Binding polypeptides with restricted diversity sequences

Abstract

The invention provides variant CDRs comprising highly restricted amino acid sequence diversity. These polypeptides provide a flexible and simple source of sequence diversity that can be used as a source for identifying novel antigen binding polypeptides. The invention also provides these polypeptides as fusion polypeptides to heterologous polypeptides such as at least a portion of phage or viral coat proteins, tags and linkers. Libraries comprising a plurality of these polypeptides are also provided. In addition, methods of and compositions for generating and using these polypeptides and libraries are provided.

1 - 25 . (canceled)  
     
     
         26 . a polypeptide comprising a variant CDRH1, H2, H3, L1, L2 and/or L3, wherein the variant CDR has a variant amino acid in at least one solvent accessible and highly diverse amino acid position, wherein the variant amino acid is encoded by a restricted codon set that encodes 10 or fewer amino acids:  
     
     
         27 . The polypeptide of  claim 26 , wherein the polypeptide comprises a variant CDRH3 comprising a variant amino acid in at least one of positions 95, 96, 97, 98, 99, 100 and 100a, numbering of positions according to the Kabat system.  
     
     
         28 . The polypeptide of  claim 26 , wherein the polypeptide comprises a variant CDRH3 comprising a variant amino acid in at least one of positions 95, 96, 97, 98, 99, 100, and a position between 100 and C-terminal sequence AMDY.  
     
     
         29 . The polypeptide of  claim 26 , wherein the variant CDRH3 comprises an insertion of one or more positions, wherein said one or more positions comprises an amino acid encoded by a restricted codon set.  
     
     
         30 . The polypeptide of  claim 26 , wherein the polypeptide comprises a variant CDRH2 comprising a variant amino acid in at least one of positions 50, 52, 53, 54, 56 and 58, numbering of positions according to the Kabat system.  
     
     
         31 . The polypeptide of  claim 26 , wherein the polypeptide comprises a variant CDRH1 comprising a variant amino acid in at least one of positions 28, 30, 31, 32 and 33, numbering of positions according to the Kabat system.  
     
     
         32 . The polypeptide of  claim 26 , wherein the polypeptide comprises a variant CDRL3 comprising a variant amino acid in at least one of positions 92, 93, 94, 95 and 97, numbering of positions according to the Kabat system.  
     
     
         33 . The polypeptide of  claim 26 , wherein the polypeptide comprises a variant CDRL2 comprising a variant amino acid in at least one of positions 51 and 54, numbering of positions according to the Kabat system.  
     
     
         34 . The polypeptide of  claim 26 , wherein the polypeptide comprises a variant CDRL1 comprising a variant amino acid in at least one of positions 29, 30, 31, 32 and 33, numbering of positions according to the Kabat system.  
     
     
         35 . The polypeptide of  claim 26 , wherein the restricted codon set is TMT, WMT, RMC, RMG, RRC, RSA, MKC, YMT, RST, KMT, SRC, MRT, WMT, or a combination thereof.  
     
     
         36 . The polypeptide of  claim 26 , wherein the codon set is TMT and/or KMT.  
     
     
         37 - 52 . (canceled)  
     
     
         53 . The polypeptide of  claim 26 , wherein the polypeptide is a heavy chain antibody variable domain.  
     
     
         54 . (canceled)  
     
     
         55 . The polypeptide of  claim 26 , further comprising a dimerization domain linked to C-terminal region of a heavy chain antibody variable domain.  
     
     
         56 . A polypeptide according to  claim 55 , wherein the dimerization domain comprises a leucine zipper domain or a sequence comprising at least one cysteine residue.  
     
     
         57 . A polypeptide according to  claim 56 , wherein the dimerization domain comprises a hinge region from an antibody and leucine zipper.  
     
     
         58 . A polypeptide according to  claim 55 , wherein the dimerization domain is a single cysteine.  
     
     
         59 - 72 . (canceled)  
     
     
         73 . A library comprising a plurality of the polypeptide of  claim 26 , and wherein the library has at least 1×10 4  distinct antibody variable domain sequences.  
     
     
         74 - 90 . (canceled)  
     
     
         91 . A method comprising: 
 a) constructing an expression vector comprising a polynucleotide sequence which encodes a light chain variable domain, a heavy chain variable domain or both of a source antibody comprising at least one, two, three, four, five or all CDRs of the source antibody selected from the group consisting of CDR L1, L2, L3, H1, H2 and H3; and    b) mutating at least one, two, three, four, five or all CDRs of the source antibody at at least one solvent accessible and highly diverse amino acid position using a restricted codon set that encodes 10 or fewer amino acids.    
     
     
         92 . The method of  claim 91 , wherein variant CDRH3 comprises an amino acid sequence: 
 (X1) n -A-M    wherein X 1  is an amino acid encoded by a restricted codon set that encodes 10 or fewer amino acids, and n=3 to 20.    
     
     
         93 . The method of  claim 91 , wherein variant CDRH2 comprises an amino acid sequence: 
 X1-I-X2-P-(X3)n-G-X4-T-X5-Y-A    wherein X1, X2, X3, X4 and/or X5 is an amino acid encoded by a restricted codon set that encodes 10 or fewer amino acids, and n=1 to 2.    
     
     
         94 . The method of  claim 91 , wherein variant CDRH1 comprises an amino acid sequence: 
 G-F-X1-I-(X2)n-I    wherein X1 and/or X2 is an amino acid encoded by a restricted codon set that encodes 10 or fewer amino acids, and n=2 to 4.    
     
     
         95 . The method of  claim 91 , wherein variant CDRL3 comprises an amino acid sequence: 
 Q-X1-(X2)n-P-X3-T-F    wherein X1 is Q or missing, and 
 X2 and/or X3 is an amino acid encoded by a restricted codon set that encodes 10 or fewer amino acids, and n=2 to 4.  
   
     
     
         96 . The method of  claim 91 , wherein variant CDRL2 comprises an amino acid sequence: 
 Y-X1-A-S-X2-L    wherein X1 and/or X2 is an amino acid encoded by a restricted codon set that encodes 10 or fewer amino acids.    
     
     
         97 . The method of  claim 91 , wherein variant CDRL1 comprises an amino acid sequence: 
 S-Q-(X1)n-V    wherein X1 is an amino acid encoded by a restricted codon set that encodes 10 or fewer amino acids, and n=3 to 5.