N-glycosylation of peptides and proteins

Patent metadata

Field	Value
Publication number	US-2016257983-A1
Application number	US-201415029730-A
Country	US
Kind code	A1
Filing date	Oct 15, 2014
Priority date	Oct 16, 2013
Publication date	Sep 8, 2016
Grant date	—

Abstract

A process for the production of a glycoconjugate by N-glycosylation of a protein or peptide comprising the sequence D/E-X—N—X—S/T, wherein each X is the same or different and is any natural amino acid other than proline, wherein the process comprises reacting the protein or peptide with a polyisoprenyl pyrophosphate of formula (I), or a salt thereof, in the presence of PglB: (I) to produce the glycoconjugate comprising the protein or peptide having a saccharide [SI] linked to the asparagine in the sequence D/E-X—N—X—S/T. Polyisoprenylpyrophosphates used as substrates in the biocatalytic process are also provided, as well as certain glycoconjugates.

First claim

1 . A process for the production of a glycoconjugate by N-glycosylation of a protein or peptide comprising the sequence D/E-X—N—X—S/T, wherein each X is the same or different and is any natural amino acid other than proline,
wherein the process comprises reacting the protein or peptide with a polyisoprenyl pyrophosphate of formula (I), or a salt thereof, in the presence of PglB:

wherein n is 1, 2, 3 or 4; and
[S1] is a monosaccharide or polysaccharide;
to produce the glycoconjugate comprising the protein or peptide having a saccharide [S1] linked to the asparagine in the sequence D/E-X—N—X—S/T.

2 . A process according to claim 1 , wherein the protein or peptide comprising the sequence D/E-X—N—X—S/T is an antibody.

3 . A process according to claim 1 , wherein n is 1, 2 or 3.

4 . A process according to claim 1 , wherein [S1] is a mono- or disaccharide.

5 . A process according to claim 1 , wherein the saccharide [S1] is modified at one or more positions.

6 . A process according to claim 1 , wherein [S1] is GlcNAc or GlcNAc-GlcNAc, and wherein [S1] is optionally further modified at one or more additional positions.

7 . A process according to claim 1 , wherein [S1] is modified to carry one or more groups R1, wherein R1 comprises a reactive group.

8 . A process according to claim 7 , wherein the reactive group is an azide, alkene or alkyne group.

9 . A process according to claim 7 , wherein R1 comprises the reactive group and a linker moiety L.

10 . A process according to claim 9 , wherein L is selected from an alkylene moiety, an oxy-alkylene- moiety and a moiety of formula —NH—C(═O)-alkylene-.

11 . A process according to claim 9 , wherein R1 is a group of formula —(C 1 -C 6 alkylene)-Y, —O—(C 1 -C 6 alkylene)-Y, —NH—C(═O)—(C 1 -C 6 alkylene)-Y, —NH—C(═O)—O—(C 1 -C 6 alkylene)-Y, —O—C(═O)—(C 1 -C 6 alkylene)-Y or —O—C(═O)—O—(C 1 -C 6 alkylene)-Y, wherein Y is a reactive group.

12 . A process according to claim 1 , which process further comprises reacting the glycoconjugate with a saccharide [S2] to produce a modified glycoconjugate comprising the protein or peptide having an extended saccharide [S1-S2] linked to the asparagine in the sequence D/E-X—N—X—S/T.

13 . A process according to claim 12 , wherein the saccharide [S2] is a mono- or polysaccharide modified to carry a group R2, wherein R2 comprises a reactive group capable of reacting with R1 or with OH.

14 . A process according to claim 13 , wherein [S2] is further modified to carry one or more labelling groups.

15 . A process according to claim 14 , wherein the labelling group is a radioactive label selected from 18-F, 64-C and 131-I.

16 . A polyisoprenyl pyrophosphate of formula (I) or a salt thereof

wherein n is 1, 2 or 3 and [S1] is a mono saccharide or polysaccharide.

17 . A process for the production of a glycoconjugate by N-glycosylation of a protein or peptide comprising the sequence D/E-X—N—X—S/T, wherein each X is the same or different and is any natural amino acid other than proline,
wherein the process comprises reacting the protein or peptide with a polyisoprenyl pyrophosphate of formula (I), or a salt thereof, in the presence of PglB:

wherein n is 1 to 7; and
[S1] is a monosaccharide or polysaccharide which is modified to carry one or more groups R1, wherein R1 comprises a reactive group;
to produce the glycoconjugate comprising the protein or peptide having a saccharide [S1] linked to the asparagine in the sequence D/E-X—N—X—S/T.

18 . A process according to claim 17 , which process further comprises reacting the glycoconjugate with a saccharide [S2] to produce a modified glycoconjugate comprising the protein or peptide having an extended saccharide [S1-S2] linked to the asparagine in the sequence D/E-X—N—X—S/T.

19 . A process according to claim 6 , wherein [S1] is modified to carry one or more groups R1, wherein R1 is a group of formula —(C 1 -C 6 alkylene)-Y, —O—(C 1 -C 6 alkylene)-Y, —NH—C(═O)—(C 1 -C 6 alkylene)-Y, —NH—C(═O)—O—(C 1 -C 6 alkylene)-Y, —O—C(═O)—(C 1 -C 6 alkylene)-Y or —O—C(═O)—O—(C 1 -C 6 alkylene)-Y, wherein Y is a reactive group.

Assignees

• Isis Innovation
• Glycovaxyn Ag

Inventors

• Davis Benjamin G
• Vijayakrishnan Balakumar
• Lui Feng
• Wacker Michael
• Faridmoayer Amirreza
• Kowarik Michael Thomas

Classifications

• C07K9/00

  Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof · CPC title

• C12P19/28

  N-glycosides · CPC title

• C07H11/04

  Phosphates; Phosphites; Polyphosphates · CPC title

• C12P19/18 Primary

  produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins · CPC title