Drug selection for lung cancer therapy using antibody-based arrays
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.
1. A method for selecting a suitable anticancer drug for the treatment of a lung tumor, the method comprising:
(a) lysing cells isolated from the lung tumor after administration of an anticancer drug, or prior to incubation with the anticancer drug, to produce a cellular extract;
(b) 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 activation state is a phosphorylation state, wherein the capture antibodies are restrained on a solid support, 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) washing and then 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 a facilitating moiety,
wherein the activation state-dependent antibodies are labeled with a first member of a signal amplification pair, and
wherein the facilitating moiety, which is glucose oxidase, generates an oxidizing agent which channels to and reacts with the first member of the signal amplification pair, which is a peroxidase in proximity to the glucose oxidase;
(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
(c) determining whether the anticancer drug is suitable or unsuitable for the treatment of the lung 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 lung tumor.
3. The method of claim 2 , wherein the circulating cells are isolated from a sample by immunomagnetic separation.
4. The method of claim 3 , wherein the sample is selected from the group consisting of whole blood, serum, plasma, sputum, bronchial lavage fluid, urine, nipple aspirate, lymph, saliva, fine needle aspirate, and combinations thereof.
5. The method of claim 2 , wherein the circulating cells are selected from the group consisting of circulating tumor cells, circulating endothelial cells, circulating endothelial progenitor cells, cancer stem cells, disseminated tumor cells, and combinations thereof.
6. The method of claim 1 , wherein the cells are isolated from tumor tissue.
7. The method of claim 1 , wherein the isolated cells are stimulated in vitro with growth factors.
8. The method of claim 7 , wherein the isolated cells are lysed following growth factor stimulation to produce the cellular extract.
9. The method of claim 1 , wherein the anticancer drug comprises an agent that interferes with the function of activated signal transduction pathway components in cancer cells.
10. The method of claim 9 , wherein the anticancer drug is selected from the group consisting of a monoclonal antibody, tyrosine kinase inhibitor, chemotherapeutic agent, radiotherapeutic agent, vaccine, and combinations thereof.
11. The method of claim 10 , wherein the monoclonal antibody is selected from the group consisting of trastuzumab (Herceptin®), alemtuzumab (Campath®), bevacizumab (Avastin®), cetuximab (Erbitux®), gemtuzumab (Mylotarg®), panitumumab (Vectibix™), rituximab (Rituxan®), tositumomab (BEXXAR®), and combinations thereof.
12. The method of claim 10 , wherein the tyrosine kinase inhibitor is selected from the group consisting of gefitinib (Iressa®), sunitinib (Sutent®), erlotinib (Tarceva®), lapatinib (GW-572016), canertinib (CI 1033), semaxinib (SU5416), vatalanib (PTK787/ZK222584), sorafenib (BAY 43-9006), imatinib mesylate (Gleevec®), leflunomide (SU101), vandetanib (ZACTIMA™; ZD6474), and combinations thereof.
13. The method of claim 10 , wherein the chemotherapeutic agent is selected from the group consisting of pemetrexed (ALIMTA®), gemcitabine (Gemzar®), sirolimus (rapamycin), rapamycin analogs, platinum compounds, carboplatin, cisplatin, satraplatin, paclitaxel (Taxol®), temsirolimus (CCI-779), everolimus (RAD001), and combinations thereof.
14. The method of claim 10 , wherein the radiotherapeutic agent is selected from the group consisting of 47 Sc, 64 Cu, 67 Cu, 89 Sr, 86 Y, 87 Y, 90 Y, 105 Rh, 111 Ag, 111 In, 117m Sn, 149 Pm, 153 Sm, 166 Ho, 177 Lu, 186 Re, 188 Re, 211 At, 212 Bi, and combinations thereof.
15. The method of claim 10 , wherein the anticancer drug is a member selected from the group consisting of carboplatin, paclitaxel (Taxol®), bevacizumab (Avastin®), pemetrexed (ALIMTA®), erlotinib (Tarceva®), gemcitabine (Gemzar®), sorafenib (BAY 43-9006), vandetanib (ZACTIMA™; ZD6474), and combinations thereof.
16. The method of claim 1 , wherein the one or more analytes comprise a plurality of signal transduction molecules.
17. The method of claim 16 , 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, and combinations thereof.
18. The method of claim 16 , wherein the plurality of signal transduction molecules is selected from the group consisting of EGFR (ErbB1), Her2 (ErbB2), Her3 (ErbB3), Her4 (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, PDGFR, cKit, Flt-3, Tie-1, Tie-2, Flt-3, cFMS, PDGFR, Abl, FTL 3, RET, Kit, HGFR, FGFR1, FGFR2, FGFR3, FGFR4, IGF-1R, and combinations thereof.
19. The method of claim 16 , wherein the plurality of signal transduction molecules is selected from the group consisting of ErbB1, ErbB2, ErbB4, and combinations thereof.
20. The method of claim 16 , wherein the plurality of signal transduction molecules is selected from the group consisting of VEGF, VEGFR-1, VEGFR-2, VEGFR-3, Eph-a, Eph-b, Eph-c, Eph-d, and combinations thereof.
21. The method of claim 16 , wherein the plurality of signal transduction molecules is selected from the group consisting of ErbB1, ErbB2, VEGFR-2, cMet, FGFR, and combinations thereof.
22. The method of claim 16 , wherein the plurality of signal transduction molecules is selected from the group consisting of VEGFR-2, VEGFR-3, Raf, PDGFR, cKit, Flt-3, Tie-1, Tie-2, and combinations thereof.
23. The method of claim 16 , wherein the plurality of signal transduction molecules is selected from the group consisting of VEGFR-1, VEGFR-2, VEGFR-3, Flt-3, CFMS, PDGFR, cKit, and combinations thereof.
24. The method of claim 1 , wherein the solid support is selected from the group consisting of glass, plastic, chips, pins, filters, beads, paper, membrane, fiber bundles, and combinations thereof.
25. The method of claim 1 , wherein the capture antibodies are restrained on the solid support in an addressable array.
26. The method of claim 1 , wherein the capture antibodies in each dilution series are serially diluted at least 2-fold.
27. The method of claim 1 , wherein the activation state-independent antibodies further comprise a detectable moiety.
28. The method of claim 27 , wherein the detectable moiety is a fluorophore.
29. The method of claim 27 , wherein the amount of the detectable moiety is correlative to the amount of one or more of the analytes.
30. The method of claim 1 , wherein the activation state-independent antibodies are directly labeled with the facilitating moiety.
31. The method of claim 1 , wherein the activation state-independent antibodies are labeled with the facilitating moiety via hybridization between an oligonucleotide conjugated to the activation state-independent antibodies and a complementary oligonucleotide conjugated to the facilitating moiety.
32. The method of claim 1 , wherein the activation state-dependent antibodies are directly labeled with the first member of the signal amplification pair.
33. The method of claim 1 , 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.
34. The method of claim 33 , wherein the first member of the binding pair is biotin.
35. The method of claim 33 , wherein the second member of the binding pair is streptavidin.
36. The method of claim 1 , wherein the oxidizing agent is hydrogen peroxide (H 2 O 2 ).
37. The method of claim 1 , wherein the peroxidase is horseradish peroxidase (HRP).
38. The method of claim 1 , wherein the second member of the signal amplification pair is a tyramide reagent.
39. The method of claim 38 , wherein the tyramide reagent is biotin-tyramide.
40. The method of claim 39 , wherein the amplified signal is generated by peroxidase oxidization of the biotin-tyramide to produce an activated tyramide.
41. The method of claim 40 , wherein the activated tyramide is directly detected.
42. The method of claim 40 , wherein the activated tyramide is detected upon the addition of a signal-detecting reagent.
43. The method of claim 42 , wherein the signal-detecting reagent is a streptavidin-labeled fluorophore.
44. The method of claim 42 , wherein the signal-detecting reagent is a combination of a streptavidin-labeled peroxidase and a chromogenic reagent.
45. The method of claim 44 , wherein the chromogenic reagent is 3,3′,5,5′-tetramethylbenzidine (TMB).
46. The method of claim 1 , wherein the lung tumor is derived from a subject with a non-small cell lung cancer (NSCLC).
47. The method of claim 46 , wherein the NSCLC is selected from the group consisting of a squamous cell carcinoma, an adenocarcinoma, a large cell carcinoma, bronchoalveolar carcinoma (BAC), and oat cell carcinoma.
48. The method of claim 1 , wherein step (b)(iii) comprises washing the plurality of detectable captured analytes prior to incubation with the second member of the signal amplification pair.