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

1. A method of increasing the anti-inflammatory window size of an IL-10 therapy in a patient suffering from an IL-10-related disease or disorder, comprising administering an effective amount to the patient 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 about 1 and about 100 amino acids in length; and

wherein scIL-10 is optionally covalently attached to a fusion partner,

thereby increasing the anti-inflammatory window size in the patient suffering from an IL-10-related disease or disorder.

2. The method of claim 1 , wherein LINKER is 5-15 amino acids in length.

3. The method of claim 1 , 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.

4. The method of claim 1 , wherein aspartic acid at position 41 is substituted on the first monomer subunit or on the second monomer subunit but not both monomer subunits.

5. The method of claim 4 , 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.

6. The method of claim 1 , wherein the amino acid substitution comprises isoleucine at position 87 to alanine (I87A).

7. The method of claim 3 , 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).

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

9. The method 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.

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

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

12. The method of claim 1 , comprising a fusion partner wherein scIL-10 is fused to a single chain Fc linker wherein the fusion protein has the sequence of Formula 2

(scIL-10)-L1-HINGE-Fc   Formula 2

wherein,

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.

13. The method of claim 1 , wherein the polypeptide comprises SEQ ID NOS: 12-21, 23-29, 31, 33, and 35-45 and 52.

14. The method of claim 1 , wherein the IL-10-related disease or disorder is selected from the group consisting of: an autoimmune disorder, a fibrotic disease, an inflammatory disease, an ischemic disease, a neurodegenerative disease, a neuropathic disease, a pain disorder, an auditory disorder, a psychiatric disorder, and cancer.