Modulation Of Antibody Effector Function By Hinge Domain Engineering

Abstract

The present invention relates to novel molecules (Fc variants) comprising at least one antigen binding region and an Fc region that further comprises a modified hinge which alters the binding of Fc to one or more Fc ligand (e.g., FcγRs) and/or modulates effector function. More specifically, this invention provides Fc variants that have modified binding affinity to one or more FcγR and/or C1q. Additionally, the Fc variants have altered antibody-dependent cell-mediated cytotoxicity (ADCC) and/or complement dependent cytotoxicity (CDC) activity. The invention further provides methods and protocols for the application of said Fc variants particularly for therapeutic purposes.

1 . A polypeptide comprising an Fc region, said Fc region having a modified human IgG1 hinge region, wherein the polypeptide binds at least C1q with a decreased affinity relative to a polypeptide having the same amino acid sequence except having a wild type human IgG1 hinge region, and wherein the modified human IgG1 hinge region comprises:
 (a) amino acid substitutions P227W and P228W; or   (b) amino acid substitutions P227G and P228G; or   (c) insertion of “GGG” between amino acid residues P227 and P228; or   (d) deletion of amino acid residues P227 and P228; or   (e) deletion of amino acid residues T223, H224, P227 and P228,   utilizing the EU index numbering system set forth in Kabat.   
     
     
         2 . The polypeptide of  claim 1 , wherein the affinity is reduced between about 10% and 100% or about 2-fold and 100-fold. 
     
     
         3 . The polypeptide of  claim 1 , wherein the polypeptide has CDC activity that is decreased at least 10%, relative to a polypeptide having the same amino acid sequence except having a wild type hinge region. 
     
     
         4 . The polypeptide of any one of  claim 1 , wherein said polypeptide binds C1q and FcγRIIIa with decreased affinity relative to a polypeptide having the same amino acid sequence except having a wild type human IgG1 hinge region. 
     
     
         5 . The polypeptide of  claim 4 , wherein the modified human IgG1 hinge region comprises:
 (a) amino acid substitutions P227W and P228W; or   (b) insertion of “GGG” between amino acid residues P227 and P228; or   (c) deletion of amino acid residues P227 and P228; or   (d) deletion of amino acid residues T223, H224, P227 and P228,   utilizing the EU index numbering system set forth in Kabat.   
     
     
         6 . The polypeptide of  claim 1 , wherein the polypeptide is an antibody. 
     
     
         7 . The polypeptide of any one of  claim 1 , wherein the polypeptide is an Fc fusion protein. 
     
     
         8 . A pharmaceutical composition comprising the polypeptide of any one of  claim 1 . 
     
     
         9 . A nucleic acid sequence encoding the polypeptide of  claim 1 . 
     
     
         10 . A host cell engineered to contain the nucleic acid sequence of  claim 9 . 
     
     
         11 . A method of decreasing the binding affinity of a polypeptide comprising an Fc region for C1q, wherein said Fc region comprises a human IgG1 hinge region, said method comprising introducing a modification into the hinge region, wherein the modification is selected from the group consisting of:
 (a) amino acid substitutions P227W and P228W;   (b) amino acid substitutions P227G and P228G;   (c) insertion of “GGG” between amino acid residues P227 and P228;   (d) deletion of amino acid residues P227 and P228; and   (e) deletion of amino acid residues T223, H224, P227 and P228,   utilizing the EU index numbering system set forth in Kabat.   
     
     
         12 . A method of decreasing the binding affinity of a polypeptide comprising an Fc region for FcγRIIIa and C1q, wherein said Fc region comprises a human IgG1 hinge region, said method comprising introducing a modification into the hinge region, wherein the modification is selected from the group consisting of:
 (a) amino acid substitutions P227W and P228W; 
 (b) insertion of “GGG” between amino acid residues P227 and P228; 
 (c) deletion of amino acid residues P227 and P228; and 
 (d) deletion of amino acid residues T223, H224, P227 and P228, 
 utilizing the EU index numbering system set forth in Kabat. 
 
     
     
         13 . The method of  claim 11 , wherein the affinity for C1q is reduced between about 10% and 100% or between about 2-fold and 100-fold, relative to the unmodified polypeptide. 
     
     
         14 . The method of  claim 12 , wherein the affinity for C1q is reduced between about 10% and 100% or between about 2-fold and 100-fold, relative to the unmodified polypeptide, and wherein affinity for FcγIIIA is reduced between about 10% and 100% or between about 2-fold and 100-fold, relative to the unmodified polypeptide. 
     
     
         15 . A method of decreasing the CDC activity of a polypeptide comprising an Fc region, wherein said Fc region comprises a human IgG1 hinge region, said method comprising introducing a modification into the hinge region, wherein the modification is selected from the group consisting of:
 (a) amino acid substitutions P227W and P228W;   (b) amino acid substitutions P227G and P228G;   (c) insertion of “GGG” between amino acid residues P227 and P228;   (d) deletion of amino acid residues P227 and P228; and   (e) deletion of amino acid residues T223, H224, P227 and P228,   utilizing the EU index numbering system set forth in Kabat.   
     
     
         16 . The method of  claim 11 , wherein the polypeptide is an antibody. 
     
     
         17 . The method of  claim 12 , wherein the polypeptide is an antibody. 
     
     
         18 . The method of  claim 15 , wherein the polypeptide is an antibody. 
     
     
         19 . The method of  claim 11 , wherein the polypeptide is an Fc fusion protein. 
     
     
         20 . The method of  claim 12 , wherein the polypeptide is an Fc fusion protein. 
     
     
         21 . The method of  claim 15 , wherein the polypeptide is an Fc fusion protein.