Fc-gamma-RIIb-SPECIFIC Fc ANTIBODY

Abstract

An objective of the present invention is to provide a polypeptide containing an Fc region having maintained or decreased binding activities towards both allotypes of FcγRIIa, types H and R, and having enhanced FcγRIIb-binding activity in comparison with a parent polypeptide; a pharmaceutical composition containing the polypeptide; an agent for treating or preventing immunological inflammatory diseases that includes the pharmaceutical composition; a production method thereof; and a method for maintaining or decreasing binding activities towards both allotypes of FcγRIIa and enhancing the FcγRIIb-binding activity. Specifically, it is found that a polypeptide containing an antibody Fc region that has an alteration of substituting Pro at position 238 (EU numbering) with Asp or Leu at position 328 (EU numbering) with Glu enhances FcγRIIb-binding activity, and maintains or decreases binding activities towards both allotypes of FcγRIIa, types H and R. It is also found that a polypeptide containing an antibody Fc region that contains an alteration of substituting Pro at position 238 (EU numbering) with Asp and several other alterations, enhances FcγRIIb-binding activity, and maintains or decreases binding activities towards both allotypes of FcγRIIa, types H and R.

1 .- 27 . (canceled) 
     
     
         28 . A mutant polypeptide comprising a mutant Fc region, wherein:
 the amino acid sequence of the mutant polypeptide is identical to the amino acid sequence of a parent polypeptide comprising an Fc region of any one of SEQ ID NOs:11-14, except for at least one amino acid mutation in the Fc region;   the FcγRIIb-binding activity of the mutant polypeptide is enhanced compared to the FcγRIIb-binding activity of the parent polypeptide;   the ratio of [dissociation constant (KD) value of the mutant polypeptide for FcγRIIa (type R)]/[KD value of the mutant polypeptide for FcγRIIb] is 1.2 or more;   the FcγRIIa (type R)-binding activity of the mutant polypeptide is the same or lower than the FcγRIIa (type R)-binding activity of the parent polypeptide; and   the FcγRIIa (type H)-binding activity of the mutant polypeptide is the same or lower than the FcγRIIa (type H)-binding activity of the parent polypeptide.   
     
     
         29 . The mutant polypeptide of  claim 28 , wherein the ratio of [KD value of the mutant polypeptide for FcγRIIa (type H)]/[KD value of the mutant polypeptide for FcγRIIb] is 4.2 or more. 
     
     
         30 . The mutant polypeptide of  claim 28 , wherein the ratio of [KD value of the parent polypeptide for FcγRIIb]/[KD value of the mutant polypeptide for FcγRIIb] is 1.6 or more. 
     
     
         31 . The mutant polypeptide of  claim 28 ,
 wherein the ratio of [KD value of X]/[KD value of Y] is 0.7 or more;   wherein X is the binding activity of the mutant polypeptide either to FcγRIIa (type R) or to FcγRIIa (type H), whichever is the stronger binding activity of the two, and Y is the binding activity of the parent polypeptide either to FcγRIIa (type R) or to FcγRIIa (type H), whichever is the stronger binding activity of the two.   
     
     
         32 . The mutant polypeptide of  claim 28 , wherein the FcγRIIIa-binding activity of the mutant polypeptide is the same or lower than the FcγRIIIa-binding activity of the parent polypeptide. 
     
     
         33 . The mutant polypeptide of  claim 28 , wherein the FcγRIa-binding activity of the mutant polypeptide is the same or lower than the FcγRIa-binding activity of the parent polypeptide. 
     
     
         34 . The mutant polypeptide of  claim 28 , wherein the at least one amino acid mutation in the Fc region includes substitution of Pro at position 238 (EU numbering) with Asp, or substitution of Leu at position 328 (EU numbering) with Glu. 
     
     
         35 . The mutant polypeptide of  claim 28 , wherein the at least one amino acid mutation in the Fc region includes substitution of Pro at position 238 (EU numbering) with Asp and one or more additional substitutions selected from:
 substitution of Gly at position 237 (EU numbering) with Tip;   substitution of Gly at position 237 (EU numbering) with Phe;   substitution of Ser at position 267 (EU numbering) with Val;   substitution of Ser at position 267 (EU numbering) with Gln;   substitution of His at position 268 (EU numbering) with Asn;   substitution of Pro at position 271 (EU numbering) with Gly;   substitution of Lys at position 326 (EU numbering) with Leu;   substitution of Lys at position 326 (EU numbering) with Gln;   substitution of Lys at position 326 (EU numbering) with Glu;   substitution of Lys at position 326 (EU numbering) with Met;   substitution of Ser at position 239 (EU numbering) with Asp;   substitution of Ser at position 267 (EU numbering) with Ala;   substitution of Leu at position 234 (EU numbering) with Trp;   substitution of Leu at position 234 (EU numbering) with Tyr;   substitution of Gly at position 237 (EU numbering) with Ala;   substitution of Gly at position 237 (EU numbering) with Asp;   substitution of Gly at position 237 (EU numbering) with Glu;   substitution of Gly at position 237 (EU numbering) with Leu;   substitution of Gly at position 237 (EU numbering) with Met;   substitution of Gly at position 237 (EU numbering) with Tyr;   substitution of Ala at position 330 (EU numbering) with Lys;   substitution of Ala at position 330 (EU numbering) with Arg;   substitution of Glu at position 233 (EU numbering) with Asp;   substitution of His at position 268 (EU numbering) with Asp;   substitution of His at position 268 (EU numbering) with Glu;   substitution of Lys at position 326 (EU numbering) with Asp;   substitution of Lys at position 326 (EU numbering) with Ser;   substitution of Lys at position 326 (EU numbering) with Thr;   substitution of Val at position 323 (EU numbering) with Ile;   substitution of Val at position 323 (EU numbering) with Leu;   substitution of Val at position 323 (EU numbering) with Met;   substitution of Tyr at position 296 (EU numbering) with Asp;   substitution of Lys at position 326 (EU numbering) with Ala;   substitution of Lys at position 326 (EU numbering) with Asn; and   substitution of Ala at position 330 (EU numbering) with Met.   
     
     
         36 . The mutant polypeptide of  claim 28 , wherein the mutant polypeptide is an antibody. 
     
     
         37 . The mutant polypeptide of  claim 28 , wherein the mutant polypeptide is an Fc fusion protein molecule. 
     
     
         38 . A pharmaceutical composition comprising the mutant polypeptide of  claim 28 . 
     
     
         39 . A method of preparing a mutant polypeptide, the method comprising:
 identifying a parent polypeptide comprising an IgG Fc region;   providing a first nucleotide sequence comprising a coding sequence encoding the parent polypeptide;   producing a second nucleotide sequence comprising a coding sequence encoding a mutant polypeptide identical to the parent polypeptide except for one or more mutations in the Fc region, the one or more mutations including one or both of: substitution of Pro at position 238 (EU numbering) with Asp and substitution of Leu at position 328 (EU numbering) with Glu;   expressing a DNA comprising the second nucleotide sequence; and   collecting a mutant polypeptide encoded by the second nucleotide sequence, wherein:
 the FcγRIIb-binding activity of the mutant polypeptide is enhanced compared to the FcγRIIb-binding activity of the parent polypeptide; 
 the FcγRIIa (type R)-binding activity of the mutant polypeptide is the same or lower than the FcγRIIa (type R)-binding activity of the parent polypeptide; and 
 the FcγRIIa (type H)-binding activity of the mutant polypeptide is the same or lower than the FcγRIIa (type H)-binding activity of the parent polypeptide. 
   
     
     
         40 . The method of  claim 39 , comprising:
 assaying the ability of the mutant polypeptide to bind to one or more of FcγRIIb, FcγRIIa (type R) or FcγRIIa (type H).   
     
     
         41 . The method of  claim 39 , comprising assaying the antigenicity of the mutant polypeptide in an animal. 
     
     
         42 . The method of  claim 39 , wherein the one or more mutations comprises substitution of Pro at position 238 (EU numbering) with Asp and one or more additional substitutions selected from:
 substitution of Gly at position 237 (EU numbering) with Tip;   substitution of Gly at position 237 (EU numbering) with Phe;   substitution of Ser at position 267 (EU numbering) with Val;   substitution of Ser at position 267 (EU numbering) with Gln;   substitution of His at position 268 (EU numbering) with Asn;   substitution of Pro at position 271 (EU numbering) with Gly;   substitution of Lys at position 326 (EU numbering) with Leu;   substitution of Lys at position 326 (EU numbering) with Gln;   substitution of Lys at position 326 (EU numbering) with Glu;   substitution of Lys at position 326 (EU numbering) with Met;   substitution of Ser at position 239 (EU numbering) with Asp;   substitution of Ser at position 267 (EU numbering) with Ala;   substitution of Leu at position 234 (EU numbering) with Trp;   substitution of Leu at position 234 (EU numbering) with Tyr;   substitution of Gly at position 237 (EU numbering) with Ala;   substitution of Gly at position 237 (EU numbering) with Asp;   substitution of Gly at position 237 (EU numbering) with Glu;   substitution of Gly at position 237 (EU numbering) with Leu;   substitution of Gly at position 237 (EU numbering) with Met;   substitution of Gly at position 237 (EU numbering) with Tyr;   substitution of Ala at position 330 (EU numbering) with Lys;   substitution of Ala at position 330 (EU numbering) with Arg;   substitution of Glu at position 233 (EU numbering) with Asp;   substitution of His at position 268 (EU numbering) with Asp;   substitution of His at position 268 (EU numbering) with Glu;   substitution of Lys at position 326 (EU numbering) with Asp;   substitution of Lys at position 326 (EU numbering) with Ser;   substitution of Lys at position 326 (EU numbering) with Thr;   substitution of Val at position 323 (EU numbering) with Ile;   substitution of Val at position 323 (EU numbering) with Leu;   substitution of Val at position 323 (EU numbering) with Met;   substitution of Tyr at position 296 (EU numbering) with Asp;   substitution of Lys at position 326 (EU numbering) with Ala;   substitution of Lys at position 326 (EU numbering) with Asn; and   substitution of Ala at position 330 (EU numbering) with Met.   
     
     
         43 . The method of  claim 39 , wherein the parent polypeptide is an IgG antibody. 
     
     
         44 . The method of  claim 39 , wherein the parent polypeptide is an Fc fusion protein molecule. 
     
     
         45 . A method of suppressing activation of B cells, mast cells, dendritic cells, and/or basophils, the method comprising administering a composition comprising the mutant polypeptide of  claim 28  to a subject in need of suppressing activation of B cells, mast cells, dendritic cells, and/or basophils. 
     
     
         46 . A method of inhibiting an immunological inflammatory disease in a subject, the method comprising administering a composition comprising the mutant polypeptide of  claim 28  to a subject suffering from or at risk of the immunological inflammatory disease. 
     
     
         47 . The method of  claim 46 , wherein the immunological inflammatory disease is an autoimmune disease caused by production of an antibody against an autoantigen of the subject. 
     
     
         48 . A method of treating a deficiency of a biologically essential protein in a subject, the method comprising:
 identifying a subject in need of treatment for a deficiency of the biologically essential protein, and   administering to the subject the mutant polypeptide of  claim 37 , wherein the fusion protein comprises the biologically essential protein.   
     
     
         49 . A method of inhibiting virus in a subject, the method comprising:
 identifying a subject in need of inhibition of infection by a virus;   providing a composition comprising the mutant polypeptide of  claim 28 , wherein the mutant polypeptide is an antibody that binds to the virus;   administering the composition to the subject.