Muscle targeting complexes and their use

Abstract

Aspects of this disclosure relate to a complex containing a muscle targeting agent covalently linked to a molecular payload. [Solution] In some embodiments, the muscle targeting agent specifically binds to internalized cell surface receptors on muscle cells. In some embodiments, the molecular payload inhibits the activity of disease alleles associated with muscle disease. In some embodiments, the molecular payload is an oligonucleotide, such as an antisense oligonucleotide or RNAi oligonucleotide.

Claims

1. A complex comprising an anti-transferrin receptor (TfR) antibody covalently linked to a molecular payload configured to modulate the expression or activity of a muscle disease gene, wherein the antibody (i) Heavy chain variable region (VH) containing an amino acid sequence at least 95% identical to SEQ ID NO: 76; and / or light chain variable region (VL) containing an amino acid sequence at least 95% identical to SEQ ID NO: 75; (ii) Heavy chain variable region (VH) containing an amino acid sequence at least 95% identical to SEQ ID NO: 69; and / or light chain variable region (VL) containing an amino acid sequence at least 95% identical to SEQ ID NO: 70; (iii) Heavy chain variable region (VH) containing an amino acid sequence at least 95% identical to SEQ ID NO: 71; and / or light chain variable region (VL) containing an amino acid sequence at least 95% identical to SEQ ID NO: 70; (iv) Heavy chain variable region (VH) containing an amino acid sequence at least 95% identical to SEQ ID NO: 72; and / or light chain variable region (VL) containing an amino acid sequence at least 95% identical to SEQ ID NO: 70; (v) Heavy chain variable region (VH) containing an amino acid sequence at least 95% identical to SEQ ID NO: 73; and / or light chain variable region (VL) containing an amino acid sequence at least 95% identical to SEQ ID NO: 74; (vi) Heavy chain variable region (VH) containing an amino acid sequence at least 95% identical to SEQ ID NO: 73; and / or light chain variable region (VL) containing an amino acid sequence at least 95% identical to SEQ ID NO: 75; (vii) Heavy chain variable region (VH) containing an amino acid sequence at least 95% identical to SEQ ID NO: 76; and / or light chain variable region (VL) containing an amino acid sequence at least 95% identical to SEQ ID NO: 74; (viii) Heavy chain variable region (VH) containing an amino acid sequence at least 95% identical to SEQ ID NO: 77; and / or light chain variable region (VL) containing an amino acid sequence at least 95% identical to SEQ ID NO: 78; (ix) Heavy chain variable region (VH) containing an amino acid sequence at least 95% identical to SEQ ID NO: 79; and / or light chain variable region (VL) containing an amino acid sequence at least 95% identical to SEQ ID NO: 80; or (x) Heavy chain variable region (VH) containing an amino acid sequence at least 95% identical to SEQ ID NO: 77; Light chain variable region (VL) containing an amino acid sequence at least 95% identical to SEQ ID NO: 80 A complex containing this.

2. Antibodies, (i) VH containing the amino acid sequence of SEQ ID NO: 76 and VL containing the amino acid sequence of SEQ ID NO: 75; (ii) VH containing the amino acid sequence of SEQ ID NO: 69 and VL containing the amino acid sequence of SEQ ID NO: 70; (iii) VH containing the amino acid sequence of SEQ ID NO: 71 and VL containing the amino acid sequence of SEQ ID NO: 70; (iv) VH containing the amino acid sequence of SEQ ID NO: 72 and VL containing the amino acid sequence of SEQ ID NO: 70; (v) VH containing the amino acid sequence of SEQ ID NO: 73 and VL containing the amino acid sequence of SEQ ID NO: 74; (vi) VH containing the amino acid sequence of SEQ ID NO: 73 and VL containing the amino acid sequence of SEQ ID NO: 75; (vii) VH containing the amino acid sequence of SEQ ID NO: 76 and VL containing the amino acid sequence of SEQ ID NO: 74; (viii) VH containing the amino acid sequence of SEQ ID NO: 77 and VL containing the amino acid sequence of SEQ ID NO: 78; (ix) VH containing the amino acid sequence of SEQ ID NO: 79 and VL containing the amino acid sequence of SEQ ID NO: 80; or (x) VH containing the amino acid sequence of SEQ ID NO: 77 and VL containing the amino acid sequence of SEQ ID NO: 80 The composite according to claim 1, comprising:

3. The complex according to claim 1 or 2, wherein the antibody is selected from the group consisting of Fab fragment, Fab' fragment, F(ab')2 fragment, scFv, Fv, and full-length IgG.

4. The complex according to claim 3, wherein the antibody is a Fab fragment.

5. Antibodies, (i) A heavy chain having at least 85% identical amino acid sequence to SEQ ID NO: 101; and / or a light chain having at least 85% identical amino acid sequence to SEQ ID NO: 90; (ii) A heavy chain having at least 85% identical amino acid sequence to SEQ ID NO: 97; and / or a light chain having at least 85% identical amino acid sequence to SEQ ID NO: 85; (iii) A heavy chain having at least 85% identical amino acid sequence to SEQ ID NO: 98; and / or a light chain having at least 85% identical amino acid sequence to SEQ ID NO: 85; (iv) A heavy chain having at least 85% identical amino acid sequence to SEQ ID NO: 99; and / or a light chain having at least 85% identical amino acid sequence to SEQ ID NO: 85; (v) A heavy chain having at least 85% identical amino acid sequence to SEQ ID NO: 100; and / or a light chain having at least 85% identical amino acid sequence to SEQ ID NO: 89; (vi) A heavy chain having at least 85% identical amino acid sequence to SEQ ID NO: 100; and / or a light chain having at least 85% identical amino acid sequence to SEQ ID NO: 90; (vii) A heavy chain having at least 85% identical amino acid sequence to SEQ ID NO: 101; and / or a light chain having at least 85% identical amino acid sequence to SEQ ID NO: 89; (viii) A heavy chain having at least 85% identical amino acid sequence to SEQ ID NO: 102; and / or a light chain having at least 85% identical amino acid sequence to SEQ ID NO: 93; (ix) A heavy chain containing at least 85% identical amino acid sequence to SEQ ID NO: 103; and / or a light chain containing at least 85% identical amino acid sequence to SEQ ID NO: 95; or (x) A heavy chain containing at least 85% identical amino acid sequence to SEQ ID NO: 102; and / or a light chain containing at least 85% identical amino acid sequence to SEQ ID NO:

95. The composite according to claim 4, comprising

6. Antibodies, (i) A heavy chain containing the amino acid sequence of SEQ ID NO: 101; and a light chain containing the amino acid sequence of SEQ ID NO: 90; (ii) A heavy chain containing the amino acid sequence of SEQ ID NO: 97; and a light chain containing the amino acid sequence of SEQ ID NO: 85; (iii) A heavy chain containing the amino acid sequence of SEQ ID NO: 98; and a light chain containing the amino acid sequence of SEQ ID NO: 85; (iv) A heavy chain containing the amino acid sequence of SEQ ID NO: 99; and a light chain containing the amino acid sequence of SEQ ID NO: 85; (v) Heavy chain containing the amino acid sequence of SEQ ID NO: 100; and light chain containing the amino acid sequence of SEQ ID NO: 89; (vi) A heavy chain containing the amino acid sequence of SEQ ID NO: 100; and a light chain containing the amino acid sequence of SEQ ID NO: 90; (vii) A heavy chain containing the amino acid sequence of SEQ ID NO: 101; and a light chain containing the amino acid sequence of SEQ ID NO: 89; (viii) A heavy chain containing the amino acid sequence of SEQ ID NO: 102; and a light chain containing the amino acid sequence of SEQ ID NO: 93; (ix) A heavy chain containing the amino acid sequence of SEQ ID NO: 103; and a light chain containing the amino acid sequence of SEQ ID NO: 95; or (x) Heavy chain containing the amino acid sequence of SEQ ID NO: 102; and light chain containing the amino acid sequence of SEQ ID NO: 95 The composite according to claim 4 or 5, comprising:

7. The complex according to any one of claims 1 to 6, wherein the antibody does not specifically bind to the transferrin binding site of the transferrin receptor, and / or the antibody does not inhibit the binding of transferrin to the transferrin receptor.

8. The conjugate according to any one of claims 1 to 7, wherein the antibody is cross-reactive with two or more extracellular epitopes of human, non-human primate, and rodent transferrin receptors.

9. The complex according to any one of claims 1 to 8, configured to promote the transferrin receptor-mediated internalization of a molecular payload into a muscle cell.

10. The complex according to any one of claims 1 to 9, wherein the molecular payload is an oligonucleotide.

11. The complex according to claim 10, wherein the oligonucleotide comprises a region complementary to a muscle disease gene having a gain-of-function disease allele.

12. The complex according to claim 10 or 11, wherein the oligonucleotide comprises at least one modified internucleoside linkage.

13. The complex according to claim 12, wherein at least one modified nucleoside linkage is a phosphorothioate linkage.

14. The complex according to any one of claims 10 to 13, wherein the oligonucleotide comprises one or more modified nucleosides.

15. The complex according to claim 14, wherein one or more modified nucleosides are 2'-modified nucleosides.

16. The complex according to any one of claims 10 to 15, wherein the oligonucleotide is a gapmer oligonucleotide that leads to RNAse H-mediated cleavage of mRNA transcripts encoded by muscle disease genes in cells.

17. The complex according to any one of claims 10 to 15, wherein the oligonucleotide is a mixmer oligonucleotide.

18. The complex according to any one of claims 10 to 15, wherein the oligonucleotide is an RNAi oligonucleotide that promotes RNAi-mediated cleavage of mRNA transcripts encoded by muscle disease genes.

19. The complex according to claim 15, wherein each 2'-modified nucleoside is selected from the group consisting of 2'-O-methyl, 2'-fluoro(2'-F), 2'-O-methoxyethyl(2'-MOE), and 2',4'-bridged nucleosides.

20. The complex according to claim 14, wherein one or more modified nucleosides are 2',4'-bridged nucleosides.

21. The complex according to any one of claims 10 to 15, wherein the oligonucleotide is a phosphorodiamidate morpholino oligomer.

22. The complex according to any one of claims 1 to 21, wherein the antibody is covalently linked to a molecular payload via a cleavable linker.

23. The complex according to claim 22, wherein the cleavable linker comprises a valine-citrulline sequence.

24. The complex according to any one of claims 1 to 23, wherein the antibody is covalently linked to a molecular payload via conjugation to a lysine residue or cysteine ​​residue of the antibody.

25. The complex according to any one of claims 1 to 24, wherein modulating the expression or activity of a muscle disease gene reduces the expression of RNA and / or protein.

26. A method for modulating the expression or activity of a muscle disease gene in a cell, comprising contacting the cell with a complex according to any one of claims 1 to 25 in an amount effective to promote the internalization of a molecular payload into the cell, wherein the cell is optionally a muscle cell.

27. The method according to claim 26, wherein the muscle disease is a disease selected from the group consisting of adult-onset Pompe disease, central nucleus myopathy (CNM), Duchenne muscular dystrophy, facioscapulohumeral muscular dystrophy (FSHD), familial hypertrophic cardiomyopathy, fibrodysplasia ossificans progressive (FOP), Friedreich's ataxia (FRDA), type II inclusion body myopathy, distal Rein myopathy, myofibril myopathy, congenital myotonia (autosomal dominant type, Thomsen disease), myotonic dystrophy type I, myotonic dystrophy type II, myotyl myopathy, oculopharyngeal muscular dystrophy, and congenital anomalous myotonia.

28. A method for treating a subject having a muscle disease, comprising administering an effective amount of the complex described in any one of claims 1 to 25 to the subject, wherein the muscle disease is optionally selected from the group consisting of adult-onset Pompe disease, central nucleus myopathy (CNM), Duchenne muscular dystrophy, facioscapulohumeral muscular dystrophy (FSHD), familial hypertrophic cardiomyopathy, fibrodysplasia ossificans progressive (FOP), Friedreich's ataxia (FRDA), type II inclusion body myopathy, distal Rein myopathy, myofibril myopathy, congenital myotonia (autosomal dominant type, Thomsen disease), myotonic dystrophy type I, myotonic dystrophy type II, myotyl myopathy, oculopharyngeal muscular dystrophy, and congenital anomalous myotonia.

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