C-Type Lectin Fold as a Scaffold for Massive Sequence Variation

Abstract

This invention provides a class of binding proteins with a range of binding specificities and affinities based upon variation at select amino acid positions within a scaffold. The variable positions may be readily modified to produce a library of binding proteins with different binding specificities and affinities. The library may be screened to identify one or more as binding a ligand of interest. Compositions comprising the binding proteins, as well as methods of using the binding proteins are also provided.

1 . A non-naturally occurring protein with binding specificity determined by a variable binding site, said protein comprising
 a scaffold comprising the amino acid sequence   
       
         
           
                 
               
                   (SEQ ID NO: 1) 
                 
                 
               
                   -Xaa 1 -Trp-Xaa 2 -Xaa 3 -Xaa 4 -Ser-Xaa 5 -Ser-Gly-Ser-Arg- 
                 
                     
                 
                   Ala-Ala-Xaa 6 -Trp-Xaa 7 -Xaa 8 -Gly-Pro-Ser-Xaa 9 -Ser- 
                 
                     
                 
                   Xaa 10 -Ala-Xaa 11 -Xaa 12 - 
                 
             
                
               
            
             
                
                
                
                
                
               
            
           
         
         wherein each of Xaa 1  to Xaa 12  is independently any amino acid residue, the side chains of which form a binding site, in whole or in part, that determines the binding specificity of the protein; and 
         at each of the Xaa 1  and Xaa 12  ends of the scaffold are polypeptides that form a superscaffold which displays said binding site in a solvent exposed portion of the protein, or 
         one of the Xaa 1  and Xaa 12  ends of the scaffold is —H and the other end is a polypeptide that forms a superscaffold which displays said binding site in a solvent exposed portion of the protein. 
       
     
     
         2 . The protein of  claim 1  wherein said scaffold polypeptide is derived from a C-type lectin fold (CTL-fold). 
     
     
         3 . The protein of  claim 2  wherein said CTL-fold is a C-type lectin-like domain (CTLD) or a MTD like domain. 
     
     
         4 . The protein of  claim 1  wherein said scaffold is in the C-terminal half of the protein. 
     
     
         5 . The protein of  claim 4  wherein said scaffold is within about 100 amino acid residues or within about 50 amino acid residues of the C-terminus of the protein. 
     
     
         6 . The protein of  claim 1  wherein said scaffold comprises 
       
         
           
                 
               
                   -A-A-L-F-G-G-X-W-X-X-T-S-X-S-G-S-R-A-A-X-W-X-X-G- 
                 
                     
                 
                   P-S-X-S-X-A-X-X-; 
                 
                     
                 
                   -X-W-X-X-T-S-X-S-G-S-R-A-A-X-W-X-X-G-P-S-X-S-X-A- 
                 
                     
                 
                   X-X-G-A-R-G-V-C-; 
                 
                     
                 
                   .A-A-L-F-G-G-X-W-X-X-T-S-X-S-G-S-R-A-A-X-W-X-X-G- 
                 
                     
                 
                   P-S-X-S-X-A-X-X-G-A-R-G-V-C-; 
                 
                   or 
                 
                     
                 
                   -X-W-X-X-T-S-X-S-G-S-R-A-A-X-W-X-X-G-P-S-X-S-X-A- 
                 
                     
                 
                   X-X-G-A-R-G-V-C-D-H-L-I-L-E. 
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         7 . The protein of  claim 7  wherein said scaffold is about 44-45 amino acid residues in length. 
     
     
         8 . A nucleic acid molecule encoding the protein of  claim 1 . 
     
     
         9 . The nucleic acid molecule of  claim 9  wherein said scaffold is all or part of a variable region (VR) operably linked to an initiation of mutagenic homing (IMH) sequence and a template region (TR). 
     
     
         10 . A method of producing a plurality of proteins with different binding specificities, said method comprising
 expressing and replicating the nucleic acid molecule of  claim 10  in a cell under conditions of mutagenic homing wherein said TR directs mutagenesis of variable residues within said scaffold.   
     
     
         11 . A method of selecting a protein with binding specificity for a molecule of interest, said method comprising
 producing a plurality of proteins in a plurality of cells by the method of  claim 11 ;   selecting proteins which bind a molecule of interest after individual contact of each of said plurality of proteins with said molecule of interest.   
     
     
         12 . The method of  claim 12  wherein said molecule of interest is a cell surface molecule. 
     
     
         13 . The method of  claim 13  wherein said molecule of interest is a cell surface molecule of a cancer or other mammalian cell or a bacterial cell surface molecule. 
     
     
         14 . The protein of  claim 1 , further comprising a label attached to said protein. 
     
     
         15 . The protein of  claim 16  wherein said label is a covalently attached, directly detectable label. 
     
     
         16 . The protein of  claim 1 , further comprising a cellular toxin or pro-drag attached to said protein. 
     
     
         17 . A method of decreasing the viability of a cancer cell, said method comprising
 covalently linking a cellular toxin or pro-drug to a protein selected by the method of  claim 14 ; and   contacting said linked protein with a cancer cell comprising a cell surface molecule which binds said protein to decrease the viability of said cell.   
     
     
         18 . The protein of  claim 19  wherein said cancer cell is in a mammalian or human subject. 
     
     
         19 . A method of detecting a bacterial cell, said method comprising
 obtaining the protein selected by the method of  claim 15 ;   contacting said protein with a bacterial cell comprising a cell surface molecule which binds said protein; and   detecting said protein on said bacterial cell.   
     
     
         20 . A method of targeting a cell expressing a cell surface molecule, said method comprising
 contacting said cell with a protein according to  claim 1  which binds said cell surface molecule.