BIOMARKERS FOR DETERMINING SENSITIVITY OF BREAST CANCER CELLS TO HER2-TARGETED THERAPY

• US 20110071042A1
• Filed: 08/10/2010
• Published: 03/24/2011
• Est. Priority Date: 05/14/2009
• Status: Active Grant

• Current Assignee: Societe des Produits Nestle SA (Nestle SA)
• Original Assignee: Prometheus Laboratories Incorporated (Merck & Co., Inc.)
• Inventors: Singh, Sharat, Lee, Tani, Kim, Phillip, Ybarrondo, Belen, Kirkland, Richard, Liu, Xinjun
• Granted Patent: US 9,726,669 B2
• Time in Patent Office: Days
• US Class Current: 506/9
• CPC Class Codes: G01N 2800/52 Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
• CPC Class Codes: G01N 33/5011 for testing antineoplastic activity
• CPC Class Codes: G01N 33/57415 of breast

Abstract

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

Claims

1. A method for determining the sensitivity of a cell to a compound that modulates HER2 activity, the method comprising:

(a) contacting the cell with the compound;

(b) lysing the cell to produce a cellular extract;

(c) determining the activation level of HER2 or p95HER2 in the cellular extract; and

(d) comparing the activation level of HER2 or p95HER2 determined in step (c) to a reference activation level of HER2 or p95HER2,

wherein the presence of a higher level of HER2 or p95HER2 activation in the cellular extract compared to the reference activation level of HER2 or p95HER2 indicates that the cell is not sensitive to the compound.

2. The method of claim 1, wherein the compound inhibits HER2 activity.

3. The method of claim 2, wherein the compound is selected from the group consisting of a monoclonal antibody, tyrosine kinase inhibitor, and combinations thereof.

4. The method of claim 3, wherein the monoclonal antibody is selected from the group consisting of trastuzumab (Herceptin®

), pertuzumab (2C4), and combinations thereof.

5. The method of claim 3, wherein the tyrosine kinase inhibitor is selected from the group consisting of gefitinib, erlotinib, pilitinib, canertinib, lapatinib, and combinations thereof.

6. The method of claim 1, wherein the reference activation level of HER2 or p95HER2 is obtained from a compound-sensitive cell that is treated with the compound.

7. The method of claim 6, wherein the compound-sensitive cell is a BT-474 cell.

8. The method of claim 6, wherein the level of HER2 activation in the cellular extract is at least 2 to 3-fold higher than the reference activation level of HER2.

9. The method of claim 6, wherein the level of p95HER2 activation in the cellular extract is at least 5-fold higher than the reference activation level of p95HER2.

10. The method of claim 6, wherein the presence of a similar or lower level of HER2 or p95HER2 activation in the cellular extract compared to the reference activation level of HER2 or p95HER2 indicates that the cell is sensitive to the compound.

11. The method of claim 1, wherein the reference activation level of HER2 or p95HER2 is obtained from a cell that is not treated with the compound.

12. The method of claim 1, wherein the method comprises determining the activation level of both HER2 and p95HER2 in the cellular extract.

13. The method of claim 1, wherein the activation level of HER2 or p95HER2 comprises a phosphorylation level of HER2 or p95HER2.

14. The method of claim 6, further comprising determining the activation level of one or more additional signal transduction molecules in the cellular extract.

15. The method of claim 14, wherein the one or more additional signal transduction molecules is selected from the group consisting of EGFR (HER1), HER3, HER4, PI3K, AKT, MEK, PTEN, SGK3, 4E-BP1, ERK2 (MAPK1), ERK1 (MAPK3), PDK1, P70S6K, GSK-3β

, Shc, IGF-1R, c-MET, c-KIT, VEGFR1, VEGFR2, VEGFR3, a receptor dimer, and combinations thereof.

16. The method of claim 15, wherein the presence of a higher level of HER3 activation in the cellular extract compared to a reference activation level of HER3 indicates that the cell is not sensitive to the compound.

17. The method of claim 16, wherein the level of HER3 activation in the cellular extract is at least 2 to 3-fold higher than the reference activation level of HER3.

18. The method of claim 15, wherein the presence of a higher level of PI3K activation in the cellular extract compared to a reference activation level of PI3K indicates that the cell is not sensitive to the compound.

19. The method of claim 15, wherein the receptor dimer is selected from the group consisting of a p95HER2/HER3 heterodimer, HER2/HER2 homodimer, HER2/HER3 heterodimer, HER1/HER2 heterodimer, HER2/HER3 heterodimer, and combinations thereof.

20. The method of claim 19, wherein the presence of a higher level of p95HER2/HER3 heterodimer activation in the cellular extract compared to a reference activation level of p95HER2/HER3 heterodimer indicates that the cell is not sensitive to the compound.

21. The method of claim 1, wherein the cell is a tumor cell.

22. The method of claim 21, wherein the tumor cell is a circulating tumor cell or a fine needle aspirate (FNA) cell obtained from a tumor.

23. The method of claim 21, wherein the tumor cell is a breast cancer cell.

24. The method of claim 1, wherein the cell is isolated from a sample.

25. The method of claim 24, wherein the sample is obtained from a subject with breast cancer.

26. The method of claim 24, wherein the sample is a whole blood, serum, plasma, or tumor tissue sample.

27. The method of claim 1, further comprising the step (e) of providing the result of the comparison obtained in step (d) to a user in a readable format.

28. The method of claim 1, wherein determining the activation level of HER2 or p95HER2 in step (c) comprises detecting a phosphorylation level of HER2 or p95HER2 in the cellular extract with an antibody specific for phosphorylated HER2 or p95HER2.

29. The method of claim 1, wherein determining the activation level of HER2 in step (c) comprises:

(i) incubating the cellular extract with a dilution series of capture antibodies specific for HER2 to form a plurality of captured receptors, wherein the capture antibodies are restrained on a solid support;

(ii) incubating the plurality of captured receptors with detection antibodies comprising activation state-independent antibodies and activation state-dependent antibodies specific for HER2 to form a plurality of detectable captured receptors,

wherein the activation state-independent antibodies are labeled with a facilitating moiety, the activation state-dependent 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 receptors 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.

30. The method of claim 1, wherein determining the activation level of p95HER2 in step (c) comprises:

(i) incubating the cellular extract with a plurality of beads specific for an extracellular domain (ECD) binding region of full-length HER2;

(ii) removing the plurality of beads from the cellular extract, thereby removing full-length HER2 to form a cellular extract devoid of full-length HER2;

(iii) incubating the cellular extract devoid of full-length HER2 with a dilution series of capture antibodies specific for an intracellular domain (ICD) binding region of full-length HER2 to form a plurality of captured receptors, wherein the capture antibodies are restrained on a solid support;

(iv) incubating the plurality of captured receptors with detection antibodies comprising activation state-independent antibodies and activation state-dependent antibodies specific for an ICD binding region of full-length HER2 to form a plurality of detectable captured receptors,

wherein the activation state-independent antibodies are labeled with a facilitating moiety, the activation state-dependent 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;

(v) incubating the plurality of detectable captured receptors with a second member of the signal amplification pair to generate an amplified signal; and

(vi) detecting the amplified signal generated from the first and second members of the signal amplification pair.

31. The method of claim 30, wherein the plurality of beads specific for an ECD binding region comprises a streptavidin-biotin pair, wherein the streptavidin is attached to the bead and the biotin is attached to an antibody.

32. The method of claim 31, wherein the antibody is specific for the ECD binding region of full-length HER2.

33. The method of claim 30, wherein the solid support is selected from the group consisting of glass, plastic, chips, pins, filters, beads, paper, membrane, fiber bundles, and combinations thereof.

34. The method of claim 30, wherein the capture antibodies are restrained on the solid support in an addressable array.

35. The method of claim 30, wherein the activation state-independent antibodies are directly labeled with the facilitating moiety.

36. The method of claim 30, wherein the activation state-dependent antibodies are directly labeled with the first member of the signal amplification pair.

37. The method of claim 30, wherein the activation state-dependent 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-dependent antibodies and a second member of the binding pair conjugated to the first member of the signal amplification pair.

38. The method of claim 37, wherein the first member of the binding pair is biotin.

39. The method of claim 37, wherein the second member of the binding pair is streptavidin.

40. The method of claim 30, wherein the facilitating moiety is glucose oxidase.

41. The method of claim 40, wherein the glucose oxidase and the activation state-independent antibodies are conjugated to a sulfhydryl-activated dextran molecule.

42. The method of claim 41, wherein the sulfhydryl-activated dextran molecule has a molecular weight of about 500 kDa.

43. The method of claim 40, wherein the oxidizing agent is hydrogen peroxide (H₂O₂).

44. The method of claim 43, wherein the first member of the signal amplification pair is a peroxidase.

45. The method of claim 44, wherein the peroxidase is horseradish peroxidase (HRP).

46. The method of claim 44, wherein the second member of the signal amplification pair is a tyramide reagent.

47. The method of claim 46, wherein the tyramide reagent is biotin-tyramide.

48. The method of claim 47, wherein the amplified signal is generated by peroxidase oxidization of the biotin-tyramide to produce an activated tyramide.

49. The method of claim 48, wherein the activated tyramide is directly detected.

50. The method of claim 48, wherein the activated tyramide is detected upon the addition of a signal-detecting reagent.

51. The method of claim 50, wherein the signal-detecting reagent is a streptavidin-labeled fluorophore.

52. The method of claim 50, wherein the signal-detecting reagent is a combination of a streptavidin-labeled peroxidase and a chromogenic reagent.

53. The method of claim 52, wherein the chromogenic reagent is 3,3′

,5,5′

-tetramethylbenzidine (TMB).