Functionalized polypeptides

Abstract

The invention provides functionalized polypeptides, especially therapeutic polypeptides (e.g., scFv), comprising a linker sequence that can be rapidly and specifically functionalized by the addition of one or functional moieties (e.g., PEG) or binding specificities (e.g., an amino acid sequence with a particular binding specificity). Such functionalized polypeptides are advantageous in that they have improved pharmacokinetic properties (e.g., improved in vivo half-life, tissue penetration and tissue residency time) over non-functionalized polypeptides. Methods for the rapid and reproducible generation of functionalized polypeptides are also provided.

We claim: 
     
         1 . A polypeptide comprising two domains connected by an amino acid linker; wherein the linker comprises two cysteines capable of forming an intrachain disulphide bond, and wherein the amino acids between the cysteines in the linker sequence form a loop when the two cysteines are disulphide bonded to one another. 
     
     
         2 . The polypeptide of  claim 1 , wherein the linker comprises the amino acid sequence set forth in SEQ ID No. 1. 
     
     
         3 . The polypeptide of  claim 1 , wherein the linker comprises the amino acid sequence set forth in SEQ ID No. 2. 
     
     
         4 . The polypeptide of  claim 1 , wherein the loop binds to a target molecule. 
     
     
         5 . The polypeptide of  claim 4 , wherein the target molecule is a PK modifier. 
     
     
         6 . The polypeptide of  claim 5 , wherein the PK modifier is serum albumin. 
     
     
         7 . The polypeptide of  claim 5 , wherein the PK modifier is a hyaluronic acid. 
     
     
         8 . The polypeptide of  claim 1 , wherein the linker comprises the amino acid sequence set forth in SEQ ID No. 3. 
     
     
         9 . The polypeptide of  claim 1 , wherein the linker comprises the amino acid sequence set forth in SEQ ID No. 4. 
     
     
         10 . The polypeptide of  claim 1 , wherein the polypeptide is an immunobinder. 
     
     
         11 . The polypeptide of  claim 10 , which is a scFv. 
     
     
         12 . The polypeptide of  claim 1 , comprising the amino acid sequence set forth in SEQ ID No. 6 or 8. 
     
     
         13 . A polypeptide of  claim 1 , wherein at least one cysteine residue in the linker is covalently linked to a functional moiety. 
     
     
         14 . The polypeptide of  claim 13 , wherein the functional moiety is PEG. 
     
     
         15 . A polypeptide of  claim 1 , wherein the two cysteine residues in the linker are covalently linked to the same functional moiety. 
     
     
         16 . The polypeptide of  claim 15 , wherein the functional moiety is PEG. 
     
     
         17 . A composition comprising the polypeptide of  claim 1  and a pharmaceutically acceptable carrier. 
     
     
         18 . An isolated nucleic acid molecule encoding the polypeptide of  claim 1 . 
     
     
         19 . An expression vector comprising the nucleic acid molecule of  claim 18 . 
     
     
         20 . A host cell comprising the expression vector of  claim 19 . 
     
     
         21 . A method producing a functionalized polypeptide comprising:
 (a) providing a library of peptide sequences;   (b) identifying from the library at least one peptide sequence that bind to a target molecule;   (c) modifying the loop region of a linker-containing polypeptide to comprise at least one peptide sequence identified in step (b), thereby producing a functionalized polypeptide.   
     
     
         22 . The method of  claim 21 , wherein said identifying step is performed using phage display, yeast display, or mRNA display.