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1. WO2020018935 - CONCEPTION DE NOVO DE COMMUTATEURS PROTÉIQUES

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We claim

1. A non-naturally occurring polypeptide comprising:

(a) a helical bundle, comprising between 2 and 7 alpha-helices; and

(b) amino acid linkers connecting each alpha helix.

2. The polypeptide of claim 1, wherein each helix is independently 18-60, 18-55, 18-50, 18-45, 22-60, 22-55, 22-50, 22-45, 25-60, 25-55, 25-50, 25-45, 28-60, 28-55, 28-50, 28-45, 32-60, 32-55, 32-50, 32-45, 35-60, 35-55, 35-50, 35-45, 38-60, 38-55, 38-50, 38-45, 40-60, 40-58, 40-55, 40-50, or 40-45 amino acids in length.

3. The polypeptide of claim 1 or 2, wherein each amino acid linker is independently between 3-10, 4-10, 5-10, 6-10, 7-10, 8-10, 9-10, 2-9, 3-9, 4-9, 5-9, 6-9, 7-9, 8-9, 2-8, 3-8, 4-8,

5-8, 6-8, 7-8, 2-7, 3-7, 4-7, 5-7, 6-7, 2-6, 3-6, 4-6, 5-6, 2-5, 3-5, 4-5, 2-4, 3-4, 2-3, or 2, 3, 4, 5, 6,

7, 8, 9, or 10 amino acids in length, not including any further functional sequences that may be fused to the linker.

4. The polypeptide of any one of claims 1 -3, wherein the polypeptide comprises one or more bioactive peptide in at least one of the alpha helices, wherein the one or more bioactive peptides are capable of selectively binding to a defined target, wherein the one or more bioactive peptides may comprise one or more bioactive peptide selected from the non-limiting group consisting of SEQ ID NO:60, 62-64, 66, 27052-27093, and 27118-27119.

5. A non-naturally occurring polypeptide comprising the polypeptide having at least 40% sequence identity, not including optional amino acid residues, along its length to the amino acid sequence of a cage polypeptide disclosed herein, or selected from the group consisting of SEQ ID NOS: 1-49, 51-52, 54-59, 61, 65, 67-14317, 27094-27117, 27120-27125, 27,278 to 27,321, and cage polypeptides listed in Table 2, Table 3, or Table 4, wherein the N-terminal and/or C-terminal 60 amino acids of the polypeptides are optional.

6. A non-naturally occurring polypeptide comprising the polypeptide having at least 40% sequence identity, not including optional amino acid residues, along its length to the amino acid sequence of a cage polypeptide listed in Table 2, Table 3, and/or Table 4.

7. The non-naturally occurring polypeptide of claim 5 or 6, wherein the polypeptide comprises the amino acid sequence having an amino acid sequence having at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, not including optional amino acid residues, along its length to the amino acid sequence elected from the group consisting of SEQ ID NOS: 1-91, SEQ ID NOS: 1-49, 51-52, 54-59, 61, 65, 67-14317, 27094-27117, 27120-27125, 27,278 to 27,321, and the cage polypeptides listed in Table 2, Table 3, and/or Table 4.

8. The non-naturally occurring polypeptide of any one of claims 5-7, further comprising one or more bioactive peptides within or replacing the latch region of the polypeptide, wherein the one or more bioactive peptides may comprise one or more bioactive peptide selected from the non-limiting group consisting of SEQ ID NO:60, 62-64, 66, 27052-27093, and 27118-27119.

9. A non-naturally occurring polypeptide comprising a polypeptide having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, not including optional amino acid residues, along its length to the amino acid sequence of a key polypeptide disclosed herein, or a key polypeptide selected from the group consisting of SEQ ID NOS: 14318-26601, 26602-27015, 27016-27050, 27,322 to 27,358, and key polypeptides listed in Table 2, Table 3, and/or Table 4.

10. The non-naturally occurring polypeptide of claim 9, wherein the polypeptide comprises an amino acid sequence having an amino acid sequence having at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, not including optional amino acid residues, along its length to the amino acid sequence of a key protein selected from the group consisting of key polypeptides listed in Table 2, Table 3, and/or Table 4.

11. A fusion protein comprising the polypeptide of any one of claims 1-8 fused to the polypeptide of any one of claims 9-10.

12. The fusion protein of claim 11, wherein the polypeptide of any one of claims 1-8 comprises a cage polypeptide and wherein the polypeptide of any one of claims 9- 10 comprises a key polypeptide, and wherein the cage polypeptide is not activated by the key polypeptide.

13. A nucleic acid encoding the polypeptide of any one of claims 1-12.

14. An expression vector comprising the nucleic acid of claim 13 operatively linked to a promoter.

15. A host cell comprising the nucleic acid of claim 13 or the expression vector of claim 14.

16. The host cell of claim 15, wherein the nucleic acid or the expression vector is integrated into a host cell chromosome.

17. The host cell of claim 15, wherein the nucleic acid or the expression vector is episomal.

18. The host cell of any one of claims 15-17, comprising:

(a) a first nucleic acid encoding the polypeptide of any one of claims 1-8 operatively linked to a first promoter; and

(b) a second nucleic acid encoding the polypeptide of any one of claims 9-10 operatively linked to a second promoter, wherein the second nucleic acid encodes a key polypeptide capable of binding to a structural region of a cage polypeptide encoded by the first nucleic acid, and wherein binding of the key polypeptide to the structural region of the cage polypeptide induces a conformational change in the cage polypeptide.

19. The host cell of claim 18, wherein the first nucleic acid comprises a plurality of first nucleic acids encoding a plurality of different cage polypeptides.

20. The host cell of claim 19, wherein the second nucleic acid comprises a plurality of second nucleic acids encoding a plurality of different key polypeptides, wherein the plurality of different key polypeptides comprise one or more key polypeptides that are capable of binding to and inducing a conformational change in only a subset of the plurality of different cage polypeptides.

21. The host cell of claim 19, wherein the second nucleic acid encodes a single key polypeptide that is capable of binding to and inducing a conformational change in each different cage polypeptide.

22. The host cell of any one of claims 15-17, comprising:

(a) a first nucleic acid encoding a fusion protein according to claim 12 operatively linked to a first promoter; and

(b) a second nucleic acid encoding a fusion protein according to claim 12 operatively linked to a second promoter, wherein:

(i) the cage polypeptide encoded by the first nucleic acid is activated by the key polypeptide encoded by the second nucleic acid;

(ii) the cage polypeptide encoded by the first nucleic acid is not activated by the key polypeptide encoded by the first nucleic acid;

(iii) the cage polypeptide encoded by the second nucleic acid is activated by the key polypeptide encoded by the first nucleic acid; and

(iv) the cage polypeptide encoded by the second nucleic acid is not activated by the key polypeptide encoded by the second nucleic acid.

23. A kit, comprising:

(a) one or more polypeptides of any one of claims 1-8;

(b) one or more polypeptides of any one of claims 9-10; and

(c) optionally, one or more fusion proteins of any one of claims 11-12.

24. A kit, comprising:

(a) a first nucleic acid encoding one or more polypeptides of any one of claims 1-8;

(b) a second nucleic acid encoding one or more polypeptides of any one of claims 9- 10; and

(c) optionally, a third nucleic acid encoding one or more fusion protein of any one of claims 11-12.

25. The kit of claim 24, wherein the first nucleic acid, the second nucleic acid, and/or the third nucleic acid comprise expression vectors.

26. A LOCKR switch comprising

(a) a cage polypeptide comprising a structural region and a latch region further comprising one or more bioactive peptides, wherein the structural region interacts with the latch region to prevent activity of the one or more bioactive peptides;

(b) an optional key polypeptide that binds to the cage structural region, thereby displacing the latch region and activating the one or more bioactive peptides; and

(c) optionally, one or more effector polypeptide(s) that bind to the one or more bioactive peptides when the one or more bioactive peptides are activated.

27. The LOCKR switch of claim 26, wherein the cage polypeptide comprises the polypeptide of any one of claims 1-8 or 11-12.

28. The LOCKR switch of claim 26 or 27, wherein the key polypeptide is present, and wherein the key polypeptide comprises the polypeptide of claim 9 or 10.

29. The LOCKR switch of any one of claims 26-28, wherein the effector polypeptide is present, and wherein the effector polypeptide comprises an effector polypeptide that selectively binds to the bioactive peptide, including but not limited to Bcl2, GFP1-10, and a protease.

30. The host cell of any one of claims 18-21, the kit of any one of claims-23-25, or the LOCKR switch of any one of claims 26-29, wherein the one or more cage polypeptide and the one or more key polypeptide comprise at least one cage polypeptide and at least one key polypeptide having an amino acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%,

70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, not including optional residues, along its length to a cage polypeptide and a key polypeptide, respectively, in the same row of Table 1, Table 2, Table 3, and/or Table 4.

31. The host cell of any one of claims 18-21, the kit of any one of claims-23-25, or the LOCKR switch of any one of claims 26-29, wherein the one or more cage polypeptide and the one or more key polypeptide comprise at least one cage polypeptide and at least one key polypeptide having an amino acid sequence having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, not including optional residues, along its length to a cage polypeptide and a key polypeptide, respectively, in the same row of Table 2, Table 3, and/or Table 4.

32. The LOCKR switch of any one of claims 26-31, wherein the one or more bioactive peptides may comprise one or more bioactive peptide selected from the non-limiting group consisting of SEQ ID NO:60, 62-64, 66, 27052-27093, and 27118-27119.

33. The host cell of any one of claims 18-21 or 30-32, the kit of any one of claims 23-25 or 30-32, or the LOCKR switch of any one of claims 26-32, wherein

(a) the one or more cage polypeptide comprise one or more cage polypeptides having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, not including optional amino acid residues, along its length to the amino acid sequence of a cage polypeptide selected from the group consisting of SEQ ID NOS: 1-49, 51-52, 54-59, 61, 65, 67-14317, 27094-27117, 27120-27125, 27,278 to 27,321, and cage polypeptides listed in Table 2, Table 3, and/or Table 4, wherein the N-terminal and/or C-terminal 60 amino acids of the polypeptides are optional; and

(b) the one or more key polypeptide comprise one or more polypeptides selected from the group consisting of a polypeptide having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, not including optional amino acid residues, along its length to the amino acid sequence of a key polypeptide selected from the group consisting of SEQ ID NOS: 14318-26601, 26602-

27015, 27016-27050, 27,322 to 27,358, and key polypeptides listed in Table 2, Table 3, and/or Table 4.

34. Use of the polypeptides, fusion proteins, nucleic acids, expression vectors, host cells, kits, and/or LOCKR switches disclosed herein to sequester bioactive peptide in the cage polypeptide, holding them in an inactive (“off’) state, until combined with the key polypeptide to induce a conformational change that activates (“on”) the bioactive peptide.