United states
Aspects of the disclosure relate to complexes comprising a muscle-targeting agent covalently linked to a molecular payload. In some embodiments, the muscle-targeting agent specifically binds to an internalizing cell surface receptor on muscle cells. In some embodiments, the molecular payload promotes the expression or activity of a functional dystrophin protein. In some embodiments, the molecular payload is an oligonucleotide, such as an antisense oligonucleotide, e.g., an oligonucleotide that causes exon skipping in a mRNA expressed from a mutant DMD allele.
1 . A method of delivering an oligonucleotide to a cell in a subject, the method comprising administering to the subject a complex comprising an anti-transferrin receptor antibody covalently linked to an oligonucleotide,
wherein the anti-transferrin receptor antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein:
the VH comprises a heavy chain complementarity determining region 1 (CDR-H1), a heavy chain complementarity determining region 2 (CDR-H2), and a heavy chain complementarity determining region 3 (CDR-H3) of a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 438, and comprises humanized framework regions, and
the VL comprises a light chain complementarity determining region 1 (CDR-L1), a light chain complementarity determining region 2 (CDR-L2), and a light chain complementarity determining region 3 (CDR-L3) of a light chain variable region comprising the amino acid sequence of SEQ ID NO: 439, and comprises humanized framework regions; and
wherein the oligonucleotide is 20 to 29 nucleotides in length and comprises a region of complementarity to a dystrophin (DMD) sequence as set forth in SEQ ID NO: 295, wherein the region of complementarity is at least 12 nucleotides in length, and wherein the oligonucleotide is a phosphorodiamidate morpholino oligomer (PMO).
2 . The method of claim 1 , wherein the oligonucleotide is an exon-skipping oligonucleotide that induces skipping of exon 44 of DMD.
3 . The method of claim 2 , wherein the oligonucleotide is 26 nucleotides in length.
4 . The method of claim 3 , wherein the region of complementarity is at least 16 nucleotides in length.
5 . The method of claim 4 , wherein the region of complementarity is complementary to a target sequence of an oligonucleotide as set forth in any one of SEQ ID NOs: 69, 71, 96, and 97.
6 . The method of claim 4 , wherein the oligonucleotide comprises the sequence of SEQ ID NO: 69 or 71.
7 . The method of claim 2 , wherein the oligonucleotide is 26 nucleotides in length, comprises the sequence of SEQ ID NO: 69 or 71, and is fully complementary to a cognate sequence of SEQ ID NO: 295.
8 . The method of claim 7 , wherein the subject has a mutated DMD allele that is associated with a dystrophinopathy and/or the subject has or is suspected of having Duchenne muscular dystrophy.
9 . The method of claim 1 , wherein the oligonucleotide is an exon-skipping oligonucleotide that induces skipping of exon 45 of DMD.
10 . The method of claim 9 , wherein the oligonucleotide is 26-29 nucleotides in length.
11 . The method of claim 10 , wherein the region of complementarity is at least 16 nucleotides in length.
12 . The method of claim 11 , wherein the region of complementarity is complementary to a target sequence of an oligonucleotide as set forth in any one of SEQ ID NOs: 123, 124, 126, 128, 129, 131, 135, 146, 148, 149, 150, 153, 155, and 260.
13 . The method of claim 11 , wherein the oligonucleotide comprises at least 22 consecutive nucleosides of the sequence of any one of SEQ ID NOs: 131, 146, 148, and 150.
14 . The method of claim 9 , wherein the oligonucleotide is 26-29 nucleotides in length, comprises 25 consecutive nucleosides of the sequence of SEQ ID NO: 131, and is fully complementary to a cognate sequence of SEQ ID NO: 295.
15 . The method of claim 14 , wherein the subject has a mutated DMD allele that is associated with a dystrophinopathy and/or the subject has or is suspected of having Duchenne muscular dystrophy.
16 . The method of claim 1 , wherein the anti-transferrin receptor antibody is covalently linked to the oligonucleotide at the 3′ end of the oligonucleotide.
17 . The method of claim 1 , wherein the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 are defined according to the Chothia numbering system.
18 . The method of claim 17 , wherein the region of complementarity is complementary to a target sequence of an oligonucleotide as set forth in any one of SEQ ID NOs: 69, 71, 96, 97, 123, 124, 126, 128, 129, 131, 135, 146, 148, 149, 150, 153, 155, and 260.
19 . The method of claim 17 , wherein the anti-transferrin receptor antibody is in the form of a full-length IgG comprising a human IgG1 heavy chain constant region, or a functional variant thereof, and a human kappa light chain constant region.
20 . The method of claim 17 , wherein the anti-transferrin receptor antibody comprises a human kappa light chain constant region and a human IgG1 heavy chain constant region comprising at least one amino acid substitution, insertion, or deletion relative to a wild-type human IgG1 heavy chain constant region.
21 . The method of claim 19 , wherein the full-length IgG comprises a heavy chain constant region comprising at least one amino acid substitution, insertion, or deletion that alters the effector function of the anti-transferrin receptor antibody, wherein the alteration of the effector function comprises reduced Fc receptor binding.
22 . The method of claim 21 , wherein the full-length IgG comprises two or more amino acid substitutions, insertions, or deletions in a CH2 domain and two or more amino acid substitutions, insertions, or deletions in a CH3 domain, relative to a full-length IgG comprising an IgG1 constant region having an amino acid sequence of SEQ ID NO: 287.
23 . The method of claim 1 , wherein the complex is formable by a process comprising reacting a first electrophile of a linker precursor compound and a thiol group of the anti-transferrin receptor antibody.
24 . The method of claim 23 , wherein the first electrophile of the linker precursor compound is a maleimide moiety, and wherein the thiol group is of a cysteine residue of the anti-transferrin receptor antibody.
25 . The method of claim 24 , wherein the maleimide moiety is present in a (maleimidomethyl)cyclohexane-1-carboxylate group of the linker precursor compound.
26 . A method of treating Duchenne muscular dystrophy in a subject in need thereof, the method comprising administering to the subject a complex comprising an anti-transferrin receptor antibody covalently linked to an oligonucleotide,
wherein the anti-transferrin receptor antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein:
the VH comprises a heavy chain complementarity determining region 1 (CDR-H1), a heavy chain complementarity determining region 2 (CDR-H2), and a heavy chain complementarity determining region 3 (CDR-H3) of a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 438, and comprises humanized framework regions, and
the VL comprises a light chain complementarity determining region 1 (CDR-L1), a light chain complementarity determining region 2 (CDR-L2), and a light chain complementarity determining region 3 (CDR-L3) of a light chain variable region comprising the amino acid sequence of SEQ ID NO: 439, and comprises humanized framework regions; and
wherein the oligonucleotide is 20 to 29 nucleotides in length and comprises a region of complementarity to a dystrophin (DMD) sequence as set forth in SEQ ID NO: 295, wherein the region of complementarity is at least 12 nucleotides in length, and wherein the oligonucleotide is a phosphorodiamidate morpholino oligomer (PMO).
27 . The method of claim 26 , wherein the oligonucleotide is an exon-skipping oligonucleotide that induces skipping of exon 44 of DMD.
28 . The method of claim 27 , wherein the oligonucleotide is 26 nucleotides in length, comprises the sequence of SEQ ID NO: 69 or 71, and is fully complementary to a cognate sequence of SEQ ID NO: 295.
29 . The method of claim 26 , wherein the oligonucleotide is an exon-skipping oligonucleotide that induces skipping of exon 45 of DMD.
30 . The method of claim 29 , wherein the oligonucleotide is 26-29 nucleotides in length, comprises 25 consecutive nucleosides of the sequence of SEQ ID NO: 131, and is fully complementary to a cognate sequence of SEQ ID NO: 295.