Surface display of whole antibodies in eukaryotes

Abstract

Methods for display of recombinant whole immunoglobulins or immunoglobulin libraries on the surface of eukaryote host cells, including yeast and filamentous fungi, are described. The methods are useful for screening libraries of recombinant immunoglobulins in eukaryote host cells to identify immunoglobulins that are specific for an antigen of interest.

What is claimed: 
     
         1 . A method for producing eukaryotic host cells that express an immunoglobulin of interest, comprising:
 (a) providing host cells that include a first nucleic acid molecule encoding a capture moiety comprising a cell surface anchoring protein fused to a binding moiety that is capable of specifically binding an immunoglobulin operably linked to a first regulatable promoter;   (b) transfecting the host cells with a plurality of nucleic acid molecules encoding a genetically diverse population of heavy and light chains of an immunoglobulin wherein at least one of the heavy or light chain encoding nucleic acid molecules is operably linked to a second regulatable promoter to produce a plurality of genetically diverse host cells capable of displaying an immunoglobulin on the surface thereof;   (c) inducing expression of the nucleic acid molecule encoding the capture moiety for a time sufficient to produce the capture moiety on the surface of the host cell; and   (d) inhibiting expression of the nucleic acid molecule encoding the capture moiety and inducing expression of the nucleic acid molecules encoding the immunoglobulins in the host cells to produce the host cells that express the immunoglobulin of interest displayed.   
     
     
         2 . The method of  claim 1  wherein, the method further includes
 (e) contacting the host cells with a detection means that specifically binds to the immunoglobulin of interest displayed on the cell surface of the host cells; and 
 (f) isolating host cells in which the detection means is bound to produce the host cells that express the immunoglobulin of interest. 
 
     
     
         3 . The method of  claim 1 , wherein the binding moiety binds the Fc region of the immunoglobulin. 
     
     
         4 . The method of  claim 1 , wherein the binding moiety is selected from the group consisting of protein A, protein A ZZ domain, protein G, and protein L. 
     
     
         5 . The method of  claim 1 , wherein the cell surface anchoring protein is a GPI protein. 
     
     
         6 . A method for producing eukaryotic host cells that express an immunoglobulin of interest, comprising:
 (a) providing a host cell that includes a first nucleic acid molecule encoding a capture moiety comprising a cell surface anchoring protein fused to a binding moiety that is capable of specifically binding an immunoglobulin operably linked to a first regulatable promoter;   (b) transfecting the host cells with a one or more nucleic acid molecules encoding the heavy and light chains of an immunoglobulin wherein at least one of the heavy or light chain encoding nucleic acid molecules is operably linked to a second regulatable promoter to generate a plurality of host cells encoding a variegated population of mutants of the immunoglobulins;   (c) inducing expression of the capture moiety for a time sufficient to produce the capture moiety on the surface of the host cells; and   (d) inhibiting expression of the capture moiety and inducing expression of the variegated population of mutants of the immunoglobulin in the host cells to produce the host cells that express the immunoglobulin of interest.   
     
     
         7 . The method of  claim 6  wherein the method further includes
 (e) contacting the host cells with a detection means that binds to the immunoglobulin of interest to identify host cells that display the immunoglobulin of interest on the surface thereof; and 
 (f) isolating the host cells that display the immunoglobulin of interest on the surface of thereof to produce the host cells that express the immunoglobulin of interest. 
 
     
     
         8 . The method of  claim 6 , wherein the binding moiety binds the Fc region of the immunoglobulin. 
     
     
         9 . The method of  claim 6 , wherein the binding moiety is selected from the group consisting of protein A, protein A ZZ domain, protein G, and protein L. 
     
     
         10 . The method of  claim 1 , wherein the cell surface anchoring protein is a GPI protein. 
     
     
         11 . A method of producing eukaryote host cells that produce an immunoglobulin having a VH domain and a VL domain and having an antigen binding site with binding specificity for an antigen of interest, the method comprising:
 (a) providing a library of eukaryote host cells displaying on their surface an immunoglobulin comprising a VH domain and a VL domain, wherein the library is created by:
 (i) providing eukaryote host cells that express a capture moiety comprising a cell surface anchoring protein fused to a moiety capable of binding to an immunoglobulin wherein expression of the capture moiety is effected by a first regulatable promoter; and 
 (ii) transfecting the host cells with a library of nucleic acid molecules encoding a genetically diverse population of immunoglobulins, wherein the VH domains of the genetically diverse population of immunoglobulins are biased for one or more VH gene families and wherein expression of at least one of the heavy or light chains of the immunoglobulins is effected by a second regulatable promoter to produce a plurality of host cells, each expressing an immunoglobulin; 
   (b) inducing expression of the capture moiety in the host cells for a time sufficient to produce the capture moiety on the surface of the host cells; and   (c) inhibiting expression of the capture moiety and inducing expression of the library of nucleic acid molecule sequences in the host cells, whereby each host cell displays an immunoglobulin at the surface thereof to produce the host cells that produce the immunoglobulin having a VH domain and a VL domain and having the antigen binding site with binding specificity for the antigen of interest.   
     
     
         12 . The method of  claim 11 , wherein the immunoglobulin comprises a synthetic human immunoglobulin VH domain and a synthetic human immunoglobulin VL domain and wherein the synthetic human immunoglobulin VH domain and the synthetic human immunoglobulin VL domain comprise framework regions and hypervariable loops, wherein the framework regions and first two hypervariable loops of both the VH domain and VL domain are essentially human germ line, and wherein the VH domain and VL domain have altered CDR3 loops. 
     
     
         13 . The method of  claim 12 , wherein in addition to having altered CDR3 loops the human synthetic immunoglobulin VH and VL domains contain mutations in other CDR loops. 
     
     
         14 . The method of  claim 12 , wherein each human synthetic immunoglobulin VH domain CDR loop is of random sequence. 
     
     
         15 . The method of  claim 12 , wherein human synthetic immunoglobulin VH domain CDR loops are of known canonical structures and incorporate random sequence elements. 
     
     
         16 . The method of  claim 11  wherein the method further includes
 (d) identifying host cells in the plurality of host cells that display immunoglobulins thereon that has a binding specificity for the antigen of interest by contacting the plurality of host cells with the antigen of interest and detecting the host cells that have the antigen of interest bound to the immunoglobulin displayed thereon to produce the host cells that produce the immunoglobulin having a VH domain and a VL domain and having the antigen binding site with binding specificity for the antigen of interest. 
 
     
     
         17 . The method of  claim 11 , wherein the antibody is selected from the group consisting of IgG, IgA, IgM, and IgE. 
     
     
         18 . The method of  claim 11 , wherein the binding moiety binds to the Fc region of the immunoglobulin. 
     
     
         19 . The method of  claim 11 , wherein the binding moiety is selected from the group consisting of protein A, protein A ZZ domain, protein G, and protein L. 
     
     
         20 . The method of  claim 11 , wherein the cell surface anchoring protein is a GPI protein. 
     
     
         21 . An immunoglobulin produced comprising the method of  claim 1 ,  6 , or  11  and derivatives thereof. 
     
     
         22 . A eukaryote host cell that expresses the immunoglobulin of  claim 21 . 
     
     
         23 . A host cell that expresses an immunoglobulin that had been identified using the method of  claim 1 ,  6 , or  11 . 
     
     
         24 . A eukaryote host cell comprising a nucleic acid molecule encoding a capture moiety comprising a cell surface anchoring protein fused to a binding moiety capable of binding an immunoglobulin operably linked to a regulatable promoter and one or more nucleic acid molecules encoding the heavy and light chains of immunoglobulins, wherein at least one of the nucleic acid molecules encoding the heavy or light chains is operably linked to a second regulatable promoter.