Multi-chain eukaryotic display vectors and uses thereof

Abstract

A eukaryotic expression vector capable of displaying a multi-chain polypeptide on the surface of a host cell is provided, such that the biological activity of the multi-chain polypeptide is exhibited at the surface of the host cell. Such a vector allows for the display of complex biologically active polypeptides, e.g., biologically active multi-chain polypeptides such as immunoglobulin Fab fragments. The present invention describes and enables the successful display of a multi-chain polypeptide on the surface of a eukaryotic host cell. Preferred vectors are described for expressing the chains of a multi-chain polypeptide in a host cell separately and independently (e.g., under separate vector control elements, and/or on separate expression vectors, thus forming a matched vector set). The use of such matched vector sets provides flexibility and versatility in the generation of eukaryotic display libraries, for example the ability to generate and to display multi-chain polypeptides by combining and recombining vectors that express variegations of the individual chains of a multi-chain polypeptide. Entire repertoires of novel chain combinations can be devised using such vector sets.

What is claimed is:  
     
         1 . A multi-chain polypeptide eukaryotic display vector comprising: 
 (a) a first polynucleotide encoding a polypeptide comprising a first chain of a biologically active multi-chain polypeptide linked to a cell surface anchor; and    (b) a second polynucleotide encoding a second chain of the multi-chain polypeptide;    wherein the vector is operable in a eukaryotic host cell to direct expression and secretion of the chains of the multi-chain polypeptide, and wherein the chains of the multi-chain polypeptide associate such that the biological activity of the multi-chain polypeptide is exhibited at the surface of the eukaryotic host cell.    
     
     
         2 . The eukaryotic display vector of  claim 1 , further comprising 
 (c) a third polynucleotide encoding a third chain of the multi-chain polypeptide.    
     
     
         3 . The eukaryotic display vector of  claim 2 , further comprising 
 (d) a fourth polynucleotide encoding a fourth chain of the multi-chain polypeptide.    
     
     
         4 . The eukaryotic display vector of  claim 1 , wherein the multi-chain polypeptide is a two-chain polypeptide.  
     
     
         5 . The eukaryotic display vector of  claim 1 , wherein the multi-chain polypeptide is a four-chain polypeptide, wherein the four-chain polypeptide is comprised of two first chains and two second chains.  
     
     
         6 . The eukaryotic display vector of  claim 1 , wherein the multi-chain polypeptide is a two-chain polypeptide selected from the group consisting of: immunoglobulin Fab fragments, the extracellular domains of T cell receptors, MHC class I molecules and MHC class II molecules.  
     
     
         7 . The eukaryotic display vector of  claim 1 , wherein the multi-chain polypeptide is an immunoglobulin (Ig) or an Ig fragment.  
     
     
         8 . The eukaryotic display vector of  claim 7 , wherein the multi-chain polypeptide is an immunoglobulin selected from the group consisting of: IgA, IgD, IgE, IgG and IgM.  
     
     
         9 . The eukaryotic display vector of  claim 8 , wherein the multi-chain polypeptide is an IgG.  
     
     
         10 . The eukaryotic display vector of  claim 7 , wherein the multi-chain polypeptide is aFab.  
     
     
         11 . The eukaryotic display vector of  claim 1 , wherein the anchor is a cell surface protein of a eukaryotic cell.  
     
     
         12 . The eukaryotic display vector of  claim 1 , wherein the anchor is a portion of a cell surface protein of a eukaryotic cell that anchors to the cell surface of the eukaryotic host cell.  
     
     
         13 . The eukaryotic display vector of  claim 1 , wherein the anchor is selected from the group consisting of: α-agglutinin, a-agglutinin components Aga1p and Aga2p, and FLO1.  
     
     
         14 . The eukaryotic display vector of  claim 1 , wherein, on expression, the first chain and the cell surface anchor are expressed as a fusion protein in the eukaryotic host cell.  
     
     
         15 . The eukaryotic display vector of  claim 1 , wherein, on expression, the first chain is linked to the cell surface anchor by means of a Jun/Fos linkage.  
     
     
         16 . The eukaryotic display vector of  claim 14 , wherein, on expression, the first chain of the multi-chain polypeptide is fused to Aga2p, wherein Aga2p is covalently linked to Aga1p, which in turn is linked to the eukaryotic host cell surface.  
     
     
         17 . The eukaryotic display vector of  claim 1 , wherein the first polynucleotide is operably linked to an Aga2p signal sequence and the second polynucleotide is operably linked to an Aga2p signal sequence.  
     
     
         18 . The eukaryotic display vector of  claim 1 , wherein the first polynucleotide is linked in frame to a polynucleotide encoding a first epitope tag, and the second polynucleotide is linked in frame to a polynucleotide encoding a second epitope tag.  
     
     
         19 . The eukaryotic display vector of  claim 1 , wherein the vector further comprises restriction endonuclease recognition sites located at the 5′ and 3′ ends of a polynucleotide segment including all of the polynucleotides encoding the chains of the multi-chain polypeptide.  
     
     
         20 . The eukaryotic display vector of  claim 1 , wherein the vector further comprises restriction endonuclease recognition sites located at the 5′ and 3′ ends of each of the polynucleotides encoding the chains of the multi-chain polypeptide.  
     
     
         21 . A vector library comprising a eukaryotic display vector according to  claim 1 .  
     
     
         22 . The vector library of  claim 21 , wherein the library comprises a heterogeneous population of multi-chain polypeptides.  
     
     
         23 . A method of displaying a biologically active multi-chain polypeptide on the surface of a eukaryotic host cell comprising utilizing the vector of  claim 1 .  
     
     
         24 . The eukaryotic display vector of  claim 1 , wherein the eukaryotic display vector is a vector selected from the group consisting of: a vector set, a dual display vector, and a yeast display vector.  
     
     
         25 . The eukaryotic display vector of  claim 24 , wherein the multi-chain polypeptide is a two-chain polypeptide, and the eukaryotic display vector is a vector set comprising a first eukaryotic vector and a second eukaryotic vector, each vector comprising a polynucleotide encoding one chain of the two-chain polypeptide.  
     
     
         26 . The eukaryotic display vector of  claim 24 , wherein the eukaryotic display vector is a vector set and wherein the multi-chain polypeptide is a three-chain polypeptide, and the vector set comprises a first eukaryotic vector, a second eukaryotic vector, and a third eukaryotic vector, each vector comprising a polynucleotide encoding one chain of the three-chain polypeptide.  
     
     
         27 . The eukaryotic display vector set of  claim 24 , wherein the eukaryotic display vector is a vector set and wherein the vector set comprises at least the following three vectors: 
 (a) a first eukaryotic vector comprising a first polynucleotide encoding the first chain of the four-chain polypeptide linked to a cell surface anchor, wherein the vector is operable in a eukaryotic host cell to direct expression and secretion of the first chain;    (b) a second eukaryotic vector comprising the first polynucleotide encoding the first chain of the four-chain polypeptide, wherein the vector is operable in a eukaryotic host cell to direct expression and secretion of the first chain; and    (c) a third eukaryotic vector comprising a second polynucleotide encoding the second chain of the four-chain polypeptide, wherein the vector is operable in a eukaryotic host cell to direct expression and secretion of the second chain, thereby forming a vector set,    wherein a eukaryotic host cell transformed with the vector set, on expression of the first and second polynucleotides, exhibits the biological activity of the four-chain polypeptide at the surface of the eukaryotic host cell.    
     
     
         28 . The eukaryotic display vector of  claim 24 , wherein the eukaryotic display vector is a dual display vector and wherein the anchor is a polypeptide operable as an anchor on the surface of a eukaryotic cell and operable as an anchor on the surface of a phage.  
     
     
         29 . The dual display vector of  claim 28 , wherein the anchor is a portion of a surface protein that anchors to the cell surface of a eukaryotic host cell and to the surface of a phage.  
     
     
         30 . The eukaryotic display vector of  claim 24 , wherein the eukaryotic display vector is a dual display vector and wherein the first polynucleotide is operably linked to a polynucleotide encoding a first signal sequence, and the second polynucleotide is operably linked to a polynucleotide encoding a second signal sequence, wherein the first signal sequence and the second signal sequence are operable in a bacterial cell and operable in a eukaryotic cell.  
     
     
         31 . The eukaryotic display vector of  claim 24 , wherein the eukaryotic display vector is a dual display vector and wherein the first polynucleotide is operably linked to a first promoter and the second polynucleotide is operably linked to a second promoter, wherein the first promoter and the second promoter are both operable in a bacterial cell and operable in a eukaryotic cell.  
     
     
         32 . A yeast Fab display vector comprising: 
 (a) a first polynucleotide encoding a first polypeptide comprising V H  and C H 1 regions of an Ig heavy chain linked to a cell surface anchor operable in yeast; and    (b) a second polynucleotide encoding a second polypeptide comprising an Ig light chain;    wherein the vector is operable in a yeast host cell to direct expression and secretion of the chains of the multi-chain polypeptide, and wherein the chains of the multi-chain polypeptide associate to form a Fab at the surface of the yeast host cell.    
     
     
         33 . The yeast Fab display vector of  claim 32 , wherein the anchor is selected from the group consisting of: α-agglutinin, a-agglutinin components Aga1p and Aga2p, and FLO1.  
     
     
         34 . The yeast Fab display vector of  claim 32 , wherein, on expression, the V H  and C H 1 regions are linked to the cell surface anchor by means of a Jun/Fos linkage.  
     
     
         35 . The yeast Fab display vector of  claim 33 , wherein, on expression, the V H  and C H 1 regions are fused to Aga2p, and the Aga2p is covalently linked to Aga1p, which in turn is linked to the yeast host cell surface.  
     
     
         36 . The yeast Fab display vector of  claim 32 , wherein the vector further comprises restriction endonuclease recognition sites located at the 5′ and 3′ ends the polynucleotides encoding the chains of the multi-chain polypeptide.  
     
     
         37 . The Fab yeast display vector of  claim 32 , wherein the vector is a vector set.  
     
     
         38 . The Fab yeast display vector of  claim 32 , wherein the vector is a dual display vector.  
     
     
         39 . The Fab yeast display vector of  claim 38 , wherein the dual display vector is operable in phage and yeast.  
     
     
         40 . A Fab yeast display library comprising a yeast display vectors according to  claim 32 .  
     
     
         41 . The Fab yeast display library of  claim 40 , wherein the yeast display library comprises a heterogeneous population of multi-chain polypeptides.  
     
     
         42 . A method of displaying a biologically active multi-chain polypeptide on the surface of a eukaryotic host cell comprising utilizing one or more vectors of  claim 32 .  
     
     
         43 . A method for displaying, on the surface of a eukaryotic host cell, a biologically active multi-chain polypeptide comprising at least two polypeptide chains, the process comprising the steps of: 
 (a) introducing into a eukaryotic host cell 
 (i) a first eukaryotic vector comprising a first polynucleotide encoding a first polypeptide chain of a biologically active multi-chain polypeptide linked to a cell surface anchor, wherein the vector is operable in a eukaryotic host cell to direct expression and secretion of the first chain; and  
 (ii) a second eukaryotic vector comprising a second polynucleotide encoding a second polypeptide chain of the multi-chain polypeptide, wherein the vector is operable in a eukaryotic host cell to direct expression and secretion of the second chain,  
    wherein a eukaryotic host cell transformed with the first eukaryotic vector and the second eukaryotic vector exhibits, on expression of the first and second polynucleotides, the biological activity of the multi-chain polypeptide at the surface of the eukaryotic host cell; and    (b) culturing the host cell under conditions suitable for expression of the first and second polynucleotides.    
     
     
         44 . The method of  claim 43 , wherein the eukaryotic host cell is selected from the group consisting of: an animal cell, a plant cell, and a fungus cell.  
     
     
         45 . The method of  claim 43 , wherein the eukaryotic host cell is selected from the group consisting of: a mammalian cell, an insect cell, and a yeast cell.  
     
     
         46 . The method of  claim 43 , wherein the eukaryotic host cell is a yeast cell.  
     
     
         47 . The method of  claim 46 , wherein the yeast cell is of a genus selected from the group consisting of: Saccharomyces, Pichia, Hansenula, Schizosaccharomyces, Kluyveromyces, Yarrowia, Debaryomyces and Candida.  
     
     
         48 . The method of  claim 47 , wherein the yeast cell is selected from the group consisting of:  Saccharomyces cerevisiae, Hansenula polymorpha, Kluyveromyces lactis, Pichia pastoris, Schizosaccharomyces pombe  and  Yarrowia lipolytica.    
     
     
         49 . The method of  claim 47 , wherein the yeast cell is  Saccharomyces cerevisiae.    
     
     
         50 . A method of displaying a biologically active multi-chain polypeptide comprising at least two polypeptide chains on the surface of a diploid eukaryotic cell, comprising: 
 (a) providing a first haploid eukaryotic cell comprising a first polynucleotide encoding a polypeptide comprising a first chain of a biologically active multi-chain polypeptide linked to a cell surface anchor;    (b) providing a second haploid eukaryotic cell, wherein the second haploid eukaryotic cell comprises a second polynucleotide encoding a polypeptide comprising a second chain of the multi-chain polypeptide;    (c) contacting the first haploid eukaryotic cell with the second haploid eukaryotic cell under conditions sufficient to permit the cells to fuse, producing a diploid eukaryotic cell; and    (d) culturing the diploid eukaryotic cell under conditions sufficient to permit expression and association of the chains of the multi-chain polypeptide, wherein the biological activity of the multi-chain polypeptide is exhibited at the surface of the diploid eukaryotic cell.    
     
     
         51 . The method of  claim 50 , wherein the first haploid eukaryotic cell and the second haploid eukaryotic cell are of opposite mating type.  
     
     
         52 . The method of  claim 50 , wherein the first haploid eukaryotic cell and the second haploid eukaryotic cell are of opposite mating type.  
     
     
         53 . A method for displaying a Fab on the surface of a diploid yeast cell comprising the steps of: 
 (a) providing a first haploid yeast cell comprising a first polynucleotide encoding a first polypeptide comprising V H  and C H 1 regions of an Ig heavy chain linked to a cell surface anchor;    (b) providing a second haploid yeast cell, wherein the second haploid yeast cell comprises a second polynucleotide encoding a second polypeptide comprising an Ig light chain;    (c) contacting the first haploid yeast cell with the second haploid yeast cell under conditions sufficient to permit the cells to fuse, producing a diploid yeast cell; and    (d) culturing the diploid yeast cell under conditions sufficient to permit expression and association of the first and second polypeptides, wherein a Fab is exhibited at the surface of the diploid yeast cell.    
     
     
         54 . The method of  claim 53 , wherein the first haploid yeast cell and the second haploid yeast cell are of opposite mating type.  
     
     
         55 . The method of  claim 54 , wherein the first and second haploid yeast cells are cells of  Saccharomyces cerevisiae.    
     
     
         56 . A method for detecting a biologically active multi-chain polypeptide comprising at least two polypeptide chains from a multi-chain polypeptide library, comprising: 
 (a) providing a first haploid eukaryotic cell population comprising a plurality of first polynucleotides each encoding a polypeptide comprising a first chain variant of a biologically active multi-chain polypeptide linked to a cell surface anchor;    (b) providing a second haploid eukaryotic cell population of opposite mating type to that of the first haploid eukaryotic cell population, wherein the second haploid eukaryotic cell population comprises a plurality of second polynucleotides each encoding a second chain variant of the multi-chain polypeptide;    (c) contacting the first haploid eukaryotic cell population with the second haploid eukaryotic cell population under conditions sufficient to permit individual cells of different mating types to fuse, producing a population of diploid eukaryotic cells;    (d) culturing the diploid eukaryotic cells under conditions sufficient to permit expression and association of the chains of the multi-chain polypeptide, wherein the biological activity of the multi-chain polypeptide is exhibited at the surface of the diploid eukaryotic cells; and    (e) detecting a particular biological activity of interest.    
     
     
         57 . The method of  claim 56 , further comprising the step: 
 (f) isolating diploid eukaryotic cells that display the biological activity.    
     
     
         58 . The method of  claim 57 , further comprising the steps of: 
 (g) repeating steps (d), (e) and (f).    
     
     
         59 . The method of  claim 57 , further comprising the steps of: 
 (g) culturing the isolated diploid eukaryotic cells of step (f) under conditions sufficient to cause the isolated diploid eukaryotic cells to undergo meiosis, producing haploid eukaryotic cells;    (h) contacting the haploid eukaryotic cells from step (g) under conditions sufficient to permit eukaryotic cells of different mating types to fuse, producing a population of diploid eukaryotic cells; and    (i) repeating steps (d), (e) and (f).    
     
     
         60 . The method of  claim 57 , further comprising the steps of: 
 (g) culturing the isolated diploid eukaryotic cells of step (f) under conditions sufficient to cause the isolated diploid eukaryotic cells to undergo meiosis, producing haploid eukaryotic cells;    (h) contacting the haploid eukaryotic cells from step (g) with one or more haploid eukaryotic cell populations selected from the group consisting of: 
 (i) the first haploid eukaryotic cell population of step (a);  
 (ii) the second haploid eukaryotic cell population of step (b);  
 (iii) a third haploid eukaryotic cell population of opposite mating type to that of the second haploid eukaryotic cell population, wherein the third haploid eukaryotic cell population comprises a plurality of first polynucleotides encoding a polypeptide comprising a first chain variant of a biologically active multi-chain polypeptide linked to a cell surface anchor; and  
 (iv) a fourth haploid eukaryotic cell population of opposite mating type to that of the first haploid eukaryotic cell population, wherein the fourth haploid eukaryotic cell population comprises a plurality of second polynucleotides each encoding a second chain variant of the multi-chain polypeptide, under conditions sufficient to permit eukaryotic cells of different mating types to fuse, producing a population of diploid eukaryotic cells; and  
 (i) repeating steps (d), (e) and (f).  
   
     
     
         61 . A method for detecting and isolating one or more multi-chain polypeptides that exhibit a biological activity of interest comprising: 
 (a) providing a eukaryotic cell wherein, on expression of a multi-chain eukaryotic display vector, the eukaryotic cell displays a multi-chain polypeptide on its surface;    (b) culturing the eukaryotic cell under conditions sufficient to permit expression of the multi-chain polypeptide; and    (c) contacting the cells with a molecule of interest; and    (d) selecting and isolating cells that exhibit a particular interaction with the molecule of interest.    
     
     
         62 . The method of  claim 61 , wherein a host cell displaying the multi-chain polypeptide exhibiting the biological activity of interest is isolated, and, optionally, is subjected to at least one additional round of selection.  
     
     
         63 . The method of  claim 61 , further comprising screening the library using a phage display screen.  
     
     
         64 . The method of  claim 61 , wherein the molecule of interest is a protein.  
     
     
         65 . The method of  claim 64 , wherein the biological activity of interest is an interaction between the multi-chain polypeptide and another molecular species and comprises a non-covalent association between the molecular species.  
     
     
         66 . The method of  claim 65 , wherein the interaction is transient.  
     
     
         67 . The method of  claim 65 , wherein the interaction is a covalent interaction.  
     
     
         68 . A method for transferring nucleic acid sequences encoding a biologically active multi-chain polypeptide between a phage display vector and a eukaryotic display vector comprising: 
 (a) obtaining a phage display vector comprising: 
 (i) a first polynucleotide encoding a polypeptide comprising a first chain of the biologically active multi-chain polypeptide, wherein the first chain is linked to a cell surface anchor, and  
 (ii) a second polynucleotide encoding a second chain of the multi-chain polypeptide,  
    wherein the phage display vector is operable in a bacterial host cell to direct expression of the chains of the multi-chain polypeptide, and wherein the chains of the multi-chain polypeptide associate such that the biological activity of the multi-chain polypeptide is exhibited at the surface of a phage comprising the phage display vector and propagating in the bacterial host cell; and    (b) inserting the first and second polynucleotides encoding the chains of the multi-chain polypeptide into a eukaryotic display vector, wherein the eukaryotic display vector is operable in a eukaryotic host cell to direct expression and secretion of the chains of the multi-chain polypeptide, and wherein the chains of the multi-chain polypeptide associate such that the biological activity of the multi-chain polypeptide is exhibited at the surface of the eukaryotic host cell.    
     
     
         69 . The method of  claim 68 , wherein the polynucleotide sequences encoding each of the chains of the multi-chain polypeptide are inserted together as a single polynucleotide into the eukaryotic display vector.  
     
     
         70 . The method of  claim 68 , wherein the polynucleotide sequences encoding each of the chains of the multi-chain polypeptide are independently inserted as separate polynucleotides into the eukaryotic display vector.  
     
     
         71 . The method of  claim 68 , wherein the transferring step (b) comprises a genetic transfer technique selected from the group consisting of: restriction digestion, PCR amplification, homologous recombination and combinations of such techniques.  
     
     
         72 . A method for transferring nucleic acid sequences encoding a biologically active Fab between a phage display vector and a eukaryotic display vector comprising: 
 (a) obtaining a phage display vector comprising: 
 (i) a first polynucleotide encoding a first polypeptide comprising V H  and C H 1 regions of an Ig heavy chain linked a cell surface anchor, and  
 (ii) a second polynucleotide encoding a second polypeptide comprising an Ig light chain,  
    wherein the phage display vector is operable in a bacterial host cell to direct expression of the first and second polypeptides, and wherein the polypeptides associate such that the biological activity of the multi-chain polypeptide is exhibited at the surface of a phage transfected with the phage display vector and propagating in the bacterial host cell; and    (b) inserting the first and second polynucleotides encoding the first and second polypeptides into a eukaryotic display vector, wherein the eukaryotic display vector is operable in a yeast host cell to direct expression and secretion of the first and second polypeptides, and wherein the polypeptides associate such that the biological activity of the Fab is exhibited at the surface of the yeast host cell.    
     
     
         73 . The method of  claim 72 , wherein the polynucleotide sequences encoding each of the polypeptides are inserted together as a single polynucleotide into the eukaryotic display vector.  
     
     
         74 . The method of  claim 72 , wherein the polynucleotide sequences encoding each of the polypeptides are independently inserted as separate polynucleotides into the eukaryotic display vector.  
     
     
         75 . The method of  claim 72 , wherein the transferring step (b) comprises a genetic transfer technique selected from the group consisting of restriction digestion, PCR amplification, homologous recombination, and combinations of such techniques.  
     
     
         76 . A eukaryotic host cell comprising a vector comprising: 
 (a) a first polynucleotide encoding a polypeptide comprising a first chain of a biologically active multi-chain polypeptide linked to a cell surface anchor; and    (b) a second polynucleotide encoding a second chain of the multi-chain polypeptide;    wherein the vector is operable in a eukaryotic host cell to direct expression and secretion of the chains of the multi-chain polypeptide, and wherein the chains of the multi-chain polypeptide associate such that the biological activity of the multi-chain polypeptide is exhibited at the surface of the eukaryotic host cell.    
     
     
         77 . The eukaryotic host cell of  claim 76 , wherein the eukaryotic host cell is selected from the group consisting of: an animal cell, a plant cell, and a fungus cell.  
     
     
         78 . The eukaryotic host cell of  claim 76 , wherein the eukaryotic host cell is selected from the group consisting of: a mammalian cell, an insect cell, and a yeast cell.  
     
     
         79 . The eukaryotic host cell of  claim 76 , wherein the eukaryotic host cell is a yeast cell.  
     
     
         80 . The eukaryotic host cell of  claim 79 , wherein the yeast cell is haploid.  
     
     
         81 . The eukaryotic host cell of  claim 79 , wherein the yeast cell is diploid.  
     
     
         82 . A pair of haploid eukaryotic cells comprising: 
 (a) a first haploid eukaryotic cell comprising a first polynucleotide encoding a polypeptide comprising a first chain of a biologically active multi-chain polypeptide linked to a cell surface anchor; and    (b) a second haploid eukaryotic cell comprising a second polynucleotide encoding a second chain of the multi-chain polypeptide.    
     
     
         83 . The haploid eukaryotic cell pair of  claim 82 , wherein the first haploid eukaryotic cell and the second haploid eukaryotic cell are of opposite mating type.  
     
     
         84 . The haploid eukaryotic cell pair of  claim 82 , wherein the multi-chain polypeptide is a two-chain polypeptide.  
     
     
         85 . The haploid eukaryotic cell pair of  claim 82 , wherein the multi-chain polypeptide is a four-chain polypeptide and wherein the four-chain polypeptide is comprised of two the first chains and two the second chains.  
     
     
         86 . The haploid eukaryotic cell pair of  claim 82 , wherein the multi-chain polypeptide is a two-chain polypeptide selected from the group consisting of immunoglobulin Fab fragments, and the extracellular domains of T cell receptors, MHC class I molecules, and MHC class II molecules.  
     
     
         87 . The haploid eukaryotic cell pair of  claim 82 , wherein the anchor is a cell surface protein of a eukaryotic cell.  
     
     
         88 . A eukaryotic host cell library comprising a plurality of diploid cells that are the fusion product of a plurality of eukaryotic host cell pairs according to  claim 82 .  
     
     
         89 . The eukaryotic host cell library of  claim 88 , wherein the plurality of diploid cells display a heterogeneous population of multi-chain polypeptides.  
     
     
         90 . A yeast cell transformed with a heterologous display vector comprising: 
 (a) a first polynucleotide encoding a polypeptide comprising a first chain of a biologically active multi-chain polypeptide linked to a cell surface anchor operable in yeast; and    (b) a second polynucleotide encoding a second chain of the multi-chain polypeptide;    wherein the vector is operable in the yeast cell to direct expression and secretion of the chains of the multi-chain polypeptide, and wherein the chains of the multi-chain polypeptide associate such that the biological activity of the multi-chain polypeptide is exhibited at the surface of the yeast cell.    
     
     
         91 . A pair of haploid yeast cells comprising: 
 (a) a first haploid yeast cell comprising a first polynucleotide encoding a polypeptide comprising a first chain of a biologically active multi-chain polypeptide linked to a cell surface anchor; and    (b) a second haploid yeast cell comprising a second polynucleotide encoding a second chain of the multi-chain polypeptide.    
     
     
         92 . The haploid yeast cell pair of  claim 91 , wherein the first haploid yeast cell and the second haploid yeast cell are of opposite mating type.  
     
     
         93 . A yeast display library comprising a population of yeast cells collectively displaying a repertoire of at least 10 7  polypeptides.  
     
     
         94 . A yeast display library comprising a population of yeast cells collectively displaying a heterogeneous population of at least 104 multi-chain polypeptides.  
     
     
         95 . The yeast display library of  claim 94 , comprising a population of yeast cells collectively displaying a heterogeneous population of at least 10 7  multi-chain polypeptides.  
     
     
         96 . The yeast display library of  claim 95 , comprising a population of yeast cells collectively displaying a heterogeneous population of at least 10 8  multi-chain polypeptides.  
     
     
         97 . A yeast display library comprising a plurality of diploid cells that are the fusion product of a plurality of yeast cell pairs according to  claim 91 .  
     
     
         98 . The yeast display library of  claim 97 , wherein the plurality of diploid cells display a heterogeneous population of Fabs.