Muscle-targeting complexes comprising an anti-transferin receptor antibody linked to an oligonucleotide and method of use thereof to induce exon skipping
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 complex comprising an anti-transferrin receptor antibody covalently linked to an exon-skipping oligonucleotide that induces skipping of exon 44 of dystrophin (DMD),
wherein the anti-transferrin receptor antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region comprise human or humanized framework regions; and 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 is 20 to 27 nucleotides in length and comprises a region of complementarity to exon 44 of a DMD transcript as set forth in SEQ ID NO: 295, wherein the region of complementarity is at least 12 nucleotides in length, wherein the oligonucleotide is a phosphorodiamidate morpholino oligomer (PMO);
wherein the oligonucleotide is covalently linked at the 5′ end or the 3′ end to the anti-transferrin receptor antibody via a linker;
wherein the oligonucleotide is configured to induce skipping of exon 44 of the DMD pre-mRNA in a muscle cell; and
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.
2. The complex of claim 1 , wherein the linker is cleavable and comprises a triazole covalently linked to a valine-citrulline sequence that further covalently links to the anti-transferrin receptor antibody.
3. The complex of claim 2 , wherein the triazole is obtained by a cycloaddition reaction between an azide and an alkyne, wherein prior to the cycloaddition reaction, the azide is covalently linked to the valine-citrulline sequence and the alkyne is provided in a bicyclononyne moiety.
4. The complex of claim 1 , wherein the nucleophile of the anti-transferrin receptor antibody is an amino group or a thiol group.
5. The complex of claim 4 , wherein the nucleophile of the anti-transferrin receptor antibody is an amino group of a lysine of the anti-transferrin receptor antibody, or a thiol group of a cysteine of the anti-transferrin receptor antibody.
6. The complex of claim 1 , wherein the first electrophile of the linker precursor compound is a maleimide moiety, and wherein the nucleophile is a thiol group of a cysteine residue of the anti-transferrin receptor antibody.
7. The complex of claim 6 , wherein the maleimide moiety is present in a (maleimidomethyl)cyclohexane-1-carboxylate group of the linker precursor compound.
8. The complex of claim 6 , 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.
9. The complex of claim 8 , wherein the nucleophile covalently attached to the oligonucleotide comprises an alkylamino group.
10. The complex of claim 9 , wherein the second electrophile of the linker precursor compound is a succinimidyl ester.
11. The complex of claim 1 , wherein the anti-transferrin receptor antibody is in the form of a Fab fragment.
12. The complex of claim 1 , wherein the anti-transferrin receptor antibody is cross-reactive with an extracellular epitope of non-human primate and/or rodent transferrin receptor.
13. The complex of claim 1 , wherein the anti-transferrin receptor antibody comprises a human IgG1 heavy chain constant region.
14. The complex of claim 13 , wherein the anti-transferrin receptor antibody further comprises a human kappa light chain constant region.
15. The complex of claim 14 , wherein the IgG1 constant region comprises at least one amino acid substitution that alters the effector function of the anti-transferrin receptor antibody.
16. The complex of claim 15 , wherein the alteration of the effector function comprises reduced Fc receptor binding.
17. The complex of claim 15 , wherein the IgG1 constant region comprises two or more amino acid substitutions in a CH2 domain and two or more amino acid substitutions in a CH3 domain, relative to an IgG1 constant region having an amino acid sequence of SEQ ID NO: 287.
18. The complex of claim 14 , wherein one or more amino acid residues in the N-terminal region of a CH2 domain of the constant region of the antibody are altered.
19. The complex of claim 18 , wherein the anti-transferrin receptor antibody binds to human TfR1 with a K D in a range of 10 −11 M to 10 −8 M.
20. The complex of claim 1 , wherein the oligonucleotide is 24, 25, or 26 nucleotides in length.
21. The complex of claim 20 , wherein the region of complementarity is at least 16 nucleotides in length.
22. The complex of claim 21 , wherein the oligonucleotide is covalently linked to a lysine residue of the anti-transferrin receptor antibody.
23. The complex of claim 21 , wherein the oligonucleotide is covalently linked to a cysteine residue of the anti-transferrin receptor antibody.
24. A composition comprising a plurality of complexes of claim 20 , wherein the average drug-to-antibody ratio (DAR) of the complexes in the composition is in a range of 1 to 5.
25. The complex of claim 1 , wherein the muscle cell is a skeletal muscle cell, cardiac muscle cell, or smooth muscle cell.
26. The complex of claim 1 , wherein the anti-transferrin receptor antibody comprises 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.
27. The complex of claim 26 , wherein the anti-transferrin receptor antibody further comprises one or more sugar or carbohydrate molecules.
28. The complex of claim 27 , 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.
29. A method of inducing skipping of exon 44 of a DMD pre-mRNA in a muscle cell of a subject expressing a DMD pre-mRNA, the method comprising administering to the subject the complex of claim 1 .
30. The method of claim 29 , wherein the oligonucleotide promotes the expression or activity of a functional dystrophin protein in the muscle cell.