Muscle targeting complexes and formulations for treatment of ankylosing muscular dystrophy

Abstract

Some aspects of the present disclosure relate to complexes and other aspects to formulations (e.g., aqueous form, lyophilized form) comprising such complexes comprising oligonucleotides covalently linked to antibodies (e.g., anti-TfR1 antibodies) (e.g., useful for targeting DMPK).

Description

[0001] Related Applications

[0002] This application claims the benefit of the following filing date under 35 U.S.C. §119(e): U.S. Provisional Application No. 63 / 331,727, filed on April 15, 2022, entitled “MUSCLE TARGETING COMPLEXES AND FORMULATIONS FOR TREATING MYOTONIC DYSTROPHY”; the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] The present application relates to targeting complexes for delivering oligonucleotide molecular payloads to cells, formulations containing such complexes, and their uses, particularly for the treatment of diseases.

[0004] References to Electronic Sequence Listings

[0005] The contents of the electronic sequence listing (D082470076WO00-SEQ-COB.xml; size: 58,346 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.

[0007] According to some aspects, the present disclosure provides complexes and formulations comprising such complexes.

[0008] According to some aspects, the formulations provided herein comprise a complex comprising an oligonucleotide covalently linked to an anti-transferrin receptor 1 (TfR1) antibody,

[0009] wherein the anti-TfR1 antibody comprises: a heavy chain complementarity determining region 1 (CDR-H1) comprising the sequence shown in SEQ ID NO: 1, 7 or 12, a heavy chain complementarity determining region 2 (CDR-H2) comprising the sequence shown in SEQ ID NO: 2, 8 or 13, a heavy chain complementarity determining region 3 (CDR-H3) comprising the sequence shown in SEQ ID NO: 3, 9 or 14, a light chain complementarity determining region 1 (CDR-L1) comprising the sequence shown in SEQ ID NO: 4, 10 or 15, a light chain complementarity determining region 2 (CDR-L2) comprising the sequence shown in SEQ ID NO: 5 or 11, and a light chain complementarity determining region 3 (CDR-H4) comprising the sequence shown in SEQ ID NO: 6 or 16. 3, CDR-L3),

[0010] 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,

[0011] And wherein the complex is formulated with tris(hydroxymethyl)aminomethane and sucrose.

[0012] According to some aspects, the formulations provided herein comprise a complex comprising formula (I): [R 1 ] n1 -R 2 The structure of each R 1 Independently comprising groups of the following formula (Ia):

[0013]

[0014] (Ia), where

[0015] R 2 Contains antibodies, and

[0016] R3 comprising an oligonucleotide comprising 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,

[0017] Where R 1 At the connection point A and R 2 covalently attached; and

[0018] Where n1 is an integer representing R 1 The number of instances of R 1 Each instance of is covalently linked to a different amino acid residue of the antibody;

[0019] wherein the complex is formulated with tris(hydroxymethyl)aminomethane and sucrose. In some embodiments, each different amino acid residue of the antibody is lysine. In some embodiments, the antibody is an anti-TfR1 antibody.

[0020] 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, 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.

[0021] In some embodiments, the formulation is in lyophilized or frozen solid form.

[0022] In some embodiments, the formulation is an aqueous solution.

[0023] In some embodiments, tris(hydroxymethyl)aminomethane is present in the aqueous solution at a concentration of 5 mM to 50 mM.

[0024] In some embodiments, sucrose is present in the aqueous solution at a concentration of 5% to 15% weight / volume (w / v %).

[0025] In some embodiments, the pH of the aqueous solution is between 6.5 and 8.5.

[0026] In some embodiments, tris(hydroxymethyl)aminomethane is present in the aqueous solution at a concentration of 25 mM, and / or the sucrose is present in the aqueous solution at a concentration of 10 w / v%, and / or the pH of the aqueous solution is 7.5.

[0027] In some embodiments, the antibody is a Fab fragment, a full-length IgG, a Fab' fragment, a F(ab') 2 fragment, scFv or Fv.

[0028] In some embodiments, the antibody is a Fab fragment.

[0029] In some embodiments, the antibody comprises a heavy chain variable region (VH) comprising an amino acid sequence at least 85% identical to SEQ ID NO: 17; and / or wherein the antibody comprises a light chain variable region (VL) comprising an amino acid sequence at least 85% 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.

[0030] In some embodiments, the antibody comprises a heavy chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 19; and / or wherein the antibody comprises a light chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 20. In some embodiments, the antibody comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 19 and / or comprises a light chain comprising an amino acid sequence of SEQ ID NO: 20.

[0031] In some embodiments, the oligonucleotide is 10 to 30 nucleotides in length.In some embodiments, the oligonucleotide comprises a nucleotide sequence having a complementary region to SEQ ID NO:22 that is at least 8 consecutive nucleotides long.

[0032] In some embodiments, the oligonucleotide comprises at least 8 consecutive nucleotides of the nucleotide sequence shown in SEQ ID NO: 21. In some embodiments, the oligonucleotide comprises the nucleotide sequence of SEQ ID NO:21.

[0033] In some embodiments, each R 1 A group comprising the following formula (Ib):

[0034]

[0035] 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, such that the oligonucleotide comprises a nucleobase sequence of CAGCGCCCACCAGUCA (SEQ ID NO: 21).

[0036] In some embodiments, each R 1 A group comprising the following formula (Ic):

[0037]

[0038] In some embodiments, the complex is present in the formulation at a concentration of 10 mg / mL to 50 mg / mL.

[0039] In some embodiments, the formulation further comprises one or more antibodies that are not covalently linked to the oligonucleotide.

[0040] In some embodiments, the average value of n1 of the complexes in the formulation is between 0.5 and 5.

[0041] According to some aspects, provided herein are 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 formulation provided herein.

[0042] 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.

[0043] In some embodiments, the complex decreases DMPK expression in the subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figures 1A to 1D Figure 2 shows the effects of the conjugate containing anti-TfR1 Fab covalently linked to ASO on the heart ( Figure 1A ), diaphragm( Figure 1B ), gastrocnemius( Figure 1C ) or tibialis anterior ( Figure 1D ) in the presence of DMPK targeting oligonucleotide (ASO).

[0045] Figures 2A to 2D The conjugate containing anti-TfR1 Fab conjugated to DMPK targeting oligonucleotide (ASO) was shown in the heart of mice ( Figure 2A ), diaphragm( Figure 2B ), tibialis anterior ( Figure 2C ) and gastrocnemius ( Figure 2D ) mice expressing both human TfR1 and two copies of a mutant human DMPK transgene containing expanded CTG repeats.

[0046] Figures 3A to 3B 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 3A Representative images of samples after in situ hybridization staining for DMPK foci and fluorescent staining of myofibers are shown (inset). Figure 3A 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 3B Quantification of DMPK foci is shown.

[0047] Figure 4 The 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").

[0048] Figure 5The 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").

[0049] Figure 6 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").

[0050] Figure 7 The 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").

[0051] Figure 8DMPK 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 calculated by one-way ANOVA (*, P < 0.05, **, P < 0.01)

[0052] Fig. 9 Shown is the x-ray diffraction (XRD) pattern of the lyophilized formulation of the anti-TfR1 Fab-ASO conjugate.

[0053] Figures 10A to 10C Shown are protein concentrations calculated from absorbance data of lyophilized formulations of anti-TfR1 Fab-ASO conjugates at 0 weeks (T0), 4 weeks (T4w), and 8 weeks (T8w) at 2 to 8°C after reconstitution. Fig. 10A The Fab concentration of the conjugates is shown in mg / ml. Fig. 10B The Fab concentration of the conjugates is shown in μM. Fig. 10C The oligonucleotide concentration of the conjugates is shown in μM.

[0054] Figures 11A to 11B The average percent recovery of lyophilized formulations of anti-TfR1 Fab-ASO conjugates at T0, T4w and T8w after reconstitution at 2 to 8°C is shown ( Fig.11A ) and drug-antibody ratio (DAR; Fig. 11B ).

[0055] Figures 12A to 12C Shown are protein concentrations calculated from absorbance data of lyophilized formulations of anti-TfR1 Fab-ASO conjugates at 0 weeks (T0), 2 weeks (T2w), 4 weeks (T4w), and 8 weeks (T8w) at 25°C after reconstitution. Fig. 12A The Fab concentration of the conjugates is shown in mg / ml. Fig. 12B The Fab concentration of the conjugates is shown in μM. Fig. 12C The oligonucleotide concentration of the conjugates is shown in μM.

[0056] Figures 13A to 13B The average recovery percentage of the lyophilized formulation of anti-TfR1 Fab-ASO conjugate at T0, T2w, T4w and T8w at 25°C is shown ( Fig.13A ) and drug-antibody ratio (DAR; Fig. 13B ).

[0057] Figures 14A to 14C Shown are protein concentrations calculated from absorbance data of lyophilized formulations of anti-TfR1 Fab-ASO conjugates at T0, T2w, T4w and T8w at 40°C after reconstitution. Fig.14A The Fab concentration of the conjugates is shown in mg / ml. Fig. 14B The Fab concentration of the conjugates is shown in μM. Fig. 14C The oligonucleotide concentration of the conjugates is shown in μM.

[0058] Figures 15A to 15B The average recovery percentage of lyophilized anti-TfR1 Fab-ASO conjugate at T0, T2w, T4w and T8w after reconstitution at 40°C is shown ( Fig.15A ) and drug-antibody ratio (DAR; Fig. 15B ).

[0059] Figures 16A to 16B Peaks 1 and 2 ( Fig.16A ) and peaks 3 and 4 ( Fig. 16B ) is the sum of the relative areas of .

[0060] Figures 17A to 17B Peaks 1 and 2 ( Fig.17A ) and peaks 3 and 4 ( Fig. 17B ) is the sum of the relative areas of .

[0061] Figures 18A to 18B Peaks 1 and 2 of the lyophilized anti-TfR1 Fab-ASO conjugate measured using size exclusion chromatography (SEC) at T0, T2w, T4w, T8w after reconstitution at 40°C are shown. Fig.18A ) and peaks 3 and 4 ( Fig.18B ) is the sum of the relative areas of .

[0062] Fig.19Shown are the percent purity of the main peak of lyophilized anti-TfR1 Fab-ASO conjugate measured using capillary electrophoresis (CE-SDS) at T0, T4w and T8w after reconstitution at 2 to 8°C.

[0063] Fig. 20 Shown are the percent purity of the main peak of the lyophilized anti-TfR1 Fab-ASO conjugate measured using CE-SDS at T0, T2w, T4w and T8w at 25°C after reconstitution.

[0064] Fig.21 Shown are the percent purity of the main peak of the lyophilized anti-TfR1 Fab-ASO conjugate measured using CE-SDS at T0, T2w, T4w and T8w at 40°C after reconstitution. DETAILED DESCRIPTION

[0065] According to some aspects, the disclosure provides a complex and a preparation comprising such a complex. In some embodiments, the complex is formulated with tris (hydroxymethyl) aminomethane and sucrose. In some embodiments, the complex is formulated into an aqueous or lyophilized (e.g., lyophilized powder) form. In some embodiments, the complex comprises an oligonucleotide covalently linked to an antibody (e.g., an oligonucleotide comprising a 5'-XYZ-3' configuration). In some embodiments, the complex comprises a muscle-targeted complex, the muscle-targeted complex comprising an oligonucleotide covalently linked to an anti-transferrin receptor 1 (TfR1) antibody (e.g., an oligonucleotide comprising a 5'-XYZ-3' configuration). In some embodiments, the complex comprises a muscle-targeted complex, the muscle-targeted complex comprising an oligonucleotide covalently linked to an anti-transferrin receptor 1 (TfR1) antibody provided in Table 2 (e.g., an oligonucleotide comprising a 5'-XYZ-3' configuration). Also provided are methods of using the complexes and formulations described herein for treating a subject with myotonic dystrophy (eg, DM1) and / or methods of reducing the expression or activity of a DMPK (eg, DMPK RNA) in a cell.

[0066] Additional aspects of the disclosure are provided below, including a description of defined terms.

[0067] definition

[0068] 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.

[0069] 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).

[0070] Antibody: As used herein, the term "antibody" refers to a polypeptide comprising at least one immunoglobulin variable domain or at least one antigenic determinant (e.g., a paratope that specifically binds to an antigen). In some embodiments, the antibody is a full-length antibody. In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a humanized antibody. However, in some embodiments, the antibody is a Fab fragment, a Fab' fragment, a F(ab') 2Fragment, Fv fragment or 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 (for example, 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. Regarding 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 γ1CH1, 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. Patent No. 5,693,780 and Kabat EA et al., (1991) supra. In some embodiments, the VH domain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or at least 99% identical to any variable chain constant region provided herein. In some embodiments, the antibody is modified, for example, by glycosylation, phosphorylation, SUMOylation (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, the antibody can be part of a larger immunoadhesion molecule, which is 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).

[0071] CDR: The term "CDR" as used herein refers to the complementarity determining region within an antibody variable sequence. A typical antibody molecule comprises a heavy chain variable region (VH) and a light chain variable region (VL), which are 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, CDR"), interspersed with more conservative regions called "framework regions" ("framework regions, FR"). 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. Methods known in the art can be used, for example, 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) to accurately identify the scope of the framework region and CDR. See, for example,

[0072] Kabat, E.A., et al. (1991) Sequences of Proteins of

[0073] Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91 - 3242; the international ImMunoGeneTics information http: / / www.imgt.org, Lefranc, M.-P. et al., Nucleic Acids Res., 27: 209 - 212 (1999); Ruiz, M. et al., Nucleic Acids Res., 28: 219 - 221 (2000); Lefranc, M.-P., Nucleic Acids Res., 29: 207 - 209

[0074] (2001); Leffanc, M.-P.. Nucleic Acids Res., 31: 307 - 310 (2003); Lefranc, M.-P. et al., In Silico Biol., 5, 0006 (2004) [Epub], 5: 45 - 60 (2005); Lefranc, M.-P et al., Nucleic Acids Res., 33: D593 - 597 (2005); Lefranc, M.-P. et al., Nucleic Acids Res., 37: D1006 - 1012 (2009); Lefranc, M.-P. et al., Nucleic Acids Res., 43: D413 - 422 (2015); Chothia et al, (1989) Nature 342: 877; Chothia, C. et al. (1987) J. Mol. Biol. 196: 901 - 917, Al - lazikani et al (1997) J. Molec. Biol. 273: 927 - 948; and Almagro, J. Mol. Recognit. 17: 132 - 143 (2004).

[0075] 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).

[0076] 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.

[0077] Table 1. CDR definitions

[0078]

[0079] ImMunoGeneTics information imgt.org, Lefranc, M.-P. et al., NucleicAcids Res., 27: 209-212 (1999)

[0080] 2 Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242

[0081] 3 Chothia et al., J. Mol. Biol. 196: 901-917 (1987))

[0082] 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.

[0083] 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.

[0084] 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.

[0085] 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) which is a serine / threonine protein kinase. Substrates of this enzyme may include myogenin, the beta 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, NM_001081562.2, NM_001288764.1, NM_001288765.1, and NM_001288766.1).

[0086] 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.

[0087] 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.

[0088] 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.

[0089] Humanized antibody: The term "humanized antibody" refers to an antibody comprising heavy and light chain variable region sequences from non-human species (e.g., mice) 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 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. WO 2005 / 123126A2 of Kasaian et al.

[0090] 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.

[0091] 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.

[0092] 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.

[0093] 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.

[0094] 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).

[0095] 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).

[0096] 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 NP_001300895.1).

[0097] 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:

[0098]

[0099] These examples are shown with phosphate groups, but any internucleoside linkages are contemplated to be between 2'-modified nucleosides.

[0100] Ranges: All ranges provided in this disclosure are inclusive of the endpoints.

[0101] Compound

[0102] Provided herein are complexes comprising targeting agents (e.g., antibodies) covalently linked to oligonucleotides. In some embodiments, complexes comprise muscle targeting antibodies covalently linked to one or more oligonucleotides. In some embodiments, oligonucleotides are antisense oligonucleotides targeting DMPK RNA for reducing expression or activity of DMPK (e.g., reducing the level of mutant or wild-type DMPK RNA, or the activity of DMPK gene product).

[0103] The complexes described herein typically comprise a linker that covalently links an antibody described herein (eg, an anti-TfR1 antibody) to an oligonucleotide (eg, an oligonucleotide comprising a 5'-XYZ-3' configuration). The linker comprises at least one covalent bond.

[0104] 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, one 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 The different amino acid residues (e.g., lysine or cysteine) of the polypeptide are covalently linked.

[0105] In some embodiments, in each complex, n1 is independently an integer (e.g., one 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 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 that is 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 that is 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 that is 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 that is at least 85% (e.g., at least 95%) identical to SEQ ID NO: 20. In some embodiments, the antibody comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 19 and / or comprises a light chain comprising an 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 In some embodiments, the antibody is a Fab fragment.

[0106] In some embodiments, the value of n1 of each or any complex (e.g., any complex of any of the compositions or formulations 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 one 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).

[0107] In some embodiments, a composition comprising a plurality of different complexes is provided (e.g., a formulation comprising tris(hydroxymethyl)aminomethane and / or sucrose as described herein). 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, such as a DMPK targeting oligonucleotide). In such embodiments, different complex types are characterized by having different numbers of oligonucleotides covalently linked to an antibody. For example, in some embodiments, a composition comprising a plurality of complexes is provided, the complex comprising formula (I): [R 1 ] n1 -R 2 The structure of each R 1 independently comprises a compound comprising an oligonucleotide (e.g., a DMPK targeting oligonucleotide), and R 2 comprising an antibody (eg, an anti-TfR1 antibody), and wherein n1 is a 1 In some embodiments, n1 is an integer of the number of examples, and different complexes of the composition may have different n1 values ​​(e.g., n1 values ​​are 1 to 27, 1 to 26, 1 to 10, 1 to 5, or 1 to 3). In some embodiments, in the complexes of the composition, n1 is independently an integer. 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, the composition described herein further comprises a complex wherein n1 is 0.

[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 2A 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, R 1 Each instance of is covalently linked to a different amino acid residue of the antibody. 1 The covalently linked amino acid comprises an ε-amino group (e.g., lysine, arginine). However, in some embodiments, 1 The covalently linked amino acid is cysteine. 1 Directly covalently linked to an amino acid residue of the antibody. However, in some embodiments, R 1 In some embodiments, R 1 It is not covalently linked to amino acid residues present in the CDR regions of the antibody.

[0110] In some embodiments, a complex provided herein (eg, a complex in a composition or formulation described herein) comprises formula (I): [R 1 ] n1 -R 2 The structure of R 1 Each instance of independently comprises a group of formula (Ia):

[0111]

[0112] Where R 3 comprising an oligonucleotide, such as an oligonucleotide comprising a 5'-XYZ-3' configuration; and R 1 At the connection point A and R 2 In some embodiments, R 2 The invention also includes antibodies comprising the sequences shown in Table 2. For example, in some embodiments, R 2 The invention comprises an 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, 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 The invention also includes an antibody 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 also comprises an antibody 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 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 also includes an antibody 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') 2In 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 thiophosphate internucleoside linkage.

[0113] In some embodiments, a complex provided herein (eg, a complex in a composition or formulation described herein) comprises formula (I): [R 1 ] n1 -R 2 structure, where each R 1 A group comprising the following formula (Ib):

[0114]

[0115] 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 n1 is an integer (e.g., one or greater) representing the number of R in each complex. 1 The number of instances, and each R 1 At the connection point A and R 2 In some embodiments, R 2 The invention also includes antibodies comprising the sequences shown in Table 2. For example, in some embodiments, R 2The invention comprises an 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, 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 The invention also includes an antibody 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 also comprises an antibody 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 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 also includes an antibody 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, scFv or Fv.

[0116] In some embodiments, a complex provided herein (eg, a complex in a composition or formulation described herein) comprises formula (I): [R 1 ] n1 -R 2 The structure of each R 1 Contains the following group of formula (Ic):

[0117]

[0118] Where R 1 At the connection point A and R 2 In some embodiments, R 2 The invention also includes antibodies comprising the sequences shown in Table 2. For example, in some embodiments, R 2 The invention comprises an 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, 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 The invention also includes an antibody 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 also comprises an antibody 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 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 also includes an antibody 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, scFv or Fv.

[0119] In some embodiments, a complex provided herein (eg, a complex in a composition or formulation described herein) comprises the structure of Formula (Id):

[0120]

[0121] 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 The invention comprises an antibody comprising a sequence shown in Table 2; wherein n1 is an integer (e.g., one or greater) representing the number of instances of a group enclosed by square brackets, wherein each instance of a group enclosed by square brackets is covalently linked to a different amino acid residue of the antibody, optionally wherein each different amino acid residue is lysine. In some embodiments, R 2 The invention comprises an 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, 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 The invention also includes an antibody 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 also comprises an antibody 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 2The invention also includes an antibody 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 also includes an antibody 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, scFv or Fv.

[0122] In some embodiments, the complex described herein comprises a structure of Formula (A):

[0123]

[0124] (A), wherein y is 0 to 15 (e.g., 3), and z is 0 to 15 (e.g., 4). In some embodiments, the amide adjacent to the antibody (e.g., anti-TfR1 antibody) shown in structure (A) is produced by a reaction with an amine (e.g., lysine ε amine) of the antibody. In some embodiments, the complex described herein comprises an anti-TfR1 antibody (e.g., anti-TfR1 Fab) covalently linked to the 5' end of an oligonucleotide (e.g., an oligonucleotide comprising a 5'-XYZ-3' configuration) via a lysine of the antibody. 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 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 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 that is 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 that is 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 that is 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 that is at least 85% (e.g., at least 95%) identical to SEQ ID NO: 20. In some embodiments, the antibody comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 19 and / or comprises a light chain comprising an 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, scFv or Fv.

[0125] Antibody

[0126] 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:

[0127]

[0128] Table 2 provides some examples of sequences of anti-TfR1 antibodies that can be used in the complexes provided herein.

[0129] Table 2. Some examples of anti-TfR1 antibody sequences

[0130]

[0131] 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).

[0132] 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).

[0133] 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).

[0134] 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).

[0135] 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.

[0136] 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.

[0137] 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.

[0138] 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. As an alternative or in addition (e.g., supplement), 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. As an alternative or in addition (e.g., supplement), 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.

[0139] 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.

[0140] Oligonucleotides

[0141] 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.

[0142] 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.

[0143] 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.

[0144] 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):

[0145]

[0146]

[0147]

[0148] In some embodiments, an oligonucleotide can comprise a complementary region to a sequence as shown below, which is an exemplary mouse DMPK gene sequence (Gene ID 13400; NM_001190490.1).

[0149]

[0150]

[0151] 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).

[0152] In some embodiments, the oligonucleotide may comprise a complementary region to a mutant form of DMPK, such as a mutant form as 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.

[0153] 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, 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 oligonucleotides target 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.

[0154] 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.

[0155] 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.

[0156] 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.

[0157] 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.

[0158] 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.

[0159] 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.

[0160] 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.

[0161] 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.

[0162] 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.

[0163] 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.

[0164] In some embodiments, an oligonucleotide described herein (eg, a DMPK targeting oligonucleotide) comprises the following 5'-XYZ-3' configuration:

[0165] 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, 1-2 2-1, 1-21-2, 2-21-1, 1-21-3, 3-20-1, 2-20-2, 1-19-4, 4-19-1, 2-19-3, 3-19-2, 1-18-5, 2-18-4, 4-18-2, 3-18-3, 1-17-6, 6-17-1.2-17-5, 5-17-2, 3-17-4, 4- 17-3, 1-16-7, 7-16-1, 2-16-6, 6-16-2, 3-16-5, 5-16-3, 4-16-4, 1-15-8.8-15-1, 2-15-7, 7-15-2, 3-15-6, 6-15-3, 4-15-5, 5-15-4, 2-14-8, 8-14-2, 3-14-7, 7 -14-3, 4-14-6. 6-14-4, 5-14-5, 3-13-8, 8-13-3, 4-13-7, 7-13-4, 5-13-6, 6-13-5, 4-12-8, 8-12-4, 5-12-7, 7-12-5. 6-12-6, 5-11-8, 8-11-5, 6-11-7 or 7-11-6.

[0166] The numbers indicate the number of nucleosides in the X, Y and Z regions, respectively, of an oligonucleotide comprising a 5'-XYZ-3' configuration.

[0167] 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)).

[0168] 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.

[0169] 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).

[0170] 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.

[0171] In some embodiments, an oligonucleotide described herein (e.g., a DMPK targeting oligonucleotide described herein) comprises a 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.

[0172] 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).

[0173] 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.

[0174] 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.

[0175] In some embodiments, the oligonucleotide comprises LLEE-(D) 8 -5'-XYZ-3' configuration of -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.

[0176] 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.

[0177] 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*dA*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.

[0178] In some embodiments, an oligonucleotide described herein (eg, a DMPK targeting oligonucleotide described herein) comprises the structure of Formula (Ie):

[0179]

[0180] 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.

[0181] 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)RA-,-C(=O)R A -,-NRAC(=O)O-,-NR A C(=O)N(R A )-, -OC(=O)-, -OC(=O)O-, -OC(=O)N(R A )-,-S(O) 2 NR A -,-NR A S(O) 2 -, or a 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.

[0182] In some embodiments, the 5' or 3' nucleoside of the oligonucleotide is of the formula -NH 2 -(CH 2 ) 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 -NH 2 -(CH 2 ) n - compound and the 5' or 3' nucleoside of the oligonucleotide. In some embodiments, the formula -NH 2 -(CH 2 ) 6-compounds are synthesized by 6-amino-1-hexanol (NH 2 -(CH 2 ) 6 The oligonucleotide is conjugated by reaction between the oligonucleotide and the 5' phosphate of the oligonucleotide.

[0183] 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.

[0184] 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.

[0185] 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.

[0186] Composition

[0187] In some embodiments, the compositions described herein comprise a complex (i.e., multiple complexes), each of which comprises an antibody (e.g., an anti-TFR1 antibody) covalently linked to one or more oligonucleotides (e.g., a DMPK targeting oligonucleotide described herein), 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. 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. The complex of the composition described herein may comprise any structure provided herein, such as a structure of formula (I) (e.g., a group comprising formula (Ia), formula (Ib), formula (Ic), or formula (Id)) or formula (A).

[0188] 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 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 of the complex 1 Independently with R 2 The different amino acid residues (e.g., lysine or cysteine) of the polypeptide are covalently linked.

[0189] 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 2, 1 to 3, 1 to 5, 1 to 10, 1 to 26, or 1 to 27. In some embodiments, the composition described herein comprises a complex having a value of 0 for n1. 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).

[0190] 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 2A 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).

[0191] preparation

[0192] The complex provided herein is formulated in a manner suitable for pharmaceutical use. In some embodiments, the complex can be delivered to the object using a preparation that minimizes degradation, promoting delivery and / or (for example, and) absorption, or providing other beneficial properties for the complex in the preparation. Therefore, in some embodiments, it has been found that the complex (for example, a complex comprising an oligonucleotide covalently linked to Fab) is formulated with tris (hydroxymethyl) aminomethane (also referred to as tromethamine or THAM) and / or sucrose for pharmaceutical use (for example, as described herein) is particularly advantageous. Therefore, in some embodiments, a preparation (for example, an aqueous solution, a lyophilized form, or a frozen form) comprising a complex together with tris (hydroxymethyl) aminomethane and / or sucrose is provided herein. Such a preparation can be appropriately prepared so that when applied to an object, whether it is applied to the direct environment of the target cell or systemically applied, a sufficient amount of the complex will enter the target muscle cell.

[0193] In some embodiments, provided herein are preparations comprising complexes (i.e., multiple complexes), the complexes comprising oligonucleotides covalently linked to antibodies (e.g., oligonucleotides comprising 5'-XYZ-3' configurations). In some embodiments, provided herein are preparations comprising complexes, wherein each complex comprises oligonucleotides covalently linked to anti-TfR1 antibodies (e.g., oligonucleotides comprising 5'-XYZ-3' configurations), optionally, wherein the antibodies of such complexes comprise CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 as shown in Table 2, and in addition, in some embodiments, wherein the complexes are formulated with tris(hydroxymethyl)aminomethane and sucrose. In some embodiments, the antibody is an anti-TfR1 antibody.

[0194] In some embodiments, a formulation comprising a complex is provided, wherein the complex comprises formula (I): [R 1 ] n1 -R 2 The structure of each R 1 independently comprises a compound comprising an oligonucleotide (eg, for example, an oligonucleotide comprising a 5'-XYZ-3' configuration), and R 2 comprising an antibody (eg, an anti-TfR1 antibody), and wherein n1 is an integer representing the number of R 1 The number of instances of .

[0195] In some embodiments, the formulations described herein comprise a complex comprising an antibody comprising CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 as shown in Table 2. In some embodiments, the antibody 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. In some embodiments, the antibody is a Fab and 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.

[0196] In some embodiments, the preparations described herein include a concentration of 1 to 50 mg / mL of the preparation of the compound described herein. In some embodiments, the preparations described herein include a concentration of 10 to 50 mg / ml or 20 to 35 mg / mL (e.g., 15 to 45 mg / mL, 20 to 40 mg / mL, 25 to 35 mg / mL, 25.5 to 34.5 mg / mL, 26 to 34 mg / mL, 27 to 33 mg / mL, 28 to 32 mg / mL, 29 to 31 mg / mL, 29.5 to 30.5 mg / mL, 10 to 40 mg / mL, 15 to 35 mg / mL, 20 to 30 mg / mL, 21 to 29 mg / mL, 21.2 to 28.8 mg / mL, 22 to 28 mg / mL, 23 to 27 mg / mL, 24 to 26 mg / mL, or 24.5 to 25.5 mg / mL). In some embodiments, the formulations described herein include a complex having a concentration of about 25 mg / mL (e.g., 25 mg / mL). In some embodiments, the formulations described herein include a complex having a concentration of about 30 mg / mL (e.g., 30 mg / mL). In some embodiments, the concentration of the complex in the formulation may vary within a range of up to 20% (e.g., + / - up to 20%, + / - up to 15%, + / - up to 10%, or + / - up to 5%) of the set value. For example, in some embodiments, the concentration of the complex in the formulation is 30 mg / mL + / - up to 15% (e.g., 30 + / - 4.5 mg / mL). In some embodiments, the concentration of the complex in the formulation is 25 mg / mL + / - up to 15% (e.g., 25 + / - 3.8 mg / mL).

[0197] In some embodiments, any one or more complexes described herein are formulated with tris(hydroxymethyl)aminomethane and sucrose in aqueous solution. In some embodiments, tris(hydroxymethyl)aminomethane is present in an aqueous solution at a concentration of 5 to 50 mM (e.g., 5 to 40 mM, 5 to 35 mM, 5 to 30 mM, 10 to 50 mM, 15 to 45 mM, 10 to 40 mM, 20 to 40 mM, 20 to 35 mM, 20 to 30 mM, 21 to 29 mM, 22 to 28 mM, 23 to 27 mM, 24 to 26 mM). In some embodiments, tris(hydroxymethyl)aminomethane is present in an aqueous solution at a concentration of about 25 mM (e.g., 25 mM). In some embodiments, sucrose is present in the aqueous solution at a concentration of 5% to 15% weight / volume (w / v%), for example, 7 to 13w / v%, 8 to 15%w / v%, 9 to 15%w / v%, 9 to 11%w / v%, 9.5 to 11%w / v%, or for example, 9 to 10w / v%, 10 to 11w / v%, 10 to 12w / v% or 8 to 12w / v%. In some embodiments, sucrose is present in the aqueous solution at a concentration of 7 to 13w / v%, 8 to 12w / v% or 9 to 11w / v%. In some embodiments, sucrose is present in the aqueous solution at a concentration of about 10w / v% (e.g., 10w / v%). In some embodiments, the pH of the aqueous solution is 6.5 to 8.5, e.g., 6.5 to 6.5, 6.7 to 6.9, 6.9 to 7.1, 7.1 to 7.3, 7.2 to 7.8, 7.3 to 7.5, 7.4 to 7.5, 7.4 to 7.6, 7.5 to 7.6; e.g., 7.0 to 8.0, or e.g., a pH of 7.0 to 7.3, 7.2 to 7.8, 7.3 to 7.5, 7.4 to 7.6, 7.5 to 7.6, 7.5 to 7.7, 7.7 to 7.9, 7.9 to 8.0, 8.0 to 8.2, 8.2 to 8.4, 8.3 to 8.4, 8.4 to 8.5, 8.5 to 8.6, or 7.3 to 7.7. In some embodiments, the pH of the aqueous solution is 7.0 to 8.0 (e.g., 7.0 to 7.8, 7.1 to 7.8, 7.2 to 7.8, 7.3 to 7.7, 7.3 to 7.5, 7.3 to 7.6, 7.4 to 7.6, or 7.4 to 7.8). In some embodiments, the pH of the aqueous solution is about 7.5 (e.g., 7.5). In some embodiments, the pH of the aqueous solution is 7.4 to 7.7. In some embodiments, the pH of the aqueous solution is 7.4 to 7.6 (e.g., 7.5 or about 7.5).

[0198] In some embodiments, any one of the formulations described herein is an aqueous solution, wherein tris(hydroxymethyl)aminomethane is present in the aqueous solution at a concentration of about 25 mM (e.g., 25 mM), wherein sucrose is present in the aqueous solution at a concentration of about 10 w / v% (e.g., 10 w / v%), and wherein the pH of the aqueous solution is about 7.5 (e.g., 7.5).

[0199] In some embodiments, any one of the formulations described herein is an aqueous solution, wherein tris(hydroxymethyl)aminomethane is present in the aqueous solution at a concentration of about 25 mM (e.g., 25 mM), wherein sucrose is present in the aqueous solution at a concentration of about 10 w / v% (e.g., 10 w / v%), wherein the complex is present in the aqueous solution at a concentration of about 20 to 35 mg / ml (e.g., 25 mg / ml or 30 mg / ml), and wherein the pH of the aqueous solution is about 7.5 (e.g., 7.5).

[0200] In some embodiments, any one or more complexes described herein are formulated with tris(hydroxymethyl)aminomethane and sucrose into a lyophilized form (e.g., a lyophilized powder). In some embodiments, the lyophilized form (e.g., a lyophilized powder) is obtained by lyophilizing any aqueous solution described herein.

[0201] In some embodiments, the lyophilized form is a lyophilized cake. In some embodiments, the lyophilized cake comprises a plurality of complexes provided herein, tris(hydroxymethyl)aminomethane, and sucrose. In some embodiments, the lyophilized cake comprises 1×10 -2 mg to 1.5 mg (e.g., 1x10 -2 mg to 1.5 mg, 4.02×10 -2 mg to 1.21 mg, 6×10 -2 mg to 1 mg, 8×10 -2or 0.3 mg to 0.5 mg) tris(hydroxymethyl)aminomethane / g cake. In some embodiments, the lyophilized cake comprises 900 mg to 1100 mg (e.g., 900 mg to 1100 mg, 989 mg to 999 mg, 950 mg to 1000 mg, 980 mg to 1000 mg, 980 mg to 990 mg, 990 mg to 1000 mg, 995 mg to 1000 mg, 992 mg to 998 mg, 994 mg to 999 mg) sucrose / g cake. In some embodiments, the lyophilized cake contains 0.5 mg to 10 mg (e.g., 0.5 mg to 10 mg, 0.5 mg to 1.5 mg, 1 mg to 2 mg, 1.5 mg to 3 mg, 1 mg to 8 mg, 0.5 mg to 6 mg, 0.5 mg to 5 mg, 1.5 mg to 3.5 mg, 1.5 mg to 4 mg, 1.5 mg to 5 mg, 2 mg to 5 mg, 2.5 mg to 5 mg, 2 mg to 3 mg, 3 mg to 4 mg, 4 mg to 5 mg) of complex / g cake.

[0202] In some embodiments, the lyophilized cake comprises 0.303 mg tris (hydroxymethyl) aminomethane / g cake, 998.6 mg sucrose / g cake, and / or (for example, and) 0.999 mg complex / g cake. In some embodiments, the lyophilized cake comprises 0.302 mg tris (hydroxymethyl) aminomethane / g cake, 997.7 mg sucrose / g cake, and / or (for example and) 1.995 mg complex / g cake. In some embodiments, the lyophilized cake comprises 0.302 mg tris (hydroxymethyl) aminomethane / g cake, 997.2 mg sucrose / g cake, and / or (for example and) 2.49 mg complex / g cake. In some embodiments, the lyophilized cake comprises 0.302 mg tris (hydroxymethyl) aminomethane / g cake, 996.7 mg sucrose / g cake, and / or (for example and) 2.99 mg complex / g cake. In some embodiments, the lyophilized cake comprises 0.302 mg tris(hydroxymethyl)aminomethane / g cake, 995.7 mg sucrose / g cake, and / or (e.g., and) 3.98 mg complex / g cake. In some embodiments, the lyophilized cake comprises 0.301 mg tris(hydroxymethyl)aminomethane / g cake, 994.7 mg sucrose / g cake, and / or (e.g., and) 4.97 mg complex / g cake.

[0203] In some embodiments, the reconstitution of the lyophilized cake (e.g., reconstitution with water) produces a solution comprising tris(hydroxymethyl)aminomethane at a concentration of 5 to 50 mM, sucrose at a concentration of 5 w / v% to 15 w / v%, and / or (e.g., and) a complex at a concentration of 10 mg / mL to 50 mg / mL. In some embodiments, 10.053 g of lyophilized cake is reconstituted with a solvent (e.g., water) to form 1 mL of a reconstitution solution, producing a solution comprising tris(hydroxymethyl)aminomethane at a concentration of 5 to 50 mM (optionally 25 mM), sucrose at a concentration of 5 w / v% to 15 w / v% (optionally 10 w / v%) and / or (e.g., and) a complex at a concentration of 10 mg / mL to 50 mg / mL. In some embodiments, about 10 g of the lyophilized cake is reconstituted with a solvent (e.g., water) to form 1 mL of a reconstituted solution, resulting in a solution comprising tris(hydroxymethyl)aminomethane at a concentration of 5 to 50 mM (optionally 25 mM), sucrose at a concentration of 5 w / v% to 15 w / v% (optionally 10 w / v%), and / or (e.g., and) a complex at a concentration of 10 mg / mL to 50 mg / mL.

[0204] In some embodiments, any one or more of the complexes described herein are formulated with tris(hydroxymethyl)aminomethane and sucrose into a frozen form (e.g., a frozen aqueous solid). In some embodiments, the frozen form (e.g., a frozen aqueous solid) is obtained by freezing any of the aqueous solutions described herein. The frozen form can be frozen to a temperature below -20°C (e.g., below -20°C, below -30°C, below -40°C, below -50°C, below -60°C, below -70°C, below -80°C, or lower).

[0205] In some embodiments, the value of n1 for each complex in the formulation is independently and optionally zero to antibody (R 2) is an integer of the number of amino acid residues (e.g., the number of lysine residues) desired or targeted for conjugation in the preparation. In some embodiments, the value of n1 for each complex in the preparation is independently and optionally selected from 0, 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 preparation is independently and optionally selected from 0, 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 preparation 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 of the complexes in the formulation is 1 to 2, 1 to 3, 1 to 5, 1 to 10, 1 to 26, or 1 to 27. In some embodiments, the formulations described herein comprise a compound of formula (I): [R 1 ] n1 -R 2 In some embodiments, the average value of n1 of the complexes of the formulation 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, in a complex herein (e.g., a complex of a formulation provided herein), R 1 Each instance of is conjugated to a different amino acid residue of the antibody. In some embodiments, each different amino acid comprises an ε-amino group (e.g., lysine, arginine). However, in some embodiments, each instance of R 1 Each different amino acid that is covalently linked is cysteine. In some embodiments, R 1 Directly covalently linked to an amino acid residue of the antibody. However, in some embodiments, R 1 Indirect covalent attachment to an amino acid of the antibody, for example, to a glycosylation site on an amino acid. In some embodiments, a formulation is provided wherein R of the complex 1The covalent linkage to the amino acid residues present in the CDR region of the antibody is present only in trace amounts, or in undetectable amounts, or not present at all. In some embodiments, there is provided a complex wherein R 1 The agent is covalently linked to amino acid residues present in the CDR regions of an antibody, such complexes being undetectable in the agent using standard detection techniques.

[0207] In some embodiments, the formulations provided herein comprise a compound of formula (I): [R 1 ] n1 -R 2 The structure of the complex, where R 1 Each instance of independently comprises a group of formula (Ia):

[0208]

[0209] Where R 3 comprising an oligonucleotide, such as an oligonucleotide comprising a 5'-XYZ-3' configuration; and R 1 At the connection point A and R 2 In some embodiments, R 2 The invention also includes antibodies comprising the sequences shown in Table 2. For example, in some embodiments, R 2 The invention comprises an 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, 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 The invention comprises an antibody 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 also comprises an antibody 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 2The invention also includes an antibody 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 also includes an antibody 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 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, 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 formulations described herein further 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). In some embodiments, the formulation further comprises tris(hydroxymethyl)aminomethane and sucrose. In some embodiments, the formulation comprises tris(hydroxymethyl)aminomethane at a concentration of 25 mM and / or (e.g., and) sucrose at a concentration of 10 w / v%, optionally wherein the pH of the formulation is 7.5. In some embodiments, the formulation comprises a complex at a concentration of 30 mg / mL.

[0210] In some embodiments, the formulations provided herein comprise a compound of formula (I): [R 1 ] n1 -R 2 A complex of the structure of wherein R 1 Each example of comprises a group of formula (Ib):

[0211]

[0212] 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 n1 is an integer (e.g., one or greater) representing the number of R in each complex. 1 The number of instances, and each R 1 At the connection point A and R 2 In some embodiments, R 2 The invention also includes antibodies comprising the sequences shown in Table 2. For example, in some embodiments, R 2The invention comprises an 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, 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 The invention comprises an antibody 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 also comprises an antibody 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 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 also includes an antibody 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 In some embodiments, the formulations described herein further comprise a fragment of formula (I): [R 1 ] n1 -R 2A complex of the structure where 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., 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, the formulation further comprises tris(hydroxymethyl)aminomethane and sucrose. In some embodiments, the formulation comprises tris(hydroxymethyl)aminomethane at a concentration of 25 mM and / or (eg, and) sucrose at a concentration of 10 w / v%, optionally wherein the formulation has a pH of 7.5. In some embodiments, the formulation comprises the complex at a concentration of 30 mg / mL.

[0213] In some embodiments, the formulations provided herein comprise a compound of formula (I): [R 1 ] n1 -R 2 A complex of structure where each R 1 A group comprising the following formula (Ic):

[0214]

[0215] Where R 1 At the connection point A and R 2 In some embodiments, R 2 The invention also includes antibodies comprising the sequences shown in Table 2. For example, in some embodiments, R 2 The invention comprises an 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, 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 2The invention comprises an antibody 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 also comprises an antibody 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 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 also includes an antibody 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 In some embodiments, the formulations described herein further comprise a fragment 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). In some embodiments, the formulation further comprises tris(hydroxymethyl)aminomethane and sucrose. In some embodiments, the formulation comprises tris(hydroxymethyl)aminomethane at a concentration of 25 mM and / or (e.g., and) sucrose at a concentration of 10 w / v%, optionally, wherein the pH of the formulation is 7.5. In some embodiments, the formulation comprises a complex at a concentration of 30 mg / mL.

[0216] In some embodiments, the formulations provided herein comprise a complex comprising the structure of Formula (Id):

[0217]

[0218] 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 sequence as shown in Table 2; wherein n1 is an integer (e.g., one or greater) representing the number of instances of a group enclosed by square brackets, wherein each instance of a group enclosed by square brackets is covalently linked to a different amino acid residue of the antibody, optionally wherein each different amino acid residue is lysine. In some embodiments, R 2 The invention comprises an 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, 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 The invention comprises an antibody 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 also comprises an antibody 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 2The invention also includes an antibody 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 also includes an antibody 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 In some embodiments, the formulations described herein further comprise a complex wherein n1 is 0. In some embodiments, the average value of n1 of the complex 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, the formulation further comprises tris(hydroxymethyl)aminomethane and sucrose. In some embodiments, the formulation comprises tris(hydroxymethyl)aminomethane at a concentration of 25 mM and / or (eg, and) sucrose at a concentration of 10 w / v%, optionally wherein the pH of the formulation is 7.5. In some embodiments, the formulation comprises the complex at a concentration of 30 mg / mL.

[0219] In some embodiments, the complex provided in the formulations described herein comprises a structure of Formula (A):

[0220]

[0221] (A), wherein y is 0 to 15 (e.g., 3), and z is 0 to 15 (e.g., 4). In some embodiments, the amide adjacent to the antibody (e.g., anti-TfR1 antibody) shown in structure (A) is produced by a reaction with an amine (e.g., lysine ε amine) of the antibody. In some embodiments, the complex described herein comprises an anti-TfR1 antibody (e.g., anti-TfR1 Fab) covalently linked to the 5' end of an oligonucleotide (e.g., an oligonucleotide comprising a 5'-XYZ-3' configuration) via a lysine of the antibody. In some embodiments, the antibody comprises the sequence 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, 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, 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 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 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 antibody comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 19 and / or a light chain comprising an 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') 2In some embodiments, the antibody is a Fab fragment. In some embodiments, the preparation further comprises tris (hydroxymethyl) aminomethane and sucrose. In some embodiments, the preparation comprises tris (hydroxymethyl) aminomethane at a concentration of 25mM and / or (e.g., and) sucrose at a concentration of 10w / v%, optionally wherein the pH of the preparation is 7.5. In some embodiments, the preparation comprises a complex at a concentration of 30mg / mL.

[0222] As described herein, in some embodiments, the formulations provided herein include sucrose. In some embodiments, sucrose is used at least in part as a lyoprotectant. In some embodiments, sucrose comes from plants, such as grass, fruit or vegetables (e.g., root vegetables (root vegetable)) sources (e.g., beets, (e.g., sugar beets (e.g., sugarcane (Saccharum spp.)))), sugarcane (e.g., beets (Beta vulgaris)), palm dates (date), sugar maple, sweet sorghum, apples, oranges, carrots, molasses, maple syrup, corn sweeteners) or animal products (e.g., honey). In some embodiments, sucrose comes from beets or sugarcane (e.g., beet sucrose, cane sucrose). In some embodiments, lyoprotectants other than sucrose can be used, such as trehalose, mannitol, lactose, polyethylene glycol or polyvinyl pyrrolidone. However, in some embodiments, a disintegration temperature regulator (e.g., dextran, polysucrose (ficoll) or gelatin) can be provided in the formulation.

[0223] In some embodiments, provided are products (eg, lyophilized formulations described herein) produced by a process comprising lyophilizing an aqueous solution of a formulation described herein (eg, an aqueous form).

[0224] In some embodiments, the pharmaceutical 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.

[0225] Uses / treatment methods

[0226] 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).

[0227] 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. 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 repeat units, about 2 to 50 repeat units, about 2 to 100 repeat units, about 50 to 1,000 repeat units, about 50 to 500 repeat units, about 50 to 250 repeat units, about 50 to 100 repeat units, about 500 to 10,000 repeat units, about 500 to 5,000 repeat units, about 500 to 2,500 repeat units, about 500 to 1,000 repeat units, or about 1,000 to 10,000 repeat units. In some embodiments, the subject may suffer from myotonic dystrophy, such as DM1. 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.

[0228] One aspect of the present disclosure includes a method, which includes administering to an object a preparation comprising an effective amount of a complex as described herein. In some embodiments, an effective amount of a pharmaceutical composition comprising a complex may be administered to an object in need of treatment, the complex comprising an antibody as described herein (e.g., Fab) covalently linked to an oligonucleotide as described herein (e.g., DMPK targeting oligonucleotide). In some embodiments, a pharmaceutical composition comprising a complex as described herein may be administered by a suitable route, which may include intravenous administration, such as as a bolus or by continuous infusion over a period of time. In some embodiments, administration may be performed by intravenous, intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarticular, intrasynovial or intrathecal routes. In some embodiments, the pharmaceutical composition may be in solid form, aqueous form or liquid form. In some embodiments, aqueous or liquid forms may be atomized or lyophilized. In some embodiments, lyophilized forms may be reconstituted with aqueous or liquid solutions.

[0229] In some embodiments, methods and / or uses for treating an object having a DMPK allele are provided, the allele may optionally contain a disease-associated repeat, the method comprising administering to the object a formulation described herein, the formulation comprising an effective amount of a complex described herein. In some embodiments, methods and / or uses for reducing the expression or activity of DMPK in a cell (e.g., a muscle cell) (e.g., reducing the level of a mutant or wild-type DMPK RNA, or the activity of a DMPK gene product) are provided, the method comprising contacting the cell with a formulation described herein comprising an effective amount of a complex described herein. In some embodiments, the method comprises administering a lyophilized form (e.g., a lyophilized powder) of a formulation described herein, including reconstructing a lyophilized form of the formulation in an aqueous solution, and administering the aqueous solution of the formulation to an object in need thereof. For example, in some embodiments, a lyophilized form of the formulation is transported and / or stored in a lyophilized form, reconstituted in a place (e.g., a health care provider place) for administering an aqueous solution of the formulation, and administered by injection or intravenous (e.g., by infusion) in a reconstituted form (e.g., as an aqueous solution).

[0230] In some embodiments, the pharmaceutical composition is administered by site-specific delivery techniques 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.

[0231] In some embodiments, a pharmaceutical composition comprising a complex containing an anti-TfR1 antibody (e.g., Fab) covalently linked to a molecular payload (e.g., a DMPK targeting oligonucleotide) is administered at an effective concentration that confers 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 treated subject (e.g., age, physical condition, health or weight), the duration of treatment, the nature of any simultaneous treatment, 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 experiments. 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.

[0232] Empirical considerations (e.g., the half-life of the complex in the subject) will generally help determine the concentration of the pharmaceutical composition for treatment. The frequency of administration can be empirically determined and adjusted to maximize the therapeutic efficacy. Any suitable method can be used to assess the efficacy of the treatment. In some embodiments, the efficacy of the treatment can be assessed as follows: by observation or evaluation of symptoms associated with myotonic dystrophy (e.g., muscle atrophy or weakness), by measurement of self-reported outcomes (e.g., mobility, self-care, daily activities, pain / discomfort, and anxiety / depression) of the subject, or by quality of life indicators (e.g., life span). In some embodiments, a pharmaceutical composition comprising a complex containing a muscle targeting agent covalently linked to a molecular payload as described herein is administered to a subject at an effective concentration sufficient to regulate 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% of the target gene activity or expression relative to a control (e.g., baseline level of gene expression before treatment).

[0233] Example

[0234] Example 1. In vivo tissue distribution of a conjugate comprising an anti-TfR1 Fab conjugated to a DMPK targeting oligonucleotide in the DM1 mouse model

[0235] A conjugate comprising an anti-TfR1 Fab conjugated to a DMPK targeting oligonucleotide (ASO) was tested in a mouse model expressing both human TfR1 and a human DMPK mutant containing expanded CUG repeats. The anti-TfR1 Fab used has the VH / VL sequence provided in Table 2. The Fab is covalently linked (conjugated by lysine) to a DMPK targeting oligonucleotide comprising a nucleobase sequence of SEQ IDNO:21 via a linker comprising a valine-citrulline sequence. The conjugate comprises the structure of the following formula (Id):

[0236]

[0237] (Id), 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), and wherein R 2 comprising an anti-TfR1 antibody provided in Table 2, and wherein in each conjugate, n1 is independently an integer from 1 to 3.

[0238] The conjugate was administered intravenously to mice at a dose equivalent to 9.7 mg / kg ASO 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; which is incorporated herein by reference), and ASO levels in tissues were plotted. Figure 1A , 1B , 1C and 1D 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.

[0239] Example 2. Sustained knockdown of toxic human DMPK in hTfR1 / DMSXL homozygous mice 4 weeks after repeated administration of anti-TfR1 Fab-ASO conjugates

[0240] The conjugates containing anti-TfR1 Fab covalently linked to DMPK targeting oligonucleotides (ASOs) as described in Example 1 (labeled in this example as "anti-TfR1 Fab-ASO conjugates") were tested in a mouse model (hTfR1 / DMSXL mice) expressing both human TfR1 and two copies of a mutant human DMPK transgene containing expanded CUG repeats. On days 0 and 7, vehicle controls (PBS) or 10 mg / kg ASO equivalent doses of anti-TfR1Fab-ASO conjugates were administered to mice. Mice were sacrificed on day 28 (four weeks after the first dose of anti-TfR1 Fab-ASO conjugates were administered) 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 (peptidylprolyl isomerase) as an internal control. DMPK expression in Figures 2A to 2D 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).

[0241] Figure 2A It was shown that anti-TfR1 Fab-ASO conjugate knocked down DMPK expression in the heart by 49% relative to PBS-treated mice. Figure 2B It was shown that anti-TfR1 Fab-ASO conjugate knocked down DMPK expression in diaphragm muscle by 40% relative to PBS-treated mice. Figure 2C 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 2D It was shown that anti-TfR1 Fab-ASO conjugate knocked down DMPK expression in gastrocnemius muscle by 44% relative to PBS-treated mice.

[0242] Figure 3A and 3B It was shown that anti-TfR1 Fab-ASO conjugate reduced DMPK foci within myofiber nuclei. Figure 3A Reduced DMPK foci were shown by in situ hybridization, and Figure 3B Quantification 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).

[0243] 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.

[0244] Example 3. Correction of splicing defects in hTfR1 / DMSXL homozygous mice by anti-TfR1 Fab-ASO conjugates

[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 ("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 set 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 the normalized percent spliced ​​in (PSI) for each spliced ​​RNA marker and was calculated using the data from heart ( Figure 4 ), diaphragm( Figure 5 ), tibialis anterior ( Figure 6 ) and gastrocnemius ( Figure 7 ) were used to calculate the composite splicing index using 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.

[0246] Figure 4It 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 that treatment with anti-TfR1 Fab-ASO conjugate corrects Mbnl2 exon 6 (E6) and Nfix E7 splicing in heart tissue of hTfR1 / DMSXL mice, but does not show correction of Ldb3 E11 splicing. Figure 4 Composite splicing index data shown are based on Ldb3E11, Mbnl2 E6, and Nfix E7 splicing data; Bin1 E11, Dtna E12, Insr E11, and Mbnl2E5 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.

[0247] Figure 5 It is shown that anti-TfR1 Fab-ASO conjugate corrects splicing in diaphragm 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 Bin1 E11, Insr E11, Ldb3 E11 and Nfix E7 splicing in diaphragm tissue of hTfR1 / DMSXL mice by treatment with anti-TfR1 Fab-ASO conjugate. Figure 5 Composite splicing index data shown are based on Bin1 E11, Insr E11, Ldb3 E11, and Nfix E7 splicing data; Dtna E12, Mbnl2 E5, Mbnl2 E6, and Ttn E313 were not included because their normalized PSI values ​​in diaphragm muscle tissue were not altered in hTfR1 / DMSXL mice relative to hTfR1 mice under the experimental conditions tested.

[0248] Figure 6 It is shown that anti-TfR1 Fab-ASO conjugate corrects splicing in tibialis anterior 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 that treatment with anti-TfR1 Fab-ASO conjugate corrects splicing of Bin1 E11, Ldb3 E11, and Nfix E7, but not Mbnl2 E6, in tibialis anterior muscle tissue of hTfR1 / DMSXL mice. Figure 6The 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 were 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.

[0249] Figure 7 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. Figure 7 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 were 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.

[0250] These results suggest that administration of anti-TfR1 Fab-ASO conjugates facilitates correction of DM1 splicing defects in cardiac and skeletal muscles.

[0251] Example 4. DMPK knockdown in non-human primate and DM1 patient myotubes

[0252] The 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 human DM1 patient myotubes (32F cells) and non-human primate (NHP) myotubes. The DM1 patient myotubes used expressed both a mutant DMPK mRNA containing 380 CUG repeats and a wild-type DMPK mRNA. The NHP myotubes used expressed only wild-type DMPK.

[0253] 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 ten days and then cDNA was synthesized using the Cells-to-Ct kit, with crude cell lysate as the source of total RNA.

[0254] 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. Figure 8 Data shown in are presented as mean DMPK expression + standard deviation relative to species-matched vehicle controls (n=4 replicates per condition).

[0255] 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) ( Figure 8 ). 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.

[0256] These results indicate that a 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 both wild-type DMPK mRNA and mutant DMPK mRNA (with expanded CUG repeats) relative to cynomolgus monkey myotubes expressing wild-type DMPK.

[0257] Example 5. Comparison of thermal stability of different exemplary formulations

[0258] Preparations comprising the conjugate described in Example 1 were prepared, comprising an anti-TfR1 Fab covalently linked to a DMPK targeting oligonucleotide (ASO). The concentration of the anti-TfR1 Fab-oligonucleotide conjugate starting material ("SM (starting material)") in a buffer containing 100 mM sodium phosphate and 100 mM sodium chloride at pH 7.4 was 33 mg / mL. For stability testing, the anti-TfR1 Fab-oligonucleotide conjugate was formulated in different buffers at a concentration of about 40 mg / mL. The buffer conditions for each of the four formulations were as follows:

[0259] Formulation buffer 1: 25 mM succinate, 10% (w / v) sucrose, pH 5.2.

[0260] Formulation buffer 2: 25 mM succinate, 10% (w / v) sucrose, pH 6.0.

[0261] Formulation buffer 3: 25 mM tris(hydroxymethyl)aminomethane, 10% (w / v) sucrose, pH 7.5 Formulation buffer 4: 100 mM sodium phosphate, 100 mM sodium chloride, pH 7.4.

[0262] use 6 centrifugal membrane filters (molecular weight cut-off of 30kDa) buffer exchange the formulation drug substance (formulation with conjugate described herein) into formulation buffers 1 to 4 as provided above. After buffer exchange, the anti-TfR1 Fab-oligonucleotide conjugate was concentrated to about 40mg / ml. An aliquot of about 150 μL from each concentrated sample was used to study the thermal stability of the conjugate in each formulation. The samples were incubated in glass bottles at -20°C, 2 to 8°C, 25°C or 40°C for 1 week ("T1"), 2 weeks ("T2"), 4 weeks ("T4") or 8 weeks ("T8") . At any temperature or time point tested, no changes in color, appearance or pH were observed in any test sample.

[0263] The thermal stability of each formulation was first tested by measuring the concentration of the conjugate in various samples at T0 (week 0), T4 and T8 and at a specific temperature. The concentration was measured using a standard BCA (Bicinchoninic Acid) assay. The BCA assay was performed using a standard procedure:

[0264] 1) Prepare Working Reagent (WR) by thoroughly mixing 20 mL BCA Reagent A with 200 μL BCA Reagent B.

[0265] 2) Prepare standard curve and dilution curve of test samples. First, dilute the test samples at 1:4, then mix 20 μL of each test sample or standard with 200 μL WR and incubate at 60°C for 10 minutes. Add the resulting solution to the first row of a 96-well plate and dilute serially. Prepare a 6-point standard curve.

[0266] 3) Measure the absorbance at 480 nm on a plate reader.

[0267] 4) The standard curve was used to determine the protein concentration of each test sample. Five dilutions of each test sample were measured.

[0268] The results of the concentration measurements for the thermal stability study are shown in Table 3 below.

[0269] Table 3.

[0270]

[0271] The concentrations shown were calculated from the mean of the measurements of 5 serial dilutions of each test sample.

[0272] The results in Table 3 show that the concentrations of Fab-oligonucleotide conjugates in Formulations 2, 3 and 4 were consistent at T0, T4 and T8. The concentrations of Fab-oligonucleotide conjugates in Formulation 1 at T4 and T8 showed reduced concentrations at lower storage temperatures compared to T0 measurements.

[0273] The thermal stability of the Fab-oligonucleotide conjugates was further investigated by measuring aggregate formation at a concentration of 40 mg / mL. After dilution to 1 mg / mL, the Fab-oligonucleotide conjugates were analyzed using a Waters ACQUITY UPLC Protein BEH SEC column ( 1.7μm,4.6x150 mm), the mobile phase was 0.2M potassium phosphate pH 6.8, 0.2M potassium chloride isocratic elution, 15% isopropanol, and the T0, T1, T2, T4 and T8 samples incubated at different specified temperatures were analyzed by size exclusion chromatography (SEC-UV). The column temperature was 30°C and a 10μg injection was used. The flow rate was 0.35mL / min and the analysis was performed at a wavelength of 280nm.

[0274] In the SEC thermal stability analysis, the percentage of high molecular weight (HMW) substances, Fab oligonucleotide conjugated monomers and low molecular weight (LMW) substances of each sample was measured. The results are shown in Table 4 below. The values ​​of starting material (SM) are shown in the top row for comparison.

[0275] Table 4.

[0276]

[0277]

[0278] The results showed that the percentage of monomer did not change significantly with increasing temperature or over time, and a slight increase in LMW species was observed over time with increasing temperature.

[0279] Overall, SEC analysis indicated that Formulations 1, 2, and 3 provided slightly improved stability at the 8 week time point relative to % monomer compared to Formulation 4. Formulation 3 showed the best stability relative to changes in % LMW species.

[0280] By using the non-reduced protein expression assay of capillary electrophoresis (CE-SDS), the thermal stability of the Fab-oligonucleotide conjugate of 40mg / mL was further studied. For the analysis by the method, the sample was diluted to 1mg / mL, then mixed with 10mM N-ethylmaleimide in PBS at 1:1. Then each of these samples of 5 μL was added to 7 μL sample buffers (Protein Express Reagent kit, Perkin Elmer CLS960008) in 96-well plates. The sample was incubated at 70 ° C for 10 minutes and then cooled on ice for 5 minutes. 32 μL of water was added to each sample well, and the plate was centrifuged for 2 minutes at 1200g before analysis. On HT Protein Express LabChip (Perkin Elmer 760499), LabChip GXII Touch HT (Perkin Elmer) was used, and the sample was analyzed using the measurement method HT High Sensitivity Antibody Analysis 200.

[0281] The results of the non-reducing CE-SDS analysis are shown below in Table 5. For comparison, the starting material showed a size of 62.4 kDa and a purity of 100.0%.

[0282] Table 5.

[0283]

[0284] Sizes are shown in kDa. Values ​​in brackets indicate percent purity.

[0285] The results showed that the purity percentage of the conjugates in Formulation 1 gradually decreased at 25°C and 40°C from T1 to T8, and the formation of new substances was observed, indicating that the conjugates in Formulation 1 were less stable at higher temperatures. The results also showed that the conjugates in Formulations 2, 3, and 4 had good purity percentages at all temperatures and time points tested. The high purity percentages of the conjugates in Formulation 3 at higher temperatures indicate that they have improved stability in Formulation 3 relative to the other formulations.

[0286] The heat stability of the Fab-oligonucleotide conjugate of 40mg / mL was further studied by using the reduced protein expression assay of capillary electrophoresis (CE-SDS).For the analysis by the method, the sample was diluted to 0.5mg / mL with PBS.Next, the sample was mixed with denaturing solution: 24.5 μL 14.2M β-mercaptoethanol (BME) was added to 700 μL protein expression sample buffers (protein expression kit, Perkin Elmer CLS960008), and 5 μL of each diluted sample was added to the 7 μL sample denaturing solution in 96 well plates subsequently.Sample was incubated at 70 ℃ for 10 minutes, then cooled on ice for 5 minutes.32 μL water was added to each sample well, and before analysis, the plate was centrifuged at 1200g for 2 minutes. The samples were analyzed on a HT ProteinExpress LabChip (Perkin Elmer 760499) using the LabChip GXII Touch HT (PerkinElmer) using the measurement method HT High Sensitivity Antibody Analysis 200.

[0287] The results of the reduced CE-SDS analysis are shown below in Table 6. The values ​​for the starting material (SM) are shown in the top row for comparison.

[0288] Table 6.

[0289]

[0290]

[0291]

[0292] Sizes are shown in kDa.

[0293] In Formulation 1, two additional peaks were observed in all samples of T2, T4, and T8, including an additional HC peak, indicating instability of the conjugate in this formulation. The overall stability of the conjugates in Formulations 2, 3, and 4 was not significantly different as observed by reduced CE-SDS.

[0294] Overall, the results of the various thermal stability tests presented herein indicate that Formulation 3 provides the best stability for the Fab-oligonucleotide conjugate.

[0295] Example 6. Comparison of freeze / thaw stability of different exemplary formulations

[0296] Preparations comprising the conjugate described in Example 1 were prepared, comprising an anti-TfR1 Fab covalently linked to a DMPK targeting oligonucleotide (ASO). The concentration of the anti-TfR1 Fab-oligonucleotide conjugate starting material ("SM") in a buffer containing 100 mM sodium phosphate and 100 mM sodium chloride at pH 7.4 was 33 mg / mL. For freeze / thaw stability testing, the anti-TfR1 Fab-oligonucleotide conjugate was formulated in different buffers at a concentration of about 40 mg / mL. The buffer conditions for each of the four formulations were as follows:

[0297] Formulation buffer 1: 25 mM succinate, 10% (w / v) sucrose, pH 5.2.

[0298] Formulation buffer 2: 25 mM succinate, 10% (w / v) sucrose, pH 6.0.

[0299] Formulation buffer 3: 25 mM tris(hydroxymethyl)aminomethane, 10% (w / v) sucrose, pH 7.5 Formulation buffer 4: 100 mM sodium phosphate, 100 mM sodium chloride, pH 7.4.

[0300] To test the freeze / thaw stability of the conjugates in each formulation, aliquots were prepared and subjected to 0, 1, 3 or 5 freeze / thaw cycles, after which time visual appearance, protein concentration and aggregate / fragment formation were assessed.

[0301] For each of Formulations 1, 2, 3, and 4, visual appearance evaluation showed that the solutions were clear, colorless, and free of visible aggregates after 0, 1, 3, and 5 freeze / thaw cycles.

[0302] BCA analysis of each formulation performed as described in Example 5 showed that the concentration of the complex did not change significantly after 5 freeze / thaw ("F / T") cycles. The results are shown below in Table 7. The values ​​for the starting material (SM) are shown in the top row for comparison.

[0303] Table 7

[0304]

[0305] Size Exclusion Chromatography (SEC) analysis of each formulation performed as described in Example 5 showed no significant differences in low molecular weight (LMW) species, monomers, and high molecular weight (HMW) species among the four formulations over 5 freeze / thaw ("F / T") cycles. The results are shown below in Table 8. The values ​​for the starting material (SM) are shown in the top row for comparison.

[0306] Table 8

[0307]

[0308] Example 7. Evaluation of the stability of lyophilized formulations of conjugates

[0309] 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 prepared and lyophilized together with tris(hydroxymethyl)aminomethane (25 mM), sucrose (10% (w / v)) at pH 7.5 and tested for stability. Prior to reconstitution, all samples at all time points and temperatures were visually identified as white cakes with uniform shapes. In addition, the samples were reconstituted in less than 1 minute and became clear and colorless with less than 10 particles. By visual analysis, all samples were considered stable for at least 8 weeks at up to 40°C before and after reconstitution.

[0310] X-ray diffraction analysis was performed to evaluate the crystallographic structure of the lyophilized composition. Approximately 20 mg of lyophilized anti-TfR1 Fab-ASO conjugate was placed in a plastic sample holder and flattened with a glass slide to ensure a flat surface. When performing this analysis, the Proto The benchtop power X-ray diffractometer is used with a fine focus copper anode metal / ceramic construction 1500 watt X-ray tube.

[0311] Measure the diffraction pattern ( Fig. 9 ) and identify the wide "halo" ( Fig. 9 ), which indicates that the substance is amorphous at time 0 (T0).

[0312] To test the absorbance of the sample, the lyophilized anti-TfR1 Fab-ASO conjugate was first reconstituted with water and then diluted to a concentration of 0.2 mg / ml using a formulation buffer (25 mM tris (hydroxymethyl) aminomethane, 10% (w / v) sucrose, pH 7.5). The absorbance at 260 nm and 280 nm was measured using a DeNovix DS11+ spectrophotometer. The readings were performed in triplicate and averaged. These results were used to calculate the concentration (Fab (mg / mL and μM) and oligonucleotide (μM)), recovery percentage, and drug-antibody ratio (DAR) at multiple time points. For all protein concentrations, recovery percentages, and DAR tests, bovine serum albumin (BSA) was used as a positive control, PBS was used as a blank and negative control for BSA, and formulation buffer was used as a blank and negative control for conjugate samples.

[0313] At 2 to 8°C, the mean concentration of Fab replicates ( Fig. 10A and Fig. 10B ) showed no significant change from time 0 (T0). In addition, there was no significant change when comparing the 4-week and 8-week time points. At 2 to 8°C, the average concentration of the replicates of oligonucleotides ( Fig. 10C ) showed no significant difference. The average recovery percentage of all samples at all time points at 2 to 8 °C ( Fig.11A ) are close to 100%. At 2 to 8°C, the average DAR ( Fig. 11B ) were close to 1.0 at all time points.

[0314] At 25°C, the mean concentration of Fab replicates was ( Fig. 12A and Fig. 12B ) showed no significant change relative to time 0 (T0), and there was no significant change when T2w, T4w and T8w were compared to each other. At 25°C, the average concentration of the replicates of oligonucleotides ( Fig. 12C ) did not show significant changes over time. The average percent recovery for all time points at 25 °C ( Fig.13A ) are close to 100%. At 25℃, the average DAR ( Fig. 13B ) were close to 1.0 at all time points.

[0315] At 40°C, the average concentration of Fab replicates ( Fig.14A and Fig. 14B ) showed no significant change relative to T0. At 40°C, the average concentration of the replicates of oligonucleotides ( Fig. 10C ) did not change significantly at any time point. The average recovery percentage of all samples at all time points at 40 °C ( Fig.15A ) is close to 100%. At 40℃, the average DAR( Fig. 15B ) were close to 1.0 at all time points.

[0316] Next, the anti-TfR1 Fab-ASO conjugate was analyzed using size exclusion chromatography (SEC). Prior to analysis, all samples were diluted to a concentration of 1.0 mg / ml using a formulation buffer (25 mM tris(hydroxymethyl)aminomethane, 10% (w / v) sucrose, pH 7.5). For the mobile phase of the chromatographic conditions, an isocratic elution containing potassium phosphate (200 mM), potassium chloride (200 mM), 5% (v / v) isopropanol (isopropanol, IPA) (pH 6.8) was used with a flow rate of 0.250 ml / min and a run time of 20 minutes. The wavelength used for measurement was 280 nm. The HPLC system used was a Dionex Ultimate 3000UPLC system with a Waters BEH SEC column ( 300x 4.6mm, 1.7μm, P / N 186005226).

[0317] At 2 to 8°C, when comparing T0, T4w and T8w, the sum of the relative areas of the first and second (main) peaks ( Fig.16A ) showed no significant difference. In addition, when comparing T0, T4w and T8w, the sum of the relative areas of the third and fourth (high molecular weight substances (HMWS)) peaks ( Fig. 16B ) also showed no significant difference.

[0318] At 25°C, when comparing T0, T2w, T4w and T8w, the sum of the relative areas of the first and second (main) peaks ( Fig.17A ) showed no significant difference. In addition, when comparing T0, T2w, T4w and T8w, the sum of the relative areas of the third and fourth (HMWS) peaks ( Fig. 17B ) also showed no significant difference.

[0319] At 40 °C, when comparing T0, T2w, T4w and T8w, the sum of the relative areas of the first and second (main) peaks ( Fig.18A ) showed no significant difference. In addition, when comparing T0, T2w, T4w and T8w, the sum of the relative areas of the third and fourth (HMWS) peaks ( Fig.18B ) also showed no significant difference.

[0320] For non-reducing capillary electrophoresis sodium dodecyl sulfate (CE-SDS) analysis, the sample was diluted to 1 mg / ml in phosphate buffered saline (PBS). The diluted sample was then further diluted 1:1 with 10 mM N-ethylmaleimide (NEM) in PBS. 5.0 μl of the resulting solution was then added to 7.0 μl of sample buffer. The sample was then incubated at 70°C for 10 minutes. After incubation, 32 μl of water was added and the sample was centrifuged at 1200 g for 2 minutes. The sample was then analyzed using HT High Sensitivity Antibody Analysis 200, HT Protein Express LabChip (Perkin Elmer, 760499) and a protein expression kit (Perkin Elmer, CLS960008). Analyzed on LabChip GXII Touch HT (Perkin Elmer).

[0321] At 2 to 8°C ( Fig.19 ), the purity of the main peak did not change significantly between T0, T4w and T8w. Fig. 20 ) and 40℃( Fig.21 ) under the conditions of T0, T2w, T4w and T8w, the purity of the main peak did not change significantly.

[0322] In order to carry out reduction CE-SDS analysis, the sample is diluted to 0.5mg / ml in PBS. 5.0μl diluted sample is added to 7.0pl sample denaturation solution (24.5μL 1M dithiothreitol (DTT) is added to 700μl protein expression sample buffer). The sample is then incubated at 70°C for 10 minutes. After incubation, 32μl water is added and the sample is centrifuged at 1200g for 2 minutes. The sample is then analyzed using HT High Sensitivity Antibody Analysis 200, HT Protein ExpressLabChip (Perkin Elmer, 760499) and protein expression kit (Perkin Elmer, CLS960008). Analyzed on LabChip GXII Touch HT (Perkin Elmer).

[0323] At 2 to 8°C (Table 9), the purity of peaks 1, 2, 3 and 4 did not change significantly between T0, T4w and T8w. At 25°C (Table 10) and 40°C (Table 11), the purity of peaks 1, 2, 3 and 4 did not change significantly between T0, T2w, T4w and T8w.

[0324] Overall, these results indicate that the lyophilized anti-TfR1 Fab-ASO conjugate is stable at all temperatures and time points tested.

[0325] Table 9. Capillary electrophoresis sodium dodecyl sulfate (CE-SDS) results at 2 to 8°C (reduced)

[0326] Time Peak 1(%) Peak 2 (%) Peak 3 (%) total(%) T0 3.05 89.97 6.09 99.11 T4w 2.87 88.40 8.42 99.69 T8h 4.12 85.15 8.41 97.68

[0327] Table 10. Capillary electrophoresis sodium dodecyl sulfate (CE-SDS) results at 25°C (reduced)

[0328] Time Peak 1(%) Peak 2 (%) Peak 3 (%) total(%) T0 3.05 89.97 6.09 99.11 T2W 3.21 88.94 7.31 99.46 T4w 2.87 87.62 9.09 99.58 T8h 4.12 85.22 8.38 97.72

[0329] Table 11. Capillary electrophoresis sodium dodecyl sulfate (CE-SDS) results at 40°C (reduced)

[0330] Time Peak 1(%) Peak 2 (%) Peak 3 (%) total(%) T0 3.05 89.97 6.09 99.11 T2W 3.21 88.80 7.34 99.35 T4w 2.91 87.80 8.75 99.46 T8h 4.14 85.20 8.41 97.75

[0331] Additional embodiments

[0332] 1. A formulation comprising a complex comprising an oligonucleotide covalently linked to an anti-transferrin receptor 1 (TfR1) antibody,

[0333] 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,

[0334] 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,

[0335] And wherein the complex is formulated with tris(hydroxymethyl)aminomethane and sucrose.

[0336] 2. Containing formula (I): [R 1 ] n1 -R 2 The preparation of the complex of structure, in which each R1 Independently comprising groups of the following formula (Ia):

[0337]

[0338] (Ia), where

[0339] R 2 Contains antibodies, and

[0340] R 3 comprising an oligonucleotide comprising 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;

[0341] Where R 1 At the connection point A and R 2 covalently attached; and

[0342] Where n1 represents R 1 An integer representing the number of instances, where R 1 Each instance of is covalently linked to a different amino acid residue of said antibody, optionally wherein each different amino acid residue is lysine;

[0343] The complex is formulated with tris(hydroxymethyl)aminomethane and sucrose,

[0344] Optionally, wherein the antibody is an anti-TfR1 antibody.

[0345] 3. The formulation of embodiment 2, wherein the antibody comprises:

[0346] A heavy chain complementary determining region 1 (CDR-H1) containing the sequence shown in SEQ ID NO:1, 7 or 12, a heavy chain complementary determining region 2 (CDR-H2) containing the sequence shown in SEQ ID NO:2, 8 or 13, a heavy chain complementary determining region 3 (CDR-H3) containing the sequence shown in SEQ ID NO:3, 9 or 14, a light chain complementary determining region 1 (CDR-L1) containing the sequence shown in SEQ ID NO:4, 10 or 15, a light chain complementary determining region 2 (CDR-L2) containing the sequence shown in SEQ ID NO:5 or 11, and a light chain complementary determining region 3 (CDR-L3) containing the sequence shown in SEQ ID NO:6 or 16.

[0347] 4. The formulation of any one of embodiments 1 to 3, wherein the formulation is in lyophilized form.

[0348] 5. The formulation of any one of embodiments 1 to 3, wherein the formulation is in a frozen solid form.

[0349] 6. The formulation of any one of embodiments 1 to 3, wherein the formulation is an aqueous solution.

[0350] 7. The formulation of embodiment 6, wherein the tris(hydroxymethyl)aminomethane is present in the aqueous solution at a concentration of 5 mM to 50 mM.

[0351] 8. The formulation of embodiment 6 or 7, wherein the sucrose is present in the aqueous solution at a concentration of 5% to 15% weight / volume (w / v%).

[0352] 9. The formulation of any one of embodiments 6 to 8, wherein the pH of the aqueous solution is 6.5 to 8.5.

[0353] 10. The formulation of any one of embodiments 6 to 9, wherein the tris(hydroxymethyl)aminomethane is present in the aqueous solution at a concentration of 25 mM, and / or the sucrose is present in the aqueous solution at a concentration of 10 w / v%, and / or the pH of the aqueous solution is 7.5.

[0354] 11. The formulation of any one of embodiments 1 to 10, wherein the antibody is a Fab fragment, a full-length IgG, a Fab' fragment, a F(ab') 2 fragment, scFv or Fv.

[0355] 12. The formulation of embodiment 11, wherein the antibody is a Fab fragment.

[0356] 13. The formulation of any one of embodiments 1 to 12, wherein the antibody comprises a heavy chain variable region (VH) comprising an amino acid sequence at least 85% identical to SEQ ID NO: 17; and / or wherein the antibody comprises a light chain variable region (VL) comprising an amino acid sequence at least 85% identical to SEQ ID NO: 18,

[0357] Optionally wherein 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.

[0358] 14. The formulation of any one of embodiments 1 to 13, wherein the antibody comprises a heavy chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 19; and / or wherein the antibody comprises a light chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 20,

[0359] Optionally, the 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.

[0360] 15. The formulation of any one of embodiments 1 to 14, wherein the oligonucleotide is 10 to 30 nucleotides in length.

[0361] 16. The formulation of any one of embodiments 1 to 15, wherein the oligonucleotide comprises a nucleotide sequence having a complementary region of at least 8 consecutive nucleotides in length to SEQ ID NO: 22.

[0362] 17. The formulation of any one of embodiments 1 to 16, wherein the oligonucleotide comprises at least 8 consecutive nucleotides of the nucleotide sequence shown in SEQ ID NO: 21, optionally wherein the oligonucleotide comprises the nucleotide sequence of SEQ ID NO: 21.

[0363] 18. The formulation of any one of embodiments 2 to 17, wherein each R 1 A group comprising the following formula (Ib):

[0364]

[0365] 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, such that the oligonucleotide comprises a nucleobase sequence of CAGCGCCCACCAGUCA (SEQ ID NO: 21).

[0366] 19. The formulation of any one of embodiments 2 to 17, wherein each R 1 A group comprising the following formula (Ic):

[0367]

[0368] 20. The formulation of any one of embodiments 1 to 19, wherein the complex is present in the formulation at a concentration of 10 mg / mL to 50 mg / mL.

[0369] 21. The formulation of any one of embodiments 1 to 20, further comprising one or more antibodies that are not covalently linked to the oligonucleotide.

[0370] 22. The formulation of embodiment 21, wherein the average value of n1 of the complexes in the formulation is 0.5 to 5.

[0371] 23. A method of reducing DMPK expression in a subject, the method comprising administering to the subject an effective amount of the formulation of any one of embodiments 1 to 22.

[0372] 24. A method of treating myotonic dystrophy in a subject, the method comprising administering to the subject an effective amount of the formulation of any one of embodiments 1 to 22.

[0373] 25. The method of embodiment 23 or embodiment 24, wherein the subject has an expansion of a disease-associated repeat of a DMPK allele associated with myotonic dystrophy.

[0374] 26. The method of embodiment 25, wherein the disease-associated repeat comprises repeating units of a CTG trinucleotide sequence.

[0375] 27. The method of any one of embodiments 23 to 26, wherein the complex reduces DMPK expression in the subject.

[0376] 28. Comprising formula (I): [R 1 ] n1 -R 2 A complex of structure where each R 1 A group comprising the following formula (Ia):

[0377]

[0378] Where R 3 an oligonucleotide comprising a structure comprising +C*+A*oG*oC*dC*dC*dC*dC*dA*dC*dC*dA*oG*oU*+C*+A (SEQ ID NO: 21), wherein +N represents an LNA (2'-4' methylene bridge) ribonucleoside, dN represents a 2'-deoxyribonucleoside, oN represents a 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;

[0379] Where R 2 comprising a Fab, and wherein the Fab comprises a CDR-H1, a CDR-H2, a CDR-H3, a CDR-L1, a CDR-L2 and a CDR-L3 selected from Table 2,

[0380] Optionally, wherein the 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,

[0381] Also optionally, wherein the 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;

[0382] Where R 1 At the connection point A and R 2 covalently linked; and wherein n1 is R 1 An integer representing the number of instances, where R 1 Each instance of is covalently linked to a different amino acid residue in Fab, optionally wherein each different amino acid residue is lysine.

[0383] 29. Comprising formula (I): [R 1 ] n1 -R 2 A complex of structure where each R 1 A group comprising the following formula (Ib):

[0384]

[0385] 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 thiophosphate internucleoside linkage; such that the oligonucleotide comprises the nucleobase sequence of CAGCGCCCACCAGUCA (SEQ ID NO: 21).

[0386] Where R 2 comprising a Fab, and wherein the Fab comprises a CDR-H1, a CDR-H2, a CDR-H3, a CDR-L1, a CDR-L2 and a CDR-L3 selected from Table 2,

[0387] Optionally, wherein the 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,

[0388] Also optionally, wherein the 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;

[0389] Where R 1 At the connection point A and R 2 covalently linked; and wherein n1 is R 1 An integer representing the number of instances of 1Each instance of is covalently linked to a different amino acid residue in Fab, optionally wherein each different amino acid residue is lysine.

[0390] 30. Comprising formula (I): [R 1 ] n1 -R 2 A complex of structure where each R 1 A group comprising the following formula (Ic):

[0391]

[0392] Where R 2 comprising a Fab comprising a CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 selected from Table 2,

[0393] Optionally, wherein R 2 comprising a Fab comprising 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,

[0394] Also optionally, wherein R 2 comprising a Fab comprising 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; wherein R 1 At the connection point A and R 2 covalent attachment;

[0395] Where n1 represents R 1 An integer representing the number of instances of 1 Each instance of is covalently linked to a different amino acid residue in Fab, optionally wherein each different amino acid residue is lysine.

[0396] 31. A complex comprising the structure of formula (Id):

[0397]

[0398] 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 phosphorothioate internucleoside linkage;

[0399] Where R 2comprising a Fab, and wherein the Fab comprises a CDR-H1, a CDR-H2, a CDR-H3, a CDR-L1, a CDR-L2 and a CDR-L3 selected from Table 2,

[0400] Optionally, wherein the 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,

[0401] Also optionally, wherein the 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;

[0402] wherein n1 is an integer 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 different amino acid residue of Fab, optionally wherein each different amino acid residue is lysine.

[0403] 32. A formulation comprising a plurality of complexes according to any one of embodiments 28 to 31, and tris(hydroxymethyl)aminomethane at a concentration of 5 to 50 mM and sucrose at a concentration of 5 w / v% to 10 w / v%, wherein the formulation is an aqueous solution and has a pH of 6.5 to 8.5, optionally wherein the concentration of the plurality of complexes is 10 mg / mL to 50 mg / mL.

[0404] 33. A lyophilized form of the formulation of embodiment 32.

[0405] 34. A product produced by a method comprising lyophilizing the formulation of embodiment 32.

[0406] 35. A frozen form of the formulation of embodiment 32.

[0407] 36. A product produced by a method comprising freezing the formulation of embodiment 32.

[0408] 37. A formulation comprising a plurality of complexes according to any one of embodiments 28 to 31, and tris(hydroxymethyl)aminomethane at a concentration of 5 to 50 mM and sucrose at a concentration of 5 w / v% to 15 w / v%, wherein the formulation is an aqueous solution and has a pH of 6.5 to 8.5, optionally wherein the concentration of the plurality of complexes is 10 mg / mL to 50 mg / mL.

[0409] 38. A formulation comprising a plurality of complexes according to any one of embodiments 28 to 31, and tris(hydroxymethyl)aminomethane at a concentration of 25 mM and sucrose at a concentration of 10 w / v%, wherein the formulation is an aqueous solution and has a pH of 7.5, optionally wherein the concentration of the plurality of complexes is 10 mg / mL to 50 mg / mL.

[0410] 39. The formulation of any of embodiments 32, 37 and 38, further comprising one or more antibodies that are not covalently linked to the oligonucleotide.

[0411] 40. The formulation of embodiment 39, wherein the average value of n1 of the complexes in the formulation is 0.5 to 5.

[0412] 41. A lyophilized form of the formulation of any one of embodiments 37 to 40.

[0413] 42. A lyophilized cake comprising a plurality of complexes according to any one of embodiments 28 to 31, tris(hydroxymethyl)aminomethane, and sucrose.

[0414] 43. The lyophilized cake of embodiment 42, comprising 4.02×10 -2 mg to 1.21 mg tris(hydroxymethyl)aminomethane / g cake; 989 mg to 999 mg sucrose / g cake; and / or 0.666 mg to 9.90 mg complex / g cake;

[0415] Optionally, wherein the lyophilized cake comprises:

[0416] (i) 0.303 mg tris(hydroxymethyl)aminomethane / g cake, 998.6 mg sucrose / g cake, and / or 0.999 mg complex / g cake;

[0417] (ii) 0.302 mg tris(hydroxymethyl)aminomethane / g cake, 997.7 mg sucrose / g cake, and / or 1.995 mg complex / g cake;

[0418] (iii) 0.302 mg tris(hydroxymethyl)aminomethane / g cake, 997.2 mg sucrose / g cake, and / or 2.49 mg complex / g cake;

[0419] (iv) 0.302 mg tris(hydroxymethyl)aminomethane / g cake, 996.7 mg sucrose / g cake, and / or 2.99 mg complex / g cake;

[0420] (v) 0.302 mg tris(hydroxymethyl)aminomethane / g cake, 995.7 mg sucrose / g cake, and / or 3.98 mg complex / g cake; or

[0421] (vi) 0.301 mg tris(hydroxymethyl)aminomethane / g cake, 994.7 mg sucrose / g cake, and / or 4.97 mg complex / g cake.

[0422] 44. The lyophilized cake of embodiment 42 or embodiment 43, wherein when about 10 g of the lyophilized cake is reconstituted in water to produce 1 mL of a solution, the solution comprises tris(hydroxymethyl)aminomethane at a concentration of 5 to 50 mM, sucrose at a concentration of 5 w / v% to 15 w / v%, and the complex at a concentration of 10 mg / mL to 50 mg / mL,

[0423] Optionally, the concentration of the tris(hydroxymethyl)aminomethane is 25 mM and / or the concentration of the sucrose is 10 w / v%.

[0424] Equivalent schemes and terminology

[0425] 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.

[0426] 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.

[0427] 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.

[0428] 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.

[0429] 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.

[0430] 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 formulation comprising a complex comprising an oligonucleotide covalently linked to an anti-transferrin receptor 1 (TfR1) antibody, 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, 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 wherein the complex is formulated with tris(hydroxymethyl)aminomethane and sucrose.

2. A preparation comprising a complex comprising formula (I): [R 1 ] n1 -R 2 The structure of each R 1 Independently comprising groups of the following formula (Ia): in R 2 Contains antibodies, and R 3 comprising an oligonucleotide comprising 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, Where R 1 At the connection point A and R 2 covalently attached; and Where n1 is an integer representing R 1 The number of instances of R 1 Each instance of is covalently linked to a different amino acid residue of said antibody, optionally wherein each different amino acid residue is lysine; The complex is formulated with tris(hydroxymethyl)aminomethane and sucrose, Optionally, wherein the antibody is an anti-TfR1 antibody.

3. The formulation of claim 2, wherein the antibody comprises: A heavy chain complementary determining region 1 (CDR-H1) containing the sequence shown in SEQ ID NO:1, 7 or 12, a heavy chain complementary determining region 2 (CDR-H2) containing the sequence shown in SEQ ID NO:2, 8 or 13, a heavy chain complementary determining region 3 (CDR-H3) containing the sequence shown in SEQ ID NO:3, 9 or 14, a light chain complementary determining region 1 (CDR-L1) containing the sequence shown in SEQ ID NO:4, 10 or 15, a light chain complementary determining region 2 (CDR-L2) containing the sequence shown in SEQ ID NO:5 or 11, and a light chain complementary determining region 3 (CDR-L3) containing the sequence shown in SEQ ID NO:6 or 16.

4. The formulation of any one of claims 1 to 3, wherein the formulation is in a lyophilized form or a frozen solid form.

5. The formulation of any one of claims 1 to 3, wherein the formulation is an aqueous solution.

6. The formulation of claim 5, wherein the tris(hydroxymethyl)aminomethane is present in the aqueous solution at a concentration of 5 mM to 50 mM.

7. The formulation of claim 5 or 6, wherein the sucrose is present in the aqueous solution at a concentration of 5% to 15% weight / volume (w / v%).

8. The formulation of any one of claims 5 to 7, wherein the pH of the aqueous solution is 6.5 to 8.

5.

9. The formulation of any one of claims 5 to 8, wherein the tris(hydroxymethyl)aminomethane is present in the aqueous solution at a concentration of 25 mM, and / or the sucrose is present in the aqueous solution at a concentration of 10 w / v%, and / or the pH of the aqueous solution is 7.

5.

10. The formulation of any one of claims 1 to 9, wherein the antibody is a Fab fragment, a full length IgG, a Fab' fragment, a F(ab')2 fragment, a scFv or a Fv.

11. The formulation of claim 10, wherein the antibody is a Fab fragment.

12. The formulation of any one of claims 1 to 11, wherein the antibody comprises a heavy chain variable region (VH) comprising an amino acid sequence at least 85% identical to SEQ ID NO: 17; and / or wherein the antibody comprises a light chain variable region (VL) comprising an amino acid sequence at least 85% identical to SEQ ID NO: 18, Optionally, the 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.

13. The formulation of any one of claims 1 to 12, wherein the antibody comprises a heavy chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 19; and / or wherein the antibody comprises a light chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 20, Optionally, the 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.

14. The formulation of any one of claims 1 to 13, wherein the oligonucleotide is 10 to 30 nucleotides in length, optionally wherein the oligonucleotide comprises a nucleotide sequence having a complementary region of at least 8 consecutive nucleotides in length to SEQ ID NO:

22.

15. The formulation of any one of claims 1 to 14, wherein the oligonucleotide comprises at least 8 consecutive nucleotides of the nucleotide sequence shown in SEQ ID NO: 21, optionally wherein the oligonucleotide comprises the nucleotide sequence of SEQ ID NO:

21.

16. The formulation of any one of claims 2 to 15, wherein each R 1 A group comprising the following formula (Ib): 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, such that the oligonucleotide comprises a nucleobase sequence of CAGCGCCCACCAGUCA (SEQ ID NO: 21).

17. The formulation of any one of claims 2 to 16, wherein each R 1 A group comprising the following formula (Ic):

18. The formulation of any one of claims 5 to 17, wherein the complex is present in the formulation at a concentration of 10 mg / mL to 50 mg / mL.

19. The formulation of any one of claims 1 to 18, further comprising one or more antibodies that are not covalently linked to the oligonucleotide.

20. The formulation of claim 19, wherein the average value of n1 of the complexes in the formulation is 0.5 to 5.

21. 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 formulation of any one of claims 1 to 20.

22. The method of claim 21, 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.

23. The method of claim 21 or claim 22, wherein the complex reduces DMPK expression in the subject.

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