Protein (poly)peptides libraries

Abstract

The present invention relates to synthetic DNA sequences which encode one or more collections of homologous proteins/(poly)peptides, and methods for generating and applying libraries of these DNA sequences. In particular, the invention relates to the preparation of a library of human-derived antibody genes by the use of synthetic consensus sequences which cover the structural repertoire of antibodies encoded in the human genome. Furthermore, the invention relates to the use of a single consensus antibody gene as a universal framework for highly diverse antibody libraries.

1 - 55 . (canceled)  
     
     
         56 . A method of preparing a library of nucleic acids, wherein each nucleic acid encodes an immunoglobulin variable domain comprising consensus framework sequences, comprising: 
 (a) identifying a plurality of immunoglobulin variable domain amino acid sequences, each comprising four consensus framework regions interspaced by three complementary determining regions CDR1, CDR2, and CDR3, wherein said consensus framework regions have been identified by the following steps:    (i) aligning three or more known human immunoglobulin sequences;    (ii) identifying the conserved framework regions of said known human immunoglobulin sequences;    (iii) comparing the amino acids at each corresponding position of said conserved framework regions; and    (iv) deducing consensus framework regions from said comparing in step (a)(iii); and    (b) synthesizing a plurality of nucleic acids encoding said plurality of immunoglobulin variable domain amino acid sequences provided in step (a), wherein each of said nucleic acids comprises DNA cleavage sites at the boundary between each consensus framework region and complementary determining region, and wherein each of said cleavage sites is unique within said nucleic acid but common to all nucleic acid sequences of said library at corresponding positions.    
     
     
         57 . The method according to  claim 56 , wherein said known human immunoglobulin sequences in step (a)(i) are human Vκ immunoglobulin sequences.  
     
     
         58 . The method according to  claim 56 , wherein said known human immunoglobulin sequences in step (a)(i) are human Vλ immunoglobulin sequences.  
     
     
         59 . The method according to  claim 56 , wherein said known human immunoglobulin sequences in step (a)(i) are human VH immunoglobulin sequences.  
     
     
         60 . The method according to  claim 57 , wherein said nucleic acids synthesized in step (b) are selected from the group consisting of Vκ1 (SEQ ID NO:42), Vκ2 (SEQ ID NO: 44), Vκ3 (SEQ ID NO: 46), and Vκ4 (SEQ ID NO: 48).  
     
     
         61 . The method according to  claim 58 , wherein said nucleic acids synthesized in step (b) are selected from the group consisting of Vλ1 (SEQ ID NO:50), Vλ2 (SEQ ID NO: 52), and Vλ3 (SEQ ID NO: 54).  
     
     
         62 . The method according to  claim 59 , wherein said nucleic acids synthesized in step (b) are selected from the group consisting of VH1A (SEQ ID NO:56), VH1B (SEQ ID NO: 58), VH2 (SEQ ID NO: 60), VH3 (SEQ ID NO: 62), VH4 (SEQ ID NO: 64), VH5 (SEQ ID NO: 66), and VH6 (SEQ ID NO: 68).  
     
     
         63 . The method according to  claim 56 , further comprising inserting said nucleic acids into an expression vector.  
     
     
         64 . The method according to  claim 63 . The method according to claim  83 , wherein said nucleic acids encoding said immunoglobulin variable domain amino acid sequences comprise codons that are frequently used in said host cell.  
     
     
         65 . The method according to  claim 64 , wherein said host cell is  E. coli.    
     
     
         66 . The method according to  claim 65 , wherein said expression vector is a phagemid vector.  
     
     
         67 . The method according to  claim 56 , wherein said CDR1 is selected from the group consisting of VH CDR1 germline sequences.  
     
     
         68 . The method according to  claim 67 , wherein said CDR1 is selected from the group consisting of VH1-12-1, VH1-13-16, VH2-31-10, VH3-13-8, VH4-11-7, CH5-12-1, and VH6-35-1.  
     
     
         69 . The method according to  claim 56 , wherein said CDR1 is selected from the group consisting of Vλ CDR1 germline sequences.  
     
     
         70 . The method according to  claim 69 , wherein said CDR1 is selected from the group consisting of VHUMLV86, DPL11, and DPL23.  
     
     
         71 . The method according to  claim 56 , wherein said CDR1 is selected from the group consisting of Vκ CDR1 germline sequences.  
     
     
         72 . The method according to  claim 71 , wherein said CDR1 is selected from the group consisting of Vκ1-14, Vκ2-6, Vκ33-1, and Vκ4-1.  
     
     
         73 . The method according to  claim 56 , wherein said CDR2 is selected from the group consisting of VH CDR2 germline sequences.  
     
     
         74 . The method according to  claim 73 , wherein said CDR2 is selected from the group consisting of VH1-12-1, VH1-13-6, VH2-31-3, VH3-13-8, VH4-11-8, VH4-31-17, VH5-12-1, and VH6-35-1.  
     
     
         75 . The method according to  claim 56 , wherein said CDR2 is selected from the group consisting of Vλ CDR2 germline sequences.  
     
     
         76 . The method according to  claim 75 , wherein said CDR2 is selected from the group consisting of DPL5, DPL12, and HUMLV318.  
     
     
         77 . The method according to  claim 56 , wherein said CDR2 is selected from the group consisting of Vκ CDR2 germline sequences.  
     
     
         78 . The method according to  claim 77 , wherein said CDR2 is selected from the group consisting of Vκ1-2, Vκ2-6, Vκ3-4, and Vκ4-1.  
     
     
         79 . The method according to  claim 56 , wherein said CDR3 is selected from random amino acid sequences.  
     
     
         80 . The method according to  claim 56  wherein said CDR3 is an amino acid sequence selected from Vκ CDR3 sequences comprising the amino acid sequence N1-Gln-N-3-N4-N-5-N6-N-7-N8-Thr, wherein: 
 N1 is an amino acid selected from the group consisting of Phe, His, Leu, Met, and Gln;    N3 is an amino acid other than Cys or Pro;    N4 is an amino acid selected from the group consisting of Asp, Gly, Asn, Ser, and Phe;    N5 is an amino acid selected from the group consisting of Asp, Gly, Asn, and Ser;    N6 is an amino acid other than Cys;    N7 is Pro or Ser; and    N8 is an amino acid other than Cys.    
     
     
         81 . The method according to  claim 56  wherein said CDR3 is an amino acid sequence selected from Vλ CDR3 sequences comprising the amino acid sequence Gln-Ser-N-3-Asp-N-5-N6-N-7-N8-N-9-N10, wherein: 
 N1 is an amino acid selected from the group consisting of Arg, Trp, or Phe;    N3 is an amino acid other than Cys or Pro;    N5 is an amino acid other then Cys or Trp;    N6 is an amino acid other than Cys or Trp;    N7 is an amino acid other than Cys or Trp;    N8 is an amino acid other than Cys or Trp or N8 is absent;    N9 is an amino acid other than Cys or Trp or N9 is absent; and    N10 is an amino acid other than Cys.