Antigen-Binding Protein Directed Against Epitope in the CH1 Domain of Human IgG Antibodies

Abstract

The present invention relates to a method for the purification of a human IgG-CH1 domain comprising molecule using an antigen-binding protein that is capable of binding to an epitope that is comprised in the CH1 domain of each of human IgG1, human IgG2, human IgG3 and human IgG4. The invention further relates to the antigen-binding proteins that can be used in the method of the invention. The framework regions of the antigen-binding proteins of the invention preferably correspond to those of antibodies naturally that are devoid of light chains as may e.g. be found in camelids. The invention further relates to nucleic acids that encode such antigen-binding proteins, to immunoadsorbent materials that comprise such proteins, and to the uses of such immunoadsorbent materials for the purification of IgG-CH1 domain containing molecules from a variety of species.

1 . A method for capturing a target molecule comprising a human IgG-CH1 domain, wherein the method comprises the steps of:
 a) contacting the target molecule with an immunoadsorbent material comprising an antigen-binding protein immobilised on a support; and,   b) capturing the target molecule with the immunoadsorbent material by specific binding of the target material to the antigen-binding protein;   wherein the antigen-binding protein: (i) is capable of binding to a single epitope present in the human IgG-CH1 domain, (ii) is cross-blocked by a VHH antigen-binding protein, (iii) comprises an immunoglobulin-derived variable domain having a complete antigen-binding site for the single epitope.   
     
     
         2 . A method according to  claim 1 , wherein the antigen-binding protein
 is naturally devoid of light chains;   b) is a variable domain of an antibody defined in a); or,   c) is a variable domain wherein the frame work sequences of a variable domain defined in b) are grafted with CDRs obtained from other sources.   
     
     
         3 . A method according to  claim 1 , wherein the target molecule is a molecule selected from the group consisting of a human or humanized IgG1 molecule, a human or humanized IgG2 molecule, a human or humanized IgG3 molecule, a human or humanized IgG4 molecule, a human or humanized IgG Fab, a human or humanized IgG F(ab′) 2 , a one armed human or humanized IgG antibody, a single chain human or humanized IgG antibody, IVIG, and human or humanized IgG, IgG1, IgG2, IgG3 or IgG4 digests. 
     
     
         4 . A method according to  claim 3 , wherein the human or humanized IgG digest is a papain or pepsin human IgG digest. 
     
     
         5 . A method according to  claim 1 , wherein the antigen-binding protein binds to an epitope of the CH1 domain which epitope involves one or more of the amino acids: a phenylalanine at position 122, none or a single cysteine at either one of positions 127 and 128, a serine or a lysine at position 156 and/or an asparagine or a serine at position 216, whereby the positions of the amino acids are based on Kabat numbering. 
     
     
         6 . A method according to  claim 1 , wherein the antigen-binding protein is a camelid VHH or camelidised VH. 
     
     
         7 . A method of purifying a target molecule, the method comprising capturing the target molecule with the immunoadsorbent material in a method of  claim 1 , wherein the method further comprises the step of:
 c) eluting the bound target molecules under conditions that decrease the affinity between the target molecules and the immunoadsorbent material, and   d) optionally, recovering the target molecule.   
     
     
         8 . The method for purifying a target molecule according to  claim 7 , wherein the antigen-binding protein does not bind to free light chains. 
     
     
         9 . The method for purifying a Fab fragment comprising a human IgG-CH1 domain without copurification of free light chains, wherein the method comprises the steps of:
 a) bringing a sample comprising the target molecule and free light chains in contact with an immunoadsorbent material comprising an antigen-binding protein immobilised on a support;   b) allowing capture of the target molecule with the immunoadsorbent material by specific binding to the antigen-binding protein; and   c) eluting the bound target molecules under conditions that decrease the affinity between the target molecules and the immunoadsorbent material, and optionally recovery of the target molecule;   wherein the antigen-binding protein is capable of binding to a single epitope, which epitope is comprised in the CH1 domain of each of human IgG1, human IgG2, human IgG3 and human IgG4, wherein the antigen-binding protein is cross-blocked by a VHH antigen-binding protein, and wherein the antigen-binding protein comprises an immunoglobulin-derived variable domain that comprises a complete antigen-binding site for the epitope.   
     
     
         10 . An in vitro method for adsorbing a target molecule comprising a human IgG-CH1 domain from a fluid, the method comprising the step of bringing an immunoadsorbent material into contact with the fluid, wherein the immunoadsorbent material comprises an antigen-binding protein immobilised onto a carrier, and wherein preferably the antigen-binding protein is immobilised onto the carrier by a covalent link. 
     
     
         11 . An isolated antigen-binding protein of  claim 1 . 
     
     
         12 . A nucleic acid comprising a nucleotide sequence encoding an antigen-binding protein according to  claim 11 . 
     
     
         13 . A host cell comprising a nucleic acid according to  claim 12 . 
     
     
         14 . A method for producing an antigen-binding protein according to  claim 11 , the method comprising the step of culturing a host cell according to  claim 12  under conditions conducive to expression of the antigen-binding protein. 
     
     
         15 . An immunoadsorbent material comprising an antigen-binding protein according to  claim 11 . 
     
     
         16 . A method of in vitro detection and/or in vitro purification of a target molecule comprising a human IgG-CH1 domain of  claim 1 . 
     
     
         17 . The method of in vitro detection and/or in vitro purification of  claim 16 , wherein the target molecule is a molecule selected from the group consisting of a human or humanized IgG1, a human or humanized IgG2, a human or humanized IgG3, a human or humanized IgG4, a human or humanized IgG Fab, a human or humanized IgG F(ab′)2, a one armed human or humanized IgG antibody, a single chain human or humanized IgG antibody, IVIG, and human or humanized IgG, IgG1, IgG2, IgG3 or IgG4 digests. 
     
     
         18 . The method of  claim 17 , wherein the digests are either papain or pepsin digests. 
     
     
         19 . The method of  claim 17 , wherein the target molecule is a human IgG or a human or humanized IgG Fab without co-binding of free light chains.