Methods for treating malignant tumors with IL-10 variant conjugates

1. A method of treating a tumor comprising administering to a patient in need thereof a therapeutically effective amount of a composition comprising a fusion protein of formula I, II, III, IV, or V

IL10-L 1 -X 1 -L 1 -X 2 -L 1 -IL10  (Formula I);

(Z) n -X 1 -L 2 -Y 2 -L 1 -IL10  (Formula II);

IL10-L 1 -Y 1 -L 2 -X 2 -(Z) n   (Formula III);

X 1 -L 2 -X 2 -L 1 -IL10  (Formula IV); or

IL10-L 1 -X 1 -L 2 -X 2   (Formula V),

wherein

“IL-10” is a monomer sequence selected from SEQ ID No: 59;

“L 1 ” is a linker of SEQ ID No: 31 or 54;

“L 2 ” is a linker of SEQ ID No: 30;

“X 1 ” is a VH region obtained from a first antibody specific for epidermal growth factor receptor (EGFR); CD3α; HIV, or Ebola;

“X 2 ” is a VL region obtained from the same antibody as X 1 ;

“Y 1 ” is a VH region obtained from a second antibody specific for EGFR, CD3α, HIV, or Ebola;

“Y 2 ” is a VL region obtained from the same antibody as Y 1 ; and

“Z” is a cytokine other than IL-10, wherein “n” is an integer of zero;

wherein the effective amount of the fusion protein reduces the tumor overexpressing EGFR.

2. The method according to claim 1 , wherein the fusion protein is SEQ ID Nos: 28-29, 35-36, 38-39, 46-47, 52, 53, 61, 63, 65, or 67.

3. The method according to claim 1 , wherein the fusion protein forms a protein complex and the protein complex is formed between SEQ ID Nos: 28 and 29; 35 and 36; 38 and 39; or 46 and 47.

4. The method according to claim 1 , wherein formula IV and V are capable of forming a fusion protein complex where the IL-10 monomer from each of formula IV and V are capable of forming a functional homodimer.

5. The method according to claim 4 , wherein formula IV is SEQ ID Nos: 35, 38, or 46.

6. The method according to claim 4 , wherein formula V is SEQ ID Nos: 36, 39, or 47.

7. The method according to claim 4 , wherein the fusion protein complex is formed between SEQ ID Nos: 35 and 36; 38 and 39, or 46 and 47.

8. The method according to claim 1 , wherein formula VI and VII are capable of forming a fusion protein complex where the IL-10 monomer from each of formula VI and VII are capable of forming a functional homodimer; and wherein the VH region and VL region pair to form a scFv.

9. The method according to claim 1 , wherein formula I is SEQ ID Nos: 33-34, 44, 52 or 53.

10. The method according to claim 1 , wherein the VH region and the VL region are from an anti-Ebola antibody; and wherein the VH region and VL region comprise a framework region with 6 CDRs.

11. The method according to claim 10 , wherein the VH region and the VL region comprise the framework regions from an anti-Ebola antibody that is engrafted with 6 CDRs from an anti-EGFR antibody.

12. The method according to claim 1 , wherein the VH region and the VL region comprise modifications that reduce the antigenicity in a subject.

13. The method according to claim 1 , wherein the composition is a pharmaceutical composition comprising a therapeutically effective amount of the fusion protein and a pharmaceutically acceptable carrier, excipient, preservative, stabilizers, or any combination thereof.

14. The method according to claim 1 , wherein the solid tumor overexpressing EGFR is lung cancer, prostate, breast, colon, head and neck, esophagogastic, liver, glioblastoma, cervix, ovarian, bladder, kidney, or pancreatic.

15. The method according to claim 1 , wherein the first antibody is an anti-ebola antibody, and the second antibody is anti-EGFR antibody.