Complexes comprising an anti-transferrin receptor antibody linked to an oligonicleotide and method of delivering oligonucleotide to a subject
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 inhibits activity of a disease allele associated with muscle disease. In some embodiments, the molecular payload is an oligonucleotide, such as an antisense oligonucleotide or RNAi oligonucleotide.
1. A complex comprising an anti-transferrin receptor antibody covalently linked to a 5′ end or a 3′ end of 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: 641, and comprises humanized framework regions,
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: 642, and comprises humanized framework regions,
wherein the oligonucleotide comprises one or more modifications and comprises an antisense strand comprising a region of complementarity of at least 15 nucleotides in length to an RNA encoded by a gene associated with a muscle disease, wherein the oligonucleotide is in the range of 15-30 nucleotides in length;
wherein the one or more modifications comprise a 2′-modified nucleoside selected from the group consisting of: a 2′-O-methyl nucleoside, a 2′-fluoro nucleoside, and combinations thereof, and/or comprise a modified backbone selected from a backbone comprising one or more phosphorothioate linkages and a phosphorodiamidate morpholino backbone.
2. The complex of claim 1 , wherein the antisense strand is 20-27 nucleotides in length.
3. The complex of claim 2 , wherein the region of complementarity is at least 18 nucleotides in length.
4. The complex of claim 2 , wherein the region of complementarity is at least 19 nucleotides in length.
5. The complex of claim 4 , wherein the oligonucleotide is double-stranded molecule and comprises the antisense strand hybridized to a sense strand.
6. The complex of claim 4 , wherein the oligonucleotide is a single stranded antisense oligonucleotide.
7. The complex of claim 5 , wherein each nucleoside of the oligonucleotide is selected from a 2′-O-methyl nucleoside and a 2′-fluoro nucleoside.
8. The complex of claim 7 , wherein the oligonucleotide comprises a modified backbone comprising one or more phosphorothioate linkages.
9. The complex of claim 6 , wherein the oligonucleotide is a phosphorodiamidate morpholino oligomer (PMO).
10. The complex of claim 1 , wherein the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 are according to the Chothia numbering system.
11. The complex of claim 10 , 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.
12. The complex of claim 10 , 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 relative to a wild-type human IgG1 heavy chain constant region.
13. The complex of claim 11 , wherein the full-length IgG comprises a heavy chain constant region comprising at least one amino acid substitution that reduces Fc receptor binding of the anti-transferrin receptor antibody.
14. The complex of claim 11 , wherein the full-length IgG comprises two or more amino acid substitutions in a CH2 domain and two or more amino acid substitutions in a CH3 domain, relative to a full-length IgG comprising an IgG1 constant region having an amino acid sequence of SEQ ID NO: 37.
15. The complex of claim 12 , wherein the anti-transferrin receptor antibody further comprises one or more sugar or carbohydrate molecules.
16. The complex of claim 15 , wherein the one or more sugar or carbohydrate molecules comprise a fucose unit.
17. The complex of claim 12 , wherein the complex is formable by a process comprising reacting a first electrophile of a linker precursor compound and a nucleophile of the anti-transferrin receptor antibody.
18. The complex of claim 17 , wherein the nucleophile of the anti-transferrin receptor antibody is a thiol group of a cysteine residue of the anti-transferrin receptor antibody.
19. The complex of claim 17 , wherein the first electrophile of the linker precursor compound is a maleimide moiety, and wherein the nucleophile of the anti-transferrin receptor antibody is a thiol-group of a cysteine residue of the anti-transferrin receptor antibody.
20. The complex of claim 19 , wherein the maleimide moiety is present in a (maleimidomethyl)cyclohexane-1-carboxylate group of the linker precursor compound.
21. The complex of claim 19 , wherein the complex is formable by a process comprising reacting a second electrophile of the linker precursor compound and a nucleophile covalently attached to the oligonucleotide.
22. The complex of claim 21 , wherein the nucleophile covalently attached to the oligonucleotide comprises an aminoalkyl group.
23. The complex of claim 22 , wherein the aminoalkyl group is a NH2-C6 group.
24. The complex of claim 21 , wherein the nucleophile covalently attached to the oligonucleotide is covalently attached to a terminal phosphate group of the oligonucleotide.
25. The complex of claim 1 , wherein the gene associated with a muscle disease is DUX4.
26. A method of delivering an oligonucleotide to a subject, the method comprising intravenously administering to the subject the complex of claim 1 .
27. The method of claim 26 , wherein the oligonucleotide is delivered to a skeletal muscle cell, a cardiac muscle cell, or a smooth muscle cell of the subject.
28. The method of claim 26 , wherein the subject is human.
29. The method of claim 28 , wherein the gene associated with a muscle disease is DUX4.
30. The method of claim 29 , wherein the subject has Facioscapulohumeral Muscular Dystrophy (FSHD).