Enhancing the circulating half-life of interleukin-2 proteins

1 . A protein comprising an interleukin-2 protein, wherein Lys 8 and Lys 9 of the interleukin-2 protein are replaced with non-lysine amino acids.

2 . A fusion protein comprising the protein of claim 1 and a carrier protein.

3 . The fusion protein of claim 2 , wherein the carrier protein is fused to the N-terminal portion of the interleukin-2 protein.

4 . The protein of claim 1 , wherein the non-lysine amino acids are hydrophobic amino acids.

5 . The protein of claim 4 , wherein the hydrophobic amino acids are selected from the group consisting of tryptophan, phenylalanine, tyrosine, methionine, glycine, alanine, leucine, isoleucine and valine.

6 . The protein of claim 1 , wherein the non-lysine amino acids are alanines.

7 . The protein of claim 1 , wherein the interleukin-2 protein is derived from a mammalian interleukin-2.

8 . The protein of claim 1 , wherein the interleukin-2 protein is derived from a human interleukin-2.

9 . The protein of claim 3 , wherein the N-terminal portion of the interleukin-2 protein comprises an O-glycosylation site.

10 . The fusion protein of claim 2 , wherein the carrier protein comprises albumin.

11 . The fusion protein of claim 2 , wherein the carrier protein comprises an immunoglobulin (Ig) moiety.

12 . The fusion protein of claim 11 , wherein the Ig moiety comprises at least a portion of an Ig heavy chain.

13 . The fusion protein of claim 12 , wherein at least one amino acid of the C-terminal portion of the Ig moiety is replaced with a hydrophobic amino acid.

14 . The fusion protein of claim 13 , wherein the C-terminal lysine residue of the Ig moiety is replaced with an alanine.

15 . The fusion protein of claim 11 , wherein the Ig moiety comprises at least the CH2 domain of an IgG2 or an IgG4 constant region.

16 . The fusion protein of claim 11 , wherein the Ig moiety comprises at least a portion of an IgG1 constant region where one or more amino acids selected from the group consisting of Leu 234 , Leu 235 , Gly 236 , Gly 237 , Asn 297 , and Pro 331 are mutated or deleted.

17 . The fusion protein of claim 11 , wherein the Ig moiety comprises at least a portion of an IgG3 constant region where one or more amino acids selected from the group consisting of Leu 281 , Leu 282 , Gly 283 , Gly 284 , Asn 344 , and Pro 378 are mutated or deleted.

18 . The fusion protein of claim 2 , further comprising a linker peptide between the carrier protein and the interleukin-2 protein.

19 . A nucleic acid molecule encoding the protein of claim 1 .

20 . An expression vector containing the nucleic acid molecule of claim 19 .

21 . A cell comprising the nucleic acid of claim 19 .

22 . A process for preparing a protein comprising maintaining the cell of claim 21 under conditions permitting expression of the protein and harvesting the expressed protein.

23 . A pharmaceutical composition comprising the protein of claim 1 and a pharmaceutically acceptable carrier.

24 . A method of treating a disease in a mammal, the method comprising the step of administering to the mammal the composition of claim 23 .

25 . The method of claim 24 , wherein the mammal is a human.

26 . The method of claim 24 , wherein the disease is selected from the group consisting of cancer, viral infection, and an immune disorder.

27 . A method of treating a patient, the method comprising administering to the patient the nucleic acid of claim 19 .

28 . A method of treating a patient, the method comprising administering to the patient the cell of claim 21 .

29 . A method of determining the extent of O-glycosylation of an immunocytokine comprising:

providing an immunocytokine which has an O-glycosylation site;

measuring the level of O-glycosylation; and

comparing the level of O-glycosylation with a control.

30 . The method of claim 29 , wherein the immunocytokine comprises an immunoglobulin (Ig) and an interleukin-2 fusion protein.

31 . The method of claim 30 , comprising measuring the extent of O-glycosylation at Thr 3 of interleukin-2.