Single chain antigen-binding polypeptides for polymer conjugation

Abstract

The present invention relates to monovalent and multivalent single-chain antigen-binding polypeptides with site-specific modifications. The provided polypeptides are capable of being covalently linked or conjugated to polyalkylene oxides at the modified sites. The resulting conjugates retain antigen binding properties and exhibit prolonged circulating time and reduced antigenicity relative to unconjugated single chain antigen binding polypeptides. Methods and compositions for making and using the single chain antigen-binding polypeptides with site-specific modifications are also provided.

1 . A single-chain antigen-binding polypeptide capable of site-specific conjugation to a polyalkylene oxide polymer, that comprises, 
 (a) a first polypeptide comprising an antigen-binding portion of a variable region of an antibody heavy or light chain;    (b) a second polypeptide comprising an antigen-binding portion of a variable region of an antibody heavy or light chain; and    (c) a peptide linker linking the first and second polypeptides,    wherein the single-chain antigen-binding polypeptide has at least one Cys residue which is capable of being conjugated to a polyalkylene oxide polymer, and has at least one antigen binding site, and    wherein the Cys residue is located at a position selected from the group consisting of:    (i) a C-terminus of the heavy chain or light chain variable region;    (ii) an N-terminus of the heavy chain or light chain variable region;    (iii) any amino acid position of the peptide linker;    (iv) both the N-terminus and C-terminus;    (v) position 2 of the linker;    (vi) both position 2 of the linker and the C-terminus; and    (iv) combinations thereof;    and wherein the single-chain antigen-binding polypeptide binds to TNF-α.    
     
     
         2 . The single-chain antigen-binding polypeptide of  claim 1  wherein the Cys residue is located at a position selected from the group consisting of position 2 of the linker, the C-terminus and combinations thereof.  
     
     
         3 . The single-chain antigen-binding polypeptide of  claim 1  wherein the first polypeptide comprises a variable region of an antibody light chain and the second polypeptide comprises a variable region of an antibody heavy chain.  
     
     
         4 . The single-chain antigen-binding polypeptide of  claim 1 , wherein the C-terminus of the second polypeptide is the native C-terminus.  
     
     
         5 . The single-chain antigen-binding polypeptide of  claim 1  wherein the peptide linker ranges in size from 2 to 18 residues  
     
     
         6 . A conjugate comprising the single-chain antigen-binding polypeptide of  claim 1 , and comprising a polyalkylene oxide polymer, wherein the polyalkylene oxide polymer is covalently linked to the single-chain antigen-binding polypeptide at a Cys residue.  
     
     
         7 . The conjugate of  claim 6  wherein the polyalkylene oxide is linked to the single-chain antigen-binding polypeptide at a Cys residue via a linker selected from the group consisting of a maleimide, vinylsulfone, thiol, orthopyridyl disulfide and a iodoactemide linker.  
     
     
         8 . The conjugate of  claim 6  wherein the polyalkylene oxide is linked to the single-chain antigen-binding polypeptide at a Cys residue via a maleimide linker.  
     
     
         9 . The conjugate of  claim 6  wherein the polyalkylene oxide ranges in size from about 5,000 to about 40,000 Daltons.  
     
     
         10 . The conjugate of  claim 6  wherein the polyalkylene oxide is a polyethylene oxide.  
     
     
         11 . The conjugate of  claim 6 , wherein the polyalkylene oxide is conjugated to at least two single-chain antigen-binding polypeptides, and each single-chain antigen-binding polypeptide is the same, or different.  
     
     
         12 . The conjugate of  claim 11  wherein the single-chain antigen-binding polypeptide is further conjugated to an additional functional moiety.  
     
     
         13 . The conjugate of  claim 12  wherein the additional functional moiety is a detectable label or tag.  
     
     
         14 . A polynucleotide encoding the single-chain antigen-binding polypeptide of  claim 1 .  
     
     
         15 . A replicable expression vector comprising the polynucleotide of  claim 14 .  
     
     
         16 . A method of producing the single-chain antigen-binding polypeptide, comprising the steps of: 
 (a) culturing a host cell comprising the expression vector of  claim 12 , and    (b) collecting the single-chain antigen-binding polypeptide expressed by the host cell.    
     
     
         17 . A method of detecting TNF-α suspected of being in a sample, comprising: 
 (a) contacting the sample with a reagent comprising the single-chain antigen-binding polypeptide of  claim 1 , and    (b) detecting whether the single-chain antigen-binding polypeptide has bound to the TNF-α.    
     
     
         18 . The method of  claim 17  wherein the single-chain antigen-binding polypeptide is covalently conjugated to at least one polyalkylene oxide polymer via a Cys residue of the single-chain antigen-binding polypeptide.  
     
     
         19 . The method of  claim 18  wherein the conjugate is anchored to a solid substrate.  
     
     
         20 . A method of treating or preventing TNF-α related toxicity in a mammal, comprising administering the single-chain antigen-binding polypeptide of  claim 1  to the mammal, wherein the single-chain antigen-binding polypeptide is administered in an amount effective to inhibit TNF-α activity in the mammal.  
     
     
         21 . The method of  claim 20  wherein the administered single-chain antigen-binding polypeptide is covalently conjugated to at least one polyalkylene oxide polymer via at least one of the Cys residues.  
     
     
         22 . The method of  claim 20  wherein the single-chain antigen-binding polypeptide is administered in an amount ranging from about 10 μg/kg to about 4,000 μg/kg.  
     
     
         23 . The method of  claim 20  wherein the single-chain antigen-binding polypeptide is administered in an amount ranging from about 20 μg/kg to about 400 μg/kg.  
     
     
         24 . A protein comprising two or more of the single-chain antigen-binding polypeptides of  claim 1 , wherein each single-chain antigen-binding polypeptide is the same or different.  
     
     
         25 . The protein of  claim 24  that is bivalent, trivalent or tetravalent.  
     
     
         26 . The protein of  claim 24  wherein the constituent single-chain antigen-binding polypeptides are noncovalently associated.  
     
     
         27 . The protein of  claim 26  herein the peptide linker of the constituent single-chain antigen-binding polypeptides range in size from 2 to 18 residues.  
     
     
         28 . The protein of  claim 24  wherein the constituent single-chain antigen-binding polypeptides are covalently linked.  
     
     
         29 . The protein of  claim 24  that is encoded as a single, multivalent protein.  
     
     
         30 . A polynucleotide encoding the protein of  claim 29.