De novo design of potent and selective interleukin mimetics

1. A non-naturally occurring polypeptide comprising domains X1, X2, X3, and X4, wherein:

(a) X1 is a peptide comprising the amino acid sequence EHALYDAL (SEQ ID NO:1);

(b) X2 is a helical-peptide of at least 8 amino acids in length;

(c) X3 is a peptide comprising the amino acid sequence YAFNFELI (SEQ ID NO:2); and

(d) X4 is a peptide comprising the amino acid sequence ITILQSWIF (SEQ ID NO:3);

wherein X1, X2, X3, and X4 may be in any order in the polypeptide;

wherein amino acid linkers may be present between any of the domains; and

wherein the polypeptide binds to IL-2 receptor βγ c heterodimer (IL-2Rβγ c ).

2. The polypeptide of claim 1 , wherein the domains are arranged N-terminal to C-terminal in an arrangement selected from the group consisting of X1-X2-X3-X4; X1-X3-X2-X4; X1-X4-X2-X3; X3-X2-X1-X4; X4-X3-X2-X1; X2-X3-X4-X1; and X2-X1-X4-X3, wherein amino acid linkers may be present between any of the domains.

3. The polypeptide of claim 1 , wherein X2 is a peptide comprising an amino acid sequence at least 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the peptide KDEAEKAKRMKEWMKRIKT (SEQ ID NO:7).

4. The polypeptide of claim 3 comprising a cysteine substitution at position 1, 2, 5, 9, 12, or 16 of SEQ NO:7.

5. The polypeptide of claim 1 , wherein the polypeptide is linked to a stabilization compound.

6. The polypeptide of claim 5 , wherein the stabilization compound is linked at a cysteine residue in the polypeptide.

7. The polypeptide of claim 6 , wherein the cysteine residue is present in X2.

8. The polypeptide of claim 6 , wherein the stabilization compound is linked to the cysteine residue via a maleimide group.

9. The polypeptide of claim 1 , wherein the polypeptide includes at least one disulfide bond.

10. A pharmaceutical composition comprising the polypeptide of claim 1 , and a pharmaceutically acceptable carrier.

11. The polypeptide of claim 1 , wherein:

X1 is a peptide comprising an amino acid sequence at least 80% identical to the peptide PKKKIQLHAEHALYDALMILNI (SEQ ID NO: 4), provided that the amino acids at positions 10-17 are identical to SEQ ID NO: 1;

X3 is a peptide comprising an amino acid sequence at least 80% identical to the peptide LEDYAFNFELILEEIARLFESG (SEQ ID NO:5), provided that the amino acids at positions 4-11 are identical to SEQ ID NO: 2; and

X4 is a peptide comprising an amino acid sequence at least 80% identical to the peptide EDEQEEMANAIITILQSWIFS (SEQ ID NO:6), provided that the amino acids at positions 12-20 are identical to SEQ ID NO: 3.

12. The polypeptide of claim 11 , wherein:

(i) X1 includes one or more of: L at residue 7, H at residue 8, and M at residue 18; and/or

(ii) X3 includes: D at residue 3, E at residue 13, and E at residue 14.

13. A non-naturally occurring polypeptide comprising domains X1, X2, X3, and X4, wherein:

X1 is a peptide comprising an amino acid sequence at least 80% identical to the peptide PKKKIQLHAEHALYDALMILNI (SEQ ID NO: 4);

X2 is a helical-peptide of at least 8 amino acids in length;

X3 is a peptide comprising an amino acid sequence at least 80% identical to the peptide LEDYAFNFELILEEIARLFESG (SEQ ID NO: 5); and

X4 is a peptide comprising an amino acid sequence at least 80% identical to the peptide EDEQEEMANAIITILQSWIFS (SEQ ID NO: 6);

wherein X1, X2, X3, and X4 may be in any order in the polypeptide;

wherein amino acid linkers may be present between any of the domains; and

wherein the polypeptide binds to IL-2 receptor βγ c heterodimer (IL-2Rβγ c ).

14. The polypeptide of claim 13 , wherein:

(i) X1 includes 1, 2, 3, 4, or all 5 of the following: L at residue 7, H at residue 8, H at residue 11, Y at residue 14; and M at residue 18; and/or

(ii) X3 includes 1, 2, 3, 4, 5, 6, 7, or all 8 of the following: D at residue 3, Y at residue 4, F at residue 6, N at residue 7, L at residue 10, I at residue 11, E at residue 13, and E at residue 14; and/or

(ii) X4 includes I at residue 19.

15. The polypeptide of claim 13 , wherein:

(i) X1 is a peptide comprising an amino acid sequence at least 80% identical to the peptide PKKKIQLHAEHALYDALMILNI (SEQ ID NO: 4), wherein

the amino acid at position 1 is P or if substituted is A, F, I, L, M, Q, R, S, or W;

the amino acid at position 2 is K or if substituted is A, D, E, G, or V;

the amino acid at position 3 is K or if substituted is D, E, F, or W;

the amino acid at position 4 is K or if substituted is D, E, N, P, R, or W;

the amino acid at position 5 is I or if substituted is D, E, H, K, L, M, or S;

the amino acid at position 6 is Q or if substituted is A, D, E, G, L, P, S, or W;

the amino acid at position 7 is L or if substituted is D, E, Q, or Y;

the amino acid at position 8 is H or if substituted is A, F, W, or Y;

the amino acid at position 9 is A or if substituted is C, F, or P;

the amino acid at position 10 is E or if substituted is C, D, F, K, or P;

the amino acid at position 11 is H or if substituted is D or F;

the amino acid at position 12 is A or if substituted is D, E, P, S, T, or V;

the amino acid at position 13 is L or if substituted is H, I, M, P, R, V, or W;

the amino acid at position 14 is Y or if substituted is F, R, or W;

the amino acid at position 15 is D or if substituted is E, N, or Y;

the amino acid at position 16 is A or if substituted is C, L, M, or S;

the amino acid at position 17 is L or if substituted is F, I, M, P, or R;

the amino acid at position 18 is M or if substituted is G, Q, or Y;

the amino acid at position 19 is I or if substituted is L, M, P, Q, or V;

the amino acid at position 20 is L or if substituted is A, K, M, Q, R, or S;

the amino acid at position 21 is N or if substituted is G, K, P, R, S, or W; and

the amino acid at position 22 is I or if substituted is D, E, K, M, N, W, or Y;

(ii) X3 is a peptide comprising an amino acid sequence at least 80% identical to the peptide LEDYAFNFELILEEIARLFESG (SEQ ID NO: 5), wherein:

the amino acid at position 1 is L or if substituted is A;

the amino acid at position 2 is E or if substituted is D, G, K, M or T;

the amino acid at position 3 is D or if substituted is E, N, or Y;

the amino acid at position 4 is Y or if substituted is C, D, G, or T;

the amino acid at position 5 is A or if substituted is F, H, S, V, W, or Y;

the amino acid at position 6 is F or if substituted is A, I, M, T, V, or Y;

the amino acid at position 7 is N or if substituted is D, K, S, or T;

the amino acid at position 8 is F or if substituted is A, C, G, L, M, S, or V;

the amino acid at position 9 is E or if substituted is C, H, K, L, R, S, T, or V;

the amino acid at position 10 is L or if substituted is F, I, M, or Y;

the amino acid at position 11 is I or if substituted is L, T, or Y;

the amino acid at position 12 is L or if substituted is F, K, M, S, or V;

the amino acid at position 13 is E or if substituted is A, D, F, G, I, N, P, Q, S, or T;

the amino acid at position 14 is E or if substituted is A, F, H, S, or V;

the amino acid at position 15 is I or if substituted is C, L, M, V, or W;

the amino acid at position 16 is A or if substituted is D, G, S, T, or V;

the amino acid at position 17 is R or if substituted is H, K, L, or N;

the amino acid at position 18 is L or if substituted is C, D, G, I, Q, R, T, or W;

the amino acid at position 19 is F or if substituted is D, M, N, or W;

the amino acid at position 20 is E or if substituted is A, C, F, G, M, S, or Y;

the amino acid at position 21 is S or if substituted is D, E, G, H, L, M, R, T, V, or W; and

the amino acid at position 22 is G or if substituted is A, D, K, N, S, or Y; and

(iii) X4 is a peptide comprising an amino acid sequence at least 80% identical to the peptide EDEQEEMANAIITILQSWIFS (SEQ ID NO: 6);

the amino acid at position 1 is E or if substituted is D, G, K, or V;

the amino acid at position 2 is D or if substituted is I, M, or S;

the amino acid at position 3 is E or if substituted is G, H, or K;

the amino acid at position 4 is Q or if substituted is E, G, I, K, R, or S;

the amino acid at position 5 is E or if substituted is A, D, G, H, S, or V;

the amino acid at position 6 is E or if substituted is C, D, G, I, M, Q, R, T, or V;

the amino acid at position 7 is M or if substituted is C, E, L, P, R, or T;

the amino acid at position 8 is A or if substituted is F, L, M, or W;

the amino acid at position 9 is N or if substituted is A, G, L, Q, R, or T;

the amino acid at position 10 is A or if substituted is C, D, E, F, H, I, or W;

the amino acid at position 11 is I or if substituted is M, N, S, V, or W;

the amino acid at position 12 is I or if substituted is K, L, S, or V;

the amino acid at position 13 is T or if substituted is C, L, M, R, or S;

the amino acid at position 14 is I or if substituted is L, P, T, or Y;

the amino acid at position 15 is L or if substituted is F, G, I, M, N, or V;

the amino acid at position 16 is Q or if substituted is H, K, or R;

the amino acid at position 17 is S or if substituted is C, F, K, W, or Y;

the amino acid at position 18 is W or if substituted is K, Q, or T;

the amino acid at position 19 is I or if substituted is C, G, or N;

the amino acid at position 20 is F or if substituted is C, G, L, or Y; and

the amino acid at position 21 is S or if substituted is A, F, G, H, or Y.

16. The polypeptide of claim 13 , wherein X4 comprises a cysteine substitution at position 3 of SEQ NO: 6.

17. The polypeptide of claim 13 , wherein X3 comprises a cysteine substitution at position 17 or 20 of SEQ ID NO: 5.

18. The polypeptide of claim 13 , wherein X2 is a peptide comprising an amino acid sequence at least 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the peptide KDEAEKAKRMKEWMKRIKT (SEQ ID NO:7).

19. The polypeptide of claim 18 , wherein X2 is a peptide comprising an amino acid sequence at least 80% identical to the peptide KDEAEKAKRMKEWMKRIKT (SEQ ID NO:7) wherein

the amino acid at position 1 is K or if substituted is A, H, L, M, R, S, or V;

the amino acid at position 2 is D or if substituted is A, E, Q, R, S, T, V, W, or Y;

the amino acid at position 3 is E or if substituted is C, G, K, L, N, Q, R, or W;

the amino acid at position 4 is A or if substituted is F, G, N, S, T, V, or Y;

the amino acid at position 5 is E or if substituted is A, G, I, M, R, V, or C;

the amino acid at position 6 is K or if substituted is C, E, L, N, R, or V;

the amino acid at position 7 is A or if substituted is C, E, I, L, S, T, V, or W;

the amino acid at position 8 is K or if substituted is H, L, M, S, T, W, or Y;

the amino acid at position 9 is R or if substituted is A, I, L, M, Q, or S;

the amino acid at position 10 is M or if substituted is A, I, S, W, or Y;

the amino acid at position 11 is K or if substituted is C, I, L, S, or V;

the amino acid at position 12 is E or if substituted is C, K, L, P, Q, R, or T;

the amino acid at position 13 is W or if substituted is A, D, H, or N;

the amino acid at position 14 is M or if substituted is A, C, G, I, L, S, T, or V;

the amino acid at position 15 is K or if substituted is A, E, G, I, L, M, R, or V;

the amino acid at position 16 is R or if substituted is G, H, L, S, T, V, or C;

the amino acid at position 17 is I or if substituted is A, L, or V;

the amino acid at position 18 is K or if substituted is A, C, D, E, G, H, I, M, or S; and

the amino acid at position 19 is T or if substituted is D, E, G, L, N, or V.

20. A non-naturally occurring polypeptide, wherein the polypeptide comprises a polypeptide at least 80% identical to the amino acid sequence selected from the group consisting of SEQ ID NOS: 90 or 181, wherein the polypeptide binds to IL-2 receptor βγ c heterodimer (IL-2Rβγ c ).

21. The polypeptide of claim 20 , wherein the polypeptide comprises a polypeptide at least 80% identical to the amino acid sequence of SEQ ID NO:90, and wherein one, two, or more of the following mutations are present and wherein numbering is relative to SEQ ID NO:181:

R50C;

E53C;

E62C;

E69C;

R73C; and/or

E82C.

22. The polypeptide of claim 20 , wherein the polypeptide comprises a polypeptide at least 80% identical to of the amino acid sequence of SEQ ID NO:181, and wherein one, two, or more of the following mutations are present

D56C;

K58C;

D59C;

R66C;

T77C;

E85C;

R50C;

E53C;

E62C;

E69C;

R73C; and/or

E82C.

23. The polypeptide of claim 20 wherein the polypeptide comprises a polypeptide at least 80% identical to the amino acid sequence of SEQ ID NO: 181.

24. The polypeptide of claim 20 wherein the polypeptide comprises a polypeptide at least 90% identical to the amino acid sequence of SEQ ID NO: 90.

25. The polypeptide of claim 20 wherein the polypeptide comprises a polypeptide at least 90% identical to the amino acid sequence of SEQ ID NO: 181.

26. The polypeptide of claim 20 wherein the polypeptide comprises a polypeptide at least 95% identical to the amino acid sequence of SEQ ID NO: 90.

27. The polypeptide of claim 20 wherein the polypeptide comprises a polypeptide at least 95% identical to the amino acid sequence of SEQ ID NO: 181.

28. The polypeptide of claim 20 wherein the polypeptide comprises a polypeptide at least 98% identical to the amino acid sequence of SEQ ID NO: 90.

29. The polypeptide of claim 20 wherein the polypeptide comprises a polypeptide at least 98% identical to the amino acid sequence of SEQ ID NO: 181.