Serum amyloid P derivatives and their preparation and use
Inventors
Willett, W. Scott • Caimi, Richard J.
Assignees
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Publication Number
US-9296800-B2
Publication Date
2016-03-29
Expiration Date
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.
Core Innovation
The invention relates to glycosylated human Serum Amyloid P (SAP) proteins with altered sialylation, where an N-linked oligosaccharide chain includes at least one branch that terminates with an α2,3-linked sialic acid moiety. The SAP protein comprises a polypeptide having the amino acid sequence of SEQ ID NO: 1 or a variant thereof, and is characterized by its glycosylation pattern and improved biological potency relative to wild-type serum-derived SAP.
The claimed glycosylated SAP proteins include α2,3-linked sialic acid moieties and have fewer α2,6-linked sialic acid moieties than naturally occurring human SAP from human serum. The document states that changing glycan sialylation is associated with increased biological potency.
The modified glycosylation results in functional activity, including inhibition of differentiation of monocytes into fibrocytes in vitro. Experimental support described in the document includes an ELISA-based fibrocyte differentiation assay using MDC, showing that recombinant α2,3-sialylated SAP is more active than α2,6 serum-derived SAP, with intermediate activity for mixed derivatives.
Claims Coverage
The document includes four independent claims covering a glycosylated human SAP protein and methods of making SAP proteins. Across these independent claims, the main inventive features are terminal α2,3 sialylation of N-linked oligosaccharide branches, reduced α2,6 sialylation relative to naturally occurring human SAP, and functional inhibition of monocyte-to-fibrocyte differentiation in vitro.
α2,3 terminal sialylated N-linked glycosylation with SEQ ID NO: 1 polypeptide
A glycosylated human Serum Amyloid P (SAP) protein comprising an N-linked oligosaccharide chain, wherein at least one branch of the N-linked oligosaccharide chain terminates with an α2,3-linked sialic acid moiety, wherein the SAP protein comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or variant thereof, and wherein the SAP protein inhibits the differentiation of monocytes into fibrocytes in vitro.
Expression in a CHO cell to obtain N-linked α2,3 terminal sialylated SAP
A method of making a human SAP protein comprising an N-linked oligosaccharide chain, wherein at least one branch of the oligosaccharide chain terminates with an α2,3-linked sialic acid moiety, the method comprising expressing a human SAP protein in a CHO cell, and isolating the human SAP protein from the cell, wherein the isolated human SAP protein comprises an N-linked oligosaccharide chain, and wherein at least one branch of the N-linked oligosaccharide chain terminates with an α2,3-linked sialic acid moiety.
Enzymatically or chemically altering provided SAP to generate N-linked α2,3 terminal sialylated glycosylated SAP
A method of making an SAP protein comprising providing an SAP protein, and enzymatically or chemically altering the SAP protein to produce a glycosylated SAP protein comprising an N-linked oligosaccharide, wherein at least one branch of the N-linked oligosaccharide chain terminates with an α2,3-linked sialic acid moiety.
Fewer α2,6 sialic acid moieties plus inhibited monocyte-to-fibrocyte differentiation for α2,3 terminal SAP
A glycosylated human Serum Amyloid P (SAP) protein comprising an N-linked oligosaccharide chain, wherein at least one branch of the N-linked oligosaccharide chain terminates with an α2,3-linked sialic acid moiety, and wherein the SAP protein has fewer α2,6-linked sialic acid moieties than naturally occurring human SAP from human serum, wherein the SAP protein comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or variant thereof, and wherein the SAP protein inhibits the differentiation of monocytes into fibrocytes in vitro.
Across the independent claims, the claimed invention centers on glycosylated human SAP having N-linked oligosaccharide branches that terminate with α2,3-linked sialic acid moieties, with reduced α2,6-linked sialic acid moieties relative to naturally occurring serum SAP, and with functional inhibition of the differentiation of monocytes into fibrocytes in vitro.
Stated Advantages
Increased biological potency versus wild-type serum-derived SAP, as reflected by improved in vitro functional potency in inhibiting differentiation of monocytes into fibrocytes.
More active α2,3-sialylated SAP than α2,6 serum-derived SAP in an ELISA-based fibrocyte differentiation assay using MDC.
Documented Applications
Therapeutic use for SAP-responsive disorders, including fibrosis.
Therapeutic use for SAP-responsive disorders including hypersensitivity, autoimmune disorders, mucositis, and inflammatory disorders.
In vitro inhibition of differentiation of monocytes into fibrocytes (fibrocyte differentiation assay using MDC).
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