25, 30) nucleotides in length and include a region complementary to a target sequence containing at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as represented by one of sequence numbers 412, 2078–2080, and 2087–2111. In some embodiments, an oligonucleotide useful for targeting DMD (e.g., for exon skipping) is 20 nucleotides long and includes a region complementary to a target sequence containing at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as represented by one of sequence numbers 412, 2078-2080, and 2087-2111. In some embodiments, an oligonucleotide useful for targeting DMD (e.g., for exon skipping) is 30 nucleotides long and includes a region complementary to a target sequence containing at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as represented by one of sequence numbers 412, 2078-2080, and 2087-2111.

[0305] In some embodiments, the oligonucleotide comprises a region complementary to a target sequence containing one or more complete or partial ESEs of DMD exon 44. In some embodiments, the oligonucleotide comprises a region complementary to a target sequence containing at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE of DMD exon 44. In some embodiments, the oligonucleotide comprises a region complementary to a target sequence containing one or more complete or partial ESEs as represented by SEQ ID NOs. 409 and 2112-2121. In some embodiments, the oligonucleotide comprises a region complementary to a target sequence containing at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as represented by any one of SEQ ID NOs. 409 and 2112-2121.

[0306] In some embodiments, the oligonucleotide comprises a region complementary to a target sequence containing at least six nucleotides (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) of one or more ESEs (e.g., two, three, four, or more adjacent ESEs) of DMD exon 44. In some embodiments, the oligonucleotide comprises a region complementary to a target sequence containing at least six nucleotides (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) of one or more ESEs (e.g., two, three, four, or more adjacent ESEs) as represented by SEQ ID NOs. 409 and 2112-2121.

[0307] In some embodiments, oligonucleotides useful for targeting DMDs (e.g., for exon skipping) are 18–35 nucleotides in length and include a region complementary to a target sequence containing at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as represented by either SEQ ID NOs. 409 and 2112–2121. In some embodiments, oligonucleotides useful for targeting DMDs (e.g., for exon skipping) are 20–30 (e.g., 20, 25, 30) nucleotides in length and include a region complementary to a target sequence containing at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as represented by either SEQ ID NOs. 409 and 2112–2121. In some embodiments, an oligonucleotide useful for targeting DMDs (e.g., for exon skipping) comprises an ESE such as that described in any one of SEQ ID NOs. 409 and 2112-2121, having a length of 20 nucleotides and containing at least 4 (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as represented in any one of SEQ ID NOs. In some embodiments, an oligonucleotide useful for targeting DMDs (e.g., for exon skipping) has a length of 30 nucleotides and comprises a region complementary to a target sequence containing at least 4 (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as represented in any one of SEQ ID NOs. 409 and 2112-2121.

[0308] In some embodiments, the oligonucleotide comprises a region complementary to a target sequence containing one or more complete or partial ESEs of DMD exon 45. In some embodiments, the oligonucleotide comprises a region complementary to a target sequence containing at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE of DMD exon 45. In some embodiments, the oligonucleotide comprises a region complementary to a target sequence containing one or more complete or partial ESEs as represented by SEQ ID NOs. 2097, 2102, 2103, and 2122-2146. In some embodiments, the oligonucleotide comprises a region complementary to a target sequence containing at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as represented by any one of SEQ ID NOs. 2097, 2102, 2103, and 2122-2146.

[0309] In some embodiments, the oligonucleotide includes a region complementary to a target sequence containing at least six nucleotides (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) of one or more ESEs (e.g., two, three, four, or more adjacent ESEs) of DMD exon 45. In some embodiments, the oligonucleotide includes a region complementary to a target sequence containing at least six nucleotides (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) of one or more ESEs (e.g., two, three, four, or more adjacent ESEs) as described in SEQ ID NOs. 2097, 2102, 2103, and 2122-2146.

[0310] In some embodiments, oligonucleotides useful for targeting DMDs (e.g., for exon skipping) are 18–35 nucleotides in length and include a region complementary to a target sequence containing at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as represented by one of sequence numbers 2097, 2102, 2103, and 2122–2146. In some embodiments, oligonucleotides useful for targeting DMDs (e.g., for exon skipping) are 20–30 (e.g., 20, 25, 30) nucleotides in length and include a region complementary to a target sequence containing at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as represented by one of sequence numbers 2097, 2102, 2103, and 2122–2146. In some embodiments, an oligonucleotide useful for targeting DMD (e.g., for exon skipping) is 20 nucleotides long and includes a region complementary to a target sequence containing at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as represented by any one of SEQ ID NOs. 2097, 2102, 2103, and 2122-2146. In some embodiments, an oligonucleotide useful for targeting DMD (e.g., for exon skipping) is 30 nucleotides long and includes a region complementary to a target sequence containing at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as represented by any one of SEQ ID NOs. 2097, 2102, 2103, and 2122-2146.

[0311] In some embodiments, the oligonucleotide comprises a region complementary to a target sequence containing one or more complete or partial ESEs of DMD exon 46. In some embodiments, the oligonucleotide comprises a region complementary to a target sequence containing at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE of DMD exon 46. In some embodiments, the oligonucleotide comprises a region complementary to a target sequence containing one or more complete or partial ESEs as represented by SEQ ID NOs. 2096 and 2147-2158. In some embodiments, the oligonucleotide comprises a region complementary to a target sequence containing at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as represented by any one of SEQ ID NOs. 2096 and 2147-2158.

[0312] In some embodiments, the oligonucleotide comprises a region complementary to a target sequence containing at least six nucleotides (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) of one or more ESEs (e.g., two, three, four, or more adjacent ESEs) of DMD exon 46. In some embodiments, the oligonucleotide comprises a region complementary to a target sequence containing at least six nucleotides (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) of one or more ESEs (e.g., two, three, four, or more adjacent ESEs) as represented by SEQ ID NOs. 2096 and 2147-2158.

[0313] In some embodiments, oligonucleotides useful for targeting DMDs (e.g., for exon skipping) are 18–35 nucleotides in length and include a region complementary to a target sequence containing at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as represented by one of SEQ ID NOs. 2096 and 2147–2158. In some embodiments, oligonucleotides useful for targeting DMDs (e.g., for exon skipping) are 20–30 (e.g., 20, 25, 30) nucleotides in length and include a region complementary to a target sequence containing at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as represented by one of SEQ ID NOs. 2096 and 2147–2158. In some embodiments, an oligonucleotide useful for targeting DMD (e.g., for exon skipping) is 20 nucleotides long and includes a region complementary to a target sequence containing at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as represented by any one of SEQ ID NOs. 2096 and 2147-2158. In some embodiments, an oligonucleotide useful for targeting DMD (e.g., for exon skipping) is 30 nucleotides long and includes a region complementary to a target sequence containing at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as represented by any one of SEQ ID NOs. 2096 and 2147-2158.

[0314] In some embodiments, the oligonucleotide comprises a region complementary to a target sequence containing one or more complete or partial ESEs of DMD exon 50. In some embodiments, the oligonucleotide comprises a region complementary to a target sequence containing at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE of DMD exon 50. In some embodiments, the oligonucleotide comprises a region complementary to a target sequence containing one or more complete or partial ESEs as represented by SEQ ID NOs. 2096 and 2160-2177. In some embodiments, the oligonucleotide comprises a region complementary to a target sequence containing at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as represented by any one of SEQ ID NOs. 2096 and 2160-2177.

[0315] In some embodiments, the oligonucleotide comprises a region complementary to a target sequence containing at least six nucleotides (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) of one or more ESEs (e.g., two, three, four, or more adjacent ESEs) of DMD exon 50. In some embodiments, the oligonucleotide comprises a region complementary to a target sequence containing at least six nucleotides (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) of one or more ESEs (e.g., two, three, four, or more adjacent ESEs) as represented by SEQ ID NOs. 2096 and 2160-2177.

[0316] In some embodiments, oligonucleotides useful for targeting DMDs (e.g., for exon skipping) are 18 to 35 nucleotides in length and include a region complementary to a target sequence containing at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as represented by any one of SEQ ID NOs. 2096 and 2160-2177. In some embodiments, oligonucleotides useful for targeting DMDs (e.g., for exon skipping) are 20 to 30 (e.g., 20, 25, 30) nucleotides in length and include a region complementary to a target sequence containing at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as represented by any one of SEQ ID NOs. 2096 and 2160-2177. In some embodiments, an oligonucleotide useful for targeting DMD (e.g., for exon skipping) is 20 nucleotides long and includes a region complementary to a target sequence containing at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as represented by any one of SEQ ID NOs. 2096 and 2160-2177. In some embodiments, an oligonucleotide useful for targeting DMD (e.g., for exon skipping) is 30 nucleotides long and includes a region complementary to a target sequence containing at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as represented by any one of SEQ ID NOs. 2096 and 2160-2177.

[0317] In some embodiments, the oligonucleotide comprises a region complementary to a target sequence containing one or more complete or partial ESEs of DMD exon 51. In some embodiments, the oligonucleotide comprises a region complementary to a target sequence containing at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE of DMD exon 51. In some embodiments, the oligonucleotide comprises a region complementary to a target sequence containing one or more complete or partial ESEs as defined in SEQ ID NOs. 402-436. In some embodiments, the oligonucleotide comprises a region complementary to a target sequence containing at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as represented by any one of SEQ ID NOs. 402-436. In some embodiments, the oligonucleotide comprises a region complementary to a target sequence containing at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as represented by SEQ ID NOs. 419.

[0318] In some embodiments, the oligonucleotide comprises a region complementary to a target sequence containing at least six nucleotides (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) of one or more ESEs (e.g., two, three, four, or more adjacent ESEs) of DMD exon 51. In some embodiments, the oligonucleotide comprises a region complementary to a target sequence containing at least six nucleotides (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) of one or more ESEs (e.g., two, three, four, or more adjacent ESEs) as represented by SEQ ID NOs. 402-436. In some embodiments, the oligonucleotide comprises a region complementary to the target sequence, which includes at least six (e.g., 6, 7, 8, 9, 10, 11, 12, 13, or 14) nucleotides of the ESE as represented by SEQ ID NOs. 418 and SEQ ID NOs. 419.

[0319] In some embodiments, oligonucleotides useful for targeting DMDs (e.g., for exon skipping) are 18 to 35 nucleotides in length and include a region complementary to a target sequence containing at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as represented by any one of SEQ ID NOs. 402 to 436. In some embodiments, oligonucleotides useful for targeting DMDs (e.g., for exon skipping) are 20 to 30 (e.g., 20, 25, 30) nucleotides in length and include a region complementary to a target sequence containing at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as represented by any one of SEQ ID NOs. 402 to 436. In some embodiments, an oligonucleotide useful for targeting DMD (e.g., for exon skipping) is 20 nucleotides long and includes a region complementary to a target sequence containing at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as represented by any one of SEQ ID NOs.402-436. In some embodiments, an oligonucleotide useful for targeting DMD (e.g., for exon skipping) is 30 nucleotides long and includes a region complementary to a target sequence containing at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as represented by any one of SEQ ID NOs.402-436.

[0320] In some embodiments, oligonucleotides useful for targeting DMDs (e.g., for exon skipping) are 20 to 30 nucleotides long (e.g., 20, 25, 30) and include a region complementary to a target sequence containing at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as represented by SEQ ID NO: 419. In some embodiments, oligonucleotides useful for targeting DMDs (e.g., for exon skipping) are 30 nucleotides long and include a region complementary to a target sequence containing at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as represented by SEQ ID NO: 419.

[0321] In some embodiments, the oligonucleotide is 20 to 30 nucleotides long (e.g., 20, 25, 30) and comprises a region complementary to the target sequence containing at least 6 (e.g., 6, 7, 8, 9, 10, 11, 12, 13, or 14) nucleotides of the ESE as represented by SEQ ID NOs. 418 and SEQ ID NOs. 419. In some embodiments, the oligonucleotide is 30 nucleotides long and comprises a region complementary to the target sequence containing at least 6 (e.g., 6, 7, 8, 9, 10, 11, 12, 13, or 14) nucleotides of the ESE as represented by SEQ ID NOs. 418 and SEQ ID NOs. 419.

[0322] Non-limiting examples of oligonucleotides useful for DMD exon 51 skipping and their target sequences are provided in SEQ ID NOs: 437-1241 and SEQ ID NOs: 1242-2046, respectively. In some embodiments, the oligonucleotide is 20-30 nucleotides in length and includes a region complementary to a target sequence containing at least 20 consecutive nucleotides from any one of SEQ ID NOs: 1242-2046. In some embodiments, the oligonucleotide is 20-30 nucleotides in length and contains at least 20 consecutive nucleotides from any one of SEQ ID NOs: 437-1241. In some embodiments, the oligonucleotide contains any one nucleotide sequence from SEQ ID NOs: 437-1241. In some embodiments, the oligonucleotide is at least 30 nucleotides long (e.g., 30, 31, 32, 33, 34, or 35) and contains any one nucleotide sequence from SEQ ID NOs: 437-1241.

[0323] In some embodiments, the oligonucleotide is 20 to 30 nucleotides in length and includes a region complementary to a target sequence containing at least 20 consecutive nucleotides from any one of the sequence numbers 1548, 1550, 1551, 1552, 1555, 1558, 1559, 1562, 1565, 1569, 1577, 1583, 1589, 1595, 1600, 1606, 1610, 1614, 1621, 1626, 1629, 1632, 1637, 1640, 1643, 1646, 1650, 1655, 1658, and 1662. In some embodiments, the oligonucleotide is 20-30 in length and contains 20 consecutive nucleotides, one of the following sequence numbers: 743, 745, 746, 747, 750, 753, 754, 757, 760, 764, 772, 778, 784, 790, 795, 801, 805, 809, 816, 821, 824, 827, 832, 835, 838, 841, 845, 850, 853, and 857. In some embodiments, the oligonucleotide comprises one of the following nucleic acid base sequences: SEQ ID NOs: 743, 745, 746, 747, 750, 753, 754, 757, 760, 764, 772, 778, 784, 790, 795, 801, 805, 809, 816, 821, 824, 827, 832, 835, 838, 841, 845, 850, 853, and 857. In some embodiments, the oligonucleotide is 30 nucleotides in length and contains one of the following nucleic acid sequences: SEQ ID NOs: 743, 745, 746, 747, 750, 753, 754, 757, 760, 764, 772, 778, 784, 790, 795, 801, 805, 809, 816, 821, 824, 827, 832, 835, 838, 841, 845, 850, 853, and 857.

[0324] In some embodiments, the oligonucleotide comprises a region complementary to a target sequence containing one or more complete or partial ESEs of DMD exon 52. In some embodiments, the oligonucleotide comprises a region complementary to a target sequence containing at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE of DMD exon 52. In some embodiments, the oligonucleotide comprises a region complementary to a target sequence containing one or more complete or partial ESEs as represented by SEQ ID NOs. 432 and 2178-2192. In some embodiments, the oligonucleotide comprises a region complementary to a target sequence containing at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as represented by any one of SEQ ID NOs. 432 and 2178-2192.

[0325] In some embodiments, the oligonucleotide includes a region complementary to a target sequence containing at least six nucleotides (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) of one or more ESEs (e.g., two, three, four, or more adjacent ESEs) of DMD exon 52. In some embodiments, the oligonucleotide includes a region complementary to a target sequence containing at least six nucleotides (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) of one or more ESEs (e.g., two, three, four, or more adjacent ESEs) as represented by SEQ ID NOs. 432 and 2178-2192.

[0326] In some embodiments, oligonucleotides useful for targeting DMDs (e.g., for exon skipping) are 18–35 nucleotides in length and include a region complementary to a target sequence containing at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as represented by either SEQ ID NOs. 432 and 2178–2192. In some embodiments, oligonucleotides useful for targeting DMDs (e.g., for exon skipping) are 20–30 (e.g., 20, 25, 30) nucleotides in length and include a region complementary to a target sequence containing at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as represented by either SEQ ID NOs. 432 and 2178–2192. In some embodiments, an oligonucleotide useful for targeting DMD (e.g., for exon skipping) is 20 nucleotides long and includes a region complementary to a target sequence containing at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as represented by one of SEQ ID NOs. 432 and 2178-2192. In some embodiments, an oligonucleotide useful for targeting DMD (e.g., for exon skipping) is 30 nucleotides long and includes a region complementary to a target sequence containing at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as represented by one of SEQ ID NOs. 432 and 2178-2192.

[0327] In some embodiments, the oligonucleotide comprises a region complementary to a target sequence containing one or more complete or partial ESEs of DMD exon 53. In some embodiments, the oligonucleotide comprises a region complementary to a target sequence containing at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE of DMD exon 53. In some embodiments, the oligonucleotide comprises a region complementary to a target sequence containing one or more complete or partial ESEs as represented by SEQ ID NOs: 416, 430, 431, 2108, 2114, 2127, and 2193-2213. In some embodiments, the oligonucleotide comprises a region complementary to a target sequence containing at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as represented by any one of SEQ ID NOs: 416, 430, 431, 2108, 2114, 2127, and 2193-2213.

[0328] In some embodiments, the oligonucleotide includes a region complementary to a target sequence containing at least six nucleotides (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) of one or more ESEs (e.g., two, three, four, or more adjacent ESEs) of DMD exon 53. In some embodiments, the oligonucleotide includes a region complementary to a target sequence containing at least six nucleotides (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) of one or more ESEs (e.g., two, three, four, or more adjacent ESEs) as described in SEQ ID NOs. 416, 430, 431, 2108, 2114, 2127, and 2193-2213.

[0329] In some embodiments, oligonucleotides useful for targeting DMDs (e.g., for exon skipping) are 18–35 nucleotides in length and include a region complementary to a target sequence containing at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as represented by one of SEQ ID NOs. 416, 430, 431, 2108, 2114, 2127, and 2193–2213. In some embodiments, oligonucleotides useful for targeting DMDs (e.g., for exon skipping) are 20–30 (e.g., 20, 25, 30) nucleotides in length and include a region complementary to a target sequence containing at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as represented by one of SEQ ID NOs. 416, 430, 431, 2108, 2114, 2127, and 2193–2213. In some embodiments, an oligonucleotide useful for targeting DMD (e.g., for exon skipping) is 20 nucleotides long and includes a region complementary to a target sequence containing at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as represented by one of SEQ ID NOs. 416, 430, 431, 2108, 2114, 2127, and 2193-2213. In some embodiments, an oligonucleotide useful for targeting DMD (e.g., for exon skipping) is 30 nucleotides long and includes a region complementary to a target sequence containing at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as represented by one of SEQ ID NOs. 416, 430, 431, 2108, 2114, 2127, and 2193-2213.

[0330] In some embodiments, the oligonucleotide comprises a region complementary to a target sequence containing one or more complete or partial ESEs of DMD exon 55. In some embodiments, the oligonucleotide comprises a region complementary to a target sequence containing at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE of DMD exon 55. In some embodiments, the oligonucleotide comprises a region complementary to a target sequence containing one or more complete or partial ESEs as represented by SEQ ID NOs. 2097, 2102, 2103, 2116, 2147, 2199, and 2214-2238. In some embodiments, the oligonucleotide comprises a region complementary to a target sequence containing at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as represented by any one of SEQ ID NOs. 2097, 2102, 2103, 2116, 2147, 2199, and 2214-2238.

[0331] In some embodiments, the oligonucleotide includes a region complementary to the target sequence containing at least six nucleotides (e.g., six, seven, eight, nine, ten, eleven, twelve, thirteen, four, or more adjacent ESEs) of one or more ESEs (e.g., two, three, four, or more adjacent ESEs) of DMD exon 55. In some embodiments, the oligonucleotide includes a region complementary to the target sequence containing at least six nucleotides (e.g., six, seven, eight, nine, ten, eleven, twelve, thirteen, fifteen, fifteen, six, seven, eight, nine, fifteen, six, seven, eight, nine, fifteen, six, six, three, four, or more adjacent ESEs) of one or more ESEs (e.g., two, three, four, or more adjacent ESEs) as represented by sequence numbers 2097, 2102, 2103, 2116, 2147, 2199, and 2214-2238.

[0332] In some embodiments, oligonucleotides useful for targeting DMDs (e.g., for exon skipping) are 18–35 nucleotides in length and include a region complementary to the target sequence containing at least 4 consecutive nucleotides (e.g., 4, 5, 6, 7, or 8 ESEs as represented by any one of SEQ ID NOs. 2097, 2102, 2103, 2116, 2147, 2199, and 2214–2238). In some embodiments, oligonucleotides useful for targeting DMDs (e.g., for exon skipping) are 20–30 nucleotides in length (e.g., 20, 25, 30) and include a region complementary to the target sequence containing at least 4 consecutive nucleotides (e.g., 4, 5, 6, 7, or 8 ESEs as represented by any one of SEQ ID NOs. 2097, 2102, 2103, 2116, 2147, 2199, and 2214–2238). In some embodiments, an oligonucleotide useful for targeting DMD (e.g., for exon skipping) is a region complementary to a target sequence having a length of 20 nucleotides and containing at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as represented by one of sequence numbers 2097, 2102, 2103, 2116, 2147, 2199, and 2214-2238. In some embodiments, an oligonucleotide useful for targeting DMD (e.g., for exon skipping) is a region complementary to a target sequence having a length of 30 nucleotides and containing at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE as represented by one of sequence numbers 2097, 2102, 2103, 2116, 2147, 2199, and 2214-2238.

[0333] In some embodiments, one of the oligonucleotides useful for targeting DMD (for example, for exon skipping) is a phosphorodiamidate morpholino oligomer (PMO).

[0334] Additional examples of oligonucleotides targeting DMD (e.g., for exon skipping) include U.S. Patent Application Publication 2013-072541, published March 21, 2013, entitled "ADENO-ASSOCIATED VIRAL VECTOR FOR EXON SKIPPING IN A GENE ENCODING A DISPENSIBLE-DOMAIN PROTEIN"; U.S. Patent Application Publication 2015-191725, published July 9, 2015, entitled "OLIGONUCLEOTIDE FOR THE TREATMENT OF MUSCULAR DYSTROPHY PATIENTS"; U.S. Patent Application Publication 2015-196670, published July 16, 2015, entitled "COMPOSITIONS AND METHODS FOR DUCHENNE MUSCULAR DYSTROPHY GENE THERAPY"; and "GENOME EDITING WITH SPLIT CAS9 EXPRESSED FROM U.S. Patent Application Publication 2017-349905, published December 7, 2017, titled "TWO VECTORS"; U.S. Patent Application Publication 2018-028554, published February 1, 2018, titled "OLIGOMERS HAVING BICYCLIC SCAFFOLD MOEITIES"; U.S. Patent Application Publication 2018-171333, published June 21, 2018, titled "ANTISENSE MOLECULES AND METHODS FOR TREATING PATHOLOGIES"; U.S. Patent Application Publication 2018-179538, published June 28, 2018, titled "ANTISENSE NUCLEIC ACIDS"; "MODIFICATION OF THE DYSTROPHIN GENE AND USES U.S. Patent Application Publication 2018-265859, published on September 20, 2018, titled "THEREOF"; U.S. Patent Application Publication 2018-369400, published on December 27, 2018, titled "NUCLEIC ACID-POLYPEPTIDE COMPOSITIONS AND METHODS OF INDUCING EXON SKIPPING";U.S. Patent Application Publication 2019-000986, published January 3, 2019, titled "NUCLEIC ACID-POLYPEPTIDE COMPOSITIONS AND METHODS OF INDUCING EXON SKIPPING"; U.S. Patent Application Publication 2019-00898, published January 10, 2019, titled "OLIGONUCLEOTIDE COMPOSITIONS AND METHODS THEREOF"; U.S. Patent Application Publication 2019-112604, published April 18, 2019, titled "METHODS AND MEANS FOR EFFICIENT SKIPPING OF EXON 45 IN DUCHENNE MUSCULAR DYSTROPHY PRE-MRNA"; "METHODS AND MEANS FOR EFFICIENT SKIPPING OF EXON 45 IN DUCHENNE MUSCULAR DYSTROPHY U.S. Patent Application Publication 2019-119679, published April 25, 2019, titled "PRE-MRNA"; U.S. Patent Application Publication 2019-127733, published May 2, 2019, titled "OLIGONUCLEOTIDE COMPOSITIONS AND METHODS THEREOF"; U.S. Patent Application Publication 2019-151476, published May 23, 2019, titled "THERAPEUTIC APPLICATIONS OF CPF1-BASED GENOME EDITING"; U.S. Patent Application Publication 2019-177723, published June 13, 2019, titled "COMPOSITIONS AND METHODS FOR TREATING DUCHENNE MUSCULAR DYSTROPHY AND RELATED DISORDERS"; "METHODS AND MEANS FOR EFFICIENT SKIPPING OF EXON U.S. Patent Application Publication 2019-177725, published on June 13, 2019, entitled "45 IN DUCHENNE MUSCULAR DYSTROPHY PRE-MRNA";U.S. Patent Application Publication 2019-209604, published July 11, 2019, titled "OLIGONUCLEOTIDES, COMPOSITIONS AND METHODS THEREOF"; U.S. Patent Application Publication 2019-249173, published August 15, 2019, titled "METHODS AND COMPOSITIONS OF BIOLOGICALLY ACTIVE AGENTS"; U.S. Patent Application Publication 2019-270994, published September 5, 2019, titled "ANTISENSE MOLECULES AND METHODS FOR TREATING PATHOLOGIES"; U.S. Patent Application Publication 2019-284556, published September 19, 2019, titled "MULTIPLE EXON SKIPPING COMPOSITIONS FOR DMD"; "ANTISENSE OLIGONUCLEOTIDES FOR INDUCING EXON SKIPPING U.S. Patent Application Publication 2019-323010, published October 24, 2019, titled "AND METHODS OF USE THEREOF"; U.S. Patent Application Publication 2019-330626, published January 31, 2019, titled "COMPOUNDS AND METHODS FOR USE IN DYSTROPHIN TRANSCRIPT"; U.S. Patent Application Publication 2019-338311, published November 7, 2019, titled "OPTIMIZED STRATEGY FOR EXON SKIPPING MODIFICATIONS USING CRISPR / CAS9 WITH TRIPLE GUIDE SEQUENCES"; U.S. Patent Application Publication 2019-359982, published November 28, 2019, titled "COMPOSITIONS FOR TREATING MUSCULAR DYSTROPHY"; "DMD REPORTER MODELS U.S. Patent Application Publication 2019-364862, published on December 5, 2019, entitled "CONTAINING HUMANIZED DUCHENNE MUSCULAR DYSTROPHY MUTATIONS";U.S. Patent Application Publication 2019-390197, published December 26, 2019, titled "OLIGONUCLEOTIDE COMPOSITIONS AND METHODS THEREOF"; U.S. Patent Application Publication 2020-040337, published February 6, 2020, titled "COMPOSITIONS FOR TREATING MUSCULAR DYSTROPHY"; U.S. Patent No. 10,287,586, registered May 14, 2019, titled "ANTISENSE MOLECULES AND METHODS FOR TREATING PATHOLOGIES"; U.S. Patent No. 10,337,003, registered July 2, 2019, titled "COMPOSITIONS FOR TREATING MUSCULAR DYSTROPHY"; "COMPOSITIONS FOR TREATING MUSCULAR U.S. Patent No. 10,364,431, registered on July 30, 2019, titled "DYSTROPHY"; U.S. Patent No. 10,450,568, registered on October 22, 2019, titled "OLIGONUCLEOTIDE COMPOSITIONS AND METHODS THEREOF"; U.S. Patent No. 10,487,106, registered on November 26, 2019, titled "ANTISENSE NUCLEIC ACIDS"; U.S. Patent No. 10,533,171, registered on January 14, 2020, titled "OLIGONUCLEOTIDE COMPRISING AN INOSINE FOR TREATING DMD"; U.S. Patent No. 10,704,060, registered on July 7, 2020, titled "RNA-GUIDED GENE EDITING AND GENE REGULATION"; "EFFECTIVE GENE THERAPY TOOLS FOR U.S. patent number 10,752,898, registered on August 25, 2020, titled "DYSTROPHIN EXON 53 SKIPPING";U.S. Patent No. 10,876,114, registered December 29, 2020, titled "METHODS AND MEANS FOR EFFICIENT SKIPPING OF AT LEAST ONE OF THE FOLLOWING EXONS OF THE HUMAN DUCHENNE MUSCULAR DYSTROPHY GENE: 43, 46, 50-53"; U.S. Patent No. 6,100,099, registered August 8, 2000, titled "TEST STRIP HAVING A DIAGONAL ARRAY OF CAPTURE SPOTS"; U.S. Patent No. 6,210,898, registered April 3, 2001, titled "METHOD OF PERFORMING IMMUNOCHROMATOGRAPHY"; "INDUCTION OF EXON SKIPPING IN EUKARYOTIC U.S. Patent No. 7,973,015, registered on July 5, 2011, titled "CELLS"; U.S. Patent No. 8,039,608, registered on October 18, 2011, titled "BIOINFORMATICALLY DETECTABLE GROUP OF NOVEL REGULATORY GENES AND USES THEREOF"; U.S. Patent No. 8,361,979, registered on January 29, 2013, titled "MEANS AND METHOD FOR INDUCING EXON-SKIPPING"; U.S. Patent No. 8,802,437, registered on August 12, 2014, titled "MEGANUCLEASE REAGENTS OF USES THEREOF FOR TREATING GENETIC DISEASES CAUSED BY FRAME SHIFT / NON SENSE MUTATIONS"; "MULTIPLE EXON SKIPPING COMPOSITIONS FOR U.S. Patent No. 8,865,883, registered on October 21, 2014, titled "DMD"; U.S. Patent No. 9,657,049, registered on May 23, 2017, titled "ENA NUCLEIC ACID PHARMACEUTICALS CAPABLE OF MODIFYING SPLICING OF MRNA PRECURSORS";U.S. Patent No. 9,657,050, registered on May 23, 2017, titled "ENA NUCLEIC ACID PHARMACEUTICALS CAPABLE OF MODIFYING SPLICING OF MRNA PRECURSORS"; U.S. Patent No. 9,988,629, registered on June 5, 2018, titled "ANTISENSE NUCLEIC ACIDS"; International Patent Publication WO2011 / 078797 A2, published on June 30, 2011, titled "ANTISENSE OLIGONUCLEOTIDES AND USES THREREOF"; International Patent Publication WO2011 / 154427 A1, published on December 15, 2011, titled "MODIFIED SNRNAS FOR USE IN THERAPY"; "PRE-MRNA SPLICE SWITCHING OR MODULATING OLIGONUCLEOTIDES COMPRISING International patent publications WO2018 / 007475 A1, published on January 11, 2018, titled "Bicycle Scaffold Moieties, With Improved Characteristics for the Treatment of Genetic Disorders"; International patent publication WO2018 / 014042 A1, published on January 18, 2018, titled "Compounds and Methods for Modulation of Dystrophy Transcrib"; International patent publication WO2018 / 017754 A1, published on January 25, 2018, titled "Therapeutic Applications of CPF1-Based Genome Editing"; and "DMD Reporter Models Containing Humanized Duschene Muscular Dystrophy International patent publication WO2018 / 107003 A1, titled "MUTATIONS," published on June 14, 2018;International patent publications include: WO2018 / 129296 A1, published on July 12, 2018, titled "OPTIMIZED STRATEGY FOR EXON SKIPPING MODIFICATIONS USING CRISPR / CAS9 WITH TRIPLE GUIDE SEQUENCES"; WO2019 / 014772 A1, published on January 24, 2019, titled "ANTISENSE OLIGONUCLEOTIDES THAT BIND TO EXON 51 OF HUMAN DYSTROPHIN PRE-MRNA"; WO2019 / 059973 A1, published on March 28, 2019, titled "EXON SKIPPING OLIGOMER CONJUGATES FOR MUSCULAR DYSTROPHY"; and "NUCLEIC ACID-POLYPEPTIDE COMPOSITIONS AND METHODS OF INDUCING EXON International patent publications WO2019 / 060775 A1, published on March 28, 2019, titled "SKIPPING"; WO2019 / 067975 A1, published on April 4, 2019, titled "COMBINATION THERAPIES FOR TREATING MUSCULAR DYSTROPHY"; WO2019 / 092507 A2, published on May 16, 2019, titled "CRISPR / CAS SYSTEMS FOR TREATMENT OF DMD"; WO2019 / 136216 A1, published on July 11, 2019, titled "THERAPEUTIC CRISPR / CAS9 COMPOSITIONS AND METHODS OF USE"; "COMPOSITIONS AND METHODS FOR CORRECTING DYSTROPHIN MUTATIONS IN HUMAN International patent publication WO2019 / 152609 A1, published on August 8, 2019, titled "CARDIOMYOCYTES"; International patent publication WO2019 / 200185 A1, published on October 17, 2019, titled "OLIGONUCLEOTIDE COMPOSITIONS AND METHODS OF USE THEREOF";International patent publications WO2019 / 215333 A1, published November 14, 2019, titled "OLIGONUCLEOTIDES CONJUGATES COMPRISING 7'-5'-ALPHA-ANOMERIC-BICYCLIC SUGAR NUCLEOSIDES"; International patent publication WO 2019 / 241385 A2, published December 19, 2019, titled "EXON SKIPPING OLIGOMERS FOR MUSCULAR DYSTROPY"; International patent publication WO2019 / 241385 A2, published December 26, 2019, titled "CORRECTION OF DYSTROPHIN EXON 43, EXON 45, OR EXON 52 DELETIONS IN DUCHENNE MUSCULAR DYSTROPHY"; and "MUSCLE" published February 6, 2020. International patent publications WO2020 / 028832 A1 titled "TARGETING COMPLEXES AND USES THEREOF FOR TREATING DYSTROPHINOPATHIES"; International patent publication WO2018 / 091544 A1 published on May 24, 2018, titled "SUBSTANCES FOR TARGETING VARIOUS SELECTED ORGANS OR TISSUES"; International patent publication WO2018 / 098480 A1 published on May 31, 2018, titled "PREVENTION OF MUSCULAR DYSTROPHY BY CRISPR / CPF1-MEDIATED GENE EDITING"; International patent publication WO1993 / 020227 A1 published on October 14, 1993, titled "METHOD OF MULTIPLEX LIGASE CHAIN ​​REACTION"; "ANTISENSE NUCLEIC International patent publication WO2013 / 100190 A1, published on July 4, 2013, titled "ACID"; International patent publication WO2013 / 163628 A2, published on October 31, 2013, titled "GENETIC CORRECTION OF MUTATED GENES";Provided in International Patent Publication WO2007 / 135105 A1, published November 29, 2007, entitled "MEANS AND METHOD FOR INDUCING EXON-SKIPPING"; International Patent Publication WO2011 / 150408 A2, published December 1, 2011, entitled "OLIGONUCLEOTIDE ANALOGUES HAVING MODIFIED INTERSUBUNIT LINKAGES AND / OR TERMINAL GROUPS"; and International Patent Publication WO2012 / 029986 A1, published March 8, 2012, entitled "ANTISENSE NUCLEIC ACID"; the contents of each are incorporated herein, in whole.

[0335] Examples of oligonucleotides for promoting DMD gene editing include: International Patent Publication WO2018053632A1, published March 29, 2018, titled "METHODS OF MODIFYING THE DYSTROPHIN GENE AND RESTORING DYSTROPHIN EXPRESSION AND USES THEREOF"; International Patent Publication WO2017049407A1, published March 30, 2017, titled "MODIFICATION OF THE DYSTROPHIN GENE AND USES THEREOF"; International Patent Publication WO2016161380A1, published October 6, 2016, titled "CRISPR / CAS-RELATED METHODS AND COMPOSITIONS FOR TREATING DUCHENNE MUSCULAR DYSTROPHY AND BECKER MUSCULAR DYSTROPHY"; and "THERAPEUTIC TARGETS FOR THE CORRECTION OF International patent publications include: WO2017095967, published on June 8, 2017, titled "THE HUMAN DYSTROPHIN GENE BY GENE EDITING AND METHODS OF USE"; WO2017072590A1, published on May 4, 2017, titled "MATERIALS AND METHODS FOR TREATMENT OF DUCHENNE MUSCULAR DYSTROPHY"; WO2018098480A1, published on May 31, 2018, titled "PREVENTION OF MUSCULAR DYSTROPHY BY CRISPR / CPF1-MEDIATED GENE EDITING"; and "RNA-Guided Systems for In Vivo Gene U.S. Patent Application Publication US20170266320A1, published September 21, 2017, titled "Editing"; International Patent Publication WO2016025469A1, published February 18, 2016, titled "PREVENTION OF MUSCULAR DYSTROPHY BY CRISPR / CAS9-MEDIATED GENE EDITING";This specification incorporates U.S. Patent Application Publication 2016 / 0201089, published on July 14, 2016, entitled "RNA-GUIDED GENE EDITING AND GENE REGULATION," and U.S. Patent Application Publication 2013 / 0145487, published on June 6, 2013, entitled "MEGANUCLEASE VARIANTS CLEAVING A DNA TARGET SEQUENCE FROM THE DYSTROPHN GENE AND USES THEREOF," the entire contents of each of these are incorporated herein. In some embodiments, oligonucleotides may have regions complementary to DMD gene sequences of multiple species, selected, for example, from human, mouse, and non-human species.

[0336] In some embodiments, the oligonucleotide may have a region complementary to a mutant DMD allele, for example, a DMD allele with at least one mutation in any of exons 1-79 of human DMD, which leads to frameshift and improper RNA splicing / processing.

[0337] In some embodiments, oligonucleotides may target lncRNA or mRNA, for example, for degradation. In some embodiments, oligonucleotides may target nucleic acids encoding proteins involved in the mismatch repair pathway, for example, MSH2, MutL-alpha, MutS-beta, MutL-alpha, for degradation. Non-limiting examples of proteins involved in the mismatch repair pathway (mRNA encoding such proteins may be targeted by the oligonucleotides described herein) are described in Iyer, R et al., "DNA triplet repeat expansion and mismatch repair," Annu Rev Biochem. 2015;84:199-226; and Schmidt MH and Pearson CE, "Disease-associated repeat instability and mismatch repair," DNA Repair (Amst). 2016 Feb;38:117-26.

[0338] In some embodiments, any one of the oligonucleotides may be in salt form, such as a sodium, potassium, or magnesium salt.

[0339] In some embodiments, a 5' or 3' nucleoside (e.g., a terminal nucleoside) of any of the oligonucleotides 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 exists between the spacer and the 5' or 3' nucleoside of the oligonucleotide. In some embodiments, a 5' or 3' nucleoside (e.g., a terminal nucleoside) of any of the oligonucleotides described herein is conjugated to a spacer which is a substituted or unsubstituted aliphatic, substituted or unsubstituted heteroaliphatic, substituted or unsubstituted carbocyclylene, substituted or unsubstituted heterocyclylene, substituted or unsubstituted arylene, substituted or unsubstituted heteroarylene, -O-, -N(R A )-, -S-, -C(=O)-, -C(=O)O-, -C(=O)NR A -, -NR A C(=O)-, -NR A C(=O)R A -, -C(=O)R A -, -NR A C(=O)O-, -NR A C(=O)N(R A )-, -OC(=O)-, -OC(=O)O-, -OC(=O)N(R A )-,-S(O)2NR A -, -NR A S(O)2-, or a combination thereof; each R A In some embodiments, the spacer is a substituted or unsubstituted alkylene, a substituted or unsubstituted heterocyclene, a substituted or unsubstituted heteroarylene, -O-, -N(R A )-, or -C(=O)N(R A )2, or a combination thereof.

[0340] In some embodiments, any one of the 5' or 3' nucleosides of the oligonucleotides described herein is of the formula -NH2-(CH2) n - is conjugated to a compound of the formula NH2-(CH2) n - exists between the compound and the 5' or 3' nucleoside of the oligonucleotide. In some embodiments, the compound of formula NH2-(CH2)6- is conjugated to the oligonucleotide by a reaction between 6-amino-1-hexanol (NH2-(CH2)6-OH) and the 5' phosphate of the oligonucleotide.

[0341] In some embodiments, oligonucleotides are conjugated to targeted agents, such as muscle targeting agents like anti-TfR antibodies, via, for example, an amine group.

[0342] a. Oligonucleotide size / arrangement Oligonucleotides may be of various different lengths, for example, depending on the format. In some embodiments, oligonucleotides are 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 nucleotides or longer. In some embodiments, oligonucleotides are 8-50 nucleotides, 8-40 nucleotides, 8-30 nucleotides, 10-15 nucleotides, 10-20 nucleotides, 15-25 nucleotides, 21-23 nucleotides, and so on.

[0343] In some embodiments, for the purposes of this disclosure, a complementary nucleic acid sequence of an oligonucleotide is specifically hybridizable to or specific to a target nucleic acid when, under conditions where the binding of the sequence to a target molecule (e.g., mRNA) interferes with the function of the target (e.g., mRNA) and causes a change in activity (e.g., inhibition of translation, alternative splicing, or exon skipping) or loss of expression (e.g., degradation of the target mRNA), and where avoidance of nonspecific binding is desired, such as under physiological conditions in the case of in vivo assays or therapeutic treatments and in vitro assays, and under conditions where the assay is performed under favorable stringency conditions. Therefore, in some embodiments, the oligonucleotide may be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to the consecutive nucleotides of the target nucleic acid. In some embodiments, the complementary nucleotide sequence does not need to be specifically hybridizable to the target nucleic acid or 100% complementary to the target sequence that is specific to the target nucleic acid.

[0344] In some embodiments, the oligonucleotide includes a region complementary to the target nucleic acid, having a length in the range of 8–15, 8–30, 8–40, or 10–50, or 5–50, or 5–40 nucleotides. In some embodiments, the region of the o...

Claims

1. A composition comprising an anti-transferrin receptor (TfR) antibody covalently linked to at least one oligonucleotide, The anti-TfR antibody is Fab and comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 101 and a light chain containing the amino acid sequence of SEQ ID NO:

90. The composition wherein the oligonucleotide is 20 to 30 nucleotides in length and complementary to at least 20 consecutive nucleotides of SEQ ID NO: 1550.

2. The composition according to claim 1, wherein each anti-TfR antibody contained in the complex is covalently linked to an average of 1 to 3 oligonucleotides.

3. The composition according to claim 1 or 2, wherein the heavy chain of the anti-TfR antibody contains N-terminal pyroglutamic acid.

4. The dissociation constant (KD) of the binding of the anti-TfR antibody to the transferrin receptor is 10. -11 M-10 -6 A composition according to any one of claims 1 to 3, which is in the range of M.

5. The oligonucleotide contains the sequence of sequence number 343 or 745, The composition according to any one of claims 1 to 4, wherein one or more thymine bases (T) in the oligonucleotide may be uridine bases (U), and one or more U may be T.

6. The composition according to any one of claims 1 to 5, wherein the oligonucleotide comprises the sequence of SEQ ID NO:

745.

7. The composition according to any one of claims 1 to 6, wherein the oligonucleotide has a length of 30 nucleotides.

8. The composition according to any one of claims 1 to 7, wherein the oligonucleotide comprises one or more modified nucleosides.

9. The composition according to any one of claims 1 to 8, wherein the oligonucleotide comprises one or more phosphorodiamidate morpholino.

10. The composition according to any one of claims 1 to 9, wherein the oligonucleotide is a phosphorodiamidate morpholino oligomer (PMO).

11. The composition according to any one of claims 1 to 10, wherein the anti-TfR antibody is covalently linked to the oligonucleotide via a cleavable linker.

12. The composition according to claim 11, wherein the cleavable linker comprises a valine-citrulline sequence.

13. The composite of the composition has the following structure (D): 【Chemistry 1】 Includes, Here, n is 3 and m is 4, where L1 is a substituted or unsubstituted aliphatic, substituted or unsubstituted heteroaliphatic, substituted or unsubstituted carbocyclylene, substituted or unsubstituted heterocyclylene, substituted or unsubstituted arylene, substituted or unsubstituted heteroarylene, -O-, -N(R Ａ ), -, -S-, -C(=O)-, -C(=O)O-, -C(=O)NR Ａ -, -NR Ａ C(=O)-, -NR Ａ C(=O)R Ａ -, -C(=O)R Ａ -, -NR Ａ C(=O)O-, -NR Ａ C(=O)N(R Ａ ), -, -OC(=O)-, -OC(=O)O-, -OC(=O)N(R Ａ ), -, -S(O) 2 NR Ａ -, -NR Ａ S(O) 2 -, or a spacer consisting of a combination thereof, where each R Ａ is hydrogen or a substituted or unsubstituted alkyl, the composition according to any one of claims 1 to 12.

14. L1 has the following structure: 【Chemistry 2】 It contains, where the piperazine moiety binds to the oligonucleotide, L2 has one of the following structures: 【Transformation 3】 The composition according to claim 13, comprising:

15. L2 has the following structure: 【Chemistry 4】 The composition according to claim 14, comprising:

16. The composition according to any one of claims 1 to 15, wherein the oligonucleotide is covalently linked via a lysine residue of the anti-TfR antibody.

17. A composition according to any one of claims 1 to 16 for use in a method for inducing skipping of dystrophin exon 51 in target muscle cells, wherein the method comprises administering the composition to the target.

18. The composition according to claim 17, wherein the subject is a human.

19. The composition according to claim 17, wherein the target is a cynomolgus macaque.

20. The composition according to any one of claims 17 to 19, wherein the subject expresses dystrophin precursor mRNA containing a frameshift mutation.

21. The composition according to any one of claims 17 to 20, wherein the composition is administered intravenously to a subject.

22. The composition according to any one of claims 17 to 21, wherein the target is one for which dystrophin exon 51 skipping is indicated.

23. The composition according to any one of claims 17 to 22, wherein the subject has Duchenne muscular dystrophy (DMD).

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