Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Glycosylation of peptides via o-linked glycosylation sequences

a glycosylation sequence and peptide technology, applied in the field of polypeptide modification by glycosylation, can solve the problems of rapid neutralization of peptides and/or allergic reactions, limited use of such polypeptides as therapeutic agents, and lack of homogeneity of the final produ

Inactive Publication Date: 2008-10-02
NOVO NORDISK AS
View PDF77 Cites 75 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides a method for specifically targeting the glycosylation sequences of a polypeptide to add modifying groups. This is achieved by introducing a mutant polypeptide with the desired glycosylation sequence and then adding the modifying group to the polypeptide using a glycosyltransferase. The invention also provides sequon polypeptides that have been modified with a polymeric modifying group. The invention further provides pharmaceutical compositions containing the polypeptide conjugates and methods of making and using them. The technical effect of the invention is the ability to specifically modify the glycosylation sequences of a polypeptide to add new groups, which can be useful in drug development and other applications.

Problems solved by technology

The lack of expression systems that can be used to manufacture polypeptides with wild-type glycosylation patterns has limited the use of such polypeptides as therapeutic agents.
It is known in the art that improperly or incompletely glycosylated polypeptides can be immunogenic, leading to rapid neutralization of the peptide and / or the development of an allergic response.
This approach has significant drawbacks, including a lack of homogeneity of the final product, and the possibility of reduced biological or enzymatic activity of the modified polypeptide.
In addition, existing glycosylation sequences may not be suitable for the attachment of a modifying group.
Such modification may, for example, cause an undesirable decrease in biological activity of the modified polypeptide.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Glycosylation of peptides via o-linked glycosylation sequences
  • Glycosylation of peptides via o-linked glycosylation sequences
  • Glycosylation of peptides via o-linked glycosylation sequences

Examples

Experimental program
Comparison scheme
Effect test

example 1

Incorporation of Glycosylation Sites into Bone Morphogenetic Protein-7 (BMP-7)

1.1. BMP-7 Sequence Information

[0709]An exemplary BMP-7 sequence is shown below (S.1).

Human Bone morphogenetic protein-7(SEQ ID NO: 164)M1STGSKQRSQNRSKTPKNQEALRMANVAENSSSDQRQACKKHELYVSFRDLGWQDWIIAPEGYAAYYCEGECAFPLNSYMNATNHAIVQTLVHFINPETVPKPCCAPTQLNAISVLYFDDSSNVILKKYRNMVVRACGCH

[0710]The N-terminal methionine may be present or absent in any BMP-7 mutant. In this example, the numbering of the amino acid residues is based on the initial unmodified sequence in which the left most residue, methionine (M), is numbered as position 1. To highlight how the mutant sequence differs in respect to the unmodified sequence, the numbering of unmodified amino acids as they appear in the mutant sequences below remains unchanged following the modification. More than one of the described sequence modifications may be present in a BMP-7 mutant of the present invention.

[0711]Preferred regions for introduction of mutations to cre...

example 2

Incorporation of Glycosylation Sequences into Neutrotrophin-3 (NT-3)

2.1. NT-3 Sequence Information

[0725]An exemplary wild-type amino acid sequence (S.2) of human NT-3 is shown below.

Human Neurotrophin-3 (SEQ ID NO: 340):

[0726]MYAEHKSHRGEYSVCDSESLWVTDKSSAIDIRGHQVTVLGEIKTGNSPVKQYFYETR CKEARPVKNGCRGIDDKHWNSQCKTSQTYVRALTSENNKLVGWRWIRIDTSCVCAL SRKIGRT

[0727]This example describes amino acid sequence mutations introducing O-linked glycosylation sequences into the wild-type NT-3 sequence S.2 (SEQ ID NO: 340) or any modified (e.g., previously mutated) version thereof. A number of mutants were created introducing O-linked glycosylation sites into 3 loop regions as well as the amino terminus.

[0728]The N-terminal methionine (M) may be present or absent in any NT-3 mutant. In this example, the numbering of the amino acid residues is based on the initial unmodified sequence in which the N-terminal residue, methionine (M), is numbered as position 1. To highlight how the mutant sequence differs wit...

example 3

Expression of Human BMP-7 and Human NT-3 Using Various Vectors and E. coli Host Cells

[0745]The BMP-7 native sequence S.1 (SEQ ID NO: 164) and the above described BMP-7 mutants C.1 to C.31 (SEQ ID NOs: 273-277, 279-283, 285-287, 289-291, 293-296, 298-305, 307-309) (Example 1) as well as the NT-3 native sequence S.2 (SEQ ID NO: 340) and the above described NT-3 mutants A.1-A.16 (SEQ ID NOs: 342, 343, 345-347, 349-351, 353-360) (Example 2) can be expressed using a variety of vectors in different E. coli host cells. Experimental results for the native sequences are summarized in Table 17, below. In addition, all BMP-7 mutants C.1 to C.31 were expressed in W3110 E. coli at 37° C. as inclusion bodies.

TABLE 17Expression of native human BMP-7 (S.1) (SEQ ID NO: 164)and native NT-3 (S.2) (SEQ ID NO: 340) in E. coliE. coli HostInductionProteinVectorCellTemperatureBMP-7pET24atrxb,gor,supp-2 DE320° C.BMP-7pET24aNovaBlue(DE3)37° C.BMP-7pET24aNovaBlue(DE3)20° C.BMP-7pcWin2W311037° C.NT-3pET24atrxb...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
covalentaaaaaaaaaa
negative chargeaaaaaaaaaa
nucleic acidaaaaaaaaaa
Login to View More

Abstract

The present invention provides sequon polypeptides with an amino acid sequence including one or more exogenous O-linked glycosylation sequence of the invention. In addition, the present invention provides methods of making polypeptide conjugates as well as methods of using such conjugates and their pharmaceutical compositions. The invention further provides libraries of sequon polypeptides, wherein each member of such library includes at least one exogenous O-linked glycosylation sequence of the invention. Also provided are methods of making and using such libraries.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]The present application claims priority to U.S. Provisional Patent Application No. 60 / 832,461 filed Jul. 21, 2006, U.S. Provisional Patent Application No. 60 / 886,616 filed Jan. 25, 2007, U.S. Provisional Patent Application No. 60 / 941,920 filed Jun. 4, 2007 and U.S. Provisional Patent Application No. 60 / 881,130 filed Jan. 18, 2007, each of which is incorporated herein by reference in their entirety for all purposes.FIELD OF THE INVENTION[0002]The invention pertains to the field of polypeptide modification by glycosylation. In particular, the invention relates to a method of preparing glycosylated polypeptides using short enzyme-recognized O-linked or S-linked glycosylation sequences.BACKGROUND OF THE INVENTION[0003]The present invention relates to glycosylation and modification of polypeptides, preferably polypeptides of therapeutic value. The administration of glycosylated and non-glycosylated polypeptides for engendering a particular phy...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(United States)
IPC IPC(8): A61K38/00C07K16/00C07K14/00C12N15/00C12N5/00C12P21/06C40B20/00C40B20/08C40B50/06C40B40/10C12N9/00C12N15/87C07H21/04
CPCA61K38/00A61K47/48092A61K47/48215A61K47/48246C12P21/005A61K47/483C07K1/047C07K1/1077C07K9/001A61K47/48284A61K47/60A61K47/549A61K47/64A61K47/643A61K47/644A61P43/00
Inventor DEFREES, SHAWN
Owner NOVO NORDISK AS
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com