Muscle targeting complexes for treatment of ankylosing muscular dystrophy and uses thereof

Abstract

Some aspects of the present disclosure relate to compositions comprising a plurality of complexes comprising an antibody (e.g., an anti-TfR1 antibody) covalently linked to one or more oligonucleotides (e.g., a DMPK targeting oligonucleotide), each oligonucleotide being covalently linked at a linkage site represented by a lysine (K) residue of the antibody. In some embodiments, the antibody comprises a heavy chain comprising a heavy chain variable region (VH) and a heavy chain constant region and a light chain comprising a light chain variable region (VL) and a light chain constant region, where at least 80% of the antibody light chain constant region (e.g., a heavy chain constant region) in the complex of the composition; the present invention relates to an antibody (e.g., 80% to 98%, 80% to 95%, 80% to 90%, 85% to 98%, 85% to 95%, 85% to 90%, 90% to 98%, 90% to 95%, 95% to 97%, or more), independently covalently linked to an oligonucleotide at a linkage site represented by K188 (based on Kabat numbering) and / or a linkage site represented by K190 (based on Kabat numbering) of a light chain constant region of the antibody.

Description

[0001] Related Applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 331,716, filed on April 15, 2022, entitled “MUSCLETARGETING COMPLEXES AND USES THEREOF FOR TREATING MYOTONIC DYSTROPHY,” under 35 U.S.C. §119(e); the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present application relates to a targeting complex for delivering an effective amount of oligonucleotide molecular payload to cells and its use, in particular to the use for disease treatment.

[0004] References to Electronic Sequence Listings

[0005] The contents of the electronic sequence listing (D082470075WO00-SEQ-COB.xml; size: 58,804 bytes; and creation date: March 22, 2023) are incorporated herein by reference in their entirety. Background Art

[0006] Myotonic dystrophy (DM) is a dominant inherited genetic disease characterized by myotonia, muscle loss or degeneration, weakened muscle function, insulin resistance, arrhythmia, smooth muscle dysfunction and nervous system abnormalities. DM is the most common form of adult-onset muscular dystrophy, with a worldwide incidence of about 1 in 8000 people worldwide. Two types of the disease have been described, i.e., myotonic dystrophy type 1 (DM1) and myotonic dystrophy type 2 (DM2). DM1 is a more common form of the disease, which is caused by repeated expansion of CTG trinucleotide repeats in the 3' non-coding region of DMPK on chromosome 19; DM2 is caused by repeated expansion of CCTG tetranucleotide repeats in the first intron of ZNF9 on chromosome 3. In DM1 patients, repetitive expansion of CTG trinucleotide repeats, which can include greater than about 50 to about 3,000 or more total repeats, results in the production of toxic RNA repeats that are capable of forming hairpin structures that bind with high affinity to essential intracellular proteins, such as muscleblind-like protein, leading to protein sequestration and the loss-of-function phenotype characteristic of the disease. There are currently no effective therapeutic agents available for DM1, other than supportive care and treatment to address disease symptoms. Summary of the invention

[0007] According to some aspects, the present disclosure provides a composition comprising a plurality of complexes. In some embodiments, the complex of the composition provided herein comprises an antibody covalently linked to one or more oligonucleotides (e.g., an anti-transferrin receptor 1 (TfR1) antibody), each oligonucleotide being covalently linked to the antibody at a bonding site represented by a lysine (K) residue of the antibody. In some embodiments, the antibody comprises a heavy chain containing a heavy chain variable region (VH) and a heavy chain constant region and a light chain containing a light chain variable region (VL) and a light chain constant region. In some embodiments, at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, 80% to 98%, 80% to 95%, 80% to 90%, 85% to 98%, 85% to 95%, 85% to 90%, 90% to 98%, 90% to 95%, 95% to 97%, or more) of the antibody light chain constant region in the complex of the composition is independently covalently linked to an oligonucleotide at the following linkage sites: the linkage site represented by K188 (based on Kabat numbering) and / or the linkage site represented by K190 (based on Kabat numbering) of the antibody light chain constant region. In some embodiments, at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, 80% to 98%, 80% to 95%, 80% to 90%, 85% to 98%, 85% to 95%, 85% to 90%, 90% to 98%, 90% to 95%, 95% to 97%, or more) of the antibody light chains in the complex of the composition are independently covalently linked to the oligonucleotide at the linkage site represented by the K residue in the sequence motif DYEKHKVYA (SEQ ID NO: 27) of the antibody light chain. In some embodiments, about 1% to 15% (e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, or about 15%) of the antibody heavy chain constant region in the complex of the composition are independently covalently linked to an oligonucleotide at the following linkage site: the linkage site represented by K213 (based on Kabat numbering) of the heavy chain constant region of the antibody.In some embodiments, about 1% to 15% (e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, or about 15%) of the antibody heavy chains in the complex of the composition are independently covalently linked to the oligonucleotide at the following linkage site: the linkage site represented by the K residue in the sequence motif VNHKPSN (SEQ ID NO: 28) of the antibody heavy chain.

[0008] According to some aspects, the disclosure provides a composition comprising a plurality of complexes comprising an antibody covalently linked to one or more oligonucleotides, each oligonucleotide being covalently linked at a linkage site represented by a lysine (K) residue of the antibody,

[0009] The antibody comprises a heavy chain comprising a heavy chain variable region (VH) and a heavy chain constant region, and a light chain comprising a light chain variable region (VL) and a light chain constant region,

[0010] wherein the oligonucleotide comprises a complementary region to DMPK RNA,

[0011] And wherein, at least 80% of the antibody light chain constant region in the complex of the composition is independently covalently linked to the oligonucleotide at the following linkage sites: the linkage site represented by K188 (based on Kabat numbering) and / or the linkage site represented by K190 (based on Kabat numbering) of the antibody light chain constant region.

[0012] According to some aspects, the disclosure provides a composition comprising a plurality of complexes comprising an antibody covalently linked to one or more oligonucleotides, each oligonucleotide being covalently linked at a linkage site represented by a lysine (K) residue of the antibody,

[0013] The antibody comprises a heavy chain comprising a heavy chain variable region (VH) and a heavy chain constant region, and a light chain comprising a light chain variable region (VL) and a light chain constant region,

[0014] wherein the oligonucleotide comprises a 5'-XYZ-3' configuration, wherein X and Z are flanking regions comprising one or more modified nucleosides, and Y is a spacer region comprising one or more 2'-deoxyribonucleosides,

[0015] And wherein, at least 80% of the antibody light chain constant region in the complex of the composition is independently covalently linked to the oligonucleotide at the following linkage sites: the linkage site represented by K188 (based on Kabat numbering) and / or the linkage site represented by K190 (based on Kabat numbering) of the antibody light chain constant region.

[0016] In some embodiments, about 1% to 15% of the antibody heavy chain constant regions in the complexes of the composition are independently covalently linked to oligonucleotides at the following linkage site: the linkage site represented by K213 (based on Kabat numbering) of the antibody heavy chain constant region.

[0017] In some embodiments, the antibody is an anti-transferrin receptor (TfR1) antibody comprising: a heavy chain complementarity determining region 1 (CDR-H1) comprising the sequence as shown in SEQ ID NO: 1, 7 or 12, a heavy chain complementarity determining region 2 (CDR-H2) comprising the sequence as shown in SEQ ID NO: 2, 8 or 13, a heavy chain complementarity determining region 3 (CDR-H3) comprising the sequence as shown in SEQ ID NO: 3, 9 or 14, a light chain complementarity determining region 1 (CDR-L1) comprising the sequence as shown in SEQ ID NO: 4, 10 or 15, a light chain complementarity determining region 2 (CDR-L2) comprising the sequence as shown in SEQ ID NO: 5 or 11, and a heavy chain complementarity determining region 3 (CDR-H4) comprising the sequence as shown in SEQ ID NO: 6 or 8. Light chain complementarity determining region 3 (CDR-L3) of the sequence shown in NO:6 or 16.

[0018] In some embodiments, the oligonucleotide comprises a 5′-XYZ-3′ configuration, wherein X and Z each comprise 4 linked nucleosides, and Y comprises 8 linked nucleosides. In some embodiments, the oligonucleotide comprises a 5′-XYZ-3′ configuration of LLEE-D8-EELL, wherein “L” represents an LNA nucleoside, “E” represents a 2′-MOE modified ribonucleoside, and “D” represents a 2′-deoxyribonucleoside.

[0019] In some embodiments, the oligonucleotide comprises a nucleobase sequence of CAGCGCCCACCAGUCA (SEQ ID NO: 21). In some embodiments, the oligonucleotide comprises the structure +C*+A*oG*oC*dG*dC*dC*dC*dA*dC*dC*dA*oG*oU*+C*+A (SEQ ID NO: 21), wherein +N represents LNA (2'-4' methylene bridge) ribonucleoside, dN represents 2'-deoxyribonucleoside, oN represents 2'-MOE modified ribonucleoside, oC represents 5-methyl-2'-MOE-cytidine, +C represents 5-methyl-2'-4'-bicyclo-cytidine (2'-4' methylene bridge), oU represents 5-methyl-2'-MOE-uridine, and * represents phosphorothioate internucleoside linkage.

[0020] According to some aspects, the present disclosure provides a composition comprising a plurality of complexes, wherein the complexes comprise formula (I): [R 1 ] n1 -R 2 The structure of

[0021] Each R 1 Independently comprising groups of formula (Ia):

[0022]

[0023] Where R 2 The invention comprises an anti-transferrin receptor (TfR1) antibody, wherein the anti-transferrin receptor (TfR1) antibody comprises a heavy chain comprising a heavy chain variable region (VH) and a heavy chain constant region, and a light chain comprising a light chain variable region (VL) and a light chain constant region; and wherein the anti-TfR1 antibody comprises: a heavy chain complementary determining region 1 (CDR-H1) comprising a sequence as shown in SEQ ID NO: 1, 7 or 12, a heavy chain complementary determining region 2 (CDR-H2) comprising a sequence as shown in SEQ ID NO: 2, 8 or 13, a heavy chain complementary determining region 3 (CDR-H3) comprising a sequence as shown in SEQ ID NO: 3, 9 or 14, a light chain complementary determining region 1 (CDR-L1) comprising a sequence as shown in SEQ ID NO: 4, 10 or 15, a light chain complementary determining region 2 (CDR-L2) comprising a sequence as shown in SEQ ID NO: 5 or 11, and a light chain complementary determining region 3 (CDR-H4) comprising a sequence as shown in SEQ ID NO: 6 or 8. NO: Light chain complementary determining region 3 (CDR-L3) of the sequence shown in 6 or 16;

[0024] Where R 3a structure comprising +C*+A*oG*oC*dG*dC*dC*dC*dA*dC*dC*dA*oG*oU*+C*+A (SEQ ID NO: 21), wherein +N represents LNA (2'-4' methylene bridge) ribonucleoside, dN represents 2'-deoxyribonucleoside, oN represents 2'-MOE modified ribonucleoside, oC represents 5-methyl-2'-MOE-cytidine, +C represents 5-methyl-2'-4'-bicyclo-cytidine (2'-4' methylene bridge), oU represents 5-methyl-2'-MOE-uridine, and * represents phosphorothioate internucleoside linkage;

[0025] Each R 1 At the junction point A, the linkage site represented by the lysine (K) residue of the anti-TfR1 antibody is connected to R 2 covalently linked, and wherein at least 80% of the light chain constant regions of the anti-TfR1 antibodies in the complex in the composition are independently linked to R at the following linkage sites: 1 Covalently linked: a linkage site represented by K188 (based on Kabat numbering) and / or a linkage site represented by K190 (based on Kabat numbering) of the light chain constant region of the anti-TfR1 antibody; and

[0026] wherein in each of the plurality of complexes, n1 is independently an integer of 1 or greater, which represents R 1 The number of instances of .

[0027] According to some aspects, the present disclosure provides a composition comprising a plurality of complexes, wherein the complexes comprise formula (I): [R 1 ] n1 -R 2 The structure of

[0028] Each R 1 Independently comprising groups of formula (Ib):

[0029]

[0030] wherein +N represents LNA (2'-4' methylene bridge) ribonucleoside, dN represents 2'-deoxyribonucleoside, oN represents 2'-MOE modified ribonucleoside, oC represents 5-methyl-2'-MOE-cytidine, +C represents 5-methyl-2'-4'-bicyclo-cytidine (2'-4' methylene bridge), oU represents 5-methyl-2'-MOE-uridine, and * represents phosphorothioate internucleoside linkage, and R 1 The oligonucleotide comprises the nucleobase sequence of CAGCGCCCACCAGUCA (SEQ ID NO: 21);

[0031] Where R 2The invention comprises an anti-transferrin receptor (TfR1) antibody, wherein the anti-transferrin receptor (TfR1) antibody comprises a heavy chain comprising a heavy chain variable region (VH) and a heavy chain constant region, and a light chain comprising a light chain variable region (VL) and a light chain constant region; and wherein the anti-TfR1 antibody comprises: a heavy chain complementary determining region 1 (CDR-H1) comprising a sequence as shown in SEQ ID NO: 1, 7 or 12, a heavy chain complementary determining region 2 (CDR-H2) comprising a sequence as shown in SEQ ID NO: 2, 8 or 13, a heavy chain complementary determining region 3 (CDR-H3) comprising a sequence as shown in SEQ ID NO: 3, 9 or 14, a light chain complementary determining region 1 (CDR-L1) comprising a sequence as shown in SEQ ID NO: 4, 10 or 15, a light chain complementary determining region 2 (CDR-L2) comprising a sequence as shown in SEQ ID NO: 5 or 11, and a light chain complementary determining region 3 (CDR-H4) comprising a sequence as shown in SEQ ID NO: 6 or 8. NO: light chain complementarity determining region 3 (CDR-L3) of the sequence shown in 6 or 16; and

[0032] Each R 1 At the junction point A, the linkage site represented by the lysine (K) residue of the anti-TfR1 antibody is connected to R 2 covalently linked, and wherein at least 80% of the light chain constant regions of the anti-TfR1 antibodies in the complex in the composition are independently linked to R at the following linkage sites: 1 Covalently linked: a linkage site represented by K188 (based on Kabat numbering) and / or a linkage site represented by K190 (based on Kabat numbering) of the light chain constant region of the anti-TfR1 antibody; and

[0033] wherein in each of the plurality of complexes, n1 is independently an integer of 1 or greater, which represents R 1 The number of instances of .

[0034] According to some aspects, the present disclosure provides a composition comprising a plurality of complexes, wherein the complexes comprise formula (I): [R 1 ] n1 -R 2 The structure of which:

[0035] Each R 1 Containing a group of formula (Ic):

[0036]

[0037] Where R 2The invention comprises an anti-transferrin receptor (TfR1) antibody, wherein the anti-transferrin receptor (TfR1) antibody comprises a heavy chain comprising a heavy chain variable region (VH) and a heavy chain constant region, and a light chain comprising a light chain variable region (VL) and a light chain constant region; and wherein the anti-TfR1 antibody comprises: a heavy chain complementary determining region 1 (CDR-H1) comprising a sequence as shown in SEQ ID NO: 1, 7 or 12, a heavy chain complementary determining region 2 (CDR-H2) comprising a sequence as shown in SEQ ID NO: 2, 8 or 13, a heavy chain complementary determining region 3 (CDR-H3) comprising a sequence as shown in SEQ ID NO: 3, 9 or 14, a light chain complementary determining region 1 (CDR-L1) comprising a sequence as shown in SEQ ID NO: 4, 10 or 15, a light chain complementary determining region 2 (CDR-L2) comprising a sequence as shown in SEQ ID NO: 5 or 11, and a light chain complementary determining region 3 (CDR-H4) comprising a sequence as shown in SEQ ID NO: 6 or 8. NO: light chain complementarity determining region 3 (CDR-L3) of the sequence shown in 6 or 16; and

[0038] Each R 1 At the junction point A, the linkage site represented by the lysine (K) residue of the anti-TfR1 antibody is connected to R 2 covalently linked, and wherein at least 80% of the light chain constant regions of the anti-TfR1 antibodies in the complex in the composition are independently linked to R at the following linkage sites: 1 Covalently linked: a linkage site represented by K188 (based on Kabat numbering) and / or a linkage site represented by K190 (based on Kabat numbering) of the light chain constant region of the anti-TfR1 antibody; and

[0039] wherein in each of the plurality of complexes, n1 is independently an integer of 1 or greater, which represents R 1 The number of instances of .

[0040] In some embodiments, 85% to 98% of the light chain constant regions of the anti-TfR1 antibodies in the complexes in the composition independently bind to R at the following linkage sites: 1 Covalently linked: the linkage site represented by K188 (based on Kabat numbering) of the light chain constant region of the anti-TfR1 antibody and / or the linkage site represented by K190 (based on Kabat numbering). In some embodiments, 95% to 97% of the light chain constant regions of the anti-TfR1 antibodies in the complex in the composition are independently linked to R at the following linkage sites: 1 Covalent linkage: the linkage site represented by K188 (based on Kabat numbering) and / or the linkage site represented by K190 (based on Kabat numbering) of the light chain constant region of the anti-TfR1 antibody.

[0041] In some embodiments, 1% to 15% of the heavy chain constant regions of the anti-TfR1 antibodies in the complex in the composition independently bind to R at the following linkage sites: 1 Covalently linked: The linkage site represented by K213 (based on Kabat numbering) of the heavy chain constant region of the anti-TfR1 antibody. In some embodiments, 4% to 8% of the heavy chain constant regions of the anti-TfR1 antibodies in the complex in the composition are independently linked to R at the following linkage sites: 1 Covalent linkage: The linkage site is represented by K213 (based on Kabat numbering) of the heavy chain constant region of the anti-TfRl antibody.

[0042] In some embodiments, the antibody is a Fab fragment, a full length IgG, a Fab' fragment, or a F(ab')2 fragment. In some embodiments, the antibody is a Fab fragment.

[0043] In some embodiments, the VH comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 17; and / or the VL comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 18. In some embodiments, the antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 17 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 18.

[0044] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:19 and the light chain comprises the amino acid sequence of SEQ ID NO:20.

[0045] In some embodiments, the composition further comprises one or more antibodies that are not covalently linked to the oligonucleotide.

[0046] In some embodiments, the average value of n1 of the complexes in the composition is between 0.5 and 5.

[0047] According to some aspects, the present disclosure provides methods of reducing DMPK expression and / or treating myotonic dystrophy in a subject, the methods comprising administering to the subject an effective amount of a composition provided herein.

[0048] In some embodiments, the subject has an expansion of a disease-associated repeat of a DMPK allele associated with myotonic dystrophy. In some embodiments, the disease-associated repeat comprises repeating units of a CTG trinucleotide sequence.

[0049] In some embodiments, the complex reduces DMPK expression in a subject. In some embodiments, reducing DMPK expression comprises reducing the level of DMPK mRNA in a muscle cell. In some embodiments, the DMPK mRNA is a mutant DMPK mRNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 Mass spectra indicating the intact mass of unlinked anti-TfR1 Fab having the sequence shown in Table 2 are shown.

[0051] Figure 2 Mass spectra are shown indicating the mass of the anti-TfR1 antibody-linker complex of the compositions provided herein after cleavage of the oligonucleotide by papain digestion.The anti-TfR1 antibodies have the sequences shown in Table 2.

[0052] Figures 3A to 3D Figure 2 shows the effects of the conjugate containing anti-TfR1 Fab covalently linked to ASO on the heart ( Figure 3A ), diaphragm( Figure 3B ), gastrocnemius( Figure 3C ) or tibialis anterior ( Figure 3D ) in the presence of DMPK targeting oligonucleotide (ASO).

[0053] Figures 4A to 4D The conjugate containing anti-TfR1 Fab conjugated to DMPK targeting oligonucleotide (ASO) was shown in the heart of mice ( Figure 4A ), diaphragm( Figure 4B ), tibialis anterior ( Figure 4C ) and gastrocnemius ( Figure 4D ) mice expressing both human TfR1 and two copies of a mutant human DMPK transgene containing expanded CTG repeats.

[0054] Figures 5A to 5B Shown are reduced DMPK foci in the nuclei of cardiac myocardial fibers in mice expressing both human TfR1 and two copies of a mutant human DMPK transgene containing expanded CTG repeats and treated with anti-TfR1 Fab conjugated to a DMPK targeting oligonucleotide (ASO). Figure 5A Representative images of samples after in situ hybridization staining for DMPK foci and fluorescent staining of myofibers are shown (inset). Figure 5A In the microscopy image shown in , the light-colored circles show the cell nuclei, and the bright spots within the nuclei show DMPK foci. Figure 5B Quantification of DMPK foci is shown.

[0055] Figure 6The splicing correction activity of a conjugate comprising an anti-TfR1 Fab covalently linked to a DMPK targeting oligonucleotide (ASO) in the heart of mice (hTfR1 / DMSXL mice) expressing both human TfR1 and two copies of a mutant human DMPK transgene containing expanded CTG repeats is shown. Composite splicing indices based on splicing of Ldb3 exon 11, Mbnl2 exon 6, and Nfix exon 7 are shown for control mice treated with vehicle control ("hTfR1-PBS"), hTfR1 / DMSXL mice treated with vehicle control ("hTfR1 / DMSXL-PBS"), and hTfR1 / DMSXL mice treated with anti-TfR1 Fab-ASO conjugates ("hTfR1 / DMSXL-conjugates").

[0056] Figure 7 The splicing correction activity of a conjugate comprising an anti-TfR1 Fab covalently linked to a DMPK targeting oligonucleotide (ASO) in the diaphragm of mice (hTfR1 / DMSXL mice) expressing both human TfR1 and two copies of a mutant human DMPK transgene containing expanded CTG repeats is shown. Composite splicing indices based on splicing of Bin1 exon 11, Insr exon 11, Ldb3 exon 11, and Nfix exon 7 are shown for control mice treated with vehicle control ("hTfR1-PBS"), hTfR1 / DMSXL mice treated with vehicle control ("hTfR1 / DMSXL-PBS"), and hTfR1 / DMSXL mice treated with anti-TfR1 Fab-ASO conjugates ("hTfR1 / DMSXL-conjugates").

[0057] Figure 8 The splicing correction activity of a conjugate comprising an anti-TfR1 Fab covalently linked to a DMPK targeting oligonucleotide (ASO) in the tibialis anterior muscle of mice (hTfR1 / DMSXL mice) expressing both human TfR1 and two copies of a mutant human DMPK transgene containing an expanded CTG repeat is shown. Composite splicing indices based on splicing of Bin1 exon 11, Ldb3 exon 11, Mbnl2 exon 6, and Nfix exon 7 are shown for control mice treated with vehicle control ("hTfR1-PBS"), hTfR1 / DMSXL mice treated with vehicle control ("hTfR1 / DMSXL-PBS"), and hTfR1 / DMSXL mice treated with an anti-TfR1 Fab-ASO conjugate ("hTfR1 / DMSXL-conjugate").

[0058] Fig. 9The splicing correction activity of a conjugate comprising an anti-TfR1 Fab covalently linked to a DMPK targeting oligonucleotide (ASO) in gastrocnemius muscle of mice (hTfR1 / DMSXL mice) expressing both human TfR1 and two copies of a mutant human DMPK transgene containing expanded CTG repeats is shown. Composite splicing indices based on splicing of Mbnl2 exon 6, Nfix exon 7, and Ttn exon 313 are shown for control mice treated with vehicle control ("hTfR1-PBS"), hTfR1 / DMSXL mice treated with vehicle control ("hTfR1 / DMSXL-PBS"), and hTfR1 / DMSXL mice treated with anti-TfR1 Fab-ASO conjugates ("hTfR1 / DMSXL-conjugates").

[0059] Fig.10 DMPK knockdown in DM1 patient myotubes and wild-type non-human primate (NHP) myotubes is shown, which is produced by incubation with a conjugate containing an anti-TfR1 Fab covalently linked to a DMPK targeting oligonucleotide (ASO). The results shown are normalized for expression in DM1 patient myotubes or NHP myotubes treated with vehicle alone. The data show the mean + standard deviation of n = 4 replicates under each condition. Data were calculated by one-way ANOVA (*, P < 0.05, **, P < 0.01). DETAILED DESCRIPTION

[0060] According to some aspects, the disclosure provides a composition comprising a plurality of complexes. In some embodiments, the complex of the composition described herein comprises an antibody covalently linked to one or more oligonucleotides (e.g., anti-transferrin receptor 1 (TfR1)), each oligonucleotide being covalently linked at a bonding site represented by a lysine (K) residue of the antibody. In some embodiments, the antibody comprises a heavy chain containing a heavy chain variable region (VH) and a heavy chain constant region and a light chain containing a light chain variable region (VL) and a light chain constant region.

[0061] In some embodiments, the antibody light chain constant region in the complex of the composition is independently covalently linked to the oligonucleotide at the following bonding sites: a bonding site represented by K188 (based on Kabat numbering) of the light chain constant region of the antibody and / or a bonding site represented by K190 (based on Kabat numbering). For example, in some embodiments, the bonding site is represented by K188 (based on Kabat numbering) of the light chain constant region of the antibody. In some embodiments, the bonding site is represented by K190 (based on Kabat numbering) of the light chain constant region of the antibody. In some embodiments, the bonding site is represented by K188 (based on Kabat numbering) and K190 (based on Kabat numbering) of the light chain constant region of the antibody. In some embodiments, at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, 80% to 98%, 80% to 95%, 80% to 90%, 85% to 98%, 85% to 95%, 85% to 90%, 90% to 98%, 90% to 95%, 95% to 97%, or more) of the antibody light chain constant region in the complex of the composition is independently covalently linked to an oligonucleotide at the following linkage sites: the linkage site represented by K188 (based on Kabat numbering) and / or the linkage site represented by K190 (based on Kabat numbering) of the antibody light chain constant region. In some embodiments, about 1% to 15% (e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, or about 15%) of the heavy chain constant region of the antibody in the complex of the composition is independently covalently linked to an oligonucleotide at the following linkage site: a linkage site represented by K213 (based on Kabat numbering) of the heavy chain constant region of the antibody. In some embodiments, both the light chain constant region and the heavy chain constant region of the antibody in the complex of the composition are independently covalently linked to an oligonucleotide at a linkage site represented by a lysine residue in the light chain constant region (e.g., K188 and / or K190) and the heavy chain constant region (e.g., K213) of the antibody.

[0062] In some embodiments, the antibody light chain in the complex of the composition is independently covalently linked to the oligonucleotide at the following bonding site: a bonding site represented by a lysine (K) residue in the sequence motif DYEKHKVYA (SEQ ID NO: 27) of the antibody light chain. For example, in some embodiments, the bonding site is represented by K at position 4 of the sequence motif DYEKHKVYA (SEQ ID NO: 27) of the antibody light chain. In some embodiments, the bonding site is represented by K at position 6 of the sequence motif DYEKHKVYA (SEQ ID NO: 27) of the antibody light chain. In some embodiments, the bonding site is represented by K at position 4 and K at position 6 of the sequence motif DYEKHKVYA (SEQ ID NO: 27) of the antibody light chain. In some embodiments, at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, 80% to 98%, 80% to 95%, 80% to 90%, 85% to 98%, 85% to 95%, 85% to 90%, 90% to 98%, 90% to 95%, 95% to 97%, or more) of the antibody light chain constant region in the complex of the composition is independently covalently linked to an oligonucleotide at the following linkage site: the linkage site represented by a lysine (K) residue in the sequence motif DYEKHKVYA (SEQ ID NO: 27) of the antibody light chain. In some embodiments, about 1% to 15% (e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, or about 15%) of the constant region of the heavy chain of the antibody in the complex of the composition are independently covalently linked to the oligonucleotide at the following linkage site: the linkage site represented by the lysine (K) residue in the motif VNHKPSN (SEQ ID NO: 28) of the antibody heavy chain. In some embodiments, the antibody light chain and heavy chain in the complex of the composition are independently covalently linked to the oligonucleotide at the linkage site represented by a lysine residue in the light chain of the antibody (e.g., K at position 4 or 6 in the motif DYEKHKVYA (SEQ ID NO: 27)) and a lysine residue in the heavy chain (e.g., K in the motif VNHKPSN (SEQ ID NO: 28)).

[0063] In some aspects, the disclosure provides methods of reducing the expression or activity of DMPK in a subject (e.g., reducing the level of mutant or wild-type DMPK RNA) and / or treating myotonic dystrophy (e.g., DM1). In some embodiments, the method comprises administering to the subject an effective amount of a composition comprising a complex described herein.

[0064] In some aspects, the disclosure also provides methods of determining a range of drug to antibody ratio (DAR) of a complex in a composition, or methods of analyzing a complex in a composition.

[0065] Additional aspects of the disclosure are provided below, including a description of defined terms.

[0066] definition

[0067] Administering: As used herein, the term "administering" or variations thereof means providing a complex to a subject (eg, to treat a condition in a subject) in a physiologically and / or (eg, and) pharmacologically usable manner.

[0068] Approximately: The term "approximately" or "about" as used herein, when applied to one or more target values, refers to a value similar to the stated reference value. In certain embodiments, the term "approximately" or "approximately" refers to a range of values ​​that fall within (greater than or less than) 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction of the stated reference value, unless otherwise stated or otherwise apparent from the context (unless such a number exceeds 100% of the possible value).

[0069] Antibody: The term "antibody" as used herein refers to a polypeptide comprising at least one immunoglobulin variable domain or at least one antigenic determinant (e.g., a paratope that specifically binds to an antigen). In some embodiments, the antibody is a full-length antibody. In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a humanized antibody. However, in some embodiments, the antibody is a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fv fragment, or a scFv fragment. In some embodiments, the antibody is a nanobody derived from a camelid antibody or a nanobody derived from a shark antibody. In some embodiments, the antibody is a double antibody. In some embodiments, the antibody comprises a framework with a human germline sequence. In another embodiment, the antibody comprises a heavy chain constant domain selected from IgG, IgG1, IgG2, IgG2A, IgG2B, IgG2C, IgG3, IgG4, IgA1, IgA2, IgD, IgM, and IgE constant domains. In some embodiments, the antibody comprises a heavy (H) chain variable region (referred to herein as VH) and / or (e.g., and) a light (L) chain variable region (referred to herein as VL). In some embodiments, the antibody comprises a constant domain, such as an Fc region. An immunoglobulin constant domain refers to a heavy chain or light chain constant domain. The amino acid sequences of human IgG heavy and light chain constant domains and their functional variations are known. With regard to the heavy chain, in some embodiments, the heavy chain of the antibodies described herein may be an alpha (α), delta (Δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain. In some embodiments, the heavy chain of the antibodies described herein may comprise a human alpha (α), delta (Δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain. In a specific embodiment, the antibodies described herein comprise human γ1 CH1, CH2, and / or (e.g., and) CH3 domains. In some embodiments, the amino acid sequence of the VH domain comprises the amino acid sequence of a human gamma (γ) heavy chain constant region, such as any known in the art. Some non-limiting examples of human constant region sequences have been described in the art, for example, see U.S. Pat. No. 5,693,780 and Kabat EA et al., (1991) supra. In some embodiments, the VH domain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or at least 99% identity with any variable chain constant region provided herein. In some embodiments, the antibody is modified, for example, by glycosylation, phosphorylation, SUMOylation and / or (e.g., and) methylation. In some embodiments, the antibody is a glycosylated antibody conjugated to one or more sugar or carbohydrate molecules.In some embodiments, one or more sugar or carbohydrate molecules are conjugated to the antibody by N-glycosylation, O-glycosylation, C-glycosylation, glycosylphosphatidylinositolization (GPI anchor attachment) and / or (e.g., and) phosphoglycosylation. In some embodiments, one or more sugar or carbohydrate molecules are monosaccharides, disaccharides, oligosaccharides or polysaccharides. In some embodiments, one or more sugar or carbohydrate molecules are branched oligosaccharides or branched polysaccharides. In some embodiments, one or more sugar or carbohydrate molecules comprise mannose units, glucose units, N-acetylglucosamine units, N-acetylgalactosamine units, galactose units, fucose units or phospholipid units. In some embodiments, the antibody is a construct comprising a polypeptide comprising one or more antigen-binding fragments of the present disclosure connected to a linker polypeptide or an immunoglobulin constant domain. The linker polypeptide comprises two or more amino acid residues connected by a peptide bond and is used to connect one or more antigen-binding moieties. Some examples of linker polypeptides have been reported (see, e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448; Poljak, RJ, et al. (1994) Structure 2: 1121-1123). In addition, an antibody may be part of a larger immunoadhesion molecule formed by covalent or non-covalent association of an antibody or antibody portion with one or more other proteins or peptides. Some examples of such immunoadhesion molecules include the use of a streptavidin core region to prepare tetrameric scFv molecules (Kipriyanov, SM, et al. (1995) Human Antibodies and Hybridomas 6:93-101), and the use of cysteine ​​residues, a marker peptide, and a C-terminal polyhistidine tag to prepare bivalent and biotinylated scFv molecules (Kipriyanov, SM, et al. (1994) Mol. Immunol. 31:1047-1058).

[0070] CDR: The term "CDR" as used herein refers to the complementary determining region within the variable sequence of an antibody. A typical antibody molecule comprises a heavy chain variable region (VH) and a light chain variable region (VL), which are generally involved in antigen binding. The VH region and the VL region can be further subdivided into hypervariable regions, also known as "complementarity determining regions" ("complementarity determining regions, CDRs"), interspersed with more conservative regions called "framework regions" ("framework regions, FRs"). Each VH and VL is generally composed of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The scope of the framework region and CDR can be precisely identified using methods known in the art, such as by the Kabat definition, the IMGT definition, the Chothia definition, the AbM definition, and / or (e.g., and) contact definition (all of which are well known in the art).

[0071] See for example,

[0072] Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIHPublication No. 91-3242; IMGT the international ImMunoGeneTics information system 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); 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., NucleicAcids Res..43:D413-422(2015); Chothia et al. al., (1989) Nature342:877; Chothia, C. etal. (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).

[0073] See also hgmp.mrc.ac.uk and bioinf.org.uk / abs. CDRs used herein may refer to CDRs defined by any method known in the art. Two antibodies having the same CDRs means that the amino acid sequences of the CDRs of the two antibodies are identical, as determined by the same method (e.g., IMGT definition).

[0074] There are three CDRs in each variable region of the heavy and light chains, referred to as CDR1, CDR2 and CDR3 for each variable region. The term "CDR set" as used herein refers to a group of three CDRs that are present in a single variable region and are capable of binding to an antigen. The exact boundaries of these CDRs have been defined differently according to different systems. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides an unambiguous residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries that define the three CDRs. These CDRs may be referred to as Kabat CDRs. Subportions of the CDRs may be designated L1, L2, and L3 or H1, H2, and H3, where "L" and "H" designate the light chain and heavy chain regions, respectively. These regions may be referred to as Chothia CDRs, which have boundaries that overlap with the Kabat CDRs. Padlan (FASEB J. 9: 133-139 (1995)) and MacCallum (J Mol Biol 262 (5): 732-45 (1996)) have described other boundaries that define CDRs that overlap with the Kabat CDRs. Other CDR boundary definitions may not strictly follow one of the above systems, but will still overlap with the Kabat CDRs. CDR overlap, although they can be shortened or lengthened based on predictions or based on experimental findings that specific residues or groups of residues or even entire CDRs do not significantly affect antigen binding. The methods used herein can utilize CDRs defined according to any one of these systems. Some examples of CDR definition systems are provided in Table 1.

[0075] Table 1. CDR definitions

[0076] <![CDATA[IMGT 1 ]]> <![CDATA[Kabat 2 ]]> <![CDATA[Chothia 3 ]]> CDR-H1 27 to 38 31 to 35 26 to 32 CDR-H2 56 to 65 50 to 65 53 to 55 CDR-H3 105 to 116 / 117 95 to 102 96 to 101 CDR-L1 27 to 38 24 to 34 26 to 32 CDR-L2 56 to 65 50 to 56 50 to 52 CDR-L3 105 to 116 / 117 89 to 97 91 to 96

[0077] 1 the international ImMunoGeneTics information imgt.org,Lefranc.M.-P.et al., Nucleic Acids Res.,27:209-212(1999)

[0078] 2Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242

[0079] 3 Chothia et al., J.Mol.Biol.196:901-917(1987))

[0080] Complementary: The term "complementary" as used herein refers to the ability to accurately pair between two nucleotides or two groups of nucleotides. In particular, complementarity is a term that characterizes the degree of binding between two nucleotides or two groups of nucleotides caused by hydrogen bond pairing. For example, if the base at one position of an oligonucleotide can hydrogen bond with the base at the corresponding position of a target nucleic acid (e.g., mRNA), the bases are considered to be complementary to each other at this 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, adenosine bases (A) are complementary to thymidine bases (T) or uracil bases (U), cytosine bases (C) are complementary to guanosine bases (G), and universal bases such as 3-nitropyrrole or 5-nitroindole can hybridize with any A, C, U or T and are considered to be complementary. Inosine (I) is also considered in the art to be a universal base, and is considered complementary to any of A, C, U, or T.

[0081] Covalently linked: As used herein, the term "covalently linked" refers to the feature of two or more molecules being linked together by at least one covalent bond. In some embodiments, two molecules can be covalently linked together by a single bond, such as a disulfide bond or a disulfide bridge, that acts as a linker between the molecules. However, in some embodiments, two or more molecules can be covalently linked together by a molecule that acts as a linker that links the two or more molecules together by multiple covalent bonds. In some embodiments, the linker can be a cleavable linker. However, in some embodiments, the linker can be a non-cleavable linker.

[0082] Disease-associated repeats: The term "disease-associated repeats" as used herein refers to a repetitive nucleotide sequence at a certain genomic position, wherein the number of units of the repetitive nucleotide sequence is associated with a genetic disease and / or (e.g., and) directly or indirectly contributes to or causes a genetic disease. The length of each repeat unit of the disease-associated repeat can be 2, 3, 4, 5 or more nucleotides. For example, in some embodiments, the disease-associated repeat is a dinucleotide repeat. In some embodiments, the disease-associated repeat is a trinucleotide repeat. In some embodiments, the disease-associated repeat is a tetranucleotide repeat. In some embodiments, the disease-associated repeat is a pentanucleotide repeat. In some embodiments, the disease-associated repeat comprises CAG repeats, CTG repeats, CUG repeats, CGG repeats, CCTG repeats, or any complementary nucleotides thereof. In some embodiments, the disease-associated repeat is in the non-coding portion of the gene. However, in some embodiments, the disease-associated repeat is in the coding region of the gene. In some embodiments, the disease-associated repeat is amplified from a normal state to a length that directly or indirectly contributes to or causes a genetic disease. In some embodiments, the disease-associated repeat is in RNA (e.g., RNA transcript). In some embodiments, the disease-associated repeat is in DNA (e.g., chromosome, plasmid). In some embodiments, the disease-associated repeats are expanded in the chromosomes of germ cells. In some embodiments, the disease-associated repeats are expanded in the chromosomes of somatic cells. In some embodiments, the disease-associated repeats are expanded to the number of repeat units associated with the congenital onset of the disease. In some embodiments, the disease-associated repeats are expanded to the number of repeat units associated with the onset of the disease in childhood. In some embodiments, the disease-associated repeats are expanded to the number of repeat units associated with the onset of the disease in adults. In DM1, the DMPK gene comprises a disease-associated repeat of the CTG unit.

[0083] DMPK: The term "DMPK" as used herein refers to a gene encoding myotonic protein kinase (also known as myotonic dystrophy protein kinase or dystrophic myotonic protein kinase) as a serine / threonine protein kinase. Substrates of this enzyme may include myogenin, the β subunit of the L-type calcium channel, and phospholemman. In some embodiments, DMPK may be a human gene (Gene ID: 1760), a non-human primate gene (e.g., Gene ID: 456139, Gene ID: 715328, Gene ID: 102125829), or a rodent gene (e.g., Gene ID: 13400). In humans, CTG repeat expansion in the 3' non-coding, untranslated region of DMPK is associated with myotonic dystrophy type I (DM1). In addition, multiple human transcript variants encoding different protein isoforms have been characterized (e.g., as annotated with the following GenBank RefSeq accession numbers: NM_001081563.2, NM_004409.4, NM 001081560.2, NM001081562.2, NM 001288764.1, NM 001288765.1, and NM 001288766.1).

[0084] DMPK allele: The term "DMPK allele" as used herein refers to any of the alternative forms (e.g., wild-type or mutant forms) of the DMPK gene. In some embodiments, the DMPK allele may encode a wild-type myotonic protein kinase that retains its normal and typical functions. In some embodiments, the DMPK allele may comprise one or more disease-associated repeat expansions. In some embodiments, a normal subject has two DMPK alleles comprising 5 to 37 repeat units. In some embodiments, the number of CTG repeat units in subjects with DM1 is from about 50 to about 3,000 or more, and the higher the number of repeats, the higher the severity of the disease. In some embodiments, a mildly affected DM1 subject has at least one DMPK allele having 50 to 150 repeat units. In some embodiments, a subject with typical DM1 has at least one DMPK allele having 100 to 1,000 or more repeat units. In some embodiments, a subject with congenital onset DM1 may have at least one DMPK allele comprising more than 2,000 repeat units.

[0085] Framework: The term "framework" or "framework sequence" used herein refers to the remaining sequence of the variable region minus CDR. Since the exact definition of CDR sequences can be determined by different systems, the meaning of framework sequences has different interpretations accordingly. The six CDRs (CDR-L1, CDR-L2 and CDR-L3 of the light chain and CDR-H1, CDR-H2 and CDR-H3 of the heavy chain) also distinguish the framework on the light chain and the heavy chain into four subregions (FR1, FR2, FR3 and FR4) on each chain, wherein CDR1 is located between FR1 and FR2, CDR2 is located between FR2 and FR3, and CDR3 is located between FR3 and FR4. In the case where a specific subregion is not designated as FR1, FR2, FR3 or FR4, the framework region mentioned by others represents the FR of the combination in the variable region of a single naturally occurring immunoglobulin chain. The FR used herein represents one of the four subregions, and a plurality of FRs represent two or more of the four subregions constituting the framework region. Human heavy chain and light chain receptor sequences are known in the art. In one embodiment, acceptor sequences known in the art can be used in the antibodies disclosed herein.

[0086] Human antibody: The term "human antibody" as used herein is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the present disclosure may contain amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo), such as in CDR, and particularly in CDR3. However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) have been grafted onto human framework sequences.

[0087] Humanized antibody: The term "humanized antibody" refers to an antibody comprising heavy and light chain variable region sequences from non-human species (e.g., mouse) but wherein at least a portion of the VH and / or (e.g., and) VL sequences have been altered to be more "human-like" (i.e., more similar to human germline variable sequences). One type of humanized antibody is a CDR-grafted antibody in which human CDR sequences are introduced into non-human VH and VL sequences to replace the corresponding non-human CDR sequences. In one embodiment, humanized anti-transferrin receptor antibodies and antigen-binding portions are provided. Such antibodies can be produced by obtaining mouse anti-transferrin receptor monoclonal antibodies using conventional hybridoma technology and then humanizing using in vitro genetic engineering, such as those disclosed in PCT Publication No. WO2005 / 123126A2 of Kasaian et al.

[0088] Kabat numbering: The terms "Kabat numbering," "Kabat definition," and "Kabat notation" are used interchangeably herein. These terms, recognized in the art, refer to a system for numbering amino acid residues in the variable regions of the heavy and light chains of an antibody or antigen-binding portion thereof that are more variable (i.e., hypervariable) than other amino acid residues (Kabat et al. (1971) Ann. NY Acad, Sci. 190:382-391 and Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 2001-0306). No.91-3242). For the heavy chain variable region, the hypervariable region of CDR1 is from amino acids 31 to 35, the hypervariable region of CDR2 is from amino acids 50 to 65, and the hypervariable region of CDR3 is from amino acids 95 to 102. For the light chain variable region, the hypervariable region of CDR1 is from amino acids 24 to 34, the hypervariable region of CDR2 is from amino acids 50 to 56, and the hypervariable region of CDR3 is from amino acids 89 to 97.

[0089] Myotonic dystrophy (DM): The term "myotonic dystrophy (DM)" as used herein refers to a genetic disease caused by mutations in the DMPK gene or the CNBP (ZNF9) gene, characterized by muscle loss, muscle weakness, and muscle function. Two types of the disease have been described, myotonic dystrophy type 1 (DM1) and myotonic dystrophy type 2 (DM2). DM1 is associated with an expansion of CTG trinucleotide repeats in the 3' noncoding region of DMPK. DM2 is associated with an expansion of CCTG tetranucleotide repeats in the first intron of ZNF9. In both DM1 and DM2, nucleotide expansion leads to toxic RNA repeats that are able to form hairpin structures that bind to key intracellular proteins (e.g., muscleblind-like proteins) with high affinity. Myotonic dystrophy, the genetic basis of the disease, and associated symptoms are described in the art (see, e.g., Thornton, CA, "Myotonic Dystrophy" Neurol Clin. (2014), 32(3): 705-719.; and Konieczny et al. "Myotonic dystrophy: candidate small molecule therapeutics" Drug Discovery Today (2017), 22: 11). In some embodiments, the subject is born with a DM1 variant known as congenital myotonic dystrophy. Symptoms of congenital myotonic dystrophy are present from birth and include weakness of all muscles, breathing problems, club feet, developmental delays, and intellectual disability. DM1 is associated with Online Mendelian Inheritance in Man (OMIM) Entry #160900. DM2 is associated with OMIM Entry #602668.

[0090] Oligonucleotide: The term "oligonucleotide" as used herein refers to an oligonucleotide compound up to 200 nucleotides in length. Some examples of oligonucleotides include, but are not limited to, RNAi oligonucleotides (e.g., siRNA, shRNA), microRNA, spacer polymers, mixed polymers, phosphorodiamidate morpholinos, peptide nucleic acids, aptamers, guide nucleic acids (e.g., Cas9 guide RNA), etc. Oligonucleotides can be single-stranded or double-stranded. In some embodiments, the oligonucleotide may include one or more modified nucleosides (e.g., 2'-O-methyl sugar modifications, purine or pyrimidine modifications). In some embodiments, the oligonucleotide may include one or more modified internucleoside linkages. In some embodiments, the oligonucleotide may include one or more phosphorothioate linkages, which may be Rp or Sp stereochemical conformations.

[0091] Complementary region: The term "complementary region" as used herein refers to a nucleotide sequence, such as an oligonucleotide, that is fully complementary to a homologous nucleotide sequence, such as a target nucleic acid, such that the two nucleotide sequences can anneal to each other under physiological conditions (e.g., in a cell). In some embodiments, the complementary region is completely complementary to the homologous nucleotide sequence of the target nucleic acid. However, in some embodiments, the complementary region is partially complementary to the homologous nucleotide sequence of the target nucleic acid (e.g., at least 80%, 90%, 95% or 99% complementary). In some embodiments, the complementary region comprises 1, 2, 3 or 4 mismatches compared to the homologous nucleotide sequence of the target nucleic acid.

[0092] Specific binding: As used herein, the term "specific binding" refers to the ability of a molecule to bind to a binding partner with a degree of affinity or avidity that enables the molecule to be used to distinguish the binding partner from a suitable control in a binding assay or other binding context. With respect to antibodies, the term "specific binding" refers to the ability of an antibody to bind to a specific antigen with a degree of affinity or avidity that enables the antibody to be used to distinguish the specific antigen from other antigens, e.g., to a degree that allows preferential targeting of certain cells (e.g., muscle cells) by binding to an antigen as described herein. In some embodiments, if the K of an antibody binding to a target is D 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 M or less, the antibody specifically binds to the target. In some embodiments, the antibody specifically binds to the transferrin receptor (eg, an epitope of the apical domain of the transferrin receptor).

[0093] Subject: The term "subject" as used herein refers to a mammal. In some embodiments, the subject is a non-human primate or a rodent. In some embodiments, the subject is a human. In some embodiments, the subject is a patient, such as a human patient suffering from or suspected of having a disease. In some embodiments, the subject is a human patient suffering from or suspected of having a disease caused by a disease-associated repeat expansion (e.g., a disease-associated repeat expansion in a DMPK allele).

[0094] Transferrin receptor: The term "transferrin receptor" (also referred to as TFRC, CD71, p90, TFR or TFR1) as used herein refers to a cell surface receptor that binds transferrin to promote internalization of iron uptake by endocytosis. In some embodiments, the transferrin receptor may be of human origin (NCBI Gene ID 7037), non-human primate origin (e.g., NCBI Gene ID 711568 or NCBI Gene ID 102136007), or rodent origin (e.g., NCBI Gene ID 22042). In addition, a variety of human transcript variants encoding different isoforms of the receptor have been characterized (e.g., as annotated with the following GenBank RefSeq accession numbers: NP_001121620.1, NP 003225.2, NP 001300894.1, and NP001300895.1).

[0095] 2'-modified nucleosides: As used herein, the terms "2'-modified nucleosides" and "2'-modified ribonucleosides" are used interchangeably and refer to nucleosides having a modified sugar moiety at the 2' position. In some embodiments, the 2'-modified nucleoside is a 2'-4' bicyclic nucleoside, wherein the 2' and 4' positions of the sugar are bridged (e.g., bridged 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., wherein the 2' position of the sugar moiety is substituted. Some non-limiting examples of 2'-modified nucleosides include: 2'-deoxy, 2'-fluoro (2'-F), 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), 2'-ON-methylacetamido (2'-O-NMA), locked nucleic acid (LNA, methylene bridged nucleic acid), ethylene bridged nucleic acid (ENA), and (S)-constrained ethyl bridged nucleic acid (cEt). In some embodiments, the 2'-modified nucleosides described herein are high-affinity modified nucleotides and oligonucleotides comprising 2'-modified nucleosides having increased affinity for a target sequence relative to an unmodified oligonucleotide. Some examples of the structures of 2'-modified nucleosides are provided below:

[0096]

[0097] These examples are shown with phosphate groups, but any internucleoside linkages are contemplated to be between 2'-modified nucleosides.

[0098] Ranges: All ranges provided in this disclosure are inclusive of the endpoints.

[0099] Compound

[0100] Provided herein are compositions comprising multiple complexes.In some embodiments, the complex comprises an antibody covalently linked to one or more oligonucleotides, each oligonucleotide being covalently linked at a bonding site represented by a lysine (K) residue of the antibody.In some embodiments, the antibody comprises a heavy chain containing a heavy chain variable region (VH) and a heavy chain constant region and a light chain containing a light chain variable region (VL) and a light chain constant region.In some embodiments, the antibody light chain constant region in the complex of the composition is independently covalently linked to the oligonucleotide at the following bonding sites: a bonding site represented by K188 (based on Kabat numbering) of the light chain constant region of the antibody and / or a bonding site represented by K190 (based on Kabat numbering).For example, in some embodiments, the bonding site is represented by K188 (based on Kabat numbering) of the light chain constant region of the antibody.In some embodiments, the bonding site is represented by K190 (based on Kabat numbering) of the light chain constant region of the antibody. In some embodiments, the linkage sites are represented by K188 (based on Kabat numbering) and K190 (based on Kabat numbering) of the light chain constant region of the antibody. In some embodiments, at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, 80% to 98%, 80% to 95%, 80% to 90%, 85% to 98%, 85% to 95%, 85% to 90%, 90% to 98%, 90% to 95%, 95% to 97%, or more) of the antibody light chain constant region in the complex of the composition is independently covalently linked to an oligonucleotide at the following linkage sites: the linkage site represented by K188 (based on Kabat numbering) and / or the linkage site represented by K190 (based on Kabat numbering) of the antibody light chain constant region. In some embodiments, the antibody heavy chain constant region in the complex of the composition is independently covalently linked to the oligonucleotide at the following linkage site: the linkage site represented by K213 (based on Kabat numbering) of the heavy chain constant region of the antibody. In some embodiments, about 1% to 15% (e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, or about 15%) of the antibody heavy chain constant region in the complex of the composition is independently covalently linked to the oligonucleotide at the following linkage site: the linkage site represented by K213 (based on Kabat numbering) of the heavy chain constant region of the antibody.

[0101] In some embodiments, the antibody light chain constant region in the complex of the composition is independently covalently linked to the oligonucleotide at the following linkage site: the linkage site represented by the lysine (K) residue in the sequence motif DYEKHKVYA (SEQ ID NO: 27) of the antibody light chain. For example, in some embodiments, the antibody is covalently linked to the oligonucleotide at the following linkage site: the linkage site represented by the K at position 4 of the sequence motif DYEKHKVYA (SEQ ID NO: 27) of the light chain. In some embodiments, the antibody is covalently linked to the oligonucleotide at the following linkage site: the linkage site represented by the K at position 6 of the sequence motif DYEKHKVYA (SEQ ID NO: 27) of the light chain. In some embodiments, the antibody is covalently linked to the oligonucleotide at the following linkage site: the linkage site represented by the K at position 4 of the sequence motif DYEKHKVYA (SEQ ID NO: 27) of the light chain and the linkage site represented by the K at position 6. In some embodiments, at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, 80% to 98%, 80% to 95%, 80% to 90%, 85% to 98%, 85% to 95%, 85% to 90%, 90% to 98%, or 90% to 95%) of the antibody light chains in the complex of the composition are independently covalently linked to the oligonucleotide at the following linkage site: the linkage site represented by a lysine (K) residue in the sequence motif DYEKHKVYA (SEQ ID NO: 27) of the antibody light chain. In some embodiments, the constant region of the antibody heavy chain in the complex of the composition is independently covalently linked to the oligonucleotide at the following linkage site: the linkage site represented by the lysine (K) residue in the sequence motif VNHKPSN (SEQ ID NO: 28) of the antibody heavy chain. In some embodiments, about 1% to 15% (e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, or about 15%) of the antibody heavy chains in the complex of the composition are independently covalently linked to the oligonucleotide at the following linkage site: the linkage site represented by the lysine (K) residue in the motif VNHKPSN (SEQ ID NO: 28) of the antibody heavy chain.

[0102] The complexes described herein generally include a linker that covalently links an antibody described herein (e.g., an anti-TfR1 antibody) to an oligonucleotide (e.g., an oligonucleotide comprising a 5'-XYZ-3' configuration) at a linkage site represented by a lysine (K) residue of the antibody. In some embodiments, in each complex, each oligonucleotide is covalently linked at a linkage site represented by a different lysine (K) residue of the antibody. The linker includes at least one covalent bond.

[0103] In some embodiments, the complex described herein comprises formula (I): [R 1 ] n1 -R 2 The structure of each R 1 independently comprises a compound comprising an oligonucleotide (eg, an oligonucleotide comprising a 5'-XYZ-3' configuration), and R 2 comprising an antibody (eg, an anti-TfR1 antibody), and wherein in each complex, n1 is independently an integer (eg, 1 or greater) representing the number of R 1 In some embodiments, each R 1 In some embodiments, each R 1 In some embodiments, R 2 The antibody (e.g., an anti-TfR1 antibody) comprises a heavy chain comprising a heavy chain variable region (VH) and a heavy chain constant region and a light chain comprising a light chain variable region (VL) and a light chain constant region. In some embodiments, each R in the complex 1 Independently with R 2 Different amino acid residues (e.g., lysine) are covalently linked.

[0104] In some embodiments, each R 1 The binding site represented by the lysine (K) residue of the antibody is 2Covalently linked. In some embodiments, the linkage site is represented by K188 (based on Kabat numbering) of the light chain constant region of the antibody. In some embodiments, the linkage site is represented by K190 (based on Kabat numbering) of the light chain constant region of the antibody. In some embodiments, the linkage site is represented by K188 (based on Kabat numbering) and K190 (based on Kabat numbering) of the light chain constant region of the antibody. In some embodiments, at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, 80% to 98%, 80% to 95%, 80% to 90%, 85% to 98%, 85% to 95%, 85% to 90%, 90% to 98%, 90% to 95%, 95% to 97%, or more) of the antibody light chain constant regions in the complex of the composition are independently bound to R at the following linkage sites: 1 Covalently linked: a linkage site represented by K188 (based on Kabat numbering) and / or a linkage site represented by K190 (based on Kabat numbering) of the light chain constant region of the antibody. In some embodiments, about 1% to 15% (e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, or about 15%) of the antibody heavy chain constant region in the complex of the composition independently binds to R at the following linkage sites: 1 Covalent Linkage: The linkage site is represented by K213 (based on Kabat numbering) of the heavy chain constant region of the antibody.

[0105] In some embodiments, each R 1 The binding site represented by the lysine (K) residue of the antibody is 2Covalently linked. In some embodiments, the linkage site is represented by K at position 4 of the sequence motif DYEKHKVYA (SEQ ID NO: 27) of the antibody light chain. In some embodiments, the linkage site is represented by K at position 6 of the sequence motif DYEKHKVYA (SEQ ID NO: 27) of the antibody light chain. In some embodiments, the linkage site is represented by K at position 4 and K at position 6 of the sequence motif DYEKHKVYA (SEQ ID NO: 27) of the antibody light chain. In some embodiments, at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, 80% to 98%, 80% to 95%, 80% to 90%, 85% to 98%, 85% to 95%, 85% to 90%, 90% to 98%, 90% to 95%, 95% to 97%, or more) of the antibody light chains in the complex of the composition independently bind to R at the following linkage sites: 1 Covalently linked: A linkage site represented by a lysine (K) residue in the sequence motif DYEKHKVYA (SEQ ID NO: 27) of an antibody light chain. In some embodiments, about 1% to 15% (e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, or about 15%) of the antibody heavy chains in the complex of the composition are independently linked to R at the following linkage sites: 1 Covalent attachment: The linkage site is represented by a lysine (K) residue in the sequence motif VNHKPSN (SEQ ID NO: 28) of the antibody heavy chain. 2 Contains anti-TfR1 Fab.

[0106] In some embodiments, in each complex, n1 is independently an integer (e.g., 1 or greater). In some embodiments, the antibody comprises a sequence as shown in Table 2. For example, in some embodiments, the antibody comprises: a heavy chain complementary determining region 1 (CDR-H1) comprising a sequence as shown in SEQ ID NO: 1, 7, or 12, a heavy chain complementary determining region 2 (CDR-H2) comprising a sequence as shown in SEQ ID NO: 2, 8, or 13, a heavy chain complementary determining region 3 (CDR-H3) comprising a sequence as shown in SEQ ID NO: 3, 9, or 14; and / or comprises: a light chain complementary determining region 1 (CDR-L1) comprising a sequence as shown in SEQ ID NO: 4, 10, or 15, a light chain complementary determining region 2 (CDR-L2) comprising a sequence as shown in SEQ ID NO: 5 or 11, and a light chain complementary determining region 3 (CDR-L3) comprising a sequence as shown in SEQ ID NO: 6 or 16. In some embodiments, the antibody comprises a heavy chain variable region (VH) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 17, and / or comprises a light chain variable region (VL) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 18. In some embodiments, the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 17 and / or comprises a VL comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, the antibody comprises a heavy chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 19, and / or comprises a light chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 20. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and / or comprises a light chain comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the antibody is a Fab fragment, a full length IgG, a Fab' fragment, a F(ab')2 fragment, a scFv, or a Fv. In some embodiments, the antibody is a Fab fragment.

[0107] In some embodiments, a plurality of different complexes comprise a common targeting agent (e.g., an antibody) and a common oligonucleotide (e.g., an oligonucleotide comprising a 5'-XYZ-3' configuration). In some such embodiments, the different complex types are characterized by having different numbers of oligonucleotides covalently attached to the antibody. For example, in some embodiments, the composition comprises a plurality of complex types, wherein each complex type comprises formula (I): [R 1 ] n1 -R 2 The structure of each R1 independently comprises a compound comprising an oligonucleotide (eg, an oligonucleotide comprising a 5'-XYZ-3' configuration) and R 2 An antibody (e.g., an anti-TfR1 antibody) is included, wherein the antibody (e.g., an anti-TfR1 antibody) includes a heavy chain containing a heavy chain variable region (VH) and a heavy chain constant region and a light chain containing a light chain variable region (VL) and a light chain constant region. In some embodiments, the value of n1 of each or any complex (e.g., any complex of any one of the compositions or methods disclosed herein) is an integer up to the number of amino acid residues (e.g., the number of lysine residues) desired or targeted for conjugation in the antibody. In some embodiments, in each complex, the value of n1 is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27. In some embodiments, in each complex, the value of n1 is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, and 26. In some embodiments, in each complex, the value of n1 is independently 1 to 27, 1 to 26, 1 to 10, 1 to 5, or 1 to 3. In some embodiments, the average value of n1 of the complexes of the composition is 1 to 5 (e.g., 1 to 5, 1 to 4, 1 to 3, 3 to 5, or 1 to 2). In some embodiments, the compositions described herein comprise a complex comprising formula (I): [R 1 ] n1 -R 2 wherein n1 is 0. In some embodiments, the average value of n1 of the complexes of the composition is 0.5 to 5 (e.g., 0.5 to 5, 1 to 5, 1 to 4, 1 to 3, 3 to 5, 0.5 to 4, 0.5 to 3, 0.5 to 2, 0.5 to 1.5, 0.5 to 1, 0.7 to 1.5, 1 to 1.6, 1 to 1.5, 1 to 1.4, 1 to 1.3, 1 to 1.2, 1.1 to 1.5, 0.8 to 2, 0.8 to 1.5, 0.8 to 1.3, 0.8 to 1.2, 0.8 to 1.1, 0.9 to 3, 0.9 to 2, 0.9 to 1.8, 0.9 to 1.6, 0.9 to 1.5, 0.9 to 1.4, 0.9 to 1.3, or 0.9 to 1.2). In some embodiments, in each complex type, n1 is independently an integer of 1 or greater, which represents the number of R 1 The invention relates to a method for preparing an aqueous solution of the present invention and wherein different composite types of the composition are characterized by having different n1 values ​​(e.g., n1 values ​​of 1 to 27, 1 to 26, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, or 1 to 3).

[0108] In some embodiments, the compositions described herein include antibodies that are not conjugated to oligonucleotides (e.g., in trace amounts) and antibodies that are conjugated to one or more oligonucleotides. In some embodiments, antibodies that are not conjugated to oligonucleotides may be referred to as comprising formula (I): 1 ] n1 -R 2 , wherein n1 is zero. Thus, in some embodiments, the composition for administration to a subject in the methods described herein comprises a compound of formula (I): [R 1 ] n1 -R 2 A compound (eg, a complex) of the structure wherein each R 1 independently comprising a group containing an oligonucleotide, R 2 wherein n1 is independently an integer of zero or greater, which reflects the number of R 1 In some embodiments, the composition wherein n1 is 1 or greater has formula (I): [R 1 ] n1 -R 2 The fraction of compounds containing this structure in which n1 is zero in the composition is less than 10%, less than 5%, less than 1%, less than 0.5%, less than 0.1%, less than 0.05% or less than 0.01% compared to all compounds of the structure. Thus, in some embodiments, the average value of n1 of the complexes in the compositions disclosed herein is from 0.5 to 5 (e.g., from 0.5 to 5, from 1 to 5, from 1 to 4, from 1 to 3, from 3 to 5, from 0.5 to 4, from 0.5 to 3, from 0.5 to 2, from 0.5 to 1.5, from 0.5 to 1, from 0.7 to 1.5, from 1 to 1.6, from 1 to 1.5, from 1 to 1.4, from 1 to 1.3, from 1 to 1.2, from 1.1 to 1.5, from 0.8 to 2, from 0.8 to 1.5, from 0.8 to 1.3, from 0.8 to 1.2, from 0.8 to 1.1, from 0.9 to 3, from 0.9 to 2, from 0.9 to 1.8, from 0.9 to 1.6, from 0.9 to 1.5, from 0.9 to 1.4, from 0.9 to 1.3, or from 0.9 to 1.2).

[0109] In some embodiments, the complex described herein comprises formula (I): [R 1 ] n1 -R 2 The structure of each R 1 Independently comprising groups of formula (Ia):

[0110]

[0111] Where R 3comprising an oligonucleotide, such as an oligonucleotide comprising a 5'-XYZ-3' configuration; R 2 An antibody comprising a heavy chain comprising a heavy chain variable region (VH) and a heavy chain constant region and a light chain comprising a light chain variable region (VL) and a light chain constant region, wherein in each complex, n1 is independently an integer (e.g., an integer of 1 or greater), which represents the number of R 1 The number of instances, and each R 1 At the connection point A, the binding site is represented by a lysine (K) residue of the antibody and R 2 In some embodiments, the linkage site is represented by K188 (based on Kabat numbering) of the light chain constant region of the antibody. In some embodiments, the linkage site is represented by K190 (based on Kabat numbering) of the light chain constant region of the antibody. In some embodiments, the linkage site is represented by K188 (based on Kabat numbering) and K190 (based on Kabat numbering) of the light chain constant region of the antibody. In some embodiments, in a composition comprising a plurality of complexes described herein, at least 80% of the antibody light chain constant regions in the complexes of the composition are independently linked to R at the following linkage sites: 1 Covalently linked: The linkage site represented by K188 (based on Kabat numbering) and / or the linkage site represented by K190 (based on Kabat numbering) of the light chain constant region of the antibody. 2 The invention comprises an anti-TfR1 antibody (eg, Fab) comprising a sequence as shown in Table 2. For example, in some embodiments, R 2 The invention comprises an anti-TfR1 antibody (e.g., Fab), wherein the anti-TfR1 antibody (e.g., Fab) comprises a heavy chain complementary determining region 1 (CDR-H1) comprising a sequence as shown in SEQ ID NO: 1, 7 or 12, a heavy chain complementary determining region 2 (CDR-H2) comprising a sequence as shown in SEQ ID NO: 2, 8 or 13, and a heavy chain complementary determining region 3 (CDR-H3) comprising a sequence as shown in SEQ ID NO: 3, 9 or 14; and / or comprises a light chain complementary determining region 1 (CDR-L1) comprising a sequence as shown in SEQ ID NO: 4, 10 or 15, a light chain complementary determining region 2 (CDR-L2) comprising a sequence as shown in SEQ ID NO: 5 or 11, and a light chain complementary determining region 3 (CDR-L3) comprising a sequence as shown in SEQ ID NO: 6 or 16. In some embodiments, R 2An anti-TfR1 antibody (e.g., Fab) comprising a heavy chain variable region (VH) comprising an amino acid sequence having at least 85% (e.g., at least 95%) identity to SEQ ID NO: 17 and / or comprising a light chain variable region (VL) comprising an amino acid sequence having at least 85% (e.g., at least 95%) identity to SEQ ID NO: 18. In some embodiments, R 2 The invention comprises an anti-TfR1 antibody (eg, Fab) comprising a VH and / or comprising a VL, wherein the VH comprises the amino acid sequence of SEQ ID NO: 17 and the VL comprises the amino acid sequence of SEQ ID NO: 18. In some embodiments, R 2 An anti-TfR1 antibody (e.g., Fab) comprising a heavy chain comprising an amino acid sequence having at least 85% (e.g., at least 95%) identity to SEQ ID NO: 19 and / or comprising a light chain comprising an amino acid sequence having at least 85% (e.g., at least 95%) identity to SEQ ID NO: 20. In some embodiments, R 2 The invention comprises an anti-TfR1 antibody (eg, Fab) comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and / or comprising a light chain comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, R 2 The antibody comprises a Fab fragment, a full-length IgG, a Fab' fragment, a F(ab')2 fragment, a scFv or a Fv. In some embodiments, R 2 In some embodiments, R 3 An oligonucleotide comprising a nucleobase sequence of CAGCGCCCACCAGUCA (SEQ ID NO: 21). 3 An oligonucleotide comprising a structure comprising +C*+A*oG*oC*dG*dC*dC*dC*dA*dC*dC*dA*oG*oU*+C*+A (SEQ ID NO: 21), wherein +N represents LNA (2'-4' methylene bridge) ribonucleoside, dN represents 2'-deoxyribonucleoside, oN represents 2'-MOE modified ribonucleoside, oC represents 5-methyl-2'-MOE-cytidine, +C represents 5-methyl-2'-4'-bicyclo-cytidine (2'-4 methylene bridge), oU represents 5-methyl-2'-MOE-uridine, and * represents a phosphorothioate internucleoside linkage. In some embodiments, R 2 Contains Fab, and each R 1 At the attachment point A, the linkage site is represented by a lysine (K) residue of the Fab and R 2covalently linked, and wherein, in a composition comprising a plurality of complexes described herein, at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, 80% to 98%, 80% to 95%, 80% to 90%, 85% to 98%, 85% to 95%, 85% to 90%, 90% to 98%, 90% to 95%, 95% to 97%, or more) of the light chain constant regions of the Fabs of the complexes in the composition independently bind to R 1 Covalently linked: a linking site represented by K188 (based on Kabat numbering) of the light chain constant region of an antibody and / or a linking site represented by K190 (based on Kabat numbering). In some embodiments, in a composition comprising a plurality of complexes described herein, about 1% to 15% (e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, or about 15%) of the antibody heavy chain constant region in the complex of the composition is independently covalently linked to an oligonucleotide at the following linking site: a linking site represented by K213 (based on Kabat numbering) of the heavy chain constant region of an antibody. In some embodiments, the composition described herein comprises a compound comprising formula (I): [R 1 ] n1 -R 2 In some embodiments, n1 is independently an integer (eg, an integer from 1 to 27, 1 to 26, 1 to 10, 1 to 5, or 1 to 3) in each complex. In some embodiments, in a composition comprising a plurality of complexes described herein, the average value of n1 of the complexes of the composition is from 0.5 to 5 (e.g., from 0.5 to 5, from 1 to 5, from 1 to 4, from 1 to 3, from 3 to 5, from 0.5 to 4, from 0.5 to 3, from 0.5 to 2, from 0.5 to 1.5, from 0.5 to 1, from 0.7 to 1.5, from 1 to 1.6, from 1 to 1.5, from 1 to 1.4, from 1 to 1.3, from 1 to 1.2, from 1.1 to 1.5, from 0.8 to 2, from 0.8 to 1.5, from 0.8 to 1.3, from 0.8 to 1.2, from 0.8 to 1.1, from 0.9 to 3, from 0.9 to 2, from 0.9 to 1.8, from 0.9 to 1.6, from 0.9 to 1.5, from 0.9 to 1.4, from 0.9 to 1.3, or from 0.9 to 1.2).

[0112] In some embodiments, the complex described herein comprises formula (I): [R 1 ] n1 -R 2The structure of each R 1 Containing a group of formula (Ib):

[0113]

[0114] wherein +N represents an LNA (2'-4' methylene bridge) ribonucleoside, dN represents a 2'-deoxyribonucleoside, oN represents a 2'-O-methoxyethyl (MOE) modified ribonucleoside, oC represents 5-methyl-2'-MOE-cytidine, +C represents 5-methyl-2'-4'-bicyclo-cytidine (2'-4' methylene bridge), oU represents 5-methyl-2'-MOE-uridine, * represents a phosphorothioate internucleoside linkage, and wherein the oligonucleotide comprises a nucleobase sequence of CAGCGCCCACCAGUCA (SEQ ID NO: 21); R 2 An antibody comprising a heavy chain comprising a heavy chain variable region (VH) and a heavy chain constant region and a light chain comprising a light chain variable region (VL) and a light chain constant region, wherein in each complex, n1 is independently an integer (e.g., an integer of 1 or greater), which represents the number of R 1 The number of instances, and each R 1 At the connection point A, the binding site is represented by a lysine (K) residue of the antibody and R 2 Covalently linked. In some embodiments, the linkage site is represented by K188 (based on Kabat numbering) of the light chain constant region of the antibody. In some embodiments, the linkage site is represented by K190 (based on Kabat numbering) of the light chain constant region of the antibody. In some embodiments, the linkage site is represented by K188 (based on Kabat numbering) and K190 (based on Kabat numbering) of the light chain constant region of the antibody. In some embodiments, in a composition comprising a plurality of complexes described herein, at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, 80% to 98%, 80% to 95%, 80% to 90%, 85% to 98%, 85% to 95%, 85% to 90%, 90% to 98%, 90% to 95%, 95% to 97%, or more) of the antibody light chain constant regions in the complexes of the composition independently bind to R at the following linkage sites: 1 Covalently linked: The linkage site represented by K188 (based on Kabat numbering) and / or the linkage site represented by K190 (based on Kabat numbering) of the light chain constant region of the antibody. 2The invention comprises an anti-TfR1 antibody (eg, Fab) comprising a sequence as shown in Table 2. For example, in some embodiments, R 2 The invention comprises an anti-TfR1 antibody (e.g., Fab), wherein the anti-TfR1 antibody (e.g., Fab) comprises a heavy chain complementary determining region 1 (CDR-H1) comprising a sequence as shown in SEQ ID NO: 1, 7 or 12, a heavy chain complementary determining region 2 (CDR-H2) comprising a sequence as shown in SEQ ID NO: 2, 8 or 13, and a heavy chain complementary determining region 3 (CDR-H3) comprising a sequence as shown in SEQ ID NO: 3, 9 or 14; and / or comprises a light chain complementary determining region 1 (CDR-L1) comprising a sequence as shown in SEQ ID NO: 4, 10 or 15, a light chain complementary determining region 2 (CDR-L2) comprising a sequence as shown in SEQ ID NO: 5 or 11, and a light chain complementary determining region 3 (CDR-L3) comprising a sequence as shown in SEQ ID NO: 6 or 16. In some embodiments, R 2 An anti-TfR1 antibody (e.g., Fab) comprising a heavy chain variable region (VH) comprising an amino acid sequence having at least 85% (e.g., at least 95%) identity to SEQ ID NO: 17 and / or comprising a light chain variable region (VL) comprising an amino acid sequence having at least 85% (e.g., at least 95%) identity to SEQ ID NO: 18. In some embodiments, R 2 The invention comprises an anti-TfR1 antibody (eg, Fab) comprising a VH and / or comprising a VL, wherein the VH comprises the amino acid sequence of SEQ ID NO: 17 and the VL comprises the amino acid sequence of SEQ ID NO: 18. In some embodiments, R 2 An anti-TfR1 antibody (e.g., Fab) comprising a heavy chain comprising an amino acid sequence having at least 85% (e.g., at least 95%) identity to SEQ ID NO: 19 and / or comprising a light chain comprising an amino acid sequence having at least 85% (e.g., at least 95%) identity to SEQ ID NO: 20. In some embodiments, R 2 The invention comprises an anti-TfR1 antibody (eg, Fab) comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and / or comprising a light chain comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, R 2 The antibody comprises a Fab fragment, a full-length IgG, a Fab' fragment, a F(ab')2 fragment, a scFv or a Fv. In some embodiments, R 2The antibody comprises a Fab fragment. In some embodiments, in each complex, n1 is independently an integer (e.g., an integer from 1 to 27, 1 to 26, 1 to 10, 1 to 5, or 1 to 3). In some embodiments, R 2 Contains Fab, and each R 1 At the attachment point A, the linkage site is represented by a lysine (K) residue of the Fab and R 2 covalently linked, and wherein, in a composition comprising a plurality of complexes described herein, at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, 80% to 98%, 80% to 95%, 80% to 90%, 85% to 98%, 85% to 95%, 85% to 90%, 90% to 98%, or 90% to 95%) of the light chain constant regions of the Fabs of the complexes in the composition independently bind to R at the following linkage sites: 1 Covalently linked: the linkage site represented by K188 (based on Kabat numbering) of the light chain constant region of Fab and / or the linkage site represented by K190 (based on Kabat numbering). In some embodiments, in a composition comprising a plurality of complexes described herein, about 1% to 15% (e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, or about 15%) of the heavy chain constant regions of the Fabs of the complexes in the composition are independently linked to R at the following linkage sites: 1 Covalent attachment: The bonding site represented by K213 (based on Kabat numbering) of the heavy chain constant region of Fab. In some embodiments, the compositions described herein comprise a compound comprising formula (I): [R 1 ] n1 -R 2, wherein n1 is 0. In some embodiments, in a composition comprising a plurality of complexes described herein, the average value of n1 of the complexes of the composition is 0.5 to 5 (e.g., 0.5 to 5, 1 to 5, 1 to 4, 1 to 3, 3 to 5, 0.5 to 4, 0.5 to 3, 0.5 to 2, 0.5 to 1.5, 0.5 to 1, 0.7 to 1.5, 1 to 1.6, 1 to 1.5, 1 to 1.4, 1 to 1.3, 1 to 1.2, 1.1 to 1.5, 0.8 to 2, 0.8 to 1.5, 0.8 to 1.3, 0.8 to 1.2, 0.8 to 1.1, 0.9 to 3, 0.9 to 2, 0.9 to 1.8, 0.9 to 1.6, 0.9 to 1.5, 0.9 to 1.4, 0.9 to 1.3, or 0.9 to 1.2).

[0115] In some embodiments, the complex described herein comprises formula (I): [R 1 ] n1 -R 2 The structure of each R 1 Containing a group of formula (Ic):

[0116]

[0117] R 2 An antibody comprising a heavy chain comprising a heavy chain variable region (VH) and a heavy chain constant region and a light chain comprising a light chain variable region (VL) and a light chain constant region, wherein in each complex, n1 is independently an integer (e.g., an integer of 1 or greater), which represents the number of R 1 The number of instances of R 1 At the connection point A, the binding site is represented by a lysine (K) residue of the antibody and R 2Covalently linked. In some embodiments, the linkage site is represented by K188 (based on Kabat numbering) of the light chain constant region of the antibody. In some embodiments, the linkage site is represented by K190 (based on Kabat numbering) of the light chain constant region of the antibody. In some embodiments, the linkage site is represented by K188 (based on Kabat numbering) and K190 (based on Kabat numbering) of the light chain constant region of the antibody. In some embodiments, in a composition comprising a plurality of complexes described herein, at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, 80% to 98%, 80% to 95%, 80% to 90%, 85% to 98%, 85% to 95%, 85% to 90%, 90% to 98%, 90% to 95%, 95% to 97%, or more) of the antibody light chain constant regions in the complexes of the composition independently bind to R at the following linkage sites: 1 Covalently linked: The linkage site represented by K188 (based on Kabat numbering) and / or the linkage site represented by K190 (based on Kabat numbering) of the light chain constant region of the antibody. 2 The invention comprises an anti-TfR1 antibody (eg, Fab) comprising a sequence as shown in Table 2. For example, in some embodiments, R 2 The invention comprises an anti-TfR1 antibody (e.g., Fab), wherein the anti-TfR1 antibody (e.g., Fab) comprises a heavy chain complementary determining region 1 (CDR-H1) comprising a sequence as shown in SEQ ID NO: 1, 7 or 12, a heavy chain complementary determining region 2 (CDR-H2) comprising a sequence as shown in SEQ ID NO: 2, 8 or 13, and a heavy chain complementary determining region 3 (CDR-H3) comprising a sequence as shown in SEQ ID NO: 3, 9 or 14; and / or comprises a light chain complementary determining region 1 (CDR-L1) comprising a sequence as shown in SEQ ID NO: 4, 10 or 15, a light chain complementary determining region 2 (CDR-L2) comprising a sequence as shown in SEQ ID NO: 5 or 11, and a light chain complementary determining region 3 (CDR-L3) comprising a sequence as shown in SEQ ID NO: 6 or 16. In some embodiments, R 2An anti-TfR1 antibody (e.g., Fab) comprising a heavy chain variable region (VH) comprising an amino acid sequence having at least 85% (e.g., at least 95%) identity to SEQ ID NO: 17 and / or comprising a light chain variable region (VL) comprising an amino acid sequence having at least 85% (e.g., at least 95%) identity to SEQ ID NO: 18. In some embodiments, R 2 The invention comprises an anti-TfR1 antibody (eg, Fab) comprising a VH and / or comprising a VL, wherein the VH comprises the amino acid sequence of SEQ ID NO: 17 and the VL comprises the amino acid sequence of SEQ ID NO: 18. In some embodiments, R 2 An anti-TfR1 antibody (e.g., Fab) comprising a heavy chain comprising an amino acid sequence having at least 85% (e.g., at least 95%) identity to SEQ ID NO: 19 and / or comprising a light chain comprising an amino acid sequence having at least 85% (e.g., at least 95%) identity to SEQ ID NO: 20. In some embodiments, R 2 The invention comprises an anti-TfR1 antibody (eg, Fab) comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and / or comprising a light chain comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, R 2 The antibody comprises a Fab fragment, a full-length IgG, a Fab' fragment, a F(ab')2 fragment, a scFv or a Fv. In some embodiments, R 2 The antibody comprises a Fab fragment. In some embodiments, in each complex, n1 is independently an integer (e.g., an integer from 1 to 27, 1 to 26, 1 to 10, 1 to 5, or 1 to 3). In some embodiments, R 2 Contains Fab, and each R 1 At the attachment point A, the linkage site is represented by a lysine (K) residue of the Fab and R 2covalently linked, and wherein, in a composition comprising a plurality of complexes described herein, at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, 80% to 98%, 80% to 95%, 80% to 90%, 85% to 98%, 85% to 95%, 85% to 90%, 90% to 98%, 90% to 95%, 95% to 97%, or more) of the light chain constant regions of the Fabs of the complexes in the composition independently bind to R 1 Covalently linked: a linkage site represented by K188 (based on Kabat numbering) of the light chain constant region of Fab and / or a linkage site represented by K190 (based on Kabat numbering). In some embodiments, in a composition comprising a plurality of complexes described herein, about 1% to 15% (e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, or about 15%) of the heavy chain constant region of the Fab of the complex in the composition is independently covalently linked to the oligonucleotide at the following linkage site: a linkage site represented by K213 (based on Kabat numbering) of the heavy chain constant region of Fab. In some embodiments, the composition described herein comprises a compound comprising formula (I): [R 1 ] n1 -R 2 , wherein n1 is 0. In some embodiments, in a composition comprising a plurality of complexes described herein, the average value of n1 of the complexes of the composition is 0.5 to 5 (e.g., 0.5 to 5, 1 to 5, 1 to 4, 1 to 3, 3 to 5, 0.5 to 4, 0.5 to 3, 0.5 to 2, 0.5 to 1.5, 0.5 to 1, 0.7 to 1.5, 1 to 1.6, 1 to 1.5, 1 to 1.4, 1 to 1.3, 1 to 1.2, 1.1 to 1.5, 0.8 to 2, 0.8 to 1.5, 0.8 to 1.3, 0.8 to 1.2, 0.8 to 1.1, 0.9 to 3, 0.9 to 2, 0.9 to 1.8, 0.9 to 1.6, 0.9 to 1.5, 0.9 to 1.4, 0.9 to 1.3, or 0.9 to 1.2).

[0118] In some embodiments, the complex described herein comprises a structure of Formula (Id):

[0119]

[0120] wherein +N represents LNA (2'-4' methylene bridge) ribonucleoside, dN represents 2'-deoxyribonucleoside, oN represents 2'-O-methoxyethyl (MOE) modified ribonucleoside, oC represents 5-methyl-2'-MOE-cytidine, +C represents 5-methyl-2'-4'-bicyclo-cytidine (2'-4' methylene bridge), oU represents 5-methyl-2'-MOE-uridine, * represents phosphorothioate internucleoside linkage, and wherein the oligonucleotide comprises a nucleobase sequence of CAGCGCCCACCAGUCA (SEQ ID NO: 21); wherein R 2Comprising an anti-TfR1 antibody (e.g., Fab), wherein the anti-TfR1 antibody (e.g., Fab) comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 selected from Table 2, optionally wherein the anti-TfR1 antibody (e.g., Fab) comprises a VH comprising the amino acid sequence of SEQ ID NO: 17 and a VL comprising the amino acid sequence of SEQ ID NO: 18, and optionally wherein the anti-TfR1 antibody (e.g., Fab) comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and a light chain comprising the amino acid sequence of SEQ ID NO: 20; and wherein in each complex, n1 is independently an integer (e.g., 1 or greater), which represents the number of instances of the group enclosed by square brackets, wherein each instance of the group enclosed by square brackets is covalently linked to a bonding site represented by a lysine (K) residue of the antibody (e.g., Fab). In some embodiments, the linkage site is represented by K188 (based on Kabat numbering) of the light chain constant region of the antibody. In some embodiments, the linkage site is represented by K190 (based on Kabat numbering) of the light chain constant region of the antibody. In some embodiments, the linkage site is represented by K188 (based on Kabat numbering) and K190 (based on Kabat numbering) of the light chain constant region of the antibody. In some embodiments, in a composition comprising a plurality of complexes described herein, at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, ... In some embodiments, R is independently covalently linked to the group enclosed by the square brackets in Formula (Id) at the following linkage sites: the linkage site represented by K188 (based on Kabat numbering) and / or the linkage site represented by K190 (based on Kabat numbering) of the light chain constant region of the antibody. 2The invention comprises an antibody (e.g., Fab) comprising a heavy chain complementary determining region 1 (CDR-H1) comprising a sequence as shown in SEQ ID NO: 1, 7 or 12, a heavy chain complementary determining region 2 (CDR-H2) comprising a sequence as shown in SEQ ID NO: 2, 8 or 13, and a heavy chain complementary determining region 3 (CDR-H3) comprising a sequence as shown in SEQ ID NO: 3, 9 or 14; and / or comprising a light chain complementary determining region 1 (CDR-L1) comprising a sequence as shown in SEQ ID NO: 4, 10 or 15, a light chain complementary determining region 2 (CDR-L2) comprising a sequence as shown in SEQ ID NO: 5 or 11, and a light chain complementary determining region 3 (CDR-L3) comprising a sequence as shown in SEQ ID NO: 6 or 16. In some embodiments, R 2 An antibody (e.g., Fab) comprising a heavy chain variable region (VH) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 17 and / or comprising a light chain variable region (VL) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 18. In some embodiments, R 2 The invention comprises an antibody (e.g., Fab) comprising a VH comprising the amino acid sequence of SEQ ID NO: 17 and / or comprising a VL comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, R 2 The invention comprises an antibody (e.g., Fab) comprising a heavy chain comprising an amino acid sequence that is at least 85% (e.g., at least 95%) identical to SEQ ID NO: 19 and / or comprising a light chain comprising an amino acid sequence that is at least 85% (e.g., at least 95%) identical to SEQ ID NO: 20. In some embodiments, R 2 The invention comprises an antibody (e.g., Fab) comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and / or comprising a light chain comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, in each complex, n1 is independently an integer (e.g., an integer from 1 to 27, 1 to 26, 1 to 10, 1 to 5, or 1 to 3). In some embodiments, R 2The invention relates to a composition comprising an antibody (e.g., Fab) covalently linked via a linkage site represented by a lysine (K) residue of the antibody (e.g., Fab), and wherein, in a composition comprising a plurality of complexes described herein, at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120%, at least 121%, at least 122%, at least 123%, at least 124%, at least 125%, at least 126%, at least 127%, at least 128%, at least 129%, at least 130%, at least 131%, at least 132%, at least 133%, at least 134%, at least 135%, at least 136%, at least 137%, at least 138%, at least 139 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, 80% to 98%, 80% to 95%, 80% to 90%, 85% to 98%, 85% to 95%, 85% to 90%, 90% to 98%, or 90% to 95%) is independently covalently linked to the group enclosed by the square brackets in Formula (Id) at the following linkage sites: the linkage site represented by K188 (based on Kabat numbering) and / or the linkage site represented by K190 (based on Kabat numbering) of the light chain constant region of the antibody. In some embodiments, in a composition comprising a plurality of complexes described herein, about 1% to 15% (e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, or about 15%) of the antibody heavy chain constant region in the complex of the composition is independently covalently linked to the group included by the square brackets in Formula (Id) at the following linkage site: the linkage site represented by K213 (based on Kabat numbering) of the heavy chain constant region of the antibody. In some embodiments, the composition described herein comprises a complex in which the value of n1 is 0. In some embodiments, in a composition comprising a plurality of complexes described herein, the average value of n1 of the complexes of the composition is from 0.5 to 5 (e.g., from 0.5 to 5, from 1 to 5, from 1 to 4, from 1 to 3, from 3 to 5, from 0.5 to 4, from 0.5 to 3, from 0.5 to 2, from 0.5 to 1.5, from 0.5 to 1, from 0.7 to 1.5, from 1 to 1.6, from 1 to 1.5, from 1 to 1.4, from 1 to 1.3, from 1 to 1.2, from 1.1 to 1.5, from 0.8 to 2, from 0.8 to 1.5, from 0.8 to 1.3, from 0.8 to 1.2, from 0.8 to 1.1, from 0.9 to 3, from 0.9 to 2, from 0.9 to 1.8, from 0.9 to 1.6, from 0.9 to 1.5, from 0.9 to 1.4, from 0.9 to 1.3, or from 0.9 to 1.2).

[0121] In some embodiments, the complex described herein comprises a structure of Formula (A):

[0122]

[0123] (A), wherein y is 0 to 15 (e.g., 3), and z is 0 to 15 (e.g., 4). In some embodiments, the antibody is an anti-TfR1 antibody (e.g., an anti-TfR1 antibody provided in Table 2), comprising a heavy chain containing a heavy chain variable region (VH) and a heavy chain constant region of each antibody and a light chain containing a light chain variable region (VL) and a light chain constant region. In some embodiments, the oligonucleotide comprises a 5'-XYZ-3' configuration and comprises the nucleotide sequence of SEQ ID NO: 21. In some embodiments, the amide adjacent to the anti-TfR1 antibody shown in the structure is produced by reaction with an amine (e.g., lysine ε amine) of the anti-TfR1 antibody. In some embodiments, the complex described herein comprises an anti-TfR1 Fab covalently linked to the 5' end of the oligonucleotide via a bonding site represented by a lysine (K) residue of the antibody. In some embodiments, the bonding site is represented by K188 (based on Kabat numbering) of the light chain constant region of the antibody. In some embodiments, the linkage site is represented by K190 (based on Kabat numbering) of the light chain constant region of the antibody. In some embodiments, the linkage site is represented by K188 (based on Kabat numbering) and K190 (based on Kabat numbering) of the light chain constant region of the antibody. In some embodiments, in a composition comprising a plurality of complexes described herein, at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%) of the antibody light chain constant region in the complex of the composition. , at least 99%, 80% to 98%, 80% to 95%, 80% to 90%, 85% to 98%, 85% to 95%, 85% to 90%, 90% to 98%, 90% to 95%, 95% to 97%, or more) is independently covalently linked to the oligonucleotide at the following linkage sites: the linkage site represented by K188 (based on Kabat numbering) and / or the linkage site represented by K190 (based on Kabat numbering) of the light chain constant region of the antibody. In some embodiments, in a composition comprising a plurality of complexes described herein, about 1% to 15% (e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, or about 15%) of the antibody heavy chain constant region in the complex of the composition is independently covalently linked to an oligonucleotide at the following linkage site: the linkage site represented by K213 (based on Kabat numbering) of the heavy chain constant region of the antibody. In some embodiments, the anti-TfR1 antibody comprises a sequence as shown in Table 2.For example, in some embodiments, the anti-TfR1 antibody comprises a heavy chain complementary determining region 1 (CDR-H1) comprising a sequence as shown in SEQ ID NO: 1, 7 or 12, a heavy chain complementary determining region 2 (CDR-H2) comprising a sequence as shown in SEQ ID NO: 2, 8 or 13, and a heavy chain complementary determining region 3 (CDR-H3) comprising a sequence as shown in SEQ ID NO: 3, 9 or 14; and / or comprises a light chain complementary determining region 1 (CDR-L1) comprising a sequence as shown in SEQ ID NO: 4, 10 or 15, a light chain complementary determining region 2 (CDR-L2) comprising a sequence as shown in SEQ ID NO: 5 or 11, and a light chain complementary determining region 3 (CDR-L3) comprising a sequence as shown in SEQ ID NO: 6 or 16. In some embodiments, the antibody comprises a heavy chain variable region (VH) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 17, and / or a light chain variable region (VL) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 18. In some embodiments, the anti-TfR1 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 17 and / or a VL comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, the anti-TfR1 antibody comprises a heavy chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 19, and / or a light chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 20. In some embodiments, the anti-TfR1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and / or a light chain comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the antibody is a Fab fragment, a full length IgG, a Fab' fragment, a F(ab')2 fragment, a scFv, or a Fv.

[0124] In some embodiments, for example, in embodiments in which the complex described herein comprises a structure of formula (A) or comprises a structure comprising a group of formula (la), (lb), (lc) or (Id), the linkage site is optionally represented by K at position 4 of the sequence motif DYEKHKVYA (SEQ ID NO: 27) of the antibody light chain of the complex described herein. In some embodiments, the linkage site is optionally represented by K at position 6 of the sequence motif DYEKHKVYA (SEQ ID NO: 27) of the antibody light chain of the complex described herein. In some embodiments, the linkage site is optionally represented by K at position 4 and / or K at position 6 of the sequence motif DYEKHKVYA (SEQ ID NO: 27) of the antibody light chain of the complex described herein. In some embodiments, in a composition comprising a plurality of complexes described herein, at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, 80% to 98%, 80% to 95%, 80% to 90%, 85% to 98%, 85% to 95%, 85% to 90%, 90% to 98%, 90% to 95%, 95% to 97%, or more) of the antibody light chains in the complexes of the composition are independently covalently linked to an oligonucleotide at the following linkage sites: NO: 27). In some embodiments, the linkage site is optionally represented by K in the sequence motif VNHKPSN (SEQ ID NO: 28) of the antibody heavy chain of the complex described herein. In some embodiments, in a composition comprising a plurality of complexes described herein, about 1% to 15% (e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, or about 15%) of the antibody heavy chains in the complexes of the composition are independently covalently linked to an oligonucleotide at the following linkage site: a linkage site represented by a K residue in the sequence motif VNHKPSN (SEQ ID NO: 28) of the antibody heavy chain.In some embodiments, in a composition comprising a plurality of complexes described herein, the average value of n1 of the complexes of the composition is from 0.5 to 5 (e.g., from 0.5 to 5, from 1 to 5, from 1 to 4, from 1 to 3, from 3 to 5, from 0.5 to 4, from 0.5 to 3, from 0.5 to 2, from 0.5 to 1.5, from 0.5 to 1, from 0.7 to 1.5, from 1 to 1.6, from 1 to 1.5, from 1 to 1.4, from 1 to 1.3, from 1 to 1.2, from 1.1 to 1.5, from 0.8 to 2, from 0.8 to 1.5, from 0.8 to 1.3, from 0.8 to 1.2, from 0.8 to 1.1, from 0.9 to 3, from 0.9 to 2, from 0.9 to 1.8, from 0.9 to 1.6, from 0.9 to 1.5, from 0.9 to 1.4, from 0.9 to 1.3, or from 0.9 to 1.2).

[0125] Linking site

[0126] Provided herein are compositions comprising multiple complexes, wherein each complex comprises an antibody covalently linked to one or more oligonucleotides. In some embodiments, the antibody comprises a heavy chain containing a heavy chain variable region (VH) and a heavy chain constant region and a light chain containing a light chain variable region (VL) and a light chain constant region. In some embodiments, each oligonucleotide is covalently linked at a bonding site represented by a lysine (K) residue of the antibody. In some embodiments, in each complex of the composition, each oligonucleotide is covalently linked at a bonding site represented by different lysine (K) residues of the antibody.

[0127] In some embodiments, the antibody light chain constant region in the complex of the composition is independently covalently linked to the oligonucleotide at the following bonding sites: a bonding site represented by K188 (based on Kabat numbering) of the light chain constant region of the antibody and / or a bonding site represented by K190 (based on Kabat numbering). For example, in some embodiments, the bonding site is represented by K188 (based on Kabat numbering) of the light chain constant region of the antibody. In some embodiments, the bonding site is represented by K190 (based on Kabat numbering) of the light chain constant region of the antibody. In some embodiments, the bonding site is represented by K188 (based on Kabat numbering) and K190 (based on Kabat numbering) of the light chain constant region of the antibody. In some embodiments, at least 80% (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 antibody light chain constant region in the complex of the composition is independently covalently linked to the oligonucleotide at the following linkage sites: the linkage site represented by K188 (based on Kabat numbering) and / or the linkage site represented by K190 (based on Kabat numbering) of the antibody light chain constant region. In some embodiments, approximately 80% to 98%, 80% to 95%, 80% to 90%, 80% to 85%, 85% to 98%, 85% to 95%, 85% to 90%, 90% to 98%, 90% to 95%, 95% to 97%, or 95% to 98% of the antibody light chain constant region in the complex of the composition is independently covalently linked to the oligonucleotide at the following linkage sites: the linkage site represented by K188 (based on Kabat numbering) and / or the linkage site represented by K190 (based on Kabat numbering) of the antibody light chain constant region. In some embodiments, about 85% to 95% (e.g., 85% to 95%, 85% to 90%, or 90% to 95%) of the antibody light chain constant region in the complex of the composition is independently covalently linked to the oligonucleotide at the following linkage site: a linkage site represented by K188 (based on Kabat numbering) of the antibody light chain constant region and / or a linkage site represented by K190 (based on Kabat numbering). In some embodiments, 90% to 95% (e.g., about 90%, about 91%, about 92%, about 93%, about 94%, or about 95%) of the antibody light chain constant region in the complex of the composition is independently covalently linked to the oligonucleotide at the following linkage site: a linkage site represented by K188 (based on Kabat numbering) of the antibody light chain constant region and / or a linkage site represented by K190 (based on Kabat numbering).It should be understood that a complex comprising the light chain constant region of an antibody covalently linked to an oligonucleotide at a linkage site represented by K188 (based on Kabat numbering) and / or a linkage site represented by K190 (based on Kabat numbering) of the light chain constant region includes: a complex comprising the antibody covalently linked to an oligonucleotide at a linkage site represented by K188 (based on Kabat numbering) of the light chain constant region of the antibody; a complex comprising the antibody covalently linked to an oligonucleotide at a linkage site represented by K190 (based on Kabat numbering) of the light chain constant region of the antibody; and / or a complex comprising the antibody covalently linked to an oligonucleotide at a linkage site represented by K188 (based on Kabat numbering) and a linkage site represented by K190 (based on Kabat numbering) of the light chain constant region of the antibody.

[0128] In some embodiments, the antibody heavy chain constant region in the complex of the composition is independently covalently linked to the oligonucleotide at the following linkage site: the linkage site represented by K213 (based on Kabat numbering) of the heavy chain constant region of each antibody. For example, in some embodiments, the linkage site is represented by K213 (based on Kabat numbering) of the heavy chain constant region of each antibody. In some embodiments, at least 1% (e.g., at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, 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%) of the antibody heavy chain constant region in the complex of the composition is independently covalently linked to the oligonucleotide at the following linkage site: the linkage site represented by K213 (based on Kabat numbering) of the heavy chain constant region of the antibody. In some embodiments, 1% to 15% (e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, or about 15%) of the antibody heavy chain constant region in the complex of the composition are independently covalently linked to an oligonucleotide at the following linkage site: the linkage site represented by K213 (based on Kabat numbering) of the heavy chain constant region of the antibody.

[0129] In some embodiments, the lysine (K) residue numbers referred to herein are based on Kabat numbering (Kabat et al. (1971) Ann. NY Acad, Sci. 190: 382-391 and Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)). The variable and constant regions of the heavy and light chains of the antibodies provided herein are numbered separately. The Kabat numbering of the light and heavy chain variable regions of antibodies is described in the art, for example, in Kabat et al. (1971) Ann. NY Acad, Sci. 190: 382-391 and Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)). of Health, Bethesda, Md. (1987) and (1991). Kabat numbering of the light chain constant region and heavy chain constant region of the antibodies provided herein can be found at imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html and imgt.org / IMGTScientificChart / Numbering / Hu_IGKCnber.html (see also Edelman, GM et al., Proc. Natl. Acad. USA, 63, 78-85 (1969). PMID: 5257969; Hieter, PA et al., Cell, 22, 197-207 (1980). PMID: 6775818; Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. 647, 662, 680, 689 (1991)).

[0130] In some embodiments, the antibody light chain in the complex of the composition is independently covalently linked to the oligonucleotide at the following bonding site: a bonding site represented by a lysine (K) residue in the sequence motif DYEKHKVYA (SEQ ID NO: 27) of the antibody light chain. For example, in some embodiments, the bonding site is represented by K at position 4 of the sequence motif DYEKHKVYA (SEQ ID NO: 27) of the antibody light chain. In some embodiments, the bonding site is represented by K at position 6 of the sequence motif DYEKHKVYA (SEQ ID NO: 27) of the antibody light chain. In some embodiments, the bonding site is represented by K at position 4 and K at position 6 of the sequence motif DYEKHKVYA (SEQ ID NO: 27) of the antibody light chain. In some embodiments, at least 80% (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 antibody light chains in the complex of the composition are independently covalently linked to the oligonucleotide at the following linkage site: the linkage site represented by a lysine (K) residue in the sequence motif DYEKHKVYA (SEQ ID NO: 27) of the antibody light chain. In some embodiments, 80% to 98%, 80% to 95%, 80% to 90%, 80% to 85%, 85% to 98%, 85% to 95%, 85% to 90%, 90% to 98%, 90% to 95%, 95% to 97%, or 95% to 98% of the antibody light chains in the complex of the composition are independently covalently linked to the oligonucleotide at the following linkage site: the linkage site represented by the lysine (K) residue in the sequence motif DYEKHKVYA (SEQ ID NO: 27) of the antibody light chain. In some embodiments, 85% to 95% (e.g., 85% to 95%, 85% to 90%, or 90% to 95%) of the antibody light chains in the complex of the composition are independently covalently linked to the oligonucleotide at the following linkage site: a linkage site represented by a lysine (K) residue in the sequence motif DYEKHKVYA (SEQ ID NO: 27) of the antibody light chain. In some embodiments, 90% to 95% (e.g., about 90%, about 91%, about 92%, about 93%, about 94%, or about 95%) of the antibody light chains in the complex of the composition are independently covalently linked to the oligonucleotide at the following linkage site: a linkage site represented by a lysine (K) residue in the sequence motif DYEKHKVYA (SEQ ID NO: 27) of the antibody light chain.It should be understood that a complex comprising an antibody light chain covalently linked to an oligonucleotide at a linkage site represented by a lysine (K) residue in the sequence motif DYEKHKVYA (SEQ ID NO: 27) of the antibody light chain includes: a complex comprising an antibody covalently linked to an oligonucleotide at a linkage site represented by K at position 4 in the sequence motif DYEKHKVYA (SEQ ID NO: 27) of the antibody light chain; a complex comprising an antibody covalently linked to an oligonucleotide at a linkage site represented by K at position 6 in the sequence motif DYEKHKVYA (SEQ ID NO: 27) of the antibody light chain; and / or a complex comprising an antibody covalently linked to an oligonucleotide at both the linkage site represented by K at position 4 and the linkage site represented by K at position 6 in the sequence motif DYEKHKVYA (SEQ ID NO: 27) of the light chain.

[0131] In some embodiments, the antibody heavy chains in the complex of the composition are independently covalently linked to the oligonucleotide at the following linkage site: a linkage site represented by a lysine (K) residue in the sequence motif VNHKPSN (SEQ ID NO: 28) of the heavy chain of the antibody. In some embodiments, about 1% to 15% (e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, or about 15%) of the antibody heavy chains in the complex of the composition are independently covalently linked to the oligonucleotide at the following linkage site: a linkage site represented by a lysine (K) residue in the sequence motif VNHKPSN (SEQ ID NO: 28) of the heavy chain of the antibody. In some embodiments, 1% to 10% (e.g., 1% to 8%, 1% to 5%, 1% to 10%, 1% to 4%, 2% to 8%, 2% to 5%, 3% to 10%, 4% to 10%, 5% to 10%, 3% to 5%, 4% to 8%, 4% to 7%, 4% to 6%, or 5% to 8%) of the antibody heavy chains in the complex of the composition are independently covalently linked to an oligonucleotide at the following linkage site: the linkage site represented by a lysine (K) residue in the sequence motif VNHKPSN (SEQ ID NO: 28) of the heavy chain of the antibody.

[0132] Antibody

[0133] In some embodiments, the complex described herein comprises an antibody that binds to human transferrin receptor 1 (TfR1). An exemplary human TfR1 amino acid sequence corresponding to NCBI sequence NP_003225.2 (transferrin receptor protein 1 isoform 1, Homo sapiens) is as follows:

[0134]

[0135] Table 2 provides some examples of sequences of anti-TfR1 antibodies that can be used in the complexes provided herein.

[0136] Table 2. Some examples of anti-TfR1 antibody sequences

[0137]

[0138]

[0139] In some embodiments, the anti-TfR1 antibody of the present disclosure comprises a heavy chain complementary determining region 1 (CDR-H1) of SEQ ID NO: 1 (according to the IMGT definition system), a heavy chain complementary determining region 2 (CDR-H2) of SEQ ID NO: 2 (according to the IMGT definition system), a heavy chain complementary determining region 3 (CDR-H3) of SEQ ID NO: 3 (according to the IMGT definition system), a light chain complementary determining region 1 (CDR-L1) of SEQ ID NO: 4 (according to the IMGT definition system), a light chain complementary determining region 2 (CDR-L2) of SEQ ID NO: 5 (according to the IMGT definition system), and a light chain complementary determining region 3 (CDR-L3) of SEQ ID NO: 6 (according to the IMGT definition system).

[0140] In some embodiments, the anti-TfR1 antibody of the present disclosure comprises a heavy chain complementary determining region 1 (CDR-H1) of SEQ ID NO: 7 (according to the Kabat definition system), a heavy chain complementary determining region 2 (CDR-H2) of SEQ ID NO: 8 (according to the Kabat definition system), a heavy chain complementary determining region 3 (CDR-H3) of SEQ ID NO: 9 (according to the Kabat definition system), a light chain complementary determining region 1 (CDR-L1) of SEQ ID NO: 10 (according to the Kabat definition system), a light chain complementary determining region 2 (CDR-L2) of SEQ ID NO: 11 (according to the Kabat definition system), and a light chain complementary determining region 3 (CDR-L3) of SEQ ID NO: 6 (according to the Kabat definition system).

[0141] In some embodiments, the anti-TfR1 antibody of the present disclosure comprises a heavy chain complementary determining region 1 (CDR-H1) of SEQ ID NO: 12 (according to the Chothia definition system), a heavy chain complementary determining region 2 (CDR-H2) of SEQ ID NO: 13 (according to the Chothia definition system), a heavy chain complementary determining region 3 (CDR-H3) of SEQ ID NO: 14 (according to the Chothia definition system), a light chain complementary determining region 1 (CDR-L1) of SEQ ID NO: 15 (according to the Chothia definition system), a light chain complementary determining region 2 (CDR-L2) of SEQ ID NO: 5 (according to the Chothia definition system), and a light chain complementary determining region 3 (CDR-L3) of SEQ ID NO: 16 (according to the Chothia definition system).

[0142] In some embodiments, the anti-TfR1 antibody of the present disclosure comprises a heavy chain variable region (VH), which comprises no more than 25 amino acid variations in the framework region compared to the VH comprising the amino acid sequence of SEQ ID NO: 17 (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). As an alternative or supplement (e.g., supplement), the anti-TfR1 antibody of the present disclosure comprises a light chain variable region (VL), which comprises no more than 25 amino acid variations in the framework region compared to the VL comprising the amino acid sequence of SEQ ID NO: 18 (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid variations).

[0143] In some embodiments, the anti-TfR1 antibody of the present disclosure comprises a VH comprising an amino acid sequence in the framework region that is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98% or 99%) identical to a VH comprising the amino acid sequence of SEQ ID NO: 17. Alternatively or additionally (e.g., supplementing), in some embodiments, the anti-TfR1 antibody of the present disclosure comprises a VL comprising an amino acid sequence in the framework region that is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98% or 99%) identical to a VL comprising the amino acid sequence of SEQ ID NO: 18.

[0144] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO: 17. Alternatively or additionally (eg, supplemented), in some embodiments, an anti-TfR1 antibody of the present disclosure comprises a VL comprising the amino acid sequence of SEQ ID NO:18.

[0145] In some embodiments, the anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising an amino acid sequence having at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98% or 99%) identity with the amino acid sequence of SEQ ID NO: 19. As an alternative or supplement (e.g., supplement), the anti-TfR1 antibody of the present disclosure comprises a light chain comprising an amino acid sequence having at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98% or 99%) identity with the amino acid sequence of SEQ ID NO: 20. In some embodiments, the anti-TfR1 antibody of the present disclosure is a Fab comprising a heavy chain comprising an amino acid sequence having at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98% or 99%) identity with the amino acid sequence of SEQ ID NO: 19. Alternatively or additionally (e.g., supplementally), the anti-TfR1 antibody of the present disclosure is a Fab comprising a light chain comprising an amino acid sequence that is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98% or 99%) identical to the amino acid sequence of SEQ ID NO: 20.

[0146] In some embodiments, the anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19. Alternatively or additionally (e.g., supplemented), the anti-TfR1 antibody of the present disclosure comprises a light chain comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the anti-TfR1 antibody of the present disclosure is a Fab comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 19. Alternatively or additionally (e.g., supplemented), the anti-TfR1 antibody of the present disclosure is a Fab comprising a light chain comprising the amino acid sequence of SEQ ID NO: 20.

[0147] In some embodiments, the anti-TfR1 antibodies provided herein may have one or more post-translational modifications. In some embodiments, N-terminal cyclization, also known as pyroglutamate (pyro-Glu) formation, may occur at the N-terminal glutamic acid (Glu) and / or glutamine (Gln) residues of the antibody during production. Therefore, it should be understood that antibodies designated as having sequences comprising N-terminal glutamic acid or glutamine residues encompass antibodies that have undergone pyroglutamate formation caused by post-translational modifications. In some embodiments, pyroglutamate formation occurs in the heavy chain sequence. In some embodiments, pyroglutamate formation occurs in the light chain sequence.

[0148] Oligonucleotides

[0149] In some embodiments, the oligonucleotides of the complexes described herein are single-stranded oligonucleotides. In some embodiments, oligonucleotides can be used for targeting DMPK (e.g., for reducing the expression or activity of DMPK RNA, such as the level of mutant or wild-type DMPK RNA). In some embodiments, oligonucleotides can be used for targeting DMPK RNA (e.g., for reducing the expression or activity of DMPK RNA, such as the level of mutant or wild-type DMPK RNA). In some embodiments, oligonucleotides include complementary regions of DMPK RNA. In some embodiments, oligonucleotides can be used to reduce the level of toxic DMPK with disease-related repeat expansion, such as in subjects suffering from or suspected of suffering from myotonic dystrophy. In some embodiments, the oligonucleotides are designed to direct RNase H-mediated degradation of a target DMPK RNA located in the nucleus of a cell, e.g., a muscle cell (e.g., a myotube) or a nervous system cell (e.g., a central nervous system (CNS) cell). In some embodiments, the oligonucleotides are designed to have desired bioavailability and / or serum stability properties. In some embodiments, the oligonucleotides are designed to have desired binding affinity properties. In some embodiments, the oligonucleotides are designed to have desired toxicity characteristics. In some embodiments, the oligonucleotides are designed to have low complement activation and / or cytokine induction properties.

[0150] In some embodiments, the DMPK targeting oligonucleotides described herein are designed to cause RNase H-mediated degradation of DMPK mRNA. It should be understood that, in some embodiments, one form of oligonucleotide (e.g., antisense oligonucleotide) can be appropriately adapted to another form (e.g., siRNA oligonucleotide) by incorporating a functional sequence (e.g., antisense strand sequence) from one form into another form.

[0151] Some examples of oligonucleotides that can be used to target DMPK are provided in the following: U.S. Patent Application Publication 20100016215A1, which was published on January 1, 2010, and is entitled Compound And Method For Treating Myotonic Dystrophy; U.S. Patent Application Publication 20130237585A1, which was published on July 19, 2010, Modulation Of Dystrophia Myotonica-Protein Kinase (DMPK) Expression; U.S. Patent Application Publication 20150064181A1, which was published on March 5, 2015, and is entitled “Antisense Conjugates For Decreasing Expression Of Dmpk”; U.S. Patent Application Publication 20150238627A1, which was published on August 27, 2015, and is entitled “Peptide-Linked Morpholino Antisense Oligonucleotides For Treatment Of Myotonic Dystrophy”; and U.S. Patent Application Publication No. 20160304877A1, published on October 20, 2016, entitled “Compounds And Methods For Modulation Of Dystrophia Myotonica-Protein Kinase (Dmpk) Expression”, the contents of each of which are incorporated herein in their entirety.

[0152] In some embodiments, the oligonucleotide may comprise a complementary region to the sequence shown below, which is an exemplary human DMPK gene sequence (Gene ID 1760; NM 001081560.2):

[0153]

[0154]

[0155]

[0156] In some embodiments, an oligonucleotide may comprise a complementary region to a sequence as shown below, which is an exemplary mouse DMPK gene sequence (Gene ID 13400; NM 001190490.1).

[0157]

[0158]

[0159] In some embodiments, the oligonucleotides may comprise complementary regions to DMPK gene sequences of multiple species (eg, selected from human, mouse, and a non-human species such as cynomolgus monkey).

[0160] In some embodiments, the oligonucleotide may comprise a complementary region to a mutant form of DMPK, such as those reported in: Botta A. et al. "The CTG repeat expansion size correlates with the splicing defects observed in muscles from myotonic dystrophy type 1 patients." J Med Genet. 2008 Oct; 45(10): 639-46.; and Machuca-Tzili L. et al. "Clinical and molecular aspects of the myotonic dystrophies: a review." Muscle Nerve. 2005 Jul; 32(1): 1-18.; the contents of each of which are incorporated herein in their entirety.

[0161] In some embodiments, the oligonucleotides provided herein are antisense oligonucleotides targeting DMPK. In some embodiments, the oligonucleotides targeting DMPK are any antisense oligonucleotides targeting DMPK, as described in U.S. Patent Application Publication US20160304877A1, which was disclosed on October 20, 2016, entitled "Compounds And Methods For Modulation Of Dystrophia Myotonica-Protein Kinase (DMPK) Expression," which is incorporated herein by reference). In some embodiments, the DMPK targeting oligonucleotide targets the DMPK gene sequence (SEQ ID NO: 24) as shown in Genbank Accession No. NM_001081560.2 or the DMPK gene sequence (SEQ ID NO: 26) as shown in Genbank Accession No. NG_009784.1. The region.

[0162] In some embodiments, the DMPK targeting oligonucleotides provided herein comprise a nucleotide sequence comprising a complementary region to a target region, wherein the target region is at least 8 consecutive nucleotides (e.g., at least 8, at least 9, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, or more consecutive nucleotides) of SEQ ID NO: 24.

[0163] In some embodiments, the DMPK targeting oligonucleotides provided herein are 10 to 35 (e.g., 10 to 35, 10 to 30, 10 to 25, 10 to 20, 10 to 15, 15 to 35, 15 to 30, 15 to 25, 15 to 20, 20 to 35, 20 to 30, 13 to 18, 14 to 17, 15 to 18, 20 to 30, 15 to 17, 27 to 30, 25 to 35, or 30 to 35) nucleotides in length. In some embodiments, the DMPK targeting oligonucleotides provided herein are 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length, optionally 15 to 30 or 16 nucleotides in length. In some embodiments, a DMPK targeting oligonucleotide provided herein is 16 nucleotides in length.

[0164] In some embodiments, the DMPK targeting oligonucleotides provided herein comprise a complementary region of at least 8 (e.g., at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more) consecutive nucleotides of DMPK RNA.

[0165] In some embodiments, the DMPK targeting oligonucleotide provided herein comprises a complementary region of at least 8 (e.g., at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more) consecutive nucleotides of the DMPK sequence shown in SEQ ID NO: 24 or 25.

[0166] In some embodiments, the DMPK targeting oligonucleotides provided herein comprise a complementary region of at least 8 (e.g., at least 8, 9, 10, 11, 12, 13, 14, 15, or 16) consecutive nucleotides of the target sequence shown in SEQ ID NO: 22 (TGACTGGTGGGCGCTG). In some embodiments, the oligonucleotides useful for targeting DMPK comprise at least 8 (e.g., at least 8, 9, 10, 11, 12, 13, 14, 15, or 16) consecutive nucleotides of the sequence shown in SEQ ID NO: 21 (CAGCGCCCACCAGUCA). In some embodiments, the oligonucleotides useful for targeting DMPK comprise the nucleotide sequence of SEQ ID NO: 21.

[0167] In some embodiments, the DMPK targeting oligonucleotide comprises a 5'-XYZ-3' configuration. An oligonucleotide comprising a 5'-XYZ-3' configuration may refer to such a chimeric antisense compound: wherein a spacer region having a plurality of nucleosides supporting RNase H cleavage is located between the flanking regions having one or more nucleosides, wherein the nucleosides comprising the spacer region are chemically different from one or more nucleosides comprising the flanking region. In some embodiments, the oligonucleotides described herein (e.g., the DMPK targeting oligonucleotides described herein) comprise a 5'-XYZ-3' configuration, wherein X and Z are flanking regions around the spacer region Y. In some embodiments, the spacer region Y comprises one or more 2'-deoxyribonucleosides. In some embodiments, each nucleoside in the spacer region Y is a 2'-deoxyribonucleoside, and the flanking region X or the flanking region Z do not comprise any 2'-deoxyribonucleosides.

[0168] In some embodiments, the spacer Y comprises a continuous segment of 6 or more 2'-deoxyribonucleosides, which can recruit RNases, such as RNase H. In some embodiments, the oligonucleotide binds to the target nucleic acid, recruits RNases at this point, and can then cut the target nucleic acid. In some embodiments, each of the flanking regions X and Z comprises one or more modified nucleosides. In some embodiments, each of the flanking regions X and Z comprises one or more high-affinity modified nucleosides, such as one to six high-affinity modified nucleosides. Some examples of high-affinity modified nucleosides include but are not limited to 2'-modified nucleosides (e.g., 2'-MOE, 2'-O-Me, 2'-F) or 2'-4'-bicyclic nucleosides (e.g., LNA, cEt, ENA). In some embodiments, the length of the flanking regions X and Z can be 1 to 20 nucleotides, 1 to 8 nucleotides, or 1 to 5 nucleotides. The flanking regions X and Z can have similar lengths or different lengths. In some embodiments, spacer Y may comprise a nucleotide sequence of 5 to 20 nucleotides, 5 to 15 nucleotides, 5 to 12 nucleotides, or 6 to 10 nucleotides in length.

[0169] In some embodiments, spacer Y comprises one or more unmodified internucleoside linkages. In some embodiments, one or both flanking regions X and Z each independently comprise a phosphorothioate internucleoside linkage (e.g., a phosphorothioate internucleoside linkage or other linkage) between at least two, at least three, at least four, at least five or more nucleotides. In some embodiments, spacer Y and both flanking regions X and Z each independently comprise a modified internucleoside linkage (e.g., a phosphorothioate internucleoside linkage or other linkage) between at least two, at least three, at least four, at least five or more nucleotides.

[0170] In some embodiments, the length of the spacer Y in the spacer polymer is 5 to 20 nucleosides. For example, the length of the spacer Y can be 5 to 20, 5 to 15, 5 to 10, 10 to 20, 10 to 15 or 15 to 20 nucleosides. In some embodiments, the length of the spacer Y is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleosides. In some embodiments, each nucleoside in the spacer Y is a 2'-deoxyribonucleoside. In some embodiments, all nucleosides in the spacer Y are 2'-deoxyribonucleosides. In some embodiments, one or more nucleosides in the spacer Y are modified nucleosides (for example, 2' modified nucleosides, such as those described herein). In some embodiments, one or more cytosines in the spacer Y are optionally 5-methyl-cytosines. In some embodiments, each cytosine in the spacer Y is 5-methyl-cytosine.

[0171] In some embodiments, the flanking region X of the oligonucleotide (X in a 5'-XYZ-3' configuration) and the flanking region Z of the oligonucleotide (Z in a 5'-XYZ-3' configuration) are independently 1 to 20 nucleosides long. For example, the flanking region X of the oligonucleotide and the flanking region Z of the oligonucleotide 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 long. In some embodiments, the flanking region X of the oligonucleotide and the flanking region Z of the oligonucleotide are independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleosides long. In some embodiments, the flanking region X of the oligonucleotide and the flanking region Z of the oligonucleotide have the same length. In some embodiments, the flanking region X of the oligonucleotide and the flanking region Z of the oligonucleotide have different lengths. In some embodiments, the flanking region X of the oligonucleotide is longer than the flanking region Z of the oligonucleotide. In some embodiments, the flanking region X of the oligonucleotide is shorter than the flanking region Z of the oligonucleotide.

[0172] In some embodiments, an oligonucleotide described herein (eg, a DMPK targeting oligonucleotide) comprises the following 5'-XYZ-3' configuration:

[0173] 5-10-5，4-12-4，3-14-3，2-16-2，1-18-1，3-10-3，2-10-2，1-10-1，2-8-2，4-6-4，3-6-3，2-6-2，4-7-4，3-7-3，2-7-2，4-8-4，3-8-3，2-8-2，1-8-1，2-9-2，1-9-1，2-10-2，1-10-1，1-12-1，1-16-1，2-15-1，1-15-2，1-14-3，3-14-1，2-14-2，1-13-4，4-13-1，2-13-3，3-13-2，1-12-5，5-12-1，2-12-4,4-12-2，3-12-3，1-11-6，6-11-1，2-11-5，5-11-2，3-11-4，4-11-3，1-17-1，2-16-1，1-16-2，1-15-3，3-15-1，2-15-2，1-14-4，4-14-1，2-14-3，3-14-2，1-13-5，5-13-1，2-13-4，4-13-2，3-13-3，1-12-6，6-12-1，2-12-5，5-12-2，3-12-4，4-12-3，1-11-7，7-11-1，2-11-6,6-11-2，3-11-5，5-11-3，4-11-4，1-18-1，1-17-2，2-17-1，1-16-3，1-16-3，2-16-2，1-15-4,4-15-1，2-15-3，3-15-2，1-14-5，5-14-1，2-14-4，4-14-2，3-14-3，1-13-6，6-13-1，2-13-5，5-13-2，3-13-4,4-13-3，1-12-7，7-12-1，2-12-6,6-12-2，3-12-5，5-12-3，1-11-8，8-11-1，2-11-7，7-11-2，3-11-6,6-11-3，4-11-5，5-11-4，1-18-1，1-17-2，2-17-1，1-16-3，3-16-1，2-16-2，1-15-4，4-15-1，2-15-3，3-15-2，1-14-5，2-14-4,4-14-2，3-14-3，1-13-6，6-13-1，2-13-5，5-13-2，3-13-4，4-13-3，1-12-7，7-12-1，2-12-6，6-12-2，3-12-5，5-12-3，1-11-8，8-11-1，2-11-7，7-11-2，3-11-6，6-11-3，4-11-5，5-11-4，1-19-1，1-18-2，2-18-1，1-17-3，3-17-1，2-17-2，1-16-4，4-16-1，2-16-3，3-16-2，1-15-5，2-15-4,4-15-2，3-15-3，1-14-6，6-14-1，2-14-5，5-14-2，3-14-4，4-14-3，1-13-7，7-13-1，2-13-6，6-13-2，3-13-5，5-13-3，4-13-4，1-12-8，8-12-1，2-12-7，7-12-2，3-12-6，6-12-3，4-12-5，5-12-4，2-11-8，8-11-2，3-11-7，7-11-3，4-11-6，6-11-4，5-11-5，1-20-1，1-19-2，2-19-1，1-18-3，3-18-1，2-18-2，1-17-4,4-17-1，2-17-3，3-17-2，1-16-5，2-16-4,4-16-2，3-16-3，1-15-6，6-15-1，2-15-5，5-15-2，3-15-4,4-15-3，1-14-7，7-14-1，2-14-6，6-14-2，3-14-5，5-14-3，4-14-4，1-13-8，8-13-1，2-13-7，7-13-2，3-13-6，6-13-3，4-13-5，5-13-4，2-12-8，8-12-2，3-12-7,7-12-3，4-12-6，6-12-4,5-12-5，3-11-8，8-11-3，4-11-7，7-11-4，5-11-6，6-11-5，1-21-1，1-20-2，2-20-1，1-20-3，3-19-1，2-19-2，1-18-4，4-18-1，2-18-3，3-18-2，1-17-5，2-17-4,4-17-2，3-17-3，1-16-6，6-16-1，2-16-5，5-16-2，3-16-4，4-16-3，1-15-7，7-15-1，2-15-6,6-15-2, 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, 2-1 8-4, 4-18-2, 3-18-3, 1-17-6, 6-17-1, 2-17-5, 5-17-2, 3-17-4, 4-17-3, 1-16-7, 7-16-1, 2-16-6, 6-16-2, 3-16-5, 5-16-3, 4-16-4, 1-15-8, 8-15-1, 2-15-7, 7-15-2, 3-15-6, 6-15-3, 4-15-5, 5- 15-4, 2-14-8, 8-14-2, 3-14-7, 7-14-3, 4-14-6, 6-14-4, 5-14-5, 3-13-8, 8-13-3, 4-13-7, 7-13-4, 5-13-6, 6-13-5, 4-12-8, 8-12-4, 5-12-7, 7-12-5, 6-12-6, 5-11-8, 8-11-5, 6-11-7, or 7-11-6.,

[0174] The numbers indicate the number of nucleosides in the X, Y and Z regions, respectively, of an oligonucleotide comprising a 5'-XYZ-3' configuration.

[0175] In some embodiments, one or more nucleosides in the flanking region X of the oligonucleotide (X in a 5'-XYZ-3' configuration) or the flanking region Z of the oligonucleotide (Z in a 5'-XYZ-3' configuration) 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), 2'-ON-methylacetamido (2'-O-NMA)).

[0176] In some embodiments, an oligonucleotide described herein (e.g., a DMPK targeting oligonucleotide described herein) comprises a 5'-XYZ-3' configuration, wherein X and Z are independently 2 to 7 (e.g., 2, 3, 4, 5, 6, or 7) nucleosides in length, and Y is 6 to 10 (e.g., 6, 7, 8, 9, or 10) nucleosides in length, wherein at least one but not all (e.g., 1, 2, 3, 4, 5, or 6) of positions 1, 2, 3, 4, 5, 6, or 7 (the 5'-most position is position 1) of X is a non-bicyclic 2'-modified nucleoside (e.g., 2'-MOE or 2'-O-Me), wherein the remaining nucleosides in both X and Z are 2'-4' bicyclic nucleosides (e.g., LNA or cEt), and wherein each nucleoside in Y is a 2' deoxyribonucleoside. In some embodiments, an oligonucleotide described herein (e.g., a DMPK targeting oligonucleotide described herein) comprises a 5'-XYZ-3' configuration, wherein X and Z are independently 2 to 7 (e.g., 2, 3, 4, 5, 6, or 7) nucleosides in length, and Y is 6 to 10 (e.g., 6, 7, 8, 9, or 10) nucleosides in length, wherein at least one but not all (e.g., 1, 2, 3, 4, 5, or 6) of positions 1, 2, 3, 4, 5, 6, or 7 (the 5'-most position is position 1) of Z is a non-bicyclic 2'-modified nucleoside (e.g., 2'-MOE or 2'-O-Me), wherein the remaining nucleosides in both X and Z are 2'-4' bicyclic nucleosides (e.g., LNA or cEt), and wherein each nucleoside in Y is a 2' deoxyribonucleoside. In some embodiments, an oligonucleotide described herein (e.g., a DMPK targeting oligonucleotide described herein) comprises a 5'-XYZ-3' configuration, wherein X and Z are independently 2 to 7 (e.g., 2, 3, 4, 5, 6, or 7) nucleosides in length, and Y is 6 to 10 (e.g., 6, 7, 8, 9, or 10) nucleosides in length, wherein at least one but not all of the 1st, 2nd, 3rd, 4th, 5th, 6th, or 7th positions (the 5'-most position is the 1st position) in X At least one but not all (e.g., 1, 2, 3, 4, 5, or 6) of positions 1, 2, 3, 4, 5, 6, or 7 (the 5'-most position is position 1) of X and Z are non-bicyclic 2'-modified nucleosides (e.g., 2'-MOE or 2'-O-Me), wherein the remaining nucleosides in both X and Z are 2'-4' bicyclic nucleosides (e.g., LNA or cEt), and wherein each nucleoside in Y is a 2' deoxyribonucleoside.

[0177] In some embodiments, an oligonucleotide described herein (e.g., a DMPK targeting oligonucleotide) is 10 to 20 nucleosides (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleosides) in length, comprises a complementary region to at least 8 consecutive nucleosides (e.g., at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or 16 consecutive nucleosides) of SEQ ID NO: 22 (TGACTGGTGGGCGCTG), and comprises a 5'-XYZ-3' configuration, wherein X comprises 3 to 5 (e.g., 3, 4, or 5) linked nucleosides, wherein at least one nucleoside in X is a 2'-modified nucleoside (e.g., 2'-MOE modified nucleoside, 2'-MOE modified nucleoside, 2'-MOE modified nucleoside, 2'-MOE modified nucleoside, 2'-MOE modified nucleoside, 2'-MOE modified nucleoside, 2'-MOE modified nucleoside, 2'-MOE modified nucleoside, 2'-MOE modified nucleoside, 2'-MOE modified nucleoside, 2'-MOE modified nucleoside, 2'-MOE modified nucleoside, 2 '-O-Me modified nucleosides, LNA, cEt or ENA); Y comprises 6 to 10 (e.g., 6, 7, 8, 9 or 10) linked 2'-deoxyribonucleosides, wherein each cytosine in Y is optionally and independently 5-methyl-cytosine; and Z comprises 3 to 5 (e.g., 3, 4 or 5) linked nucleosides, wherein at least one nucleoside in Z is a 2'-modified nucleoside (e.g., 2'-MOE modified nucleosides, 2'-O-Me modified nucleosides, LNA, cEt or ENA).

[0178] In some embodiments, an oligonucleotide described herein (e.g., a DMPK targeting oligonucleotide described herein) comprises at least 8 consecutive nucleosides (e.g., at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or 16 consecutive nucleosides) of a nucleotide sequence of SEQ ID NO: 21 (CAGCGCCCACCAGUCA), and comprises a 5'-XYZ-3' configuration, wherein X comprises 3 to 5 (e.g., 3, 4, or 5) linked nucleosides, wherein at least one nucleoside in X is a 2'-modified nucleoside (e.g., a 2'-MOE modified nucleoside). In some embodiments, each thymine base (T) of the nucleotide sequence of the antisense oligonucleotide can be independently and optionally replaced by a uracil base (U), and each U can be independently and optionally replaced by a T.

[0179] In some embodiments, an oligonucleotide described herein (e.g., a DMPK targeting oligonucleotide described herein) comprises the nucleotide sequence of SEQ ID NO: 21, and comprises a 5'-XYZ-3' configuration, wherein X comprises 3 to 5 (e.g., 3, 4, or 5) linked nucleosides, wherein at least one nucleoside in X is a 2'-modified nucleoside (e.g., a 2'-MOE modified nucleoside, a 2'-O-Me modified nucleoside, LNA, cEt, or ENA); Y comprises 6 to 10 (e.g., 6, 7, 8, 9, or 10) linked 2'-deoxyribonucleosides, wherein each cytosine in Y is optionally and independently a 5-methyl-cytosine; and Z comprises 3 to 5 (e.g., 3, 4, or 5) linked nucleosides, wherein at least one nucleoside in Z is a 2'-modified nucleoside (e.g., a 2'-MOE modified nucleoside, a 2'-O-Me modified nucleoside, LNA, cEt, or ENA). In some embodiments, each thymine base (T) of the nucleotide sequence of the antisense oligonucleotide can be independently and optionally replaced by a uracil base (U), and each U can be independently and optionally replaced by a T.

[0180] In some embodiments, X comprises at least one 2'-4' bicyclic nucleoside (e.g., LNA, cEt, or ENA) and at least one non-bicyclic 2'-modified nucleoside (e.g., a 2'-MOE modified nucleoside or a 2'-O-Me modified nucleoside), and / or (e.g., and) Z comprises at least one 2'-4' bicyclic nucleoside (e.g., LNA, cEt, or ENA) and at least one non-bicyclic 2'-modified nucleoside (e.g., a 2'-MOE modified nucleoside or a 2'-O-Me modified nucleoside).

[0181] In some embodiments, the 2'-4' bicyclic nucleoside is selected from LNA, cEt, and ENA nucleosides. In some embodiments, the non-bicyclic 2'-modified nucleoside is a 2'-MOE modified nucleoside or a 2'-OMe modified nucleoside.

[0182] In some embodiments, the nucleosides of oligonucleotides are linked together by phosphorothioate internucleoside linkages, phosphodiester internucleoside linkages or their combinations. In some embodiments, oligonucleotides only comprise phosphorothioate internucleoside linkages connecting each nucleoside (i.e., oligonucleotides comprise complete phosphorothioate backbones). In some embodiments, oligonucleotides comprise at least one phosphorothioate internucleoside linkage. In some embodiments, oligonucleotides comprise a mixture of phosphorothioate internucleoside linkages and phosphodiester internucleoside linkages. In some embodiments, oligonucleotides only comprise phosphorothioate internucleoside linkages connecting every pair of 2'-deoxyribonucleosides, and a mixture of phosphorothioate internucleoside linkages and phosphodiester internucleoside linkages connecting the remaining nucleosides.

[0183] In some embodiments, the oligonucleotide comprises a 5'-XYZ-3' configuration of LLEE-(D)8-EELL, wherein "E" is a 2'-MOE modified ribonucleoside; "L" is LNA; "D" is a 2'-deoxyribonucleoside; and "10" or "8" is the number of 2'-deoxyribonucleosides in Y, and wherein the oligonucleotide comprises phosphorothioate internucleoside linkages, phosphodiester internucleoside linkages, or a combination thereof.

[0184] In some embodiments, each cytidine (e.g., 2'-modified cytidine) in X and / or Z of the oligonucleotide is optionally and independently 5-methyl-cytidine, and / or each uridine (e.g., 2'-modified uridine) in X and / or Z is optionally and independently 5-methyl-uridine.

[0185] In some embodiments, an oligonucleotide described herein (eg, a DMPK targeting oligonucleotide described herein) comprises a 5'-XYZ-3' configuration and comprises a nucleobase sequence of CAGCGCCCACCAGUCA (SEQ ID NO: 21). In some embodiments, an oligonucleotide described herein (e.g., a DMPK targeting oligonucleotide described herein) comprises a structure of +C*+A*oG*oC*dG*dC*dC*dC*dA*dC*dC*dA*oG*oU*+C*+A (SEQ ID NO: 21), wherein +N represents LNA (2'-4' methylene bridge) ribonucleoside, dN represents 2'-deoxyribonucleoside, oN represents 2'-O-methoxyethyl (MOE) modified ribonucleoside, oC represents 5-methyl-2'-MOE-cytidine, +C represents 5-methyl-2'-4'-bicyclo-cytidine (2'-4' methylene bridge), oU represents 5-methyl-2'-MOE-uridine, and * represents a phosphorothioate internucleoside linkage.

[0186] In some embodiments, an oligonucleotide described herein (eg, a DMPK targeting oligonucleotide described herein) comprises the structure of Formula (Ie):

[0187]

[0188] In some embodiments, an oligonucleotide described herein (eg, a DMPK targeting oligonucleotide described herein) can be in the form of a salt, for example, a sodium salt, a potassium salt, or a magnesium salt.

[0189] In some embodiments, the 5' or 3' nucleoside (e.g., terminal nucleoside) of an oligonucleotide is optionally conjugated to an amine group via a spacer. In some embodiments, the spacer comprises an aliphatic moiety. In some embodiments, the spacer comprises a polyethylene glycol moiety. In some embodiments, there is a phosphodiester linkage between the spacer and the 5' or 3' nucleoside of the oligonucleotide. In some embodiments, the 5' or 3' nucleoside (e.g., terminal nucleoside) of an oligonucleotide described herein is covalently linked to a spacer, and the spacer is a substituted or unsubstituted aliphatic, substituted or unsubstituted heteroaliphatic, substituted or unsubstituted carbocyclylene, substituted or unsubstituted heterocyclylene, substituted or unsubstituted arylene, substituted or unsubstituted heteroarylene, -O-, -N(R A )-, -S-, -C(=O)-, -C(=O)O-, -C(=O)NR A -,-NR A C(=O)-,、-NR A C(=O)R A -,-C(=O)R A -,-NR A C(=O)O-,-NR A C(=O)N(R A )-, -OC(=O)-, -OC(=O)O-, -OC(=O)N(R A )-,-S(O)2NR A , -NR A S(O)2-, or a combination thereof; each R A is independently hydrogen or substituted or unsubstituted alkyl. In certain embodiments, the spacer is substituted or unsubstituted alkylene, substituted or unsubstituted heterocyclylene, substituted or unsubstituted heteroarylene, -O-, -N(R A )-or-C(=O)N(R A )2, or a combination thereof.

[0190] In some embodiments, the 5' or 3' nucleoside of the oligonucleotide is in the formula -NH2-(CH2) n -compound conjugation, wherein n is an integer from 1 to 12. In some embodiments, n is 6, 7, 8, 9, 10, 11 or 12. In some embodiments, in the formula -NH2-(CH2) n - There is a phosphodiester linkage between the compound and the 5' or 3' nucleoside of the oligonucleotide. In some embodiments, the compound of formula -NH2-(CH2)6- is conjugated to the oligonucleotide via a reaction between 6-amino-1-hexanol (NH2-(CH2)6-OH) and the 5' phosphate of the oligonucleotide.

[0191] In some embodiments, the oligonucleotide is conjugated to a targeting agent, such as a muscle targeting agent (eg, an anti-TfR1 antibody), such as via an amine group of a lysine of the targeting agent.

[0192] In some embodiments, it is understood that methylation of the nucleobase uracil at the C5 position forms thymine. Therefore, in some embodiments, a nucleotide or nucleoside having a C5 methylated uracil (or 5-methyl-uracil) can be equivalently identified as a thymine nucleotide or nucleoside.

[0193] In some embodiments, any one or more thymine bases (T) in any oligonucleotide provided herein may independently and optionally be a uracil base (U), and / or any one or more U in the oligonucleotide provided herein (e.g., the oligonucleotide shown in SEQ ID NO: 21) may independently and optionally be T.

[0194] Composition

[0195] In some embodiments, the compositions described herein include a complex (i.e., multiple complexes), each complex comprising an antibody (e.g., an anti-TFR1 antibody) covalently linked to one or more oligonucleotides (e.g., a DMPK targeting oligonucleotide described herein) at a linkage site represented by a lysine (K) residue of the antibody, wherein the antibody comprises a heavy chain containing a heavy chain variable region (VH) and a heavy chain constant region and a light chain containing a light chain variable region (VL) and a light chain constant region. In some embodiments, the antibody of such a complex comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 as shown in Table 2.

[0196] In some embodiments, the antibody light chain constant region in the complex of the composition is independently covalently linked to the oligonucleotide at the following bonding sites: a bonding site represented by K188 (based on Kabat numbering) of the light chain constant region of the antibody and / or a bonding site represented by K190 (based on Kabat numbering). For example, in some embodiments, the bonding site is represented by K188 (based on Kabat numbering) of the light chain constant region of the antibody. In some embodiments, the bonding site is represented by K190 (based on Kabat numbering) of the light chain constant region of the antibody. In some embodiments, the bonding site is represented by K188 (based on Kabat numbering) and K190 (based on Kabat numbering) of the light chain constant region of the antibody. In some embodiments, at least 80% (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, In some embodiments, the antibody heavy chain constant region in the complex of the composition is independently covalently linked to the oligonucleotide at the following linkage site: the linkage site represented by K188 (based on Kabat numbering) of the light chain constant region of the antibody and / or the linkage site represented by K190 (based on Kabat numbering). In some embodiments, the antibody heavy chain constant region in the complex of the composition is independently covalently linked to the oligonucleotide at the following linkage site: the linkage site represented by K213 (based on Kabat numbering) of the heavy chain constant region of the antibody. In some embodiments, about 1% to 15% (e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, or about 15%) of the antibody heavy chain constant region in the complex of the composition is independently covalently linked to an oligonucleotide at the following linkage site: the linkage site represented by K213 (based on Kabat numbering) of the heavy chain constant region of the antibody. In some embodiments, the antibody light chain in the complex of the composition is independently covalently linked to an oligonucleotide at the following linkage site: the linkage site represented by a lysine (K) residue in the sequence motif DYEKHKVYA (SEQ ID NO: 27) of the antibody light chain. For example, in some embodiments, the antibody is covalently linked to the oligonucleotide at the following linkage site: the linkage site represented by K at position 4 in the sequence motif DYEKHKVYA (SEQ ID NO: 27) of the light chain of the antibody.In some embodiments, the antibody is covalently linked to the oligonucleotide at the following linkage sites: the linkage site represented by K at position 6 in the sequence motif DYEKHKVYA (SEQ ID NO: 27) of the light chain of the antibody. In some embodiments, the antibody is covalently linked to the oligonucleotide at the following linkage sites: the linkage site represented by K at position 4 and the linkage site represented by K at position 6 in the sequence motif DYEKHKVYA (SEQ ID NO: 27) of the light chain of the antibody. In some embodiments, at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, 80% to 98%, 80% to 95%, 80% to 90%, 85% to 98%, 85% to 95%, 85% to 90%, 90% to 98%, 90% to 95%, 95% to 97% or more) of the antibody light chains in the complex of the composition are independently covalently linked to the oligonucleotide at the following linkage site: the linkage site represented by the lysine (K) residue in the sequence motif DYEKHKVYA (SEQ ID NO: 27) of the antibody light chain. In some embodiments, the antibody heavy chains in the complex of the composition are independently covalently linked to the oligonucleotide at the following linkage site: a linkage site represented by a lysine (K) residue in the sequence motif VNHKPSN (SEQ ID NO: 28) of the heavy chain of the antibody. In some embodiments, about 1% to 15% (e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, or about 15%) of the antibody heavy chains in the complex of the composition are independently covalently linked to the oligonucleotide at the following linkage site: a linkage site represented by a lysine (K) residue in the motif VNHKPSN (SEQ ID NO: 28) of the heavy chain of the antibody. In some embodiments, the antibody is an anti-TfR1 Fab.

[0197] In some embodiments, the compositions described herein comprise a complex (ie, a plurality of complexes), wherein each complex comprises formula (I): [R 1 ] n1 -R 2 The structure of each R 1 independently comprises a compound containing an oligonucleotide (e.g., a DMPK targeting oligonucleotide described herein) and R 2 Covalently linked, where R 2The invention comprises an antibody (e.g., an anti-TfR1 antibody) comprising a heavy chain comprising a heavy chain variable region (VH) and a heavy chain constant region and a light chain comprising a light chain variable region (VL) and a light chain constant region. 1 The binding site represented by the lysine (K) residue of the antibody is 2 In some embodiments, at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, 80% to 98%, 80% to 95%, 80% to 90%, 85% to 98%, 85% to 95%, 85% to 90%, 90% to 98%, 90% to 95%, 95% to 97%, or more) of the antibody light chain constant region in the complex of the composition are independently bound to R at the following linkage sites: 1 Covalently linked: a linkage site represented by K188 (based on Kabat numbering) and / or a linkage site represented by K190 (based on Kabat numbering) of the light chain constant region of the antibody. In some embodiments, about 1% to 15% (e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, or about 15%) of the antibody heavy chain constant region in the complex of the composition independently binds to R at the following linkage sites: 1 Covalently linked: The linkage site represented by K213 (based on Kabat numbering) of the heavy chain constant region of the antibody. In some embodiments, at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, 80% to 98%, 80% to 95%, 80% to 90%, 85% to 98%, 85% to 95%, 85% to 90%, 90% to 98%, 90% to 95%, 95% to 97% or more) of the antibody light chains in the complex of the composition are independently linked to R at the following linkage sites: 1Covalently linked: A linkage site represented by a lysine (K) residue in the sequence motif DYEKHKVYA (SEQ ID NO: 27) of the light chain of an antibody. In some embodiments, about 1% to 15% (e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, or about 15%) of the antibody heavy chains in the complex of the composition are independently linked to R at the following linkage sites: 1 Covalent attachment: The linkage site represented by the lysine (K) residue in the motif VNHKPSN (SEQ ID NO: 28) of the heavy chain of the antibody. In some embodiments, in each complex, n1 is independently an integer of 1 or greater, which represents the number of R 1 The number of instances of .

[0198] In some embodiments, the value of n1 for the complexes in the composition is independently and optionally from one to as high as an antibody (e.g., R 2 In some embodiments, the value of n1 for each complex in the composition is independently and optionally selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27. In some embodiments, the value of n1 for each complex in the composition is independently and optionally selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, and 26. In some embodiments, the value of n1 for each complex in the composition is independently and optionally selected from an integer of 1 to 27, 1 to 26, 1 to 10, 1 to 5, or 1 to 3. In some embodiments, the average value of n1 for the complexes of the composition is 1 to 3, 1 to 5, 1 to 10, 1 to 26, or 1 to 27. In some embodiments, the composition described herein comprises Complex I having a value of n1 of 0. In some embodiments, the average value of n1 of the complexes of the composition is from 0.5 to 5 (e.g., from 0.5 to 5, from 1 to 5, from 1 to 4, from 1 to 3, from 3 to 5, from 0.5 to 4, from 0.5 to 3, from 0.5 to 2, from 0.5 to 1.5, from 0.5 to 1, from 0.7 to 1.5, from 1 to 1.6, from 1 to 1.5, from 1 to 1.4, from 1 to 1.3, from 1 to 1.2, from 1.1 to 1.5, from 0.8 to 2, from 0.8 to 1.5, from 0.8 to 1.3, from 0.8 to 1.2, from 0.8 to 1.1, from 0.9 to 3, from 0.9 to 2, from 0.9 to 1.8, from 0.9 to 1.6, from 0.9 to 1.5, from 0.9 to 1.4, from 0.9 to 1.3, or from 0.9 to 1.2).

[0199] In some embodiments, the compositions described herein include antibodies that are not conjugated to oligonucleotides (e.g., in trace amounts) and antibodies that are conjugated to one or more oligonucleotides. In some embodiments, antibodies that are not conjugated to oligonucleotides may be referred to as comprising formula (I): 1 ] n1 -R 2 A compound of the structure of wherein n1 is zero. Thus, in some embodiments, the composition for administration to a subject in the methods described herein comprises a compound of formula (I): [R 1 ] n1 -R 2 A compound (eg, a complex) of the structure wherein each R 1 independently comprising a group containing an oligonucleotide, R 2 comprising an antibody, and n1 is independently an integer of zero or greater, which reflects the number of R 1 In some embodiments, compared to all compounds having the structure in which n1 is 1 or greater, the compounds in which n1 is zero include compounds of formula (I): [R 1 ] n1 -R 2 The fraction of compounds having the structure of is less than 10%, less than 5%, less than 1%, less than 0.5%, less than 0.1%, less than 0.05% or less than 0.01%. Thus, in some embodiments, the average value of n1 of the complexes in the compositions disclosed herein is from 0.5 to 5 (e.g., from 0.5 to 5, from 1 to 5, from 1 to 4, from 1 to 3, from 3 to 5, from 0.5 to 4, from 0.5 to 3, from 0.5 to 2, from 0.5 to 1.5, from 0.5 to 1, from 0.7 to 1.5, from 1 to 1.6, from 1 to 1.5, from 1 to 1.4, from 1 to 1.3, from 1 to 1.2, from 1.1 to 1.5, from 0.8 to 2, from 0.8 to 1.5, from 0.8 to 1.3, from 0.8 to 1.2, from 0.8 to 1.1, from 0.9 to 3, from 0.9 to 2, from 0.9 to 1.8, from 0.9 to 1.6, from 0.9 to 1.5, from 0.9 to 1.4, from 0.9 to 1.3, or from 0.9 to 1.2).

[0200] In some embodiments, the compositions described herein comprise a compound of formula (I): [R 1 ] n1 -R 2 A complex of a structure of wherein each R in the complex of the composition provided herein 1 Independently comprising groups of formula (Ia):

[0201]

[0202] Where R 3comprising an oligonucleotide, for example comprising an oligonucleotide comprising a 5'-XYZ-3' configuration; R 2 An antibody comprising a heavy chain comprising a heavy chain variable region (VH) and a heavy chain constant region, and a light chain comprising a light chain variable region (VL) and a light chain constant region, wherein in each complex, n1 is independently an integer (e.g., an integer of 1 or greater) representing the number of R 1 The number of instances, and each R 1 At the connection point A, the binding site is represented by a lysine (K) residue of the antibody and R 2 Covalently linked. In some embodiments, the antibody light chain constant region in the complex of the composition is independently covalently linked to the oligonucleotide at the following linkage sites: a linkage site represented by K188 (based on Kabat numbering) of the light chain constant region of the antibody and / or a linkage site represented by K190 (based on Kabat numbering). For example, in some embodiments, the linkage site is represented by K188 (based on Kabat numbering) of the light chain constant region of the antibody. In some embodiments, the linkage site is represented by K190 (based on Kabat numbering) of the light chain constant region of the antibody. In some embodiments, the linkage site is represented by K188 (based on Kabat numbering) and K190 (based on Kabat numbering) of the light chain constant region of the antibody. In some embodiments, at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, 80% to 98%, 80% to 95%, 80% to 90%, 85% to 98%, 85% to 95%, 85% to 90%, 90% to 98%, 90% to 95%, 95% to 97%, or more) of the constant regions of the antibody light chains in the complex of the composition independently bind to R at the following linkage sites: 1 Covalently linked: a linking site represented by K188 (based on Kabat numbering) of the light chain constant region of the antibody and / or a linking site represented by K190 (based on Kabat numbering). In some embodiments, about 1% to 15% (e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, or about 15%) of the antibody heavy chain constant region in the complex of the composition is independently covalently linked to the oligonucleotide at the following linking site: a linking site represented by K213 (based on Kabat numbering) of the heavy chain constant region of the antibody. In some embodiments, R 2The invention comprises an anti-TfR1 antibody (eg, Fab) comprising a sequence as shown in Table 2. For example, in some embodiments, R 2 The invention comprises an anti-TfR1 antibody (e.g., Fab), wherein the anti-TfR1 antibody (e.g., Fab) comprises: a heavy chain complementary determining region 1 (CDR-H1) comprising the sequence shown in SEQ ID NO: 1, 7, or 12, a heavy chain complementary determining region 2 (CDR-H2) comprising the sequence shown in SEQ ID NO: 2, 8, or 13, and a heavy chain complementary determining region 3 (CDR-H3) comprising the sequence shown in SEQ ID NO: 3, 9, or 14; and / or a light chain complementary determining region 1 (CDR-L1) comprising the sequence shown in SEQ ID NO: 4, 10, or 15, a light chain complementary determining region 2 (CDR-L2) comprising the sequence shown in SEQ ID NO: 5 or 11, and a light chain complementary determining region 3 (CDR-L3) comprising the sequence shown in SEQ ID NO: 6 or 16. In some embodiments, R 2 An anti-TfR1 antibody (e.g., Fab) comprising a heavy chain variable region (VH) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 17, and / or a light chain variable region (VL) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 18. In some embodiments, R 2 The invention comprises an anti-TfR1 antibody (eg, Fab) comprising a VH comprising the amino acid sequence of SEQ ID NO: 17 and / or a VL comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, R 2 An anti-TfR1 antibody (e.g., Fab) comprising a heavy chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 19 and / or a light chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 20. In some embodiments, R 2 The invention comprises an anti-TfR1 antibody (eg, Fab) comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and / or a light chain comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, R 2 The invention comprises an antibody (e.g., Fab) that is a Fab fragment, a full-length IgG, a Fab' fragment, a F(ab')2 fragment, a scFv, or a Fv. In some embodiments, R 2 In some embodiments, R 3An oligonucleotide comprising a nucleobase sequence of CAGCGCCCACCAGUCA (SEQ ID NO: 21). 3 An oligonucleotide comprising a structure comprising +C*+A*oG*oC*dG*dC*dC*dC*dA*dC*dC*dA*oG*oU*+C*+A (SEQ ID NO: 21), wherein +N represents LNA (2'-4' methylene bridge) ribonucleoside, dN represents 2'-deoxyribonucleoside, oN represents 2'-MOE modified ribonucleoside, oC represents 5-methyl-2'-MOE-cytidine, +C represents 5-methyl-2'-4'-bicyclo-cytidine (2'-4' methylene bridge), oU represents 5-methyl-2'-MOE-uridine, and * represents a phosphorothioate internucleoside linkage. In some embodiments, in each complex, n1 is independently an integer (e.g., an integer from 1 to 27, 1 to 26, 1 to 10, 1 to 5, or 1 to 3). In some embodiments, the compositions described herein comprise a compound of formula (I): [R 1 ] n1 -R 2 , wherein n1 is 0. In some embodiments, the average value of n1 of the complexes of the composition is 0.5 to 5 (e.g., 0.5 to 5, 1 to 5, 1 to 4, 1 to 3, 3 to 5, 0.5 to 4, 0.5 to 3, 0.5 to 2, 0.5 to 1.5, 0.5 to 1, 0.7 to 1.5, 1 to 1.6, 1 to 1.5, 1 to 1.4, 1 to 1.3, 1 to 1.2, 1.1 to 1.5, 0.8 to 2, 0.8 to 1.5, 0.8 to 1.3, 0.8 to 1.2, 0.8 to 1.1, 0.9 to 3, 0.9 to 2, 0.9 to 1.8, 0.9 to 1.6, 0.9 to 1.5, 0.9 to 1.4, 0.9 to 1.3, or 0.9 to 1.2).

[0203] In some embodiments, the compositions described herein comprise a compound of formula (I): [R 1 ] n1 -R 2 A complex of a structure of wherein each R in the complex of the composition provided herein 1 Examples include groups of formula (Ib):

[0204]

[0205] wherein +N represents LNA (2'-4' methylene bridge) ribonucleoside, dN represents 2'-deoxyribonucleoside, oN represents 2'-O-methoxyethyl (MOE) modified ribonucleoside, oC represents 5-methyl-2'-MOE-cytidine, +C represents 5-methyl-2'--4'--bicyclo-cytidine (2'-4' methylene bridge), oU represents 5-methyl-2'-MOE-uridine, * represents phosphorothioate internucleoside linkage, and wherein the oligonucleotide comprises a nucleobase sequence of CAGCGCCCACCAGUCA (SEQ ID NO: 21); wherein R 2 An antibody comprising a heavy chain comprising a heavy chain variable region (VH) and a heavy chain constant region, and a light chain comprising a light chain variable region (VL) and a light chain constant region, wherein in each complex, n1 is independently an integer (e.g., an integer of 1 or greater), which represents the number of R 1 The number of instances, and each R 1 At the connection point A, the binding site is represented by a lysine (K) residue of the antibody and R 2 In some embodiments, the antibody light chain constant region in the complex of the composition is independently linked to R at the following linkage sites: 1 Covalently linked: A linkage site represented by K188 (based on Kabat numbering) of the light chain constant region of an antibody and / or a linkage site represented by K190 (based on Kabat numbering). For example, in some embodiments, the linkage site is represented by K188 (based on Kabat numbering) of the light chain constant region of an antibody. In some embodiments, the linkage site is represented by K190 (based on Kabat numbering) of the light chain constant region of an antibody. In some embodiments, the linkage site is represented by K188 (based on Kabat numbering) and K190 (based on Kabat numbering) of the light chain constant region of an antibody. In some embodiments, at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, 80% to 98%, 80% to 95%, 80% to 90%, 85% to 98%, 85% to 95%, 85% to 90%, 90% to 98%, 90% to 95%, 95% to 97%, or more) of the constant regions of the antibody light chains in the complex of the composition independently bind to R at the following linkage sites: 1Covalently linked: a linkage site represented by K188 (based on Kabat numbering) and / or a linkage site represented by K190 (based on Kabat numbering) of the light chain constant region of the antibody. In some embodiments, about 1% to 15% (e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, or about 15%) of the antibody heavy chain constant region in the complex of the composition independently binds to R at the following linkage sites: 1 Covalent attachment: The linkage site represented by K213 (based on Kabat numbering) of the heavy chain constant region of the antibody. 2 The invention comprises an anti-TfR1 antibody (eg, Fab) comprising a sequence as shown in Table 2. For example, in some embodiments, R 2 The invention comprises an anti-TfR1 antibody (e.g., Fab), wherein the anti-TfR1 antibody (e.g., Fab) comprises: a heavy chain complementary determining region 1 (CDR-H1) comprising the sequence shown in SEQ ID NO: 1, 7, or 12, a heavy chain complementary determining region 2 (CDR-H2) comprising the sequence shown in SEQ ID NO: 2, 8, or 13, and a heavy chain complementary determining region 3 (CDR-H3) comprising the sequence shown in SEQ ID NO: 3, 9, or 14; and / or a light chain complementary determining region 1 (CDR-L1) comprising the sequence shown in SEQ ID NO: 4, 10, or 15, a light chain complementary determining region 2 (CDR-L2) comprising the sequence shown in SEQ ID NO: 5 or 11, and a light chain complementary determining region 3 (CDR-L3) comprising the sequence shown in SEQ ID NO: 6 or 16. In some embodiments, R 2 An anti-TfR1 antibody (e.g., Fab) comprising a heavy chain variable region (VH) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 17, and / or a light chain variable region (VL) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 18. In some embodiments, R 2 The invention comprises an anti-TfR1 antibody (eg, Fab) comprising a VH comprising the amino acid sequence of SEQ ID NO: 17 and / or a VL comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, R 2An anti-TfR1 antibody (e.g., Fab) comprising a heavy chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 19 and / or a light chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 20. In some embodiments, R 2 The invention comprises an anti-TfR1 antibody (eg, Fab) comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and / or a light chain comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, R 2 The invention comprises an antibody which is a Fab fragment, a full-length IgG, a Fab' fragment, a F(ab')2 fragment, a scFv or a Fv. In some embodiments, R 2 The invention further comprises an antibody which is a Fab fragment. In some embodiments, in each complex, n1 is independently an integer (e.g., an integer from 1 to 27, 1 to 26, 1 to 10, 1 to 5, or 1 to 3). In some embodiments, the composition described herein further comprises a compound of formula (I): [R 1 ] n1 -R 2 , wherein n1 is 0. In some embodiments, the average value of n1 of the complexes of the composition is 0.5 to 5 (e.g., 0.5 to 5, 1 to 5, 1 to 4, 1 to 3, 3 to 5, 0.5 to 4, 0.5 to 3, 0.5 to 2, 0.5 to 1.5, 0.5 to 1, 0.7 to 1.5, 1 to 1.6, 1 to 1.5, 1 to 1.4, 1 to 1.3, 1 to 1.2, 1.1 to 1.5, 0.8 to 2, 0.8 to 1.5, 0.8 to 1.3, 0.8 to 1.2, 0.8 to 1.1, 0.9 to 3, 0.9 to 2, 0.9 to 1.8, 0.9 to 1.6, 0.9 to 1.5, 0.9 to 1.4, 0.9 to 1.3, or 0.9 to 1.2).

[0206] In some embodiments, the compositions described herein comprise a compound of formula (I): [R 1 ] n1 -R 2 A complex of a structure of wherein each R 1 Examples include groups of formula (Ic):

[0207]

[0208] Where R 2An antibody comprising a heavy chain comprising a heavy chain variable region (VH) and a heavy chain constant region, and a light chain comprising a light chain variable region (VL) and a light chain constant region, wherein in each complex, n1 is independently an integer (e.g., an integer of 1 or greater), which represents the number of R 1 The number of instances of R 1 At the connection point A, the binding site is represented by a lysine (K) residue of the antibody and R 2 In some embodiments, the antibody light chain constant region in the complex of the composition is independently linked to R at the following linkage sites: 1 Covalently linked: A linkage site represented by K188 (based on Kabat numbering) of the light chain constant region of an antibody and / or a linkage site represented by K190 (based on Kabat numbering). For example, in some embodiments, the linkage site is represented by K188 (based on Kabat numbering) of the light chain constant region of an antibody. In some embodiments, the linkage site is represented by K190 (based on Kabat numbering) of the light chain constant region of an antibody. In some embodiments, the linkage site is represented by K188 (based on Kabat numbering) and K190 (based on Kabat numbering) of the light chain constant region of an antibody. In some embodiments, at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, 80% to 98%, 80% to 95%, 80% to 90%, 85% to 98%, 85% to 95%, 85% to 90%, 90% to 98%, 90% to 95%, 95% to 97%, or more) of the constant regions of the antibody light chains in the complex of the composition independently bind to R at the following linkage sites: 1 Covalently linked: a linkage site represented by K188 (based on Kabat numbering) and / or a linkage site represented by K190 (based on Kabat numbering) of the light chain constant region of the antibody. In some embodiments, about 1% to 15% (e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, or about 15%) of the antibody heavy chain constant region in the complex of the composition independently binds to R at the following linkage sites: 1 Covalent attachment: The linkage site represented by K213 (based on Kabat numbering) of the heavy chain constant region of the antibody. 2 The invention comprises an anti-TfR1 antibody (eg, Fab) comprising a sequence as shown in Table 2. For example, in some embodiments, R 2The invention comprises an anti-TfR1 antibody (e.g., Fab), wherein the anti-TfR1 antibody (e.g., Fab) comprises: a heavy chain complementary determining region 1 (CDR-H1) comprising the sequence shown in SEQ ID NO: 1, 7, or 12, a heavy chain complementary determining region 2 (CDR-H2) comprising the sequence shown in SEQ ID NO: 2, 8, or 13, and a heavy chain complementary determining region 3 (CDR-H3) comprising the sequence shown in SEQ ID NO: 3, 9, or 14; and / or a light chain complementary determining region 1 (CDR-L1) comprising the sequence shown in SEQ ID NO: 4, 10, or 15, a light chain complementary determining region 2 (CDR-L2) comprising the sequence shown in SEQ ID NO: 5 or 11, and a light chain complementary determining region 3 (CDR-L3) comprising the sequence shown in SEQ ID NO: 6 or 16. In some embodiments, R 2 An anti-TfR1 antibody (e.g., Fab) comprising a heavy chain variable region (VH) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 17, and / or a light chain variable region (VL) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 18. In some embodiments, R 2 The invention comprises an anti-TfR1 antibody (eg, Fab) comprising a VH comprising the amino acid sequence of SEQ ID NO: 17 and / or a VL comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, R 2 An anti-TfR1 antibody (e.g., Fab) comprising a heavy chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 19 and / or a light chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 20. In some embodiments, R 2 The invention comprises an anti-TfR1 antibody (eg, Fab) comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and / or a light chain comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, R 2 The invention comprises an antibody which is a Fab fragment, a full-length IgG, a Fab' fragment, a F(ab')2 fragment, a scFv or a Fv. In some embodiments, R 2The invention further comprises an antibody which is a Fab fragment. In some embodiments, in each complex, n1 is independently an integer (e.g., an integer from 1 to 27, 1 to 26, 1 to 10, 1 to 5, or 1 to 3). In some embodiments, the composition described herein further comprises a compound of formula (I): [R 1 ] n1 -R 2 , wherein n1 is 0. In some embodiments, the average value of n1 of the complexes of the composition is 0.5 to 5 (e.g., 0.5 to 5, 1 to 5, 1 to 4, 1 to 3, 3 to 5, 0.5 to 4, 0.5 to 3, 0.5 to 2, 0.5 to 1.5, 0.5 to 1, 0.7 to 1.5, 1 to 1.6, 1 to 1.5, 1 to 1.4, 1 to 1.3, 1 to 1.2, 1.1 to 1.5, 0.8 to 2, 0.8 to 1.5, 0.8 to 1.3, 0.8 to 1.2, 0.8 to 1.1, 0.9 to 3, 0.9 to 2, 0.9 to 1.8, 0.9 to 1.6, 0.9 to 1.5, 0.9 to 1.4, 0.9 to 1.3, or 0.9 to 1.2).

[0209] In some embodiments, the compositions described herein comprise a complex comprising the structure of Formula (Id):

[0210]

[0211] wherein +N represents an LNA (2'-4' methylene bridge) ribonucleoside, dN represents a 2'-deoxyribonucleoside, oN represents a 2'-O-methoxyethyl (MOE) modified ribonucleoside, oC represents 5-methyl-2'-MOE-cytidine, +C represents 5-methyl-2'-4'-bicyclo-cytidine (2'-4' methylene bridge), oU represents 5-methyl-2'-MOE-uridine, * represents a phosphorothioate internucleoside linkage, and wherein the oligonucleotide comprises a nucleobase sequence of CAGCGCCCACCAGUCA (SEQ ID NO: 21), wherein R 2Comprising an anti-TfR1 antibody (e.g., Fab), wherein the anti-TfR1 antibody (e.g., Fab) comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 selected from Table 2, optionally wherein the anti-TfR1 antibody (e.g., Fab) comprises a VH comprising the amino acid sequence of SEQ ID NO: 17 and a VL comprising the amino acid sequence of SEQ ID NO: 18, and optionally wherein the anti-TfR1 antibody (e.g., Fab) comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and a light chain comprising the amino acid sequence of SEQ ID NO: 20; and wherein in each complex, n1 is independently an integer (e.g., 1 or greater) representing the number of instances of the group enclosed by square brackets, wherein each instance of the group enclosed by square brackets is covalently linked to a bonding site represented by a lysine (K) residue of the antibody (e.g., Fab). In some embodiments, the antibody light chain constant region in the complex of the composition is independently covalently linked to the group included in the square brackets in Formula (Id) at the following bonding sites: a bonding site represented by K188 (based on Kabat numbering) of the light chain constant region of the antibody and / or a bonding site represented by K190 (based on Kabat numbering). For example, in some embodiments, the bonding site is represented by K188 (based on Kabat numbering) of the light chain constant region of the antibody. In some embodiments, the bonding site is represented by K190 (based on Kabat numbering) of the light chain constant region of the antibody. In some embodiments, the bonding site is represented by K188 (based on Kabat numbering) and K190 (based on Kabat numbering) of the light chain constant region of the antibody. In some embodiments, at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 80% to ...0%, at least 81% to 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 80% to 80%, at least 80% to 80% The light chain constant region of the antibody (0% to 95%, 80% to 90%, 85% to 98%, 85% to 95%, 85% to 90%, 90% to 98%, 90% to 95%, 95% to 97% or more) is independently covalently linked to the group enclosed by square brackets in Formula (Id) at the following linkage sites: the linkage site represented by K188 (based on Kabat numbering) and / or the linkage site represented by K190 (based on Kabat numbering) of the light chain constant region of the antibody.In some embodiments, about 1% to 15% (e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, or about 15%) of the antibody heavy chain constant region in the complex of the composition is independently covalently linked to the group in the square brackets in Formula (Id) at the following linkage site: the linkage site represented by K213 (based on Kabat numbering) of the heavy chain constant region of the antibody. In some embodiments, R. 2 The invention comprises an anti-TfR1 antibody (eg, Fab) comprising the sequence shown in Table 2. For example, in some embodiments, R 2 The invention comprises an antibody (e.g., Fab) comprising: a heavy chain complementary determining region 1 (CDR-H1) comprising the sequence shown in SEQ ID NO: 1, 7, or 12, a heavy chain complementary determining region 2 (CDR-H2) comprising the sequence shown in SEQ ID NO: 2, 8, or 13, and a heavy chain complementary determining region 3 (CDR-H3) comprising the sequence shown in SEQ ID NO: 3, 9, or 14; and / or a light chain complementary determining region 1 (CDR-L1) comprising the sequence shown in SEQ ID NO: 4, 10, or 15, a light chain complementary determining region 2 (CDR-L2) comprising the sequence shown in SEQ ID NO: 5 or 11, and a light chain complementary determining region 3 (CDR-L3) comprising the sequence shown in SEQ ID NO: 6 or 16. In some embodiments, R 2 An antibody (e.g., Fab) comprising a heavy chain variable region (VH) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 17, and / or a light chain variable region (VL) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 18. In some embodiments, R 2 The invention comprises an antibody (e.g., Fab) comprising a VH comprising the amino acid sequence of SEQ ID NO: 17 and / or a VL comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, R 2 The invention also includes an antibody (e.g., Fab) comprising a heavy chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 19 and / or a light chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 20. In some embodiments, R 2An antibody (e.g., Fab) comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and / or a light chain comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, in each complex, n1 is independently an integer (e.g., an integer from 1 to 27, 1 to 26, 1 to 10, 1 to 5, or 1 to 3). In some embodiments, in each complex, n1 is independently an integer of 1 or greater. In some embodiments, the compositions described herein further comprise a complex wherein n1 is 0. In some embodiments, the average value of n1 of the complexes of the composition is from 0.5 to 5 (e.g., from 0.5 to 5, from 1 to 5, from 1 to 4, from 1 to 3, from 3 to 5, from 0.5 to 4, from 0.5 to 3, from 0.5 to 2, from 0.5 to 1.5, from 0.5 to 1, from 0.7 to 1.5, from 1 to 1.6, from 1 to 1.5, from 1 to 1.4, from 1 to 1.3, from 1 to 1.2, from 1.1 to 1.5, from 0.8 to 2, from 0.8 to 1.5, from 0.8 to 1.3, from 0.8 to 1.2, from 0.8 to 1.1, from 0.9 to 3, from 0.9 to 2, from 0.9 to 1.8, from 0.9 to 1.6, from 0.9 to 1.5, from 0.9 to 1.4, from 0.9 to 1.3, or from 0.9 to 1.2).

[0212] In some embodiments, the compositions described herein comprise a complex comprising the structure of Formula (A):

[0213]

[0214] (A), wherein y is 0 to 15 (e.g., 3) and z is 0 to 15 (e.g., 4). In some embodiments, the antibody is an anti-TfR1 antibody (e.g., an anti-TfR1 antibody provided in Table 2), comprising a heavy chain comprising a heavy chain variable region (VH) and a heavy chain constant region, and a light chain comprising a light chain variable region (VL) and a light chain constant region. In some embodiments, the oligonucleotide comprises a 5'-XYZ-3' configuration and comprises the nucleobase sequence of SEQ ID NO: 21. In some embodiments, the amide adjacent to the antibody shown in the structure is produced by reaction with an amine of the antibody (e.g., lysine ε amine). In some embodiments, the complex described herein comprises an anti-TfR1 Fab covalently linked to the 5' end of the oligonucleotide via a bonding site represented by a lysine (K) residue of the antibody. In some embodiments, the antibody light chain constant region in the complex of the composition is independently covalently linked to the oligonucleotide at the following bonding sites: a bonding site represented by K188 (based on Kabat numbering) of the light chain constant region of the antibody and / or a bonding site represented by K190 (based on Kabat numbering). For example, in some embodiments, the bonding site is represented by K188 (based on Kabat numbering) of the light chain constant region of the antibody. In some embodiments, the bonding site is represented by K190 (based on Kabat numbering) of the light chain constant region of the antibody. In some embodiments, the bonding site is represented by K188 (based on Kabat numbering) and K190 (based on Kabat numbering) of the light chain constant region of the antibody. In some embodiments, at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, 80% to 98%, 80% to 95%, 80% to 90%, 85% to 98%, 85% to 95%, 85% to 90%, 90% to 98%, 90% to 95%, 95% to 97% or more) of the constant regions of the antibody light chains in the complex of the composition are independently covalently linked to the oligonucleotide at the following linkage sites: the linkage site represented by K188 (based on Kabat numbering) and / or the linkage site represented by K190 (based on Kabat numbering) of the antibody light chain constant region. In some embodiments, about 1% to 15% (e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, or about 15%) of the antibody heavy chain constant region in the complex of the composition are independently covalently linked to an oligonucleotide at the following linkage site: the linkage site represented by K213 (based on Kabat numbering) of the heavy chain constant region of the antibody.In some embodiments, the anti-TfR1 antibody comprises a sequence as shown in Table 2. For example, in some embodiments, the antibody comprises a heavy chain complementary determining region 1 (CDR-H1) comprising the sequence shown in SEQ ID NO: 1, 7 or 12, a heavy chain complementary determining region 2 (CDR-H2) comprising the sequence shown in SEQ ID NO: 2, 8 or 13, a heavy chain complementary determining region 3 (CDR-H3) comprising the sequence shown in SEQ ID NO: 3, 9 or 14; and / or comprises a light chain complementary determining region 1 (CDR-L1) comprising the sequence shown in SEQ ID NO: 4, 10 or 15, a light chain complementary determining region 2 (CDR-L2) comprising the sequence shown in SEQ ID NO: 5 or 11, and a light chain complementary determining region 3 (CDR-L3) comprising the sequence shown in SEQ ID NO: 6 or 16. In some embodiments, the antibody comprises a heavy chain variable region (VH) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 17, and / or comprises a light chain variable region (VL) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 18. In some embodiments, the anti-TfR1 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 17 and / or comprises a VL comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, the anti-TfR1 antibody comprises a heavy chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 19 and / or comprises a light chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 20. In some embodiments, the anti-TfR1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and / or comprises a light chain comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the antibody is a Fab fragment, a full length IgG, a Fab' fragment, a F(ab')2 fragment, a scFv, or a Fv.

[0215] In some embodiments, for example, in embodiments where the composition comprises a complex comprising a structure of Formula (A) or a structure comprising a group of Formula (Ia), (Ib), (Ic), or (Id), the linkage site is optionally represented by K at position 4 of the sequence motif DYEKHKVYA (SEQ ID NO: 27) of the light chain of the antibody of the complex described herein. In some embodiments, the linkage site is optionally represented by K at position 6 of the sequence motif DYEKHKVYA (SEQ ID NO: 27) of the light chain of the antibody of the complex described herein. In some embodiments, the linkage site is optionally represented by K at position 4 and / or K at position 6 of the sequence motif DYEKHKVYA (SEQ ID NO: 27) of the light chain of the antibody of the complex described herein. In some embodiments, in a composition comprising a plurality of complexes described herein, at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, 80% to 98%, 80% to 95%, 80% to 90%, 85% to 98%, 85% to 95%, 85% to 90%, 90% to 98%, 90% to 95%, 95% to 97% or more) of the constant regions of the antibody light chains in the complexes of the composition are independently covalently linked to an oligonucleotide at the following linkage site: a light chain constant region of an antibody comprising the sequence motif DYEKHKVYA (SEQ ID NO: 27). In some embodiments, the linkage site is optionally represented by K in the sequence motif VNHKPSN (SEQ ID NO: 28) of the heavy chain of an antibody of a complex described herein. In some embodiments, in a composition comprising a plurality of complexes described herein, about 1% to 15% (e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, or about 15%) of the antibody heavy chains in the complexes of the composition are independently covalently linked to an oligonucleotide at the following linkage site: a linkage site represented by a K residue in the motif VNHKPSN (SEQ ID NO: 28) of the heavy chain of an antibody.In some embodiments, in a composition comprising a plurality of complexes described herein, the average value of n1 of the complexes of the composition is from 0.5 to 5 (e.g., from 0.5 to 5, from 1 to 5, from 1 to 4, from 1 to 3, from 3 to 5, from 0.5 to 4, from 0.5 to 3, from 0.5 to 2, from 0.5 to 1.5, from 0.5 to 1, from 0.7 to 1.5, from 1 to 1.6, from 1 to 1.5, from 1 to 1.4, from 1 to 1.3, from 1 to 1.2, from 1.1 to 1.5, from 0.8 to 2, from 0.8 to 1.5, from 0.8 to 1.3, from 0.8 to 1.2, from 0.8 to 1.1, from 0.9 to 3, from 0.9 to 2, from 0.9 to 1.8, from 0.9 to 1.6, from 0.9 to 1.5, from 0.9 to 1.4, from 0.9 to 1.3, or from 0.9 to 1.2).

[0216] In some embodiments, the composition comprising the complex (i.e., multiple complexes) is formulated in a manner suitable for the methods described herein. In some embodiments, a composition comprising a muscle-targeted complex is delivered to a subject using a formulation that minimizes degradation, promotes delivery and / or (e.g., and) uptake, or provides additional beneficial properties for the complex in the formulation. Therefore, in some embodiments, a composition comprising a complex (e.g., multiple complexes comprising oligonucleotides covalently linked to Fab) is formulated with one or more pharmaceutically acceptable carriers and / or excipients. In some embodiments, a composition comprising a muscle-targeted complex (e.g., a complex comprising oligonucleotides covalently linked to Fab) is formulated with one or more pharmaceutically acceptable carriers and / or excipients in an aqueous solution. In some embodiments, a composition comprising multiple complexes and one or more pharmaceutically acceptable carriers and / or excipients may be lyophilized (e.g., for storage). In some embodiments, a lyophilized composition may be reconstituted (e.g., reconstituted with water) to be applied to a subject. In some embodiments, a composition comprising multiple complexes may be frozen (e.g., for storage). In some embodiments, a frozen composition may be thawed before being applied to a subject, for example, to produce an aqueous solution. The composition (e.g., in an aqueous solution, in a frozen composition, or in a lyophilized composition) can be suitably prepared so that when it is administered to a subject, whether administered into the immediate environment of the target cells or administered systemically, a sufficient amount of the complex is able to enter the target muscle cells.

[0217] In some embodiments, the composition is formulated to be compatible with its intended route of administration. Some examples of routes of administration include parenteral administration, such as intravenous, intradermal, subcutaneous administration. Typically, the route of administration is intravenous or subcutaneous.

[0218] Uses / treatment methods

[0219] As described herein, a complex comprising an anti-TfR1 antibody (e.g., Fab) covalently linked to a molecular payload (e.g., a DMPK targeting oligonucleotide) is effective in treating a subject with myotonic dystrophy (e.g., DM1). In some embodiments, the complex comprises a molecular payload that is an oligonucleotide, such as an oligonucleotide that promotes expression or activity reduction of DMPK (e.g., a mutant or wild-type DMPK RNA level reduction).

[0220] In some embodiments, the subject can be a human subject, a non-human primate subject, a rodent subject, or any suitable mammalian subject. In some embodiments, the subject can suffer from myotonic dystrophy, such as DM1. In some embodiments, the subject has a DMPK allele, which can optionally include a disease-associated repeat, such as a CTG trinucleotide repeat expansion. In some embodiments, the subject may have a DMPK allele having an expanded disease-associated repeat comprising about 2 to 10 repeating units, about 2 to 50 repeating units, about 2 to 100 repeating units, about 50 to 1,000 repeating units, about 50 to 500 repeating units, about 50 to 250 repeating units, about 50 to 100 repeating units, about 500 to 10,000 repeating units, about 500 to 5,000 repeating units, about 500 to 2,500 repeating units, about 500 to 1,000 repeating units, or about 1,000 to 10,000 repeating units. In some embodiments, the subject suffers from symptoms of DM1, such as muscle atrophy, muscle loss, excessive daytime sleepiness, or cognitive delay. In some embodiments, the subject does not suffer from symptoms of DM1. In some embodiments, the subject suffers from congenital myotonic dystrophy. In some embodiments, the subject is ambulatory. In some embodiments, the subject is non-ambulatory.

[0221] One aspect of the disclosure includes methods comprising administering to a subject an effective amount of a complex described herein. In some embodiments, an effective amount of a pharmaceutical composition comprising a complex comprising a muscle targeting agent covalently linked to a molecular payload may be administered to a subject in need of treatment.

[0222] In some embodiments, a pharmaceutical composition comprising a complex as described herein may 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, the pharmaceutical composition may be in solid form, aqueous form, or liquid form. In some embodiments, the aqueous or liquid form may be atomized or lyophilized. In some embodiments, the lyophilized form may be reconstituted with an aqueous or liquid solution.

[0223] Compositions for intravenous administration may include 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.). For intravenous injection, water-soluble antibodies can be administered by a drip method, by which a pharmaceutical preparation comprising an 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. Intramuscular preparations, such as sterile preparations in the form of suitable soluble salts of antibodies, may be dissolved in a pharmaceutical excipient such as water for injection, 0.9% saline, or 5% glucose solution and administered.

[0224] In some embodiments, a pharmaceutical composition comprising a complex containing a muscle targeting agent covalently linked to a molecular payload is administered by site-specific or local delivery techniques. Some examples of these techniques include implantable reservoir sources of the complex, local delivery catheters, site-specific carriers, direct injection, or direct application.

[0225] In some embodiments, a pharmaceutical composition comprising a complex containing a muscle targeting agent covalently linked to a molecular payload is administered at an effective concentration to confer a therapeutic effect on the subject. As recognized by those skilled in the art, the effective amount varies according to the severity of the disease, the unique characteristics of the subject being treated (e.g., age, physical condition, health or weight), the duration of treatment, the nature of any simultaneous treatments, the route of administration, and related factors. These related factors are known to those skilled in the art and can be resolved only by routine experimentation. In some embodiments, the effective concentration is the maximum dose that is considered safe for the patient. In some embodiments, the effective concentration will be the lowest possible concentration that provides maximum efficacy.

[0226] Empirical considerations, such as the half-life of the complex in the subject, will generally help determine the concentration of the pharmaceutical composition to use for treatment. The frequency of administration can be empirically determined and adjusted to maximize the effectiveness of the treatment.

[0227] The effectiveness of the treatment can be assessed using any suitable method. In some embodiments, the effectiveness of the treatment can be assessed by evaluation of observations of symptoms associated with myotonic dystrophy (e.g., muscle atrophy or weakness), by measurement of self-reported outcomes of the subject (e.g., mobility, self-care, daily activities, pain / discomfort, and anxiety / depression), or by quality of life indicators (e.g., life expectancy).

[0228] In some embodiments, a pharmaceutical composition described herein comprising a complex comprising a muscle targeting agent covalently linked to a molecular payload is administered to a subject at an effective concentration sufficient to modulate the activity or expression of a target gene by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% relative to a control (e.g., baseline level of gene expression prior to treatment).

[0229] Example

[0230] Example 1. Peptide mapping to determine the conjugation site of the oligonucleotide on the anti-TfR1 Fab-oligonucleotide conjugate

[0231] Peptide mapping was performed to determine the conjugation site of an anti-TfR1 conjugate comprising an anti-TfR1 Fab having the heavy and light chain sequences shown in Table 2, covalently linked to an antisense oligonucleotide (ASO) targeting DMPK via a linker comprising a valine-citrulline sequence (conjugated via lysine). The DMPK targeting ASO comprises a 5'-XYZ-3' configuration and comprises the nucleobase sequence of SEQ ID NO: 21. The conjugate comprises the structure of formula (Id):

[0232]

[0233] wherein +N represents an LNA (2'-4' methylene bridge) ribonucleoside, dN represents a 2'-deoxyribonucleoside, oN represents a 2'-O-methoxyethyl (MOE) modified ribonucleoside, oC represents 5-methyl-2'-MOE-cytidine, +C represents 5-methyl-2'-4'-bicyclo-cytidine (2'-4' methylene bridge), oU represents 5-methyl-2'-MOE-uridine, * represents a phosphorothioate internucleoside linkage, and wherein the oligonucleotide comprises a nucleobase sequence of CAGCGCCCACCAGUCA (SEQ ID NO: 21), and wherein R 2 is an anti-TfR1 Fab provided in Table 2, and wherein in each conjugate, n1 is independently an integer from 1 to 3.

[0234] Papain digestion

[0235] Oligonucleotides were cleaved from anti-TfR1 conjugates by digestion with papain. Prior to digestion, Thermo Scientific immobilized papain (P / N: 20341) was activated by adding 0.5 mL of 50% slurry to 4 mL of freshly prepared papain activation buffer (20 mM sodium phosphate, 10 mM EDTA, 20 mM cysteine, pH 7.0). The papain resin was mixed thoroughly and centrifuged at 3000 rcf for 5 minutes to pull out the resin. The supernatant was discarded and a second repeated wash was performed. After two washes, papain was resuspended to 50% slurry by adding 250 μL papain activation buffer. The anti-TfR1 conjugates that had undergone buffer were exchanged into papain digestion buffer (20 mM sodium phosphate, 10 mM EDTA, pH 7.0) using a Thermo Scientific Zeba desalting column (P / N: 89882). Before adding the conjugate, the column was balanced 3 times with 0.3mL buffer. The conjugate of 40 μL at a concentration of at least 8mg / mL was added to 100 μL of balanced immobilized papain resin. The sample was incubated with 1400rpm shaking for 1 hour at 40°C. After incubation, the papain resin was removed by adding each sample to a filter tube and centrifuging briefly until all liquid samples were thoroughly filtered. Another group of Zeba columns were balanced with LC-MS grade water (3 × 300 μμL). 100 μL of papain-digested conjugates were added to each column, and rotated at 1500rcf for 3 minutes to perform buffer exchange. 40 μg of material was diluted to 50 μL volumes with LC-MS water. Complete digestion of oligonucleotides was observed in all samples.

[0236] Mass spectra of intact mass of unlinked anti-TfR1 antibody Figure 1 The intact mass of the unlinked antibody determined by mass spectrometry was 47968.73 Da, and was consistent with the theoretical mass of an antibody with a post-translational modification (PTM) of 47968.01 Da (the theoretical mass of an antibody without a PTM is 47986.03 Da), which converts the first residue (Q) of the heavy chain into pyroglutamic acid. Figure 2The mass spectrum of the mass of the anti-TfR1 antibody-joint complex of the composition provided herein after cutting oligonucleotides by papain digestion is shown in. Two main peaks were observed, corresponding to a linker to antibody ratio (linker to antibody ratio, LAR) of 1 ("LAR1") and a LAR of 2 ("LAR2"). Next to each peak, the mass of the anti-TfR1 antibody-joint complex is shown, which is equal to the mass of the intact antibody (with pyroglutamic acid PTM) (47968.01Da) plus the mass of the joint (about 927.07Da / joint). The theoretical mass of the anti-TfR1 antibody-joint complex with LAR of 1 is 48895.08. The theoretical mass of the anti-TfR1 antibody-joint complex with LAR of 2 is 49822.15. Figure 1 The data shown in indicate that the anti-TfR1 conjugates of the tested compositions mainly include conjugates in which the antibody of the conjugate is linked to one DMPK-targeting ASO molecule and conjugates in which the antibody of the conjugate is linked to two DMPK-targeting ASO molecules.

[0237] Chymotrypsin digestion

[0238] The anti-TfR1 linker conjugate with the cleaved oligonucleotide was digested with immobilized chymotrypsin. 15 μL of Thermo SMART chymotrypsin resin (kit P / N: 60109104MBLPH) was added to an Eppendorf tube. 150 μL of chymotrypsin Smart digestion buffer was added. 40 μg of anti-TfR1 linker conjugate in the prepared 50 μL sample was added after papain digestion. The heating block was set to 70°C and mixed at 1400 rpm and incubated for 2.5 hours. After incubation, the papain resin was removed by adding each sample to a filter tube and briefly centrifuged until all liquid samples were thoroughly filtered. A stock solution of 500 mM tris(2-carboxyethyl)phosphine (TCEP) in water was prepared. The stock solution was added to the digested sample to a final concentration of 20 mM TCEP and incubated at room temperature for 30 minutes to reduce disulfides. The sample was concentrated by speedvac until the total sample volume was approximately 100 μL. Then 25 μL peptide samples (about 10 ug total peptides) were injected into a mass spectrometer for peptide mapping analysis. There was 100% sequence coverage in all chymotrypsin digests (data not shown), and papain-cleaved joints were identified in all conjugate groups. Oligonucleotides were then quantified according to the heavy and light chains of the antibody.

[0239] The summary of the percentage of occupancy of the oligonucleotide of each joint site is shown in Table 7 below.The hotspot joint sites are lysine 188 (K188) and lysine 190 (K190) in the light chain constant region in the composition based on Kabat numbering.In general, about 96% of the lysine residues corresponding to K188 and K190 are covalently linked to the oligonucleotide.Since 4% to 8% of the lysine residues corresponding to K213 are covalently linked to the oligonucleotide, some linkages are observed at the joint site lysine 213 (K213) based on Kabat numbering in the heavy chain constant region in the composition.A small amount of modification to other lysines is observed at very low levels (<1%).

[0240] Table 7.

[0241]

[0242] *Hotspot is K188 and / or K190

[0243] **Based on Kabat numbering

[0244] Example 2. In vivo tissue distribution of a conjugate comprising an anti-TfR1 Fab conjugated to a DMPK targeting oligonucleotide in the DM1 mouse model

[0245] A conjugate comprising an anti-TfR1 Fab covalently linked to a DMPK targeting oligonucleotide (ASO) as described in Example 1 (labeled in this example as "anti-TfR1 Fab-ASO conjugate") was tested in a mouse model expressing both human TfR1 and a human DMPK mutant containing expanded CUG repeats.

[0246] The conjugates were administered intravenously to mice at doses equivalent to 9.7 mg / kg ASO each on days 0 and 7. Tissue exposure of ASO was tested by hybrid ELISA (Burki et al., Nucleic Acid Ther. 2015 Oct; 25(5): 275-84; incorporated herein by reference), and ASO levels in tissues were plotted. Figure 3A , 3B , 3C and 3D show the amount of ASO in the heart, diaphragm, gastrocnemius or tibialis anterior muscle two weeks after the first injection, respectively. These results indicate that conjugates comprising anti-TfR1 antibodies (e.g., anti-TfR1 Fabs having VH and VL sequences provided in Table 2) are capable of delivering oligonucleotides (e.g., DMPK targeting ASOs) to a variety of muscle tissues after intravenous administration.

[0247] Example 3. Sustained knockdown of toxic human DMPK in hTfR1 / DMSXL homozygous mice 4 weeks after repeated administration of anti-TfR1 Fab-ASO conjugates

[0248] A conjugate comprising an anti-TfR1 Fab covalently linked to a DMPK targeting oligonucleotide (ASO) as described in Example 1 (labeled "anti-TfR1 Fab-ASO conjugate" in this example) was tested in a mouse model (hTfR1 / DMSXL mouse) expressing both human TfR1 and two copies of a mutant human DMPK transgene containing expanded CUG repeats.

[0249] On days 0 and 7, vehicle control (PBS) or 10 mg / kg ASO equivalent dose of anti-TfR1 Fab-ASO conjugate was administered to mice. Mice were sacrificed on day 28 (4 weeks after the first dose of anti-TfR1 Fab-ASO conjugate) and tissues were collected. RNA was extracted and selected tissue samples were fixed, paraffin-embedded and sectioned, followed by in situ hybridization. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) of RNA samples was performed to measure human DMPK and mouse Ppib (peptidyl prolyl isomerase) as internal controls. DMPK expression in Figures 4A to 4D The data are shown as geometric mean + / - standard deviation (n=6 to 9). The significance was assessed by Student's t-test (****P<0.0001).

[0250] Figure 4A It was shown that anti-TfR1 Fab-ASO conjugate knocked down DMPK expression in the heart by 49% relative to PBS-treated mice. Figure 4B It was shown that anti-TfR1 Fab-ASO conjugate knocked down DMPK expression in diaphragm muscle by 40% relative to PBS-treated mice. Figure 4C It was shown that anti-TfR1 Fab-ASO conjugate knocked down DMPK expression in tibialis anterior muscle by 49% relative to PBS-treated mice. Figure 4D It was shown that anti-TfR1 Fab-ASO conjugate knocked down DMPK expression in gastrocnemius muscle by 44% relative to PBS-treated mice.

[0251] Figure 5A and 5B It was shown that anti-TfR1 Fab-ASO conjugate reduced DMPK foci within myofiber nuclei. Figure 5A Reduced DMPK foci were shown by in situ hybridization, and Figure 5BQuantification of DMPK foci in fluorescence microscopy images is shown, demonstrating that the conjugate reduced foci area by 49%. Data are presented as mean + / - standard deviation (n=7). Significance was assessed by t-test (*P<0.05).

[0252] These results demonstrate that administration of anti-TfR1 Fab-ASO conjugates results in robust, sustained knockdown of human toxic DMPK in cardiac and skeletal muscle.

[0253] Example 4. Correction of splicing defects in hTfR1 / DMSXL homozygous mice by anti-TfR1 Fab-ASO conjugates

[0254] A conjugate comprising an anti-TfR1 Fab covalently linked to a DMPK targeting oligonucleotide (ASO) as described in Example 2 (labeled in this example as "anti-TfR1 Fab-ASO conjugate") was tested in a mouse model ("hTfR1 / DMSXL") that expresses both human TfR1 and two copies of a mutant human DMPK transgene containing expanded CUG repeats. These mice are known to exhibit splicing defects consistent with those observed in patients with DM1 (Huguet, et al. (2012) PLOS Genetics 8(11): e1003043). On days 0 and 7, mice were administered a vehicle control ("hTfR1 / DMSXL-PBS") or a 10 mg / kg ASO equivalent dose of anti-TfR1 Fab-ASO conjugate ("hTfR1 / DMSXL-conjugate"). Mice expressing only human TfR1 but not mutant human DMPK transgenes (hTfR1 mice) and mice treated with PBS ("hTfR1-PBS") were used as another control to determine the extent of the splicing phenotype in hTfR1 / DMSXL mice and to assess the extent of the effect of the conjugate on splicing. Mice were sacrificed on day 28 (four weeks after administration of the first dose of anti-TfR1 Fab-ASO conjugate), tissues were collected and RNA was extracted. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed to measure exon inclusion in a panel of RNAs known to be mis-spliced ​​during DM1 progression in humans and mice (Nakamori, et al. (2013) Ann. Neurol. 74(6): 862-872; Huguet, et al. (2012) PLOS Genetics 8(11): e1003043). Exon inclusion was calculated as percent splicedin (PSI) normalized to each spliced ​​RNA marker and was calculated using the data from heart ( Figure 6 ), diaphragm( Figure 7 ), tibialis anterior ( Figure 8 ) and gastrocnemius ( Fig. 9 ) were used to calculate the composite splicing index based on the normalized PSI values ​​of the splicing markers in . The composite splicing index was calculated as previously described (Tanner MK, et al. (2021) Nucleic Acids Res. 49: 2240-2254) and is shown as mean / -standard deviation.

[0255] Figure 6 It is shown that anti-TfR1 Fab-ASO conjugate corrects splicing in hTfR1 / DMSXL mouse heart tissue, as shown in the composite splicing index data. The normalized PSI values ​​used to generate the composite splicing index data show correction of Mbnl2 exon 6 (E6) and Nfix E7 splicing in hTfR1 / DMSXL mouse heart tissue by treatment with anti-TfR1 Fab-ASO conjugate, but not correction of Ldb3 E11 splicing. Figure 6 The composite splicing index data shown in are based on Ldb3E11, Mbnl2 E6, and Nfix E7 splicing data; Bin1 E11, Dtna E12, Insr E11, and Mbnl2 E5 were not included because their normalized PSI values ​​in cardiac tissue were not altered in hTfR1 / DMSXL mice relative to hTfR1 mice under the experimental conditions tested.

[0256] Figure 7 Anti-TfR1 Fab-ASO conjugates are shown to correct splicing in hTfR1 / DMSXL mouse diaphragm tissue, as shown in the composite splicing index data. Normalized PSI values ​​used to generate the composite splicing index data show correction of Bin1 E11, Insr E11, Ldb3 E11, and Nfix E7 splicing in hTfR1 / DMSXL mouse diaphragm tissue by treatment with anti-TfR1 Fab-ASO conjugates. Figure 7 The composite splicing index data shown in are based on Bin1 E11, Insr E11, Ldb3 E11, and Nfix E7 splicing data; Dtna E12, Mbnl2 E5, Mbnl2 E6, and Ttn E313 are not included because their normalized PSI values ​​in diaphragm tissue were not altered in hTfR1 / DMSXL mice relative to hTfR1 mice under the experimental conditions tested.

[0257] Figure 8Anti-TfR1 Fab-ASO conjugates are shown to correct splicing in hTfR1 / DMSXL mouse tibialis anterior muscle tissue, as shown in the composite splicing index data. The normalized PSI values ​​used to generate the composite splicing index data show correction of Bin1 E11, Ldb3 E11, and Nfix E7 splicing in hTfR1 / DMSXL mouse tibialis anterior muscle tissue by treatment with anti-TfR1 Fab-ASO conjugates, but not correction of Mbnl2 E6 splicing. Figure 8 The composite splicing index data shown in are based on Bin1 E11, Ldb3 E11, Mbnl2 E6, and Nfix E7 splicing data; Dtna E12, Insr E11, Mbnl2 E5, and Ttn E313 are not included because their normalized PSI values ​​in tibialis anterior muscle tissue were not altered in hTfR1 / DMSXL mice relative to hTfR1 mice under the experimental conditions tested.

[0258] Fig. 9 It is shown that anti-TfR1 Fab-ASO conjugate corrects splicing in gastrocnemius muscle tissue of hTfR1 / DMSXL mice, as shown in the composite splicing index data. The normalized PSI values ​​used to generate the composite splicing index data show the correction of Mbnl2 E6, Nfix E7 and TtnE313 splicing in gastrocnemius muscle tissue of hTfR1 / DMSXL mice by treatment with anti-TfR1 Fab-ASO conjugate. Fig. 9 The composite splicing index data shown in are based on Mbnl2 E6, Nfix E7, and Ttn E313 splicing data; Bin1 E11, Dtna E12, Insr E11, Ldb3 E11, and Mbnl2 E5 are not included because their normalized PSI values ​​in gastrocnemius muscle tissue were not altered in hTfR1 / DMSXL mice relative to hTfR1 mice under the experimental conditions tested.

[0259] These results suggest that administration of anti-TfR1 Fab-ASO conjugates facilitates the correction of DM1 splicing defects in cardiac and skeletal muscles.

[0260] Example 5. DMPK knockdown in non-human primate and DM1 patient myotubes

[0261] The conjugate comprising an anti-TfR1 Fab covalently linked to a DMPK targeting oligonucleotide (ASO) as described in Example 2 (labeled in this example as "anti-TfR1 Fab-ASO conjugate") was tested in human DM1 patient myotubes (32F cells) and non-human primate (NHP) myotubes. The DM1 patient myotubes used expressed both wild-type DMPK mRNA and a mutant DMPK mRNA containing 380 CUG repeats. The NHP myotubes used expressed only wild-type DMPK.

[0262] DM1 patient cells or NHP cells were seeded into 96-well plates in growth medium at a density of 50,000 cells / well and allowed to recover overnight. The next day, the growth medium was changed to low serum differentiation medium and the cells were treated with conjugates at concentrations equivalent to 125nM, 250nM, or 500nM ASO. The cells were incubated for 10 days and then cDNA was synthesized using the Cells-to-Ct kit, with crude cell lysate as the source of total RNA.

[0263] The cDNA was used to assess total DMPK knockdown using Taqman PCR. Data were normalized to PPIB expression and expressed using 2 -ΔΔCt The knockdown of DMPK compared to PBS-treated controls ("Vehicle") was determined. Fig.10 Data shown in are presented as mean DMPK expression + standard deviation relative to species-matched vehicle controls (n=4 replicates per condition).

[0264] The results showed that when treated at physiologically relevant concentrations, anti-TfR1 Fab-ASO conjugates achieved knockdown of DMPK expression in both normal NHP myotubes and DM1 patient myotubes, with a higher degree of knockdown of DMPK expression in DM1 patient cells (expressing both wild-type DMPK mRNA and DMPK mRNA containing 380 CUG repeats) compared to NHP cells (expressing only wild-type DMPK mRNA) ( Fig.10 ). At an ASO equivalent concentration of 125 nM, the conjugate achieved approximately 40% DMPK knockdown in NHP myotubes and approximately 65% ​​DMPK knockdown in DM1 patient myotubes relative to the vehicle-only control. At an ASO equivalent concentration of 250 nM, the conjugate achieved approximately 45% DMPK knockdown in NHP myotubes and approximately 80% DMPK knockdown in DM1 patient myotubes relative to the vehicle-only control. At an ASO equivalent concentration of 500 nM, the conjugate achieved approximately 60% DMPK knockdown in NHP myotubes and approximately 90% DMPK knockdown in DM1 patient myotubes relative to the vehicle-only control.

[0265] These results indicate that the conjugate comprising an anti-TfR1 Fab covalently linked to a DMPK targeting oligonucleotide can achieve a greater degree of DMPK knockdown in human myotubes expressing wild-type DMPK mRNA and mutant DMPK mRNA (with expanded CUG repeats) relative to cynomolgus monkey myotubes expressing wild-type DMPK.

[0266] Additional embodiments

[0267] 1. A composition comprising a plurality of complexes comprising an antibody covalently linked to one or more oligonucleotides, each oligonucleotide being covalently linked at a linkage site represented by a lysine (K) residue of the antibody,

[0268] wherein the antibody comprises a heavy chain comprising a heavy chain variable region (VH) and a heavy chain constant region, and a light chain comprising a light chain variable region (VL) and a light chain constant region,

[0269] wherein the oligonucleotide comprises a complementary region to DMPK RNA,

[0270] And wherein at least 80% of the antibody light chain constant region in the complex of the composition is independently covalently linked to the oligonucleotide at the following linkage sites: the linkage site represented by K188 (based on Kabat numbering) and / or the linkage site represented by K190 (based on Kabat numbering) of the light chain constant region of the antibody.

[0271] 2. A composition comprising a plurality of complexes comprising an antibody covalently linked to one or more oligonucleotides, each oligonucleotide being covalently linked at a linkage site represented by a lysine (K) residue of the antibody,

[0272] wherein the antibody comprises a heavy chain comprising a heavy chain variable region (VH) and a heavy chain constant region, and a light chain comprising a light chain variable region (VL) and a light chain constant region,

[0273] wherein the oligonucleotide comprises a 5'-XYZ-3' configuration, wherein X and Z are flanking regions comprising one or more modified nucleosides, and Y is a spacer region comprising one or more 2'-deoxyribonucleosides,

[0274] And wherein at least 80% of the antibody light chain constant region in the complex of the composition is independently covalently linked to the oligonucleotide at the following linkage sites: the linkage site represented by K188 (based on Kabat numbering) and / or the linkage site represented by K190 (based on Kabat numbering) of the light chain constant region of the antibody.

[0275] 3. The composition of embodiment 1 or embodiment 2, wherein approximately 1% to 15% of the antibody heavy chain constant region in the complex of the composition is independently covalently linked to the oligonucleotide at the following linkage site: the linkage site represented by K213 (based on Kabat numbering) of the heavy chain constant region of the antibody.

[0276] 4. A composition comprising a plurality of complexes comprising an antibody covalently linked to one or more oligonucleotides, each oligonucleotide being covalently linked at a linkage site represented by a lysine (K) residue of the antibody,

[0277] wherein the antibody comprises a heavy chain and a light chain,

[0278] wherein the oligonucleotide comprises a complementary region to DMPK RNA,

[0279] And wherein at least 80% of the antibody light chains in the complex of the composition are independently covalently linked to the oligonucleotide at the following linkage site: the linkage site represented by the lysine (K) residue in the sequence motif DYEKHKVYA (SEQ ID NO: 27) of the antibody light chain.

[0280] 5. A composition comprising a plurality of complexes comprising an antibody covalently linked to one or more oligonucleotides, each oligonucleotide being covalently linked at a linkage site represented by a lysine (K) residue of the antibody,

[0281] wherein the antibody comprises a heavy chain and a light chain,

[0282] wherein the oligonucleotide comprises a 5'-XYZ-3' configuration, wherein X and Z are flanking regions comprising one or more modified nucleosides, and Y is a spacer region comprising one or more 2'-deoxyribonucleosides,

[0283] And wherein at least 80% of the antibody light chains in the complex of the composition are independently covalently linked to the oligonucleotide at the following linkage site: the linkage site represented by the lysine (K) residue in the sequence motif DYEKHKVYA (SEQ ID NO: 27) of the antibody light chain.

[0284] 6. The composition of embodiment 4 or embodiment 5, wherein approximately 1% to 15% of the antibody heavy chains in the complex of the composition are independently covalently linked to the oligonucleotide at the following linkage site: a linkage site represented by a lysine (K) residue in the sequence motif VNHKPSN (SEQ ID NO: 28) of the heavy chain of the antibody.

[0285] 7. The composition of any one of embodiments 1 to 6, wherein the antibody is an anti-transferrin receptor (TfR1) antibody, comprising: a heavy chain complementary determining region 1 (CDR-H1) comprising the sequence shown in SEQ ID NO: 1, 7 or 12, a heavy chain complementary determining region 2 (CDR-H2) comprising the sequence shown in SEQ ID NO: 2, 8 or 13, a heavy chain complementary determining region 3 (CDR-H3) comprising the sequence shown in SEQ ID NO: 3, 9 or 14, a light chain complementary determining region 1 (CDR-L1) comprising the sequence shown in SEQ ID NO: 4, 10 or 15, a light chain complementary determining region 2 (CDR-L2) comprising the sequence shown in SEQ ID NO: 5 or 11, and a light chain complementary determining region 3 (CDR-L3) comprising the sequence shown in SEQ ID NO: 6 or 16.

[0286] 8. The composition of any one of embodiments 1 to 7, wherein the oligonucleotide comprises a 5'-XYZ-3' configuration, wherein X and Z each comprise 4 linked nucleosides, and Y comprises 8 linked nucleosides.

[0287] 9. The composition of any one of embodiments 1 to 8, wherein the oligonucleotide comprises a 5'-XYZ-3' configuration of LLEE-D8-EELL, wherein "L" represents an LNA nucleoside, "E" represents a 2'-MOE modified ribonucleoside, and "D" represents a 2'-deoxyribonucleoside.

[0288] 10. The composition of any one of embodiments 1 to 9, wherein the oligonucleotide comprises the nucleobase sequence of CAGCGCCCACCAGUCA (SEQ ID NO: 21).

[0289] 11. The composition of any one of embodiments 1 to 10, wherein the oligonucleotide comprises the structure +C*+A*oG*oC*dG*dC*dC*dC*dA*dC*dC*dA*oG*oU*+C*+A (SEQ ID NO: 21), wherein +N represents LNA (2'-4' methylene bridge) ribonucleoside, dN represents 2'-deoxyribonucleoside, oN represents 2'-MOE modified ribonucleoside, oC represents 5-methyl-2'-MOE-cytidine, +C represents 5-methyl-2'-4'-bicyclo-cytidine (2'-4' methylene bridge), oU represents 5-methyl-2'-MOE-uridine, and * represents phosphorothioate internucleoside linkages.

[0290] 12. A composition comprising a plurality of complexes, wherein the complexes comprise formula (I): [R 1 ] n1 -R 2 The structure of each R1 Independently comprising groups of formula (Ia):

[0291]

[0292] Where R 2 Comprising an anti-transferrin receptor (TfR1) antibody, the antibody comprising a heavy chain comprising a heavy chain variable region (VH) and a heavy chain constant region, and a light chain comprising a light chain variable region (VL) and a light chain constant region; and wherein the anti-TfR1 antibody comprises: a heavy chain complementary determining region 1 (CDR-H1) comprising the sequence shown in SEQ ID NO: 1, 7 or 12, a heavy chain complementary determining region 2 (CDR-H2) comprising the sequence shown in SEQ ID NO: 2, 8 or 13, a heavy chain complementary determining region 3 (CDR-H3) comprising the sequence shown in SEQ ID NO: 3, 9 or 14, a light chain complementary determining region 1 (CDR-L1) comprising the sequence shown in SEQ ID NO: 4, 10 or 15, a light chain complementary determining region 2 (CDR-L2) comprising the sequence shown in SEQ ID NO: 5 or 11, and a light chain complementary determining region 3 (CDR-L3) comprising the sequence shown in SEQ ID NO: 6 or 16;

[0293] R 3 a structure comprising +C*+A*oG*oC*dG*dC*dC*dC*dA*dC*dC*dA*oG*oU*+C*+A (SEQ ID NO: 21), wherein +N represents LNA (2'-4' methylene bridge) ribonucleoside, dN represents 2'-deoxyribonucleoside, oN represents 2'-MOE modified ribonucleoside, oC represents 5-methyl-2'-MOE-cytidine, +C represents 5-methyl-2'-4'-bicyclo-cytidine (2'-4' methylene bridge), oU represents 5-methyl-2'-MOE-uridine, and * represents phosphorothioate internucleoside linkage;

[0294] Each R 1 At the connection point A, the linkage site represented by the lysine (K) residue of the anti-TfR1 antibody is connected to R 2 covalently linked, and wherein at least 80% of the light chain constant regions of the anti-TfR1 antibodies of the complex in the composition independently bind to R at the following linkage sites: 1 Covalently linked: a linkage site represented by K188 (based on Kabat numbering) and / or a linkage site represented by K190 (based on Kabat numbering) of the light chain constant region of the anti-TfR1 antibody; and

[0295] wherein in each of the plurality of complexes, n1 is independently an integer of 1 or greater, which represents R 1 The number of instances of .

[0296] 13. A composition comprising a plurality of complexes, wherein the complexes comprise formula (I): [R 1 ] n1 -R 2 The structure of each R 1 Independently comprising groups of formula (Ib):

[0297]

[0298] wherein +N represents LNA (2'-4' methylene bridge) ribonucleoside, dN represents 2'-deoxyribonucleoside, oN represents 2'-MOE modified ribonucleoside, oC represents 5-methyl-2'-MOE-cytidine, +C represents 5-methyl-2'-4'-bicyclo-cytidine (2'-4' methylene bridge), oU represents 5-methyl-2'-MOE-uridine, and * represents phosphorothioate internucleoside linkage, and R 1 The oligonucleotide comprises the nucleobase sequence of CAGCGCCCACCAGUCA (SEQ ID NO: 21);

[0299] R 2 Comprising an anti-transferrin receptor (TfR1) antibody, the anti-transferrin receptor (TfR1) antibody comprising a heavy chain comprising a heavy chain variable region (VH) and a heavy chain constant region, and a light chain comprising a light chain variable region (VL) and a light chain constant region; and wherein the anti-TfR1 antibody comprises: a heavy chain complementary determining region 1 (CDR-H1) comprising the sequence shown in SEQ ID NO: 1, 7 or 12, a heavy chain complementary determining region 2 (CDR-H2) comprising the sequence shown in SEQ ID NO: 2, 8 or 13, a heavy chain complementary determining region 3 (CDR-H3) comprising the sequence shown in SEQ ID NO: 3, 9 or 14, a light chain complementary determining region 1 (CDR-L1) comprising the sequence shown in SEQ ID NO: 4, 10 or 15, a light chain complementary determining region 2 (CDR-L2) comprising the sequence shown in SEQ ID NO: 5 or 11, and a light chain complementary determining region 3 (CDR-L3) comprising the sequence shown in SEQ ID NO: 6 or 16; and

[0300] Each R 1 At the connection point A, the linkage site represented by the lysine (K) residue of the anti-TfR1 antibody is connected to R 2 covalently linked, and wherein at least 80% of the light chain constant regions of the anti-TfR1 antibodies of the complex in the composition independently bind to R at the following linkage sites: 1Covalently linked: a linkage site represented by K188 (based on Kabat numbering) and / or a linkage site represented by K190 (based on Kabat numbering) of the light chain constant region of the anti-TfR1 antibody; and

[0301] wherein in each of the plurality of complexes, n1 is independently an integer of 1 or greater, which represents R 1 The number of instances of .

[0302] 14. A composition comprising a plurality of complexes, wherein the complexes comprise formula (I): [R 1 ] n1 -R 2 The structure of each R 1 A group comprising formula (Ic):

[0303]

[0304] R 2 Comprising an anti-transferrin receptor (TfR1) antibody, the anti-transferrin receptor (TfR1) antibody comprising a heavy chain comprising a heavy chain variable region (VH) and a heavy chain constant region, and a light chain comprising a light chain variable region (VL) and a light chain constant region; and wherein the anti-TfR1 antibody comprises: a heavy chain complementary determining region 1 (CDR-H1) comprising the sequence shown in SEQ ID NO: 1, 7 or 12, a heavy chain complementary determining region 2 (CDR-H2) comprising the sequence shown in SEQ ID NO: 2, 8 or 13, a heavy chain complementary determining region 3 (CDR-H3) comprising the sequence shown in SEQ ID NO: 3, 9 or 14, a light chain complementary determining region 1 (CDR-L1) comprising the sequence shown in SEQ ID NO: 4, 10 or 15, a light chain complementary determining region 2 (CDR-L2) comprising the sequence shown in SEQ ID NO: 5 or 11, and a light chain complementary determining region 3 (CDR-L3) comprising the sequence shown in SEQ ID NO: 6 or 16; and

[0305] Each R 1 At the connection point A, the linkage site represented by the lysine (K) residue of the anti-TfR1 antibody is connected to R 2 covalently linked, and wherein at least 80% of the light chain constant regions of the anti-TfR1 antibodies of the complex in the composition independently bind to R at the following linkage sites: 1 Covalently linked: a linkage site represented by K188 (based on Kabat numbering) and / or a linkage site represented by K190 (based on Kabat numbering) of the light chain constant region of the anti-TfR1 antibody; and

[0306] wherein in each of the plurality of complexes, n1 is independently an integer of 1 or greater, which represents R 1 The number of instances of .

[0307] 15. The composition of any one of embodiments 12 to 14, wherein 85% to 98% of the light chain constant regions of the anti-TfR1 antibodies in the complex in the composition independently bind to R at the following bonding sites: 1 Covalent linkage: the bonding site represented by K188 (based on Kabat numbering) and / or the bonding site represented by K190 (based on Kabat numbering) of the light chain constant region of the anti-TfR1 antibody.

[0308] 16. The composition of any one of embodiments 12 to 15, wherein 95% to 97% of the light chain constant regions of the anti-TfR1 antibodies in the complex in the composition independently bind to R at the following bonding sites: 1 Covalent linkage: the linkage site represented by K188 (based on Kabat numbering) and / or the linkage site represented by K190 (based on Kabat numbering) of the light chain constant region of the anti-TfR1 antibody.

[0309] 17. The composition of any one of embodiments 12 to 16, wherein 1% to 15% of the heavy chain constant regions of the anti-TfR1 antibodies in the complex in the composition independently bind to R at the following bonding sites: 1 Covalent linkage: The bonding site represented by K213 (based on Kabat numbering) of the heavy chain constant region of the anti-TfR1 antibody.

[0310] 18. The composition of any one of embodiments 12 to 17, wherein 4% to 8% of the heavy chain constant regions of the anti-TfR1 antibodies in the complex in the composition independently bind to R at the following bonding sites: 1 Covalent linkage: The bonding site represented by K213 (based on Kabat numbering) of the heavy chain constant region of the anti-TfR1 antibody.

[0311] 19. The composition of any one of embodiments 1 to 18, wherein the antibody is a Fab fragment, a full-length IgG, a Fab' fragment, or a F(ab')2 fragment.

[0312] 20. The composition of any one of embodiments 1 to 19, wherein the antibody is a Fab fragment.

[0313] 21. The composition of any one of embodiments 1 to 20, wherein the VH comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 17; and / or wherein the VL comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 18.

[0314] 22. The composition of any one of embodiments 1 to 21, wherein the antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 17 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 18.

[0315] 23. The composition of any one of embodiments 1 to 22, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 19, and the light chain comprises the amino acid sequence of SEQ ID NO: 20.

[0316] 24. The composition of any one of embodiments 1 to 23, further comprising one or more antibodies that are not covalently linked to the oligonucleotide.

[0317] 25. The composition of embodiment 24, wherein the average value of n1 of the complexes in the composition is 0.5 to 5.

[0318] 26. A method of reducing DMPK expression in a subject, the method comprising administering to the subject an effective amount of the composition of any one of embodiments 1 to 25.

[0319] 27. A method of treating myotonic dystrophy in a subject, the method comprising administering to the subject an effective amount of the composition of any one of embodiments 1 to 25.

[0320] 28. The method of embodiment 26 or embodiment 27, wherein the subject has an expansion of a disease-associated repeat of a DMPK allele associated with myotonic dystrophy.

[0321] 29. The method described in embodiment 28, wherein the disease-associated repeat comprises repeating units of a CTG trinucleotide sequence.

[0322] 30. The method of any one of embodiments 26 to 29, wherein the complex reduces DMPK expression in the subject.

[0323] 31. The method of embodiment 30, wherein reducing DMPK expression comprises reducing the level of DMPK mRNA in muscle cells, optionally wherein the DMPK mRNA is a mutant DMPK mRNA.

[0324] Equivalent schemes and terminology

[0325] The disclosure described illustratively herein can be appropriately practiced in the absence of any one or more elements, one or more limitations not specifically disclosed herein. Thus, for example, in each case herein, any one of the terms "comprising / including", "consisting essentially of" and "consisting of" can be replaced with any one of the other two terms. The terms and expressions adopted are used as descriptive rather than restrictive terms, and the use of such terms and expressions is not intended to exclude any equivalent form or part thereof of the features shown and described, but it should be recognized that various modifications can be made within the scope of the disclosure. Therefore, it should be understood that although the disclosure has been specifically disclosed through some preferred embodiments, optional features, modifications and changes of the concepts disclosed herein may be adopted by those skilled in the art, and such modifications and changes are considered to be within the scope of the disclosure.

[0326] In addition, where features or aspects of the disclosure are described in terms of Markush groups or other alternative groupings, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group or other grouping.

[0327] It will be appreciated that in some embodiments, reference may be made to the sequences shown in the sequence listing when describing the structure of an oligonucleotide or other nucleic acid. In such embodiments, the actual oligonucleotide or other nucleic acid may have one or more substituted nucleotides (e.g., RNA counterparts of DNA nucleotides or DNA counterparts of RNA nucleotides) and / or (e.g., and) one or more modified nucleotides and / or (e.g., and) one or more modified internucleoside linkages and / or (e.g., and) one or more other modifications compared to the specified sequence while retaining complementary properties that are substantially identical or similar to the specified sequence.

[0328] Unless otherwise specified herein or clearly contradictory to the context, nouns modified by quantifiers will be interpreted as one / kind or more / kinds in the context of describing the present invention (especially in the context of the appended claims). Unless otherwise specified, the terms "comprising", "having", "including" and "containing" will be interpreted as open terms (i.e., meaning "including but not limited to"). Unless otherwise specified herein, the recording of the value range herein is only intended to be used as a shorthand method for referring to each individual value falling within the range, and each individual value is incorporated into the specification as if it is recorded separately herein. Unless otherwise specified, the range of values ​​herein includes their endpoints (e.g., the range of X to Y includes values ​​X and Y). It should be understood that the recording of the value from X to Y herein indicates that the specific value falls within the range of X to Y. Unless otherwise specified herein or clearly contradictory to the context in other cases, all methods described herein can be performed in any suitable order. Unless otherwise specified, the use of any and all examples or exemplary language (such as "for example") provided herein is only intended to better illustrate the present invention, and does not limit the scope of the present invention. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0329] Several embodiments of the invention are described herein. Variations of those embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description.

[0330] The inventor expects that the technician adopts such changes when appropriate, and the inventor hopes to practice the present invention in a manner other than specifically described herein. Therefore, as allowed by applicable law, the present invention includes all modifications and equivalents of the subject matter recorded in the appended claims. In addition, unless otherwise specified in this article or in other cases clearly contradictory with the context, the present invention covers any combination of the above-mentioned elements in all possible changes. Those skilled in the art will recognize or only use routine experiments to determine many equivalents of the specific embodiments of the invention described herein. Such equivalents are intended to be covered by the appended claims.

Claims

1. A composition comprising a plurality of complexes comprising an antibody covalently linked to one or more oligonucleotides, each oligonucleotide being covalently linked at a linkage site represented by a lysine (K) residue of the antibody, wherein the antibody comprises a heavy chain comprising a heavy chain variable region (VH) and a heavy chain constant region, and a light chain comprising a light chain variable region (VL) and a light chain constant region, wherein the oligonucleotide comprises a complementary region to DMPK RNA, And among them, At least 80% of the antibody light chain constant region in the complex of the composition is independently covalently linked to the oligonucleotide at the following linkage sites: the linkage site represented by K188 (based on Kabat numbering) and / or the linkage site represented by K190 (based on Kabat numbering) of the light chain constant region of the antibody.

2. A composition comprising a plurality of complexes comprising an antibody covalently linked to one or more oligonucleotides, each oligonucleotide being covalently linked at a linkage site represented by a lysine (K) residue of the antibody, wherein the antibody comprises a heavy chain comprising a heavy chain variable region (VH) and a heavy chain constant region, and a light chain comprising a light chain variable region (VL) and a light chain constant region, wherein the oligonucleotide comprises a 5'-XYZ-3' configuration, wherein X and Z are flanking regions comprising one or more modified nucleosides, and Y is a spacer region comprising one or more 2'-deoxyribonucleosides, And among them, At least 80% of the antibody light chain constant region in the complex of the composition is independently covalently linked to the oligonucleotide at the following linkage sites: the linkage site represented by K188 (based on Kabat numbering) and / or the linkage site represented by K190 (based on Kabat numbering) of the light chain constant region of the antibody.

3. The composition of claim 1 or claim 2, wherein approximately 1% to 15% of the antibody heavy chain constant region in the complex of the composition is independently covalently linked to an oligonucleotide at the following linkage site: the linkage site represented by K213 (based on Kabat numbering) of the heavy chain constant region of the antibody.

4. The composition of any one of claims 1 to 3, wherein the antibody is an anti-transferrin receptor (TfR1) antibody, comprising: a heavy chain complementary determining region 1 (CDR-H1) comprising the sequence shown in SEQ ID NO: 1, 7 or 12, a heavy chain complementary determining region 2 (CDR-H2) comprising the sequence shown in SEQ ID NO: 2, 8 or 13, a heavy chain complementary determining region 3 (CDR-H3) comprising the sequence shown in SEQ ID NO: 3, 9 or 14, a light chain complementary determining region 1 (CDR-L1) comprising the sequence shown in SEQ ID NO: 4, 10 or 15, a light chain complementary determining region 2 (CDR-L2) comprising the sequence shown in SEQ ID NO: 5 or 11, and a light chain complementary determining region 3 (CDR-L3) comprising the sequence shown in SEQ ID NO: 6 or 16.

5. The composition of any one of claims 1 to 4, wherein the oligonucleotide comprises a 5′-XYZ-3′ configuration, wherein X and Z each comprise 4 linked nucleosides, and Y comprises 8 linked nucleosides, optionally wherein the oligonucleotide comprises a 5′-XYZ-3′ configuration of LLEE-D8-EELL, wherein “L” represents an LNA nucleoside, “E” represents a 2′-MOE modified ribonucleoside, and “D” represents a 2′-deoxyribonucleoside.

6. The composition of any one of claims 1 to 5, wherein the oligonucleotide comprises a nucleobase sequence of CAGCGCCCACCAGUCA (SEQ ID NO: 21), optionally wherein the oligonucleotide comprises the structure +C*+A*oG*oC*dG*dC*dC*dC*dA*dC*dC*dA*oG*oU*+C*+A (SEQ ID NO: 21), wherein +N represents LNA (2'-4' methylene bridge) ribonucleoside, dN represents 2'-deoxyribonucleoside, oN represents 2'-MOE modified ribonucleoside, oC represents 5-methyl-2'-MOE-cytidine, +C represents 5-methyl-2'-4'-bicyclo-cytidine (2'-4' methylene bridge), oU represents 5-methyl-2'-MOE-uridine, and * represents a phosphorothioate internucleoside linkage.

7. A composition comprising a plurality of complexes, wherein the complexes comprise formula (I): [R 1 ] n1 -R 2 The structure of each R 1 Independently comprising groups of formula (Ia): Where R 2 Comprising an anti-transferrin receptor (TfR1) antibody, the anti-transferrin receptor (TfR1) antibody comprising a heavy chain comprising a heavy chain variable region (VH) and a heavy chain constant region, and a light chain comprising a light chain variable region (VL) and a light chain constant region; and wherein the anti-TfR1 antibody comprises: a heavy chain complementary determining region 1 (CDR-H1) comprising the sequence shown in SEQ ID NO: 1, 7 or 12, a heavy chain complementary determining region 2 (CDR-H2) comprising the sequence shown in SEQ ID NO: 2, 8 or 13, a heavy chain complementary determining region 3 (CDR-H3) comprising the sequence shown in SEQ ID NO: 3, 9 or 14, a light chain complementary determining region 1 (CDR-L1) comprising the sequence shown in SEQ ID NO: 4, 10 or 15, a light chain complementary determining region 2 (CDR-L2) comprising the sequence shown in SEQ ID NO: 5 or 11, and a light chain complementary determining region 3 (CDR-L3) comprising the sequence shown in SEQ ID NO: 6 or 16; Where R 3 a structure comprising +C*+A*oG*oC*dG*dC*dC*dC*dA*dC*dC*dA*oG*oU*+C*+A (SEQ ID NO: 21), wherein +N represents LNA (2'-4' methylene bridge) ribonucleoside, dN represents 2'-deoxyribonucleoside, oN represents 2'-MOE modified ribonucleoside, oC represents 5-methyl-2'-MOE-cytidine, +C represents 5-methyl-2'-4'-bicyclo-cytidine (2'-4' methylene bridge), oU represents 5-methyl-2'-MOE-uridine, and * represents phosphorothioate internucleoside linkage; Each R 1 At the connection point A, the linkage site represented by the lysine (K) residue of the anti-TfR1 antibody is connected to R 2 covalently linked, and wherein at least 80% of the anti-TfR1 antibody light chain constant region in the complex of the composition independently binds to R at the following bonding sites 1 Covalently linked: a linkage site represented by K188 (based on Kabat numbering) and / or a linkage site represented by K190 (based on Kabat numbering) of the light chain constant region of the anti-TfR1 antibody; and wherein in each of the plurality of complexes, n1 is independently an integer of 1 or greater, which represents R 1 The number of instances of .

8. A composition comprising a plurality of complexes, wherein the complexes comprise formula (I): [R 1 ] n1 -R 2 The structure of each R 1 Independently comprising groups of formula (Ib): wherein +N represents LNA (2'-4' methylene bridge) ribonucleoside, dN represents 2'-deoxyribonucleoside, oN represents 2'-MOE modified ribonucleoside, oC represents 5-methyl-2'-MOE-cytidine, +C represents 5-methyl-2'-4'-bicyclo-cytidine (2'-4' methylene bridge), oU represents 5-methyl-2'-MOE-uridine, and * represents phosphorothioate internucleoside linkage, and R 1 The oligonucleotide comprises the nucleobase sequence of CAGCGCCCACCAGUCA (SEQ ID NO: 21); Where R 2 Comprising an anti-transferrin receptor (TfR1) antibody, the anti-transferrin receptor (TfR1) antibody comprising a heavy chain comprising a heavy chain variable region (VH) and a heavy chain constant region, and a light chain comprising a light chain variable region (VL) and a light chain constant region; and wherein the anti-TfR1 antibody comprises: a heavy chain complementary determining region 1 (CDR-H1) comprising the sequence shown in SEQ ID NO: 1, 7 or 12, a heavy chain complementary determining region 2 (CDR-H2) comprising the sequence shown in SEQ ID NO: 2, 8 or 13, a heavy chain complementary determining region 3 (CDR-H3) comprising the sequence shown in SEQ ID NO: 3, 9 or 14, a light chain complementary determining region 1 (CDR-L1) comprising the sequence shown in SEQ ID NO: 4, 10 or 15, a light chain complementary determining region 2 (CDR-L2) comprising the sequence shown in SEQ ID NO: 5 or 11, and a light chain complementary determining region 3 (CDR-L3) comprising the sequence shown in SEQ ID NO: 6 or 16; and Each R 1 At the connection point A, the linkage site represented by the lysine (K) residue of the anti-TfR1 antibody is connected to R 2 covalently linked, and wherein at least 80% of the anti-TfR1 antibody light chain constant region in the complex of the composition independently binds to R at the following bonding sites 1 Covalently linked: a linkage site represented by K188 (based on Kabat numbering) and / or a linkage site represented by K190 (based on Kabat numbering) of the light chain constant region of the anti-TfR1 antibody; and wherein in each of the plurality of complexes, n1 is independently an integer of 1 or greater, which represents R 1 The number of instances of .

9. A composition comprising a plurality of complexes, wherein the complexes comprise formula (I): [R 1 ] n1 -R 2 The structure of which: Each R 1 Containing a group of formula (Ic): Where R 2 Comprising an anti-transferrin receptor (TfR1) antibody, the anti-transferrin receptor (TfR1) antibody comprising a heavy chain comprising a heavy chain variable region (VH) and a heavy chain constant region, and a light chain comprising a light chain variable region (VL) and a light chain constant region; and wherein the anti-TfR1 antibody comprises: a heavy chain complementary determining region 1 (CDR-H1) comprising the sequence shown in SEQ ID NO: 1, 7 or 12, a heavy chain complementary determining region 2 (CDR-H2) comprising the sequence shown in SEQ ID NO: 2, 8 or 13, a heavy chain complementary determining region 3 (CDR-H3) comprising the sequence shown in SEQ ID NO: 3, 9 or 14, a light chain complementary determining region 1 (CDR-L1) comprising the sequence shown in SEQ ID NO: 4, 10 or 15, a light chain complementary determining region 2 (CDR-L2) comprising the sequence shown in SEQ ID NO: 5 or 11, and a light chain complementary determining region 3 (CDR-L3) comprising the sequence shown in SEQ ID NO: 6 or 16; and Each R 1 At the connection point A, the linkage site represented by the lysine (K) residue of the anti-TfR1 antibody is connected to R 2 covalently linked, and wherein at least 80% of the anti-TfR1 antibody light chain constant region in the complex of the composition independently binds to R at the following bonding sites 1 Covalently linked: a linkage site represented by K188 (based on Kabat numbering) and / or a linkage site represented by K190 (based on Kabat numbering) of the light chain constant region of the anti-TfR1 antibody; and wherein in each of the plurality of complexes, n1 is independently an integer of 1 or greater, which represents R 1 The number of instances of .

10. The composition of any one of claims 7 to 9, wherein: (a) 85% to 98% of the anti-TfR1 antibody light chain constant region in the complex of the composition independently binds to R at the following bonding sites: 1 Covalently linked: a linkage site represented by K188 (based on Kabat numbering) and / or a linkage site represented by K190 (based on Kabat numbering) of the light chain constant region of the anti-TfR1 antibody; or (b) 95% to 97% of the anti-TfR1 antibody light chain constant region in the complex of the composition independently binds to R at the following bonding sites: 1 Covalent linkage: the bonding site represented by K188 (based on Kabat numbering) and / or the bonding site represented by K190 (based on Kabat numbering) of the light chain constant region of the anti-TfR1 antibody.

11. The composition of any one of claims 7 to 10, wherein: (a) 1% to 15% of the heavy chain constant region of the anti-TfR1 antibody in the complex of the composition independently binds to R at the following bonding sites: 1 Covalently linked: the bonding site represented by K213 (based on Kabat numbering) of the heavy chain constant region of the anti-TfR1 antibody; or (b) 4% to 8% of the heavy chain constant region of the anti-TfR1 antibody in the complex of the composition independently binds to R at the following bonding sites: 1 Covalent linkage: The bonding site represented by K213 (based on Kabat numbering) of the heavy chain constant region of the anti-TfR1 antibody.

12. The composition of any one of claims 1 to 11, wherein the antibody is a Fab fragment, a full-length IgG, a Fab' fragment, or a F(ab')2 fragment.

13. The composition of any one of claims 1 to 12, wherein the antibody is a Fab fragment.

14. The composition of any one of claims 1 to 13, wherein the VH comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 17; and / or wherein the VL comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 18, optionally wherein the antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 17 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:

18.

15. The composition of any one of claims 1 to 14, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 19 and the light chain comprises the amino acid sequence of SEQ ID NO:

20.

16. The composition of any one of claims 1 to 15, further comprising one or more antibodies not covalently linked to the oligonucleotide. The composition of claim 16 , wherein the average value of n1 of the complexes in the composition is 0.5 to 5.

18. A method of reducing DMPK expression and / or treating myotonic dystrophy in a subject, the method comprising administering to the subject an effective amount of the composition of any one of claims 1 to 17.

19. The method of claim 18, wherein the subject has an expansion of a disease-associated repeat of a DMPK allele associated with myotonic dystrophy, optionally wherein the disease-associated repeat comprises repeating units of a CTG trinucleotide sequence.

20. The method of claim 18 or claim 19, wherein the complex reduces DMPK expression in the subject, optionally wherein reducing DMPK expression comprises reducing the level of DMPK mRNA in muscle cells, and further optionally wherein the DMPK mRNA is a mutant DMPK mRNA.

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