Enrichment method for variant proteins with altered binding properties

Abstract

A method for selecting novel proteins such as growth hormone and antibody fragment variants having altered binding properties for their respective receptor molecules is provided. The method comprises fusing a gene encoding a protein of interest to the carboxy terminal domain of the gene III coat protein of the filamentous phage M13. The gene fusion is mutated to form a library of structurally related fusion proteins that are expressed in low quantity on the surface of a phagemid particle. Biological selection and screening are employed to identify novel ligands useful as drug candidates. Disclosed are preferred phagemid expression vectors and selected human growth hormone variants.

1 . A method for selecting novel binding polypeptides comprising:
 (a) constructing a replicable expression vector comprising a transcription regulatory element operably linked to a gene fusion encoding a fusion protein wherein the gene fusion comprises a first gene encoding a polypeptide, and a second gene encoding at least a portion of a phage coat protein;   (b) mutating the vector at one or more selected positions within the first gene thereby forming a family of related plasmids;   (c) transforming suitable host cells with the plasmids;   (d) infecting the transformed host cells with a helper phage having a gene encoding the phage coat protein;   (e) culturing the transformed infected host cells under conditions suitable for forming recombinant phagemid particles containing at least a portion of the plasmid and capable of transforming the host, the conditions adjusted so that no more than a minor amount of phagemid particles display more than one copy of the fusion protein on the surface of the particle;   (f) contacting the phagemid particles with a target molecule so that at least a portion of the phagemid particles bind to the target molecule; and   (g) separating the phagemid particles that bind from those that do not.   
     
     
         2 . The method of  claim 1  further comprising infecting a suitable host cells with the phagemid particles that bind and repeating steps (d) through (g). 
     
     
         3 . The method of  claim 2  wherein the steps are repeated one or more times. 
     
     
         4 . The method of  claim 1  wherein the expression vector further comprises a secretory signal sequence. 
     
     
         5 . The method of  claim 1  wherein the transcription regulatory element is a promoter system selected from the group; lac Z, pho A, tryptophan, tac, λ PL , bacteriophage T7, and combinations thereof. 
     
     
         6 . The method of  claim 1  wherein the first gene encodes a mammalian protein. 
     
     
         7 . The method of  claim 6  wherein the protein is selected from the group; growth hormone, human growth hormone (hGH), des-N methionyl human growth hormone, bovine growth hormone, parathyroid hormone, thyroxine, insulin A-chain, insulin B-chain, proinsulin, relaxin A-chain, relaxin B-chain, prorelaxin, follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), leutinizing hormone (LH), glycoprotein hormone receptors, calcitonin, glucagon, factor VIII, an antibody, lung surfactant, urokinase, streptokinase, human tissue-type plasminogen activator (t-PA), bombesin, factor IX, thrombin, hemopoietic growth factor, tumor necrosis factor-alpha and -beta, enkephalinase, human serum albumin, mullerian-inhibiting substance, mouse gonadotropin-associated peptide, β-lactamase, tissue factor protein, inhibin, activin, vascular endothelial growth factor, integrin receptors, thrombopoietin, protein A or D, rheumatoid factors, NGF-β, platelet-growth factor, transforming growth factor; TGF-alpha and TGF-beta, insulin-like growth factor-I and -II, insulin-like growth factor binding proteins, CD-4, DNase, latency associated peptide, erythropoietin, HER2 ligands, osteoinductive factors, interferon-alpha, -beta, and -gamma, colony stimulating factors (CSFs), M-CSF, GM-CSF, and G-CSF, interleukins (ILs), IL-1, IL-2, IL-3, IL-4, superoxide dismutase; decay accelerating factor, viral antigen, HIV envelope proteins GP120 and GP140, atrial natriuretic peptides A, B, or C, or immuno globulins, and fragments of the above-listed proteins. 
     
     
         8 . The method of  claim 7  wherein the protein is a human protein. 
     
     
         9 . The method of  claim 8  wherein the protein comprises more than about 100 amino acid residues. 
     
     
         10 . The method of  claim 1  wherein the protein comprises a plurality of rigid secondary structures displaying amino acids capable of interacting with the target, and the mutations are primarily produced at positions corresponding to codons encoding the amino acids. 
     
     
         11 . The method of  claim 10  wherein the rigid secondary structures comprise structures selected from the group; α-(3.6 13 ) helix, 3 10  helix,   4.4 16 ) helix, parallel and anti-parallel β-pleated sheets, reverse turns, and non-ordered structures. 
     
     
         12 . The method of  claim 10  wherein the mutations are produced at more than one codon. 
     
     
         13 . The method of  claim 12  wherein the mutations are produced on more than one rigid secondary structure. 
     
     
         14 . The method of  claim 1  wherein the helper phage is selected from the group M13KO7, M13R408, M13-VCS, and Phi X 174. 
     
     
         15 . The method of  claim 14  wherein the helper phage is M13KO7 and the coat protein is the M13 phage gene III coat protein. 
     
     
         16 . The method of  claim 15  wherein the host is  E. coli.    
     
     
         17 . The method of  claim 16  wherein the plasmid is under tight control of the transcription regulatory element. 
     
     
         18 . The method of  claim 17  wherein the amount is less than about 1%. 
     
     
         19 . The method of  claim 18  wherein the amount is less than 20% the amount of phagemid particles displaying a single copy of the fusion protein. 
     
     
         20 . The method of  claim 19  wherein the amount is less than 10%. 
     
     
         21 . The method of  claim 1  further comprising in step (a), inserting a DNA triplet, encoding an mRNA suppressible terminator codon between said first gene encoding a polypeptide, and said second gene encoding at least a portion of a phage coat protein. 
     
     
         22 . The method of  claim 21  wherein said mRNA suppressible terminator codon is selected from the following: UAG (amber), UAA (ocher) and UGA (opel). 
     
     
         23 . The method of  claim 22  wherein said suppressible mutation results in the detectable production of a fusion polypeptide containing sadi polypeptide and said coat protein when said expression vector is grown in a suppressor host cell; and, when grown in a non-suppressor host cell said polypeptide is synthesized substantially without fusion to said phage coat protein. 
     
     
         24 . A human growth hormone variant wherein hGH amino acids 172, 174, 176 and 178 respectively are as a group sequentially selected from one of the following: (1) R,S,F,R; (2) R,A,Y,R; (3) K,T,Y,K; (4) R,S,Y,R; (5) K,A,Y,R; (6) R,F,F,R; (7) K,Q,Y,R; (8) R,T,Y,H; (9) Q,R,Y,R; (10) K,K,Y,K; (11) R,S,F,S; and (12) K,S,N,R. 
     
     
         25 . A phagemid comprising a replicable expression vector comprising a transcription regulatory element operably linked to a gene fusion encoding a fusion protein wherein the gene fusion comprises a first gene encoding a polypeptide, and a second gene encoding at least a portion of a phage coat protein, wherein a DNA triplet codon encoding an mRNA suppressible terminator codon selected from UAG, UAA and UGA is inserted between the fused ends of the first and second genes, or is substituted for an amino acid encoding triplet codon adjacent to the gene fusion junction. 
     
     
         26 . The phagemid of  claim 25  wherein said first gene encodes a mammalian protein. 
     
     
         27 . The phagemid of  claim 26  wherein the protein is selected from the group: growth hormone, human growth hormone (hGH), des-N-methionyl human growth hormone, bovine growth hormone, parathyroid hormone, thyroxine, insulin A-chain, insulin B-chain, proinsulin, relaxin A-chain, relaxin B-chain, prorelaxin, follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), leutinizing hormone (LH), glycoprotein hormone receptors, calcitonin, glucagon, factor VIII, an antibody, lung surfactant, urokinase, streptokinase, human tissue-type plasminogen activator (t-PA), bombesin, factor IX, thrombin, hemopoietic growth factor, tumor necrosis factor-alpha and -beta, enkephalinase, human serum albumin, mullerian-inhibiting substance, mouse gonadotropin-associated peptide, β-lactamase, tissue factor protein, inhibin, activin, vascular endothelial growth factor, integrin receptors, thrombopoietin, protein A or D, rheumatoid factors, NGF-β platelet-growth factor, transforming growth factor; TGF-alpha and TGF-beta, insulin-like growth-I and -II, insulin-like growth factor binding proteins, CD-4, DNase, latency associated peptide, erythropoietin, osteoinductive factors, interferon-alpha, -beta, and -gamma, colony stimulating factors (CSFs), M-CSF, GM-CSF, and G-CSF, interleukins (ILs), IL-1, IL-2, IL-3, IL-4, superoxide dismutase; decay accelerating factor, viral antigen, HIV envelope proteins GP120 and GP140, atrial natriuretic peptides A, B or C immuno globulins, and fragments of the above-listed proteins. 
     
     
         28 . The phagemid of  claim 27  wherein said protein is a human protein. 
     
     
         29 . The phagemid of  claim 28  wherein the protein comprises more than about 100 amino acid residues. 
     
     
         30 . The phagemid of  claim 25  wherein said protein comprises a plurality of rigid secondary structures displaying amino acids capable of interacting with the target. 
     
     
         31 . The phagemid of  claim 30  wherein said rigid secondary structures comprises structures selected from the group; α-(3.6 13 ) helix, 3 10  helix, π-(4.4 16 ) helix, parallel and anti-parallel β-pleated sheets, reverse turns, and non-ordered structures. 
     
     
         32 . The phagemid of  claim 25  wherein the helper phage is selected from the group M13KO7, M13R408, M13-VCS, and Phi X 174. 
     
     
         33 . The phagemid of  claim 32  wherein the helper phage is M13KO7 and the coat protein is the M13 phage gene III coat protein. 
     
     
         34 . The phagemid of  claim 33  wherein the host is the  E. coli  wild type or suppressor type. 
     
     
         35 . The phagemid of  claim 34  wherein the plasmid is under tight control of the transcription regulatory element. 
     
     
         36 . The phagemid of  claim 35  wherein the number of phagemid particles displaying more than one copy of the fusion protein on the surface of the particles is less than 1%. 
     
     
         37 . The phagemid of  claim 36  wherein said number of phagemid particles is less than about 10%. 
     
     
         38 . The phagemid of  claim 37  wherein the number of phagemid particles is less than about 20%. 
     
     
         39 . A human growth variant wherein hGH amino acids 10, 14, 18, and 21 respectively are as a group sequentially selected from one of the following:
 (1) H,G,N,N; (2) A,W,D,N; (3) F,S,F,L; (4) Y,T,V,N and (5) I,N,I,N.   
     
     
         40 . A human growth variant wherein hGH amino acids 174 is serine and 176 is tyrosine and hGH amino acids 167, 171, 175 and 179 respectively are as a group sequentially selected from one of the following: (1) N,S,T,T; (2) E,S,T,I; (3) K,S,T,L; (4) N,N,T,T; (5) R,D,I,I; and (6) N,S,T,Q. 
     
     
         41 . A method for selecting novel binding polypeptides comprising
 (a) constructing a replicable expression vector comprising a transcription regulatory element operably linked to DNA encoding a protein of interest containing one or more subunits, wherein the DNA encoding at least one of the subunits is fused to the DNA encoding at least a portion of a phage coat protein;   (b) mutating the DNA encoding the protein of interest at one or more selected positions thereby forming a family of related vectors;   (c) transforming suitable host cells with the vectors;   (d) infecting the transformed host cells with a helper phage having a gene encoding the phage coat protein;   (e) culturing the transformed infected host cells under conditions suitable for forming recombinant phagemid particles containing at least a portion of the plasmid and capable of transforming the host, the conditions adjusted so that no more than a minor amount of phagemid particles display more than one copy of the fusion protein on the surface of the particle;   (f) contacting the phagemid particles with a target molecule so that at least a portion of the phagemid particles bind to the target molecule; and   (g) separating the phagemid particles that bind from those that do not.   
     
     
         42 . The method of  claim 41  wherein the expression vector further comprises a secretory signal sequence operably linked to the DNA encoding each subunit of the protein of interest. 
     
     
         43 . The method of  claim 42  wherein the protein of interest is a mammalian protein. 
     
     
         44 . The method of  claim 43  wherein the protein of interest is selected from the group; insulin, relaxin, follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), leutinizing hormone (LH), glycoprotein hormone receptors, monoclonal and polyclonal antibodies, lung surfactant, integrin receptors, insulin-like growth factor-I and -II, and fragments of the above-listed proteins. 
     
     
         45 . The method of  claim 44  wherein the protein of interest is a humanized antibody. 
     
     
         46 . The method of  claim 45  wherein the protein of interest is a humanized Fab fragment capable of binding to the HER-2 receptor (human epidermal growth factor receptor-2). 
     
     
         47 . A human growth hormone (hGH) variant wherein hGH amino acid glutamate 174  is replaced by serine 174  and phenylalanine 176  is replaced by tyrosine 176  and one or more of the eight naturally occurring hGH amino acids F10, M14, H18, H21, R167, D171, T175 and I179 are replaced by another natural amino acid. 
     
     
         48 . The hGH variant of  claim 47  wherein the eight naturally occurring hGH amino acids F10, M14, H18, H21, R167, D171, T175 and I179 respectively are as a group replaced with a corresponding amino acid sequentially selected from one of the following groups: 
       
         
           
                 
                 
               
                   (1) 
                   H, G, N, N, N, S, T, T; 
                 
                     
                 
                   (2) 
                   H, G, N, N, E, S, T, I; 
                 
                     
                 
                   (3) 
                   H, G, N, N, N, N, T, T; 
                 
                     
                 
                   (4) 
                   A, W, D, N, N, S, T, T; 
                 
                     
                 
                   (5) 
                   A, W, D, N, E, S, T, I; 
                 
                     
                 
                   (6) 
                   A, W, D, N, N, N, T, T; 
                 
                     
                 
                   (7) 
                   F, S, F, L, N, S, T, T; 
                 
                     
                 
                   (8) 
                   F, S, F, L, E, S, T, I; 
                 
                     
                 
                   (9) 
                   F, S, F, L, N, N, T, T. 
                 
                     
                 
                   (10) 
                   H, G, N, N, N, S, T, N; 
                 
                     
                 
                   (11) 
                   A, N, D, A, N, N, T, N; 
                 
                     
                 
                   (12) 
                   F, S, F, G, H, S, T, T; 
                 
                     
                 
                   (13) 
                   H, Q, T, S, A, D, N, S; 
                 
                     
                 
                   (14) 
                   H, G, N, N, N, A, T, T; 
                 
                     
                 
                   (15) 
                   F, S, F, L, S, D, T, T; 
                 
                     
                 
                   (16) 
                   A, S, T, N, R, D, T, I; 
                 
                     
                 
                   (17) 
                   Q, Y, N, N, H, S, T, T; 
                 
                     
                 
                   (18) 
                   W, G, S, S, R, D, T, I; 
                 
                     
                 
                   (19) 
                   F, L, S, S, K, N, T, V; 
                 
                     
                 
                   (20) 
                   W, N, N, S, H, S, T, T; 
                 
                     
                 
                   (21) 
                   A, N, A, S, N, S, T, T; 
                 
                     
                 
                   (22) 
                   P, S, D, N, R, D, T, I; 
                 
                     
                 
                   (23) 
                   H, G, N, N, N, N, T, S; 
                 
                     
                 
                   (24) 
                   F, S, T, G, R, D, T, I; 
                 
                     
                 
                   (25) 
                   M, T, S, N, Q, S, T, T; 
                 
                     
                 
                   (26) 
                   F, S, F, L, T, S, T, S; 
                 
                     
                 
                   (27) 
                   A, W, D, N, R, D, T, I; 
                 
                     
                 
                   (28) 
                   A, W, D, N, H, S, T, N; 
                 
                     
                 
                   (29) 
                   M, Q, M, N, N, S, T, T; 
                 
                     
                 
                   (30) 
                   H, Y, D, H, R, D, T, T; 
                 
                     
                 
                   (31) 
                   L, N, S, H, R, D, T, I; 
                 
                     
                 
                   (32) 
                   L, N, S, H, T, S, T, T; 
                 
                     
                 
                   (33) 
                   A, W, D, N, N, A, T, T; 
                 
                     
                 
                   (34) 
                   F, S, T, G, R, D, T, I; 
                 
                     
                 
                   (35) 
                   A, W, D, N, R, D, T, I; 
                 
                     
                 
                   (36) 
                   I, Q, E, H, N, S, T, T; 
                 
                     
                 
                   (37) 
                   F, S, L, A, N, S, T, V; 
                 
                     
                 
                   (38) 
                   F, S, F, L, K, D, T, T; 
                 
                     
                 
                   (39) 
                   M, A, D, N, N, S, T, T; 
                 
                     
                 
                   (40) 
                   A, W, D, N, S, S, V, T; 
                 
                     
                 
                   (41) 
                   H, Q, Y, S, R, D, T, I. 
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         49 . The method of  claim 48  wherein said human growth hormone variant (11) further contains leucine 15  replaced by arginine 15  and lysine 168  replaced by arginine 168 . 
     
     
         50 . The method of  claim 48  wherein said human growth hormone variant
 (40) further contains phenylalanine 176      
     
     
         51 . A method for selecting novel binding polypeptides comprising:
 (a) constructing a replicable expression vector comprising a transcription regulatory element operably linked to a gene fusion encoding a fusion protein wherein the gene fusion comprises a first gene encoding a polypeptide operable connected to a linking amino acid sequence, and a second gene encoding at least a portion of a phage coat protein;   (b) mutating the vector at one or more selected positions within the amino acid linking sequence of the first gene thereby forming a family of related plasmids;   (c) transforming suitable host cells with the plasmids;   (d) infecting the transformed host cells with a helper phage having a gene encoding the phage coat protein;   (e) culturing the transformed infected host cells under conditions suitable for forming recombinant phagemid particles containing at least a portion of the plasmid and capable of transforming the host, the conditions adjusted so that no more than a minor amount of phagemid particles display more than one copy of the fusion protein on the surface of the particle;   (f) contacting the phagemid particles with a target molecule so that at least a portion of the phagemid particles bind to the target molecule; and   (g) contacting the bound phagemid particles with a protease capable of hydrolysing the linking a amino acid sequence of at least a portion of the bound phagemid particles, and   (h) isolating the hydrolyzed phagemid particles.   
     
     
         52 . The method of  claim 51  further comprising infecting suitable host cells with the hydrolyzed phagemid particles and repeating steps (d) through (h).