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 α 2,6 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 α 2,6 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  or  2 , 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 α 2,6 linkage, and wherein said polypeptide having a reduced binding to an Fc activating receptor as compared to an unpurified antibody preparation. 
     
     
         4 . The isolated polypeptide of  claim 3 , wherein the Fc activating receptor is selected from the group consisting of FcγRIIA, FcγRIIC and FcγRIIIA. 
     
     
         5 . The polypeptide of any one of  claims 1 - 4  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 α 2,6 linkage as compared to an unpurified antibody. 
     
     
         6 . The polypeptide of any one of  claims 1 - 5 , having an increased anti-inflammatory activity in vitro. 
     
     
         7 . The polypeptide of any one  claims 1 - 6 , having an increased anti-inflammatory activity in vivo. 
     
     
         8 . The polypeptide of any one of  claims 1 - 7 , derived from a naturally occurring antibody source. 
     
     
         9 . The polypeptide of any one of  claims 1 - 7 , derived from a recombinant antibody source. 
     
     
         10 . The polypeptide of any one of  claims 1 - 7 , wherein said unmodified antibody comprises IVIG. 
     
     
         11 . The polypeptide of any one of  claims 1 - 7 , 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. 
     
     
         12 . The polypeptide of any one of  claims 1 - 11 , modified by treatment with α2-6 sialyltransferase. 
     
     
         13 . The polypeptide of any one of  claims 1 - 12 , which is purified. 
     
     
         14 . A pharmaceutical formulation comprising the polypeptide of any one of  claims 1 - 13  in combination with a suitable carrier or diluent. 
     
     
         15 . A method of modulating properties of a polypeptide comprising an Fc region comprising altering the sialylation of the polysaccharide chain of the Fc region. 
     
     
         16 . A method of  claim 15 , wherein said properties comprise a higher anti-inflammatory activity than an unpurified antibody. 
     
     
         17 . The method of  claim 15  or  16 , 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 a 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 α 2,6 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 α 2,6 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 α 2,6 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 α 2,6 linkage. 
 
     
     
         18 . The method of any one of  claims 15 - 17 , wherein said unpurified source of the polypeptide containing at least one Fc region comprises a human unpurified IgG antibody. 
     
     
         19 . The method of any one of  claims 15 - 18 , 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. 
     
     
         20 . The method of  claim 19 , wherein the expression system comprises modified host expression systems capable of N-linked glycosylation selected from the group consisting of bacterial, fungal, plant, vertebrate and invertebrate expression systems, and any combinations thereof. 
     
     
         21 . The method of any one of  claims 15 - 20 , 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 α 2,6 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 α 2,6 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 α 2,6 linkage. 
     
     
         22 . The method of  claim 21 , wherein said removal is achieved by physical or chemical methods. 
     
     
         23 . The method of  claim 21  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. 
     
     
         24 . The method of  claim 23 , 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. 
     
     
         25 . The method of any one of  claims 15 - 24 , 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 α 2,6 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 α 2,6 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 α 2,6 linkage. 
     
     
         26 . The method of  claim 25  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. 
     
     
         27 . The method of  claim 26 , 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. 
     
     
         28 . The method of any one of  claims 25 - 27  wherein said enrichment is achieved by a chemical reaction with an enzyme creating α 2,6 linkages between the carbohydrate attached to the polypeptide containing least one Fc region and a terminal sialic acid. 
     
     
         29 . The method of  claim 28 , wherein the enzyme is α-(2,6) sialyltransferase. 
     
     
         30 . A method of treating an inflammatory disease comprising administering to a patient a therapeutically effective dose of the polypeptide of any one of  claims 1 - 14 . 
     
     
         31 . The method of  claim 30 , wherein the inflammatory disease is selected from the group consisting of arthritis, thrombocytopenia, and nephritis.