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  • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
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  • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
  • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
  • A61K47/6807—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug or compound being a sugar, nucleoside, nucleotide, nucleic acid, e.g. RNA antisense
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  • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
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  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
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  • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
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Definitions

• the present application relates to targeting complexes for delivering oligonucleotide molecular payloads to cells, formulations comprising such complexes, and uses thereof, particularly uses relating to treatment of disease.
• Myotonic dystrophy is a dominantly inherited genetic disease that is characterized by myotonia, muscle loss or degeneration, diminished muscle function, insulin resistance, cardiac arrhythmia, smooth muscle dysfunction, and neurological abnormalities.
• DM is the most common form of adult-onset muscular dystrophy, with a worldwide incidence of about 1 in 8000 people worldwide.
• DM1 the more common form of the disease, results from a repeat expansion of a CTG trinucleotide repeat in the 3' noncoding region of DMPK on chromosome 19; DM2 results from a repeat expansion of a CCTG tetranucleotide repeat in the first intron of ZNF9 on chromosome 3.
• the repeat expansion of a CTG trinucleotide repeat which may comprise greater than about 50 to about 3,000 or more total repeats, leads to generation of toxic RNA repeats capable of forming hairpin structures that bind essential intracellular proteins, e.g. muscleblind-like proteins, with high affinity resulting in protein sequestration and the loss-of-function phenotypes that are characteristic of the disease.
• no effective therapeutic for DM1 is currently available.
• the present disclosure provides complexes and formulations comprising such complexes.
• a formulation provided herein comprises complexes that comprise an oligonucleotide covalently linked to an anti-transferrin receptor 1 (TfRl) antibody, wherein the anti-TfRl antibody comprises: a heavy chain complementarity determining region 1 (CDR-H1) comprising a sequence as set forth in SEQ ID NOs: 1, 7, or 12, a heavy chain complementarity determining region 2 (CDR-H2) comprising a sequence as set forth in SEQ ID NOs: 2, 8, or 13, a heavy chain complementarity determining region 3 (CDR-H3) comprising a sequence as set forth in SEQ ID NOs: 3, 9, or 14, a light chain complementarity determining region 1 (CDR-L1) comprising a sequence as set forth in SEQ ID NOs: 4, 10, or 15, a light chain complementarity determining region 2 (CDR-L2) comprising a sequence as set forth in SEQ ID NOs: 5 or 11, and a light chain complementarity determining region 3
• CDR-H1 compris
• a formulation provided herein comprises complexes comprising a structure of formula (I): [ R 1 ] >, i -R 2 , wherein each R 1 independently comprises a group of the formula (la):
• R 2 comprises an antibody
• R 3 comprises an oligonucleotide comprising a 5’-X-Y-Z-3’ configuration, wherein X and Z are flanking regions comprising one or more modified nucleosides and Y is a gap region comprising one or more 2’ -deoxyribonucleosides
• R 1 is covalently linked to R 2 at attachment point A
• nl is an integer representing the number of instances of R 1 , wherein each instance of R 1 is covalently linked to a different amino acid residue of the antibody; wherein the complexes are formulated with tris(hydroxymethyl)aminomethane and sucrose.
• each different amino acid residue of the antibody is a lysine.
• the antibody is an anti-TfRl antibody.
• the antibody comprises a heavy chain complementarity determining region 1 (CDR-H1) comprising a sequence as set forth in SEQ ID NOs: 1, 7, or 12, a heavy chain complementarity determining region 2 (CDR-H2) comprising a sequence as set forth in SEQ ID NOs: 2, 8, or 13, a heavy chain complementarity determining region 3 (CDR-H3) comprising a sequence as set forth in SEQ ID NOs: 3, 9, or 14, a light chain complementarity determining region 1 (CDR-L1) comprising a sequence as set forth in SEQ ID NOs: 4, 10, or 15, a light chain complementarity determining region 2 (CDR-L2) comprising a sequence as set forth in SEQ ID NOs: 5 or 11, and a light chain complementarity determining region 3 (CDR-L3) comprising a sequence as set forth in SEQ ID NOs: 6 or 16.
• the formulation is in a lyophilized form or a frozen solid form.
• the tris(hydroxymethyl)aminomethane is present in the aqueous solution at a concentration in the range of 5 mM to 50 mM.
• the sucrose is present in the aqueous solution at a concentration in the range of 5 % to 15 % weight per volume (w/v%).
• the aqueous solution has a pH in the range of 6.5 to 8.5.
• 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 ⁇ IN% and/or the aqueous solution is at a pH of 7.5.
• the antibody is a Fab fragment, a full-length IgG, a Fab' fragment, a F(ab')2 fragment, an scFv, or an Fv.
• the antibody is a Fab fragment.
• 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.
• VL light chain variable region
• 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.
• 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 the amino acid sequence of SEQ ID NO: 19 and/or a light chain comprising the amino acid sequence of SEQ ID NO: 20.
• the oligonucleotide is 10-30 nucleotides in length. In some embodiments, the oligonucleotide comprises a nucleotide sequence having a region of complementarity of at least 8 consecutive nucleotides in length to SEQ ID NO: 22.
• the oligonucleotide comprises at least 8 consecutive nucleotides of a nucleotide sequence as set forth in SEQ ID NO: 21. In some embodiments, the oligonucleotide comprises the nucleotide sequence of SEQ ID NO: 21.
• each R 1 comprises a group of the formula (lb): wherein +N represents an LNA (2’-4’ methylene bridge) ribonucleoside, dN represents a 2’ -deoxyribonucleoside, oN represents a 2’-O-methoxyethyl (MOE) modified ribonucleoside, oC represents a 5-methyl-2’-MOE-cytidine, +C represents a 5-methyl-2’-4’- bicyclic-cytidine (2 ’-4’ methylene bridge), oU represents a 5-methyl-2’-MOE-uridine, * represents a phosphorothioate internucleoside linkage, such that the oligonucleotide comprises a nucleobase sequence of CAGCGCCCACCAGUCA (SEQ ID NO: 21).
• each R 1 comprises a group of the formula (Ic):
• the complexes are present in the formulation at a concentration in the range of 10 mg/mL to 50 mg/mL.
• the formulation further comprises one or more antibodies that are not covalently linked to an oligonucleotide.
• the average value of nl of complexes in the formulation is in the range of 0.5 to 5.
• methods of reducing DMPK expression and/or treating myotonic dystrophy in a subject comprising administering to the subject an effective amount of a formulation provided herein.
• the subject has an expansion of a disease-associated repeat of a DMPK allele that is associated with myotonic dystrophy.
• the disease-associated repeat comprises repeating units of a CTG trinucleotide sequence.
• the complexes reduce DMPK expression in the subject.
• FIGs. 1A-1D show the amount of DMPK- targeting oligonucleotide (ASO) in the heart (FIG. 1A), diaphragm (FIG. IB), gastrocnemius (FIG. 1C), or tibialis anterior (FIG. ID), respectively, after administration of conjugates containing an anti-TfRl Fab covalently linked to the ASO.
• ASO DMPK- targeting oligonucleotide
• FIGs. 2A-2D show the ability of conjugates containing an anti-TfRl Fab conjugated to a DMPK-targeting oligonucleotide (ASO) to knock down human DMPK RNA in the heart (FIG. 2A), diaphragm (FIG. 2B), tibialis anterior (FIG. 2C) and gastrocnemius (FIG. 2D) of mice expressing both human TfRl and two copies of a mutant human DMPK transgene that harbors expanded CTG repeats.
• ASO DMPK-targeting oligonucleotide
• FIGs. 3A-3B show reduced DMPK foci in nuclei of cardiac muscle fibers in mice expressing both human TfRl and two copies of a mutant human DMPK transgene that harbors expanded CTG repeats and treated with anti-TfRl Fab conjugated to DMPK-targeting oligonucleotide (ASO).
• FIG. 3A shows representative images of samples following in situ hybridization staining for DMPK foci and fluorescence staining of myofibers (inset panels). In the microscopy images shown in FIG. 3A, the light rounded shapes show cell nuclei, and the bright puncta within the nuclei show DMPK foci.
• FIG. 3B shows quantification of DMPK foci.
• FIG. 4 shows the splicing correction activity of conjugates containing an anti- TfRl Fab covalently linked to a DMPK-targeting oligonucleotide (ASO) in the heart of mice expressing both human TfRl and two copies of a mutant human DMPK transgene that harbors expanded CTG repeats (hTfRl/DMSXL mice).
• ASO DMPK-targeting oligonucleotide
• FIG. 5 shows the splicing correction activity of conjugates containing an anti- TfRl Fab covalently linked to a DMPK-targeting oligonucleotide (ASO) in the diaphragm of mice expressing both human TfRl and two copies of a mutant human DMPK transgene that harbors expanded CTG repeats (hTfRl/DMSXL mice).
• ASO DMPK-targeting oligonucleotide
• FIG. 6 shows the splicing correction activity of conjugates containing an anti- TfRl Fab covalently linked to a DMPK-targeting oligonucleotide (ASO) in the tibialis anterior of mice expressing both human TfRl and two copies of a mutant human DMPK transgene that harbors expanded CTG repeats (hTfRl/DMSXL mice).
• ASO DMPK-targeting oligonucleotide
• FIG. 7 shows the splicing correction activity of conjugates containing an anti- TfRl Fab covalently linked to a DMPK-targeting oligonucleotide (ASO) in the gastrocnemius of mice expressing both human TfRl and two copies of a mutant human DMPK transgene that harbors expanded CTG repeats (hTfRl/DMSXL mice).
• ASO DMPK-targeting oligonucleotide
• Composite splicing indices based on splicing of Mbnl2 exon 6, Nfix exon 7, and Tin exon 313 are shown for control mice treated with vehicle control (“hTfRl - PBS”), hTfRl/DMSXL mice treated with vehicle control (“hTfRl/DMSXL - PBS”), and hTfRl/DMSXL mice treated with anti-TfRl Fab-ASO conjugate (“hTfRl/DMSXL - Conjugate”).
• FIG. 8 shows DMPK knockdown in DM1 patient myotubes and wild-type nonhuman primate (NHP) myotubes resulting from incubation with conjugates containing an anti- TfRl Fab covalently linked to a DMPK-targeting oligonucleotide (ASO).
• FIG. 9 shows x-ray diffraction (XRD) pattern of a lyophilized formulation of anti-TfRl Fab-ASO conjugates.
• FIGs. 10A-10C show protein concentrations calculated from absorbance data of a lyophilized formulation of anti-TfRl Fab-ASO conjugates after reconstitution at 0 weeks (TO), 4 weeks (T4w), and 8 weeks (T8w), at 2-8°C.
• FIG. 10A shows concentration of the Fab of the conjugates in mg/ml.
• FIG. 10B shows concentration of the Fab of the conjugates in pM.
• FIG. 10C shows concentration of the oligonucleotide of the conjugates in pM.
• FIGs. 11A-11B show the average percent recovery (FIG. 11 A) and Drug Antibody Ratio (DAR; FIG. 11B) for a lyophilized formulation of anti-TfRl Fab-ASO conjugates after reconstitution at TO, T4w, and T8w, at 2-8°C.
• FIGs. 12A-12C show protein concentrations calculated from absorbance data of a lyophilized formulation of anti-TfRl Fab-ASO conjugates after reconstitution at 0 weeks (TO), 2 weeks (T2w), 4 weeks (T4w), and 8 weeks (T8w), at 25°C.
• FIG. 12A shows concentration of the Fab of the conjugates in mg/ml.
• FIG. 12B shows concentration of the Fab of the conjugates in
• FIG. 12C shows concentration of the oligonucleotide of the conjugates in
• FIGs. 13A-13B show the average percent recovery (FIG. 13A) and Drug Antibody Ratio (DAR; FIG. 13B) for a lyophilized formulation of anti-TfRl Fab-ASO conjugates after reconstitution at TO, T2w, T4w, and T8w, at 25°C.
• DAR Drug Antibody Ratio
• FIGs. 14A-14C show protein concentrations calculated from absorbance data of a lyophilized formulation of anti-TfRl Fab-ASO conjugates after reconstitution at TO, T2w, T4w, and T8w, at 40°C.
• FIG. 14A shows concentration of the Fab of the conjugates in mg/ml.
• FIG. 14B shows concentration of the Fab of the conjugates in pM.
• FIG. 14C shows concentration of the oligonucleotide of the conjugates in pM.
• FIGs. 15A-15B show the average percent recovery (FIG. 15A) and Drug Antibody Ratio (DAR; FIG. 15B) of lyophilized anti-TfRl Fab-ASO conjugates after reconstitution at TO, T2w, T4w, and T8w, at 40°C.
• DAR Drug Antibody Ratio
• FIGs. 16A-16B show the sum of the relative area of peaks 1 and 2 (FIG. 16A) and peaks 3 and 4 (FIG. 16B) measured using size-exclusion chromatography (SEC) of lyophilized anti-TfRl Fab-ASO conjugates after reconstitution at TO, T4w, and T8w, at 2-8°C.
• FIGs. 17A-17B show the sum of the relative area of peaks 1 and 2 (FIG. 17A) and peaks 3 and 4 (FIG. 17B) measured using size-exclusion chromatography (SEC) of lyophilized anti-TfRl Fab-ASO conjugates after reconstitution at TO, T2w, T4w, and T8w, at 25°C.
• FIGs. 18A-18B show the sum of the relative area of peaks 1 and 2 (FIG. 18A) and peaks 3 and 4 (FIG. 18B) measured using size-exclusion chromatography (SEC) of lyophilized anti-TfRl Fab-ASO conjugates after reconstitution at TO, T2w, T4w, T8w, at 40°C.
• SEC size-exclusion chromatography
• FIG. 19 shows percent purity of the main peak of lyophilized anti-TfRl Fab- ASO conjugates after reconstitution measured using capillary electrophoresis (CE-SDS) at TO, T4w, and T8w, at 2-8 °C.
• CE-SDS capillary electrophoresis
• FIG. 20 shows percent purity of the main peak of lyophilized anti-TfRl Fab- ASO conjugates after reconstitution measured using CE-SDS at TO, T2w, T4w, and T8w, at 25°C.
• FIG. 21 shows percent purity of the main peak of lyophilized anti-TfRl Fab- ASO conjugates after reconstitution measured using CE-SDS at TO, T2w, T4w, and T8w, at 40°C.
• the present disclosure provides complexes and formulations comprising such complexes.
• the complexes are formulated with tris(hydroxymethyl)aminomethane and sucrose.
• the complexes are formulated as aqueous or lyophilized (e.g., lyophilized powder) forms.
• a complex comprises an oligonucleotide (e.g., an oligonucleotide comprising a 5’-X-Y-Z-3’ configuration) covalently linked to an antibody.
• a complex comprises a muscle-targeting complex comprising an oligonucleotide (e.g., an oligonucleotide comprising a 5’-X-Y-Z-3’ configuration) covalently linked to an antitransferrin receptor 1 (TfRl) antibody.
• a complex comprises a muscletargeting complex comprising an oligonucleotide (e.g., an oligonucleotide comprising a 5’-X- Y-Z-3’ configuration) covalently linked to the anti-transferrin receptor 1 (TfRl) antibody provided in Table 2.
• DM1 myotonic dystrophy
• DMPK e.g., a DMPK RNA
• Administering means to provide a complex to a subject in a manner that is physiologically and/or (e.g., and) pharmacologically useful (e.g., to treat a condition in the subject).
• an antibody refers to a polypeptide that includes at least one immunoglobulin variable domain or at least one antigenic determinant, e.g., paratope that specifically binds to an antigen.
• an antibody is a full- length antibody.
• an antibody is a chimeric antibody.
• an antibody is a humanized antibody.
• an antibody is a Fab fragment, a Fab’ fragment, a F(ab')2 fragment, a Fv fragment or a scFv fragment.
• an antibody is a nanobody derived from a camelid antibody or a nanobody derived from shark antibody.
• an antibody is a diabody.
• an antibody comprises a framework having a human germline sequence.
• an antibody comprises a heavy chain constant domain selected from the group consisting of IgG, IgGl, IgG2, IgG2A, IgG2B, IgG2C, IgG3, IgG4, IgAl, IgA2, IgD, IgM, and IgE constant domains.
• an antibody comprises a heavy (H) chain variable region (abbreviated herein as VH), and/or (e.g., and) a light (L) chain variable region (abbreviated herein as VL).
• an antibody comprises a constant domain, e.g., an Fc region.
• An immunoglobulin constant domain refers to a heavy or light chain constant domain. Human IgG heavy chain and light chain constant domain amino acid sequences and their functional variations are known.
• the heavy chain of an antibody described herein can be an alpha (a), delta (A), epsilon (E), gamma (y) or mu (p) heavy chain.
• the heavy chain of an antibody described herein can comprise a human alpha (a), delta (A), epsilon (E), gamma (y) or mu (p) heavy chain.
• an antibody described herein comprises a human gamma 1 CHI, CH2, and/or (e.g., and) CH3 domain.
• the amino acid sequence of the VH domain comprises the amino acid sequence of a human gamma (y) heavy chain constant region, such as any known in the art. Non-limiting examples of human constant region sequences have been described in the art, e.g., see U.S. Pat. No. 5,693,780 and Kabat E A et al., (1991) supra.
• the VH domain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or at least 99% identical to any of the variable chain constant regions provided herein.
• an antibody is modified, e.g., modified via glycosylation, phosphorylation, sumoylation, and/or (e.g., and) methylation.
• an antibody is a glycosylated antibody, which is conjugated to one or more sugar or carbohydrate molecules.
• the one or more sugar or carbohydrate molecule are conjugated to the antibody via N-glycosylation, O-glycosylation, C-glycosylation, glypiation (GPI anchor attachment), and/or (e.g., and) phosphoglycosylation.
• the one or more sugar or carbohydrate molecule are monosaccharides, disaccharides, oligosaccharides, or glycans. In some embodiments, the one or more sugar or carbohydrate molecule is a branched oligosaccharide or a branched glycan. In some embodiments, the one or more sugar or carbohydrate molecule includes a mannose unit, a glucose unit, an N-acetylglucosamine unit, an N-acetylgalactosamine unit, a galactose unit, a fucose unit, or a phospholipid unit.
• an antibody is a construct that comprises a polypeptide comprising one or more antigen binding fragments of the disclosure linked to a linker polypeptide or an immunoglobulin constant domain.
• Linker polypeptides comprise two or more amino acid residues joined by peptide bonds and are used to link one or more antigen binding portions. Examples of linker polypeptides have been reported (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994) Structure 2: 1121-1123).
• an antibody may be part of a larger immunoadhesion molecule, formed by covalent or noncovalent association of the antibody or antibody portion with one or more other proteins or peptides.
• immunoadhesion molecules include use of the streptavidin core region to make a tetrameric scFv molecule (Kipriyanov, S. M., et al. (1995) Human Antibodies and Hybridomas 6:93-101) and use of a cysteine residue, a marker peptide and a C-terminal polyhistidine tag to make bivalent and biotinylated scFv molecules (Kipriyanov, S. M., et al. (1994) Mol. Immunol. 31:1047-1058).
• CDR refers to the complementarity determining region within antibody variable sequences.
• a typical antibody molecule comprises a heavy chain variable region (VH) and a light chain variable region (VL), which are usually involved in antigen binding.
• VH and VL regions can be further subdivided into regions of hypervariability, also known as “complementarity determining regions” (“CDR”), interspersed with regions that are more conserved, which are known as “framework regions” (“FR”).
• CDR complementarity determining regions
• FR framework regions
• Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
• the extent of the framework region and CDRs can be precisely identified using methodology known in the art, for example, by the Kabat definition, the IMGT definition, the Chothia definition, the AbM definition, and/or (e.g., and) the contact definition, all of which are well known in the art. See, e.g., Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242; IMGT®, the international ImMunoGeneTics information system® http://www.imgt.org, Lefranc, M.-P.
• a CDR may refer to the CDR defined by any method known in the art. Two antibodies having the same CDR means that the two antibodies have the same amino acid sequence of that CDR as determined by the same method, for example, the IMGT definition.
• CDR1 There are three CDRs in each of the variable regions of the heavy chain and the light chain, which are designated CDR1, CDR2 and CDR3, for each of the variable regions.
• CDR set refers to a group of three CDRs that occur in a single variable region capable of binding the 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 defining the three CDRs.
• CDRs may be referred to as Kabat CDRs.
• Sub-portions of CDRs may be designated as LI, L2 and L3 or Hl, H2 and H3 where the "L” and the "H” designates the light chain and the heavy chains regions, respectively.
• These regions may be referred to as Chothia CDRs, which have boundaries that overlap with Kabat CDRs.
• Other boundaries defining CDRs overlapping with the Kabat CDRs have been described by Padlan (FASEB J. 9:133-139 (1995)) and MacCallum (J Mol Biol 262(5):732-45 (1996)).
• CDR boundary definitions may not strictly follow one of the above systems, but will nonetheless overlap with the Kabat CDRs, although they may be shortened or lengthened in light of prediction or experimental findings that particular residues or groups of residues or even entire CDRs do not significantly impact antigen binding.
• the methods used herein may utilize CDRs defined according to any of these systems. Examples of CDR definition systems are provided in Table 1.
• Complementary refers to the capacity for precise pairing between two nucleotides or two sets of nucleotides.
• complementary is a term that characterizes an extent of hydrogen bond pairing that brings about binding between two nucleotides or two sets of nucleotides. For example, if a base at one position of an oligonucleotide is capable of hydrogen bonding with a base at the corresponding position of a target nucleic acid (e.g., an mRNA), then the bases are considered to be complementary to each other at that position.
• a target nucleic acid e.g., an mRNA
• Base pairings may include both canonical Watson-Crick base pairing and non-Watson-Crick base pairing (e.g., Wobble base pairing and Hoogsteen base pairing).
• adenosine-type bases are complementary to thymidine-type bases (T) or uracil- type bases (U)
• cytosine-type bases are complementary to guanosine-type bases (G)
• universal bases such as 3 -nitropyrrole or 5-nitroindole can hybridize to and are considered complementary to any A, C, U, or T.
• Inosine (I) has also been considered in the art to be a universal base and is considered complementary to any A, C, U or T.
• Covalently linked refers to a characteristic of two or more molecules being linked together via at least one covalent bond.
• two molecules can be covalently linked together by a single bond, e.g., a disulfide bond or disulfide bridge, that serves as a linker between the molecules.
• two or more molecules can be covalently linked together via a molecule that serves as a linker that joins the two or more molecules together through multiple covalent bonds.
• a linker may be a cleavable linker.
• a linker may be a non-cleavable linker.
• Disease-associated-repeat refers to a repeated nucleotide sequence at a genomic location for which the number of units of the repeated nucleotide sequence is correlated with and/or (e.g., and) directly or indirectly contributes to, or causes, genetic disease.
• Each repeating unit of a disease associated repeat may be 2, 3, 4, 5 or more nucleotides in length.
• a disease associated repeat is a dinucleotide repeat.
• a disease associated repeat is a trinucleotide repeat.
• a disease associated repeat is a tetranucleotide repeat.
• a disease associated repeat is a pentanucleotide repeat.
• the disease-associated-repeat comprises CAG repeats, CTG repeats, CUG repeats, CGG repeats, CCTG repeats, or a nucleotide complement of any thereof.
• a disease-associated-repeat is in a non-coding portion of a gene.
• a disease-associated-repeat is in a coding region of a gene.
• a disease-associated-repeat is expanded from a normal state to a length that directly or indirectly contributes to, or causes, genetic disease.
• a disease-associated-repeat is in RNA (e.g., an RNA transcript). In some embodiments, a disease-associated-repeat is in DNA (e.g., a chromosome, a plasmid). In some embodiments, a disease-associated-repeat is expanded in a chromosome of a germline cell. In some embodiments, a disease-associated-repeat is expanded in a chromosome of a somatic cell. In some embodiments, a disease-associated-repeat is expanded to a number of repeating units that is associated with congenital onset of disease.
• a disease- associated-repeat is expanded to a number of repeating units that is associated with childhood onset of disease. In some embodiments, a disease-associated-repeat is expanded to a number of repeating units that is associated with adult onset of disease.
• the DMPK gene comprises a disease-associated repeat of CTG units.
• DMPK refers to a gene that encodes myotonin-protein kinase (also known as myotonic dystrophy protein kinase or dystrophia myotonica protein kinase), a serine/threonine protein kinase. Substrates for this enzyme may include myogenin, the beta-subunit of the L-type calcium channels, and phospholemman.
• DMPK may be a human (Gene ID: 1760), non-human primate (e.g., Gene ID: 456139, Gene ID: 715328, Gene ID: 102125829), or rodent gene (e.g., Gene ID: 13400).
• DM1 myotonic dystrophy type I
• multiple human transcript variants e.g., as annotated under 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 have been characterized that encode different protein isoforms.
• DMPK allele refers to any one of alternative forms (e.g., wild-type or mutant forms) of a DMPK gene.
• a DMPK allele may encode for wild-type myotonin-protein kinase that retains its normal and typical functions.
• a DMPK allele may comprise one or more disease- associated-repeat expansions.
• normal subjects have two DMPK alleles comprising in the range of 5 to 37 repeat units.
• the number of CTG repeat units in subjects having DM1 is in the range of about 50 to about 3,000 or more, with higher numbers of repeats leading to an increased severity of disease.
• mildly affected DM1 subjects have at least one DMPK allele having in the range of 50 to 150 repeat units.
• subjects with classic DM1 have at least one DMPK allele having in the range of 100 to 1,000 or more repeat units.
• subjects having DM1 with congenital onset may have at least one DMPK allele comprising more than 2,000 repeat units.
• Framework refers to the remaining sequences of a variable region minus the CDRs. Because the exact definition of a CDR sequence can be determined by different systems, the meaning of a framework sequence is subject to correspondingly different interpretations.
• the six CDRs also divide the framework regions on the light chain and the heavy chain into four sub-regions (FR1, FR2, FR3 and FR4) on each chain, in which CDR1 is positioned between FR1 and FR2, CDR2 between FR2 and FR3, and CDR3 between FR3 and FR4.
• a framework region represents the combined FRs within the variable region of a single, naturally occurring immunoglobulin chain.
• a FR represents one of the four sub-regions, and FRs represents two or more of the four sub-regions constituting a framework region.
• Human heavy chain and light chain acceptor sequences are known in the art. In one embodiment, the acceptor sequences known in the art may be used in the antibodies disclosed herein.
• Human antibody is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences.
• the human antibodies of the disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3.
• the term "human antibody”, as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
• Humanized antibody refers to antibodies which comprise heavy and light chain variable region sequences from a non-human species (e.g., a mouse) but in which at least a portion of the VH and/or (e.g., and) VL sequence has been altered to be more "human-like", i.e., more similar to human germline variable sequences.
• a non-human species e.g., a mouse
• VH and/or VL sequence e.g., and
• VL sequence e.g., and VL sequence has been altered to be more "human-like", i.e., more similar to human germline variable sequences.
• One type of humanized antibody is a CDR-grafted antibody, in which human CDR sequences are introduced into non-human VH and VL sequences to replace the corresponding nonhuman CDR sequences.
• humanized anti-transferrin receptor antibodies and antigen binding portions are provided.
• Such antibodies may be generated by obtaining murine anti-transferrin receptor monoclonal antibodies using traditional hybridoma technology followed by humanization using in vitro genetic engineering, such as those disclosed in Kasaian et al PCT publication No. WO 2005/123126 A2.
• Kabat numbering The terms "Kabat numbering", “Kabat definitions and “Kabat labeling” are used interchangeably herein. These terms, which are recognized in the art, refer to a system of numbering amino acid residues which are more variable (i.e. hypervariable) than other amino acid residues in the heavy and light chain variable regions of an antibody, or an antigen binding portion thereof (Kabat et al. (1971) Ann. NY Acad, Sci. 190:382-391 and, Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242).
• the hypervariable region ranges from amino acid positions 31 to 35 for CDR1, amino acid positions 50 to 65 for CDR2, and amino acid positions 95 to 102 for CDR3.
• the hypervariable region ranges from amino acid positions 24 to 34 for CDR1, amino acid positions 50 to 56 for CDR2, and amino acid positions 89 to 97 for CDR3.
• Myotonic dystrophy refers to a genetic disease caused by mutations in the DMPK gene or CNBP (ZNF9) gene that is characterized by muscle loss, muscle weakening, and muscle function. Two types of the disease, myotonic dystrophy type 1 (DM1) and myotonic dystrophy type 2 (DM2), have been described. DM1 is associated with an expansion of a CTG trinucleotide repeat in the 3' non-coding region of DMPK. DM2 is associated with an expansion of a CCTG tetranucleotide repeat in the first intron of ZNF9.
• DM1 and DM2 the nucleotide expansions lead to toxic RNA repeats capable of forming hairpin structures that bind critical intracellular proteins, e.g., muscleblind-like proteins, with high affinity.
• Myotonic dystrophy the genetic basis for the disease, and related symptoms are described in the art (see, e.g. Thornton, C.A., “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).
• subjects are born with a variation of DM1 called congenital myotonic dystrophy.
• DM1 is associated with Online Mendelian Inheritance in Man (OMIM) Entry # 160900.
• DM2 is associated with OMIM Entry # 602668.
• oligonucleotide refers to an oligomeric nucleic acid compound of up to 200 nucleotides in length.
• oligonucleotides include, but are not limited to, RNAi oligonucleotides (e.g., siRNAs, shRNAs), microRNAs, gapmers, mixmers, phosphorodiamidate morpholinos, peptide nucleic acids, aptamers, guide nucleic acids (e.g., Cas9 guide RNAs), etc.
• Oligonucleotides may be single- stranded or double-stranded.
• an oligonucleotide may comprise one or more modified nucleosides (e.g., 2'-O-methyl sugar modifications, purine or pyrimidine modifications).
• an oligonucleotide may comprise one or more modified intemucleoside linkage.
• an oligonucleotide may comprise one or more phosphorothioate linkages, which may be in the Rp or Sp stereochemical conformation.
• Region of complementarity refers to a nucleotide sequence, e.g., of an oligonucleotide, that is sufficiently complementary to a cognate nucleotide sequence, e.g., of a target nucleic acid, such that the two nucleotide sequences are capable of annealing to one another under physiological conditions (e.g., in a cell).
• a region of complementarity is fully complementary to a cognate nucleotide sequence of target nucleic acid.
• a region of complementarity is partially complementary to a cognate nucleotide sequence of target nucleic acid (e.g., at least 80%, 90%, 95% or 99% complementarity). In some embodiments, a region of complementarity contains 1, 2, 3, or 4 mismatches compared with a cognate nucleotide sequence of a target nucleic acid.
• the term “specifically binds” 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 an appropriate control in a binding assay or other binding context.
• the term, “specifically binds”, refers to the ability of the antibody to bind to a specific antigen with a degree of affinity or avidity, compared with an appropriate reference antigen or antigens, that enables the antibody to be used to distinguish the specific antigen from others, e.g., to an extent that permits preferential targeting to certain cells, e.g., muscle cells, through binding to the antigen, as described herein.
• an antibody specifically binds to a target if the antibody has a KD for binding the target of at least about 10’ 4 M, 10’ 5 M, 10’ 6 M, IO’ 7 M, 10’ 8 M, 10’ 9 M, IO’ 10 M, 10’ 11 M, 10 12 M, 10’ 13 M, or less.
• an antibody specifically binds to the transferrin receptor, e.g., an epitope of the apical domain of transferrin receptor.
• Subject refers to a mammal.
• a subject is non-human primate, or rodent.
• a subject is a human.
• a subject is a patient, e.g., a human patient that has or is suspected of having a disease.
• the subject is a human patient who has or is suspected of having a disease resulting from a disease-associated-repeat expansion, e.g., in a DMPK allele.
• Transferrin receptor As used herein, the term, “transferrin receptor” (also known as TFRC, CD71, p90, TFR, or TFR1) refers to an internalizing cell surface receptor that binds transferrin to facilitate iron uptake by endocytosis.
• a transferrin receptor may be of human (NCBI Gene ID 7037), non-human primate (e.g., NCBI Gene ID 711568 or NCBI Gene ID 102136007), or rodent (e.g., NCBI Gene ID 22042) origin.
• multiple human transcript variants have been characterized that encoded different isoforms of the receptor (e.g., as annotated under GenBank RefSeq Accession Numbers: NP_001121620.1, NP_003225.2, NP_001300894.1, and NP_001300895.1).
• 2’-modified nucleoside As used herein, the terms “2’-modified nucleoside” and “2’ -modified ribonucleoside” are used interchangeably and refer to a nucleoside having a sugar moiety modified at the 2’ position. In some embodiments, the 2’ -modified nucleoside is a 2’-4’ bicyclic nucleoside, where the 2’ and 4’ positions of the sugar are bridged (e.g., via a methylene, an ethylene, or a (S)-constrained ethyl bridge).
• the 2’- modified nucleoside is a non-bicyclic 2’-modified nucleoside, e.g., where the 2’ position of the sugar moiety is substituted.
• Non-limiting examples of 2’-modified nucleosides include: 2’- deoxy, 2’-fluoro (2’-F), 2’-O-methyl (2’-0-Me), 2’-O-methoxyethyl (2’-M0E), 2’-O- aminopropyl (2’-O-AP), 2’-O-dimethylaminoethyl (2’-0-DMA0E), 2’-O- dimethylaminopropyl (2’-O-DMAP), 2’-O-dimethylaminoethyloxyethyl (2’-O-DMAEOE), 2’- O-N-methylacetamido (2’-0-NMA), locked nucleic acid (LNA, methylene-bridged nucleic acid), ethylene-bridged nucleic
• the 2’ -modified nucleosides described herein are high-affinity modified nucleotides and oligonucleotides comprising the 2’-modified nucleosides have increased affinity to a target sequences, relative to an unmodified oligonucleotide. Examples of structures of 2’-modified nucleosides are provided below:
• a complex that comprise a targeting agent, e.g., an antibody, covalently linked to an oligonucleotide.
• a complex comprises a muscle-targeting antibody covalently linked to one or more oligonucleotides.
• the oligonucleotide is an antisense oligonucleotide that targets a DMPK RNA to reduce expression or activity of DMPK (e.g., reduce the level of a mutant or wild-type DMPK RNA, or the activity of a DMPK gene product).
• Complexes described herein generally comprise a linker that covalently links an antibody (e.g., an anti-TfRl antibody) described herein to an oligonucleotide (e.g., an oligonucleotide comprising a 5’-X-Y-Z-3’ configuration).
• a linker comprises at least one covalent bond.
• complexes described herein comprise a structure of formula (I): [ R 1 ] >, i -R 2 , in which each R 1 independently comprises a compound comprising an oligonucleotide (e.g., an oligonucleotide comprising a 5’-X-Y-Z-3’ configuration) and R 2 comprises an antibody (e.g., an anti-TfRl antibody), and wherein in each complex nl is independently an integer (e.g., one or greater) representing the number of instances of R 1 in each complex.
• each R 1 independently comprises a group comprising an oligonucleotide.
• each R 1 independently comprises a group that comprises additional elements in addition to an oligonucleotide.
• R 2 comprises an antibody (e.g., an anti-TfRl antibody) comprising a heavy chain comprising a heavy chain variable region (VH) and a heavy chain constant region, and a light chain comprising a light chain variable region (VL) and a light chain constant region.
• each R 1 of a complex is independently covalently linked to a different amino acid residue (e.g., lysine or cysteine) of R 2 .
• the antibody comprises a sequence as set forth in Table 2.
• the antibody comprises a heavy chain complementarity determining region 1 (CDR-H1) comprising a sequence as set forth in SEQ ID NOs: 1, 7, or 12, a heavy chain complementarity determining region 2 (CDR-H2) comprising a sequence as set forth in SEQ ID NOs: 2, 8, or 13, a heavy chain complementarity determining region 3 (CDR-H3) comprising a sequence as set forth in SEQ ID NOs: 3, 9, or 14; and/or comprises a light chain complementarity determining region 1 (CDR-L1) comprising a sequence as set forth in SEQ ID NOs: 4, 10, or 15, a light chain complementarity determining region 2 (CDR- L2) comprising a sequence as set forth in SEQ ID NOs: 5 or 11, and a light chain complementarity determining region 3 (CDR-H1) comprising a sequence as set forth in SEQ ID NOs: 1, 7, or 12, a heavy chain complementarity determining region 2 (CDR-H
• the antibody comprises a heavy chain variable region (VH) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 17 and/or comprises a light chain variable region (VL) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 18.
• VH heavy chain variable region
• VL light chain variable region
• 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.
• the antibody comprises a heavy chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 19 and/or comprises a light chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 20.
• the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and/or comprises a light chain comprising the amino acid sequence of SEQ ID NO: 20.
• the antibody is a Fab fragment, a full- length IgG, a Fab' fragment, a F(ab')2 fragment, an scFv, or an Fv.
• the antibody is a Fab fragment.
• the value of nl of each or any complex is an integer up to the number of amino acid residues in the antibody to which conjugation is desired or targeted (e.g., the number of lysine residues).
• the value of nl in each complex the value of nl 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.
• the value of nl in each complex the value of nl 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.
• compositions described herein comprise complexes that comprise a structure of formula (I): [R ⁇ ni-R 2 , wherein nl is 0.
• the average value of nl of complexes of the composition is in the range of 0.5 to 5 (e.g., 0.5-5, 1-5, 1-4, 1-3, 3-5, 0.5-4, 0.5-3, 0.5-2, 0.5-1.5, 0.5-1, 0.7-1.5, 1-1.6, 1-1.5, 1-1.4, 1-1.3, 1-1.2, 1.1-1.5, 0.8-2, 0.8- 1.5, O.8-1.3, 0.8-1.2, 0.8-1.1, 0.9-3, 0.9-2, 0.9-1.8, 0.9-1.6, 0.9-1.5, 0.9-1.4, 0.9-1.3, or 0.9-1.2).
• nl is independently an integer of one or greater representing the number of instances of R 1 in each complex of the complex type, and in which the different complex types of the composition are characterized by having different nl values (e.g., nl values in the range of 1-27, 1-26, 1-25, 1-20, 1-15, 1-10, 1-5, or 1-3).
• compositions are provided (e.g., formulations comprising tris(hydroxymethyl)aminomethane and/or sucrose, as described herein) that comprise a plurality of different complexes.
• the plurality of different complexes comprise a common targeting agent (e.g. an antibody) and a common oligonucleotide (e.g., an oligonucleotide comprising a 5’-X-Y-Z-3’ configuration, such as a DMPK-targeting oligonucleotide).
• a common targeting agent e.g. an antibody
• a common oligonucleotide e.g., an oligonucleotide comprising a 5’-X-Y-Z-3’ configuration, such as a DMPK-targeting oligonucleotide.
• different complex types are characterized by having different numbers of oligonucleotides covalently linked to an antibody.
• compositions comprise a plurality of complexes comprising a structure of formula (I): [R ⁇ ni-R 2 , in which each R 1 independently comprises a compound comprising an oligonucleotide (e.g., a DMPK-targeting oligonucleotide) and R 2 comprises an antibody (e.g., anti-TfRl antibody), and in which nl is an integer representing the number of instances of R 1 in a complex, and in which different complexes of the composition may have different nl values (e.g., nl values in the range of 1- 27, 1-26, 1-10, 1-5, or 1-3).
• nl is an integer representing the number of instances of R 1 in a complex
• different complexes of the composition may have different nl values (e.g., nl values in the range of 1- 27, 1-26, 1-10, 1-5, or 1-3).
• nl is independently an integer.
• the average value of nl of complexes of the composition is in the range of 0.5 to 5 (e.g., 0.5-5, 1-5, 1-4, 1-3, 3-5, 0.5-4, 0.5-3, 0.5-2, 0.5- 1.5, 0.5-1, 0.7-1.5, 1-1.6, 1-1.5, 1-1.4, 1-1.3, 1-1.2, 1.1-1.5, 0.8-2, 0.8-1.5, O.8-1.3, 0.8-1.2, 0.8- 1.1, 0.9-3, 0.9-2, 0.9-1.8, 0.9-1.6, 0.9-1.5, 0.9-1.4, 0.9-1.3, or 0.9-1.2).
• compositions described herein comprise complexes in which nl is 0.
• a composition described herein comprises antibody that is not conjugated to an oligonucleotide (e.g., in trace amounts) and antibody conjugated to one or more oligonucleotides.
• antibody that is not conjugated to an oligonucleotide may be referred to as a compound comprising a structure of formula (I): [R 'JHI-R 2 , for which nl is zero.
• a composition for administration to a subject in the methods described herein comprises compounds (e.g., complexes) comprising a structure of formula (I): [R 'JHI -R 2 , for which each R 1 independently comprises a group comprising an oligonucleotide, R 2 comprises an antibody and nl is independently an integer of zero or greater that reflects the number of instances of R 1 in each compound (e.g., complex).
• the fraction of compounds comprising a structure of formula (I): [R ⁇ ni-R 2 , in a composition, for which nl is zero, compared with all compounds of that structure in the composition for which nl is one or greater, 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%.
• the average value of nl of complexes in a composition disclosed herein is in the range of 0.5 to 5 (e.g., 0.5-5, 1-5, 1-4, 1-3, 3-5, 0.5-4, 0.5-3, 0.5-2, 0.5-1.5, 0.5-1, 0.7-1.5, 1-1.6, 1-1.5, 1-1.4, 1-1.3, 1-1.2, 1.1-1.5, 0.8-2, 0.8-1.5, O.8-1.3, 0.8-1.2, 0.8-1.1, 0.9-3, 0.9-2, 0.9-1.8, 0.9-1.6, 0.9-1.5, 0.9-1.4, 0.9-1.3, or 0.9-1.2).
• 0.5-5, 1-5, 1-4, 1-3, 3-5, 0.5-4, 0.5-3, 0.5-2 0.5-1.5, 0.5-1, 0.7-1.5, 1-1.6, 1-1.5, 1-1.4, 1-1.3, 1-1.2, 1.1-1.5, 0.8-2, 0.8-1.5, O.8-1.3, 0.8-1.2, 0.8-1.1, 0.9-3, 0.9-2,
• each instance of R 1 in a complex is covalently linked to a different amino acid residue of the antibody.
• an amino acid to which R 1 is covalently linked comprises an s-amino group (e.g., lysine, arginine).
• an amino acid to which R 1 is covalently linked is a cysteine.
• R 1 is directly covalently linked to an amino acid residue of the antibody.
• R 1 is indirectly covalently linked to an amino acid of the antibody, e.g., covalently linked to a glycosylation site on the amino acid.
• R 1 is not covalently linked to an amino acid residue residing in a CDR region of the antibody.
• complexes provided herein comprise a structure of formula (I): [ R 1 J >, i -R 2 , in which each instance of R 1 independently comprises a group of the formula (la):
• R 3 comprises an oligonucleotide, e.g., an oligonucleotide comprising a 5’-X-Y-Z-3’ configuration; and R 1 is covalently linked (e.g., indirectly or directly linked, e.g., directly linked) to R 2 at attachment point A.
• R 2 comprises an antibody comprising a sequence as set forth in Table 2.
• R 2 comprises an antibody comprising a heavy chain complementarity determining region 1 (CDR- Hl) comprising a sequence as set forth in SEQ ID NOs: 1, 7, or 12, a heavy chain complementarity determining region 2 (CDR-H2) comprising a sequence as set forth in SEQ ID NOs: 2, 8, or 13, a heavy chain complementarity determining region 3 (CDR-H3) comprising a sequence as set forth in SEQ ID NOs: 3, 9, or 14; and/or comprising a light chain complementarity determining region 1 (CDR-L1) comprising a sequence as set forth in SEQ ID NOs: 4, 10, or 15, a light chain complementarity determining region 2 (CDR-L2) comprising a sequence as set forth in SEQ ID NOs: 5, or 11, and a light chain complementarity determining region 3 (CDR-L3) comprising a sequence as set forth in SEQ ID NO: 6 or 16.
• CDR- Hl heavy chain complementarity determining region 1
• CDR-H2 comprising
• R 2 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 comprising a light chain variable region (VL) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 18.
• R 2 comprises an antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 17 and/or comprising a VL comprising the amino acid sequence of SEQ ID NO: 18.
• R 2 comprises 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 comprising a light chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 20.
• R 2 comprises an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and/or comprising a light chain comprising the amino acid sequence of SEQ ID NO: 20.
• R 2 comprises an antibody that is a Fab fragment, a full-length IgG, a Fab' fragment, a F(ab')2 fragment, an scFv, or an Fv.
• R 3 comprises an oligonucleotide comprising a nucleobase sequence of CAGCGCCCACCAGUCA (SEQ ID NO: 21). In some embodiments, R 3 comprises an oligonucleotide comprising a structure of +C*+A*oG*oC*dG*dC*dC*dC*dA*dC*dC*dA*oG*oU*+C*+A (SEQ ID NO: 21), wherein +N represents an ENA (2’ -4’ methylene bridge) ribonucleoside, dN represents a 2’- deoxyribonucleoside, oN represents a 2’ -MOE modified ribonucleoside, oC represents a 5- methyl-2’-MOE-cytidine, +C represents a 5-methyl-2’-4’-bicyclic-cytidine (2 ’-4’ methylene bridge), oU represents a 5-methyl-2’-MOE-uridine, and
• complexes provided herein comprise a structure of formula (I): [ R 1 J >, i -R 2 , in which each R 1 comprises a group of the formula (lb): wherein +N represents an LNA (2’-4’ methylene bridge) ribonucleoside, dN represents a 2’- deoxyribonucleoside, oN represents a 2’-O-methoxyethyl (MOE) modified ribonucleoside, oC represents a 5-methyl-2’-MOE-cytidine, +C represents a 5-methyl-2’-4’-bicyclic-cytidine (2’- 4’ methylene bridge), oU represents a 5-methyl-2’-MOE-uridine, * represents a phosphorothioate internucleoside linkage, and wherein the oligonucleotide comprises a nucleobase sequence of CAGC
• R 2 comprises an antibody comprising a sequence as set forth in Table 2.
• R 2 comprises an antibody comprising a heavy chain complementarity determining region 1 (CDR-H1) comprising a sequence as set forth in SEQ ID NOs: 1, 7, or 12, a heavy chain complementarity determining region 2 (CDR-H2) comprising a sequence as set forth in SEQ ID NOs: 2, 8, or 13, a heavy chain complementarity determining region 3 (CDR-H3) comprising a sequence as set forth in SEQ ID NOs: 3, 9, or 14; and/or comprising a light chain complementarity determining region 1 (CDR-L1) comprising a sequence as set forth in SEQ ID NOs: 4, 10, or 15, a light chain complementarity determining region 2 (CDR- L2) comprising a sequence as set forth in SEQ ID NOs: 5, or 11, and a light chain complementarity determining region 3 (CDR-L3) comprising a sequence as set forth in SEQ ID NO: 6 or
• R 2 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 comprising a light chain variable region (VL) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 18.
• R 2 comprises an antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 17 and/or comprising a VL comprising the amino acid sequence of SEQ ID NO: 18.
• R 2 comprises 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 comprising a light chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 20.
• R 2 comprises an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and/or comprising a light chain comprising the amino acid sequence of SEQ ID NO: 20.
• R 2 comprises an antibody that is a Fab fragment, a full-length IgG, a Fab' fragment, a F(ab')2 fragment, an scFv, or an Fv.
• complexes provided herein comprise a structure of formula (I): [ R 1 J >, i - R 2 , in which each R 1 comprises a group of the formula (Ic):
• R 2 comprises an antibody comprising a sequence as set forth in Table 2.
• R 2 comprises an antibody comprising a heavy chain complementarity determining region 1 (CDR-H1) comprising a sequence as set forth in SEQ ID NOs: 1, 7, or 12, a heavy chain complementarity determining region 2 (CDR-H2) comprising a sequence as set forth in SEQ ID NOs: 2, 8, or 13, a heavy chain complementarity determining region 3 (CDR-H3) comprising a sequence as set forth in SEQ ID NOs: 3, 9, or 14; and/or comprising a light chain complementarity determining region 1 (CDR-L1) comprising a sequence as set forth in SEQ ID NOs: 4, 10, or 15, a light chain complementarity determining region 2 (CDR- L2) comprising a sequence as set forth in SEQ ID NOs: 5, or 11, and a light chain complementarity determining region 3 (CDR-H1) comprising a sequence as set forth in SEQ ID NOs: 1, 7, or 12, a heavy chain complementarity
• R 2 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 comprising a light chain variable region (VL) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 18.
• R 2 comprises an antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 17 and/or comprising a VL comprising the amino acid sequence of SEQ ID NO: 18.
• R 2 comprises 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 comprising a light chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 20.
• R 2 comprises an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and/or comprising a light chain comprising the amino acid sequence of SEQ ID NO: 20.
• R 2 comprises an antibody that is a Fab fragment, a full-length IgG, a Fab' fragment, a F(ab')2 fragment, an scFv, or an Fv.
• complexes provided herein comprise a structure of the formula (Id):
• oligonucleotide comprises a nucleobase sequence of CAGCGCCCACCAGUCA (SEQ ID NO: 21); wherein R 2 comprises an antibody comprising a sequence as set forth in Table 2; wherein nl is an integer (e.g., one or greater) representing the number of instances of the group enclosed by square brackets, wherein each
• R 2 comprises an antibody comprising a heavy chain complementarity determining region 1 (CDR-H1) comprising a sequence as set forth in SEQ ID NOs: 1, 7, or 12, a heavy chain complementarity determining region 2 (CDR-H2) comprising a sequence as set forth in SEQ ID NOs: 2, 8, or 13, a heavy chain complementarity determining region 3 (CDR-H3) comprising a sequence as set forth in SEQ ID NOs: 3, 9, or 14; and/or comprising a light chain complementarity determining region 1 (CDR-L1) comprising a sequence as set forth in SEQ ID NOs: 4, 10, or 15, a light chain complementarity determining region 2 (CDR-L2) comprising a sequence as set forth in SEQ ID NOs: 5, or 11, and a light chain complementarity determining region 3 (CDR-L3) comprising a sequence as set forth in SEQ ID NO: 6 or 16.
• CDR-H1 heavy chain complementarity determining region 1
• CDR-H2 comprising a
• R 2 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 comprising a light chain variable region (VL) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 18.
• R 2 comprises an antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 17 and/or comprising a VL comprising the amino acid sequence of SEQ ID NO: 18.
• R 2 comprises 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 comprising a light chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 20.
• R 2 comprises an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and/or comprising a light chain comprising the amino acid sequence of SEQ ID NO: 20.
• R 2 comprises an antibody that is a Fab fragment, a full-length IgG, a Fab' fragment, a F(ab')2 fragment, an scFv, or an Fv.
• complexes described herein comprise a structure of formula (A):
• a complex described herein comprises an anti-TfRl antibody (e.g., an anti-TfRl Fab) covalently linked via a lysine of the antibody to the 5’ end of an oligonucleotide (e.g., an oligonucleotide comprising a 5’-X-Y-Z-3’ configuration).
• the antibody comprises a sequence as set forth in Table 2.
• the antibody comprises a heavy chain complementarity determining region 1 (CDR-H1) comprising a sequence as set forth in SEQ ID NOs: 1, 7, or 12, a heavy chain complementarity determining region 2 (CDR- H2) comprising a sequence as set forth in SEQ ID NOs: 2, 8, or 13, a heavy chain complementarity determining region 3 (CDR-H3) comprising a sequence as set forth in SEQ ID NOs: 3, 9, or 14; and/or comprises a light chain complementarity determining region 1 (CDR-L1) comprising a sequence as set forth in SEQ ID NOs: 4, 10, or 15, a light chain complementarity determining region 2 (CDR-L2) comprising a sequence as set forth in SEQ ID NOs: 5, or 11, and a light chain complementarity determining region 3 (CDR-L3) comprising a sequence as set forth in SEQ ID NO: 6 or 16.
• CDR-H1 comprising a sequence as set forth in SEQ
• the antibody comprises a heavy chain variable region (VH) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 17 and/or comprises a light chain variable region (VL) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 18.
• VH heavy chain variable region
• VL light chain variable region
• 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.
• the antibody comprises a heavy chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 19 and/or comprises a light chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 20.
• the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and/or comprises a light chain comprising the amino acid sequence of SEQ ID NO: 20.
• the antibody is a Fab fragment, a full-length IgG, a Fab' fragment, a F(ab')2 fragment, an scFv, or an Fv.
• complexes described herein comprise an antibody that binds human transferrin receptor 1 (TfRl).
• TfRl transferrin receptor 1
• An example human TfRl amino acid sequence, corresponding to NCBI sequence NP_003225.2 (transferrin receptor protein 1 isoform 1, homo sapiens) is as follows:
• Table 2 provides examples of sequences of an anti-TfRl antibody useful in the complexes provided herein.
• the anti-TfRl antibody of the present disclosure comprises a heavy chain complementarity determining region 1 (CDR-H1) of SEQ ID NO: 1 (according to the IMGT definition system), a heavy chain complementarity determining region 2 (CDR-H2) of SEQ ID NO: 2 (according to the IMGT definition system), a heavy chain complementarity determining region 3 (CDR-H3) of SEQ ID NO: 3 (according to the IMGT definition system), a light chain complementarity determining region 1 (CDR-L1) of SEQ ID NO: 4 (according to the IMGT definition system), a light chain complementarity determining region 2 (CDR-L2) of SEQ ID NO: 5 (according to the IMGT definition system), and a light chain complementarity determining region 3 (CDR-L3) of SEQ ID NO: 6 (according to the IMGT definition system).
• CDR-H1 heavy chain complementarity determining region 1
• CDR-H2 heavy chain complementarity determining
• the anti-TfRl antibody of the present disclosure comprises a heavy chain complementarity determining region 1 (CDR-H1) of SEQ ID NO: 7 (according to the Kabat definition system), a heavy chain complementarity determining region 2 (CDR-H2) of SEQ ID NO: 8 (according to the Kabat definition system), a heavy chain complementarity determining region 3 (CDR-H3) of SEQ ID NO: 9 (according to the Kabat definition system), a light chain complementarity determining region 1 (CDR-L1) of SEQ ID NO: 10 (according to the Kabat definition system), a light chain complementarity determining region 2 (CDR-L2) of SEQ ID NO: 11 (according to the Kabat definition system), and a light chain complementarity determining region 3 (CDR-L3) of SEQ ID NO: 6 (according to the Kabat definition system).
• CDR-H1 heavy chain complementarity determining region 1
• CDR-H2 heavy chain complementarity determining region 2
• the anti-TfRl antibody of the present disclosure comprises a heavy chain complementarity determining region 1 (CDR-H1) of SEQ ID NO: 12 (according to the Chothia definition system), a heavy chain complementarity determining region 2 (CDR-H2) of SEQ ID NO: 13 (according to the Chothia definition system), a heavy chain complementarity determining region 3 (CDR-H3) of SEQ ID NO: 14 (according to the Chothia definition system), a light chain complementarity determining region 1 (CDR-L1) of SEQ ID NO: 15 (according to the Chothia definition system), a light chain complementarity determining region 2 (CDR-L2) of SEQ ID NO: 5 (according to the Chothia definition system), and a light chain complementarity determining region 3 (CDR-L3) of SEQ ID NO: 16 (according to the Chothia definition system).
• CDR-H1 heavy chain complementarity determining region 1
• CDR-H2 heavy chain complementarity determining
• the anti-TfRl antibody of the present disclosure comprises a heavy chain variable region (VH) containing no more than 25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) in the framework regions as compared with the VH comprising the amino acid sequence of SEQ ID NO: 17.
• VH heavy chain variable region
• the anti-TfRl antibody of the present disclosure comprises a light chain variable region (VL) containing no more than 25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) in the framework regions as compared with the VL comprising the amino acid sequence of SEQ ID NO: 18.
• VL light chain variable region
• the anti-TfRl antibody of the present disclosure comprises a VH comprising an amino acid sequence that is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regions to the VH comprising the amino acid sequence of SEQ ID NO: 17.
• the anti-TfRl antibody of the present disclosure comprises a VL comprising an amino acid sequence that is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regions to the VL comprising the amino acid sequence of SEQ ID NO: 18.
• the anti-TfRl antibody of the present disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO: 17.
• the anti-TfRl antibody of the present disclosure comprises a VL comprising the amino acid sequence of SEQ ID NO: 18.
• the anti-TfRl antibody of the present disclosure comprises a heavy chain comprising an amino acid sequence least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO: 19.
• the anti-TfRl antibody of the present disclosure comprises a light chain comprising an amino acid sequence least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO: 20.
• the anti-TfRl antibody of the present disclosure is a Fab that comprises a heavy chain comprising an amino acid sequence least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO: 19.
• the anti-TfRl antibody of the present disclosure is a Fab that comprises a light chain comprising an amino acid sequence least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO: 20.
• the anti-TfRl antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19. Alternatively or in addition (e.g., in addition), the anti-TfRl antibody of the present disclosure comprises a light chain comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the anti-TfRl antibody of the present disclosure is a Fab that comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 19. Alternatively or in addition (e.g., in addition), the anti-TfRl antibody of the present disclosure is a Fab that comprises a light chain comprising the amino acid sequence of SEQ ID NO: 20.
• the anti-TfRl antibody provided herein may have one or more post-translational modifications.
• N-terminal cyclization also called pyroglutamate formation (pyro-Glu)
• pyro-Glu N-terminal cyclization
• Glu N-terminal Glutamate
• Gin Glutamine residues during production.
• an antibody specified as having a sequence comprising an N-terminal glutamate or glutamine residue encompasses antibodies that have undergone pyroglutamate formation resulting from a post-translational modification.
• pyroglutamate formation occurs in a heavy chain sequence.
• pyroglutamate formation occurs in a light chain sequence.
• an oligonucleotide of the complexes described herein is a single stranded oligonucleotide.
• the oligonucleotide is useful for targeting DMPK (e.g., for reducing expression or activity of a DMPK RNA, such as the level of a mutant or wild-type DMPK RNA).
• an oligonucleotide that is useful for targeting DMPK RNAs e.g., for reducing expression or activity of a DMPK RNA, such as the level of a mutant or wild-type DMPK RNA).
• the oligonucleotide comprises a region of complementarity to a DMPK RNA.
• the oligonucleotide is useful for reducing levels of toxic DMPK having disease- associated repeat expansions, e.g., in a subject having or suspected of having myotonic dystrophy.
• the oligonucleotide is designed to direct RNAse H mediated degradation of the target DMPK RNA residing in the nucleus of cells, e.g., muscle cells (e.g., myotubes) or cells of the nervous system (e.g., central nervous system (CNS) cells).
• CNS central nervous system
• the oligonucleotide is designed to have desirable bioavailability and/or serumstability properties. In some embodiments, the oligonucleotide is designed to have desirable binding affinity properties. In some embodiments, the oligonucleotide is designed to have desirable toxicity profiles. In some embodiments, the oligonucleotide is designed to have low- complement activation and/or cytokine induction properties.
• DMPK-targeting oligonucleotides described herein are designed to caused RNase H mediated degradation of DMPK mRNA. It should be appreciated that, in some embodiments, oligonucleotides in one format (e.g., antisense oligonucleotides) may be suitably adapted to another format (e.g., siRNA oligonucleotides) by incorporating functional sequences (e.g., antisense strand sequences) from one format to the other format. [000100] Examples of oligonucleotides useful for targeting DMPK are provided in US
• oligonucleotides may comprise a region of complementarity to a sequence set forth as follows, which is an example human DMPK gene sequence (Gene ID 1760; NM_001081560.2): AGGGGGGCTGGACCAAGGGGTGGGGAGAAGGGGAGGAGGCCTCGGCCGGCCGCA GAGAGAAGTGGCCAGAGAGGCCCAGGGGACAGCCAGGGACAGGCAGACATGCAG CCAGGGCTCCAGGGCCTGGACAGGGGCTGCCAGGCCCTGTGACAGGAGGACCCCG AGCCCCCGGCCCGGGGAGGGGCCATGGTGCTGCCTGTCCAACATGTCAGCCGAGG TGCGGCTGAGGCGGCTCCAGCAGCTGGTGTTGGACCCGGGCTTCCTGGGGCTGGA GCCCCTGCTCGACCTTCCTGGGCGTCCACCAGGAGCTGGGCGCCTCCGAACTGG CCCAGGACAAGTACGTGGCCGACTTCTTGCAGTGGGCGGAGCCCATCGTGGTGAG GCTGGACAAGTACGTGGCCGACT
• oligonucleotides may comprise a region of complementarity to a sequence set forth as follows, which is an example mouse DMPK gene sequence (Gene ID 13400; NM_001190490.1).
• an oligonucleotide may comprise a region of complementarity to DMPK gene sequences of multiple species, e.g., selected from human, mouse and non-human species (e.g., cynomolgus monkey).
• the oligonucleotide may comprise a region of complementarity to a mutant form of DMPK, for example, 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(l): 1-18.; the contents of each of which are incorporated herein by reference in their entireties.
• an oligonucleotide provided herein is an antisense oligonucleotide targeting DMPK.
• the oligonucleotide targeting DMPK is any one of the antisense oligonucleotides targeting DMPK as described in US Patent Application Publication US20160304877A1, published on October 20, 2016, entitled “Compounds And Methods For Modulation Of Dystrophia Myotonica-Protein Kinase (DMPK) Expression,” incorporated herein by reference).
• the DMPK targeting oligonucleotide targets a region of the DMPK gene sequence as set forth in Genbank accession No. NM_001081560.2 (SEQ ID NO: 24) or as set forth in Genbank accession No. NG_009784.1 (SEQ ID NO: 26).
• a DMPK targeting oligonucleotide comprises a nucleotide sequence comprising a region complementary to a target region that is at least 8 continuous 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 continuous nucleotides) of SEQ ID NO: 24.
• a DMPK targeting oligonucleotide provided herein is 10-35 (e.g., 10-35, 10-30, 10-25, 10-20, 10-15, 15-35, 15-30, 15-25, 15-20, 20-35, 20-30, 13- 18, 14-17, 15-18, 20-30, 15-17, 27-30, 25-35, or 30-35) nucleotides in length.
• a DMPK targeting oligonucleotide provided herein is 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 15-30, or 16 nucleotides in length.
• a DMPK targeting oligonucleotide provided herein is 16 nucleotides in length.
• a DMPK targeting oligonucleotide provided herein comprises a region of complementarity 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 to a DMPK RNA.
• a DMPK targeting oligonucleotide provided herein comprises a region of complementarity 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 to a DMPK sequence as set forth in SEQ ID NO: 24 or 25.
• a DMPK targeting oligonucleotide provided herein comprises a region of complementarity of at least 8 (e.g., at least 8, 9, 10, 11, 12, 13, 14, 15, or 16) consecutive nucleotides to a target sequence as set forth in SEQ ID NO: 22 (TGACTGGTGGGCGCTG).
• an oligonucleotide useful for targeting DMPK comprises at least 8 (e.g., at least 8, 9, 10, 11, 12, 13, 14, 15, or 16) consecutive nucleotides of a sequence as set forth in SEQ ID NO: 21 (CAGCGCCCACCAGUCA).
• an oligonucleotide useful for targeting DMPK comprises the nucleotide sequence of SEQ ID NO: 21.
• the DMPK targeting oligonucleotide comprises a 5’-X- Y-Z-3’ configuration.
• An oligonucleotide comprising a 5’-X-Y-Z-3’ configuration can refer to a chimeric antisense compound in which a gap region having a plurality of nucleosides that support RNase H cleavage is positioned between flanking regions having one or more nucleosides, wherein the nucleosides comprising the gap region are chemically distinct from the nucleoside or nucleosides comprising the flanking regions.
• an oligonucleotide described herein comprises a 5'-X-Y-Z-3' configuration, with X and Z as flanking regions around a gap region Y.
• the gap region Y comprises one or more 2’-deoxyribonucleosides.
• each nucleoside in the gap region Y is a 2’ -deoxyribonucleoside, and neither the flanking region X nor the flanking region Z contains any 2’-deoxyribonucleosides.
• the gap region Y comprises a continuous stretch of 6 or more 2’-deoxyribonucleosides, which are capable of recruiting an RNAse, such as RNAse H.
• the oligonucleotide binds to the target nucleic acid, at which point an RNAse is recruited and can then cleave the target nucleic acid.
• the flanking regions X and Z each comprise one or more modified nucleosides.
• flanking regions X and Z each comprise one or more high-affinity modified nucleosides, e.g., one to six high-affinity modified nucleosides.
• 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).
• the flanking regions X and Z may be of 1-20 nucleotides, 1-8 nucleotides, or 1-5 nucleotides in length.
• the flanking regions X and Z may be of similar length or of dissimilar lengths.
• the gap region Y may comprise a nucleotide sequence of 5-20 nucleotides, 5-15 nucleotides, 5-12 nucleotides, or 6-10 nucleotides in length.
• the gap region Y comprises one or more unmodified intemucleoside linkages.
• one or both flanking regions X and Z each independently comprise phosphorothioate intemucleoside linkages (e.g., phosphorothioate intemucleoside linkages or other linkages) between at least two, at least three, at least four, at least five or more nucleotides.
• the gap region Y and two flanking regions X and Z each independently comprise modified intemucleoside linkages (e.g., phosphorothioate intemucleoside linkages or other linkages) between at least two, at least three, at least four, at least five or more nucleotides.
• the gap region Y in the gapmer is 5-20 nucleosides in length.
• the gap region Y may be 5-20, 5-15, 5-10, 10-20, 10-15, or 15-20 nucleosides in length.
• the gap region Y is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleosides in length.
• each nucleoside in the gap region Y is a 2’ -deoxyribonucleoside. In some embodiments, all nucleosides in the gap region Y are 2 ’-deoxyribonucleosides. In some embodiments, one or more of the nucleosides in the gap region Y is a modified nucleoside (e.g., a 2’ modified nucleoside such as those described herein). In some embodiments, one or more cytosines in the gap region Y are optionally 5-methyl-cytosines. In some embodiments, each cytosine in the gap region Y is a 5-methyl-cytosine.
• flanking region X of the oligonucleotide (X in the 5'-X-Y-Z-3' configuration) and the flanking region Z of the oligonucleotide (Z in the 5'-X-Y-Z- 3' configuration) are independently 1-20 nucleosides long.
• the flanking region X of the oligonucleotide and the flanking region Z of the oligonucleotide may be independently
• 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 are of the same length.
• flanking region X of the oligonucleotide and the flanking region Z of the oligonucleotide are of 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.
• an oligonucleotide described herein comprises a 5'-X-Y-Z-3' configuration of 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-
• the numbers indicate the number of nucleosides in X, Y, and Z regions, respectively, in an oligonucleotide comprising the 5’-X-Y-Z-3’ configuration.
• one or more nucleosides in the flanking region X of the oligonucleotide (X in the 5'-X-Y-Z-3' configuration) or the flanking region Z of the oligonucleotide (Z in the 5'-X-Y-Z-3' configuration) are modified nucleosides (e.g., high- affinity modified nucleosides).
• the modified nucleoside e.g., high- affinity modified nucleosides
• the 2’- modified nucleoside is a 2’-4’ bicyclic nucleoside or a non-bicyclic 2’-modified nucleoside.
• 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’-0-Me), 2’-O-methoxyethyl (2’-M0E), 2’-O-aminopropyl (2’-O-AP), 2’-O- dimethylaminoethyl (2’-O-DMAOE), 2’-O-dimethylaminopropyl (2’-O-DMAP), 2’-O- dimethylaminoethyloxyethyl (2’-O-DMAEOE), or 2
• an oligonucleotide described herein comprises a 5'-X-Y-Z-3' configuration, wherein X and Z are independently 2-7 (e.g., 2, 3, 4, 5, 6, or 7) nucleosides in length and Y is 6-10 (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 in X (the 5’-most position is position 1) is a non-bicyclic 2’- modified nucleoside (e.g., 2’-M0E or 2’-0-Me), wherein the rest of the nucleosides in both X and Z are 2’-4’ bicyclic nucleosides (e.g., LNA or cEt), and wherein each nucleoside in Y
• an oligonucleotide described herein (e.g., a DMPK targeting oligonucleotide described herein)comprises a 5'-X-Y-Z-3' configuration, wherein X and Z are independently 2-7 (e.g., 2, 3, 4, 5, 6, or 7) nucleosides in length and Y is 6-10 (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 in Z (the 5’-most position is position 1) is a non- bicyclic 2’-modified nucleoside (e.g., 2’-M0E or 2’-0-Me), wherein the rest of the nucleosides in both X and Z are 2’-4’ bicyclic nucleosides (e.g., LNA or cEt), and wherein each nucleoside in Y is a
• an oligonucleotide described herein comprises a 5'-X- Y-Z-3' configuration, wherein X and Z are independently 2-7 (e.g., 2, 3, 4, 5, 6, or 7) nucleosides in length and Y is 6-10 (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 in X and at least one of positions but not all (e.g., 1, 2, 3, 4, 5, or 6) of positions 1, 2, 3, 4, 5, 6, or 7 in Z (the 5’- most position is position 1) is a non-bicyclic 2’-modified nucleoside (e.g., 2’-M0E or 2’-O- Me), wherein the rest of the nucleosides in both X and Z are 2’-4’
• X and Z are independently 2-7 (e.g., 2, 3, 4, 5, 6, or 7) nucleoside
• an oligonucleotide described herein is 10-20 nucleosides (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleosides) in length, comprises a region of complementarity 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’-X-Y-Z-3’ configuration, wherein X comprises 3-5 (e.g., 3, 4, or 5) linked nucleosides, wherein at least one of the nucleosides in X is a 2’-modified nucleoside (e.g., 2’- MOE modified nucleoside, 2’-0-Me modified nucleoside, LNA, cEt
• an 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 consecutive nucleosides) of the nucleotide sequence of SEQ ID NO: 21 (CAGCGCCCACCAGUCA), and comprises a 5’-X-Y-Z-3’ configuration, wherein X comprises 3-5 (e.g., 3, 4, or 5) linked nucleosides, wherein at least one of the nucleosides in X is a 2’ -modified nucleoside (e.g., 2’ -MOE modified nucleoside, 2’-0-Me modified nucleoside, LNA, cEt, or ENA); Y comprises 6-10 (e.g., 6, 7, 8, 9, or 10) linked
• each thymine base (T) of the nucleotide sequence of the antisense oligonucleotide may independently and optionally be replaced with a uracil base (U), and each U may independently and optionally be replaced with a T.
• an oligonucleotide described herein comprises the nucleotide sequence of SEQ ID NO: 21 and comprises a 5’-X-Y-Z-3’ configuration, wherein X comprises 3-5 (e.g., 3, 4, or 5) linked nucleosides, wherein at least one of the nucleosides in X is a 2’ -modified nucleoside (e.g., 2’-M0E modified nucleoside, 2’-0-Me modified nucleoside, LNA, cEt, or ENA); Y comprises 6-10 (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 3-5 (e.g., 3, 4, or 5) linked nucleosides, wherein at least one
• each thymine base (T) of the nucleotide sequence of the antisense oligonucleotide may independently and optionally be replaced with a uracil base (U), and each U may independently and optionally be replaced with a T.
• 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., 2’-M0E modified nucleoside or 2’-0-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., 2’-M0E modified nucleoside or 2’-0-Me modified nucleoside).
• the 2’-4’ bicyclic nucleoside is selected from LNA, cEt, and ENA nucleosides.
• the non-bicyclic 2’-modified nucleoside is a 2’- MOE modified nucleoside or a 2’-OMe modified nucleoside.
• the nucleosides of the oligonucleotides are joined together by phosphorothioate intemucleoside linkages, phosphodiester intemucleoside linkages or a combination thereof.
• the oligonucleotide comprises only phosphorothioate internucleoside linkages joining each nucleoside (i.e., the oligonucleotide comprises a fully phosphorothioate backbone).
• the oligonucleotide comprises at least one phosphorothioate intemucleoside linkage.
• the oligonucleotide comprises a mix of phosphorothioate and phosphodiester internucleoside linkages. In some embodiments, the oligonucleotide comprises only phosphorothioate intemucleoside linkages joining each pair of 2’ -deoxyribonucleosides and a mix of phosphorothioate and phosphodiester intemucleoside linkages joining the remaining nucleosides.
• the oligonucleotide comprises a 5’-X-Y-Z-3’ configuration of LLEE-(D)s-EELL, wherein “E” is a 2’-M0E modified ribonucleoside; “L” is LNA; “D” is a 2’ -deoxy ribonucleoside; and “10” or “8” is the number of 2’- deoxyribonucleosides in Y, and wherein the oligonucleotide comprises phosphorothioate intemucleoside linkages, phosphodiester intemucleoside linkages or a combination thereof.
• each cytidine (e.g., a 2’ -modified cytidine) in X and/or Z of the oligonucleotide is optionally and independently a 5-methyl-cytidine
• each uridine (e.g., a 2’ -modified uridine) in X and/or Z is optionally and independently a 5-methyl-uridine.
• an oligonucleotide described herein comprises a 5’-X-Y-Z-3’ configuration and comprises a nucleobase sequence of CAGCGCCCACCAGUCA (SEQ ID NO: 21).
• an oligonucleotide described herein comprises a structure of +C*+A*oG*oC*dG*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’-O-methoxyethyl (MOE) modified ribonucleoside, oC represents a 5-methyl-2’-MOE-cytidine, +C represents a 5-methyl-2’-4’-bicyclic-cytidine (2’- 4’ methylene bridge), oU represents a 5-methyl-2’-MOE-uridine, * represents a phosphorothioate in
• an oligonucleotide described herein e.g., a DMPK targeting oligonucleotide described herein
• can be in salt form e.g., as sodium, potassium, or magnesium salts.
• the 5’ or 3’ nucleoside (e.g., terminal nucleoside) of the oligonucleotide is conjugated to an amine group, optionally via a spacer.
• the spacer comprises an aliphatic moiety.
• the spacer comprises a polyethylene glycol moiety.
• a phosphodiester linkage is present between the spacer and the 5’ or 3’ nucleoside of the oligonucleotide.
• the 5’ or 3’ nucleoside of the oligonucleotide is conjugated to a compound of the formula -NH2-(CH2)n-, wherein n is an integer from 1 to 12. In some embodiments, n is 6, 7, 8, 9, 10, 11, or 12.
• a phosphodiester linkage is present between the compound of the formula NH2-(CH2) n - and the 5’ or 3’ nucleoside of the oligonucleotide.
• a compound of the formula NH2- (CH2)6- is conjugated to the oligonucleotide via a reaction between 6-amino-l -hexanol (NH2- (CH 2 ) 6 -OH) and the 5’ phosphate of the oligonucleotide.
• the oligonucleotide is conjugated to a targeting agent, e.g., a muscle targeting agent such as an anti-TfRl antibody, e.g., via an amine group of a lysine of the targeting agent.
• a targeting agent e.g., a muscle targeting agent such as an anti-TfRl antibody
• nucleobase uracil at the C5 position forms thymine.
• a nucleotide or nucleoside having a C5 methylated uracil (or 5-methyl-uracil) may be equivalently identified as a thymine nucleotide or nucleoside.
• any one or more of the thymine bases (T’s) in any one of the oligonucleotides provided herein may independently and optionally be uracil bases (U’s), and/or any one or more of the U’s in the oligonucleotides provided herein (e.g., the oligonucleotide as set forth in SEQ ID NO: 21) may independently and optionally be T’s.
• compositions described herein comprise complexes (z.e., a plurality of complexes), each of which complex comprises an antibody (e.g., anti-TFRl 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.
• an antibody e.g., anti-TFRl antibody
• oligonucleotides e.g., a DMPK-targeting oligonucleotide described herein
• the antibody of such complexes comprises a CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 as set forth in Table 2.
• Complexes of a composition described herein can comprise any structure provided herein, e.g., a structure of formula (I) (e.g., comprising a group of the formula (la), formula (lb), formula (Ic), or formula (Id)) or formula (A).
• compositions described herein comprise complexes (z.e., a plurality of complexes) wherein each complex comprises a structure of formula (I): [ R 1 ]n i - R 2 , in which each R 1 independently comprises a compound comprising an oligonucleotide (e.g., a DMPK-targeting oligonucleotide described herein) and is covalently linked to R 2 , wherein R 2 comprises an antibody (e.g., anti-TfRl antibody) comprising a heavy chain comprising a heavy chain variable region (VH) and a heavy chain constant region, and a light chain comprising a light chain variable region (VL) and a light chain constant region.
• each R 1 of a complex is independently covalently linked to a different amino acid residue (e.g., lysine or cysteine) of R 2 .
• the value of nl of complexes in the composition is independently and optionally an integer from one up to the number of amino acid residues to which conjugation is desired or targeted (e.g., the number of lysine residues) in the antibody (e.g., an antibody comprised within R 2 ).
• the value of nl of 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.
• the value of nl of 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.
• the value of nl of each complex in the composition is independently selected and optionally from an integer in the range of 1 to 27, 1 to 26, 1 to 10, 1 to 5, or 1 to 3.
• the average value of nl of complexes of the composition is in the range of 1 to 2, 1 to 3, 1 to 5, 1 to 10, 1 to 26, or 1 to 27.
• compositions described herein comprise complexes in which the value of nl is 0.
• the average value of nl of complexes of the composition is in the range of 0.5 to 5 (e.g., 0.5-5, 1-5, 1-4, 1-3, 3-5, 0.5-4, 0.5-3, 0.5-2, 0.5-1.5, 0.5-1, 0.7-1.5, 1- 1.6, 1-1.5, 1-1.4, 1-1.3, 1-1.2, 1.1-1.5, 0.8-2, 0.8-1.5, O.8-1.3, 0.8-1.2, 0.8-1.1, 0.9-3, 0.9-2, 0.9- 1.8, 0.9-1.6, 0.9-1.5, 0.9-1.4, 0.9-1.3, or 0.9-1.2).
• a composition described herein comprises antibody that is not conjugated to an oligonucleotide (e.g., in trace amounts) and antibody conjugated to one or more oligonucleotides.
• antibody that is not conjugated to an oligonucleotide may be referred to as a compound comprising a structure of formula (I): for which nl is zero.
• a composition for administration to a subject in the methods described herein comprises compounds (e.g., complexes) comprising a structure of formula (I): [ 1 ]>, i -R 2 , for which each R 1 independently comprises a group comprising an oligonucleotide, R 2 comprises an antibody and nl is independently an integer of zero or greater that reflects the number of instances of R 1 in each compound (e.g., complex).
• the fraction of compounds comprising a structure of formula (I): [R ⁇ ni-R 2 , in a composition, for which nl is zero, compared with all compounds of that structure in the composition for which nl is one or greater, 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%.
• the average value of nl of complexes in a composition disclosed herein is in the range of 0.5 to 5 (e.g., 0.5-5, 1-5, 1-4, 1-3, 3-5, 0.5-4, 0.5-3, 0.5-2, 0.5-1.5, 0.5-1, 0.7-1.5, 1-1.6, 1-1.5, 1-1.4, 1-1.3, 1-1.2, 1.1-1.5, 0.8-2, 0.8-1.5, O.8-1.3, 0.8-1.2, 0.8-1.1, 0.9-3, 0.9-2, 0.9-1.8, 0.9-1.6, 0.9-1.5, 0.9-1.4, 0.9-1.3, or 0.9-1.2).
• 0.5-5, 1-5, 1-4, 1-3, 3-5, 0.5-4, 0.5-3, 0.5-2 0.5-1.5, 0.5-1, 0.7-1.5, 1-1.6, 1-1.5, 1-1.4, 1-1.3, 1-1.2, 1.1-1.5, 0.8-2, 0.8-1.5, O.8-1.3, 0.8-1.2, 0.8-1.1, 0.9-3, 0.9-2,
• Complexes provided herein are formulated in a manner suitable for pharmaceutical use.
• complexes can be delivered to a subject using a formulation that minimizes degradation, facilitates delivery and/or (e.g., and) uptake, or provides another beneficial property to complexes in the formulation.
• formulating complexes e.g., complexes comprising an oligonucleotide covalently linked with a Fab
• tris(hydroxymethyl)aminomethane also known as tromethamine or THAM
• sucrose is particularly advantageous for pharmaceutical use, e.g., as described herein.
• formulations e.g., aqueous solutions, lyophilized forms, or frozen forms
• formulations comprising complexes together with tris(hydroxymethyl)aminomethane and/or sucrose.
• Such formulations can be suitably prepared such that when administered to a subject, either into the immediate environment of a target cell or systemically, a sufficient amount of the complexes enter target muscle cells.
• formulations are provided herein that comprise complexes (/'. ⁇ ?., a plurality of complexes) that comprise an oligonucleotide (e.g., an oligonucleotide comprising a 5’-X-Y-Z-3’ configuration) covalently linked to an antibody.
• an oligonucleotide e.g., an oligonucleotide comprising a 5’-X-Y-Z-3’ configuration
• a formulation comprising complexes, in which each complex comprises an oligonucleotide (e.g., an oligonucleotide comprising a 5’-X-Y-Z-3’ configuration) covalently linked to an anti-TfRl antibody, optionally wherein the antibody of such complexes comprises a CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 as set forth in Table 2, and further, in some embodiments, wherein the complexes are formulated with tris(hydroxymethyl)aminomethane and sucrose.
• the antibody is an anti-TfRl antibody.
• formulations comprise complexes comprising a structure of formula (I): [ R 1 ] >, i -R 2 , in which each R 1 independently comprises a compound comprising an oligonucleotide (e.g., e.g., an oligonucleotide comprising a 5’-X-Y- Z-3’ configuration) and R 2 comprises an antibody (e.g., anti-TfRl antibody), and in which nl is an integer representing the number of instances of R 1 in the complex.
• oligonucleotide e.g., e.g., an oligonucleotide comprising a 5’-X-Y- Z-3’ configuration
• R 2 comprises an antibody (e.g., anti-TfRl antibody)
• nl is an integer representing the number of instances of R 1 in the complex.
• formulations described herein comprise complexes comprising an antibody that comprises a CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 as set forth in Table 2.
• 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.
• 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.
• a formulation described herein comprises complexes described herein at a concentration of between 1-50 mg of the complex per mL of the formulation. In some embodiments, a formulation described herein comprises complexes at a concentration of 10-50 mg/ml or 20-35 mg/mL (e.g., 15-45 mg/mL, 20-40 mg/mL, 25-35 mg/mL, 25.5-34.5 mg/mL, 26-34 mg/mL, 27-33 mg/mL, 28-32 mg/mL, 29-31 mg/mL, 29.5- 30.5 mg/mL, 10-40 mg/mL, 15-35 mg/mL, 20-30 mg/mL, 21-29 mg/mL, 21.2-28.8 mg/mL, 22-28 mg/mL, 23-27 mg/mL, 24-26 mg/mL, or 24.5-25.5 mg/mL).
• 10-50 mg/ml or 20-35 mg/mL e.g., 15-45 mg/mL, 20-40 mg
• a formulation described herein comprises complexes at a concentration of approximately 25 mg/mL (e.g., 25 mg/mL). In some embodiments, a formulation described herein comprises complexes at a concentration of approximately 30 mg/mL (e.g., 30 mg/mL). In some embodiments, the concentration of complexes in a formulation may vary by up to 20% (e.g., +/- up to 20%, +/- up to 15%, +/- up to 10%, or +/- up to 5%) of a set value. For example, in some embodiments, the concentration of complexes in the formulation is 30 mg/mL +/- up to 15% (e.g., 30 +/- 4.5 mg/mL). In some embodiments, the concentration of complexes in the formula is 25 mg/mL +/- up to 15% (e.g., 25 +/- 3.8 mg/mL).
• any one or a plurality of the complexes described herein is formulated with tris(hydroxymethyl)aminomethane and sucrose in an aqueous solution.
• the tris(hydroxymethyl)aminomethane is present in the aqueous solution at a concentration in the range of 5-50 mM (e.g., 5-40 mM, 5-35 mM, 5-30 mM, 10-50 mM, 15- 45 mM, 10-40 mM, 20-40 mM, 20-35 mM, 20-30 mM, 21-29 mM, 22-28 mM, 23-27 mM, 24- 26 mM).
• the tris(hydroxymethyl)aminomethane is present in the aqueous solution at a concentration of approximately 25 mM (e.g., 25 mM).
• the sucrose is present in the aqueous solution at a concentration in the range of 5 % to 15 % weight per volume (w/v%), for example, 7-13 ⁇ IN%, 8-15% ⁇ IN%, 9-15% ⁇ IN%, 9-11% w/v%, 9.5-11% w/v%, or for example, in the range of 9-10 w/v%, 10-11 I/N%, 10-12 ⁇ IN%, or 8-12 IIN%.
• the sucrose is present in the aqueous solution at a concentration in the range of 7-13 ⁇ IN%, 8-12 ⁇ IN%, or 9-11 ⁇ IN%. In some embodiments, the sucrose is present in the aqueous solution at a concentration of approximately 10 ⁇ IN% (e.g., 10 w/v%).
• the aqueous solution has a pH in the range of 6.5 to 8.5, for example, 6.5-6.5, 6.7-6.9, 6.9-7.1, 7.1-7.3, 7.2-7.8, 7.3-7.5, 7.4-7.5, 7.4-7.6, 7.5-7.6; for example, 7.0-8.0, or for example, in the pH range of 7.0-7.3, 7.2-7.8, 7.3-7.5, 7.4-7.6, 7.5-7.6, 7.5-7.7, 7.7-7.9, 7.9-8.0, 8.0-8.2, 8.2-8.4, 8.3-8.4, 8.4-8.5, 8.5-8.6, or 7.3-7.7.
• the aqueous solution has a pH in the range of 7.0-8.0 (e.g., 7.0-7.8, 7.1-7.8, 7.2- 7.8, 7.3-7.7, 7.3-7.5, 7.3-7.6, 7.4-7.6, or 7.4-7.8). In some embodiments, the aqueous solution has a pH of approximately 7.5 (e.g., 7.5). In some embodiments, the aqueous solution has a pH in the range of 7.4-7.7. In some embodiments, the aqueous solution has a pH in the range of 7.4-7.6 (e.g., 7.5, or about 7.5).
• 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 approximately 25 mM (e.g., 25 mM), wherein sucrose is present in the aqueous solution at a concentration of approximately 10 w/v% (e.g., 10 w/v%), and wherein the aqueous solution is at a pH of approximately 7.5 (e.g., 7.5).
• 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 approximately 25 mM (e.g., 25 mM), wherein sucrose is present in the aqueous solution at a concentration of approximately 10 w/v% (e.g., 10 w/v%), wherein complexes are present in the aqueous solution at a concentration of approximately 20-35 mg/ml (e.g., 25 mg/ml or 30 mg/ml) and wherein the aqueous solution is at a pH of approximately 7.5 (e.g., 7.5).
• tris(hydroxymethyl)aminomethane is present in the aqueous solution at a concentration of approximately 25 mM (e.g., 25 mM)
• sucrose is present in the aqueous solution at a concentration of approximately 10 w/v% (e.g., 10 w/v%)
• any one or a plurality of the complexes described herein is formulated with tris(hydroxymethyl)aminomethane and sucrose in a lyophilized form (e.g., lyophilized powder).
• a lyophilized form e.g., lyophilized powder
• the lyophilized form is obtained by lyophilization of any one of the aqueous solutions described herein.
• a lyophilized form is a lyophilized cake.
• a lyophilized cake comprises a plurality of complexes provided herein, tris(hydroxymethyl)aminomethane, and sucrose.
• a lyophilized cake comprises IxlO -2 mg-1.5 mg (e.g., IxlO -2 mg-1.5 mg, 4.02xl0 -2 mg-1.21 mg, 6xl0 -2 mg-1 mg, 8xl0 -2 mg-1 mg, 0.1 mg-1 mg, 0.05 mg-0.2 mg, 0.05 mg-0.3 mg, 0.05 mg-0.4 mg, 0.1 mg-0.2 mg, 0.1 mg-0.3 mg, 0.1 mg-0.4 mg, 0.1 mg-0.5 mg, 0.2 mg-0.3 mg, 0.2 mg-0.4 mg, 0.2 mg-0.4 mg, 0.3 mg-0.4 mg, or 0.3 mg-0.5 mg) tris(hydroxymethyl)aminomethane per g of cake.
• IxlO -2 mg-1.5 mg e.g., IxlO -2 mg-1.5 mg, 4.02xl0 -2 mg-1.21 mg, 6xl0 -2 mg-1 mg, 8xl0 -2 mg-1 mg
• 0.1 mg-1 mg 0.05 mg-0.2 mg, 0.05 mg
• a lyophilized cake comprises 900 mg-1100 mg (e.g., 900 mg-1100 mg, 989 mg-999 mg, 950 mg- 1000 mg, 980 mg- 1000 mg, 980 mg-990 mg, 990 mg- 1000 mg, 995 mg- 1000 mg, 992 mg-998 mg, 994 mg-999 mg) sucrose per g of cake.
• 900 mg-1100 mg e.g., 900 mg-1100 mg, 989 mg-999 mg, 950 mg- 1000 mg, 980 mg- 1000 mg, 980 mg-990 mg, 990 mg- 1000 mg, 995 mg- 1000 mg, 992 mg-998 mg, 994 mg-999 mg
• a lyophilized cake comprises 0.5 mg- 10 mg (e.g., 0.5 mg- 10 mg, 0.5 mg-1.5 mg, 1 mg-2 mg, 1.5 mg-3 mg, 1 mg-8 mg, 0.5 mg-6 mg, 0.5 mg-5 mg, 1.5 mg-3.5 mg, 1.5 mg-4 mg, 1.5 mg-5 mg, 2 mg-5 mg, 2.5 mg-5 mg, 2 mg-3mg, 3 mg-4 mg, 4 mg-5 mg) of complexes per g of cake.
• a lyophilized cake comprises 0.303 mg tris(hydroxymethyl)aminomethane per g of cake, 998.6 mg sucrose per g of cake, and/or (e.g., and) 0.999 mg complexes per g of cake.
• a lyophilized cake comprises 0.302 mg tris(hydroxymethyl)aminomethane per g of cake, 997.7 mg sucrose per g of cake, and/or (e.g., and) 1.995 mg complexes per g of cake. In some embodiments, a lyophilized cake comprises 0.302 mg tris(hydroxymethyl)aminomethane per g of cake, 997.2 mg sucrose per g of cake, and/or (e.g., and) 2.49 mg complexes per g of cake.
• a lyophilized cake comprises 0.302 mg tris(hydroxymethyl)aminomethane per g of cake, 996.7 mg sucrose per g of cake, and/or (e.g., and) 2.99 mg complexes per g of cake. In some embodiments, a lyophilized cake comprises 0.302 mg tris(hydroxymethyl)aminomethane per g of cake, 995.7 mg sucrose per g of cake, and/or (e.g., and) 3.98 mg complexes per g of cake.
• a lyophilized cake comprises 0.301 mg tris(hydroxymethyl)aminomethane per g of cake, 994.7 mg sucrose per g of cake, and/or (e.g., and) 4.97 mg complexes per g of cake.
• reconstitution of a lyophilized cake results in a solution comprising tris(hydroxymethyl)aminomethane at a concentration of 5 to 50 mM, sucrose at a concentration of 5 ⁇ IN% to 15 ⁇ IN%, and/or (e.g., and) complexes at a concentration of 10 mg/mL to 50 mg/mL.
• reconstitution of 10.053 g of a lyophilized cake with a solvent (e.g., water) to form 1 mL of reconstituted solution results in a solution comprising tris(hydroxymethyl)aminomethane at a concentration of 5 to 50 mM (optionally 25 mM), sucrose at a concentration of 5 ⁇ IN% to 15 ⁇ IN% (optionally 10 w/v%), and/or (e.g., and) complexes at a concentration of 10 mg/mL to 50 mg/mL.
• a solvent e.g., water
• reconstitution of about 10 g of a lyophilized cake with a solvent (e.g., water) to form 1 mL of reconstituted solution results in a solution comprising tris(hydroxymethyl)aminomethane at a concentration of 5 to 50 mM (optionally 25 mM), sucrose at a concentration of 5 ⁇ IN% to 15 ⁇ IN% (optionally 10 w/v%), and/or (e.g., and) complexes at a concentration of 10 mg/mL to 50 mg/mL.
• a solvent e.g., water
• any one or a plurality of the complexes described herein is formulated with tris(hydroxymethyl)aminomethane and sucrose in a frozen form (e.g., a frozen aqueous solid).
• the frozen form e.g., frozen aqueous solid
• a frozen form may be frozen to a temperature of less than -20 °C (e.g., less than -20 °C, less than -30 °C, less than -40 °C, less than -50 °C, less than -60 °C, less than -70 °C, less than -80 °C, or lower).
• the value of nl of each complex in the formulation is independently and optionally an integer from zero up to the number of amino acid residues to which conjugation is desired or targeted (e.g., the number of lysine residues) in the antibody (R 2 ).
• the value of nl of each complex in the formulation 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.
• the value of nl of each complex in the formulation 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.
• the value of nl of each complex in the formulation is independently selected and optionally from an integer in the range of 1 to 27, 1 to 26, 1 to 10, 1 to 5, or 1 to 3.
• the average value of nl of complexes in the formulation is in the range of 1 to 2, 1 to 3, 1 to 5, 1 to 10, 1 to 26 or 1 to 27.
• formulations described herein comprise complexes that comprise a structure of formula (I): [R ⁇ ni-R 2 , wherein nl is 0.
• the average value of nl of complexes of the formulation is in the range of 0.5 to 5 (e.g., 0.5-5, 1-5, 1-4, 1-3, 3-5, 0.5-4, 0.5-3, 0.5-2, 0.5-1.5, 0.5-1, 0.7-1.5, 1-1.6, 1-1.5, 1-1.4, 1-1.3, 1-1.2, 1.1-1.5, 0.8-2, 0.8-1.5, O.8-1.3, 0.8-1.2, 0.8-1.1, 0.9-3, 0.9-2, 0.9-1.8, 0.9- 1.6, 0.9-1.5, 0.9-1.4, 0.9-1.3, or 0.9-1.2).
• each instance of R 1 in a complex herein is conjugated to a different amino acid residue of the antibody.
• each different amino acid comprises an s-amino group (e.g., lysine, arginine).
• each different amino acid to which R 1 is covalently linked is a cysteine.
• R 1 is directly covalently linked to an amino acid residue of the antibody.
• R 1 is indirectly covalently linked to an amino acid of the antibody, e.g., covalently linked to a glycosylation site on the amino acid.
• formulations are provided in which complexes for which R 1 is covalently linked to an amino acid residue residing in a CDR region of the antibody are present in only trace amounts, or in undetectable amount, or not at all. In some embodiments, formulations are provided in which complexes for which R 1 is covalently linked to an amino acid residue residing in a CDR region of the antibody are not detectable in the formulation using standard detection techniques.
• formulations provided herein comprise complexes that comprise a structure of formula (I): [R ’JHI-R 2 , in which each instance of R 1 independently comprises a group of the formula (la): in which R 3 comprises an oligonucleotide, e.g., an oligonucleotide comprising a 5’-X-Y-Z-3’ configuration; and R 1 is covalently linked to R 2 at attachment point A.
• R 2 comprises an antibody comprising a sequence as set forth in Table 2.
• R 2 comprises an antibody comprising a heavy chain complementarity determining region 1 (CDR-H1) comprising a sequence as set forth in SEQ ID NOs: 1, 7, or 12, a heavy chain complementarity determining region 2 (CDR-H2) comprising a sequence as set forth in SEQ ID NOs: 2, 8, or 13, a heavy chain complementarity determining region 3 (CDR-H3) comprising a sequence as set forth in SEQ ID NOs: 3, 9, or 14; and/or comprising a light chain complementarity determining region 1 (CDR-L1) comprising a sequence as set forth in SEQ ID NOs: 4, 10, or 15, a light chain complementarity determining region 2 (CDR- L2) comprising a sequence as set forth in SEQ ID NOs: 5, or 11, and a light chain complementarity determining region 3 (CDR-L3) comprising a sequence as set forth in SEQ ID NO: 6 or 16.
• CDR-H1 heavy chain complementarity determining region 1
• CDR-H2 comprising a
• R 2 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 comprising a light chain variable region (VL) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 18.
• R 2 comprises an antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 17 and/or comprising a VL comprising the amino acid sequence of SEQ ID NO: 18.
• R 2 comprises 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 comprising a light chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 20.
• R 2 comprises an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and/or comprising a light chain comprising the amino acid sequence of SEQ ID NO: 20.
• R 2 comprises an antibody that is a Fab fragment, a full-length IgG, a Fab' fragment, a F(ab')2 fragment, an scFv, or an Fv.
• R 3 comprises an oligonucleotide comprising a nucleobase sequence of CAGCGCCCACCAGUCA (SEQ ID NO: 21). In some embodiments, R 3 comprises an oligonucleotide comprising a structure of +C*+A*oG*oC*dG*dC*dC*dC*dA*dC*dC*dA*oG*oU*+C*+A (SEQ ID NO: 21), wherein +N represents an LNA (2’ -4’ methylene bridge) ribonucleoside, dN represents a 2’- deoxyribonucleoside, oN represents a 2’ -MOE modified ribonucleoside, oC represents a 5- methyl-2’-MOE-cytidine, +C represents a 5-methyl-2’-4’-bicyclic-cytidine (2 ’-4’ methylene bridge), oU represents a 5-methyl-2’-MOE-uridine, and
• nl is independently an integer (e.g., an integer in the range of 1-27, 1-26, 1-10, 1-5, or 1-3).
• formulations described herein further comprise complexes that comprise a structure of formula (I): [R ⁇ ni-R 2 , wherein nl is 0.
• the average value of nl of complexes of the composition is in the range of 0.5 to 5 (e.g., 0.5-5, 1-5, 1-4, 1-3, 3-5, 0.5-4, 0.5-3, 0.5-2, 0.5-1.5, 0.5-1, 0.7-1.5, 1-1.6, 1-1.5, 1-1.4, 1-1.3, 1-1.2, 1.1-1.5, 0.8-2, 0.8-1.5, O.8-1.3, 0.8-1.2, 0.8-1.1, 0.9-3, 0.9-2, 0.9-1.8, 0.9-1.6, 0.9-1.5, 0.9-1.4, 0.9-1.3, or 0.9-1.2).
• formulations further comprise tris(hydroxymethyl)aminomethane and sucrose.
• formulations comprise tris(hydroxymethyl)aminomethane at a concentration of 25 mM and/or (e.g., and) sucrose at a concentration of 10 ⁇ IN%, optionally wherein the formulation has a pH of 7.5. In some embodiments, formulations comprise complexes at a concentration of 30 mg/mL.
• formulations provided herein comprise complexes that comprise a structure of formula (I): [R ⁇ ni-R 2 , in which each instance of R 1 in a complex of a formulation provided herein comprises a group of the formula (lb): wherein +N represents an LNA (2’-4’ methylene bridge) ribonucleoside, dN represents a 2’- deoxyribonucleoside, oN represents a 2’-O-methoxyethyl (MOE) modified ribonucleoside, oC represents a 5-methyl-2’-MOE-cytidine, +C represents a 5-methyl-2’-4’-bicyclic-cytidine (2’- 4’ methylene bridge), oU represents a 5-methyl-2’-MOE-uridine, * represents a phosphorothioate internucleoside linkage, and wherein the oligonucleotide comprises a nucleobase sequence of CAGCGCCCACC
• R 2 comprises an antibody comprising a sequence as set forth in Table 2.
• R 2 comprises an antibody comprising a heavy chain complementarity determining region 1 (CDR-H1) comprising a sequence as set forth in SEQ ID NOs: 1, 7, or 12, a heavy chain complementarity determining region 2 (CDR-H2) comprising a sequence as set forth in SEQ ID NOs: 2, 8, or 13, a heavy chain complementarity determining region 3 (CDR-H3) comprising a sequence as set forth in SEQ ID NOs: 3, 9, or 14; and/or comprising a light chain complementarity determining region 1 (CDR-L1) comprising a sequence as set forth in SEQ ID NOs: 4, 10, or 15, a light chain complementarity determining region 2 (CDR- L2) comprising a sequence as set forth in SEQ ID NOs: 5, or 11, and a light chain complementarity determining region 3 (CDR-L3) comprising a sequence as set forth in SEQ ID NO: 6 or
• R 2 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 comprising a light chain variable region (VL) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 18.
• R 2 comprises an antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 17 and/or comprising a VL comprising the amino acid sequence of SEQ ID NO: 18.
• R 2 comprises 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 comprising a light chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 20.
• R 2 comprises an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and/or comprising a light chain comprising the amino acid sequence of SEQ ID NO: 20.
• R 2 comprises an antibody that is a Fab fragment, a full-length IgG, a Fab' fragment, a F(ab')2 fragment, an scFv, or an Fv.
• formulations described herein further comprise complexes that comprise a structure of formula (I): wherein nl is 0.
• the average value of nl of complexes of the composition is in the range of 0.5 to 5 (e.g., 0.5-5, 1-5, 1-4, 1-3, 3-5, 0.5-4, 0.5-3, 0.5-2, 0.5-1.5, 0.5-1, 0.7-1.5, 1-1.6, 1-1.5, 1-1.4, 1-1.3, 1-1.2, 1.1-1.5, 0.8-2, 0.8-1.5, O.8-1.3, 0.8-1.2, 0.8-1.1, 0.9-3, 0.9-2, 0.9-1.8, 0.9-1.6, 0.9-1.5, 0.9-1.4, 0.9-1.3, or 0.9-1.2).
• formulations further comprise tris(hydroxymethyl)aminomethane and sucrose.
• formulations comprise tris(hydroxymethyl)aminomethane at a concentration of 25 mM and/or (e.g., and) sucrose at a concentration of 10 ⁇ IN%, optionally wherein the formulation has a pH of 7.5.
• formulations comprise complexes at a concentration of 30 mg/mL.
• formulations provided herein comprise complexes that comprise a structure of formula (I): [R ⁇ ni-R 2 , in which each R 1 comprises a group of the formula (Ic):
• R 2 comprises an antibody comprising a sequence as set forth in Table 2.
• R 2 comprises an antibody comprising a heavy chain complementarity determining region 1 (CDR-H1) comprising a sequence as set forth in SEQ ID NOs: 1, 7, or 12, a heavy chain complementarity determining region 2 (CDR-H2) comprising a sequence as set forth in SEQ ID NOs: 2, 8, or 13, a heavy chain complementarity determining region 3 (CDR-H3) comprising a sequence as set forth in SEQ ID NOs: 3, 9, or 14; and/or comprising a light chain complementarity determining region 1 (CDR-L1) comprising a sequence as set forth in SEQ ID NOs: 4, 10, or 15, a light chain complementarity determining region 2 (CDR- L2) comprising a sequence as set forth in SEQ ID NOs: 5, or 11, and a light chain complementarity determining region 3 (CDR-H1) comprising a sequence as set forth in SEQ ID NOs: 1, 7, or 12, a heavy chain complementarity
• R 2 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 comprising a light chain variable region (VL) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 18.
• R 2 comprises an antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 17 and/or comprising a VL comprising the amino acid sequence of SEQ ID NO: 18.
• R 2 comprises 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 comprising a light chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 20.
• R 2 comprises an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and/or comprising a light chain comprising the amino acid sequence of SEQ ID NO: 20.
• R 2 comprises an antibody that is a Fab fragment, a full-length IgG, a Fab' fragment, a F(ab')2 fragment, an scFv, or an Fv.
• formulations described herein further comprise complexes that comprise a structure of formula (I): wherein nl is 0.
• the average value of nl of complexes of the composition is in the range of 0.5 to 5 (e.g., 0.5-5, 1-5, 1-4, 1-3, 3-5, 0.5-4, 0.5-3, 0.5-2, 0.5-1.5, 0.5-1, 0.7-1.5, 1-1.6, 1-1.5, 1-1.4, 1-1.3, 1-1.2, 1.1-1.5, 0.8-2, 0.8-1.5, O.8-1.3, 0.8-1.2, 0.8-1.1, 0.9-3, 0.9-2, 0.9-1.8, 0.9-1.6, 0.9-1.5, 0.9-1.4, 0.9-1.3, or 0.9-1.2).
• formulations further comprise tris(hydroxymethyl)aminomethane and sucrose.
• formulations comprise tris(hydroxymethyl)aminomethane at a concentration of 25 mM and/or (e.g., and) sucrose at a concentration of 10 ⁇ IN%, optionally wherein the formulation has a pH of 7.5.
• formulations comprise complexes at a concentration of 30 mg/mL.
• formulations provided herein comprise complexes that comprise a structure of the formula (Id):
• oligonucleotide comprises a nucleobase sequence of CAGCGCCCACCAGUCA (SEQ ID NO: 21); wherein R 2 comprises an antibody comprising a sequence as set forth in Table 2; wherein nl is an integer (e.g., one or greater) representing the number of instances of the group enclosed by square brackets, wherein each
• R 2 comprises an antibody comprising a heavy chain complementarity determining region 1 (CDR-H1) comprising a sequence as set forth in SEQ ID NOs: 1, 7, or 12, a heavy chain complementarity determining region 2 (CDR-H2) comprising a sequence as set forth in SEQ ID NOs: 2, 8, or 13, a heavy chain complementarity determining region 3 (CDR-H3) comprising a sequence as set forth in SEQ ID NOs: 3, 9, or 14; and/or comprising a light chain complementarity determining region 1 (CDR-L1) comprising a sequence as set forth in SEQ ID NOs: 4, 10, or 15, a light chain complementarity determining region 2 (CDR-L2) comprising a sequence as set forth in SEQ ID NOs: 5, or 11, and a light chain complementarity determining region 3 (CDR-L3) comprising a sequence as set forth in SEQ ID NO: 6 or 16.
• CDR-H1 heavy chain complementarity determining region 1
• CDR-H2 comprising a
• R 2 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 comprising a light chain variable region (VL) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 18.
• R 2 comprises an antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 17 and/or comprising a VL comprising the amino acid sequence of SEQ ID NO: 18.
• R 2 comprises 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 comprising a light chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 20.
• R 2 comprises an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and/or comprising a light chain comprising the amino acid sequence of SEQ ID NO: 20.
• R 2 comprises an antibody that is a Fab fragment, a full-length IgG, a Fab' fragment, a F(ab')2 fragment, an scFv, or an Fv.
• formulations described herein further comprise complexes in which nl is 0.
• the average value of nl of complexes of the composition is in the range of 0.5 to 5 (e.g., 0.5-5, 1-5, 1-4, 1-3, 3-5, 0.5-4, 0.5-3, 0.5-2, 0.5-1.5, 0.5-1, 0.7-1.5, 1-1.6, 1-1.5, 1-1.4, 1-1.3, 1-1.2, 1.1-1.5, 0.8-2, 0.8- 1.5, O.8-1.3, 0.8-1.2, 0.8-1.1, 0.9-3, 0.9-2, 0.9-1.8, 0.9-1.6, 0.9-1.5, 0.9-1.4, 0.9-1.3, or 0.9-1.2).
• formulations further comprise tris(hydroxymethyl)aminomethane and sucrose.
• formulations comprise tris(hydroxymethyl)aminomethane at a concentration of 25 mM and/or (e.g., and) sucrose at a concentration of 10 ⁇ IN%, optionally wherein the formulation has a pH of 7.5.
• formulations comprise complexes at a concentration of 30 mg/mL.
• complexes provided in the formulations described herein comprise a structure of formula (A): oligonucleotide
• a complex described herein comprises an anti-TfRl antibody (e.g., an anti-TfRl Fab) covalently linked via a lysine of the antibody to the 5’ end of an oligonucleotide (e.g., an oligonucleotide comprising a 5’-X-Y-Z-3’ configuration).
• the antibody comprises a sequence as set forth in Table 2.
• the antibody comprises a heavy chain complementarity determining region 1 (CDR-H1) comprising a sequence as set forth in SEQ ID NOs: 1, 7, or 12, a heavy chain complementarity determining region 2 (CDR- H2) comprising a sequence as set forth in SEQ ID NOs: 2, 8, or 13, a heavy chain complementarity determining region 3 (CDR-H3) comprising a sequence as set forth in SEQ ID NOs: 3, 9, or 14; and/or comprises a light chain complementarity determining region 1 (CDR-L1) comprising a sequence as set forth in SEQ ID NOs: 4, 10, or 15, a light chain complementarity determining region 2 (CDR-L2) comprising a sequence as set forth in SEQ ID NOs: 5, or 11, and a light chain complementarity determining region 3 (CDR-L3) comprising a sequence as set forth in SEQ ID NO: 6 or 16.
• CDR-H1 comprising a sequence as set forth in SEQ
• the antibody comprises a heavy chain variable region (VH) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 17 and/or comprises a light chain variable region (VL) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 18.
• VH heavy chain variable region
• VL light chain variable region
• 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.
• the antibody comprises a heavy chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 19 and/or comprises a light chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 20.
• the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and/or comprises a light chain comprising the amino acid sequence of SEQ ID NO: 20.
• the antibody is a Fab fragment, a full-length IgG, a Fab' fragment, a F(ab')2 fragment, an scFv, or an Fv.
• the antibody is a Fab fragment.
• formulations further comprise tris(hydroxymethyl)aminomethane and sucrose.
• formulations comprise tris(hydroxymethyl)aminomethane at a concentration of 25 mM and/or (e.g., and) sucrose at a concentration of 10 ⁇ IN%, optionally wherein the formulation has a pH of 7.5.
• formulations comprise complexes at a concentration of 30 mg/mL.
• sucrose serves at least in part as a lyoprotectant.
• the sucrose is from a plant, e.g., grass, fruit, or vegetable (e.g., root vegetable) source (e.g., beet (e.g., sugar beet, for example, Saccharum spp.)), sugarcane (e.g., Beta vulgaris), dates, sugar maple, sweet sorghum, apples, oranges, carrots, molasses, maple syrup, com sweeteners) or animal product (e.g., honey).
• a plant e.g., grass, fruit, or vegetable (e.g., root vegetable) source
• beet e.g., sugar beet, for example, Saccharum spp.
• sugarcane e.g., Beta vulgaris
• dates e.g., sugar maple, sweet sorghum, apples, oranges, carrots, molasses, maple syrup, com sweeteners
• honey e.g., honey
• the sucrose is from beet or sugarcane (e.g., beet sucrose, sugarcane sucrose).
• a lyoprotectant other than sucrose may be used, e.g., trehalose, mannitol, lactose, polyethylene glycol, or polyvinyl pyrrolidone.
• a collapse temperature modifier e.g., dextran, ficoll, or gelatin
• a formulation may be provided in a formulation.
• a product e.g., lyophilized formulation described herein
• a process comprising lyophilizing an aqueous solution of a formulation (e.g., aqueous form) described herein.
• a pharmaceutical composition is formulated to be compatible with its intended route of administration.
• routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, administration.
• the route of administration is intravenous or subcutaneous.
• complexes comprising an anti-TfRl antibody (e.g., a Fab) covalently linked to a molecular payload (e.g., a DMPK targeting oligonucleotide) as described herein are effective in treating a subject having a myotonic dystrophy, e.g., DM1.
• a molecular payload e.g., a DMPK targeting oligonucleotide
• complexes comprise a molecular payload that is an oligonucleotide, e.g., an oligonucleotide that facilitates reduced expression or activity of DMPK (e.g., reduced level of a mutant or wild-type DMPK RNA).
• a subject may be a human subject, a non-human primate subject, a rodent subject, or any suitable mammalian subject.
• a subject may have myotonic dystrophy.
• a subject has a DMPK allele, which may optionally contain a disease-associated repeat, e.g., a CTG trinucleotide repeat expansion.
• a subject may have a DMPK allele with an expanded disease- associated-repeat that comprises about 2-10 repeat units, about 2-50 repeat units, about 2-100 repeat units, about 50-1,000 repeat units, about 50-500 repeat units, about 50-250 repeat units, about 50-100 repeat units, about 500-10,000 repeat units, about 500-5,000 repeat units, about 500-2,500 repeat units, about 500-1,000 repeat units, or about 1,000-10,000 repeat units.
• a subject may have myotonic dystrophy, such as DM1.
• a subject is suffering from symptoms of DM1, e.g. muscle atrophy, muscle loss, excessive daytime sleepiness or cognitive delay.
• a subject is not suffering from symptoms of DM1.
• subjects have congenital myotonic dystrophy.
• a subject is ambulant. In some embodiments, a subject is non-ambulant.
• An aspect of the disclosure includes methods involving administering to a subject a formulation comprising an effective amount of complex(es) as described herein.
• an effective amount of a pharmaceutical composition that comprises complex(es) comprising an antibody (e.g., Fab) described herein covalently linked to an oligonucleotide (e.g., a DMPK targeting oligonucleotide) described herein can be administered to a subject in need of treatment.
• a pharmaceutical composition comprising complex(es) as described herein may be administered by a suitable route, which may include intravenous administration, e.g., as a bolus or by continuous infusion over a period of time.
• administration may be performed by intravenous, intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra-articular, intrasynovial, or intrathecal routes.
• a pharmaceutical composition may be in solid form, aqueous form, or a liquid form.
• an aqueous or liquid form may be nebulized or lyophilized.
• a lyophilized form may be reconstituted with an aqueous or liquid solution.
• methods of and/or uses for reducing the expression or activity of DMPK e.g., reducing the level of a mutant or wild-type DMPK RNA, or the activity of a DMPK gene product
• a cell e.g., a muscle cell
• the method comprises administering a lyophilized form (e.g. , lyophilized powder) of the formulation described herein, comprising reconstituting a lyophilized form of the formulation in an aqueous solution, and administering the aqueous solution of the formulation to a subject in need thereof.
• a lyophilized form of the formulation is shipped and/or stored in the lyophilized form, reconstituted at a location for administering the aqueous solution of the formulation (e.g., healthcare provider location), and administered in the reconstituted form (e.g., as an aqueous solution) by injection or intravenously, e.g., by infusion.
• a pharmaceutical composition is administered via sitespecific or local delivery techniques.
• these techniques include implantable depot sources of the complex, local delivery catheters, site specific carriers, direct injection, or direct application.
• a pharmaceutical composition that comprises a complex comprising a an anti-TfRl antibody (e.g., a fab) covalently linked to a molecular payload (e.g., a DMPK targeting oligonucleotide) is administered at an effective concentration that confers therapeutic effect on a subject.
• Effective amounts vary, as recognized by those skilled in the art, depending on the severity of the disease, unique characteristics of the subject being treated, e.g. age, physical conditions, health, or weight, the duration of the treatment, the nature of any concurrent therapies, the route of administration and related factors. These related factors are known to those in the art and may be addressed with no more than routine experimentation.
• an effective concentration is the maximum dose that is considered to be safe for the patient. In some embodiments, an effective concentration will be the lowest possible concentration that provides maximum efficacy.
• Empirical considerations e.g. the half-life of the complex(es) in a subject, generally will contribute to determination of the concentration of pharmaceutical composition that is used for treatment.
• the frequency of administration may be empirically determined and adjusted to maximize the efficacy of the treatment.
• the efficacy of treatment may be assessed using any suitable methods. In some embodiments, the efficacy of treatment may be assessed by evaluation or observation of symptoms associated with a myotonic dystrophy, e.g. muscle atrophy or muscle weakness, through measures of a subject’s self-reported outcomes, e.g.
• a pharmaceutical composition that comprises a complex comprising a muscle-targeting agent covalently linked to a molecular payload described herein is administered to a subject at an effective concentration sufficient to modulate activity or expression of a target gene by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% relative to a control, e.g. baseline level of gene expression prior to treatment.
• Example 1 In vivo tissue distribution of conjugates containing anti-TfRl Fab conjugated to a DMPK-targeting oligonucleotide in DM1 mouse model
• Conjugates comprising an anti-TfRl Fab conjugated to a DMPK-targeting oligonucleotide (ASO) were tested in a mouse model that expresses both human TfRl and a human DMPK mutant that harbors expanded CUG repeats.
• the anti-TfRl Fab used has the VH/VE sequences provided in Table 2.
• the Fab was covalently linked (through lysine conjugation) via a linker comprising a valine-citrulline sequence to a DMPK targeting oligonucleotide comprising a nucleobase sequence of SEQ ID NO: 21.
• the conjugate comprises a structure of formula (Id): (Id), wherein +N represents an LNA (2’ -4’ methylene bridge) ribonucleoside, dN represents a 2’-deoxyribonucleoside, oN represents a 2’-O-methoxyethyl (MOE) modified ribonucleoside, oC represents a 5-methyl-2’-MOE-cytidine, +C represents a 5-methyl-2’-4’-bicyclic-cytidine (2’-4’ methylene bridge), oU represents a 5-methyl-2’-MOE-uridine, * represents a phosphorothioate internucleoside linkage, and wherein the oligonucleotide comprises a nucleobase sequence of CAGCGCCCACCAGUCA (SEQ ID NO: 21), and in which R 2 comprises the anti-TfRl antibody provided in Table 2, and wherein in each conjugate nl is independently an integer of 1-3.
• FIGs. 1A, IB, 1C, and ID show the amount of ASO in the heart, diaphragm, gastrocnemius, or tibialis anterior, respectively, two weeks after the first injection.
• conjugates comprising an anti-TfRl antibody (e.g., an anti-TfRl Fab having the VH and VL sequences provided in Table 2) are capable of delivering an oligonucleotide (e.g., a DMPK- targeting ASO) to various muscle tissues following intravenous administration.
• an anti-TfRl antibody e.g., an anti-TfRl Fab having the VH and VL sequences provided in Table 2
• an oligonucleotide e.g., a DMPK- targeting ASO
• Example 2 Sustained knockdown of toxic human DMPK in hTfRl/DMSXL homozygous mice at 4 weeks after repeat dosing of anti-TfRl Fab-ASO conjugates
• Conjugates (labeled in this Example as “Anti-TfRl Fab-ASO conjugate”) as described in Example 1, containing an anti-TfRl Fab covalently linked to a DMPK-targeting oligonucleotide (ASO) were tested in a mouse model that expresses both human TfRl and two copies of a mutant human DMPK transgene that harbors expanded CUG repeats (hTfRl/DMSXL mice). Mice were administered either vehicle control (PBS) or 10 mg/kg ASO-equivalent dose of anti-TfRl Fab-ASO conjugate at days 0 and 7.
• PBS vehicle control
• ASO-equivalent dose of anti-TfRl Fab-ASO conjugate at days 0 and 7.
• FIG. 2A shows that anti-TfRl Fab-ASO conjugate knocked down DMPK expression in heart by 49% relative to PBS-treated mice.
• FIG. 2B shows that anti-TfRl Fab- ASO conjugate knocked down DMPK expression in diaphragm by 40% relative to PBS-treated mice.
• FIG. 2C shows that anti-TfRl Fab-ASO conjugate knocked down DMPK expression in tibialis anterior by 49% relative to PBS-treated mice.
• FIG. 2D shows that anti-TfRl Fab-ASO conjugate knocked down DMPK expression in gastrocnemius by 44% relative to PBS-treated mice.
• FIGs. 3A and 3B show that anti-TfRl Fab-ASO conjugate reduced DMPK foci within nuclei of myofibers.
• FIG. 3 A shows reduced DMPK foci by in situ hybridization
• Conjugates (labeled in this Example as “Anti-TfRl Fab-ASO conjugate”) as described in Example 1, containing an anti-TfRl Fab covalently linked to a DMPK-targeting oligonucleotide (ASO) were tested in a mouse model (“hTfRl/DMSXL”) that expresses both human TfRl and two copies of a mutant human DMPK transgene that harbors expanded CUG repeats. These mice are known to display splicing defects that are consistent with those observed in patients afflicted with DM1 (Huguet, et al. (2012) PLOS Genetics 8(11): el003043).
• mice were administered either vehicle control (“hTfRl/DMSXL - PBS”) or 10 mg/kg ASO-equivalent dose of anti-TfRl Fab-ASO conjugate (“hTfRl/DMSXL - Conjugate”) on days 0 and 7.
• Mice expressing only the human TfRl but not the mutant human DMPK transgene (hTfRl mice) and treated with PBS (“hTfRl - PBS”) were used as another control to define the extent of the splicing phenotype in hTfRl/DMSXL mice and assess the magnitude of the effect of the conjugate on splicing.
• RT-qPCR Reverse transcription-quantitative polymerase chain reaction
• Exon inclusion was calculated as normalized percent spliced in (PSI) for each splicing RNA marker, and composite splicing indices were calculated using the normalized PSI values from splicing markers in heart (FIG. 4), diaphragm (FIG. 5), tibialis anterior (FIG. 6), and gastrocnemius (FIG. 7).
• Composite splicing indices were calculated as previously described (Tanner MK, et al. (2021) Nucleic Acids Res. 49:2240-2254), and are shown as mean +/- standard deviation. [000175] FIG.
• Bini El l, Dtna E12, Insr El l, and Mbnl2 E5 were not included because their normalized PSI values in heart tissue were not changed in hTfRl/DMSXL mice relative to hTfRl mice under the experimental conditions tested.
• FIG. 5 shows that anti-TfRl Fab-ASO conjugate corrected splicing in diaphragm tissue of hTfRl/DMSXL mice, as demonstrated by composite splicing index data.
• the normalized PSI values used to generate the composite splicing index data showed correction of Bini El l, Insr El l, Ldb3 El l and Nfix E7 splicing in diaphragm tissue of hTfRl/DMSXL mice by treatment with anti-TfRl Fab-ASO conjugate.
• FIG. 6 shows that anti-TfRl Fab-ASO conjugate corrected splicing in tibialis anterior tissue of hTfRl/DMSXL mice, as demonstrated by composite splicing index data.
• the normalized PSI values used to generate the composite splicing index data showed correction of Bini El l, Ldb3 El l, and Nfix E7 splicing in tibialis anterior tissue of hTfRl/DMSXL mice by treatment with anti-TfRl Fab-ASO conjugate, but did not show correction of Mbnl2 E6 splicing.
• FIG. 7 shows that anti-TfRl Fab-ASO conjugate corrected splicing in gastrocnemius tissue of hTfRl/DMSXL mice, as demonstrated by composite splicing index data.
• the normalized PSI values used to generate the composite splicing index data showed correction of Mbnl2 E6, Nfix E7, and Ttn E313 splicing in gastrocnemius tissue of hTfRl/DMSXL mice by treatment with anti-TfRl Fab-ASO conjugate.
• Bini El l, Dtna E12, Insr El l, Ldb3 El l, and Mbnl2 E5 were not included because their normalized PSI values in gastrocnemius tissue were not changed in hTfRl/DMSXL mice relative to hTfRl mice under the experimental conditions tested.
• Example 4 DMPK knockdown in non-human primate and DM1 patient myotubes
• Conjugates (labeled in this Example as “Anti-TfRl Fab-ASO conjugate”) as described in Example 1, containing an anti-TfRl Fab covalently linked to a DMPK-targeting oligonucleotide (ASO) were tested in human DM1 patient myotubes (32F cells) and in non- human primate (NHP) myotubes.
• the DM1 patient myotubes used express both a mutant DMPK mRNA containing 380 CUG repeats and a wild-type DMPK mRNA.
• the NHP myotubes used express only wild-type DMPK.
• DM1 patient cells or NHP cells were seeded at a density of 50,000 cells per well in 96 well plates in growth medium and were allowed to recover overnight. The following day, the growth medium was changed to a low-serum differentiation medium and the cells were treated with conjugates at a concentration equivalent to 125 nM, 250 nM, or 500 nM ASO. The cells were incubated for ten days, then cDNA was synthesized using the Cells-to-Ct kit with crude cell lysates as the source of total RNA.
• the anti-TfRl Fab-ASO conjugates achieved knockdown of DMPK expression in both normal NHP myotubes and DM1 patient myotubes, with greater knockdown of DMPK expression in DM1 patient cells (expressing both DMPK mRNA containing 380 CUG repeats and wild-type DMPK mRNA) compared to NHP cells (expressing only wild-type DMPK mRNA) when treated at physiologically relevant concentrations (FIG. 8).
• the conjugates achieved approximately 40% DMPK knockdown relative to vehicle-only control in NHP myotubes, and approximately 65% DMPK knockdown in DM1 patient myotubes.
• the conjugates achieved approximately 45% DMPK knockdown relative to vehicle-only control in NHP myotubes, and approximately 80% DMPK knockdown in DM1 patient myotubes.
• the conjugates achieved approximately 60% DMPK knockdown relative to vehicle-only control in NHP myotubes, and approximately 90% DMPK knockdown in DM1 patient myotubes.
• conjugates containing anti-TfRl Fab covalently linked to a DMPK-targeting oligonucleotide can achieve greater knockdown of DMPK 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.
• Example 5 Comparison of Thermal Stability of Different Exemplary Formulations
• Formulations comprising conjugates as described in Example 1, containing an anti-TfRl Fab covalently linked to a DMPK-targeting oligonucleotide (ASO) were prepared.
• the anti-TfRl Fab-oligonucleotide conjugates starting material (“SM”) was at a concentration of 33 mg/mL in a buffer containing 100 mM sodium phosphate and 100 mM sodium chloride at pH 7.4.
• SM mM sodium phosphate
• the anti-TfRl Fab-oligonucleotide conjugates were formulated in different buffers at a concentration of approximately 40 mg/mL.
• the buffer conditions in each of the four formulations were as follows:
• Formulation buffer 1 25 mM succinate, 10% (w/v) sucrose, pH 5.2.
• Formulation buffer 2 25 mM succinate, 10% (w/v) sucrose, pH 6.0.
• 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.
• formulation drug substance (formulation with conjugates described herein) was buffer exchanged into formulation buffers 1-4 as provided above using Vivaspin® 6 centrifugal membrane filters (30kDa molecular weight cutoff).
• the anti-TfRl Fab-oligonucleotide conjugates were concentrated to approximately 40 mg/ml. Aliquots of approximately 150 pL from each of the concentrated samples were used to study thermal stability of the conjugates in each formulation. The samples were incubated for 1 week (“T 1”), 2 weeks (“T2”), 4 weeks (“T4”), or 8 weeks (“T8”) at -20 °C, 2-8 °C, 25 °C, or 40 °C in glass vials.
• test samples were first diluted 1:4, then 20pL of each test sample or standard was thoroughly mixed with 200pL of WR and incubated at 60°C for 10 minutes. The resulting solution was added to the first row of a 96-well plate and serially diluted. A 6-point standard curve was prepared.
• HMW high molecular weight
• LMW low molecular weight
• Results of the non-reduced CE-SDS analysis are shown in Table 5 below.
• the starting material showed a size of 62.4 kDa with 100.0% purity.
• Samples were incubated at 70 °C for 10 minutes, then cooled on ice for 5 minutes. 32 pL of water was added to each sample well, and the plate was centrifuged at 1200 g for 2 minutes prior to analysis. Samples were analyzed on an HT Protein Express LabChip (Perkin Elmer 760499), using a LabChip GXII Touch HT (Perkin Elmer), using measurement method HT High Sensitivity Antibody Analysis 200.
• Results of the reduced CE-SDS analysis are shown in Table 6 below. Values for the starting material (SM) are shown for comparison in the top row.
• ⁇ Sizes are shown in kDa.
• Example 6 Comparison of Freeze/Thaw Stability of Different Exemplary Formulations
• Formulations comprising conjugates as described in Example 1, containing an anti-TfRl Fab covalently linked to a DMPK-targeting oligonucleotide (ASO) were prepared.
• the anti-TfRl Fab-oligonucleotide conjugates starting material (“SM”) was at a concentration of 33 mg/mE in a buffer containing 100 mM sodium phosphate and 100 mM sodium chloride at pH 7.4.
• SM DMPK-targeting oligonucleotide
• the anti-TfRl Fab-oligonucleotide conjugates were formulated in different buffers at a concentration of approximately 40 mg/mL.
• the buffer conditions in each of the four formulations were as follows:
• Formulation buffer 1 25 mM succinate, 10% (w/v) sucrose, pH 5.2.
• Formulation buffer 2 25 mM succinate, 10% (w/v) sucrose, pH 6.0.
• 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.
• SEC Size exclusion chromatography
• Conjugates (labeled in this Example as “Anti-TfRl Fab-ASO conjugate”) as described in Example 1, containing an anti-TfRl Fab covalently linked to a DMPK-targeting oligonucleotide (ASO) were prepared and lyophilized with tris(hydroxymethyl)aminomethane (25mM), sucrose (10%(w/v)), at a pH of 7.5 and tested for stability. Prior to reconstitution all samples at all timepoints and temperatures were visually identified as a white cake and uniform in shape. Additionally, the samples reconstituted in less than 1 minute, and became clear and colorless with fewer than 10 particles. All samples were considered stable by visual analysis pre- and post-reconstitution up to 40° for at least 8 weeks.
• X-ray diffraction analysis was performed to evaluate the crystallographic structure of the lyophilized composition. Approximately 20 mg of lyophilized anti-TfRl Fab- ASO conjugates was placed in a plastic sample holder and flattened with a glass slide to ensure a flat surface. When performing this analysis, a Proto AXRD® Benchtop Power X-ray Diffractometer was used with a fine focus Cu-anode metal/ceramic construction 1500-Watt x- ray tube.
• the lyophilized anti-TfRl Fab-ASO conjugates were first reconstituted with water and then diluted using formulation buffer (25mM tris(hydroxymethyl)aminomethane, 10%(w/v) sucrose, pH 7.5) to a concentration of 0.2 mg/ml. Absorbance was measured at 260 nm and 280 nm using a DeNovix DS 11+ spectrophotometer. Readings were performed in triplicate and averaged. These results were used to calculate the concentration (Fab (mg/mL and pM) and oligonucleotide (pM)), percent recovery, and Drug Antibody Ratio (DAR), at multiple timepoints.
• formulation buffer 25mM tris(hydroxymethyl)aminomethane, 10%(w/v) sucrose, pH 7.5
• DAR Drug Antibody Ratio
• bovine serum albumin (BSA) was used as a positive control
• PBS was used as a blank and negative control for BSA
• formulation buffer was used as a blank and negative control for the conjugates samples.
• the anti-TfRl Fab-ASO conjugates were analyzed using size-exclusion chromatograph (SEC). All samples were diluted to a concentration of 1.0 mg/ml using formulation buffer (25mM tris(hydroxymethyl)aminomethane, 10%(w/v) sucrose, pH 7.5) prior to analysis. For the mobile phase of the chromatographic conditions an isocratic elution was used containing potassium phosphate (200 mM), potassium chloride (200 mM), 5 % (v/v) isopropanol (IPA) at a pH of 6.8, at a flow rate of 0.250 ml/min using a 20 minute run time. The wavelength used for measurements was 280 nm.
• the high-performance liquid chromatography system used was the Dionex Ultimate 3000 UPLC System, with a Waters BEH SEC column (200 A, 300 x 4.6 mm, 1.7 pm, P/N 186005226).
• CE- SDS capillary electrophoresis sodium dodecyl sulfate
• samples were diluted to 0.5 mg/ml in PBS. 5.0 pl of the diluted sample was added to 7.0 pl of sample denaturing solution (24.5 pL of IM dithiothreitol (DTT) added to 700 pL Protein Express Sample Buffer). The samples were then incubated at 70°C for 10 minutes. After incubation, 32 pl of water was added and the samples were centrifuged at 1200 g for 2 minutes. The samples were then analyzed using HT High Sensitivity Antibody Analysis 200, using HT Protein Express LabChip (Perkin Elmer, 760499) with Protein Express Reagent Kit (Perkin Elmer, CLS960008). Analysis was performed on a LabChip GXII Touch HT (Perkin Elmer).
• a formulation comprising complexes that comprise an oligonucleotide covalently linked to an anti-transferrin receptor 1 (TfRl) antibody, wherein the anti-TfRl antibody comprises: a heavy chain complementarity determining region 1 (CDR-H1) comprising a sequence as set forth in SEQ ID NOs: 1, 7, or 12, a heavy chain complementarity determining region 2 (CDR-H2) comprising a sequence as set forth in SEQ ID NOs: 2, 8, or 13, a heavy chain complementarity determining region 3 (CDR-H3) comprising a sequence as set forth in SEQ ID NOs: 3, 9, or 14, a light chain complementarity determining region 1 (CDR-L1) comprising a sequence as set forth in SEQ ID NOs: 4, 10, or 15, a light chain complementarity determining region 2 (CDR-L2) comprising a sequence as set forth in SEQ ID NOs: 5 or 11, and a light chain complementarity determining region 3 (CDR-L3) comprising a sequence
• a formulation comprising complexes comprising a structure of formula (I): [R ⁇ ni-R 2 , wherein each R 1 independently comprises a group of the formula (la):
• R 2 comprises an antibody
• R 3 comprises an oligonucleotide comprising a 5’-X-Y-Z-3’ configuration, wherein X and Z are flanking regions comprising one or more modified nucleosides and Y is a gap region comprising one or more 2’ -deoxyribonucleosides; wherein R 1 is covalently linked to R 2 at attachment point A; and wherein nl is an integer representing the number of instances of R 1 , wherein each instance of R 1 is covalently linked to a different amino acid residue of the antibody, optionally wherein each different amino acid residue is a lysine; wherein the complexes are formulated with tris(hydroxymethyl)aminomethane and sucrose, optionally wherein the antibody is an anti-TfRl antibody.
• a heavy chain complementarity determining region 1 comprising a sequence as set forth in SEQ ID NOs: 1, 7, or 12
• a heavy chain complementarity determining region 2 comprising a sequence as set forth in SEQ ID NOs: 2, 8, or 13
• a heavy chain complementarity determining region 3 comprising a sequence as set forth in SEQ ID NOs: 3, 9, or 14
• a light chain complementarity determining region 1 comprising a sequence as set forth in SEQ ID NOs: 4, 10, or 15, a light chain complementarity determining region 2 (CDR-L2) comprising a sequence as set forth in SEQ ID NOs: 5 or 11, and a light chain complementarity determining region 3 (CDR-L3) comprising a sequence as set forth in SEQ ID NOs: 6 or 16.
• any one of embodiments 1-12 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, optionally wherein 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.
• VH heavy chain variable region
• VL light chain variable region
• any one of embodiments 1-13 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, optionally wherein 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.
• oligonucleotide comprises a nucleotide sequence having a region of complementarity of at least 8 consecutive nucleotides in length to SEQ ID NO: 22.
• oligonucleotide comprises at least 8 consecutive nucleotides of a nucleotide sequence as set forth in SEQ ID NO: 21, optionally wherein the oligonucleotide comprises the nucleotide sequence of SEQ ID NO: 21.
• each R 1 comprises a group of the formula (lb): wherein +N represents an LNA (2’-4’ methylene bridge) ribonucleoside, dN represents a 2’- deoxyribonucleoside, oN represents a 2’-O-methoxyethyl (MOE) modified ribonucleoside, oC represents a 5-methyl-2’-MOE-cytidine, +C represents a 5-methyl-2’-4’-bicyclic-cytidine (2’- 4’ methylene bridge), oU represents a 5-methyl-2’-MOE-uridine, * represents a phosphorothioate internucleoside linkage, such that the oligonucleotide comprises a nucleobase sequence of CAGCGCCCACCAGUCA (SEQ ID NO: 21). 19. The formulation of any one of embodiments 2-17, wherein each R 1 comprises a group of the formula (Ic):
• a method of reducing DMPK expression in a subject comprising administering to the subject an effective amount of the formulation of any one of embodiments 1-22.
• 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 1-22.
• a complex comprising a structure of formula (I): [R ⁇ ni-R 2 , wherein each R 1 comprises a group of the formula (la):
• R 3 comprises an oligonucleotide comprising a structure of +C*+A*oG*oC*dG*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 a 5- methyl-2’-MOE-cytidine, +C represents a 5-methyl-2’-4’-bicyclic-cytidine (2 ’-4’ methylene bridge), oU represents a 5-methyl-2’-MOE-uridine, and * represents a phosphorothioate intemucleoside linkage; wherein R 2 comprises a Fab, and wherein the Fab comprises a CDR-H
• a complex comprising a structure of formula (I): [R ⁇ ni-R 2 , wherein each R 1 comprises a group of the formula (lb): wherein +N represents an LNA (2’-4’ methylene bridge) ribonucleoside, dN represents a 2’ -deoxyribonucleoside, oN represents a 2’-O-methoxyethyl (MOE) modified ribonucleoside, oC represents a 5-methyl-2’-MOE-cytidine, +C represents a 5-methyl-2’-4’- bicyclic-cytidine (2 ’-4’ methylene bridge), oU represents a 5-methyl-2’-MOE-uridine, * represents a phosphorothioate internucleoside linkage, such that the oligonucleotide comprises a nucleobase sequence of CAGCGCCCACCAGUCA (SEQ ID NO: 21); wherein R 2 comprises a Fab, and wherein the Fab comprises
• a complex comprising a structure of formula (I): [R ⁇ ni-R 2 , wherein R 1 comprises a group of the formula (Ic):
• R 2 comprises a Fab comprising a CDR-H1, a CDR-H2, a CDR-H3, a CDR-L1, a CDR-L2, and a CDR-L3 selected from Table 2, optionally wherein R 2 comprises 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, further optionally wherein R 2 comprises 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 is covalently linked to R 2 at attachment point A; wherein nl is an integer representing the number of instances of R 1 , wherein each instance of R 1 is covalently linked to a different amino acid residue of the Fab, optionally wherein each different amino acid residue is a lysine.
• +N represents an LNA (2’-4’ methylene bridge) ribonucleoside
• dN represents a 2’ -deoxyribonucleoside
• oN represents a 2’-O-methoxyethyl (MOE) modified ribonucleoside
• oC represents a 5-methyl-2’-MOE-cytidine
• +C represents a 5-methyl-2’-4’- bicyclic-cytidine (2 ’-4’ methylene bridge)
• oU represents a 5-methyl-2’-MOE-uridine
• * represents a phosphorothioate internucleoside linkage
• R 2 comprises a Fab
• the Fab comprises a CDR-H1, a CDR-H2, a CDR-H3, a CDR-E1, a CDR-E2, and a CDR-E3 selected from Table 2, optionally wherein the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 17 and
• a formulation comprising a plurality of complexes of any one of embodiments 28-31, 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 is at a pH of 6.5 to 8.5, optionally wherein the plurality of complexes are at a concentration of 10 mg/mL to 50 mg/mL.
• a formulation comprising a plurality of complexes of any one of embodiments 28-31, and tris(hydroxymethyl)aminomethane at a concentration of 5 to 50 mM, and sucrose at a concentration of 5 ⁇ IN% to 15 ⁇ IN%, wherein the formulation is an aqueous solution and is at a pH of 6.5 to 8.5, optionally wherein the plurality of complexes are at a concentration of
• a formulation comprising a plurality of complexes of any one of embodiments 28-31, and tris(hydroxymethyl)aminomethane at a concentration of 25 mM, and sucrose at a concentration of 10 ⁇ IN%, wherein the formulation is an aqueous solution and is at a pH of 7.5, optionally wherein the plurality of complexes are at a concentration of 10 mg/mL to
• the lyophilized cake of embodiment 42 comprising 4.02xl0 -2 mg-1.21 mg tris(hydroxymethyl)aminomethane per g of cake; 989 mg-999 mg sucrose per g of cake; and/or 0.666 mg-9.90 mg complexes per g of cake; optionally wherein the lyophilized cake comprises:
• the lyophilized cake of embodiment 42 or embodiment 43 wherein when about 10 g of the lyophilized cake are 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 ⁇ IN% to 15 ⁇ IN%, and complexes at a concentration of 10 mg/mL to
• the tris(hydroxymethyl)aminomethane is at a concentration of 25 mM and/or the sucrose is at a concentration of 10 ⁇ IN%.
• sequences presented in the sequence listing may be referred to in describing the structure of an oligonucleotide or other nucleic acid.
• the actual oligonucleotide or other nucleic acid may have one or more alternative nucleotides (e.g., an RNA counterpart of a DNA nucleotide or a DNA counterpart of an RNA nucleotide) and/or (e.g., and) one or more modified nucleotides and/or (e.g., and) one or more modified intemucleoside linkages and/or (e.g., and) one or more other modification compared with the specified sequence while retaining essentially same or similar complementary properties as the specified sequence.

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