Single-domain antigen-binding proteins that bind mammalian igg

Abstract

The present application relates to antigen-binding proteins that are capable of binding to mammalian IgG. The frame-work regions of the antigen-binding proteins of the application preferably correspond to those of antibodies naturally that are devoid of light chains as may e.g. be found in camelids. The application further relates to nucleic acids that encode such antigen-binding proteins, to immunoadsorbent materials that comprise such proteins, to the uses of such immunoadsorbent materials for the purification of mammalian IgG antibodies and for therapeutic apheresis.

1 . An antigen-binding protein comprising an amino acid sequence that comprises 4 framework regions, FRI to FR4, and 3 complementarity determining regions, CDR1 to CDR3, that are operably linked in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein:
 a) the CDR1 has an amino acid sequence selected from the group consisting of SEQ ID No's: 1-49 or an amino acid sequence that differs from SEQ ID No's: 1-49 in one or two of the amino acid residues;   b) the CDR2 has an amino acid sequence having at least 80% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID No's: 50-98; and,   c) the CDR3 is an amino acid sequence having at least 80% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID No's: 99-147; and,   wherein each of the framework regions has at least 50% amino acid identity with the framework amino acid sequence of any one of SEQ ID No's: 148-196, and wherein the antigen-binding protein specifically binds to the Fe domain of a mammalian IgG molecule.   
     
     
         2 . An antigen-binding protein according to  claim 1 , wherein the antigen binding protein has one or more properties selected from the group consisting of:
 a) the antigen-binding protein binds the human IgG molecule with a binding affinity of at least 10-7 M as analyzed by BiaCore using polyclonal human IgG;   b) the antigen-binding protein is obtainable by expression in yeast at an expression level of at least 0.5 g/L of yeast culture;   c) the antigen-binding protein has a dynamic binding capacity of at least 2 mg human IgG/ml resin, when coupled to NHS activated carrier at a density of 20 mg antigen-binding protein per ml NHS resin and using a flow-rate of 150 cm/h;   d) human IgG bound to the antigen-binding protein when coupled to NHS resin as defined in c) is recovered from the antigen-binding protein with a yield of at least 90% using 0.1 M glycine, pH 2.0;   e) human IgG bound to the antigen-binding protein when coupled to NHS resin as defined in c) is recovered from the antigen-binding protein with a yield of at least 70% using 0.1 M glycine pH 3.0; and,   f) the antigen-binding protein when coupled to NHS resin as defined in c) retains a residual dynamic binding capacity of at least 70% after cleaning-in-place cycles, wherein in each cleaning-in-place cycle the antigen-binding protein coupled to NHS resin is contacted for 15 minutes with 0.05 M NaOH and 0.5 M NaCl at a flow rate of 150 cm/h.   
     
     
         3 . An antigen-binding protein according to any one of the preceding claims, wherein the mammalian IgG is a human IgG molecule. 
     
     
         4 . An antigen-binding protein according to any one of the preceding claims, wherein the human IgG is an IgG1, IgG2, IgG3 or IgG4 molecule. 
     
     
         5 . An antigen-binding protein according to any one of the preceding claims, wherein the antigen-binding protein binds to the Fe domain of human IgG and does not bind to an IgG molecule of murine or bovine origin. 
     
     
         6 . An antigen-binding protein according to  claim 5 , wherein:
 a) the CDR1 has an amino acid sequence selected from the group consisting of SEQ ID No's: 1-15, 17-25, 31-36, 38, 43 and 44 and amino acid sequences that differs from SEQ ID No's: 1-15, 17-25, 31-36, 38, 43 and 44 in no more than 4 amino acid residues;   b) the CDR2 has an amino acid sequence selected from the group consisting of SEQ ID No's: 50-64, 66-74, 80-85, 87, 92 and 93 and an amino acid sequences that differs from SEQ ID No's: 50-64, 66-74, 80-85, 87, 92 and 93 in no more than 6 amino acid residues; and,   c) the CDR3 has an amino acid sequence selected from the group consisting of SEQ ID No's: 99-113, 115-123, 129-134, 136, 141 and 142 and an amino acid sequences that differs from SEQ ID No's: 99-113, 115-123, 129-134, 136, 141 and 142 in no more than 6 amino acid residues.   
     
     
         7 . An antigen-binding protein according to  claim 6 , wherein the antigen-binding protein has an amino acid sequence selected from the group consisting of SEQ ID No's 148-162, 164-172, 178-183, 185, 190 and 191. 
     
     
         8 . An antigen-binding protein according to  claim 5 , wherein the antigen-binding protein further does not bind to an IgG molecule of caprine origin. 
     
     
         9 . An antigen-binding protein according to  claim 8 , wherein:
 a) the CDR1 has an amino acid sequence selected from the group consisting of SEQ ID No's: 1-15, 17-25, 31-36, 38 and 44 and amino acid sequences that differs from SEQ ID No's: 1-15, 17-25, 31-36, 38 and 44 in no more than 4 amino acid residues;   b) the CDR2 has an amino acid sequence selected from the group consisting of SEQ ID No's: 50-64, 66-74, 80-85, 87 and 93 and an amino acid sequences that differs from SEQ ID No's: 50-64, 66-74, 80-85, 87 and 93 in no more than 6 amino acid residues; and,   c) the CDR3 has an amino acid sequence selected from the group consisting of SEQ ID No's: 99-113, 115-123, 129-134, 136 and 142 and an amino acid sequences that differs from SEQ ID No's: 99-113, 115-123, 129-134, 136 and 142 in no more than 6 amino acid residues.   
     
     
         10 . An antigen-binding protein according to  claim 9 , wherein the antigen-binding protein has an amino acid sequence selected from the group consisting of SEQ ID No's 148-162, 164-172, 178-183, 185 and 191. 
     
     
         11 . An antigen-binding protein according to  claim 8 , wherein the antigen-binding protein further does not bind to an IgG molecule that originates from rat, syrian hamster, guinea pig, dog, cat or sheep. 
     
     
         12 . An antigen-binding protein according to  claim 11 , wherein:
 a) the CDR1 has an amino acid sequence selected from the group consisting of SEQ ID No's: 1-15 and amino acid sequences that differs from SEQ ID No's: 1-15 in no more than 4 amino acid residues;   b) the CDR2 has an amino acid sequence selected from the group consisting of SEQ ID No's: 50-64 and an amino acid sequences that differs from SEQ ID No's: 50-64 in no more than 6 amino acid residues; and,   c) the CDR3 has an amino acid sequence selected from the group consisting of SEQ ID No's: 99-113 and an amino acid sequences that differs from SEQ ID No's: 99-113 in no more than 6 amino acid residues.   
     
     
         13 . An antigen-binding protein according to  claim 12 , wherein the antigen-binding protein has an amino acid sequence selected from the group consisting of SEQ ID No's 148-162. 
     
     
         14 . An antigen-binding protein according to  claim 11 , wherein the antigen binding protein has one or more properties selected from the group consisting of:
 a) the antigen-binding protein binds the human IgG molecule with a binding affinity of at least 5 nM as analyzed by BiaCore using polyclonal human IgG;   b) human IgG bound to the antigen-binding protein when coupled to reference NHS carrier is recovered from the antigen-binding protein with a yield of at least 99% using 0.1 M glycine, pH 3.0;   c) human IgG bound to the antigen-binding protein when coupled to reference NHS carrier is recovered from the antigen-binding protein with a yield of at least 95% using 0.1 M glycine, pH 4.0;   d) human IgG bound to the antigen-binding protein when coupled to reference NHS carrier is recovered from the antigen-binding protein with a yield of at least 99% using 0.1-0.2 M arginine, pH 3.0;   e) retains a residual dynamic binding capacity of at least 90, 95 or 100% after 100 cleaning-in-place cycles, wherein in each cleaning-in-place cycle the antigen-binding protein coupled to reference NHS carrier is contacted for 15 minutes with 0.1 M NaOH at a flow rate of 150 cm/h; and,   f) retains a residual dynamic binding capacity of at least 80% after 40 cleaning-in-place cycles, wherein in each cleaning-in-place cycle the antigen-binding protein coupled to reference NHS carrier is contacted for 15 minutes with 0.2 M NaOH at a flow rate of 150 cm/h.   
     
     
         15 . An antigen-binding protein according to  claim 14 , wherein:
 a) the CDR1 has an amino acid sequence selected from the group consisting of SEQ ID No's: 1-9 and amino acid sequences that differs from SEQ ID No's: 1-9 in no more than 4 amino acid residues;   b) the CDR2 has an amino acid sequence selected from the group consisting of SEQ ID No's: 50-58 and an amino acid sequences that differs from SEQ ID No's: 50-58 in no more than 6 amino acid residues; and,   c) the CDR3 has an amino acid sequence selected from the group consisting of SEQ ID No's: 99-107 and an amino acid sequences that differs from SEQ ID No's: 99-107 in no more than 6 amino acid residues.   
     
     
         16 . An antigen-binding protein according to  claim 15 , wherein the antigen-binding protein has an amino acid sequence selected from the group consisting of SEQ ID No's 148-156. 
     
     
         17 . An antigen-binding protein according to  claim 11 , wherein the antigen binding protein has one or more properties selected from the group consisting of:
 a) the antigen-binding protein binds the human IgG molecule with a binding affinity of at least 3 nM as analyzed by BiaCore using polyclonal human IgG; and,   b) the antigen-binding protein is obtainable by expression in yeast at an expression level of at least 1.2 g/L of yeast culture.   
     
     
         18 . An antigen-binding protein according to  claim 17 , wherein:
 a) the CDR1 has an amino acid sequence selected from the group consisting of SEQ ID No's: 10-15 and amino acid sequences that differs from SEQ ID No's: 10-15 in no more than 4 amino acid residues;   b) the CDR2 has an amino acid sequence selected from the group consisting of SEQ ID No's: 59-64 and an amino acid sequences that differs from SEQ ID No's: 59-64 in no more than 6 amino acid residues; and   c) the CDR3 has an amino acid sequence selected from the group consisting of SEQ ID No's: 108-113 and an amino acid sequences that differs from SEQ ID No's: 108-113 in no more than 6 amino acid residues.   
     
     
         19 . An antigen-binding protein according to  claim 18 , wherein the antigen-binding protein has an amino acid sequence selected from the group consisting of SEQ ID No's 157-162. 
     
     
         20 . An antigen-binding protein according to  claim 1  or  2 , wherein the antigen-binding protein binds to the Fe domain of an IgG molecule from at least two different species selected from the group consisting of human, murine, and bovine. 
     
     
         21 . An antigen-binding protein according to  claim 20 , wherein:
 a) the CDR1 has an amino acid sequence selected from the group consisting of SEQ ID No's: 16, 26-30, 37, 42 and 47-49 and amino acid sequences that differs from SEQ ID No's: 16, 26-30, 37, 42 and 47-49 in no more than 4 amino acid residues;   b) the CDR2 has an amino acid sequence selected from the group consisting of SEQ ID No's: 65, 75-79, 86, 91 and 96-98 and an amino acid sequences that differs from SEQ ID No's: 65, 75-79, 86, 91 and 96-98 in no more than 6 amino acid residues; and,   c) the CDR3 has an amino acid sequence selected from the group consisting of SEQ ID No's: 114, 124-128, 135, 140 and 145-147 and an amino acid sequences that differs from SEQ ID No's: 114, 124-128, 135, 140 and 145-147 in no more than 6 amino acid residues.   
     
     
         22 . An antigen-binding protein according to  claim 21 , wherein the antigen-binding protein has an amino acid sequence selected from the group consisting of SEQ ID No's 163, 173-177, 184, 189 and 194-196. 
     
     
         23 . An antigen-binding protein according to  claim 20 , wherein the antigen-binding protein binds to the Fe domain of an IgG molecule from a human, murine, bovine, rat, rabbit, dog, cat, swine, sheep, monkey, donkey, and horse. 
     
     
         24 . An antigen-binding protein according to  claim 23 , wherein:
 a) the CDR1 has an amino acid sequence selected from the group consisting of SEQ ID No's: 16, 28-30, 37, 42 and 47-49 and amino acid sequences that differs from SEQ ID No's: 16, 28-30, 37, 42 and 47-49 in no more than 4 amino acid residues;   b) the CDR2 has an amino acid sequence selected from the group consisting of SEQ ID No's: 65, 77-79, 86, 91 and 96-98 and an amino acid sequences that differs from SEQ ID No's: 65, 77-79, 86, 91 and 96-98 in no more than 6 amino acid residues; and,   c) the CDR3 has an amino acid sequence selected from the group consisting of SEQ ID No's: 114, 126-128, 135, 140 and 145-147 and an amino acid sequences that differs from SEQ ID No's: 114, 126-128, 135, 140 and 145-147 in no more than 6 amino acid residues.   
     
     
         25 . An antigen-binding protein according to  claim 21 , wherein the antigen-binding protein has an amino acid sequence selected from the group consisting of SEQ ID No's 163, 175-177, 184, 189 and 194-196. 
     
     
         26 . An antigen-binding protein according to  claim 23 , wherein the antigen-binding protein binds to the Fe domain of an IgG molecule from all mammalian species. 
     
     
         27 . An antigen-binding protein according to  claim 26 , wherein the antigen binding protein has one or more properties selected from the group consisting of:
 a) the antigen-binding protein binds the human IgG molecule with a binding affinity of at least 20 nM as analyzed by BiaCore using polyclonal human IgG;   b) the antigen-binding protein is obtainable by expression in yeast at an expression level of at least 2.5 g/L of yeast culture; and,   c) human IgG bound to the antigen-binding protein when coupled to reference NHS carrier is recovered from the antigen-binding protein with a yield of at least 99% using 0.1 M glycine, pH 3.0 or 0.2 M arginine, pH 3.0.   
     
     
         28 . An antigen-binding protein according to  claim 27 , wherein:
 a) the CDR1 has an amino acid sequence selected from the group consisting of SEQ ID No's: 16, 28-30 and 48 and amino acid sequences that differs from SEQ ID No's: 16, 28-30 and 48 in no more than 4 amino acid residues;   b) the CDR2 has an amino acid sequence selected from the group consisting of SEQ ID No's: 65, 77-79 and 97 and an amino acid sequences that differs from SEQ ID No's: 65, 77-79 and 97 in no more than 6 amino acid residues; and,   c) the CDR3 has an amino acid sequence selected from the group consisting of SEQ ID No's: 114, 126-128 and 146 and an amino acid sequences that differs from SEQ ID No's: 114, 126-128 and 146 in no more than 6 amino acid residues.   
     
     
         29 . An antigen-binding protein according to  claim 28 , wherein the antigen-binding protein has the amino acid sequence selected from the group consisting of SEQ ID No's 163, 175-177 and 195. 
     
     
         30 . A multivalent antigen-binding protein comprising the amino acid sequences of at least two antigen-binding proteins according to any one of  claims 1 - 29 . 
     
     
         31 . An antigen-binding protein according to any one  claims 1 - 30 , wherein the antigen-binding protein is part of a fusion protein further comprising an amino acid sequence of a therapeutic protein or peptide. 
     
     
         32 . A nucleic acid comprising a nucleotide sequence encoding an antigen-binding protein according to any one of the preceding claims. 
     
     
         33 . A nucleic acid according to  claim 32 , wherein the nucleotide sequence is operably linked to a promoter and optionally other regulatory elements. 
     
     
         34 . A host cell comprising a nucleic acid according to  claim 31  or  32 . 
     
     
         35 . A host cell according to  claim 34  wherein the host cell is a yeast, preferably  Saccharomyces cerevisiae.    
     
     
         36 . A method for producing an antigen-binding protein according to any one of  claims 1 - 31 , the method comprising the steps of:
 a) culturing a host cell according to  claim 34  or  35  under conditions conducive to expression of the antigen-binding protein; and optionally   b) purifying the antigen-binding protein from at least one of the host cell and the culture medium.   
     
     
         37 . A composition comprising an antigen-binding protein according to any one of  claims 1 - 31 . 
     
     
         38 . An immunoadsorbent material comprising an antigen-binding protein according to any one of  claims 1 - 31 . 
     
     
         39 . An immunoadsorbent material according to  claim 38 , wherein the antigen-binding protein is immobilized onto a carrier, wherein preferably the antigen-binding protein is immobilised onto the carrier by a covalent link. 
     
     
         40 . An immunoadsorbent material according to  claim 38  or  39 , wherein the carrier comprises Sepharose. 
     
     
         41 . Use of an antigen-binding protein according to any one of  claims 1 - 31  for the detection and/or purification of a mammalian IgG molecule. 
     
     
         42 . A method for the purification of a mammalian IgG molecule, the method comprising the steps of:
 a) bringing a sample comprising a mammalian IgG molecule in contact with an immunoadsorbent material according to any one of  claims 38 - 40  under conditions that allow binding of the mammalian IgG molecule to the immunoadsorbent material;   b) optionally, performing a washing step;   c) eluting the bound mammalian IgG molecule under conditions that decrease the affinity between the mammalian IgG molecule and the immunoadsorbent material; and,   c) optionally, further processing mammalian IgG molecule.   
     
     
         43 . A method for removing, depleting or inactivating mammalian IgG in a body fluid, in which an immunoadsorbent material according to any one of  claims 38 - 40  is brought into extracorporeal contact with the body fluid of a subject, preferably a human subject. 
     
     
         44 . Use of an antigen-binding proteins according to any one of  claims 1 - 31 , for extracorporeal removal of or depletion of mammalian IgG in a subject's body fluid of a subject, preferably a human subject.