Compositions for modulating IL-10 immunostimulatory and anti-inflammatory properties

1. A single chain IL-10 (scIL-10) polypeptide comprising an amino acid sequence arrangement from N-terminus to C-terminus in accordance with Formula 1:

(first monomer subunit)-LINKER-(second monomer subunit)   Formula 1

wherein the first monomer subunit and the second monomer subunit may be independently selected from: SEQ ID NO: 1; or SEQ ID NO: 1 comprising at least one amino acid substitution, wherein the at least one amino acid substitution is at an amino acid position of SEQ ID NO: 1 that is selected from: amino acid position 22, amino acid position 41, amino acid position 87, and any combination thereof with the proviso that at least one of the first monomer subunit or the second monomer subunit comprises at least one amino acid substitution;

wherein LINKER is an amino acid linker of between 1 and 15 amino acids in length; wherein the amino acid substitutions comprise the substitution of amino acids of scIL-10 that interface with IL-10R1, IL-10R2 or amino acids that interface with both IL-10R1 and IL-10R2; and

wherein the scIL-10 polypeptide is optionally covalently attached to a fusion partner.

2. The polypeptide of claim 1 , wherein the amino acid substitutions comprise substitutions of amino acids selected from: methionine at position 22 and aspartic acid at position 41 or any combination thereof.

3. The polypeptide of claim 2 , wherein aspartic acid at position 41 is substituted on the first monomer subunit or on the second monomer subunit but not both monomer subunits.

4. The polypeptide of claim 3 , wherein methionine at position 22 is substituted on only one monomer subunit that is not the same monomer subunit comprising the substitution of aspartic acid at position 41.

5. The polypeptide of claim 1 , wherein the amino acid substitution comprises isoleucine at position 87.

6. The polypeptide of claim 1 , wherein the amino acid substitutions are selected from: methionine at position 22 to alanine (M22A); aspartic acid at position 41 to asparagine (D41N); aspartic acid at position 41 to alanine (D41A); aspartic acid at position 41 to phenylalanine (D41F).

7. The polypeptide of claim 1 , comprising a fusion partner wherein scIL-10 is fused to the hinge region IgG1.

8. The polypeptide of claim 1 , comprising a fusion partner wherein scIL-10 is fused to a modified hinge region if IgG1 wherein the modification to the hinge region is the deletion of between 1 and 10 amino acids from the hinge region of IgG1.

9. The polypeptide of claim 1 , comprising a fusion partner wherein scIL-10 is fused to the hinge region of IgG1 via a mucin linker.

10. The polypeptide of claim 9 , wherein the mucin linker comprises an amino acid sequence that is a tandem repeat of MUC20.

11. The scIL-10 polypeptide of claim 1 , comprising a fusion partner wherein the fusion protein comprises an amino acid sequence of Formula 2

(scIL-10)-L1-HINGE-Fc   Formula 2

wherein,

(scIL-10) has the amino acid sequence of Formula 1;

L1 is a linker having the following arrangement from amino-terminus to carboxy-terminus:

L2-CL-L3-CH1-L4 or L2-CH1-L3-CL-L4

wherein,

L2 and L4 are independently polypeptide linkers or are independently absent;

L3 is a polypeptide linker;

CL is a constant region polypeptide of an immunoglobulin light chain; and

CH1 is a constant region polypeptide from a CH1 domain of an immunoglobulin heavy chain;

HINGE is a hinge sequence of an immunoglobulin or is absent with the proviso that if HINGE is absent, L4 is present; and

Fc is the carboxy-terminus of an immunoglobulin or any active fragment or derivative thereof.

12. The polypeptide of claim 11 , wherein CL, CH1, HINGE and Fc are at least 90% identical to the CL, CH1, hinge and Fc regions respectively of human IgG1.

13. The polypeptide of claim 11 , wherein L3 is a polypeptide linker having the amino acid sequence (GGGGS)n (SEQ ID NO: 54) wherein n is 1-5.

14. The polypeptide of claim 11 , wherein L2 is present and is a polypeptide linker having the amino acid sequence (GGGGS)n (SEQ ID NO: 54) wherein n is 1-5.

15. The polypeptide of claim 11 , wherein L4 is present and is a polypeptide linker having the amino acid sequence (GGGGS)n (SEQ ID NO: 54) wherein n is 1-5.

16. The polypeptide of claim 11 , wherein HINGE and L2 are present and L4 is absent.

17. The polypeptide of claim 11 , wherein HINGE, L2 and L4 are present.

18. The polypeptide of claim 11 , wherein HINGE is absent and L4 is present.

19. The polypeptide of claim 11 , wherein HINGE is absent and L2 and L4 are present.

20. A dimerized complex comprising the polypeptide of claim 11 wherein L1 is a linker having the following arrangement from amino-terminus to carboxy-terminus: L2-CL-L3-CH1-L4.

21. The polypeptide of claim 1 , selected from the group consisting of: SEQ ID NOs: 20-21 and SEQ ID NOS: 37-44.

22. The polypeptide of claim 1 , selected from the group consisting of: SEQ ID NOS: 17, 18 and 19.