Muscle-targeting complexes comprising an anti-transferrin receptor antibody linked to an oligonucleotide
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 expression or activity of DUX4. 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 binds in the range of C89 to F760 of human transferrin receptor protein 1 (TfR1) having an amino acid sequence as set forth in SEQ ID NO: 1;
wherein the oligonucleotide comprises one or more modifications and a region of complementarity of at least 12 nucleotides in length to the nucleotide sequence as set forth in SEQ ID NO: 52, 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: 2′-O-methyl nucleoside, a 2′-fluoro nucleoside, a 2′-O-methoxyethyl nucleoside, and 2′,4′-bridged nucleosides, and combinations thereof, and/or comprise a modified backbone selected from a backbone comprising one or more phosphorothioate linkages, a phosphorodiamidate morpholino backbone, and a peptide nucleic acid (PNA) backbone.
2. The complex of claim 1 , wherein the region of complementarity is at least 16 nucleotides in length.
3. The complex of claim 1 , wherein the region of complementarity is at least 17 nucleotides in length.
4. The complex of claim 1 , wherein the region of complementarity is at least 18 nucleotides in length.
5. The complex of claim 1 , wherein the region of complementarity is at least 19 nucleotides in length.
6. The complex of claim 1 , wherein the region of complementarity is at least 20 nucleotides in length.
7. The complex of claim 1 , wherein the oligonucleotide is a phosphorodiamidate morpholino oligomer (PMO).
8. The complex of claim 1 , wherein the oligonucleotide comprises a modified backbone comprising one or more phosphorothioate linkages.
9. The complex of claim 1 , wherein the anti-transferrin receptor antibody further comprises one or more sugar or carbohydrate molecules.
10. The complex of claim 9 , wherein the one or more sugar or carbohydrate molecules comprise a mannose unit, a glucose unit, an N-acetylglucosamine unit, an N-acetylgalactosamine unit, a galactose unit, a fucose unit, a phospholipid unit, or combinations thereof.
11. The complex of claim 1 , wherein the oligonucleotide is covalently linked to a lysine in the anti-transferrin receptor antibody via a cleavable linker.
12. The complex of claim 11 , wherein the cleavable linker comprises a valine-citrulline sequence.
13. The complex of claim 12 , wherein the complex is obtained by a cycloaddition reaction between an azide and an alkyne to form a triazole.
14. The complex of claim 13 , wherein, prior to the cycloaddition reaction, the azide is covalently linked to the valine-citrulline sequence of the cleavable linker that is covalently linked to the oligonucleotide and the alkyne is provided in a bicyclononyne moiety that further covalently links to the anti-transferrin receptor antibody.
15. The complex of claim 11 , wherein the cleavable linker further comprises one or more polyethylene glycol units.
16. The complex of claim 1 , wherein the anti-transferrin receptor antibody is in the form of a ScFv, Fab fragment, Fab′ fragment, F(ab′)2 fragment, or Fv fragment.
17. The complex of claim 1 , wherein the anti-transferrin receptor antibody is in the form of a Fab fragment.
18. The complex of claim 1 , wherein the oligonucleotide is a double-stranded molecule comprising the single strand hybridized to a complementary strand.
19. A complex comprising a muscle-targeting agent covalently linked to a 5′ end or a 3′ end of an oligonucleotide,
wherein the muscle targeting agent binds in the range of C89 to F760 of human transferrin receptor protein 1 (TfR1) having an amino acid sequence as set forth in SEQ ID NO: 1;
wherein the oligonucleotide comprises one or more modifications and comprises a region of complementarity of at least 12 nucleotides in length to an RNA encoded by a gene associated with a disease associated with muscle weakness, 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: 2′-O-methyl nucleoside, a 2′-fluoro nucleoside, a 2′-O-methoxyethyl nucleoside, and 2′,4′-bridged nucleosides, and combinations thereof, and/or comprise a modified backbone selected from a backbone comprising one or more phosphorothioate linkages, a phosphorodiamidate morpholino backbone, and a peptide nucleic acid (PNA) backbone.
20. The complex of claim 19 , wherein the muscle-targeting agent comprises an anti-transferrin receptor antibody comprising a CDRH1, a CDRH2, a CDRH3, a CDRL1, a CDRL2, and a CDRL3 of an antibody selected from the group consisting of: OKT9, M11, M23, M27, B84, 7A4, 8A2, 15D2, 10D11, 7B10, 15G11, 16G5, 13C3, 16G4, 16F6, 7G7, 4C2, 1B12, 13D4, 8D3, OX26, DF1513, 1A1B2, 66IG10, MEM-189, JF0956, 29806, TFRC/1818, 1E6, 66Ig10, TFRC/1059, Q1/71, 23D10, 13E4, TFRC/1149, ER-MP21, YTA74.4, BU54, 2B6, RI7 217, BA120g, LUCA31, B3/25, T58/30, R17 217.1.3, 5E9C11, BE0023, BK19.9, B3/25, T56/14, and T58/1.
21. The complex of claim 19 , wherein the muscle-targeting agent comprises an anti-transferrin receptor antibody comprising a CDRH1, a CDRH2, a CDRH3, a CDRL1, a CDRL2, and a CDRL3 of an antibody selected from the group consisting of: OKT9, M11, M23, M27, B84, 7A4, 8A2, 15D2, 10D11, 7B10, 15G11, 16G5, 13C3, 16G4, 16F6, 7G7, 4C2, 1B12, 13D4, OX26, DF1513, 1A1B2, 66IG10, JF0956, 29806, TFRC/1818, 1E6, 66Ig10, TFRC/1059, Q1/71, 13E4, TFRC/1149, BA120g, LUCA31, B3/25, 5E9C11, BK19.9, and T58/1.
22. The complex of claim 21 , wherein the anti-transferrin receptor antibody is a human antibody or humanized antibody.
23. The complex of claim 22 , wherein the anti-transferrin receptor antibody further comprises one or more sugar or carbohydrate molecules.
24. The complex of claim 23 , wherein the one or more sugar or carbohydrate molecules comprise a mannose unit, a glucose unit, an N-acetylglucosamine unit, an N-acetylgalactosamine unit, a galactose unit, a fucose unit, a phospholipid unit, or combinations thereof.
25. The complex of claim 19 , wherein the muscle-targeting agent comprises a muscle targeting peptide.
26. The complex of claim 25 , wherein the muscle-targeting peptide comprises 10 to 20 amino acids.
27. The complex of claim 25 , wherein the muscle-targeting peptide is bicyclic.
28. The complex of claim 19 , wherein the oligonucleotide comprises a gap region flanked both 5′ and 3′ by flanking regions, wherein each flanking region comprises one or more 2′-modified nucleosides selected from the group consisting of: a 2′-O-methyl nucleoside, a 2′-fluoro nucleoside, a 2′-O-methoxyethyl nucleoside, a 2′,4′-bridged nucleoside, and combinations thereof, and wherein each nucleoside of the gap region is a 2′-deoxyribonucleoside.
29. The complex of claim 19 wherein the oligonucleotide comprises a modified backbone comprising one or more phosphorothioate linkages.
30. The complex of claim 19 , wherein the disease associated with muscle weakness is a muscular dystrophy.