Claims for Patent: 8,163,499

Summary for Patent: 8,163,499

Title:	Drug selection for breast cancer therapy using antibody-based arrays
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.
Inventor(s):	Singh; Sharat (Rancho Santa Fe, CA), Harvey; Jeanne (Livermore, CA), Kim; Phillip (Irvine, CA), Liu; Xinjun (San Diego, CA), Liu; Limin (San Diego, CA), Barham; Robert (San Marcos, CA), Neri; Bruce (Carlsbad, CA)
Assignee:	Prometheus Laboratories Inc. (San Diego, CA)
Application Number:	12/511,017
Patent Claims:	1. A method for selecting a suitable anticancer drug for the treatment of a breast tumor, the method comprising: (a) isolating cells of a breast tumor after administration of an anticancer drug, or prior to incubation with an anticancer drug; (b) lysing the isolated 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 breast tumor is derived from a subject with ductal carcinoma or lobular carcinoma. 3. The method of claim 2, wherein the ductal carcinoma is invasive ductal carcinoma or ductal carcinoma in situ. 4. The method of claim 2, wherein the lobular carcinoma is invasive lobular carcinoma or lobular carcinoma in situ. 5. The method of claim 1, wherein the cells comprise circulating cells of the breast tumor. 6. The method of claim 5, wherein the circulating cells are isolated from a sample by immunomagnetic separation. 7. The method of claim 6, wherein the sample is selected from the group consisting of whole blood, serum, plasma, ductal lavage fluid, nipple aspirate, lymph, bone marrow aspirate, urine, saliva, fine needle aspirate, and combinations thereof. 8. The method of claim 5, 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. 9. The method of claim 1, wherein the cells are isolated from tumor tissue. 10. The method of claim 9, wherein the tumor tissue is primary tumor tissue or metastatic tumor tissue. 11. The method of claim 9, wherein the cells are isolated from tumor tissue as a fine needle aspirate sample. 12. The method of claim 1, wherein the isolated cells are stimulated in vitro with growth factors. 13. 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. 14. The method of claim 13, wherein the monoclonal antibody is selected from the group consisting of trastuzumab (Herceptin.RTM.), alemtuzumab (Campath.RTM.), bevacizumab (Avastin.RTM.), cetuximab (Erbitux.RTM.), gemtuzumab (Mylotarg.RTM.), panitumumab (Vectibix.TM.), rituximab (Rituxan.RTM.), tositumomab (BEXXAR.RTM.), and combinations thereof. 15. The method of claim 13, wherein the tyrosine kinase inhibitor is selected from the group consisting of gefitinib (Iressa.RTM.), sunitinib (Sutent.RTM.), erlotinib (Tarceva.RTM.), lapatinib (Tykerb.RTM.), canertinib (CI 1033), semaxinib (SU5416), vatalanib (PTK787/ZK222584), sorafenib (BAY 43-9006), imatinib mesylate (Gleevec.RTM.), leflunomide (SU101), vandetanib (ZACTIMA.TM.; ZD6474), and combinations thereof. 16. The method of claim 13, wherein the chemotherapeutic agent is selected from the group consisting of pemetrexed (ALIMTA.RTM.), gemcitabine (Gemzar.RTM.), sirolimus (rapamycin), rapamycin analogs, platinum compounds, carboplatin, cisplatin, satraplatin, paclitaxel (Taxol.RTM.), docetaxel (Taxotere.RTM.), temsirolimus (CCI-779), everolimus (RAD001), and combinations thereof. 17. The method of claim 13, wherein the hormonal therapeutic agent is selected from the group consisting of aromatase inhibitors, selective estrogen receptor modulators, steroids, finasteride, gonadotropin-releasing hormone agonists, pharmaceutically acceptable salts thereof, stereoisomers thereof, derivatives thereof, analogs thereof, and combinations thereof. 18. The method of claim 13, wherein the radiotherapeutic agent is selected from the group consisting of .sup.47Sc, .sup.64Cu, .sup.67Cu, .sup.89Sr, .sup.86Y, .sup.87Y, .sup.90Y, .sup.105Rh, .sup.111Ag, .sup.111In, .sup.117Sn, .sup.149Pm, .sup.153Sm, .sup.166Ho, .sup.177Lu, .sup.186Re, .sup.188Re, .sup.211At, .sup.212Bi, and combinations thereof. 19. The method of claim 1, wherein the one or more analytes comprise a plurality of signal transduction molecules. 20. The method of claim 19, 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. 21. The method of claim 19, 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. 22. The method of claim 19, 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. 23. 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. 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 activation state-dependent antibodies are directly labeled with the first member of the signal amplification pair. 27. 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. 28. The method of claim 27, wherein the first member of the binding pair is biotin. 29. The method of claim 27, wherein the second member of the binding pair is streptavidin. 30. The method of claim 1, wherein the sulfhydryl-activated dextran molecule has a molecular weight of 500 kDa. 31. The method of claim 1, wherein the oxidizing agent is hydrogen peroxide (H.sub.2O.sub.2). 32. The method of claim 31, wherein the first member of the signal amplification pair is a peroxidase. 33. The method of claim 32, wherein the peroxidase is horseradish peroxidase (HRP). 34. The method of claim 32, wherein the second member of the signal amplification pair is a tyramide reagent. 35. The method of claim 34, wherein the tyramide reagent is biotin-tyramide. 36. The method of claim 35, wherein the amplified signal is generated by peroxidase oxidization of the biotin-tyramide to produce an activated tyramide. 37. The method of claim 36, wherein the activated tyramide is directly detected. 38. The method of claim 36, wherein the activated tyramide is detected upon the addition of a signal-detecting reagent. 39. The method of claim 38, wherein the signal-detecting reagent is a streptavidin-labeled fluorophore. 40. The method of claim 38, wherein the signal-detecting reagent is a combination of a streptavidin-labeled peroxidase and a chromogenic reagent. 41. The method of claim 40, wherein the chromogenic reagent is 3,3',5,5'-tetramethylbenzidine (TMB). 42. A method for identifying the response of a breast tumor to treatment with an anticancer drug, the method comprising: (a) isolating cells of a breast tumor after administration of an anticancer drug, or prior to incubation with an anticancer drug; (b) lysing the isolated 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. 43. The method of claim 42, wherein the breast tumor is derived from a subject with ductal carcinoma or lobular carcinoma. 44. The method of claim 42, wherein the cells comprise circulating cells of the breast tumor. 45. The method of claim 42, wherein the cells are isolated from tumor tissue. 46. The method of claim 42, wherein the tumor tissue is primary tumor tissue or metastatic tumor tissue. 47. The method of claim 45, wherein the cells are isolated from tumor tissue as a fine needle aspirate sample. 48. The method of claim 42, 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. 49. The method of claim 42, wherein the one or more analytes comprise a plurality of signal transduction molecules. 50. The method of claim 49, 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. 51. The method of claim 49, 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. 52. The method of claim 49, 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. 53. The method of claim 42, wherein the activation state is selected from the group consisting of a phosphorylation state, ubiquitination state, complexation state, and combinations thereof. 54. A method for predicting the response of a subject having a breast tumor to treatment with an anticancer drug, the method comprising: (a) isolating cells of a breast tumor after administration of an anticancer drug, or prior to incubation with an anticancer drug; (b) lysing the isolated 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) predicting the likelihood that the subject will respond 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. 55. The method of claim 54, wherein the breast tumor is derived from a subject with ductal carcinoma or lobular carcinoma. 56. The method of claim 54, wherein the cells comprise circulating cells of the breast tumor. 57. The method of claim 54, wherein the cells are isolated from tumor tissue. 58. The method of claim 57, wherein the tumor tissue is primary tumor tissue or metastatic tumor tissue. 59. The method of claim 57, wherein the cells are isolated from tumor tissue as a fine needle aspirate sample. 60. The method of claim 54, 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. 61. The method of claim 54, wherein the one or more analytes comprise a plurality of signal transduction molecules. 62. The method of claim 61, 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. 63. The method of claim 61, 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. 64. The method of claim 61, 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. 65. The method of claim 54, wherein the activation state is selected from the group consisting of a phosphorylation state, ubiquitination state, complexation state, and combinations thereof. 66. A method for detecting the presence of a truncated receptor, said method comprising: (a) incubating a cellular extract with a plurality of beads specific for an extracellular domain (ECD) binding region of a full-length receptor; (b) removing said plurality of beads from said cellular extract, thereby removing said full-length receptor to form a cellular extract devoid of said full-length receptor; (c) incubating said cellular extract devoid of said full-length receptor with a plurality of capture antibodies, wherein said plurality of capture antibodies is specific for an intracellular domain (ICD) binding region of said truncated receptor and wherein said plurality of capture antibodies is restrained on a solid support to form a plurality of captured truncated receptors; (d) incubating the plurality of captured truncated receptors with detection antibodies specific for the corresponding truncated receptors to form a plurality of detectable captured truncated receptors; (e) incubating the plurality of detectable captured truncated receptors with first and second members of a signal amplification pair to generate an amplified signal; and (f) detecting an amplified signal generated from the first and second members of the signal amplification pair. 67. The method of claim 66, wherein said truncated receptor is p95ErbB2. 68. The method of claim 66, wherein said full-length receptor is ErbB2 (HER-2). 69. The method of claim 66, wherein said plurality of beads specific for an extracellular domain (ECD) binding region comprise a streptavidin-biotin pair, wherein the streptavidin is attached to the bead and the biotin is attached to an antibody. 70. The method of claim 69, wherein said antibody is specific for said ECD binding region of said full-length receptor. 71. The method of claim 66, wherein the cellular extract is produced by lysing circulating cells of a breast tumor. 72. The method of claim 66, wherein the cellular extract is produced by lysing cells isolated from tumor tissue. 73. The method of claim 72, wherein the tumor tissue is primary tumor tissue or metastatic tumor tissue. 74. The method of claim 72, wherein the cells are isolated from tumor tissue as a fine needle aspirate sample. 75. The method of claim 66, wherein an activation state of said plurality of detectable captured truncated receptors is interrogated. 76. The method of claim 75, wherein said activation state is selected from the group consisting of a phosphorylation state, ubiquitination state, complexation state, and combinations thereof. 77. A method for detecting the presence of a truncated receptor, said method comprising: (a) incubating a cellular extract with a plurality of beads specific for an extracellular domain (ECD) binding region of a full-length receptor; (b) removing said plurality of beads from said cellular extract, thereby removing said full-length receptor to form a cellular extract devoid of said full-length receptor; (c) incubating said cellular extract devoid of said full-length receptor with a plurality of capture antibodies, wherein said plurality of capture antibodies is specific for an intracellular domain (ICD) binding region of said truncated receptor and wherein said plurality of capture antibodies is restrained on a solid support to form a plurality of captured truncated receptors; (d) incubating the plurality of captured truncated receptors with detection antibodies comprising a plurality of activation state-independent antibodies and a plurality of activation state-dependent antibodies specific for the corresponding truncated receptors to form a plurality of detectable captured truncated 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; (e) incubating the plurality of detectable captured truncated receptors with a second member of the signal amplification pair to generate an amplified signal; and (f) detecting the amplified signal generated from the first and second members of the signal amplification pair. 78. The method of claim 77, wherein said truncated receptor is p95ErbB2. 79. The method of claim 77, wherein said full-length receptor is ErbB2 (HER-2). 80. The method of claim 77, wherein said plurality of beads specific for an extracellular domain (ECD) binding region comprise a streptavidin-biotin pair, wherein the streptavidin is attached to the bead and the biotin is attached to an antibody. 81. The method of claim 80, wherein said antibody is specific for said ECD binding region of said full-length receptor. 82. The method of claim 77, wherein the cellular extract is produced by lysing circulating cells of a breast tumor. 83. The method of claim 77, wherein the cellular extract is produced by lysing cells isolated from tumor tissue. 84. The method of claim 83, wherein the tumor tissue is primary tumor tissue or metastatic tumor tissue. 85. The method of claim 83, wherein the cells are isolated from tumor tissue as a fine needle aspirate sample. 86. The method of claim 77, wherein the facilitating moiety is glucose oxidase. 87. The method of claim 86, wherein the glucose oxidase and the activation state-independent antibodies are conjugated to a sulfhydryl-activated dextran molecule. 88. The method of claim 87, wherein the sulfhydryl-activated dextran molecule has a molecular weight of 500 kDa.

Details for Patent 8,163,499

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
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