Fc RECEPTOR-BINDING POLYPEPTIDES WITH MODIFIED EFFECTOR FUNCTIONS

Abstract

Disclosed are processes for producing a variant polypeptide (e.g. antibodies) having modified binding characteristics for human Fc gamma receptor IIA (CD32A) leading to increased inhibition of proinflammatory mediators while retaining binding to a target antigen via its Fv portion, which processes comprise altering the polypeptides by substitution of at least two amino acid residues at EU position 325, 326 or 328 of a human IgG CH2 region for a sequence selected from SAAF, SKAF, NAAF and NKAF. The polypeptides that can be generated according to the methods of the invention are highly variable, and they can include antibodies and fusion proteins that contain an Fc region or a biologically active portion thereof.

What is claimed is: 
     
         1 . An altered polypeptide comprising at least an FcγR binding portion of an Fc region wherein the polypeptide comprises at least one mutation compared to a starting polypeptide and wherein at least one mutation is selected from a group consisting of a substitution at EU amino acid positions 325, 326 and 328. 
     
     
         2 . The altered polypeptide of  claim 1 , wherein the altered polypeptide elicits a modified Fc gamma receptor activity. 
     
     
         3 . The altered polypeptide of  claim 1 , wherein the altered polypeptide is an antibody or fragment thereof or a fusion protein. 
     
     
         4 . The altered polypeptide of  claim 1 , wherein the FcγR binding portion or the Fc region is derived from a mouse, rat or human antibody. 
     
     
         5 . The altered polypeptide of  claim 4 , wherein the FcγR binding portion comprises a complete Fc region. 
     
     
         6 . The altered polypeptide of  claim 2 , wherein the modified Fc gamma receptor activity is the inhibition of the release of proinflammatory mediators. 
     
     
         7 . The altered polypeptide of  claim 2 , wherein said Fc gamma receptor is the human CD32A. 
     
     
         8 . The altered polypeptide of  claim 3 , wherein said antibody is human IgG1 isotype, human IgG2 isotype, human IgG3 isotype or human IgG4 isotype. 
     
     
         9 . The altered polypeptide of  claim 8 , comprising a mutation selected from the group consisting of the amino acid residue at EU position 325 is substituted with serine; the amino acid residue at EU position 326 is substituted with alanine; the amino acid residue at EU position 328 is substituted with phenylalanine; and combinations thereof. 
     
     
         10 . The altered polypeptide of  claim 9 , wherein said heavy chain constant region comprises two or more amino acid substitutions with an amino acid residue that is different from the corresponding amino acid residue in an unaltered antibody, wherein said substitutions occur at two or more amino acid residues selected from EU positions 325, 326 and 328 of said heavy chain constant region. 
     
     
         11 . The altered polypeptide of  claim 10 , wherein EU position 326 of the heavy chain constant region is substituted with alanine and EU position 328 of the heavy chain constant region is substituted with phenylalanine. 
     
     
         12 . The altered polypeptide of  claim 9 , wherein EU positions 325-328 of the heavy chain constant region consist of a sequence selected from SAAF (SEQ ID NO: 86), SKAF (SEQ ID NO: 87) and NKAF (SEQ ID NO: 89). 
     
     
         13 . The altered polypeptide of  claim 1 , wherein said polypeptide binds to a target selected from a toll-like receptor (TLR), MD2 accessory protein, soluble TLR4, the TLR4/MD2 complex, both soluble TLR4 and the TLR4/MD2 complex, TLR2, and CD14. 
     
     
         14 . A method of activating ICAM signaling, said method comprising binding human FcγR CD32A with an altered polypeptide comprising at least an FcγR binding portion of an Fc region wherein the polypeptide comprises at least one mutated amino acid residue of the heavy chain constant region selected from one or more of the amino acid residues that correspond to EU positions 325, 326 and 328 with an amino acid residue that is different from the corresponding amino acid residue in an unaltered polypeptide, wherein said altered polypeptide elicits an inhibition of proinflammatory mediators release by ligation with human FcγR CD32A while retaining binding to antigen via its Fv region as compared to an unaltered polypeptide. 
     
     
         15 . The method of  claim 14 , wherein said altered polypeptide is an altered antibody. 
     
     
         16 . The method of  claim 15 , wherein the altered antibody comprises a mutation selected from the group consisting of the amino acid residue at EU position 325 is substituted with serine; the amino acid residue at EU position 326 is substituted with alanine; the amino acid residue at EU position 328 is substituted with phenylalanine; and combinations thereof. 
     
     
         17 . The method of  claim 15 , wherein said heavy chain constant region of said altered antibody contains two or more substitutions, with an amino acid residue that is different from the corresponding amino acid residue in an unaltered antibody, wherein said substitutions occur at two or more amino acid residues selected from residues that correspond to EU positions 325, 326 and 328. 
     
     
         18 . The method of  claim 15 , wherein the residue at EU position 326 of said altered antibody is substituted with alanine and the residue at EU position 328 of said altered antibody is substituted with phenylalanine. 
     
     
         19 . The method of  claim 15 , wherein residues at EU positions 325-328 of said altered antibody consist of a sequence selected from SAAF (SEQ ID NO: 86), SKAF (SEQ ID NO: 87) and NKAF (SEQ ID NO: 89). 
     
     
         20 . The method of  claim 15 , wherein said altered antibody to a target selected from a toll-like receptor (TLR), MD2 accessory protein, soluble TLR4, the TLR4/MD2 complex, both soluble TLR4 and the TLR4/MD2 complex, TLR2, and CD14. 
     
     
         21 . An isolated polypeptide comprising a gamma Fc (γFc) region, wherein residues at EU positions 325-328 of said region consist of an amino acid motif selected from SAAF (SEQ ID NO: 86) and NKAF (SEQ ID NO: 89).