WO2023283629A1 - Muscle targeting complexes and formulations thereof for treating facioscapulohumeral muscular dystrophy - Google Patents

Muscle targeting complexes and formulations thereof for treating facioscapulohumeral muscular dystrophy Download PDF

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WO2023283629A1
WO2023283629A1 PCT/US2022/073546 US2022073546W WO2023283629A1 WO 2023283629 A1 WO2023283629 A1 WO 2023283629A1 US 2022073546 W US2022073546 W US 2022073546W WO 2023283629 A1 WO2023283629 A1 WO 2023283629A1
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seq
amino acid
cdr
sequence
antibody
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PCT/US2022/073546
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French (fr)
Inventor
Timothy Weeden
Scott Hilderbrand
Sean SPRING
Peiyi SHEN
Cody A. DESJARDINS
Romesh R. SUBRAMANIAN
Mohammed T. QATANANI
Brendan QUINN
John NAJIM
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Dyne Therapeutics, Inc.
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Publication of WO2023283629A1 publication Critical patent/WO2023283629A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2881—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD71
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the present application relates to targeting complexes for delivering molecular payloads (e.g., oligonucleotides) to cells, formulations comprising such complexes, and uses thereof, particularly uses relating to treatment of disease.
  • molecular payloads e.g., oligonucleotides
  • MDLs Muscular dystrophies
  • FSHD Facioscapulohumeral muscular dystrophy
  • DUX4 double homeobox 4
  • the DUX4 gene which encodes the DUX4 protein, is located in the D4Z4 repeat region on chromosome 4 and is typically expressed only in fetal development, after which it is repressed by hypermethylation of the D4Z4 repeats which surround and compact the DUX4 gene.
  • Two types of FSHD, Type 1 and Type 2 have been described.
  • Type 1 which accounts for about 95% of cases, is associated with deletions of D4Z4 repeats on chromosome 4.
  • FSHD1 FSHD1
  • FSHD1 FSHD1
  • complexes and formulations comprising such complexes.
  • complexes provided herein are formulated with histidine (e.g ., L-histidine) and sucrose.
  • complexes provided herein are formulated as aqueous or lyophilized (e.g., lyophilized powder) forms.
  • complexes provided herein are formulated as frozen forms.
  • complexes provided herein comprise a phosphorodiamidate morpholino oligomer (PMO) covalently linked to an antibody.
  • PMO phosphorodiamidate morpholino oligomer
  • complexes provided herein comprise a muscle-targeting complex comprising a PMO covalently linked to an anti-transferrin receptor 1 (TfRl) antibody.
  • the anti-TfRl antibody has undergone pyroglutamate formation resulting from a post-translational modification.
  • a complex comprises a muscle-targeting complex comprising a PMO covalently linked to the anti-transferrin receptor 1 (TfRl) antibody, e.g., having a sequence as set forth in Table 2.
  • the PMO targets a DUX4 RNA (e.g., in the 3’UTR region of a DUX4 RNA).
  • DUX4 expression e.g., protein and/or RNA
  • activity in a cell e.g., a muscle cell.
  • complexes comprising a structure of formula (I): [ R 1 J n i -R 2 , wherein each R 1 comprises a group of the formula (la):
  • R 3 comprises a phosphorodiamidate morpholino oligomer (PMO) comprising a region of complementarity of at least 8 consecutive nucleotides to the nucleotide sequence as set forth in SEQ ID NO: 22; wherein R 2 comprises a Fab, and wherein the Fab comprises a CDR-H1, a CDR-H2, a CDR-H3, a CDR-F1, a CDR-F2, and a CDR-F3 selected from Table 2.
  • PMO phosphorodiamidate morpholino oligomer
  • R 1 is covalently linked to R 2 at attachment point A; and wherein nl is an integer of one or greater 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.
  • the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 17 and a VF comprising the amino acid sequence of SEQ ID NO: 18.
  • the Fab comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and a light chain comprising the amino acid sequence of SEQ ID NO: 20,
  • each different amino acid residue is a lysine.
  • the region of complementarity is at least 16 nucleotides in length.
  • the PMO comprises at least 8 consecutive nucleotides of the nucleotide sequence as set forth in SEQ ID NO: 21.
  • the PMO comprises at least 16 consecutive nucleotides of the nucleotide sequence as set forth in SEQ ID NO: 21.
  • compositions comprising complexes that comprise a phosphorodiamidate morpholino oligomer (PMO) covalently linked to an anti transferrin receptor 1 (TfRl) antibody, wherein the PMO comprises a nucleotide sequence having a region of complementarity of at least 8 consecutive nucleotides to the sequence set forth as SEQ ID NO: 22, wherein 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,
  • formulations comprising complexes comprising a structure of formula [R'j n i -R 2 , wherein each R 1 independently comprises a group of the formula (la):
  • R 2 comprises an antibody
  • R 3 comprises a phosphorodiamidate morpholino oligomer (PMO), wherein R 1 is covalently linked to R 2 at attachment point A; wherein the PMO comprises a nucleotide sequence having a region of complementarity of at least 8 consecutive nucleotides to the sequence set forth as SEQ ID NO: 22, wherein nl is an integer of one or greater 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; and wherein the complexes are formulated with histidine and sucrose.
  • PMO phosphorodiamidate morpholino oligomer
  • each different amino acid residue is a lysine.
  • the antibody is an anti-TfRl antibody.
  • the average value of nl of complexes in the formulation is in the range of 0.5 to 5.
  • 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
  • the formulation is in a lyophilized form, an aqueous solution, or a frozen solid form.
  • the formulation is in an aqueous solution and the histidine is present in the aqueous solution at a concentration in the range of 10 mM to 50 mM.
  • the formulation is in an aqueous solution and the sucrose is present in the aqueous solution at a concentration in the range of 5 % to 15 % weight per volume (w/v%).
  • the formulation is in an aqueous solution and the aqueous solution has a pH in the range of 5.0 to 7.0.
  • the formulation is in an aqueous solution and the histidine is present in the aqueous solution at a concentration of 25 mM, and/or the sucrose is present in the aqueous solution at a concentration of 10 w/v%, and/or the aqueous solution is at a pH of 6.0.
  • 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.
  • 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 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.
  • the antibody 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.
  • the PMO comprises a nucleotide sequence that is 15-30 nucleotides in length.
  • the PMO comprises a nucleotide sequence having a region of complementarity of at least 20 consecutive nucleotides to the sequence set forth as SEQ ID NO: 22.
  • the PMO comprises at least 8 consecutive nucleotides of a nucleotide sequence as set forth in SEQ ID NO: 21.
  • the PMO comprises the nucleotide sequence as set forth in SEQ ID NO: 21.
  • each R 1 comprises a group of the formula (lb):
  • each R 1 comprises a group of the formula (Ic):
  • the complexes are present in the formulation at a concentration in the range between 10 mg/mL to 50 mg/mL.
  • aspects of the present disclosure provide methods of reducing DUX4 expression or activity in a cell or subject, the method comprising contacting the cell with or administering to the subject the formulation described herein with an effective amount of the complex for promoting internalization of the molecular payload in the cell.
  • the cell is a muscle cell.
  • the complex reduces DUX4 protein level.
  • FSHD facioscapulohumeral muscular dystrophy
  • R 2 comprises a Fab
  • the Fab comprises a CDR-H1, a CDR-H2, a CDR-H3, a CDR-L1, a CDR-L2, and a CDR-L3 selected from Table 2, wherein -pN indicates a base position of a phosphorodiamidate morpholino oligomer, wherein -p reflects a phosphorodiamidate linkage, and wherein N corresponds to a nucleobase of adenine (A), cytosine (C), guanine (G), or thymine (T), such that the oligonucleotide PMO has a nucleobase sequence of GGGC ATTTT AAT ATATCTCTGAACT (SEQ ID NO: 21); wherein R 1 is covalently linked to R 2 at attachment point A; and wherein nl is an integer of one or greater representing the number of instances of R 1 , wherein each instance of R 1 is covalently linked
  • the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 17 and a VL comprising the amino acid sequence of SEQ ID NO: 18.
  • the Fab comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and a light chain comprising the amino acid sequence of SEQ ID NO: 20.
  • each different amino acid residue is a lysine.
  • Some aspects of the present disclosure provide complexes comprising a structure of formula (I): [R'j ni -R 2 , wherein each 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, wherein R 1 is covalently linked to R 2 at attachment point A; wherein nl is an integer of one or greater 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.
  • 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.
  • 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.
  • each different amino acid residue is a lysine.
  • R 2 comprises a Fab
  • the Fab comprises a CDR-H1, a CDR-H2, a CDR-H3, a CDR-L1, a CDR-L2, and a CDR-L3 selected from Table 2, wherein -pN indicates a base position of a phosphorodiamidate morpholino oligomer, wherein -p reflects a phosphorodiamidate linkage, and wherein N corresponds to a nucleobase of adenine (A), cytosine (C), guanine (G), or thymine (T), such that the oligonucleotide PMO has a nucleobase sequence of GGGC ATTTT AAT ATATCTCTGAACT (SEQ ID NO: 21); wherein R 1 is covalently linked to R 2 at attachment point A; and wherein nl is an integer of one or greater representing the number of instances of R 1 , wherein each instance of R 1 is covalently linked
  • the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 17 and a VL comprising the amino acid sequence of SEQ ID NO: 18.
  • the Fab comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and a light chain comprising the amino acid sequence of SEQ ID NO: 20.
  • each different amino acid residue is a lysine.
  • FIG. 1 shows data illustrating that a composition comprising conjugates comprising the anti-TfRl Fab having the VH/VL sequences shown in Table 2, in which the Fab is covalently linked (through lysine conjugation) via a linker comprising a valine-citrulline sequence to a DUX4-targeting oligonucleotide (SEQ ID NO: 21), inhibited DUX4 expression in C6 (AB1080) immortalized FSHD1 cells, as indicated by decreased mRNA expression of MDB3L2, TRIM43, and ZSCAN4.
  • the conjugates showed superior activity compared with unconjugated DUX4- targeting oligonucleotide in inhibiting DUX4 expression.
  • the present disclosure provides complexes and formulations comprising such complexes.
  • the complexes are formulated with histidine (e.g., L-histidine) and sucrose.
  • the complexes are formulated as aqueous or lyophilized (e.g., lyophilized powder) forms.
  • a complex comprises a phosphorodiamidate morpholino oligomer (PMO) covalently linked to an antibody.
  • a complex comprises a muscle-targeting complex comprising a PMO covalently linked to an anti-transferrin receptor 1 (TfRl) antibody.
  • TfRl anti-transferrin receptor 1
  • a complex comprises a muscle-targeting complex comprising a PMO covalently linked to the anti transferrin receptor 1 (TfRl) antibody shown in Table 2.
  • the PMO targets a DUX4 RNA (e.g., in the 3’UTR region of a DUX4 RNA (e.g., as set forth in NM_001293798.2)).
  • FSHD facioscapulohumeral muscular dystrophy
  • 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 (D), epsilon (e), gamma (g) or mu (m) heavy chain.
  • the heavy chain of an antibody described herein can comprise a human alpha (a), delta (D), epsilon (e), gamma (g) or mu (m) 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 (g) heavy chain constant region, such as any known in the art.
  • 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 ah, (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, R, 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 Rabat 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., Rabat, 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,
  • 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.
  • Rabat Rabat 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 Rabat CDRs.
  • Sub-portions of CDRs may be designated as LI, L2 and L3 or HI, 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 Rabat CDRs.
  • Other boundaries defining CDRs overlapping with the Rabat 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 Rabat 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.
  • DUX4 refers to a gene that encodes double homeobox 4, a protein which is generally expressed during fetal development and in the testes of adult males.
  • DUX4 may be a human (Gene ID: 100288687), non human primate (e.g., Gene ID: 750891, Gene ID: 100405864), or rodent gene (e.g., Gene ID: 306226).
  • expression of the DUX4 gene outside of fetal development and the testes is associated with facioscapulohumeral muscular dystrophy.
  • Facioscapulohumeral muscular dystrophy As used herein, the term “facioscapulohumeral muscular dystrophy (FSHD)” refers to a genetic disease caused by mutations in the DUX4 gene or SMCHD1 gene that is characterized by muscle mass loss and muscle atrophy, primarily in the muscles of the face, shoulder blades, and upper arms. Two types of the disease, Type 1 and Type 2, have been described. Type 1 is associated with deletions in D4Z4 repeat regions on chromosome 4 allelic variant 4qA which contains the DUX4 gene. Type 2 is associated with mutations in the SMCHD1 gene.
  • Type 1 and Type 2 FSHD are characterized by aberrant production of the DUX4 protein after fetal development outside of the testes. Facioscapulohumeral dystrophy, the genetic basis for the disease, and related symptoms are described in the art (see, e.g., Campbell, A.E., et al.,
  • FSHD Type 1 is associated with Online Mendelian Inheritance in Man (OMIM) Entry # 158900.
  • FSHD Type 2 is associated with OMIM Entry # 158901.
  • 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 e.g., and VL sequence
  • 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.
  • Rabat numbering The terms "Rabat numbering", “Rabat definitions and “Rabat 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 (Rabat et al. (1971) Ann. NY Acad, Sci. 190:382-391 and,
  • 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.
  • Morpholinos As used herein, the term “morpholino”, also referred to as a “phosphorodiamidate morpholino oligomer”, refers to a molecular structure that contains nucleobases attached to a backbone of methylenemorpholine rings linked through a phosphorodiamidate group.
  • the oligonucleotide may be a morpholino- based compounds. Morpholino-based oligomeric compounds are described in Dwaine A. Braasch and David R. Corey, Biochemistry, 2002, 41(14), 4503-4510); Genesis, volume 30, issue 3, 2001; Heasman, J., Dev.
  • the morpholino-based oligomeric compound is a phosphorodiamidate morpholino oligomer (PMO) (e.g., as described in Iverson, Curr. Opin. Mol. Ther., 3:235-238, 2001; and Wang et al., J.
  • PMO phosphorodiamidate morpholino oligomer
  • 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'-0-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 K D for binding the target of at least about 10 4 M, 10 5 M, 10 6 M, 10 7 M, 10 8 M, 10 9 M, 10 10 M, 10 11 M, 10 12 M, 10 13 M, or less.
  • 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 FSHD.
  • 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).
  • a complex that comprise a targeting agent, e.g., an antibody, covalently linked to an oligonucleotide.
  • a complex comprises a muscle targeting antibody (e.g., an anti-TfRl antibody) covalently linked to an oligonucleotide.
  • the oligonucleotide is a PMO.
  • the oligonucleotide is an antisense oligonucleotide that targets a DUX4 RNA.
  • Complexes described herein generally comprise a linker that covalently links an antibody (e.g., any one of the anti-TfRl antibodies described herein) to one or more oligonucleotides (e.g., a PMO).
  • a linker comprises at least one covalent bond.
  • complexes provided herein comprise a structure of formula (I): [ R 1 J n i -R 2 , in which each R 1 independently comprises a compound comprising an oligonucleotide (e.g., a PMO) and R 2 comprises an antibody (e.g., anti-TfRl antibody), and in which nl is an integer (e.g., of one or greater) representing the number of instances of R 1 in the complex.
  • nl is independently an integer (e.g., of zero or greater) representing the number of instances of R1 in each complex.
  • each R 1 independently comprises a group comprising an oligonucleotide. In some embodiments, each R 1 independently comprises a group that comprises additional elements in addition to an oligonucleotide.
  • 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 .
  • R 2 comprises an anti-TfRl Fab.
  • nl is independently an integer of zero or greater. In some embodiments, in each complex, nl is independently an integer of one or greater. In some embodiments, nl is an integer of one or greater. In some embodiments, 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 ID NOs: 1, 7, or 12
  • 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 from one 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). In some embodiments, the value of nl is selected from 1, 2, 3, 4, 5, 6, 7, 8,
  • the value of nl is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 and 26. In some embodiments, the value of nl is in the range of 1-27, 1-26, 1-10, 1-5, or 1-3. In some embodiments, 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. In some embodiments, 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.
  • nl in each complex, is independently in the range of 1-27, 1-26, 1-10, 1-5, or 1-3. In some embodiments, 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, or 1-2). In some embodiments, compositions described herein comprise complexes that comprise a structure of formula (I): [R'j 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., 0.5-1, 1-5, 1-4, 1-3, 1-2, 2-4, 3-5, 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, 0.8-1.3, 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, 0.9-1.2, 1-4.6, 1-4.5, 1-4.4, 1-4.3, 1-4.2, 1-3.5, 1-2.5, 1.1-5, 1.1-4.5, 1.1-4, 1.1- 3.5, 1.1-3, 1.1-2.5, 1.1-2.2, 1.2-5, 1.2-4.5, 1.2-4, 1.2-3.5, 1.2-3, 1.2-3, 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 histidine and/or sucrose, as described herein) that comprise a plurality of different complexes.
  • the plurality of different complexes comprises a common targeting agent (e.g., an antibody) and a common oligonucleotide (e.g., PMO).
  • a common targeting agent e.g., an antibody
  • a common oligonucleotide e.g., PMO
  • different complex types are characterized by having different numbers of oligonucleotides covalently linked to an antibody.
  • compositions comprise a plurality of complex types in which each complex type comprises a structure of formula (I): [ R 1 J n i -R 2 , in which each R 1 independently comprises a compound comprising an oligonucleotide (e.g., a PMO) and R 2 comprises an antibody (e.g., anti-TfRl antibody), and in which in each complex type 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).
  • nl values e.g., nl values in the range of 1-27, 1-26
  • each different complex types of the composition have different nl values in the range of 1-27, 1-26, 1-25, 1-20, 1-15, 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., 0.5-1, 1-5, 1-4, 1-3, 1-2, 2-4, 3-5, 0.5-5, 1-5, 1-4, 1-3, 3-5, 0.5-4, 0.5-3, 0.5-2, 0.5-
  • compositions described herein comprise complexes in which nl is 0.
  • compositions are provided herein (e.g., formulations comprising histidine and/or sucrose as described herein) that comprise unconjugated antibody (e.g., in trace amounts) and antibody conjugated to one or more oligonucleotides.
  • an “unconjugated antibody” refers to an antibody that is not conjugated to an oligonucleotide.
  • unconjugated antibody may be referred to as a compound comprising a structure of formula (I): [R'j n i -R 2 , for which nl is zero.
  • compositions are provided (e.g., formulations as described herein) that comprise compounds (e.g., complexes) comprising a structure of formula (I): [R'j n i -R 2 , for which each R 1 independently comprises a group comprising an oligonucleotide, R 2 comprises an antibody and nl is an integer of zero or greater that reflects the number of instances of R 1 in the complex. In some embodiments, 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'j n i -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 of the composition is in the range of 0.5 to 5 (e.g., 0.5-1, 1-5, 1-4, 1-3, 1-2, 2-4, 3-5, 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, 0.8-1.3, 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,
  • each instance of R 1 in a complex is conjugated to a different amino acid residue of the antibody. In some embodiments, 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 e-amino group (e.g., lysine, arginine). In some embodiments, each different amino acid comprises an e-amino group (e.g., lysine, arginine). However, in some embodiments, an amino acid to which R 1 is covalently linked is a cysteine. In some embodiments, 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. However, in some embodiments, 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'j n 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., a phosphorodiamidate morpholino oligomer (PMO); and R 1 is covalently linked (e.g., indirectly or directly linked, e.g., directly linked) to R 2 at attachment point A.
  • nl is independently an integer (e.g., of one or greater) representing the number of instances of R 1 in each complex.
  • 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 2 comprises an antibody that is a Fab fragment.
  • R 3 comprises an oligonucleotide, e.g., a phosphorodiamidate morpholino oligomer (PMO) comprising the base sequence of
  • R 2 comprises a Fab and each R 1 is covalently linked at attachment point A to a different amino acid residue of the Fab, optionally wherein each different amino acid residue is a lysine.
  • nl is independently an integer (e.g., an integer in the range of 1- 27, 1-26, 1-10, 1-5, or 1-3).
  • complexes provided herein comprise a structure of formula (I): [ R 1 J n i -R 2 , in which each R 1 comprises a group of the formula (lb):
  • R 1 is covalently linked (e.g., indirectly or directly linked, e.g., directly linked) to R 2 at attachment point A, wherein -p reflects a phosphorodiamidate linkage, and wherein N corresponds to a nucleobase of adenine (A), cytosine (C), guanine (G), or thymine (T), such that the PMO comprises a base sequence of GGGC ATTTT A AT AT AT CTCT G A ACT (SEQ ID NO: 21).
  • nl is independently an integer (e.g., of one or greater) representing the number of instances of R 1 in each complex, and each 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
  • 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 2 comprises an antibody that is a Fab fragment.
  • nl is independently an integer (e.g., an integer in the range of 1-27, 1-26, 1-10, 1-5, or 1-3).
  • R 2 comprises a Fab and each R 1 is covalently linked at attachment point A to a different amino acid residue of the Fab, optionally wherein each different amino acid residue is a lysine.
  • complexes provided herein comprise a structure of formula (I): [ R 1 J n i -R 2 , in which each R 1 comprises a group of the formula (Ic):
  • R 1 is covalently linked (e.g., indirectly or directly linked, e.g., directly linked) to R 2 at attachment point A.
  • nl is independently an integer (e.g., of one or greater) representing the number of instances of R 1 in each complex, wherein each 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- 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 a comprising 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 2 comprises an antibody that is a Fab fragment.
  • nl is independently an integer (e.g., an integer in the range of 1-27, 1-26, 1-10, 1-5, or 1-3).
  • R 2 comprises a Fab and each R 1 is covalently linked at attachment point A to a different amino acid residue of the Fab, optionally wherein each different amino acid residue is a lysine.
  • complexes provided herein comprise a structure of the formula (Id):
  • -pN indicates a base position of a phosphorodiamidate morpholino oligomer (PMO); wherein -p reflects a phosphorodiamidate linkage, and wherein N corresponds to a nucleobase of adenine (A), cytosine (C), guanine (G), or thymine (T), such that the PMO has a base sequence of GGGCATTTTAATATATCTCTGAACT (SEQ ID NO: 21); wherein R 2 comprises an antibody comprising a sequence as set forth in Table 2; wherein in each complex nl is independently an integer (e.g., of one or greater) representing the number of instances of the group enclosed by square brackets, wherein each instance of the group enclosed by square brackets is covalently linked to a different amino acid residue of the antibody (e.g., a Fab), optionally wherein each different amino acid residue is a lysine.
  • PMO phosphorodiamidate morpholino oligomer
  • R 2 comprises an antibody (e.g., a Fab) 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
  • R 2 comprises an antibody (e.g., a Fab) comprising a heavy chain variable region (VH) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 17 and/or comprising a light chain variable region (VL) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 18.
  • VH heavy chain variable region
  • VL light chain variable region
  • R 2 comprises an antibody (e.g., a Fab) comprising a VH comprising the amino acid sequence of SEQ ID NO: 17 and/or comprising a VL comprising the amino acid sequence of SEQ ID NO:
  • R 2 comprises an antibody (e.g., a Fab) comprising a heavy chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 19 and/or 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 (e.g., a Fab) comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and/or comprising a light chain comprising the amino acid sequence of SEQ ID NO: 20.
  • nl is independently an integer (e.g., an integer in the range of 1- 27, 1-26, 1-10, 1-5, or 1-3).
  • R 2 comprises an antibody (e.g., a Fab) that is covalently linked via different amino acid residue of the antibody (e.g., Fab), optionally wherein each different amino acid residue is a lysine.
  • complexes described herein comprise a structure of: oligonucleotide
  • the antibody is an anti-TfRl antibody (e.g., the anti-TfRl antibody provided in Table 2).
  • the oligonucleotide is a PMO and comprises the nucleotide sequence of SEQ ID NO: 21.
  • the amide shown adjacent to the anti-TfRl antibody in the structure results from a reaction with an amine of the anti-TfRl antibody, such as a lysine epsilon amine.
  • a complex described herein comprises an anti-TfRl Fab covalently linked via a lysine of the Fab to the 5’ end of a PMO.
  • 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- Ll) 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 ID NOs: 1, 7, or 12
  • 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 provided herein comprise an antibody that binds human transferrin receptor 1 (TfRl).
  • TfRl human transferrin receptor 1 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 Rabat definition system), a heavy chain complementarity determining region 2 (CDR-H2) of SEQ ID NO: 8 (according to the Rabat definition system), a heavy chain complementarity determining region 3 (CDR-H3) of SEQ ID NO: 9 (according to the Rabat definition system), a light chain complementarity determining region 1 (CDR-L1) of SEQ ID NO: 10 (according to the Rabat definition system), a light chain complementarity determining region 2 (CDR-L2) of SEQ ID NO: 11 (according to the Rabat definition system), and a light chain complementarity determining region 3 (CDR-L3) of SEQ ID NO: 6 (according to the Rabat 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 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 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 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.
  • the anti-TfRl antibody of the present disclosure is a Fab that comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19.
  • the anti-TfRl antibody of the present disclosure comprises a light chain comprising the amino acid sequence of SEQ ID NO: 20.
  • 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 a DUX4 RNA (e.g., for reducing DUX4 expression or activity).
  • an oligonucleotide useful for targeting a DUX4 RNA (e.g., for reducing DUX4 expression or activity) comprises a region of complementarity to a DUX4 RNA.
  • the oligonucleotide may be designed to cause degradation of an mRNA.
  • the oligonucleotide may be designed to block translation of an mRNA.
  • an oligonucleotide may be designed to cause degradation and block translation of an mRNA.
  • an oligonucleotide useful for targeting a DUX4 RNA is 15-40 (e.g., 15-40, 15-35, 15-30, 15-25, 15-20, 20-40, 20-35, 20-30, 20-25, 22-40, 22-35, 22-30, 22-28, 22-25, 24-40, 24-35, 24-30, 24-28, 24-25, 25- 40, 25-35, 25-30, 25-28, 30-40, 30-35, or 35-40) nucleotides in length.
  • an oligonucleotide useful for targeting a DUX4 RNA is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length, optionally 15-30, 24, or 25 nucleotides in length.
  • an oligonucleotide useful for targeting a DUX4 RNA 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, or 30) consecutive nucleotides to a DUX4 RNA as set forth in SEQ ID NO: 23.
  • an oligonucleotide useful for targeting a DUX4 RNA comprises a region of complementarity of at least 8 (e.g., at least 8, 9, 10, 11, 12, 13,
  • an oligonucleotide useful for targeting a DUX4 RNA 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, or 25) consecutive nucleotides to a target sequence as set forth in SEQ ID NO: 22 ( AGTT C AG AG AT AT ATT A A A AT GCCC ) .
  • an oligonucleotide useful for targeting a DUX4 RNA comprises at least 8 (e.g., at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) consecutive nucleotides of a sequence as set forth in SEQ ID NO: 21 (GGGCATTTTAATATATCTCTGAACT).
  • an oligonucleotide useful for targeting a DUX4 RNA comprises 24 or 25 consecutive nucleotides of a sequence as set forth in SEQ ID NO: 21.
  • an oligonucleotide useful for targeting a DUX4 RNA comprises the nucleotide sequence of SEQ ID NO: 21.
  • any one of the oligonucleotides provided herein is a PMO.
  • 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 may independently and optionally be T’s.
  • compositions described herein comprise complexes (i.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., an 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., an 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 (i.e., a plurality of complexes) wherein each complex comprises a structure of formula (I): [R'j n i -R 2 , in which each R 1 independently comprises a compound comprising an oligonucleotide (e.g., an 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. In some embodiments, the average value of nl of complexes of the composition is in the range of 1 to 2,
  • 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., 0.5-1, 1-5, 1-4, 1-3, 1-2, 2-4, 3-5, 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, 0.8-1.3, 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, 0.9-1.2, 1-4.6, 1-4.5, 1-4.4, 1-4.3, 1-4.2, 1-3.5, 1-2.5, 1.1-5, 1.1-4.5, 1.1-4,
  • 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'j ni -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 1 J n 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'j n i -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., 0.5-1, 1-5, 1-4, 1-3, 1-2, 2-4, 3-5, 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, 0.8-1.3, 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, 0.9-1.2, 1-4.6, 1-4.5, 1-4.4, 1-4.3, 1-4.2, 1-3.5, 1-2.5, 1.1-5, 1.1-4.5, 1.1-4, 1.1-3.5, 1.1-3, 1.1-2.5, 1.1-2.2, 1.2-5, 1.2-4.5, 1.2-4, 1.2-3.5,
  • 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 a PMO covalently linked with a Fab
  • histidine and/or sucrose is particularly advantageous for pharmaceutical use, e.g., as described herein.
  • formulations e.g., aqueous solutions, lyophilized forms
  • formulations comprising complexes together with histidine and/or sucrose in frozen forms.
  • formulations described herein comprise complexes (e.g., a plurality of complexes comprising a PMO covalently linked with a Fab), histidine, and sucrose.
  • formulations comprising muscle-targeting complexes are formulated with histidine and/or sucrose in aqueous solutions.
  • formulation comprising a plurality of the complexes, histidine, and sucrose can be lyophilized (e.g., for storage).
  • the lyophilized formulation may be reconstituted (e.g., with water) for administration to a subject.
  • 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 ( i.e ., a plurality of complexes), each of which complex comprises a phosphorodiamidate morpholino oligomer (PMO) covalently linked to an antibody.
  • PMO phosphorodiamidate morpholino oligomer
  • a formulation comprising complexes, in which each complex comprises a phosphorodiamidate morpholino oligomer (PMO) 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 histidine (e.g ., L-histidine) and sucrose.
  • the antibody is an anti-TfRl antibody.
  • formulations comprise complexes of the formula [ R 1 J n i -R 2 , in which each R 1 independently comprises a compound comprising an oligonucleotide (e.g., a PMO) and R 2 comprises an antibody (e.g., anti-TfRl antibody), and in which nl is an integer of one or greater representing the number of instances of R 1 in the complex.
  • each R 1 independently comprises a compound comprising an oligonucleotide (e.g., a PMO) and R 2 comprises an antibody (e.g., anti-TfRl antibody), and in which nl is an integer of one or greater representing the number of instances of R 1 in the complex.
  • formulations comprise a plurality of complexes wherein each complex comprises a structure of formula (I): [ R 1 J n i -R 2 , in which each R 1 independently comprises a compound comprising an oligonucleotide (e.g., a PMO) and R 2 comprises an antibody (e.g., anti-TfRl antibody), and in which in each complex nl is an integer independently of one or greater representing the number of instances of R 1 in each 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.
  • the value of nl of each complex in the formulation 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 (R 2 ).
  • the value of nl of each complex in the formulation 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 formulation 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 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. In some embodiments, the average value of nl of complexes of the formulation is in the range of 1 to 3, 1 to 5, 1 to 10, 1 to 26, or 1 to 27.
  • a formulation 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 antibody may be referred to as a compound comprising a structure of formula (I): [R'j n i -R 2 , for which nl is zero.
  • formulations comprise compounds (e.g., complexes) comprising a structure of formula (I): [R ⁇ ni -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'j n i -R 2 , in a formulation, for which nl is zero, compared with all compounds of that structure in the formulation 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 of the formulation is in the range of 0.5 to 5 (e.g., 0.5-1, 1-5, 1-4, 1-3, 1-2, 2-4, 3-5, 0.5-5, 1-5, 1-4, 1-3, 3-5, 0.5-4, 0.5-3, 0.5-2, 0.5-
  • 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 e-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'j n i -R 2 , in which each instance of R 1 in a complex of a formulation provided herein independently comprises a group of the formula (la):
  • R 3 comprises an oligonucleotide, e.g., a phosphorodiamidate morpholino oligomer (PMO); wherein R 1 is covalently linked (e.g., indirectly or directly linked, e.g., directly linked) to R 2 at attachment point A.
  • nl is independently an integer (e.g., of one or greater) representing the number of instances of R 1 in each complex.
  • 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 2 comprises an antibody that is a Fab fragment.
  • R 3 comprises an oligonucleotide, e.g., a phosphorodiamidate morpholino oligomer (PMO) comprising the base sequence of
  • R 2 comprises an antibody a Fab and each R 1 is covalently linked at attachment point A to a different amino acid residue of the antibody Fab, optionally wherein each different amino acid residue is a lysine.
  • 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 provided herein comprise complexes that comprise a structure of formula (I): [ R 1 J n i -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., 0.5-1, 1-5, 1-4, 1-3, 1-2, 2-4, 3-5, 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, 0.8-1.3, 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, 0.9-1.2, 1-4.6, 1-4.5, 1-4.4, 1-4.3, 1-4.2, 1-3.5, 1-2.5, 1.1-5, 1.1-4.5, 1.1-4, 1.1-3.5, 1.1-3, 1.1-2.5, 1.1-2.2, 1.2-5, 1.2-4.5, 1.2-4,
  • formulations provided herein comprise complexes that comprise a structure of formula (I): [R'j n i -R 2 , in which each instance of R 1 in a complex of a formulation provided herein comprises a group of the formula (lb):
  • R 1 is covalently linked (e.g., indirectly or directly linked, e.g., directly linked) to R 2 at attachment point A; wherein -p reflects a phosphorodiamidate linkage, and wherein N corresponds to a nucleobase of adenine (A), cytosine (C), guanine (G), or thymine (T), such that the PMO has a base sequence of GGGC ATTTT A AT ATCTCTGAACT (SEQ ID NO: 21).
  • nl is independently an integer (e.g., of one or greater) representing the number of instances of R 1 in each complex, and each 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 2 comprises an antibody that is a Fab fragment.
  • nl is independently an integer (e.g., an integer in the range of 1-27, 1-26, 1-10, 1-5, or 1-3).
  • R 2 comprises a Fab and each R 1 is covalently linked at attachment point A to a different amino acid residue of the Fab, optionally wherein each different amino acid residue is a lysine.
  • formulations provided herein comprise complexes that comprise a structure of formula (I): [ R 1 J n i -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., 0.5-1, 1-5, 1-4, 1-3, 1-2, 2-4, 3-5, 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, 0.8-1.3, 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-
  • formulations provided herein comprise complexes that comprise a structure of formula (I): [R'j n i -R 2 , in which each instance of R 1 in a complex of a formulation provided herein comprises a group of the formula (Ic):
  • R 1 is covalently linked (e.g., indirectly or directly linked, e.g., directly linked) to R 2 at attachment point A.
  • formulations provided herein comprise complexes that comprise a structure of formula (I): [R'j n i -R 2 , in which each instance of R 1 in a complex of a formulation provided herein is:
  • R 1 is covalently linked (e.g., indirectly or directly linked, e.g., directly linked) to R 2 at attachment point A.
  • nl is independently an integer (e.g., of one or greater) representing the number of instances of R 1 in each complex.
  • 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 2 comprises an antibody that is a Fab fragment.
  • nl is independently an integer (e.g., an integer in the range of 1-27, 1-26, 1-10, 1-5, or 1-3).
  • R 2 comprises a Fab and each R 1 is covalently linked at attachment point A to a different amino acid residue of the Fab, optionally wherein each different amino acid residue is a lysine.
  • 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., 0.5-1, 1-5, 1-4, 1-3, 1-2, 2-4, 3-5, 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, 0.8-1.3, 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, 0.9- 1.2, 1-4.6, 1-4.5, 1-4.4, 1-4.3, 1-4.2, 1-3.5, 1-2.5, 1.1-5, 1.1-4.5, 1.1-4, 1.1-3.5, 1.1-3, 1.1-2.5,
  • formulations provided herein comprise complexes that comprise a structure of formula (Id):
  • -pN indicates a base position of a phosphorodiamidate morpholino oligomer (PMO); wherein -p reflects a phosphorodiamidate linkage, and wherein N corresponds to a nucleobase of adenine (A), cytosine (C), guanine (G), or thymine (T), such that the PMO has a base sequence of GGGCATTTTAATATATCTCTGAACT (SEQ ID NO: 21); wherein R 2 comprises an antibody comprising a sequence as set forth in Table 2; wherein in each complex nl is independently an integer (e.g., of one or greater) representing the number of instances of the group enclosed by square brackets, wherein each instance of the group enclosed by square brackets is covalently linked to a different amino acid residue of the antibody (e.g., Fab), optionally wherein each different amino acid residue is a lysine.
  • PMO phosphorodiamidate morpholino oligomer
  • R 2 comprises an antibody (e.g., a Fab) comprising a sequence as set forth in Table 2.
  • R 2 comprises an antibody (e.g., Fab) 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-F1) 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
  • R 2 comprises an antibody (e.g., a Fab) comprising a heavy chain variable region (VH) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 17 and/or comprising a light chain variable region (VL) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 18.
  • R 2 comprises an antibody (e.g., a Fab) comprising a VH comprising the amino acid sequence of SEQ ID NO: 17 and/or comprising a VL comprising the amino acid sequence of SEQ ID NO: 18.
  • R 2 comprises an antibody (e.g., a Fab) comprising a heavy chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 19 and/or 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 (e.g., a Fab) comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and/or comprising a light chain comprising the amino acid sequence of SEQ ID NO: 20.
  • nl is independently an integer (e.g., an integer in the range of 1-27, 1-26, 1-10, 1-5, or 1-3).
  • R 2 comprises an antibody (e.g., a Fab) that is covalently linked via different amino acid residue of the antibody (e.g., Fab), optionally wherein each different amino acid residue is a lysine.
  • 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., 0.5-1, 1-5, 1-4, 1-3, 1-2, 2-4, 3-5, 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, 0.8-1.3, 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, 0.9-1.2, 1-4.6, 1-4.5, 1-4.4, 1-4.3, 1-4.2, 1-3.5, 1-2.5, 1.1-5, 1.1-4.5, 1.1-4, 1.1-3.5, 1.1-3, 1.1-2.5, 1.1-2.2, 1.2-5, 1.2-4.5, 1.2-4,
  • complexes provided in the formulations described herein comprise a structure of formula (A): oligonucleotide antibod .y1
  • the antibody is an anti-TfRl antibody (e.g., the anti-TfRl antibody provided in Table 2).
  • the oligonucleotide is a PMO and comprises the base sequence of SEQ ID NO: 21.
  • the amide shown adjacent to the anti-TfRl antibody in the structure (A) results from a reaction with an amine of the anti-TfRl antibody, such as a lysine epsilon amine.
  • a complex described herein comprises an anti-TfRl Fab covalently linked via a lysine of the Fab to the 5’ end of a PMO.
  • 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- Ll) 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 ID NOs: 1, 7, or 12
  • 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.
  • a formulation comprising complexes described herein wherein a concentration of the complexes in the formulation therein is between 1-50 mg/mL of the complex, optionally 10-50 mg/ml or 20-35 mg/mL (e.g., 1-10 mg/mL, 10-15 mg/mL, 15-20 mg/mL, 20-22 mg/mL, 22-24 mg/ml, 24-26 mg/ml, 24-25 mg/ml, 25-26 mg/ml, 22-25 mg/mL, 25-27 mg/mL, 27-29 mg/mL, 29-30 mg/mL, 25-30 mg/mL, 29-31 mg/ml, 30-31 mg/ml, 31-32 mg/ml, 30-32 mg/mL, 32-33 mg/ml, 32-35 mg/mL, 30-35 mg/mL, 35-40 mg/mL, 40-45 mg/mL, 45-50 mg/mL), optionally approximately 25 mg/mL (e.g., 25 mg/mL, 10-50 mg/ml or
  • any one or a plurality of the complexes described herein is formulated with the histidine (e.g ., L-histidine) and the sucrose in a lyophilized form (e.g., lyophilized powder).
  • histidine e.g ., L-histidine
  • sucrose in a lyophilized form (e.g., lyophilized powder).
  • any one or a plurality of the complexes described herein is formulated with the histidine (e.g., L-histidine) and the sucrose in an aqueous solution.
  • the histidine e.g., L-histidine
  • the histidine is present in the aqueous solution at a concentration in the range of 10-50 mM, 10-20 mM, 20 mM to 30 mM, or 20 mM to 40 mM, e.g., 20-22 mM, 22-24 mM, 24-25 mM, 25-26 mM, 24-26 mM, 26-27 mM, 24-27 mM, 27-28 mM, 28-29 mM, 29-30 mM, 27-30 mM, approximately 22-27 mM, approximately 23-26 mM, approximately 24-26 mM, approximately 26-28 mM, approximately 28-30 mM, approximately 30-32 mM, approximately 32-35 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, 8-15% w/v%, 9-15% w/v%, 9-11% w/v%, 9.5-11% w/v%, or for example, in the range of 5-6 w/v%, 6-7 w/v%, 7-8 w/v%, 8-9 w/v%, 9-10 w/v%, 10-11 w/v%, 11-12% w/v%, 10-12 w/v%, 12-13% w/v%, 13-14% w/v%, 12-14 w/v%, 14-15 w/v%, or 8-12 w/v%.
  • w/v% weight per volume
  • the sucrose is present in the aqueous solution at a concentration in the range of 8- 12 w/v% (e.g., 10 w/v%).
  • the aqueous solution has a pH in the range of 5.0 to 7.0, for example, 5.0-5.2, 5.2-5.4, 5.4-5.6, 5.6-5.8, 5.8-6.0, 5.9-6.0, 5.9-6.1, 6.0-6.1; for example, 5.5 to 6.5, or for example, in the pH range of 5.5-5.8, 5.8-6.0, 5.9-6.1, 6.0-6.1, 6.0-6.2, 6.2-6.4, 6.4-6.5, 6.5-6.7, 6.7-6.8, 6.8-6.9, 6.9-7.0, 7.0-7.1, or 5.8-6.2.
  • the aqueous solution has a pH in the range of 5.8-6.2 (e.g., 5.8-6.0, 5.8-6.1, 5.9-6.1). In some embodiments, the aqueous solution has a pH in the range of 5.9-6.2. In some embodiments, the aqueous solution has a pH in the range of 6.0-6.1 (e.g., about 6.0, or 6.0).
  • a formulation e.g., in aqueous solution
  • aqueous solution comprising one or a plurality of complexes, histidine, and sucrose
  • any one of the formulations described herein is an aqueous solution, wherein the histidine (e.g., L-histidine) is present in the aqueous solution at a concentration of 25 mM, wherein the sucrose is present in the aqueous solution at a concentration of 10 w/v%, and wherein the aqueous solution is at a pH of about 6.0 (e.g., 6.0, 5.9-6.1).
  • the histidine e.g., L-histidine
  • sucrose is present in the aqueous solution at a concentration of 10 w/v%
  • the aqueous solution is at a pH of about 6.0 (e.g., 6.0, 5.9-6.1).
  • a formulation e.g., in aqueous solution
  • a formulation described herein comprising a plurality of complexes, histidine and sucrose
  • the histidine e.g., L- histidine
  • sucrose is present in the aqueous solution at a concentration of 10 w/v%
  • the pH of about 6.0 e.g., 6.0, 5.9-6.1
  • the concentration of complexes in the formulation is 10-50 mg/ml or 20- 35 mg/mL (e.g ., 1-10 mg/mL, 10-15 mg/mL, 15-20 mg/mL, 20-22 mg/mL, 22-24 mg/ml, 24-26 mg/ml, 22-25 mg/mL, 25-27 mg/mL, 27-29 mg/mL, 29-31 mg/ml, 29-30 mg/mL, 30-31 mg/ml, 31-
  • 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.
  • 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.
  • any one or a plurality of the complexes described herein is formulated with histidine 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 product e.g., lyophilized formulation described herein
  • a process comprising lyophilizing an aqueous solution of a formulation (e.g., in aqueous form) described herein.
  • any one or a plurality of the complexes described herein is formulated with histidine 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).
  • a product e.g., frozen formulation described herein
  • a process comprising freezing an aqueous solution of a formulation (e.g., in 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., Fab) covalently linked to a molecular payload (e.g., oligonucleotide, e.g., phosphorodiamidate morpholino oligomer (PMO)) as described herein are effective in treating FSHD.
  • a molecular payload e.g., oligonucleotide, e.g., phosphorodiamidate morpholino oligomer (PMO)
  • PMO phosphorodiamidate morpholino oligomer
  • complexes are effective in treating Type 1 FSHD.
  • complexes are effective in treating Type 2 FSHD.
  • FSHD is associated with deletions in D4Z4 repeat regions on chromosome 4 which contain the DUX4 gene.
  • FSHD is associated with mutations in the SMCHD1 gene.
  • a subject may be a human subject, a non-human primate subject, a rodent subject, or any suitable mammalian subject.
  • the non human primate subject is a cynomolgus monkey.
  • the subject is human.
  • the subject is a human subject that is between 2-60 (e.g., 2-60, 2-50, 2- 40, 2-30, 2-20, 2-10) years of age. In some embodiments, the subject is a human subject that is between 5-30 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) years old. In some embodiments, the subject is a human subject that is between 5-12 (e.g., 5, 6, 7, 8, 9, 10, 11, or 12) years of age.
  • the subject is a human subject that is between 4-16 (e.g., 4-16, 5-16, 6-16, 7-16, 8-16, 9-16, 10-16, 11-16, 12- 16, 13-16, 14-16, 15-16, 4-15, 5-15, 6-15, 7-15, 8-15, 9-15, 10-15, 11-15, 12-15, 13-15, 14-15, 4-14, 5-14, 6-14, 7-14, 8-14, 9-14, 10-14, 11-14, 12-14, 13-14, 4-13, 5-13, 6-13, 7-13, 8-13, 9- 13, 10-13, 11-13, 12-13, 4-12, 5-12, 6-12, 7-12, 8-12, 9-12, 10-12, 11-12, 4-11, 5-11, 6-11, 7-11, 8-11, 9-16, 10-11, 4-10, 5-10, 6-10, 7-10, 8-10, 9-10, 4-9, 5-9, 6-9, 7-9, 8-9, 4-9, 5-9, 6-9, 7-9, 8-9, 4-8, 5-8, 6-8, 7-8, 4-7,
  • the subject is a human subject that is about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 years of age.
  • a subject may have myotonic dystrophy.
  • a subject has elevated expression of the DUX4 gene outside of fetal development and the testes.
  • the subject has facioscapulohumeral muscular dystrophy of Type 1 or Type 2.
  • the subject having FSHD has mutations in the SMCHD1 gene.
  • the subject having FSHD has deletion mutations in D4Z4 repeat regions on chromosome 4.
  • a subject is ambulant. In some embodiments, a subject is non-ambulant.
  • An aspect of the disclosure includes methods and/or uses 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., anti-TfRl antibody, anti-TfRl Fab) described herein covalently linked to an oligonucleotide (e.g., antisense oligonucleotide targeting a mutant allele of DUX4) described herein can be administered to a subject in need of treatment.
  • a pharmaceutical composition is administered systemically.
  • 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. In some embodiments, administration may be performed by intravenous, intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra- articular, intrasynovial, or intrathecal routes. In some embodiments, a pharmaceutical composition comprising complex(es) as described herein is administered by infusion (e.g., intravenous infusion). In some embodiments, 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 nebulized or lyophilized form may be reconstituted with an aqueous or liquid solution.
  • FSHD facioscapulohumeral muscular dystrophy
  • a cell e.g., muscle cell
  • the method comprising contacting the cell with or administering to the subject the formulation described herein with an effective amount of the complex(es) for promoting internalization of the molecular payload in the cell.
  • the method comprises administering a lyophilized form (e.g., lyophilized powder) of a formulation comprising a plurality of complexes 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 comprising a complex or plurality of complexes 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 location for administering the aqueous solution of the formulation e.g., healthcare provider location
  • administered in the reconstituted form e.g., as an aqueous solution
  • a pharmaceutical composition that comprises complex(es) comprising an anti-TfRl antibody covalently linked to a molecular payload is administered via site- specific 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 an anti-TfRl antibody (e.g., a Fab) covalently linked to a molecular payload (e.g., oligonucleotide, e.g., phosphorodiamidate morpholino oligomer (PMO)) is administered at an effective concentration that confers therapeutic effect on a subject.
  • an anti-TfRl antibody e.g., a Fab
  • a molecular payload e.g., oligonucleotide, e.g., phosphorodiamidate morpholino oligomer (PMO)
  • 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.
  • 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.
  • the efficacy of treatment may be assessed by evaluation or observation of symptoms associated with FSHD including muscle mass loss and muscle atrophy, primarily in the muscles of the face, shoulder blades, and upper arms.
  • 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 Effects of conjugates containing an anti-TfRl Fab covalently linked to a DUX4-targeting oligonucleotide in FSHD patient-derived immortalized myoblasts
  • the anti-TfRl Fab comprising the VH and VL sequences shown in Table 2 was covalently linked (through lysine conjugation) via a linker comprising a Valine-Citmlline sequence to a DUX4 -targeting oligonucleotide (SEQ ID NO: 21) to achieve enhanced muscle delivery of the oligonucleotide.
  • the oligonucleotide is a PMO and targets a sequence in the 3’- UTR of the DUX4 transcript.
  • the activity of the conjugate was evaluated in the C6(AB 1080) immortalized FSHD1 cell line, which has significant levels of surface TfRl expression and activation of DUX4 transcriptome markers (MBD3L2, TRIM43, ZSCAN4). It was demonstrated that receptor-mediated delivery of the PMO (SEQ ID NO: 21) by the anti-TfRl Fab into muscle cells resulted in approximately 75% reduction of DUX4 transcriptome biomarkers at an 8 nM PMO concentration, whereas equivalent unconjugated PMO shows no significant biomarker reduction compared to vehicle treated cells (FIG. 1). The results show that conjugating with anti-TfRl Fab enhances delivery of therapeutic oligonucleotides to muscle cells for the treatment of FSHD.
  • the term ‘unconjugated’ indicates that the oligonucleotide was not conjugated to an antibody.
  • C6 immortalized FSHD myoblasts were seeded to a density of 45,000 cells/well on a 96-well plate (ThermoFisher Scientific) in Skeletal Growth Media (CAT# C- 23060, Promocell) with Supplementary mix (C-39365, Promocell) and 1% Penstrep (15140-122, Gibco). Growth media was replaced with differentiation media, NbActiv4 (Brainbits) and 1% Pen/Strep (Gibco), 24 hours later.
  • the cells were treated with unconjugated DUX4-targeting oligonucleotide, the conjugate at a PMO concentration of 8 nM, vehicle in technical replicates for 4 hours prior to washout with 1XPBS (10010023, Gibco) one time.
  • 1XPBS 10010023, Gibco
  • TRIM43 Hs00299174_m 1
  • MBD3F2 Hs00544743_ml
  • ZSCAN4 Hs00537549_ml
  • RPF13A Hs04194366_gl
  • Example 2 Freeze-Thaw Stress Study of Exemplary anti-TfRl Fab covalently linked to a DUX4-targeting Oligonucleotide at Various Concentrations
  • the anti-TfRl Fab comprising the VH and VL sequences shown in Table 2 was covalently linked (through lysine conjugation) via a linker comprising a Valine-Citrulline sequence to a DUX4 -targeting oligonucleotide of SEQ ID NO. 21 (“anti-TfRl Fab-DUX4- targeting oligonucleotide conjugate”), at the concentration of 13.1 lmg/mL.
  • the conjugate was supplied into the formulation buffer of 25 mM Histidine, 10% Sucrose, at pH 6.0 (“Formulation 1”). This conjugate was concentrated to approximately 20 mg/mL, 30 mg/mL, and 40 mg/mL using Vivaspin 6 centrifugal membrane filters (30,000 mwco). The concentration was measured using the standard BCA method at 480nm.
  • a F/T (freeze/thaw) study was performed by taking an aliquot of approximately 200 pL from each of the concentrated conjugates and incubating at - 80°C for 3 hours for each cycle, and thawing thoroughly at room temperature for 1-2 hours. A total of 5 cycles was performed and aliquots of 60 pL each sample after each of cycles 0, 1, 3, and 5 were taken for visual analysis. No change in color and appearance was observed in any of the test samples at different specified concentrations of the conjugate, for each of the cycles 0, 1, 3, and 5, as shown in Table 3. *C/C indicates that the sample was clear and colorless.
  • a formulation comprising complexes that comprise a phosphorodiamidate morpholino oligomer (PMO) covalently linked to an anti-transferrin receptor 1 (TfRl) antibody, wherein the PMO comprises a nucleotide sequence having a region of complementarity of at least 8 consecutive nucleotides to the sequence set forth as SEQ ID NO: 22, wherein 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 complement
  • a formulation comprising: complexes of the formula [R'j n i -R 2 , wherein each R 1 independently comprises a group of the formula:
  • R 2 comprises an antibody
  • R 3 is a phosphorodiamidate morpholino oligomer (PMO), wherein R 1 is covalently linked to R 2 at attachment point A; wherein the PMO comprises a nucleotide sequence having a region of complementarity of at least 8 consecutive nucleotides to the sequence set forth as SEQ ID NO: 22, wherein nl is an integer of one or greater 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; and wherein the complexes are formulated with histidine and sucrose, optionally wherein the antibody is an anti-TfRl antibody, and optionally wherein the average value of nl of complexes in the formulation is in the range of 1 to 5.
  • PMO phosphorodiamidate morpholino oligomer
  • 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 comprising a sequence as set forth in SEQ ID NOs: 5, or 11
  • a light chain complementarity determining region 3 comprising a sequence as set forth in SEQ ID NO: 6 or 16.
  • 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 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 1-12 wherein the antibody comprises a heavy chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 19; and/or wherein the antibody comprises a light chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 20, optionally wherein the antibody 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.
  • the PMO comprises a nucleotide sequence that is 15-30 nucleotides in length.
  • each R 1 comprises a group of formula:
  • R 1 is covalently linked to R 2 at attachment point A; wherein -p reflects a phosphorodiamidate linkage, and wherein N corresponds to a nucleobase of adenine (A), cytosine (C), guanine (G), or thymine (T); such that the PMO has a nucleobase sequence of GGGC ATTTT A AT AT AT CTCT G A ACT (SEQ ID NO: 21).
  • each R 1 comprises a group of formula:
  • a method of reducing DUX4 expression or activity in a cell or subject comprising contacting the cell with or administering to the subject the formulation of any one of embodiments 1-19 with an effective amount of the complex for promoting internalization of the molecular payload in the cell, optionally wherein the cell is a muscle cell.
  • 21 The method of embodiment 20, wherein the complex reduces DUX4 protein level.
  • 22 A method of treating a subject having one or more deletions of a D4Z4 repeat in chromosome 4 that is associated with facioscapulohumeral muscular dystrophy (FSHD), comprising administering to the subject the formulation of any one of embodiments 1-19.
  • FSHD facioscapulohumeral muscular dystrophy
  • a complex comprising a structure of formula [R'j n i -R 2 , wherein each R 1 comprises a group of the formula: wherein R 2 comprises a Fab, and wherein the Fab comprises a CDR-H1, a CDR-H2, a CDR-H3, a CDR-L1, a CDR-L2, and a CDR-L3 selected from Table 2, optionally wherein the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 17 and a VL comprising the amino acid sequence of SEQ ID NO: 18, further optionally wherein the Fab comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and a light chain comprising the amino acid sequence of SEQ ID NO: 20, wherein -pN- indicates a base position of a phosphorodiamidate morpholino oligomer, wherein -p reflects a phosphorodiamidate linkage, and wherein N corresponds to a nucleobase of adenine
  • a complex comprising a structure of formula [R'j n i -R 2 , wherein each R 1 comprises a group of the formula: wherein 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 of one or greater 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
  • a formulation comprising a plurality of complexes of embodiment 23 or embodiment 24, and histidine at a concentration of 25 mM, and sucrose at a concentration of 10 w/v%, wherein the formulation is an aqueous solution and is at a pH of 6.0.
  • 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 intemucleotide 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.

Abstract

Aspects of the disclosure relate to complexes and other aspects relate to formulations (e.g., aqueous, lyophilized forms) comprising such complexes (e.g., a complex of the exemplary formula shown below) comprising a phosphorodiamidate morpholino oligomer (e.g., oligonucleotide useful for targeting DUX4) covalently linked to an antibody (e.g., anti-TfRl antibody). In some embodiments, the complexes are formulated with histidine (e.g., L-histidine) and sucrose at a specified pH (e.g., about 5.0 to 7.0). Also provided are uses of these formulations for treating a subject having one or more deletions of a D4Z4 repeat in chromosome 4 that is associated with facioscapulohumeral muscular dystrophy (FSHD). (I)

Description

MUSCLE TARGETING COMPLEXES AND FORMULATIONS THEREOF FOR TREATING FACIOSCAPULOHUMERAL MUSCULAR DYSTROPHY
RELATED APPLICATIONS
[0001] This application claims the benefit under 35 U.S.C § 119(e) of the filing date of U.S. Provisional Application No. 63/220,430, entitled “MUSCLE TARGETING COMPLEXES AND FORMULATIONS THEREOF FOR TREATING FACIOSCAPULOHUMERAL MUSCULAR DYSTROPHY”, filed July 9, 2021, the contents of which are incorporated herein by reference in their entirety.
FIELD OF THE INVENTION
[0002] The present application relates to targeting complexes for delivering molecular payloads (e.g., oligonucleotides) to cells, formulations comprising such complexes, and uses thereof, particularly uses relating to treatment of disease.
REFERENCE TO ELECTRONIC SEQUENCE LISTING [0003] The contents of the electronic sequence listing (D082470062WO00-SEQ-CBD.xml; Size: 23,326 bytes; and Date of Creation: July 7, 2022) is herein incorporated by reference in its entirety.
BACKGROUND OF INVENTION
[0004] Muscular dystrophies (MDs) are a group of diseases characterized by the progressive weakness and loss of muscle mass. These diseases are caused by mutations in genes which encode proteins needed to form healthy muscle tissue. Facioscapulohumeral muscular dystrophy (FSHD) is a dominantly inherited type of MD which primarily affects muscles of the face, shoulder blades, and upper arms. Other symptoms of FSHD include abdominal muscle weakness, retinal abnormalities, hearing loss, and joint pain and inflammation. FSHD is the most prevalent of the nine types of MD affecting both adults and children, with a worldwide incidence of about 1 in 8,300 people. FSHD is caused by aberrant production of double homeobox 4 (DUX4), a protein whose function is unknown. The DUX4 gene, which encodes the DUX4 protein, is located in the D4Z4 repeat region on chromosome 4 and is typically expressed only in fetal development, after which it is repressed by hypermethylation of the D4Z4 repeats which surround and compact the DUX4 gene. Two types of FSHD, Type 1 and Type 2 have been described. Type 1, which accounts for about 95% of cases, is associated with deletions of D4Z4 repeats on chromosome 4. Unaffected individuals generally have more than 10 repeats arrayed in the subtelomeric region of chromosome 4, whereas the most common form of FSHD (FSHD1) is caused by a contraction of the array to fewer than 10 repeats, associated with decreased epigenetic repression and variegated expression of DUX4 in skeletal muscle. Two allelic variants of chromosome 4q (4qA and 4qB) exist in the region distal to D4Z4. 4qA is in cis with a functional polyadenylation consensus site. Contractions on 4qA alleles are pathogenic because the DUX4 transcript is polyadenylated and translated into stable protein. Type 2 FSHD, which accounts for about 5% of cases, is associated with mutations of the SMCHD1 gene on chromosome 18. Besides supportive care and treatments to address the symptoms of the disease, there are no effective therapies for FSHD.
SUMMARY OF INVENTION
[0005] According to some aspects, the present disclosure provides complexes and formulations comprising such complexes. In some embodiments, complexes provided herein are formulated with histidine ( e.g ., L-histidine) and sucrose. In some embodiments, complexes provided herein are formulated as aqueous or lyophilized (e.g., lyophilized powder) forms. In some embodiments, complexes provided herein are formulated as frozen forms. In some embodiments, complexes provided herein comprise a phosphorodiamidate morpholino oligomer (PMO) covalently linked to an antibody. In some embodiments, complexes provided herein comprise a muscle-targeting complex comprising a PMO covalently linked to an anti-transferrin receptor 1 (TfRl) antibody. In some embodiments, the anti-TfRl antibody has undergone pyroglutamate formation resulting from a post-translational modification. In some embodiments, a complex comprises a muscle-targeting complex comprising a PMO covalently linked to the anti-transferrin receptor 1 (TfRl) antibody, e.g., having a sequence as set forth in Table 2. In some embodiments, the PMO targets a DUX4 RNA (e.g., in the 3’UTR region of a DUX4 RNA). Also provided are methods of using these complexes and formulations described herein for treating a subject having one or more deletions of a D4Z4 repeat in chromosome 4 that is associated with facioscapulohumeral muscular dystrophy (FSHD), and/or methods for reducing DUX4 expression (e.g., protein and/or RNA) or activity in a cell (e.g., a muscle cell). [0006] Some aspects of the present disclosure provide complexes comprising a structure of formula (I): [ R 1 J n i -R2, wherein each R1 comprises a group of the formula (la):
Figure imgf000005_0001
(la), wherein R3 comprises a phosphorodiamidate morpholino oligomer (PMO) comprising a region of complementarity of at least 8 consecutive nucleotides to the nucleotide sequence as set forth in SEQ ID NO: 22; wherein R2 comprises a Fab, and wherein the Fab comprises a CDR-H1, a CDR-H2, a CDR-H3, a CDR-F1, a CDR-F2, and a CDR-F3 selected from Table 2. wherein R1 is covalently linked to R2 at attachment point A; and wherein nl is an integer of one or greater representing the number of instances of R1, wherein each instance of R1 is covalently linked to a different amino acid residue of the Fab.
[0007] In some embodiments, the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 17 and a VF comprising the amino acid sequence of SEQ ID NO: 18.
[0008] In some embodiments, the Fab comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and a light chain comprising the amino acid sequence of SEQ ID NO: 20,
[0009] In some embodiments, each different amino acid residue is a lysine.
[00010] In some embodiments, the region of complementarity is at least 16 nucleotides in length.
[00011] In some embodiments, the PMO comprises at least 8 consecutive nucleotides of the nucleotide sequence as set forth in SEQ ID NO: 21.
[00012] In some embodiments, the PMO comprises at least 16 consecutive nucleotides of the nucleotide sequence as set forth in SEQ ID NO: 21.
[00013] Other aspects of the present disclosure provide formulations comprising complexes that comprise a phosphorodiamidate morpholino oligomer (PMO) covalently linked to an anti transferrin receptor 1 (TfRl) antibody, wherein the PMO comprises a nucleotide sequence having a region of complementarity of at least 8 consecutive nucleotides to the sequence set forth as SEQ ID NO: 22, wherein 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, and wherein the complexes are formulated with histidine and sucrose.
[00014] Other aspects of the present disclosure provide formulations comprising complexes comprising a structure of formula [R'jn i-R2, wherein each R1 independently comprises a group of the formula (la):
Figure imgf000006_0001
(la),
R2 comprises an antibody; and
R3 comprises a phosphorodiamidate morpholino oligomer (PMO), wherein R1 is covalently linked to R2 at attachment point A; wherein the PMO comprises a nucleotide sequence having a region of complementarity of at least 8 consecutive nucleotides to the sequence set forth as SEQ ID NO: 22, wherein nl is an integer of one or greater representing the number of instances of R1, wherein each instance of R1 is covalently linked to a different amino acid residue of the antibody; and wherein the complexes are formulated with histidine and sucrose.
[00015] In some embodiments, each different amino acid residue is a lysine.
[00016] In some embodiments, the antibody is an anti-TfRl antibody.
[00017] In some embodiments, the average value of nl of complexes in the formulation is in the range of 0.5 to 5. [00018] In some embodiments, 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.
[00019] In some embodiments, the formulation is in a lyophilized form, an aqueous solution, or a frozen solid form.
[00020] In some embodiments, the formulation is in an aqueous solution and the histidine is present in the aqueous solution at a concentration in the range of 10 mM to 50 mM.
[00021] In some embodiments, the formulation is in an aqueous solution and the sucrose is present in the aqueous solution at a concentration in the range of 5 % to 15 % weight per volume (w/v%).
[00022] In some embodiments, the formulation is in an aqueous solution and the aqueous solution has a pH in the range of 5.0 to 7.0.
[00023] In some embodiments, the formulation is in an aqueous solution and the histidine is present in the aqueous solution at a concentration of 25 mM, and/or the sucrose is present in the aqueous solution at a concentration of 10 w/v%, and/or the aqueous solution is at a pH of 6.0. [00024] In some embodiments, the antibody is a Fab fragment, a full-length IgG, a Fab' fragment, a F(ab')2 fragment, an scFv, or an Fv.
[00025] In some embodiments, the antibody is a Fab fragment.
[00026] In some embodiments, the antibody comprises a heavy chain variable region (VH) comprising an amino acid sequence at least 85% identical to SEQ ID NO: 17; and/or wherein the antibody comprises a light chain variable region (VL) comprising an amino acid sequence at least 85% identical to SEQ ID NO: 18.
[00027] In some embodiments, the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 17 and a VL comprising the amino acid sequence of SEQ ID NO: 18. [00028] In some embodiments, the antibody comprises a heavy chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 19; and/or wherein the antibody comprises a light chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 20. [00029] In some embodiments, the antibody 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.
[00030] In some embodiments, the PMO comprises a nucleotide sequence that is 15-30 nucleotides in length.
[00031] In some embodiments, the PMO comprises a nucleotide sequence having a region of complementarity of at least 20 consecutive nucleotides to the sequence set forth as SEQ ID NO: 22.
[00032] In some embodiments, the PMO comprises at least 8 consecutive nucleotides of a nucleotide sequence as set forth in SEQ ID NO: 21.
[00033] In some embodiments, the PMO comprises the nucleotide sequence as set forth in SEQ ID NO: 21.
[00034] In some embodiments, each R1 comprises a group of the formula (lb):
Figure imgf000008_0001
(lb), in which -pN indicates a base position of a phosphorodiamidate morpholino oligomer (PMO), wherein -p reflects a phosphorodiamidate linkage, wherein N corresponds to a nucleobase of adenine (A), cytosine (C), guanine (G), or thymine (T); such that the PMO has a nucleobase sequence of GGGC ATTTT AATAT ATCTCTGA ACT (SEQ ID NO: 21), and wherein R1 is covalently linked to R2 at attachment point A. [00035] In some embodiments, each R1 comprises a group of the formula (Ic):
Figure imgf000009_0001
(Ic).
[00036] In some embodiments, the complexes are present in the formulation at a concentration in the range between 10 mg/mL to 50 mg/mL.
[00037] Other aspects of the present disclosure provide methods of reducing DUX4 expression or activity in a cell or subject, the method comprising contacting the cell with or administering to the subject the formulation described herein with an effective amount of the complex for promoting internalization of the molecular payload in the cell.
[00038] In some embodiments, the cell is a muscle cell.
[00039] In some embodiments, the complex reduces DUX4 protein level.
[00040] Other aspects of the present disclosure provide methods of treating a subject having one or more deletions of a D4Z4 repeat in chromosome 4 that is associated with facioscapulohumeral muscular dystrophy (FSHD), comprising administering to the subject the formulation described herein.
[00041] Some aspects of the present disclosure provide complexes comprising a structure of formula (I) [R'jn i-R2, wherein each R1 comprises a group of the formula (lb):
Figure imgf000010_0001
(lb), wherein R2 comprises a Fab, and wherein the Fab comprises a CDR-H1, a CDR-H2, a CDR-H3, a CDR-L1, a CDR-L2, and a CDR-L3 selected from Table 2, wherein -pN indicates a base position of a phosphorodiamidate morpholino oligomer, wherein -p reflects a phosphorodiamidate linkage, and wherein N corresponds to a nucleobase of adenine (A), cytosine (C), guanine (G), or thymine (T), such that the oligonucleotide PMO has a nucleobase sequence of GGGC ATTTT AAT ATATCTCTGAACT (SEQ ID NO: 21); wherein R1 is covalently linked to R2 at attachment point A; and wherein nl is an integer of one or greater representing the number of instances of R1, wherein each instance of R1 is covalently linked to a different amino acid residue of the Fab.
[00042] In some embodiments, the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 17 and a VL comprising the amino acid sequence of SEQ ID NO: 18.
[00043] In some embodiments, the Fab comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and a light chain comprising the amino acid sequence of SEQ ID NO: 20.
[00044] In some embodiments, each different amino acid residue is a lysine. [00045] Some aspects of the present disclosure provide complexes comprising a structure of formula (I): [R'jni-R2, wherein each R1 comprises a group of the formula (Ic):
Figure imgf000011_0001
(Ic), wherein R2 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, wherein R1 is covalently linked to R2 at attachment point A; wherein nl is an integer of one or greater representing the number of instances of R1, wherein each instance of R1 is covalently linked to a different amino acid residue of the Fab.
[00046] In some embodiments, R2 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. [00047] In some embodiments, R2 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. [00048] In some embodiments, each different amino acid residue is a lysine.
[00049] Some aspects of the present disclosure provide complexes comprising a structure of formula (Id):
Figure imgf000012_0001
(Id), wherein R2 comprises a Fab, and wherein the Fab comprises a CDR-H1, a CDR-H2, a CDR-H3, a CDR-L1, a CDR-L2, and a CDR-L3 selected from Table 2, wherein -pN indicates a base position of a phosphorodiamidate morpholino oligomer, wherein -p reflects a phosphorodiamidate linkage, and wherein N corresponds to a nucleobase of adenine (A), cytosine (C), guanine (G), or thymine (T), such that the oligonucleotide PMO has a nucleobase sequence of GGGC ATTTT AAT ATATCTCTGAACT (SEQ ID NO: 21); wherein R1 is covalently linked to R2 at attachment point A; and wherein nl is an integer of one or greater representing the number of instances of R1, wherein each instance of R1 is covalently linked to a different amino acid residue of the Fab.
[00050] In some embodiments, the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 17 and a VL comprising the amino acid sequence of SEQ ID NO: 18.
[00051] In some embodiments, the Fab comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and a light chain comprising the amino acid sequence of SEQ ID NO: 20.
[00052] In some embodiments, each different amino acid residue is a lysine.
BRIEF DESCRIPTION OF THE DRAWINGS [00053] FIG. 1 shows data illustrating that a composition comprising conjugates comprising the anti-TfRl Fab having the VH/VL sequences shown in Table 2, in which the Fab is covalently linked (through lysine conjugation) via a linker comprising a valine-citrulline sequence to a DUX4-targeting oligonucleotide (SEQ ID NO: 21), inhibited DUX4 expression in C6 (AB1080) immortalized FSHD1 cells, as indicated by decreased mRNA expression of MDB3L2, TRIM43, and ZSCAN4. The conjugates showed superior activity compared with unconjugated DUX4- targeting oligonucleotide in inhibiting DUX4 expression.
DETAILED DESCRIPTION OF INVENTION [00054] According to some aspects, the present disclosure provides complexes and formulations comprising such complexes. In some embodiments, the complexes are formulated with histidine (e.g., L-histidine) and sucrose. In some embodiments, the complexes are formulated as aqueous or lyophilized (e.g., lyophilized powder) forms. In some embodiments, a complex comprises a phosphorodiamidate morpholino oligomer (PMO) covalently linked to an antibody. In some embodiments, a complex comprises a muscle-targeting complex comprising a PMO covalently linked to an anti-transferrin receptor 1 (TfRl) antibody. In some embodiments, a complex comprises a muscle-targeting complex comprising a PMO covalently linked to the anti transferrin receptor 1 (TfRl) antibody shown in Table 2. In some embodiments, the PMO targets a DUX4 RNA (e.g., in the 3’UTR region of a DUX4 RNA (e.g., as set forth in NM_001293798.2)). Also provided are methods of using these complexes and formulations described herein for treating a subject having one or more deletions of a D4Z4 repeat in chromosome 4 that is associated with facioscapulohumeral muscular dystrophy (FSHD), and/or methods for reducing DUX4 expression (e.g., protein and/or RNA) or activity in a cell (e.g., a muscle cell).
[00055] Further aspects of the disclosure, including a description of defined terms, are provided below.
DEFINITIONS
[00056] Administering: As used herein, the terms “administering” or “administration” 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).
[00057] Approximately: As used herein, the term “approximately” or “about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term “approximately” or “about” refers to a range of values that fall within 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value). [00058] Antibody: As used herein, the term “antibody” refers to a polypeptide that includes at least one immunoglobulin variable domain or at least one antigenic determinant, e.g., paratope that specifically binds to an antigen. In some embodiments, an antibody is a full-length antibody. In some embodiments, an antibody is a chimeric antibody. In some embodiments, an antibody is a humanized antibody. However, in some embodiments, an antibody is a Fab fragment, a Fab’ fragment, a F(ab')2 fragment, a Fv fragment or a scFv fragment. In some embodiments, an antibody is a nanobody derived from a camelid antibody or a nanobody derived from shark antibody. In some embodiments, an antibody is a diabody. In some embodiments, an antibody comprises a framework having a human germline sequence. In another embodiment, 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. In some embodiments, 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). In some embodiments, 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. With respect to the heavy chain, in some embodiments, the heavy chain of an antibody described herein can be an alpha (a), delta (D), epsilon (e), gamma (g) or mu (m) heavy chain. In some embodiments, the heavy chain of an antibody described herein can comprise a human alpha (a), delta (D), epsilon (e), gamma (g) or mu (m) heavy chain. In a particular embodiment, an antibody described herein comprises a human gamma 1 CHI, CH2, and/or (e.g., and) CH3 domain. In some embodiments, the amino acid sequence of the VH domain comprises the amino acid sequence of a human gamma (g) 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 ah, (1991) supra. In some embodiments, the VH domain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or at least 99% identical to any of the variable chain constant regions provided herein. In some embodiments, an antibody is modified, e.g., modified via glycosylation, phosphorylation, sumoylation, and/or (e.g., and) methylation. In some embodiments, an antibody is a glycosylated antibody, which is conjugated to one or more sugar or carbohydrate molecules. In some embodiments, 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.
In some embodiments, 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. In some embodiments, 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, R, et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994) Structure 2:1121-1123). Still further, 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. Examples of such 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).
[00059] CDR: As used herein, the term "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. The 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”). 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 Rabat 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., Rabat, 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. et al., Nucleic Acids Res., 27:209-212 (1999); Ruiz, M. et al., Nucleic Acids Res., 28:219-221 (2000); Lefranc, M.-P, Nucleic Acids Res., 29:207-209 (2001); Lefranc, M.-P, Nucleic Acids Res., 31:307-310 (2003); Lefranc, M.-P. et al., In Silico Biol., 5, 0006 (2004) [Epub], 5:45-60 (2005); Lefranc, M.-P. et al., Nucleic Acids Res., 33:D593-597 (2005); Lefranc, M.-P. et al., Nucleic Acids Res., 37:D1006-1012 (2009); Lefranc, M.-P. et al., Nucleic Acids Res., 43:D413-422 (2015); Chothia et al., (1989) Nature 342:877; Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917, Al-lazikani et al (1997) J. Molec. Biol. 273:927-948; and Almagro, J. Mol. Recognit. 17:132-143 (2004). See also bioinf.org.uk/abs. As used herein, 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.
[00060] 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. The term "CDR set" as used herein 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 Rabat (Rabat 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. These CDRs may be referred to as Rabat CDRs. Sub-portions of CDRs may be designated as LI, L2 and L3 or HI, 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 Rabat CDRs. Other boundaries defining CDRs overlapping with the Rabat CDRs have been described by Padlan (FASEB J. 9:133-139 (1995)) and MacCallum (J Mol Biol 262(5):732-45 (1996)). Still other CDR boundary definitions may not strictly follow one of the above systems, but will nonetheless overlap with the Rabat 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.
Table 1. CDR Definitions
Figure imgf000016_0001
1 IMGT®, the international ImMunoGeneTics information system®, imgt.org, Lefranc, M.-P. et al., Nucleic Acids Res., 27:209-212 (1999)
2 Rabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242
3 Chothia et al., J. Mol. Biol. 196:901-917 (1987)) [00061] Complementary: As used herein, the term “complementary” refers to the capacity for precise pairing between two nucleotides or two sets of nucleotides. In particular, 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. 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). For example, in some embodiments, for complementary base pairings, adenosine-type bases (A) are complementary to thymidine-type bases (T) or uracil-type bases (U), that cytosine-type bases (C) are complementary to guanosine-type bases (G), and that 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.
[00062] Covalently linked: As used herein, the term “covalently linked” refers to a characteristic of two or more molecules being linked together via at least one covalent bond. In some embodiments, 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. However, in some embodiments, 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. In some embodiments, a linker may be a cleavable linker. However, in some embodiments, a linker may be a non-cleavable linker.
[00063] DUX4: As used herein, the term “DUX4” refers to a gene that encodes double homeobox 4, a protein which is generally expressed during fetal development and in the testes of adult males. In some embodiments, DUX4 may be a human (Gene ID: 100288687), non human primate (e.g., Gene ID: 750891, Gene ID: 100405864), or rodent gene (e.g., Gene ID: 306226). In humans, expression of the DUX4 gene outside of fetal development and the testes is associated with facioscapulohumeral muscular dystrophy. In addition, multiple human transcript variants (e.g., as annotated under GenBank RefSeq Accession Numbers: NM_001293798.2, NM_001306068.2, NM_001363820.1) have been characterized that encode different protein isoforms.
[00064] Facioscapulohumeral muscular dystrophy (FSHD): As used herein, the term “facioscapulohumeral muscular dystrophy (FSHD)” refers to a genetic disease caused by mutations in the DUX4 gene or SMCHD1 gene that is characterized by muscle mass loss and muscle atrophy, primarily in the muscles of the face, shoulder blades, and upper arms. Two types of the disease, Type 1 and Type 2, have been described. Type 1 is associated with deletions in D4Z4 repeat regions on chromosome 4 allelic variant 4qA which contains the DUX4 gene. Type 2 is associated with mutations in the SMCHD1 gene. Both Type 1 and Type 2 FSHD are characterized by aberrant production of the DUX4 protein after fetal development outside of the testes. Facioscapulohumeral dystrophy, the genetic basis for the disease, and related symptoms are described in the art (see, e.g., Campbell, A.E., et al.,
“Facioscapulohumeral dystrophy: Activating an early embryonic transcriptional program in human skeletal muscle” Human Mol Genet. (2018); and Tawil, R. “Facioscapulohumeral muscular dystrophy” Handbook Clin. Neurol. (2018), 148: 541-548.) FSHD Type 1 is associated with Online Mendelian Inheritance in Man (OMIM) Entry # 158900. FSHD Type 2 is associated with OMIM Entry # 158901.
[00065] Framework: As used herein, the term "framework" or "framework sequence" 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 (CDR-L1, CDR-L2, and CDR-L3 of light chain and CDR-H1, CDR-H2, and CDR-H3 of heavy chain) 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. Without specifying the particular sub-regions as FR1, FR2, FR3 or FR4, a framework region, as referred by others, represents the combined FRs within the variable region of a single, naturally occurring immunoglobulin chain. As used herein, 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.
[00066] Human antibody: The term "human antibody", as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the 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. However, the term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. [00067] Humanized antibody: The term "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. 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. In one embodiment, 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.
[00068] Rabat numbering: The terms "Rabat numbering", "Rabat definitions and "Rabat 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 (Rabat et al. (1971) Ann. NY Acad, Sci. 190:382-391 and,
Rabat, 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). For the heavy chain variable region, 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. For the light chain variable region, 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.
[00069] Morpholinos: As used herein, the term “morpholino”, also referred to as a “phosphorodiamidate morpholino oligomer”, refers to a molecular structure that contains nucleobases attached to a backbone of methylenemorpholine rings linked through a phosphorodiamidate group. In some embodiments, the oligonucleotide may be a morpholino- based compounds. Morpholino-based oligomeric compounds are described in Dwaine A. Braasch and David R. Corey, Biochemistry, 2002, 41(14), 4503-4510); Genesis, volume 30, issue 3, 2001; Heasman, J., Dev. Biol., 2002, 243, 209-214; Nasevicius et al., Nat. Genet., 2000, 26, 216-220; Facerra et al., Proc. Natl. Acad. Sci., 2000, 97, 9591-9596; and U.S. Pat. No. 5,034,506, issued Jul. 23, 1991. In some embodiments, the morpholino-based oligomeric compound is a phosphorodiamidate morpholino oligomer (PMO) (e.g., as described in Iverson, Curr. Opin. Mol. Ther., 3:235-238, 2001; and Wang et al., J. Gene Med., 12:354-364, 2010; the disclosures of which are incorporated herein by reference in their entireties). [00070] Oligonucleotide: As used herein, the term “oligonucleotide” refers to an oligomeric nucleic acid compound of up to 200 nucleotides in length. Examples of 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. In some embodiments, an oligonucleotide may comprise one or more modified nucleosides (e.g., 2'-0-methyl sugar modifications, purine or pyrimidine modifications). In some embodiments, an oligonucleotide may comprise one or more modified intemucleoside linkage. In some embodiments, an oligonucleotide may comprise one or more phosphorothioate linkages, which may be in the Rp or Sp stereochemical conformation.
[00071] Region of complementarity: As used herein, the term “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). In some embodiments, a region of complementarity is fully complementary to a cognate nucleotide sequence of target nucleic acid. However, in some embodiments, 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.
[00072] Specifically binds: As used herein, 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. With respect to an antibody, 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.
In some embodiments, an antibody specifically binds to a target if the antibody has a KD for binding the target of at least about 104 M, 105 M, 106 M, 107 M, 108 M, 109 M, 10 10 M, 10 11 M, 10 12 M, 10 13 M, or less. In some embodiments, an antibody specifically binds to the transferrin receptor, e.g., an epitope of the apical domain of transferrin receptor.
[00073] Subject: As used herein, the term “subject” refers to a mammal. In some embodiments, a subject is non-human primate, or rodent. In some embodiments, a subject is a human. In some embodiments, a subject is a patient, e.g., a human patient that has or is suspected of having a disease. In some embodiments, the subject is a human patient who has or is suspected of having FSHD.
[00074] 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. In some embodiments, 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. In addition, 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).
[00075] Ranges: All ranges provided in the present disclosure are inclusive of the end points.
Complexes
[00076] Provided herein are complexes that comprise a targeting agent, e.g., an antibody, covalently linked to an oligonucleotide. In some embodiments, a complex comprises a muscle targeting antibody (e.g., an anti-TfRl antibody) covalently linked to an oligonucleotide. In some embodiments, the oligonucleotide is a PMO. In some embodiments, the oligonucleotide is an antisense oligonucleotide that targets a DUX4 RNA.
[00077] Complexes described herein generally comprise a linker that covalently links an antibody (e.g., any one of the anti-TfRl antibodies described herein) to one or more oligonucleotides (e.g., a PMO). A linker comprises at least one covalent bond.
[00078] In some embodiments, complexes provided herein (e.g., in compositions or formulations described herein) comprise a structure of formula (I): [ R 1 J n i -R2, in which each R1 independently comprises a compound comprising an oligonucleotide (e.g., a PMO) and R2 comprises an antibody (e.g., anti-TfRl antibody), and in which nl is an integer (e.g., of one or greater) representing the number of instances of R1 in the complex. In some embodiments, in each complex nl is independently an integer (e.g., of zero or greater) representing the number of instances of R1 in each complex. In some embodiments, each R1 independently comprises a group comprising an oligonucleotide. In some embodiments, each R1 independently comprises a group that comprises additional elements in addition to an oligonucleotide. In some embodiments, R2 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. In some embodiments, each R1 of a complex is independently covalently linked to a different amino acid residue (e.g., lysine or cysteine) of R2. In some embodiments, R2 comprises an anti-TfRl Fab. [00079] In some embodiments, in each complex, nl is independently an integer of zero or greater. In some embodiments, in each complex, nl is independently an integer of one or greater. In some embodiments, nl is an integer of one or greater. In some embodiments, the antibody comprises a sequence as set forth in Table 2. For example, in some embodiments, 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. In some embodiments, the antibody comprises a heavy chain variable region (VH) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 17 and/or comprises a light chain variable region (VL) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 18. In some embodiments, the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 17 and/or comprises a VL comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, the antibody comprises a heavy chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 19 and/or comprises a light chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 20. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and/or comprises a light chain comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the antibody is a Fab fragment, a full- length IgG, a Fab' fragment, a F(ab')2 fragment, an scFv, or an Fv. In some embodiments, the antibody is a Fab fragment.
[00080] In some embodiments, the value of nl of each or any complex (e.g., any complex in any of the compositions or formulations disclosed herein) is an integer from one 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). In some embodiments, the value of nl is selected from 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27. In some embodiments, the value of nl is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 and 26. In some embodiments, the value of nl is in the range of 1-27, 1-26, 1-10, 1-5, or 1-3. In some embodiments, 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. In some embodiments, 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. In some embodiments, in each complex, the value of nl is independently in the range of 1-27, 1-26, 1-10, 1-5, or 1-3. In some embodiments, 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, or 1-2). In some embodiments, compositions described herein comprise complexes that comprise a structure of formula (I): [R'jni-R2, wherein nl is 0. In some embodiments, the average value of nl of complexes of the composition is in the range of 0.5 to 5 (e.g., 0.5-1, 1-5, 1-4, 1-3, 1-2, 2-4, 3-5, 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, 0.8-1.3, 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, 0.9-1.2, 1-4.6, 1-4.5, 1-4.4, 1-4.3, 1-4.2, 1-3.5, 1-2.5, 1.1-5, 1.1-4.5, 1.1-4, 1.1- 3.5, 1.1-3, 1.1-2.5, 1.1-2.2, 1.2-5, 1.2-4.5, 1.2-4, 1.2-3.5, 1.2-3, 1.2-2.5, 1.2-2.2, 1.3-5, 1.3-4.5, 1.3-4, 1.3-3.5, 1.3-3, 1.3-2.5, 1.3-2.2, 1.4-5, 1.4-4.5, 1.4-4, 1.4-3.5, 1.4-3, 1.4-2.5, 1.4-2.2, 1.5-5, 1.5-4.5, 1.5-4, 1.5-3.5, 1.5-3, 1.5-2.5, 1.5-2.2, 1.6-3, 1.6-2.5, 1.6-2.2, 1.7-3, 1.7-2.5, 1.7-2.2, 1.8- 3, 1.8-2.5, or 1.8-2.2). In some embodiments, in each complex type nl is independently an integer of one or greater representing the number of instances of R1 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).
[00081] In some embodiments, compositions are provided (e.g., formulations comprising histidine and/or sucrose, as described herein) that comprise a plurality of different complexes.
In some embodiments, the plurality of different complexes comprises a common targeting agent (e.g., an antibody) and a common oligonucleotide (e.g., PMO). In such embodiments, different complex types are characterized by having different numbers of oligonucleotides covalently linked to an antibody. For example, in some embodiments, compositions are provided that comprise a plurality of complex types in which each complex type comprises a structure of formula (I): [ R 1 J n i -R2, in which each R1 independently comprises a compound comprising an oligonucleotide (e.g., a PMO) and R2 comprises an antibody (e.g., anti-TfRl antibody), and in which in each complex type nl is independently an integer of one or greater representing the number of instances of R1 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). In some embodiments, each different complex types of the composition have different nl values in the range of 1-27, 1-26, 1-25, 1-20, 1-15, 1-10, 1-5, or 1-3. In some embodiments, in complexes of a composition nl is independently an integer. In some embodiments, the average value of nl of complexes of the composition is in the range of 0.5 to 5 (e.g., 0.5-1, 1-5, 1-4, 1-3, 1-2, 2-4, 3-5, 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, 0.8-1.3, 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, 0.9-1.2, 1-4.6, 1-4.5, 1-4.4, 1-4.3, 1-
4.2, 1-3.5, 1-2.5, 1.1-5, 1.1-4.5, 1.1-4, 1.1-3.5, 1.1-3, 1.1-2.5, 1.1-2.2, 1.2-5, 1.2-4.5, 1.2-4, 1.2-
3.5, 1.2-3, 1.2-2.5, 1.2-2.2, 1.3-5, 1.3-4.5, 1.3-4, 1.3-3.5, 1.3-3, 1.3-2.5, 1.3-2.2, 1.4-5, 1.4-4.5,
1.4-4, 1.4-3.5, 1.4-3, 1.4-2.5, 1.4-2.2, 1.5-5, 1.5-4.5, 1.5-4, 1.5-3.5, 1.5-3, 1.5-2.5, 1.5-2.2, 1.6-3, 1.6-2.5, 1.6-2.2, 1.7-3, 1.7-2.5, 1.7-2.2, 1.8-3, 1.8-2.5, or 1.8-2.2). In some embodiments, compositions described herein comprise complexes in which nl is 0.
[00082] In some embodiments, compositions are provided herein (e.g., formulations comprising histidine and/or sucrose as described herein) that comprise unconjugated antibody (e.g., in trace amounts) and antibody conjugated to one or more oligonucleotides. As used herein, an “unconjugated antibody” refers to an antibody that is not conjugated to an oligonucleotide. In some embodiments, unconjugated antibody may be referred to as a compound comprising a structure of formula (I): [R'jn i-R2, for which nl is zero. Accordingly, in some embodiments, compositions are provided (e.g., formulations as described herein) that comprise compounds (e.g., complexes) comprising a structure of formula (I): [R'jn i-R2, for which each R1 independently comprises a group comprising an oligonucleotide, R2 comprises an antibody and nl is an integer of zero or greater that reflects the number of instances of R1 in the complex. In some embodiments, nl is independently an integer of zero or greater that reflects the number of instances of R1 in each compound (e.g., complex). In some embodiments, the fraction of compounds comprising a structure of formula (I): [R'jn i-R2, 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%. As such, in some embodiments, the average value of nl of complexes of the composition is in the range of 0.5 to 5 (e.g., 0.5-1, 1-5, 1-4, 1-3, 1-2, 2-4, 3-5, 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, 0.8-1.3, 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,
0.9-1.2, 1-4.6, 1-4.5, 1-4.4, 1-4.3, 1-4.2, 1-3.5, 1-2.5, 1.1-5, 1.1-4.5, 1.1-4, 1.1-3.5, 1.1-3, 1.1-
2.5, 1.1-2.2, 1.2-5, 1.2-4.5, 1.2-4, 1.2-3.5, 1.2-3, 1.2-2.5, 1.2-2.2, 1.3-5, 1.3-4.5, 1.3-4, 1.3-3.5, 1.3-3, 1.3-2.5, 1.3-2.2, 1.4-5, 1.4-4.5, 1.4-4, 1.4-3.5, 1.4-3, 1.4-2.5, 1.4-2.2, 1.5-5, 1.5-4.5, 1.5-4,
1.5-3.5, 1.5-3, 1.5-2.5, 1.5-2.2, 1.6-3, 1.6-2.5, 1.6-2.2, 1.7-3, 1.7-2.5, 1.7-2.2, 1.8-3, 1.8-2.5, or 1.8-2.2).
[00083] In some embodiments, each instance of R1 in a complex is conjugated to a different amino acid residue of the antibody. In some embodiments, each instance of R1 in a complex is covalently linked to a different amino acid residue of the antibody. In some embodiments, an amino acid to which R1 is covalently linked comprises an e-amino group (e.g., lysine, arginine). In some embodiments, each different amino acid comprises an e-amino group (e.g., lysine, arginine). However, in some embodiments, an amino acid to which R1 is covalently linked is a cysteine. In some embodiments, each different amino acid to which R1 is covalently linked is a cysteine. In some embodiments, R1 is directly covalently linked to an amino acid residue of the antibody. However, in some embodiments, R1 is indirectly covalently linked to an amino acid of the antibody, e.g., covalently linked to a glycosylation site on the amino acid.
[00084] In some embodiments, R1 is not covalently linked to an amino acid residue residing in a CDR region of the antibody.
[00085] In some embodiments, complexes provided herein (e.g., in compositions or formulations described herein) comprise a structure of formula (I): [R'jn i-R2, in which each instance of R1 independently comprises a group of the formula (la):
Figure imgf000025_0001
(la), in which R3 comprises an oligonucleotide, e.g., a phosphorodiamidate morpholino oligomer (PMO); and R1 is covalently linked (e.g., indirectly or directly linked, e.g., directly linked) to R2 at attachment point A. In some embodiments, in each complex nl is independently an integer (e.g., of one or greater) representing the number of instances of R1 in each complex. In some embodiments, R2 comprises an antibody comprising a sequence as set forth in Table 2. For example, in some embodiments, R2 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. In some embodiments, R2 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. In some embodiments, R2 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. In some embodiments, R2 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. In some embodiments, R2 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. In some embodiments, R2 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. In some embodiments, R2 comprises an antibody that is a Fab fragment. In some embodiments, R3 comprises an oligonucleotide, e.g., a phosphorodiamidate morpholino oligomer (PMO) comprising the base sequence of
GGGC ATTTT A AT AT AT CTCT G A ACT (SEQ ID NO: 21). In some embodiments, R2 comprises a Fab and each R1 is covalently linked at attachment point A to a different amino acid residue of the Fab, optionally wherein each different amino acid residue is a lysine. In some embodiments, in each complex nl is independently an integer (e.g., an integer in the range of 1- 27, 1-26, 1-10, 1-5, or 1-3).
[00086] In some embodiments, complexes provided herein (e.g., in compositions or formulations described herein) comprise a structure of formula (I): [ R 1 J n i -R2, in which each R1 comprises a group of the formula (lb):
Figure imgf000026_0001
(lb), in which -pN indicates a base position of a phosphorodiamidate morpholino oligomer (PMO);
R1 is covalently linked (e.g., indirectly or directly linked, e.g., directly linked) to R2 at attachment point A, wherein -p reflects a phosphorodiamidate linkage, and wherein N corresponds to a nucleobase of adenine (A), cytosine (C), guanine (G), or thymine (T), such that the PMO comprises a base sequence of GGGC ATTTT A AT AT AT CTCT G A ACT (SEQ ID NO: 21). In some embodiments, in each complex nl is independently an integer (e.g., of one or greater) representing the number of instances of R1 in each complex, and each R1 is covalently linked to R2 at attachment point A. In some embodiments, R2 comprises an antibody comprising a sequence as set forth in Table 2. For example, in some embodiments, R2 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. In some embodiments, R2 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. In some embodiments, R2 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. In some embodiments, R2 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. In some embodiments, R2 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. In some embodiments, R2 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. In some embodiments, R2 comprises an antibody that is a Fab fragment. In some embodiments, in each complex nl is independently an integer (e.g., an integer in the range of 1-27, 1-26, 1-10, 1-5, or 1-3). In some embodiments, R2 comprises a Fab and each R1 is covalently linked at attachment point A to a different amino acid residue of the Fab, optionally wherein each different amino acid residue is a lysine. [00087] In some embodiments, complexes provided herein (e.g., in compositions or formulations described herein) comprise a structure of formula (I): [ R 1 J n i -R2, in which each R1 comprises a group of the formula (Ic):
Figure imgf000028_0001
(Ic), wherein R1 is covalently linked (e.g., indirectly or directly linked, e.g., directly linked) to R2 at attachment point A. In some embodiments, in each complex nl is independently an integer (e.g., of one or greater) representing the number of instances of R1 in each complex, wherein each R1 is covalently linked to R2 at attachment point A. In some embodiments, R2 comprises an antibody comprising a sequence as set forth in Table 2. For example, in some embodiments, R2 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. In some embodiments, R2 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. In some embodiments, R2 comprises an antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 17 and/or a comprising VL comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, R2 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. In some embodiments, R2 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. In some embodiments, R2 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. In some embodiments, R2 comprises an antibody that is a Fab fragment. In some embodiments, in each complex nl is independently an integer (e.g., an integer in the range of 1-27, 1-26, 1-10, 1-5, or 1-3). In some embodiments, R2 comprises a Fab and each R1 is covalently linked at attachment point A to a different amino acid residue of the Fab, optionally wherein each different amino acid residue is a lysine.
[00088] In some embodiments, complexes provided herein (e.g., in compositions or formulations described herein) comprise a structure of the formula (Id):
Figure imgf000029_0001
(Id), in which -pN indicates a base position of a phosphorodiamidate morpholino oligomer (PMO); wherein -p reflects a phosphorodiamidate linkage, and wherein N corresponds to a nucleobase of adenine (A), cytosine (C), guanine (G), or thymine (T), such that the PMO has a base sequence of GGGCATTTTAATATATCTCTGAACT (SEQ ID NO: 21); wherein R2 comprises an antibody comprising a sequence as set forth in Table 2; wherein in each complex nl is independently an integer (e.g., of one or greater) representing the number of instances of the group enclosed by square brackets, wherein each instance of the group enclosed by square brackets is covalently linked to a different amino acid residue of the antibody (e.g., a Fab), optionally wherein each different amino acid residue is a lysine. In some embodiments, R2 comprises an antibody (e.g., a Fab) comprising a sequence as set forth in Table 2. For example, in some embodiments, R2 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. In some embodiments, R2 comprises an antibody (e.g., a Fab) comprising a heavy chain variable region (VH) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 17 and/or comprising a light chain variable region (VL) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 18. In some embodiments,
R2 comprises an antibody (e.g., a Fab) comprising a VH comprising the amino acid sequence of SEQ ID NO: 17 and/or comprising a VL comprising the amino acid sequence of SEQ ID NO:
18. In some embodiments, R2 comprises an antibody (e.g., a Fab) comprising a heavy chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 19 and/or comprising a light chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 20. In some embodiments, R2 comprises an antibody (e.g., a Fab) comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and/or comprising a light chain comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, in each complex nl is independently an integer (e.g., an integer in the range of 1- 27, 1-26, 1-10, 1-5, or 1-3). In some embodiments, R2 comprises an antibody (e.g., a Fab) that is covalently linked via different amino acid residue of the antibody (e.g., Fab), optionally wherein each different amino acid residue is a lysine. [00089] In some embodiments, complexes described herein comprise a structure of: oligonucleotide
HN antibody
Figure imgf000031_0001
(A), wherein y is 0-15 (e.g., 3) and z is 0-15 (e.g., 4). In some embodiments, the antibody is an anti-TfRl antibody (e.g., the anti-TfRl antibody provided in Table 2). In some embodiments, the oligonucleotide is a PMO and comprises the nucleotide sequence of SEQ ID NO: 21. In some embodiments, the amide shown adjacent to the anti-TfRl antibody in the structure results from a reaction with an amine of the anti-TfRl antibody, such as a lysine epsilon amine. In some embodiments, a complex described herein comprises an anti-TfRl Fab covalently linked via a lysine of the Fab to the 5’ end of a PMO. In some embodiments, the antibody comprises a sequence as set forth in Table 2. For example, in some embodiments, 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- Ll) 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. In some embodiments, the antibody comprises a heavy chain variable region (VH) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 17 and/or comprises a light chain variable region (VL) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 18. In some embodiments, the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 17 and/or comprises a VL comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, the antibody comprises a heavy chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 19 and/or comprises a light chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 20. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and/or comprises a light chain comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the antibody is a Fab fragment, a full- length IgG, a Fab' fragment, a F(ab')2 fragment, an scFv, or an Fv.
Antibodies
[00090] In some embodiments, complexes provided herein comprise an antibody that binds human transferrin receptor 1 (TfRl). An example human transferrin receptor 1 amino acid sequence, corresponding to NCBI sequence NP_003225.2 (transferrin receptor protein 1 isoform 1, homo sapiens) is as follows:
MMDQ ARS AF S NLF GGEPLS YTRF S LARQ VDGDN S H VEMKLA VDEEEN ADNNTKAN VT KPKRC S GS IC Y GTIA VIVFFLIGFMIG YLG Y C KG VEPKTECERLAGTES P VREEPGEDFP A ARRLYWDDLKRKLS EKLDS TDFT GTIKLLNEN S Y VPRE AGS QKDENL ALY VEN QFREF KLSKVWRDQHFVKIQVKDSAQNSVIIVDKNGRLVYLVENPGGYVAYSKAATVTGKLV H ANF GTKKDFEDL YTP VN GS IVI VRAGKITF AEKV AN AES LN AIG VLI YMD QTKFPIVN A ELS FF GH AHLGT GDP YTPGFPS FNHT QFPPS RS S GLPNIP V QTIS RA A AEKLF GNMEGDCP S D WKTDS T CRM VT S ES KN VKLT V S N VLKEIKILNIFG VIKGFVEPDH Y V V V G AQRD A W GPG A AKS G V GT ALLLKLAQMFS DM VLKDGF QPS RS IIF AS WS AGDF GS V G ATE WLEG Y LS S LHLKAFT YINLDKA VLGT S NFKV S AS PLLYTLIEKTMQN VKHP VT GQFLY QDS NW A SKVEKLTLDNAAFPFLAYSGIPAVSFCFCEDTDYPYLGTTMDTYKELIERIPELNKVARA A AE V AGQFVIKLTHD VELNLD YERYN S QLLS FVRDLN Q YR ADIKEMGLS LQ WLY S ARG DFFRAT S RLTTDF GN AEKTDRFVMKKLNDR VMR VE YHFLS P Y V S PKES PFRH VFW GS G S HTLP ALLENLKLRKQNN G AFNETLFRN QL AL ATWTIQG A AN ALS GD VWDIDNEF (SEQ ID NO: 24).
[00091] Table 2 provides examples of sequences of an anti-TfRl antibody useful in the complexes provided herein.
Table 2. Examples of anti-TfRl antibody sequences
Figure imgf000032_0001
Figure imgf000033_0001
[00092] In some embodiments, 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). [00093] In some embodiments, 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 Rabat definition system), a heavy chain complementarity determining region 2 (CDR-H2) of SEQ ID NO: 8 (according to the Rabat definition system), a heavy chain complementarity determining region 3 (CDR-H3) of SEQ ID NO: 9 (according to the Rabat definition system), a light chain complementarity determining region 1 (CDR-L1) of SEQ ID NO: 10 (according to the Rabat definition system), a light chain complementarity determining region 2 (CDR-L2) of SEQ ID NO: 11 (according to the Rabat definition system), and a light chain complementarity determining region 3 (CDR-L3) of SEQ ID NO: 6 (according to the Rabat definition system). [00094] In some embodiments, 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).
[00095] In some embodiments, 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. Alternatively or in addition (e.g., in addition), 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.
[00096] In some embodiments, 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. Alternatively or in addition (e.g., in addition), in some embodiments, 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.
[00097] In some embodiments, the anti-TfRl antibody of the present disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO: 17. Alternatively or in addition (e.g., in addition), in some embodiments, the anti-TfRl antibody of the present disclosure comprises a VL comprising the amino acid sequence of SEQ ID NO: 18.
[00098] In some embodiments, 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. In some embodiments, 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. Alternatively or in addition (e.g., in addition), 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. 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 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.
[00099] In some embodiments, the anti-TfRl antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19. In some embodiments, the anti-TfRl antibody of the present disclosure is a Fab that 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. 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.
[000100] In some embodiments, the anti-TfRl antibody provided herein may have one or more post-translational modifications. In some embodiments, N-terminal cyclization, also called pyroglutamate formation (pyro-Glu), may occur in the antibody at N-terminal Glutamate (Glu) and/or Glutamine (Gin) residues during production. As such, it should be appreciated that 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. In some embodiments, pyroglutamate formation occurs in a heavy chain sequence. In some embodiments, pyroglutamate formation occurs in a light chain sequence.
Oligonucleotides
[000101] In some embodiments, an oligonucleotide of the complexes described herein is a single stranded oligonucleotide. In some embodiments, the oligonucleotide is useful for targeting a DUX4 RNA (e.g., for reducing DUX4 expression or activity). In some embodiments, an oligonucleotide useful for targeting a DUX4 RNA (e.g., for reducing DUX4 expression or activity) comprises a region of complementarity to a DUX4 RNA. In some embodiments, the oligonucleotide may be designed to cause degradation of an mRNA. In some embodiments, the oligonucleotide may be designed to block translation of an mRNA. In some embodiments, an oligonucleotide may be designed to cause degradation and block translation of an mRNA.
[000102] An example human DUX4 gene sequence (NM_001293798.2) (SEQ ID NO: 23) is provided below.
ATGGCCCTCCCGACACCCTCGGACAGCACCCTCCCCGCGGAAGCCCGGGGACGAGG
ACGGCGACGGAGACTCGTTTGGACCCCGAGCCAAAGCGAGGCCCTGCGAGCCTGCT TTGAGCGGAACCCGTACCCGGGCATCGCCACCAGAGAACGGCTGGCCCAGGCCATC
GGCATTCCGGAGCCCAGGGTCCAGATTTGGTTTCAGAATGAGAGGTCACGCCAGCT
GAGGCAGCACCGGCGGGAATCTCGGCCCTGGCCCGGGAGACGCGGCCCGCCAGAA
GGCCGGCGAAAGCGGACCGCCGTCACCGGATCCCAGACCGCCCTGCTCCTCCGAGC
CTTTGAGAAGGATCGCTTTCCAGGCATCGCCGCCCGGGAGGAGCTGGCCAGAGAGA
CGGGCCTCCCGGAGTCCAGGATTCAGATCTGGTTTCAGAATCGAAGGGCCAGGCAC
CCGGGACAGGGTGGCAGGGCGCCCGCGCAGGCAGGCGGCCTGTGCAGCGCGGCCC
CCGGCGGGGGTCACCCTGCTCCCTCGTGGGTCGCCTTCGCCCACACCGGCGCGTGG
GGAACGGGGCTTCCCGCACCCCACGTGCCCTGCGCGCCTGGGGCTCTCCCACAGGG
GGCTTTCGTGAGCCAGGCAGCGAGGGCCGCCCCCGCGCTGCAGCCCAGCCAGGCCG
CGCCGGCAGAGGGGATCTCCCAACCTGCCCCGGCGCGCGGGGATTTCGCCTACGCC
GCCCCGGCTCCTCCGGACGGGGCGCTCTCCCACCCTCAGGCTCCTCGCTGGCCTCCG
CACCCGGGCAAAAGCCGGGAGGACCGGGACCCGCAGCGCGACGGCCTGCCGGGCC
CCTGCGCGGTGGCACAGCCTGGGCCCGCTCAAGCGGGGCCGCAGGGCCAAGGGGT
GCTTGCGCCACCCACGTCCCAGGGGAGTCCGTGGTGGGGCTGGGGCCGGGGTCCCC
AGGTCGCCGGGGCGGCGTGGGAACCCCAAGCCGGGGCAGCTCCACCTCCCCAGCCC
GCGCCCCCGGACGCCTCCGCCTCCGCGCGGCAGGGGCAGATGCAAGGCATCCCGGC
GCCCTCCCAGGCGCTCCAGGAGCCGGCGCCCTGGTCTGCACTCCCCTGCGGCCTGCT
GCTGGATGAGCTCCTGGCGAGCCCGGAGTTTCTGCAGCAGGCGCAACCTCTCCTAG
AAACGGAGGCCCCGGGGGAGCTGGAGGCCTCGGAAGAGGCCGCCTCGCTGGAAGC
ACCCCTCAGCGAGGAAGAATACCGGGCTCTGCTGGAGGAGCTTTAGGACGCGGGGT
CTAGGCCCGGTGAGAGACTCCACACCGCGGAGAACTGCCATTCTTTCCTGGGCATC
CCGGGGATCCCAGAGCCGGCCCAGGTACCAGCAGACCTGCGCGCAGTGCGCACCCC
GGCTGACGTGCAAGGGAGCTCGCTGGCCTCTCTGTGCCCTTGTTCTTCCGTGAAATT
CTGGCTGAATGTCTCCCCCCACCTTCCGACGCTGTCTAGGCAAACCTGGATTAGAGT
TACATCTCCTGGATGATTAGTTCAGAGATATATTAAAATGCCCCCTCCCTGTGGATC
CTATAG
[000103] In some embodiments, an oligonucleotide useful for targeting a DUX4 RNA (e.g., for reducing DUX4 expression or activity) is 15-40 (e.g., 15-40, 15-35, 15-30, 15-25, 15-20, 20-40, 20-35, 20-30, 20-25, 22-40, 22-35, 22-30, 22-28, 22-25, 24-40, 24-35, 24-30, 24-28, 24-25, 25- 40, 25-35, 25-30, 25-28, 30-40, 30-35, or 35-40) nucleotides in length. In some embodiments, an oligonucleotide useful for targeting a DUX4 RNA is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length, optionally 15-30, 24, or 25 nucleotides in length. [000104] In some embodiments, an oligonucleotide useful for targeting a DUX4 RNA (e.g., for reducing DUX4 expression or activity) 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, or 30) consecutive nucleotides to a DUX4 RNA as set forth in SEQ ID NO: 23. In some embodiments, an oligonucleotide useful for targeting a DUX4 RNA (e.g., for reducing DUX4 expression or activity) 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, or 30) consecutive nucleotides to the 3’UTR of a DUX4 RNA as set forth in SEQ ID NO: 23. In some embodiments, an oligonucleotide useful for targeting a DUX4 RNA (e.g., for reducing DUX4 expression or activity) 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, or 25) consecutive nucleotides to a target sequence as set forth in SEQ ID NO: 22 ( AGTT C AG AG AT AT ATT A A A AT GCCC ) . In some embodiments, an oligonucleotide useful for targeting a DUX4 RNA (e.g., for reducing DUX4 expression or activity) comprises at least 8 (e.g., at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) consecutive nucleotides of a sequence as set forth in SEQ ID NO: 21 (GGGCATTTTAATATATCTCTGAACT). In some embodiments, an oligonucleotide useful for targeting a DUX4 RNA (e.g., for reducing DUX4 expression or activity) comprises 24 or 25 consecutive nucleotides of a sequence as set forth in SEQ ID NO: 21. In some embodiments, an oligonucleotide useful for targeting a DUX4 RNA (e.g., for reducing DUX4 expression or activity) comprises the nucleotide sequence of SEQ ID NO: 21. In some embodiments, any one of the oligonucleotides provided herein is a PMO.
[000105] In some embodiments, it should be appreciated that methylation of the nucleobase uracil at the C5 position forms thymine. Thus, in some embodiments, a nucleotide or nucleoside having a C5 methylated uracil (or 5 -methyl-uracil) may be equivalently identified as a thymine nucleotide or nucleoside.
[000106] In some embodiments, any one or more of the thymine bases (T’s) in any one of the oligonucleotides provided herein (e.g., the oligonucleotide as set forth in SEQ ID NO: 21) may independently and optionally be uracil bases (U’s), and/or any one or more of the U’s in the oligonucleotides provided herein may independently and optionally be T’s.
Compositions
[000107] In some embodiments, compositions described herein comprise complexes (i.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., an oligonucleotide described herein), wherein the antibody comprises a heavy chain comprising a heavy chain variable region (VH) and a heavy chain constant region, and a light chain comprising a light chain variable region (VL) and a light chain constant region. In some embodiments, the antibody of such 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).
[000108] In some embodiments, compositions described herein comprise complexes (i.e., a plurality of complexes) wherein each complex comprises a structure of formula (I): [R'jn i-R2, in which each R1 independently comprises a compound comprising an oligonucleotide (e.g., an oligonucleotide described herein) and is covalently linked to R2, wherein R2 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. In some embodiments, each R1 of a complex is independently covalently linked to a different amino acid residue (e.g., lysine or cysteine) of R2.
[000109] In some embodiments, 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 R2). In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, compositions described herein comprise complexes in which the value of nl is 0. In some embodiments, the average value of nl of complexes of the composition is in the range of 0.5 to 5 (e.g., 0.5-1, 1-5, 1-4, 1-3, 1-2, 2-4, 3-5, 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, 0.8-1.3, 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, 0.9-1.2, 1-4.6, 1-4.5, 1-4.4, 1-4.3, 1-4.2, 1-3.5, 1-2.5, 1.1-5, 1.1-4.5, 1.1-4, 1.1- 3.5, 1.1-3, 1.1-2.5, 1.1-2.2, 1.2-5, 1.2-4.5, 1.2-4, 1.2-3.5, 1.2-3, 1.2-2.5, 1.2-2.2, 1.3-5, 1.3-4.5, 1.3-4, 1.3-3.5, 1.3-3, 1.3-2.5, 1.3-2.2, 1.4-5, 1.4-4.5, 1.4-4, 1.4-3.5, 1.4-3, 1.4-2.5, 1.4-2.2, 1.5-5, 1.5-4.5, 1.5-4, 1.5-3.5, 1.5-3, 1.5-2.5, 1.5-2.2, 1.6-3, 1.6-2.5, 1.6-2.2, 1.7-3, 1.7-2.5, 1.7-2.2, 1.8-
3, 1.8-2.5, or 1.8-2.2).
[000110] In some embodiments, 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. In some embodiments, antibody that is not conjugated to an oligonucleotide may be referred to as a compound comprising a structure of formula (I): [R'jni-R2, for which nl is zero. Accordingly, in some embodiments, a composition for administration to a subject in the methods described herein comprises compounds (e.g., complexes) comprising a structure of formula (I): [ R 1 J n i -R2, for which each R1 independently comprises a group comprising an oligonucleotide, R2 comprises an antibody and nl is independently an integer of zero or greater that reflects the number of instances of R1 in each compound (e.g., complex). In some embodiments, the fraction of compounds comprising a structure of formula (I): [R'jn i-R2, 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%. As such, in some embodiments, the average value of nl of complexes in a composition disclosed herein is in the range of 0.5 to 5 (e.g., 0.5-1, 1-5, 1-4, 1-3, 1-2, 2-4, 3-5, 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, 0.8-1.3, 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, 0.9-1.2, 1-4.6, 1-4.5, 1-4.4, 1-4.3, 1-4.2, 1-3.5, 1-2.5, 1.1-5, 1.1-4.5, 1.1-4, 1.1-3.5, 1.1-3, 1.1-2.5, 1.1-2.2, 1.2-5, 1.2-4.5, 1.2-4, 1.2-3.5, 1.2-3, 1.2-2.5, 1.2-2.2, 1.3-5, 1.3-4.5, 1.3-4, 1.3-3.5, 1.3-3, 1.3-2.5, 1.3-2.2, 1.4-5, 1.4-4.5, 1.4-4, 1.4-3.5, 1.4-3, 1.4-2.5, 1.4-2.2, 1.5-5, 1.5-4.5, 1.5-4, 1.5-3.5, 1.5-3, 1.5-2.5, 1.5-2.2, 1.6-3, 1.6- 2.5, 1.6-2.2, 1.7-3, 1.7-2.5, 1.7-2.2, 1.8-3, 1.8-2.5, or 1.8-2.2).
Formulations
[000111] Complexes provided herein are formulated in a manner suitable for pharmaceutical use. In some embodiments, 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. Accordingly, in some embodiments, it has been found that formulating complexes (e.g., complexes comprising a PMO covalently linked with a Fab) with histidine and/or sucrose is particularly advantageous for pharmaceutical use, e.g., as described herein. Thus, in some embodiments, provided herein are formulations (e.g., aqueous solutions, lyophilized forms) comprising complexes together with histidine and/or sucrose. In some embodiments, provided herein are formulations comprising complexes together with histidine and/or sucrose in frozen forms. In some embodiments, formulations described herein comprise complexes (e.g., a plurality of complexes comprising a PMO covalently linked with a Fab), histidine, and sucrose. In some embodiments, formulations comprising muscle-targeting complexes (e.g., complexes comprising a PMO covalently linked with a Fab) are formulated with histidine and/or sucrose in aqueous solutions. In some embodiments, formulation comprising a plurality of the complexes, histidine, and sucrose can be lyophilized (e.g., for storage). In some embodiments, the lyophilized formulation may be reconstituted (e.g., with water) for administration to a subject. 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. [000112] In some embodiments, formulations are provided herein that comprise complexes ( i.e ., a plurality of complexes), each of which complex comprises a phosphorodiamidate morpholino oligomer (PMO) covalently linked to an antibody. In some embodiments, provided herein is a formulation comprising complexes, in which each complex comprises a phosphorodiamidate morpholino oligomer (PMO) 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 histidine ( e.g ., L-histidine) and sucrose. In some embodiments, the antibody is an anti-TfRl antibody.
[000113] In some embodiments, formulations are provided that comprise complexes of the formula [ R 1 Jn i -R2, in which each R1 independently comprises a compound comprising an oligonucleotide (e.g., a PMO) and R2 comprises an antibody (e.g., anti-TfRl antibody), and in which nl is an integer of one or greater representing the number of instances of R1 in the complex. In some embodiments, formulations are provided that comprise a plurality of complexes wherein each complex comprises a structure of formula (I): [ R 1 J n i -R2, in which each R1 independently comprises a compound comprising an oligonucleotide (e.g., a PMO) and R2 comprises an antibody (e.g., anti-TfRl antibody), and in which in each complex nl is an integer independently of one or greater representing the number of instances of R1 in each complex. [000114] In some embodiments, 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. In some embodiments, the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 17 and a VL comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, the antibody is a Fab and comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and a light chain comprising the amino acid sequence of SEQ ID NO: 20.
[000115] In some embodiments, the value of nl of each complex in the formulation 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 (R2). In some embodiments, the value of nl of each complex in the formulation is independently and optionally selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27. In some embodiments, the value of nl of each complex in the formulation is independently and optionally selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 and 26. In some embodiments, the value of 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. In some embodiments, the average value of nl of complexes of the formulation is in the range of 1 to 3, 1 to 5, 1 to 10, 1 to 26, or 1 to 27.
[000116] In some embodiments, a formulation described herein comprises antibody that is not conjugated to an oligonucleotide (e.g., in trace amounts) and antibody conjugated to one or more oligonucleotides. In some embodiments, antibody that is not conjugated to an oligonucleotide antibody may be referred to as a compound comprising a structure of formula (I): [R'jn i-R2, for which nl is zero. Accordingly, in some embodiments, formulations are provided that comprise compounds (e.g., complexes) comprising a structure of formula (I): [R^ni-R2, for which each R1 independently comprises a group comprising an oligonucleotide, R2 comprises an antibody and nl is independently an integer of zero or greater that reflects the number of instances of R1 in each compound (e.g., complex). In some embodiments, the fraction of compounds comprising a structure of formula (I): [R'jn i-R2, in a formulation, for which nl is zero, compared with all compounds of that structure in the formulation 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%.
In some embodiments, the average value of nl of complexes of the formulation is in the range of 0.5 to 5 (e.g., 0.5-1, 1-5, 1-4, 1-3, 1-2, 2-4, 3-5, 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, 0.8-1.3, 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, 0.9-1.2, 1-4.6, 1-4.5, 1-4.4, 1-4.3, 1-
4.2, 1-3.5, 1-2.5, 1.1-5, 1.1-4.5, 1.1-4, 1.1-3.5, 1.1-3, 1.1-2.5, 1.1-2.2, 1.2-5, 1.2-4.5, 1.2-4, 1.2-
3.5, 1.2-3, 1.2-2.5, 1.2-2.2, 1.3-5, 1.3-4.5, 1.3-4, 1.3-3.5, 1.3-3, 1.3-2.5, 1.3-2.2, 1.4-5, 1.4-4.5, 1.4-4, 1.4-3.5, 1.4-3, 1.4-2.5, 1.4-2.2, 1.5-5, 1.5-4.5, 1.5-4, 1.5-3.5, 1.5-3, 1.5-2.5, 1.5-2.2, 1.6-3, 1.6-2.5, 1.6-2.2, 1.7-3, 1.7-2.5, 1.7-2.2, 1.8-3, 1.8-2.5, or 1.8-2.2).
[000117] In some embodiments, each instance of R1 in a complex herein (e.g., a complex of a formulation provided herein) is conjugated to a different amino acid residue of the antibody. In some embodiments, each different amino acid comprises an e-amino group (e.g., lysine, arginine). However, in some embodiments, each different amino acid to which R1 is covalently linked is a cysteine. In some embodiments, R1 is directly covalently linked to an amino acid residue of the antibody. However, in some embodiments, R1 is indirectly covalently linked to an amino acid of the antibody, e.g., covalently linked to a glycosylation site on the amino acid. In some embodiments, formulations are provided in which complexes for which R1 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 R1 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. [000118] In some embodiments, formulations provided herein comprise complexes that comprise a structure of formula (I): [R'jn i-R2, in which each instance of R1 in a complex of a formulation provided herein independently comprises a group of the formula (la):
Figure imgf000042_0001
(la), in which R3 comprises an oligonucleotide, e.g., a phosphorodiamidate morpholino oligomer (PMO); wherein R1 is covalently linked (e.g., indirectly or directly linked, e.g., directly linked) to R2 at attachment point A. In some embodiments, in each complex nl is independently an integer (e.g., of one or greater) representing the number of instances of R1 in each complex. In some embodiments, R2 comprises an antibody comprising a sequence as set forth in Table 2.
For example, in some embodiments, R2 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. In some embodiments, R2 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. In some embodiments, R2 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. In some embodiments, R2 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. In some embodiments, R2 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. In some embodiments, R2 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. In some embodiments, R2 comprises an antibody that is a Fab fragment. In some embodiments, R3 comprises an oligonucleotide, e.g., a phosphorodiamidate morpholino oligomer (PMO) comprising the base sequence of
GGGC ATTTT A AT AT AT CTCT G A ACT (SEQ ID NO: 21). In some embodiments, R2 comprises an antibody a Fab and each R1 is covalently linked at attachment point A to a different amino acid residue of the antibody Fab, optionally wherein each different amino acid residue is a lysine. In some embodiments, in each complex nl is independently an integer (e.g., an integer in the range of 1-27, 1-26, 1-10, 1-5, or 1-3). In some embodiments, formulations provided herein comprise complexes that comprise a structure of formula (I): [ R 1 J n i -R2, wherein nl is 0.
In some embodiments, the average value of nl of complexes of the composition is in the range of 0.5 to 5 (e.g., 0.5-1, 1-5, 1-4, 1-3, 1-2, 2-4, 3-5, 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, 0.8-1.3, 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, 0.9-1.2, 1-4.6, 1-4.5, 1-4.4, 1-4.3, 1-4.2, 1-3.5, 1-2.5, 1.1-5, 1.1-4.5, 1.1-4, 1.1-3.5, 1.1-3, 1.1-2.5, 1.1-2.2, 1.2-5, 1.2-4.5, 1.2-4,
1.2-3.5, 1.2-3, 1.2-2.5, 1.2-2.2, 1.3-5, 1.3-4.5, 1.3-4, 1.3-3.5, 1.3-3, 1.3-2.5, 1.3-2.2, 1.4-5, 1.4- 4.5, 1.4-4, 1.4-3.5, 1.4-3, 1.4-2.5, 1.4-2.2, 1.5-5, 1.5-4.5, 1.5-4, 1.5-3.5, 1.5-3, 1.5-2.5, 1.5-2.2,
1.6-3, 1.6-2.5, 1.6-2.2, 1.7-3, 1.7-2.5, 1.7-2.2, 1.8-3, 1.8-2.5, or 1.8-2.2).
[000119] In some embodiments, formulations provided herein comprise complexes that comprise a structure of formula (I): [R'jn i-R2, in which each instance of R1 in a complex of a formulation provided herein comprises a group of the formula (lb):
Figure imgf000043_0001
(lb), in which -pN indicates a base position of a phosphorodiamidate morpholino oligomer (PMO),
R1 is covalently linked (e.g., indirectly or directly linked, e.g., directly linked) to R2 at attachment point A; wherein -p reflects a phosphorodiamidate linkage, and wherein N corresponds to a nucleobase of adenine (A), cytosine (C), guanine (G), or thymine (T), such that the PMO has a base sequence of GGGC ATTTT A AT AT ATCTCTGAACT (SEQ ID NO: 21).
In some embodiments, in each complex nl is independently an integer (e.g., of one or greater) representing the number of instances of R1 in each complex, and each R1 is covalently linked to R2 at attachment point A. In some embodiments, R2 comprises an antibody comprising a sequence as set forth in Table 2. For example, in some embodiments, R2 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. In some embodiments, R2 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. In some embodiments, R2 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. In some embodiments, R2 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. In some embodiments, R2 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. In some embodiments, R2 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. In some embodiments, R2 comprises an antibody that is a Fab fragment. In some embodiments, in each complex nl is independently an integer (e.g., an integer in the range of 1-27, 1-26, 1-10, 1-5, or 1-3). In some embodiments, R2 comprises a Fab and each R1 is covalently linked at attachment point A to a different amino acid residue of the Fab, optionally wherein each different amino acid residue is a lysine. In some embodiments, formulations provided herein comprise complexes that comprise a structure of formula (I): [ R 1 J n i -R2, wherein nl is 0. In some embodiments, the average value of nl of complexes of the composition is in the range of 0.5 to 5 (e.g., 0.5-1, 1-5, 1-4, 1-3, 1-2, 2-4, 3-5, 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, 0.8-1.3, 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, 0.9-1.2, 1-4.6, 1-4.5, 1-4.4, 1-4.3, 1-4.2, 1-3.5, 1-2.5, 1.1-5, 1.1-4.5, 1.1-4, 1.1-3.5, 1.1-3, 1.1-2.5, 1.1-2.2, 1.2-5, 1.2-4.5, 1.2-4, 1.2-3.5, 1.2-3, 1.2-2.5, 1.2-2.2, 1.3-5, 1.3-4.5, 1.3-4, 1.3-3.5, 1.3-3, 1.3-2.5, 1.3-2.2, 1.4-5, 1.4-4.5, 1.4-4, 1.4-3.5, 1.4-3, 1.4-2.5, 1.4-2.2, 1.5-5, 1.5-
4.5, 1.5-4, 1.5-3.5, 1.5-3, 1.5-2.5, 1.5-2.2, 1.6-3, 1.6-2.5, 1.6-2.2, 1.7-3, 1.7-2.5, 1.7-2.2, 1.8-3, 1.8-2.5, or 1.8-2.2).
[000120] In some embodiments, formulations provided herein comprise complexes that comprise a structure of formula (I): [R'jn i-R2, in which each instance of R1 in a complex of a formulation provided herein comprises a group of the formula (Ic):
Figure imgf000045_0001
(Ic), wherein R1 is covalently linked (e.g., indirectly or directly linked, e.g., directly linked) to R2 at attachment point A.
[000121] In some embodiments, formulations provided herein comprise complexes that comprise a structure of formula (I): [R'jn i-R2, in which each instance of R1 in a complex of a formulation provided herein is:
Figure imgf000046_0001
(Ic), wherein R1 is covalently linked (e.g., indirectly or directly linked, e.g., directly linked) to R2 at attachment point A. In some embodiments, in each complex nl is independently an integer (e.g., of one or greater) representing the number of instances of R1 in each complex. In some embodiments, R2 comprises an antibody comprising a sequence as set forth in Table 2. For example, in some embodiments, R2 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. In some embodiments, R2 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. In some embodiments, R2 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. In some embodiments, R2 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. In some embodiments, R2 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. In some embodiments, R2 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. In some embodiments, R2 comprises an antibody that is a Fab fragment. In some embodiments, in each complex nl is independently an integer (e.g., an integer in the range of 1-27, 1-26, 1-10, 1-5, or 1-3). In some embodiments, R2 comprises a Fab and each R1 is covalently linked at attachment point A to a different amino acid residue of the Fab, optionally wherein each different amino acid residue is a lysine. In some embodiments, formulations described herein further comprise complexes that comprise a structure of formula (I): [R^ni-R2, wherein nl is 0. In some embodiments, the average value of nl of complexes of the composition is in the range of 0.5 to 5 (e.g., 0.5-1, 1-5, 1-4, 1-3, 1-2, 2-4, 3-5, 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, 0.8-1.3, 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, 0.9- 1.2, 1-4.6, 1-4.5, 1-4.4, 1-4.3, 1-4.2, 1-3.5, 1-2.5, 1.1-5, 1.1-4.5, 1.1-4, 1.1-3.5, 1.1-3, 1.1-2.5,
1.1-2.2, 1.2-5, 1.2-4.5, 1.2-4, 1.2-3.5, 1.2-3, 1.2-2.5, 1.2-2.2, 1.3-5, 1.3-4.5, 1.3-4, 1.3-3.5, 1.3-3, 1.3-2.5, 1.3-2.2, 1.4-5, 1.4-4.5, 1.4-4, 1.4-3.5, 1.4-3, 1.4-2.5, 1.4-2.2, 1.5-5, 1.5-4.5, 1.5-4, 1.5- 3.5, 1.5-3, 1.5-2.5, 1.5-2.2, 1.6-3, 1.6-2.5, 1.6-2.2, 1.7-3, 1.7-2.5, 1.7-2.2, 1.8-3, 1.8-2.5, or 1.8- 2.2). [000122] In some embodiments, formulations provided herein comprise complexes that comprise a structure of formula (Id):
Figure imgf000048_0001
(Id), in which -pN indicates a base position of a phosphorodiamidate morpholino oligomer (PMO); wherein -p reflects a phosphorodiamidate linkage, and wherein N corresponds to a nucleobase of adenine (A), cytosine (C), guanine (G), or thymine (T), such that the PMO has a base sequence of GGGCATTTTAATATATCTCTGAACT (SEQ ID NO: 21); wherein R2 comprises an antibody comprising a sequence as set forth in Table 2; wherein in each complex nl is independently an integer (e.g., of one or greater) representing the number of instances of the group enclosed by square brackets, wherein each instance of the group enclosed by square brackets is covalently linked to a different amino acid residue of the antibody (e.g., Fab), optionally wherein each different amino acid residue is a lysine. In some embodiments, R2 comprises an antibody (e.g., a Fab) comprising a sequence as set forth in Table 2. For example, in some embodiments, R2 comprises an antibody (e.g., Fab) 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-F1) 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. In some embodiments, R2 comprises an antibody (e.g., a Fab) comprising a heavy chain variable region (VH) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 17 and/or comprising a light chain variable region (VL) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 18. In some embodiments, R2 comprises an antibody (e.g., a Fab) comprising a VH comprising the amino acid sequence of SEQ ID NO: 17 and/or comprising a VL comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, R2 comprises an antibody (e.g., a Fab) comprising a heavy chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 19 and/or comprising a light chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 20. In some embodiments, R2 comprises an antibody (e.g., a Fab) comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and/or comprising a light chain comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, in each complex nl is independently an integer (e.g., an integer in the range of 1-27, 1-26, 1-10, 1-5, or 1-3). In some embodiments, R2 comprises an antibody (e.g., a Fab) that is covalently linked via different amino acid residue of the antibody (e.g., Fab), optionally wherein each different amino acid residue is a lysine. In some embodiments, formulations described herein further comprise complexes in which nl is 0. In some embodiments, the average value of nl of complexes of the composition is in the range of 0.5 to 5 (e.g., 0.5-1, 1-5, 1-4, 1-3, 1-2, 2-4, 3-5, 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, 0.8-1.3, 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, 0.9-1.2, 1-4.6, 1-4.5, 1-4.4, 1-4.3, 1-4.2, 1-3.5, 1-2.5, 1.1-5, 1.1-4.5, 1.1-4, 1.1-3.5, 1.1-3, 1.1-2.5, 1.1-2.2, 1.2-5, 1.2-4.5, 1.2-4,
1.2-3.5, 1.2-3, 1.2-2.5, 1.2-2.2, 1.3-5, 1.3-4.5, 1.3-4, 1.3-3.5, 1.3-3, 1.3-2.5, 1.3-2.2, 1.4-5, 1.4- 4.5, 1.4-4, 1.4-3.5, 1.4-3, 1.4-2.5, 1.4-2.2, 1.5-5, 1.5-4.5, 1.5-4, 1.5-3.5, 1.5-3, 1.5-2.5, 1.5-2.2,
1.6-3, 1.6-2.5, 1.6-2.2, 1.7-3, 1.7-2.5, 1.7-2.2, 1.8-3, 1.8-2.5, or 1.8-2.2).
[000123] In some embodiments, complexes provided in the formulations described herein comprise a structure of formula (A): oligonucleotide
Figure imgf000049_0001
antibod .y1
(A), wherein y is 0-15 (e.g., 3) and z is 0-15 (e.g., 4). In some embodiments, the antibody is an anti-TfRl antibody (e.g., the anti-TfRl antibody provided in Table 2). In some embodiments, the oligonucleotide is a PMO and comprises the base sequence of SEQ ID NO: 21. In some embodiments, the amide shown adjacent to the anti-TfRl antibody in the structure (A) results from a reaction with an amine of the anti-TfRl antibody, such as a lysine epsilon amine. In some embodiments, a complex described herein comprises an anti-TfRl Fab covalently linked via a lysine of the Fab to the 5’ end of a PMO. In some embodiments, the antibody comprises a sequence as set forth in Table 2. For example, in some embodiments, 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- Ll) 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. In some embodiments, the antibody comprises a heavy chain variable region (VH) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 17 and/or comprises a light chain variable region (VL) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 18. In some embodiments, the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 17 and/or comprises a VL comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, the antibody comprises a heavy chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 19 and/or comprises a light chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 20. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and/or comprises a light chain comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the antibody is a Fab fragment, a full- length IgG, a Fab' fragment, a F(ab')2 fragment, an scFv, or an Fv.
[000124] In some embodiments, provided is a formulation comprising complexes described herein wherein a concentration of the complexes in the formulation therein is between 1-50 mg/mL of the complex, optionally 10-50 mg/ml or 20-35 mg/mL (e.g., 1-10 mg/mL, 10-15 mg/mL, 15-20 mg/mL, 20-22 mg/mL, 22-24 mg/ml, 24-26 mg/ml, 24-25 mg/ml, 25-26 mg/ml, 22-25 mg/mL, 25-27 mg/mL, 27-29 mg/mL, 29-30 mg/mL, 25-30 mg/mL, 29-31 mg/ml, 30-31 mg/ml, 31-32 mg/ml, 30-32 mg/mL, 32-33 mg/ml, 32-35 mg/mL, 30-35 mg/mL, 35-40 mg/mL, 40-45 mg/mL, 45-50 mg/mL), optionally approximately 25 mg/mL (e.g., 25 mg/mL) or approximately 30 mg/mL (e.g., 30 mg/mL). [000125] In some embodiments, any one or a plurality of the complexes described herein is formulated with the histidine ( e.g ., L-histidine) and the sucrose in a lyophilized form (e.g., lyophilized powder).
[000126] In some embodiments, any one or a plurality of the complexes described herein is formulated with the histidine (e.g., L-histidine) and the sucrose in an aqueous solution. In some embodiments, the histidine (e.g., L-histidine) is present in the aqueous solution at a concentration in the range of 10-50 mM, 10-20 mM, 20 mM to 30 mM, or 20 mM to 40 mM, e.g., 20-22 mM, 22-24 mM, 24-25 mM, 25-26 mM, 24-26 mM, 26-27 mM, 24-27 mM, 27-28 mM, 28-29 mM, 29-30 mM, 27-30 mM, approximately 22-27 mM, approximately 23-26 mM, approximately 24-26 mM, approximately 26-28 mM, approximately 28-30 mM, approximately 30-32 mM, approximately 32-35 mM, approximately 35-40 mM, 40-45 mM, 45-50 mM, approximately 25 mM, or optionally, 25 mM. In some embodiments, 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, 8-15% w/v%, 9-15% w/v%, 9-11% w/v%, 9.5-11% w/v%, or for example, in the range of 5-6 w/v%, 6-7 w/v%, 7-8 w/v%, 8-9 w/v%, 9-10 w/v%, 10-11 w/v%, 11-12% w/v%, 10-12 w/v%, 12-13% w/v%, 13-14% w/v%, 12-14 w/v%, 14-15 w/v%, or 8-12 w/v%. In some embodiments, the sucrose is present in the aqueous solution at a concentration in the range of 8- 12 w/v% (e.g., 10 w/v%). In some embodiments, the aqueous solution has a pH in the range of 5.0 to 7.0, for example, 5.0-5.2, 5.2-5.4, 5.4-5.6, 5.6-5.8, 5.8-6.0, 5.9-6.0, 5.9-6.1, 6.0-6.1; for example, 5.5 to 6.5, or for example, in the pH range of 5.5-5.8, 5.8-6.0, 5.9-6.1, 6.0-6.1, 6.0-6.2, 6.2-6.4, 6.4-6.5, 6.5-6.7, 6.7-6.8, 6.8-6.9, 6.9-7.0, 7.0-7.1, or 5.8-6.2. In some embodiments, the aqueous solution has a pH in the range of 5.8-6.2 (e.g., 5.8-6.0, 5.8-6.1, 5.9-6.1). In some embodiments, the aqueous solution has a pH in the range of 5.9-6.2. In some embodiments, the aqueous solution has a pH in the range of 6.0-6.1 (e.g., about 6.0, or 6.0).
[000127] In some embodiments, provided is a formulation (e.g., in aqueous solution) described herein comprising one or a plurality of complexes, histidine, and sucrose, In some embodiments, any one of the formulations described herein is an aqueous solution, wherein the histidine (e.g., L-histidine) is present in the aqueous solution at a concentration of 25 mM, wherein the sucrose is present in the aqueous solution at a concentration of 10 w/v%, and wherein the aqueous solution is at a pH of about 6.0 (e.g., 6.0, 5.9-6.1).
[000128] In some embodiments, provided is a formulation (e.g., in aqueous solution) described herein comprising a plurality of complexes, histidine and sucrose, wherein the histidine (e.g., L- histidine) is present in the aqueous solution at a concentration of 25 mM, wherein the sucrose is present in the aqueous solution at a concentration of 10 w/v%, and wherein the pH of about 6.0 (e.g., 6.0, 5.9-6.1), and the concentration of complexes in the formulation is 10-50 mg/ml or 20- 35 mg/mL ( e.g ., 1-10 mg/mL, 10-15 mg/mL, 15-20 mg/mL, 20-22 mg/mL, 22-24 mg/ml, 24-26 mg/ml, 22-25 mg/mL, 25-27 mg/mL, 27-29 mg/mL, 29-31 mg/ml, 29-30 mg/mL, 30-31 mg/ml, 31-32 mg/ml, 25-30 mg/mL, 30-32 mg/mL, 32-35 mg/mL, 30-35 mg/mL, 35-40 mg/mL, 40-45 mg/mL, 45-50 mg/mL), optionally 25 mg/mL or 30 mg/mL. .
[000129] As described herein, in some embodiments, formulations provided herein comprise sucrose. In some embodiments, sucrose serves at least in part as a lyoprotectant. In some embodiments, 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). In some embodiments, the sucrose is from beet or sugarcane (e.g., beet sucrose, sugarcane sucrose). In some embodiments, a lyoprotectant other than sucrose may be used, e.g., trehalose, mannitol, lactose, polyethylene glycol, or polyvinyl pyrrolidone. However, in some embodiments, a collapse temperature modifier (e.g., dextran, ficoll, or gelatin) may be provided in a formulation.
[000130] In some embodiments, any one or a plurality of the complexes described herein is formulated with histidine and sucrose in a lyophilized form (e.g., lyophilized powder). In some embodiments, the lyophilized form (e.g., lyophilized powder) is obtained by lyophilization of any one of the aqueous solutions described herein.
[000131] In some embodiments, provided is a product (e.g., lyophilized formulation described herein), produced by a process comprising lyophilizing an aqueous solution of a formulation (e.g., in aqueous form) described herein.
[000132] In some embodiments, any one or a plurality of the complexes described herein is formulated with histidine and sucrose in a frozen form (e.g., a frozen aqueous solid). In some embodiments, the frozen form (e.g., frozen aqueous solid) is obtained by freezing of any one of the aqueous solutions described herein. 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).
[000133] In some embodiments, provided is a product (e.g., frozen formulation described herein), produced by a process comprising freezing an aqueous solution of a formulation (e.g., in aqueous form) described herein.
[000134] In some embodiments, a pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, administration. Typically, the route of administration is intravenous or subcutaneous. Methods of Use / Treatment
[000135] Complexes comprising an anti-TfRl antibody (e.g., Fab) covalently linked to a molecular payload (e.g., oligonucleotide, e.g., phosphorodiamidate morpholino oligomer (PMO)) as described herein are effective in treating FSHD. In some embodiments, complexes are effective in treating Type 1 FSHD. In some embodiments, complexes are effective in treating Type 2 FSHD. In some embodiments, FSHD is associated with deletions in D4Z4 repeat regions on chromosome 4 which contain the DUX4 gene. In some embodiments, FSHD is associated with mutations in the SMCHD1 gene.
[000136] In some embodiments, a subject may be a human subject, a non-human primate subject, a rodent subject, or any suitable mammalian subject. In some embodiments, the non human primate subject is a cynomolgus monkey. In some embodiments, the subject is human.
In some embodiments, the subject is a human subject that is between 2-60 (e.g., 2-60, 2-50, 2- 40, 2-30, 2-20, 2-10) years of age. In some embodiments, the subject is a human subject that is between 5-30 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) years old. In some embodiments, the subject is a human subject that is between 5-12 (e.g., 5, 6, 7, 8, 9, 10, 11, or 12) years of age. In some embodiments, the subject is a human subject that is between 4-16 (e.g., 4-16, 5-16, 6-16, 7-16, 8-16, 9-16, 10-16, 11-16, 12- 16, 13-16, 14-16, 15-16, 4-15, 5-15, 6-15, 7-15, 8-15, 9-15, 10-15, 11-15, 12-15, 13-15, 14-15, 4-14, 5-14, 6-14, 7-14, 8-14, 9-14, 10-14, 11-14, 12-14, 13-14, 4-13, 5-13, 6-13, 7-13, 8-13, 9- 13, 10-13, 11-13, 12-13, 4-12, 5-12, 6-12, 7-12, 8-12, 9-12, 10-12, 11-12, 4-11, 5-11, 6-11, 7-11, 8-11, 9-16, 10-11, 4-10, 5-10, 6-10, 7-10, 8-10, 9-10, 4-9, 5-9, 6-9, 7-9, 8-9, 4-9, 5-9, 6-9, 7-9, 8-9, 4-8, 5-8, 6-8, 7-8, 4-7, 5-7, 6-7, 4-6, 5-6, or 4-5) years of age. In some embodiments, the subject is a human subject that is about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 years of age. In some embodiments, a subject may have myotonic dystrophy. In some embodiments, a subject has elevated expression of the DUX4 gene outside of fetal development and the testes.
In some embodiments, the subject has facioscapulohumeral muscular dystrophy of Type 1 or Type 2. In some embodiments, the subject having FSHD has mutations in the SMCHD1 gene.
In some embodiments, the subject having FSHD has deletion mutations in D4Z4 repeat regions on chromosome 4. In some embodiments, a subject is ambulant. In some embodiments, a subject is non-ambulant.
[000137] An aspect of the disclosure includes methods and/or uses involving administering to a subject a formulation comprising an effective amount of complex(es) as described herein. In some embodiments, an effective amount of a pharmaceutical composition that comprises complex(es) comprising an antibody (e.g., anti-TfRl antibody, anti-TfRl Fab) described herein covalently linked to an oligonucleotide (e.g., antisense oligonucleotide targeting a mutant allele of DUX4) described herein can be administered to a subject in need of treatment. In some embodiments, a pharmaceutical composition is administered systemically. In some embodiments, 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. In some embodiments, administration may be performed by intravenous, intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra- articular, intrasynovial, or intrathecal routes. In some embodiments, a pharmaceutical composition comprising complex(es) as described herein is administered by infusion (e.g., intravenous infusion). In some embodiments, a pharmaceutical composition may be in solid form, aqueous form, or a liquid form. In some embodiments, an aqueous or liquid form may be nebulized or lyophilized. In some embodiments, a nebulized or lyophilized form may be reconstituted with an aqueous or liquid solution. In some embodiments, provided are methods of and/or uses for treating a subject having one or more deletions of a D4Z4 repeat in chromosome 4 that is associated with facioscapulohumeral muscular dystrophy (FSHD), comprising administering to the subject the formulation described herein with an effective amount of the complex(es).
[000138] In some embodiments, provided are methods of and/or uses for reducing DUX4 expression or activity in a cell (e.g., muscle cell) or subject, the method comprising contacting the cell with or administering to the subject the formulation described herein with an effective amount of the complex(es) for promoting internalization of the molecular payload in the cell. In some embodiments, the method comprises administering a lyophilized form (e.g., lyophilized powder) of a formulation comprising a plurality of complexes 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. For example, in some embodiments, a lyophilized form of the formulation comprising a complex or plurality of complexes 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.
[000139] In some embodiments, a pharmaceutical composition that comprises complex(es) comprising an anti-TfRl antibody covalently linked to a molecular payload (e.g., anti-TfRl antibody linked to PMO) is administered via site- specific or local delivery techniques. Examples of these techniques include implantable depot sources of the complex, local delivery catheters, site specific carriers, direct injection, or direct application. [000140] In some embodiments, a pharmaceutical composition that comprises a complex comprising an anti-TfRl antibody (e.g., a Fab) covalently linked to a molecular payload (e.g., oligonucleotide, e.g., phosphorodiamidate morpholino oligomer (PMO)) 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. In some embodiments, 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. [000141] 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.
[000142] 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 FSHD including muscle mass loss and muscle atrophy, primarily in the muscles of the face, shoulder blades, and upper arms. In some embodiments, 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.
EXAMPLES
Example 1. Effects of conjugates containing an anti-TfRl Fab covalently linked to a DUX4-targeting oligonucleotide in FSHD patient-derived immortalized myoblasts [000143] The anti-TfRl Fab comprising the VH and VL sequences shown in Table 2 was covalently linked (through lysine conjugation) via a linker comprising a Valine-Citmlline sequence to a DUX4 -targeting oligonucleotide (SEQ ID NO: 21) to achieve enhanced muscle delivery of the oligonucleotide. The oligonucleotide is a PMO and targets a sequence in the 3’- UTR of the DUX4 transcript. The activity of the conjugate was evaluated in the C6(AB 1080) immortalized FSHD1 cell line, which has significant levels of surface TfRl expression and activation of DUX4 transcriptome markers (MBD3L2, TRIM43, ZSCAN4). It was demonstrated that receptor-mediated delivery of the PMO (SEQ ID NO: 21) by the anti-TfRl Fab into muscle cells resulted in approximately 75% reduction of DUX4 transcriptome biomarkers at an 8 nM PMO concentration, whereas equivalent unconjugated PMO shows no significant biomarker reduction compared to vehicle treated cells (FIG. 1). The results show that conjugating with anti-TfRl Fab enhances delivery of therapeutic oligonucleotides to muscle cells for the treatment of FSHD.
[000144] As used in this Example, the term ‘unconjugated’ indicates that the oligonucleotide was not conjugated to an antibody.
Experimental Procedures
Cell culture and test article treatment
[000145] C6 (AB1080) immortalized FSHD myoblasts were seeded to a density of 45,000 cells/well on a 96-well plate (ThermoFisher Scientific) in Skeletal Growth Media (CAT# C- 23060, Promocell) with Supplementary mix (C-39365, Promocell) and 1% Penstrep (15140-122, Gibco). Growth media was replaced with differentiation media, NbActiv4 (Brainbits) and 1% Pen/Strep (Gibco), 24 hours later. The cells were treated with unconjugated DUX4-targeting oligonucleotide, the conjugate at a PMO concentration of 8 nM, vehicle in technical replicates for 4 hours prior to washout with 1XPBS (10010023, Gibco) one time. Conditioned differentiation media was immediately added back to wells and the cells were harvested 5 days later for downstream analyses.
RNA extraction and qPCR
[000146] Total RNA was extracted from cell monolayers with the RNeasy 96 Kit (Qiagen) in accordance with the manufacturer's instructions. The RNA was quantified with the Biotek Plate Reader and diluted to 50 ng per sample with Nuclease-Free Water (Qiagen) and reverse transcribed with qScript cDNA SuperMix (QuantaBio). Gene expression was analyzed by qPCR with specific TaqMan assays (ThermoFisher) by measuring levels of TRIM43 (Hs00299174_m 1 ), MBD3F2 (Hs00544743_ml), ZSCAN4 (Hs00537549_ml) and RPF13A (Hs04194366_gl) transcripts. Two-step amplification reactions and fluorescence measurements for Ct determination were conducted on a QuantS tudio 7 instrument (Thermo Scientific). Fog fold changes in the expression of transcripts of interest were calculated according to the 2-DD( method using RPF13A as the reference gene and cells exposed to vehicle as the control group. Data are expressed as means ± S.D.
[000147] Example 2. Freeze-Thaw Stress Study of Exemplary anti-TfRl Fab covalently linked to a DUX4-targeting Oligonucleotide at Various Concentrations [000148] The anti-TfRl Fab comprising the VH and VL sequences shown in Table 2 was covalently linked (through lysine conjugation) via a linker comprising a Valine-Citrulline sequence to a DUX4 -targeting oligonucleotide of SEQ ID NO. 21 (“anti-TfRl Fab-DUX4- targeting oligonucleotide conjugate”), at the concentration of 13.1 lmg/mL. The conjugate was supplied into the formulation buffer of 25 mM Histidine, 10% Sucrose, at pH 6.0 (“Formulation 1”). This conjugate was concentrated to approximately 20 mg/mL, 30 mg/mL, and 40 mg/mL using Vivaspin 6 centrifugal membrane filters (30,000 mwco). The concentration was measured using the standard BCA method at 480nm. A F/T (freeze/thaw) study was performed by taking an aliquot of approximately 200 pL from each of the concentrated conjugates and incubating at - 80°C for 3 hours for each cycle, and thawing thoroughly at room temperature for 1-2 hours. A total of 5 cycles was performed and aliquots of 60 pL each sample after each of cycles 0, 1, 3, and 5 were taken for visual analysis. No change in color and appearance was observed in any of the test samples at different specified concentrations of the conjugate, for each of the cycles 0, 1, 3, and 5, as shown in Table 3. *C/C indicates that the sample was clear and colorless.
Table 3. F/T stress study of conjugates in formulation samples at Various Conjugate Concentrations
Figure imgf000057_0001
[000149] Example 3. SEC-UV Analysis of Exemplary anti-TfRl Fab conjugated to a DUX4-targeting Oligonucleotide at Various Concentrations Following Freeze-Thaw Stress Study of Conjugates
[000150] Following the freeze-thaw stress study of the anti-TfRl Fab-DUX4-targeting oligonucleotide conjugate in Formulation 1 as disclosed in Example 2, SEC-UV analysis was measured after cycles 0, 1, 3, and 5. SEC-UV was conducted via standard analytical methods, as described as follows:
[000151] Sample preparation: All samples were diluted to 1 mg/mL concentration prior to analysis [000152] Chromatographic conditions: Waters ACQUITY UPLC Protein BEH SEC Column 125 A, 1.7 pm, 4.6 x 150 mm + guard; Mobile phase: 0.2 M potassium phosphate pH 6.8, 0.2 M KC1 - Isocratic elution, 15% IPA; Flow rate: 0.35 mL/min, Column temperature 30 °C, Sample and Blank Injection: 10 pg; HPLC System: Dionex Ultimate-3000 UPLC system [000153] 10 pg injection from 1 mg/mL working solution
[000154] For the samples of the anti-TfRl Fab-DUX4-targeting oligonucleotide conjugates at concentrations (conjugate in formulation) of 20 mg/ml, 30 mg/ml, and 40 mg/ml, after cycles 0, 1, 3, and 5, no significant change was observed in the SEC-UV peaks of these test samples and starting material (anti-TfRl Fab-DUX4-targeting oligonucleotide conjugates in 25 mM histidine, 10% sucrose, pH 6.0). These SEC-UV results indicate that Formulation 1 (25 mM Histidine, 10% Sucrose, at pH 6.0) of the anti-TfRl Fab-DUX4-targeting oligonucleotide conjugate shows good stability.
ADDITIONAL EMBODIMENTS
1. A formulation comprising complexes that comprise a phosphorodiamidate morpholino oligomer (PMO) covalently linked to an anti-transferrin receptor 1 (TfRl) antibody, wherein the PMO comprises a nucleotide sequence having a region of complementarity of at least 8 consecutive nucleotides to the sequence set forth as SEQ ID NO: 22, wherein 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 NO: 6, and wherein the complexes are formulated with histidine and sucrose.
2. A formulation comprising: complexes of the formula [R'jn i-R2, wherein each R1 independently comprises a group of the formula:
Figure imgf000059_0001
R2 comprises an antibody; and
R3 is a phosphorodiamidate morpholino oligomer (PMO), wherein R1 is covalently linked to R2 at attachment point A; wherein the PMO comprises a nucleotide sequence having a region of complementarity of at least 8 consecutive nucleotides to the sequence set forth as SEQ ID NO: 22, wherein nl is an integer of one or greater representing the number of instances of R1, wherein each instance of R1 is covalently linked to a different amino acid residue of the antibody, optionally wherein each different amino acid residue is a lysine; and wherein the complexes are formulated with histidine and sucrose, optionally wherein the antibody is an anti-TfRl antibody, and optionally wherein the average value of nl of complexes in the formulation is in the range of 1 to 5.
3. The formulation of embodiment 2, wherein 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 NO: 6 or 16.
4. The formulation of any one of embodiments 1-3, wherein the formulation is in a lyophilized form. 5. The formulation of any one of embodiments 1-3, wherein the formulation is in an aqueous solution.
6. The formulation of embodiment 5, wherein the histidine is present in the aqueous solution at a concentration in the range of 10 mM to 50 mM.
7. The formulation of embodiment 5 or 6, wherein the sucrose is present in the aqueous solution at a concentration in the range of 5 % to 15 % weight per volume (w/v%).
8. The formulation of any one of embodiments 5 to 7, wherein the aqueous solution has a pH in the range of 5.0 to 7.0.
9. The formulation of any one of embodiments 5-8, wherein the histidine is present in the aqueous solution at a concentration of 25 mM, and/or the sucrose is present in the aqueous solution at a concentration of 10 w/v%, and/or the aqueous solution is at a pH of 6.0.
10. The formulation of any one of embodiments 1-9, wherein the antibody is a Fab fragment, a full-length IgG, a Fab' fragment, a F(ab')2 fragment, an scFv, or an Fv.
11. The formulation of embodiments 10, wherein the antibody is a Fab fragment.
12. The formulation of any one of embodiments 1-11, wherein the antibody comprises a heavy chain variable region (VH) comprising an amino acid sequence at least 85% identical to SEQ ID NO: 17; and/or wherein the antibody comprises a light chain variable region (VL) comprising an amino acid sequence at least 85% identical to SEQ ID NO: 18, optionally wherein 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.
13. The formulation of any one of embodiments 1-12, wherein the antibody comprises a heavy chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 19; and/or wherein the antibody comprises a light chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 20, optionally wherein the antibody 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. 14. The formulation of any one of embodiments 1-13, wherein the PMO comprises a nucleotide sequence that is 15-30 nucleotides in length.
15. The formulation of any one of embodiments 1-14, wherein the PMO comprises a nucleotide sequence having a region of complementarity of at least 20 consecutive nucleotides to the sequence set forth as SEQ ID NO: 22.
16. The formulation of any one of embodiments 1-15, wherein the PMO comprises at least 8 consecutive nucleotides of a nucleotide sequence as set forth in SEQ ID NO: 21, optionally wherein the PMO comprises the nucleotide sequence as set forth in SEQ ID NO: 21.
17. The formulation of any one of embodiments 2-16, wherein each R1 comprises a group of formula:
Figure imgf000061_0001
, in which -pN- indicates a base position of a phosphorodiamidate morpholino oligomer (PMO), R1 is covalently linked to R2 at attachment point A; wherein -p reflects a phosphorodiamidate linkage, and wherein N corresponds to a nucleobase of adenine (A), cytosine (C), guanine (G), or thymine (T); such that the PMO has a nucleobase sequence of GGGC ATTTT A AT AT AT CTCT G A ACT (SEQ ID NO: 21). 18. The formulation of any one of embodiments 2-16, wherein each R1 comprises a group of formula:
Figure imgf000062_0001
19. The formulation of any one of embodiments 1-18, wherein the complexes are present in the formulation at a concentration in the range between 10 mg/mL to 50 mg/mL.
20. A method of reducing DUX4 expression or activity in a cell or subject, the method comprising contacting the cell with or administering to the subject the formulation of any one of embodiments 1-19 with an effective amount of the complex for promoting internalization of the molecular payload in the cell, optionally wherein the cell is a muscle cell.
21. The method of embodiment 20, wherein the complex reduces DUX4 protein level. 22. A method of treating a subject having one or more deletions of a D4Z4 repeat in chromosome 4 that is associated with facioscapulohumeral muscular dystrophy (FSHD), comprising administering to the subject the formulation of any one of embodiments 1-19.
23. A complex comprising a structure of formula [R'jn i-R2, wherein each R1 comprises a group of the formula:
Figure imgf000063_0001
wherein R2 comprises a Fab, and wherein the Fab comprises a CDR-H1, a CDR-H2, a CDR-H3, a CDR-L1, a CDR-L2, and a CDR-L3 selected from Table 2, optionally wherein the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 17 and a VL comprising the amino acid sequence of SEQ ID NO: 18, further optionally wherein the Fab comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and a light chain comprising the amino acid sequence of SEQ ID NO: 20, wherein -pN- indicates a base position of a phosphorodiamidate morpholino oligomer, wherein -p reflects a phosphorodiamidate linkage, and wherein N corresponds to a nucleobase of adenine (A), cytosine (C), guanine (G), or thymine (T) , such that the oligonucleotide PMO has a nucleobase sequence of GGGC ATTTT A AT AT AT CTCT G A ACT (SEQ ID NO: 21); R1 is covalently linked to R2 at attachment point A; and wherein nl is an integer of one or greater representing the number of instances of R1, wherein each instance of R1 is covalently linked to a different amino acid residue of the Fab, optionally wherein each different amino acid residue is a lysine. 24. A complex comprising a structure of formula [R'jn i-R2, wherein each R1 comprises a group of the formula:
Figure imgf000064_0001
wherein R2 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 R2 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 R2 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 R1 is covalently linked to R2 at attachment point A; wherein nl is an integer of one or greater representing the number of instances of R1, wherein each instance of R1 is covalently linked to a different amino acid residue of the Fab, optionally wherein each different amino acid residue is a lysine. 25. A formulation comprising a plurality of complexes of embodiment 23 or embodiment 24, and histidine at a concentration of 25 mM, and sucrose at a concentration of 10 w/v%, wherein the formulation is an aqueous solution and is at a pH of 6.0.
26. A lyophilized form of the formulation of embodiment 25.
27. A product produced by a process comprising lyophilizing the formulation of embodiment 25.
28. A frozen form of the formulation of embodiment 25.
29. A product produced by a process comprising freezing the formulation of embodiment 25.
EQUIVALENTS AND TERMINOLOGY
[000155] The disclosure illustratively described herein suitably can be practiced in the absence of any element or elements, limitation or limitations that are not specifically disclosed herein. Thus, for example, in each instance herein any of the terms “comprising”, “consisting essentially of’, and “consisting of’ may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the disclosure. Thus, it should be understood that although the present disclosure has been specifically disclosed by preferred embodiments, optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this disclosure.
[000156] In addition, where features or aspects of the disclosure are described in terms of Markush groups or other grouping of alternatives, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group or other group.
[000157] It should be appreciated that, in some embodiments, sequences presented in the sequence listing may be referred to in describing the structure of an oligonucleotide or other nucleic acid. In such embodiments, the actual oligonucleotide or other nucleic acid may have one or more 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 intemucleotide 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.
[000158] The use of the terms “a” and “an” and “the” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non- claimed element as essential to the practice of the invention.
[000159] Embodiments of this invention are described herein. Variations of those embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. [000160] The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

CLAIMS What is claimed is:
1. A complex comprising a structure of formula (I): [R'jni-R2, wherein each R1 comprises a group of the formula (la):
Figure imgf000067_0001
(la), wherein R3 comprises a phosphorodiamidate morpholino oligomer (PMO) comprising a region of complementarity of at least 8 consecutive nucleotides to the nucleotide sequence as set forth in SEQ ID NO: 22; wherein R2 comprises a Fab, and wherein the Fab comprises a CDR-H1, a CDR-H2, a CDR-H3, a CDR-F1, a CDR-F2, and a CDR-F3 selected from Table 2, optionally wherein the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 17 and a VF comprising the amino acid sequence of SEQ ID NO: 18, further optionally wherein the Fab comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and a light chain comprising the amino acid sequence of SEQ ID NO: 20, wherein R1 is covalently linked to R2 at attachment point A; and wherein nl is an integer of one or greater representing the number of instances of R1, wherein each instance of R1 is covalently linked to a different amino acid residue of the Fab, optionally wherein each different amino acid residue is a lysine.
2. The complex of claim 1, wherein the region of complementarity is at least 16 nucleotides in length.
3. The complex of claim 1 or claim 2, wherein the PMO comprises at least 8 consecutive nucleotides of the nucleotide sequence as set forth in SEQ ID NO: 21, optionally wherein the PMO comprises at least 16 consecutive nucleotides of the nucleotide sequence as set forth in SEQ ID NO: 21.
4. A formulation comprising complexes that comprise a phosphorodiamidate morpholino oligomer (PMO) covalently linked to an anti-transferrin receptor 1 (TfRl) antibody, wherein the PMO comprises a nucleotide sequence having a region of complementarity of at least 8 consecutive nucleotides to the sequence set forth as SEQ ID NO: 22, wherein 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, and wherein the complexes are formulated with histidine and sucrose.
5. A formulation comprising complexes comprising a structure of formula [R'jn i-R2, wherein each R1 independently comprises a group of the formula (la):
Figure imgf000068_0001
(la),
R2 comprises an antibody; and
R3 comprises a phosphorodiamidate morpholino oligomer (PMO), wherein R1 is covalently linked to R2 at attachment point A; wherein the PMO comprises a nucleotide sequence having a region of complementarity of at least 8 consecutive nucleotides to the sequence set forth as SEQ ID NO: 22, wherein nl is an integer of one or greater representing the number of instances of R1, wherein each instance of R1 is covalently linked to a different amino acid residue of the antibody, optionally wherein each different amino acid residue is a lysine; and wherein the complexes are formulated with histidine and sucrose, optionally wherein the antibody is an anti-TfRl antibody, and optionally wherein the average value of nl of complexes in the formulation is in the range of 0.5 to 5.
6. The formulation of claim 5, wherein 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.
7. The formulation of any one of claims 4 to 6, wherein the formulation is in a lyophilized form, an aqueous solution, or a frozen solid form.
8. The formulation of claim 7, wherein the formulation is in an aqueous solution and wherein the histidine is present in the aqueous solution at a concentration in the range of 10 mM to 50 mM.
9. The formulation of claim 7 or 8, wherein the formulation is in an aqueous solution and wherein the sucrose is present in the aqueous solution at a concentration in the range of 5 % to 15 % weight per volume (w/v%).
10. The formulation of any one of claims 7 to 9, wherein the formulation is in an aqueous solution and wherein the aqueous solution has a pH in the range of 5.0 to 7.0.
11. The formulation of any one of claims 7 to 10, wherein the formulation is in an aqueous solution and wherein the histidine is present in the aqueous solution at a concentration of 25 mM, and/or the sucrose is present in the aqueous solution at a concentration of 10 w/v%, and/or the aqueous solution is at a pH of 6.0.
12. The formulation of any one of claims 4 to 11, wherein the antibody is a Fab fragment, a full-length IgG, a Fab' fragment, a F(ab')2 fragment, an scFv, or an Fv.
13. The formulation of claim 12, wherein the antibody is a Fab fragment.
14. The formulation of any one of claims 4 to 13, 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 a VL comprising the amino acid sequence of SEQ ID NO: 18.
15. The formulation of any one of claims 4 to 14, 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 a light chain comprising the amino acid sequence of SEQ ID NO: 20.
16. The formulation of any one of claims 4 to 15, wherein the PMO comprises a nucleotide sequence that is 15-30 nucleotides in length.
17. The formulation of any one of claims 4 to 16, wherein the PMO comprises a nucleotide sequence having a region of complementarity of at least 20 consecutive nucleotides to the sequence set forth as SEQ ID NO: 22.
18. The formulation of any one of claims 4 to 17, wherein the PMO comprises at least 8 consecutive nucleotides of a nucleotide sequence as set forth in SEQ ID NO: 21, optionally wherein the PMO comprises the nucleotide sequence as set forth in SEQ ID NO: 21.
19. The formulation of any one of claims 5 to 18, wherein each R1 comprises a group of the formula (lb):
Figure imgf000071_0001
(lb), in which -pN indicates a base position of a phosphorodiamidate morpholino oligomer (PMO), wherein -p reflects a phosphorodiamidate linkage, wherein N corresponds to a nucleobase of adenine (A), cytosine (C), guanine (G), or thymine (T); such that the PMO has a nucleobase sequence of GGGC ATTTT AATAT ATCTCTGA ACT (SEQ ID NO: 21), and wherein R1 is covalently linked to R2 at attachment point A.
20. The formulation of any one of claims 5 to 18, wherein each R1 comprises a group of the formula (Ic):
Figure imgf000072_0001
21. The formulation of any one of claims 4 to 19, wherein the complexes are present in the formulation at a concentration in the range between 10 mg/mL to 50 mg/mL.
22. A method of reducing DUX4 expression or activity in a cell or subject, the method comprising contacting the cell with or administering to the subject the formulation of any one of claims 4 to 21 with an effective amount of the complex for promoting internalization of the molecular payload in the cell, optionally wherein the cell is a muscle cell.
23. The method of claim 22, wherein the complex reduces DUX4 protein level.
24. A method of treating a subject having one or more deletions of a D4Z4 repeat in chromosome 4 that is associated with facioscapulohumeral muscular dystrophy (FSHD), comprising administering to the subject the formulation of any one of claims 5 to 23.
25. A complex comprising a structure of formula (I) [R'jni-R2, wherein each R1 comprises a group of the formula (lb):
Figure imgf000073_0001
(lb), wherein R2 comprises a Fab, and wherein the Fab comprises a CDR-H1, a CDR-H2, a CDR-H3, a CDR-L1, a CDR-L2, and a CDR-L3 selected from Table 2, optionally wherein the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 17 and a VL comprising the amino acid sequence of SEQ ID NO: 18, further optionally wherein the Fab comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and a light chain comprising the amino acid sequence of SEQ ID NO: 20, wherein -pN indicates a base position of a phosphorodiamidate morpholino oligomer, wherein -p reflects a phosphorodiamidate linkage, and wherein N corresponds to a nucleobase of adenine (A), cytosine (C), guanine (G), or thymine (T), such that the oligonucleotide PMO has a nucleobase sequence of GGGC ATTTT AAT ATATCTCTGAACT (SEQ ID NO: 21); wherein R1 is covalently linked to R2 at attachment point A; and wherein nl is an integer of one or greater representing the number of instances of R1, wherein each instance of R1 is covalently linked to a different amino acid residue of the Fab, optionally wherein each different amino acid residue is a lysine.
26. A complex comprising a structure of formula (I): [R'jni-R2, wherein each R1 comprises a group of the formula (Ic):
Figure imgf000074_0001
(Ic), wherein R2 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 R2 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 R2 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 R1 is covalently linked to R2 at attachment point A; wherein nl is an integer of one or greater representing the number of instances of R1, wherein each instance of R1 is covalently linked to a different amino acid residue of the Fab, optionally wherein each different amino acid residue is a lysine.
27. A complex comprising a structure of formula (Id):
Figure imgf000075_0001
(Id), wherein R2 comprises a Fab, and wherein the Fab comprises a CDR-H1, a CDR-H2, a CDR-H3, a CDR-L1, a CDR-L2, and a CDR-L3 selected from Table 2, optionally wherein the Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 17 and a VL comprising the amino acid sequence of SEQ ID NO: 18, further optionally wherein the Fab comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and a light chain comprising the amino acid sequence of SEQ ID NO: 20, wherein -pN indicates a base position of a phosphorodiamidate morpholino oligomer, wherein -p reflects a phosphorodiamidate linkage, and wherein N corresponds to a nucleobase of adenine (A), cytosine (C), guanine (G), or thymine (T), such that the oligonucleotide PMO has a nucleobase sequence of GGGC ATTTT AAT ATATCTCTGAACT (SEQ ID NO: 21); wherein R1 is covalently linked to R2 at attachment point A; and wherein nl is an integer of one or greater representing the number of instances of R1, wherein each instance of R1 is covalently linked to a different amino acid residue of the Fab, optionally wherein each different amino acid residue is a lysine.
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