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WO2003087761A2 - Profilage de molecules d'une maladie et reponse therapeutique utilisant des anticorps phospho-specifiques - Google Patents

Profilage de molecules d'une maladie et reponse therapeutique utilisant des anticorps phospho-specifiques Download PDF

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WO2003087761A2
WO2003087761A2 PCT/US2003/010573 US0310573W WO03087761A2 WO 2003087761 A2 WO2003087761 A2 WO 2003087761A2 US 0310573 W US0310573 W US 0310573W WO 03087761 A2 WO03087761 A2 WO 03087761A2
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signal transduction
disease
protein
patient
phospho
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WO2003087761A3 (fr
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Katherine Crosby
Bradley Smith
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Cell Signaling Technology Inc
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Cell Signaling Technology Inc
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57515—Immunoassay; Biospecific binding assay; Materials therefor for cancer of the breast
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • G01N2333/91205—Phosphotransferases in general
    • G01N2333/9121—Phosphotransferases in general with an alcohol group as acceptor (2.7.1), e.g. general tyrosine, serine or threonine kinases
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the invention relates generally to signaling proteins and antibodies, and their use to characterize and monitor disease.
  • the regulation of proteins by secondary modification represents an important cellular mechanism for regulating most aspects of cellular organization and control, including growth, development, homeostasis, and cellular communication.
  • protein phosphorylation plays a critical role in the etiology of many pathological conditions and diseases, including cancer, developmental disorders, autoimmune diseases, and diabetes.
  • protein modification it is not yet well understood at the molecular level. The reasons for this lack of understanding are, first, that the cellular modification system is extraordinarily complex, and second, that the technology necessary to unravel its complexity has not yet been fully developed.
  • the complexity of protein modification on a proteome-wide scale derives from three factors: the large number of modifying proteins, e.g. kinases, encoded in the genome, the much larger number of sites on substrate proteins that are modified by these enzymes, and the dynamic nature of protein expression during growth, development, disease states, and aging.
  • the human genome encodes, for example, over 520 different protein kinases, making them the most abundant class of enzymes known. See Hunter, Nature 411: 355-65 (2001).
  • Each of these kinases phosphorylates specific serine, threonine, or tyrosine residues located within distinct amino acid sequences, or motifs, contained within different protein substrates.
  • kinases phosphorylate many different proteins: it is estimated that one-third of all proteins encoded by the human genome are phosphorylated, and many are phosphorylated at multiple sites by different kinases. See Graves et al., Pharmacol. Ther. 82: 111-21 (1999). Many of these phosphorylation sites regulate critical biological processes and may prove to be important diagnostic or therapeutic targets for molecular medicine. For example, of the more than 100 dominant oncogenes identified to date, 46 are protein kinases. See Hunter, supra.
  • IressaTM an inhibitor of EGFR
  • Gleevec® an inhibitor of BCR/ABL
  • CML CML
  • the successful development, demonstration of efficacy, approval, and use of such targeted drugs will often depend in large part upon the ability of the clinician to determine the activation status of the specific protein that the drug is targeted against.
  • the development of the HER2 inhibitor Herceptin® required the ability to select patients on the basis of their HER2/neu expression (Baselga J. et al. Semin Oncol 1999 26(4 Suppl 12): 78-83).
  • HerceptestTM diagnostic assay only predicts a successful patient response in approximately 30% of the cases when Herceptin® is used as a single agent (Leyland-Jones. Lancet Oncol (2002) Mar;3(3): 137-44). This low predictive rate is observed even though all of the patients treated are judged to be over- expressing HER2/neu, demonstrating the significant limitations of this type of diagnostic assay and the need for identifying better biomarkers of responsiveness to therapies like Herceptin®. Given the complexity of most signaling pathways, downstream pathway markers may prove to be the most predictive of a patient's potential responsiveness to a therapeutic (whether targeted or a general chemotherapeutic), or of disease progression and outcome prognosis.
  • HER2/neu and erb-B family signal transduction suggests that downstream pathway markers may be more predictive of patient response to Herceptin® therapy than merely detecting the activity of HER2.
  • single-target therapies may not be efficacious in patients having diseases characterized by multiple signaling protein anomalies.
  • Recent results with Gleevec® further indicate that patients will develop resistance to single agent-targeted therapeutics (Sawyers, Science 294(5548): 1834 (2001)).
  • Successful, long-term treatment of such patients will likely require combination therapies targeting multiple signaling pathways and multiple signaling proteins. Identifying the most efficacious therapies for such patients will require a more detailed knowledge of the signaling pathways underlying the patient's disease than is currently available.
  • cDNA gene arrays have been widely used to profile the genetic states of large numbers of genes in various diseases including cancer (Bertucci F. et al., Lab Invest 2003 83(3): 305-16).
  • these methods have several shortcomings.
  • gene expression does not necessarily correlate to protein expression, nor does protein expression itself provide an accurate readout of protein activity, in vivo. Indeed, the activity of many proteins, including signal transduction molecules, is modulated by post-translational modification, such as phosphorylation. Therefore, gene arrays are inherently limited in their power to predict cellular response to a therapy or progression of a disease.
  • Figure 1 Pathway profiling in LNCaP human prostrate cell line model system.
  • Figure 2 Pathway activation in 54 human breast cancer patients.
  • A cluster analysis of pathway activation.
  • B cluster analysis of patients based upon pathway activation.
  • Figure 3 Pathway activation in 46 human glioma cancer patients.
  • A multi-dimensional plot analysis of pathway activation.
  • B cluster analysis of patients based upon pathway activation.
  • the present invention provides, in part, a powerfully informative new method for identifying the most relevant signal transduction pathway biomarkers of disease progression, or therapeutic responsiveness, using panels of phospho-specific antibodies in assays of cellular content, such as IHC, flow cytometry, reversed phase assays, and the like.
  • the invention also provides, in part, a powerful new method for utilizing correlated biomarker(s) to predict patient response to a therapeutic composition having efficacy against a disease involving altered signal transduction by employing one or more phospho-specific antibodies to detect activation status of such biomarker(s) in cellular assays. Kits for carrying out the methods of the invention are also provided.
  • utilizing a panel of phospho- specific antibodies to profile signal transduction pathway activation in cellular samples from a plurality of patients having a particular disease coupled with determining correlations among activation statuses of multiple proteins in a pathway and a given outcome (e.g. disease progression, therapeutic responsiveness, survival, etc.) enables the identification of the most relevant and statistically-significant biomarkers of the given outcome.
  • a given outcome e.g. disease progression, therapeutic responsiveness, survival, etc.
  • the method employs a panel of phospho-specific antibodies in a cellular assay (e.g immunofluorescence, IHC, etc.) of a plurality of patient samples in order to identify those signal transduction proteins whose phosphorylation statuses are most highly correlated with an outcome (e.g. therapeutic responsiveness), thus identifying the most relevant biomarkers of the outcome.
  • a cellular assay e.g immunofluorescence, IHC, etc.
  • the invention also provides, in part, a powerful new method for utilizing biomarker(s) correlated to therapeutic response (or disease outcome) to predict patient response to a therapeutic composition (or provide a prognosis of outcome) by using one or more phospho-specific antibodies to detect activation status of such biomarker(s) in a cellular assay. Kits for carrying out these methods are also provided.
  • panels of activation state-specific antibodies in cellular assays of a plurality of patient samples with known outcomes coupled with determining correlations with outcome is a powerfully informative technique having several advantages overcoming the limitations of prior methodologies.
  • the use of panels of antibodies enables the profiling of multiple signaling pathways and multiple signaling proteins in a given disease, thereby identifying multiple signaling events underlying a disease.
  • single signaling proteins appear to have limited utility as biomarkers or predictors of an outcome. See Leyland-Jones, supra.; Sawyers, supra.
  • Cellular analysis and in particular IHC and flow cytometry, is an accepted clinical procedure (advantageous for clinical/prognostic assays), and enables examination of protein activity at the cell or tissue level (as opposed to protein expression; see Levine, supra.), including the ability to rapidly analyze multiple sequential tissue slices or cells in parallel.
  • particular cells having activated proteins can be identified, and can, therefore, be directly compared to normal cells to identify differences in in vivo signaling.
  • protein localization within a cell may be determined, in addition to phosphorylation status. Protein localization plays a large role in the regulation of protein function, and may be very important to elucidating molecular bases underlying disease.
  • Profiling signaling pathway activation in cellular samples from a plurality of patients having a known outcome provides the statistical power necessary to identify relevant signaling events across patient groups, or patient subsets. Correlating multiple protein activation states with a particular outcome ensures that identified biomarkers are, in fact, relevant to that outcome, and thus may be exploited as the best predictive biomarkers. In contrast, the predictive power of individual proteins whose activity is implicated in a disease, but has not been correlated with an outcome or validated as a good biomarker for that outcome, is dubious. See Leyland-Jones, supra.; Sawyers, supra.
  • the invention provides a method for identifying protein biomarkers of patient responsiveness (or resistance) to a therapeutic composition having efficacy against a disease involving altered signal transduction, comprising the steps of: (a) obtaining cellular samples from a plurality of patients having the disease, the tissue samples comprising samples from patients (i) treated with the therapeutic composition, (ii) responsive to the therapeutic composition, and (iii) non- responsive (or resistant) to the therapeutic composition; (b) utilizing a panel of phospho-specific antibodies in a cellular assay to detect the phosphorylation statuses of a plurality of signal transduction proteins in the cellular samples; and (c) determining correlations between the phosphorylation statuses of the signal transduction proteins detected in step (b) and responsiveness to the therapeutic composition, wherein one or more significant correlation(s) identifies one or more signal transduction protein(s) as biomarker(s) of patient responsiveness to said therapeutic composition.
  • the method described above was employed to identify four novel biomarkers (ERK, estrogen receptor (ER)(Serine118), mTOR, and AKT) useful for predicting breast cancer responsiveness to EGFR or HER2 inhibitors, and four novel biomarkers (PTEN, EGFR, AKT and ERK) useful for predicting glioma cancer.
  • the invention provides a method for identifying protein biomarkers useful in disease prognosis, comprising the steps of: (a) obtaining cellular samples from a plurality of patients having a disease involving altered signal transduction, the cellular samples comprising (i) samples from patients having negative and positive disease outcomes, and/or (ii) samples from patients having early-stage and advanced disease; (b) utilizing a panel of phospho-specific antibodies in a cellular assay to detect the phosphorylation statuses of a plurality of signal transduction proteins in the cellular samples; and (c) determining correlations between the phosphorylation statuses of the signal transduction proteins detected in step (b) and progression or outcome of the disease in the patients, wherein one or more significant correlation(s) identifies one or more signal transduction protein(s) as biomarker(s) useful in disease prognosis. Certain preferred embodiments of the method are described in more detail below.
  • biomarkers identified as the most relevant to a given outcome such as therapeutic response, may be utilized, according to the present invention, to predict or select patients likely to have that outcome. For example, patient response to a therapeutic composition (such as Gleevec®, Iressa®, or a chemotherapeutic) having efficacy against a disease involving altered signal transduction may be predicted utilizing such biomarkers.
  • a therapeutic composition such as Gleevec®, Iressa®, or a chemotherapeutic
  • biomarkers that are correlated to therapeutic responsiveness in a group of patients having a certain disease will enable presently-unavailable levels of predictive accuracy and avoid prescription of therapeutics to patients who will not respond (compare low Herceptest® predictive rates (-30%) to almost 100% predictive power of exemplary correlated biomarkers identified hereunder and demonstrated in Example 4 (patients found to have a given set of over-expressed or phosphorylated proteins would either relapse or have stable disease or be disease free following therapy, depending on protein expression and phosphorylation).
  • the invention provides a method for predicting patient response to a therapeutic composition having efficacy against a disease involving altered signal transduction, comprising the steps of: (a) obtaining at least one cellular sample from a candidate patient having, or a risk of, the disease; (b) utilizing one or more phospho-specific antibodies in a cellular assay to detect the phosphorylation status, in the cellular sample, of one or more signal transduction protein(s) that is/are a correlated biomarker(s) of responsiveness to the therapeutic composition; and (c) determining whether the patient is likely to respond to, or resist, the therapeutic by comparing the phosphorylation status(es) detected in step (b) with a reference biomarker phosphorylation profile characteristic of patients responsive to, or resistant to, the therapeutic composition.
  • kits of the present invention enable heretofore unavailable predictive and diagnostic assays pertaining to diseases characterized by signal transduction changes or anomalies, and will be of great value in elucidating the molecular mechanisms of diseases, accelerating drug discovery and approval, and assisting clinicians to prescribe appropriate therapeutics.
  • All references cited herein are hereby incorporated herein by reference.
  • therapeutic composition means any composition of one or more therapeutic compounds, either alone or together (such as in a cocktail of multiple therapeutics); the term encompasses all types of therapeutics, including, but not limited to, small molecule inhibitors, antibody inhibitors, anti-sense or peptide inhibitors, or otherwise, whether “targeted therapeutics” directed to a single protein (such as IressaTM, Gleevec®, and Herceptin®) or agents having more broad activity, such as chemotherapeutic agents;
  • disease involving altered signal transduction means a disease or condition in which altered activity (relative to the non-disease state) of one or more signal transduction proteins is relevant to the genesis and progression of the disease;
  • signal transduction protein means any protein, or peptide fragment thereof, which acts to transmit a signal within a cell (or into a cell) when activated (or deactivated) by post-translational modification (for example, phosphorylation).
  • Signal transduction pathways, or cascades, comprising exemplary signal transduction proteins presently known have been extensively described (See, e.g. Hunter T., Cell 100 ⁇ 1): W$-27 (2000); Cell Signaling Technology, Inc., 2002 Annual Catalog, Pathway Diagrams pgs. 232-253), and include but are not limited to the MAP kinase, AKT, NfkB, WNT, and PKC signaling pathways and their members.
  • cellular sample means any biological sample from an organism containing one or more cell(s), including single cells of any origin, tissue or biopsy samples, or a lysate of any of the foregoing;
  • cellular assay means any assay of cellular protein activity and content, including whole or fixed-cell assays, or cell lysate assays; the terms encompasses, but is not limited to, immunohistochemical (IHC) assays, flow cytometric (FC) assays, immunofluorescent (IF) assays, capture-and-detection assays, and reversed phase assays;
  • IHC immunohistochemical
  • FC flow cytometric
  • IF immunofluorescent
  • phospho-specific antibody means an antibody, whether polyclonal or monoclonal, that binds to a target protein only when phosphorylated at a particular residue or site, and does not substantially bind to the protein when not phosphorylated at that residue or site, or to proteins other than the target protein; the term encompasses humanized antibodies, antibody binding fragments, recombinant antibodies, and the like, as described in more detail in the specification below;
  • protein-specific antibody means an antibody, whether polyclonal or monoclonal, that binds to an unphosphorylated target protein and does not substantially bind to proteins other than the target protein, and is therefore suitable for detecting the presence of the protein in a sample; the term encompasses humanized antibodies, antibody binding fragments, recombinant antibodies, and the like, as described in more detail in the specification below; “correlated biomarker” means a signal transduction protein whose activity (i.e.
  • phosphorylation state is significantly correlated with a particular outcome in a group of patients having a disease (for example, therapeutic responsiveness, outcome, prognosis, etc.) and serves, therefore, as a relevant biomarker of the given outcome; the correlation may be either positive or negative and the protein may be activated, or de-activated, by phosphorylation; the term is used interchangeably with "biomarker";
  • responsiveness as used with respect to a therapeutic composition means either positive responsiveness, non-responsiveness, or resistance;
  • panel with respect to antibodies means two or more antibodies;
  • reaction(s) suitable for detecting binding of antibodies means any material or compound, chemical or biological, suitable for detecting the binding of an antibody to its target; the term encompasses, but is not limited to, fluorescent labels, radio-labels, luminescent reactions, secondary antibodies, and the like; as discussed in more detail in “Cellular Assays” and “Kits” below.
  • biomarker or set of biomarkers
  • cluster analysis means a statistical method to group variables together based upon how they correlate; in the present disclosure, cluster analysis refers to a method to group multiple signal transduction protein biomarkers according to how they are expressed or activated in a particular group of patients, for example, patients having disease mediated through one receptor, as opposed to a second receptor; the cluster analysis may group patients according to the activation or phosphorylation of signaling proteins as well as other molecular biomarkers such as the expression, cellular localization or cleavage of signaling molecules.
  • the methods of the invention are applicable to any disease or condition, whether in humans or animals, involving and/or arising, in whole or in part, from altered signal transduction.
  • Cellular signaling pathways are well known in the art (see, e.g., Hunter T., Cell 100(1): 113- 27 (2000); Cell Signaling Technology, Inc., 2002 Catalogue, Pathway Diagrams pgs. 232-253). Accordingly, diseases involving or characterized by altered signal transduction may be readily identified, for example, by profiling signal transduction proteins and pathways in diseased tissue with panels of phospho-specific antibodies, as taught herein, and then comparing the pathway activation with normal (non- diseased) tissue pathway activation.
  • the disease is a cancer, and in a one preferred embodiment, the disease is breast cancer.
  • Other cancers within the scope of the present invention include, but are not limited to, gliomas, lung cancer, colon cancer and prostate cancer. Specific signaling pathway alterations have been described for many cancers, including loss of PTEN and resulting activation of AKT signaling in prostate cancer (Whang YE. Proc Natl Acad Sci U S A 1998 Apr.
  • cardiovascular disease has been shown to involve hypertrophy of the cardiac cells involving multiple pathways such as the PKC family (Malhotra A. Mol Cell Biochem 2001 Sep;225(1-):97-107).
  • Inflammatory diseases such as rheumatory arthritis is known to involve the chemokine receptors and disrupted downstream signaling (D'Ambrosio D. J Immunol Methods 2003 Feb;273(1-2):3-13).
  • the invention is not limited to diseases presently known to involve altered cellular signaling, but includes diseases subsequently shown to involve signaling alterations or anomalies.
  • the methods (and kits) of the invention may be employed to examine and profile phosphorylation status of any signaling pathway, and any signal transduction protein within such pathways, or collections of such proteins.
  • Single or multiple distinct pathways may be profiled (sequentially or simultaneously), or subsets of proteins within a single pathway or across multiple pathways may be examined (again, sequentially or simultaneously).
  • Exemplary signaling pathways include the following pathways and their protein members: The MAP kinase pathway including ras, raf, MEK, ERK and elk; the AKT pathway including PI-3-kinase, PDK1 , AKT and bad; the NfkB pathway including IKKs, IkB and NfkB: the PKC pathway including PI-3-kinase, various PKC isoforms and various PKC substrates such as MARCKS; the WNT pathway including frizzled receptors, beta-catenin, APC and other co-factors and TCF (see Cell Signaling Technology, Inc. 2002 Catalog pages 231-279 and Hunter T., supra.)
  • Exemplary types of signaling proteins within the scope of the present invention include, but are not limited to, kinases, kinase substrates (i.e. phosphorylated substrates), phosphatases, phosphatase substrates, binding proteins (such as 14-3-3), receptor ligands and receptors (cell surface receptor tyrosine kinases and nuclear receptors)).
  • Kinases and protein binding domains for example, have been well described (see, e.g., Cell Signaling Technology, Inc., 2002 Catalogue "The Human Protein Kinases” and "Protein Interaction Domains" pgs. 254-279).
  • the invention encompasses signal transduction proteins having other post- translationally modifications (e.g. acetylation, glycosylation) identified as relevant to a particular disease (see “Antibodies” section below).
  • post- translationally modifications e.g. acetylation, glycosylation
  • the correlated biomarkers being assayed are members of the MAP kinase, AKT, NFkB, WNT, and/or PKC signaling pathways.
  • the MAP kinase pathway includes the ras oncogene that is activated in a wide range of cancers (see Cell Signaling Technology, Inc. Catalog, supra, at pages 231-279 and Hunter T, supra, and references therein).
  • the AKT pathway is the central cell survival pathway that is activated by such oncogenic events as overexpression of an upstream receptor tyrosine kinase such as EGFR (ibid) or loss of an upstream regulatory protein such as PTEN (ibid).
  • the NfkB pathway mediates complex cellular response including cell proliferation as well as cell apoptosis all of which are involved in disease (ibid).
  • Activation of the WNT pathway occurs downstream of the APC mutations that are a common cause of colon cancer (ibid).
  • PKC pathway activation is thought to play a role in diseases such as cardiovascular disease and diabetes (ibid).
  • the invention is not limited to presently elucidated signaling pathways and signal transduction proteins, and encompasses signaling pathways and proteins subsequently identified.
  • compositions having efficacy against a disease involving altered signal transduction.
  • Such compositions may include a single therapeutic compound, or multiple therapeutic compounds (such as in a cocktail).
  • Cocktails may include compounds of differing types, for example broad-spectrum chemotherapeutic agents together with targeted small molecule inhibitors.
  • the compositions may include components other than the therapeutic(s), such as stabilizers, buffers, and the like, which formulations are well known in the art and are outside the scope of the present invention.
  • Therapeutics within the scope of the present invention include, but are not limited to, small molecule inhibitors, antibody inhibitors (including humanized or chimeric antibodies), anti-sense or peptide inhibitors.
  • the therapeutic may be a "targeted therapeutic" directed to a single signaling protein (such as IressaTM, a small molecule inhibitor which targets EGFR) ), Gleevec® , a small molecule inhibitor which targets BCR-ABL, PDGFR and c-kit), and Herceptin® , a humanized monoclonal antibody which targets HER2).
  • the therapeutic may be a compound having a more broad spectrum of activity, such as chemotherapeutic agents like taxol, cisplatin and methatrexate.
  • the methods of the invention pertain to a therapeutic composition comprising at least one targeted therapeutic.
  • the targeted therapeutic is a kinase inhibitor.
  • the methods of the invention pertain to a therapeutic composition comprising at least one chemotherapeutic.
  • biomarkers of patient responsiveness to targeted therapeutics and/or chemotherapeutic compositions will be of great value in ensuring that the right patients get the right therapy, and in avoiding unwanted side-effects in patients receiving a therapy to which they will not respond.
  • the methods and kits of the invention may employ virtually any phospho-specific antibody capable of detecting a desired signal transduction protein when phosphorylated at a particular residue or site.
  • Phospho-specific antibodies are widely commercially available (e.g. from Cell Signaling Technology, Inc.; BioSource, Inc.; Santa Cruz Biotechnology, Inc.; Upstate Biotechnology, Inc.), and may also be produced by techniques well known in the art (see below).
  • panels of phospho-specific antibodies are employed. Such panels may include any collection of two or more phospho-specific antibodies to detect the phosphorylation statuses of two or more target signal transduction proteins. The particular number of antibodies selected for the panel will depend on the signal transduction proteins, pathway or pathways for which profiling is desired.
  • phospho-specific antibodies against all known signaling protein members of a given pathway will be employed, however, less than all of the members may be examined.
  • the panel may include phospho-specific antibodies to multiple proteins in two or more distinct pathways. The phosphorylation profile of multiple complete signaling pathways may also be examined.
  • the panel comprises two to five phospho-specific antibodies.
  • the panel comprises five to ten phospho-specific antibodies.
  • the panel comprises ten to twenty phospho-specific antibodies.
  • the panel comprises twenty or more phospho-specific antibodies.
  • the antibodies in a given panel may used sequentially, in tandem, or simultaneously to detect activation statuses of the various targets.
  • the panel of phospho-specific antibodies employed comprises at least one protein that is a member of the MAP kinase, AKT, NFkB, WNT, and/or PKC signaling pathways.
  • Panels of phospho-specific antibodies used may also include additional non-phospho-specific antibodies or reagents.
  • other modification-specific antibodies may be included, such as acetylation- or nitrosylation-specific antibodies, to detect activation of signal transduction targets having such modifications.
  • Control antibodies may also be included, for example, protein-specific antibodies that detect merely the presence of a given signal transduction protein (not its modification status), or site-specific antibodies that detect a target in its unphosphorylated form.
  • phospho-specific antibodies to one or more signal transduction biomarkers correlated with response to the therapeutic are employed.
  • a single phospho-specific antibody may be used to detect the phosphorylation status of a single correlated biomarker, for example, if only one such biomarker has been identified as relevant to the disease for which therapy is being considered.
  • two or more (i.e. multliple) phospho- specific antibodies against two or more correlated biomarkers being examined may be employed.
  • the particular number of antibodies selected for predicting patient response in a given case will depend on the number of signal transduction proteins that have been identified as relevant, correlated biomarkers of patient responsiveness to the particular therapeutic composition in a particular disease.
  • One or multiple biomarkers may be identified as relevant predictors of patient response to a particular therapeutic composition for a particular disease. For example, as described in the Examples below, two correlated biomarkers of breast cancer patient responsiveness to EGFR-direct therapeutics were identified, and two correlated biomarkers predict response of such patients to HER2-directed therapeutics.
  • Phospho-specific antibodies employed to predict patient response may be against a single or multiple correlated biomarker(s) in one pathway (e.g. MAPK pathway) or may be against correlated biomarkers from differing pathways (e.g. MAPK pathway and PKC pathway). Additional phospho-specific antibodies may also be employed
  • a single phospho-specific protein against a single correlated biomarker is employed to predict patient response to a therapeutic composition (having activity against a disease involving altered signal transduction.
  • two or more phospho-specific antibodies against two or more correlated biomarkers are employed.
  • two to five phospho-specific antibodies against two to five correlated biomarkers are employed.
  • five to ten phospho-specific antibodies against five to ten correlated biomarkers are employed.
  • Phospho-specific antibodies may be use to detect phosphorylation of correlated biomarkers in the examined cellular sample sequentially, in. tandem, or simultaneously to detect activation statuses of the various targets.
  • the phospho-specific antibodies employed comprise at least one target (i.e. the correlated biomarker) that is a member of the MAP kinase, AKT, NFkB, WNT, and/or PKC signaling pathways.
  • the methods and kits of the invention for predicting patient response to a therapeutic composition are not limited to presently known signal transduction proteins or pathways, and may be beneficially employed using antibodies to subsequently identified signaling proteins whose phosphorylation is correlated to therapeutic response in a given disease.
  • phospho-specific antibodies against ERK, estrogen receptor (ER)(Ser118), mTOR and AKT are employed to select patients likely to respond to EGFR inhibitors (both ERK and ER(Ser118) phosphorylated, but not mTOR and AKT) or HER2 inhibitors (ERK, ER(Ser118), mTOR, and AKT are all activated).
  • non-phospho-specific antibodies or reagents, or phospho-specific antibodies to targets other than correlated biomarkers may also be employed in the predictive methods of the invention.
  • modification-specific antibodies may be included, such as acetylation- or nitrosylation-specific antibodies, to detect activation of signal transduction targets having such modifications.
  • Control antibodies may also be included, for example, protein-specific antibodies that detect merely the presence of a given signal transduction protein (not its modification status), or site-specific antibodies that detect a target in its unphosphorylated form.
  • Additional phospho-specific antibodies may also be employed (sequentially or simulataneously) to profile the phosphorylation status of additional signal transduction proteins that are not correlated biomarkers, if such additional pathway activation information is desired.
  • Conditions suitable for the binding of antibodies to their signal transduction protein targets are well known in the art, and described in more detail in "Cellular Assays" below.
  • kits of the invention are not limited to the use of whole antibodies, but include equivalent molecules, such as protein binding domains or nucleic acid aptamers, which bind, in a phospho- specific manner, to essentially the same phosphorylated epitope to which the particular phospho-specific antibodies bind. See, e.g., Neuberger et al., Nature 312: 604 (1984). Such equivalent non-antibody reagents may be suitably employed in the methods of the invention further described below.
  • antibody refers to all types of immunoglobulins, including IgG, IgM, IgA, IgD, and IgE, including F ab or antigen-recognition fragments thereof.
  • the antibodies may be monoclonal or polyclonal and may be of any species of origin, including (for example) mouse, rat, rabbit, horse, or human, or may be chimeric antibodies. See, e.g., M. Walker et al., Molec. Immunol. 26: 403-11 (1989); Morrision et al., Proc. Nat'l. Acad. Sci. 81: 6851 (1984); Neuberger et al., Nature 312: 604 (1984)).
  • the antibodies may be recombinant monoclonal antibodies produced according to the methods disclosed in U.S. Pat. No. 4,474,893 (Reading) or U.S. Pat. No. 4,816,567 (Cabilly et al.)
  • the antibodies may also be chemically constructed by specific antibodies made according to the method disclosed in U.S. Pat. No. 4,676,980 (Segel ef a/.)
  • Polyclonal antibodies useful in the practice of the methods and kits of the invention may be produced according to standard techniques by immunizing a suitable animal (e.g., rabbit, goat, etc.) with an antigen encompassing the phosphorylated residue or site to which specificity is desired), collecting immune serum from the animal, separating the polyclonal antibodies from the immune serum, and screening for phospho-epitope specificity in accordance with known procedures.
  • a suitable animal e.g., rabbit, goat, etc.
  • an antigen encompassing the phosphorylated residue or site to which specificity is desired
  • separating the polyclonal antibodies from the immune serum and screening for phospho-epitope specificity in accordance with known procedures.
  • ANTIBODIES A LABORATORY MANUAL, Chapter 5, p. 75-76, Harlow & Lane Eds., Cold Spring Harbor Laboratory (1988); Czernik, Methods In Enzymology, 201: 264-283 (1991); Merrifield, J. Am. Chem. Soc
  • Monoclonal antibodies suitable for use in the methods and kits of the invention may be produced in a hybridoma cell line according to the well-known technique of Kohler and Milstein. Nature 265: 495-97 (1975); Kohler and Milstein, Eur. J. Immunol. 6: 511 (1976); see also, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel et al. Eds. (1989).
  • Monoclonal antibodies so produced are highly specific, and improve the selectivity and specificity of the therapeutic-response predictive and methods provided by the invention.
  • a solution containing the appropriate antigen i.e. a desired phospho-epitope of a signal transduction protein
  • the animal is sacrificed and spleen cells obtained.
  • the spleen cells are then immortalized by fusing them with myeloma cells, typically in the presence of polyethylene glycol, to produce hybridoma cells.
  • Rabbit fusion hybridomas for example, may be produced as described in U.S Patent No. 5,675,063, C. Knight, Issued October 7, 1997.
  • the hybridoma cells are then grown in a suitable selection media, such as hypoxanthine- aminopterin-thymidine (HAT), and the supernatant screened for monoclonal antibodies having the desired specificity (against the signal transduction protein) by standard techniques. See e.g. Czernik, supra.
  • a suitable selection media such as hypoxanthine- aminopterin-thymidine (HAT)
  • HAT hypoxanthine- aminopterin-thymidine
  • the secreted antibody may be recovered from tissue culture supernatant by conventional methods such as precipitation, ion exchange or affinity chromatography, or the like.
  • Monoclonal Fab fragments may also be produced in Escherichia coli by recombinant techniques known to those skilled in the art. See, e.g., W. Huse, Science 246: 1275-81 (1989); Mul ⁇ inax ef al., Proc. Nat'l Acad. Sci. 87: 8095 (1990). If monoclonal antibodies of one isotype are preferred for a particular application, particular isotypes can be prepared directly, by selecting from the initial fusion, or prepared secondarily, from a parental hybridoma secreting a monoclonal antibody of different isotype by using the sib selection technique to isolate class-switch variants (Steplewski, et al., Proc. Nat'l. Acad. Sci., 82: 8653 (1985); Spira et al., J. Immunol. Methods, 74: 307 (1984)).
  • Cellular samples to be analyzed in the method of the invention may consist of tissue samples taken during the course of surgery, biopsies taken for the sake of patient diagnosis, ductal lavages, fine needle aspirants, blood, serum, urine or other fluid samples or skin, hair follicle or scrapings taken for clinical analysis.
  • Fresh samples may be analyzed by immunohistochemical or immunofluorescent methods on whole cells or by reverse-phase array methods on lysates prepared from the patient samples.
  • Tissue samples may be dispersed, enabling a flow cytometric analysis.
  • the samples may be frozen or fixed using fixation methods well known in the art as described below in the examples.
  • the fixed cells may be paraffin-embedded or used in flow cytometric analyses.
  • the cells derived may also be analyzed as cell smears in which fresh or fixed cells are placed on slides.
  • Suitable cellular samples from a subject include tissue or tumor samples, individual or multiple cell samples, fine needle aspirate, ductal lavage, bone marrow sample, ascites fluid, urine, lymphatic, or blood samples containing one or more cells, or lysates of the foregoing.
  • the analysis of the tissue or cell samples may be done by standard immunohistochemical methods well known in the art as described in the examples. This analysis may be done manually or by automatic cell staining instruments. The detection of the bound antibodies may be done with solid substrates or with fluorescent labels. Scoring of the stained tissues or cells may be done manually or by automatic analysis. The fixed cells may be analyzed by flow cytometry using multiple antibodies following standard methods well known in the art.
  • the cellular sample will be a tumor sample from a cancer patient, for example, a breast cancer patient.
  • multiple tissue samples are prepared as a tissue microarray for IHC-based staining and analysis. Construction of tissue microarrays is well known in the art (Zhang D. et al. Mod Pathol (2003) Jan;16(1):79-85).
  • Phosphorylation status(es) in a cellular sample are examined, in accordance with the methods and kits of the invention, using phospho- specific antibodies in a cellular assay, namely, any assay suitable for detecting in vivo protein activity in a particular cell.
  • suitable cellular assays include the following preferred assays: immunhisto- chemistry (IHC), flow cytometry (FC), immunofluorescence (IF) (all of which are whole cell or tissue-based staining assays), and capture-and- detection (e.g. ELISA), or reversed phase assays (which are cell-lysate based assays).
  • cellular analysis of protein acitivation has many advantages. Methods like IHC and FC are well-used and accepted clinical procedures, and thus are highly-desirable assay formats for clinical and prognostic assays.
  • Cellular assays enable examination of protein activity at the cell or tissue level (as opposed to genetic or protein expression level; see Levine, supra.), including the ability to rapidly analyze multiple sequential tissue slices or cells in parallel.
  • particular cells having activated proteins can be identified, and can, therefore, be directly compared to normal cells to identify differences in in vivo signaling.
  • protein localization (which plays a significant role in protein function) within a cell may be determined, in addition to phosphorylation status.
  • Immunohistochemical staining using tissues may be carried out according to well known techniques. See, e.g., ANTIBODIES: A LABORATORY MANUAL, Chapter 10, Harlow & Lane Eds., Cold Spring Harbor Laboratory (1988). Briefly, paraffin-embedded tissue (e.g. tumor tissue) is prepared for immunohistochemical staining by deparaffinizing tissue sections with xylene followed by ethanol; hydrating in water then PBS; unmasking antigen by heating slide in sodium citrate buffer; incubating sections in hydrogen peroxide; blocking in blocking solution; incubating slide in primary antibody (i.e. phospho-specific antibodies against signal transduction proteins) and secondary antibody; and finally detecting using ABC avidin/biotin method according to manufacturer's instructions.
  • tissue either diseased (e.g. a tumor biopsy) or normal
  • ANTIBODIES A LABORATORY MANUAL, Chapter 10, Harlow & Lane Eds., Cold Spring Harbor Laboratory (1988).
  • Flow cytometry assay may also be employed to determine the activation status of signal transduction proteins and correlated biomarkers. For example, bone marrow cells or peripheral blood cells from patients may be analyzed by flow cytometry for biomarkers of therapeutic response or disease progression, as well as for other markers identifying various hematopoietic cell types. In this manner, activation status of malignant cells may be specifically characterized.
  • Flow cytometry may be carried out according to standard methods. See, e.g. Chow et al., Cytometry (Communications in Clinical Cytometry) 46: 72-78 (2001 ).
  • the following protocol for cytometric analysis may be employed: fixation of the cells with 1% paraformaldehyde for 10 minutes at 37 °C followed by permeabilization in 90% methanol for 30 minutes on ice. Cells may then be stained with the primary phospho-specific antibody or antibodies, washed and labeled with a fluorescent-labeled secondary antibody. Alternatively, the cells may be stained with a fluorescent-labeled primary antibody. The cells would then be analyzed on a flow cytometer (e.g. a Beckman Coulter EPICS-XL) according to the specific protocols of the instrument used. Immunoassay formats and variations thereof which may be useful for carrying out the methods disclosed herein are well known in the art. See generally E.
  • Monoclonal antibodies may be used, for example, in a "two-site” or “sandwich” assay, with a single cell line serving as a source for both the labeled monoclonal antibody and the bound monoclonal antibody.
  • assays are described in U.S. Pat. No. 4,376,110.
  • concentration of detectable reagent should be sufficient such that the binding of phosphorylated target is detectable compared to background.
  • the biomarkers may be analyzed in an ELISA or reverse-phase array format.
  • a capture antibody for each biomarker is affixed to a solid substrate such as a plastic ELISA plate, nitrocellulose membrane or bead.
  • the patient lysate is incubated with the labeled substrate allowing for the capture of the biomarker proteins to the substrate via the capture antibodies.
  • the substrate is then washed.
  • the captured proteins are then detected using a second antibody specific for each protein.
  • the bound detection antibody may be detected by a labeled secondary antibody or by labeling (fluorescent or enzyme) the detection antibody.
  • lysates of patient samples are fixed to a solid substrate in predetermined locations.
  • the fixed sample is then incubated with the antibodies.
  • the bound antibodies are detected by various detection methods such as a secondary detection antibodies or by prelabeling the antibodies with fluorescent labels.
  • Phospho-specific antibodies employed in the methods of the invention may be conjugated to a solid support suitable for a diagnostic assay (e.g., beads, plates, slides or wells formed from materials such as latex or polystyrene) in accordance with known techniques, such as precipitation.
  • Antibodies or equivalent binding reagents may likewise be conjugated to detectable groups such as radiolabels (e.g., 35 S, 125 l, 131 l), enzyme labels (e.g., horseradish peroxidase, alkaline phosphatase), and fluorescent labels (e.g., fluorescein) in accordance with known techniques.
  • detectable groups such as radiolabels (e.g., 35 S, 125 l, 131 l), enzyme labels (e.g., horseradish peroxidase, alkaline phosphatase), and fluorescent labels (e.g., fluorescein) in accordance with known techniques.
  • phospho-specific antibodies employed in cellular assays may be optimized for use in other clinically-suitable applications, for example bead-based multiplex-type assays, such as IGEN, LuminexTM and/or BioplexTM assay formats, or otherwise optimized for antibody arrays formats.
  • Any suitable software or algorithm for calculating correlations between the activation (i.e. phosphorylation) of a given signal transduction protein or collection of such proteins may be employed in methods of the invention in identifying relevant biomarkers of disease progression, outcome, prognosis, and/or therapeutic responsiveness.
  • correlations may be calculated by standard methods, such as the Pearson's tests or Chi-Squared tests. Such methods are well known in the art (see Introduction to Biostatistics, Sokal and Rohlf). The analysis may be done manually or using statistical software such as SYSTAT.
  • correlations are determined by performing cluster analysis of protein activity and at least one outcome of interest, such as disease outcome (i.e. survival, death) or therapeutic response or resistance.
  • Cluster analysis uses various statistical methods ranging from simple Pearson correlations to sophisticated mathematical models such as unsupervised learning sets. The outcomes of cluster analysis are often unpredicted by simple analysis of the data. This is especially true of complex data sets such as the data generated by screening hundreds of patient samples on a tissue micro-array using multiple antibodies. Therefore, cluster analysis is a more powerful and informative method than single protein activity correlations that have been previously attempted. As shown in the examples, novel combinations of biomarkers are identified by cluster analysis.
  • cluster analysis of MAP kinase, AKT, STAT, WNT and other pathway activity in breast cancer patients identified phosphorylation of ERK, ER, mTOR and AKT as sufficient to segregate patients into two groups corresponding to EGFR or HER2 expression. Similarily, cluster analysis of AKT, ERK and STAT biomarkers surveyed in Herceptin treated patient samples revealed that the combination of phosphorylated AKT and phosphorylated S6 ribosomal protein predicts patient response and survival to Herceptin combination therapy.
  • Such correlation analysis enables identification of the best (most highly correlated) biomarkers of disease progression, outcome, or therapeutic responsiveness. Exploitation of such correlated biomarkers in, e.g. predicting therapeutic response of a patient in order to make a treatment determination for that patient, avoids the limitations of present assays based on markers of questionable power (See HerceptestTM discussion above). Identified correlations may be positive or negative: that is, the phosphorylation/activation of a particular biomarker may be negatively correlated with survival, meaning it is associated with non- survival. Similarily, activation or phosphorylation of a particular biomarker may predict a positive patient response or a negative response to a given theratpeutic.
  • the effect of the activation of a given biomarker on a patient response to therapy will depend on the biology of the tumor and the target(s) of the therapy. If the therapy is targeting one oncogene and the patient's tumor is being driven by another oncogene, then it is unlikely that the patient will response well to the therapy. The inverse is true as well.
  • the mostly highly correlated signaling proteins are selected as the best biomarkers of a given outcome.
  • Significant correlations are generally identified as those having a P value of less than 0.05 which means that there is a greater than 95% confidence level that the correlations are not occurring by chance.
  • a P value of less than 0.05 indicates that the frequency distribution observed among biomarkers in a subpopulation of patients, for example the patients that response well to a therapy, is significantly different than the frequency distributions observed in the overall population.
  • This conclusion provides the basis for making predictions on how a defined subpopulation of patients will respond to a drug for example.
  • Insignificant correlations indicate proteins not useful as biomarkers as described herein.
  • proteins having significant correlations, though not selected as the most highly, and therefore best and most relevant, biomarkers of an outcome may be used, in addition to the best biomarkers, to provide additional information on pathway activation in a given patient.
  • Correlations may also be determined to identify differences in signaling activity among patient subsets. For example, among breast cancer patients, certain patients will have disease mediated by EGFR signaling, and other patients will have disease mediated by HER2 signaling. Cluster analysis of grouped patient subsets can identify what downstream pathways are activated in subsets of patients. Patients that have tumors driven by AKT pathway activation may response best to drugs such as Rapamycin which targets the mTOR protein which is downstream of AKT. Alternatively, patients that have tumors driven by ERK may benefit most by treatment with ERK pathway inhibitors such as MEK inhibitors. Cluster analysis with multiple biomarkers is required for such an analysis given the complex networks that compose the cellular signaling that drives most tumorigenesis.
  • kits for carrying out the methods disclosed herein.
  • the invention provides a kit predicting patient response to a therapeutic composition having efficacy against a disease involving altered signal transduction, comprising (a) one or more phospho-specific antibodies against one or more signal transduction protein(s) that is/are a correlated biomarker(s) of responsiveness to the therapeutic composition, and (b) one or more additional reagent(s) suitable for detecting binding of the antibodies to the signal transduction protein(s) in a cellular assay.
  • the therapeutic composition comprises at least one kinase inhibitor or chemotherapeutic.
  • the kit comprises a plurality of phospho-specific antibodies and protein-specific antibodies against a plurality of correlated biomarkers.
  • kits may be used, for example, by a clinician or physician as an aid to selecting an appropriate therapy for a particular patient, for example, a breast cancer patient under consideration for EGFR- or HER2- inhibitor therapy.
  • novel biomarkers of breast cancer responsiveness to EGFR inhibitors and HER2 inhibitors have now been identified.
  • the invention provides a kit for selecting a breast cancer patient likely to respond to a therapeutic composition targeting EGFR or HER2, comprising (a) phospho-specific antibodies against ERK, ER(Ser118), mTOR, and AKT, and (b) one or more additional reagent(s) suitable for detecting binding of these antibodies to their targets in a cellular assay.
  • a therapeutic composition targeting EGFR or HER2 comprising (a) phospho-specific antibodies against ERK, ER(Ser118), mTOR, and AKT, and (b) one or more additional reagent(s) suitable for detecting binding of these antibodies to their targets in a cellular assay.
  • Activation of both ERK and ER at Serine 118), but not mTOR and AKT, in a cellular sample from the breast cancer patient identifies the patient as having HER2- mediated cancer and thus likely to respond to a HER2-inhibitor.
  • the invention provides a kit for prognosis of disease outcome in a patient having a disease involving altered signal transduction, comprising (a) one or more phospho-specific antibodies against one or more signal transduction protein(s) that is/are a correlated biomarker(s) of outcome or progression of the disease, and (b) one or more additional reagent(s) suitable for detecting binding of said antibodies to the signal transduction protein(s) in a cellular assay.
  • kits may be used, for example, by a clinician or physician in predicting whether a given patient will survive or present with an aggressive form of a disease, and thus, will aid in determining an appropriately aggressive or passive treatment strategy.
  • the invention provides a kit for identifying protein biomarkers of disease outcome or patient responsiveness to a therapeutic composition having efficacy against a disease involving altered signal transduction, comprising (a) a panel of phospho-specific antibodies against a plurality of signal transduction proteins, and (b) one or more additional reagent(s) suitable for detecting binding of the antibodies to said signal transduction protein(s) in a cellular assay.
  • the cellular assay comprises an immunohistochemical (IHC), flow cytometric, immunofluorescent, capture-and-detection, or reversed phase assay, and the kit is optimized for staining or analyzing at least one cellular sample from a patient.
  • the kit comprises phospho-specific antibodies against one or more members of the MAP kinase, AKT, NFkB, WNT, and/or PKC signaling pathways.
  • Reagents suitable for detecting binding of the antibodies may, for example, be a second antibody conjugated to a detectable group or label.
  • the kit may include an appropriate assay container, for example, a microtiter plate, slide, etc.
  • the reagents may also include ancillary agents such as buffering agents and protein stabilizing agents, e.g., polysaccharides and the like.
  • the kit may further include, where necessary, other members of the signal-producing system of which system the detectable group is a member (e.g., enzyme substrates), agents for reducing background interference in a test, control reagents, apparatus for conducting a test, and the like. For example, blocking reagents and/or positive and negative controls may be included.
  • test kit may be packaged in any suitable manner, typically with all elements in a single container along with a sheet of printed instructions for carrying out the test. Methods and reagents for carrying out and detecting antibody- protein binding reactions are well known in the art, as described in "Antibodies and Arrays" above.
  • Immunohistochemical (IHC) analysis of paraffin-embedded samples is the most common method for analyzing the pathology of diseased tissues. Determining the molecular pathology of a tumor in order to identify relevant biomarkers of outcome may be accomplished using the methods of the present invention with IHC analysis of paraffin- embedded tissues. IHC analysis of patient tissue samples with phospho- specific antibodies to downstream signaling molecules may be used, for example, to prescreen patients for inclusion in a clinical trial, to follow patients during treatment and to detect resistance to the targeted therapeutic.
  • Tissue micro-arrays are a well- established method to rapidly and uniformly stain large numbers of tissue samples (Zhang et al., Mod. Pathol. 16(1): 79-85 (2003), and may be prepared using commercially available Beecher instruments.
  • Custom tissue microarrays containing tissue samples from breast cancer patients were obtained commercially (Clinomics, Inc.).
  • the microarrays contained human breast cancer tissues obtained from standard biopsy procedures from patients, subsequently fixed in formallin.
  • the tissue was paraffin-embedded following standard procedures (see ANTIBODIES, A LABORATORY MANUAL, supra.).
  • cultured human LNCaP prostate cancer cells were grown in cell culture and treated with the PI-3-kinase inhibitor LY294002 (LY). The cells were then washed, spun down and the cell pellet was fixed and embedded in paraffin. For IHC staining, 2-4 micron thick slices were cut from the paraffin blocks using a microtome and placed on glass slides.
  • the sections were then de-paraffinized with xylene and ethanol.
  • the tissues were microwaved for 10 minutes in an citrate pH 6.5 buffer for antigen retrieval, or 30 minutes in a pressure cooker for tissue arrays. After a 10 minute incubation in 0.3%H2O2, the sections were blocked in 5% goat serum for 1 hour.
  • the LNCaP cell slides were then stained with AKT, phospho-AKT, phospho-PKD1 , phospho-GSK3, phospho-FKHR, phospho-mTOR, phospho-S6 ribosomal protein and cleaved caspase 3 antibodies (Cell Signaling Technology, Inc.) for 2 hours at room temperature or overnight at 4°C.
  • the breast cancer tissue slides were stained with a variety of receptor tyrosine kinase antibodies and downstream signaling protein phospho-specific antibodies (Cell Signaling Technology, Inc.) as listed in the tables and figures below. After 3 washes in PBS, the slides were then probed with a secondary antibody labeled with biotin.
  • the slides were further developed with a avidin-biotin-HRP reagent (ABC kit) following standard manufacturer procedures.
  • the slides were developed using a HRP substrate, either DAB or NovaRedTM and counterstained with hematoxylin. Positive staining for antibody staining was scored (0-3 or positive-negative) based upon staining intensity, number of cells stained and correct localization of stain. The frequencies of scores were tabulated and the Chi-Squared tests of significance were calculated using standard statistical methods.
  • the cluster analysis was done using well- known publicly available clustering programs such as Cluster and Treeview.
  • AKT pathway activation and it's inhibition can be demonstrated by IHC with phospho-specific antibodies using the PTEN-negative LNCaP cell line. Because these cells are PTEN deficient, the AKT pathway is constitutively activated.
  • the results show that LY inhibition of this pathway is reflected in the loss of staining or phosphorylation of proteins downstream of PI-3-kinase including AKT, PDK1 , GSK3, FKHR, mTOR and S6 ribosomal protein.
  • induction of cleavage of caspase 3 and cellular apoptosis can be observed.
  • the results of the immunohistochemical study of the breast tumor section arrays were first analyzed for correlations between activation states of proteins and pathological indices including tumor grade and lymph node status (results not shown). Such correlations would indicate the relevance of activation of those proteins with that specific tumor as well as add novel prognostic information. Phosphorylation of ERK and estrogen receptor significantly negatively correlated with tumor grade and lymph node status. Based upon this data, progression of disease may be predicted by monitoring ERK and estrogen receptor phosphorylation. These results further indicate the power of an IHC analysis using panels of phospho-specific antibodies to provide new prognostic information for breast cancer patients. The immunohistochemical results were then analyzed for statistically significant correlations between activation states of the proteins.
  • a cluster analysis was performed using HER2 and EGFR overexpression and ERK pathway (phospho-ERK and phospho-ER (Ser118)) activation or AKT pathway (phospho-AKT and phospho-mTOR) activation.
  • the results of this cluster analysis clearing segregate patients into two groups; one expresses just EGFR and not HER2 and has both the ERK and AKT pathways activated.
  • the other patient group expresses primarily HER2 and just has the ERK pathway activated. Therefore, the patients may be classified into an EGFR signaling group and a HER2 signaling group based on the identified biomarkers.
  • biomarkers may now be beneficially employed to directly indicate what breast cancer patients would respond best to an EGFR inhibitor (such as IressaTM) or a HER2 inhibitor (such as Herceptin®).
  • Figure 3A is a multidimensional plot, similar based upon a cluster analysis that outlines the relative correlations between the various signaling molecules observed in the patients.
  • PTEN loss leads to AKT activation and downstream activation of AKT substrates such as FKHR and mTOR.
  • ERK activation which correlates with EGFR activation; not shown
  • S6 phosphorylation which also is regulated by AKT activation.
  • Figure 2B presents the corresponding glioma patient cluster analysis based upon the data described in Figure 2A.
  • Glioma patients may be grouped according to PTEN presence, EGFR expression and ERK and AKT activation. These biomarkers for differing patient groups translate directly into differing treatment regimes: Patients that express PTEN and EGFR and have active downstream signaling would be the best candidates for EGFR inhibitors (such as IressaTM). In contrast, patients that lack PTEN and have active AKT signaling would be most likely to respond to AKT pathway inhibitors (such as rapamycin).
  • EGFR inhibitors such as IressaTM
  • AKT pathway inhibitors such as rapamycin
  • Predictive biomarkers of drug response may be identified, according to the methods of the invention, by analyzing tissue samples from patients before therapy and then to compare those results with patient response or survival after therapy. This method was employed with tissue samples from breast cancer patients in order to identify correlated biomarkers predictive of patient response to the HER2- inhibitor, Herceptin®.
  • tissue micro-array of 250 breast cancer patients, all treated with a combination of Herceptin (HER2 inhibitor), radiation and chemo-therapies, was commercially obtained (Clinomics, Inc.). The treatment regimes are typical of most therapies prescribed to breast cancer patients at this time.
  • the tissue micro-array was analyzed with panels of antibodies to receptor tyrosine kinases (HER2, EGFR and IGFR), ligands to HER2 and EGFR (NDF, TGF-alpha) and downstream proteins (phosphorylation of AKT, ERK, S6 ribosomal protein, STAT3). The conditions used were as described for Example 1 above.
  • the IHC was scored 0 to 3.
  • the IHC was scored positive or negative for the sake of simplicity. It will be recognized that some statistical power is lost in going to the simpler scoring system, and more accurate (e.g. automated) scoring methodologies may be employed as they are developed in the future.
  • Table 1 presents the results of the analysis of signaling protein phosphorylation compared to patient survival. From the Chi-Squared statistical analysis AKT activation and IGFR expression significantly correlate with patient survival; patients that have AKT activated (score of 1 ,2 or 3) have a much lower survival than patients that don't have any AKT phosphorylation. Likewise, patients that overexpress IGFR (score of 3) are far more likely to die than patients whose tumors don't overexpress IGFR.
  • biomarkers may be employed to assist in patient prognosis as well as predict patients likely to respond to Herceptin® therapy. Comparing these results with the results obtained with the single biomarker analysis indicates that biomarkers of predictive power may be obtained by considering combinations of multiple biomarkers as opposed to single markers.
  • Table 3 presents the results of the comparison of protein phosphorylation and patient response considering the single biomarkers. None of the comparisons were statistically significant. However, when an upstream receptor tyrosine kinase is added to the comparison (Table 4) significant correlations are identified. The combination of EGFR expression and ERK phosphorylation and the combination of IGFR expression and S6 ribosomal protein phosphorylation were identified as statistically significant biomarkers of patient response to therapy. Patients that were EGFR negative but phospho-ERK positive did poorly. Likewise, patients that were IGFR and phospho-S6 positive relapse 92% of the time. Combining three biomarkers gave the most predictive power (Table 4)
  • the methods of the invention are further exemplified by analysis of samples collected from 7 breast cancer patients before treatment with Herceptin® in combination with chemotherapy (see Table 6). The samples were then analyzed by IHC as described above. Expression of EGFR and IGFR and phosphorylation of AKT, S6 ribosomal protein and ERK were determined. The results indicate that patients who express IGFR and have active downstream signaling (patients #4-7) do not responsd to the therapy. In contrast, patients that either don't express IGFR (patient #2) or express IGFR but don't have active downstream AKT and S6 signaling (patients #1 and #3) do respond. These results are consistent with the tissue micro-array results from the previous examples above. Patient response to a targeted therapy may only be predicted by the use of a combination of molecular biomarkers, detected using phospho-specific antibodies in accordance with the methods disclosed herein.
  • Table 1 Analysis of phospho-protein biomarkers and breast cancer patient survival following Herceptin® combination therapy.
  • Table 2. Analysis of combinations of phospho-protein biomarkers and breast cancer patient survival following Herceptin® combination therapy.
  • Table 3 Analysis of phosphorylation of a single protein and breast cancer patient response to Herceptin® combination therapy.
  • Table 4 Analysis of phosphorylation of a single protein and expression of an receptor tyrosine kinase and breast cancer patient response to Herceptin® combination therapy.
  • Table 5 Analysis of phosphorylation of multiple proteins and breast cancer patient response to Herceptin® combination therapy.
  • Table 6 Analysis of phosphorylation of downstream signaling proteins, expression of receptor tyrosine kinases and breast cancer patient response to Herceptin® combination therapy.
  • Table 4 Analysis of receptor and downstream protein activation versus response in patients following therapy. Analysis on tissue array samples for which clinical and Herceptest data was available and who over- expressed HER2/neu.
  • Table 5 Analysis of ligand and receptor expression and downstream protein activation versus patient response in patients following therapy. Analysis on tissue array samples for which clinical and Herceptest data was available and who over-expressed HER2/neu.

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Abstract

La présente invention concerne des procédés d'identification des biomarqueurs les plus pertinents de la voie de transduction du signal de l'évolution d'une maladie, du résultat thérapeutique ou de la sensibilité thérapeutique faisant intervenir des anticorps phospho-spécifiques dans des analyses cellulaires de manière à identifier les protéines dont l'activité est en corrélation avec le résultat thérapeutique pertinent (p.ex. la sensibilité thérapeutique). L'invention concerne également un procédé d'utilisation d'un(des) biomarqueur(s) corrélé(s) de manière à prédire la réponse du patient à une composition thérapeutique efficace contre une maladie impliquant une transduction de signal altérée. A cet effet, on utilise un ou plusieurs anticorps phospho-spécifique(s) afin de détecter l'état d'activation de tel(s) biomarqueur(s) dans des analyses cellulaires. Font également l'objet de cette invention des kits permettant de mettre en oeuvre les procédés de cette invention.
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WO2006052249A1 (fr) * 2004-11-08 2006-05-18 The Regents Of The University Of California Procedes impliquant la voie de signalisation pi3k/akt dans des gliomes et des cancers de la prostate
WO2005085863A3 (fr) * 2004-02-06 2006-08-24 Applied Genomics Inc Correlation entre biomarqueurs et composes dans le diagnostic et le traitement du cancer
WO2008009004A3 (fr) * 2006-07-13 2008-10-30 Cell Signaling Technology Inc Réactifs pour la détection de la phosphorylation des protéines dans les voies de signalisation
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WO2005085863A3 (fr) * 2004-02-06 2006-08-24 Applied Genomics Inc Correlation entre biomarqueurs et composes dans le diagnostic et le traitement du cancer
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WO2008009004A3 (fr) * 2006-07-13 2008-10-30 Cell Signaling Technology Inc Réactifs pour la détection de la phosphorylation des protéines dans les voies de signalisation
WO2008008998A3 (fr) * 2006-07-13 2008-12-24 Cell Signaling Technology Inc Réactifs pour la détection de phosphorylation de protéines dans les chemins de signalisation
US9285369B2 (en) 2006-09-21 2016-03-15 Nestec S.A. Antibody-based arrays for detecting multiple signal transducers in rare circulating cells
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US10473640B2 (en) 2006-09-21 2019-11-12 Société des Produits Nestlé S.A. Drug selection for gastric cancer therapy using antibody-based arrays
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US8658388B2 (en) 2006-09-21 2014-02-25 Nestec S.A. Antibody-based arrays for detecting multiple signal transducers in rate circulating cells
EP2132572A4 (fr) * 2007-03-20 2010-04-14 Apocell Inc Evaluation de médicaments ciblant rtk
US8609349B2 (en) 2008-02-25 2013-12-17 Nestec S.A. Drug selection for breast cancer therapy using antibody-based arrays
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US9274116B2 (en) 2008-02-25 2016-03-01 Nestec S.A. Drug selection for breast cancer therapy using antibody-based arrays
WO2009108637A1 (fr) * 2008-02-25 2009-09-03 Prometheus Laboratories, Inc. Sélection d’un médicament pour le traitement du cancer du sein à partir des matrices d’anticorps
EP2618146A3 (fr) * 2008-02-25 2013-11-06 Nestec S.A. Sélection d'un médicament pour la thérapie du cancer du sein à partir des matrices d'anticorps
US10436786B2 (en) 2008-02-25 2019-10-08 Société des Produits Nestlé S.A. Methods for detecting truncated receptors using antibody-based arrays
US8163499B2 (en) 2008-02-25 2012-04-24 Prometheus Laboratories Inc. Drug selection for breast cancer therapy using antibody-based arrays
CN102016581A (zh) * 2008-02-25 2011-04-13 普罗米修斯实验室股份有限公司 用抗体阵列选择乳腺癌治疗药物
US9719995B2 (en) 2011-02-03 2017-08-01 Pierian Holdings, Inc. Drug selection for colorectal cancer therapy using receptor tyrosine kinase profiling
US10401364B2 (en) 2011-02-03 2019-09-03 Soiété Des Produits Nestlé S.A. Drug selection for colorectal cancer therapy using receptor tyrosine kinase profiling
US9664683B2 (en) 2011-09-02 2017-05-30 Pierian Holdings, Inc. Profiling of signal pathway proteins to determine therapeutic efficacy
US10914728B2 (en) 2016-10-24 2021-02-09 Novo Nordisk A/S Bioassay for insulin formulations

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