Polypeptides

Abstract

The present disclosure relates to a class of engineered polypeptides having a binding affinity for albumin. It also relates to new methods and uses that exploit binding by these and other compounds to albumin in different contexts, some of which have significance for treatment or diagnosis of disease in mammals including humans.

1 - 84 . (canceled) 
     
     
         85 . An albumin binding polypeptide comprising an amino acid sequence selected from: 
       
         
           
                 
               
                   i) 
                 
                   (SEQ ID NO: 204) 
                 
                   LAX 3 AKX 6 X 7 ANX 10 ELDX 14 YGVSDFYKRLIX 26 KAKTVEGVEALKX 39 X 40   
                 
                     
                 
                   ILX 43 X 44 LP 
                 
             
                
                
                
                
                
               
            
           
         
         wherein independently of each other 
         X 3  is selected from E, S, Q and C; 
         X 6  is selected from E, S and C; 
         X 7  is selected from A and S; 
         X 10  is selected from A, S and R; 
         X 14  is selected from A, S, C and K; 
         X 26  is selected from D and E; 
         X 39  is selected from D and E; 
         X 40  is selected from A and E; 
         X 43  is selected from A and K; 
         X 44  is selected from A, S and E; 
         L in position 45 is present or absent; and 
         P in position 46 is present or absent; and 
         ii) an amino acid sequence which has at least 95% identity to the sequence defined in i), provided that X 7  is neither L, E nor D. 
       
     
     
         86 . The albumin binding polypeptide according to  claim 85 , wherein X 6  is E. 
     
     
         87 . The albumin binding polypeptide according to  claim 85 , wherein X 3  is S. 
     
     
         88 . The albumin binding polypeptide according to  claim 85 , wherein X 3  is E. 
     
     
         89 . The albumin binding polypeptide according to  claim 85 , wherein X 7  is A. 
     
     
         90 . The albumin binding polypeptide according to  claim 85 , wherein X 14  is S. 
     
     
         91 . The albumin binding polypeptide according to  claim 85 , wherein X 14  is C. 
     
     
         92 . The albumin binding polypeptide according to  claim 85 , wherein X 10  is A. 
     
     
         93 . The albumin binding polypeptide according to  claim 85 , wherein X 10  is S. 
     
     
         94 . The albumin binding polypeptide according to  claim 85 , wherein X 26  is D. 
     
     
         95 . The albumin binding polypeptide according to  claim 85 , wherein X 26  is E. 
     
     
         96 . The albumin binding polypeptide according to  claim 85 , wherein X 39  is D. 
     
     
         97 . The albumin binding polypeptide according to  claim 85 , wherein X 39  is E. 
     
     
         98 . The albumin binding polypeptide according to  claim 85 , wherein X 40  is A. 
     
     
         99 . The albumin binding polypeptide according to  claim 85 , wherein X 43  is A. 
     
     
         100 . The albumin binding polypeptide according to  claim 85 , wherein X 44  is A. 
     
     
         101 . The albumin binding polypeptide according to  claim 85 , wherein X 44  is S. 
     
     
         102 . The albumin binding polypeptide according to  claim 85 , wherein L in position 45 is present. 
     
     
         103 . The albumin binding polypeptide according to  claim 85 , wherein P in position 46 is present. 
     
     
         104 . The albumin binding polypeptide according to  claim 85 , which binds to albumin such that the k off  value of the interaction is at most 5×10 −5  s −1 . 
     
     
         105 . The albumin binding polypeptide according to  claim 104 , which binds to albumin such that the k off  value of the interaction is at most 5×10 −6  s −1 . 
     
     
         106 . The albumin binding polypeptide according to  claim 85 , whose amino acid sequence is selected from any one of SEQ ID NO:1-144 and SEQ ID NO:164-203. 
     
     
         107 . The albumin binding polypeptide according to  claim 106 , whose amino acid sequence is selected from any one of SEQ ID NO:4-5, SEQ ID NO:7-8, SEQ ID NO:10-11, SEQ ID NO:13-14, SEQ ID NO:16-17, SEQ ID NO:19-20, SEQ ID NO:22-23, SEQ ID NO:25-26, SEQ ID NO:28-29, SEQ ID NO:31-32, SEQ ID NO:34-35, SEQ ID NO:37-38, SEQ ID NO:41-42, SEQ ID NO:49-50, SEQ ID NO:164-170 and SEQ ID NO:192-203. 
     
     
         108 . The albumin binding polypeptide according to  claim 106 , whose amino acid sequence is selected from any one of SEQ ID NO:1-144. 
     
     
         109 . The albumin binding polypeptide according to  claim 108 , whose amino acid sequence is selected from any one of SEQ ID NO:4-5, SEQ ID NO:7-8, SEQ ID NO:10-11, SEQ ID NO:13-14, SEQ ID NO:16-17, SEQ ID NO:19-20, SEQ ID NO:22-23, SEQ ID NO:25-26, SEQ ID NO:28-29, SEQ ID NO:31-32, SEQ ID NO:34-35, SEQ ID NO:37-38, SEQ ID NO:41-42 and SEQ ID NO:49-50. 
     
     
         110 . The albumin binding polypeptide according to  claim 85 , further comprising one or more additional amino acid residues positioned at the N- and/or the C-terminal of the sequence defined in i). 
     
     
         111 . The albumin binding polypeptide according to  claim 110 , in which the additional amino acids comprise at least one serine residue at the N-terminal of the polypeptide. 
     
     
         112 . The albumin binding polypeptide according to  claim 110 , in which the additional amino acids comprise a glycine residue at the N-terminal of the polypeptide. 
     
     
         113 . The albumin binding polypeptide according to  claim 110 , in which the additional amino acids comprise a cysteine residue at the N-terminal of the polypeptide. 
     
     
         114 . The albumin binding polypeptide according to  claim 110 , in which the additional amino acids comprise a lysine residue at the C-terminal of the polypeptide. 
     
     
         115 . The albumin binding polypeptide according to  claim 110 , in which the additional amino acids comprise a glycine residue at the C-terminal of the polypeptide. 
     
     
         116 . The albumin binding polypeptide according to  claim 110 , in which the additional amino acids comprise a cysteine residue at the C-terminal of the polypeptide. 
     
     
         117 . The albumin binding polypeptide according to  claim 110 , whose amino acid sequence is selected from any one of SEQ ID NO:145-150 and SEQ ID NO:162-163. 
     
     
         118 . The albumin binding polypeptide according to  claim 85 , comprising no more than two cysteine residues. 
     
     
         119 . The albumin binding polypeptide according to  claim 118 , comprising no more than one cysteine residue. 
     
     
         120 . The albumin binding polypeptide according to  claim 85 , which binds to human serum albumin. 
     
     
         121 . A fusion protein or conjugate comprising
 i) a first moiety consisting of the albumin binding polypeptide according to  claim 85 ; and   ii) a second moiety consisting of a polypeptide having a desired biological activity.   
     
     
         122 . The fusion protein or conjugate according to  claim 121 , in which the second moiety having a desired biological activity is a therapeutically active polypeptide. 
     
     
         123 . The fusion protein or conjugate according to  claim 122 , in which the second moiety having a desired biological activity is selected from the group consisting of human endogenous enzymes, hormones, growth factors, chemokines, cytokines and lymphokines. 
     
     
         124 . The fusion protein or conjugate according to  claim 123 , in which the second moiety is selected from the group consisting of interleukin-2 (IL-2), glucagon-like peptide 1 (GLP-1), brain natriuretic peptide (BNP), interleukin-1 receptor antagonist (IL-1-RA), keratinocyte growth factor (KGF), ancestim, growth hormone (GH), granulocyte-colony stimulating factor (G-CSF), cytotoxic T lymphocyte-associated protein 4 (CTLA-4), myostatin, Factor VII, Factor VIII and Factor IX. 
     
     
         125 . The fusion protein or conjugate according to  claim 122 , in which the second moiety having a desired biological activity is a non-human biologically active protein, selected from the group consisting of bacterial toxins, enzymes and activating proteins. 
     
     
         126 . The fusion protein or conjugate according to  claim 121 , in which the second moiety having a desired biological activity is a binding polypeptide capable of selective interaction with a target molecule. 
     
     
         127 . The fusion protein or conjugate according to  claim 126 , in which the binding polypeptide is selected from the group consisting of antibodies and fragments and domains thereof substantially retaining antibody binding activity; microbodies, maxybodies, avimers, other small disulfide-bonded proteins; binding proteins derived from a scaffold selected from the group consisting of staphylococcal protein A and domains thereof, other three helix domains, lipocalins, ankyrin repeat domains, cellulose binding domains, y crystallines, green fluorescent protein, human cytotoxic T lymphocyte-associated antigen 4, protease inhibitors, Kunitz domains, PDZ domains, SH3 domains, peptide aptamers, staphylococcal nuclease, tendamistats, fibronectin type III domain, transferrin, zinc fingers and conotoxins. 
     
     
         128 . The fusion protein or conjugate according to  claim 127 , in which said target molecule is selected from the group consisting of amyloid B (AB) peptide of Alzheimer's disease; other disease-associated amyloid peptides; toxins, bacterial toxins, snake venoms; blood clotting factors, von Willebrand factor; interleukins, interleukin-13 (IL-13); myostatin; pro-inflammatory factors, tumor necrosis factor alpha (TNF-α), TNF-α receptor, IL-1, IL-23 and IL-8; complement factors, complement component 3 (C3), C5; hypersensitivity mediators, histamine, immunoglobulin E (IgE); tumor-related antigens, cluster of differentiation molecule 19 (CD19), CD20, CD22, CD30, CD33, CD40, CD52, CD70, oncogene MET (cMet), epidermal growth factor receptor 1 (HER1), HER2, HER3, HER4, carbonic anhydrase IX (CAIX), carcinoembryonic antigen (CEA), IL-2 receptor, mucin 1 (MUC1), prostate-specific membrane antigen (PSMA), tumor-associated glycoprotein 72 (TAG-72), granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), growth hormone (GH), insulin and somatostatin. 
     
     
         129 . The fusion protein or conjugate according to  claim 121 , comprising a further moiety consisting of a polypeptide having a further, desired biological activity, which may be the same as or different from that of the second moiety. 
     
     
         130 . The fusion protein or conjugate according to  claim 129 , wherein the second moiety is a therapeutically active polypeptide, and the further moiety is a binding polypeptide capable of selective interaction with a target molecule. 
     
     
         131 . The fusion protein or conjugate according to  claim 121 , in which the second moiety is conjugated to the albumin binding polypeptide via the thiol group of any cysteine residue present at position X 14  of the polypeptide. 
     
     
         132 . The albumin binding polypeptide according to  claim 85 , further comprising a cytotoxic agent. 
     
     
         133 . The albumin binding polypeptide according to  claim 132 , wherein the cytotoxic agent is selected from calicheamycin, auristatin, doxorubicin, maytansinoid, taxol, ecteinascidin, geldanamycin, methotrexate and their derivatives, and combinations thereof. 
     
     
         134 . The albumin binding polypeptide according to  claim 85  further comprising a label. 
     
     
         135 . The albumin binding polypeptide according to  claim 134 , in which said label is selected from the group consisting of fluorescent dyes and metals, chromophoric dyes, chemiluminescent compounds and bioluminescent proteins, enzymes, radionuclides and particles. 
     
     
         136 . The albumin binding polypeptide according to  claim 135 , comprising a chelating environment provided by a polyaminopolycarboxylate chelator conjugated to the albumin binding polypeptide via a thiol group of a cysteine residue or an amine group of a lysine residue. 
     
     
         137 . The albumin binding polypeptide according to  claim 136 , wherein the polyaminopolycarboxylate chelator is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid or a derivative thereof. 
     
     
         138 . The albumin binding polypeptide according to  claim 137 , wherein the 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid derivative is 1,4,7,10-tetraazacyclododecane-1,4,7-tris-acetic acid-10-maleimidoethylacetamide. 
     
     
         139 . The albumin binding polypeptide according to  claim 136 , wherein the polyaminopolycarboxylate chelator is diethylenetriaminepentaacetic acid or derivatives thereof. 
     
     
         140 . A polynucleotide encoding the albumin binding polypeptide according to  claim 85 . 
     
     
         141 . A method of producing a polypeptide, comprising expressing the polynucleotide according to  claim 140 . 
     
     
         142 . An expression vector comprising the polynucleotide according to  claim 140 . 
     
     
         143 . A host cell comprising the expression vector according to  claim 142 . 
     
     
         144 . A method of producing the albumin binding polypeptide according to  claim 85  by non-biological peptide synthesis using amino acids and/or amino acid derivatives having protected reactive side-chains, the non-biological peptide synthesis comprising
 step-wise coupling of the amino acids and/or the amino acid derivatives to form the albumin binding polypeptide; 
 deprotecting the protected reactive side-chains of the albumin binding polypeptide; and 
 folding of the albumin binding polypeptide in aqueous solution. 
 
     
     
         145 . The method of producing a polypeptide according to  claim 144 , further comprising
 conjugating the albumin binding polypeptide with a therapeutically active polypeptide.   
     
     
         146 . The fusion protein or conjugate according to  claim 121 , further comprising a cytotoxic agent. 
     
     
         147 . The fusion protein or conjugate according to  claim 146 , wherein the cytotoxic agent is selected from calicheamycin, auristatin, doxorubicin, maytansinoid, taxol, ecteinascidin, geldanamycin, methotrexate and their derivatives, and combinations thereof. 
     
     
         148 . The fusion protein or conjugate according to  claim 121  further comprising a label. 
     
     
         149 . The fusion protein or conjugate according to  claim 148 , in which said label is selected from the group consisting of fluorescent dyes and metals, chromophoric dyes, chemiluminescent compounds and bioluminescent proteins, enzymes, radionuclides and particles. 
     
     
         150 . The fusion protein or conjugate according to  claim 149 , comprising a chelating environment provided by a polyaminopolycarboxylate chelator conjugated to the albumin binding polypeptide via a thiol group of a cysteine residue or an amine group of a lysine residue. 
     
     
         151 . The fusion protein or conjugate according to  claim 150 , wherein the polyaminopolycarboxylate chelator is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid or a derivative thereof. 
     
     
         152 . The fusion protein or conjugate according to  claim 151 , wherein the 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid derivative is 1,4,7,10-tetraazacyclododecane-1,4,7-tris-acetic acid-10-maleimidoethylacetamide. 
     
     
         153 . The fusion protein or conjugate according to  claim 150 , wherein the polyaminopolycarboxylate chelator is diethylenetriaminepentaacetic acid or derivatives thereof. 
     
     
         154 . A polynucleotide encoding the fusion protein according to  claim 121 . 
     
     
         155 . A method of producing a polypeptide, comprising expressing the polynucleotide according to  claim 154 . 
     
     
         156 . An expression vector comprising the polynucleotide according to  claim 154 . 
     
     
         157 . A host cell comprising the expression vector according to  claim 156 .