Derivatives of growth hormone and related proteins

Abstract

The growth hormone supergene family comprises greater than 20 structurally related cytokines and growth factors. A general method is provided for creating site-specific, biologically active conjugates of these proteins. The method involves adding cysteine residues to non-essential regions of the proteins or substituting cysteine residues for non-essential amino acids in the proteins using site-directed mutagenesis and then covalently coupling a cysteine-reactive polymer or other type of cysteine-reactive moiety to the proteins via the added cysteine residue. Disclosed herein are preferred sites for adding cysteine residues or introducing cysteine substitutions into the proteins, and the proteins and protein derivatives produced thereby.

1 . A cysteine variant of a member of the GH supergene family comprising a cysteine residue substituted for an amino acid selected from the group consisting of an amino acid in the loop regions, an amino acid near the ends of the alpha helices, an amino acid proximal to the first amphipathic helix, and an amino acid distal to the final amphipathic helix or wherein the cysteine residue is added at the N-terminus or C-terminus of the proteins. 
     
     
         2 . A cysteine variant of a member of the GH supergene family comprising a cysteine residue introduced between two amino acids in the loop regions, the ends of the alpha helices, proximal to the first amphipathic helix, or distal to the final amphipathic helix. 
     
     
         3 . A cysteine variant according to  claim 1  wherein the amino acid substituted for is part of an N- or O-linked glycosylation site. 
     
     
         4 . A cysteine variant according to  claim 2  wherein the cysteine residue is introduced between two amino acids in an N-linked glycosylation site or adjacent to an amino acid in an N-linked or O-linked glycosylation site. 
     
     
         5 . A cysteine variant according to  claim 1  wherein the member of the GH supergene family is selected from the group consisting of growth hormone, prolactin, placental lactogen, erythropoietin, thrombopoietin, interleukin-2, interleukin-3, interleukin-4, interleukin-5, interleukin-6, interleukin-7, interleukin-9, interleukin-10, interleukin-11, interleukin-12 (p35 subunit), interleukin-13, interleukin-15, oncostatin M, ciliary neurotrophic factor, leukemia inhibitory factor, alpha interferon, beta interferon, gamma interferon, omega interferon, tau interferon, granulocyte-colony stimulating factor, granulocyte-macrophage colony stimulating factor, cardiotrophin-1 and macrophage colony stimulating factor. 
     
     
         6 . A cysteine variant according to  claim 1  wherein the amino acid substituted for is in the A-B loop, the B-C loop, the C-D loop or D-E loop of interferon/interferon-10-like members of the GH supergene family. 
     
     
         7 . A cysteine variant according to  claim 2  wherein the loop region is the A-B loop, the B-C loop, the C-D loop or D-E loop of interferon/interferon-10-like members of the GH supergene family. 
     
     
         8 . A cysteine variant according to  claim 1  wherein the added cysteine is PEGylated. 
     
     
         9 . A cysteine variant according to  claim 1  wherein the member of the GH supergene family is growth hormone. 
     
     
         10 . The cysteine variant according to  claim 9  wherein the substituted for amino acid is selected from the group consisting of amino acids located at the N-terminal end of the A-B loop, the B-C loop, the C-D loop, the first three or last three amino acids in the A, B, C and D helices and the amino acids proximal to helix A and distal to helix D. 
     
     
         11 . The cysteine variant according to  claim 10  wherein the substituted for amino acid is selected from the group consisting of F1, T3, P5, E33, A34, K38, E39, Q40, S43, Q46, N47, P48, Q49, T50, S51, S55, T60, A98, N99, S100, G104, A105, S106, E129, D130, G131, S132, P133, T135, G136, Q137, K140, Q141, T142, S144, K145, D147, T148, N149, S150, H151, N152, D153, S184, E186, G187, S188, and G190. 
     
     
         12 . A cysteine variant according to  claim 1  wherein the member of the GH supergene family is erythropoietin. 
     
     
         13 . The cysteine variant according to  claim 12  wherein the substituted for amino acid is selected from the group consisting of amino acids located in the A-B loop, the B-C loop, the C-D loop, the amino acids proximal to helix A and distal to helix D and the N- or C-terminus. 
     
     
         14 . A cysteine variant according to  claim 13  wherein the substituted for amino acid is selected from the group consisting of serine-126, N24, I25, T26, N38, I39, T40, N83, S84, A1, P2, P3, R4, D8, S9, T27, G28, A30, E31, H32, S34, N36, D43, T44, K45, N47, A50, K52, E55, G57, Q58, G77, Q78, A79, Q86, W88, E89, T107, R110, A111, G113, A114, Q115, K116, E117, A118, S120, P121, P122, D123, A124, A125, A127, A128, T132, K154, T157, G158, E159, A160, T163, G164, D165, R166 and S85. 
     
     
         15 . A cysteine variant according to  claim 2  wherein the member of the GH supergene family is selected from the group consisting of growth hormone, erythropoietin, alpha interferon, and granulocyte-colony stimulating factor. 
     
     
         16 . A cysteine variant according to  claim 15  wherein the cysteine is introduced into the region selected from the group consisting of amino acids located at the N-terminal end of the A-B loop, the B-C loop, the C-D loop, the first three or last three amino acids in the A, B, C and D helices and the amino acids proximal to helix A and distal to helix D. 
     
     
         17 .- 22 . (canceled) 
     
     
         23 . A cysteine variant according to  claim 2  wherein the added cysteine is PEGylated.