High throughput generation and screening of fully human antibody repertoire in yeast

Abstract

Compostions, kits and methods are provided for generating highly diverse libraries of proteins such as antibodies via homologous recombination in vivo, and screening these libraries against protein, peptide and nucleic acid targets using a two-hybrid method in yeast. The method for screening a library of tester proteins against a target protein or peptide comprises: expressing a library of tester proteins in yeast cells, each tester protein being a fusion protein comprised of a first polypeptide subunit whose sequence varies within the library, a second polypeptide subunit whose sequence varies within the library independently of the first polypeptide, and a linker peptide which links the first and second polypeptide subunits; expressing one or more target fusion proteins in the yeast cells expressing the tester proteins, each of the target fusion proteins comprising a target peptide or protein; and selecting those yeast cells in which a reporter gene is expressed, the expression of the reporter gene being activated by binding of the tester fusion protein to the target fusion protein.

1 . A kit, comprising: 
 a first and second populations of haploid yeast cells of opposite mating types,    the first population of haploid yeast cells comprising a library of tester expression vectors for the library of tester fusion proteins, each of the tester expression vector comprising a first transcription sequence encoding either an activation domain or a DNA binding domain of a transcription activator, 
 a first nucleotide sequence encoding a first polypeptide subunit,  
 a second nucleotide sequence encoding a second polypeptide subunit, and  
 a linker sequence encoding a linker peptide that links the first nucleotide sequence and the second nucleotide sequence, wherein the first polypeptide subunit, the linker peptide, the second polypeptide are expressed as a tester fusion protein with either the activation domain or the DNA binding domain of the transcription activator;  
   the second population of haploid yeast cells comprises a target expression vector for a target fusion protein, the target expression vector comprising 
 a second transcription sequence encoding either the activation domain or the DNA binding domain of the transcription activator which is not expressed by the library of tester expression vectors, and  
   a target sequence encoding the target protein or peptide, wherein the target protein or peptide is expressed as a fusion protein with either the activation domain or the DNA binding domain of the transcription activator which is not expressed by the library of tester expression vectors, and either the first or second population of haploid yeast cells comprises a reporter construct comprising a reporter gene whose expression is under transcriptional control of the transcription activator.    
     
     
         2 . The kit of  claim 1 , wherein the second population of haploid yeast cells comprises a plurality of target expression vectors, each of the target expression vectors encoding 
 a second transcription sequence encoding either the activation domain or the DNA binding domain of the transcription activator which is not expressed by the library of tester expression vectors; and    a target sequence encoding the target protein or peptide.    
     
     
         3 . The kit of  claim 1 , wherein the haploid yeast cells of opposite mating types are α and  a  type strains of yeast.  
     
     
         4 . The kit of  claim 1 , wherein the first polypeptide subunit comprises an antibody heavy-chain variable region, and the second polypeptide subunit comprises an antibody light-chain variable region.  
     
     
         5 . The method of  claim 1 , wherein the protein encoded by the reporter gene is selected from the group consisting of β-galactosidase, α-galactosidase, luciferase, β-glucuronidase, chloramphenicol acetyl transferase, secreted embryonic alkaline phosphatase, green fluorescent protein, enhanced blue fluorescent protein, enhanced yellow fluorescent protein, and enhanced cyan fluorescent protein.  
     
     
         6 . The kit of  claim 1 , wherein the diversity of the tester fusion proteins encoded by the library of tester expression vectors is at least 1×10 6 .  
     
     
         7 . The kit of  claim 1 , wherein the diversity of the tester fusion proteins encoded by the library of tester expression vectors is at least 1×10 7 .  
     
     
         8 . The kit of  claim 1 , wherein the diversity of the tester fusion proteins encoded by the library of tester expression vectors is at least 1×10 8 .  
     
     
         9 . The kit of  claim 1 , wherein the target fusion protein comprises an antigen associated with a disease state.  
     
     
         10 . The kit of  claim 1 , wherein the target fusion protein comprises a tumor-surface antigen.  
     
     
         11 . The kit of  claim 1 , wherein the target fusion protein comprises a human growth factor selected from the group consisting of epidermal growth factors, transferrin, insulin-like growth factor, transforming growth factors, interleukin-1, and interleukin-2.  
     
     
         12 . The kit of  claim 1 , wherein the first polypeptide subunit and the second polypeptide subunit are encoded by variable regions of immunoglobulin genes of a human, non-human primates, or rodent.  
     
     
         13 . The kit of  claim 1 , wherein the first polypeptide subunit and the second polypeptide subunit are encoded respectively by a heavy-chain variable region and a light-chain variable region of a human immunoglobulin gene.  
     
     
         14 . The kit of  claim 1 , wherein the first polypeptide subunit is encoded by a heavy-chain variable region of a first human immunoglobulin gene, and the second polypeptide subunit is encoded by a light chain variable region of a second human immunoglobulin gene different from the first human immunoglobulin gene.  
     
     
         15 . A kit, comprising: 
 a first and second populations of haploid yeast cells of opposite mating types,    the first population of haploid yeast cells comprising a library of tester expression vectors for the library of tester fusion proteins, each of the tester expression vector comprising a first transcription sequence encoding either an activation domain or a DNA binding domain of a transcription activator, 
 a first nucleotide sequence encoding a first polypeptide subunit,  
 a second nucleotide sequence encoding a second polypeptide subunit, and  
 a linker sequence encoding a linker peptide that links the first nucleotide sequence and the second nucleotide sequence, wherein the first polypeptide subunit, the linker peptide, the second polypeptide are expressed as a tester fusion protein with either the activation domain or the DNA binding domain of the transcription activator,  
   wherein either the first or second population of haploid yeast cells comprises a reporter construct comprising a reporter gene whose expression is under transcriptional control of the transcription activator.    
     
     
         16 . The kit of  claim 15 , wherein the second population of haploid yeast cells comprises 
 a target expression vector for a target fusion protein, the target expression vector comprising a second transcription sequence encoding either the activation domain or the DNA binding domain of the transcription activator which is not expressed by the library of tester expression vectors.    
     
     
         17 . The kit of  claim 15 , wherein the diversity of the tester fusion proteins encoded by the library of tester expression vectors is at least 1×10 6 .  
     
     
         18 . The kit of  claim 15 , wherein the diversity of the tester fusion proteins encoded by the library of tester expression vectors is at least 1×10 7 .  
     
     
         19 . The kit of  claim 15 , wherein the diversity of the tester fusion proteins encoded by the library of tester expression vectors is at least 1×10 8 .  
     
     
         20 . The kit of  claim 15 , wherein the first polypeptide subunit comprises an antibody heavy-chain variable region, and the second polypeptide subunit comprises an antibody light-chain variable region.  
     
     
         21 . The kit of  claim 15 , wherein the first polypeptide subunit and the second polypeptide subunit are encoded by variable regions of immunoglobulin genes of a human, non-human primates, or rodent.  
     
     
         22 . The kit of  claim 15 , wherein the first polypeptide subunit and the second polypeptide subunit are encoded respectively by a heavy-chain variable region and a light-chain variable region of a human immunoglobulin gene.  
     
     
         23 . The kit of  claim 15 , wherein the first polypeptide subunit is encoded by a heavy-chain variable region of a first human immunoglobulin gene, and the second polypeptide subunit is encoded by a light chain variable region of a second human immunoglobulin gene different from the first human immunoglobulin gene.  
     
     
         24 . The kit of  claim 15 , wherein the haploid yeast cells of opposite mating types are  α  and  a  type strains of yeast.  
     
     
         25 . The kit of  claim 15 , wherein the transcription activator is a transcription activator having separable DNA-binding and transcription activation domains.  
     
     
         26 . The kit of  claim 15 , wherein the transcription activator is selected from the group consisting of GAL4, GCN4, and ADR1 transcription activator.  
     
     
         27 . A method for generating a library of yeast expression vectors, comprising: 
 a) transforming into yeast cells 
 i) a linearized yeast expression vector having a 5′- and 3′-terminus sequence at a first site of linearization; and  
 ii) a library of first insert nucleotide sequences that are linear, double stranded, each of the first insert sequences comprising a first nucleotide sequence encoding a first polypeptide subunit, a 5′- and 3′-flanking sequence at the ends of the first insert sequence which are sufficiently homologous to the 5′- and 3′-terminus sequences of the vector at the first site of linearization, respectively, to enable homologous recombination to occur;  
   b) having homologous recombination occur between the linearized yeast expression vector and the first insert sequence in the transformed yeast cells, such that the first insert sequence is included in the expression vector;    c) isolating from the transformed yeast cells the expression vectors that contain the library of the first insert sequences;    d) linearizing the expression vectors containing the library of the first insert sequences to generate a 5′- and 3′-terminus sequence at a second site of linearization;    e) transforming into yeast cells 
 i) the linearized expression vectors in step d), and  
 ii) a library of second insert nucleotide sequences that are linear, double stranded, each of the second insert sequences comprising a second nucleotide sequence encoding a second polypeptide subunit, a 5′- and 3′-flanking sequence at the ends of the second insert sequence which are sufficiently homologous to the 5′- and 3′-terminus sequences of the vector at the second site of linearization, respectively, to enable homologous recombination to occur; and  
   f) having homologous recombination occur between the linearized yeast expression vector at the second linearization site and the second insert sequence in the transformed yeast cells, such that the second insert sequence is included in the vector and the first and second nucleotide sequences are linked by a linker sequence;    wherein    the expression vector expresses the first polypeptide subunit, the second polypeptide subunit, and the linker polypeptide as a single fusion protein in the transformed yeast cells by the library of yeast expression vectors; and    the first and second nucleotide sequences each independently varies within the library of expression vectors.    
     
     
         28 . The method of  claim 27 , wherein the 5′- or 3′-flanking sequence of the first or the second insert nucleotide sequence is between 30-120 bp in length.  
     
     
         29 . The method of  claim 27 , wherein the 5′- or 3′-flanking sequence of the first or the second insert nucleotide sequence is between 40-90 bp in length.  
     
     
         30 . The method of  claim 27 , wherein the 5′- or 3′-flanking sequence of the first or the second insert nucleotide sequence is between 60-80 bp in length.  
     
     
         31 . The method of  claim 27 , wherein the linker sequence is between 30-120 bp in length.  
     
     
         32 . The method of  claim 27 , wherein the linker sequence is between 45-102 bp in length.  
     
     
         33 . The method of  claim 27 , wherein the linker sequence is between 45-63 bp in length.  
     
     
         34 . The method of  claim 27 , wherein the linker sequence comprises a nucleotide sequence encoding an amino acid sequence of Gly-Gly-Gly-Gly-Ser in 3 or 4 tandem repeats.  
     
     
         35 . The method of  claim 27 , wherein the 5′- and 3′-flanking sequences at the ends of the first or second insert nucleotide sequence comprise a 5′- and 3′-site-specific recombination site, respectively, that are recognized by a site-specific recombinase.  
     
     
         36 . The method of  claim 35 , wherein one of the 5′- and 3′-site-specific recombination sites is coliphase P1 loxP, and the other is a mutant loxP sequence.  
     
     
         37 . The method of  claim 35 , wherein the 5′- and 3′-site-specific recombination sites are each independently selected from the group consisting of SEQ ID Nos 1-13.  
     
     
         38 . The method of  claim 35 , wherein the site-specific recombinase is CRE recombinase.  
     
     
         39 . The method of  claim 35 , further comprising: 
 causing site-specific recombination between the members of the library of the yeast expression vectors at the 5′- and 3′-recombination sites, the recombination resulting in exchange of the first or second nucleotide sequences between the members of the library of the yeast expression vectors.    
     
     
         40 . The method of  claim 39 , wherein the recombination is caused by expression of a recombinase that is inducibly controlled in the yeast cells.  
     
     
         41 . The method of  claim 40 , wherein the 5′- and 3′-recombination sites are different loxP sequences, and the recombination is caused by inducible expression of CRE recombinase in the yeast cells.  
     
     
         42 . The method of  claim 27 , wherein the diversity of the first or second polypeptide subunit encoded by the library of yeast expression vectors is at least 10 3 .  
     
     
         43 . The method of  claim 27 , wherein the diversity of the first or second polypeptide subunit encoded by the library of yeast expression vectors is at least 10 3 .  
     
     
         44 . The method of  claim 27 , wherein the diversity of the first or second polypeptide subunit encoded by the library of yeast expression vectors is at least 10 5 .  
     
     
         45 . The method of  claim 27 , wherein the diversity of the fusion proteins encoded by the library of yeast expression vectors is at least 1×10 8 .  
     
     
         46 . The method of  claim 27 , wherein the diversity of the fusion proteins encoded by the library of yeast expression vectors is at least 1×10 10 .  
     
     
         47 . The method of  claim 27 , wherein the diversity of the fusion proteins encoded by the library of yeast expression vectors is at least 1×10 12 .  
     
     
         48 . The method of  claim 27 , wherein each of the expression vectors further comprises a sequence encoding an affinity tag.  
     
     
         49 . The method of  claim 48 , wherein the affinity tag is selected from the group consisting of a polyhistidine tag, polyarginine tag, glutathione-S-transferase, maltose binding protein, staphylococcal protein A tag, and an EE-epitope tag.  
     
     
         50 . The method of  claim 48 , wherein the first nucleotide sequence and the second nucleotide sequence respectively encode a heavy chain variable region and a light chain variable region of immunoglobulin genes of a human, non-human primates, or rodent.  
     
     
         51 . The method of  claim 48 , wherein the first nucleotide sequence and the second nucleotide sequence respectively encode a heavy chain variable region and a light chain variable region of a human immunoglobulin gene.  
     
     
         52 . The method of  claim 48 , the linearized yeast expression vector further comprising: a transcription sequence encoding an activation domain or a DNA binding domain of a transcription activator.  
     
     
         53 . The method of  claim 52 , wherein the transcription sequence is capable of being expressed as a fusion protein with the single fusion protein comprising the antibody heavy chain variable region, the antibody light chain variable region, and the linker polypeptide.  
     
     
         54 . The method of  claim 52 , wherein the transcription activator is a transcription activator having separable DNA-binding and transcription activation domains.  
     
     
         55 . The method of  claim 52 , wherein the transcription activator is selected from the group consisting of GAL4, GCN4, and ADR1 transcription activator.