Polypeptides With Enhanced Anti-Inflammatory And Decreased Cytotoxic Properties And Relating Methods

Abstract

The invention provides a polypeptide containing at least one IgG Fc region, wherein said at least one IgG Fc region is glycosylated with at least one galactose moiety connected to a respective terminal sialic acid moiety by a α2,6 linkage, and wherein said polypeptide having a higher anti-inflammatory activity as compared to an unpurified antibody.

What is claimed is: 
     
         1 . An isolated polypeptide containing at least one IgG Fc region, having altered properties compared to an unpurified antibody preparation, wherein sialylation of the isolated polypeptide is higher than the sialylation of the unpurified antibody preparation. 
     
     
         2 . The isolated polypeptide of  claim 1 , wherein said at least one IgG Fc region is glycosylated with at least one galactose moiety connected to a respective terminal sialic acid moiety by a α2,6 linkage, and wherein said polypeptide having a higher anti-inflammatory activity as compared to an unpurified antibody preparation. 
     
     
         3 . The isolated polypeptide of  claim 1 , wherein said at least one IgG Fc region is glycosylated with at least one galactose moiety connected to a respective terminal sialic acid moiety by a α2,6 linkage, and wherein said polypeptide having a reduced binding to an Fc activating receptor selected from the group consisting of Fc?RIIA, Fc?RIIC and Fc?RIIIA, as compared to an unpurified antibody preparation. 
     
     
         4 . The isolated polypeptide of  claim 1  comprising a human IgG1, IgG2, IgG3 or IgG4 Fc region, said polypeptide having a higher content of the at least one galactose moiety connected to the respective terminal sialic acid moiety by a α2,6 linkage as compared to an unpurified antibody. 
     
     
         5 . The isolated polypeptide of  claim 1 , derived either from a naturally occurring antibody source or a recombinant antibody source. 
     
     
         6 . The isolated polypeptide of  claim 1 , wherein said unmodified antibody comprises IVIG. 
     
     
         7 . The isolated polypeptide of  claim 1  produced from a recombinant source and lacking Fab region, wherein said at least one IgG Fc region is glycolylated with two galactose moieties. 
     
     
         8 . The isolated polypeptide of  claim 1  encoded by a nucleic acid sequence comprising SEQ ID NO: 1. 
     
     
         9 . The isolated polypeptide of  claim 1 , derived from a cell line having an enhanced activity of creating α2,6 linkages between at least one galactose moiety and a respective terminal sialic acid in a protein's polysaccharide chain. 
     
     
         10 . The isolated polypeptide of  claim 1 , modified by treatment with α2-6 sialyltransferase. 
     
     
         11 . A method of modulating properties of a polypeptide comprising an Fc region comprising altering the sialylation of the polysaccharide chain of the Fc region. 
     
     
         12 . A method of  claim 11 , wherein said properties comprise a higher anti-inflammatory activity than an unpurified antibody. 
     
     
         13 . The method of  claim 11 , wherein the step of altering sialylation comprises:
 providing an unpurified source of the polypeptide containing at least one Fc region, said unpurified source of the polypeptide containing at least one Fc region comprising plurality of the polypeptides containing at least one Fc region having a polysaccharide chain comprising a terminal sialic acid connected to a galactose moiety through a α2,6 linkage, and a plurality of the polypeptides containing at least one Fc region lacking a polysaccharide chain comprising a terminal sialic acid connected to a galactose moiety through the α2,6 linkage; and   increasing the ratio of the plurality of the polypeptides containing at least one Fc region having the polysaccharide chain comprising the terminal sialic acid connected to the galactose moiety through the α2,6 linkage to the plurality of the polypeptide containing at least one Fc region lacking the polysaccharide chain comprising the terminal sialic acid connected to the galactose moiety through the α2,6 linkage.   
     
     
         14 . The method of  claim 11 , wherein the unpurified source of the polypeptide containing at least one Fc region is provided from expressing a vector comprising a nucleic acid sequence in an expression system, wherein said nucleic acid sequence is translated into an IgG antibody. 
     
     
         15 . The method of  claim 11 , wherein the step of increasing the ratio of the plurality of the polypeptides containing at least one Fc region having the polysaccharide chain comprising the terminal sialic acid connected to the galactose moiety through the α2,6 linkage to the plurality of the polypeptide containing at least one Fc region lacking the polysaccharide chain comprising the terminal sialic acid connected to the galactose moiety through the α2,6 linkage is achieved through a removal of the polypeptides containing at least one Fc region lacking the polysaccharide chain comprising the terminal sialic acid connected to the galactose moiety through the α2,6 linkage. 
     
     
         16 . The method of  claim 15  wherein said removal is achieved by a method selected from the group consisting of HPLC, lectin affinity chromatography, high pH anion exchange chromatography, and any combination thereof. 
     
     
         17 . The method of  claim 16 , wherein the lectin affinity chromatography is performed using a lectin having a lower affinity to α2,6 linkages than to α2,3 linkages between the galactose moiety and the terminal sialic acid. 
     
     
         18 . The method of  claim 15 , wherein the step of increasing the ratio of the plurality of the polypeptides containing at least one Fc region having the polysaccharide chain comprising the terminal sialic acid connected to the galactose moiety through the α2,6 linkage to the plurality of the polypeptide containing at least one Fc region lacking the polysaccharide chain comprising the terminal sialic acid connected to the galactose moiety through the α2,6 linkage is achieved through an enrichment of said unpurified source of the polypeptide containing at least one Fc region having the polysaccharide chain comprising the terminal sialic acid connected to the galactose moiety through the α2,6 linkage. 
     
     
         19 . The method of  claim 18  wherein said enrichment is achieved by a method selected from the group consisting of HPLC, lectin affinity chromatography, high pH anion exchange chromatography, and any combination thereof. 
     
     
         20 . The method of  claim 19 , wherein the lectin affinity chromatography is performed using a lectin having a higher affinity to α2,6 linkages than to α2,3 linkages between the galactose moiety and the terminal sialic acid. 
     
     
         21 . The method of  claim 18 , wherein said enrichment is achieved by a chemical reaction with an enzyme creating α2,6 linkages between the carbohydrate attached to the polypeptide containing least one Fc region and a terminal sialic acid. 
     
     
         22 . A method of treating an inflammatory disease selected from the group consisting of arthritis, thrombocytopenia, and nephritis comprising administering to a patient a therapeutically effective dose of the polypeptide of  claim 1 . 
     
     
         23 . A method of treating an inflammatory disease comprising administering to a subject in need thereof a therapeutic composition comprising a plurality of isolated polypeptides, each containing at least one IgG Fc region, wherein
 a first portion of the respective Fc regions comprises respective carbohydrate chains having galactose moieties connected to respective terminal sialic acid moieties by 2,6 linkage;   a dose of the therapeutic composition is smaller than a dose of a second composition which comprises a plurality of isolated polypeptides, each containing at least one IgG Fc region, having a second portion of the respective Fc regions comprising respective carbohydrate chains having galactose moieties connected to respective terminal sialic acid moieties by 2,6 linkage; and either   the first portion is greater than the second portion, whereby the dose of the therapeutic composition and the dose of the second composition suppress inflammation to substantially the same extent, or   the first portion is greater than the second portion, whereby the therapeutic composition suppresses inflammation to a greater extent than an equal dose of the second composition.   
     
     
         24 . A composition comprising glycoproteins containing an Fc region wherein the composition has been formulated to contain sialylated glycoproteins in an amount sufficient to achieve an immunosuppressive activity in a mammal. 
     
     
         25 . The composition of  claim 24 , wherein the composition comprises sialylated glycoproteins in an amount of about 5% or more. 
     
     
         26 . The composition of  claim 24 , wherein the composition comprises sialylated glycoproteins in an amount of about 10% or more. 
     
     
         27 . The composition of  claim 24 , wherein the composition comprises sialylated glycoproteins in an amount of about 30% or more. 
     
     
         28 . The composition of  claim 24 , wherein the composition comprises sialylated glycoproteins in an amount of about 5% to about 30%. 
     
     
         29 . The composition of  claim 24 , wherein the sialylated glycoproteins comprise one or more terminal sialic acid residues or analogues thereof. 
     
     
         30 . The composition of  claim 29 , wherein the terminal sialic acid residue(s) is linked to the glycoprotein by an alpha 2,6 linkage. 
     
     
         31 . An IVIG derived composition formulated to contain sialylated Fc containing glycoproteins in an amount of about 5% to about 30% and wherein the sialylated glycoproteins comprise one or more terminal sialic acid residues linked to the glycoprotein by an alpha 2,6 linkage. 
     
     
         32 . A recombinant Fc glycoprotein, or fragment thereof, comprising at least one terminal sialic acid residue(s), or analogue(s) thereof, linked to the glycoprotein by an alpha 2,6 linkage. 
     
     
         33 . A recombinant Fc glycoprotein comprising an N-linked carbohydrate at Asn 297 wherein in the carbohydrate has a biantennary GlnNac2, Man3, GlcNAc2, Gal2 structure having one or more terminal sialic acid residue(s) linked by an alpha 2,6 linkage. 
     
     
         34 . An Fc containing glycoprotein of any of the above claims wherein the Fc region is IgG or a subclass thereof. 
     
     
         35 . A pharmaceutical preparation comprising the glycoproteins of  claim 24 . 
     
     
         36 . A method of treating an inflammatory disorder in a subject using the pharmaceutical preparation of  claim 35 .