Drug selection for breast cancer therapy using antibody-based arrays

Patent metadata

Field	Value
Publication number	US-9274116-B2
Application number	US-201314091114-A
Country	US
Kind code	B2
Filing date	Nov 26, 2013
Priority date	Feb 25, 2008
Publication date	Mar 1, 2016
Grant date	Mar 1, 2016

Abstract

The present invention provides compositions and methods for detecting the activation states of components of signal transduction pathways in tumor cells. Information on the activation states of components of signal transduction pathways derived from use of the invention can be used for cancer diagnosis, prognosis, and in the design of cancer treatments.

First claim

What is claimed is:

1. A method for selecting a suitable anticancer drug for the treatment of a breast tumor, the method comprising:
(a) incubating cells isolated from a breast tumor with an anticancer drug in vitro;
(b) lysing the incubated cells to produce a cellular extract;
(c) detecting an activation state of one or more analytes in the cellular extract using an assay comprising a plurality of dilution series of capture antibodies specific for the one or more analytes, wherein the capture antibodies are restrained on a solid support, and wherein the assay comprises:
(i) incubating the cellular extract with the 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 plurality of activation state-independent antibodies and a plurality of activation state-dependent antibodies specific for the corresponding analytes to form a plurality of detectable captured analytes,
wherein the activation state-independent antibodies are labeled with glucose oxidase, wherein the glucose oxidase and the activation state-independent antibodies are conjugated to a sulfhydryl-activated dextran molecule, wherein the activation state-dependent antibodies are labeled with a first member of a signal amplification pair, and wherein the glucose oxidase 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 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
(d) determining whether the anticancer drug is suitable or unsuitable for the treatment of the breast tumor by comparing the activation state detected for the one or more analytes with a reference activation profile generated in the absence of the anticancer drug.

2. The method of claim 1 , wherein the cells comprise circulating cells of the breast tumor.

3. The method of claim 1 , wherein the cells are isolated from tumor tissue.

4. The method of claim 1 , wherein the anticancer drug is selected from the group consisting of a monoclonal antibody, tyrosine kinase inhibitor, chemotherapeutic agent, hormonal therapeutic agent, radiotherapeutic agent, vaccine, and combinations thereof.

5. The method of claim 1 , wherein the one or more analytes comprise a plurality of signal transduction molecules.

6. The method of claim 5 , wherein the plurality of signal transduction molecules is selected from the group consisting of receptor tyrosine kinases, non-receptor tyrosine kinases, tyrosine kinase signaling cascade components, nuclear hormone receptors, nuclear receptor coactivators, nuclear receptor repressors, and combinations thereof.

7. The method of claim 5 , wherein the plurality of signal transduction molecules is selected from the group consisting of EGFR (ErbB1), HER-2 (ErbB2), p95ErbB2, HER-3 (ErbB3), HER-4 (ErbB4), Raf, SRC, Mek, NFkB-IkB, mTor, PI3K, VEGF, VEGFR-1, VEGFR-2, VEGFR-3, Eph-a, Eph-b, Eph-c, Eph-d, cMet, FGFR, cKit, Flt-3, Tie-1, Tie-2, Flt-3, cFMS, PDGFRA, PDGFRB, Abl, FTL 3, RET, Kit, HGFR, FGFR1, FGFR2, FGFR3, FGFR4, IGF-1R, ER, PR, NCOR, AIB1, and combinations thereof.

8. The method of claim 5 , wherein the plurality of signal transduction molecules is selected from the group consisting of ErbB1, ErbB2, p95ErbB2, ErbB3, ErbB4, VEGFR-1, VEGFR-2, VEGFR-3, ER, PR, and combinations thereof.

9. The method of claim 1 , wherein the cells are isolated from the breast tumor of a subject who has not received treatment with the anticancer drug.

10. The method of claim 1 , wherein the reference activation profile provides the activation state for the one or more analytes in cells of the breast tumor prior to treatment with the anticancer drug.

11. The method of claim 1 , wherein the activation state is selected from the group consisting of a phosphorylation state, ubiquitination state, complexation state, and combinations thereof.

12. The method of claim 1 , wherein the sulfhydryl-activated dextran molecule has a molecular weight of about 500 kDa.

13. The method of claim 1 , wherein the oxidizing agent is hydrogen peroxide (H 2 O 2 ).

14. The method of claim 13 , wherein the first member of the signal amplification pair is a peroxidase.

15. The method of claim 14 , wherein the peroxidase is horseradish peroxidase (HRP).

16. The method of claim 14 , wherein the second member of the signal amplification pair is a tyramide reagent.

17. A method for identifying the response of a breast tumor to treatment with an anticancer drug, the method comprising:
(a) incubating cells isolated from a breast tumor with an anticancer drug in vitro;
(b) lysing the incubated cells to produce a cellular extract;
(c) detecting an activation state of one or more analytes in the cellular extract using an assay comprising a plurality of dilution series of capture antibodies specific for the one or more analytes, wherein the capture antibodies are restrained on a solid support, and wherein the assay comprises:
(i) incubating the cellular extract with the 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 plurality of activation state-independent antibodies and a plurality of activation state-dependent antibodies specific for the corresponding analytes to form a plurality of detectable captured analytes,
wherein the activation state-independent antibodies are labeled with glucose oxidase, wherein the glucose oxidase and the activation state-independent antibodies are conjugated to a sulfhydryl-activated dextran molecule, wherein the activation state-dependent antibodies are labeled with a first member of a signal amplification pair, and wherein the glucose oxidase 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 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
(d) identifying the breast tumor as responsive or non-responsive to treatment with the anticancer drug by comparing the activation state detected for the one or more analytes with a reference activation profile generated in the absence of the anticancer drug.

18. The method of claim 17 , wherein the cells comprise circulating cells of the breast tumor.

19. The method of claim 17 , wherein the cells are isolated from tumor tissue.

20. The method of claim 17 , wherein the anticancer drug is selected from the group consisting of a monoclonal antibody, tyrosine kinase inhibitor, chemotherapeutic agent, hormonal therapeutic agent, radiotherapeutic agent, vaccine, and combinations thereof.

21. The method of claim 17 , wherein the one or more analytes comprise a plurality of signal transduction molecules.

22. The method of claim 21 wherein the plurality of signal transduction molecules is selected from the group consisting of receptor tyrosine kinases, non-receptor tyrosine kinases, tyrosine kinase signaling cascade components, nuclear hormone receptors, nuclear receptor coactivators, nuclear rec

Assignees

• Nestec Sa

Inventors

• Singh Sharat
• Harvey Jeanne
• Kim Phillip
• Liu Xinjun
• Liu Limin
• Barham Robert
• Neri Bruce

Classifications

• G01N33/57515 Primary

  of the breast · CPC title

• G01N33/5011

  for testing antineoplastic activity · CPC title

• G01N2800/60

  Complex ways of combining multiple protein biomarkers for diagnosis · CPC title

• G01N2800/52

  Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis · CPC title

• G01N33/57415 Primary

  Physics · mapped topic