Modified polypeptides

Abstract

The present invention relates to polypeptides with reduced immune response including reduced allergenicity having one or more amino acid residues being substituted with other amino acid residues and/or having coupled one or more polymeric molecules in the vicinity of the polypeptides metal binding site, a method for preparing modified polypeptides of the invention, the use of the polypeptide for reducing the immunogenicity and allergenicity and compositions comprising the polypeptide.

What is claimed is:  
     
         1 . A polypeptide with reduced immune response, having one or more amino acid residues modified, wherein the C alpha -atoms of the amino acid residues are located less than 15 Å from a ligand bound to the polypeptide.  
     
     
         2 . The polypeptide of  claim 1 , wherein the polypeptide has reduced allergenicity.  
     
     
         3 . The polypeptide of  claim 1 , wherein the C beta -atom of the amino acid residues is located closer to the ligand than the C alpha -atom.  
     
     
         4 . The polypeptide of  claim 1 , wherein the C alpha -atoms of the amino acid residues are located less than 10 Å from the ligand and the amino acid residues have an accessibility of at least 15%.  
     
     
         5 . The polypeptide of  claim 1 , wherein the ligand is a metal or metal ion.  
     
     
         6 . The polypeptide of  claim 1 , wherein the polypeptide is modified by substitution of amino acid residues.  
     
     
         7 . The polypeptide of  claim 1 , wherein the modified polypeptide is selected from a diverse library of variants.  
     
     
         8 . The polypeptide of  claim 6 , wherein the substituting amino acids contain amino groups in the form of Lysine residues(s), or carboxylic groups in the form of Aspartic acid or Glutamic acid residues, or SH-groups in the form of Cysteine residues.  
     
     
         9 . The polypeptide of  claim 6 , wherein the modification(s) is(are) prepared by a conservative substitution of an amino acid residue, such as an Arginine to Lysine substitution or Aspargine to Aspartate/Glutamate or a Glutamine to Aspartate/Glutamate substitution or Threonine/Serine to Cysteine.  
     
     
         10 . The polypeptide of  claim 1 , wherein the polypeptide is modified by coupling one or more polymeric molecules to the polypeptide, thereby providing a polypeptide-polymer conjugate.  
     
     
         11 . The polypeptide of  claim 10 , wherein the parent polypeptide moiety of the conjugate has a molecular weight from 1 to 1000 kDa.  
     
     
         12 . The polypeptide of  claim 10 , wherein the polymeric molecules coupled to the polypeptide have a molecular weight from 0.1 to 100 kDa.  
     
     
         13 . The polypeptide of  claim 1 , wherein the polypeptide or parent polypeptide is an enzyme selected from the group of Oxidoreductases, including laccases and Superoxide dismutase (SOD); Hydrolases, including carbohydrases, amylases, proteases, especially subtilisins; Transferases, including Transglutaminases (TGases); Isomerases, including Protein disulfide Isomerases (PDI); Lyases, including Pectate lyases.  
     
     
         14 . The polypeptide of  claim 13 , wherein the polypeptide or parent polypeptide is PD498, Savinase®, BPN′, Amylase, Proteinase K, Proteinase R, Subtilisin DY, Lion Y, Rennilase®, JA16, Alcalase®.  
     
     
         15 . The polypeptide of  claim 14 , wherein the polypeptide or parent polypeptide of the conjugate is a PD498 variant with one or more of the following substitutions: The amino acid residues in position 86, 87, 7, 47, 51, 219, 12, 218, 10, 11, 53, 28, 1, 65, 61, 63, 67, 60, 69, 55, 44, 45, 111, 115, 109, 215, 200, 202, 170, 268, 250, 152, 254, 136, 269, 246, 141 is substituted with K, D, E, or C, preferably R250K, R250D, R250E, R250C.  
     
     
         16 . The polypeptide of  claim 14 , wherein the polypeptide or parent polypeptide is a BPN′ variant with one or more of the following substitutions: The amino acid residues in position 77, 2, 5, 43, 214, 206, 22, 215, 14, 17, 9, 36, 211, 195, 197, 154, 163, 247, 265, 251, 143, 127, 260, 131, 128, 243 is substituted with K, D, E, or C, preferably R247K, R247D, R247E, R247C.  
     
     
         17 . The polypeptide of  claim 14 , wherein the polypeptide or parent polypeptide is a Savinase® variant with one or more of the following substitutions: The amino acid residues in position 75, 2, 42, 208, 200, 14, 22, 17, 189, 241, 125, 125, 141, 245, 259, 237, 254, 157 is substituted with K, D, E, or C, preferably R241K, R241D, R241E, R241C.  
     
     
         18 . The polypeptide of  claim 14 , wherein the polypeptide or parent polypeptide is an amylase variant with one or more of the following substitutions: The amino acid residue in position 124, 126, 128, 159, 160, 166, 185, 186, 189, 190, 193, 194, 195, 196, 198, 201, 202, 203, 209, 210, 214, 242, 244, 247, 296, 298, 299, 302, 303, 304, 306, 307, 308, 310, 311, 314, 345, 347, 405, 406, 407, 408, 409, 433, 434, 435, 436, 437, 475, 476, 477, 478 is substituted with K, D, E, or C.  
     
     
         19 . The polypeptide of  claim 10 , wherein the polymeric molecule is selected from natural or synthetic homo- and heteropolymers, selected from the group of the synthetic polymeric molecules including Branched PEGs, poly-vinyl alcohol (PVA), poly-carboxyl acids, poly-(vinylpyrolidone) and poly-D,L-amino acids, or natural occurring polymeric molecules including dextrans, including carboxymethyl-dextrans, and celluloses such as methylcellulose, carboxymethylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, and hydrolysates of chitosan, starches, such as hydroxyethyl-starches, hydroxypropyl-starches, glycogen, agarose, guar gum, inulin, pullulans, xanthan gums, carrageenin, pectin and alginic acid.  
     
     
         20 . The polypeptide of  claim 19 , wherein the modified polypeptide is savinase variant R241KbPEG1000 or R241KbPEG2000.  
     
     
         21 . The polypeptide of  claim 1 , wherein the modified polypeptide is savinase variants R241Q, R241E, R241H or R241K.  
     
     
         22 . A method for preparing polypeptides with reduced immune response comprising the steps of: 
 (a) identifying amino acid residues located on the surface of the 3-dimensional structure of the parent polypeptide in question,    (b) selecting target amino acid residues on the surface of the 3-dimensional structure of the parent polypeptide to be modified,    (c) substituting one or more amino acid residues selected in step b) with other amino acid residues, and/or    (d) coupling polymeric molecules to the amino acid residues in step b)and/or step c).    
     
     
         23 . The method of  claim 22 , wherein the C alpha -atoms of the amino acid residues are located less than 15 Å from the ligand bound to said polypeptide.  
     
     
         24 . The method of  claim 22 , wherein the C beta -atoms of the amino acid residues are located closer to the ligand than the C alpha -atom.  
     
     
         25 . The method of  claim 22 , wherein the C alpha -atoms of the amino acid residues are located less than 10 Å from the ligand and the amino acid residues have an accessibility of at least 15%.  
     
     
         26 . The method of  claim 22 , wherein the identification of amino acid residues located on the surface on the polypeptide referred to in step (a) are performed by a computer program analyzing the 3-dimensional structure of the parent polypeptide in question.  
     
     
         27 . The method of  claim 22 , wherein step (b) comprises selecting arginine or lysine residues on the surface of the parent polypeptide.  
     
     
         28 . The method of  claim 27 , wherein one or more arginine residues identified in step b) is (are) substituted with a lysine residue(s) in step (c).  
     
     
         29 . A composition comprising a modified polypeptide of  claim 1  and further comprising ingredients used in industrial products.  
     
     
         30 . The composition of  claim 29 , wherein the industrial product is a detergent, such as a laundry, dish wash or hard surface cleaning product, including bio-film products or a food or feed product or a textile product.  
     
     
         31 . An enzyme selected from the group consisting of carbohydrases, lipases, oxidoreductases, transferases, isomerases and lyases with reduced immune response, said enzyme having one or more amino acid residues modified, wherein the C alpha -atoms of said amino acid residues are located less than 15 Å from a ligand bound to said enzyme.  
     
     
         32 . The enzyme of  claim 31 , wherein the enzyme has reduced allergenicity.  
     
     
         33 . The enzyme of  claim 31 , wherein the C beta -atom of the amino acid residues is located closer to the ligand than the C alpha -atom.  
     
     
         34 . The enzyme of  claim 31 , wherein the C alpha -atoms of the amino acid residues are located less than 10 Å from the ligand and said amino acid residues have an accessibility of at least 15%.  
     
     
         35 . The enzyme of  claim 31 , wherein the ligand is a metal or metal ion.  
     
     
         36 . The enzyme of  claim 31 , wherein the enzyme is modified by substitution of amino acid residues.  
     
     
         37 . The enzyme of  claim 31 , wherein the modified enzyme has been selected from a diverse library of variants.  
     
     
         38 . The enzyme of  claim 36 , wherein the substituting amino acids contain amino groups in the form of lysine residues(s), carboxylic groups in the form of aspartic acid or glutamic acid residues, or SH-groups in the form of cysteine residues.  
     
     
         39 . The enzyme of  claim 36 , wherein the modification(s) is (are) a conservative substitution of an amino acid residue.  
     
     
         40 . The enzyme of  claim 39 , wherein the conservative substitution is a substitution of arginine to lysine, aspargine to aspartate/glutamate, glutamine to aspartate/glutamate or threonine/serine to cysteine.  
     
     
         41 . The enzyme of  claim 31 , wherein the enzyme is modified by coupling one or more polymeric molecules to said enzyme, thereby providing an enzyme-polymer conjugate.  
     
     
         42 . The enzyme of  claim 41 , wherein the parent enzyme moiety of the conjugate has a molecular weight from 1 to 1000 kDa, preferred 4 to 100 kDa, more preferred 12 to 60 kDa.  
     
     
         43 . The enzyme of  claim 41 , wherein the polymeric molecules coupled to the enzyme have a molecular weight from 0.1 to 100, preferably 0.1 to 60 kDa, more preferably 0.3-5 kDa, most preferably 1 to 2 kDa.  
     
     
         44 . The enzyme of  claim 41 , wherein the polymeric molecule is selected from the group comprising a natural or synthetic homo- and heteropolymers, selected from the group of the synthetic polymeric molecules including Branched PEGs, polyvinyl alcohol (PVA), poly-carboxyl acids, poly-(vinylpyrolidone) and poly-D,L-amino acids, or natural occurring polymeric molecules including dextrans, including carboxymethyl-dextrans, and celluloses such as methylcellulose, carboxymethylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, and hydrolysates of chitosan, starches, such as hydroxyethyl-starches, hydroxypropyl-starches, glycogen, agarose, guar gum, inulin, pullulans, xanthan gums, carrageenin, pectin and alginic acid.  
     
     
         45 . The enzyme of  claim 31 , wherein the enzyme or parent enzyme is an amylase variant comprising one or more substitutions at positions 124, 126, 128, 159, 160, 166, 185, 186, 189, 190, 193, 194, 195, 196, 198, 201, 202, 203, 209, 210, 214, 242, 244, 247, 296, 298, 299, 302, 303, 304, 306, 307, 308, 310, 311, 314, 345, 347, 405, 406, 407, 408, 409, 433, 434, 435, 436, 437, 475, 476, 477, 478 with C, D, E, or K.  
     
     
         46 . A composition, comprising an enzyme of  claim 31  and ingredients used in industrial products.  
     
     
         47 . The composition of  claim 46 , wherein the industrial product is a detergent, such as a laundry, dish wash or hard surface cleaning product, including bio-film products or a food or feed product or a textile product.  
     
     
         48 . The composition of  claim 47 , further comprising ingredients used in personal care products, especially skin care products.  
     
     
         48 . A pharmaceutical composition, comprising an enzyme of  claim 31  ingredients used in pharmaceuticals.  
     
     
         49 . A method for preparing enzymes with reduced immune response comprising the steps of: 
 (a) identifying amino acid residues located on the surface of the 3-dimensional structure of the parent enzyme in question,    (b) selecting target amino acid residues on the surface of said 3-dimensional structure of said parent enzyme to be modified,    (c) substituting one or more amino acid residues selected in step (b) with other amino acid residues, and/or    (d) coupling polymeric molecules to the amino acid residues in step (b)and/or step (c).    
     
     
         50 . The method of  claim 49 , wherein the C alpha -atoms of the amino acid residues are located less than 15 Å from the ligand bound to said enzyme.  
     
     
         51 . The method of  claim 49  or  50 , wherein the C beta -atoms of the amino acid residues are located closer to the ligand than the C alpha -atom.  
     
     
         52 . The method of any of claims  49 - 51 , wherein the C alpha -atoms of the amino acid residues are located less than 10 Å from the ligand and said amino acid residues have an accessibility of at least 15%, preferable at least 20%, more preferably at least 30%.  
     
     
         53 . The method of any of claims  49 - 52 , wherein the identification of amino acid residues located on the surface on the enzyme referred to in step (a) are performed by a computer program analyzing the 3-dimensional structure of the parent enzyme in question.  
     
     
         54 . The method of any of claims  49 - 53 , wherein step (b) comprises selecting arginine or lysine residues on the surface of the parent enzyme.  
     
     
         55 . The method of  claim 54 , wherein one or more arginine residues identified in step (b) is (are) substituted with a lysine residue(s) in step (c).  
     
     
         56 . Use of the enzyme of any of claims  31 - 45  for reducing the allergenicity of industrial products.  
     
     
         57 . Use of the enzyme of any of claims  31 - 45  for reducing the immunogenicity of pharmaceuticals.