Muscle-targeting complex and its use for treating myotonic dystrophy

Abstract

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

Description

Technical Field

[0001] Related Applications This application claims priority under 35 U.S.C § 119(e) to U.S. Provisional Application No. 63 / 143,827, filed January 30, 2021, entitled "MUSCLE TARGETING COMPLEXES AND USES THEREOF FOR TREATING MYOTONIC DYSTROPHY"; U.S. Provisional Application No. 63 / 069,075, filed August 23, 2020, entitled "MUSCLE TARGETING COMPLEXES AND USES THEREOF FOR TREATING MYOTONIC DYSTROPHY"; and U.S. Provisional Application No. 63 / 055,749, filed July 23, 2020, entitled "MUSCLE TARGETING COMPLEXES AND USES THEREOF FOR TREATING MYOTONIC DYSTROPHY"; the contents of each of which are hereby incorporated by reference in their entirety.

[0002] Field of the Invention This application relates to targeted complexes for delivering molecular payloads (e.g., oligonucleotides) to cells and their use, particularly for use in treating diseases.

[0003] Reference to a Sequence Listing Submitted as a Text File via EFS-WEB This application contains a Sequence Listing. This was submitted in ASCII format from EFS-Web and is hereby incorporated by reference in its entirety. The ASCII copy created on July 8, 2021, is named D082470038WO00-SEQ-ZJG and is 268,942 bytes in size. <00,00090>

Background Art

[0004] Background of the Invention Myotonic dystrophy (DM) is an autosomal dominant disorder characterized by myotonia, muscle loss or degeneration, muscle function decline, insulin resistance, cardiac arrhythmias, smooth muscle dysfunction, and neurological abnormalities. DM is the most common form of adult-onset muscular dystrophy, with a worldwide incidence of approximately 1 in 8,000 people. Two types of the disease have been reported: myotonic dystrophy type 1 (DM1) and myotonic dystrophy type 2 (DM2). The more common form of the disease, DM1, is caused by an expansion of a CTG trinucleotide repeat in the 3' untranslated region of DMPK on chromosome 19; DM2 is caused by an expansion of a CCTG tetranucleotide repeat in the first intron of ZNF9 on chromosome 3. In DM1 patients, the expansion of CTG trinucleotide repeats, which can contain from about 50 to about 3,000+ total repeats, leads to the production of toxic RNA repeats that can form hairpin structures that bind with high affinity to essential intracellular proteins, such as muscleblind-like proteins, resulting in the phenotypes of protein sequestration and loss of function that are characteristic of the disease. Except for supportive therapies and treatments to address the symptoms of the disease, no effective treatments for DM1 are currently available. SUMMARY OF THE INVENTION

[0005] SUMMARY OF THE INVENTION In several aspects, this disclosure provides complexes that target muscle cells for the purpose of delivering molecular payloads to those cells. In some embodiments, the complexes provided herein are particularly useful for delivering molecular payloads that inhibit the expression or activity of DMPK alleles containing extended disease-associated repeats, for example, in subjects having or suspected of having myotonic dystrophy. Accordingly, in some embodiments, the complexes provided herein include a muscle targeting agent (e.g., a muscle-targeting antibody) that specifically binds to a receptor on the surface of muscle cells for the purpose of delivering the molecular payload to muscle cells. In some embodiments, the complex may be taken up into the cell via receptor-mediated internalization, and the molecular payload may be released into the cell to perform its function. For example, a complex modified to deliver an oligonucleotide may release the oligonucleotide so that the oligonucleotide can inhibit mutant DMPK expression in muscle cells. In some embodiments, the oligonucleotide is released by endosomal cleavage of a covalent linker connecting the oligonucleotide and the muscle targeting agent of the complex.

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

[0007] In some embodiments, the antibody includes: (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 antibody does not inhibit the binding of transferrin to the transferrin receptor. In some embodiments, the antibody is cross-reactive to two or more extracellular epitopes of the transferrin receptor in humans, non-human primate animals, and rodent animals. In some embodiments, the complex is configured to facilitate the internalization of molecular payloads into muscle cells mediated by the transferrin receptor.

[0012] In some embodiments, the molecular payload is an oligonucleotide. In some embodiments, the oligonucleotide comprises at least 15 consecutive nucleotides of sequence numbers 148-383 and 621-638, where one or more thymidine bases (T) in the oligonucleotide may optionally be uridine bases (U), and / or one or more U may optionally be T. In some embodiments, the oligonucleotide comprises a sequence containing one of sequence numbers 159, 162, 172, 174, 180, 182, 188, 190, 195, 196, 201, 203, 212, 215, 218, 222, 248, and 264, where one or more U in the oligonucleotide may optionally be T. In some embodiments, the oligonucleotide comprises a region complementary to at least 15 consecutive nucleotides of one of sequence numbers 384-619.

[0013] In some embodiments, oligonucleotides mediate RNAse H-mediated cleavage of DMPK mRNA transcripts.

[0014] In some embodiments, the oligonucleotide comprises the formula 5'-XYZ-3', where X and Z are flanking regions containing one or more 2'-modified nucleosides selected from the group consisting of 2'-O-methyl, 2'-fluoro, 2'-O-methoxyethyl, and 2',4'-bridged nucleosides, and where Y is a gap region, and each nucleoside in Y is a 2'-deoxyribonucleoside.

[0015] In some embodiments, the oligonucleotide comprises one or more phosphorothioate nucleoside linkages.

[0016] 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.

[0017] In some embodiments, the antibody is ligated to the molecular payload via conjugation to a lysine or cysteine ​​residue of the antibody.

[0018] In some embodiments, reducing expression involves reducing the RNA level of DMPK, optionally the RNA level reduced herein is in the nucleus of a cell, and optionally the cell is a muscle cell. In some embodiments, DMPK is encoded from an allele containing disease-associated repeats.

[0019] Another aspect of this disclosure relates to a method for reducing the expression of DMPK in cells, the method comprising contacting cells with the complex disclosed herein in an amount effective to promote the internalization of the molecular payload in the cells, where optionally the cells are muscle cells.

[0020] Another aspect of this disclosure relates to a method for treating a subject having an extension of disease-associated repeats of a DMPK allele associated with myotonic dystrophy, the method comprising administering an effective amount of the complex disclosed herein to the subject. In some embodiments, the disease-associated repeats comprise repeating units of a CTG trinucleotide sequence. In some embodiments, the complex is administered intravenously to the subject. [Brief explanation of the drawing]

[0021] Simple description of the drawing [Figure 1] Figure 1 shows a non-limiting schematic diagram illustrating the effect of transfecting Hepa1-6 cells with antisense oligonucleotides targeting DMPK (ASO300) on DMPK expression levels compared to vehicle transfection.

[0022] [Figure 2A] Figure 2A shows a non-limiting schematic diagram of the HIL-HPLC trace obtained during purification of a muscle-targeting complex containing an anti-transferrin receptor antibody covalently linked to a DMPK antisense oligonucleotide.

[0023] [Figure 2B] Figure 2B shows an unrestricted image of the SDS-PAGE analysis of the muscle targeting complex.

[0024] [Figure 3] A non-limiting schematic diagram is drawn showing the ability of the muscle-targeted RI7 217 Fab antibody-oligonucleotide (DTX-C-008) containing ASO300 to reduce DMPK expression levels.

[0025] [Figure 4A-B]Figures 4A–4E illustrate a non-limiting schematic diagram showing the ability of the muscle-targeted RI7 217 Fab antibody-oligonucleotide complex (DTX-C-008) containing ASO300 to reduce DMPK expression levels in mouse muscle tissue in vivo, compared to vehicle treatment, treatment with naked ASO300, or treatment with the control untargeted complex (DTX-C-007). (N=3 C57Bl / 6 WT mice) [Figure 4C-D] Figures 4A–4E illustrate a non-limiting schematic diagram showing the ability of the muscle-targeted RI7 217 Fab antibody-oligonucleotide complex (DTX-C-008) containing ASO300 to reduce DMPK expression levels in mouse muscle tissue in vivo, compared to vehicle treatment, treatment with naked ASO300, or treatment with the control untargeted complex (DTX-C-007). (N=3 C57Bl / 6 WT mice) [Figure 4E] Figures 4A–4E illustrate a non-limiting schematic diagram showing the ability of the muscle-targeted RI7 217 Fab antibody-oligonucleotide complex (DTX-C-008) containing ASO300 to reduce DMPK expression levels in mouse muscle tissue in vivo, compared to vehicle treatment, treatment with naked ASO300, or treatment with the control untargeted complex (DTX-C-007). (N=3 C57Bl / 6 WT mice)

[0026] [Figure 5A] Figures 5A-5B illustrate a non-limiting schematic diagram showing the tissue selectivity of the muscle-targeted RI7 217 Fab antibody-oligonucleotide (DTX-C-008) containing ASO300. The muscle-targeted conjugate containing ASO300 (DTX-C-008) did not reduce DMPK expression levels in mouse brain or spleen tissue in vivo compared to vehicle treatment, treatment with naked ASO300, or treatment with the control non-targeted conjugate (DTX-C-007). (N=3 C57Bl / 6 WT mice) [Figure 5B]Figures 5A-5B illustrate a non-limiting schematic diagram showing the tissue selectivity of the muscle-targeted RI7 217 Fab antibody-oligonucleotide (DTX-C-008) containing ASO300. The muscle-targeted conjugate containing ASO300 (DTX-C-008) did not reduce DMPK expression levels in mouse brain or spleen tissue in vivo compared to vehicle treatment, treatment with naked ASO300, or treatment with the control non-targeted conjugate (DTX-C-007). (N=3 C57Bl / 6 WT mice)

[0027] [Figure 6A] Figures 6A–6F illustrate a non-limiting schematic diagram showing the ability of the muscle-targeted RI7 217 Fab antibody-oligonucleotide conjugate (DTX-C-008) containing ASO300 to reduce DMPK expression levels in mouse muscle tissue in vivo, compared to vehicle treatment, treatment with naked ASO300, or treatment with the control untargeted conjugate (DTX-C-007). (N=5 C57Bl / 6 WT mice) [Figure 6B-C] Figures 6A–6F illustrate a non-limiting schematic diagram showing the ability of the muscle-targeted RI7 217 Fab antibody-oligonucleotide conjugate (DTX-C-008) containing ASO300 to reduce DMPK expression levels in mouse muscle tissue in vivo, compared to vehicle treatment, treatment with naked ASO300, or treatment with the control untargeted conjugate (DTX-C-007). (N=5 C57Bl / 6 WT mice) [Figure 6D-E] Figures 6A–6F illustrate a non-limiting schematic diagram showing the ability of the muscle-targeted RI7 217 Fab antibody-oligonucleotide conjugate (DTX-C-008) containing ASO300 to reduce DMPK expression levels in mouse muscle tissue in vivo, compared to vehicle treatment, treatment with naked ASO300, or treatment with the control untargeted conjugate (DTX-C-007). (N=5 C57Bl / 6 WT mice) [Figure 6F]Figures 6A–6F illustrate a non-limiting schematic diagram showing the ability of the muscle-targeted RI7 217 Fab antibody-oligonucleotide complex (DTX-C-008) containing ASO300 to reduce DMPK expression levels in mouse muscle tissue in vivo, compared to vehicle treatment, treatment with naked ASO300, or treatment with a control untargeted complex (DTX-C-007). (N=5 C57Bl / 6 WT mice)

[0028] [Figure 7A] Figures 7A-7L illustrate a non-limiting schematic diagram showing the ability of a muscle-targeting antibody-oligonucleotide complex (DTX-C-012) containing ASO300 covalently linked to an anti-hTfR antibody to reduce DMPK expression levels in cynomolgus monkey muscle tissue in vivo, compared to vehicle treatment (physiological saline) and naked DMPK ASO (ASO300). (N=3 male cynomolgus monkeys) [Figure 7B-C] Figures 7A-7L illustrate a non-limiting schematic diagram showing the ability of a muscle-targeting antibody-oligonucleotide complex (DTX-C-012) containing ASO300 covalently linked to an anti-hTfR antibody to reduce DMPK expression levels in cynomolgus monkey muscle tissue in vivo, compared to vehicle treatment (physiological saline) and naked DMPK ASO (ASO300). (N=3 male cynomolgus monkeys) [Figure 7D-E] Figures 7A-7L illustrate a non-limiting schematic diagram showing the ability of a muscle-targeting antibody-oligonucleotide complex (DTX-C-012) containing ASO300 covalently linked to an anti-hTfR antibody to reduce DMPK expression levels in cynomolgus monkey muscle tissue in vivo, compared to vehicle treatment (physiological saline) and naked DMPK ASO (ASO300). (N=3 male cynomolgus monkeys) [Figure 7F-G]Figures 7A-7L illustrate a non-limiting schematic diagram showing the ability of a muscle-targeting antibody-oligonucleotide complex (DTX-C-012) containing ASO300 covalently linked to an anti-hTfR antibody to reduce DMPK expression levels in cynomolgus monkey muscle tissue in vivo, compared to vehicle treatment (physiological saline) and naked DMPK ASO (ASO300). (N=3 male cynomolgus monkeys) [Figure 7H-I] Figures 7A-7L illustrate a non-limiting schematic diagram showing the ability of a muscle-targeting antibody-oligonucleotide complex (DTX-C-012) containing ASO300 covalently linked to an anti-hTfR antibody to reduce DMPK expression levels in cynomolgus monkey muscle tissue in vivo, compared to vehicle treatment (physiological saline) and naked DMPK ASO (ASO300). (N=3 male cynomolgus monkeys) [Figure 7J-K] Figures 7A-7L illustrate a non-limiting schematic diagram showing the ability of a muscle-targeting antibody-oligonucleotide complex (DTX-C-012) containing ASO300 covalently linked to an anti-hTfR antibody to reduce DMPK expression levels in cynomolgus monkey muscle tissue in vivo, compared to vehicle treatment (physiological saline) and naked DMPK ASO (ASO300). (N=3 male cynomolgus monkeys) [Figure 7L] Figures 7A-7L illustrate a non-limiting schematic diagram showing the ability of a muscle-targeting antibody-oligonucleotide complex (DTX-C-012) containing ASO300 covalently linked to an anti-hTfR antibody to reduce DMPK expression levels in cynomolgus monkey muscle tissue in vivo, compared to vehicle treatment (physiological saline) and naked DMPK ASO (ASO300). (N=3 male cynomolgus monkeys)

[0029] [Figure 8A]Figures 8A-8B illustrate a non-limiting schematic diagram showing the ability of a muscle-targeting antibody-oligonucleotide complex (DTX-C-012) containing ASO300 covalently linked to an anti-hTfR antibody to reduce DMPK expression levels in cynomolgus monkey smooth muscle tissue in vivo, compared to vehicle treatment (physiological saline) and naked DMPK ASO (ASO300). (N=3 male cynomolgus monkeys) [Figure 8B] Figures 8A-8B illustrate a non-limiting schematic diagram showing the ability of a muscle-targeting antibody-oligonucleotide complex (DTX-C-012) containing ASO300 covalently linked to an anti-hTfR antibody to reduce DMPK expression levels in cynomolgus monkey smooth muscle tissue in vivo, compared to vehicle treatment (physiological saline) and naked DMPK ASO (ASO300). (N=3 male cynomolgus monkeys)

[0030] [Figure 9A] Figures 9A–9D illustrate a non-limiting schematic diagram showing the tissue selectivity of a muscle-targeting antibody-oligonucleotide (DTX-C-012) containing ASO300 covalently linked to an anti-hTfR antibody. The muscle-targeting complex containing DMPK-ASO did not reduce DMPK expression levels in the kidney, brain, or spleen tissue of cynomolgus monkeys in vivo compared to vehicle treatment. (N=3 male cynomolgus monkeys) [Figure 9B-C] Figures 9A–9D illustrate a non-limiting schematic diagram showing the tissue selectivity of a muscle-targeting antibody-oligonucleotide (DTX-C-012) containing ASO300 covalently linked to an anti-hTfR antibody. The muscle-targeting complex containing DMPK-ASO did not reduce DMPK expression levels in the kidney, brain, or spleen tissue of cynomolgus monkeys in vivo compared to vehicle treatment. (N=3 male cynomolgus monkeys) [Figure 9D]Figures 9A–9D illustrate a non-limiting schematic diagram showing the tissue selectivity of a muscle-targeting antibody-oligonucleotide (DTX-C-012) containing ASO300 covalently linked to an anti-hTfR antibody. The muscle-targeting complex containing DMPK-ASO did not reduce DMPK expression levels in the kidney, brain, or spleen tissue of cynomolgus monkeys in vivo compared to vehicle treatment. (N=3 male cynomolgus monkeys)

[0031] [Figure 10] Figure 10 shows normalized DMPK mRNA tissue expression levels across several tissue types in cynomolgus monkeys. (N=3 male cynomolgus monkeys)

[0032] [Figure 11] Figures 11A-11B illustrate a non-limiting schematic diagram showing the ability of a muscle-targeted RI7 217 Fab antibody-oligonucleotide conjugate (DTX-C-008) containing ASO300 to reduce DMPK expression levels in mouse muscle tissue in vivo for up to 28 days after DTX-C-008 administration, compared to vehicle treatment (physiological saline) and naked DMPK ASO (ASO300).

[0033] [Figure 12] Figure 12 shows that a single dose of a muscle-targeting conjugate (DTX-C-012) containing ASO300 covalently linked to an anti-hTfR antibody is safe and tolerable in cynomolgus monkeys. (N=3 male cynomolgus monkeys)

[0034] [Figure 13A]Figures 13A-13B illustrate a non-limiting schematic diagram showing the ability of muscle-targeted RI7 217 Fab antibody-oligonucleotide conjugate (DTX-C-008) containing ASO300 to reduce DMPK expression levels in mouse muscle tissue in vivo for up to 12 weeks after DTX-C-008 administration, compared to vehicle treatment (PBS) and control IgG2a Fab antibody-oligonucleotide conjugate (DTX-C-007) and naked DMPK ASO (ASO300). (N=5 C57Bl / 6 WT mice) [Figure 13B] Figures 13A-13B illustrate a non-limiting schematic diagram showing the ability of muscle-targeted RI7 217 Fab antibody-oligonucleotide conjugate (DTX-C-008) containing ASO300 to reduce DMPK expression levels in mouse muscle tissue in vivo for up to 12 weeks after DTX-C-008 administration, compared to vehicle treatment (PBS) and control IgG2a Fab antibody-oligonucleotide conjugate (DTX-C-007) and naked DMPK ASO (ASO300). (N=5 C57Bl / 6 WT mice)

[0035] [Figure 14A] Figures 14A-14B show a non-limiting schematic diagram illustrating the ability of the muscle-targeting RI7 217 Fab antibody-oligonucleotide complex (DTX-C-008) containing ASO300 to target nuclear mutant DMPK RNA in a mouse model. (N=6 mice) [Figure 14B] Figures 14A-14B show a non-limiting schematic diagram illustrating the ability of the muscle-targeting RI7 217 Fab antibody-oligonucleotide complex (DTX-C-008) containing ASO300 to target nuclear mutant DMPK RNA in a mouse model. (N=6 mice)

[0036] [Figure 15]Figures 15A-15B illustrate a non-limiting schematic diagram showing the dose-dependent reduction in actin expression levels and functional grade of myotonia by a muscle-targeted RI7 217 Fab antibody-ASO complex (DTX-actin) containing actin-targeting oligonucleotides. (N=2 HSALR mice)

[0037] [Figure 16A-B] Figures 16A–16C show a non-limiting schematic diagram demonstrating that a muscle-targeted RI7 217 Fab antibody-oligonucleotide complex (DTX-C-008) containing ASO300 can significantly shorten the elongated QTc interval in a mouse model used to verify the functional modification of arrhythmias in a DM1 cardiac model. (N=10 mice) [Figure 16C] Figures 16A–16C show a non-limiting schematic diagram demonstrating that a muscle-targeted RI7 217 Fab antibody-oligonucleotide complex (DTX-C-008) containing ASO300 can significantly shorten the elongated QTc interval in a mouse model used to verify the functional modification of arrhythmias in a DM1 cardiac model. (N=10 mice)

[0038] [Figure 17A] Figures 17A-17B illustrate a non-limiting schematic diagram showing that a muscle-targeting antibody-oligonucleotide complex (DTX-C-012) containing the ASO300 antisense oligonucleotide covalently linked to an anti-hTfR antibody can reduce DMPK expression levels and modify the splicing of the DMPK-specific target gene (Bin1) in human cells from DM1 patients. (N=3) [Figure 17B] Figures 17A-17B illustrate a non-limiting schematic diagram showing that a muscle-targeting antibody-oligonucleotide complex (DTX-C-012) containing the ASO300 antisense oligonucleotide covalently linked to an anti-hTfR antibody can reduce DMPK expression levels and modify the splicing of the DMPK-specific target gene (Bin1) in human cells from DM1 patients. (N=3)

[0039] [Figure 18A-B] Figures 18A–18C illustrate non-limiting schematic diagrams showing the dose-response of selected antisense oligonucleotides in DMPK knockdown in human DM1 myotubes. ASO300 was used as a control. All tested oligonucleotides showed DMPK knockdown activity. Statistical analysis: One-way ANOVA with Tukey's HSD post-hoc test for naked ASO300 treatment; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 18C] Figures 18A–18C illustrate non-limiting schematic diagrams showing the dose-response of selected antisense oligonucleotides in DMPK knockdown in human DM1 myotubes. ASO300 was used as a control. All tested oligonucleotides showed DMPK knockdown activity. Statistical analysis: One-way ANOVA with Tukey's HSD post-hoc test for naked ASO300 treatment; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0040] [Figure 19] Figures 19A–19B show non-limiting schematic diagrams illustrating the dose-response of selected antisense oligonucleotides in DMPK knockdown in non-human primate (NHP) DM1 myotubes. ASO300 was used as a control. All tested oligonucleotides showed DMPK knockdown activity.

[0041] [Figure 20] Figure 20 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.

[0042] [Figure 21]Figures 21A–21F show the binding of humanized anti-TfR Fab to human TfR1 (hTfR1) or cynomolgus monkey TfR1 (cTfR1) as measured by ELISA. Figure 21A shows the binding of the humanized 3M12 variant to hTfR1. Figure 21B shows the binding of the humanized 3M12 variant to cTfR1. Figure 21C shows the binding of the humanized 3A4 variant to hTfR1. Figure 21D shows the binding of the humanized 3A4 variant to cTfR1. Figure 21E shows the binding of the humanized 5H12 variant to hTfR1. Figure 21F shows the binding of the humanized 5H12 variant to hTfR1.

[0043] [Figure 22] Figure 22 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.

[0044] [Figure 23A-C] Figures 23A–23F 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 23A shows the binding of the humanized 3M12 variant alone or in conjugate with a DMPK-targeted oligo to hTfR1. The respective EC50 values ​​are also shown. Figure 23B shows the binding of the humanized 3M12 variant alone or in conjugate with a DMPK-targeted oligo to cTfR1. The respective EC50 values ​​are also shown. Figure 23C shows the binding of the humanized 3A4 variant alone or in conjugate with a DMPK-targeted oligo to hTfR1. The respective EC50 values ​​are also shown. [Figure 23D-F]Figures 23A–23F 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 23D 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 23E 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 23F 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.

[0045] [Figure 24] Figure 24 shows DMPK expression in RD cells treated with DMPK-targeted oligonucleotides compared to cells treated with PBS. The treatment duration was 3 days. DMPK-targeted oligonucleotides were delivered to cells as free oligonucleotides (gymnotic incorporation, "free") or using transfection reagents ("trans").

[0046] [Figure 25] Figure 25 shows DMPK expression in RD cells treated with conjugates containing the indicated humanized anti-TfR antibody, conjugated to DMPK-targeted oligonucleotide (ASO300), at various concentrations. The treatment duration was 3 days. ASO300 delivered using a transfection agent (labeled "Trans") was used as a control.

[0047] [Figure 26] Figure 26 shows the results of Atp2a1 splicing correction using an anti-TfR1 antibody-oligonucleotide conjugate (Ab-ASO) in a DM1 HSA-LR mouse model, measured in the gastrocnemius muscle. The anti-TfR antibody used was RI7 217, and the oligonucleotide targets human skeletal muscle actin.

[0048] [Figure 27-1] Figure 27 shows splicing modifications of DM1 in over 30 different RNAs, measured in the gastrocnemius muscle of HSA-LR mice treated with an anti-TfR1 antibody-oligonucleotide (Ab-ASO) conjugate or saline. The anti-TfR antibody used was RI7 217, and the oligonucleotide targets human skeletal muscle actin. [Figure 27-2] Figure 27 shows splicing modifications of DM1 in over 30 different RNAs, measured in the gastrocnemius muscle of HSA-LR mice treated with an anti-TfR1 antibody-oligonucleotide (Ab-ASO) conjugate or saline. The anti-TfR antibody used was RI7 217, and the oligonucleotide targets human skeletal muscle actin. [Figure 27-3] Figure 27 shows splicing modifications of DM1 in over 30 different RNAs, measured in the gastrocnemius muscle of HSA-LR mice treated with an anti-TfR1 antibody-oligonucleotide (Ab-ASO) conjugate or saline. The anti-TfR antibody used was RI7 217, and the oligonucleotide targets human skeletal muscle actin.

[0049] [Figure 28] Figure 28 shows splicing abnormalities in the quadriceps, gastrocnemius, or tibialis anterior muscles of HSA-LR mice treated with anti-TfR1 antibody-oligonucleotide conjugates (Ab-ASO) or saline. The data represent complex splicing abnormalities measured in more than 30 RNAs, as shown in Figure 27.

[0050] [Figure 29]Figure 29 shows the myotonicity grades measured in the quadriceps, gastrocnemius, and tibialis anterior muscles of HSA-LR mice treated with saline, unconjugated oligonucleotides (ASOs), or anti-TfR1 antibody-oligonucleotide conjugates (Ab-ASOs). Myotonicity was measured by electromyography (EMG) and graded as 0, 1, 2, or 3 based on the frequency of myotonic discharges.

[0051] [Figure 30A] Figures 30A–30E show the in vivo activity of conjugates containing specified anti-TfR Fabs (control, 3M12 VH3 / VK2, 3M12 VH4 / VK3, and 3A4 VH3 N54S / VK4) conjugated to DMPK-targeted oligonucleotides in reducing DMPK mRNA expression in mice expressing human TfR1 (hTfR1 knock-in mice). Figure 30A shows the experimental design (e.g., IV dose, administration frequency). DMPK mRNA levels were measured 14 days after the initial administration in the tibialis anterior muscle (Figure 30B), gastrocnemius muscle (Figure 30C), heart (Figure 30D), and diaphragm (Figure 30E) of mice. [Figure 30B-C] Figures 30A–30E show the in vivo activity of conjugates containing specified anti-TfR Fabs (control, 3M12 VH3 / VK2, 3M12 VH4 / VK3, and 3A4 VH3 N54S / VK4) conjugated to DMPK-targeted oligonucleotides in reducing DMPK mRNA expression in mice expressing human TfR1 (hTfR1 knock-in mice). Figure 30A shows the experimental design (e.g., IV dose, administration frequency). DMPK mRNA levels were measured 14 days after the initial administration in the tibialis anterior muscle (Figure 30B), gastrocnemius muscle (Figure 30C), heart (Figure 30D), and diaphragm (Figure 30E) of mice. [Figure 30D-E]Figures 30A–30E show the in vivo activity of conjugates containing specified anti-TfR Fabs (control, 3M12 VH3 / VK2, 3M12 VH4 / VK3, and 3A4 VH3 N54S / VK4) conjugated to DMPK-targeted oligonucleotides in reducing DMPK mRNA expression in mice expressing human TfR1 (hTfR1 knock-in mice). Figure 30A shows the experimental design (e.g., IV dose, administration frequency). DMPK mRNA levels were measured 14 days after the initial administration in the tibialis anterior muscle (Figure 30B), gastrocnemius muscle (Figure 30C), heart (Figure 30D), and diaphragm (Figure 30E) of mice.

[0052] [Figure 31A-B] Figures 31A-31C show that a conjugate containing an anti-TfR antibody conjugated to a DMPK-targeted oligonucleotide modified splicing and reduced focus in CM-DM1-32F primary cells expressing DMPK mutant mRNA containing 380 CUG repeats. Figure 31A shows that the conjugate reduced mutant DMPK mRNA expression. Figure 31B shows that the conjugate modified BIN1 exon 11 splicing. [Figure 31C] Figures 31A–31C show that a conjugate containing an anti-TfR antibody conjugated to a DMPK-targeted oligonucleotide modified splicing and reduced focus in CM-DM1-32F primary cells expressing DMPK mutant mRNA containing 380 CUG repeats. Figure 31C shows images and image quantifications from fluorescence in situ hybridization (FISH) analysis, demonstrating that the conjugate reduced nuclear focus formed by mutant DMPK mRNA. In the microscopic images shown in the upper panel of Figure 31C, bright round shapes represent cell nuclei, and bright spots within the nuclei of DM1 cells (the three microscopic panels on the right) represent CUG focus.

[0053] [Figure 32]Figure 32 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.

[0054] [Figure 33] Figure 33 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.

[0055] [Figure 34] Figure 34 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.

[0056] [Figure 35A-1] Figures 35A–35B show splicing corrections for over 30 different RNAs known to be mis-spliced ​​in DM1 patients, measured in the tibialis anterior muscle (Figure 35A) or quadriceps muscle (Figure 35B) of HSA-LR mice treated with a single dose of anti-TfR antibody-oligonucleotide (Ab-ASO) conjugate or saline. The anti-TfR antibody used was RI7 217 Fab, and the oligonucleotide targets human skeletal actin (ACTA1). [Figure 35A-2]Figures 35A–35B show splicing corrections for over 30 different RNAs known to be mis-spliced ​​in DM1 patients, measured in the tibialis anterior muscle (Figure 35A) or quadriceps muscle (Figure 35B) of HSA-LR mice treated with a single dose of anti-TfR antibody-oligonucleotide (Ab-ASO) conjugate or saline. The anti-TfR antibody used was RI7 217 Fab, and the oligonucleotide targets human skeletal actin (ACTA1). [Figure 35A-3] Figures 35A–35B show splicing corrections for over 30 different RNAs known to be mis-spliced ​​in DM1 patients, measured in the tibialis anterior muscle (Figure 35A) or quadriceps muscle (Figure 35B) of HSA-LR mice treated with a single dose of anti-TfR antibody-oligonucleotide (Ab-ASO) conjugate or saline. The anti-TfR antibody used was RI7 217 Fab, and the oligonucleotide targets human skeletal actin (ACTA1). [Figure 35B-1] Figures 35A–35B show splicing corrections for over 30 different RNAs known to be mis-spliced ​​in DM1 patients, measured in the tibialis anterior muscle (Figure 35A) or quadriceps muscle (Figure 35B) of HSA-LR mice treated with a single dose of anti-TfR antibody-oligonucleotide (Ab-ASO) conjugate or saline. The anti-TfR antibody used was RI7 217 Fab, and the oligonucleotide targets human skeletal actin (ACTA1). [Figure 35B-2] Figures 35A–35B show splicing corrections for over 30 different RNAs known to be mis-spliced ​​in DM1 patients, measured in the tibialis anterior muscle (Figure 35A) or quadriceps muscle (Figure 35B) of HSA-LR mice treated with a single dose of anti-TfR antibody-oligonucleotide (Ab-ASO) conjugate or saline. The anti-TfR antibody used was RI7 217 Fab, and the oligonucleotide targets human skeletal actin (ACTA1). [Figure 35B-3] Figures 35A–35B show splicing corrections for over 30 different RNAs known to be mis-spliced ​​in DM1 patients, measured in the tibialis anterior muscle (Figure 35A) or quadriceps muscle (Figure 35B) of HSA-LR mice treated with a single dose of anti-TfR antibody-oligonucleotide (Ab-ASO) conjugate or saline. The anti-TfR antibody used was RI7 217 Fab, and the oligonucleotide targets human skeletal actin (ACTA1).

[0057] [Figure 36A-B] Figures 36A-36C show the EMG myotonicity grades in the quadriceps (Figure 36A), gastrocnemius (Figure 36B), and tibialis anterior (Figure 36C) muscles of HSA-LR mice treated with vehicle, a single dose of unconjugated ASO, or a single dose of anti-TfR1 antibody-ASO conjugate (Ab-ASO). The anti-TfR1 antibody used was RI7 217 Fab. [Figure 36C] Figures 36A–36C show the EMG myotonicity grades in the quadriceps (Figure 36A), gastrocnemius (Figure 36B), and tibialis anterior (Figure 36C) muscles of HSA-LR mice treated with vehicle, a single dose of unconjugated ASO, or a single dose of anti-TfR1 antibody-ASO conjugate (Ab-ASO). The anti-TfR1 antibody used was RI7 217 Fab, and its oligonucleotide targets human skeletal muscle actin (ACTA1).

[0058] [Figure 37] Figure 37 shows human ACTA1 expression, as measured by qPCR, in HSALR DM1 mice compared to vehicle-treated mice after a single dose of naked ASO or a dose equivalent of anti-TFR1 antibody-ASO conjugate (Ab-ASO). The anti-TfR1 antibody used was RI7 217 Fab, and the oligonucleotide targets human skeletal muscle actin (ACTA1).

[0059] [Figure 38A-B]Figures 38A–38C show ACTA1 expression in the quadriceps (Figure 38A), gastrocnemius (Figure 38B), and tibialis anterior (Figure 38C) muscles of HSALR DM1 mice after a single dose of 10 mg / kg naked ASO, 20 mg / kg naked ASO, or dose equivalent of an anti-TFR antibody-ASO conjugate (Ab-ASO), compared to vehicle-treated mice. The anti-TfR1 antibody used was RI7 217 Fab, and the oligonucleotide targets human skeletal muscle actin (ACTA1). (*p<0.05; ***p<0.001) [Figure 38C] Figures 38A–38C show ACTA1 expression in the quadriceps (Figure 38A), gastrocnemius (Figure 38B), and tibialis anterior (Figure 38C) muscles of HSALR DM1 mice after a single dose of 10 mg / kg naked ASO, 20 mg / kg naked ASO, or dose equivalent of an anti-TFR antibody-ASO conjugate (Ab-ASO), compared to vehicle-treated mice. The anti-TfR1 antibody used was RI7 217 Fab, and the oligonucleotide targets human skeletal muscle actin (ACTA1). (*p<0.05; ***p<0.001) [Modes for carrying out the invention]

[0060] 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 inhibits the expression or activity of a target gene in muscle cells, for example, in subjects having or suspected of having a rare muscle disease. For example, in some embodiments, the complex is provided to target a DMPK allele containing an extended disease-related repeat and to treat subjects having DM1. In some embodiments, the complex provided herein may contain an oligonucleotide that inhibits the expression of a DMPK allele containing an extended disease-related repeat. As another example, the complex may contain an oligonucleotide that interferes with the binding of disease-related DMPK mRNA to muscleblind-like proteins (e.g., MBNL1, 2, and / or (e.g., and) 3), thereby reducing the toxic effects of the disease-related DMPK allele. In some embodiments, a synthetic nucleic acid payload (e.g., a DNA or RNA payload) expressing one or more proteins that reduce the toxic effects of disease-associated DMPK alleles can be used. In some embodiments, the complex may include a molecular payload of synthetic cDNA and / or (e.g., and) synthetic mRNA expressing one or more Muscleblind-like proteins (e.g., MBNL1, 2, and / or (e.g., and) 3) or fragments thereof, for example. In some embodiments, the complex may include a molecular payload such as a guide molecule (e.g., guide RNA) that can program nucleic acids to target sequences in or near the disease-associated repeats of DMPK with a nucleic acid-programmable nuclease (e.g., Cas9). In some embodiments, a portion or all of the disease-associated repeat sequences can be cleaved from the DMPK gene using such a nucleic acid-programmable nuclease.

[0061] Further aspects of this disclosure, including the definition of terms, are provided below.

[0062] I. Definition To administer (give): As used herein, the term “administer” or “give” means to provide a complex to a subject in a physiologically and / or pharmacologically useful manner (for example, to treat a disease in the subject).

[0063] about: When 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.

[0064] antibody: As used herein, the term “antibody” refers to a polypeptide comprising at least one immunoglobulin variable domain or at least one antigenic determinant, e.g., a paratope that specifically binds to an antigen. In some embodiments, the antibody is a full-length antibody. In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a humanized antibody. However, in some embodiments, the antibody is a Fab fragment, Fab', F(ab')2 fragment, Fv fragment, or 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 noncovalent 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).

[0065] 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.

[0066] 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 provided in Table 1. Table 1. CDR definition [Table 1] 1 IMGT®, the international ImMunoGeneTics information system®, imgt.org, Lefranc, M.-P. et al., Nucleic Acids Res., 27:209-212 (1999) 2 Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242 3 Chothia et al., J. Mol. Biol. 196:901-917 (1987))

[0067] CDR-grafted antibodies: The term "CDR-conjugated 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.

[0068] Chimeric antibodies: 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.

[0069] Complementary: As used herein, the term “complementary” refers to the capacity for accurate pairing between two nucleotides or 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 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 at that position. Base pairing may encompass 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 universal bases such as 3-nitropyrrole or 5-nitroindole can hybridize with any of A, C, U, or T and are considered complementary to them. Inosine (I) is also considered a universal base in the art and is considered complementary to any of A, C, U, or T.

[0070] Conserved amino acid substitutions: As used herein, “conservative amino acid substitution” refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein to which the 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.

[0071] Covalently linked: As used herein, the term “covalently linked” refers to the characteristic of two or more molecules 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 acting as a linker, which binds 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.

[0072] Cross-reaction: As used herein, and in the context of targeting agents (e.g., antibodies), the term “cross-reacting” 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.

[0073] Disease-related recurrence: As used herein, the term “disease-associated repeat” refers to a repeating nucleotide sequence at a location in the genome in which several units of a repeating nucleotide sequence correlate with and / or (for example, and) directly or indirectly contribute to or cause a genetic disease. Each repeating unit of a disease-associated repeat may be 2, 3, 4, 5 or more nucleotides in length. For example, in some embodiments, a disease-associated repeat is a dinucleotide repeat. In some embodiments, a disease-associated repeat is a trinucleotide repeat. In some embodiments, a disease-associated repeat is a tetranucleotide repeat. In some embodiments, a disease-associated repeat is a pentanucleotide repeat. In some embodiments, a disease-associated repeat includes a CAG repeat, CTG repeat, CUG repeat, CGG repeat, CCTG repeat, or a nucleotide complement of any of these. In some embodiments, a disease-associated repeat is located in the non-coding region of a gene. However, in some embodiments, a disease-associated repeat is located in the coding region of a gene. In some embodiments, a disease-associated repeat is extended from a normal state to a length that directly or indirectly contributes to or causes a genetic disease. In some embodiments, disease-associated repeats are located in RNA (e.g., RNA transcripts). In some embodiments, disease-associated repeats are located in DNA (e.g., chromosomes, plasmids). In some embodiments, disease-associated repeats are extended to the chromosomes of germline cells. In some embodiments, disease-associated repeats are extended to the chromosomes of somatic cells. In some embodiments, disease-associated repeats are extended to numerous repeat units associated with congenital onset. In some embodiments, disease-associated repeats are extended to numerous repeat units associated with childhood onset of the disease. In some embodiments, disease-associated repeats are extended to numerous repeat units associated with adult onset of the disease.

[0074] DMPK: As used herein, the term “DMPK” refers to the gene encoding myotonin protein kinase (also known as myotonic dystrophy protein kinase or dystrophia myotonica protein kinase), which is a serine / threonine protein kinase. Substrates of this enzyme may include myogenin, the beta subunit of L-type calcium channels, and phosphoremann. In some embodiments, DMPK may be a human (Gene ID: 1760), a non-human primate (e.g., Gene ID: 456139, Gene ID: 715328), or a rodent gene (e.g., Gene ID: 13400). In humans, CTG repeat expansion in the 3' uncoding untranslated region of DMPK is associated with myotonic dystrophy type I (DM1). In addition, several human transcript variants encoding various isoforms of the receptor have been characterized (for example, those annotated with GenBank RefSeq accessions: NM_001081563.2, NM_004409.4, NM_001081560.2, NM_001081562.2, NM_001288764.1, NM_001288765.1, and NM_006128).

[0075] DMPK allergen: As used herein, the term “DMPK allele” refers to one of the alternative forms of the DMPK gene (e.g., wild-type or mutant form). In some embodiments, a DMPK allele may encode a wild-type myotonin protein kinase that retains its normal and typical function. In some embodiments, a DMPK allele may contain one or more disease-associated repeat extensions. In some embodiments, a normal subject has two DMPK alleles containing repeat units ranging from 5 to 37. In some embodiments, the number of CTG repeat units in a subject with DM1 ranges from about 50 to about 3,000+, with a higher number of repeats indicating greater disease severity. In some embodiments, a subject with mild DM1 has at least one DMPK allele containing repeat units ranging from 50 to 150. In some embodiments, a subject with classical DM1 has at least one DMPK allele containing repeat units ranging from 100 to 1,000 or more. In some embodiments, a subject with congenital DM1 may have at least one DMPK allele containing more than 2,000 repeat units.

[0076] 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.

[0077] Human antibodies: When used herein, the term “human antibody” is intended to encompass antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of this disclosure may include, for example, amino acid residues not encoded by human germline immunoglobulin sequences (mutations introduced, for example, 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 antibody” 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.

[0078] 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.).

[0079] Internalized cell surface receptors: As used herein, the term “internalizing cell surface receptor” refers, for example, to a cell surface receptor that is internalized by a cell in response to an external stimulus (e.g., a ligand that binds to the receptor). In some embodiments, the internalizing cell surface receptor is internalized by endocytosis. In some embodiments, the internalizing cell surface receptor is internalized by clathrin-mediated endocytosis. However, in some embodiments, the internalizing cell surface receptor is internalized by clathrin-independent pathways, such as phagocytosis, macropinocytosis, caveolae- and raft-mediated uptake, or clathrin-independent constitutive endocytosis. In some embodiments, the internalizing cell surface receptor comprises an intracellular domain, a transmembrane domain, and / or (e.g., and), an extracellular domain, which optionally further comprises a ligand-binding domain. In some embodiments, the cell surface receptor becomes internalized by the cell after ligand binding. In some embodiments, the ligand may be a muscle targeting agent or a muscle targeting antibody. In some embodiments, the internalizing cell surface receptor is a transferrin receptor.

[0080] Isolated antibodies: When used herein, "isolated antibody" is intended to refer to an antibody 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, an isolated antibody may be substantially free of other cellular material and / or (for example, and) chemicals.

[0081] 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, from amino acid positions 50-65 for CDR2, and from 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.

[0082] 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.

[0083] Muscle targeting agents: 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.

[0084] Muscle targeting antibodies: As used herein, the term “muscle-targeting antibody” refers to a muscle-targeting agent that is an antibody that specifically binds to an antigen found in or on muscle cells. In some embodiments, a muscle-targeting antibody specifically binds to an antigen on a muscle cell that facilitates the internalization of the muscle-targeting antibody (and any associated molecular payload) into the muscle cell. In some embodiments, a muscle-targeting antibody specifically binds to an internalized cell surface receptor present on the muscle cell. In some embodiments, a muscle-targeting antibody is an antibody that specifically binds to a transferrin receptor.

[0085] Myotonic dystrophy (DM): As used herein, the term “myotonic dystrophy (DM)” refers to a genetic disorder caused by mutations in the DMPK or CNBP (ZNF9) gene, characterized by muscle loss, muscle weakness, and impaired muscle function. Two types of myotonic dystrophy have been reported: myotonic dystrophy type 1 (DM1) and myotonic dystrophy type 2 (DM2). DM1 is associated with an expansion of the CTG trinucleotide repeat in the 3' non-coding region of DMPK. DM2 is associated with an expansion of the CCTG tetranucleotide repeat in the first intron of ZNF9. In both DM1 and DM2, the nucleotide expansion leads to toxic RNA repeats that can form hairpin structures that bind with high affinity to important intracellular proteins, e.g., muscleblind-like proteins. Myotonic dystrophy, its genetic basis, and associated symptoms have been reported in the art (see, for example, Thornton, CA, "Myotonic Dystrophy" Neurol Clin. (2014), 32(3): 705-719; and Konieczny et al. "Myotonic dystrophy: candidate small molecule therapeutics" Drug Discovery Today (2017), 22:11). In some aspects, subjects are born with a variation of DM1 called congenital myotonic dystrophy. Symptoms of congenital myotonic dystrophy are present from birth and include general muscle weakness, respiratory distress, clubfoot, developmental delay, and intellectual disability. DM1 is associated with Online Mendelian Inheritance in Man (OMIM) entry #160900. DM2 is associated with OMIM entry #602668.

[0086] Oligonucleotides: As used herein, the term “oligonucleotide” refers to an oligomeric nucleic acid compound with a length of up to 200 nucleotides. Examples of oligonucleotides include, but are not limited to, RNAi oligonucleotides (e.g., siRNA, shRNA), microRNAs, gapmers, mixmers, phosphorodiamidite morpholinos, 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.

[0087] Recombinant antibodies: When used herein, the term “recombinant human antibody” 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.).

[0088] Complementary areas: 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.

[0089] Binds specifically: As used herein, the term “specifically binds” refers to the ability of a molecule to bind to a binding partner to a degree of affinity or binding activity that the molecule can be used to distinguish the binding partner from a suitable control in a binding assay or other binding context. With respect to antibodies, the term “specifically binds” refers to the ability of an antibody to bind to a specific antigen to a degree of affinity or binding activity (for example, to the extent that it allows preferential targeting to a cell (e.g., muscle cells) through binding to the antigen, as described herein,) compared to a suitable reference antigen, or an antigen that the antibody can be used to distinguish a particular antigen from other antigens. In some embodiments, when the antibody binds to the target, at least about 10-4 M, 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, 10 -13 M or less than this amount of K D When having, the antibody specifically binds to the target. In some embodiments, the antibody specifically binds to an epitope of the tip domain of the transferrin receptor, for example, the transferrin receptor.

[0090] Subject: As used herein, the term "subject" refers to a mammal. In some embodiments, the subject is a non-human primate or a rodent. In some embodiments, the subject is a human. In some embodiments, the subject is a patient suspected of having or having a disease, for example, a human patient. In some embodiments, the subject is a human patient suspected of having or having a disease related to repeat expansion, for example, a disease caused by the DMPK allele.

[0091] 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., those annotated with GenBank RefSeq accessions: NP_001121620.1, NP_003225.2, NP_001300894.1, and NP_001300895.1).

[0092] 2' Modified nucleoside: As used herein, the terms “2'-modified nucleoside” and “2'-modified ribonucleoside” are used interchangeably 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:

[0093] [ka]

[0094] II. Complex 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.

[0095] 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 targeting disease-associated repeats in muscle cells.

[0096] In some embodiments, the complex comprises a muscle targeting agent, such as an anti-transferrin receptor antibody, covalently linked to a molecular payload, such as a disease-associated repeat, such as a DMPK allele, on an antisense oligonucleotide.

[0097] 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.

[0098] 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.

[0099] 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.

[0100] 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.

[0101] 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.

[0102] i. Muscle targeting antibodies 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.

[0103] 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.

[0104] 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 receptor 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 the 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 Pharmacol. Exp. Ther., 292:1048-1052).

[0105] 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.

[0106] 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).

[0107] 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)

[0108] 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).

[0109] 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: MMDQARSAFSNLFGGEPLSYTRFSLARQVDGDNHVEMKLAADEEENADNNMKASVRKPKRFNGRLCFAAIALVIFFLIGFMSGYLGYCKRVEQKEECVKLAETEETDKSETMETEDVPTSSRLYWADLKTLLSEKLNSIEFADTIKQLSQNTYTPREAGSQKDESLAYYIENQFHEFKFSKVWRDEHYV KIQVKSSIGQNMVTIVQSNGNLDPVESPEGYVAFSKPTEVSGKLVHANFGTKKDFEELSYSVNGSLVIVRAGEITFAEKVANAQSFNAIGVLIYMDKNKFPVVEADLALFGHAHLGTGDPYTPGFPSFNHTQFPPSQSSGLPNIPVQTISRAAAEKLFGKMEGSCPARWNIDSSCKLELSQNQNVKLIVKN VLKERRILNIFGVIKGYEEPDRYVVVGAQRDALGAGVAAKSSVGTGLLLKLAQVFSDMISKDGFRPSRSIIFASWTAGDFGAVGATEWLEGYLSSLHLKAFTYINLDKVVLGTSNFKVSASPLLYTLMGKIMQDVKHPVDGKSLYRDSNWISKVEKLSFDNAAYPFLAYSGIPAVSFCFCEDADYPYLGTR LDTYEALTQKVPQLNQMVRTAAEVAGQLIIKLTHDVELNLDYEMYNSKLLSFMKDLNQFKTDIRDMGLSLQWLYSARGDYFRATSRLTTDFHNAEKTNRFVMREINDRIMKVEYHFLSPYVSPRESPFRHIFWGSGSHTLSALVENLKLRQKNITAFNETLFRNQLALATWTIQGVANALSGDIWNIDNEF (Sequence ID 108)

[0110] In some embodiments, the anti-transferrin receptor antibody comprises the following amino acid segments of the receptor: It binds to FVKIQVKDSAQNSVIIVDKNGRLVYLVENPGGYVAYSKAATVTGKLVHANFGTKKDFEDLYTPVNGSIVIVRAGKITFAEKVANAESLNAIGVLIYMDQTKFPIVNAELSFFGHAHLGTGDPYTPGFPSFNHTQFPPSRSSGLPNIPVQTISRAAAEKLFGNMEGDCPSDWKTDSTCRMVTSESKNVKLTVSNVLKE (SEQ ID NO: 109) and does not inhibit the binding interaction between the transferrin receptor and transferrin and / or (for example, and) human hemochromatosis protein (also known as HFE). In some embodiments, the anti-transferrin receptor antibodies described herein do not bind to the epitope of SEQ ID NO: 109.

[0111] 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, such as 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, for example, recombinant DNA techniques. Examples of antibody production and methodological additions are also known in the art (see, for example, Harlow et al., "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory, 1988).

[0112] 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 the international patent application publication WO2014065661, published on May 1, 2014, entitled "Modified antibody, antibody-conjugate and process for the preparation thereof".

[0113] 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) of an immunoglobulin molecule. Non-limiting examples of human constant regions are described in the art. For example, see Kabat EA et al., (1991) above.

[0114] 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.

[0115] In some embodiments, humanized antibodies that bind to transferrin receptors 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 to 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 human transferrin receptors. In some embodiments, the humanized anti-TfR antibodies described herein bind to amino acid segments of human or non-human primate transferrin receptors, such as those provided in SEQ ID NOs. 105-108. In some embodiments, the humanized anti-TfR antibodies described herein bind to amino acid segments corresponding to amino acids 90-96 of the human transferrin receptor shown in SEQ ID NO. 105, which are not present in the tip domain of the transferrin receptor. In some embodiments, the humanized anti-TfR antibodies described herein bind to TfR1 but not to TfR2.

[0116] 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.

[0117] 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] * The mutation site follows the Kabat numbering of each VH sequence containing the mutation.

[0118] 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 CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, the same as CDR-H1, CDR-H2, and CDR-H3 in any one of the anti-TfR antibodies provided in Table 2, and comprises a humanized heavy chain variable region and / or (for example, and) a humanized light chain variable region.

[0119] A humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the recipient's complementarity-determining region (CDR) are replaced with residues from the CDR of a non-human species (donor antibody), such as mouse, rat, or rabbit, having the desired specificity, affinity, and volume. In some embodiments, Fv framework region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. Furthermore, the humanized antibody may also include residues that are not found on the recipient antibody or on the transferred CDR or framework sequence, but are included to further improve and optimize antibody performance. Generally, a humanized antibody will contain substantially all of at least one, typically two, variable domains, where all or substantially all of the CDR region corresponds to that of a non-human immunoglobulin, and all or substantially all of the FR region corresponds to that of the human immunoglobulin consensus sequence. Optimally, the humanized antibody will also contain at least some portion (Fc) of the immunoglobulin constant region or domain, typically that of the human immunoglobulin. Antibodies may have modified Fc regions as described in WO99 / 58572. Other forms of humanized antibodies have one or more CDRs (one, two, three, four, five, or six) that are modified from the original antibody. These are also referred to as one or more CDRs derived from one or more CDRs from the original antibody. Affinity maturation may also be involved in humanized antibodies.

[0120] Humanized antibodies and methods for producing them are known, for example, Almagro et al., Front. Biosci. 13:1619-1633 (2008); Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. 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 As described in 36:43-60 (2005); Osbourn et al., Methods 36:61-68 (2005); and Klimka et al., Br.J. Cancer, 83:252-260 (2000). All of these contents 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 these contents are incorporated herein by reference.

[0121] In some embodiments, the humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising one or more amino acid variations (e.g., in the VH framework region) compared to any one of the VHs listed in Table 2, and / or (e.g., and), a humanized VL comprising one or more amino acid variations (e.g., in the VL framework region) compared to any one of the VLs listed in Table 2.

[0122] In some embodiments, the humanized anti-TfR antibodies of this disclosure include a humanized VH containing only 25 amino acid variations (for example, only 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) compared to the VH of any 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 in addition (for example, in addition), the humanized anti-TfR antibodies of this disclosure include humanized VLs containing only 25 amino acid variations (for example, only 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) compared to any one VL of any of the anti-TfR antibodies listed in Table 2 (for example, any one of SEQ ID NOs: 18, 44, and 62).

[0123] In some embodiments, the humanized anti-TfR antibody of this disclosure comprises a humanized VH having at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical amino acid sequence in the framework region to any VH of any of the anti-TfR antibodies listed in Table 2 (e.g., any one of SEQ ID NOs. 17, 22, 26, 43, 61, 65, and 68). Alternatively or in addition (e.g., in addition), the humanized anti-TfR antibody of this disclosure comprises a humanized VL having at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical amino acid sequence in the framework region to any VL of any of the anti-TfR antibodies listed in Table 2 (e.g., any one of SEQ ID NOs. 18, 44, and 62).

[0124] In some embodiments, the humanized anti-TfR antibody of the present disclosure comprises humanized VH including CDR-H1 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO: 1, CDR-H2 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 19, or SEQ ID NO: 23, and CDR-H3 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO: 3, and contains only 25 amino acid variations in the framework region (for example, only 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) compared to VH as represented by SEQ ID NO: 17, SEQ ID NO: 22, or SEQ ID NO: 26. Alternatively or in addition (for example, in addition), the anti-TfR antibody of the present disclosure comprises a humanized VL 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 contains only 25 amino acid variations in the framework region (for example, only 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) compared to a VL as represented by SEQ ID NO: 18.

[0125] In some embodiments, the humanized anti-TfR antibody of the present disclosure comprises a humanized VH having CDR-H1 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO: 1, CDR-H2 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 19, or SEQ ID NO: 23, and CDR-H3 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO: 3, which is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework region to the VH as represented by SEQ ID NO: 17, SEQ ID NO: 22, or SEQ ID NO: 26. 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: 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), 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 any one of SEQ ID NOs: 18.

[0126] In some embodiments, the humanized anti-TfR antibody of the present disclosure comprises humanized VH including CDR-H1 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO: 7, CDR-H2 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO: 8, SEQ ID NO: 20, or CDR-H2 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO: 20, and CDR-H3 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO: 9, and contains only 25 amino acid variations in the framework region (for example, only 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) compared to VH as represented by SEQ ID NO: 17, SEQ ID NO: 22, or SEQ ID NO: 26. 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: 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), and contains only 25 amino acid variations in the framework region (for example, only 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) compared to a VL as represented by SEQ ID NO: 18.

[0127] In some embodiments, the humanized anti-TfR antibody of the present disclosure comprises a humanized VH having CDR-H1 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO: 7, CDR-H2 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO: 8, SEQ ID NO: 20, or SEQ ID NO: 24, and CDR-H3 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO: 9, 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 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: 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 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 any one of SEQ ID NOs: 18.

[0128] In some embodiments, the humanized anti-TfR antibody of the present disclosure comprises humanized VH including humanized VH comprising CDR-H1 (according to the Chothia definition system) having the amino acid sequence of SEQ ID NO: 12, CDR-H2 (according to the Chothia definition system) having the amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 21, or SEQ ID NO: 25, and CDR-H3 (according to the Chothia definition system) having the amino acid sequence of SEQ ID NO: 14, and contains only 25 amino acid variations in the framework region (for example, only 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) compared to VH as represented by SEQ ID NO: 17, SEQ ID NO: 22, or SEQ ID NO: 26. Alternatively or in addition (for example, in addition), the humanized anti-TfR antibody of the present disclosure comprises a humanized VL including a humanized VL comprising

[0129] In some embodiments, the humanized anti-TfR antibody of the present disclosure comprises a humanized VH having CDR-H1 (according to the Chothia definition system) having the amino acid sequence of SEQ ID NO: 12, CDR-H2 (according to the Chothia definition system) having the amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 21, or SEQ ID NO: 25, and CDR-H3 (according to the Chothia definition system) having the amino acid sequence of SEQ ID NO: 14, which is at least 75% (for example, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework region to the VH as represented by SEQ ID NO: 17, SEQ ID NO: 22, or SEQ ID NO: 26. Alternatively or in addition (for example, in addition), the anti-TfR antibody of the present disclosure comprises a humanized VL 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 as represented by any one of SEQ ID NOs: 18.

[0130] In some embodiments, the humanized anti-TfR antibody of the present disclosure comprises humanized VH including CDR-H1 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO: 27, CDR-H2 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO: 28, and CDR-H3 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO: 29, and contains only 25 amino acid variations in the framework region (for example, only 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) compared to VH as represented by SEQ ID NO: 43. Alternatively or in addition (for example, in addition), the humanized anti-TfR antibody of the present disclosure comprises a humanized VL 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), and contains only 25 amino acid variations in the framework region (for example, only 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) compared to a VL as represented by SEQ ID NO: 44.

[0131] In some embodiments, the humanized anti-TfR antibody of the present disclosure comprises humanized VH including CDR-H1 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO: 27, CDR-H2 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO: 28, and CDR-H3 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO: 29, which are at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to VH as represented by 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: 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 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.

[0132] In some embodiments, the humanized anti-TfR antibody of the present disclosure comprises humanized VH including CDR-H1 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO: 33, CDR-H2 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO: 34, and CDR-H3 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO: 35, and contains only 25 amino acid variations in the framework region (for example, only 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) compared to VH as represented by SEQ ID NO: 43. Alternatively or in addition (for example, in addition), the humanized anti-TfR antibody of the present disclosure comprises a humanized VL 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 contains only 25 amino acid variations in the framework region (for example, only 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) compared to a VL as represented by SEQ ID NO: 44.

[0133] In some embodiments, the humanized anti-TfR antibody of the present disclosure comprises humanized VH including CDR-H1 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO: 33, CDR-H2 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO: 34, and CDR-H3 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO: 35, which are at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to VH as represented by 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.

[0134] In some embodiments, the humanized anti-TfR antibody of the present disclosure comprises humanized VH including CDR-H1 (according to the Chothia definition system) having the amino acid sequence of SEQ ID NO: 38, CDR-H2 (according to the Chothia definition system) having the amino acid sequence of SEQ ID NO: 39, and CDR-H3 (according to the Chothia definition system) having the amino acid sequence of SEQ ID NO: 40, and contains only 25 amino acid variations in the framework region (for example, only 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) compared to VH as represented by SEQ ID NO: 43. Alternatively or in addition (for example, in addition), the humanized anti-TfR antibody of the present disclosure comprises a humanized VL having CDR-L1 (according to the Chothia definition system) having the amino acid sequence of SEQ ID NO: 41, CDR-L2 (according to the Chothia definition system) having the amino acid sequence of SEQ ID NO: 31, and CDR-L3 (according to the Chothia definition system) having the amino acid sequence of SEQ ID NO: 42, and contains only 25 amino acid variations in the framework region (for example, only 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) compared to a VL as represented by SEQ ID NO: 44.

[0135] In some embodiments, the humanized anti-TfR antibody of the present disclosure comprises humanized VH including CDR-H1 (according to the Chothia definition system) having the amino acid sequence of SEQ ID NO: 38, CDR-H2 (according to the Chothia definition system) having the amino acid sequence of SEQ ID NO: 39, and CDR-H3 (according to the Chothia definition system) having the amino acid sequence of SEQ ID NO: 40, which in the framework region is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to VH as represented by SEQ ID NO: 43. Alternatively or in addition (for example, in addition), the humanized anti-TfR antibody of the present disclosure comprises a humanized VL 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 to the VL as represented by SEQ ID NO: 44 in the framework region.

[0136] In some embodiments, the humanized anti-TfR antibody of the present disclosure comprises humanized VH including CDR-H1 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO: 45, SEQ ID NO: 63, or SEQ ID NO: 66, CDR-H2 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO: 46, and CDR-H3 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO: 47, and contains only 25 amino acid variations in the framework region (for example, only 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) compared to VH as represented by SEQ ID NO: 61, SEQ ID NO: 65, or SEQ ID NO: 68. 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: 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), and contains only 25 amino acid variations in the framework region (for example, only 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) compared to a VL as represented by SEQ ID NO: 62.

[0137] In some embodiments, the humanized anti-TfR antibody of the present disclosure comprises a humanized VH having CDR-H1 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO: 45, SEQ ID NO: 63, or SEQ ID NO: 63, CDR-H2 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO: 46, and CDR-H3 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO: 47, and is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework region to the VH as represented by SEQ ID NO: 61, SEQ ID NO: 65, and SEQ ID NO: 68. 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: 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 to the VL as represented by SEQ ID NO: 62 in the framework region.

[0138] In some embodiments, the humanized anti-TfR antibody of the present disclosure comprises humanized VH including CDR-H1 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO: 51, SEQ ID NO: 64, or SEQ ID NO: 67, CDR-H2 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO: 52, and CDR-H3 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO: 53, and contains only 25 amino acid variations in the framework region (for example, only 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) compared to VH as represented by SEQ ID NO: 61, SEQ ID NO: 65, and SEQ ID NO: 68. 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: 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 contains only 25 amino acid variations in the framework region (for example, only 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) compared to a VL as represented by SEQ ID NO: 62.

[0139] In some embodiments, the humanized anti-TfR antibody of the present disclosure comprises a humanized VH having CDR-H1 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO: 51, SEQ ID NO: 64, or SEQ ID NO: 67, CDR-H2 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO: 52, and CDR-H3 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO: 53, which is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework region to the VH as represented by SEQ ID NO: 61, SEQ ID NO: 65, and SEQ ID NO: 68. 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: 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 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: 62.

[0140] In some embodiments, the humanized anti-TfR antibody of the present disclosure comprises humanized VH including CDR-H1 (according to the Chothia definition system) having the amino acid sequence of SEQ ID NO: 56, CDR-H2 (according to the Chothia definition system) having the amino acid sequence of SEQ ID NO: 57, and CDR-H3 (according to the Chothia definition system) having the amino acid sequence of SEQ ID NO: 58, and contains only 25 amino acid variations in the framework region (for example, only 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) compared to VH as represented by SEQ ID NO: 61, SEQ ID NO: 65, and SEQ ID NO: 68. Alternatively or in addition (for example, in addition), the humanized anti-TfR antibody of the present disclosure comprises a humanized VL having CDR-L1 (according to the Chothia definition system) having the amino acid sequence of SEQ ID NO: 59, CDR-L2 (according to the Chothia definition system) having the amino acid sequence of SEQ ID NO: 49, and CDR-L3 (according to the Chothia definition system) having the amino acid sequence of SEQ ID NO: 60, and contains only 25 amino acid variations in the framework region (for example, only 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) compared to a VL as represented by SEQ ID NO: 62.

[0141] In some embodiments, the humanized anti-TfR antibody of the present disclosure comprises humanized VH including CDR-H1 (according to the Chothia definition system) having the amino acid sequence of SEQ ID NO: 56, CDR-H2 (according to the Chothia definition system) having the amino acid sequence of SEQ ID NO: 57, and CDR-H3 (according to the Chothia definition system) having the amino acid sequence of SEQ ID NO: 58, which are at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework region to VH as represented by SEQ ID NO: 61, SEQ ID NO: 65, and SEQ ID NO: 68. 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: 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 to the VL as represented by SEQ ID NO: 62 in the framework region.

[0142] 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] * The mutation location follows the Kabat numbering of each VH sequence containing the mutation. ** CDRs using the Kabat numbering system are shown in bold.

[0143] 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 when compared to each humanized VH provided in Table 3, it contains one or more amino acid variations (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) in the framework region. Alternatively or in addition (e.g., in addition), 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 when compared to each humanized VL provided in Table 3, it contains one or more amino acid variations (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) in the framework region.

[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: 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.

[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: 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.

[0146] 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.

[0147] 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.

[0148] 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.

[0149] 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.

[0150] 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.

[0151] 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.

[0152] 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.

[0153] 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.

[0154] In some embodiments, the humanized anti-TfR antibodies described herein are full-length IgG that may contain heavy chain constant regions and light chain constant regions from a human antibody. In some embodiments, the heavy chain of any of the anti-TfR antibodies described herein may include a heavy chain constant region (CH) or a portion thereof (e.g., CH1, CH2, CH3, or a combination thereof). The heavy chain constant region may be of any preferred origin, e.g., human, mouse, rat, or rabbit. In one specific example, the heavy chain constant region is from human IgG (gamma heavy chain), e.g., IgG1, IgG2, or IgG4. An example of a human IgG1 constant region is given below: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(Sequence ID 81)

[0155] In some embodiments, the heavy chain of any of the anti-TfR antibodies described herein comprises a mutant human IgG1 constant region. For example, the introduction of 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 Fcγ receptor binding (Bruhns, P., et al. (2009) and Xu, D. et al. (2000)). Mutant human IgG1 constant regions are provided below (mutations are in bold and underlined): [ka] (Sequence ID 176)

[0156] In some embodiments, the light chain constant region of any of the anti-TfR antibodies described herein may be any light chain constant region known in the art. In some examples, this may be a kappa light chain or a lambda light chain. In some embodiments, the light chain constant region is a kappa light chain, the sequence of which is provided below: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(Sequence ID 83)

[0157] 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.

[0158] 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 only 25 amino acid variations compared to SEQ ID NO: 81 or SEQ ID NO: 82 (for example, only 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation). 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 a heavy chain constant region as represented by SEQ ID NO: 81. In some embodiments, the anti-TfR antibody described herein comprises a heavy chain including one of the VH variants listed in Table 3 and a heavy chain constant region as represented by SEQ ID NO: 82.

[0159] 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 only 25 amino acid variations compared to SEQ ID NO: 83 (for example, only 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation). 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 as represented by SEQ ID NO: 83.

[0160] 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] * The mutation location follows the Kabat numbering of each VH sequence containing the mutation. ** CDRs using the Kabat numbering system are shown in bold, and VH / VL titles are underlined.

[0161] In some embodiments, the humanized anti-TfR antibody of the present disclosure comprises a heavy chain containing only 25 amino acid variations (for example, only 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) compared to the heavy chain as represented by any one of SEQ ID NOs. Alternatively or in addition (for example, in addition), the humanized anti-TfR antibody of the present disclosure comprises a light chain containing only 25 amino acid variations (for example, only 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) compared to the light chain represented by any one of SEQ ID NOs. 85, 89, 90, 93, and 95.

[0162] 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 in addition (e.g., in addition), 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. Alternatively or in addition (for example, in addition), 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.

[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: 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.

[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: 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.

[0165] 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.

[0166] In some embodiments, the humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising 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 comprising 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 present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 88 and a light chain comprising the amino acid sequence of SEQ ID NO: 89.

[0167] In some embodiments, the humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising 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 comprising 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 present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 88 and a light chain comprising the amino acid sequence of SEQ ID NO: 90.

[0168] In some embodiments, the humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising 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 comprising 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 present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 91 and a light chain comprising the amino acid sequence of SEQ ID NO: 89.

[0169] In some embodiments, the humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising 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 comprising 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 present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 91 and a light chain comprising the amino acid sequence of SEQ ID NO: 90.

[0170] In some embodiments, the humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising 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 comprising 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 present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 92 and a light chain comprising the amino acid sequence of SEQ ID NO: 93.

[0171] In some embodiments, the humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising 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 comprising 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 present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 94 and a light chain comprising the amino acid sequence of SEQ ID NO: 95.

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

[0173] 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 may be produced by pepsin or papain digestion of the antibody molecule, and the Fab' fragment may be produced by reducing the disulfide crosslinks of the F(ab')2 fragment. In some embodiments, the heavy chain constant region on the Fab fragment of the anti-TfR1 antibody described herein is as follows: Contains the amino acid sequence ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT (Sequence ID 96).

[0174] In some embodiments, the humanized anti-TfR antibody described herein comprises a heavy chain comprising one of the VH variants listed in Table 3 and a heavy chain containing 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 VH variants listed in Table 3 and a heavy chain containing a heavy chain constant region with only 25 amino acid variations compared to SEQ ID NO: 96 (for example, only 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation). In some embodiments, the humanized anti-TfR antibody described herein comprises a heavy chain comprising one of the VH variants listed in Table 3 and a heavy chain containing a heavy chain constant region as represented by SEQ ID NO: 96.

[0175] 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 only 25 amino acid variations compared to SEQ ID NO: 83 (for example, only 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation). 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 as represented by SEQ ID NO: 83.

[0176] 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] [Table 5-3] [Table 5-4] * The mutation location follows the Kabat numbering of each VH sequence containing the mutation. ** CDRs using the Kabat numbering system are shown in bold, and VH / VL titles are underlined.

[0177] In some embodiments, the humanized anti-TfR antibody of the present disclosure comprises a heavy chain containing only 25 amino acid variations (for example, only 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) compared to the heavy chain as represented by any one of SEQ ID NOs. Alternatively or in addition (for example, in addition), the humanized anti-TfR antibody of the present disclosure comprises a light chain containing only 25 amino acid variations (for example, only 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) compared to the light chain represented by any one of SEQ ID NOs. 85, 89, 90, 93, and 95.

[0178] 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 in addition (e.g., in addition), 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 in addition (e.g., in addition), 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.

[0179] 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 (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: 97, and a light chain having the amino acid sequence of SEQ ID NO: 85.

[0180] 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.

[0181] 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: 99, 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: 99, and a light chain having the amino acid sequence of SEQ ID NO: 85.

[0182] 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.

[0183] 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.

[0184] In some embodiments, the humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising 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 comprising 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 present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 101 and a light chain comprising the amino acid sequence of SEQ ID NO: 89.

[0185] In some embodiments, the humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising 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 comprising 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 present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 101 and a light chain comprising the amino acid sequence of SEQ ID NO: 90.

[0186] In some embodiments, the humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising an amino acid sequence that is at least x0% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 102, and / or (e.g., and) a light chain comprising an amino acid sequence 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 present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 102 and a light chain comprising the amino acid sequence of SEQ ID NO: 93.

[0187] It should be noted that in the original text, the "x0%" in the description of SEQ ID NO: 102 in item seems to be an incorrect expression. It is likely a typo and should be "80%" as in other similar descriptions. The translation is based on the corrected understanding.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: 103, 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: 95. In some embodiments, the humanized anti-TfR antibody of the Disclosure comprises a heavy chain having the amino acid sequence of SEQ ID NO: 103, and a light chain having the amino acid sequence of SEQ ID NO: 95.

[0188] 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: 102, 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: 95. In some embodiments, the humanized anti-TfR antibody of the Disclosure comprises a heavy chain having the amino acid sequence of SEQ ID NO: 102, and a light chain having the amino acid sequence of SEQ ID NO: 95.

[0189] 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 (e.g., Fab, Fab', F(ab')2, Fv), single-chain antibodies, bispecific antibodies, or nanobodies. In some embodiments, the humanized anti-TfR antibody described herein is 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, or a portion thereof, e.g., the Fc portion, as represented by SEQ ID NO: 81 or SEQ ID NO: 82) at either the N-terminus or the C-terminus.

[0190] 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).

[0191] 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 light and heavy chain assembly, or to change antibody stability (e.g., increased or decreased), or to facilitate linker conjugation.

[0192] 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).

[0193] 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.

[0194] 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.

[0195] 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).

[0196] 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 C1q 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.

[0197] 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.

[0198] 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.

[0199] 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".

[0200] 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 (e.g., and) light chain sequences. In some embodiments, the anti-TfR1 antibody described herein includes either one of the VH and VL sequences, either one of the IgG heavy chain and light chain sequences, or either one of the Fab heavy chain and light chain sequences described herein, and further includes a signal peptide (e.g., an N-terminal signal peptide). In some embodiments, the signal peptide includes the amino acid sequence MGWSCIILFLVATATGVHS (SEQ ID NO: 104).

[0201] Other known anti-transferrin receptor antibodies Any other suitable anti-transferrin receptor antibody known in the art may be used as a muscle targeting agent in the conjugate disclosed herein. Examples of known anti-transferrin receptor antibodies (including relevant references and binding epitopes) are listed in Table 6. In some embodiments, the anti-transferrin receptor antibody comprises the complementarity determining region (CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3) of any of the anti-transferrin receptor antibodies provided herein, for example, the anti-transferrin receptor antibodies listed in Table 6.

[0202] Table 6 - List of anti-transferrin receptor antibody clones, including relevant references and binding epitope information. [Table 6-1] [Table 6-2] [Table 6-3]

[0203] 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 6. 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 6. 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 6. 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 6. 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 6.

[0204] 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 6. 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.

[0205] Consequently, 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 6), insofar as immunospecific binding to the transferrin receptor (for example, human transferrin receptor) is maintained (for example, substantially, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% compared to the binding of the original antibody from which it is derived). In some embodiments, the 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 6), insofar as immunospecific binding to the transferrin receptor (for example, human transferrin receptor) is maintained (for example, substantially, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% compared to the binding of the original antibody from which it is derived). In some embodiments, the amino 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 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 6), insofar as immunospecific binding to the transferrin receptor (for example, human transferrin receptor) is maintained (for example, substantially maintained by, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 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 extended by 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 6), insofar as immunospecific binding to the transferrin receptor (for example, human transferrin receptor) is maintained (for example, substantially maintained by, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 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 6), as long as immunospecific binding to the transferrin receptor (for example, human transferrin receptor) is maintained (for example, substantially maintained compared to the binding of the original antibody from which it is derived, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). In some embodiments, the carboxyl 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 6), insofar as immunospecific binding to the transferrin receptor (for example, human transferrin receptor) is maintained (for example, substantially, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% compared to the binding of the original antibody from which it is derived).For example, any of the methods described in the art, including binding assays and conditions, can be used to determine whether immunospecific binding to a transferrin receptor (e.g., human transferrin receptor) is maintained.

[0206] 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 6. For example, an antibody may contain one or more CDR sequences from any of the anti-transferrin receptor antibodies selected from Table 6 that contain up to 5, 4, 3, 2, or 1 amino acid residue variation 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 6), insofar as immunospecific binding to the transferrin receptor (e.g., human transferrin receptor) is maintained (e.g., substantially, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% compared to the binding of the original antibody from which it is derived). In some embodiments, any of the amino acid variations in any of the CDRs provided herein 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 6. 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 6.

[0207] 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 6. 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 6.

[0208] 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 6. 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 6.

[0209] In some embodiments, an anti-transferrin receptor antibody that specifically binds to a transferrin receptor (e.g., human transferrin receptor) comprises a light chain variable VL domain of any of the anti-transferrin receptor antibodies selected from Table 6, 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., 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 6. 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 6. 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 any one of the anti-transferrin receptor antibodies selected from Table 6. In some embodiments, the light chain variable framework region derived from the above amino acid sequence consists of the above amino acid sequence, except for the presence of substitutions, deletions, and / or (for example, and) insertions, preferably up to 10 substitutions. In some embodiments, the light chain variable framework region derived from the above amino acid sequence consists of the above 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.

[0210] In some embodiments, anti-transferrin receptor antibodies that specifically bind to the transferrin receptor are shown in the table. 6 The selected anti-transferrin receptor antibody comprises CDR-L1, CDR-L2, and CDR-L3 of any of the anti-transferrin receptor antibodies, such as one of the anti-transferrin receptor antibodies selected from. In some embodiments, the antibody further comprises one, two, three, or all four VL framework regions derived from the VL of a human or primate antibody. The primate or human light chain framework 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 has a light chain complementarity determining region that is identical to any of the antibodies provided herein (e.g., Table 6 The amino acids in the light chain complementarity-determining region of any of the anti-transferrin receptor antibodies selected from may be the same number or substantially the same number of amino acids. In some embodiments, the amino acid residues in the light chain framework region of primates or humans are as follows: 6 The antitransferrin receptor antibody is derived from a natural primate or human antibody light chain framework region having at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, or at least 99% (or more) identity with any of the light chain framework regions of any of the antitransferrin receptor antibodies, such as one of the antitransferrin receptor antibodies selected from the range of antibodies. In some embodiments, the antitransferrin 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 antitransferrin receptor antibody further comprises one, two, three, or all four VL framework regions derived from the human light chain variable lambda subfamily.

[0211] In some embodiments, any of the anti-transferrin receptor antibodies provided herein further comprises a light chain variable domain including 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 is a table 6 It 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 antibodies, such as any one of the anti-transferrin receptor antibodies selected from.

[0212] 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 6.

[0213] 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 6, 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, 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.

[0214] 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)).

[0215] Table 7 provides heavy and light chain CDRs of antibodies according to various definition systems. Various definition systems, such as the Kabat definition, Chothia definition, and / or Contact definition, are described. For example, see (for example, 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 7. Heavy and light chain CDRs of mouse transferrin receptor antibodies. [Table 7]

[0216] Heavy chain variable domain (VH) and light chain variable domain sequences are also provided:

[0217] VH QVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGEINPTNGRTNYIEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARGTRAYHYWGQGTSVTVSS(Sequence ID 124)

[0218] VL DIQMTQSPASLSVSVGETVTITCRASDNLYSNLAWYQQKQGKSPQLLVYDATNLADGVPSRFSGSGSGTQYSLKINSLQSEDFGTYYCQHFWGTPLTFGAGTKLELK(Sequence ID 125)

[0219] In some aspects, the transferrin receptor antibodies of this disclosure include CDR-H1, CDR-H2, and CDR-H3, which are the same as CDR-H1, CDR-H2, and CDR-H3 shown in Table 7. Alternatively or in addition (for example, in addition), the transferrin receptor antibodies of this disclosure include CDR-L1, CDR-L2, and CDR-L3, which are the same as CDR-L1, CDR-L2, and CDR-L3 shown in Table 7.

[0220] In some embodiments, the transferrin receptor antibodies of the present disclosure comprise CDR-H1, CDR-H2, and CDR-H3, which together contain only 5 amino acid variations (e.g., only 5, 4, 3, 2, or 1 amino acid variation) compared to CDR-H1, CDR-H2, and CDR-H3 shown in Table 7. "Together" means that the total number of amino acid variations in all three heavy chain CDRs is within the defined range. 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, which together contain only 5 amino acid variations (e.g., only 5, 4, 3, 2, or 1 amino acid variation) compared to CDR-L1, CDR-L2, and CDR-L3 shown in Table 7.

[0221] 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 7. 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 7.

[0222] 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 7. 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 7. 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 7, 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).

[0223] 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 7 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 7 by at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) in combination.

[0224] 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.

[0225] In some embodiments, the transferrin receptor antibody of the present disclosure comprises a VH containing only 25 amino acid variations (e.g., only 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) compared to VH as 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 containing only 1 only 5 amino acid variations (e.g., only 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, only 5, 4, 3, 2, or 1 amino acid variation) compared to VL as represented by SEQ ID NO: 125.

[0226] 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: 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 as represented by SEQ ID NO: 125.

[0227] In some embodiments, the transferrin receptor antibodies of the present disclosure are humanized antibodies (e.g., humanized variants of antibodies). In some embodiments, the transferrin receptor antibodies of the present disclosure 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 7, and include a humanized heavy chain variable region and / or (e.g., and) a humanized light chain variable region.

[0228] 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.

[0229] In some embodiments, humanization is achieved by conjugating a CDR (e.g., as shown in Table 7) into the IGKV1-NL1*01 and IGHV1-3*01 human variable domains. In some embodiments, the anti-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 VL as represented by SEQ ID NO: 125), and / or (e.g., and), 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 VH as represented by SEQ ID NO: 124). In some embodiments, the anti-transferrin receptor antibody of the present disclosure is a humanized variant comprising amino acid substitutions at all of positions 9, 13, 17, 18, 40, 45, and 70 (compared to VL as represented by SEQ ID NO: 125), and / or (for example, and), amino acid substitutions at all of positions 1, 5, 7, 11, 12, 20, 38, 40, 44, 66, 75, 81, 83, 87, and 108 (compared to VH as represented by SEQ ID NO: 124).

[0230] In some embodiments, the anti-transferrin receptor antibody of the present disclosure is a humanized antibody containing residues at positions 43 and 48 of VL as represented by SEQ ID NO: 125. Alternatively or in addition (for example, in addition), the anti-transferrin receptor antibody of the present disclosure is a humanized antibody containing residues at positions 48, 67, 69, 71, and 73 of VH as represented by SEQ ID NO: 124.

[0231] The VH and VL amino acid sequences of examples of humanized antibodies that may be used in accordance with this disclosure are provided below:

[0232] Humanized VH EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQRLEWIGEINPTNGRTNYIEKFKSRATLTVDKSASTAYMELSSLRSEDTAVYYCARGTRAYHYWGQGTMVTVSS(Sequence ID 128)

[0233] Humanized VL DIQMTQSPSSLSASVGDRVTITCRASDNLYSNLAWYQQKPGKSPKLLVYDATNLADGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQHFWGTPLTFGQGTKVEIK(Sequence ID 129)

[0234] In some embodiments, the anti-transferrin receptor antibody of the present disclosure comprises VH, which comprises the amino acid sequence of SEQ ID NO: 128. Alternatively or in addition (for example, in addition), the anti-transferrin receptor antibody of the present disclosure comprises VL, which comprises the amino acid sequence of SEQ ID NO: 129.

[0235] In some embodiments, the anti-transferrin receptor antibody of the present disclosure comprises a VH containing only 25 amino acid variations (e.g., only 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) compared to VH as represented by SEQ ID NO: 128. Alternatively or in addition (e.g., in addition), the anti-transferrin receptor antibody of the present disclosure comprises a VL containing only 15 amino acid variations (e.g., only 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) compared to VL as represented by SEQ ID NO: 129.

[0236] In some embodiments, the anti-transferrin receptor antibody of the present disclosure comprises a VH having at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) the same amino acid sequence as VH as represented by SEQ ID NO: 128. Alternatively or in addition (e.g., in addition), the anti-transferrin receptor antibody of the present disclosure comprises a VL having at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) the same amino acid sequence as VL as represented by SEQ ID NO: 129.

[0237] In some embodiments, the anti-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 as represented by SEQ ID NO: 125), and / or (for example, and), one or more amino acid substitutions at positions 48, 67, 69, 71, and 73 (compared to VH as represented by SEQ ID NO: 124). In some embodiments, the anti-transferrin receptor antibody of the present disclosure is a humanized variant comprising S43A and / or (for example, and), V48L mutations (compared to VL as represented by SEQ ID NO: 125), and / or (for example, and), one or more A67V, L69I, V71R, and K73T mutations (compared to VH as represented by SEQ ID NO: 124).

[0238] In some embodiments, the anti-transferrin receptor antibodies of the present disclosure are humanized variants comprising one or more amino acid substitutions at positions 9, 13, 17, 18, 40, 43, 48, 45, and 70 (compared to VL as represented by SEQ ID NO: 125), and / or (for 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 as represented by SEQ ID NO: 124).

[0239] 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.

[0240] 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.

[0241] 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)

[0242] 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)

[0243] 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.

[0244] Examples of the heavy and light chain amino acid sequences of the described anti-transferrin receptor antibodies are provided below:

[0245] Heavy chain (VH + human IgG1 constant region) QVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGEINPTNGRTNYIEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARGTRAYHYWGQGTSVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(Sequence ID 132)

[0246] Lightweight chain (VL + Kappa lightweight chain) DIQMTQSPASLSVSVGETVTITCRASDNLYSNLAWYQQKQGKSPQLLVYDATNLADGVPSRFSGSGSGTQYSLKINSLQSEDFGTYYCQHFWGTPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence ID 133)

[0247] Heavy chain (humanized VH + human IgG1 constant region) EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQRLEWIGEINPTNGRTNYIEKFKSRATLTVDKSASTAYMELSSLRSEDTAVYYCARGTRAYHYWGQGTMVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(Sequence ID 134)

[0248] Light chain (humanized VL + kappa light chain) DIQMTQSPSSLSASVGDRVTITCRASDNLYSNLAWYQQKPGKSPKLLVYDATNLADGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQHFWGTPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence ID 135)

[0249] In some embodiments, the anti-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 anti-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 anti-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 anti-transferrin receptor antibody described herein comprises a light chain having the amino acid sequence of SEQ ID NO: 133.

[0250] In some embodiments, the anti-transferrin receptor antibody of the present disclosure comprises a heavy chain containing only 25 amino acid variations (e.g., only 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) compared to the heavy chain as represented by SEQ ID NO: 132. Alternatively or in addition (e.g., in addition), the anti-transferrin receptor antibody of the present disclosure comprises a light chain containing only 15 amino acid variations (e.g., only 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) compared to the light chain as represented by SEQ ID NO: 133.

[0251] In some embodiments, the anti-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 anti-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 anti-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 anti-transferrin receptor antibody described herein comprises a light chain having the amino acid sequence of SEQ ID NO: 135.

[0252] In some embodiments, the anti-transferrin receptor antibody of the present disclosure comprises a heavy chain containing only 25 amino acid variations (e.g., only 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) compared to the heavy chain of a humanized antibody as represented by SEQ ID NO: 134. Alternatively or in addition (e.g., in addition), the anti-transferrin receptor antibody of the present disclosure comprises a light chain containing only 15 amino acid variations (e.g., only 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) compared to the light chain of a humanized antibody as represented by SEQ ID NO: 135.

[0253] In some embodiments, anti-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 anti-transferrin receptor antibodies described herein are provided below:

[0254] Heavy chain FAB (VH + some human IgG1 constant region) QVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGEINPTNGRTNYIEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARGTRAYHYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP(Sequence ID 136)

[0255] Heavy chain FAB (humanized VH + some human IgG1 constant region) EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQRLEWIGEINPTNGRTNYIEKFKSRATLTVDKSASTAYMELSSLRSEDTAVYYCARGTRAYHYWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP(Sequence ID 137)

[0256] 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.

[0257] 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.

[0258] The anti-transferrin receptor antibodies described herein may be in any form of antibody, 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 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 anti-transferrin receptor antibodies described herein are scFv condensed with a constant region (e.g., the human IgG1 constant region as represented by SEQ ID NO: 130).

[0259] 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 cell suspension culture (e.g., CHO-K1 cells Cat. No. 85051005 derived from the European Collection of Animal Cell Culture).

[0260] 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, antibodies identified as having a sequence containing an N-terminal glutamate or glutamine residue should be understood to encompass antibodies that have undergone pyroglutamate formation resulting from post-translational modification. In some embodiments, pyroglutamate formation occurs in the heavy chain sequence. In some embodiments, pyroglutamate formation occurs in the light chain sequence.

[0261] 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.

[0262] 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).

[0263] 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.

[0264] 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).

[0265] 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.

[0266] 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.

[0267] 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 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).

[0268] 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 C1q binding and / or (e.g., 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 (e.g., and) to increase the antibody's affinity for the Fcγ receptor. This approach is described in further international publication WO 00 / 42072.

[0269] 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.

[0270] 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.

[0271] 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 subclass of any isotype. The antibody may 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.

[0272] 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 mentioned 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 sensor." Biomol This is described in Eng 2002;18:269-72; 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.

[0273] 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."

[0274] 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; this entire content is incorporated by reference thereto. Here, a 12-amino acid peptide with 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.

[0275] 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 which is incorporated herein by reference. Nonspecific cell binders were selected by pre-incubating random 12-mer peptide phage display libraries with a mixture of non-muscle cell types. Following rounds of selection, the 12-amino acid peptide TARGEHKEEELI (SEQ ID NO: 140) appeared most frequently. Consequently, in some embodiments, muscle targeting agents contain the amino acid sequence TARGEHKEEELI (SEQ ID NO: 140).

[0276] 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 a high degree of hydrophobic amino acid properties (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 are characterized 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 to date (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 approximately 2–25 amino acids, approximately 2–20 amino acids, approximately 2–15 amino acids, approximately 2–10 amino acids, or approximately 2–5 amino acids. The muscle-targeting peptides may contain naturally occurring amino acids, such as cysteine ​​and alanine, or amino acids that do not exist naturally, or modified amino acids.Amino acids that do not exist in nature 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).

[0277] 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 that binds to an internalized cell surface receptor expressed by muscle cells, for example, transferrin. 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.

[0278] 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, AC and Levy, M. "Aptamers and aptamer targeted delivery" RNA biology, 2009, 6:3, 316-20.; Germer, K. et al. "RNA aptamers and their therapeutic and diagnostic applications." Int. J. Biochem. Mol. Biol. 2013; 4:27-40). In some embodiments, muscle-targeted aptamers have been disclosed to date (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.

[0279] 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.

[0280] 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.

[0281] 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.

[0282] 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).

[0283] 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.

[0284] B. Molecular payload Several aspects of this disclosure provide molecular payloads for modulating biological outcomes, such as the transcription of DNA 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-related muscle cells), proteins (e.g., proteins that bind to nucleic acids or proteins in disease-related muscle cells), or small molecules (e.g., small molecules that modulate the function of nucleic acids or proteins in disease-related muscle cells). In some embodiments, the molecular payload is an oligonucleotide comprising a chain having a region complementary to a DMPK allele containing a disease-related repeat extension. 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.

[0285] i. oligonucleotides Any preferred oligonucleotide may be used as a molecular payload as described herein. In some embodiments, the oligonucleotide may be designed to induce mRNA degradation (e.g., the oligonucleotide may be a degradation-inducing gapmer, siRNA, ribozyme, or aptamer). In some embodiments, the oligonucleotide may be designed to block the translation of mRNA (e.g., the oligonucleotide may be a translation-blocking mixmer, siRNA, or aptamer). In some embodiments, the oligonucleotide may be designed to induce mRNA degradation and thereby block its translation. In some embodiments, the oligonucleotide may be a guide nucleic acid (e.g., guide RNA) for directing the activity of an enzyme (e.g., a gene editing enzyme). Other examples of oligonucleotides are provided herein. In some embodiments, it should be understood that by incorporating a functional sequence (e.g., an antisense strand sequence) from one format into the other format, an oligonucleotide of one format (e.g., an antisense oligonucleotide) may be suitably adapted to another format (e.g., an siRNA oligonucleotide).

[0286] Examples of oligonucleotides useful for targeting DMPK include U.S. Patent Application Publication No. 20100016215A1, published January 1, 2010, entitled "Compound And Method For Treating Myotonic Dystrophy"; U.S. Patent Application Publication No. 20130237585A1, published July 19, 2010, entitled "Modulation Of Dystrophia Myotonica-Protein Kinase (DMPK) Expression"; U.S. Patent Application Publication No. 20150064181A1, published March 5, 2015, entitled "Antisense Conjugates For Decreasing Expression Of Dmpk"; U.S. Patent Application Publication No. 20150238627A1, published August 27, 2015, entitled "Peptide-Linked Morpholino Antisense Oligonucleotides For Treatment Of Myotonic Dystrophy"; and "Compounds And This is provided in U.S. Patent Application Publication 20160304877A1, published on October 20, 2016, entitled “Methods For Modulation Of Dystrophia Myotonica-Protein Kinase (Dmpk) Expression,” and the entire contents of each of these are incorporated herein by reference.

[0287] Examples of oligonucleotides for promoting DMPK gene editing include U.S. Patent Application Publication No. 20170088819A1, published on March 3, 2017, entitled "Genetic Correction Of Myotonic Dystrophy Type 1," and International Patent Application Publication No. WO18002812A1, published on April 1, 2018, entitled "Materials And Methods For Treatment Of Myotonic Dystrophy Type 1 (DM1) And Other Related Disorders," the entire contents of each of these publications are incorporated herein by reference.

[0288] In some embodiments, the oligonucleotide may have a region complementary to the following sequence, which is an example of the human DMPK gene sequence (Gene ID 1760; NM_001081560.2):

[0289] In some embodiments, the oligonucleotide may have a region complementary to the sequence shown below, which is an example of the mouse DMPK gene sequence (Gene ID 13400;NM_001190490.1):

[0290] In some embodiments, the oligonucleotide may have a region complementary to a mutant of DMPK, for example, the mutant reported in Botta A. et al. "The CTG repeat expansion size correlates with the splicing defects observed in muscles from myotonic dystrophy type 1 patients." J Med Genet. 2008 Oct;45(10):639-46.; and Machuca-Tzili L. et al. "Clinical and molecular aspects of the myotonic dystrophies: a review." Muscle Nerve. 2005 Jul;32(1):1-18. (The entire contents of each of these are incorporated herein).

[0291] 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.

[0292] In some embodiments, the oligonucleotides provided herein are antisense oligonucleotides that target DMPK. In some embodiments, oligonucleotide targeting is one of the antisense oligonucleotides that target DMPK (e.g., Gapmer) described in U.S. Patent Application Publication US20160304877A1, published October 20, 2016, entitled "Compounds And Methods For Modulation Of Dystrophia Myotonica-Protein Kinase (DMPK) Expression," which is incorporated herein by reference. In some embodiments, the DMPK-targeting oligonucleotide targets a region of the DMPK gene sequence represented by Genbank accession number NM_001081560.2 (SEQ ID NO: 131) or Genbank accession number NG_009784.1.

[0293] In some embodiments, the DMPK-targeted oligonucleotide comprises a nucleotide sequence comprising a region complementary to a target region which is at least 10 consecutive nucleotides in SEQ ID NO: 131 (for example, at least 10, at least 12, at least 14, at least 16, or more consecutive nucleotides).

[0294] In some embodiments, DMPK-targeted oligonucleotides include a gapmer motif. “Gapmer” refers to a chimeric antisense compound in which an internal region having multiple nucleotides supporting RNase H cleavage is located between an external region having one or more nucleotides, where the nucleotides containing the internal region are chemically distinct from the nucleotides or the nucleotides containing the external region. The internal region may be referred to as the “gap segment,” and the external region as the “wing segment.” In some embodiments, DMPK-targeted oligonucleotides include one or more modified nucleotides and / or (for example, and) one or more intermodified nucleotide links. In some embodiments, the internucleotide links are phosphorothioate links. In some embodiments, the oligonucleotide includes a complete phosphorothioate backbone. In some embodiments, the oligonucleotide is a DNA gapmer with a cET end (for example, 3-10-3; cET-DNA-cET). In some embodiments, the DMPK-targeted oligonucleotide comprises one or more 6'-(S)-CH3 bicyclic nucleotides, one or more β-D-2'-deoxyribonucleotides, and / or (for example, and) one or more 5-methylcytosine nucleotides.

[0295] In some embodiments, the DMPK-targeted oligonucleotide is a gapmer having the formula 5'-XYZ-3', with X and Z as wing segments and a gap segment Y. In some embodiments, the DMPK-targeted oligonucleotide is a gapmer having the formula 5'-4-8-4-3'. In some embodiments, the DMPK-targeted oligonucleotide is a gapmer having the formula 5'-5-10-5-3'. In some embodiments, the DMPK-targeted oligonucleotide is a gapmer having the formula 5'-3-10-3-3'. In some embodiments, the DMPK-targeted oligonucleotide is a gapmer comprising one or more 5-methylcytosine nucleotide, 2'OMe nucleotide, 2'fluoronucleotide, LNA, and / or (for example, and) 2'-O-methoxyethyl (2'-MOE) nucleotide. In some embodiments, the DMPK-targeted oligonucleotide is a gapmer comprising one or more modified nucleotide linkages (for example, phosphorothioate linkages). In some embodiments, the DMPK-targeted oligonucleotide is a gapmer comprising a complete phosphorothioate backbone.

[0296] In some embodiments, any one of the DMPK-targeted oligonucleotides may be in salt form, for example, as a sodium, potassium, or magnesium salt.

[0297] 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 These are independently hydrogen or substituted or unsubstituted alkyl. In certain embodiments, the spacer is substituted or unsubstituted alkylene, substituted or unsubstituted heterocyclene, substituted or unsubstituted heteroarylene, -O-, -N(R A )-, or -C(=O)N(R A )2, or a combination thereof.

[0298] 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.

[0299] In some embodiments, oligonucleotides are conjugated to targeted agents, such as muscle targeting agents like anti-TfR antibodies, via, for example, an amine group.

[0300] 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.

[0301] 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 normal function of the target (e.g., mRNA) causing loss of activity (e.g., inhibition of translation) or loss of expression (e.g., degradation of 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.

[0302] 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 oligonucleotide complementary to the target nucleic acid has a length of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides. In some embodiments, the complementary region is complementary to at least 8 consecutive nucleotides of the target nucleic acid. In some embodiments, the oligonucleotide may contain one, two, or three base mismatches compared to a portion of the target nucleic acid's sequence of nucleotides. In some embodiments, the oligonucleotide may have up to three mismatches over 15 bases, or up to two mismatches over 10 bases.

[0303] In some embodiments, the oligonucleotide comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive nucleotides of a sequence containing any one of sequence numbers 148-383 and 621-638. In some embodiments, the oligonucleotide comprises a sequence containing any one of sequence numbers 148-383 and 621-638. In some embodiments, the oligonucleotide comprises a sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, or 97% sequence identity with at least 12 or at least 15 consecutive nucleotides of any one of sequence numbers 148-383 and 621-638.

[0304] In some embodiments, the oligonucleotide comprises a sequence that targets a DMPK sequence containing any one of sequence numbers 384-619. In some embodiments, the oligonucleotide comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides (e.g., a sequence of nucleotides) that are complementary to a DMPK sequence containing any one of sequence numbers 384-619. In some embodiments, the oligonucleotide comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, or 97% complementary to at least 12 or at least 15 sequence of nucleotides from any one of sequence numbers 384-619.

[0305] In some embodiments, the oligonucleotide is complementary to any one of the target sequences of the oligonucleotides provided herein (e.g., oligonucleotides listed in Table 8 or Table 17) (e.g., at least 85%, at least 90%, at least 95%, or 100%). In some embodiments, such target sequences are 100% complementary to the oligonucleotides listed in Table 8 or Table 17.

[0306] In some embodiments, one or more thymine bases (T) on any one of the oligonucleotides provided herein (for example, the oligonucleotides listed in Table 8 or Table 17) may optionally be uracil bases (U), and / or one or more U may optionally be T.

[0307] b. Oligonucleotide modification: The oligonucleotides described herein may be modified, including, for example, modified sugar moieties, modified nucleoside linkages, modified nucleotides, and / or combinations thereof. In addition, in some embodiments, oligonucleotides may exhibit one or more of the following properties: non-mediating of alternative splicing; non-immunostimulant; nuclease-resistant; having improved cellular uptake compared to unmodified oligonucleotides; non-toxic to cells or mammals; having improved exit into endosomes within cells; minimizing TLR stimulation; or evading pattern recognition receptors. Any modified chemical properties or formats of the oligonucleotides described herein may be combined with each other. For example, 1, 2, 3, 4, 5, or more different types of modifications may be contained within the same oligonucleotide.

[0308] In some embodiments, specific nucleotide modifications may be used to make the oligonucleotide into which the modification is incorporated more resistant to nuclease digestion than the native oligodeoxynucleotide or oligoribonucleotide molecule; these modified oligonucleotides remain intact for longer periods than unmodified oligonucleotides. Specific examples of modified oligonucleotides include those with modified backchains, such as phosphorothioates, phosphotriesters, methylphosphonates, short-chain alkyl or cycloalkyl sugar linkages, or modified nucleoside linkages such as short-chain heteroatom or heterocyclic sugar linkages. Consequently, the oligonucleotides of this disclosure can be stabilized against nucleolysis by modification, such as the incorporation of nucleotide modifications.

[0309] In some embodiments, the oligonucleotide may be an oligonucleotide with a length of up to 50 nucleotides or up to 100 nucleotides, where 2-10, 2-15, 2-16, 2-17, 2-18, 2-19, 2-20, 2-25, 2-30, 2-40, 2-45 nucleotides, or more, are modified nucleotides. The oligonucleotide may be an oligonucleotide with a length of 8-30 nucleotides, where 2-10, 2-15, 2-16, 2-17, 2-18, 2-19, 2-20, 2-25, 2-30 nucleotides are modified nucleotides. The oligonucleotide may be an oligonucleotide with a length of 8-15 nucleotides, where 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 2-11, 2-12, 2-13, 2-14 nucleotides are modified nucleotides. Optionally, any oligonucleotide may be modified except for nucleotides 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Oligonucleotide modifications are further described herein.

[0001] c. Modified nucleoside In some embodiments, the oligonucleotides described herein comprise at least one nucleoside modified at the 2' position of a sugar. In some embodiments, the oligonucleotide comprises at least one 2'-modified nucleoside. In some embodiments, all nucleosides on the oligonucleotide are 2'-modified nucleosides.

[0002] In some embodiments, the oligonucleotides described herein include one or more non-bicyclic 2'-modified nucleotides, for example, modified nucleosides of 2'-deoxy, 2'-fluoro (2'-F), 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-O--N-methylacetamide (2'-O--NMA).

[0003] In some embodiments, the oligonucleotides described herein comprise one or more 2'-4' bicyclic nucleosides, wherein the ribose ring in the nucleoside comprises a crosslinking moiety connecting two atoms in the ring (e.g., a methylene (LNA) crosslink, an ethylene (ENA) crosslink, or a (S)-restricted ethyl (cEt) crosslink connecting a 2'-O atom to a 4'-C atom). An example of LNA is described in the international patent application publication WO / 2008 / 043753, published on April 17, 2008, entitled “RNA Antagonist Compounds For The Modulation Of PCSK9” (the contents of which are incorporated herein by reference in their entirety). Other modifications that may be used in the oligonucleotides disclosed herein include ethylene-crosslinked nucleic acids (ENAs). ENAs include, but are not limited to, 2'-O,4'-C-ethylene-crosslinked nucleic acids. Examples of ENA are provided in the international patent publication WO 2005 / 042777, published on 12 May 2005 and titled "APP / ENA Antisense"; Morita et al., Nucleic Acid Res., Suppl 1:241-242, 2001; Surono et al., Hum. Gene Ther., 15:749-757, 2004; Koizumi, Curr. Opin. Mol. Ther., 8:144-149, 2006; and Horie et al., Nucleic Acids Symp. Ser(Oxf), 49:171-172, 2005, the entirety of which disclosures are incorporated herein by reference. Examples of cEt are provided in U.S. Patents 7,101,993; 7,399,845 and 7,569,686, each of which is incorporated herein by reference in whole.

[0004] In some embodiments, the oligonucleotides include modified nucleosides disclosed in one of the following U.S. patents or patent application publications: U.S. Patent 7,399,845, issued July 15, 2008, titled "6-Modified Bicyclic Nucleic Acid Analogs"; U.S. Patent 7,741,457, issued June 22, 2010, titled "6-Modified Bicyclic Nucleic Acid Analogs"; U.S. Patent 8,022,193, issued September 20, 2011, titled "6-Modified Bicyclic Nucleic Acid Analogs"; U.S. Patent 7,569,686, issued August 4, 2009, titled "Compounds And Methods For Synthesis Of Bicyclic Nucleic Acid Analogs"; U.S. Patent 7,335,765, issued February 26, 2008, titled "Novel Nucleoside And Oligonucleotide" "Analogues"; U.S. Patent 7,314,923, issued January 1, 2008, titled "Novel Nucleoside And Oligonucleotide Analogues"; U.S. Patent 7,816,333, issued October 19, 2010, titled "Oligonucleotide Analogues And Methods Utilizing The Same"; and U.S. Publication No. 2011 / 0009471, currently U.S. Patent 8,957,201, issued February 17, 2015, titled "Oligonucleotide Analogues And Methods Utilizing The Same". For all purposes, the entire contents of each of these are incorporated herein by reference.

[0005] In some embodiments, the oligonucleotide contains at least one modified nucleoside, resulting in an increased Tm of the oligonucleotide in the range of 1°C, 2°C, 3°C, 4°C, or 5°C compared to an oligonucleotide without at least one modified nucleoside. The oligonucleotide may contain multiple modified nucleosides in total, resulting in an increased Tm of the oligonucleotide in the range of 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, or above, compared to an oligonucleotide without modified nucleosides.

[0006] Oligonucleotides may contain a mix of different types of nucleosides. For example, oligonucleotides may contain a mix of 2'-deoxyribonucleosides or ribonucleosides and 2'-fluoro-modified nucleosides. Oligonucleotides may contain a mix of deoxyribonucleosides or ribonucleosides and 2'-O-Me-modified nucleosides. Oligonucleotides may contain a mix of 2'-fluoro-modified nucleosides and 2'-O-Me-modified nucleosides. Oligonucleotides may contain a mix of 2'-4' bicyclic nucleosides and 2'MOE, 2'-fluoro, or 2'-O-Me-modified nucleosides. Oligonucleotides may contain a mix of non-bicyclic 2'-modified nucleosides (e.g., 2'-MOE, 2'-fluoro, or 2'-O-Me) and 2'-4' bicyclic nucleosides (e.g., LNA, ENA, cEt).

[0007] Oligonucleotides may contain alternating nucleosides of different types. For example, oligonucleotides may contain alternating 2'-deoxyribonucleosides or ribonucleosides and 2'-fluoro-modified nucleosides. Oligonucleotides may contain alternating deoxyribonucleosides or ribonucleosides and 2'-O-Me-modified nucleosides. Oligonucleotides may contain alternating 2'-fluoro-modified nucleosides and 2'-O-Me-modified nucleosides. Oligonucleotides may contain alternating 2'-4' bicyclic nucleosides and 2'-MOE, 2'-fluoro, or 2'-O-Me-modified nucleosides. Oligonucleotides may include alternating non-bicyclic 2'-modified nucleosides (e.g., 2'-MOE, 2'-fluoro, or 2'-O-Me) and 2'-4' bicyclic nucleosides (e.g., LNA, ENA, cEt).

[0008] In some embodiments, the oligonucleotides described herein include a 5'-vinylphosphonic acid modification, one or more unbase residues, and / or one or more reversed unbase residues.

[0009] d. Internucleoside linkage / main chain In some embodiments, the oligonucleotide may contain phosphorothioate or other modified nucleoside linkages. In some embodiments, the oligonucleotide contains phosphorothioate nucleoside linkages. In some embodiments, the oligonucleotide contains phosphorothioate nucleoside linkages between at least two nucleotides. In some embodiments, the oligonucleotide contains phosphorothioate nucleoside linkages between all nucleotides. For example, in some embodiments, the oligonucleotide contains modified nucleoside linkages at the 5' or 3' ends of the nucleotide sequence, at the first, second, and / or (for example, and) third nucleoside linkages.

[0010] Phosphorus-containing linkages that may be used include, but are not limited to, normal 3'-5' linkages, their 2'-5' linkage analogs, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotryesters, aminoalkyl phosphotryesters, methyl and other alkylphosphonates (including 3'-alkylene phosphonates and chiral phosphonates), phosphinates, phosphoramidates (including 3'-aminophosphoramidates and aminoalkylphosphoramidates), thionophosphoramidates, thionoalkyl phosphonates, thionoalkyl phosphotryesters, and boranophosphorates, and those having opposite polarity (where adjacent pairs of nucleoside units are 3'-5' to 5'-3' or (connected from 2'-5' to 5'-2') and encompassing; US Patent No. 3,687,808; No. 4,469,863; No. 4,476,301; No. 5,023,243; No. 5,177,196; No. 5,188,897; No. 5,264,423; No. 5,276,019; No. 5,278,302; No. 5,286,717; No. 5,321,131; No. 5,39 See issues 9,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050.

[0011] In some embodiments, the oligonucleotide may have a heteroatom backbone such as a methylene (methylimino) or MMI backbone; an amide backbone (see De Mesmaeker et al. Ace.Chem.Res. 1995, 28:366-374); a morpholino backbone (see Summerton and Weller, U.S. Patent No. 5,034,506); or a peptide nucleic acid (PNA) backbone (where the phosphodiester backbone of the oligonucleotide is replaced by a polyamide backbone, and the nucleotide is directly or indirectly bonded to the aza nitrogen atom of the polyamide backbone, see Nielsen et al., Science 1991, 254,1497).

[0310] e. Stereospecific oligonucleotides In some embodiments, the phosphorus atoms between nucleotides of the oligonucleotide are chiral, and the properties of the oligonucleotide are tuned based on the stereochemistry of the chiral phosphorus atoms. In some embodiments, suitable methods may be used to synthesize P-chiral oligonucleotide analogs in a stereocontrolled manner (e.g., as described in Oka N, Wada T, Stereocontrolled synthesis of oligonucleotide analogs containing chiral internucleotidic phosphorus atoms. Chem Soc Rev. 2011 Dec;40(12):5829-43). In some embodiments, phosphorothioate-containing oligonucleotides are provided, comprising nucleoside units linked together by either substantially all Sp phosphorothioate intersugar linkages or substantially all Rp phosphorothioate intersugar linkages. In some embodiments, such phosphorothioate oligonucleotides having substantially chiral pure intersugar linkages are prepared by enzymatic or chemical synthesis, for example, as described in U.S. Patent 5,587,261 issued December 12, 1996 (the contents of which are incorporated herein by reference in their entirety). In some embodiments, chiral-controlled oligonucleotides provide selective cleavage patterns for target nucleic acids. For example, in some embodiments, chiral-controlled oligonucleotides provide a single cleavage site within a complementary sequence of nucleic acids, as described, for example, in U.S. Patent Application Publication 20170037399 A1, titled "CHIRAL DESIGN," published on February 2, 2017 (the contents of which are incorporated herein by reference in their entirety).

[0311] f. morpholino In some embodiments, oligonucleotides may be morpholino-based compounds. Morpholino-based oligomeric compounds are described in Dwaine A. Braasch and David R. Corey, Biochemistry, 2002, 41(14), 4503-4510; Genesis, volume 30, issue 3, 2001; Heasman, J., Dev. Biol., 2002, 243, 209-214; Naseviius et al., Nat. Genet., 2000, 26, 216-220; Lacerra et al., Proc. Natl. Acad. Sci., 2000, 97, 9591-9596; and U.S. Patent No. 5,034,506 issued July 23, 1991. In some embodiments, morpholino-based oligomeric compounds are phosphorodiamidate morpholino oligomers (PMOs) (for example, as described in Iverson, Curr. Opin. Mol. Ther., 3:235-238, 2001; and Wang et al., J. Gene Med., 12:354-364, 2010; these disclosures are incorporated herein by reference in their entirety).

[0312] g. Peptide nucleic acid (PNA) In some embodiments, both the sugar and nucleoside linkages (back chain) of the nucleotide unit of the oligonucleotide are replaced with novel groups. In some embodiments, the base unit is maintained for hybridization with a suitable nucleic acid target compound. One such oligomeric compound, an oligonucleotide mimetic shown to have excellent hybridization properties, is called a peptide nucleic acid (PNA). In PNA compounds, the sugar-back chain of the oligonucleotide is replaced with an amide-containing back chain, e.g., an aminoethylglycine back chain. The nucleic acid base is retained and is directly or indirectly bonded to the aza nitrogen atom of the amide portion of the back chain. Representative publications reporting the preparation of PNA compounds include, but are not limited to, U.S. Patents 5,539,082; 5,714,331; and 5,719,262 (each incorporated herein by reference). Further teachings of PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.

[0313] h.Gapmer In some embodiments, the oligonucleotides described herein are gapmers. Gapmer oligonucleotides generally have the formula 5'-XYZ-3', with X and Z as flanking regions surrounding a gap region Y. In some embodiments, the flanking region X of formula 5'-XYZ-3' is also referred to as the X region, flanking sequence X, 5' wing region X, or 5' wing segment. In some embodiments, the flanking region Z of formula 5'-XYZ-3' is also referred to as the Z region, flanking sequence Z, 3' wing region Z, or 3' wing segment. In some embodiments, the gap region Y of formula 5'-XYZ-3' is also referred to as the Y region, Y segment, or gap segment Y. In some embodiments, each nucleoside in the gap region Y is a 2'-deoxyribonucleoside, and neither the 5' wing region X nor the 3' wing region Z contains any 2'-deoxyribonucleoside.

[0314] In some embodiments, the Y region is a sequence of nucleotides, e.g., a region of six or more DNA nucleotides, capable of recruiting an RNAse such as RNAse H. In some embodiments, a gapmer binds to a target nucleic acid, in which case an RNAse is recruited and then cleaved from the target nucleic acid. In some embodiments, the Y region is flanked at both the 5' and 3' ends by regions X and Z containing high-affinity modified nucleosides, e.g., one to six high-affinity modified nucleosides. Examples of high-affinity modified nucleosides include, but are not limited to, 2'-modified nucleosides (e.g., 2'-MOE, 2'O-Me, 2'-F) or 2'-4' bicyclic nucleosides (e.g., LNA, cEt, ENA). In some embodiments, the flanking sequences X and Z may be 1 to 20 nucleotides, 1 to 8 nucleotides, or 1 to 5 nucleotides in length. The flanking sequences X and Z may be of similar or dissimilar lengths. In some embodiments, the gap segment Y may be a nucleotide sequence having a length of 5–20 nucleotides, 5–15, 12 nucleotides, or 6–10 nucleotides.

[0012] In some embodiments, the gap region of the gapmer oligonucleotide may contain, in addition to DNA nucleotides, modified nucleotides known to be acceptable for efficient RNase H action, such as C4'-substituted nucleotides, acyclic nucleotides, and arabino-type nucleotides. In some embodiments, the gap region contains one or more unmodified internucleoside links. In some embodiments, one or both flanking regions each independently contain one or more phosphorothioate internucleoside links (e.g., phosphorothioate internucleoside links or other links) between at least two, at least three, at least four, at least five, or more nucleotides. In some embodiments, the gap region and the two flanking regions each independently contain modified internucleoside links (e.g., phosphorothioate internucleoside links or other links) between at least two, at least three, at least four, at least five, or more nucleotides.

[0315] Gapmers can be produced using appropriate methods. Representative U.S. patents, U.S. patent publications, and PCT publications teaching the preparation of gapmers are U.S. Patent Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,6 No. 23,065; No. 5,652,355; No. 5,652,356; No. 5,700,922; No. 5,898,031; No. 7,015,315; No. 7,1 No. 01,993; No. 7,399,845; No. 7.432,250; No. 7,569,686; No. 7,683,036; No. 7,750,131; No. 8,5 No. 80,756; No. 9,045,754; No. 9,428,534; No. 9,695,418; No. 10,017,764; No. 10,260,069; No. 9,428,534; No. 8,580,756; U.S. Patent Publications US20050074801, US20090221685, US200902869 This includes, but is not limited to, Patent Nos. 69, US20100197762, and US20110112170; PCT Publications W02004069991; W02005023825; W02008049085 and W02009090182; and European Patent No. EP2,149,605. Each of these is incorporated herein by reference in its entirety.

[0316] In some aspects, the gapmer has a length of 10–40 nucleosides. For example, the gapmer may have a length of 10–40, 10–35, 10–30, 10–25, 10–20, 10–15, 15–40, 15–35, 15–30, 15–25, 15–20, 20–40, 20–35, 20–30, 20–25, 25–40, 25–35, 25–30, 30–40, 30–35, or 35–40 nucleosides. In some embodiments, the gapmer has a length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleosides.

[0317] In some embodiments, the gap region Y on the gapmer has a length of 5–20 nucleosides. For example, the gap region Y may have a length of 5–20, 5–15, 5–10, 10–20, 10–15, or 15–20 nucleosides. In some embodiments, the gap region Y has a length of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleosides. In some embodiments, each nucleoside in the gap region Y is a 2'-deoxyribonucleoside. In some embodiments, all nucleosides in the gap region Y are 2'-deoxyribonucleosides. In some embodiments, one or more nucleosides in the gap region Y are modified nucleosides (for example, 2'-modified nucleosides, e.g., those described herein). In some embodiments, one or more cytosines in gap region Y are optionally 5-methylcytosine. In some embodiments, each cytosine in gap region Y is 5-methylcytosine.

[0318] In some embodiments, the 5' wing region of a gapmer (X in the 5'-XYZ-3' formula) and the 3' wing region of a gapmer (Z in the 5'-XYZ-3' formula) are independently 1 to 20 nucleoside lengths. For example, the 5' wing region of a gapmer (X in the 5'-XYZ-3' formula) and the 3' wing region of a gapmer (Z in the 5'-XYZ-3' formula) can independently be 1 to 20, 1 to 15, 1 to 10, 1 to 7, 1 to 5, 1 to 3, 1 to 2, 2 to 5, 2 to 7, 3 to 5, 3 to 7, 5 to 20, 5 to 15, 5 to 10, 10 to 20, 10 to 15, or 15 to 20 nucleoside lengths. In some embodiments, the 5' wing region of the gapmer (X in the 5'-XYZ-3' formula) and the 3' wing region of the gapmer (Z in the 5'-XYZ-3' formula) are independently the lengths of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleosides. In some embodiments, the 5' wing region of the gapmer (X in the 5'-XYZ-3' formula) and the 3' wing region of the gapmer (Z in the 5'-XYZ-3' formula) are the same length. In some embodiments, the 5' wing region of the gapmer (X in the 5'-XYZ-3' formula) and the 3' wing region of the gapmer (Z in the 5'-XYZ-3' formula) are of different lengths. In some embodiments, the 5' wing region of gapmer (X in the 5'-XYZ-3' equation) is longer than the 3' wing region of gapmer (Z in the 5'-XYZ-3' equation). In some embodiments, the 5' wing region of gapmer (X in the 5'-XYZ-3' equation) is shorter than the 3' wing region of gapmer (Z in the 5'-XYZ-3' equation).

[0319] In some aspects, gapmer is 5-10-5, 4-12-4, 3-14-3, 2-16-2, 1-18-1, 3-10-3, 2-10-2, 1-10-1, 2-8-2, 4-6-4, 3-6-3, 2-6-2, 4-7-4, 3-7-3, 2-7-2, 4-8-4, 3-8-3, 2-8-2, 1-8-1, 2-9-2, 1-9-1, 2-10-2, 1-10-1, 1-12-1, 1-16-1, 2-15-1, 1-15-2, 1-14-3, 3-14-1, 2-14-2, 1-13-4, 4-13-1, 2-13-3, 3-13-2, 1-12- 5, 5-12-1, 2-12-4, 4-12-2, 3-12-3, 1-11-6, 6-11-1, 2-11-5, 5-11-2, 3-11-4, 4-11-3, 1-17-1, 2-16-1, 1-16-2, 1-15-3, 3-15-1, 2-15-2, 1-14-4, 4-14 -1, 2-14-3, 3-14-2, 1-13-5, 5-13-1, 2-13-4, 4-13-2, 3-13-3, 1-12-6, 6-12-1, 2-12-5, 5-12-2, 3-12-4, 4-12-3, 1-11-7, 7-11-1, 2-11-6, 6-11-2, 3-1 1-5, 5-11-3, 4-11-4, 1-18-1, 1-17-2, 2-17-1, 1-16-3, 1-16-3, 2-16-2, 1-15-4, 4-15-1, 2-15-3, 3-15-2, 1-14-5, 5-14-1, 2-14-4, 4-14-2, 3-14-3, 1- 13-6, 6-13-1, 2-13-5, 5-13-2, 3-13-4, 4-13-3, 1-12-7, 7-12-1, 2-12-6, 6-12-2, 3-12-5, 5-12-3, 1-11-8, 8-11-1, 2-11-7, 7-11-2, 3-11-6, 6-11-3, 4 -11-5, 5-11-4, 1-18-1, 1-17-2, 2-17-1, 1-16-3, 3-16-1, 2-16-2, 1-15-4, 4-15-1, 2-15-3, 3-15-2, 1-14-5, 2-14-4, 4-14-2, 3-14-3, 1-13-6, 6-13-1, 2-13-5, 5-13-2, 3-13-4, 4-13-3, 1-12-7, 7-12-1, 2-12-6, 6-12-2, 3-12-5, 5-12-3, 1-11-8, 8-11-1, 2-11-7, 7-11-2, 3-11-6, 6-11-3, 4-11-5, 5-11-4,1-19-1、1-18-2、2-18-1、1-17-3、3-17-1、2-17-2、1-16-4、4-16-1、2-16-3、3-16-2、1-15-5、2-15-4、4-15-2、3-15-3、1-14-6、6-14-1、2-14-5、5-14-2、3-14-4、4-14-3、1-13-7、7-13-1、2-13-6、6-13-2、3-13-5、5-13-3、4-13-4、1-12-8、8-12-1、2-12-7、7-12-2、3-12-6、6-12-3、4-12-5、5-12-4、2-11-8、8-11-2、3-11-7、7-11-3、4-11-6、6-11-4、5-11-5、1-20-1、1-19-2、2-19-1、1-18-3、3-18-1、2-18-2、1-17-4、4-17-1、2-17-3、3-17-2、1-16-5、2-16-4、4-16-2、3-16-3、1-15-6、6-15-1、2-15-5、5-15-2、3-15-4、4-15-3、1-14-7、7-14-1、2-14-6、6-14-2、3-14-5、5-14-3、4-14-4、1-13-8、8-13-1、2-13-7、7-13-2、3-13-6、6-13-3、4-13-5、5-13-4、2-12-8、8-12-2、3-12-7、7-12-3、4-12-6、6-12-4、5-12-5、3-11-8、8-11-3、4-11-7、7-11-4、5-11-6、6-11-5、1-21-1、1-20-2、2-20-1、1-20-3、3-19-1、2-19-2、1-18-4、4-18-1、2-18-3、3-18-2、1-17-5、2-17-4、4-17-2、3-17-3、1-16-6、6-16-1、2-16-5、5-16-2、3-16-4、4-16-3、1-15-7、7-15-1、2-15-6、6-15-2、3-15-5、5-15-3、4-15-4、1-14-8、8-14-1、2-14-7、7-14-2、3-14-6、6-14-3、4-14-5、5-14-4、2-13-8、8-13-2、3-13-7、7-13-3、4-13-6、6-13-4、5-13-5、1-12-10、10-12-1、2-12-9、9-12-2、3-12-8、8-12-3、4-12-7、7-12-4、5-12-6、6-12-5、4-11-8, 8-11-4, 5-11-7, 7-11-5, 6-11-6, 1-22-1, 1-21-2, 2-21-1, 1-21-3, 3-20-1, 2-20-2, 1-19-4, 4-19-1, 2-19-3, 3-19-2, 1-18-5, 2-18-4, 4-18-2, 3-18-3, 1-17-6, 6-17-1, 2-17-5, 5-17-2, 3-17-4, 4-17-3, 1-16-7, 7-16-1, 2-16-6, 6-16-2, 3-16-5, 5-16-3, 4-16-4, 1 Includes 5'-XYZ-3' of -15-8, 8-15-1, 2-15-7, 7-15-2, 3-15-6, 6-15-3, 4-15-5, 5-15-4, 2-14-8, 8-14-2, 3-14-7, 7-14-3, 4-14-6, 6-14-4, 5-14-5, 3-13-8, 8-13-3, 4-13-7, 7-13-4, 5-13-6, 6-13-5, 4-12-8, 8-12-4, 5-12-7, 7-12-5, 6-12-6, 5-11-8, 8-11-5, 6-11-7, or 7-11-6. The numbers indicate the nucleoside numbers in the X, Y, and Z regions of the 5'-XYZ-3' gapmer.

[0320] In some embodiments, one or more nucleosides in the 5' wing region of the gapmer (X in the 5'-XYZ-3' formula) or the 3' wing region of the gapmer (Z in the 5'-XYZ-3' formula) are modified nucleotides (e.g., high-affinity modified nucleosides). In some embodiments, the modified nucleoside (e.g., high-affinity modified nucleosides) is a 2' modified nucleoside. In some embodiments, the 2' modified nucleoside is a 2'-4' bicyclic nucleoside or a non-bicyclic 2' modified nucleoside. In some embodiments, high affinity modified nucleosides are 2'-4' bicyclic nucleosides (e.g., LNA, cEt, or ENA) or non-bicyclic 2'-modified nucleosides (e.g., 2'-fluoro(2'-F), 2'-O-methyl(2'-O-Me), 2'-O-methoxyethyl(2'-MOE), 2'-O-aminopropyl(2'-O-AP), 2'-O-dimethylaminoethyl(2'-O-DMAOE), 2'-O-dimethylaminopropyl(2'-ODMAP), 2'-O-dimethylaminoethyloxyethyl(2'-O-DMAEOE), or 2'-ON-methylacetamide(2'-O-NMA)).

[0321] In some embodiments, one or more nucleosides in the 5' wing region of the gapmer (X in the 5'-XYZ-3' formula) are high-affinity modified nucleosides. In some embodiments, each nucleoside in the 5' wing region of the gapmer (X in the 5'-XYZ-3' formula) is a high-affinity modified nucleoside. In some embodiments, one or more nucleosides in the 3' wing region of the gapmer (Z in the 5'-XYZ-3' formula) are high-affinity modified nucleosides. In some embodiments, each nucleoside in the 3' wing region of the gapmer (Z in the 5'-XYZ-3' formula) is a high-affinity modified nucleoside. In some embodiments, one or more nucleosides in the 5' wing region of the gapmer (X in the 5'-XYZ-3' formula) are high-affinity modified nucleosides, and one or more nucleosides in the 3' wing region of the gapmer (Z in the 5'-XYZ-3' formula) are high-affinity modified nucleosides. In some embodiments, each nucleoside in the 5' wing region of the gapmer (X in the 5'-XYZ-3' formula) is a high-affinity modified nucleoside, and each nucleoside in the 3' wing region of the gapmer (Z in the 5'-XYZ-3' formula) is a high-affinity modified nucleoside.

[0322] In some embodiments, the 5' wing region of the gapmer (X in the 5'-XYZ-3' formula) contains the same high-affinity nucleoside as the 3' wing region of the gapmer (Z in the 5'-XYZ-3' formula). For example, the 5' wing region (X in the 5'-XYZ-3' formula) and the 3' wing region (Z in the 5'-XYZ-3' formula) of the gapmer may contain one or more non-bicyclic 2'-modified nucleosides (e.g., 2'-MOE or 2'-O-Me). In another example, the 5' wing region (X in the 5'-XYZ-3' formula) and the 3' wing region (Z in the 5'-XYZ-3' formula) of the gapmer may contain one or more 2'-4' bicyclic nucleosides (e.g., LNA or cEt). In some embodiments, each nucleoside in the 5' wing region of the gapmer (X in the 5'-XYZ-3' formula) and the 3' wing region of the gapmer (Z in the 5'-XY-Z-3' formula) is a non-bicyclic 2' modified nucleoside (e.g., 2'-MOE or 2'-O-Me). In some embodiments, each nucleoside in the 5' wing region of the gapmer (X in the 5'-XYZ-3' formula) and the 3' wing region of the gapmer (Z in the 5'-XYZ-3' formula) is a 2'-4' bicyclic nucleoside (e.g., LNA or cEt).

[0323] In some embodiments, the gapmer comprises a 5'-XYZ-3' configuration, where X and Z are independently nucleosides of length 1–7 (e.g., 1, 2, 3, 4, 5, 6, or 7), Y is a nucleoside of length 6–10 (e.g., 6, 7, 8, 9, or 10), each nucleoside of X and Z is a non-bicyclic 2' modified nucleoside (e.g., 2'-MOE or 2'-O-Me), and each nucleoside of Y is a 2' deoxyribonucleoside. In some embodiments, the gapmer comprises a 5'-XYZ-3' configuration, where X and Z are independently nucleosides of length 1–7 (e.g., 1, 2, 3, 4, 5, 6, or 7), Y is a nucleoside of length 6–10 (e.g., 6, 7, 8, 9, or 10), each nucleoside of X and Z is a 2'-4' bicyclic nucleoside (e.g., LNA or cEt), and each nucleoside of Y is a 2' deoxyribonucleoside. In some embodiments, the 5' wing region of the gapmer (X in the 5'-XYZ-3' formula) contains high-affinity nucleosides that are different from those in the 3' wing region of the gapmer (Z in the 5'-XYZ-3' formula). For example, the 5' wing region of a gapmer (X in the 5'-XYZ-3' formula) may contain one or more non-bicyclic 2'-modified nucleosides (e.g., 2'-MOE or 2'-O-Me), and the 3' wing region of a gapmer (Z in the 5'-XYZ-3' formula) may contain one or more 2'-4' bicyclic nucleosides (e.g., LNA or cEt). In another example, the 3' wing region of a gapmer (Z in the 5'-XYZ-3' formula) may contain one or more non-bicyclic 2'-modified nucleosides (e.g., 2'-MOE or 2'-O-Me), and the 5' wing region of a gapmer (X in the 5'-XYZ-3' formula) may contain one or more 2'-4' bicyclic nucleosides (e.g., LNA or cEt).

[0324] In some embodiments, the gapmer comprises a 5'-XYZ-3' configuration, where X and Z are independently nucleosides of length 1–7 (e.g., 1, 2, 3, 4, 5, 6, or 7), Y is a nucleoside of length 6–10 (e.g., 6, 7, 8, 9, or 10), each nucleoside of X is a non-bicyclic 2' modified nucleoside (e.g., 2'-MOE or 2'-O-Me), each nucleoside of Z is a 2'-4' bicyclic nucleoside (e.g., LNA or cEt), and each nucleoside of Y is a 2'-deoxyribonucleoside. In some embodiments, the gapmer comprises a 5'-XYZ-3' configuration, where X and Z are independently nucleosides of length 1–7 (e.g., 1, 2, 3, 4, 5, 6, or 7), Y is a nucleoside of length 6–10 (e.g., 6, 7, 8, 9, or 10), each nucleoside of X is a 2'-4' bicyclic nucleoside (e.g., LNA or cEt), each nucleoside of Z is a non-bicyclic 2' modified nucleoside (e.g., 2'MOE or 2'-O-Me), and each nucleoside of Y is a 2'-deoxyribonucleoside.

[0325] In some embodiments, the 5' wing region of the gapmer (X in the 5'-XYZ-3' formula) includes one or more non-bicyclic 2'-modified nucleosides (e.g., 2'-MOE or 2'-OMe) and one or more 2'-4' bicyclic nucleosides (e.g., LNA or cEt). In some embodiments, the 3' wing region of the gapmer (Z in the 5'-XYZ-3' formula) includes one or more non-bicyclic 2'-modified nucleosides (e.g., 2'-MOE or 2'-O-Me) and one or more 2'-4' bicyclic nucleosides (e.g., LNA or cEt). In some embodiments, both the 5' wing region of the gapmer (X in the 5'-XYZ-3' formula) and the 3' wing region of the gapmer (Z in the 5'-XYZ-3' formula) contain one or more non-bicyclic 2'-modified nucleosides (e.g., 2'-MOE or 2'-OMe) and one or more 2'-4' bicyclic nucleosides (e.g., LNA or cEt).

[0326] In some embodiments, the gapmer comprises a 5'-XYZ-3' configuration, where X and Z are independently nucleosides of length 2-7 (e.g., 2, 3, 4, 5, 6, or 7), Y is a nucleoside of length 6-10 (e.g., 6, 7, 8, 9, or 10), where at least one (e.g., 1, 2, 3, 4, 5, 6, or 7) of positions 1, 2, 3, 4, 5, 6, or 7 of X (the highest 5' position being position 1) is a non-bicyclic 2' modified nucleoside (e.g., 2'-MOE or 2'-O-Me), the remainder of the nucleosides in both X and Z are 2'-4' bicyclic nucleosides (e.g., LNA or cEt), and each nucleoside in Y is a 2' deoxyribonucleoside. In some embodiments, the gapmer comprises a 5'-XYZ-3' configuration, where X and Z are independently nucleosides of length 2-7 (e.g., 2, 3, 4, 5, 6, or 7), Y is a nucleoside of length 6-10 (e.g., 6, 7, 8, 9, or 10), at least one (e.g., 1, 2, 3, 4, 5, 6, or 7) of Z (the most 5' position being position 1) but not all of them are non-bicyclic 2' modified nucleosides (e.g., 2'-MOE or 2'-O-Me), the remainder of the nucleosides in both X and Z are 2'-4' bicyclic nucleosides (e.g., LNA or cEt), and each nucleoside in Y is a 2' deoxyribonucleoside.In some embodiments, the gapmer comprises a 5'-XYZ-3' configuration, where X and Z are independently nucleosides of length 2-7 (e.g., 2, 3, 4, 5, 6, or 7), and Y is a nucleoside of length 6-10 (e.g., 6, 7, 8, 9, or 10), with X having at least one (e.g., 1, 2, 3, 4, 5, 6, or 7) but not all of them, and Z having at least one (e.g., 1, 2, 3, 4, 5, or 6) of the 5' position. Not all of the positions (e.g., 1, 2, 3, 4, 5, or 6) but at least one of positions 1, 2, 3, 4, 5, 6, or 7 is a non-bicyclic 2' modified nucleoside (e.g., 2'-MOE or 2'-O-Me), the rest of the nucleosides in both X and Z are 2'-4' bicyclic nucleosides (e.g., LNA or cEt), and each nucleoside in Y is a 2' deoxyribonucleoside.

[0327] Non-bicyclic 2'-modified nucleosides (e.g., 2'-MOE or 2'-O-Me) and 2'-4' bicyclic nucleosides (e.g., LNA or cEt) in the mix in the 5'-wing region of the gapmer (X in the 5'-X-Y-Z-3' form) and / or the 3'-wing region of the gapmer (Z in the 5'-X-Y-Z-3' form) non-limiting examples of the gapmer configuration are as follows: BBB-(D)n-BBBAA; KKK-(D)n-KKKAA; LLL-(D)n-LLLAA; BBB-(D)n-BBBEE; KKK-(D)n-KKKEE; LLL-(D)n-LLLEE; BBB-(D)n-BBBAA; KKK-(D)n-KKKAA; LLL-(D)n-LLLAA; BBB-(D)n-BBBEE; KKK-(D)n-KKKEE; LLL-(D)n-LLLEE; BBB-(D)n-BBBAAA; KKK-(D)n-KKKAAA; LLL-(D)n-LLLAAA; BBB-(D)n-BBBEEE; KKK-(D)n-KKKEEE; LLL-(D)n-LLLEEE; BBB-(D)n-BBBAAA; KKK-(D)n-KKKAAA; LLL-(D)n-LLLAAA; BBB-(D)n-BBBEEE; KKK-(D)n-KKKEEE; LLL-(D)n-LLLEEE; BABA-(D)n-ABAB; KAKA-(D)n-AKAK; LALA-(D)n-ALAL; BEBE-(D)n-EBEB; KEKE-(D)n-EKEK; LELE-(D)n-ELEL; BABA-(D)n-ABAB; KAKA-(D)n-AKAK; LALA-(D)n-ALAL; BEBE-(D)n-EBEB; KEKE-(D)n-EKEK; LELE-(D)n-ELEL; ABAB-(D)n-ABAB; AKAK-(D)n-AKAK; ALAL-(D)n-ALAL; EBEB-(D)n-EBEB; EKEK-(D)n-EKEK; ELEL-(D)n-ELEL; ABAB-(D)n-ABAB; AKAK-(D)n-AKAK; ALAL-(D)n-ALAL; EBEB-(D)n-EBEB; EKEK-(D)n-EKEK; ELEL-(D)n-ELEL; AABB-(D)n-BBAA; BBAA-(D)n-AABB; AAKK-(D)n-KKAA; AALL-(D)n-LLAA; EEBB-(D)n-BBEE; EEKK-(D)n-KKEE;EELL-(D)n-LLEE;AABB-(D)n-BBAA;AAKK-(D)n-KKAA;AALL-(D)n-LLAA;EEBB-(D)n-BBEE;EEKK-(D)n-KKEE;EELL-(D)n-LLEE;BBB-(D)n-BBA;KKK-(BBA; D)n-KKA;LLL-(D)n-LLA;BBB-(D)n-BBE;KKK-(D)n-KKE;LLL-(D)n-LLE;BBB-(D)n-BBA;KKK-(D)n-KKA;LLL-(D)n-LLA;BBB-(D)n-BBE; E;LLL-(D)n-LLE;BBB-(D)n-BBA;KKK-(D)n-KKA;LLL-(D)n-LLA;BBB-(D)n-BBE;KKK-(D)n-KKE;LLL-(D)n-LLE;ABBB-(D)n-BBBA;ACC-KK-KKA; ALLL-(D)n-LLLA;EBBB-(D)n-BBBE;EKKK-(D)n-KKKE;ELLL-(D)n-LLLE;ABBB-(D)n-BBBA;AKKK-(D)n-KKKA;ALLL-(D)n-LLLA;EBBB-(D)n-BBBE;EKKK -(D)n-CCKE;ELLL-(D)n-LLLE;ABBB-(D)n-BBBAA;ACC-(D)n-CCKAA;ALLL-(D)n-LLLAA;EBBB-(D)n-BBBEE;ECC-(D)n-CCKEE;ELLL-(D)n-LLLEE;ACC-(D)n-LLLAA; BBB-(D)n-BBBAA;ACC-(D)n-CCKAA;ALLL-(D)n-LLLAA;EBBB-(D)n-BBBEE;CCK-(D)n-CCKEE;ELLL-(D)n-LLLEE;AABBB-(D)n-BBB;AACCC-(D)n-CCKK ;AALLL-(D)n-LLL;EEBBB-(D)n-BBB;EEKKK-(D)n-KKK;EELLL-(D)n-LLL;AABBB-(D)n-BBB;AACKK-(D)n-KKK;AALLL-(D)n-LLL; KKK-(D)n-KKK;EELLL-(D)n-LLL;AABBB-(D)n-BBBA;AACKK-(D)n-KKKA;AALLL-(D)n-LLLA;EEBBB-(D)n-BBBE;EEKK-(D)n-KKKE;EELLL-(D)n-LLLE;AABBB-(D)n-BBBA;AAKKK-(D)n-KKKA;AALLL-(D)n-LLLA;EEBBB-(D)n-BBBE;EEKKK-(D)n-KKKE;EELLL-(D)n-LLLE;ABBAABB-(D)n-BB;AKKAA KK-(D)n-KK;ALLAALLL-(D)n-LL;EBBEEBB-(D)n-BB;EKKEEKK-(D)n-KK;ELLEELL-(D)n-LL;ABBAABB-(D)n-BB;AKKAAKK-(D)n-KK;ALLAALL-( D)n-LL;EBBEEBB-(D)n-BB;EKKEEKK-(D)n-KK;ELLEELL-(D)n-LL;ABBABB-(D)n-BBB;AKKAKK-(D)n-KKK;ALLALLL-(D)n-LLL;EBBEBB-(D)n-B BB;EKKEKK-(D)n-KKK;ELLELL-(D)n-LLL;ABBABB-(D)n-BBB;AKKAKK-(D)n-KKK;ALLALL-(D)n-LLL;EBBEBB-(D)n-BBB;EKKEKK-(D)n-KKK;ELL ELL-(D)n-LLL;EEEK-(D)n-EEEEEEEE;EEK-(D)n-EEEEEEEEE;EK-(D)n-EEEEEEEEEE;EK-(D)n-EEEKK;K-(D)n-EEEKEKE;K-(D)n-EEEKEKEE;K- (D)n-EEKEK;EK-(D)n-EEEEKEKE;EK-(D)n-EEEKEK;EEK-(D)n-KEEKE;EK-(D)n-EEKEK;EK-(D)n-KEEK;EEK-(D)n-EEEKEK;EK-(D)n-KEEEKEE;E This includes K-(D)n-EEKEKE; EK-(D)n-EEEKEKE; and EK-(D)n-EEEEKEK; "A" nucleosides include 2'-modified nucleosides; "B" represents 2'-4' bicyclic nucleosides; "K" represents --bound ethyl nucleosides (cEt); "L" represents LNA nucleosides; "E" represents 2'-MOE modified ribonucleosides; "D" represents 2'-deoxyribonucleosides; and "n" represents the length of the gap segment (Y in the 5'-XYZ-3' configuration), which is an integer between 1 and 20.

[0328] In some embodiments, any one of the gapmers described herein contains one or more modified nucleoside linkages (e.g., phosphorothioate linkages) in each of the X, Y, and Z regions. In some embodiments, each internucleoside linkage in any one of the gapmers described herein is a phosphorothioate linkage. In some embodiments, each of the X, Y, and Z regions independently contains a mix of phosphorothioate linkages and phosphodiester linkages. In some embodiments, each internucleoside linkage in gap region Y is a phosphorothioate linkage, the 5' wing region X contains a mix of phosphorothioate linkages and phosphodiester linkages, and the 3' wing region Z contains a mix of phosphorothioate linkages and phosphodiester linkages.

[0329] i.Mixmer In some embodiments, the oligonucleotides described herein may be mixmers or may contain mixmer sequence patterns. Generally, a mixmer is an oligonucleotide containing both naturally occurring and non-naturally occurring nucleosides, or an oligonucleotide containing two different types of non-naturally occurring nucleosides, typically in an alternating pattern. Mixmers generally have a higher binding affinity than unmodified oligonucleotides and bind specifically to target molecules; for example, they may be used to block binding sites on target molecules. Generally, mixmers do not recruit RNases to target molecules and therefore do not promote cleavage of target molecules. Oligonucleotides that are not capable of recruiting RNase H are described; see, for example, WO2007 / 112754 or WO2007 / 112753.

[0330] In some embodiments, the mixmer includes or consists of a repeating pattern of nucleoside analogs and naturally occurring nucleosides, or a repeating pattern of one type of nucleoside analog and another type of nucleoside analog. However, the mixmer does not need to include a repeating pattern and instead may include any configuration of modified nucleosides and naturally occurring nucleosides, or any configuration of one type of modified nucleoside and another type of modified nucleoside. The repeating pattern may, for example, consist of modified nucleosides such as LNA every two or three nucleosides, with the remaining nucleosides being naturally occurring nucleosides such as DNA, or 2'-substituted nucleoside analogs such as 2'MOE or 2'-fluoro analogs, or any other modified nucleosides described herein. It is recognized that repeating patterns of modified nucleosides, such as LNA units, may be combined with modified nucleosides at fixed positions—for example, at the 5' or 3' end.

[0331] In some embodiments, the mixmer does not contain regions of naturally occurring nucleosides, such as DNA nucleosides, that are more than five, more than four, more than three, or more than two consecutive nucleosides. In some embodiments, the mixmer includes at least a region consisting of at least two consecutive modified nucleosides, such as at least two consecutive LNAs. In some embodiments, the mixmer includes at least a region consisting of at least three consecutive modified nucleosides, such as at least three consecutive LNAs.

[0332] In some embodiments, the mixmer does not contain regions of nucleoside analogs such as LNAs that are consecutive in numbers of more than 7, more than 6, more than 5, more than 4, more than 3, or more than 2. In some embodiments, the LNA units may be replaced with other nucleoside analogs such as the nucleoside analogs referred to herein.

[0333] The mixmer may be designed to contain a mixture of affinity-enhancing modified nucleosides, such as LNA nucleosides and 2'-O-Me nucleosides, in non-limiting examples. In some embodiments, the mixmer contains modified nucleoside linkages (e.g., phosphorothioate nucleoside linkages or other linkages) between at least two, at least three, at least four, at least five, or more nucleosides.

[0334] The mixmer may be produced using any preferred method. Representative U.S. patents, U.S. patent publications, and PCT publications teaching the preparation of the mixmer include U.S. Patent Publications US20060128646, US20090209748, US20090298916, US20110077288, and US20120322851, and U.S. Patent No. 7687617.

[0335] In some embodiments, the mixmer comprises one or ...

Claims

1. A complex comprising an anti-transferrin receptor (TfR) antibody covalently linked to an oligonucleotide targeting DMPK, The aforementioned oligonucleotides have a length of 15 to 20 nucleotides. Furthermore, The antibody comprises a heavy chain variable region (VH) having at least 95% the same amino acid sequence as SEQ ID NO: 76, and a light chain variable region (VL) having at least 95% the same amino acid sequence as SEQ ID NO:

75. Here, the VH and VL of the antibody are, (i) Heavy chain complementarity determination region 1 (CDR-H1) as described in SEQ ID NO: 33, heavy chain complementarity determination region 2 (CDR-H2) as described in SEQ ID NO: 34, and heavy chain complementarity determination region 3 (CDR-H3) as described in SEQ ID NO: 35; light chain complementarity determination region 1 (CDR-L1) as described in SEQ ID NO: 36, light chain complementarity determination region 2 (CDR-L2) as described in SEQ ID NO: 37, and light chain complementarity determination region 3 (CDR-L3) as described in SEQ ID NO: 32; (ii) CDR-H1 as described in Sequence ID No. 27, CDR-H2 as described in Sequence ID No. 28, and CDR-H3 as described in Sequence ID No. 29; CDR-L1 as described in Sequence ID No. 30, CDR-L2 as described in Sequence ID No. 31, and CDR-L3 as described in Sequence ID No. 32; or (iii) CDR-H1 as described in Sequence ID No. 38, CDR-H2 as described in Sequence ID No. 39, and CDR-H3 as described in Sequence ID No. 40; CDR-L1 as described in Sequence ID No. 41, CDR-L2 as described in Sequence ID No. 31, and CDR-L3 as described in Sequence ID No. 42 including, The aforementioned composite.

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

75.

3. Antibodies are Fab fragment, Fab' fragment, F(ab') ２ The complex according to claim 1 or 2, selected from the group consisting of fragments, scFv, Fv, and full-length IgG.

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

5. The complex according to any one of claims 1 to 4, wherein the antibody is a Fab fragment and comprises a heavy chain having at least 85% the same amino acid sequence as SEQ ID NO: 101; and / or a light chain having at least 85% the same amino acid sequence as SEQ ID NO:

90.

6. The complex according to claim 3 or 4, wherein the antibody is a Fab fragment and 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:

90.

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

8. The complex according to any one of claims 1 to 7, wherein the oligonucleotide comprises at least 15 consecutive nucleotides from any one of sequence numbers 148 to 383 and 621 to 638, wherein one or more thymidine bases (T) in the oligonucleotide may optionally be uridine bases (U), and / or one or more U may optionally be T.

9. The complex according to claims 1 to 8, wherein the oligonucleotide comprises at least 15 consecutive nucleotides from one of sequence numbers 159, 162, 172, 174, 180, 182, 188, 190, 195, 196, 201, 203, 212, 215, 218, 222, 228, 232, 248, 264, and 266, wherein one or more thymidine bases (T) in the oligonucleotide may optionally be uridine bases (U), and / or one or more U may optionally be T.

10. The complex according to any one of claims 1 to 9, wherein the oligonucleotide comprises at least 15 consecutive nucleotides of sequence number 222.

11. The complex according to any one of claims 1 to 9, wherein the oligonucleotide comprises at least 15 consecutive nucleotides of sequence number 228.

12. The complex according to any one of claims 1 to 9, wherein the oligonucleotide comprises at least 15 consecutive nucleotides of sequence number 232.

13. The complex according to any one of claims 1 to 9, wherein the oligonucleotide comprises at least 15 consecutive nucleotides of sequence number 266.

14. The complex according to any one of claims 1 to 7, wherein the oligonucleotide comprises a region complementary to at least 15 consecutive nucleotides of any one of sequence numbers 384 to 619.

15. The complex according to any one of claims 1 to 7, wherein the oligonucleotide comprises a region complementary to at least 15 consecutive nucleotides of any one of sequence numbers 395, 398, 408, 410, 416, 418, 424, 426, 431, 432, 437, 439, 448, 451, 454, 458, 464, 468, 484, 500, and 502.

16. The complex according to any one of claims 1 to 7, wherein the oligonucleotide comprises a region complementary to at least 15 consecutive nucleotides of sequence number 458.

17. The complex according to any one of claims 1 to 7, wherein the oligonucleotide comprises a region complementary to at least 15 consecutive nucleotides of sequence number 464.

18. The complex according to any one of claims 1 to 7, wherein the oligonucleotide comprises a region complementary to at least 15 consecutive nucleotides of sequence number 468.

19. The complex according to any one of claims 1 to 7, wherein the oligonucleotide comprises a region complementary to at least 15 consecutive nucleotides of SEQ ID NO:

502.

20. The complex according to any one of claims 1 to 19, wherein the oligonucleotide comprises the formula 5'-X-Y-Z-3', where X and Z are flanking regions comprising one or more 2'-modified nucleosides selected from the group consisting of 2'-O-methyl, 2'-fluoro, 2'-O-methoxyethyl, and 2',4'-bridged nucleosides, and where Y is a gap region, and each nucleoside in Y is a 2'-deoxyribonucleoside.

21. The complex according to any one of claims 1 to 20, wherein the oligonucleotide comprises one or more phosphorothioate nucleoside linkages, wherein each nucleoside linkage of the oligonucleotide is optionally a phosphorothioate linkage.

22. The complex according to any one of claims 1 to 21, wherein the oligonucleotide is in salt form, and optionally the salt is a sodium, potassium, or magnesium salt.

23. The complex according to any one of claims 1 to 22, wherein the antibody is covalently linked to an oligonucleotide via a cleavable linker.

24. The complex according to any one of claims 1 to 22, wherein the antibody is covalently linked to the oligonucleotide via an inclementable linker.

25. The complex according to claim 23, wherein the cleavable linker comprises a valine-citrulline sequence.

26. The composite according to claim 24, wherein the non-cleavable linker comprises an optionally substituted alkyl group.

27. ​​The complex according to any one of claims 1 to 26, wherein the oligonucleotide is covalently linked to the antibody via a cysteine ​​residue of the antibody.

28. The complex according to any one of claims 1 to 26, wherein an oligonucleotide is covalently linked to the antibody via a lysine residue of the antibody.

29. A complex for use in a method of treating a disease or condition that can be improved or defended against by reducing DMPK expression in cells, wherein the method comprises bringing the cells into contact with the complex, according to any one of claims 1 to 28.

30. The complex according to claim 29, wherein the cell comprises a DMPK allele containing a disease-associated repeat.

31. The complex according to claim 29 or 30, wherein reducing DMPK expression is a reduction in DMPK RNA levels.

32. The complex according to any one of claims 29 to 31, wherein the reduced RNA level is that of the nucleus of a cell.

33. The complex according to any one of claims 29 to 32, wherein the cells are muscle cells.

34. A complex for use in a method of treating a subject having disease-associated repetitions of a DMPK allele associated with myotonic dystrophy, the complex according to any one of claims 1 to 33, wherein the method comprises administering the complex to the subject.

35. The complex according to claim 34, wherein the myotonic dystrophy is myotonic dystrophy type 1 (DM1).

36. The composite according to claim 34 or 35, wherein the subject is a human.

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