Profiling of signal pathway proteins to determine therapeutic efficacy

Patent metadata

Field	Value
Publication number	US-9664683-B2
Application number	US-201414194323-A
Country	US
Kind code	B2
Filing date	Feb 28, 2014
Priority date	Sep 2, 2011
Publication date	May 30, 2017
Grant date	May 30, 2017

Abstract

The present invention provides methods for detecting, measuring and quantitating the activation states of components of the PI3K signaling pathway in cells such as tumor cells. In particular embodiments, the present invention enable the determination of tumor adaptation to anticancer therapy. Accordingly, the present invention provides methods for improved cancer therapy selection/adjustment and disease monitoring.

First claim

What is claimed is:

1. A method for selecting a treatment for a subject having or suspected of having a solid tumor cancer, the method comprising:
(a) measuring the level of dimerization of at least two receptor tyrosine kinases (RTKs) analytes, wherein measuring comprises:
(i) incubating a cellular extract obtained from the subject with one or a plurality of dilution series of capture antibodies to form a plurality of captured analytes;
(ii) incubating the plurality of captured analytes with detection antibodies comprising a first or a plurality of first activation state-independent antibodies and a second or a plurality of second activation state-independent antibodies specific for a first member and a second member, respectively, of a dimerized pair of analytes to form a plurality of detectable captured dimerized analytes, wherein the first activation state-independent antibodies are labeled with a facilitating moiety, the second activation state-independent antibodies are labeled with a first member of a signal amplification pair, and the facilitating moiety generates an oxidizing agent which channels to and reacts with the first member of the signal amplification pair;
(iii) incubating the plurality of detectable captured dimerized analytes with a second member of the signal amplification pair to generate an amplified signal; and
(iv) detecting the amplified signal generated from the first and second members of the signal amplification pair; and

(b) selecting an anticancer drug by comparing the dimerization of the at least two RTKs to a reference dimerization profile of the same two RTK wherein the reference dimerization profile is generated in the absence of the anticancer drug.

2. The method of claim 1 , further comprises calibrating the level of dimerization of at least two RTKs against a standard curve generated for said at least two RTKs.

3. The method of claim 1 , wherein the cellular extract is isolated from a subject having cancer after administration of an anticancer drug.

4. The method of claim 1 , wherein the cellular extract is contacted with an anticancer drug.

5. The method of claim 1 , wherein the anticancer drug is selected from the group consisting of a PI3K modulating compound, a RTK modulating compound, or a combination thereof.

6. The method of claim 1 , wherein said at least two RTKs is a member selected form the group consisting of a HER1/HER2 dimer, a HER1/HER3 dimer, a HER2/HER3 dimer, a HER2/HER2 dimer, a HER2/HER4 dimer, a p95HER2/HER3 dimer, and a p95HER2/HER2 dimer.

7. The method of claim 1 , wherein the cellular extract is isolated from a subject having or suspected of having a cancer selected from the group consisting of breast, lung, pancreatic, colorectal, or gastric cancer.

8. The method of claim 1 , wherein the first activation state-independent antibodies are directly labeled with the facilitating moiety.

9. The method of claim 1 , wherein the second activation state-independent antibodies are directly labeled with the first member of the signal amplification pair.

10. The method of claim 1 , wherein the second activation state-independent antibodies are labeled with the first member of the signal amplification pair via binding between a first member of a binding pair conjugated to the activation state-independent antibodies and a second member of the binding pair conjugated to the first member of the signal amplification pair.

11. The method of claim 1 , wherein the first member of the binding pair is biotin and/or the second member of the binding pair is streptavidin.

12. The method of claim 1 , wherein the facilitating moiety is glucose oxidase.

13. The method of claim 12 , wherein the glucose oxidase and the activation state-independent antibodies are conjugated to a sulfhydryl-activated dextran molecule.

14. The method of claim 13 , wherein the sulfhydryl-activated dextran molecule has a molecular weight of about 500 kDa.

15. The method of claim 1 , wherein the capture antibodies are on a solid support, selected from the group consisting of glass, plastic, chips, pins, filters, beads, paper, membrane, fiber bundles, and combinations thereof.

16. The method of claim 15 , wherein the capture antibodies are restrained on the solid support in an addressable array.

17. The method of claim 1 , wherein the first member of the signal amplification pair is a peroxidase.

18. The method of claim 17 , wherein the peroxidase is horseradish peroxidase (HRP).

19. The method of claim 17 , wherein the second member of the signal amplification pair is a tyramide reagent.

20. The method of claim 19 , wherein the tyramide reagent is biotin-tyramide.

21. The method of claim 20 , wherein the amplified signal is generated by peroxidase oxidization of the biotin-tyramide to produce an activated tyramide.

22. The method of claim 21 , wherein the activated tyramide is directly detected.

23. The method of claim 21 , wherein the activated tyramide is detected upon the addition of a signal-detecting reagent.

24. The method of claim 23 , wherein the signal-detecting reagent is a streptavidin-labeled fluorophore.

25. The method of claim 23 , wherein the signal-detecting reagent is a combination of a streptavidin-labeled peroxidase and a chromogenic reagent.

26. The method of claim 25 , wherein the chromogenic reagent is 3,3′,5,5′-tetramethylbenzidine (TMB).

27. A method for selecting a treatment for a subject having or suspected of having a solid tumor cancer, the method comprising:
(a) measuring the level of a PI3K complex activation, wherein said PI3K complex comprises i) dimerization of at least two receptor tyrosine kinases (RTKs) analytes; ii) a PI3K p85 subunit and a PI3K p110 subunit, said measuring comprises:
(i) incubating a cellular extract obtained from the subject with one or a plurality of dilution series of capture antibodies to form a plurality of captured analytes;
(ii) incubating the plurality of captured analytes with (1) first detection antibodies comprising a first or a plurality of first activation state-independent antibodies specific for one member of a dimerized receptor tyrosine kinase pair or a PI3K p110 subunit; and (2) second detection antibodies comprising either second or a plurality of second activation state-independent antibodies specific for one member of a dimerized receptor tyrosine kinase pair, a PI3K p85 or a PI3K p110 subunit or activation state-dependent antibodies specific for a PI3K p85 subunit and/or a PI3K p110 subunit to form a plurality of detectable captured dimerized and complexed analytes,

wherein the first detection antibodies are labeled with a facilitating moiety, the second detection antibodies are labeled with a first member of a signal amplification pair, and the facilitating moiety generates an oxidizing agent which channels to and reacts with the first member of the signal amplification pair;
(iii) incubating the plurality of detectable captured dimerized analytes with a second member of the signal amplification pair to generate an amplified signal; and
detecting the amplified signal generated from the first and second members of the signal amplification pair; and

(b) selecting an anticancer drug by comparing the level of the PI3K complex activation to a reference PI3K complex activation profile wherein the reference dimerization profile is generated in the absence of the anticancer drug.

Assignees

• Nestec Sa
• Pierian Holdings Inc

Inventors

• Singh Sharat
• Kim Phillip

Classifications

• G01N33/5759 Primary

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

• G01N33/57492 Primary

  Physics · mapped topic

• G01N33/5011 Primary

  for testing antineoplastic activity · CPC title

• G01N33/5041

  involving analysis of members of signalling pathways · CPC title

• G01N33/542

  with steric inhibition or signal modification, e.g. fluorescent quenching · CPC title