Humanized immunobinders of cell-surface antigens

Abstract

The invention provides methods for identifying immunobinders, such as scFv antibodies, capable of specifically binding to cell surface antigens, and compositions identified according to said methods.

What is claimed is: 
     
         1 . A humanized immunobinder capable of binding to an antigen of interest, wherein the immunobinder comprises:
 a variable heavy chain comprising three heavy chain complementarity determining regions (CDRs) and four framework region sequences having at least 90% identity to the four framework region sequences of SEQ ID NO: 4, wherein the framework region sequences have one or more amino acid residues selected from the group consisting of: Threonine (T) at position 24 (AHo numbering), Valine (V) at position 25 (AHo numbering), Glycine or Alanine (G/A) at position 56 (AHo numbering), Lysine (K) at position 82 (AHo numbering), Threonine (T) at position 84 (AHo numbering), Valine (V) at position 89 (AHo numbering) and Arginine (R) at position 108 (AHo numbering), and   a variable light chain comprising three light chain CDRs and a light chain framework sequence,   
       wherein the heavy chain and light chain CDRs are from an immunobinder derived from a B-cell clone identified by:
 (a) providing a plurality of B-cells; 
 (b) contacting the plurality of B-cells with the antigen of interest, wherein the antigen of interest comprises a first sortable label, and staining the plurality of B-cells with anti-IgG antibodies comprising a second sortable label and anti-IgM antibodies comprising a third sortable label; 
 (c) separating from the plurality of B-cells one or more B-cells that can specifically bind to the antigen of interest using a cell sorter, wherein the presence of the first and second sortable label, but not the third sortable label, in a complex is indicative of the binding of a B-cell to an antigen of interest, thereby identifying a B-cell clone that binds to the antigen of interest. 
 
     
     
         2 . The humanized immunobinder of  claim 1 , wherein the plurality of B-cells is provided in a plurality of lymphocytes. 
     
     
         3 . The humanized immunobinder of  claim 1 , wherein the plurality of B-cells are stained prior to being contacted with the antigen of interest in step (b). 
     
     
         4 . The humanized immunobinder of  claim 1 , wherein the plurality of B-cells are stained after being contacted with the antigen of interest in step (b). 
     
     
         5 . The humanized immunobinder of  claim 1 , wherein identification of the B-cell clone comprises clonally isolating the B-cells obtained in step (c), optionally followed by clonal expansion of the clonally isolated cells. 
     
     
         6 . The humanized immunobinder of  claim 5 , further comprising isolating a nucleic acid molecule encoding an immunobinder from the B-cell clone that binds to an antigen of interest. 
     
     
         7 . The humanized immunobinder of  claim 6 , further comprising producing an immunobinder capable of binding to an antigen of interest, wherein the immunobinder is produced by introducing the nucleic acid molecule into an expression environment such that the encoded immunobinder is produced. 
     
     
         8 . The humanized immunobinder of  claim 1 , wherein the one or more separated B-cells are cultivated under suitable conditions so that antibodies are secreted into the culture medium. 
     
     
         9 . The humanized immunobinder of  claim 8 , wherein the antibodies are tested for specific binding to the antigen of interest. 
     
     
         10 . The humanized immunobinder of  claim 8 , wherein the antibodies are mouse, rabbit, rat, hamster, sheep, chicken, camel, goat, human antibodies. 
     
     
         11 . The humanized immunobinder of  claim 1 , wherein the sortable labels are fluorescent labels. 
     
     
         12 . The humanized immunobinder of  claim 1 , wherein the antigen of interest is a soluble antigen. 
     
     
         13 . The humanized immunobinder of  claim 1 , wherein the B-cells are B-cells from a rabbit. 
     
     
         14 . The humanized immunobinder of  claim 13 , wherein the rabbit was immunized with an antigen of interest. 
     
     
         15 . The humanized immunobinder of  claim 1 , wherein the immunobinder is an antibody. 
     
     
         16 . The humanized immunobinder of  claim 15 , wherein the antibody is a full-length immunoglobulin, Fab or scFv. 
     
     
         17 . The humanized immunobinder of  claim 1 , wherein the antigen of interest is expressed from an exogenous gene. 
     
     
         18 . The humanized immunobinder of  claim 1 , wherein the variable light chain framework regions have sequences that have at least 90% identity to the framework region sequences of SEQ ID NO: 2.