Receptor Tyrosine Kinase Signaling Pathway Analysis For Diagnosis And Therapy

Abstract

The invention provides a method for determining the activation status of receptor tyrosine kinase (RTK) pathways in either cell samples or patient samples by measuring receptor dimerization and relative amounts of protein-protein complexes or activated effector proteins that are characteristic of an RTK pathway. The invention also provides a method of using such status information to select patients responsive to pathway-specific drugs, and more particularly, to methods for measuring ErbB receptors and receptor complexes and using such information to select patients responsive to ErbB pathway-specific drugs. Preferably, methods of the invention are implemented by using sets of binding compounds having releasable molecular tags that are specific for multiple components of one or more complexes formed in RTK activation. After binding, molecular tags are released and separated from the assay mixture for analysis.

1 . A method of profiling one or more components of a signaling pathway comprising one or more cell surface receptors, cell surface receptor complexes, signaling complexes, or effector proteins, the method comprising the steps of:
 providing for each of the one or more components at least one cleaving probe specific for a first antigenic determinant and having a cleavage-inducing moiety with an effective proximity;   providing one or more binding compounds specific for a second antigenic determinant of each of the one or more components, such that each binding compound has one or more molecular tags each attached thereto by a cleavable linkage, and such that the one or more molecular tags attached to different binding compounds have different separation characteristics so that upon separation molecular tags from different binding compounds form distinct peaks in a separation profile;   mixing the cleaving probes, the binding compounds, and the one or more components such that cleaving probes specifically bind to the first antigenic determinants and binding compounds specifically bind to the second antigenic determinants and such that cleavable linkages of the binding compounds are within the effective proximity of cleavage-inducing moieties of the cleaving probes so that molecular tags are released; and   separating and identifying the released molecular tags to determine the presence or absence or the amount of the one or more components of the signaling pathway.   
     
     
         2 . The method of  claim 1  wherein said step of mixing includes generating an active species by said cleavage-inducing moiety, the active species cleaving said cleavable linkages within said effective proximity. 
     
     
         3 . The method of  claim 2  wherein whenever said one or more components includes a cell surface receptor complex or a signaling complex, said steps of providing include providing a cleaving probe specific for a first protein of the cell surface receptor complex or the signaling complex and providing one or more binding compounds specific for a second protein of the cell surface receptor complex or signaling complex. 
     
     
         4 . The method of  claim 3  wherein whenever said one or more components includes an effector protein that has a post-translational modification site, said steps of providing include providing a second binding compound specific for the post-translational modification site, wherein the post-translational modification site is different from said second antigenic determinant. 
     
     
         5 . The method according to  claims 1 ,  2 ,  3 , or  4  wherein said signaling pathway is an mTOR pathway, a Ras-MAPK pathway, or a PI3K-Akt pathway. 
     
     
         6 . The method of  claim 2  wherein said cleavage-inducing moiety is a photosensitizer and said active species is singlet oxygen. 
     
     
         7 . The method of  claim 2  wherein said separation characteristic is electrophoretic mobility and wherein said separation profile is an electropherogram. 
     
     
         8 . A method of determining disease status of a patient suffering from a disease characterized by aberrant expression of one or more components of a signaling pathway comprising one or more cell surface receptors, cell surface receptor complexes, signaling complexes, or effector proteins, the method comprising the steps of:
 measuring directly in a patient sample an amount of each of one or more components of the signaling pathway;   comparing each such amount to its corresponding amount in a reference sample; and   correlating differences in the amounts from the patient sample and the respective corresponding amounts from the reference sample to the disease status the patient;   wherein the step of measuring includes the steps of:   providing for each of the one or more components at least one cleaving probe specific for a first antigenic determinant and having a cleavage-inducing moiety with an effective proximity;   providing one or more binding compounds specific for a second antigenic determinant of each of the one or more components, such that each binding compound has one or more molecular tags each attached thereto by a cleavable linkage, and such that the one or more molecular tags attached to different binding compounds have different separation characteristics so that upon separation molecular tags from different binding compounds form distinct peaks in a separation profile;   mixing the cleaving probes, the binding compounds, and the one or more components such that cleaving probes specifically bind to the first antigenic determinants and binding compounds specifically bind to the second antigenic determinants and such that cleavable linkages of the binding compounds are within the effective proximity of cleavage-inducing moieties of the cleaving probes so that molecular tags are released; and   separating and identifying the released molecular tags to determine the presence or absence or the amount of the one or more components of the signaling pathway.   
     
     
         9 . The method of  claim 8  wherein said step of mixing includes generating an active species by said cleavage-inducing moiety, the active species cleaving said cleavable linkages within said effective proximity. 
     
     
         10 . The method of  claim 9  wherein whenever said one or more components includes a cell surface receptor complex or a signaling complex, said steps of providing include providing a cleaving probe specific for a first protein of the cell surface receptor complex or the signaling complex and providing one or more binding compounds specific for a second protein of the cell surface receptor complex or signaling complex. 
     
     
         11 . The method of  claim 10  wherein whenever said one or more components includes an effector protein that has a post-translational modification site, said steps of providing include providing a second binding compound specific for the post-translational modification site, wherein the post-translational modification site is different from said second antigenic determinant. 
     
     
         12 . The method according to  claims 8 ,  9 ,  10 , or  11  wherein said signaling pathway is an mTOR pathway, a Ras-MAPK pathway, or a PI3K-Akt pathway. 
     
     
         13 . The method of  claim 12  wherein said cleavage-inducing moiety is a photosensitizer and said active species is singlet oxygen. 
     
     
         14 . The method of  claim 13  wherein said separation characteristic is electrophoretic mobility and wherein said separation profile is an electropherogram. 
     
     
         15 . A method of selecting a patient responsive to a pathway-specific drug of an mTOR pathway comprising components of one or more cell surface receptors, cell surface receptor complexes, signaling complexes, or effector proteins, the method comprising the steps of:
 measuring directly in a patient sample an amount of each of one or more components of the mTOR pathway;   comparing each such amount to its corresponding amount in a reference sample; and   correlating differences in the amounts from the patient sample and the respective corresponding amounts from the reference sample to the responsiveness of the patient to the pathway-specific drug;   wherein the step of measuring includes the steps of:   providing for each of the one or more components at least one cleaving probe specific for a first antigenic determinant and having a cleavage-inducing moiety with an effective proximity;   providing one or more binding compounds specific for a second antigenic determinant of each of the one or more components, such that each binding compound has one or more molecular tags each attached thereto by a cleavable linkage, and such that the one or more molecular tags attached to different binding compounds have different separation characteristics so that upon separation molecular tags from different binding compounds form distinct peaks in a separation profile;   mixing the cleaving probes, the binding compounds, and the one or more components such that cleaving probes specifically bind to the first antigenic determinants and binding compounds specifically bind to the second antigenic determinants and such that cleavable linkages of the binding compounds are within the effective proximity of cleavage-inducing moieties of the cleaving probes so that molecular tags are released; and   separating and identifying the released molecular tags to determine the presence or absence or the amount of the one or more components of the mTOR pathway.   
     
     
         16 . The method of  claim 15  wherein said step of mixing includes generating an active species by said cleavage-inducing moiety, the active species cleaving said cleavable linkages within said effective proximity. 
     
     
         17 . The method of  claim 16  wherein whenever said one or more components includes a cell surface receptor complex or a signaling complex, said steps of providing include providing a cleaving probe specific for a first protein of the cell surface receptor complex or the signaling complex and providing one or more binding compounds specific for a second protein of the cell surface receptor complex or signaling complex. 
     
     
         18 . The method of  claim 17  wherein whenever said one or more components includes an effector protein that has a post-translational modification site, said steps of providing include providing a second binding compound specific for the post-translational modification site, wherein the post-translational modification site is different from said second antigenic determinant. 
     
     
         19 . The method of  claim 18  wherein said cleavage-inducing moiety is a photosensitizer and said active species is singlet oxygen. 
     
     
         20 . The method of  claim 19  wherein said separation characteristic is electrophoretic mobility and wherein said separation profile is an electropherogram. 
     
     
         21 . The method of  claim 20  wherein said pathway specific drug is selected from the group consisting of rapamycin, FK506, CCI-779, LY294002, RAD001, AR23573. 
     
     
         22 . A method of selecting a patient responsive to a pathway-specific drug of a PI3K-Akt pathway comprising components of one or more cell surface receptors, cell surface receptor complexes, signaling complexes, or effector proteins, the method comprising the steps of:
 measuring directly in a patient sample an amount of each of one or more components of the PI3K-Akt pathway;   comparing each such amount to its corresponding amount in a reference sample; and   correlating differences in the amounts from the patient sample and the respective corresponding amounts from the reference sample to the responsiveness of the patient to the pathway-specific drug;   wherein the step of measuring includes the steps of:   providing for each of the one or more components at least one cleaving probe specific for a first antigenic determinant and having a cleavage-inducing moiety with an effective proximity;   providing one or more binding compounds specific for a second antigenic determinant of each of the one or more components, such that each binding compound has one or more molecular tags each attached thereto by a cleavable linkage, and such that the one or more molecular tags attached to different binding compounds have different separation characteristics so that upon separation molecular tags from different binding compounds form distinct peaks in a separation profile;   mixing the cleaving probes, the binding compounds, and the one or more components such that cleaving probes specifically bind to the first antigenic determinants and binding compounds specifically bind to the second antigenic determinants and such that cleavable linkages of the binding compounds are within the effective proximity of cleavage-inducing moieties of the cleaving probes so that molecular tags are released; and   separating and identifying the released molecular tags to determine the presence or absence or the amount of the one or more components of the PI3K-Akt pathway.   
     
     
         23 . The method of  claim 22  wherein said step of mixing includes generating an active species by said cleavage-inducing moiety, the active species cleaving said cleavable linkages within said effective proximity. 
     
     
         24 . The method of  claim 23  wherein whenever said one or more components includes a cell surface receptor complex or a signaling complex, said steps of providing include providing a cleaving probe specific for a first protein of the cell surface receptor complex or the signaling complex and providing one or more binding compounds specific for a second protein of the cell surface receptor complex or signaling complex. 
     
     
         25 . The method of  claim 24  wherein whenever said one or more components includes an effector protein that has a post-translational modification site, said steps of providing include providing a second binding compound specific for the post-translational modification site, wherein the post-translational modification site is different from said second antigenic determinant. 
     
     
         26 . The method of  claim 25  wherein said cleavage-inducing moiety is a photosensitizer and said active species is singlet oxygen. 
     
     
         27 . The method of  claim 26  wherein said separation characteristic is electrophoretic mobility and wherein said separation profile is an electropherogram. 
     
     
         28 . The method of  claim 27  wherein said pathway specific drug is selected from the group consisting of Cetuximab (Erbitux), Trastuzumab (Herceptin), h-R3 (TheraCIM), ABX-EGF, MDX-447, ZD-1839 (Iressa), OSI-774 (Tarceva), PKI 166, GW2016, CI-1033, EKB-569, EMD 72000, and 4D4 Mab. 
     
     
         29 . A method of selecting a patient responsive to one or more pathway-specific drugs, the method comprising the steps of:
 measuring in a patient sample an amount of each of one or more cell surface receptors, cell surface receptor complexes, signaling complexes, and post-translational modifications thereof;   comparing each such amount to its corresponding amount in a reference sample; and   correlating differences in the amounts from the patient sample and the respective corresponding amounts from the reference sample to the responsiveness of the patient to one or more pathway-specific drugs.   
     
     
         30 . The method of  claim 29  wherein said patient sample is a fixed tissue sample or a frozen tissue sample. 
     
     
         31 . The method of  claim 29  wherein said one or more cell surface receptors and cell surface receptor complexes are one or more receptor tyrosine kinases and receptor tyrosine kinase complexes and wherein said disease is a cancer. 
     
     
         32 . The method of  claim 31  wherein said one or more cell surface receptors and cell surface receptor complexes are one or more ErbB receptors and ErbB receptor complexes. 
     
     
         33 . The method of  claim 32  wherein said one or more ErbB receptors are one or more of Her1, Her2, or Her3 and said one or more ErbB receptor complexes are one or more of Her1-Her2 heterodimers, Her1-Her3 heterodimers, Her2-Her2 homodimers, or Her2-Her3 heterodimers. 
     
     
         34 . The method of  claim 29  wherein said step of correlating includes forming a response index that is a function of one or more of said amounts of said one or more cell surface receptors, cell surface receptor complexes, signaling complexes, and post-translational modifications thereof, the response index having a value that is predictive of a patient's response to one or more pathway-specific drugs. 
     
     
         35 . A method of determining an activation status of a receptor tyrosine kinase of cells in a sample, the method comprising the step of simultaneously measuring (a) expression of a receptor tyrosine kinase dimer, and (b) expression of at least one signaling complex selected from the group consisting of Her1//HShc, Grb2//Sos, Her1//Grb7, Her1//RasGAP, Grb2//Shc, Her2//Shc, 14-3-3//Bad, Her3//PI3K, Her3//Shc, Her3//Grb7, Her3//Shc, Her3//Grb7, Her2//Shc, YAP//Her4, Her3//PI3K, Her3//Shc, Her3//Grb7, YAP//Her4, YAP//Her4, IGF-1R//PI3K, IGF-1R//Shc; IGFR//IRS1; VEGFR//Shc; VEGFR//PI(3)K; VEGFR//Src; VEGFR//FRS2, VEGFR//Shc; VEGFR//Src; VEGFR//FRS2, PDGFRa//Crk, PDGFR//Grb2; PDGFR//Grb7; PDGFR//Nck; PDGFR//Shc; PDGFR//STAT5, PDGFRa//Crk, PDGFRb//GAP, PDGFR//Grb2; PDGFR//Grb7; PDGFR//Nck; PDGFR//Shc, PDGFR//Shp2; PDGFR//RasGAP, DGFR//STAT5, PDGFRb//GAP, PDGFR//Grb2; PDGFR//Grb7; PDGFR//Nck; PDGFR//Shc, PDGFR//Shp2, DGFR//RasGAP; PDGFR//STAT5, Kit//Shp-1; Kit//p85PI(3)K; Kit//Grb2; Kit//CRKL, FGFR//PLCg1; FGFR//Crk; FGFR//FRS2; FGFR//Shp2; FGFR//Shb, Trk//p75NTR; Trk//PI(3)K. 
     
     
         36 . The method of  claim 35  wherein said receptor tyrosine kinase dimer is a Her1-Her1 dimer and said signaling complex is selected from the group consisting of Her1//Shc, Grb2//Sos, Her1//Grb7, Her1//RasGAP. 
     
     
         37 . The method of  claim 35  wherein said receptor tyrosine kinase dimer is a Her1-Her2 dimer and said signaling complex is selected from the group consisting of Her1//Shc, Grb2//Shc, Her2//Shc, Grb2//Sos, 14-3-3//Bad, Her1//RasGAP. 
     
     
         38 . The method of  claim 35  wherein said receptor tyrosine kinase dimer is a Her1-Her3 dimer and said signaling complex is selected from the group consisting of Her3//PI3K, Her3//Shc, Her3//Grb7, Her1//Shc, Grb2//Sos, 14-3-3//Bad, Her1//RasGAP. 
     
     
         39 . The method of  claim 35  wherein said receptor tyrosine kinase dimer is a Her1-Her4 dimer and said signaling complex is selected from the group consisting of Her3//PI3K, Her1//Shc, Grb2//Sos, Her1//RasGAP. 
     
     
         40 . The method of  claim 35  wherein said receptor tyrosine kinase dimer is a Her2-Her3 dimer and said signaling complex is selected from the group consisting of Her3//PI3K, Her3//Shc, Her3//Grb7, Grb2//Shc, Her2//Shc, Grb2//Sos, 14-3-3//Bad, Her1//RasGAP. 
     
     
         41 . A method of determining an activation status of a receptor tyrosine kinase of cells in a sample, the method comprising the step of simultaneously measuring (a) expression of a receptor tyrosine kinase dimer, and (b) expression of at least one activated effector protein. 
     
     
         42 . The method of  claim 41  wherein said receptor tyrosine kinase dimer is a Her1-Her1 dimer and said effector protein is selected from the group consisting of Ras-GTP; Raf; Mek; Erk1/2; Jnk1/2; STAT3; PLCg; and PKC. 
     
     
         43 . The method of  claim 41  wherein said receptor tyrosine kinase dimer is a Her2-Her3 dimer and said effector protein is selected from the group consisting of Ras-GTP; Raf; Mek; Erk1/2; Rsk; PLCg; PKC; PI(3)K; Akt; IkB; p70s6k; GSK3b; mTor; Bad; and STAT3. 
     
     
         44 . The method of  claim 41  wherein said receptor tyrosine kinase dimer is a Her1-Her1 homodimer and wherein said activated effector protein is a phosphorylated Erk1/2 protein, a phosphorylated Jnk protein, or a phosphorylated p38 protein. 
     
     
         45 . The method of  claim 41  wherein said receptor tyrosine kinase dimer is a Her2-Her3 heterodimer and wherein said activated effector protein is a phosphorylated Akt protein, a phosphorylated Erk1/2 protein, a phosphorylated Jnk protein, or a phosphorylated p38 protein.