Serum amyloid p derivatives and their preparation and use

Abstract

One aspect of the present invention relates to the surprising discovery that modification of a glycan structure on a human SAP polypeptide can increase the biological activity of the SAP polypeptide relative to a corresponding sample of wild-type SAP isolated from human serum. The disclosure provides both variant human SAP polypeptides and methods for making the same. In particular, the present invention provides methods and compositions for in vitro and in vivo addition, deletion, or modification of sugar residues to produce SAP polypeptides, such as a human SAP polypeptide, having a desired glycosylation pattern.

We claim: 
     
         1 . A glycosylated human Serum Amyloid P (SAP) polypeptide comprising an N-linked oligosaccharide chain, wherein at least one branch of the oligosaccharide chain terminates with a α2,3-linked sialic acid moiety. 
     
     
         2 . A glycosylated human SAP polypeptide comprising an N-linked oligosaccharide chain, wherein the oligosaccharide chain has at least 50% fewer α2,6-linked sialic acid moieties than wild-type SAP isolated from human serum. 
     
     
         3 . The glycosylated human SAP polypeptide of  claim 1 , wherein all branches of the oligosaccharide chain terminate with α2,3-linked sialic acid moieties. 
     
     
         4 . The glycosylated human SAP polypeptide of  claim 1 , wherein the oligosaccharide chain is substantially free of α2,6-linked sialic acid moieties. 
     
     
         5 . The glycosylated human SAP polypeptide of  claim 1 , wherein the polypeptide comprises an amino acid sequence at least 95% identical to SEQ ID NO: 1. 
     
     
         6 . The glycosylated human SAP polypeptide of  claim 1 , wherein the polypeptide is a fusion protein comprising an SAP domain and one or more heterologous domains. 
     
     
         7 . The glycosylated human SAP polypeptide of  claim 1 , wherein the polypeptide comprises one or more modified amino acid residues. 
     
     
         8 . The glycosylated human SAP polypeptide of  claim 7 , wherein the one or more modified amino acid residues comprise a PEGylated amino acid, a prenylated amino acid, an acetylated amino acid, a biotinylated amino acid, and/or an amino acid conjugated to an organic derivatizing agent. 
     
     
         9 . The glycosylated human SAP polypeptide of  claim 1 , wherein the SAP polypeptide has an IC 50  for inhibiting the differentiation of monocytes into fibrocytes in vitro that is less than one-half that of a corresponding sample of wild-type SAP isolated from human serum. 
     
     
         10 . A pharmaceutical preparation suitable for use in a mammal comprising the human SAP polypeptide of  claim 1  and a pharmaceutically acceptable carrier. 
     
     
         11 . A method of treating or preventing a-disorder or condition in a patient, the method comprising administering to a patient in need thereof a therapeutically effective amount of the SAP polypeptide of  claim 1 , wherein the disorder or condition is selected from a fibrotic or fibroproliferative disorder or condition, a hypersensitivity disorder or condition, an autoimmune disorder or condition, an inflammatory disorder or condition, and mucositis. 
     
     
         12 . A method of making a human SAP polypeptide, comprising:
 i) expressing a human SAP polypeptide in a CHO cell; and   ii) isolating the human SAP polypeptide from the cell.   
     
     
         13 . The method of  claim 12 , wherein the isolated SAP polypeptide comprises an N-linked oligosaccharide chain, and wherein at least one branch of the oligosaccharide chain terminates with a α2,3-linked sialic acid moiety. 
     
     
         14 . The method  claim 12 , wherein the isolated SAP polypeptide comprises an N-linked oligosaccharide chain, and wherein the oligosaccharide chain has at least 50% fewer α2,6-linked sialic acid moieties than wild-type SAP isolated from human serum. 
     
     
         15 . The method of  claim 12 , wherein the isolated human SAP polypeptide has an IC 50  for inhibiting the differentiation of monocytes into fibrocytes in vitro that is less than one-half that of a corresponding sample wild-type SAP isolated from human serum. 
     
     
         16 . The method of  claim 12 , further comprising enzymatically or chemically altering the isolated SAP polypeptide to produce an SAP polypeptide having a modified oligosaccharide chain. 
     
     
         17 . The method of  claim 16 , wherein the process of enzymatically or chemically altering the isolated SAP polypeptide removes one or more terminal α2,6-linked sialic acid moieties from the oligosaccharide chain. 
     
     
         18 . The method of  claim 16 , wherein the process of enzymatically or chemically altering the isolated SAP polypeptide replaces one or more terminal α2,6-linked sialic acid moieties on the oligosaccharide chain with one or more α2,3-linked sialic acid moieties. 
     
     
         19 . A method of making an SAP polypeptide, comprising:
 i) providing an SAP polypeptide, and   ii) enzymatically or chemically altering the SAP polypeptide to produce a glycosylated SAP polypeptide comprising an N-linked oligosaccharide.   
     
     
         20 . The method of  claim 19 , wherein the N-linked oligosaccharide chain has at least 50% fewer α2,6-linked sialic acid moieties than a wild-type human SAP protein isolated from human serum. 
     
     
         21 . The method of  claim 19 , wherein at least one branch of the oligosaccharide chain terminates in an α2,3-linked sialic acid moiety. 
     
     
         22 . The human SAP polypeptide of  claim 19 , wherein the glycosylated SAP polypeptide has an IC 50  for inhibiting the differentiation of monocytes into fibrocytes in vitro that is less than one-half that of a corresponding sample wild-type SAP isolated from human serum. 
     
     
         23 . A human SAP polypeptide prepared by a process comprising:
 i) expressing a SAP polypeptide in a CHO cell; and   ii) isolating the SAP polypeptide from the cell.   
     
     
         24 . The human SAP polypeptide of  claim 23 , wherein the SAP polypeptide has an IC 50  for inhibiting the differentiation of monocytes into fibrocytes in vitro that is less than one-half that of a corresponding sample of wild-type SAP isolated from human serum. 
     
     
         25 . A CHO cell comprising a nucleic acid encoding an exogenous SAP polypeptide. 
     
     
         26 . A human SAP polypeptide having an IC 50  for inhibiting the differentiation of monocytes into fibrocytes in vitro that is less than one-half that of a corresponding sample of wild-type SAP isolated from human serum.