Identification and Engineering of Antibodies with Variant Fc Regions and Methods of Using Same

Abstract

The present invention relates to molecules, particularly polypeptides, more particularly immunoglobulins (e.g., antibodies), comprising a variant Fc region, wherein said variant Fc region comprises at least one amino acid modification relative to a wild-type Fc region, which variant Fc region binds FcγRIIIA and/or FcγRIIA with a greater affinity, relative to a comparable molecule comprising the wild-type Fc region. The molecules of the invention are particularly useful in preventing, treating, or ameliorating one or more symptoms associated with a disease, disorder, or infection. The molecules of the invention are particularly useful for the treatment or prevention of a disease or disorder where an enhanced efficacy of effector cell function (e.g., ADCC) mediated by FcγR is desired, e.g., cancer, infectious disease, and in enhancing the therapeutic efficacy of therapeutic antibodies the effect of which is mediated by ADCC.

What is claimed is: 
     
         1 . A method for producing a tetrameric FcγR complex, wherein said tetrameric complex has an enhanced affinity for an Fc region, relative to the affinity of a monomeric FcγR for the Fc region, comprising:
 (a) producing a fusion protein, such that a 15 amino acid AVITAG sequence is operably linked to the soluble region of FcγR; 
 (b) biotinylating the protein produced in step (a); and 
 (c) mixing the biotinylated protein produced in step (b) with streptaividn conjugated to a detectable substance in an appropriate molar ratio, such that a tetrameric FcγR complex is formed, 
 wherein the streptaividn is conjugated to a detectable substance. 
 
     
     
         2 . The tetrameric FcγR complex produced by the method of  claim 1 . 
     
     
         3 . The tetrameric FcγR complex of  claim 2 , wherein the complex binds an Fc region with an 8-fold higher affinity than a monomeric FcγR binds the Fc region. 
     
     
         4 . The tetrameric FcγR complex of  claim 2 , wherein the complex binds an Fc region with a 10-fold higher affinity than a monomeric FcγR binds the Fc region. 
     
     
         5 . The method of  claim 1 , wherein the tetrameric FcγR complex is a tetrameric FcγRIIIA complex, and the soluble region used in step (a) is the soluble region of FcγRIIIA. 
     
     
         6 . The method of  claim 1 , wherein the tetrameric FcγR complex is a tetrameric FcγRIIB complex, and the soluble region used in step (a) is the soluble region of FcγRIIB. 
     
     
         7 . The method of  claim 1 , wherein the detectable substance is an enzyme, a fluorescent material, a bioluminescent material, or a radioactive material. 
     
     
         8 . The method of  claim 1 , wherein the detectable substance is phycoerythin. 
     
     
         9 . The method of  claim 1 , wherein the 15 amino acid AVITAG sequence is operably linked to the C-terminus of the soluble FcγR. 
     
     
         10 . The method of  claim 1 , wherein the protein produced in step (a) is biotinylated enzymatically. 
     
     
         11 . The method of  claim 1 , wherein the protein produced in step (a) is biotinylated with  E. coli  BirA enzyme. 
     
     
         12 . The method of  claim 1 , wherein the biotinylated protein produced in step (a) is mixed with streptavidin in a 1:5 molar ratio. 
     
     
         13 . A method for monitoring the binding of a tetrameric FcγR to an Fc region, said method comprising the steps of:
 (i) contacting a polypeptide comprising an Fc region with the tetrameric FcγR complex of  claim 2 ; and 
 (ii) measuring the amount of the tetrameric FcγR complex interacting with said Fc region by detecting said detectable substance. 
 
     
     
         14 . The method of  claim 13 , wherein said detectable substance is detected by fluorescence activated cell sorting (FACS), or by a radioimmunoassay, or by an ELISA assay. 
     
     
         15 . The method of  claim 13 , wherein said polypeptide comprising an Fc region is expressed on the surface of a cell. 
     
     
         16 . The method of  claim 13 , wherein said polypeptide comprising an Fc region is captured on a surface.