Anti-transferrin receptor (TfR) antibodies and uses thereof

Abstract

Aspects of the present disclosure relate to antibodies that bind to a transferrin receptor (e.g., transferrin receptor 1), and conjugates that include the antibodies covalently linked to a molecular payload. Methods of using the antibodies are also provided.

Description

[Technical Field]

[0001] Related Applications This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63 / 143,825, entitled "ANTI-TRANSFERRIN RECEPTOR (TFR) ANTIBODY AND USES THEREOF," filed January 30, 2021, U.S. Provisional Patent Application No. 63 / 069,071, entitled "ANTI-TRANSFERRIN RECEPTOR (TFR) ANTIBODY AND USES THEREOF," filed August 23, 2020, and U.S. Provisional Patent Application No. 63 / 055,721, entitled "ANTI-TRANSFERRIN RECEPTOR (TFR) ANTIBODY AND USES THEREOF," filed July 23, 2020, the contents of each of which are incorporated herein by reference in their entirety.

[0002] FIELD OF THE INVENTION The present application relates to novel anti-transferrin receptor (TfR) antibodies and uses of the antibodies.

[0003] Reference to a sequence listing submitted as a text file via EFS-WEB This application contains a Sequence Listing, which has been submitted via EFS-Web in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy created on July 8, 2021, is named D082470037WO00-SEQ-DWY and is 120,479 bytes in size. [Background technology]

[0004] background The transferrin receptor (TfR) is a dimeric transmembrane glycoprotein receptor involved in iron transport. In humans, two transferrin receptors, transferrin receptor 1 (TfR1) and transferrin receptor 2 (TfR2), have been characterized. TfR has been shown to be overexpressed in cancer cells with higher metastatic potential. TfR1 has been shown to be expressed on endothelial cells of the blood-brain barrier and can be used to enable the delivery of large molecules into the brain. Summary of the Invention

[0005] The present disclosure is based, at least in part, on the development of humanized antibodies (anti-TfR antibodies) that bind to the transferrin receptor. In some embodiments, the anti-TfR antibodies described herein selectively bind to human or non-human primate (NHP) transferrin receptor 1 (TfR1) with high specificity and affinity (e.g., in the subnanomolar to nanomolar range). In some embodiments, the anti-TfR antibodies described herein are useful for targeting tissues and / or cells (e.g., and) that express TfR1. In some embodiments, the anti-TfR antibodies provided herein are used for detecting TfR1 in cells or tissues. In some embodiments, the anti-TfR antibodies provided herein are used in diagnostic, therapeutic, or research applications. In some embodiments, the anti-TfR antibodies described herein are used to deliver a molecular payload to target cells or tissues (e.g., cells or tissues that express TfR1).

[0006] Thus, in some aspects, conjugates (e.g., diagnostic or therapeutic agents) are provided that include an anti-TfR antibody conjugated (e.g., covalently conjugated) to a molecular payload. In some embodiments, the anti-TfR antibody is used to deliver the conjugated molecular payload to cells or tissues (e.g., muscle or brain) that express TfR1 to diagnose and / or (e.g., and) treat a disease (e.g., a muscular or neurological disease). In some aspects, the present disclosure provides data demonstrating that the anti-TfR antibodies described herein have superior activity in delivering molecular payloads to target cells (e.g., muscle cells) compared to other known anti-TfR antibodies.

[0007] One aspect of the disclosure relates to an antibody that binds to the human transferrin receptor (TfR), the antibody comprising: (i) a heavy chain variable region (VH) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 76; and / or a light chain variable region (VL) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 75; (ii) a heavy chain variable region (VH) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 69; and / or a light chain variable region (VL) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 70; (iii) a heavy chain variable region (VH) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 71; and / or a light chain variable region (VL) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 70; (iv) a heavy chain variable region (VH) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 72; and / or a light chain variable region (VL) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 70; (v) a heavy chain variable region (VH) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 73; and / or a light chain variable region (VL) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 74; (vi) a heavy chain variable region (VH) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 73; and / or a light chain variable region (VL) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 75; (vii) a heavy chain variable region (VH) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 76; and / or a light chain variable region (VL) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 74; (viii) a heavy chain variable region (VH) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 77; and / or a light chain variable region (VL) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 78; (ix) a heavy chain variable region (VH) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 79; and / or a light chain variable region (VL) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 80; or (x) a heavy chain variable region (VH) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 77; a light chain variable region (VL) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 80.

[0008] In some embodiments, the antibody comprises: (i) a VH comprising the amino acid sequence of SEQ ID NO: 76 and a VL comprising the amino acid sequence of SEQ ID NO: 75; (ii) a VH comprising the amino acid sequence of SEQ ID NO: 69 and a VL comprising the amino acid sequence of SEQ ID NO: 70; (iii) a VH comprising the amino acid sequence of SEQ ID NO: 71 and a VL comprising the amino acid sequence of SEQ ID NO: 70; (iv) a VH comprising the amino acid sequence of SEQ ID NO: 72 and a VL comprising the amino acid sequence of SEQ ID NO: 70; (v) a VH comprising the amino acid sequence of SEQ ID NO: 73 and a VL comprising the amino acid sequence of SEQ ID NO: 74; (vi) a VH comprising the amino acid sequence of SEQ ID NO: 73 and a VL comprising the amino acid sequence of SEQ ID NO: 75; (vii) a VH comprising the amino acid sequence of SEQ ID NO: 76 and a VL comprising the amino acid sequence of SEQ ID NO: 74; (viii) a VH comprising the amino acid sequence of SEQ ID NO: 77 and a VL comprising the amino acid sequence of SEQ ID NO: 78; (ix) a VH comprising the amino acid sequence of SEQ ID NO: 79 and a VL comprising the amino acid sequence of SEQ ID NO: 80; or (x) VH comprising the amino acid sequence of SEQ ID NO: 77 and VL comprising the amino acid sequence of SEQ ID NO: 80.

[0009] In some embodiments, the antibody is selected from the group consisting of a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an scFv, an Fv, and a full-length IgG. In some embodiments, the antibody is a Fab fragment.

[0010] In some embodiments, the antibody comprises: (i) a heavy chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 101; and / or a light chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 90; (ii) a heavy chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 97; and / or a light chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 85; (iii) a heavy chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 98; and / or a light chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 85; (iv) a heavy chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 99; and / or a light chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 85; (v) a heavy chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 100; and / or a light chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 89; (vi) a heavy chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 100; and / or a light chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 90; (vii) a heavy chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 101; and / or a light chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 89; (viii) a heavy chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 102; and / or a light chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 93; (ix) a heavy chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 103; and / or a light chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 95; or (x) a heavy chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 102; and / or a light chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 95.

[0011] In some embodiments, the antibody comprises: (i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 101 and a light chain comprising the amino acid sequence of SEQ ID NO: 90; (ii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 97 and a light chain comprising the amino acid sequence of SEQ ID NO: 85; (iii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 98 and a light chain comprising the amino acid sequence of SEQ ID NO: 85; (iv) a heavy chain comprising the amino acid sequence of SEQ ID NO: 99 and a light chain comprising the amino acid sequence of SEQ ID NO: 85; (v) a heavy chain comprising the amino acid sequence of SEQ ID NO: 100 and a light chain comprising the amino acid sequence of SEQ ID NO: 89; (vi) a heavy chain comprising the amino acid sequence of SEQ ID NO: 100 and a light chain comprising the amino acid sequence of SEQ ID NO: 90; (vii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 101 and a light chain comprising the amino acid sequence of SEQ ID NO: 89; (viii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 102 and a light chain comprising the amino acid sequence of SEQ ID NO: 93; (ix) a heavy chain comprising the amino acid sequence of SEQ ID NO: 103 and a light chain comprising the amino acid sequence of SEQ ID NO: 95; or (x) a heavy chain comprising the amino acid sequence of SEQ ID NO: 102 and a light chain comprising the amino acid sequence of SEQ ID NO: 95.

[0012] In some embodiments, the equilibrium dissociation constant (K) for binding of the antibody to the transferrin receptor is D ) is 10 -11 M to 10 -6In some embodiments, the antibody does not specifically bind to the transferrin binding site of the transferrin receptor and / or the antibody does not inhibit binding of transferrin to the transferrin receptor. In some embodiments, the antibodies are cross-reactive with two or more extracellular epitopes of human, non-human primate, and rodent transferrin receptors.

[0013] Another aspect of the present disclosure relates to a conjugate comprising an antibody covalently linked to a molecular payload. In some embodiments, the molecular payload is a diagnostic or therapeutic agent. In some embodiments, the molecular payload is an oligonucleotide, a polypeptide, or a small molecule. In some embodiments, the antibody and the molecular payload are linked via a linker. In some embodiments, the linker comprises a valine-citrulline sequence.

[0014] Another aspect of the present disclosure relates to compositions comprising the antibodies or conjugates disclosed herein. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier.

[0015] Yet another aspect of the present disclosure relates to a method for delivering a molecular payload to a cell, the method comprising contacting the cell with a complex or composition disclosed herein. In some embodiments, the cell is a muscle cell. In some embodiments, the cell is in vitro. In some embodiments, the cell is in a subject. In some embodiments, the subject is a human.

[0016] Another aspect of the present disclosure relates to a method of delivering a molecular payload to muscle in a subject, the method comprising administering to the subject an effective amount of a conjugate disclosed herein. In some embodiments, the administration is intravenous.

[0017] Another aspect of the present disclosure relates to a method of treating a disease, the method comprising administering to a subject an effective amount of a complex or composition disclosed herein, wherein the molecular payload is a therapeutic agent. In some embodiments, the muscle disease is a rare muscle disease or muscle atrophy. [Brief explanation of the drawings]

[0018] [Figure 1A-F] Figures 1A-1F show the binding of humanized anti-TfR Fabs to human TfR1 (hTfR1) or cynomolgus monkey TfR1 (cTfR1) as measured by ELISA. Figure 1A shows the binding of humanized 3M12 variants to hTfR1. Figure 1B shows the binding of humanized 3M12 variants to cTfR1. Figure 1C shows the binding of humanized 3A4 variants to hTfR1. Figure 1D shows the binding of humanized 3A4 variants to cTfR1. Figure 1E shows the binding of humanized 5H12 variants to hTfR1. Figure 1F shows the binding of humanized 5H12 variants to hTfR1.

[0019] [Figure 2] Figure 2 shows the quantified cellular uptake of anti-TfR Fab conjugates into rhabdomyosarcoma (RD) cells. The molecular payload in the tested conjugates was a DMPK-targeting oligonucleotide, and conjugate uptake was promoted by the indicated anti-TfR Fab. Conjugates with a negative control Fab (anti-mouse TfR) or a positive control Fab (anti-human TfR1) were also included in the assay. Cells were incubated with the indicated conjugates at a concentration of 100 nM for 4 hours. Cell uptake was measured by mean Cypher5e fluorescence.

[0020] [Figure 3A-C]Figures 3A-3F show the binding of oligonucleotide-conjugated or unconjugated humanized anti-TfR Fabs to human TfR1 (hTfR1) and cynomolgus monkey TfR1 (cTfR1) as measured by ELISA. Figure 3A shows the binding of humanized 3M12 variants, alone or in conjugate with a DMPK-targeted oligo, to hTfR1. The respective EC50 values ​​are also shown. Figure 3B shows the binding of humanized 3M12 variants, alone or in conjugate with a DMPK-targeted oligo, to cTfR1. The respective EC50 values ​​are also shown. Figure 3C shows the binding of humanized 3A4 variants, alone or in conjugate with a DMPK-targeted oligo, to hTfR1. The respective EC50 values ​​are also shown. [Figure 3D-F] Figures 3A-3F show the binding of oligonucleotide-conjugated or unconjugated humanized anti-TfR Fabs to human TfR1 (hTfR1) and cynomolgus monkey TfR1 (cTfR1) as measured by ELISA. Figure 3D shows the binding of the humanized 3A4 variant, alone or in conjugate with a DMPK-targeted oligo, to cTfR1. The respective EC50 values ​​are also shown. Figure 3E shows the binding of the humanized 5H12 variant, alone or in conjugate with a DMPK-targeted oligo, to hTfR1. The respective EC50 values ​​are also shown. Figure 3F shows the binding of the humanized 5H12 variant, alone or in conjugate with a DMPK-targeted oligo, to cTfR1. The respective EC50 values ​​are also shown.

[0021] [Figure 4] Figure 4 shows DMPK expression in RD cells treated with various concentrations of conjugates containing the indicated humanized anti-TfR Fab antibodies conjugated to a DMPK-targeting oligonucleotide (ASO300). The duration of treatment was 3 days. ASO300 delivered using a transfection agent (labeled "Trans") was used as a control.

[0022] [Figure 5] FIG. 5 shows the serum stability over time of linkers used to connect anti-TfR antibodies to molecular payloads (e.g., oligonucleotides) in various species after intravenous administration.

[0023] [Figure 6] Figure 6 shows data demonstrating that conjugates containing the indicated anti-TfR Fabs (3M12 VH3 / VK2, 3M12 VH4 / VK3, and 3A4 VH3 N54S / VK4) conjugated to DMD exon skipping oligonucleotides resulted in enhanced exon skipping in DMD patient myotubes compared to naked DMD exon skipping oligos.

[0024] [Figure 7A] Figures 7A-7E show the in vivo activity of conjugates containing the indicated anti-TfR Fabs (control, 3M12 VH3 / VK2, 3M12 VH4 / VK3, and 3A4 VH3 N54S / VK4) conjugated to DMPK-targeting oligonucleotides in reducing DMPK mRNA expression in mice expressing human TfR1 (hTfR1 knock-in mice). Figure 7A shows the experimental design (e.g., IV dose, dosing frequency). DMPK mRNA levels were measured in the tibialis anterior muscle (Figure 7B), gastrocnemius muscle (Figure 7C), heart (Figure 7D), and diaphragm (Figure 7E) of mice 14 days after the first dose. [Figure 7B-C] Figures 7A-7E show the in vivo activity of conjugates containing the indicated anti-TfR Fabs (control, 3M12 VH3 / VK2, 3M12 VH4 / VK3, and 3A4 VH3 N54S / VK4) conjugated to DMPK-targeting oligonucleotides in reducing DMPK mRNA expression in mice expressing human TfR1 (hTfR1 knock-in mice). Figure 7A shows the experimental design (e.g., IV dose, dosing frequency). DMPK mRNA levels were measured in the tibialis anterior muscle (Figure 7B), gastrocnemius muscle (Figure 7C), heart (Figure 7D), and diaphragm (Figure 7E) of mice 14 days after the first dose. [Figure 7D-E]Figures 7A-7E show the in vivo activity of conjugates containing the indicated anti-TfR Fabs (control, 3M12 VH3 / VK2, 3M12 VH4 / VK3, and 3A4 VH3 N54S / VK4) conjugated to DMPK-targeting oligonucleotides in reducing DMPK mRNA expression in mice expressing human TfR1 (hTfR1 knock-in mice). Figure 7A shows the experimental design (e.g., IV dose, dosing frequency). DMPK mRNA levels were measured in the tibialis anterior muscle (Figure 7B), gastrocnemius muscle (Figure 7C), heart (Figure 7D), and diaphragm (Figure 7E) of mice 14 days after the first dose.

[0025] [Figure 8] Figure 8 shows ELISA measurements of the binding of anti-TfR Fab 3M12 VH4 / Vk3 to recombinant human (circles), cynomolgus monkey (squares), mouse (upward-pointing triangles), or rat (downward-pointing triangles) TfR1 proteins over a Fab concentration range of 230 pM to 500 nM. The results demonstrate that the anti-TfR Fab is reactive with human and cynomolgus monkey TfR1. No binding to mouse or rat recombinant TfR1 was observed. Data are presented as relative fluorescence units normalized to baseline.

[0026] [Figure 9] Figure 9 shows the results of an ELISA testing the affinity of anti-TfR Fab 3M12 VH4 / Vk3 for recombinant human TfR1 or TfR2 over a Fab concentration range of 230 pM to 500 nM. Data are presented as relative fluorescence units normalized to baseline. The results demonstrate that the Fab does not bind to recombinant human TfR2.

[0027] [Figure 10] FIG. 10 shows the serum stability of the linker used to connect anti-TfR Fab 3M12 VH4 / Vk3 to the control antisense oligonucleotide over 72 hours of incubation in PBS or in rat, mouse, cynomolgus monkey or human serum. DETAILED DESCRIPTION OF THE INVENTION

[0028] Detailed Description of Specific Embodiments The present disclosure is based, at least in part, on the development of humanized anti-TfR antibodies, examples of which are listed in Table 3, that have demonstrated high binding affinity and specificity for human TfR. Also provided are uses of anti-TfR antibodies and variants thereof in research, diagnostic / detection, and therapeutic applications. In some embodiments, the anti-TfR antibodies described herein are used to deliver molecular payloads (e.g., oligonucleotides, peptides, small molecules) to target cells or tissues that express TfR. In some embodiments, the molecular payload to be delivered is conjugated to the anti-TfR antibody and is delivered to target cells or tissues that express TfR via receptor internalization. Exemplary tissues that express TfR and can be targeted using the anti-TfR antibodies described herein include, but are not limited to, the brain, muscle, adrenal gland, appendix, bone marrow, colon, duodenum, endometrium, esophagus, fat, gallbladder, heart, kidney, liver, lung, lymph node, ovary, pancreas, placenta, prostate, salivary gland, skin, small intestine, spleen, stomach, testis, thyroid, and bladder. In some embodiments, such an approach has beneficial effects in muscle cells and across the blood-brain barrier, which has proven difficult to achieve. In some aspects, the present disclosure provides data demonstrating that the anti-TfR antibodies described herein have superior activity in delivering molecular payloads to target cells (e.g., muscle cells) compared to other known anti-TfR antibodies.

[0029] Thus, the present disclosure provides conjugates comprising any one of the anti-TfR1 antibodies covalently linked to a molecular payload. In some embodiments, the conjugates are particularly useful for delivering molecular payloads that inhibit target gene expression or activity in muscle cells, e.g., in subjects with or suspected of having a rare muscle disease or muscle atrophy (e.g., as listed in Table 6). For example, in some embodiments, the conjugates are particularly useful for delivering drugs to the brain to treat neurological diseases (e.g., as listed in Table 7).

[0030] Further aspects of the disclosure, including a description of defined terms, are provided below.

[0031] I. Definition Administering: As used herein, the term "administering" or "administration" means providing a conjugate to a subject in a physiologically and / or (e.g., and) pharmacologically useful manner (e.g., treating a condition in the subject).

[0032] Approximately: As used herein, the term "approximately" or "about," when applied to one or more values ​​of interest, refers to a value similar to a stated reference value. In certain embodiments, the term "approximately" or "about" refers to a value that falls within a range of plus or minus (more than or less than) 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the stated reference value, unless otherwise stated or clear from the context (except where such number exceeds 100% of the possible values).

[0033] Antibody: As used herein, the term "antibody" refers to a polypeptide that includes at least one immunoglobulin variable domain or at least one antigenic determinant, e.g., a paratope, that specifically binds to an antigen. In some embodiments, the antibody is a full-length antibody. In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a humanized antibody. However, in some embodiments, the antibody is a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an Fv fragment, or an scFv fragment. In some embodiments, the antibody is a nanobody derived from a camelid antibody or a nanobody derived from a shark antibody. In some embodiments, the antibody is a bispecific antibody. In some embodiments, the antibody comprises a framework with human germline sequences. In another embodiment, the antibody comprises a heavy chain constant region selected from the group consisting of the constant regions of IgG, IgG1, IgG2, IgG2A, IgG2B, IgG2C, IgG3, IgG4, IgA1, IgA2, IgD, IgM, and IgE. In some embodiments, an antibody comprises a heavy (H) chain variable region (abbreviated herein as VH) and / or (for example, and), a light (L) chain variable region (abbreviated herein as VL). In some embodiments, an antibody comprises a constant region, for example, an Fc region. An immunoglobulin constant region refers to a heavy or light chain constant region. Human IgG heavy and light chain constant region amino acid sequences and their functional variations are known. With respect to the heavy chain, in some embodiments, the heavy chain of an antibody described herein can be an alpha (α), delta (Δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain. In some embodiments, the heavy chain of an antibody described herein can comprise a human alpha (α), delta (Δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain. In specific embodiments, the antibody described herein comprises a human gamma 1 CH1 domain, CH2 domain, and / or (for example, and), CH3 domain. In some embodiments, the amino acid sequence of the VH domain comprises the amino acid sequence of a human gamma (γ) heavy chain constant region, such as any sequence known in the art.Non-limiting examples of human constant region sequences are described in the art; see, e.g., U.S. Patent No. 5,693,780 and Kabat EA et al. (1991), supra. In some embodiments, the VH domain comprises an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or at least 99% identical to any of the variable chain constant regions provided herein. In some embodiments, the antibody is modified (e.g., modified via glycosylation, phosphorylation, sumoylation, and / or (e.g., and) methylation). In some embodiments, the antibody is a glycosylated antibody conjugated to one or more sugar or carbohydrate molecules. In some embodiments, one or more sugar or carbohydrate molecules are conjugated to the antibody via N-glycosylation, O-glycosylation, C-glycosylation, glypiation (GPI anchor attachment), and / or (e.g., and) phosphoglycosylation. In some embodiments, the one or more sugar or carbohydrate molecules are monosaccharides, disaccharides, oligosaccharides, or glycans. In some embodiments, the one or more sugar or carbohydrate molecules are branched oligosaccharides or branched glycans. In some embodiments, the one or more sugar or carbohydrate molecules comprise a mannose unit, a glucose unit, an N-acetylglucosamine unit, an N-acetylgalactosamine unit, a galactose unit, a fucose unit, or a phospholipid unit. In some embodiments, the antibody is a construct comprising a polypeptide comprising one or more antigen-binding fragments of the present disclosure linked to a linker polypeptide or an immunoglobulin constant region. The linker polypeptide comprises two or more amino acid residues linked by a peptide bond and is used to link one or more antigen-binding moieties. Examples of linker polypeptides have been reported (see, for example, Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, RJ, et al. (1994) Structure 2:1121-1123).Furthermore, an antibody may be part of a larger immunoadhesion molecule formed by covalent or noncovalent association of one or more other proteins or peptides with the antibody or antibody portion. Examples of such immunoadhesion molecules include the use of streptavidin core regions to generate tetrameric scFv molecules (Kipriyanov, SM, et al. (1995) Human Antibodies and Hybridomas 6:93-101), and the use of cysteine ​​residues, marker peptides, and C-terminal polyhistidine tags to generate bivalent and biotinylated scFv molecules (Kipriyanov, SM, et al. (1994) Mol. Immunol. 31:1047-1058).

[0034] CDR: As used herein, the term "CDR" refers to a complementarity-determining region within an antibody variable sequence. A typical antibody molecule comprises a heavy chain variable region (VH) and a light chain variable region (VL), which are typically involved in antigen binding. The VH and VL regions can be further subdivided into hypervariable regions, also known as "complementarity-determining regions" ("CDRs"), interspersed with more conserved regions known as "framework regions" ("FRs"). Each VH and VL typically consists of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The extent of the framework regions and CDRs can be precisely identified using methodologies known in the art, such as the Kabat definition, the IMGT definition, the Chothia definition, the AbM definition, and / or (for example, and) the contact definition, all of which are well known in the art.For example, Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242; IMGT (registered trademark), the international ImMunoGeneTics information system (registered trademark) http: / / www.imgt.org, Lefranc, M.-P. et al., Nucleic Acids. Res.,27:209-212(1999);Ruiz,M.et al.,Nucleic Acids Res.,28:219-221(2000);Lefranc,M.-P.,Nucleic Acids Res.,29:207-209(2001);Lefranc,M.-P.,Nucleic Acids Res.,31:307-310(2003);Lefranc,M.-P.et al.,In Silico Biol.,5,0006(2004)[Epub],5:45-60(2005);Lefranc,M.-P.et al.,Nucleic Acids Res.,33:D593-597(2005);Lefranc,M.-P.et al.,Nucleic Acids Res.,37:D1006-1012(2009);Lefranc,M.-P.et al.,Nucleic Acids Res.,43:D413-422(2015);Chothia et al.,(1989)Nature 342:877;Chothia,C.et al. (1987) J. Mol. Biol. 196:901-917, Al-lazikani et al. al (1997) J. Molec. Biol. 273:927-948; and Almagro, J. Mol. Recognit. 17:132-143 (2004). See also hgmp.mrc.ac.uk and bioinf.org.uk / abs. As used herein, CDRs may refer to CDRs defined by any method known in the art.Two antibodies having the same CDRs means that the two antibodies have the same amino acid sequence of their CDRs when determined by the same method, for example, the IMGT definition.

[0035] There are three CDRs in each of the heavy and light chain variable regions, designated CDR1, CDR2, and CDR3 for each variable region. The term "CDR set" as used herein refers to a group of three CDRs occurring in a single variable region capable of binding to an antigen. The exact boundaries of these CDRs have been defined differently according to various systems. The system described by Kabat (Kabat et al., Sequence of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides an unambiguous residue numbering system applicable to any antibody variable region, but also provides precise residue boundaries defining the three CDRs. These CDRs are sometimes referred to as Kabat CDRs. Sub-portions of the CDRs are sometimes designated L1, L2, and L3, or H1, H2, and H3, where "L" and "H" designate the light chain and heavy chain regions, respectively. These regions are sometimes referred to as Chothia CDRs, whose boundaries overlap with the Kabat CDRs. Other boundaries defining CDRs that overlap with the Kabat CDRs are described by Padlan (FASEB J. 9:133-139 (1995)) and MacCallum (J Mol Biol 262(5):732-45(1996)). Still other CDR boundary definitions may not strictly adhere to one of the above systems, but may still overlap with the Kabat CDRs, and may be shortened or extended in light of predictions or experimental findings that a particular residue or group of residues, or even an entire CDR, does not significantly affect antigen binding. The methods used herein may utilize CDRs defined according to any of these systems. Examples of CDR definition systems are shown in Table 1. Table 1. CDR definition [Table 1]

[0036] CDR-grafted antibody: The term "CDR-grafted antibody" refers to an antibody that contains heavy and light chain variable region sequences from one species but in which one or more sequences of the CDR regions of its VH and / or (e.g., and) VL have been replaced with CDR sequences of another species, such as an antibody having murine heavy and light chain variable regions but in which one or more of the murine CDRs (e.g., CDR3) have been replaced with human CDR sequences.

[0037] Chimeric antibody: The term "chimeric antibody" refers to an antibody that contains heavy and light chain variable region sequences from one species and constant region sequences from another species, such as an antibody having murine heavy and light chain variable regions linked to human constant regions.

[0038] Complementary: As used herein, the term "complementary" refers to the ability for precise pairing between two nucleotides or two pairs of nucleotides. In particular, complementary is a term that characterizes the degree of hydrogen bond pairing that results in binding between two nucleotides or two pairs of nucleotides. For example, if the base of an oligonucleotide at a certain position can hydrogen bond with the base of a target nucleic acid (e.g., mRNA) at the corresponding position, then the bases are considered to be complementary to each other at that position. Base pairing may include both standard Watson-Crick base pairing and non-Watson-Crick base pairing (e.g., Wobble base pairing and Hoogsteen base pairing). For example, in some embodiments, for complementary base pairing, an adenosine-type base (A) is complementary to a thymidine-type base (T) or a uracil-type base (U), a cytosine-type base (C) is complementary to a guanosine-type base (G), and a universal base such as 3-nitropyrrole or 5-nitroindole can hybridize with any A, C, U, or T. Inosine (I) is also considered a universal base in the art and is considered complementary to any A, C, U, or T.

[0039] Conservative amino acid substitution: As used herein, "conservative amino acid substitution" refers to an amino acid substitution that does not change the relative charge or size characteristics of the protein in which the amino acid substitution is made.Variants can be prepared according to methods for modifying polypeptide sequences known to those skilled in the art, and can be found, for example, in references that summarize such methods, such as Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Fourth Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2012, or Current Protocols in Molecular Biology, FMA Usubel, et al., eds., John Wiley & Sons, Inc., New York.Conservative amino acid substitutions include those made to amino acids in the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

[0040] Covalently linked (or linked): As used herein, the term "covalently linked (or linked)" refers to the characteristic of two or more molecules being linked together via at least one covalent bond. In some embodiments, two molecules may be covalently linked together by a single bond (e.g., a disulfide bond or disulfide bridge) that acts as an intermolecular linker. However, in some embodiments, two or more molecules may be covalently linked together via a molecule that acts as a linker that connects two or more molecules together through multiple covalent bonds. In some embodiments, the linker may be a cleavable linker. However, in some embodiments, the linker may be a non-cleavable linker.

[0041] Cross-reactive: As used herein, and in the context of targeting agents (e.g., antibodies), the term "cross-reactive" refers to the property of an agent that can specifically bind to more than one antigen of the same type or class (e.g., multiple homologous, paralogous, or orthologous antigens) with similar affinity or avidity. For example, in some embodiments, an antibody that is cross-reactive to a similar type or class of human and non-human primate antigens (e.g., human transferrin receptor and non-human primate transferrin receptor) is capable of binding to a human antigen and a non-human primate antigen with similar affinity or avidity. In some embodiments, the antibody is cross-reactive to a similar type or class of human antigen and rodent antigen. In some embodiments, the antibody is cross-reactive to a similar type or class of rodent antigen and non-human primate antigen. In some embodiments, the antibody is cross-reactive to a similar type or class of human antigen, non-human primate antigen, and rodent antigen.

[0042] Framework: As used herein, the term "framework" or "framework sequence" refers to the remaining sequence of the variable region minus the CDRs. Because the exact definition of the CDR sequence can be determined by various systems, the meaning of the framework sequence is interpreted accordingly. The six CDRs (CDR-L1, CDR-L2, and CDR-L3 of the light chain and CDR-H1, CDR-H2, and CDR-H3 of the heavy chain) also divide the framework regions on the light and heavy chains into four subregions (FR1, FR2, FR3, and FR4) on each chain, where CDR1 is located between FR1 and FR2, CDR2 is located between FR2 and FR3, and CDR3 is located between FR3 and FR4. When a framework region that does not specify a specific subregion as FR1, FR2, FR3, or FR4 is referred to by others, it represents the combined FR(s) in the variable region of a single naturally occurring immunoglobulin chain. As used herein, FR refers to one of the four subregions, and FR(s) refers to two or more of the four subregions containing framework regions. Human heavy and light chain acceptor sequences are known in the art. In one embodiment, acceptor sequences known in the art may be used in the antibodies disclosed herein.

[0043] Human antibody: The term "human antibody," as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the present disclosure may include, for example, in the CDRs, particularly CDR3, amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody," as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0044] Humanized antibody: The term "humanized antibody" refers to an antibody that contains heavy and light chain variable region sequences from a non-human species (e.g., mouse), but in which at least a portion of the VH and / or (e.g., and) VL sequences have been altered to be more "human-like," i.e., more similar to human germline variable sequences. One type of humanized antibody is a CDR-grafted antibody in which human CDR sequences are introduced onto non-human VH and VL sequences to replace the corresponding non-human CDR sequences. In one embodiment, humanized anti-TfR antibodies and antigen-binding portions are provided. Such antibodies may be produced by obtaining a murine anti-transferrin receptor monoclonal antibody using conventional hybridoma technology, followed by humanization using in vitro genetic engineering (such as that disclosed in PCT Publication No. WO 2005 / 123126 A2 to Kasaian et al.).

[0045] Isolated antibody: As used herein, an "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds to the transferrin receptor is substantially free of antibodies that specifically bind to antigens other than the transferrin receptor). However, an isolated antibody that specifically binds to the transferrin receptor complex may have cross-reactivity to other antigens, such as transferrin receptor molecules from other species. Moreover, an isolated antibody may be substantially free of other cellular material and / or (by way of example and) chemicals.

[0046] Molecular payload: As used herein, the term "molecular payload" refers to a molecule or species that functions to modulate a biological outcome. In some embodiments, the molecular payload is linked or otherwise associated with an anti-TFR antibody. 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, modulate the expression of a protein, or modulate the activity of a protein. In some embodiments, the molecular payload is an oligonucleotide comprising a strand having a region complementary to a target gene.

[0047] Oligonucleotide: As used herein, the term "oligonucleotide" refers to an oligomeric nucleic acid compound up to 200 nucleotides in length. Examples of oligonucleotides include, but are not limited to, RNAi oligonucleotides (e.g., siRNA, shRNA), microRNA, gapmers, mixmers, phosphorodiamidates, morpholinos, peptide nucleic acids, aptamers, guide nucleic acids (e.g., Cas9 guide RNA), and the like. Oligonucleotides may be single-stranded or double-stranded. In some embodiments, oligonucleotides may contain one or more modified nucleotides (e.g., 2'-O-methyl sugar modifications, purine or pyrimidine modifications). In some embodiments, oligonucleotides may contain one or more modified internucleotide linkages. In some embodiments, oligonucleotides may contain one or more phosphorothioate linkages, which may be in an Rp or Sp stereochemical configuration.

[0048] Recombinant antibody: The term "recombinant human antibody," as used herein, refers to any human antibody that is prepared, expressed, created, or isolated by recombinant means, e.g., an antibody expressed using a recombinant expression vector transfected into a host cell (as described in more detail in this disclosure), an antibody isolated from a recombinant combinatorial human antibody library (Hoogenboom HR, (1997) TIB Tech. 15:62-70; Azzazy H., and Highsmith WE, (2002) Clin. Biochem. 35:425-445; Gavilondo JV, and Larrick JW (2002) BioTechniques 29:128-145; Hoogenboom H., and Chames P. (2000) Immunology Today 21:371-378), or an antibody isolated from a human immunoglobulin gene transgenic animal (e.g., a mouse) (see, e.g., Taylor, LD, et al. (See, e.g., Kellermann SA, and Green LL (2002) Current Opinion in Biotechnology 13:593-597; Little M. et al (2000) Immunology Today 21:364-370), or any other means involving splicing of human immunoglobulin gene sequences with other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when human Ig sequence transgenic animals are used, in vivo somatic mutagenesis), such that the amino acid sequences of the VH and VL regions of the recombinant antibodies, while derived from and related to human germline VH and VL sequences, are sequences that may not naturally occur within the germline repertoire of human antibodies in vivo.One aspect of the present disclosure provides fully human antibodies capable of binding to the human transferrin receptor that can be generated using techniques well known in the art, such as, but not limited to, techniques using human Ig phage libraries (e.g., those disclosed in PCT Publication No. WO 2005 / 007699 A2 to Jermutus et al.).

[0049] Region of Complementarity: As used herein, the term "region of complementarity" refers to a nucleotide sequence (e.g., a nucleotide sequence of an oligonucleotide) that is sufficiently complementary to a cognate nucleotide sequence (e.g., a nucleotide sequence of a target nucleic acid) such that the two nucleotide sequences are capable of annealing to each other under physiological conditions (e.g., in a cell). In some embodiments, the region of complementarity is fully complementary to the cognate nucleotide sequence of the target nucleic acid. However, in some embodiments, the region of complementarity is only partially complementary (e.g., at least 80%, 90%, 95%, or 99% complementary) to the cognate nucleotide sequence of the target nucleic acid. In some embodiments, the region of complementarity contains one, two, three, or four mismatches compared to the cognate nucleotide sequence of the target nucleic acid.

[0050] Specific binding: As used herein, the term "specific binding" refers to the ability of a molecule to bind to a binding partner in a binding assay or other binding context, with a degree of affinity or avidity that can be used to distinguish the binding partner from an appropriate control. With respect to an antibody, the term "specific binding" refers to the ability of an antibody to bind to a specific antigen, relative to an appropriate reference antigen, or an antigen that can be used to distinguish the specific antigen from other antigens, with a degree of affinity or avidity (e.g., that allows preferential targeting to certain cells (e.g., muscle cells) through binding to the antigen, as described herein). In some embodiments, the antibody binds to the target with at least about 10 -4 M, 10 -5 M, 10-6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, 10 -13 M or less K D In some embodiments, the antibody specifically binds to the transferrin receptor.

[0051] Subject: As used herein, the term "subject" refers to a mammal. In some embodiments, the subject is a non-human primate animal or a rodent. In some embodiments, the subject is a human. In some embodiments, the subject is a patient, e.g., a human patient, having or suspected of having a disease. In some embodiments, the subject is a human patient having or suspected of having a disease caused by a disease-associated repeat expansion in a DMPK allele, e.g., a human patient.

[0052] Transferrin receptor: As used herein, the term "transferrin receptor" (also known as TFRC, CD71, p90, TFR, or TFR1) refers to an internalized cell surface receptor that binds to transferrin to facilitate iron uptake by endocytosis. In some embodiments, the transferrin receptor may originate from humans (NCBI Gene ID 7037), non-human primates (e.g., NCBI Gene ID 711568 or NCBI Gene ID 102136007), or rodents (e.g., NCBI Gene ID 22042). In addition, multiple human transcript variants encoding different isoforms of the receptor have been characterized (e.g., those annotated with GenBank RefSeq accession numbers: NP_001121620.1, NP_003225.2, NP_001300894.1, and NP_001300895.1).

[0053] An example of a human transferrin receptor amino acid sequence, corresponding to the NCBI sequence NP_003225.2 (transferrin receptor protein 1 isoform 1, homo sapiens), is as follows: (SEQ ID NO: 105)

[0054] An example of a primate non-human transferrin receptor amino acid sequence, corresponding to the NCBI sequence NP_001244232.1 (Transferrin receptor protein 1, Macaca mulatta), is as follows: MMDQARSAFSNLFGGEPLSYTRFSLARQVDGDNSHVEMKLGVDEEENTDNNTKPNGTKPKRCGGNICYGTIAVIIFFLIGFMIGYLGYCKGVEPKTECERLAGTESPAREEPEEDFPAAPRLYWDDLKRKLSEKLDTTDFTSTIKLLNENLYVPREAGSQKDENLALYIENQFREFKLSKVWRDQHFVKI QVKDSAQNSVIIVDKNGGLVYLVENPGGYVAYSKAATVTGKLVHANFGTKKDFEDLDSPVNGSIVIVRAGKITFAEKVANAESLNAIGVLIYMDQTKFPIVKADLSFFGHAHLGTGDPYTPGFPSFNHTQFPPSQSSGLPNIPVQTISRAAAEKLFGNMEGDCPSDWKTDSTCKMVTSENKSVKLTVSNV LKETKILNIFGVIKGFVEPDHYVVVGAQRDAWGPGAAKSSVGTALLLKLAQMFSDMVLKDGFQPSRSIIFASWSAGDFGSVGATEWLEGYLSSLHLKAFTYINLDKAVLGTSNFKVSASPLLYTLIEKTMQDVKHPVTGRSLYQDSNWASKVEKLTLDNAAFPFLAYSGIPAVSFCFCEDTDYPYLGTTM DTYKELVERIPELNKVARAAAEVAGQFVIKLTHDTELNLDYERYNSQLLLFLRDLNQYRADVKEMGLSLQWLYSARGDFFRATSRLTTDFRNAEKRDKFVMKKLNDRVMRVEYYFLSPYVSPKESPFRHVFWGSGSHTLSALLESLKLRRQNNSAFNETLFRNQLALATWTIQGAANALSGDVWDIDNEF (SEQ ID NO: 106)

[0055] An example of a primate non-human transferrin receptor amino acid sequence corresponding to the NCBI sequence XP_005545315.1 (transferrin receptor protein 1, Macaca fascicularis) is as follows: (SEQ ID NO: 107)

[0056] 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: (SEQ ID NO: 108)

[0057] 2'-modified nucleoside: As used herein, the terms "2'-modified nucleoside" and "2'-modified ribonucleoside" are used interchangeably and refer to a nucleoside having a sugar moiety modified at the 2'-position. In some embodiments, the 2'-modified nucleoside is a 2'-4' bicyclic nucleoside, in which the 2' and 4' positions of the sugar are bridged (e.g., by a methylene, ethylene, or (S)-constrained ethyl bridge). In some embodiments, the 2'-modified nucleoside is a non-bicyclic 2'-modified nucleoside, e.g., in which the 2' position of the sugar moiety is substituted. Non-limiting examples of 2'-modified nucleosides include the following: 2'-deoxy, 2'-fluoro (2'-F), 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), 2'-ON-methylacetamide (2'-O-NMA), locked nucleic acids (LNA, methylene-bridged nucleic acids), ethylene-bridged nucleic acids (ENA), and (S)-constrained ethyl-bridged nucleic acids (cEt). In some embodiments, the 2'-modified nucleosides described herein are high-affinity modified nucleotides, and oligonucleotides comprising the 2'-modified nucleotides have increased affinity for target sequences compared to unmodified oligonucleotides. Examples of structures of 2'-modified nucleosides are provided below: [ka]

[0058] II. Anti-TfR antibody In some embodiments, an agent that binds to the transferrin receptor, e.g., an anti-TfR antibody, can target muscle cells and / or mediate transport of the agent across the blood-brain barrier (e.g., and). The transferrin receptor is an internalized cell surface receptor that transports transferrin across the cell membrane and participates in the regulation and homeostasis of intracellular iron levels. Some aspects of the present disclosure provide transferrin receptor-binding proteins capable of binding to the transferrin receptor. An antibody that binds, e.g., specifically binds, to the transferrin receptor may be internalized into the cell upon binding to the transferrin receptor, e.g., through receptor-mediated endocytosis.

[0059] In some embodiments, provided herein are humanized antibodies that bind to the transferrin receptor with high specificity and affinity. In some embodiments, the humanized anti-TfR antibodies described herein specifically bind to any extracellular epitope of the transferrin receptor, or to an epitope that becomes exposed to the antibody. In some embodiments, the humanized anti-TfR antibodies provided herein specifically bind to transferrin receptor from humans, non-human primates, mice, rats, etc. In some embodiments, the humanized anti-TfR antibodies provided herein bind to the human transferrin receptor. In some embodiments, the humanized anti-TfR antibodies described herein bind to an amino acid segment of the human or non-human primate transferrin receptor, such as those provided in SEQ ID NOs: 105-108. In some embodiments, the humanized anti-TfR antibodies described herein bind to an amino acid segment corresponding to amino acids 90-96 of the human transferrin receptor set forth in SEQ ID NO: 105, which is not in the apical domain of the transferrin receptor. In some embodiments, the humanized anti-TfR antibodies described herein bind to TfR1 but not TfR2.

[0060] In some embodiments, the anti-TfR antibodies described herein (e.g., 3M12 and humanized variants) bind to an epitope in TfR1, wherein the epitope comprises residues from amino acids 258-291 and / or 358-381 of SEQ ID NO: 105. In some embodiments, the anti-TfR antibodies described herein (e.g., 3M12 and humanized variants) bind to an epitope comprising amino acid residues from amino acids 258-291 and 358-381 of SEQ ID NO: 105. In some embodiments, the anti-TfR antibodies described herein (e.g., 3M12 and humanized variants) bind to an epitope comprising one or more of residues K261, S273, Y282, T362, S368, S370, and K371 of human TfR1 set forth in SEQ ID NO: 105. In some embodiments, the anti-TfR antibodies described herein (e.g., 3M12 and humanized variants) bind to an epitope comprising residues K261, S273, Y282, T362, S368, S370, and K371 of human TfR1, as set forth in SEQ ID NO: 105.

[0061] 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 -13 In some embodiments, the anti-TfR antibodies described herein bind to TfR1 (e.g., human or non-human primate TfR1) with a binding affinity (e.g., designated Kd) of 1 M or less. In some embodiments, the anti-TfR antibodies described herein bind to TfR1 with a Kd in the sub-nanomolar range. In some embodiments, the anti-TfR antibodies described herein selectively bind to transferrin receptor 1 (TfR1) but not transferrin receptor 2 (TfR2). In some embodiments, the anti-TfR antibodies described herein (e.g., 10 -7 M, 10 -8 M, 10 -9M, 10 -10 M, 10 -11 M, 10 -12 M, 10 -13 The anti-TfR antibodies bind to human TfR1 and cynomolgus monkey TfR1 (with a Kd of M or less), but not to mouse TfR1. The affinity and binding kinetics of anti-TfR antibodies can be tested using any suitable method, including, but not limited to, biosensor technology (e.g., OCTET or BIACORE). In some embodiments, the binding of any one of the anti-TfR antibodies described herein does not compete with or inhibit transferrin binding to TfR1. In some embodiments, the binding of any one of the anti-TfR antibodies described herein does not compete with or inhibit HFE-beta2-microglobulin binding to TfR1.

[0062] The anti-TfR antibodies described herein are humanized antibodies. The CDR and variable region amino acid sequences of the murine monoclonal anti-TfR antibodies from which the humanized anti-TfR antibodies described herein are derived are provided in Table 2. Table 2. Mouse monoclonal anti-TfR antibodies [Table 2-1] [Table 2-2] [Table 2-3]

[0063] In some embodiments, an anti-TfR antibody of the disclosure is a humanized variant of any one of the anti-TfR antibodies provided in Table 2. In some embodiments, an anti-TfR antibody of the disclosure comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 that are the same as the CDR-H1, CDR-H2, and CDR-H3 of any one of the anti-TfR antibodies provided in Table 2, and comprises a humanized heavy chain variable region and / or (by way of example and) a humanized light chain variable region.

[0064] Humanized antibodies are human immunoglobulins (recipient antibody) in which residues from the recipient's complementarity-determining regions (CDRs) are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. In some embodiments, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the imported CDR or framework sequences, but may also include residues incorporated to further refine and optimize antibody performance. In general, humanized antibodies will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. A humanized antibody will optimally also comprise at least a portion of an immunoglobulin (typically a human immunoglobulin) constant region or domain (Fc). The antibody may have an Fc region modified as described in WO 99 / 58572. Other forms of humanized antibodies have one or more CDRs (1, 2, 3, 4, 5, 6) that are altered with respect to the original antibody, also referred to as one or more CDRs derived from one or more CDRs from the original antibody. Humanized antibodies may also be affinity matured.

[0065] Humanized antibodies and methods for making them are known, see, e.g., Almagro et al., Front. Biosci. 13:1619-1633 (2008); Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005); Padlan et al., Mol. Immunol. 28:489-498 (1991); Dall'Acqua et al., Methods 36:43-60 (2005); Osbourn et al., Methods 36:61-68 (2005); and Klimka et al., Br. J. Cancer, 83:252-260 (2000), the contents of all of which are incorporated herein by reference. Human framework regions that can be used for humanization are described, for example, in Sims et al. J. Immunol. 151:2296 (1993); Carter et al. Proc. Natl. Acad. Sci. USA 89:4285 (1992); Presta et al. J. Immunol. 151:2623 (1993); Almagro et al., Front. Biosci. 13:1619-1633 (2008)); Baca et al., J. Biol. Chem. 272:10678-10684 (1997); and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996), the contents of all of which are incorporated herein by reference.

[0066] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH (e.g., in the VH framework region) that comprises one or more amino acid variations compared to any one of the VHs listed in Table 2, and / or a humanized VL (e.g., in the VL framework region) that comprises one or more amino acid variations compared to any one of the VLs listed in Table 2 (e.g., and).

[0067] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH that contains no more than 25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) compared to the VH of any of the anti-TfR antibodies listed in Table 2 (e.g., any one of SEQ ID NOs: 17, 22, 26, 43, 61, 65, and 68). Alternatively or additionally (e.g., additionally), the humanized anti-TfR antibodies of the present disclosure include a humanized VL that contains no more than 25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) compared to the VL of any one of the anti-TfR antibodies listed in Table 2 (e.g., any one of SEQ ID NOs: 18, 44, and 62).

[0068] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising an amino acid sequence that is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regions to the VH of any of the anti-TfR antibodies listed in Table 2 (e.g., any one of SEQ ID NOs: 17, 22, 26, 43, 61, 65, and 68). Alternatively or additionally (e.g., additionally), in some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VL comprising an amino acid sequence that is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regions to the VL of any of the anti-TfR antibodies listed in Table 2 (e.g., any one of SEQ ID NOs: 18, 44, and 62).

[0069] In some embodiments, a humanized anti-TfR antibody of the disclosure comprises a CDR-H1 having the amino acid sequence of SEQ ID NO: 1 (according to the IMGT definition system), a CDR-H2 having the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 19, or SEQ ID NO: 23 (according to the IMGT definition system), and a CDR-H3 having the amino acid sequence of SEQ ID NO: 3 (according to the IMGT definition system), and a humanized VH containing no more than 25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) in the framework regions compared to the VH set forth in SEQ ID NO: 17, SEQ ID NO: 22, or SEQ ID NO: 26. Alternatively or additionally (for example, additionally), the anti-TfR antibodies of the present disclosure include a CDR-L1 having the amino acid sequence of SEQ ID NO: 4 (according to the IMGT definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 5 (according to the IMGT definition system), a CDR-L3 having the amino acid sequence of SEQ ID NO: 6 (according to the IMGT definition system), and a humanized VL containing no more than 25 amino acid variations (for example, no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) in the framework regions compared to the VL set forth in SEQ ID NO: 18.

[0070] In some embodiments, a humanized anti-TfR antibody of the disclosure comprises a humanized VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 1 (according to the IMGT definition system), a CDR-H2 having the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 19, or SEQ ID NO: 23 (according to the IMGT definition system), and a CDR-H3 having the amino acid sequence of SEQ ID NO: 3 (according to the IMGT definition system), and which is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regions to the VH set forth in SEQ ID NO: 17, SEQ ID NO: 22, or SEQ ID NO: 26. Alternatively or additionally (for example, additionally), a humanized anti-TfR antibody of the present disclosure comprises a humanized VL comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 4 (according to the IMGT definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 5 (according to the IMGT definition system), and a CDR-L3 having the amino acid sequence of SEQ ID NO: 6 (according to the IMGT definition system), and is at least 75% (for example, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regions to a VL set forth in any one of SEQ ID NOs: 18.

[0071] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a CDR-H1 having the amino acid sequence of SEQ ID NO:7 (according to the Kabat definition system), a CDR-H2 having the amino acid sequence of SEQ ID NO:8, SEQ ID NO:20, or SEQ ID NO:24 (according to the Kabat definition system), and a CDR-H3 having the amino acid sequence of SEQ ID NO:9 (according to the Kabat definition system), and a humanized VH containing no more than 25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) in the framework regions compared to the VH set forth in SEQ ID NO:17, SEQ ID NO:22, or SEQ ID NO:26. Alternatively or additionally (for example, additionally), a humanized anti-TfR antibody of the present disclosure comprises a CDR-L1 having the amino acid sequence of SEQ ID NO: 10 (according to the Kabat definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 11 (according to the Kabat definition system), and a CDR-L3 having the amino acid sequence of SEQ ID NO: 6 (according to the Kabat definition system), and includes a humanized VL containing no more than 25 amino acid variations (for example, no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) in the framework regions compared to the VL set forth in SEQ ID NO: 18.

[0072] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO:7 (according to the Kabat definition system), a CDR-H2 having the amino acid sequence of SEQ ID NO:8, SEQ ID NO:20, or SEQ ID NO:24 (according to the Kabat definition system), and a CDR-H3 having the amino acid sequence of SEQ ID NO:9 (according to the Kabat definition system), and which is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regions to the VH set forth in SEQ ID NO:17, SEQ ID NO:22, or SEQ ID NO:26. Alternatively or additionally (for example, additionally), the humanized anti-TfR antibody of the present disclosure comprises a humanized VL comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 10 (according to the Kabat definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 11 (according to the Kabat definition system), and a CDR-L3 having the amino acid sequence of SEQ ID NO: 6 (according to the Kabat definition system), and is at least 75% (for example, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regions to a VL set forth in any one of SEQ ID NOs: 18.

[0073] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a CDR-H1 having the amino acid sequence of SEQ ID NO: 12 (according to the Chothia definition system), a CDR-H2 having the amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 21, or SEQ ID NO: 25 (according to the Chothia definition system), a CDR-H3 having the amino acid sequence of SEQ ID NO: 14 (according to the Chothia definition system), and a humanized VH containing no more than 25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) in the framework regions compared to the VH set forth in SEQ ID NO: 17, SEQ ID NO: 22, or SEQ ID NO: 26. Alternatively or additionally (for example, additionally), a humanized anti-TfR antibody of the present disclosure comprises a CDR-L1 having the amino acid sequence of SEQ ID NO: 15 (according to the Chothia definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 5 (according to the Chothia definition system), and a CDR-L3 having the amino acid sequence of SEQ ID NO: 16 (according to the Chothia definition system), and includes a humanized VL containing no more than 25 amino acid variations (for example, no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) in the framework regions compared to the VL set forth in SEQ ID NO: 18.

[0074] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 12 (according to the Chothia definition system), a CDR-H2 having the amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 21, or SEQ ID NO: 25 (according to the Chothia definition system), and a CDR-H3 having the amino acid sequence of SEQ ID NO: 14 (according to the Chothia definition system), and which is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regions to the VH set forth in SEQ ID NO: 17, SEQ ID NO: 22, or SEQ ID NO: 26. Alternatively or additionally (for example, additionally), the anti-TfR antibody of the present disclosure comprises a humanized VL comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 15 (according to the Chothia-defined system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 5 (according to the Chothia-defined system), and a CDR-L3 having the amino acid sequence of SEQ ID NO: 16 (according to the Chothia-defined system), which is at least 75% (for example, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regions to a VL set forth in any one of SEQ ID NOs: 18.

[0075] In some embodiments, a humanized anti-TfR antibody of the disclosure comprises a humanized VH that comprises a CDR-H1 having the amino acid sequence of SEQ ID NO: 27 (according to the IMGT definition system), a CDR-H2 having the amino acid sequence of SEQ ID NO: 28 (according to the IMGT definition system), and a CDR-H3 having the amino acid sequence of SEQ ID NO: 29 (according to the IMGT definition system), and that contains no more than 25 amino acid variations in the framework regions compared to the VH set forth in SEQ ID NO: 43 (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations). Alternatively or additionally (for example, additionally), a humanized anti-TfR antibody of the present disclosure comprises a CDR-L1 having the amino acid sequence of SEQ ID NO: 30 (according to the IMGT definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 31 (according to the IMGT definition system), a CDR-L3 having the amino acid sequence of SEQ ID NO: 32 (according to the IMGT definition system), and a humanized VL containing no more than 25 amino acid variations (for example, no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) in the framework regions compared to the VL set forth in SEQ ID NO: 44.

[0076] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 27 (according to the IMGT definition system), a CDR-H2 having the amino acid sequence of SEQ ID NO: 28 (according to the IMGT definition system), and a CDR-H3 having the amino acid sequence of SEQ ID NO: 29 (according to the IMGT definition system), and is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regions to the VH set forth in SEQ ID NO: 43. Alternatively or additionally (for example, additionally), the humanized anti-TfR antibody of the present disclosure comprises a humanized VL comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 30 (according to the IMGT definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 31 (according to the IMGT definition system), and a CDR-L3 having the amino acid sequence of SEQ ID NO: 32 (according to the IMGT definition system), and is at least 75% (for example, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regions to the VL set forth in SEQ ID NO: 44.

[0077] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH that includes a CDR-H1 having the amino acid sequence of SEQ ID NO: 33 (according to the Kabat definition system), a CDR-H2 having the amino acid sequence of SEQ ID NO: 34 (according to the Kabat definition system), and a CDR-H3 having the amino acid sequence of SEQ ID NO: 35 (according to the Kabat definition system), and that contains no more than 25 amino acid variations in the framework regions compared to the VH set forth in SEQ ID NO: 43 (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations). Alternatively or additionally (for example, additionally), the humanized anti-TfR antibodies of the present disclosure include a CDR-L1 having the amino acid sequence of SEQ ID NO: 36 (according to the Kabat definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 37 (according to the Kabat definition system), and a CDR-L3 having the amino acid sequence of SEQ ID NO: 32 (according to the Kabat definition system), and a humanized VL containing no more than 25 amino acid variations (for example, no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) in the framework regions compared to the VL set forth in SEQ ID NO: 44.

[0078] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 33 (according to the Kabat definition system), a CDR-H2 having the amino acid sequence of SEQ ID NO: 34 (according to the Kabat definition system), and a CDR-H3 having the amino acid sequence of SEQ ID NO: 35 (according to the Kabat definition system), and is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regions to the VH set forth in SEQ ID NO: 43. Alternatively or in addition (for example, in addition), the humanized anti-TfR antibody of the present disclosure comprises a humanized VL comprising a CDR-L1 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO: 36, a CDR-L2 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO: 37, and a CDR-L3 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO: 32, which is at least 75% (for example, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regions to the VL as set forth in SEQ ID NO: 44.

[0079] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH that comprises a CDR-H1 having the amino acid sequence of SEQ ID NO: 38 (according to the Chothia definition system), a CDR-H2 having the amino acid sequence of SEQ ID NO: 39 (according to the Chothia definition system), and a CDR-H3 having the amino acid sequence of SEQ ID NO: 40 (according to the Chothia definition system), and that contains no more than 25 amino acid variations in the framework regions compared to the VH set forth in SEQ ID NO: 43 (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations). Alternatively or additionally (for example, additionally), a humanized anti-TfR antibody of the present disclosure comprises a CDR-L1 having the amino acid sequence of SEQ ID NO: 41 (according to the Chothia definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 31 (according to the Chothia definition system), and a CDR-L3 having the amino acid sequence of SEQ ID NO: 42 (according to the Chothia definition system), and includes a humanized VL containing no more than 25 amino acid variations (for example, no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) in the framework regions compared to the VL set forth in SEQ ID NO: 44.

[0080] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 38 (according to the Chothia definition system), a CDR-H2 having the amino acid sequence of SEQ ID NO: 39 (according to the Chothia definition system), and a CDR-H3 having the amino acid sequence of SEQ ID NO: 40 (according to the Chothia definition system), which is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regions to the VH set forth in SEQ ID NO: 43. Alternatively or additionally (for example, additionally), a humanized anti-TfR antibody of the present disclosure comprises a humanized VL comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 41 (according to the Chothia-defined system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 31 (according to the Chothia-defined system), and a CDR-L3 having the amino acid sequence of SEQ ID NO: 42 (according to the Chothia-defined system), which is at least 75% (for example, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regions to the VL set forth in SEQ ID NO: 44.

[0081] In some embodiments, a humanized anti-TfR antibody of the disclosure comprises a CDR-H1 having the amino acid sequence of SEQ ID NO:45, SEQ ID NO:63, or SEQ ID NO:66 (according to the IMGT definition system), a CDR-H2 having the amino acid sequence of SEQ ID NO:46 (according to the IMGT definition system), and a CDR-H3 having the amino acid sequence of SEQ ID NO:47 (according to the IMGT definition system), and a humanized VH containing no more than 25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) in the framework regions compared to the VH set forth in SEQ ID NO:61, SEQ ID NO:65, or SEQ ID NO:68. Alternatively or additionally (for example, additionally), a humanized anti-TfR antibody of the present disclosure comprises a CDR-L1 having the amino acid sequence of SEQ ID NO: 48 (according to the IMGT definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 49 (according to the IMGT definition system), and a CDR-L3 having the amino acid sequence of SEQ ID NO: 50 (according to the IMGT definition system), and includes a humanized VL containing no more than 25 amino acid variations (for example, no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) in the framework regions compared to the VL set forth in SEQ ID NO: 62.

[0082] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO:45, SEQ ID NO:63, or SEQ ID NO:66 (according to the IMGT definition system), a CDR-H2 having the amino acid sequence of SEQ ID NO:46 (according to the IMGT definition system), and a CDR-H3 having the amino acid sequence of SEQ ID NO:47 (according to the IMGT definition system), and which is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regions to the VH set forth in SEQ ID NO:61, SEQ ID NO:65, or SEQ ID NO:68. Alternatively or additionally (for example, additionally), a humanized anti-TfR antibody of the present disclosure comprises a humanized VL comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 48 (according to the IMGT definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 49 (according to the IMGT definition system), and a CDR-L3 having the amino acid sequence of SEQ ID NO: 50 (according to the IMGT definition system), and which is at least 75% (for example, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regions to the VL set forth in SEQ ID NO: 62.

[0083] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a CDR-H1 having the amino acid sequence of SEQ ID NO:51, SEQ ID NO:64, or SEQ ID NO:67 (according to the Kabat definition system), a CDR-H2 having the amino acid sequence of SEQ ID NO:52 (according to the Kabat definition system), and a CDR-H3 having the amino acid sequence of SEQ ID NO:53 (according to the Kabat definition system), and a humanized VH containing no more than 25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) in the framework regions compared to the VH set forth in SEQ ID NO:61, SEQ ID NO:65, or SEQ ID NO:68. Alternatively or additionally (for example, additionally), the humanized anti-TfR antibodies of the present disclosure include a humanized VL that comprises a CDR-L1 having the amino acid sequence of SEQ ID NO: 54 (according to the Kabat definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 55 (according to the Kabat definition system), and a CDR-L3 having the amino acid sequence of SEQ ID NO: 50 (according to the Kabat definition system), and that contains no more than 25 amino acid variations (for example, no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) in the framework regions compared to the VL set forth in SEQ ID NO: 62.

[0084] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO:51, SEQ ID NO:64, or SEQ ID NO:67 (according to the Kabat definition system), a CDR-H2 having the amino acid sequence of SEQ ID NO:52 (according to the Kabat definition system), and a CDR-H3 having the amino acid sequence of SEQ ID NO:53 (according to the Kabat definition system), and which is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regions to the VH set forth in SEQ ID NO:61, SEQ ID NO:65, or SEQ ID NO:68. Alternatively or additionally (for example, additionally), the humanized anti-TfR antibody of the present disclosure comprises a humanized VL comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 54 (according to the Kabat definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 55 (according to the Kabat definition system), and a CDR-L3 having the amino acid sequence of SEQ ID NO: 50 (according to the Kabat definition system), and is at least 75% (for example, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regions to the VL set forth in SEQ ID NO: 62.

[0085] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH that comprises a CDR-H1 having the amino acid sequence of SEQ ID NO: 56 (according to the Chothia definition system), a CDR-H2 having the amino acid sequence of SEQ ID NO: 57 (according to the Chothia definition system), and a CDR-H3 having the amino acid sequence of SEQ ID NO: 58 (according to the Chothia definition system), and that contains no more than 25 amino acid variations in the framework regions (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) compared to the VH set forth in SEQ ID NO: 61, SEQ ID NO: 65, or SEQ ID NO: 68. Alternatively or additionally (for example, additionally), a humanized anti-TfR antibody of the present disclosure comprises a CDR-L1 having the amino acid sequence of SEQ ID NO: 59 (according to the Chothia definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 49 (according to the Chothia definition system), and a CDR-L3 having the amino acid sequence of SEQ ID NO: 60 (according to the Chothia definition system), and includes a humanized VL containing no more than 25 amino acid variations (for example, no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) in the framework regions compared to the VL set forth in SEQ ID NO: 62.

[0086] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 56 (according to the Chothia definition system), a CDR-H2 having the amino acid sequence of SEQ ID NO: 57 (according to the Chothia definition system), and a CDR-H3 having the amino acid sequence of SEQ ID NO: 58 (according to the Chothia definition system), and is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regions to the VH set forth in SEQ ID NO: 61, SEQ ID NO: 65, or SEQ ID NO: 68. Alternatively or additionally (for example, additionally), a humanized anti-TfR antibody of the present disclosure comprises a humanized VL comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 59 (according to the Chothia definition system), a CDR-L2 having the amino acid sequence of SEQ ID NO: 49 (according to the Chothia definition system), and a CDR-L3 having the amino acid sequence of SEQ ID NO: 60 (according to the Chothia definition system), which is at least 75% (for example, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regions to the VL set forth in SEQ ID NO: 62.

[0087] 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]

[0088] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising CDR-H1, CDR-H2, and CDR-H3 of any one of the anti-TfR antibodies provided in Table 2, and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) amino acid variations compared to the respective humanized VH provided in Table 3. Alternatively or additionally (e.g., additionally), a humanized anti-TfR antibody of the present disclosure comprises a humanized VL comprising CDR-L1, CDR-L2, and CDR-L3 of any one of the anti-TfR antibodies provided in Table 2, and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) amino acid variations compared to the respective humanized VL provided in Table 3.

[0089] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 69, and / or a humanized VL comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 70. In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising the amino acid sequence of SEQ ID NO: 69 and a humanized VL comprising the amino acid sequence of SEQ ID NO: 70.

[0090] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 71, and / or a humanized VL comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to (e.g., and) SEQ ID NO: 70. In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising the amino acid sequence of SEQ ID NO: 71 and a humanized VL comprising the amino acid sequence of SEQ ID NO: 70.

[0091] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 72, and / or a humanized VL comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 70. In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising the amino acid sequence of SEQ ID NO: 72 and a humanized VL comprising the amino acid sequence of SEQ ID NO: 70.

[0092] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 73, and / or a humanized VL comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to (e.g., and) SEQ ID NO: 74. In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising the amino acid sequence of SEQ ID NO: 73 and a humanized VL comprising the amino acid sequence of SEQ ID NO: 74.

[0093] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 73, and / or a humanized VL comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to (e.g., and) SEQ ID NO: 75. In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising the amino acid sequence of SEQ ID NO: 73 and a humanized VL comprising the amino acid sequence of SEQ ID NO: 75.

[0094] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 76, and / or a humanized VL comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to (e.g., and) SEQ ID NO: 74. In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising the amino acid sequence of SEQ ID NO: 76 and a humanized VL comprising the amino acid sequence of SEQ ID NO: 74.

[0095] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 76, and / or a humanized VL comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to (e.g., and) SEQ ID NO: 75. In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising the amino acid sequence of SEQ ID NO: 76 and a humanized VL comprising the amino acid sequence of SEQ ID NO: 75.

[0096] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 77, and / or a humanized VL comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to (e.g., and) SEQ ID NO: 78. In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising the amino acid sequence of SEQ ID NO: 77 and a humanized VL comprising the amino acid sequence of SEQ ID NO: 78.

[0097] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 79, and / or a humanized VL comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to (e.g., and) SEQ ID NO: 80. In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising the amino acid sequence of SEQ ID NO: 79 and a humanized VL comprising the amino acid sequence of SEQ ID NO: 80.

[0098] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 77, and / or a humanized VL comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to (e.g., and) SEQ ID NO: 80. In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a humanized VH comprising the amino acid sequence of SEQ ID NO: 77 and a humanized VL comprising the amino acid sequence of SEQ ID NO: 80.

[0099] In some embodiments, the humanized anti-TfR antibodies described herein are full-length IgGs, which may include heavy and light chain constant regions from a human antibody. In some embodiments, the heavy chain of any of the anti-TfR antibodies described herein may include a heavy chain constant region (CH) or a portion thereof (e.g., CH1, CH2, CH3, or a combination thereof). The heavy chain constant region may belong to any suitable source, e.g., human, mouse, rat, or rabbit. In a specific example, the heavy chain constant region is from a human IgG (gamma heavy chain), e.g., IgG1, IgG2, or IgG4. An example of a human IgG1 constant region is provided below: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 81)

[0100] 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 the LALA mutation on the CH2 domain of human IgG1 (a mutant derived from mAb b12 mutated to replace the lower hinge residues Leu234 and Leu235 with Ala234 and Ala235) is known to reduce Fcγ receptor binding (Bruhns, P., et al. (2009) and Xu, D. et al. (2000)). The mutant human IgG1 constant region is given below (mutations are bolded and underlined): [ka]

[0101] In some embodiments, the light chain of any of the anti-TfR antibodies described herein may further comprise a light chain constant region (CL), which may be any CL known in the art. In some examples, the CL is a kappa light chain. In other examples, the CL is a lambda light chain. In some embodiments, the CL is a kappa light chain, the sequence of which is given below: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 83)

[0102] 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.

[0103] In some embodiments, the humanized anti-TfR antibodies described herein comprise a heavy chain comprising any one of a VH or any variant thereof listed in Table 3, and a heavy chain constant region that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 81 or SEQ ID NO: 82. In some embodiments, the humanized anti-TfR antibodies described herein comprise a heavy chain comprising any one of a VH or any variant thereof listed in Table 3, and a heavy chain constant region that contains no more than 25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) compared to SEQ ID NO: 81 or SEQ ID NO: 82. In some embodiments, the humanized anti-TfR antibodies described herein comprise a heavy chain comprising any one of the VHs listed in Table 3 or any variant thereof, and a heavy chain constant region set forth in SEQ ID NO: 81. In some embodiments, the humanized anti-TfR antibodies described herein comprise a heavy chain comprising any one of the VHs listed in Table 3 or any variant thereof, and a heavy chain constant region set forth in SEQ ID NO: 82.

[0104] In some embodiments, the humanized anti-TfR antibodies described herein comprise a light chain comprising any one of the VLs listed in Table 3 or any variant thereof, and a light chain constant region that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 83. In some embodiments, the humanized anti-TfR antibodies described herein comprise a light chain comprising any one of the VLs listed in Table 3 or any variant thereof, and a light chain constant region that contains 25 or fewer amino acid variations (e.g., 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid variations) compared to SEQ ID NO: 83. In some embodiments, the humanized anti-TfR antibodies described herein comprise a light chain comprising any one of the VLs listed in Table 3 or any variant thereof, and a light chain constant region set forth in SEQ ID NO: 83.

[0105] 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]

[0106] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a heavy chain containing no more than 25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) compared to the heavy chain set forth in any one of SEQ ID NOs: 84, 86, 87, 88, 91, 92, and 94. Alternatively or additionally (e.g., additionally), a humanized anti-TfR antibody of the present disclosure comprises a light chain that contains no more than 25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) compared to the light chain set forth in any one of SEQ ID NOs: 85, 89, 90, 93, and 95.

[0107] In some embodiments, the humanized anti-TfR antibodies described herein comprise a heavy chain comprising an amino acid sequence at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to any one of SEQ ID NOs: 84, 86, 87, 88, 91, 92, and 94. Alternatively or additionally (e.g., additionally), the humanized anti-TfR antibodies described herein comprise a light chain comprising an amino acid sequence at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to any one of SEQ ID NOs: 85, 89, 90, 93, and 95. In some embodiments, the anti-TfR antibodies described herein comprise a heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 84, 86, 87, 88, 91, 92, and 94. Alternatively or additionally (for example, additionally), the anti-TfR antibodies described herein comprise a light chain comprising the amino acid sequence of any one of SEQ ID NOs: 85, 89, 90, 93 and 95.

[0108] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 84, and / or a light chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to (e.g., and) SEQ ID NO: 85. In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 84 and a light chain comprising the amino acid sequence of SEQ ID NO: 85.

[0109] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 86, and / or a light chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to (e.g., and) SEQ ID NO: 85. In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 86 and a light chain comprising the amino acid sequence of SEQ ID NO: 85.

[0110] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 87, and / or a light chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to (e.g., and) SEQ ID NO: 85. In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 87 and a light chain comprising the amino acid sequence of SEQ ID NO: 85.

[0111] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 88, and / or a light chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to (e.g., and) SEQ ID NO: 89. In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 88 and a light chain comprising the amino acid sequence of SEQ ID NO: 89.

[0112] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 88, and / or a light chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to (e.g., and) SEQ ID NO: 90. In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 88 and a light chain comprising the amino acid sequence of SEQ ID NO: 90.

[0113] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 91, and / or a light chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to (e.g., and) SEQ ID NO: 89. In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 91 and a light chain comprising the amino acid sequence of SEQ ID NO: 89.

[0114] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 91, and / or a light chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to (e.g., and) SEQ ID NO: 90. In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 91 and a light chain comprising the amino acid sequence of SEQ ID NO: 90.

[0115] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 92, and / or a light chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to (e.g., and) SEQ ID NO: 93. In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 92 and a light chain comprising the amino acid sequence of SEQ ID NO: 93.

[0116] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 94, and / or a light chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to (e.g., and) SEQ ID NO: 95. In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 94 and a light chain comprising the amino acid sequence of SEQ ID NO: 95.

[0117] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 92, and / or a light chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to (e.g., and) SEQ ID NO: 95. In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 92 and a light chain comprising the amino acid sequence of SEQ ID NO: 95.

[0118] In some embodiments, the anti-TfR antibody is a Fab fragment, Fab' fragment, or F(ab')2 fragment of an intact antibody (full-length antibody). Antigen-binding fragments of intact antibodies (full-length antibodies) can be prepared by conventional methods (e.g., recombinantly or by digesting the heavy chain constant region of a full-length IgG using an enzyme such as papain). For example, F(ab')2 fragments can be produced by pepsin or papain digestion of an antibody molecule, and Fab fragments can be generated by reducing the disulfide bridges of an F(ab')2 fragment. In some embodiments, the heavy chain constant region of the Fab fragment of an anti-TfR1 antibody described herein comprises the amino acid sequence ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT (SEQ ID NO: 96).

[0119] In some embodiments, the humanized anti-TfR antibodies described herein comprise a heavy chain comprising any one of a VH listed in Table 3 or any variant thereof, and a heavy chain constant region that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 96. In some embodiments, the humanized anti-TfR antibodies described herein comprise a heavy chain comprising any one of a VH listed in Table 3 or any variant thereof, and a heavy chain constant region that contains 25 or fewer amino acid variations (e.g., 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid variations) compared to SEQ ID NO: 96. In some embodiments, the humanized anti-TfR antibodies described herein comprise a heavy chain comprising any one of a VH listed in Table 3 or any variant thereof, and a heavy chain constant region set forth in SEQ ID NO:96.

[0120] In some embodiments, the humanized anti-TfR antibodies described herein comprise a light chain comprising any one of the VLs listed in Table 3 or any variant thereof, and a light chain constant region that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 83. In some embodiments, the humanized anti-TfR antibodies described herein comprise a light chain comprising any one of the VLs listed in Table 3 or any variant thereof, and a light chain constant region that contains 25 or fewer amino acid variations (e.g., 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid variations) compared to SEQ ID NO: 83. In some embodiments, the humanized anti-TfR antibodies described herein comprise a light chain comprising any one of the VLs listed in Table 3 or any variant thereof, and a light chain constant region set forth in SEQ ID NO: 83.

[0121] 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]

[0122] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a heavy chain containing no more than 25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) compared to the heavy chain set forth in any one of SEQ ID NOs: 97-103. Alternatively or additionally (e.g., additionally), a humanized anti-TfR antibody of the present disclosure comprises a light chain that contains no more than 25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) compared to the light chain set forth in any one of SEQ ID NOs: 85, 89, 90, 93, and 95.

[0123] In some embodiments, the humanized anti-TfR antibodies described herein comprise a heavy chain comprising an amino acid sequence at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to any one of SEQ ID NOs: 97-103. Alternatively or additionally (e.g., additionally), the humanized anti-TfR antibodies described herein comprise a light chain comprising an amino acid sequence at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to any one of SEQ ID NOs: 85, 89, 90, 93, and 95. In some embodiments, the anti-TfR antibodies described herein comprise a heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 97-103. Alternatively or additionally (e.g., additionally), the anti-TfR antibodies described herein comprise a light chain comprising the amino acid sequence of any one of SEQ ID NOs: 85, 89, 90, 93, and 95.

[0124] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 97, and / or a light chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to (e.g., and) SEQ ID NO: 85. In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 97 and a light chain comprising the amino acid sequence of SEQ ID NO: 85.

[0125] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 98, and / or a light chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to (e.g., and) SEQ ID NO: 85. In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 98 and a light chain comprising the amino acid sequence of SEQ ID NO: 85.

[0126] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 99, and / or a light chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to (e.g., and) SEQ ID NO: 85. In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 99 and a light chain comprising the amino acid sequence of SEQ ID NO: 85.

[0127] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 100, and / or a light chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to (e.g., and) SEQ ID NO: 89. In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 100 and a light chain comprising the amino acid sequence of SEQ ID NO: 89.

[0128] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 100, and / or a light chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to (e.g., and) SEQ ID NO: 90. In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 100 and a light chain comprising the amino acid sequence of SEQ ID NO: 90.

[0129] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 101, and / or a light chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to (e.g., and) SEQ ID NO: 89. In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 101 and a light chain comprising the amino acid sequence of SEQ ID NO: 89.

[0130] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 101, and / or a light chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to (e.g., and) SEQ ID NO: 90. In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 101 and a light chain comprising the amino acid sequence of SEQ ID NO: 90.

[0131] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 102, and / or a light chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to (e.g., and) SEQ ID NO: 93. In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 102 and a light chain comprising the amino acid sequence of SEQ ID NO:93.

[0132] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 103, and / or a light chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to (e.g., and) SEQ ID NO: 95. In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 103 and a light chain comprising the amino acid sequence of SEQ ID NO: 95.

[0133] In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 102, and / or a light chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to (e.g., and) SEQ ID NO: 95. In some embodiments, a humanized anti-TfR antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 102 and a light chain comprising the amino acid sequence of SEQ ID NO: 95.

[0134] In some embodiments, the humanized anti-TfR receptor antibodies described herein can be in any antibody form, including, but not limited to, intact (i.e., full-length) antibodies, antigen-binding fragments thereof (such as Fab, Fab', F(ab')2, and Fv), single-chain antibodies, bispecific antibodies, or nanobodies. In some embodiments, the humanized anti-TfR antibodies described herein are scFvs. In some embodiments, the humanized anti-TfR antibodies described herein are scFv-Fabs (e.g., scFvs fused to a portion of a constant region). In some embodiments, the anti-TfR receptor antibodies described herein are scFvs fused at either the N-terminus or the C-terminus to a constant region (e.g., the human IgG1 constant region set forth in SEQ ID NO: 81 or SEQ ID NO: 82, or a portion thereof, such as an Fc portion).

[0135] In some embodiments, conservative mutations may be introduced into an antibody sequence (e.g., a CDR or framework sequence) at a position where the residue is unlikely to be involved in interactions with the target antigen (e.g., transferrin receptor) as determined, e.g., based on a crystal structure. In some embodiments, one or more mutations (e.g., amino acid substitutions) are introduced into the Fc region (e.g., in the CH2 domain (residues 231-340 of human IgG1), and / or (e.g., in the CH3 domain (residues 341-447 of human IgG1), and / or (e.g., in the hinge region), numbered according to the Kabat numbering system (e.g., EU index of Kabat)) of an anti-TfR antibody described herein to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or (e.g., and) antigen-dependent cellular cytotoxicity.

[0136] In some embodiments, one or more mutations (e.g., amino acid substitutions) are introduced into the hinge region of the Fc region (CH1 domain) such that the number of cysteine ​​residues in the hinge region can be varied (e.g., increased or decreased), e.g., as described in U.S. Patent No. 5,677,425. The number of cysteine ​​residues in the hinge region of the CH1 domain can be altered, e.g., to facilitate assembly of the light and heavy chains, or to alter (e.g., increase or decrease) the stability of the antibody, or to facilitate linker conjugation.

[0137] In some embodiments, one or more mutations (e.g., amino acid substitutions) are introduced into the Fc region of a muscle-targeting antibody described herein (e.g., in the CH2 domain (residues 231-340 of human IgG1), and / or (e.g., and) in the CH3 domain (residues 341-447 of human IgG1), and / or (e.g., and) in the hinge region, numbered according to the Kabat numbering system (e.g., EU index of Kabat)) to increase or decrease the affinity of the antibody for an Fc receptor (e.g., an activated Fc receptor) on the surface of an effector cell. Mutations in the Fc region of an antibody that increase or decrease the affinity of the antibody for an Fc receptor, and techniques for introducing such mutations into an Fc receptor or fragment thereof, are known to those of skill in the art. Examples of mutations in the Fc receptor of an antibody that can be made to alter the affinity of the antibody to the Fc receptor are described, for example, in Smith P et al., (2012) PNAS 109:6181-6186, U.S. Patent No. 6,737,056, and International Publication Nos. WO 02 / 060919; WO 98 / 23289; and WO 97 / 34631, which are incorporated herein by reference.

[0138] In some embodiments, one or more amino acid mutations (i.e., substitutions, insertions, or deletions) are introduced into an IgG constant region or FcRn-binding fragment thereof (preferably an Fc or hinge-Fc domain fragment) to alter (e.g., increase or decrease) the half-life of the antibody in vivo. See, e.g., International Publication Nos. WO 02 / 060919; WO 98 / 23289; and WO 97 / 34631; and U.S. Patent Nos. 5,869,046, 6,121,022, 6,277,375, and 6,165,745, for mutations that may alter (e.g., increase or decrease) the half-life of an antibody in vivo.

[0139] In some embodiments, one or more amino acid mutations (i.e., substitutions, insertions, or deletions) are introduced into the IgG constant region or FcRn-binding fragment thereof (preferably, the Fc or hinge-Fc domain fragment) to decrease the half-life of the anti-TfR antibody in vivo. In some embodiments, one or more amino acid mutations (i.e., substitutions, insertions, or deletions) are introduced into the IgG constant region or FcRn-binding fragment thereof (preferably, the Fc or hinge-Fc domain fragment) to increase the half-life of the antibody in vivo. In some embodiments, the antibody may have one or more amino acid mutations (e.g., substitutions) in the second constant (CH2) domain (residues 231-340 of human IgG1) and / or (for example, and) in the third constant (CH3) domain (residues 341-447 of human IgG1) numbered according to the EU index of Kabat (Kabat EA et al. (1991) supra). In some embodiments, the IgG1 constant region of the antibodies described herein comprises a methionine (M) to tyrosine (Y) substitution at position 252, a serine (S) to threonine (T) substitution at position 254, and a threonine (T) to glutamic acid (E) substitution at position 256, numbered according to the EU index as in Kabat. See U.S. Patent No. 7,658,921, which is incorporated herein by reference. Mutant IgGs of this type, termed "YTE mutants," have been shown to exhibit a four-fold increased half-life compared to the wild-type version of the same antibody (see Dall'Acqua WF et al., (2006) J Biol Chem 281:23514-24). In some embodiments, the antibody comprises an IgG constant region comprising one, two, three or more amino acid substitutions of amino acid residues at positions 251-257, 285-290, 308-314, 385-389, and 428-436, numbered according to the EU index as in Kabat.

[0140] In some embodiments, one or more amino acid substitutions are introduced into the IgG constant region Fc region to alter the effector function(s) of the anti-TfR receptor antibody. The effector ligand with altered affinity can be, for example, an Fc receptor or the C1 component of complement. This approach is described in further detail in U.S. Patent Nos. 5,624,821 and 5,648,260. In some embodiments, deletion or inactivation of the constant region domain (through point mutation or other means) can reduce binding of circulating antibodies to Fc receptors, thereby increasing tumor localization. See, e.g., U.S. Patent Nos. 5,585,097 and 8,591,886 for a description of mutations that delete or inactivate constant regions, thereby increasing tumor localization. In some embodiments, one or more amino acid substitutions may be introduced into the Fc region of an antibody described herein to remove potential glycosylation sites in the Fc region, which may reduce binding to Fc receptors (see, e.g., Shields RL et al., (2001) J Biol Chem 276:6591-604).

[0141] In some embodiments, one or more amino acid residues in the constant region of an anti-TfR antibody described herein can be replaced with a different amino acid residue such that the antibody can have altered C1q binding and / or (for example, and) reduced or eliminated complement-dependent cytotoxicity (CDC). This approach is described in further detail in U.S. Pat. No. 6,194,551 (Idusogie et al.). In some embodiments, one or more amino acid residues in the N-terminal region of the CH2 domain of an antibody described herein are altered to thereby alter the antibody's ability to fix complement. This approach is further described in International Publication No. WO 94 / 29351. In some embodiments, the Fc region of an antibody described herein is modified to increase the antibody's ability to mediate antibody-dependent cellular cytotoxicity (ADCC) to cells and / or (for example, and) increase the antibody's affinity for Fcγ receptors. This approach is further described in International Publication No. WO 00 / 42072.

[0142] In some embodiments, the heavy and / or (by way of example) light chain variable domain(s) sequence(s) of the antibodies provided herein can be used to generate, for example, CDR-grafted, chimeric, humanized, or composite human antibodies, or antigen-binding fragments, as described elsewhere herein. As will be understood by one of skill in the art, any variant, CDR-grafted, chimeric, humanized, or composite antibody derived from any of the antibodies provided herein may be useful in the compositions and methods described herein and will retain the ability to specifically bind to the transferrin receptor, such that the variant, CDR-grafted, chimeric, humanized, or composite antibody may have at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or more binding to the transferrin receptor compared to the original antibody from which it was derived.

[0143] In some embodiments, the antibodies provided herein contain mutations that confer desired properties to the antibody. For example, to avoid potential complications due to Fab-arm exchange, which is known to occur in native IgG4 mAbs, the antibodies provided herein may contain the stabilizing "Adair" mutation (Angal S., et al., "A single amino acid substitution abolishes the heterogeneity of chimeric mouse / human (IgG4) antibody," Mol Immunol 30, 105-108; 1993), in which serine 228 (EU numbering; residue 241 Kabat numbering) is converted to proline, resulting in an IgG1-like hinge sequence. Consequently, any of the antibodies may include the stabilizing "Adair" mutation.

[0144] In some embodiments, the antibody is modified (e.g., modified via glycosylation, phosphorylation, sumoylation, and / or (e.g., and) methylation). In some embodiments, the antibody is a glycosylated antibody conjugated to one or more sugar or carbohydrate molecules. In some embodiments, the one or more sugar or carbohydrate molecules are conjugated to the antibody via N-glycosylation, O-glycosylation, C-glycosylation, glypiation (GPI anchor attachment), and / or (e.g., and) phosphoglycosylation. In some embodiments, the one or more sugar or carbohydrate molecules are monosaccharides, disaccharides, oligosaccharides, or glycans. In some embodiments, the one or more sugar or carbohydrate molecules are branched oligosaccharides or branched glycans. In some embodiments, the one or more sugar or carbohydrate molecules include a mannose unit, a glucose unit, an N-acetylglucosamine unit, an N-acetylgalactosamine unit, a galactose unit, a fucose unit, or a phospholipid unit. In some embodiments, there are about 1-10, about 1-5, about 5-10, about 1-4, about 1-3, or about 2 sugar molecules. In some embodiments, the glycosylated antibody is fully or partially glycosylated. In some embodiments, the antibody is glycosylated by chemical reaction or by enzymatic means. In some embodiments, the antibody is glycosylated in vitro or inside a cell (optionally deficient in an enzyme (e.g., glycosyltransferase) in the N- or O-glycosylation pathway). In some embodiments, the antibody is functionalized with a sugar or carbohydrate molecule as described in International Patent Application Publication WO2014065661, published May 1, 2014, entitled "Modified antibody, antibody-conjugate and process for the preparation thereof."

[0145] In some embodiments, any one of the anti-TfR1 antibodies described herein can include a signal peptide (e.g., an N-terminal signal peptide) on the heavy and / or (e.g., and) light chain sequence. In some embodiments, the anti-TfR1 antibodies described herein include any one of the VH and VL sequences, any one of the IgG heavy and light chain sequences, or any one of the Fab heavy and light chain sequences described herein, and further include a signal peptide (e.g., an N-terminal signal peptide). In some embodiments, the signal peptide includes the amino acid sequence MGWSCIILFLVATATGVHS (SEQ ID NO: 104).

[0146] III. Generation of anti-TfR antibodies Antibodies capable of binding to the TfR described herein can be produced by any method known in the art, see, for example, Harlow and Lane, (1998) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York.

[0147] In some embodiments, antibodies specific to a target antigen (e.g., TfR) can be produced by conventional hybridoma technology. The full-length target antigen or a fragment thereof can be coupled to a carrier protein, such as KLH, as needed, and used to immunize a host animal that produces antibodies that bind to the antigen. The route and schedule of immunization of the host animal generally follows established and conventional techniques for antibody stimulation and production, as further described herein. General techniques for producing mouse, humanized, and human antibodies are known in the art and are described herein. It is contemplated that any mammalian subject, including humans, or antibody-producing cells therefrom can be engineered to serve as the basis for the production of mammalian, including human hybridoma cell lines. Typically, the host animal is inoculated intraperitoneally, intramuscularly, orally, subcutaneously, intraplantarly, and / or (for example, intradermally) with an amount of immunogen, including the amounts described herein.

[0148] If desired, the antibody of interest (monoclonal or polyclonal) (e.g., produced by a hybridoma) may be sequenced, and the polynucleotide sequence may then be cloned into a vector for expression or propagation. The sequence encoding the antibody of interest may be maintained within a vector in a host cell, which may then be propagated and frozen for future use. Alternatively, the polynucleotide sequence may be used to "humanize" the antibody or for genetic engineering to improve affinity (affinity maturation) or other properties of the antibody. For example, if the antibody is to be used in human clinical trials and treatments, the constant region may be modified to more closely resemble human constant regions to avoid an immune response. It may be desirable to genetically engineer the antibody sequence to obtain greater affinity and greater efficacy for the target antigen. It will be apparent to those skilled in the art that one or more polynucleotide changes can be made to an antibody while still maintaining its binding specificity for the target antigen.

[0149] In other embodiments, fully human antibodies can be obtained by using commercially available mice that have been modified to express specific human immunoglobulin proteins. Transgenic animals designed to produce a more desirable (e.g., fully human) or stronger immune response can also be used to generate humanized or human antibodies. Examples of such technologies are the Xenomouse® from Amgen (Fremont, CA), the HuMAb-Mouse® and TC Mouse® from Medarex, Inc. (Princeton, NJ), or the H2L2 mouse from Harbour Antibodies BV (The Netherlands). In another alternative, antibodies can be produced recombinantly by phage display or yeast technology. See, e.g., U.S. Patent Nos. 5,565,332; 5,580,717; 5,733,743; and 6,265,150; and Winter et al., (1994) Annu. Rev. Immunol 12:433-455. Alternatively, phage display technology (McCafferty et al., (1990) Nature 348:552-553) can be used to produce human antibodies and antibody fragments in vitro, from immunoglobulin variable (V) domain gene repertoires from unimmunized donors.

[0150] Antigen-binding fragments of intact antibodies (full-length antibodies) can be prepared via routine methods, for example, F(ab')2 fragments can be produced by pepsin digestion of the antibody molecule, and Fab' fragments can be generated by reducing the disulfide bridges of the F(ab')2 fragment. Genetically engineered antibodies, such as humanized antibodies, chimeric antibodies, single-chain antibodies, and bispecific antibodies, can be produced, for example, through conventional recombinant techniques. In one example, DNA encoding a monoclonal antibody specific to a target antigen can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the monoclonal antibody). Hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA is placed into one or more expression vectors and transfected into host cells such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, human HEK293 cells, or myeloma cells that do not produce immunoglobulin protein, resulting in the synthesis of the monoclonal antibody in the recombinant host cells. For example, see PCT Publication No. 87 / 04462. The DNA can then be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains for the homologous murine sequences (Morrison et al., (1984) Proc. Nat. Acad. Sci., 81:6851), or by covalently linking all or part of the coding sequence for a non-immunoglobulin polypeptide to the immunoglobulin coding sequence. In that manner, genetically engineered antibodies, e.g., "chimeric" or "hybrid" antibodies, with binding specificity for a target antigen can be prepared.

[0151] Single-chain antibodies can be prepared recombinantly by linking a nucleotide sequence encoding a heavy chain variable region with a nucleotide sequence encoding a light chain variable region, preferably incorporating a flexible linker between the two variable regions.

[0152] Alternatively, techniques described for the production of single-chain antibodies (U.S. Pat. Nos. 4,946,778 and 4,704,692) can be adapted to generate phage or yeast scFv libraries, and TfR-specific scFv clones can be identified from the libraries using routine procedures. Positive clones can be subjected to further screening to identify those with high TfR-binding affinity.

[0153] The antibody obtained and described herein can be characterized by methods known in the art.For example, one method is to identify the epitope that antigen binds, or "epitope mapping".There are many methods known in the art for mapping and characterizing the location of epitopes on proteins, including solving the crystal structure of antibody-antigen complex, competitive assay, gene fragment expression assay, and synthetic peptide-based assay, as described in, for example, Chapter 11 of Harlow and Lane, Using Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1999. In one example, epitope mapping can be achieved using H / D-Ex (hydrogen-deuterium exchange) coupled with proteolysis and mass spectrometry. In an additional example, epitope mapping can be used to determine antibody-binding sequences. Epitopes can be linear epitopes, i.e., contained in a single stretch of amino acids, or conformational epitopes formed by steric interactions of amino acids that are not necessarily contained in a single stretch (linear sequence of the primary structure). Peptides of various lengths (e.g., at least 4-6 amino acids) can be isolated or synthesized (e.g., recombinantly) and used in antibody binding assays. In another example, antibody-binding epitopes can be determined in systematic screening by using overlapping peptides derived from the target antigen sequence and determining antibody binding. In gene fragment expression assays, the open reading frame encoding the target antigen is fragmented randomly or by specific gene constructs, and the reactivity of the expressed fragments of the antigen with the antibody being tested is determined. Gene fragments can be generated, for example, by PCR, and then transcribed and translated into proteins in vitro in the presence of radioactive amino acids. Antibody binding to the radiolabeled antigen fragments is then determined by immunoprecipitation and gel electrophoresis. Specific epitopes can also be identified by using large libraries of random peptide sequences displayed on the surface of phage particles (phage libraries). Alternatively, defined libraries of overlapping peptide fragments can be tested for binding to test antibodies in simple binding assays. In additional examples, mutagenesis of the antigen-binding domain, domain swapping experiments, and alanine substitution mutagenesis can be performed to identify residues required, sufficient, and / or (e.g., and) necessary for epitope binding. Alternatively, to determine whether an antibody binds to the same epitope as another antibody, a competition assay can be performed using another antibody known to bind to the same antigen. Competition assays are well known to those skilled in the art.

[0154] In some examples, anti-TfR antibodies are prepared by recombinant techniques, as exemplified below. Nucleic acids encoding the heavy and light chains of the anti-TfR antibodies described herein can be cloned into an expression vector, with each nucleotide sequence operably linked to an appropriate promoter. In one example, the nucleotide sequences encoding the heavy and light chains are each operably linked to a separate promoter. Alternatively, the nucleotide sequences encoding the heavy and light chains can be operably linked to a single promoter so that both the heavy and light chains are expressed from the same promoter. If necessary, an internal ribosome entry site (IRES) can be inserted between the heavy and light chain coding sequences.

[0155] In some instances, the nucleotide sequences encoding the two chains of an antibody can be cloned into two vectors, which can be introduced into the same or different cells. If the two chains are expressed in different cells, each can be isolated from the host cell in which it is expressed, and the isolated heavy and light chains can be mixed and incubated under appropriate conditions to allow antibody formation.

[0156] Generally, the nucleic acid sequence encoding one or all chains of an antibody can be cloned into a suitable expression vector, operably linked to a suitable promoter, using methods known in the art. For example, the nucleotide sequence and the vector can be contacted with a restriction enzyme under appropriate conditions to create complementary ends on each molecule that can pair with each other and be ligated together with a ligase. Alternatively, synthetic nucleic acid linkers can be ligated to the ends of the gene. These synthetic linkers contain nucleic acid sequences that correspond to specific restriction sites in the vector. The selection of expression vectors / promoters depends on the type of host cell used to produce the antibody.

[0157] A variety of promoters can be used to express the antibodies described herein, including, but not limited to, viral LTRs, such as the cytomegalovirus (CMV) immediate early promoter, Rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR, simian virus 40 (SV40) early promoter, E. coli UV promoter, and herpes simplex TK virus promoter.

[0158] Regulatable promoters can also be used, including those that use the lac repressor from Escherichia coli as a transcriptional regulator to regulate transcription from the lac operator with a mammalian promoter [Brown, M. et al., Cell, 49:603-612 (1987)] or the tetracycline repressor (tetR) [Gossen, M., and Bujard, H., Proc. Natl. Acad. Sci. USA, 89:5547-555115 (1992); Yao, F. et al., Human Gene Therapy, 9:1939-1950 (1998); Shockelt, P., et al., Proc. Natl. Acad. Sci. USA, 92:6522-6526 (1995)]. Other systems include FK506 dimers, VP16 or p65 using astroradiol, RU486, diphenol murislerone, or rapamycin. Inducible systems are available from Invitrogen, Clontech, and Ariad, among others.

[0159] Regulatable promoters containing repressors with operons can be used. In one embodiment, the lac repressor from Escherichia coli can function as a transcriptional regulator to control transcription from mammalian cell promoters with the lac operator [M. Brown et al., Cell, 49:603-612 (1987)]; Gossen and Bujard (1992); [M. Gossen et al., Natl. Acad. Sci. USA, 89:5547-5551 (1992)]. The tetracycline repressor (tetR) and transcription activator (VP16) were combined with a minimal promoter, tetO, derived from the human cytomegalovirus (hCMV) promoter to create a tetR-tet operator system that controls gene expression in mammalian cells. In one embodiment, a tetracycline-inducible switch is used. The tetracycline repressor (tetR) alone, rather than a tetR-mammalian cell transcription factor fusion derivative, can function as a potent transregulator to control gene expression in mammalian cells when the tetracycline operator is appropriately positioned downstream of the TATA element of the CMVIE promoter (Yao et al., Human Gene Therapy). One particular advantage of this tetracycline-inducible switch is that it does not require the use of tetracycline repressor-mammalian cell transactivator or repressor fusion proteins, which may be toxic to cells in some cases, to achieve its regulatable effect (Gossen et al., Natl. Acad. Sci. USA, 89:5547-5551 (1992); Shockett et al., Proc. Natl. Acad. Sci. USA, 92:6522-6526 (1995)).

[0160] Additionally, vectors can include, for example, some or all of the following: a selectable marker gene, such as a neomycin gene for stable or transient transfectant selection in mammalian cells; an enhancer / promoter sequence from the immediate early gene of human CMV for high-level transcription; a transcription termination and RNA processing signal from SV40 for mRNA stability; an internal ribosome binding site (IRES), a versatile multiple cloning site; and T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA. Suitable vectors and methods for creating vectors containing transgenes are well known and available in the art. Examples of polyadenylation signals useful for practicing the methods described herein include, but are not limited to, the human collagen I polyadenylation signal, the human collagen II polyadenylation signal, and the SV40 polyadenylation signal.

[0161] One or more vectors (e.g., expression vectors) containing nucleic acids encoding any of the antibodies may be introduced into host cells suitable for producing the antibodies. The host cells may be cultured under conditions suitable for expression of the antibody or any of its polypeptide chains. Such antibodies or their polypeptide chains may be recovered by cultured cells (e.g., from the cells or culture supernatant) using conventional methods, such as affinity purification. If desired, the antibody polypeptide chains may be incubated under appropriate conditions for a suitable period of time to allow for antibody production.

[0162] In some embodiments, the method for preparing the antibodies described herein includes a recombinant expression vector encoding both the heavy and light chains of the anti-TfR antibody, as also described herein. The recombinant expression vector can be introduced into a suitable host cell (e.g., dhfr-CHO cells) by conventional methods, such as calcium phosphate-mediated transfection. Positively transformed host cells can be selected and cultured under appropriate conditions to allow expression of the two polypeptide chains that form the antibody, which can be recovered from the cells or culture medium. If necessary, the two chains recovered from the host cells can be incubated under appropriate conditions to allow antibody formation. In some embodiments, the host cells used to express the anti-TfR antibodies described herein are CHO-S cells (e.g., Thermo Fisher catalog #R80007).

[0163] In one example, two recombinant expression vectors are provided, one encoding the heavy chain of an anti-TfR antibody and the other encoding the light chain of an anti-TfR antibody, and both recombinant expression vectors can be introduced into suitable host cells (e.g., dhfr-CHO cells) by conventional methods, such as calcium phosphate-mediated transfection.

[0164] Alternatively, each expression vector can be introduced into a suitable host cell. Positive transformants can be selected and cultured under appropriate conditions that allow for the expression of the antibody polypeptide chains. If two expression vectors are introduced into the same host cell, the antibody produced therein can be recovered from the host cell or culture medium. If necessary, the polypeptide chains can be recovered from the host cell or culture medium and then incubated under appropriate conditions that allow for antibody formation. If two expression vectors are introduced into different host cells, each of them can be recovered from the corresponding host cell or corresponding culture medium. The two polypeptide chains can then be incubated under conditions that are suitable for antibody formation.

[0165] Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells, and recover the antibody from the culture medium. For example, some antibodies can be isolated by affinity chromatography using matrices coupled with Protein A or Protein G.

[0166] In some embodiments, any one of the anti-TfR antibodies described herein is produced by recombinant DNA technology in Chinese hamster ovary (CHO) cell suspension culture, optionally in CHO-K1 cell (e.g., CHO-K1 cells from the European Collection of Animal Cell Culture, Cat. No. 85051005) suspension culture.

[0167] In some embodiments, the antibodies provided herein may have one or more post-translational modifications. In some embodiments, N-terminal cyclization, also referred to as pyroglutamic acid formation (pyroGlu), may occur in antibodies at N-terminal glutamic acid (Glu) and / or glutamine (Gln) residues during production. Therefore, it should be understood that an antibody identified as having a sequence containing an N-terminal glutamic acid or glutamine residue encompasses antibodies that have undergone pyroglutamic acid formation due to post-translational modification. In some embodiments, pyroglutamic acid formation occurs in the heavy chain sequence. In some embodiments, pyroglutamic acid formation occurs in the light chain sequence.

[0168] IV. Complex In some embodiments, the humanized anti-TfR antibodies described herein can be used to deliver a molecular payload to a target cell or tissue (e.g., a cell or tissue expressing TfR). Accordingly, some embodiments of the present disclosure provide a molecular payload as a conjugate comprising any one of the humanized anti-TfR antibodies described herein (e.g., humanized 3-A4, 3-M12, or 5-H12 in IgG or Fab form as provided in Tables 4 and 5). The conjugates described herein can be used in a variety of applications, e.g., diagnostic or therapeutic applications.

[0169] In some embodiments, the complex comprises an anti-TfR antibody (e.g., an antisense oligonucleotide) covalently linked to an oligonucleotide. In some embodiments, the complexes described herein may be used to modulate the activity or function of at least one gene, protein, and / or (e.g., and) nucleic acid. In some embodiments, the molecular payload present with the complex is responsible for modulating the gene, protein, and / or (e.g., and) nucleic acid. The molecular payload may be a small molecule, protein, nucleic acid, oligonucleotide, or any molecular entity capable of modulating the activity or function of a gene, protein, and / or (e.g., and) nucleic acid in a cell. In some embodiments, the molecular payload is an oligonucleotide that targets disease-associated repeats in muscle cells.

[0170] A. Molecular Payload Some aspects of the present disclosure provide molecular payloads that can be linked to any one of the anti-TfR antibodies described herein to modulate a biological outcome (e.g., transcription of a DNA sequence, expression of a protein, or activity of a protein). In some embodiments, such molecular payloads can target muscle cells, e.g., via specific binding to a nucleic acid or protein in muscle cells delivered to the muscle cells by the linked anti-TfR antibody. It should be understood that various types of molecular payloads can be used in accordance with the present disclosure. For example, a molecular payload can include or consist of an oligonucleotide (e.g., an antisense oligonucleotide), a peptide (e.g., a peptide that binds to a nucleic acid or protein in a muscle cell associated with a disease), a protein (e.g., a protein that binds to a nucleic acid or protein in a muscle cell associated with a disease), or a small molecule (e.g., a small molecule that modulates the function of a nucleic acid or protein in a muscle cell associated with a disease).

[0171] In some embodiments, the molecular payload is an oligonucleotide comprising a strand having a region complementary to a gene provided in Table 6. Table 6. List of muscle diseases and corresponding genes. [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4]

[0172] In some embodiments, the molecular payload is an agent for treating neurological disorders. As used herein, "neurological disorders" refers to diseases or disorders that affect the CNS and / or (for example, and) have a pathogenesis in the CNS. Examples of CNS diseases or disorders include, but are not limited to, neuropathy, amyloidosis, cancer, eye diseases or disorders, viral or microbial infection, inflammation, ischemia, neurodegenerative diseases, stroke, behavioral disorders, and lysosomal storage diseases. For the purposes of this application, the CNS will be understood to include the eye, which is usually isolated from the rest of the body by the blood-retinal barrier. Specific examples of neurological disorders include neurodegenerative diseases (including, but not limited to, Lewy body disease, post-polio syndrome, Shy-Drager syndrome, olivopontocerebellar atrophy, Parkinson's disease, multiple system atrophy, striatonigral degeneration, tauopathies (including, but not limited to, Alzheimer's disease and supranuclear palsy), prion diseases (bovine spongiform encephalopathy, scrapie, Creutzfeldt-Jakob syndrome, kuru, Gerstmann-Sträussler-Scheinker disease, chronic wasting disease, and fatal familial insomnia), bulbar palsies, motor neuron diseases, and heterogeneous degenerative disorders of the nervous system (Canavan disease, Huntington's disease, neuronal ceroid lipofuscinosis, Alexander disease, Tourette's syndrome, Menkes kinky syndrome, etc.). Neurological disorders, including but not limited to, neurodegenerative disorders (e.g., neurodegenerative disorders including, but not limited to, neuropathies ... Table 7. Examples of Neurological Disorder Drugs and Conditions Treated [Table 7]

[0173] In some embodiments, at least one (e.g., at least two, at least three, at least four, at least five, at least ten) molecular payloads (e.g., oligonucleotides) are linked to any one of the anti-TfR antibodies described herein. In some embodiments, all of the molecular payloads attached to the anti-TfR antibody are the same, e.g., target the same gene. In some embodiments, all of the molecular payloads attached to the anti-TfR antibody are different, e.g., the molecular payloads may target different portions of the same target gene, or the molecular payloads may target at least two different target genes. In some embodiments, the anti-TfR antibodies described herein may be attached to some molecular payloads that are the same and some that are different.

[0174] The present disclosure also provides compositions comprising a plurality of conjugates, wherein at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) of the conjugates comprise an anti-TfR antibody linked to an equal number of molecular payloads (e.g., oligonucleotides).

[0175] Exemplary molecular payloads are described in further detail herein; however, it should be understood that the exemplary molecular payloads provided herein are not meant to be limiting.

[0176] i. Oligonucleotides Any suitable oligonucleotide may be used as a molecular payload as described herein. In some embodiments, the oligonucleotide may be designed to cause degradation of mRNA (e.g., the oligonucleotide may be a gapmer, siRNA, ribozyme, or aptamer that causes degradation). In some embodiments, the oligonucleotide may be designed to block translation of mRNA (e.g., the oligonucleotide may be a mixmer, siRNA, or aptamer that blocks translation). In some embodiments, the oligonucleotide may be designed to cause degradation of mRNA to block its translation. In some embodiments, the oligonucleotide may be a guide nucleic acid (e.g., a guide RNA) to direct the activity of an enzyme (e.g., a gene editing enzyme). Other examples of oligonucleotides are provided herein. It should be understood that in some embodiments, an oligonucleotide of one format (e.g., an antisense oligonucleotide) may be suitably adapted to another format (e.g., an siRNA oligonucleotide) by incorporating a functional sequence (e.g., an antisense strand sequence) from one format into the other format. In some embodiments, the oligonucleotide may comprise a region of complementarity to a target gene provided in Table 6.

[0177] In some embodiments, the oligonucleotide may target lncRNA or mRNA, for example, for degradation. In some embodiments, the oligonucleotide may target nucleic acids encoding proteins involved in the mismatch repair pathway, for example, MSH2, MutL alpha, MutS beta, MutL alpha, for example, for degradation. Non-limiting examples of proteins involved in the mismatch repair pathway (the mRNA encoding such proteins may be targeted by the oligonucleotides described herein) are described in Iyer, RR et al., "DNA triplet repeat expansion and mismatch repair," Annu Rev Biochem. 2015;84:199-226; and Schmidt MH and Pearson CE, "Disease-associated repeat instability and mismatch repair," DNA Repair (Amst). 2016 Feb;38:117-26.

[0178] In some embodiments, any one of the oligonucleotides may be in a salt form, for example, as a sodium, potassium, or magnesium salt.

[0179] In some embodiments, the 5' or 3' nucleoside (e.g., the terminal nucleoside) of any one of the oligonucleotides described herein is conjugated to an amine group, optionally via a spacer. In some embodiments, the spacer comprises an aliphatic moiety. In some embodiments, the spacer comprises a polyethylene glycol moiety. In some embodiments, a phosphodiester linkage is present between the spacer and the 5' or 3' nucleoside of the oligonucleotide. In some embodiments, the 5' or 3' nucleoside (e.g., the terminal nucleoside) of any one of the oligonucleotides described herein is conjugated to a spacer, which is a substituted or unsubstituted aliphatic, substituted or unsubstituted heteroaliphatic, substituted or unsubstituted carbocyclylene, substituted or unsubstituted heterocyclylene, substituted or unsubstituted arylene, substituted or unsubstituted heteroarylene, -O-, -N(R A )-, -S-, -C(=O)-, -C(=O)O-, -C(=O)NR A -, -NR A C(=O)-, -NR A C(=O)R A -, -C(=O)R A -, -NR A C(=O)O-, -NR A C(=O)N(R A )-, -OC(=O)-, -OC(=O)O-, -OC(=O)N(R A )-, -S(O)NR A -, -NR A S(O)2-, or a combination thereof; each R A are independently hydrogen or substituted or unsubstituted alkyl. In certain embodiments, the spacer is a substituted or unsubstituted alkylene, a substituted or unsubstituted heterocyclylene, a substituted or unsubstituted heteroarylene, -O-, -N(R A )-, or -C(=O)N(R A )2, or a combination thereof.

[0180] In some embodiments, the 5' or 3' nucleoside of any one of the oligonucleotides described herein has the formula -NH2-(CH2) n -, where n is an integer from 1 to 12. In some embodiments, n is 6, 7, 8, 9, 10, 11, or 12. In some embodiments, the phosphodiester linkage is conjugated to a compound of the formula NH2-(CH2) n 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 reaction between 6-amino-1-hexanol (NH2-(CH2)6-OH) and the 5' phosphate of the oligonucleotide.

[0181] In some embodiments, the oligonucleotide is conjugated to a targeting agent, e.g., a muscle-targeting agent such as an anti-TfR antibody, e.g., via an amine group.

[0182] Oligonucleotide size / sequence Oligonucleotides may be of a variety of different lengths, e.g., depending on the format. In some embodiments, the 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 in length, or longer. In some embodiments, the oligonucleotides are 8-50 nucleotides in length, 8-40 nucleotides in length, 8-30 nucleotides in length, 10-15 nucleotides in length, 10-20 nucleotides in length, 15-25 nucleotides in length, 21-23 nucleotides in length, etc.

[0183] In some embodiments, for purposes of the present disclosure, a complementary nucleic acid sequence of an oligonucleotide is specifically hybridizable to or specific for a target nucleic acid when the binding of said sequence to the 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 the target mRNA) and when there is a sufficient degree of complementarity to avoid non-specific binding of said sequence to non-target sequences under conditions where avoidance of non-specific binding is desired, e.g., physiological conditions in the case of in vivo assays or therapeutic treatments and in the case of in vitro assays, and the assay is performed under suitable conditions of stringency. Thus, in some embodiments, an 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 consecutive nucleotides of a target nucleic acid. In some embodiments, a complementary nucleotide sequence need not be 100% complementary to the sequence of the target to be specifically hybridizable to or specific for a target nucleic acid.

[0184] In some embodiments, the oligonucleotide comprises a region of complementarity to the target nucleic acid that ranges from 8 to 15, 8 to 30, 8 to 40, or 10 to 50, or 5 to 50, or 5 to 40 nucleotides in length. In some embodiments, the region of complementarity of the oligonucleotide to the target nucleic acid is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length. In some embodiments, the region of complementarity is complementary to at least 8 consecutive nucleotides of the target nucleic acid. In some embodiments, the oligonucleotide may contain 1, 2, or 3 base mismatches compared to a portion of consecutive nucleotides in the target nucleic acid. In some embodiments, the oligonucleotide may have up to 3 mismatches over 15 bases, or up to 2 mismatches over 10 bases.

[0185] In some embodiments, the oligonucleotide is complementary (e.g., at least 85%, at least 90%, at least 95%, or 100%) to the target sequence of any one of the oligonucleotides provided herein. In some embodiments, such target sequence is 100% complementary to the oligonucleotides provided herein.

[0186] In some embodiments, any one or more of the thymine bases (T) on any one of the oligonucleotides provided herein can optionally be uracil bases (U), and / or any one or more of the U can optionally be T.

[0187] b. Oligonucleotide Modifications: The oligonucleotides described herein may be modified, including, for example, modified sugar moieties, modified internucleoside linkages, modified nucleotides, and / or (for example, and) combinations thereof.In addition, in some embodiments, the oligonucleotide may exhibit one or more of the following properties: not mediating alternative splicing; not immunostimulating; nuclease-resistant; have improved cellular uptake compared to unmodified oligonucleotides; not toxic to cells or mammals; have improved endosomal exit in cells; minimize TLR stimulation; or avoid pattern recognition receptors.Any of the modified chemistries or formats of the oligonucleotides described herein can be combined with each other.For example, 1, 2, 3, 4, 5, or more different types of modifications can be included in the same oligonucleotide.

[0188] In some embodiments, specific nucleotide modifications can be used to make the oligonucleotides that incorporate the modifications more resistant to nuclease digestion than native oligodeoxynucleotide or oligoribonucleotide molecules; these modified oligonucleotides remain intact for longer than unmodified oligonucleotides.Specific examples of modified oligonucleotides include those that include modified backbones, such as phosphorothioates, phosphotriesters, methylphosphonates, short-chain alkyl or cycloalkyl intersugar linkages, or modified internucleoside linkages, such as short-chain heteroatom or heterocyclic intersugar linkages.As a result, the oligonucleotides of the present disclosure can be stabilized against nucleolytic degradation by modifications, such as incorporating nucleotide modifications.

[0189] In some embodiments, the oligonucleotide may be up to 50 nucleotides or up to 100 nucleotides in length, with 2-10, 2-15, 2-16, 2-17, 2-18, 2-19, 2-20, 2-25, 2-30, 2-40, 2-45, or more nucleotides of the oligonucleotide being modified nucleotides. The oligonucleotide may be 8-30 nucleotides in length, with 2-10, 2-15, 2-16, 2-17, 2-18, 2-19, 2-20, 2-25, or 2-30 nucleotides of the oligonucleotide being modified nucleotides. The oligonucleotide may be 8-15 nucleotides in length, with 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 2-11, 2-12, 2-13, or 2-14 nucleotides of the oligonucleotide being modified nucleotides. Optionally, the oligonucleotide may be modified at every nucleotide except 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Oligonucleotide modifications are further described herein.

[0190] C. modified nucleoside In some embodiments, the oligonucleotides described herein comprise at least one nucleoside modified at the 2' position of the sugar. In some embodiments, the oligonucleotide comprises at least one 2'-modified nucleoside. In some embodiments, all of the nucleosides on the oligonucleotide are 2'-modified nucleosides. In some embodiments, the oligonucleotides described herein include one or more non-bicyclic 2'-modified nucleosides, such as 2'-deoxy, 2'-fluoro (2'-F), 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'-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-methylacetamido (2'-O-NMA) modified nucleosides.

[0191] In some embodiments, the oligonucleotides described herein comprise one or more 2'-4' bicyclic nucleosides, wherein the ribose ring in the nucleoside comprises a bridging moiety connecting two atoms in the ring (e.g., connecting the 2'-O atom to the 4'-C atom via a methylene (LNA) bridge, an ethylene (ENA) bridge, or an (S)-constrained ethyl (cEt) bridge). Examples of LNAs are described in International Patent Application Publication WO / 2008 / 043753, published April 17, 2008, entitled "RNA Antagonist Compounds For The Modulation Of PCSK9," the contents of which are incorporated herein by reference in their entirety. Examples of ENAs are provided in International Patent Publication No. WO 2005 / 042777, published May 12, 2005, entitled "APP / ENA Antisense"; Morita et al., Nucleic Acid Res., Suppl 1:241-242, 2001; Surono et al., Hum. Gene Ther., 15:749-757, 2004; Koizumi, Curr. Opin. Mol. Ther., 8:144-149, 2006; and Horie et al., Nucleic Acids Symp. Ser(Oxf), 49:171-172, 2005, the disclosures of which are incorporated herein by reference in their entireties. Examples of cEt are provided in US Patents 7,101,993; 7,399,845 and 7,569,686, each of which is incorporated herein by reference in its entirety.

[0192] In some embodiments, the oligonucleotide comprises a modified nucleoside disclosed in one of the following U.S. patents or published patent applications: U.S. Patent 7,399,845, issued July 15, 2008, entitled "6-Modified Bicyclic Nucleic Acid Analogs"; U.S. Patent 7,741,457, issued June 22, 2010, entitled "6-Modified Bicyclic Nucleic Acid Analogs"; U.S. Patent 8,022,193, issued September 20, 2011, entitled "6-Modified Bicyclic Nucleic Acid Analogs"; U.S. Patent 7,569,686, issued August 4, 2009, entitled "Compounds And Methods For Synthesis Of Bicyclic Nucleic Acid Analogs"; U.S. Patent 7,335,765, issued February 26, 2008, entitled "Novel Nucleoside And Oligonucleotide U.S. Patent No. 7,314,923, issued January 1, 2008, entitled "Novel Nucleoside And Oligonucleotide Analogues"; U.S. Patent No. 7,816,333, issued October 19, 2010, entitled "Oligonucleotide Analogues And Methods Utilizing The Same" and U.S. Publication No. 2011 / 0009471, now U.S. Patent No. 8,957,201, issued February 17, 2015, entitled "Oligonucleotide Analogues And Methods Utilizing The Same," the entire contents of each of which are incorporated herein by reference for all purposes. In some embodiments, the oligonucleotide comprises at least one modified nucleoside that results in an increase in the Tm of the oligonucleotide in the range of 1° C., 2° C., 3° ​​C., 4° C., or 5° C. compared to an oligonucleotide without the at least one modified nucleoside.The oligonucleotide may have multiple modified nucleosides that result in a total increase in the 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 more, compared to an oligonucleotide that does not have the modified nucleosides.

[0193] Oligonucleotides may contain a mixture of different types of nucleosides. For example, oligonucleotides may contain a mixture of 2'-deoxyribonucleosides or ribonucleosides and 2'-fluoro-modified nucleosides. Oligonucleotides may contain a mixture of deoxyribonucleosides or ribonucleosides and 2'-O-Me-modified nucleosides. Oligonucleotides may contain a mixture of 2'-fluoro-modified nucleosides and 2'-O-Me-modified nucleosides. Oligonucleotides may contain a mixture of 2'-4' bicyclic nucleosides and 2'MOE, 2'-fluoro, or 2'-O-Me-modified nucleosides. Oligonucleotides may contain a mixture of non-bicyclic 2'-modified nucleosides (e.g., 2'-MOE, 2'-fluoro, or 2'-O-Me) and 2'-4' bicyclic nucleosides (e.g., LNA, ENA, cEt). Oligonucleotides can contain different types of alternating nucleosides.For example, oligonucleotides can contain alternating 2'-deoxyribonucleosides or ribonucleosides and 2'-fluoro modified nucleosides.Oligonucleotides can contain alternating deoxyribonucleosides or ribonucleosides and 2'-O-Me modified nucleosides.Oligonucleotides can contain alternating 2'-fluoro modified nucleosides and 2'-O-Me modified nucleosides.Oligonucleotides can contain alternating 2'-4' bicyclic nucleosides and 2'-MOE, 2'-fluoro, or 2'-O-Me modified nucleosides. Oligonucleotides can contain 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). In some embodiments, the oligonucleotides described herein contain 5'-vinylphosphonate modifications, one or more abasic residues, and / or one or more inverted abasic residues.

[0194] d. Internucleoside linkage / backbone In some embodiments, oligonucleotides may contain phosphorothioate or other modified internucleoside linkages. In some embodiments, oligonucleotides include phosphorothioate internucleoside linkages. In some embodiments, oligonucleotides include phosphorothioate internucleoside linkages between at least two nucleotides. In some embodiments, oligonucleotides include phosphorothioate internucleoside linkages between all nucleotides. For example, in some embodiments, oligonucleotides include modified internucleoside linkages at the first, second, and / or (for example, and) third internucleoside linkages at the 5' or 3' end of the nucleotide sequence. Phosphorus-containing linkages that may be used include, but are not limited to, normal 3'-5' linkages, 2'-5' linked analogs thereof, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates (including 3' alkylene phosphonates and chiral phosphonates), phosphinates, phosphoramidates (including 3'-amino phosphoramidates and aminoalkyl phosphoramidates), thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates, as well as those of reverse polarity (where adjacent pairs of nucleoside units are 3'-5' to 5'-3' or 2'-5' to 5'-2'); U.S. Patent Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,39 See Nos. 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. In some embodiments, oligonucleotides may have heteroatom backbones such as methylene (methylimino) or MMI backbones; amide backbones (see De Mesmaeker et al. Ace. Chem. Res. 1995, 28:366-374); morpholino backbones (see Summerton and Weller, U.S. Pat. No. 5,034,506); or peptide nucleic acid (PNA) backbones (in which the phosphodiester backbone of the oligonucleotide is replaced by a polyamide backbone and the nucleotides are linked directly or indirectly to aza nitrogen atoms of the polyamide backbone; see Nielsen et al., Science 1991, 254, 1497).

[0195] e. stereospecific oligonucleotides In some embodiments, the internucleotide phosphorus atom of the oligonucleotide is chiral, and the properties of the oligonucleotide are adjusted based on the configuration of the chiral phosphorus atom. In some embodiments, suitable methods can be used to synthesize P-chiral oligonucleotide analogs in a stereocontrolled manner (e.g., as described in Oka N, Wada T, Stereocontrolled synthesis of oligonucleotide analogs containing chiral internucleotidic phosphorus atoms. Chem Soc Rev. 2011 Dec; 40(12):5829-43). In some embodiments, phosphorothioate-containing oligonucleotides are provided that contain 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 with substantially chiral pure intersugar linkages are prepared by enzymatic synthesis or chemical synthesis, for example, as described in U.S. Patent No. 5,587,261, issued December 12, 1996, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the chiral-controlled oligonucleotide provides a selective cleavage pattern for the target nucleic acid. For example, in some embodiments, the chiral-controlled oligonucleotide provides a single cleavage site within the complementary sequence of the nucleic acid, as described, for example, in U.S. Patent Application Publication No. 20170037399 A1, entitled "CHIRAL DESIGN," published February 2, 2017, the contents of which are incorporated herein by reference in their entirety. f. Morpholino In some embodiments, the oligonucleotide may be a morpholino-based compound.Morpholino-based oligomeric compounds are described in Dwaine A. Braasch and David R. Corey, Biochemistry, 2002, 41(14), 4503-4510; Genesis, volume 30, issue 3, 2001; Heasman, J., Dev. Biol., 2002, 243, 209-214; Nasevicius et al., Nat. Genet., 2000, 26, 216-220; Lacerra et al., Proc. Natl. Acad. Sci., 2000, 97, 9591-9596; and U.S. Patent No. 5,034,506, issued July 23, 1991. In some embodiments, the morpholino-based oligomeric compound is a phosphorodiamidate morpholino oligomer (PMO) (e.g., as described in Iverson, Curr. Opin. Mol. Ther., 3:235-238, 2001; and Wang et al., J. Gene Med., 12:354-364, 2010; the disclosures of which are incorporated herein by reference in their entireties).

[0196] g. Peptide nucleic acid (PNA) In some embodiments, both the sugar and the internucleoside linkage (backbone) of the nucleotide unit of the oligonucleotide are replaced with novel groups. In some embodiments, the base unit is maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an oligonucleotide mimic that has been shown to have excellent hybridization properties, is called a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of an oligonucleotide is replaced with an amide-containing backbone, such as an aminoethylglycine backbone. The nucleobases are retained and are directly or indirectly linked to the aza nitrogen atoms of the amide portion of the backbone. Representative publications reporting the preparation of PNA compounds include, but are not limited to, U.S. Patent Nos. 5,539,082; 5,714,331; and 5,719,262 (each of which is incorporated herein by reference). Further teachings on PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500. h.Gapmers 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 gap region Y. In some embodiments, flanking region X of the formula 5'-XYZ-3' is also referred to as X region, flanking sequence X, 5' wing region X, or 5' wing segment. In some embodiments, flanking region Z of the formula 5'-XYZ-3' is also referred to as Z region, flanking sequence Z, 3' wing region Z, or 3' wing segment. In some embodiments, gap region Y of the formula 5'-XYZ-3' is also referred to as Y region, Y segment, or gap segment Y. In some embodiments, each nucleoside in gap region Y is a 2'-deoxyribonucleoside, and neither 5' wing region X nor 3' wing region Z contains a 2'-deoxyribonucleoside. In some embodiments, the Y region is a contiguous stretch of nucleotides, e.g., a region of six or more DNA nucleotides, which can recruit an RNAse, such as RNAse H. In some embodiments, the gapmer binds to the target nucleic acid, at which point an RNAse can be recruited and then cleave the target nucleic acid. In some embodiments, the Y region is flanked on both the 5' and 3' ends by regions X and Z comprising high-affinity modified nucleosides, e.g., 1 to 6 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 can be 1 to 20 nucleotides, 1 to 8 nucleotides, or 1 to 5 nucleotides in length. The flanking sequences X and Z can be of similar or dissimilar lengths. In some embodiments, the gap segment Y can be a nucleotide sequence between 5 and 20 nucleotides, between 5 and 15, 12 nucleotides, or between 6 and 10 nucleotides in length.

[0197] In some embodiments, the gap region of a gapmer oligonucleotide may contain, in addition to DNA nucleotides, modified nucleotides known to be permissive 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 linkages. In some embodiments, one or both flanking regions each independently contain one or more phosphorothioate internucleoside linkages (e.g., phosphorothioate internucleoside linkages or other linkages) 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 linkages (e.g., phosphorothioate internucleoside linkages or other linkages) between at least two, at least three, at least four, at least five, or more nucleotides. Gapmers can be produced using suitable methods. Representative U.S. patents, U.S. patent publications, and PCT publications that teach the preparation of gapmers include 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 80,756; 9,045,754; 9,428,534; 9,695,418; 10,017,764; 10,260,069; 9,428,534; 8,580,756; U.S. Patent Publication Nos. US20050074801, US20090221685; US200902869 69, US20100197762, and US20110112170; PCT Publication Nos. WO2004069991; WO2005023825; WO2008049085 and WO2009090182; and European Patent No. EP2,149,605, each of which is incorporated herein by reference in its entirety. In some embodiments, the gapmer is 10 to 40 nucleosides in length. For example, gapmers can be 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 length. In some embodiments, the gapmer is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleosides in length.

[0198] In some embodiments, gap region Y on the gapmer is 5 to 20 nucleosides in length. For example, gap region Y can be 5 to 20, 5 to 15, 5 to 10, 10 to 20, 10 to 15, or 15 to 20 nucleosides in length. In some embodiments, gap region Y is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleosides in length. In some embodiments, each nucleoside in gap region Y is a 2'-deoxyribonucleoside. In some embodiments, all nucleosides in gap region Y are 2'-deoxyribonucleosides. In some embodiments, one or more of the nucleosides in gap region Y are modified nucleosides (e.g., 2'-modified nucleosides such as 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 a 5-methylcytosine.

[0199] In some embodiments, the 5' wing region of the gapmer (X of the formula 5'-XYZ-3') and the 3' wing region of the gapmer (Z of the formula 5'-XYZ-3') are independently 1 to 20 nucleosides in length. For example, the 5' wing region of the gapmer (X of the formula 5'-XYZ-3') and the 3' wing region of the gapmer (Z of the formula 5'-XYZ-3') can independently be 1 to 20, 1 to 15, 1 to 10, 1 to 7, 1 to 5, 1 to 3, 1 to 2, 2 to 5, 2 to 7, 3 to 5, 3 to 7, 5 to 20, 5 to 15, 5 to 10, 10 to 20, 10 to 15, or 15 to 20 nucleosides in length. In some embodiments, the 5' wing region of the gapmer (X of the formula 5'-XYZ-3') and the 3' wing region of the gapmer (Z of the formula 5'-XYZ-3') are independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleosides in length. In some embodiments, the 5' wing region of the gapmer (X of the formula 5'-XYZ-3') and the 3' wing region of the gapmer (Z of the formula 5'-XYZ-3') are the same length. In some embodiments, the 5' wing region of the gapmer (X of the formula 5'-XYZ-3') and the 3' wing region of the gapmer (Z of the formula 5'-XYZ-3') are different lengths. In some embodiments, the 5' wing region of the gapmer (X in the 5'-XYZ-3' formula) is longer than the 3' wing region of the gapmer (Z in the 5'-XYZ-3' formula). In some embodiments, the 5' wing region of the gapmer (X in the 5'-XYZ-3' formula) is shorter than the 3' wing region of the gapmer (Z in the 5'-XYZ-3' formula).

[0200] In some embodiments, the gapmers are 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 and 5'-XYZ-3', including 5'-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 number of nucleosides in the X, Y, and Z regions of the 5'-XYZ-3' gapmer.

[0201] 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 nucleosides (e.g., high-affinity modified nucleosides). In some embodiments, the modified nucleosides (e.g., high-affinity modified nucleosides) are 2'-modified nucleosides. In some embodiments, the 2'-modified nucleosides are 2'-4' bicyclic nucleosides or non-bicyclic 2'-modified nucleosides. In some embodiments, the high affinity modified nucleoside is a 2'-4' bicyclic nucleoside (e.g., LNA, cEt, or ENA) or a non-bicyclic 2'-modified nucleoside (e.g., 2'-fluoro (2'-F), 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA)).

[0202] In some embodiments, one or more nucleosides in the 5' wing region of the gapmer (X of the formula 5'-XYZ-3') are high-affinity modified nucleosides. In some embodiments, each nucleoside in the 5' wing region of the gapmer (X of the formula 5'-XYZ-3') is a high-affinity modified nucleoside. In some embodiments, one or more nucleosides in the 3' wing region of the gapmer (Z of the formula 5'-XYZ-3') are high-affinity modified nucleosides. In some embodiments, each nucleoside in the 3' wing region of the gapmer (Z of the formula 5'-XYZ-3') is a high-affinity modified nucleoside. In some embodiments, one or more nucleosides in the 5' wing region of the gapmer (X of the formula 5'-XYZ-3') are high-affinity modified nucleosides and one or more nucleosides in the 3' wing region of the gapmer (Z of the formula 5'-XYZ-3') are high-affinity modified nucleosides. In some embodiments, each nucleoside in the 5' wing region of the gapmer (X of the formula 5'-XYZ-3') is a high-affinity modified nucleoside and each nucleoside in the 3' wing region of the gapmer (Z of the formula 5'-XYZ-3') is a high-affinity modified nucleoside.

[0203] In some embodiments, the 5' wing region of the gapmer (X of the 5'-XYZ-3' formula) contains the same high-affinity nucleosides as the 3' wing region of the gapmer (Z of the 5'-XYZ-3' formula). For example, the 5' wing region of the gapmer (X of the 5'-XYZ-3' formula) and the 3' wing region of the gapmer (Z of the 5'-XYZ-3' formula) can contain one or more non-bicyclic 2'-modified nucleosides (e.g., 2'-MOE or 2'-O-Me). In another example, the 5' wing region of the gapmer (X of the 5'-XYZ-3' formula) and the 3' wing region of the gapmer (Z of the 5'-XYZ-3' formula) can 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 formula 5'-XYZ-3') and the 3' wing region of the gapmer (Z in the formula 5'-XYZ-3') 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 formula 5'-XYZ-3') and the 3' wing region of the gapmer (Z in the formula 5'-XYZ-3') is a 2'-4' bicyclic nucleoside (e.g., LNA or cEt).

[0204] In some embodiments, the gapmer comprises a 5'-XYZ-3' configuration, wherein X and Z are independently 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleosides in length, Y is 6 to 10 (e.g., 6, 7, 8, 9, or 10) nucleosides in length, 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 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleosides in length, Y is 6 to 10 (e.g., 6, 7, 8, 9, or 10) nucleosides in length, 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 of the 5'-XYZ-3' formula) comprises a different high affinity nucleoside than the 3' wing region of the gapmer (Z of the 5'-XYZ-3' formula). For example, the 5' wing region of the gapmer (X in the 5'-XYZ-3' formula) can include one or more non-bicyclic 2'-modified nucleosides (e.g., 2'-MOE or 2'-O-Me), and the 3' wing region of the gapmer (Z in the 5'-XYZ-3' formula) can include one or more 2'-4' bicyclic nucleosides (e.g., LNA or cEt). In another example, the 3' wing region of the gapmer (Z in the 5'-XYZ-3' formula) can include one or more non-bicyclic 2'-modified nucleosides (e.g., 2'-MOE or 2'-O-Me), and the 5' wing region of the gapmer (X in the 5'-XYZ-3' formula) can include one or more 2'-4' bicyclic nucleosides (e.g., LNA or cEt).

[0205] In some embodiments, the gapmer comprises a 5'-XYZ-3' configuration, wherein X and Z are independently 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleosides in length, Y is 6 to 10 (e.g., 6, 7, 8, 9, or 10) nucleosides in length, 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, wherein X and Z are independently 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleosides in length, Y is 6 to 10 (e.g., 6, 7, 8, 9, or 10) nucleosides in length, 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.

[0206] In some embodiments, the 5' wing region of the gapmer (X of the 5'-XYZ-3' formula) comprises one or more non-bicyclic 2'-modified nucleosides (e.g., 2'-MOE or 2'-O-Me) and one or more 2'-4' bicyclic nucleosides (e.g., LNA or cEt). In some embodiments, the 3' wing region of the gapmer (Z of the 5'-XYZ-3' formula) comprises one or more non-bicyclic 2'-modified nucleosides (e.g., 2'-MOE or 2'-O-Me) and one or more 2'-4' bicyclic nucleosides (e.g., LNA or cEt). In some embodiments, both the 5' wing region of the gapmer (X of the formula 5'-XYZ-3') and the 3' wing region of the gapmer (Z of the formula 5'-XYZ-3') comprise one or more non-bicyclic 2'-modified nucleosides (e.g., 2'-MOE or 2'-O-Me) and one or more 2'-4' bicyclic nucleosides (e.g., LNA or cEt).

[0207] In some embodiments, the gapmer comprises a 5'-XYZ-3' configuration, wherein X and Z are independently 2 to 7 (e.g., 2, 3, 4, 5, 6, or 7) nucleosides in length, Y is 6 to 10 (e.g., 6, 7, 8, 9, or 10) nucleosides in length, at least one but not all (e.g., 1, 2, 3, 4, 5, or 6) of positions 1, 2, 3, 4, 5, 6, or 7 of X (the 5'-most position is position 1) is a non-bicyclic 2'-modified nucleoside (e.g., 2'-MOE or 2'-O-Me), the remainder of the nucleosides of both X and Z are 2'-4' bicyclic nucleosides (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, wherein X and Z are independently 2 to 7 (e.g., 2, 3, 4, 5, 6, or 7) nucleosides in length, Y is 6 to 10 (e.g., 6, 7, 8, 9, or 10) nucleosides in length, at least one but not all (e.g., 1, 2, 3, 4, 5, or 6) of positions 1, 2, 3, 4, 5, 6, or 7 of Z (the 5'-most position is position 1) is a non-bicyclic 2'-modified nucleoside (e.g., 2'-MOE or 2'-O-Me), the remainder of the nucleosides of both X and Z are 2'-4' bicyclic nucleosides (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 2 to 7 (e.g., 2, 3, 4, 5, 6, or 7) nucleosides in length, Y is 6 to 10 (e.g., 6, 7, 8, 9, or 10) nucleosides in length, and at least one, but not all (e.g., 1, 2, 3, 4, 5, 6, or 7) of positions 1, 2, 3, 4, 5, 6, or 7 of X and Z (the 5'-most position) are 5'-XYZ-3'. At least one of positions 1, 2, 3, 4, 5, 6, or 7, but not all (e.g., 1, 2, 3, 4, 5, or 6) of X and Z is 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.

[0208] Non-cyclic 2'-modified nucleosides (e.g., 2'-MOE or 2'-O-Me) and 2'-4'-bicyclic nucleosides (e.g., LNA or cEt) in the 5'-wing region (X in the 5'-X-Y-Z-3' format) and / or 3'-wing region (Z in the 5'-X-Y-Z-3' format) of a gapmer. Non-limiting examples of gapmer constructs having such mixtures 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-CCKKK" ;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 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-EEEEEEEEEE;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-EEEEKEK;EK-(D)n-KEEEKEE;E "A" nucleosides include 2'-modified nucleosides; "B" represents a 2'-4' bicyclic nucleoside; "K" represents a constrained ethyl nucleoside (cEt); "L" represents an LNA nucleoside; "E" represents a 2'-MOE modified ribonucleoside; "D" represents a 2' deoxyribonucleoside; and "n" represents the length of the gap segment (Y in the 5'-XYZ-3' configuration) and is an integer between 1 and 20.

[0209] In some embodiments, any one of the gapmers described herein comprises 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 comprises a mix of phosphorothioate and phosphodiester linkages. In some embodiments, each internucleoside linkage in gap region Y is a phosphorothioate linkage, 5' wing region X comprises a mix of phosphorothioate and phosphodiester linkages, and 3' wing region Z comprises a mix of phosphorothioate and phosphodiester linkages.

[0210] i.Mixmers In some embodiments, the oligonucleotides described herein may be mixmers or may include mixmer sequence patterns. Generally, mixmers are oligonucleotides containing both naturally occurring nucleosides and non-naturally occurring nucleosides, or oligonucleotides containing two different types of non-naturally occurring nucleosides, typically in an alternating pattern. Mixmers generally have higher binding affinity than unmodified oligonucleotides and specifically bind to target molecules, for example, they may be used to block binding sites on target molecules. Generally, mixmers do not allow RNase to be recruited to target molecules, and therefore do not promote cleavage of target molecules. Such oligonucleotides that are not capable of recruiting RNase H have been described, see, for example, WO2007 / 112754 or WO2007 / 112753.

[0211] In some embodiments, a mixmer comprises 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, a mixmer need not comprise a repeating pattern, but instead can comprise any arrangement of modified nucleosides and naturally occurring nucleosides, or any arrangement of one type of modified nucleoside and another type of modified nucleoside. Illustratively, the repeating pattern may be such that every third or third nucleoside is a modified nucleoside, such as LNA, 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 nucleoside 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' termini.

[0212] In some embodiments, a mixmer does not contain a region of more than five, more than four, more than three, or more than two consecutive naturally occurring nucleosides, such as DNA nucleosides. In some embodiments, a mixmer contains at least a region consisting of at least two consecutive modified nucleosides, such as at least two consecutive LNAs. In some embodiments, a mixmer contains at least a region consisting of at least three consecutive modified nucleosides, such as at least three consecutive LNAs.

[0213] In some embodiments, the mixmer does not contain a region of more than 7, more than 6, more than 5, more than 4, more than 3, or more than 2 consecutive nucleoside analogs, such as LNA. In some embodiments, the LNA units may be replaced with other nucleoside analogs, such as those mentioned herein.

[0214] Mixmers may be designed to contain a mixture of affinity-enhancing modified nucleosides, such as, in non-limiting examples, LNA nucleosides and 2'-O-Me nucleosides. In some embodiments, mixmers contain modified internucleoside linkages (e.g., phosphorothioate internucleoside linkages or other linkages) between at least two, at least three, at least four, at least five, or more nucleosides.

[0215] Mixmers may be produced using any suitable method. Representative U.S. patents, U.S. patent publications, and PCT publications that teach the preparation of mixmers include U.S. Patent Publication Nos. US20060128646, US20090209748, US20090298916, US20110077288, and US20120322851, and U.S. Patent No. 7,687,617.

[0216] In some embodiments, a mixmer comprises one or more morpholino nucleosides. For example, in some embodiments, a mixmer may comprise morpholino nucleosides mixed (e.g., in a staggered manner) with one or more other nucleosides (e.g., DNA, RNA nucleosides) or modified nucleosides (e.g., LNA, 2'-O-Me nucleosides).

[0217] In some embodiments, mixmers are useful for splice correcting or exon skipping, as reported, for example, in Touznik A., et al., LNA / DNA mixmer-based antisense oligonucleotides correct alternative splicing of the SMN2 gene and restore SMN protein expression in type 1 SMA fibroblasts, Scientific Reports, volume 7, Article number: 3672 (2017), and Chen S. et al., Synthesis of a Morpholino Nucleic Acid (MNA)-Uridine Phosphoramidite, and Exon Skipping Using MNA / 2'-O-Methyl Mixmer Antisense Oligonucleotide, Molecules 2016, 21, 1582 (the contents of each of which are incorporated herein by reference).

[0218] j.RNA interference (RNAi) In some embodiments, the oligonucleotides provided herein may be in the form of small interfering RNA (siRNA), also known as small interfering RNA or silencing RNA. siRNAs are a class of double-stranded RNA molecules, typically about 20-25 base pairs in length, that target nucleic acids (e.g., mRNA) for degradation via the RNA interference (RNAi) pathway in cells. The specificity of an siRNA molecule may be determined by the binding of the antisense strand to its target RNA. Effective siRNA molecules are generally less than 30-35 base pairs in length to avoid triggering nonspecific RNA interference pathways in cells via the interferon response, although longer siRNAs may also be effective. In some embodiments, siRNA molecules 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 or more base pairs in length. In some embodiments, the siRNA molecule is 8-30 base pairs in length, 10-15 base pairs in length, 10-20 base pairs in length, 15-25 base pairs in length, 19-21 base pairs in length, 21-23 base pairs in length.

[0219] After selecting a suitable target RNA sequence, siRNA molecules comprising the nucleotide sequence complementary to all or part of the target sequence, i.e., antisense sequence, can be designed and prepared using suitable methods (see, for example, PCT Publication No. WO2004 / 016735; and US Patent Publication No. 2004 / 0077574 and 2008 / 0081791). siRNA molecules can be double-stranded (i.e., dsRNA molecules comprising an antisense strand and a complementary sense strand that hybridizes to form dsRNA) or single-stranded (i.e., ssRNA molecules comprising only an antisense strand). siRNA molecules can comprise a double-stranded, asymmetric double-stranded, hairpin, or asymmetric hairpin secondary structure with self-complementary sense and antisense strands.

[0220] In some embodiments, the antisense strand of the siRNA molecule is 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 or more nucleotides in length. In some embodiments, the antisense strand is 8-50 nucleotides in length, 8-40 nucleotides in length, 8-30 nucleotides in length, 10-15 nucleotides in length, 10-20 nucleotides in length, 15-25 nucleotides in length, 19-21 nucleotides in length, or 21-23 nucleotides in length.

[0221] In some embodiments, the sense strand of the siRNA molecule is 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 or more nucleotides in length. In some embodiments, the sense strand is 8-50 nucleotides in length, 8-40 nucleotides in length, 8-30 nucleotides in length, 10-15 nucleotides in length, 10-20 nucleotides in length, 15-25 nucleotides in length, 19-21 nucleotides in length, or 21-23 nucleotides in length.

[0222] In some embodiments, siRNA molecule comprises antisense strand, which comprises the region of complementarity with target region on target mRNA.In some embodiments, the region of complementarity is 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 target region on target mRNA.In some embodiments, the target region is the region of a contiguous nucleotide on target mRNA.In some embodiments, to be specifically hybridizable or specific for target RNA sequence, complementary nucleotide sequence does not need to be 100% complementary to that of its target.

[0223] In some embodiments, the siRNA molecule comprises an antisense strand that includes a region of complementarity to a target RNA sequence, wherein the region of complementarity ranges from 8 to 15, 8 to 30, 8 to 40, or 10 to 50, or 5 to 50, or 5 to 40 nucleotides in length. In some embodiments, the region of complementarity is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length. In some embodiments, the complementary region is complementary to at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25 or more consecutive nucleotides of the target RNA sequence.In some embodiments, the siRNA molecule comprises a nucleotide sequence that contains 1, 2, 3, 4 or 5 bases or less mismatch compared with the consecutive nucleotide portion of the target RNA sequence.In some embodiments, the siRNA molecule comprises a nucleotide sequence that has up to 3 mismatches per 15 bases or up to 2 mismatches per 10 bases.

[0224] In some embodiments, the siRNA molecule comprises an antisense strand comprising a nucleotide sequence that is complementary (e.g., at least 85%, at least 90%, at least 95%, or 100%) to the target RNA sequence of the oligonucleotide provided herein. In some embodiments, the siRNA molecule comprises an antisense strand comprising a nucleotide sequence that is at least 85%, at least 90%, at least 95%, or 100% identical to the oligonucleotide provided herein. In some embodiments, the siRNA molecule comprises an antisense strand comprising at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, or more consecutive nucleotides of the oligonucleotide provided herein.

[0225] Double-stranded siRNA may comprise sense RNA strands and antisense RNA strands of the same length or different lengths.Double-stranded siRNA molecules can also be assembled from a single oligonucleotide of stem-loop structure (where the self-complementary sense and antisense regions of siRNA molecules are linked using nucleic acid-based or non-nucleic acid-based linker(s)), and circular single-stranded RNA (where circular RNA can be processed either in vivo or in vitro to produce active siRNA molecules that can mediate RNAi) with a stem that comprises two or more loop structures and self-complementary sense and antisense strands.Therefore, small hairpin RNA (shRNA) molecules are also contemplated herein.These molecules contain a specific antisense sequence in addition to a reverse complementary (sense) sequence, and are typically separated by a spacer or loop sequence. Cleavage of the spacer or loop (optionally with additional processing steps that may result in the addition or removal of one, two, three, or more nucleotides from the 3'-end and / or (for example, and) the 5'-end of one or both strands) provides the single-stranded RNA molecule and its reverse complement so that they can anneal to form a dsRNA molecule. The spacer can be long enough to allow the antisense and sense sequences to anneal to form a double-stranded structure (or stem) prior to cleavage of the spacer (and optionally with subsequent processing steps that may result in the addition or removal of one, two, three, four, or more nucleotides from the 3'-end and / or (for example, and) the 5'-end of one or both strands). The spacer sequence can be an unrelated nucleotide sequence located between two complementary nucleotide sequence regions that, once annealed to form the double-stranded nucleic acid, comprise the shRNA.

[0226] The overall length of siRNA molecule can vary from about 14 nucleotides to about 100 nucleotides according to the type of siRNA molecule designed.Generally, between about 14 and about 50 of these nucleotides are complementary to RNA target sequence, that is, constitute the specific antisense sequence of siRNA molecule.For example, when siRNA is double-stranded siRNA or single-stranded siRNA, its length can vary from about 14 nucleotides to about 50 nucleotides, while when siRNA is shRNA or circular molecule, its length can vary from about 40 nucleotides to about 100 nucleotides.

[0227] An siRNA molecule may include a 3' overhang at one end of the molecule, and the other end may be blunt or may also have an overhang (5' or 3'). When an siRNA molecule includes overhangs at both ends of the molecule, the lengths of the overhangs may be the same or different. In one embodiment, an siRNA molecule of the present disclosure includes a 3' overhang of about 1 to about 3 nucleotides on both ends of the molecule. In some embodiments, an siRNA molecule includes a 3' overhang of about 1 to about 3 nucleotides on the sense strand. In some embodiments, an siRNA molecule includes a 3' overhang of about 1 to about 3 nucleotides on the antisense strand. In some embodiments, an siRNA molecule includes a 3' overhang of about 1 to about 3 nucleotides on both the sense strand and the antisense strand.

[0228] In some embodiments, the siRNA molecule comprises one or more modified nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more). In some embodiments, the siRNA molecule comprises one or more modified nucleotides and / or (e.g., and) one or more modified internucleotide linkages. In some embodiments, the modified nucleotide is a modified sugar moiety (e.g., a 2'-modified nucleotide). In some embodiments, the siRNA molecule comprises one or more 2'-modified nucleotides, such as 2'-deoxy, 2'-fluoro (2'-F), 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA). In some embodiments, each nucleotide of the siRNA molecule is a modified nucleotide (e.g., a 2'-modified nucleotide). In some embodiments, the siRNA molecule comprises one or more phosphorodiamidate morpholinos. In some embodiments, each nucleotide of the siRNA molecule is a phosphorodiamidate morpholino.

[0229] In some embodiments, the siRNA molecule contains phosphorothioate or other modified internucleoside linkages. In some embodiments, the siRNA molecule comprises phosphorothioate internucleoside linkages. In some embodiments, the siRNA molecule comprises phosphorothioate internucleoside linkages between at least two nucleotides. In some embodiments, the siRNA molecule comprises phosphorothioate internucleoside linkages between all nucleotides. For example, in some embodiments, the siRNA molecule comprises modified internucleoside linkages at the first, second, and / or (for example, and) third internucleoside linkages at the 5' or 3' end of the siRNA molecule.

[0230] In some embodiments, the modified internucleotide linkage is a phosphorus-containing linkage. In some embodiments, phosphorus-containing linkages that can be used include phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates, including 3' alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates, including 3'-aminophosphoramidates and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates, their 2'-5' linked analogs, and reverse polarity nucleotides in which adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Patents including, but not limited to, those having U.S. Patent Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399, See Nos. 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.

[0231] Any of the modified chemistries or formats of the siRNA molecules described herein can be combined with each other, for example, one, two, three, four, five, or more different types of modifications can be included on the same siRNA molecule.

[0232] In some embodiments, the antisense strand comprises one or more modified nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more). In some embodiments, the antisense strand comprises one or more modified nucleotides and / or (e.g., and) one or more modified internucleotide linkages. In some embodiments, the modified nucleotide comprises a modified sugar moiety (e.g., a 2'-modified nucleotide). In some embodiments, the antisense strand contains one or more 2'-modified nucleotides, such as 2'-deoxy, 2'-fluoro (2'-F), 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA). In some embodiments, each nucleotide in the antisense strand is a modified nucleotide (e.g., a 2'-modified nucleotide). In some embodiments, the antisense strand contains one or more phosphorodiamidate morpholinos. In some embodiments, the antisense strand is a phosphorodiamidate morpholino oligomer (PMO).

[0233] In some embodiments, the antisense strand contains phosphorothioate or other modified internucleotide linkages. In some embodiments, the antisense strand contains phosphorothioate internucleoside linkages. In some embodiments, the antisense strand contains phosphorothioate internucleoside linkages between at least two nucleotides. In some embodiments, the antisense strand contains phosphorothioate internucleoside linkages between all nucleotides. For example, in some embodiments, the antisense strand contains modified internucleotide linkages at the first, second, and / or (for example, and) third internucleoside linkages at the 5' or 3' end of the siRNA molecule. In some embodiments, the modified internucleotide linkage is a phosphorus-containing linkage.In some embodiments, phosphorus-containing linkages that may be used include phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates, including 3' alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates, including 3'-amino phosphoramidates and aminoalkyl phosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates, 2'-5' linked analogs thereof, as well as nucleoside units having opposite polarity, where adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Patents including, but not limited to, those having U.S. Patent Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399, See Nos. 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.

[0234] Any of the antisense strand modified chemistries or formats described herein can be combined with each other, for example, one, two, three, four, five, or more different types of modifications can be included on the same antisense strand.

[0235] In some embodiments, the sense strand comprises one or more modified nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more). In some embodiments, the sense strand comprises one or more modified nucleotides and / or (e.g., and) one or more modified internucleotide linkages. In some embodiments, the modified nucleotide is a modified sugar moiety (e.g., a 2'-modified nucleotide). In some embodiments, the sense strand comprises one or more 2'-modified nucleotides, such as 2'-deoxy, 2'-fluoro (2'-F), 2'-O-methyl (2'-O-Me), 2'O-methoxyethyl (2'-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA). In some embodiments, each nucleotide in the sense strand is a modified nucleotide (e.g., a 2'-modified nucleotide). In some embodiments, the sense strand comprises one or more phosphorodiamidate morpholinos. In some embodiments, the antisense strand is a phosphorodiamidate morpholino oligomer (PMO). In some embodiments, the sense strand contains phosphorothioate or other modified internucleotide linkages. In some embodiments, the sense strand comprises phosphorothioate internucleoside linkages. In some embodiments, the sense strand comprises phosphorothioate internucleoside linkages between at least two nucleotides. In some embodiments, the sense strand comprises phosphorothioate internucleoside linkages between all nucleotides. For example, in some embodiments, the sense strand comprises a modified internucleotide linkage at the first, second, and / or (for example, and) third internucleoside linkage at the 5' or 3' end of the sense strand.

[0236] In some embodiments, the modified internucleotide linkage is a phosphorus-containing linkage. In some embodiments, phosphorus-containing linkages that can be used include phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates, including 3' alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates, including 3'-aminophosphoramidates and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates, their 2'-5' linked analogs, and reverse polarity nucleotides in which adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Patents including, but not limited to, those having U.S. Patent Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399, See Nos. 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.

[0237] Any of the sense strand modification chemistries or formats described herein can be combined with each other, for example, one, two, three, four, five, or more different types of modifications can be included on the same sense strand.

[0238] In some embodiments, the antisense or sense strand of the siRNA molecule comprises a modification that enhances or reduces RNA-induced silencing complex (RISC) loading. In some embodiments, the antisense strand of the siRNA molecule comprises a modification that enhances RISC loading. In some embodiments, the sense strand of the siRNA molecule comprises a modification that reduces RISC loading and reduces off-target effects. In some embodiments, the antisense strand of the siRNA molecule comprises a 2'-O-methoxyethyl (2'-MOE) modification. As described in Song et al., (2017) Mol Ther Nucleic Acids 9:242-250, the entire contents of which are incorporated herein by reference, the addition of a 2'-O-methoxyethyl (2'-MOE) group at the cleavage site improves both the specificity and silencing activity of siRNA by facilitating the directed RNA-induced silencing complex (RISC) loading of the modified strand. In some embodiments, the antisense strand of the siRNA molecule contains a 2'-OMe-phosphorodithioate modification, which increases RISC loading, as described in Wu et al. (2014) Nat Commun 5:3459, which is incorporated by reference in its entirety.

[0239] In some embodiments, the sense strand of the siRNA molecule contains a 5' morpholino, which reduces RISC loading of the sense strand and improves antisense strand selection and RNAi activity, as described in Kumar et al., (2019) Chem Commun (Camb) 55(35):5139-5142, the entire contents of which are incorporated herein by reference. In some embodiments, the sense strand of the siRNA molecule is modified with a synthetic RNA-like high-affinity nucleotide analog, locked nucleic acid (LNA). This reduces RISC loading of the sense strand and further enhances antisense strand incorporation into RISC, as described in Elman et al., (2005) Nucleic Acids Res. 33(1):439-447, the entire contents of which are incorporated herein by reference. In some embodiments, the sense strand of the siRNA molecule comprises a 5'unlocked nucleic acid (UNA) modification, which reduces the RISC loading of the sense strand and improves the silencing potency of the antisense strand, as described in Snead et al., (2013) Mol Ther Nucleic Acids 2(7):e103, the entirety of which is incorporated herein by reference. In some embodiments, the sense strand of the siRNA molecule comprises a 5-nitroindole modification, which reduces the RNAi potency of the sense strand and reduces off-target effects, as described in Zhang et al., (2012) Chembiochem 13(13):1940-1945, the entirety of which is incorporated herein by reference. In some embodiments, the sense strand contains 2'-O' methyl (2'-O-Me) modifications, which reduce RISC loading and off-target effects of the sense strand, as described in Zheng et al., FASEB (2013) 27(10):4017-4026, which is incorporated herein by reference in its entirety.In some embodiments, the sense strand of the siRNA molecule is completely substituted with morpholino, 2'-MOE, or 2'-O-Me residues and is not recognized by RISC, as described in Kole et al., (2012) Nature reviews. Drug Discovery 11(2):125-140, the entire contents of which are incorporated herein by reference. In some embodiments, the antisense strand of the siRNA molecule contains a 2'-MOE modification, and the sense strand contains a 2'-O-Me modification (see, for example, Song et al., (2017) Mol Ther Nucleic Acids 9:242-250). In some embodiments, at least one (e.g., at least two, at least three, at least four, at least five, or at least 10) siRNA molecule is linked (e.g., covalently) to a muscle-targeting agent. In some embodiments, the muscle-targeting agent may comprise or consist of a nucleic acid (e.g., DNA or RNA), a peptide (e.g., an antibody), a lipid (e.g., a microvesicle), or a sugar moiety (e.g., a polysaccharide). In some embodiments, the muscle targeting agent is an antibody. In some embodiments, the muscle targeting agent is an anti-transferrin receptor antibody (e.g., any one of the anti-TfR antibodies provided herein). In some embodiments, the muscle targeting agent can be linked to the 5' end of the sense strand of the siRNA molecule. In some embodiments, the muscle targeting agent can be linked to the 3' end of the sense strand of the siRNA molecule. In some embodiments, the muscle targeting agent can be linked internally to the sense strand of the siRNA molecule. In some embodiments, the muscle targeting agent can be linked to the 5' end of the antisense strand of the siRNA molecule. In some embodiments, the muscle targeting agent can be linked to the 3' end of the antisense strand of the siRNA molecule. In some embodiments, the muscle targeting agent can be linked internally to the antisense strand of the siRNA molecule.

[0240] k. microRNA (miRNA) In some embodiments, the oligonucleotide may be a microRNA (miRNA). MicroRNAs (also referred to as "miRNAs") are small, non-coding RNAs that belong to a class of regulatory molecules that control gene expression by binding to complementary sites on target RNA transcripts. Typically, miRNAs are generated from large RNA precursors (called pri-miRNAs), which are processed in the nucleus into approximately 70-nucleotide pre-miRNAs that fold into imperfect stem-loop structures. These pre-miRNAs typically undergo additional processing steps in the cytoplasm, where mature miRNAs, 18-25 nucleotides in length, are excised from one side of the pre-miRNA hairpin by the RNase III enzyme Dicer.

[0241] As used herein, miRNA encompasses fragments of pri-miRNA, pre-miRNA, mature miRNA, or variants thereof that retain the biological activity of mature miRNA. In one embodiment, the size range of miRNA can be from 21 nucleotides to 170 nucleotides. In one embodiment, the size range of miRNA is from 70 nucleotides to 170 nucleotides in length. In another embodiment, mature miRNAs that are 21 nucleotides to 25 nucleotides in length can be used.

[0242] l. Aptamer In some embodiments, the oligonucleotides provided herein may be in the form of an aptamer. Generally, in the context of molecular payloads, an aptamer is any nucleic acid that specifically binds to a target, such as a small molecule, protein, or nucleic acid, in a cell. In some embodiments, the aptamer is a DNA aptamer or an RNA aptamer. In some embodiments, the nucleic acid aptamer is single-stranded DNA or RNA (ssDNA or ssRNA). It should be understood that single-stranded nucleic acid aptamers may form helix and / or (for example, and) loop structures. The nucleic acids forming nucleic acid aptamers may include naturally occurring nucleotides, modified nucleotides, naturally occurring nucleotides with hydrocarbon linkers (e.g., alkylene) or polyether linkers (e.g., PEG linkers) inserted between one or more nucleotides, modified nucleotides with hydrocarbon or PEG linkers inserted between one or more nucleotides, or combinations thereof. Exemplary publications and patents describing aptamers and methods of producing aptamers include, by way of example, Lorsch and Szostak, 1996; Jayasena, 1999; U.S. Patent Nos. 5,270,163; 5,567,588; 5,650,275; 5,670,637; 5,683,867; 5,696,249; 5,789,157; ​​5,843,653; 5,864,026; 5,989,823; 6,569,630; 8,318,438 and PCT application WO 99 / 31275, each of which is incorporated herein by reference.

[0243] m. ribozyme In some embodiments, the oligonucleotide provided herein can be in the form of ribozyme.Ribozyme (ribonucleic acid enzyme) is a molecule, typically an RNA molecule, that can carry out specific biochemical reactions similar to the action of protein enzymes.Ribozyme is a molecule with catalytic activity that includes the ability to cleave specific phosphodiester bonds in the RNA molecules (such as mRNA, RNA-containing substrates, lncRNA, and ribozyme itself) that they hybridize with.

[0244] Ribozymes can take on one of several physical structures, one of which is called a "hammerhead." Hammerhead ribozymes consist of a conserved nine-base catalytic core, a double-stranded stem and loop structure (stem-loop II), and two regions complementary to the target RNA flanking regions surrounding the catalytic core. The flanking regions enable the ribozyme to specifically bind to the target RNA by forming double-stranded stems I and III. Cleavage occurs in cis (i.e., cleavage of the same RNA molecule containing the hammerhead motif) or trans (cleavage of an RNA substrate other than that containing the ribozyme) following a specific ribonucleotide triplet by transesterification of a 3',5'-phosphodiester to a 2',3'-cyclic phosphodiester. Without wishing to be bound by theory, this catalytic activity is believed to require the presence of a specific, highly conserved sequence in the catalytic region of the ribozyme.

[0245] Modifications in ribozyme structures also encompass the substitution or replacement of various non-core portions of molecules with non-nucleotide molecules.For example, Benseler et al. (J.Am.Chem.Soc.(1993)115:8483-8484) disclosed a hammerhead-like molecule, in which two base pairs of stem II and all four nucleotides of loop II were replaced with non-nucleoside linkers based on hexaethylene glycol, propanediol, bis(triethylene glycol) phosphate, tris(propanediol) bisphosphate, or bis(propanediol) phosphate.Ma et al. (Biochem.(1993)32:1751-1758; Nucleic Acids Res.(1993)21:2585-2589) replaced the 6-nucleotide loop of the TAR ribozyme hairpin with a non-nucleotide linker based on ethylene glycol. Thomson et al. (Nucleic Acids Res. (1993) 21:5600-5603) replaced loop II with linear non-nucleotide linkers of 13, 17, and 19 atoms in length.

[0246] Ribozyme oligonucleotides can be prepared using well-known methods (see, for example, PCT Publications WO9118624; WO9413688; WO9201806; and WO92 / 07065; and U.S. Patents 5,436,143 and 5,650,502) or purchased from commercial sources (e.g., US Biochemicals), and, if desired, can incorporate nucleotide analogs to increase the resistance of the oligonucleotide to degradation by cellular nucleases. Ribozymes can be synthesized in any known manner, for example, by using a commercially available synthesizer (e.g., manufactured by Applied Biosystems, Inc. or Milligen). Ribozymes can also be produced in recombinant vectors by conventional means. See Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory (Current Edition). Ribozyme RNA sequences can be synthesized in conventional ways, for example, by using RNA polymerases such as T7 or SP6.

[0247] n. Guide nucleic acid In some embodiments, the oligonucleotide is a guide nucleic acid, for example, a guide RNA (gRNA) molecule. Generally, a guide RNA is a short synthetic RNA composed of (1) a scaffold sequence that binds to a nucleic acid programmable DNA binding protein (napDNAbp), such as Cas9, and (2) a nucleotide spacer portion that defines a DNA target sequence (for example, a genomic DNA target) to which the gRNA binds to bring the nucleic acid programmable DNA binding protein close to the DNA target sequence. In some embodiments, the napDNAbp is a nucleic acid programmable protein that forms a complex with (for example, by binding to or associating with) one or more RNA(s) that target the nucleic acid programmable protein to a target DNA sequence (for example, a target genomic DNA sequence). In some embodiments, a nucleic acid programmable nuclease, when in a complex with an RNA, is sometimes referred to as a nuclease:RNA complex. A guide RNA can exist as a complex of two or more RNAs or as a single RNA molecule.

[0248] A guide RNA (gRNA) that exists as a single RNA molecule is sometimes referred to as a single guide RNA (sgRNA), although gRNA is also used to refer to a guide RNA that exists either as a single molecule or as a complex of two or more molecules. Typically, a gRNA that exists as a single RNA species contains two domains: (1) a domain that shares homology with the target nucleic acid (i.e., directs the Cas9 complex to bind to the target); and (2) a domain that binds to the Cas9 protein. In some embodiments, domain (2) corresponds to a sequence known as tracrRNA and contains a stem-loop structure. In some embodiments, domain (2) is identical to or homologous to tracrRNA as provided in Jinek et al., Science 337:816-821 (2012), the entire contents of which are incorporated herein by reference.

[0249] In some embodiments, the gRNA comprises two or more of domains (1) and (2) and is sometimes referred to as an extended gRNA. For example, the extended gRNA will bind to two or more Cas9 proteins and bind to the target nucleic acid in two or more distinct regions, as described herein. The gRNA comprises a nucleotide sequence complementary to the target site that mediates the binding of the nuclease / RNA complex to the target site, providing sequence specificity for the nuclease:RNA complex.In some embodiments, the RNA-programmable nuclease is a (CRISPR-related system) Cas9 endonuclease, e.g., Cas9 (Csn1) from Streptococcus pyogenes (see, e.g., "Complete genome sequence of an M1 strain of Streptococcus pyogenes" by Ferretti, JJ, McShan WM, Ajdic DJ, Savic DJ, Savic G., Lyon K., Primeaux C., Sezate S., Suvorov AN, Kenton S., Lai HS, Lin SP, Qian Y., Jia HG, Najar FZ, Ren Q., Zhu H., Song L., White J., Yuan X., Clifton SW, Roe BA, McLaughlin See RE, Proc. Natl. Acad. Sci. USA 98:4658-4663 (2001); "CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III," Deltcheva E., Chylinski K., Sharma CM, Gonzales K., Chao Y., Pirzada ZA, Eckert MR, Vogel J., Charpentier E., Nature 471:602-607 (2011); and "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity," Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna JA, Charpentier E. Science 337:816-821 (2012), the entire contents of each of which are incorporated herein by reference.

[0250] o. multimers In some embodiments, molecular payloads may comprise a multimer (e.g., a concatemer) of two or more oligonucleotides connected by a linker. Thus, in some embodiments, the oligonucleotide loading of the complex / conjugate can be increased beyond the available linking sites on the targeting agent (e.g., available thiol sites or amine sites on an antibody), or can be otherwise tailored to reach a specific payload loading amount. The oligonucleotides in the multimer can be the same or different (e.g., targeting different genes, or different sites on the same gene, or their products).

[0251] In some embodiments, a multimer comprises two or more oligonucleotides linked together by a cleavable linker. However, in some embodiments, a multimer comprises two or more oligonucleotides linked together by a non-cleavable linker. In some embodiments, a multimer comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more oligonucleotides linked together. In some embodiments, a multimer comprises 2-5, 2-10, or 4-20 oligonucleotides linked together.

[0252] In some embodiments, a multimer comprises two or more oligonucleotides linked end-to-end (in a linear configuration). In some embodiments, a multimer comprises two or more oligonucleotides linked end-to-end via an oligonucleotide-based linker (e.g., a poly-dT linker, an abasic linker). In some embodiments, a multimer comprises the 5' end of one oligonucleotide linked to the 3' end of another oligonucleotide. In some embodiments, a multimer comprises the 3' end of one oligonucleotide linked to the 3' end of another oligonucleotide. In some embodiments, a multimer comprises the 5' end of one oligonucleotide linked to the 5' end of another oligonucleotide. Still further, in some embodiments, a multimer may comprise a branched structure comprising multiple oligonucleotides linked together by a branched linker.

[0253] Further examples of multimers that may be used in the conjugates provided herein are described, for example, in U.S. Patent Application No. 2015 / 0315588 A1, published November 5, 2015, entitled "Methods of Delivering Multiple Targeting Oligonucleotides to a Cell Using Cleavable Linkers"; U.S. Patent Application No. 2015 / 0247141 A1, published September 3, 2015, entitled "Multimeric Oligonucleotide Compounds"; U.S. Patent Application No. US 2011 / 0158937 A1, published June 30, 2011, entitled "Immunostimulatory Oligonucleotide Multimers"; and U.S. Patent Application No. US 2011 / 0158937 A1, published December 2, 1997, entitled "Triplex-Forming Antisense Oligonucleotides Having Abasic Linkers, Targeting Nucleic Acids Comprising Mixed Sequences of Purines and No. 5,693,773 entitled Pyrimidines, the contents of each of which are incorporated herein by reference in their entirety.

[0254] o. Splice-altering oligonucleotides In some embodiments, the oligonucleotides of the present disclosure (e.g., antisense oligonucleotides, including morpholinos) target splicing. In some embodiments, the oligonucleotides target splicing by inducing exon skipping and restoring the reading frame within the gene. As a non-limiting example, the oligonucleotides may induce skipping of exons encoding frameshift mutations and / or exons encoding premature stop codons (e.g., and). In some embodiments, the oligonucleotides may induce exon skipping by blocking spliceosome recognition of splice sites. In some embodiments, exon skipping results in a truncated but functional protein compared to a reference protein (e.g., the truncated but functional DMD protein described below). In some embodiments, the oligonucleotides promote the inclusion of a specific exon (e.g., exon 7 of the SMN2 gene described below). In some embodiments, the oligonucleotides may induce exon inclusion by targeting splice site inhibitory sequences. RNA splicing has been implicated in muscle diseases, including Duchenne muscular dystrophy (DMD) and spinal muscular atrophy (SMA).

[0255] Alterations (e.g., deletions, point mutations, and duplications) in the gene encoding dystrophin (DMD) cause DMD. These alterations can lead to frameshift mutations and / or (e.g., and) nonsense mutations. In some embodiments, the oligonucleotides of the present disclosure promote skipping of one or more DMD exons (e.g., exon 8, exon 43, exon 44, exon 45, exon 50, exon 51, exon 52, exon 53, and / or (e.g., and) exon 55), resulting in a functional truncated protein. For examples, see U.S. Patent No. 8,486,907, published July 16, 2013, and U.S. Patent No. 20140275212, published September 18, 2014.

[0256] In SMA, there is a loss of functional SMN1. Although the SMN2 gene is a paralog to SMN1, alternative splicing of the SMN2 gene primarily leads to skipping of exon 7 and the subsequent production of a truncated SMN protein that cannot compensate for the loss of SMN1. In some embodiments, the oligonucleotides of the present disclosure promote the inclusion of SMN2 exon 7. In some embodiments, the oligonucleotides are antisense oligonucleotides that target SMN2 splice site inhibitory sequences (see, e.g., U.S. Patent No. 7,838,657, published November 23, 2010).

[0257] ii. Small molecules: Any suitable small molecule may be used as a molecular payload, as described herein.

[0258] iii. Peptides / proteins Any suitable peptide or protein may be used as a molecular payload as described herein. In some embodiments, the protein is an enzyme (e.g., acid alpha-glucosidase, e.g., encoded by the GAA gene). These peptides or proteins may be produced, synthesized, and / or (e.g., and) derivatized using several methodologies, e.g., phage-displayed peptide libraries, one-bead-one-compound peptide libraries, or position-scanning synthetic peptide combinatorial libraries. Exemplary methodologies have been characterized in the art and are incorporated by reference (Gray, B.P., and Brown, K.C., "Combinatorial Peptide Libraries: Mining for Cell-Binding Peptides," Chem Rev. 2014, 114:2, 1020-1081; Samoylova, T.I., and Smith, B.F., "Elucidation of muscle-binding peptides by phage display screening," Muscle Nerve, 1999, 22:4, 460-6).

[0259] iv. Nucleic Acid Contracts Any suitable gene expression construct may be used as a molecular payload as described herein. In some embodiments, the gene expression construct may be a vector or a cDNA fragment. In some embodiments, the gene expression construct may be messenger RNA (mRNA). In some embodiments, the mRNA used herein may be modified mRNA, e.g., as described in U.S. Patent 8,710,200, issued April 24, 2014, entitled "Engineered nucleic acids encoding a modified erythropoietin and their expression." In some embodiments, the mRNA may include a 5' methyl cap. In some embodiments, the mRNA may include a polyA tail, optionally up to 160 nucleotides in length. The gene expression construct may encode a protein sequence deficient in a muscle disease. In some embodiments, the gene expression construct may be expressed, e.g., overexpressed, in the nucleus of a muscle cell. In some embodiments, the gene expression construct may encode a gene deficient in a muscle disease. In some embodiments, the gene expression construct encodes a protein comprising at least one zinc finger. In some embodiments, the gene expression construct encodes a protein that binds to a gene in Table 6. In some embodiments, the gene expression construct encodes a protein that leads to reduced expression of a protein (e.g., a mutant protein) encoded by a gene in Table 6. In some embodiments, the gene expression construct encodes a gene-editing enzyme.Additional examples of nucleic acid constructs that may be used as molecular payloads are provided in International Patent Application Publication WO2017152149A1, published September 19, 2017, entitled "CLOSED-ENDED LINEAR DUPLEX DNA FOR NON-VIRAL GENE TRANSFER"; U.S. Patent No. 8,853,377B2, issued October 7, 2014, entitled "MRNA FOR USE IN TREATMENT OF HUMAN GENETIC DISEASES"; and U.S. Patent No. US8822663B2, issued September 2, 2014, entitled "ENGINEERED NUCLEIC ACIDS AND METHODS OF USE THEREOF," the contents of each of which are incorporated herein by reference in their entirety.

[0260] v. Detectable Labels / Diagnostic Agents Any suitable detectable label or diagnostic agent can be used as the molecular payload of the present disclosure. "Diagnostic agent" refers to an agent that is used for diagnostic purposes, for example, by detecting another molecule in cells or tissues. In some embodiments, the diagnostic agent is an agent that targets (for example, binds to) a biomarker (for example, a nucleic acid biomarker, a protein biomarker, or a metabolite biomarker) known to be associated with a disease in a subject, and generates a detectable signal that can be used to determine the presence / absence of the biomarker and thus diagnose the disease. For example, the diagnostic agent can be, without limitation, an antibody or an antisense nucleic acid.

[0261] In some embodiments, the diagnostic agent contains a detectable label. A detectable label refers to a moiety having at least one element, isotope, or structural or functional group incorporated therein that allows for detection of the molecule (e.g., a protein or polypeptide, or other entity) to which the diagnostic agent binds. In some embodiments, detectable labels fall into one (or more) of five classes: a) agents containing an isotopic moiety that may be a radioisotope or a heavy isotope, including, but not limited to, 2H, 3H, 13C, 14C, 15N, 18F, 31P, 32P, 35S, 67Ga, 76Br, 99mTc (Tc-99m), 111In, 123I, 125I, 131I, 153Gd, 169Yb, and 186Re; b) enzymes (e.g., Western a) an agent containing an immunological moiety, which may be an antibody or antigen, optionally conjugated to a fluorescent protein (e.g., horseradish peroxidase); b) an agent containing a colored, luminescent, phosphorescent, or fluorescent moiety (e.g., the fluorescently labeled fluorescein isothiocyanate (FITC)); c) an agent containing one or more photoaffinity moieties; and e) an agent that is a ligand for one or more known binding partners (e.g., biotin-streptavidin, His-NiTNAFK506-FKBP). In some embodiments, the detectable label comprises a radioisotope. In some embodiments, the detectable label comprises a fluorescent moiety. In some embodiments, the detectable label comprises a dye, e.g., a fluorescent dye, e.g., fluorescein isothiocyanate, Texas Red, rhodamine, Cy3, Cy5, Cy5.5, Alexa 647, and derivatives. In some embodiments, the detectable label comprises biotin. In some embodiments, the detectable molecule is a fluorescent polypeptide (e.g., GFP or a derivative thereof, such as enhanced GFP (EGFP)), or a luciferase (e.g., firefly luciferase, Renilla luciferase, or Gaussia luciferase). In some embodiments, the detectable label may react with a suitable substrate (e.g., luciferin) to generate a detectable signal.Non-limiting examples of fluorescent proteins include GFP and its derivatives, proteins containing chromophores that emit light of various colors (such as red, yellow, and cyan fluorescent proteins), etc. Exemplary fluorescent proteins include, by way of example, Sirius, Azurite, EBFP2, TagBFP, mTurquoise, ECFP, Cerulean, TagCFP, mTFP1, mUkG1, mAG1, AcGFP1, TagGFP2, EGFP, mWasabi, EmGFP, TagYPF, EYFP, Topaz, SYFP2, Venus, Citrine, mKO, mKO2, mOrange, mOrange2, TagRFP, TagRFP-T, mStrawberry, mRuby, mCherry, mRaspberry, mKate2, mPlum, mNeptune, T-Sapphire, mAmetrine, and mKeima. See, e.g., Chalfie, M. and Kain, S.R. (eds.) Green fluorescent protein: properties, applications, and protocols (Methods of biochemical analysis, v. 47, Wiley-Interscience, and Hoboken, NJ, 2006, and / or (e.g., and), Chudakov, D.M., et al., Physiol Rev. 90(3):1103-63, 2010, which are incorporated herein by reference for a discussion of GFP and countless other fluorescent or luminescent proteins. In some embodiments, the detectable label comprises a dark quencher, e.g., a substance that absorbs excitation energy from a fluorophore and dissipates the energy as heat.

[0262] B. Linker The conjugates described herein generally include a linker connecting any one of the anti-TfR antibodies described herein to the molecular payload. The linker includes at least one covalent bond. In some embodiments, the linker may be a single bond, such as a disulfide bond or disulfide bridge, connecting the anti-TfR antibody to the molecular payload. However, in some embodiments, the linker may connect any one of the anti-TfR antibodies described herein to the molecular payload through multiple covalent bonds. In some embodiments, the linker may be a cleavable linker. However, in some embodiments, the linker may be a non-cleavable linker. Linkers are generally stable in vitro and in vivo and may be stable in certain cellular environments. In addition, linkers generally do not negatively affect the functional properties of either the anti-TfR antibody or the molecular payload. Examples and methods for the synthesis of linkers are known in the art (see, for example, Kline, T. et al., "Methods to Make Homogenous Antibody Drug Conjugates," Pharmaceutical Research, 2015, 32:11, 3480-3493; Jain, N. et al., "Current ADC Linker Chemistry," Pharm Res. 2015, 32:11, 3526-3540; McCombs, JR and Owen, SC, "Antibody Drug Conjugates: Design and Selection of Linker, Payload and Conjugation Chemistry," AAPS J. 2015, 17:2, 339-351).

[0263] The precursor of the linker will typically contain two different reactive species that can attach to both the anti-TfR antibody and the molecular payload. In some embodiments, the two different reactive species may be nucleophiles and / or (for example, and) electrophiles. In some embodiments, the linker is connected to the anti-TfR antibody via conjugation to a lysine or cysteine ​​residue of the anti-TfR antibody. In some embodiments, the linker is connected to the cysteine ​​residue of the anti-TfR antibody via a maleimide-containing linker, where optionally, the maleimide-containing linker comprises a maleimidocaproyl or maleimidomethylcyclohexane-1-carboxylate group. In some embodiments, the linker is connected to the cysteine ​​residue of the anti-TfR antibody or a thiol-functionalized molecular payload via a 3-arylpropionitrile functional group. In some embodiments, the linker is connected to the lysine residue of the anti-TfR antibody. In some embodiments, the linker is connected to the anti-TfR antibody and / or the molecular payload (by way of example and not limitation) via an amide bond, a carbamate bond, a hydrazide, a triazole, a thioether, or a disulfide bond.

[0264] i. Cleavable linker The cleavable linker may be a protease-sensitive linker, a pH-sensitive linker, or a glutathione-sensitive linker, which are generally only cleavable intracellularly and are preferably stable in an extracellular environment, e.g., outside of a muscle cell.

[0265] Protease-sensitive linkers are cleavable by protease enzyme activity. These linkers typically contain peptide sequences and may be 2-10 amino acids, about 2-5 amino acids, about 5-10 amino acids, about 10 amino acids, about 5 amino acids, about 3 amino acids, or about 2 amino acids in length. In some embodiments, the peptide sequence may contain naturally occurring amino acids, such as cysteine, alanine, or non-naturally occurring or modified amino acids. Non-naturally occurring amino acids include β-amino acids, homo-amino acids, proline derivatives, 3-substituted alanine derivatives, linear core amino acids, N-methyl amino acids, and other amino acids known in the art. In some embodiments, the protease-sensitive linker contains a valine-citrulline or alanine-citrulline dipeptide sequence. In some embodiments, the protease-sensitive linker may be cleaved by lysosomal proteases, such as cathepsin B, and / or endosomal proteases (e.g., α- and β-amino acids).

[0266] A pH-sensitive linker is a covalent linkage that is readily degraded in high or low pH environments. In some embodiments, the pH-sensitive linker may be cleaved at a pH in the range of 4 to 6. In some embodiments, the pH-sensitive linker comprises a hydrazone or a cyclic acetal. In some embodiments, the pH-sensitive linker is cleaved in an endosome or lysosome.

[0267] In some embodiments, the glutathione-sensitive linker comprises a disulfide moiety. In some embodiments, the glutathione-sensitive linker is cleaved by a disulfide exchange reaction with glutathione species inside a cell. In some embodiments, the disulfide moiety further comprises at least one amino acid, for example, a cysteine ​​residue.

[0268] In some embodiments, the linker is a Val-cit linker (e.g., as described in U.S. Patent 6,214,345, incorporated herein by reference). In some embodiments, prior to conjugation, the val-cit linker has the following structure: [ka]

[0269] In some embodiments, after conjugation, the val-cit linker has the following structure: [ka]

[0270] In some embodiments, the Val-cit linker is attached to a reactive chemical moiety (e.g., SPAAC for click chemistry conjugation). In some embodiments, prior to click chemistry conjugation, the val-cit linker attached to the reactive chemical moiety (e.g., SPAAC for click chemistry conjugation) has the following structure: [ka] wherein n is any number from 0 to 10. In some embodiments, n is 3.

[0271] In some embodiments, a val-cit linker attached to a reactive chemical moiety (e.g., a SPAAC for click chemistry conjugation) is conjugated (e.g., via a different chemical moiety) to a molecular payload (e.g., an oligonucleotide). In some embodiments, the val-cit linker attached to a reactive chemical moiety (e.g., a SPAAC for click chemistry conjugation) and conjugated to a molecular payload (e.g., an oligonucleotide) has the following structure (before click chemistry conjugation): [ka] wherein n is any number from 0 to 10. In some embodiments, n is 3.

[0272] In some embodiments, after conjugation to a molecular payload (e.g., an oligonucleotide), the val-cit linker has the following structure: [ka] wherein n is any number from 0 to 10, and m is any number from 0 to 10. In some embodiments, n is 3 and m is 4.

[0273] ii. Non-cleavable linker In some embodiments, a non-cleavable linker may be used. Generally, a non-cleavable linker cannot be easily degraded in a cellular or physiological environment. In some embodiments, the non-cleavable linker comprises an optionally substituted alkyl group, where the substitution may include halogen, hydroxyl group, oxygen species, and other common substitutions. In some embodiments, the linker may comprise an optionally substituted alkyl, an optionally substituted alkylene, an optionally substituted arylene, a heteroarylene, a peptide sequence comprising at least one unnatural amino acid, a truncated glycan, an enzymatically non-degradable sugar(s), an azide, an alkyne-azide, a peptide sequence comprising an LPXT sequence, a thioether, a biotin, a biphenyl, a repeating unit of polyethylene glycol or an equivalent compound, an acid ester, an acid amide, a sulfamide, and / or (for example, and), an alkoxy-amine linker. In some embodiments, sortase-mediated ligation is performed to link an anti-TfR antibody comprising an LPXT sequence to (G) nThese may be used to covalently link to molecular payloads containing the sequence (see, for example, Proft T. Sortase-mediated protein ligation: an emerging biotechnology tool for protein modification and immobilization. Biotechnol Lett. 2010, 32(1):1-10).

[0274] In some embodiments, the linker may comprise a substituted alkylene, an optionally substituted alkenylene, an optionally substituted alkynylene, an optionally substituted cycloalkylene, an optionally substituted cycloalkenylene, an optionally substituted arylene, an optionally substituted heteroarylene further comprising at least one heteroatom selected from N, O, and S; an optionally substituted heterocyclylene further comprising at least one heteroatom selected from N, O, and S; an imino, an optionally substituted nitrogen species, an optionally substituted oxygen species O, an optionally substituted sulfur species, or a poly(alkylene oxide), such as polyethylene oxide or polypropylene oxide.

[0275] iii. Linker conjugation In some embodiments, the linker is connected to the anti-TfR antibody and / or (for example, and) the molecular payload via a phosphate, thioether, ether, carbon-carbon, carbamate, or amide bond. In some embodiments, the linker is connected to the oligonucleotide via a phosphate or phosphorothioate group, for example, a phosphate at the end of the oligonucleotide backbone. In some embodiments, the linker is connected to the anti-TfR antibody via a lysine or cysteine ​​residue present on the anti-TfR antibody.

[0276] In some embodiments, the linker is connected to the anti-TfR antibody and / or (for example, and) molecular payload via a cycloaddition reaction between an azide and an alkyne to form a triazole, where the azide and alkyne may be located on the anti-TfR antibody, the molecular payload, or the linker. In some embodiments, the alkyne may be a cyclic alkyne, for example, a cyclooctyne. In some embodiments, the alkyne may be a bicyclononyne (also known as bicyclo[6.1.0]nonyne or BCN) or a substituted bicyclononyne. In some embodiments, the cyclooctane is as described in International Patent Application Publication No. WO2011136645, entitled "Fused Cyclooctyne Compounds and Their Use in Metal-free Click Reactions," published November 3, 2011. In some embodiments, the azide may be an azide-containing sugar or carbohydrate molecule. In some embodiments, the azide may be 6-azido-6-deoxygalactose or 6-azido-N-acetylgalactosamine. In some embodiments, the azide-containing sugar or carbohydrate molecule is as described in International Patent Application Publication No. WO2016170186, published October 27, 2016, entitled "Process For The Modification Of A Glycoprotein Using A Glycosyltransferase That Is Or Is Derived From A β(1,4)-N-Acetylgalactosaminyltransferase."In some embodiments, the cycloaddition reaction between an azide and an alkyne to form a triazole (wherein the azide and alkyne may be located on the anti-TfR antibody, the molecular payload, or the linker) is as described in International Patent Application Publication No. WO2014065661, published May 1, 2014, entitled "Modified antibody, antibody-conjugate and process for the preparation thereof"; or International Patent Application Publication No. WO2016170186, published October 27, 2016, entitled "Process For The Modification Of A Glycoprotein Using A Glycosyltransferase That Is Or Is Derived From A β(1,4)-N-Acetylgalactosaminyltransferase."

[0277] In some embodiments, the linker further comprises a spacer, e.g., a polyethylene glycol spacer or an acyl / carbamoyl sulfamide spacer, e.g., a HydraSpace™ spacer. In some embodiments, the spacer is as described in Verkade, JMM et al., "A Polar Sulfamide Spacer Significantly Enhances the Manufacturability, Stability, and Therapeutic Index of Antibody-Drug Conjugates," Antibodies, 2018, 7, 12.

[0278] In some embodiments, the linker is connected to the anti-TfR antibody and / or (for example, and) the molecular payload via a Diels-Alder reaction between a dienophile and a diene / hetero-diene, where the dienophile and diene / hetero-diene may be located on the anti-TfR antibody, the molecular payload, or the linker. In some embodiments, the linker is connected to the anti-TfR antibody and / or (for example, and) the molecular payload via other pericyclic reactions, for example, an ene reaction. In some embodiments, the linker is connected to the anti-TfR antibody and / or (for example, and) the molecular payload via an amide, thioamide, or sulfonamide coupling reaction. In some embodiments, the linker is connected to the anti-TfR antibody and / or (for example, and) the molecular payload via a condensation reaction to form an oxime group, hydrazone group, or semicarbazide group present between the linker and the anti-TfR antibody and / or (for example, and) the molecular payload.

[0279] In some embodiments, the linker is attached to the anti-TfR antibody and / or (by way of example and) the molecular payload by a conjugate addition reaction between a nucleophile (e.g., an amine group or a hydroxyl group) and an electrophile (e.g., a carboxylic acid or an aldehyde). In some embodiments, the nucleophile may be present on the linker and the electrophile may be present on the anti-TfR antibody or the molecular payload prior to the reaction between the linker and the anti-TfR antibody or the molecular payload. In some embodiments, the electrophile may be present on the linker and the nucleophile may be present on the anti-TfR antibody or the molecular payload prior to the reaction between the linker and the anti-TfR antibody or the molecular payload. In some embodiments, the electrophile may be an azide, pentafluorophenyl, silicon center, carbonyl, carboxylic acid, anhydride, isocyanate, thioisocyanate, succinimidyl ester, sulfosuccinimidyl ester, maleimide, alkyl halide, alkyl pseudohalide, epoxide, episulfide, aziridine, aryl, activated phosphorus center, and / or (by way of example only) activated sulfur center. In some embodiments, the nucleophile may be an optionally substituted alkene, an optionally substituted alkyne, an optionally substituted aryl, an optionally substituted heterocyclyl, a hydroxyl group, an amino group, an alkylamino group, an anilide group, or a thiol group.

[0280] In some embodiments, a val-cit linker attached to a reactive chemical moiety (e.g., SPAAC for click chemistry conjugation) is conjugated to an anti-TfR antibody with the following structure: [ka] wherein m is any number from 0 to 10. In some embodiments, m is 4.

[0281] In some embodiments, a val-cit linker attached to a reactive chemical moiety (e.g., SPAAC for click chemistry conjugation) is conjugated to an anti-TfR antibody having the structure: [ka] wherein m is any number from 0 to 10. In some embodiments, m is 4.

[0282] In some embodiments, the val-cit linker attached to a reactive chemical moiety (e.g., SPAAC for click chemistry conjugation) and conjugated to an anti-TfR antibody has the following structure: [ka] wherein n is any number from 0 to 10, and m is any number from 0 to 10. In some embodiments, n is 3, and / or (for example, and) m is 4.

[0283] In some embodiments, the val-cit linker connecting the antibody and the molecular payload has the following structure: [ka] wherein n is any number from 0 to 10, and m is any number from 0 to 10. In some embodiments, n is 3, and / or (for example, and) m is 4. In some embodiments, n is 3, and / or (for example, and) m is 4. In some embodiments, X is NH (for example, NH from the amine group of lysine), S (for example, S from the thiol group of cysteine), or O of an antibody (for example, O from the hydroxyl group of serine, threonine, or tyrosine).

[0284] In some embodiments, the conjugates described herein have the following structure: [ka] wherein n is any number from 0 to 10, and m is any number from 0 to 10. In some embodiments, n is 3, and / or (for example, and) m is 4.

[0285] In structural formulas (A), (B), (C), and (D), L, in some embodiments, is a substituted or unsubstituted aliphatic, substituted or unsubstituted heteroaliphatic, substituted or unsubstituted carbocyclylene, substituted or unsubstituted heterocyclylene, substituted or unsubstituted arylene, substituted or unsubstituted heteroarylene, -O-, -N(R A )-, -S-, -C(=O)-, -C(=O)O-, -C(=O)NR A -, -NR A C(=O)-, -NR A C(=O)R A -, -C(=O)R A -, -NR A C(=O)O-, -NR A C(=O)N(R A )-, -OC(=O)-, -OC(=O)O-, -OC(=O)N(R A )-, -S(O)NR A -, -NR A In some embodiments, L is a spacer that is: [ka] wherein the piperazine moiety is linked to the oligonucleotide and L2 is [ka] is.

[0286] In some embodiments, L1 is [ka] where the piperazine moiety is linked to the oligonucleotide.

[0287] In some embodiments, L1 is [ka] is.

[0288] In some embodiments, L1 is linked to the 5' phosphate of the oligonucleotide.

[0289] In some embodiments, L1 is optional (eg, does not have to be present).

[0290] In some embodiments, any one of the conjugates described herein has the structure: [ka] In the formula, n is 0 to 15 (for example, 3), and m is 0 to 15 (for example, 4).

[0291] C. Examples of antibody-molecular payload conjugates Further provided herein are non-limiting examples of conjugates comprising any one of the anti-TfR antibodies described herein covalently linked to any of the molecular payloads (e.g., oligonucleotides) described herein. In some embodiments, the anti-TfR antibody (e.g., any one of the anti-TfR antibodies provided in Table 2) is covalently linked to the molecular payload (e.g., an oligonucleotide) via a linker. Any of the linkers described herein may be used. In some embodiments, when the molecular payload is an oligonucleotide, the linker is linked to the 5' end, 3' end, or internally of the oligonucleotide. In some embodiments, the linker is linked to the anti-TfR antibody via a thiol-reactive linkage (e.g., via a cysteine ​​in the anti-TfR antibody). In some embodiments, the linker (e.g., a Val-cit linker) is linked to the antibody (e.g., an anti-TfR antibody described herein) via an amine group (e.g., via a lysine in the antibody).

[0292] An example structure of a conjugate comprising an anti-TfR antibody covalently linked to a molecular payload via a Val-cit linker is provided below: [ka] wherein the linker is linked to the antibody via a thiol-reactive linkage (eg, via a cysteine ​​in the antibody).

[0293] Another example of the structure of a conjugate comprising an anti-TfR antibody covalently linked to a molecular payload via a Val-cit linker is provided below: [ka] wherein n is a number between 0 and 10, m is a number between 0 and 10, and the linker is linked to the antibody via an amine group (e.g., on a lysine residue) and / or (for example, and) the linker is linked to the oligonucleotide (e.g., at the 5' end, 3' end, or internally). In some embodiments, the linker is linked to the antibody via lysine, the linker is linked to the oligonucleotide at the 5' end, n is 3, and m is 4. In some embodiments, the molecular payload is an oligonucleotide comprising a sense strand and an antisense strand, and the linker is linked to the sense strand or the antisense strand at the 5' end or the 3' end.

[0294] It should be understood that antibodies can be linked to molecular payloads with various stoichiometries, a property that may be referred to as the drug-antibody ratio (DAR), where "drug" is the molecular payload. In some embodiments, one molecular payload is linked to the antibody (DAR=1). In some embodiments, two molecular payloads are linked to the antibody (DAR=2). In some embodiments, three molecular payloads are linked to the antibody (DAR=3). In some embodiments, four molecular payloads are linked to the antibody (DAR=4). In some embodiments, a mixture of different conjugates, each having a different DAR, is provided. In some embodiments, the average DAR of the conjugates in such a mixture may range from 1 to 3, 1 to 4, 1 to 5, or more. The DAR may be increased by conjugating molecular payloads to various sites on the antibody and / or by (for example, and) conjugating multimers to one or more sites on the antibody. For example, a DAR of 2 may be achieved by conjugating a single molecular payload to two different sites on an antibody, or by conjugating a dimeric molecular payload to a single site on an antibody.

[0295] In some embodiments, the conjugates described herein comprise an anti-TfR antibody described herein (e.g., an antibody in Tables 2-5) covalently linked to a molecular payload. In some embodiments, the conjugates described herein comprise an anti-TfR antibody described herein (e.g., an antibody in Tables 2-5) covalently linked to a molecular payload via a linker (e.g., a Val-cit linker). In some embodiments, the linker (e.g., a Val-cit linker) is linked to the antibody (e.g., an anti-TfR antibody described herein) via a thiol-reactive linkage (e.g., via a cysteine ​​in the antibody). In some embodiments, the linker (e.g., a Val-cit linker) is linked to the antibody (e.g., an anti-TfR antibody described herein) via an amine group (e.g., via a lysine in the antibody).

[0296] In some embodiments, in any one of the example conjugates described herein, the molecular payload is an oligonucleotide comprising a region of complementarity of at least 15 nucleotides to any one of the gene target sequences described herein.

[0297] In some embodiments, the conjugates described herein comprise an anti-TfR antibody covalently linked to a molecular payload, wherein the anti-TfR antibody comprises CDR-H1, CDR-H2, and CDR-H3 that are the same as CDR-H1, CDR-H2, and CDR-H3 shown in Table 2; and CDR-L1, CDR-L2, and CDR-L3 that are the same as CDR-L1, CDR-L2, and CDR-L3 shown in Table 2.

[0298] In some embodiments, the conjugates described herein comprise an anti-TfR antibody covalently linked to a molecular payload, wherein the anti-TfR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:69, SEQ ID NO:71, or SEQ ID NO:72, and a VL comprising the amino acid sequence of SEQ ID NO:70.

[0299] In some embodiments, the conjugates described herein comprise an anti-TfR antibody covalently linked to a molecular payload, wherein the anti-TfR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 73 or SEQ ID NO: 76 and a VL comprising the amino acid sequence of SEQ ID NO: 74.

[0300] In some embodiments, the conjugates described herein comprise an anti-TfR antibody covalently linked to a molecular payload, wherein the anti-TfR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 73 or SEQ ID NO: 76, and a VL comprising the amino acid sequence of SEQ ID NO: 75.

[0301] In some embodiments, the conjugates described herein comprise an anti-TfR antibody covalently linked to a molecular payload, wherein the anti-TfR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 77 and a VL comprising the amino acid sequence of SEQ ID NO: 78.

[0302] In some embodiments, the conjugates described herein comprise an anti-TfR antibody covalently linked to a molecular payload, wherein the anti-TfR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:77 or SEQ ID NO:79, and a VL comprising the amino acid sequence of SEQ ID NO:80.

[0303] In some embodiments, the conjugates described herein comprise an anti-TfR antibody covalently linked to a molecular payload, wherein the anti-TfR antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:84, SEQ ID NO:86, or SEQ ID NO:87, and a light chain comprising the amino acid sequence of SEQ ID NO:85.

[0304] In some embodiments, the conjugates described herein comprise an anti-TfR antibody covalently linked to a molecular payload, wherein the anti-TfR antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:88 or SEQ ID NO:91, and a light chain comprising the amino acid sequence of SEQ ID NO:89.

[0305] In some embodiments, the conjugates described herein comprise an anti-TfR antibody covalently linked to a molecular payload, wherein the anti-TfR antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:88 or SEQ ID NO:91, and a light chain comprising the amino acid sequence of SEQ ID NO:90.

[0306] In some embodiments, the conjugates described herein comprise an anti-TfR antibody covalently linked to a molecular payload, wherein the anti-TfR antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:92 or SEQ ID NO:94, and a light chain comprising the amino acid sequence of SEQ ID NO:95.

[0307] In some embodiments, the conjugates described herein comprise an anti-TfR antibody covalently linked to a molecular payload, wherein the anti-TfR antibody 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.

[0308] In some embodiments, the conjugates described herein comprise an anti-TfR antibody covalently linked to a molecular payload, wherein the anti-TfR antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:97, SEQ ID NO:98, or SEQ ID NO:99, and a VL comprising the amino acid sequence of SEQ ID NO:85.

[0309] In some embodiments, the conjugates described herein comprise an anti-TfR antibody covalently linked to a molecular payload, wherein the anti-TfR antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 100 or SEQ ID NO: 101, and a light chain comprising the amino acid sequence of SEQ ID NO: 89.

[0310] In some embodiments, the conjugates described herein comprise an anti-TfR antibody covalently linked to a molecular payload, wherein the anti-TfR antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 100 or SEQ ID NO: 101, and a light chain comprising the amino acid sequence of SEQ ID NO: 90.

[0311] In some embodiments, the conjugates described herein comprise an anti-TfR antibody covalently linked to a molecular payload, wherein the anti-TfR antibody 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.

[0312] In some embodiments, the conjugates described herein comprise an anti-TfR antibody covalently linked to a molecular payload, wherein the anti-TfR antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 102 or SEQ ID NO: 103, and a light chain comprising the amino acid sequence of SEQ ID NO: 95.

[0313] In some embodiments, the conjugate described herein comprises an anti-TfR antibody covalently linked via a lysine to the 5' end of an oligonucleotide, wherein the anti-TfR antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 84 and a light chain comprising the amino acid sequence of SEQ ID NO: 85, and the conjugate has the structure: [ka] In the formula, n is 3 and m is 4.

[0314] In some embodiments, a conjugate described herein comprises an anti-TfR antibody covalently linked via a lysine to the 5' end of an oligonucleotide, wherein the anti-TfR antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 86 and a light chain comprising the amino acid sequence of SEQ ID NO: 85, and wherein the conjugate has the structure: [ka] In the formula, n is 3 and m is 4.

[0315] In some embodiments, the conjugate described herein comprises an anti-TfR antibody covalently linked via a lysine to the 5' end of an oligonucleotide, wherein the anti-TfR antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 87 and a light chain comprising the amino acid sequence of SEQ ID NO: 85, and the conjugate has the structure: [ka] In the formula, n is 3 and m is 4.

[0316] In some embodiments, the conjugate described herein comprises an anti-TfR antibody covalently linked via a lysine to the 5' end of an oligonucleotide, wherein the anti-TfR antibody 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, and the conjugate has the structure: [ka] In the formula, n is 3 and m is 4.

[0317] In some embodiments, a conjugate described herein comprises an anti-TfR antibody covalently linked via a lysine to the 5' end of an oligonucleotide, wherein the anti-TfR antibody 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, and wherein the conjugate has the structure: [ka] In the formula, n is 3 and m is 4.

[0318] In some embodiments, a conjugate described herein comprises an anti-TfR antibody covalently linked via a lysine to the 5' end of an oligonucleotide, wherein the anti-TfR antibody 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, and wherein the conjugate has the structure: [ka] In the formula, n is 3 and m is 4.

[0319] In some embodiments, a conjugate described herein comprises an anti-TfR antibody covalently linked via a lysine to the 5' end of an oligonucleotide, wherein the anti-TfR antibody 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, and wherein the conjugate has the structure: [ka] In the formula, n is 3 and m is 4.

[0320] In some embodiments, the conjugate described herein comprises an anti-TfR antibody covalently linked via a lysine to the 5' end of an oligonucleotide, wherein the anti-TfR antibody 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, and the conjugate has the structure: [ka] In the formula, n is 3 and m is 4.

[0321] In some embodiments, a conjugate described herein comprises an anti-TfR antibody covalently linked via a lysine to the 5' end of an oligonucleotide, wherein the anti-TfR antibody 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, and wherein the conjugate has the structure: [ka] In the formula, n is 3 and m is 4.

[0322] In some embodiments, the conjugate described herein comprises an anti-TfR antibody covalently linked via a lysine to the 5' end of an oligonucleotide, wherein the anti-TfR antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 92 and a light chain comprising the amino acid sequence of SEQ ID NO: 95, and the conjugate has the structure: [ka] In the formula, n is 3 and m is 4.

[0323] In some embodiments, the conjugate described herein comprises an anti-TfR Fab covalently linked via a lysine to the 5' end of an oligonucleotide, wherein the anti-TfR Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 69 and a VL comprising the amino acid sequence of SEQ ID NO: 70, and the conjugate has the structure: [ka] In the formula, n is 3 and m is 4.

[0324] In some embodiments, the conjugate described herein comprises an anti-TfR Fab covalently linked via a lysine to the 5' end of an oligonucleotide, wherein the anti-TfR Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 71 and a VL comprising the amino acid sequence of SEQ ID NO: 70, and the conjugate has the structure: [ka] In the formula, n is 3 and m is 4.

[0325] In some embodiments, the conjugate described herein comprises an anti-TfR Fab covalently linked via a lysine to the 5' end of an oligonucleotide, wherein the anti-TfR Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 72 and a VL comprising the amino acid sequence of SEQ ID NO: 70, and the conjugate has the structure: [ka] In the formula, n is 3 and m is 4.

[0326] In some embodiments, the conjugate described herein comprises an anti-TfR Fab covalently linked via a lysine to the 5' end of an oligonucleotide, wherein the anti-TfR Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 73 and a VL comprising the amino acid sequence of SEQ ID NO: 74, and the conjugate has the structure: [ka] In the formula, n is 3 and m is 4.

[0327] In some embodiments, the conjugate described herein comprises an anti-TfR Fab covalently linked via a lysine to the 5' end of an oligonucleotide, wherein the anti-TfR Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 73 and a VL comprising the amino acid sequence of SEQ ID NO: 75, and the conjugate has the structure: [ka] In the formula, n is 3 and m is 4.

[0328] In some embodiments, the conjugate described herein comprises an anti-TfR Fab covalently linked via a lysine to the 5' end of an oligonucleotide, wherein the anti-TfR Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 76 and a VL comprising the amino acid sequence of SEQ ID NO: 74, and the conjugate has the structure: [ka] In the formula, n is 3 and m is 4.

[0329] In some embodiments, the conjugate described herein comprises an anti-TfR Fab covalently linked via a lysine to the 5' end of an oligonucleotide, wherein the anti-TfR Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 76 and a VL comprising the amino acid sequence of SEQ ID NO: 75, and the conjugate has the structure: [ka] In the formula, n is 3 and m is 4.

[0330] In some embodiments, the conjugate described herein comprises an anti-TfR Fab covalently linked via a lysine to the 5' end of an oligonucleotide, wherein the anti-TfR Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 77 and a VL comprising the amino acid sequence of SEQ ID NO: 78, and the conjugate has the structure: [ka] In the formula, n is 3 and m is 4.

[0331] In some embodiments, the conjugate described herein comprises an anti-TfR Fab covalently linked via a lysine to the 5' end of an oligonucleotide, wherein the anti-TfR Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 79 and a VL comprising the amino acid sequence of SEQ ID NO: 80, and the conjugate has the structure: [ka] In the formula, n is 3 and m is 4.

[0332] In some embodiments, the conjugate described herein comprises an anti-TfR Fab covalently linked via a lysine to the 5' end of an oligonucleotide, wherein the anti-TfR Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 77 and a VL comprising the amino acid sequence of SEQ ID NO: 80, and the conjugate has the structure: [ka] In the formula, n is 3 and m is 4.

[0333] In some embodiments, the conjugate described herein comprises an anti-TfR Fab covalently linked via a lysine to the 5' end of an oligonucleotide, wherein the anti-TfR Fab comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 97 and a light chain comprising the amino acid sequence of SEQ ID NO: 85, and the conjugate has the structure: [ka] In the formula, n is 3 and m is 4.

[0334] In some embodiments, the conjugate described herein comprises an anti-TfR Fab covalently linked via a lysine to the 5' end of an oligonucleotide, wherein the anti-TfR Fab comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 98 and a light chain comprising the amino acid sequence of SEQ ID NO: 85, and the conjugate has the structure: [ka] In the formula, n is 3 and m is 4.

[0335] In some embodiments, the conjugate described herein comprises an anti-TfR Fab covalently linked via a lysine to the 5' end of an oligonucleotide, wherein the anti-TfR Fab comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 99 and a light chain comprising the amino acid sequence of SEQ ID NO: 85, and the conjugate has the structure: [ka] In the formula, n is 3 and m is 4.

[0336] In some embodiments, the conjugate described herein comprises an anti-TfR Fab covalently linked via a lysine to the 5' end of an oligonucleotide, wherein the anti-TfR Fab comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 100 and a light chain comprising the amino acid sequence of SEQ ID NO: 89, and the conjugate has the structure: [ka] In the formula, n is 3 and m is 4.

[0337] In some embodiments, the conjugate described herein comprises an anti-TfR Fab covalently linked via a lysine to the 5' end of an oligonucleotide, wherein the anti-TfR Fab comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 100 and a light chain comprising the amino acid sequence of SEQ ID NO: 90, and the conjugate has the structure: [ka] In the formula, n is 3 and m is 4.

[0338] In some embodiments, the conjugate described herein comprises an anti-TfR Fab covalently linked via a lysine to the 5' end of an oligonucleotide, wherein the anti-TfR Fab 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, and the conjugate has the structure: [ka] In the formula, n is 3 and m is 4.

[0339] In some embodiments, the conjugate described herein comprises an anti-TfR Fab covalently linked via a lysine to the 5' end of an oligonucleotide, wherein the anti-TfR Fab 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, and the conjugate has the structure: [ka] In the formula, n is 3 and m is 4.

[0340] In some embodiments, the conjugate described herein comprises an anti-TfR Fab covalently linked via a lysine to the 5' end of an oligonucleotide, wherein the anti-TfR Fab 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, and the conjugate has the structure: [ka] In the formula, n is 3 and m is 4.

[0341] In some embodiments, the conjugate described herein comprises an anti-TfR Fab covalently linked via a lysine to the 5' end of an oligonucleotide, wherein the anti-TfR Fab comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 103 and a light chain comprising the amino acid sequence of SEQ ID NO: 95, and the conjugate has the structure: [ka] In the formula, n is 3 and m is 4.

[0342] In some embodiments, the conjugate described herein comprises an anti-TfR Fab covalently linked via a lysine to the 5' end of an oligonucleotide, wherein the anti-TfR Fab comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 102 and a light chain comprising the amino acid sequence of SEQ ID NO: 95, and the conjugate has the structure: [ka] In the formula, n is 3 and m is 4.

[0343] In some embodiments, in any one of the example conjugates described herein, L1 is any one of the spacers described herein.

[0344] In some embodiments, L1 is [ka] wherein the piperazine moiety is linked to the oligonucleotide and L2 is [ka] is.

[0345] In some embodiments, L1 is [ka] where the piperazine moiety is linked to the oligonucleotide.

[0346] In some embodiments, L1 is [ka] is.

[0347] In some embodiments, L1 is linked to the 5' phosphate of the oligonucleotide.

[0348] In some embodiments, L1 is optional (eg, does not have to be present).

[0349] IV. Formulations The anti-TfR antibodies or conjugates provided herein may be formulated in any suitable manner. Generally, the antibodies or conjugates provided herein are formulated in a manner suitable for pharmaceutical use. For example, the antibodies or conjugates may be delivered to a subject using a formulation that minimizes degradation, facilitates delivery and / or (for example, and) uptake, or provides another beneficial property to the conjugate in the formulation. In some embodiments, provided herein are compositions comprising an antibody or conjugate and a pharmaceutically acceptable carrier. Such compositions may be suitably formulated so that a sufficient amount of the conjugate can enter the target muscle cell when administered either into the environment surrounding the target cell of a subject or into the subject's systemic environment. In some embodiments, the antibody or conjugate is formulated in a buffer solution such as phosphate-buffered saline, in a liposome, in a micellar structure, or in a capsid.

[0350] It should be understood that in some embodiments, a composition may individually include one or more components of the conjugates provided herein (e.g., an anti-TfR antibody, a linker, a molecular payload, or a precursor molecule of any one of these).

[0351] In some embodiments, the antibody or conjugate is formulated in water or an aqueous solution (e.g., pH-adjusted water). In some embodiments, the antibody or conjugate is formulated in a basic buffered aqueous solution (e.g., PBS). In some embodiments, a formulation as disclosed herein includes an excipient. In some embodiments, the excipient confers improved stability, improved absorption, improved solubility, and / or (e.g., and) therapeutic enhancement of the active ingredient to the composition. In some embodiments, the excipient is a buffer (e.g., sodium citrate, sodium phosphate, Tris base, or sodium hydroxide) or a vehicle (e.g., a buffer solution, petrolatum, dimethyl sulfoxide, or mineral oil).

[0352] In some embodiments, the complex or a component thereof (e.g., an oligonucleotide or an antibody) is lyophilized to extend its shelf life and then brought into solution prior to use (e.g., administration to a subject). Consequently, the excipient in a composition comprising the complex or a component thereof described herein may be a lyoprotectant (e.g., mannitol, lactose, polyethylene glycol, or polyvinylpyrrolidone) or a collapse temperature modifier (e.g., dextran, ficoll, or gelatin).

[0353] In some embodiments, the pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral administration, such as intravenous administration, intradermal administration, and subcutaneous administration. Typically, the route of administration is intravenous or subcutaneous administration.

[0354] Pharmaceutical compositions suitable for use in injections include sterile aqueous solutions (wherein they are soluble in water) or dispersions, and sterile powders for the extemporaneous preparation of sterile injection solutions or dispersions. Carriers can be, for example, solvents or dispersion media containing water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), and suitable mixtures thereof. In some embodiments, the formulations include isotonic agents, such as sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride in the composition. Sterile injection solutions can be prepared by incorporating the required amount of the complex with one or a combination of the above-listed ingredients in the selected solvent, and then optionally filtering and sterilizing.

[0355] In some embodiments, the composition may contain at least about 0.1% or more of the complex or its components, although the percentage of the active ingredient(s) may be between about 1% and about 80% or more by weight or volume of the total composition. Factors such as solubility, bioavailability, biological half-life, route of administration, shelf life of the product, and other pharmacological considerations will be taken into account by those skilled in the art when preparing such pharmaceutical formulations. Therefore, various dosages and treatment regimens may be desired.

[0356] V. How to Use Some aspects of the present disclosure provide various uses of the anti-TfR antibodies, antibody fragments or variants, nucleic acids encoding them, and conjugates described herein, including in research, diagnostic methods, detection methods, and therapeutic methods. In some embodiments, the anti-TfR antibodies described herein are used to deliver a molecular payload (e.g., a diagnostic or therapeutic agent) to a target cell or tissue that expresses the transferrin receptor. In some embodiments, the target cell is a muscle cell. In some embodiments, the target tissue is muscle. In some embodiments, the target tissue is the brain. To deliver the molecular payload, the anti-TfR antibody may be conjugated (e.g., covalently conjugated) to the molecular payload to form a conjugate.

[0357] a. Diagnostic and detection methods Also provided herein are uses of any one of the above-described antibodies, antigen-binding fragments, polynucleotides, vectors, or cells, as well as any suitable means in diagnostic and / or (by way of example) detection methods. Antibodies or antigen-binding fragments are suitable, for example, for use in immunoassays, in which they can be used in liquid phase or bound to a solid-phase support. Examples of immunoassays that can utilize antibodies or antigen-binding fragments are competitive and non-competitive immunoassays, in either direct or indirect formats. Examples of such immunoassays are enzyme-linked immunoassays (ELISAs), radioimmunoassays (RIAs), sandwich (immunometric assays), flow cytometry, Western blot assays, immunoprecipitation assays, immunohistochemistry, immunomicroscopy, lateral flow immunochromatographic assays, and proteomics arrays. Antigens and antibodies or antigen-binding fragments can be attached to many different solid supports (e.g., carriers, membranes, columns, proteomic arrays, etc.). Examples of well-known solid support materials include glass, polystyrene, polyvinyl chloride, polyvinylidene difluoride, polypropylene, polyethylene, polycarbonate, dextran, nylon, amylose, natural and modified cellulose (such as nitrocellulose), polyacrylamide, agarose, and magnetite. Such supports can be either immobilized or suspended in solution (e.g., as beads).

[0358] In some embodiments, any one of the anti-TfR antibodies provided herein is useful for detecting the presence of transferrin receptor in a biological sample. The term "detecting," as used herein, encompasses quantitative or qualitative detection. In some embodiments, the biological sample includes cells or tissues, such as blood, CSF, and BBB-containing tissues. The biological sample can be in vitro (e.g., cultured) or in vivo (e.g., in a subject). The present disclosure also contemplates the use of any one of the anti-TfR antibodies described herein in research applications (e.g., as a reagent for immunoassays such as Western blotting, immunostaining, ELISA, and / or FACS).

[0359] In some embodiments, anti-TfR antibodies are provided for use in diagnostic or detection methods. In some aspects, methods for detecting the presence of transferrin receptor in a biological sample are provided. In some embodiments, the method comprises contacting a biological sample with an anti-TfR antibody as described herein under conditions that allow binding of the anti-TfR antibody to the transferrin receptor, and detecting whether a complex is formed between the anti-TfR antibody and the transferrin receptor. Such methods may be in vitro or in vivo methods. In some embodiments, the anti-TfR antibody is used to select subjects eligible for treatment with an anti-TfR antibody, for example, when the transferrin receptor is a biomarker for patient selection.

[0360] Exemplary disorders that may be diagnosed using the anti-TfR antibodies described herein include disorders involving immature red blood cells, due to the fact that transferrin receptors are expressed on reticulocytes and are therefore detectable by any of the antibodies of the invention. Such disorders include anemias and other disorders resulting from reduced levels of reticulocytes, or congenital polycythemia or neoplastic polycythemia vera, where, by way of example, an elevated red blood cell count due to reticulocyte hyperproliferation leads to thickening of the blood and concomitant physiological symptoms.

[0361] In some embodiments, labeled anti-TfR antibodies are used to detect the presence / level of transferrin receptor in biological samples. Labels include, but are not limited to, directly detected labels or moieties (such as fluorescent, chromophoric, electron-dense, chemiluminescent, and radioactive labels) and indirectly detected moieties, such as enzymes or ligands, for example, through enzymatic reactions or molecular interactions. Exemplary labels include, but are not limited to, radioisotopes 32P, 14C, 125I, 3H, and 131, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferases such as firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidase (saccharide oxidase), and the like. oxidases, such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases (such as uricase and xanthine oxidase) coupled to enzymes that use hydrogen peroxide to oxidize dye precursors (such as HRP, lactoperoxidase, or microperoxidase), biotin / avidin, spin labels, bacteriophage labels, stable free radicals, and the like. In some embodiments, the detectable label is an agent suitable for detecting transferrin receptors in cells in vitro, which may be a radioactive molecule, a radiopharmaceutical, or an iron oxide particle. Radioactive molecules suitable for in vivo imaging include, but are not limited to, 122 I, 123 I, 124 I, 125 I, 131 I, 18 F, 75 Br, 76 Br, 77 Br, 211 At,225 Ac, 177 Lu, 153 Sm, 186 Re, 188 Re, 67 Cu, 213 Bi, 212 Bi, 212 Pb, and 67 Exemplary radiopharmaceuticals suitable for in vivo imaging include: 111 Inoxyquinoline, 131 I sodium iodide, 99 mTc Mebrofenin, and 99 mTc Red Blood Cells, 123 I sodium iodide, 99 mTc Examethadime, 99 mTc macroaggregated albumin, 99 mTc medronate, 99 mTc mertiatide, 99 mTc Oxidronate, 99 mTc Pentetate, 99 mTc pertechnetate, 99 mTc sestamibi, 99 mTc sulfur colloid, 99 These include mTc tetrofosmin, thallium-201, or xenon-133.

[0362] In certain embodiments, the anti-TfR antibodies described herein can be used to deliver a detectable label to a target cell or tissue (e.g., to a muscle cell or across the blood-brain barrier to the brain) for visualization of the cell or tissue (e.g., by fluorescence microscopy or magnetic resonance imaging (MRI)). Any of the detectable labels described herein can be used for this purpose.

[0363] In some embodiments, the anti-TfR antibody used in the diagnostic or detection method lacks or has reduced effector function. In some embodiments, the anti-TfR antibody used in the diagnostic / detection method is engineered to have no or reduced effector function (e.g., by using a Fab, modifying the Ig backbone, introducing one or more Fc mutations to reduce or eliminate effector function, and / or (e.g., and) modifying the glycosylation state of the antibody).

[0364] Various techniques are available for determining binding to the transferrin receptor. One such assay is an enzyme-linked immunosorbent assay (ELISA) to confirm binding ability to the human transferrin receptor (and brain antigens). According to this assay, a plate coated with an antigen (e.g., recombinant transferrin receptor) is incubated with a sample containing an anti-TfR antibody, and binding of the antibody to the antigen of interest is determined.

[0365] To conduct a diagnostic assay in vivo, a suitable amount of an anti-TfR antibody conjugated to a label (e.g., an imaging or contrast agent) can be administered to a subject requiring testing. The presence of the labeled antibody can be detected based on a signal emitted from the label by routine methods. Assays for assessing the uptake of systemically administered antibodies and other biological activities of antibodies are known to those skilled in the art.

[0366] To conduct scientific research assays, anti-TfR antibodies can be used to study the biological activity of transferrin receptors and / or (for example) to detect the presence of transferrin receptors intracellularly. For example, a suitable amount of anti-TfR antibody can be contacted with a sample suspected of producing transferrin receptors (for example, a new cell type not previously identified as producing transferrin receptors). The antibody and sample can be incubated under suitable conditions for a suitable period of time to allow binding of the antibody to the transferrin receptor antigen. Such an interaction can then be detected via routine methods, for example, ELISA, histological staining, or FACS.

[0367] b. Treatment method The anti-TfR antibodies described herein can be used to deliver molecular payloads that are therapeutic agents (e.g., oligonucleotides, peptides / proteins, nucleic acid constructs, etc.). In some aspects, the present disclosure also provides conjugates comprising an anti-TfR antibody covalently linked to a molecular payload for use in treating disease.

[0368] In some aspects, a conjugate comprising an anti-TfR antibody covalently linked to a molecular payload as described herein is effective to treat a muscle disease (e.g., a rare muscle disease or muscle atrophy). In some embodiments, the conjugate is effective to treat a rare muscle disease provided in Table 6. In some embodiments, the muscle disease is associated with a disease allele, e.g., a disease allele for a particular muscle disease may comprise a genetic alteration in the corresponding gene listed in Table 6.

[0369] In some embodiments, the conjugate is effective to treat muscle atrophy associated with the activity of one or more genes listed in Table 6 under the "Muscle Atrophy Gene Targets" section. In some embodiments, the muscle atrophy results from chronic illness, including AIDS, congestive heart failure, cancer, chronic obstructive pulmonary disease, and renal failure, or muscle disuse.

[0370] In another aspect, a conjugate comprising an anti-TfR antibody covalently linked to a molecular payload as described herein is effective for treating a neurological disease. In some embodiments, the neurological disease includes, but is not limited to, neuropathy, amyloidosis, cancer, ocular disease or disorder, viral or microbial infection, inflammation, ischemia, neurodegenerative disease, stroke, behavioral disorder, and lysosomal storage disease. For purposes of this application, the CNS will be understood to include the eye, which is normally isolated from the rest of the body by the blood-retinal barrier. Specific examples of neurological disorders include, but are not limited to, neurodegenerative diseases (including, but not limited to, Lewy body disease, post-polio syndrome, Shy-Drager syndrome, olivopontocerebellar atrophy, Parkinson's disease, multiple system atrophy, striatonigral degeneration), tauopathies (including, but not limited to, Alzheimer's disease and supranuclear palsy), prion diseases (including, but not limited to, bovine spongiform encephalopathy, scrapie, Creutzfeldt-Jakob syndrome, kuru, Gerstmann-Sträussler-Scheinker disease, chronic wasting disease, and fatal familial insomnia), bulbar palsies, motor neuron diseases, and heterogeneous degenerative disorders of the nervous system (including, but not limited to, Canavan disease, Huntington's disease, neuronal ceroid lipofuscinosis, Alexander disease, Tourette's syndrome, Menkes kinky syndrome, and others). and Unverricht-Lundborg syndrome), dementia (including, but not limited to, Pick's disease and spinocerebellar ataxia), cancer (including, but not limited to, cancer of the CNS, brain metastases resulting from cancer elsewhere in the body). In some embodiments, for treating a neurological disease, the conjugate comprises an anti-TfR antibody described herein conjugated to a drug for treating a neurological disease (e.g., a drug listed in Table 7).

[0371] In some embodiments, the subject may be a human subject, a non-human primate animal subject, a rodent animal subject, or any suitable mammalian animal subject. In some embodiments, the subject may have a muscle disease provided in Table 6. In some embodiments, the subject may have or be at risk for developing muscle atrophy.

[0372] Aspects of the present disclosure include methods involving administering to a subject an effective amount of a conjugate as described herein. In some embodiments, an effective amount of a pharmaceutical composition comprising a conjugate comprising an anti-TfR antibody covalently linked to a molecular payload can be administered to a subject in need of treatment. In some embodiments, a pharmaceutical composition comprising a conjugate as described herein can be administered by a suitable route, which may include intravenous administration, for example, as a bolus or by continuous infusion over a period of time. In some embodiments, intravenous administration can be by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarticular, intrasynovial, or intrathecal routes. In some embodiments, the pharmaceutical composition can be in solid, aqueous, or liquid form. In some embodiments, the aqueous or liquid form can be sprayed or lyophilized. In some embodiments, the sprayed or lyophilized form can be reconstituted with an aqueous or liquid solution.

[0373] Compositions for intravenous administration may contain a variety of carriers, such as vegetable oils, dimethylacetamide, dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (glycerol, propylene glycol, liquid polyethylene glycol, etc.). Water-soluble antibodies for intravenous injection may be administered by infusion, whereby a pharmaceutical formulation containing the antibody and a physiologically acceptable excipient is infused. Physiologically acceptable excipients may include, for example, 5% dextrose, 0.9% saline, Ringer's solution, or other suitable excipients. For intramuscular preparations, for example, a sterile formulation of a suitable soluble salt form of the antibody may be dissolved and administered in a pharmaceutical excipient such as water for injection, 0.9% saline, or 5% glucose solution.

[0374] In some embodiments, pharmaceutical compositions comprising a conjugate comprising an anti-TfR antibody covalently linked to a molecular payload are administered via site-specific or localized delivery techniques, examples of which include an implanted depot source of the conjugate, a localized delivery catheter, a site-specific carrier, direct injection, or direct application.

[0375] In some embodiments, a pharmaceutical composition comprising a conjugate comprising an anti-TfR antibody covalently linked to a molecular payload is administered at an effective concentration to confer a therapeutic effect on the subject. As will be recognized by those skilled in the art, the effective amount will vary depending on the severity of the disease, the specific characteristics of the subject being treated, such as age, physical condition, health, or weight, the duration of treatment, the nature of any concomitant treatments, the route of administration, and related factors. These related factors are known to those skilled in the art and can be addressed with minimal routine experimentation. In some embodiments, the effective concentration is the maximum dose deemed safe for the patient. In some embodiments, the effective concentration will be the lowest feasible concentration that provides maximum efficacy.

[0376] Empirical considerations, such as the half-life of the conjugate in the subject, will generally contribute to det...

Claims

1. 1. An antibody that binds to the human transferrin receptor (TfR), comprising: a heavy chain variable region (VH) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 76; and a light chain variable region (VL) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 75, wherein the VH and VL of the antibody are: (i) heavy chain complementarity determining region 1 (CDR-H1) as set forth in SEQ ID NO: 27, heavy chain complementarity determining region 2 (CDR-H2) as set forth in SEQ ID NO: 28, and heavy chain complementarity determining region 3 (CDR-H3) as set forth in SEQ ID NO: 29; and light chain complementarity determining region 1 (CDR-L1) as set forth in SEQ ID NO: 30, light chain complementarity determining region 2 (CDR-L2) as set forth in SEQ ID NO: 31, and light chain complementarity determining region 3 (CDR-L3) as set forth in SEQ ID NO: 32; (ii) CDR-H1 as set forth in SEQ ID NO: 33, CDR-H2 as set forth in SEQ ID NO: 34, and CDR-H3 as set forth in SEQ ID NO: 35; and CDR-L1 as set forth in SEQ ID NO: 36, CDR-L2 as set forth in SEQ ID NO: 37, and CDR-L3 as set forth in SEQ ID NO: 32; or (iii) CDR-H1 as represented by SEQ ID NO: 38, CDR-H2 as represented by SEQ ID NO: 39, and CDR-H3 as represented by SEQ ID NO: 40; and CDR-L1 as represented by SEQ ID NO: 41, CDR-L2 as represented by SEQ ID NO: 31, and CDR-L3 as represented by SEQ ID NO:

42. The antibody comprising:

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

75.

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

4. The antibody of claim 1 or 2, wherein the antibody is a Fab fragment.

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

90.

6. The antibody of any one of claims 1 to 5, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 101; and a light chain comprising the amino acid sequence of SEQ ID NO:

90.

7. An antibody described in any one of claims 1 to 6, comprising a heavy chain containing N-terminal pyroglutamic acid.

8. A conjugate comprising the antibody of any one of claims 1 to 7 covalently linked to a molecular payload.

9. The conjugate of claim 8 , wherein the molecular payload comprises a diagnostic or therapeutic agent.

10. The conjugate of claim 8 or 9, wherein the molecular payload comprises an oligonucleotide, a polypeptide, or a small molecule.

11. The conjugate of any one of claims 8 to 10, wherein the antibody and the molecular payload are covalently linked via a cleavable linker.

12. 12. The conjugate of claim 11, wherein the cleavable linker comprises a valine-citrulline sequence.

13. The conjugate of any one of claims 8 to 10, wherein the antibody and the molecular payload are covalently linked via a non-cleavable linker.

14. 14. The conjugate of claim 13, wherein the non-cleavable linker comprises an optionally substituted alkyl group.

15. The conjugate of any one of claims 8 to 14, wherein the molecular payload is covalently linked to the antibody via a lysine residue of the antibody.

16. The conjugate of any one of claims 8 to 14, wherein the molecular payload is covalently linked to the antibody via a cysteine ​​residue of the antibody.

17. A composition comprising an antibody according to any one of claims 1 to 7 or a complex according to any one of claims 8 to 16.

18. 18. The composition of claim 17, wherein the composition further comprises a pharmaceutically acceptable carrier.

19. A method for delivering a molecular payload to a cell, comprising contacting the cell in vitro with a complex according to any one of claims 8 to 16 or a composition according to claim 17 or 18.

20. 20. The method of claim 19, wherein the cell is a muscle cell.

21. A conjugate according to any one of claims 8 to 16 or a composition according to claim 17 or 18 for use in a method of in vivo diagnosis, comprising: The method includes contacting the complex or composition with a cell in the subject. The complex or composition comprising:

22. 22. The complex or composition of claim 21, wherein the cell is a muscle cell.

23. 23. The complex or composition of claim 21 or 22, wherein the subject is a human.

24. 19. A conjugate according to any one of claims 8 to 16 or a composition according to claim 17 or 18 for use in a method for treating a disease or condition that can be ameliorated or prevented by delivery of a molecular payload to muscle of a subject, comprising: The method comprises administering the complex or composition to a subject. The complex or composition comprising:

25. 25. The complex or composition of claim 24, wherein the administration is intravenous administration.

26. A conjugate according to any one of claims 8 to 16 or a composition according to claim 17 or 18 for use in a method for treating a disease, comprising: The method comprises administering the complex or composition to a subject, wherein the molecular payload is a therapeutic agent.

27. 27. The complex or composition of claim 26, wherein the disease is a muscle disease and the molecular payload is a drug for treating the muscle disease.

28. 28. The complex or composition of claim 26 or 27, wherein the muscle disease is a rare muscle disease or muscle atrophy.

29. The complex or composition according to any one of claims 24 to 28, wherein the subject is a human.

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