Method of detecting cancer based on glycan biomarkers

Patent metadata

Field	Value
Publication number	US-9766245-B2
Application number	US-201414147446-A
Country	US
Kind code	B2
Filing date	Jan 3, 2014
Priority date	Jan 4, 2013
Publication date	Sep 19, 2017
Grant date	Sep 19, 2017

Abstract

The present invention provides a method for labeling or detecting a protein with certain glycosyl groups. The methods are particularly useful for detecting cancer cells comprising the detected glycosyl groups. The present invention further provides labeling agents and detection agents, labeled proteins and mixtures, and kits and arrays thereof.

First claim

What is claimed is:

1. A glycan comprising:
1) a first glycosyl group comprising fucose-α(1-2)-galactose; and
2) a second glycosyl group covalently linked to the first glycosyl group, wherein the second glycosyl group is covalently linked to the first glycosyl group at the C-3 position of the galactose on the first glycosyl group, and wherein the second glycosyl group comprises a reactive group (R), wherein linking the second glycosyl group to the first glycosyl group involves reacting a labeling agent with a glycosyltransferase, wherein the labeling agent is a compound of the formula:

wherein R is a substituent selected from the group consisting of a straight chain or branched C 1 -C 12 carbon chain bearing a carbonyl group, an azide group, a straight chain or branched C 1 -C 12 carbon chain bearing an azide group, a straight chain or branched C 1 -C 12 carbon chain bearing an alkyne, or a straight chain or branched C 1 -C 12 carbon chain bearing an alkene.

2. The glycan of claim 1 , wherein the glycan is attached to a glycoprotein or glycolipid.

3. The glycan of claim 1 , further comprising a detection agent covalently linked to the second glycosyl group, wherein the detection agent is covalently linked to the second glycosyl group via a reaction between a coupling moiety on the detection agent and the reactive group, and wherein the detection agent is selected from the group consisting of a fluorescent reagent, an enzymatic reagent capable of converting substrates calorimetrically or fluorometrically, a fluorescent and luminescent probe, a metal-binding probe, a protein-binding probe, a probe for antibody-based binding, a radioactive probe, a photocaged probe, a spin-label, spectroscopic probe, a heavy-atom containing probe, poly(ethylene)glycol-containing probe and poly(propylene)glycol-containing probe.

4. The glycan of claim 1 , wherein the glycan has the formula of

5. The glycan of claim 1 , wherein the glycosyltransferase is specific for the fucose-α(1-2)-galactose group.

6. The glycan of claim 1 , wherein the glycosyl transferase is a bacteria homologue of the human blood group A antigen glycosyltransferase (BgtA) or a variant or fragment thereof.

7. The glycan of claim 1 , wherein the glycosyltransferase is a human blood group A antigen glycosyltransferase or a variant or fragment thereof.

8. A reaction mixture for generating the glycan of claim 4 comprising
1) fucose-α(1-2)-galactose;
2) a labeling agent of the formula:

wherein R is a reactive group,
wherein R is a substituent selected from the group consisting of a straight chain or branched C 1 -C 12 carbon chain bearing a carbonyl group, an azide group, a straight chain or branched C 1 -C 12 carbon chain bearing an azide group, a straight chain or branched C 1 -C 12 carbon chain bearing an alkyne, or a straight chain or branched C 1 -C 12 carbon chain bearing an alkene;
wherein the labeling agent is recognized by the glycosyltransferase.

9. The mixture of claim 8 , wherein the glycan is attached to a glycoprotein or glycolipid.

10. The reaction mixture of claim 8 , further comprising a detection agent, and wherein the detection agent is selected from the group consisting of a fluorescent reagent, enzymatic reagent capable of converting substrates calorimetrically or fluorometrically, fluorescent and luminescent probe, metal-binding probe, protein-binding probe, probe for antibody-based binding, radioactive probe, photocaged probe, spin-label, spectroscopic probe, a heavy-atom containing probe, poly(ethylene)glycol-containing probe and poly(propylene)glycol-containing probe.

11. The reaction mixture of claim 8 , wherein labeling agent is placed on a solid support.

12. A method for labeling a glycan comprising fucose-α(1-2)-galactose, the method comprising: reacting the fucose-α(1-2)-galactose with a labeling agent in the presence of a glycosyltransferase, wherein the labeling agent is a compound of the formula:

wherein R is a reactive group selected from the group consisting of a straight chain or branched C 1 -C 12 carbon chain bearing a carbonyl group, an azide group, a straight chain or branched C 1 -C 12 carbon chain bearing an azide group, a straight chain or branched C 1 -C 12 carbon chain bearing an alkyne, or a straight chain or branched C 1 -C 12 carbon chain bearing an alkene; wherein the labeling agent is recognized by the glycosyltransferase;
wherein the reactive group is capable of reacting with a detection agent; and

wherein the glycosyltransferase is specific for the glycosyl group labeling agent.

Assignees

• California Inst Of Techn

Inventors

• Hsieh-Wilson Linda C
• Yi Wen
• Chaubard Jean-Luc

Classifications

• G01N33/5756

  involving tumour-associated glycolinkage [TAG] · CPC title

• G01N33/5759 Primary

  involving compounds localised on the membrane of tumour or cancer cells · CPC title

• G01N2400/02

  involving antibodies to sugar part of glycoproteins · CPC title

• G01N33/533

  with fluorescent label · CPC title

• C12P19/18

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