Construction of diverse synthetic peptide and polypeptide libraries

Abstract

The present invention concerns the design and construction of diverse peptide and polypeptide libraries. In particular, the invention concerns methods of analytical database design for creating datasets using multiple relevant parameters as filters, and methods for generating sequence diversity by directed multisyntheses oligonucleotide synthesis. The present methods enable the reduction of large complex annotated databases to simpler datasets of related sequences, based upon relevant single or multiple key parameters that can be individually directly defined. The methods further enable the creation of diverse libraries based on this approach, using multisynthetic collections of discrete and degenerate oligonucleotides to capture the diverse collection of sequences, or portions thereof.

1 - 18 . (canceled) 
     
     
         19 . A method of making a library of antibody heavy or light chains, comprising the steps of
 (a) aligning a plurality of antibody heavy or light chain sequences comprising one or more CDR sequence motifs;   (b) creating a first dataset by applying a predetermined combination of two or more filters to said antibody heavy or light chain sequences;   (c) analyzing said first dataset for positional amino acid usage frequency within at least one of said CDR sequence motifs;   (d) creating a second dataset characterized by a minimum threshold amino acid usage frequency at one or more amino acid positions within said CDR sequence motif; and   (e) synthesizing an antibody heavy or light chain library designed with the aid of the first and second datasets identified.   
     
     
         20 . The method of  claim 20  wherein said library is synthesized by generating a discrete number of defined or degenerate oligonucleotides such that only defined amino acids are generated. 
     
     
         21 . The method of  claim 20  wherein the diversity of the physical library produced exceeds the diversity of a library which is a physical representation of the datasets identified. 
     
     
         22 . The method of  claim 22  wherein at least one amino acid not meeting the minimum threshold amino acid usage frequency is also synthesized to provide said diversity. 
     
     
         23 . The method of  claim 20  wherein the diversity of the physical library produced is less than the diversity of a library which is a physical representation of the datasets identified. 
     
     
         24 . The method of  claim 24  wherein not all amino acids meeting the minimum threshold amino acid usage frequency are synthesized. 
     
     
         25 . The method of  claim 20  wherein said CDRs are cloned into a scaffold of framework sequences. 
     
     
         26 . The method of  claim 26  wherein said framework sequences are the most frequently used framework sequences in the database comprising said CDRs. 
     
     
         27 . The method of  claim 20  wherein said physical library is expressed using a prokaryotic or eukaryotic expression system. 
     
     
         28 . The method of  claim 20  wherein said physical library is expressed and displayed using a phagemid display, mRNA display, microbial cell display, mammalian cell display, microbead display technique, antibody array, or display based on protein-DNA linkage. 
     
     
         29 . The method of  claim 20  wherein said library is screened for one or more chemical and/or biological properties of its members: 
     
     
         30 . The method of  claim 30  wherein said biological property is selected from the group consisting of half-life, potency, efficacy, binding affinity, and immunogenicity. 
     
     
         31 . The method of  claim 20  comprising the introduction of amino acid side-chain diversity at one or more amino acid positions. 
     
     
         32 . The method of  claim 32  wherein said amino acid side-chain diversity is introduced by providing amino acid residues with at least two different side-chain chemical functionalities at said amino acid position or positions. 
     
     
         33 . The method of  claim 33  wherein at least 30% to at least 50% of all amino acid chemistries are represented at each amino acid position. 
     
     
         34 . The method of  claim 33  wherein said side-chain diversity is introduced by using combinatorial degenerate oligonucleotide synthesis. 
     
     
         35 - 41 . (canceled)