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

Patent metadata

Field	Value
Publication number	US-2016176950-A1
Application number	US-201514624483-A
Country	US
Kind code	A1
Filing date	Feb 17, 2015
Priority date	Apr 5, 2006
Publication date	Jun 23, 2016
Grant date	—

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.

First claim

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 glycosylated 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 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 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 inflamm

Assignees

• Univ Rockefeller

Inventors

• Ravetch Jeffrey
• Nimmerjahn Falk
• Kaneko Yoshikatsu

Classifications

• A61P37/00

  Drugs for immunological or allergic disorders · CPC title

• A61P29/00

  Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID] · CPC title

• C07K2317/41

  Glycosylation, sialylation, or fucosylation · CPC title

• C07K2317/71

  Decreased effector function due to an Fc-modification · CPC title

• C07K16/18

  against material from animals or humans · CPC title