Quantitative control of sialylation and specific mono-sialylation

Patent metadata

Field	Value
Publication number	US-2016102333-A1
Application number	US-201514970734-A
Country	US
Kind code	A1
Filing date	Dec 16, 2015
Priority date	Jul 5, 2013
Publication date	Apr 14, 2016
Grant date	—

Abstract

The present disclosure is directed to the use of certain glycosyltransferase variants having N-terminal truncation deletions. It was found that the combination of two different truncation variants of human β-galactoside-α-2,6-sialyltransferase I (hST6Gal-I) exhibited different specific sialyltransferase enzymatic activities. In one example, under conditions wherein the first variant Δ89 hST6Gal-I catalyzed formation of bi-sialylated target molecules the second variant Δ108 hST6Gal-I catalyzed formation of mono-sialylated target molecules. Thus, disclosed are variants of mammalian glycosyltransferase, nucleic acids encoding the same, methods and means for recombinantly producing the variants of mammalian glycosyltransferase and use thereof, particularly for sialylating in a quantitatively controlled manner terminal acceptor groups of glycan moieties being part of glycoproteins such as immunoglobulins.

First claim

1 . A composition comprising an aqueous buffer permitting glycosyltransferase enzymatic activity, the composition further comprising:
(a) a glycosylated target molecule, the target molecule being selected from a glycoprotein and a glycolipid, the target molecule comprising a plurality of antennae, at least two of the antennae each having as terminal structure a β-D-galactosyl-1,4-N-acetyl-β-D-glucosamine moiety with a hydroxyl group at the C6 position in the galactosyl residue; (b) N-terminally truncated human β-galactoside-α-2,6-sialyltransferase I of SEQ ID NO:2 (Δ89 hST6Gal-I); (c) N-terminally truncated human β-galactoside-α-2,6-sialyltransferase I of SEQ ID NO:3 (Δ108 hST6Gal-I); (d) cytidine-5′-monophospho-N-acetylneuraminic acid as donor compound for a sialyltransferase-catalyzed reaction.

2 . The composition according to claim 1 , wherein the target molecule is a glycoprotein selected from the group consisting of a glycosylated cell surface protein, a glycosylated protein signaling molecule, a glycosylated immunoglobulin, and a glycosylated protein of viral origin.

3 . The composition according to claim 1 , wherein each of Δ89 hST6Gal-I and Δ108 hST6Gal-I is present in a pre-determined amount.

4 . The composition according to claim 3 , wherein each amount of Δ89 hST6Gal-I and Δ108 hST6Gal-I has a pre-determined enzymatic activity.

5 . A method for producing in vitro a sialylated target molecule with a controlled quantity of sialyl residues added to one or more antennal terminal structure(s) of the target molecule, the target molecule being selected from a glycoprotein and a glycolipid, the target molecule comprising a plurality of antennae, at least two of the antennae each having as terminal structure a β-D-galactosyl-1,4-N-acetyl-β-D-glucosamine moiety with a hydroxyl group at the C6 position in the galactosyl residue, the method comprising the steps of
(a) providing a composition according to claim 3 ;
(b) incubating the composition of step (a) under conditions permitting glycosyltransferase enzymatic activity and for a pre-determined time interval, thereby forming terminal antennal N-acetylneuraminyl-α2,6-β-D-galactosyl-1,4-N-acetyl-β-D-glucosamine residue(s), wherein Δ89 hST6Gal-I catalyzes formation of a bi-sialylated target molecule and Δ108 hST6Gal-I catalyzes formation of a mono-sialylated target molecule;
thereby producing in vitro the sialylated target molecule with a controlled quantity of sialyl residues added to one or more antennal terminal structure(s) of the target molecule.

6 . The method according to claim 5 , wherein the target molecule is incubated with Δ89 hST6Gal-I and Δ108 hST6Gal-I simultaneously in the same vessel and under the same conditions.

7 . The method according to claim 6 , wherein Δ89 hST6Gal-I catalyzes formation of a bi- or higher sialylated target molecule, and Δ108 hST6Gal-I catalyzes formation of a mono-sialylated target molecule.

8 . The method according to claim 7 , wherein a higher amount of Δ108 hST6Gal-I enzymatic activity relative to the amount of Δ89 hST6Gal-I enzymatic activity results in an increased likelihood of formation of a mono-sialylated target molecule compared to the likelihood of formation of a bi- or higher sialylated target molecule.

9 . The method according to claim 5 , wherein the target molecule is contains no sialyl residue as antennal terminal structure.

10 . The method according to claim 9 , wherein the target molecule is a monoclonal antibody of the IgG class, specifically selected from the group consisting of IgG1, IgG2, IgG3 and an IgG4.

11 . A preparation of glycosylated target molecules, the target molecules being immunoglobulin molecules of the IgG class, wherein the amounts of mono- and bi-sialylated target molecules in the preparation are controlled quantities, the preparation being obtained by a method according to claim 5 .

12 . A method for producing in vitro a sialylated target molecule with a single sialyl residue added to one antennal terminal structure of the target molecule, the method comprising the steps of
(a) providing a composition comprising
i. the target molecule, the target molecule being selected from a glycoprotein and a glycolipid, the target molecule comprising a plurality of antennae, at least two of the antennae each having as terminal structure a β-D-galactosyl-1,4-N-acetyl-β-D-glucosamine moiety with a hydroxyl group at the C6 position in the galactosyl residue;
ii. N-terminally truncated human β-galactoside-α-2,6-sialyltransferase I of SEQ ID NO:3 (Δ108 hST6Gal-I);
iii. cytidine-5′-monophospho-N-acetylneuraminic acid as donor compound for a sialyltransferase-catalyzed reaction;
(b) incubating the composition of step (a) under conditions permitting glycosyltransferase enzymatic activity, thereby forming per target molecule a single terminal antennal N-acetylneuraminyl-α2,6-β-D-galactosyl-1,4-N-acetyl-β-D-glucosamine residue; thereby producing in vitro the sialylated target molecule with a single sialyl residue added to one antennal terminal structure of the target molecule.

13 . The method according to claim 12 , wherein the target molecule provided in step (a) is free of α2,6 sialylated terminal antennal residues.

Assignees

• Roche Diagnostics Operations

Inventors

• Czabany Tibor
• Engel Alfred
• Greif Michael
• Jung Christine
• Luley Christiane
• Malik Sebastian
• Mueller Rainer
• Nidetzky Bernd
• Ribitsch Doris
• Schmoelzer Katharina
• Schwab Helmut
• Sobek Harald
• Suppmann Bernhard
• Thomann Marco
• Zitzenbacher Sabine

Classifications

• C12P21/005 Primary

  Glycopeptides, glycoproteins · CPC title

• C07K16/00

  Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies · CPC title

• C07K2317/14

  Specific host cells or culture conditions, e.g. components, pH or temperature · CPC title

• C12Y204/99001

  Beta-galactoside alpha-2,6-sialyltransferase (2.4.99.1) · CPC title

• C12N9/1081

  transferring other glycosyl groups (2.4.99) · CPC title