Methods for generating polynucleotides having desired characteristics by iterative selection and recombination

Abstract

A method for DNA reassembly after random fragmentation, and its application to mutagenesis of nucleic acid sequences by in vitro or in vivo recombination is described. In particular, a method for the production of nucleic acid fragments or polynucleotides encoding mutant proteins is described. The present invention also relates to a method of repeated cycles of mutagenesis, shuffling and selection which allow for the directed molecular evolution in vitro or in vivo of proteins.

1 - 40 . (canceled)  
     
     
         41 . A method for producing a library of nucleic acids encoding a plurality of modified antigen binding sites, wherein the modified antigen binding sites are derived from a first nucleic acid comprising a sequence encoding a first antigen binding site, the method comprising: (a) providing a first nucleic acid encoding a first antigen binding site; (b) providing a set of mutagenic oligonucleotides that encode naturally-occurring amino acid variants at a plurality of targeted codons in the first nucleic acid; and (c) using the set of mutagenic oligonucleotides to generate a set of antigen binding site-encoding variant nucleic acids encoding a range of amino acid variations at each amino acid codon that was mutagenized, thereby producing a library of nucleic acids encoding a plurality of modified antigen binding sites.  
     
     
         42 . The method of  claim 41 , wherein step (b) provides a set of mutagenic oligonucleotides that encode all nineteen naturally-occurring amino acid variants for each targeted codon, thereby generating all 19 possible natural amino acid changes at each amino acid codon mutagenized.  
     
     
         43 . The method of  claim 41 , further comprising expressing the set of variant antigen binding site-encoding nucleic acids such that antigen binding site-encoding polypeptides encoded by the variant nucleic acids are expressed.  
     
     
         44 . The method of  claim 41 , wherein the set of mutagenic oligonucleotides comprises a 19-fold degenerate mutagenic oligonucleotide for each codon to be mutagenized, wherein each of the 19-fold degenerate mutagenic oligonucleotides comprises a homologous first sequence and a degenerate triplet second sequence.  
     
     
         45 . The method of  claim 41 , wherein the antigen binding site comprises a single stranded antigen binding polypeptide, a Fab fragment, an Fc fragment, a Fd fragment, a F(ab′) 2  fragment, a Fv fragment or a complementarity determining region (CDR).  
     
     
         46 . The method of  claim 45 , wherein the antigen binding site polypeptide further comprises an antibody polypeptide or further comprises an antigen binding site of a T cell receptor (TCR) or a receptor.  
     
     
         47 . The method of  claim 46 , wherein the antigen binding site polypeptide of a T cell receptor further comprises a T cell receptor or the antigen binding site of a receptor further comprises a receptor.  
     
     
         48 . The method of  claim 41 , wherein the nucleic acid of step (a) is derived from a nucleic acid encoding a mammalian polypeptide.  
     
     
         49 . The method of  claim 48 , wherein the mammalian polypeptide comprises a human polypeptide.  
     
     
         50 . The method of  claim 48 , wherein the mammalian polypeptide is an antibody.  
     
     
         51 . The method of  claim 41 , wherein the nucleic acid of step (a) is derived from a human nucleic acid encoding an antigen binding site.  
     
     
         52 . The method of  claim 51 , wherein the nucleic acid of step (a) is derived from a phage comprising a human nucleic acid sequence encoding an antigen binding site, wherein the phage expresses the antigen binding site.  
     
     
         53 . The method of  claim 51 , wherein the nucleic acid of step (a) is derived from a non-human mammal comprising a human nucleic acid sequence encoding an antigen binding site, wherein the non-human mammal expresses the antigen binding site.  
     
     
         54 . The method of  claim 53 , wherein the non-human mammal is a transgenic non-human mammal.  
     
     
         55 . The method of  claim 54 , wherein the transgenic non-human mammal is a mouse.  
     
     
         56 . The method of  claim 41 , wherein at least two amino acid codons in the antigen binding site are mutagenized.  
     
     
         57 . The method of  claim 56 , wherein all the amino acid codons in the antigen binding site are mutagenized.  
     
     
         58 . The method of  claim 46 , wherein all the amino acid codons in the antibody polypeptide are mutagenized or all the amino acid codons in the T cell receptor (TCR) or the receptor are mutagenized.  
     
     
         59 . The method of  claim 41 , wherein a degenerate mutagenic oligonucleotide comprises a first homologous sequence, a degenerate triplet second sequence, and a third homologous sequence, or wherein each degenerate oligonucleotide comprises a first homologous sequence, a plurality of degenerate triplets second sequences, and a third homologous sequence.  
     
     
         60 . The method of  claim 43 , further comprising screening the expressed antigen binding site polypeptide for its ability to specifically bind an antigen.  
     
     
         61 . The method of  claim 60 , comprising screening the expressed antigen binding site polypeptide for its ability to specifically bind an antigen capable of being specifically bound by the first antigen binding site polypeptide.  
     
     
         62 . The method of  claim 61 , comprising identifying an antigen binding site variant by its increased antigen binding affinity or increased antigen binding specificity or by its decreased antigen binding affinity or decreased antigen binding specificity as compared to the affinity or specificity of the first antigen binding site to the antigen.  
     
     
         63 . The method of  claim 41 , further comprising mutagenizing the first nucleic acid of step (a) by a method comprising an optimized directed evolution system.  
     
     
         64 . The method of  claim 41 , further comprising mutagenizing the first nucleic acid of step (a) by a method comprising a synthetic ligation reassembly.  
     
     
         65 . The method of  claim 43 , comprising screening the expressed antigen binding site polypeptide for its ability to specifically bind an antigen by a method comprising expression of the expressed antigen binding site polypeptide in a solid phase.  
     
     
         66 . The method of  claim 65 , comprising screening the expressed antigen binding site polypeptide for its ability to specifically bind an antigen by a method comprising use of an ELISA.  
     
     
         67 . The method of  claim 43 , comprising screening the expressed antigen binding site polypeptide for its ability to specifically bind an antigen by a method comprising phage display of the antigen binding site polypeptide or by a method comprising expression of the expressed antigen binding site polypeptide in a liquid phase.  
     
     
         68 . The method of  claim 41 , wherein the set of progeny antigen binding site-encoding variant nucleic acids is generated by amplifying the nucleic acid of step (a) by a polymerase-based amplification using a plurality of oligonucleotides.  
     
     
         69 . The method of  claim 68 , wherein the amplification comprises a polymerase chain reaction (PCR).  
     
     
         70 . A library of nucleic acids encoding a plurality of modified antigen binding sites, wherein the modified antigen binding sites are derived from a first nucleic acid comprising a sequence encoding a first antigen binding site, made by a method of  claim 41 .  
     
     
         71 . A method for producing from a library of variant antibodies from a template antibody, the method comprising: (a) providing a first nucleic acid encoding the template antibody; (b) providing a set of mutagenic oligonucleotides that encode naturally-occurring amino acid variants at a plurality of targeted codons in the first nucleic acid; and c) using the set of mutagenic oligonucleotides to generate a set of antibody-encoding variant nucleic acids encoding a range of amino acid variations at each amino acid codon that was mutagenized, thereby producing a library of nucleic acids encoding a plurality of variant antibodies.  
     
     
         72 . The method of  claim 71 , wherein step (b) provides a set of mutagenic oligonucleotides that encode all nineteen naturally-occurring amino acid variants for each targeted codon, thereby generating all 19 possible natural amino acid changes at each amino acid codon mutagenized.  
     
     
         73 . The method of  claim 71 , wherein the antibody is selected from the group consisting of polypeptides comprising a Fab fragment, an Fd fragment, an Fc fragment, a F(ab′) 2  fragment, a Fv fragment and a complementarity determining region (CDR).  
     
     
         74 . The method of  claim 71 , wherein the plurality of oligonucleotides comprises a degenerate oligonucleotide for each codon to be mutagenized, wherein each of the degenerate oligonucleotides comprises a homologous first sequence and a degenerate triplet second sequence.  
     
     
         75 . The method of  claim 71 , wherein the set of progeny polynucleotides encoding antibodies is generated by amplifying the nucleic acid of step (a) using a plurality of oligonucleotides.  
     
     
         76 . A library of variant antibodies derived from a template antibody made by a method of  claim 71 .  
     
     
         77 . A method for producing from a library of variant T cell receptors (TCRs) from a template T cell receptor (TCR), the method comprising: (a) providing a first nucleic acid encoding the template T cell receptor; (b) providing a set of mutagenic oligonucleotides that encode naturally-occurring amino acid variants at a plurality of targeted codons in the first nucleic acid; and c) using the set of mutagenic oligonucleotides to generate a set of T cell receptor (TCR)-encoding variant nucleic acids encoding a range of amino acid variations at each amino acid codon that was mutagenized, thereby producing a library of nucleic acids encoding a plurality of variant T cell receptors (TCRs).  
     
     
         78 . A library of variant T cell receptors (TCRs) derived from a template T cell receptor (TCR) made by a method of  claim 77 .  
     
     
         79 . A method of making a set of nucleic acids encoding a set of antigen binding site variants comprising the steps of: (a) providing a template nucleic acid encoding an antigen-binding polypeptide; (b) providing a plurality of oligonucleotides that encode all nineteen naturally-occurring amino acid variants at a single amino acid residue of the antigen-binding polypeptide; and (c) generating a set of progeny antigen binding site-encoding variant nucleic acids encoding a non-stochastic range of single amino acid substitutions at each amino acid codon that was mutagenized, whereby all 19 possible natural amino acid changes are generated at each amino acid codon mutagenized, thereby making a set of nucleic acids encoding a set of antigen binding site variants.  
     
     
         80 . The method of  claim 79 , further comprising expressing the set of progeny antigen binding site-encoding polynucleotides such that antigen binding site-encoding polypeptides encoded by the progeny polynucleotides are expressed.  
     
     
         81 . The method of  claim 79 , wherein the plurality of oligonucleotides comprises a set of degenerate oligonucleotides and each of the degenerate oligonucleotides comprises a homologous first sequence and a degenerate triplet second sequence.  
     
     
         82 . The method of  claim 79 , wherein the antigen binding site-encoding polypeptide comprises a single stranded antigen binding polypeptide.  
     
     
         83 . The method of  claim 79 , wherein the antigen binding site-encoding polypeptide comprises an antibody polypeptide or the antigen binding site-encoding polypeptide comprises an antigen binding site of a T cell receptor (TCR) or a receptor.  
     
     
         84 . The method of  claim 83 , wherein the antigen binding site of a T cell receptor further comprises a T cell receptor or the antigen-binding site of a receptor further comprises a receptor.  
     
     
         85 . The method of  claim 79 , wherein the nucleic acid of step (a) is derived from a nucleic acid encoding a mammalian antibody polypeptide.  
     
     
         86 . The method of  claim 85 , wherein the nucleic acid of step (a) is derived from a human nucleic acid.  
     
     
         87 . The method of  claim 79 , wherein at least two amino acid codons in the antigen binding site are mutagenized and a set of degenerate oligonucleotides that encode all nineteen naturally-occurring amino acid variants are provided for each amino acid codon mutagenized.  
     
     
         88 . The method of  claim 79 , wherein all the amino acid codons in the antigen binding site are mutagenized and a set of degenerate oligonucleotides that encode all nineteen naturally-occurring amino acid variants are provided for each amino acid codon mutagenized.  
     
     
         89 . The method of  claim 83 , wherein all the amino acid codons in the antibody polypeptide are mutagenized or all the amino acid codons in the antigen binding site of the T cell receptor (TCR) or the receptor are mutagenized.  
     
     
         90 . The method of  claim 79 , wherein a degenerate oligonucleotide comprises a first homologous sequence, a degenerate triplet second sequence, and a homologous third sequence or each degenerate oligonucleotide comprises a first homologous sequence, a degenerate triplet second sequence, and a homologous third sequence.  
     
     
         91 . The method of  claim 80 , further comprising screening an expressed antigen binding site-encoding polypeptide for its ability to specifically bind an antigen.  
     
     
         92 . The method of  claim 80 , comprising screening the expressed antigen binding site-encoding polypeptide for its ability to specifically bind an antigen capable of being specifically bound by the first antigen binding site.  
     
     
         93 . The method of  claim 92 , comprising identifying an antigen binding site variant by its increased antigen binding affinity or antigen binding specificity to the antigen as compared to the affinity or specificity of the antigen binding site encoded by the nucleic acid of step (a).  
     
     
         94 . The method of  claim 79 , further comprising mutagenizing the template nucleic acid by a method comprising an optimized directed evolution system.  
     
     
         95 . The method of  claim 79 , further comprising mutagenizing the template nucleic acid by a method comprising a synthetic ligation reassembly.  
     
     
         96 . The method of  claim 79 , comprising screening the expressed antigen binding site-encoding polypeptide for its ability to specifically bind an antigen by an ELISA.  
     
     
         97 . The method of  claim 79 , wherein the set of variant nucleic acids is generated by performing amplification reactions on the nucleic acid of step (a) using the set of oligonucleotides to generate a set of variant nucleic acids encoding nineteen amino acid substitution variants at a single amino acid residue of the antigen-binding polypeptide.  
     
     
         98 . The method of  claim 97 , wherein the amplification comprises a polymerase-based amplification.  
     
     
         99 . The method of  claim 98 , wherein polymerase-based amplification comprises a polymerase chain reaction (PCR).  
     
     
         100 . The method of  claim 79 , wherein the set of variant nucleic acids comprises 10 10  members, 10 5  members, or 10 3  members.  
     
     
         101 . A method of making a set of antibody variants comprising the steps of: (a) providing a nucleic acid encoding an antibody; (b) providing a plurality of oligonucleotides; (c) generating a non-stochastic range of single amino acid substitutions at each amino acid codon, whereby all 19 possible natural amino acid changes are generated at each amino acid codon mutagenized, thereby generating a set of variant nucleic acids; and (d) expressing the set of variant nucleic acids such that the antibody variants encoded by the variant nucleic acids are expressed.  
     
     
         102 . The method of  claim 101 , wherein the antibody is selected from the group consisting of polypeptides comprising a Fab fragment, a Fd fragment, an Fc fragment, a F(ab′) 2  fragment, a Fv fragment and a complementarity determining region (CDR).  
     
     
         103 . The method of  claim 101 , wherein the plurality of oligonucleotides comprises a set of degenerate oligonucleotides that encode all nineteen naturally-occurring amino acid variants at a single amino acid residue of the antibody, wherein each of the degenerate oligonucleotides comprises a homologous first sequence and a degenerate triplet second sequence.  
     
     
         104 . The method of  claim 101 , wherein generating a non-stochastic range of single amino acid substitutions comprises performing amplification reactions on the nucleic acid of step (a) using the set of oligonucleotides to generate a set of variant nucleic acids encoding nineteen amino acid substitution variants at a single amino acid residue of the antibody.  
     
     
         105 . A method of identifying a variant of an antigen binding site comprising the steps of: (a) providing a nucleic acid encoding an antigen binding site; (b) providing a set of oligonucleotides that encode all nineteen naturally-occurring amino acid variants at all residues of the antigen-binding site; (c) incorporating the sequence of the oligonucleotides of step (b) into the nucleic acid of step (a) to generate a set of variant nucleic acids encoding nineteen amino acid substitution variants at each residue of the antigen binding site; (d) expressing each of the variant nucleic acids as polypeptides and measuring the variant's affinity to the antigen; and (e) identifying a variant of the antigen binding site by its increased or decreased antigen binding specificity as compared to the antigen binding affinity of the antigen binding site encoded by the nucleic acid of step (a).  
     
     
         106 . The method of  claim 105 , wherein the variant nucleic acids are expressed using in vitro transcription/translation or the variant nucleic acids are expressed using phage display.  
     
     
         107 . The method of  claim 105 , wherein the set of oligonucleotides comprises a set of degenerate oligonucleotides that encode all nineteen naturally-occurring amino acid variants at a single amino acid residue of the antibody, wherein each of the degenerate oligonucleotides comprises a homologous first sequence and a degenerate triplet second sequence.  
     
     
         108 . The method of  claim 106 , wherein the antigen binding site comprises an antibody or the antigen binding site comprises an antigen binding site of a T cell receptor or a receptor.  
     
     
         109 . The method of  claim 108 , wherein the antibody is selected from the group consisting of polypeptides comprising a Fab fragment, an Fd fragment, an Fc fragment, a F(ab′) 2  fragment, a Fv fragment and a complementarity determining region (CDR).  
     
     
         110 . The method of  claim 105 , wherein incorporating the sequence of the oligonucleotides of step (b) into the nucleic acid of step (a) is accomplished by an amplification reaction using the oligonucleotides as primers.