WO2007041502A2 - Procedes pour la determination de la sensibilite a la therapie cancereuse - Google Patents

Procedes pour la determination de la sensibilite a la therapie cancereuse Download PDF

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Publication number
WO2007041502A2
WO2007041502A2 PCT/US2006/038451 US2006038451W WO2007041502A2 WO 2007041502 A2 WO2007041502 A2 WO 2007041502A2 US 2006038451 W US2006038451 W US 2006038451W WO 2007041502 A2 WO2007041502 A2 WO 2007041502A2
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Prior art keywords
her2
herl
dimer
cancer cell
her3
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PCT/US2006/038451
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English (en)
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WO2007041502A3 (fr
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Chris Petropoulos
Mike Bates
Colombe Chappey
Sharat Singh
Ali Mukherjee
Mengxiang Tang
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Monogram Biosciences
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Publication of WO2007041502A3 publication Critical patent/WO2007041502A3/fr

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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
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57492—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds localized on the membrane of tumor or cancer cells

Definitions

  • the present invention relates generally to biomarkers, and more particularly, to the use of ErbB cell surface receptor complexes, such as dimers and oligomers, as biomarkers for determining responsiveness of a cancer to anticancer therapy including a Her2-acting agent, particularly trastuzumab therapy.
  • ErbB cell surface receptor complexes such as dimers and oligomers
  • a biomarker is generally a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacological responses to a therapeutic intervention. See Atkinson et al., 2001, Clin. Pharmacol. Ther. 69:89-95. Biomarkers vary widely in nature, ease of measurement, and correlation with physiological states of interest. See, e.g., Frank et al., 2003, Nature Reviews Drug Discovery 2:566-580. It is widely believed that the development of new validated biomarkers will lead both to significant reductions in healthcare and drug development costs and to significant improvements in treatment for a wide variety of diseases and conditions. Thus, a great deal of effort has been directed to using new technologies to find new classes of biomarkers. See, e.g., Petricoin et al., 2002, Nature Reviews Drug Discovery, 1:683-695; and Sidransky, 2002, Nature Reviews Cancer 2:210-219.
  • the invention provides a method for determining whether a cancer cell is likely to respond to treatment with a Her2-acting agent.
  • the methods comprise detecting a biomarker or combination of biomarkers associated with responsiveness to treatment with a Her2-acting agent as described hereinafter, and determining whether the cancer cell is likely to respond to treatment with the Her2-acting agent.
  • the invention provides a method for determining whether a cancer or cancer cell is likely to respond to treatment with a Her2 -acting agent, comprising determining whether the cancer or cancer cell is likely to respond to treatment with a score for the cancer or cancer cell according to a formula of the invention as described hereinafter, wherein the score indicates the probability that the cancer or cancer cell is likely to respond to treatment with a Her2-acting agent.
  • the Her2-acting agent is trastuzumab.
  • the invention provides a method for determining whether a cancer or cancer cell is likely to respond to treatment with a Her2-acting agent, comprising determining a score for the cancer cell determined according to a formula of the invention as described hereinafter, wherein the score indicates that the subject is likely to respond to treatment with a Her2-acting agent, hi certain preferred embodiments, the Her2-acting agent is trastuzumab.
  • the invention provides a method for determining whether a subject with cancer is likely to respond to treatment with a Her2-acting agent, comprising determining a Diagnostic Index for a cell in a biological sample from the subject's cancer according to a formula of the invention as described hereinafter, wherein the Diagnostic Index indicates the probability that the subject is likely to respond to treatment with a Her2-acting agent.
  • the Her2-acting agent is trastuzumab.
  • the invention provides a method for determining whether a subject with cancer is likely to respond to treatment with a Her2-acting agent, comprising determining a balanced dimer score for a cell in a biological sample from the subject's cancer determined according to a formula of the invention as described hereinafter, wherein the balanced dimer score indicates that the subject is likely to respond to treatment with a Her2-acting agent.
  • the Her2-acting agent is trastuzumab.
  • the invention provides methods of treating a subject with cancer.
  • the methods comprise determining that the subject is afflicted with a cancer comprising a cancer cell that is likely to respond to treatment with a Her2- acting agent according to a method of the invention, and administering an effective amount of a Her2-acting agent to the subject.
  • the methods comprise deterrnining that a subject is afflicted with a cancer comprising a cancer cell that is likely to respond to treatment with a Her2-acting agent according to a method of the invention, then advising a medical professional of the treatment option of administering to the subject an effective amount of a Her2-acting agent.
  • the Her2- acting agent is trastuzumab.
  • the cancer is breast cancer.
  • Figures 1 A-IF provide diagrams illustrating the use of releasable molecular tags to measure receptor dimer populations.
  • Figures 1G-1H provide diagrams illustrating the use of releasable molecular tags to measure cell surface receptor complexes in fixed tissue specimens.
  • Figures 2A-2E provide diagrams illustrating an embodiment of the method of the invention for profiling relative amounts of dimers of a plurality of receptor types.
  • Figures 3 A-3D provide diagrams illustrating methods for attaching molecular tags to antibodies.
  • Figures 4A-4B present representative electropherograms showing expression of total Her2 and Herl-Her2 dimers in patient samples.
  • Figures 5 presents a representative electropherogram showing expression of total Her2 and Her2-Her3 dimers in patient samples.
  • Figures 6A-6B present representative electropherograms showing expression of total Herl, Herl-Herl dimers, and phosphorylated Herl inpatient samples.
  • Figure 7 presents a representative electropherogram showing expression of Herl- Her3 dimers in patient samples.
  • Figure 8 presents representative data showing amounts of total Herl expression (HIT), Herl-Herl dimer expression (Hl 1 dimers per cell), phosphorylated Herl, internal control expression, and % Tumor cells observed in the samples by immunohistochemical analysis.
  • Figure 9 shows a computer system in accordance with the present invention.
  • Figure 10 shows the disribution of different biomarkers in patients responsive and non-responsive to treatment with a Her2-acting agent.
  • IT represents total Herl
  • 1 ID represents Her I/Her 1 dimers
  • 2T represents total Her2
  • 2P represents phosphorylated Her2
  • 3T represents total Her3
  • 12D represents Herl/Her2 dimers
  • 23D represents Her2/Her3 dimers
  • 13D represents Herl/Her3 dimers
  • PTEN represents PTEN
  • 22D represents HEr2/Her2 dimers
  • 12+23 represents Herl/Her2 dimers + Her2/Her3 dimers
  • 13+ 23 represents Herl/Her3 dimers + Her2/Her3 dimers
  • 12+13 represents Herl/Her2 dimers + Herl/Her3 dimers
  • sum represents Herl/Her2 dimers + Herl/Her3 dimers + Her2/Her3 dimers
  • sum - PTEN represents Herl/Her2 dimers + Herl/Her3 dimers + Her2/Her3 dimers
  • Sum/PTEN
  • Figures 1 IA-D present classification trees showing the ability of different biomarkers to distinguish responders from nonresponders.
  • the amount of Herl/Her2 +Herl/Her3 dimers is the first distinguishing trait
  • log (unnormalized PTEN RFUs) is the second distinguishing trait.
  • the score calculated accoding to the first aspect of Formula II is the first distinguishing trait
  • log (normalized PTEN RFUs) is the second.
  • log (Herl/Her2) +log (Herl/Her3) is the first distinguishing trait
  • log (normalized PTEN RFUs) is the second.
  • Figure HD log (unnormalized PTEN RFUs) is the first distinguishing trait
  • log (Herl/Her2) +log (Herl/Her3) is the second.
  • Figure 12 presents the distribution of total Her3, Herl/Her3 dimers, and Her2/Her3 dimers observed in Group 1 patient samples.
  • Figure 13 presents a Kaplan-Meier curve showing the ability of Formula I to signficantly distinguish patients who tend to survive from patients who tend to die quickly.
  • Figure 14 presents a Kaplan-Meier curve showing that grouping patients labeled as complete or partial response or stable disease versus progessive disease also distinguishes patients who tend to survive from patients who tend to die quickly.
  • the term “about 5 ⁇ g/kg” means a range of from 4.5 ⁇ g/kg to 5.5 ⁇ g/kg.
  • “about 1 hour” means a range of from 48 minutes to 72 minutes.
  • Antibody means an immunoglobulin that specifically binds to, and is thereby defined as complementary with, a particular spatial and polar organization of another molecule.
  • the antibody can be monoclonal, polyclonal, or recombinant and can be prepared by techniques that are well known in the art such as immunization of a host and collection of sera (polyclonal) or by preparing continuous hybrid cell lines and collecting the secreted protein (monoclonal), or by cloning and expressing nucleotide sequences or mutagenized versions thereof coding at least for the amino acid sequences required for specific binding of natural antibodies.
  • Antibodies may include a complete immunoglobulin or fragment thereof, which immunoglobulins include the various classes and isotypes, such as IgA, IgD, IgE, IgGl, IgG2a, IgG2b and IgG3, IgM, etc. Fragments thereof may include Fab, Fv and F(ab')2, Fab', and the like. Antibodies may also be single-chain antibodies, or an antigen-binding fragment thereof, chimeric antibodies, humanized antibodies, or any other antibody derivative known to one of skill in the art that retains binding activity that is specific for a particular binding site.
  • aggregates, polymers, and conjugates of immunoglobulins or their fragments can be used where appropriate so long as binding affinity for a particular binding site is maintained.
  • Guidance in the production and selection of antibodies and antibody derivatives for use in immunoassays can be found in readily available texts and manuals, e.g., Harlow and Lane, 1988, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York; Howard and Bethell, 2001, Basic Methods in Antibody Production and Characterization, CRC Press; Wild, ed., 1994, The Immunoassay Handbook, Stockton Press, New York.
  • Antibody binding composition means a molecule or a complex of molecules that comprises one or more antibodies, or antigen-binding fragment, and derives its binding specificity from such antibody or antibody fragment.
  • Antibody binding compositions include, but are not limited to, (i) antibody pairs in which a first antibody binds specifically to a target molecule and a second antibody binds specifically to a constant region of the first antibody; a biotinylated antibody that binds specifically to a target molecule and a streptavidin protein, which protein is derivatized with moieties such as molecular tags or photosensitizers, or the like, via a biotin moiety; (ii) antibodies specific for a target molecule and conjugated to a polymer, such as dextran, which, in turn, is derivatized with moieties such as molecular tags or photosensitizers, either directly by covalent bonds or indirectly via streptavidin-biotin linkages; (iii) antibodies specific for a
  • fragment in the phrase “antigen-binding antibody fragment” refers to a peptide or polypeptide comprising an amino acid sequence of at least about 5 contiguous amino acid residues, at least about 10 contiguous amino acid residues, at least about 15 contiguous amino acid residues, at least about 20 contiguous amino acid residues, at least about 25 contiguous amino acid residues, at least about 40 contiguous amino acid residues, at least about 50 contiguous amino acid residues, at least about 60 contiguous amino residues, at least about 70 contiguous amino acid residues, at least about 80 contiguous amino acid residues, at least about 90 contiguous amino acid residues, at least about 100 contiguous amino acid residues, at least about 110 contiguous amino acid residues, or at least about 120 contiguous amino acid residues, of the amino acid sequence of another polypeptide, e.g., an antibody that preferentially binds an ErbB receptor.
  • another polypeptide e.g., an antibody that preferentially binds an
  • Antigenic determinant means a site on the surface of a molecule, usually a protein, to which a single antibody molecule binds.
  • a protein has several or many different antigenic determinants and reacts with antibodies of many different specificities.
  • a preferred antigenic determinant is a phosphorylation site of a protein.
  • a "binding compound,” as used herein, refers to an antibody binding composition, an antibody, a peptide, a peptide or non-peptide ligand for a cell surface receptor, a protein, an oligonucleotide, an oligonucleotide analog, such as a peptide nucleic acid, a lectin, or any other molecular entity that is capable of specifically binding to a target protein or molecule or stable complex formation with an analyte of interest, such as a complex of proteins.
  • a binding compound which can be represented by the formula below, comprises one or more molecular tags attached to a binding moiety.
  • Binding moiety means any molecule to which molecular tags can be directly or indirectly attached that is capable of specifically binding to an analyte.
  • Binding moieties include, but are not limited to, antibodies, antibody binding compositions, peptides, proteins, nucleic acids, and organic molecules having a molecular weight of up to about 1000 daltons and containing atoms selected from the group consisting of hydrogen, carbon, oxygen, nitrogen, sulfur, and phosphorus.
  • binding moieties are antibodies or antibody binding compositions.
  • Cancer and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia.
  • cancers include squamous cell cancer, lung cancer, e.g., small-cell lung cancer or non-small cell lung cancer; gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial carcinoma, salivary gland carcinoma, kidney cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancer.
  • lung cancer e.g., small-cell lung cancer or non-small cell lung cancer
  • gastrointestinal cancer pancreatic cancer
  • glioblastoma glioblastoma
  • cervical cancer ovarian cancer
  • liver cancer bladder cancer
  • hepatoma hepatoma
  • breast cancer colon cancer
  • colorectal cancer endometrial carcinoma
  • salivary gland carcinoma kidney cancer
  • prostate cancer vulval cancer
  • thyroid cancer hepatic carcinoma and various types of head and neck cancer.
  • Cancer cell refers to a cell that is cancerous, as defined above.
  • a "cleavable linkage,” as used herein, refers to a chemical linking group that may be cleaved under conditions that do not degrade the structure or affect detection characteristics of a molecular tag connected with the cleavable linkage.
  • a "cleavage-inducing moiety,” or “cleaving agent,” as used herein, is a group that produces an active species that is capable of cleaving a cleavable linkage, preferably by oxidation.
  • the active species is a chemical species that exhibits short-lived activity so that its cleavage-inducing effects are only in the proximity of the site of its generation.
  • a "cleaving probe,” as used herein, refers to a reagent that comprises a cleavage- inducing moiety as defined herein and an antibody binding composition, an antibody, a peptide, a peptide or non-peptide ligand for a cell surface receptor, a protein, such as biotin or streptavidin, an oligonucleotide, an oligonucleotide analog, such as a peptide nucleic acid, a lectin, or any other molecular entity that is capable of specifically binding to a target protein or molecule or stable complex formation with an analyte of interest, such as a complex of proteins.
  • “Complex” as used herein means an assemblage or aggregate of molecules in direct or indirect contact with one another.
  • "contact,” or more particularly, “direct contact” in reference to a complex of molecules, or in reference to specificity or specific binding means two or more molecules are close enough so that attractive noncovalent interactions, such as Van der Waal forces, hydrogen bonding, ionic and hydrophobic interactions, and the like, dominate the interaction of the molecules.
  • a complex of molecules is stable in that under assay conditions the complex is thermodynamically more favorable than a non-aggregated, or non- complexed, state of its component molecules.
  • “complex” usually refers to a stable aggregate of two or more proteins, and is equivalently referred to as a "protein-protein complex.” Most typically, a “complex” refers to a stable aggregate of two proteins.
  • Dimers in reference to cell surface membrane receptors means a complex of two or more membrane-bound receptor proteins that may be the same or different. Dimers of identical receptors are referred to as “homodimers” and dimers of different receptors are referred to as “heterodimers.” Dimers usually consist of two receptors in contact with one another. Dimers may be created in a cell surface membrane by passive processes, such as Van der Waal interactions, and the like, as described above in the definition of "complex,” or dimers may be created by active processes, such as by ligand-induced dimerization, covalent linkages, interaction with intracellular components, or the like. See, e.g., Schlessinger, 2000, Cell 103:211-225. As used herein, the term “dimer” is understood to refer to "cell surface membrane receptor dimer,” unless understood otherwise from the context.
  • Disease status includes, but is not limited to, the following features: likelihood of contracting a disease, presence or absence of a disease, prognosis of disease severity, and likelihood that a patient, or cancer or cancer cell obtained from a patient or subject, will respond to treatment by a particular therapeutic agent that acts through a receptor complex.
  • disease status further includes detection of precancerous or cancerous cells or tissues, the selection of patients that are likely to respond to treatment by a therapeutic agent that acts through one or more receptor complexes, such as one or more receptor dimers, and the ameliorative effects of treatment with such therapeutic agents.
  • disease status in reference to Her receptor complexes means likelihood that a cancer patient will respond to treatment with a Her, or ErbB, dimer-acting drug.
  • disease status in reference to Her receptor complexes refers to the likelihood that a cancer patient will respond to treatment with a Her2-acting drug.
  • such cancer patient is a breast cancer patient and such Her2-acting drugs include trastuzumab (Herceptin ® ).
  • Diagnostic Index refers to a number that reflects the likelihood that a cancer or cancer cell will respond to treatment with a Her2-acting agent.
  • a threshold Diagnostic Index is determined for each appropriate formula of the invention that distinguishes cancers or cancer cells that are likely to respond to treatment with a Her2 -acting agent from cancers or cancer cells that are not likely to respond to treatment.
  • the Diagnostic Index is determined for each appropriate formula of the invention such that cancers or cancer cells likely to respond to treatment with a Her2-acting agent are significantly more likely to respond to treatment with a Her2- acting agent than cancers or cancer cells not likely to respond to treatment with a Her2- acting agent.
  • the Diagnostic Index is a number between 0 and 1.
  • cancers or cancer cells that have a Diagnostic Index above the threshold Diagnostic Index are likely to respond to treatment with a Her2-acting agent, while cancers or cancer cells that have a Diagnostic Index below the threshold Diagnostic Index are likely to respond to treatment with a Her2- acting agent.
  • cancers or cancer cells that have a Diagnostic Index above the threshold Diagnostic Index are significantly more likely to respond to treatment with a Her2-acting agent than cancers or cancer cells that have a Diagnostic Index below the threshold Diagnostic Index.
  • Effective protein refers to an intracellular protein that is a component of a signal transduction pathway and that may be chemically altered resulting in the acquisition or loss of an activity or property. Such chemical alteration may include any post-translational modification known to one of skill in the art as well as processing by proteinases.
  • effector proteins are chemically modified by phosphorylation and acquire protein kinase activity as a result of such phosphorylation.
  • effector proteins are chemically modified by phosphorylation and lose protein kinase activity as a result of such phosphorylation.
  • effector proteins are chemically modified by phosphorylation and lose the ability to form stable complexes with particular proteins as a result of such phosphorylation.
  • exemplary effector proteins include, but are not limited to, mTOR proteins, TSC proteins, Akt proteins, Erk proteins, p38 proteins, and Jnk proteins.
  • post-translational modifications of effector proteins an effector protein may have one or more sites, referred to herein as a "post-translational modification site," which are characteristic amino acids of the effector protein where a post-translational modification may be attached or ⁇ emoved in the course of a signal transduction event.
  • ErbB receptor or "Her receptor” is a receptor protein tyrosine kinase which belongs to the ErbB receptor family and includes EGFR ("Herl"), ErbB2 ("Her2"), ErbB3 ("Her3") and ErbB4 ("Her4") receptors.
  • the ErbB receptor generally comprises an extracellular domain, which may bind an ErbB ligand; a lipophilic transmembrane domain; a conserved intracellular tyrosine kinase domain; and a carboxyl-terrninal signaling domain harboring several tyrosine residues which can be phosphorylated.
  • the ErbB receptor may comprise a native ErbB receptor sequence or an amino acid sequence variant thereof.
  • the ErbB receptor comprises a native human ErbB receptor sequence.
  • ErbB receptor includes truncated versions of Her receptors, including but not limited to, EGFRvIII and p95Her2 as discussed in Chu et ai, 1997, Biochem. J. 324:855-861 and Xia et al, 2004, Oncogene 23:646-653.
  • ErbBl "epidermal growth factor receptor,” “EGFR,” and “Herl” are used interchangeably herein and refer to native EGFR, and allelic variants thereof, as disclosed, for example, in Carpenter et ai, 1987, Ann. Rev. Biochem. 56:881-914, including such variants as, for example, a deletion mutant EGFR as in Humphrey et al, 1987, P.N.A.S. USA 87:4207-4211. Unless indicated otherwise, the terms “ ErbBl” " EGFR " and “Herl” when used herein refer to the human protein.
  • Herl The gene encoding Herl is referred to herein as "erbBl.”
  • antibodies which bind to Herl include MAb 579 (ATCC CRL RB 8506), MAb 455 (ATCC CRL HB8507), MAb 225 (ATCC CRL 8508), MAb 528 (ATCC CRL 8509) and variants thereof, such as chimerized 225 (C225) and reshaped human 225 (H225) as disclosed in, for example, U.S. Patent No.4,943,533, and International Patent Publication WO 96/40210.
  • ErbB3 and Her3 are used interchangeably herein and refer to native Her3, and allelic variants thereof, as described, for example, in U.S. Pat. Nos. 5,183,884 and 5,480,968 as well as Kraus et al, 1989, P.N.A.S. (USA) 86:9193-9197. Unless indicated otherwise, the terms “ErbB3” and “Her3” when used herein refer to the human protein. The gene encoding Her3 is referred to herein as "erbB3.” Examples of antibodies which bind Her3 include, for example, the 8B8 antibody (ATCC HB 12070) as described in, for example, U.S. Pat. No. 5,968,511.
  • ErbB4 and Her4 are used interchangeably herein and refer to native Her4, and allelic variants thereof, as described, for example, in E.P. Pat. App. No. 599,274; Plowman et al, 1993, P.N.A.S. (USA) 90:1746-1750; and Plowman et al, 1993, Nature 366:473-475, including such variants as, e.g., the Her4 isoforms disclosed in International Patent Publication No. WO 99/19488. Unless indicated otherwise, the terms "ErbB4" and "Her4" when used herein refer to the human protein. The gene encoding Her4 is referred to herein as "erbB4.”
  • a "Her2-acting agent,” as used herein, refers to a compound that can inhibit a biological activity of Her2. Such biological activities include, but are not limited to, dimerization, autophosphorylation, phosphorylation of another receptor, signal transduction, and the like. Exemplary Her2-acting agents include, but are not limited to, the large molecules 4D5 and trastuzumab and small molecules such as AEE-788 and lapatinib.
  • IGF-IR insulin-like growth factor- 1 receptor
  • IGF-IR refers to a human receptor tyrosine kinase, or homolog or variant thereof, such as those disclosed in Ullrich et al, 1986, EMBOJ., 5:2503-2512 and Steele-Perkins et al, 1988, J. Biol Chem. 263:11486-11492.
  • an isolated polypeptide or protein in reference to a polypeptide or protein means substantially separated from the components of its natural environment.
  • an isolated polypeptide or protein is a composition that consists of at least eighty percent of the polypeptide or protein identified by sequence on a weight basis as compared to components of its natural environment; more preferably, such composition consists of at least ninety- five percent of the polypeptide or protein identified by sequence on a weight basis as compared to components of its natural environment; and still more preferably, such composition consists of at least ninety-nine percent of the polypeptide or protein identified by sequence on a weight basis as compared to components of its natural environment.
  • an isolated polypeptide or protein is a homogeneous composition that can be resolved as a single spot after conventional separation by two- dimensional gel electrophoresis based on molecular weight and isoelectric point.
  • Protocols for such analysis by conventional two-dimensional gel electrophoresis are well known to one of ordinary skill in the art, such as, e.g., the procedures described by Hames and Rickwood, eds., 1981, Gel Electrophoresis of Proteins: A Practical Approach, IRL Press, Oxford; Scopes, 1982, Protein Purification, Springer- Verlag, New York; and Rabilloud, ed., 2000, Proteome Research: Two-Dimensional Gel Electrophoresis and Identification Methods, Springer- Verlag, Berlin.
  • kits refers to any delivery system for delivering materials or reagents for carrying out a method of the invention.
  • delivery systems include systems that allow for the storage, transport, or delivery of reaction reagents (e.g., probes, enzymes, etc. in the appropriate containers) and/or supporting materials ⁇ e.g., buffers, written instructions for performing the assay etc.) from one location to another.
  • reaction reagents e.g., probes, enzymes, etc. in the appropriate containers
  • supporting materials e.g., buffers, written instructions for performing the assay etc.
  • kits include one or more enclosures (e.g., boxes) containing the relevant reaction reagents and/or supporting materials.
  • Such contents may be delivered to the intended recipient together or separately.
  • a first container may contain an enzyme for use in an assay, while a second container may contain probes.
  • a cancer cell that is likely to respond to treatment with trastuzumab as determined according to a method of the invention has an increased probability of responding to treatment with trastuzumab relative to a reference cancer cell, for example, a cancer cell with a probability of responding to treatment that is the average cancer cell's probability of responding to treatment with trastuzumab.
  • the average cancer cell's probability of responding to treatment with trastuzumab is the average response observed for a statistically significant number of cancer cells treated with trastuzumab.
  • a subject is administered a therapy as described herein to "manage” a disorder so as to prevent or slow the progression or worsening of the disorder.
  • a subject is administered a therapy as described herein to "manage” a disorder so as to lengthen of the life of the subject over his or her theoretical life expectancy without being administered therapy for the disorder.
  • a "molecular tag,” as used herein, refers to a molecule that can be distinguished from other molecules based on one or more physical, chemical, or optical differences among the molecules being separated, including but not limited to, electrophoretic mobility, molecular weight, shape, solubility, pKa, hydrophobicity, charge, charge/mass ratio, polarity, or the like.
  • molecular tags in a plurality or set differ in electrophoretic mobility and optical detection characteristics and can be separated by electrophoresis.
  • molecular tags in a plurality or set may differ in molecular weight, shape, solubility, pKa, hydrophobicity, charge, polarity, and can be separated by normal phase or reverse phase HPLC, ion exchange HPLC, capillary electrochromatography, mass spectroscopy, gas phase chromatography, or like technique.
  • the term "monoclonal antibody” refers to an antibody that is derived from a single cellular clone, including any eukaryotic, prokaryotic, or phage clone, and is not dependent upon the method by which it is produced. Therefore, a “monoclonal antibody” can refer to a composition comprising a population of antibodies that each bind to a single epitope wherein said composition lacks antibodies that bind a different epitope than the single epitope to which the population of antibodies bind. It is noted, of course, that in certain instances, a single epitope is present in a polypeptide at multiple positions. In such instances, although the monoclonal antibody may bind to multiple positions, it is, nonetheless, still considered to be binding to a single epitope.
  • Percent identical means that in an optimal alignment between the two sequences, the candidate sequence is identical to the reference sequence in a number of subunit positions equivalent to the indicated percentage, the subunits being nucleotides for polynucleotide comparisons or amino acids for polypeptide comparisons.
  • an "optimal alignment" of sequences being compared is one that maximizes matches between subunits and minimizes the number of gaps employed in constructing an alignment. Percent identities may be determined with commercially available implementations of algorithms described by Needleman and Wunsch, 1970, J. MoI. Biol.
  • candidate sequence may be a component or segment of a larger polypeptide or polynucleotide and that such comparisons for the purpose computing percentage identity is to be carried out with respect to the relevant component or segment.
  • the terms “prevent”, “preventing” and “prevention” refer to the impedition of the recurrence or onset of a disorder or one or more symptoms of a disorder in a subject.
  • Phosphatidylinositol-3 kinase protein or equivalently a “PI3K protein,” refers to a human intracellular protein of the set of human proteins described under NCBI accession numbers NP_852664, NP 852556, and NP_852665, and homologs or variants thereof, and proteins having amino acid sequences substantially identical thereto.
  • Platinum-derived growth factor receptor or "PDGFR” means a human receptor tyrosine kinase protein that is substantially identical to PDGFR ⁇ or PDGFR ⁇ , or homologs or variants thereof, as described in Heldin et al, 1999, Physiological Reviews 79:1283-1316.
  • the invention includes determining the status of cancers, pre-cancerous conditions, f ⁇ brotic or sclerotic conditions by measuring one or more dimers of the following group: PDGFR ⁇ homodimers, PDGFR ⁇ homodimers, and PDGFR ⁇ -PDGFR ⁇ heterodimers.
  • f ⁇ brotic conditions include lung or kidney fibrosis
  • sclerotic conditions include atherosclerosis.
  • Cancers include, but are not limited to, breast cancer, colorectal carcinoma, glioblastoma, and ovarian carcinoma.
  • Reference to "PDGFR” alone is understood to mean “PDGFR ⁇ ” or "PDGFR ⁇ .”
  • a "polypeptide” refers to a class of compounds composed of amino acid residues chemically bonded together by amide linkages with elimination of water between the carboxy group of one amino acid and the amino group of another amino acid.
  • a polypeptide is a polymer of amino acid residues, which may contain a large number of such residues. Peptides are similar to polypeptides, except that, generally, they are comprised of a lesser number of amino acids. Peptides are sometimes referred to as oligopeptides. There is no clear-cut distinction between polypeptides and peptides. For convenience, in this disclosure and claims, the term “polypeptide” will be used to refer generally to peptides and polypeptides.
  • the amino acid residues may be natural, i.e., one of the twenty amino acids ordinarily found in human proteins, or non-natural. Further, a polypeptide may be expressed by an organism or synthesized synthetically.
  • Protein refers to a polypeptide, usually synthesized by a biological cell, folded into a defined three-dimensional structure. Proteins are generally from about 5,000 to about 5,000,000 or more in molecular weight, more usually from about 5,000 to about 1,000,000 molecular weight, and may include posttranslational modifications, such acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, farnesylation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination,
  • Proteins include, by way of illustration and not limitation, cytokines or interleukins, enzymes such as, e.g., kinases, proteases, galactosidases and so forth, protamines, histones, albumins, immunoglobulins, scleroproteins, phosphoproteins, mucoproteins, chromoproteins, lipoproteins, nucleoproteins, glycoproteins, T-cell receptors, other receptors, proteoglycans, and the like,
  • PTEN refers to a human lipid phosphatase that converts phosphatidylinositol 3,4,5-t ⁇ iphosphate (PIP3) into phosphatidylinositol 4,5-diphosphate (PIP2) and that is substantially identical to nucleotide sequences, or variants thereof, described in Li et ah, 1997, Science 275:1943- 1947 and Steck et al, 1997, Nature Genetics 15:356-362.
  • Reference sample means one or more cell, xenograft, or tissue samples that are representative of a normal or non-diseased state to which measurements on patient samples are compared to determine whether a receptor complex is present in excess or in reduced amount in the patient sample.
  • the nature of the reference sample is a matter of design choice for a particular assay and may be derived or determined from normal tissue of the patient him- or herself, or from tissues from a healthy individual or a population of healthy individuals.
  • values relating to amounts of receptor complexes in reference samples are obtained under essentially identical experimental conditions as corresponding values for patient samples being tested.
  • Reference samples may be from the same kind of tissue as that the patient sample, or it may be from different tissue types, and the population from which reference samples are obtained may be selected for characteristics that match those of the patient, such as age, sex, race, and the like.
  • amounts of receptor complexes on patient samples are compared to corresponding values of reference samples that have been previously tabulated and are provided as average ranges, average values with standard deviations, or like representations.
  • Receptor complex means a complex that comprises at least one cell surface membrane receptor.
  • Receptor complexes may include a dimer of cell surface membrane receptors, or one or more intracellular proteins, such as adaptor proteins, that form links in the various signaling pathways.
  • intracellular proteins that may be part of a receptor complex includes, but is not limit to, PDK proteins, Grb2 proteins, Grb7 proteins, She proteins, and Sos proteins, Src proteins, CbI proteins, PLC ⁇ proteins, Shp2 proteins, GAP proteins, Nek proteins, Vav proteins, and Crk proteins.
  • receptor complexes include PI3K or She proteins.
  • Receptor tyrosine kinase refers to a human receptor protein having intracellular kinase activity and being selected from the RTK family of proteins, such as those described in Schlessinger, 2000, Cell 103: 211-225 and Blume-Jensen and Hunter, supra.
  • Receptor tyrosine kinase dimer refers to a complex in a cell surface membrane comprising two receptor tyrosine kinase proteins.
  • a receptor tyrosine kinase dimer may comprise two covalently linked receptor tyrosine kinase proteins. Exemplary RTK dimers are listed in Table 1.
  • RTK dimers of particular interest are Her receptor dimers and VEGFR dimers.
  • "Responsiveness,” to “respond” to treatment, and other forms of this verb, as used herein, refer to the reaction of a cancer cell to treatment with a Her2-acting agent.
  • a cancer cell responds to treatment with a Her2-acting agent if growth of a tumor comprising the cancer cell is retarded about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more.
  • a cancer cell responds to treatment with a Her2-acting agent if a tumor comprising the cancer cell shrinks by about 5%, 10%, 20%, 30%, 40%, 50% or more as determined by any appropriate measure, e.g., by mass or volume.
  • a cancer cell responds to treatment with a Her2-acting agent if a patient with a tumor comprising the cancer cell experiences a life expectancy extended by about 5%, 10%, 20%, 30%, 40%, 50% or more beyond the life expectancy predicted if no treatment is administered.
  • sample or "tissue sample” or “patient sample” or “patient cell or tissue sample” or “specimen” each refers to a collection of similar cells obtained from a tissue of a subject or patient.
  • the source of the tissue sample may be solid tissue as from a fresh, frozen and/or preserved organ or tissue sample or biopsy or aspirate; blood or any blood constituents; bodily fluids such as cerebral spinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid; or cells from any time in gestation or development of the subject.
  • the tissue sample may contain compounds which are not naturally intermixed with the tissue in nature such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, or the like.
  • tissue samples or patient samples are fixed, particularly conventional formalin-fixed paraffin-embedded samples.
  • samples are typically used in an assay for receptor complexes in the form of thin sections, e.g. 3-10 ⁇ m thick, of fixed tissue mounted on a microscope slide, or equivalent surface.
  • samples also typically undergo a conventional re-hydration procedure, and optionally, an antigen retrieval procedure as a part of, or preliminary to, assay measurements.
  • Separatation profile in reference to the separation of molecular tags means a chart, graph, curve, bar graph, or other representation of signal intensity data versus a parameter related to the molecular tags, such as retention time, mass, or the like, that provides a readout, or measure, of the number of molecular tags of each type produced in an assay.
  • a separation profile may be an electropherogram, a chromatogram, an electrochromatogram, amass spectrogram, or like graphical representation of data depending on the separation technique employed.
  • a "peak” or a "band” or a "zone” in reference to a separation profile means a region where a separated compound is concentrated.
  • electrophoretic resolution is "electrophoretic resolution," which may be taken as the distance between adjacent peak maximums divided by four times the larger of the two standard deviations of the peaks.
  • adjacent peaks have a resolution of at least 1.0, and more preferably, at least 1.5, and most preferably, at least 2.0.
  • the desired resolution may be obtained by selecting a plurality of molecular tags whose members have electrophoretic mobilities that differ by at least a peak-resolving amount, such quantity depending on several factors well known to those of ordinary skill, including signal detection system, nature of the fluorescent moieties, the diffusion coefficients of the tags, the presence or absence of sieving matrices, nature of the electrophoretic apparatus, e.g. presence or absence of channels, length of separation channels, and the like. Electropherograms may be analyzed to associate features in the data with the presence, absence, or quantities of molecular tags using analysis programs, such as disclosed in U.S. Patent Application Publication 2003/0170734 Al.
  • SHC (standing for "Src homology 2/ ⁇ -collagen-related”) means any one of a family of adaptor proteins (66, 52, and 46 kDalton) in RTK signaling pathways substantially identical to those described in Pelicci et al, 1992, Cell 70: 93-104, or variants or homologs thereof.
  • SHC means the human versions of such adaptor proteins.
  • a “signaling pathway” or “signal transduction pathway,” as used herein, refers to a series of molecular events usually beginning with the interaction of cell surface receptor and/or receptor dimer with an extracellular ligand or with the binding of an intracellular molecule to a phosphorylated site of a cell surface receptor. Such beginning event then triggers a series of further molecular interactions or events, wherein the series of such events or interactions results in a regulation of gene expression, for example, by regulation of transcription in the nucleus of a cell, or by regulation of the processing or translation of rnRNA transcripts.
  • signaling pathway means either the Ras- MAPK pathway, the PI3K-Akt pathway, or an mTOR pathway.
  • Ras-MAPK pathway means a signaling pathway that includes the phosphorylation of a MAPK protein subsequent to the formation of a Ras-GTP complex.
  • PBK- Akt pathway means a signaling pathway that includes the phosphorylation of an Akt protein by a PI3K protein.
  • mTOR pathway means a signaling pathway comprising one or more of the following entities; an mTOR protein, a PDK protein, an Akt protein, an S6K1 protein, an FKBP protein, including an FKBP12 protein, a TSCl protein, a TSC2 protein, a p70S6K protein, a raptor protein, a rheb protein, a PDK protein, a 4E-BP1 protein, wherein each of the proteins may be phosphorylated at a post-translational modification site.
  • mTOR pathways may also include the following complexes: FKBP12//mTOR, raptor//mTOR, raptor//4E-BPl, raptor//S6Kl, raptor//4E-BPl//mTOR, raptor//S6Kl//mTOR.
  • FKBP12//mTOR raptor//mTOR
  • raptor//4E-BPl raptor///S6Kl
  • raptor//4E-BPl//mTOR raptor//S6Kl//mTOR.
  • “Specific” or “specificity” in reference to the binding of one molecule to another molecule, such as a binding compound, or probe, for a target analyte or complex means the recognition, contact, and formation of a stable complex between the probe and target, together with substantially less recognition, contact, or complex formation of the probe with other molecules.
  • “specific” in reference to the binding of a first molecule to a second molecule means that to the extent the first molecule recognizes and forms a complex with another molecules in a reaction or sample, it forms the largest number of the complexes with the second molecule. In certain embodiments, this largest number is at least fifty percent of all such complexes form by the first molecule.
  • Specific binding include antibody-antigen interactions, enzyme- substrate interactions, formation of duplexes or triplexes among polynucleotides and/or oligonucleotides, receptor-ligand interactions, and the like.
  • Specific binding include antibody-antigen interactions, enzyme- substrate interactions, formation of duplexes or triplexes among polynucleotides and/or oligonucleotides, receptor-ligand interactions, and the like.
  • "Spectrally resolvable" in reference to a plurality of fluorescent labels means that the fluorescent emission bands of the labels are sufficiently distinct, i.e. sufficiently non- overlapping, that molecular tags to which the respective labels are attached can be distinguished on the basis of the fluorescent signal generated by the respective labels by standard photodetection systems, e.g.
  • substantially identical in reference to proteins or amino acid sequences of proteins in a family of related proteins that are being compared means either that one protein has an amino acid sequence that is at least fifty percent identical to the other protein or that one protein is an isoform or splice variant of the same gene as the other protein.
  • substantially identical means one protein, or amino acid sequence thereof, is at least eighty percent identical to the other protein, or amino acid sequence thereof.
  • substantially no refers to an undetectable amount, as measured by the eTagTM assay.
  • the terms “subject” and “patient” are used interchangeably.
  • the terms “subject” and “subjects” refer to an animal, preferably a mammal including a non-primate ⁇ e.g., a cow, pig, horse, donkey, goat, camel, cat, dog, guinea pig, rat, mouse, sheep) and a primate ⁇ e.g., a monkey, such as a cynomolgous monkey, gorilla, chimpanzee, and a human), preferably a human.
  • the subject is a subject with cancer, for example, ovarian cancer.
  • Treatment refers to the administration of a Her2-acting agent to impede growth of a cancer, to cause a cancer to shrink by weight or volume, to extend the expected survival time of the subject, and the like.
  • the invention provides methods for determining whether a cancer is likely to respond to treatment with a Her2-acting agent.
  • the methods comprise determining a probability that the cancer will respond to treatment with the Her2-acting agent based on application of a formula of the invention to one or more biomarkers associated with responsiveness and/or non- responsiveness to treatment with the Her2-acting agent as described herein.
  • the biomarkers associated with responsiveness to treatment with the Her2- acting agent comprise the presence and/or amount of expression of one or more of Herl- Herl dimers, Herl-Her2 dimers, Herl-Her3 dimers, and/or Her2-Her3 dimers and/or presence and/or amount of Her 1 phosphorylation and/or Her2 phosphorylation, or any combination thereof.
  • the presence and/or amount of the biomarker(s) positively correlate with responsiveness to treatment of a cancer or cancer cell with a Her2-acting agent.
  • the likelihood that the cancer will respond to treatment with the Her2-acting agent is presented as a Diagnostic Index.
  • the methods comprise detecting the number of one or more types of Her-1 containing dimers (Herl-Herl, Herl-Her2, Herl-Her3) per cell.
  • the embodiments presented below comprise detecting whether the number or numbers of Herl -containing dimers per cell is greater than a particular value, whereby, if greater, this indicates that the cell or cancer is likely to respond to a Her2-acting agent. It is to be understood that the invention also encompasses methods whereby if the number or numbers of such Herl -containing dimers per cell is determined to be less than or equal to the particular value presented in such embodiments, then this indicates that the cell or cancer is not likely to respond to the Her2- acting agent.
  • the present invention also encompasses a method that comprises detecting the number of Herl-Her2 dimers per cell such that if the number is about 0 this indicates that the cell is not likely to respond to a Her2-acting agent.
  • the invention provides a method for determining whether a subject with cancer is likely to respond to treatment with a Her2-acting agent that comprises detecting on the cancer cell substantially no Herl-Her2 dimer and substantially no Herl-Her3 dimer, wherein detection of substantially no Herl-Her2 dimer and substantially no Herl-H.er3 dimer indicates that the Her2-positive cancer cell is likely to respond to treatment with the Her2-acting agent.
  • the invention provides a method for determining whether a Her2-positive cancer cell is not likely to respond to treatment with a Her2-acting agent, comprising detecting Herl-Her2 dimer and Herl-Her3 dimer on the cancer cell, wherein Herl-Her2 dimer and Herl-Her3 dimer on the cancer cell indicates that the Her2-positive cancer cell is not likely to respond to treatment with the Her2-acting agent.
  • the invention provides a method for determining whether a Her2-positive cancer cell is likely to respond to treatment with a Her2-acting agent, comprising: determining log(Herl-Her2 dimer concentration) + log(Herl-Her3 dimer concentration) + log(Her2-Her3 dimer concentration) - log(phosphatase and tensin homologue ("PTEN") concentration in normalized RFUs), such that if value determined is less than or equal to about 2.091, or if the value determined for log(PTEN concentration in nomralized RFUs) is greater than about 2.779, the Her2-positive cancer cell is likely to respond to treatment with the Her2-acting agent.
  • PTEN tensin homologue
  • the invention provides a method for determining whether a Her2-positive cancer cell is not likely to respond to treatment with a Her2-acting agent, comprising: determining log(Herl-Her2 dimer concentration) + log(Herl-Her3 dimer concentration) + log(Her2-Her3 dimer concentration) - log(PTEN concentration in normalized RFUs), such that if value determined is greater than about 2.091, or if the value determined for log(PTEN concentration in nomralized RFUs) is greater than about 2.779, the Her2-positive cancer cell is not likely to respond to treatment with the Her2- acting agent.
  • the invention provides a method for determining whether a Her2-positive cancer cell is likely to respond to treatment with a Her2-acting agent, comprising: detecting whether the cancer cell comprises Herl-Her2 dimer, Herl-Her3 dimer, Her2-Her3 dimer and/or PTEN, wherein if 1) the cancer cell exhibits substantially no Herl-Her2 dimer a score of 1 is provided, 2) if the cancer cells exhibits substantially no Herl-Her3 dimer a score of 1 is provided, 3) if the cancer cell exhibits substantially no Her2-Her3 dimer a score of 1 is provided, and 4) if the cancer cell exhibits an amount of PTEN corresponding to greater than about 600 normalized RFUs a score of 1 is provided, such that if a total score of greater than or equal to 2 is provided, the Her2- positive cancer cell is likely to respond to treatment with the Her2-acting agent.
  • the methods further comprise detecting whether the cancer cell comprises Her 1 -Her 1 dimer, wherein if the cancer cell exhibits substantially no Herl-Herl dimer a score of 1 is provided, such that if a total score of greater than or equal to 3 is provided, the Her2 -positive cancer cell is likely to respond to treatment with the Her2-acting agent.
  • the invention provides a method for determining whether a Her2-positive cancer cell is not likely to respond to treatment with a Her2-acting agent, comprising: detecting whether the cancer cell comprises Herl-Her2 dimer, Herl-Her3 dimer, Her2-Her3 dimer and/or PTEN, wherein if 1) the cancer cell exhibits substantially no Herl-Her2 dimer a score of 1 is provided, 2) if the cancer cells exhibits substantially no Herl-Her3 dimer a score of 1 is provided, 3) if the cancer cell exhibits substantially no Her2-Her3 dimer a score of 1 is provided, and 4) if the cancer cell exhibits an amount of PTEN corresponding to greater than about 600 normalized RFUs a score of 1 is provided, such that if a total score of less than 2 is provided, the Her2-positive cancer cell is not likely to respond to treatment with the Her2-acting agent.
  • the methods further comprise detecting whether the cancer cell comprises Herl-Herl dimer, wherein if the cancer cell exhibits substantially no Herl-Herl dimer a score of 1 is provided, such that if a total score of less than 3 is provided, the Her2-positive cancer cell is not likely to respond to treatment with the Her2-acting agent.
  • the Her2-acting agent is trastuzumab, 4D5, AEE-788, and lapatinib. In a preferred embodiment, the Her2-acting agent is trastuzumab.
  • substantially no Herl-Her2 dimer and substantially no Herl-Her3 is detected on the cancer cell.
  • the amount of PTEN is detected that corresponds to greater than about 600 normalized RFUs.
  • Herl-Her2 dimer and Herl-Her3 dimer is detected on the cancer cell.
  • an amount of PTEN is detected that corresponds to less than about 600 normalized RFUs.
  • detecting comprises contacting the cancer cell with a binding compound having a molecular tag attached thereto by a cleavable linkage, and a cleaving probe having a cleavage inducing-moiety and detecting whether and what molecular tag is released.
  • the binding compound and the cleaving probe each specifically binds one of either: Herl or Her2, or Herl or Her3.
  • the cleaving probe and the binding probe do not both bind the same receptor.
  • the cleavage-inducing moiety cleaves the cleavable linker so that the molecular tag is released.
  • the molecular tag released if Herl -Her 2 dimer is present is distinguishable from the molecular tag released if Herl-Her3 dimer is present.
  • activating the cleavage-inducing moiety cleaves the cleavable linker.
  • the binding compound specifically binds a Herl epitope.
  • the binding compound comprises an antibody or antigen-binding fragment.
  • the binding compound specifically binds a Herl ligand binding site.
  • the binding compound comprises a Herl ligand.
  • the binding compound specifically binds a Her2 epitope. In certain embodiments, the binding compound specifically binds a Her2 ligand binding site. In certain embodiments, the binding compound comprises a Her2 ligand. In certain embodiments, the cleaving probe specifically binds a Herl epitope.
  • the binding compound specifically binds a Her3 epitope. In certain embodiments, the binding compound specifically binds a Her3 ligand binding site. In certain embodiments.the binding compound comprises a Her3 ligand. In certain embodiments, the cleaving probe specifically binds a Herl epitope.
  • the cleaving probe comprises an antibody or antigen- binding fragment, hi certain embodiments, the cleaving probe specifically binds a Herl ligand binding site. In certain embodiments, the cleaving probe comprises a Herl ligand. [0095] In certain embodiments, the cleaving probe specifically binds a Her2 epitope. In certain embodiments, the cleaving probe specifically binds a Her2 ligand binding site. In certain embodiments, the cleaving probe comprises a Her2 ligand. In certain embodiments, the cleaving probe specifically binds a Her3 epitope. In certain embodiments, the cleaving probe specifically binds a Her3 ligand binding site. In certain embodiments, the cleaving probe comprises a Her3 ligand.
  • the methods comprise detecting the amount of
  • the methods comprise detecting the amount of Herl-Herl dimers on a cancer cell present on a cancer cell, then detecting the amount of Herl-Her3 dimers on the cancer cell. In certain embodiments, the methods comprise detecting the amount of Herl-Her2 dimers on a cancer cell present on a cancer cell, then detecting the amount of Herl-Her3 dimers on the cancer cell. In certain embodiments, the methods comprise detecting the amount of Herl-Her3 dimers on a cancer cell present on a cancer cell, then detecting the amount of Herl-Herl dimers on the cancer cell.
  • the methods comprise detecting the amount of Herl-Her3 dimers on a cancer cell present on a cancer cell, then detecting the amount of Herl-Her2 dimers on the cancer cell.
  • the methods comprise detecting the amount of Herl-Her2 dimers on a cancer cell present on a cancer cell, then detecting the amount of Herl-Herl dimers on the cancer cell, hi certain embodiments, the methods comprise detecting the amount of Herl-Her2 dimers on a cancer cell present on a cancer cell, then detecting the amount of Herl-Her3 dimers on the cancer cell.
  • the methods comprise detecting the amount of Herl-Herl dimers on a cancer cell present on a cancer cell, then detecting the amount of Her2-Her3 dimers on the cancer cell. In certain embodiments, the methods comprise detecting the amount of Herl-Her3 dimers on a cancer cell present on a cancer cell, then detecting the amount of Her2-Her3 dimers on the cancer cell. In certain embodiments, the methods comprise detecting the amount of Herl-Her2 dimers on a cancer cell present on a cancer cell, then detecting the amount of Her2-Her3 dimers on the cancer cell.
  • the methods further comprise detecting the total amount of Herl, Her2, and/or Her3, or any combination thereof, subsequent to detecting the amount of Herl-Herl dimers, Herl-Her2 dimers, and/or Herl-Her3 dimers, or any combination thereof.
  • the invention provides a method of using ErbB cell surface receptor complexes as biomarkers for the status of a disease or other physiological conditions in a biological organism, particularly a cancer status in a human.
  • ErbB receptor complexes are measured directly from patient samples; that is, measurements are made without culturing, formation of xenografts, or the use of like techniques, that require extra labor and expense and that may introduce artifacts and/or noise into the measurement process,
  • measurements of one or more receptor complexes are made directly on tissue lysates of frozen patient samples or on sections of fixed patient samples.
  • one or more ErbB receptor complexes are measured in sections of formalin-fixed paraffin-embedded (FFPE) samples.
  • FFPE formalin-fixed paraffin-embedded
  • one or more ErbB receptor complexes are measured on a single biopsy obtained from a subject. More preferably, one or more ErbB receptor complexes are measured on a plurality of biopsies obtained from a subject. In certain embodiments, one or more ErbB receptor complexes are measured on two, three, four, five, six, seven, eight, nine, ten or more biopsies obtained from a subject.
  • the invention provides an indirect measurement of ErbB receptor phosphorylation through the measurement of complexes that depend on such posttranslational modifications for their formation.
  • the invention provides an indirect measurement of ErbB receptor dimerization and/or activation through measurement of phosphorylation of one or more members of an ErbB dimer.
  • a plurality of ErbB receptor complexes are simultaneously measured in the same assay reaction mixture.
  • such complexes are measured using binding compounds having one or more molecular tags releasably attached, such that after binding to a protein in a complex, the molecular tags may be released and separated from the reaction, or assay, mixture for detection and/or quantification.
  • the invention provides a method for determining a disease status of a patient comprising: measuring an amount of each of one or more ErbB receptor dimers in a patient sample; comparing each such amount to its corresponding amount from a reference sample; and correlating differences in the amounts from the patient sample and the respective corresponding amounts from the reference sample to the presence or severity of a disease condition in the patient.
  • the step of measuring comprising the steps of: (i) providing one or more binding compounds specific for a protein of each of the one or more receptor dimers, 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; (ii) mixing the binding compounds and the one or more complexes such that binding compounds specifically bind to their respective receptor dimers to form detectable complexes; (iii) cleaving the cleavable linkage of each binding compound forming detectable complexes, and (iv) separating and identifying the released molecular tags to determine the presence or absence or the amount of the one or more receptor dimers.
  • the step of measuring the amounts of one or more types of ErbB receptor dimer comprising the following steps: (i) providing for each of the one or more types of receptor dimer a cleaving probe specific for a first receptor in each of the one or more receptor dimers, each cleaving probe having a cleavage-inducing moiety with an effective proximity; (ii) providing one or more binding compounds specific for a second receptor of each of the one or more receptor dimers, 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; (iii) mixing the cleaving probes, the binding compounds, and the one or more types of receptor dimers such that cleaving probes specifically bind to first receptors of the receptor dimers and binding compounds specifically bind to the second receptors of the receptor
  • a biological sample which comprises a mixed cell population suspected of containing the rare cell of interest is obtained from a patient.
  • a sample is then prepared by mixing the biological specimen with magnetic particles which are coupled to a biospecific ligand specifically reactive with an antigen on the rare cell that is different from or not found on blood cells (referred to herein as a "capture antigen"), so that other sample components may be substantially removed.
  • the sample is subjected to a magnetic field which is effective to separate cells labeled with the magnetic particles, including the rare cells of interest, if any are present in the specimen.
  • the cell population so isolated is then analyzed using molecular tags conjugated to binding moieties specific for biomarkers to determine the presence and/or number of rare cells.
  • the magnetic particles used in this method are colloidal magnetic nanoparticles.
  • such rare cell populations are circulating epithelial cells, which may be isolated from patient's blood using epithelial-specific capture antigens such as, for example, those disclosed in Hayes et al., 2002, International J. Oncol. 21 :1111-1117; Soria et a/., 1999, Clin. Can. Res. 5:971-975; Ady et al., 2004, British J. Cancer 90:443-448; each of which are hereby incorporated by reference in its entirety.
  • monoclonal antibody BerEP4 (Dynal A.S., Oslo, Norway) may be used to capture human epithelial cells with magnetic particles.
  • the invention provides a method for determining a cancer status of a patient comprising the following steps: (i) immunomagnetically isolating , a patient sample comprising circulating epithelial cells by contacting a sample of patient blood with one or more antibody compositions, each antibody composition being specific for a capture antigen and being attached to a magnetic particle; (ii) measuring an amount of each of one or more ErbB receptor complexes in the patient sample; comparing each such amount to its corresponding amount from a reference sample; and correlating differences in the amounts from the patient sample and the respective corresponding amounts from the reference sample to the presence or severity of a cancer condition in the patient.
  • the step of measuring comprises the steps of: (i) providing one or more binding compounds specific for a protein of each of the one or more ErbB receptor complexes, 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; (ii) mixing the binding compounds and the one or more ErbB receptor complexes such that binding compounds specifically bind to their respective proteins of the one or more ErbB receptor complexes to form detectable complexes; (iii) cleaving the cleavable linkage of each binding compound forming detectable complexes, and (iv) separating and identifying the released molecular tags to determine the presence or absence or the amount of the one or more ErbB receptor complexes.
  • ErbB receptor complexes comprising the following steps: (i) providing for each of the one or more ErbB receptor complexes a cleaving probe specific for a first protein in each of the one or more ErbB receptor complexes, each cleaving probe having a cleavage- inducing moiety with an effective proximity; (ii) providing one or more binding compounds specific for a second protein of each of the one or more ErbB receptor complexes, 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; (iii) mixing the cleaving probes, the binding compounds, and the one or more complexes such that cleaving probes specifically bind to first proteins of the ErbB receptor complexes and binding compounds specifically bind to the second proteins of the ErbB receptor complexes and such
  • Biomarkers of the invention include dimers and oligomers of the following receptors. Table 1. Exemplary Receptor Complexes of Cell Surface Membranes
  • Such drugs are referred to herein as "dimer-acting" drugs, or "ErbB dimer-acting” drugs.
  • the number, type, formation, and/or dissociation of receptor dimers in the cells of a patient being treated, or whose treatment is contemplated, have a bearing on the effectiveness or suitability of using a particular ErbB dimer-acting drug.
  • the following ErbB receptor dimers are biomarkers related to the indicated drugs.
  • the invention provides biomarkers for monitoring the effect on disease status of an ErbB dimer-acting drug, comprising detecting the presence and/or amount of one or more biomarker associated with the ErbB dimer-acting drug.
  • the dimer-acting drugs listed in Table 2 are described and characterized by, for example, Traxler, 2002, Expert Opin. Ther. Targets 7: 215-234; Baselga, ed., 2002, Oncology Biotherapeutics 2:1-36; Nam et ah, 2003, Current Drug Targets 4:159- 179; and Seymour, 2001, Current Drug Targets 2:117-133; each of which is hereby incorporated by reference in its entirety.
  • the invention relates to Her2-acting agents, as defined above.
  • the Her2-acting agent can be any such agent known to one of skill in the art, without limitation.
  • the Her2-acting agent is selected from the group consisting of 4D5, trastuzumab, AEE-788, and lapatinib.
  • the Her2-acting agent is trastuzumab (Herceptin ® ). See, e.g., Goldenberg, 1999, Clin Ther. 21:309-18; and Shak, 1999, Semin Oncol. 26:71-7.
  • Samples containing molecular complexes suitable for use as biomarkers may come from a wide variety of sources for use with the present invention to relate receptor complexes populations to disease status or health status, including cell cultures, animal or plant tissues, patient biopsies, or the like.
  • samples are human patient samples.
  • Samples are prepared for assays of the invention using conventional techniques, which may depend on the source from which a sample is taken.
  • tissue samples that may be used include, but are not limited to, breast, prostate, ovary, colon, lung, endometrium, stomach, salivary gland or pancreas.
  • the tissue sample can be obtained by a variety of procedures including, but not limited to surgical excision, aspiration or biopsy.
  • the tissue may be fresh or frozen.
  • assays of the invention are carried out on tissue samples that have been fixed and embedded in paraffin or the like; therefore, in such embodiments a step of deparaffination can be carried out.
  • a tissue sample may be fixed (i.e., preserved) by conventional methodology. See, e.g., Lee G.
  • tissue sample is first fixed and is then dehydrated through an ascending series of alcohols, infiltrated and embedded with paraffin or other sectioning media so that the tissue sample may be sectioned.
  • tissue sample may be embedded and processed in paraffin by conventional methodology according to conventional techniques described by the references provided above.
  • paraffin examples include, but are not limited to, Paraplast, Broloid, and Tissuemay.
  • sections may have a thickness in a range from about three microns to about twelve microns, and preferably, a thickness in a range of from about 5 microns to about 10 microns.
  • a section may have an area of from about 10 mm 2 to about 1 cm 2 .
  • slide adhesives include, but are not limited to, silane, gelatin, poly-L-lysine and the like.
  • the paraffin embedded sections may be attached to positively charged slides and/or slides coated with poly-L-lysine.
  • the tissue sections are generally deparaffinized and rehydrated to water prior to detection of biomarkers.
  • the tissue sections may be deparaffinized by several conventional standard methodologies. For example, xylenes and a gradually descending series of alcohols may be used according to conventional techniques described by the references provided above. Alternatively, commercially available deparaffmizing non-organic agents such as Hemo- De® (CMS, Houston, Tex.) may be used.
  • samples may be prepared by conventional cell lysis techniques (e.g., 0.14 M NaCl, 1.5 mM MgCl 2 , 10 mM Tris-Cl (pH 8.6), 0.5% Nonidet P-40, and protease and/or phosphatase inhibitors as required).
  • sample preparation may also include a tissue disaggregation step, such as, for example, crushing, mincing, grinding, sonication, or the like.
  • an enrichment step may be carried out prior to conducting an assay for surface receptor dimer populations.
  • Immunomagnetic isolation or enrichment may be carried out using a variety of techniques and materials known in the art, as disclosed in the following representative references that are incorporated by reference: U.S. Patent Nos. 6,365,362; 5,646,001; 5,998,224; 5,665,582; 6,048,515; 5,508,164; 5,691,208; 4,452,773; and 4,375,407; Radbruch et al, 1994, Methods in Cell Biology, Vol. 42, Ch.
  • the preferred magnetic particles for use in carrying out this invention are particles that behave as colloids. Such particles are characterized by their sub-micron particle size, which is generally less than about 200 nanometers (nm) (0.20 microns), and their stability to gravitational separation from solution for extended periods of time. In addition to the many other advantages, this size range makes them essentially invisible to analytical techniques commonly applied to cell analysis. Particles within the range of 90- 150 nm and having between 70-90% magnetic mass are also contemplated for use in the present invention. Suitable magnetic particles are composed of a crystalline core of superparamagnetic material surrounded by molecules which are bonded, e.g., physically absorbed or covalently attached, to the magnetic coie and which confer stabilizing colloidal properties.
  • the coating material should preferably be applied in an amount effective to prevent non specific interactions between biological macromolecules found in the sample and the magnetic cores.
  • biological macromolecules may include sialic acid residues on the surface of non-target cells, lectins, glyproteins and other membrane components.
  • the material should contain as much magnetic mass/nanoparticle as possible.
  • the size of the magnetic crystals comprising the core is sufficiently small that they do not contain a complete magnetic domain.
  • the size of the nanoparticles is sufficiently small such that their Brownian energy exceeds their magnetic moment. As a consequence, North Pole, South Pole alignment and subsequent mutual attraction/repulsion of these colloidal magnetic particles does not appear to occur even in moderately strong magnetic fields, contributing to their solution stability.
  • magnetic particles should be separable in high magnetic gradient external field separators. That characteristic facilitates sample handling and provides economic advantages over the more complicated internal gradient columns loaded with ferromagnetic beads or steel wool.
  • Magnetic particles having the above-described properties can be prepared by modification of base materials described in U.S. Pat. Nos. 4,795,698, 5,597,531 and 5,698,271, which patents are incorporated by reference in their entireties.
  • a wide variety of separation techniques may be employed that can distinguish molecules based on one or more physical, chemical, or optical differences among molecules being separated including but not limited to electrophoretic mobility, molecular weight, shape, solubility, pKa, hydrophobicity, charge, charge/mass ratio, polarity, or the like.
  • molecular tags in a plurality or set differ in electrophoretic mobility and optical detection characteristics and are separated by electrophoresis.
  • molecular tags in a plurality or set may differ in molecular weight, shape, solubility, pKa, hydrophobicity, charge, polarity, and are separated by normal phase or reverse phase HPLC, ion exchange HPLC, capillary electrochromatography, mass spectroscopy, gas phase chromatography, or like technique.
  • the number of molecular tags in a plurality may vary depending on several factors including the mode of separation employed, the labels used on the molecular tags for detection, the sensitivity of the binding moieties, the efficiency with which the cleavable linkages are cleaved, and the like.
  • the number of molecular tags in a plurality for measuring populations of receptor dimers is in the range of from 2 to 20.
  • the size of the plurality may be in the range of from 2 to 18, 2 to 16, 2 to 14, 2 o 12, 2 to 10, 2 to 8, 2 to 6, 2 to 4, or 2 to 3.
  • Receptor dimers may be detected in assays having homogeneous formats or a non-homogeneous, e.g., heterogeneous, formats.
  • a homogeneous format no step is required to separate binding compounds specifically bound to target complexes from unbound binding compounds.
  • homogeneous formats employ reagent pairs comprising (i) one or more binding compounds with releasable molecular tags and (ii) at least one cleaving probe that is capable of generating an active species that reacts with and releases molecular tags within an effective proximity of the cleaving probe.
  • Receptor dimers may also be detected by assays employing a heterogeneous format.
  • Heterogeneous techniques normally involve a separation step, where intracellular complexes having binding compounds specifically bound are separated from unbound binding compounds, and optionally, other sample components, such as proteins, membrane fragments, and the like. Separation can be achieved in a variety of ways, such as employing a reagent bound to a solid support that distinguishes between complex-bound and unbound binding compounds.
  • the solid support may be a vessel wall, e.g., microtiter well plate well, capillary, plate, slide, beads, including magnetic beads, liposomes, or the like.
  • the primary characteristics of the solid support are that it (1) permits segregation of the bound and unbound binding compounds and (2) does not interfere with the formation of the binding complex, or the other operations in the determination of receptor dimers. Usually, in fixed samples, unbound binding compounds are removed simply by washing.
  • a sample may be combined with a solvent into which the molecular tags are to be released.
  • the solvent may include any additional reagents for the cleavage.
  • the solvent conveniently may be a separation buffer, e.g. an electrophoretic separation medium.
  • the medium may be irradiated with light of appropriate wavelength to release the molecular tags into the buffer.
  • assay reaction conditions include salt concentrations (e.g., required for specific binding) that degrade separation performance when molecular tags are separated on the basis of electrophoretic mobility.
  • an assay buffer is replaced by a separation buffer, or medium, prior to release and separation of the molecular tags.
  • Assays employing releasable molecular tags and cleaving probes can be made in many different formats and configurations depending on the complexes that are detected or measured.
  • One of skill in the art can, guided by the present disclosure, routinely select the numbers and specificities of particular binding compounds and cleaving probes for use in the methods of the invention.
  • Figs. IA and IB the use of releasable molecular tags to measure dimers of cell surface membranes is shown diagrammatically in Figs. IA and IB.
  • Binding compounds (100) having molecular tags "mTi” and “m T 2 " and cleaving probe (102) having photosensitizer "PS” are combined with biological cells (104).
  • Binding compounds having molecular tag "mT 1 " are specific for cell surface receptors Ri (106) and binding compounds having molecular tag “mT 2 " are specific for cell surface receptors R 2 (108).
  • Cell surface receptors Ri and R 2 are present as monomers, e.g. (106) and (108), and as dimers (110) in cell surface membrane (112).
  • binding compounds and cleaving probes each comprise an antibody binding composition, which permits the molecular tags and cleavage-inducing moiety to be specifically targeted to membrane components.
  • antibody binding compositions are monoclonal antibodies.
  • binding buffers may comprise buffers used in conventional ELISA techniques, or the like.
  • the only molecular tags released are those on binding compounds that form stable complexes with Rj-R 2 dimers and a cleaving probe.
  • Released molecular tags (126) are removed from the assay mixture and separated (128) in accordance with a separation characteristic so that a distinct peak (130) is formed in a separation profile (132). In accordance with the invention, such removal and separation may be the same step.
  • the binding buffer may be removed and replaced with a buffer more suitable for separation, i.e.
  • binding buffers typically have salt concentrations that may degrade the performance of some separation techniques, such as capillary electrophoresis, for separating molecular tags into distinct peaks.
  • exchange of buffers may be accomplished by membrane filtration.
  • Reagents (1122) of the invention comprise (i) cleaving probes (1108), first binding compound (1106), and second binding compound (1107), wherein first binding compound (1106) is specific for protein (1102) and second binding compound (1107) is specific for protein (1104) at a different antigenic determinant than that cleaving probe (1108) is specific for.
  • cleaving probe (1108) is activated to produce active species that cleave the cleavable linkages of the molecular tags within the effective proximity of the photosensitizer.
  • molecular tags are released from monomers of protein (1104) that have both reagents (1107) and (1108) attached and from heterodimers that have reagent (1108) attached and either or both of reagents (1106) and (1107) attached. Released molecular tags (1123) are separated, and peaks (1118 and 1124) in a separation profile (1126) are correlated to the amounts of the released molecular tags.
  • relative peak heights, or areas may reflect (i) the differences in affinity of the first and second binding compounds for their respective antigenic determinants, and/or (ii) the presence or absence of the antigenic determinant that the binding compound is specific for.
  • the later situation is important whenever a binding compound is used to monitor the post-translational state of a protein, such as, for example, a phosphorylation state.
  • Homodimers may be measured as illustrated in Fig. ID.
  • an assay may comprise three reagents (1128): cleaving probes (1134), first binding compound (1130), and second binding compound (1132).
  • First binding compound (1130) and cleaving probe (1134) are constructed to be specific for the same antigenic determinant (1135) on protein (1138) that exists (1140) in a sample as either a homodimer (1136) or a monomer (1138).
  • reagents (1128) are combined with a sample under conditions that promote the formation of stable complexes between the reagents and their respective targets, multiple complexes (1142 through 1150) form in the assay mixture.
  • cleaving probe (1134) and binding compound (1130) are specific for the same antigenic determinant (1135), four different combinations (1144 through 1150) of reagents may form complexes with homodimers. Of the complexes in the assay mixture, only those (1143) with both a cleaving probe (1134) and at least one binding compound will contribute released molecular tags (1151) for separation and detection (1154).
  • the size of peak (1153) is proportional to the amount of homodimer in the assay mixture, while the size of peak (1152) is proportional to the total amount of protein (1138) in the assay mixture, both in monomelic form (1142) or in homodimeric form (1146 and 1148). Fig.
  • IE illustrates the analogous measurements for cell surface receptors that form heterodimers in cell surface membrane (1161).
  • dimers may be measured in either lysates of cells or tissues, or in fixed samples whose membranes have been permeabilized or removed by the fixing process.
  • binding compounds may be specific for either extracellular or intracellular domains of cell surface membrane receptors.
  • releasable molecular tags may also be used for the simultaneous detection or measurement of multiple dimers and intracellular complexes in a cellular sample.
  • Cells (160) which may be from a sample from in vitro cultures or from a specimen of patient tissue, are lysed (172) to render accessible molecular complexes associated with the cell membrane, and/or post-translational modification sites, such as phosphorylation sites, within the cytoplasmic domains of the membrane molecules. After lysing, the resulting lysate (174) is combined with assay reagents (176) that include multiple cleaving probes (175) and multiple binding compounds (177).
  • Assay conditions are selected (178) that allow reagents (176) to specifically bind to their respective targets, so that upon activation cleavable linkages within the effective proximity (180) of the cleavage-inducing moieties are cleaved and molecular tags are released (182).
  • the released molecular tags are separated (184) and identified in a separation profile (186), such as an electropherogram, and based on the number and quantities of molecular tags measured, a profile is obtained of the selected molecular complexes in the cells of the sample.
  • FIGs. IG and IH illustrate an embodiment of the invention for measuring receptor complexes in fixed or frozen tissue samples.
  • Fixed tissue sample (1000) e.g. a formalin-fixed paraffin-embedded sample
  • Enlargement (1007) shows portion (1008) of section (1004) on portion (1014) of microscope slide (1006).
  • Receptor dimer molecules (1018) are illustrated as embedded in the remnants of membrane structure (1016) of the fixed sample.
  • cleaving probe and binding compounds are incubated with the fixed sample so that they bind to their target molecules.
  • cleaving probes (1012) (illustrated in the figure as an antibody having a photosensitizer (“PS") attached) and first binding compound (1010)(illustrated as an antibody having molecular tag "mTi” attached) specifically bind to receptor (1011) common to all of the dimers shown
  • second binding compound (1017)(with “mT 2 ") specifically binds to receptor (1015)
  • third binding compound (1019)(with "mT 3 ”) specifically binds to receptor (1013).
  • buffer (1024) After washing to remove binding compounds and cleaving probe that are not specifically bound to their respective target molecules, buffer (1024) (referred to as "illumination buffer” in the figure) is added.
  • buffer (1024) may be contained on section (1004), or a portion thereof, by creating a hydrophobic barrier on slide (1006), e.g. with a wax pen.
  • buffer (1024) now containing release molecular tags (1025) is transferred to a separation device, such as a capillary electrophoresis instrument, for separation (1028) and identification of the released molecular tags in, for example, electropherogram (1030).
  • a separation device such as a capillary electrophoresis instrument
  • Figs. IG and IH may be normalized by including measurements on cellular or tissue targets that are representative of the total cell number in the sample and/or the numbers of particular subtypes of cells in the sample. Such tissue targets are referred to herein as "tissue indicators.”
  • tissue indicators are referred to herein as "tissue indicators.”
  • the additional measurement may be preferred, or even necessary, because of the cellular and tissue heterogeneity in patient samples, particularly tumor samples, which may comprise substantial fractions of normal cells. For example, in Fig.
  • values for the total amount of receptor (1011) may be given as a ratio of the following two measurements: area of peak (1030) of molecular tag CmT 1 ") and the area of a peak corresponding to a molecular tag correlated with a cellular or tissue component common to all the cells in the sample, e.g., tubulin, or the like.
  • area of peak (1030) of molecular tag CmT 1 a peak corresponding to a molecular tag correlated with a cellular or tissue component common to all the cells in the sample.
  • cytokeratin or similar markers may be used.
  • detection methods based on releasable molecular tags may include an additional step of providing a binding compound (with a distinct molecular tag) specific for a normalization protein, such as, e.g., tubulin.
  • FIGS 2A-2E illustrate another embodiment of the invention for profiling dimerization among a plurality of receptor types.
  • Figure 2A outlines the basic steps of such an assay.
  • Cell membranes (200) that are to be tested for dimers of cell surface receptors are combined with sets of binding compounds (202) and (204) and cleaving probe (206).
  • Membrane fractions (200) contain three different types of monomer receptor molecules ("1 ,” “2,” and “3") in its cell membrane which associate to form three different heterodimers: 1-2, 1-3, and 2-3. This arrangement reflects some of the dimers that can form between, for example, Herl, Her2, and Her3.
  • Three antibody reagents (202) and (204) are combined with membrane fraction (200), each of the antibody reagents having binding specificity for one of the three receptor molecules, where antibody (206) is specific for receptor molecule 1, antibody (204) is specific for receptor molecule 2, and antibody (202) is specific for receptor molecule 3.
  • the antibody for the first receptor molecule is covalently coupled to a photosensitizer molecule, labeled PS.
  • the antibodies for the second and third receptor molecules are linked to two different tags, labeled T 2 and T 3 , respectively, through a linkage that is cleavable by an active species generated by the photosensitizer moiety. [0136] After mixing, the antibodies are allowed to bind (208) to molecules on the surface of the membranes.
  • the photosensitizer is activated (210), cleaving the linkage between tags and antibodies that are within an actionable distance from a sensitizer molecule, thereby releasing tags into the assay medium.
  • Material from the reaction is then separated (212), e.g., by capillary electrophoresis, as illustrated.
  • the tags T 2 and T 3 are released, and separation by electrophoresis will reveal two bands corresponding to these tags. Because the tags are designed to have a known electrophoretic mobility, each of the bands can be uniquely assigned to one of the tags used in the assay.
  • Fig. 2A provides a table listing five different assay combinations.
  • Fig. 2C are the illustrative results for each assay composition.
  • Assay I represents the results from the complete assay, as described in Figure 2A.
  • Assay II the antibody specific for receptor molecule 1, which is linked to the photosensitizer, is omitted.
  • This assay yields no signal, indicating that the T 2 and T 3 signals obtained in Assay I require the photosensitizer reagent.
  • Assay V shows that the tag signals require the presence of the membranes.
  • Assays HI and IV show that each tagged reagent does not require the presence of the other to be cleaved.
  • the first combination comprises a photosensitizer coupled to the antibody specific for monomer number 1, and is the same combination used in the illustration of Figure 2A-2C, and has the same dimer population as in Figure 2C.
  • the second combination comprises a photosensitizer coupled to the antibody specific for monomer number 2, and the population profile yields the same number for heterodimer 1 -2, plus a value for heterodimer 2-3.
  • the third combination comprises a photosensitizer coupled to the antibody specific for monomer number 3, and the population profile yields the same number for heterodimer 1-3 and 2-3 as obtained from the first two combinations.
  • a preferred embodiment for measuring relative amounts of receptor dimers containing a common component receptor is described below, In this assay design, two different receptor dimers (“1-2" and “2-3") each having a common component, "2,” may be measured ratiometrically with respect to the common component.
  • An assay design is shown for measuring receptor heterodimer comprising receptor "1" and receptor "2” and receptor heterodimer comprising receptor "2" and receptor "3".
  • a key feature of this embodiment is that cleaving probe be made specific for the common receptor of the pair of heterodimers.
  • Binding compound specific for receptor "2" provides a signal related to the total amount of receptor “2” in the assay, whereas binding compound specific for receptor "1” and binding compound specific for receptor “3” provide signals related only to the amount of receptor “1” and receptor “3” present as heterodimers with receptor “2,” respectively.
  • This design may be generalized to more than two receptor complexes that contain a common component by simply adding binding compounds specific for the components of the additional complexes.
  • each different binding compound has one or more molecular tags attached through cleavable linkages.
  • the nature of the binding compound, cleavable linkage and molecular tag may vary widely.
  • a binding compound may comprise an antibody binding composition, an antibody, a peptide, a peptide or non- peptide ligand for a cell surface receptor, a protein, an oligonucleotide, an oligonucleotide analog, such as a peptide nucleic acid, a lectin, or any other molecular entity that is capable of specifically binding to a target protein or molecule or stable complex formation with an analyte of interest, such as a complex of proteins.
  • a binding compound which can be represented by the formula below, comprises one or more molecular tags attached to a binding moiety.
  • cleavable linkage L
  • E a molecular tag
  • cleavable linkage L
  • L may be an oxidation-labile linkage, and more preferably, it is a linkage that may be cleaved by singlet oxygen.
  • the moiety "-(L-E)k" indicates that a single binding compound may have multiple molecular tags attached via cleavable linkages.
  • k is an integer greater than or equal to one, but in other embodiments, k may be greater than several hundred, e.g. 100 to 500, or k is greater than several hundred to as many as several thousand, e.g. 500 to 5000.
  • each of the plurality of different types of binding compound has a different molecular tag, E.
  • Cleavable linkages e.g. oxidation-labile linkages
  • molecular tags, E are attached to B by way of conventional chemistries.
  • B is an antibody binding composition that specifically binds to a target, such as a predetermined antigenic determinant of a target protein, such as a cell surface receptor.
  • a target protein such as a cell surface receptor.
  • Such compositions are readily formed from a wide variety of commercially available antibodies, either monoclonal and polyclonal, specific for proteins of interest.
  • antibodies specific for epidermal growth factor receptors are disclosed in U.S. Patent Nos. 5,677,171; 5,772,997; 5,968,511; 5,480,968; 5,811,098, each of which are incorporated by reference in its entirety.
  • U.S. Patent No. 6,488,390 hereby incorporated by reference in its entirety, discloses antibodies specific for a G-protein coupled receptor, CCR4.
  • Patent 5,599,681 discloses antibodies specific for phosphorylation sites of proteins.
  • Commercial vendors such as Cell Signaling Technology (Beverly, MA), Biosource International (Camarillo, CA), and Upstate (Charlottesville, VA), also provide monoclonal and polyclonal antibodies specific for many receptors.
  • Cleavable linkage, L can be virtually any chemical linking group that may be cleaved under conditions that do not degrade the structure or affect detection characteristics of the released molecular tag, E.
  • cleavable linkage L
  • cleavable linkage L
  • cleavable linkage L
  • cleavage agent generated by the cleaving probe that acts over a short distance so that only cleavable linkages in the immediate proximity of the cleaving probe are cleaved.
  • an agent must be activated by making a physical or chemical change to the reaction mixture so that the agent produces a short lived active species that diffuses to a cleavable linkage to effect cleavage.
  • the cleavage agent is preferably attached to a binding moiety, such as an antibody, that targets prior to activation the cleavage agent to a particular site in the proximity of a binding compound with releasable molecular tags.
  • a cleavage agent is referred to herein as a "cleavage-inducing moiety," which is discussed more fully below.
  • Cleavable linkages may not only include linkages that are labile to reaction with a locally acting reactive species, such as hydrogen peroxide, singlet oxygen, or the like, but also linkages that are labile to agents that operate throughout a reaction mixture, such as base-labile linkages, photocleavable linkages, linkages cleavable by reduction, linkages cleaved by oxidation, acid-labile linkages, peptide linkages cleavable by specific proteases, and the like.
  • cleavable reagent systems may be employed with the invention.
  • a disulfide linkage maybe introduced between an antibody binding composition and a molecular tag using a heterofunctional agent such as N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), succinimidyloxycarbonyl-o:-methyl- ⁇ -(2-pyridyldithio)toluene (SMPT), or the like, available from vendors such as Pierce Chemical Company (Rockford, IL).
  • SPDP N-succinimidyl 3-(2-pyridyldithio)propionate
  • SMPT succinimidyloxycarbonyl-o:-methyl- ⁇ -(2-pyridyldithio)toluene
  • Pierce Chemical Company Pierce Chemical Company (Rockford, IL).
  • Disulfide bonds introduced by such linkages can be broken by treatment with a reducing agent, such as dithiothreitol (DTT), dithioerythritol (DTE), 2-mercaptoethanol, sodium borohydride, or the like.
  • a reducing agent such as dithiothreitol (DTT), dithioerythritol (DTE), 2-mercaptoethanol, sodium borohydride, or the like.
  • Typical concentrations of reducing agents to effect cleavage of disulfide bonds are in the range of from 10 to 100 mM.
  • An oxidatively labile linkage may be introduced between an antibody binding composition and a molecular tag using the homobifunctional NHS ester cross-linking reagent, disuccinimidyl tartarate (DST)(available from Pierce) that contains central cis-diols that are susceptible to cleavage with sodium periodate (e.g., 15 mM periodate at physiological pH for 4 hours).
  • DST disuccinimidyl tartarate
  • Linkages that contain esterified spacer components may be cleaved with strong nucleophilic agents, such as hydroxylamine, e.g., 0.1 N hydroxylamine, pH 8.5, for 3-6 hours at 37 0 C.
  • Such spacers can be introduced by a homobifunctional cross-linking agent such as ethylene glycol bis(succinirnidylsuccinate)(EGS) available from Pierce (Rockford, IL).
  • a base labile linkage can be introduced with a sulfone group.
  • Homobifunctional cross-linking agents that can be used to introduce sulfone groups in a cleavable linkage include bis[2-(succinimidyloxycarbonyloxy)ethyl]sulfone (BSOCOES), and 4,4-difluoro-3,3-dinitrophenylsulfone (DFDNPS).
  • Exemplary basic conditions for cleavage include 0.1 M sodium phosphate, adjusted to pH 11.6 by addition of Tris base, containing 6 M urea, 0.1% SDS, and 2 mM DTT, with incubation at 37 0 C for 2 hours.
  • Photocleavable linkages also include those disclosed in U.S. Patent No. 5,986,076.
  • L When L is oxidation labile, L may be a thioether or its selenium analog; or an olefin, which contains carbon-carbon double bonds, wherein cleavage of a double bond to an oxo group, releases the molecular tag, E.
  • Illustrative oxidation labile linkages are disclosed in U.S. Patent Nos. 6,627,400 and 5,622,929 and in published U.S. Patent Application Nos. 2002/0013126 and 2003/0170915; each of which is hereby incorporated herein by reference in its entirety.
  • Molecular tag, E in the present invention may comprise an electrophone tag as described in the following references when separation of pluralities of molecular tags are carried out by gas chromatography or mass spectrometry: See, e.g., Zhang et al., 2002, Bioconjugate Chem. 13:1002-1012; Giese, 1983, Anal. Chem. 2:165-168; and U.S. Patent Nos. 4,650,750; 5,360,819; 5,516,931 ; and 5,602,273, each of which is hereby incorporated by reference in its entirety.
  • Molecular tag, E is preferably a water-soluble organic compound that is stable with respect to the active species, especially singlet oxygen, and that includes a detection or reporter group.
  • E may vary widely in size and structure.
  • E has a molecular weight in the range of from about 50 to about 2500 daltons, more preferably, from about 50 to about 1500 daltons. Preferred structures of E are described more fully below.
  • E may comprise a detection group for generating an electrochemical, fluorescent, or chromogenic signal. In embodiments employing detection by mass, E may not have a separate moiety for detection purposes. Preferably, the detection group generates a fluorescent signal.
  • Molecular tags within a plurality are selected so that each has a unique separation characteristic and/or a unique optical property with respect to the other members of the same plurality.
  • the chromatographic or electrophoretic separation characteristic is retention time under set of standard separation conditions conventional in the art, e.g., voltage, column pressure, column type, mobile phase, electrophoretic separation medium, or the like.
  • the optical property is a fluorescence property, such as emission spectrum, fluorescence lifetime, fluorescence intensity at a given wavelength or band of wavelengths, or the like.
  • the fluorescence property is fluorescence intensity.
  • each molecular tag of a plurality may have the same fluorescent emission properties, but each will differ from one another by virtue of a unique retention time.
  • two or more of the molecular tags of a plurality may have identical migration, or retention, times, but they will have unique fluorescent properties, e.g. spectrally resolvable emission spectra, so that all the members of the plurality are distinguishable by the combination of molecular separation and fluorescence measurement,
  • released molecular tags are detected by electrophoretic separation and the fluorescence of a detection group.
  • molecular tags having substantially identical fluorescence properties have different electrophoretic mobilities so that distinct peaks in an electropherogram are formed under separation conditions.
  • pluralities of molecular tags of the invention are separated by conventional capillary electrophoresis apparatus, either in the presence or absence of a conventional sieving matrix.
  • Exemplary capillary electrophoresis apparatus include Applied Biosystems (Foster City, CA) models 310, 3100 and 3700; Beckman (Fullerton, CA) model P/ACE MDQ; Araersham Biosciences (Sunnyvale, CA) MegaBACE 1000 or 4000; SpectruMedix genetic analysis system; and the like.
  • Electrophoretic mobility is proportional to q/M 2/3 , where q is the charge on the molecule and M is the mass of the molecule. Desirably, the difference in mobility under the conditions of the determination between the closest electrophoretic labels will be at least about 0.001, usually 0.002, more usually at least about 0.01, and maybe 0.02 or more.
  • the electrophoretic mobilities of molecular tags of a plurality differ by at least one percent, and more preferably, by at least a percentage in the range of from 1 to 10 percent.
  • Molecular tags are identified and quantified by analysis of a separation profile, or more specifically, an electropherogram, and such values are correlated with the amounts and kinds of receptor dimers present in a sample.
  • the molecular tags are detected or identified by recording fluorescence signals and migration times (or migration distances) of the separated compounds, or by constructing a chart of relative fluorescent and order of migration of the molecular tags (e.g., as an electropherogram).
  • the presence, absence, and/or amounts of molecular tags are measured by using one or more standards as disclosed by published U.S. Patent Application No. 2003/0170734A1, which is hereby incorporated by reference in its entirety.
  • Pluralities of molecular tags may also be designed for separation by chromatography based on one or more physical characteristics that include but are not limited to molecular weight, shape, solubility, pKa, hydrophobicity, charge, polarity, or the like, e.g. as disclosed in published U.S. Patent Application No. 2003/0235832, which hereby is incorporated by reference in its entirety.
  • a chromatographic separation technique is selected based on parameters such as column type, solid phase, mobile phase, and the like, followed by selection of a plurality of molecular tags that may be separated to form distinct peaks or bands in a single operation.
  • HPLC technique is selected for use in the invention, including the number of molecular tags to be detected (i.e., the size of the plurality), the estimated quantities of each molecular tag that will be generated in the assays, the availability and ease of synthesizing molecular tags that are candidates for a set to be used in multiplexed assays, the detection modality employed, and the availability, robustness, cost, and ease of operation of HPLC instrumentation, columns, and solvents. Generally, columns and techniques are favored that are suitable for analyzing limited amounts of sample and that provide the highest resolution separations.
  • An exemplary HPLC instrumentation system suitable for use with the present invention is the Agilent 1100 Series HPLC system (Agilent Technologies, Palo Alto, CA).
  • molecular tag, E is (M, D), where M is a mobility- modifying moiety and D is a detection moiety.
  • the notation "(M, D)” is used to indicate that the ordering of the M and D moieties may be such that either moiety can be adjacent to the cleavable linkage, L. That is, "B-L-(M, D)" designates binding compound of either of two forms: “B-L-M-D" or "B-L-D-M.”
  • Detection moiety may be a fluorescent label or dye, a chromogenic label or dye, an electrochemical label, or the like.
  • D is a fluorescent dye.
  • Exemplary fluorescent dyes for use with the invention include water-soluble rhodamine dyes, fluoresceins, 4,7-dichlorofluorcsceins, benzoxanthene dyes, and energy transfer dyes, as disclosed in the following references: Anonymous, 2002, Handbook of Molecular Probes and Research Reagents, 8 th ed., Molecular Probes, Eugene, OR; U.S. Patent Nos.
  • D is a fluorescein or a fluorescein derivative.
  • binding compounds comprise a biotinylated antibody (300) as a binding moiety.
  • Molecular tags are attached to binding moiety (300) by way of avidin or strep tavidin bridge (306).
  • binding moiety (300) is first reacted with a target complex, after which avidin or streptavidin is added (304) to form antibody-biotin-avidin complex (305).
  • avidin or streptavidin is added (304) to form antibody-biotin-avidin complex (305).
  • biotinylated molecular tags (310) to form binding compound (312).
  • binding compounds comprise an antibody (314) derivatized with a multi-functional moiety (316) that contains multiple functional groups (318) that are reacted (320) molecular tag precursors to give a final binding compound having multiple molecular tags (322) attached.
  • exemplary multi-functional moieties include aminodextran, and like materials.
  • each of the binding compounds is separately derivatized by a different molecular tag, it is pooled with other binding compounds to form a plurality of binding compounds.
  • each different kind of binding compound is present in a composition in the same proportion; however, proportions may be varied as a design choice so that one or a subset of particular binding compounds are present in greater or lower proportion depending on the desirability or requirements for a particular embodiment or assay.
  • Factors that may affect such design choices include, but are not limited to, antibody affinity and avidity for a particular target, relative prevalence of a target, fluorescent characteristics of a detection moiety of a molecular tag, and the like.
  • a cleavage-inducing moiety, or cleaving agent is a group that produces an active species that is capable of cleaving a cleavable linkage, preferably by oxidation.
  • the active species is a chemical species that exhibits short-lived activity so that its cleavage-inducing effects are only in the proximity of the site of its generation. Either the active species is inherently short lived, so that it will not create significant background because beyond the proximity of its creation, or a scavenger is employed that efficiently scavenges the active species, so that it is not available to react with cleavable linkages beyond a short distance from the site of its generation.
  • Illustrative active species include singlet oxygen, hydrogen peroxide, NADH, and hydroxyl radicals, phenoxy radical, superoxide, and the like.
  • Illustrative quenchers for active species that cause oxidation include polyenes, carotenoids, vitamin E, vitamin C, amino acid-pyrrole N-conjugates of tyrosine, histidine, and glutathione, and the like. See, e.g. Beutner et ah, 2000, Meth. Enzymol. 319:226-241.
  • cleavable linkages preferably are within about 1000 nm, and preferably within about 20-200 nm, of a bound cleavage-inducing moiety. More preferably, for photosensitizer cleavage-inducing moieties generating singlet oxygen, cleavable linkages are within about 20-100 nm of a photosensitizer in a receptor complex.
  • cleavage- inducing moiety cleavage- inducing moiety
  • cleavable linkage cleave enough molecular tag to generate a detectable signal
  • effective proximity One of ordinary skill in the art will recognize that the effective proximity of a particular sensitizer may depend on the details of a particular assay design and may be determined or modified by routine experimentation.
  • a sensitizer is a compound that can be induced to generate a reactive intermediate, or species, usually singlet oxygen.
  • a sensitizer used in accordance with the invention is a photosensitizer.
  • Other sensitizers included within the scope of the invention are compounds that on excitation by heat, light, ionizing radiation, or chemical activation will release a molecule of singlet oxygen.
  • the best known members of this class of compounds include the endoperoxides such as 1,4- biscarboxyethyl- 1 ,4-naphthalene endoperoxide, 9, 10-diphenylanthracene-9, 10- endoperoxide and 5,6,11 ,12-tetraphenyl naphthalene 5,12-endoperoxide. Heating or direct absorption of light by these compounds releases singlet oxygen. Further sensitizers are disclosed by Di Mascio et al, 1994, FEBS Lett. 355:287 (peroxidases and oxygenases); and Kanofsky, 1983, J.Biol. Chem.
  • Attachment of a binding agent to the cleavage-inducing moiety may be direct or indirect, covalent or non-covalent, and can be accomplished by well-known techniques commonly available in the literature. See, e.g., Ichiro Chibata, 1978, Immobilized Enzymes, Halsted Press, New York; and Cuatrecasas, 1970, J. Biol. Chem. 245:3059.
  • the preferred cleavage-inducing moiety in accordance with the present invention is a photosensitizer that produces singlet oxygen.
  • photosensitizer refers to a light-adsorbing molecule that when activated by light converts molecular oxygen into singlet oxygen.
  • Photosensitizers may be attached directly or indirectly, via covalent or non-covalent linkages, to the binding agent of a class-specific reagent.
  • Guidance for constructing such compositions, particularly for antibodies as binding agents available in the literature, e.g. in the fields of photodynamic therapy, immunodiagnostics, and the like. Exemplary guidance may be found in Ullman et al., 1994, Proc. Natl.
  • a large variety of light sources are available to photo-activate photosensitizers to generate singlet oxygen. Both polychromatic and monochromatic sources may be used as long as the source is sufficiently intense to produce enough singlet oxygen in a practical time duration.
  • the length of the irradiation depends on the nature of the photosensitizer, the nature of the cleavable linkage, the power of the source of irradiation, and its distance from the sample, and so forth. In general, the period for irradiation may be less than about a microsecond to as long as about 10 minutes, usually in the range of about one millisecond to about 60 seconds.
  • the intensity and length of irradiation should be sufficient to excite at least about 0.1% of the photosensitizer molecules, usually at least about 30% of the photosensitizer molecules and preferably, substantially all of the photosensitizer molecules.
  • Exemplary light sources include, by way of illustration and not limitation, lasers such as, e.g., helium-neon lasers, argon lasers, YAG lasers, He/Cd lasers, and ruby lasers; photodiodes; mercury, sodium and xenon vapor lamps; incandescent lamps such as, e.g., tungsten and tungsten/halogen; fiashlamps; and the like.
  • the photoactivation device is an array of light emitting diodes (LEDs) mounted in housing that permits the simultaneous illumination of all the wells in a 96-well plate.
  • LEDs light emitting diodes
  • a suitable LED for use in the present invention is a high power GaAIAs IR emitter, such as model OD-880W manufactured by OPTO DIODE CORP. (Newbury Park, CA).
  • photosensitizers that may be utilized in the present invention are those that have the above properties and those disclosed by U.S. Patent Nos. 5,536,834, 5,763,602, 5,565,552, 5,709,994, 5,340,716, 5,516,636, 6,251,581, and 6,001,673; published European Patent Application No. 0484027; Martin et al, 1990, Methods Enzymol. 186:635-645; and Yarmush et a/., 1993, Crit. Rev. Therapeutic Drug Carrier Syst. 10:197-252.
  • a photosensitizer may be associated with a solid phase support by being covalently or non-covalently attached to the surface of the support or incorporated into the body of the support.
  • the photosensitizer is associated with the support in an amount necessary to achieve the necessary amount of singlet oxygen.
  • the amount of photosensitizer is determined empirically according to routine methods.
  • a photosensitizer is incorporated into a latex particle to form photosensitizer beads, e.g. as disclosed by U.S. Patent Nos. 5,709,994 and 6,346,384; and International Patent Publication No. WO 01/84157.
  • photosensitizer beads may be prepared by covalently attaching a photosensitizer, such as rose bengal, to 0.5 micron latex beads by means of chloromethyl groups on the latex to provide an ester linking group, as described in J. Amer. Chem. Soc, 97:3741 (1975).
  • a photosensitizer beads is illustrated in Fig. 3C and 3D. As described in Fig.
  • complexes (230) are formed after combining reagents (1122) with a sample.
  • This reaction may be carried out, for example, in a conventional 96-well or 384-well micro titer plate, or the like, having a filter membrane that forms one wall, e.g. the bottom, of the wells that allows reagents to be removed by the application of a vacuum.
  • This allows the convenient exchange of buffers, if the buffer required for specific binding of binding compounds is different that the buffer required for either singlet oxygen generation or separation. For example, in the case of antibody-based binding compounds, a high salt buffer is required.
  • a cleaving probe instead of attaching a photosensitizer directly to a binding compound, such as an antibody, a cleaving probe comprises two components: antibody (232) derivitized with a capture moiety, such as biotin (indicated in Fig. 3C as "bio") and photosensitizer bead (338) whose surface is derivatized with an agent (234) that specifically binds with the capture moiety, such as avidin or streptavidin.
  • Photosensitizer beads are then captured (236) by photosensitizer beads by way of the capture moiety, such as biotin (bio).
  • a buffer exchange also serves to remove unbound binding compounds, which leads to an improved signal.
  • photosensitizer beads (338) are illuminated (240) so that singlet oxygen is generated (242) and molecular tags are released (244). See Figure 3D.
  • Such released molecular tags (346) are then separated to form separation profile (352) and dimers are quantified ratiometrically from peaks (348) and (350). See Figure 3D.
  • Photosensitizer beads may be used in either homogeneous or heterogeneous assay formats.
  • a cleaving probe may comprise a primary haptenated antibody and a secondary anti-hapten binding protein derivatized with multiple photosensitizer molecules.
  • a preferred primary haptenated antibody is a biotinylated antibody
  • preferred secondary anti-hapten binding proteins may be either an anti- biotin antibody or streptavidin.
  • Other combinations of such primary and secondary reagents are well known in the art. Exemplary combinations of such reagents are taught by Haugland, 2002, Handbook of Fluorescent Probes and Research Reagents, Ninth Edition, Molecular Probes, Eugene, OR.
  • binding compounds having releasable tags (“mTi” and “mT 2 ”) > and primary antibody derivatized with biotin are specifically bound to different epitopes of receptor dimer in membrane.
  • Biotin-specific binding protein e.g. streptavidin
  • Biotin-specific binding protein is attached to biotin bringing multiple photosensitizers into effective proximity of binding compounds.
  • Biotin-specific binding protein may also be an anti-biotin antibody, and photosensitizers may be attached via free amine group on the protein by conventional coupling chemistries, e.g., Hermanson (supra).
  • An exemplary photosensitizer for such use is an NHS ester of methylene blue prepared as disclosed in published European Patent Application 0510688.
  • a combination of the assay components is made, including the sample being tested, the binding compounds, and optionally the cleaving probe.
  • assay components may be combined in any order. In certain applications, however, the order of addition may be relevant. For example, one may wish to monitor competitive binding, such as in a quantitative assay. Or one may wish to monitor the stability of an assembled complex. In such applications, reactions may be assembled in stages, and may require incubations before the complete mixture has been assembled, or before the cleaving reaction is initiated.
  • the amounts of each reagent can generally be determined empirically.
  • the amount of sample used in an assay will be determined by the predicted number of target complexes present and the means of separation and detection used to monitor the signal of the assay.
  • the amounts of the binding compounds and the cleaving probe can be provided in molar excess relative to the expected amount of the target molecules in the sample, generally at a molar excess of at least about 1.5, more desirably about 10- fold excess, or more.
  • the concentration used may be higher or lower, depending on the affinity of the binding agents and the expected number of target molecules present on a single cell. Where one is determining the effect of a chemical compound on formation of oligomeric cell surface complexes, the compound may be added to the cells prior to, simultaneously with, or after addition of the probes, depending on the effect being monitored.
  • the assay mixture can be combined and incubated under conditions that provide for binding of the probes to the cell surface molecules, usually in an aqueous medium, generally at a physiological pH (comparable to the pH at which the cells are cultures), maintained by a buffer at a concentration in the range of about 10 to 200 mM.
  • a physiological pH common to the pH at which the cells are cultures
  • Conventional buffers may be used, as well as other conventional additives as necessary, such as salts, growth medium, stabilizers, etc.
  • Physiological and constant temperatures are normally employed. Incubation temperatures normally range from about 4° to 7O 0 C, usually from about 15° to 45 0 C, more usually about 25° to 37 0 C.
  • the mixture can be treated to activate the cleaving agent to cleave the tags from the binding compounds that are within the effective proximity of the cleaving agent, releasing the corresponding tag from the cell surface into solution.
  • the nature of this treatment will depend on the mechanism of action of the cleaving agent. For example, where a photosensitizer is employed as the cleaving agent, activation of cleavage can comprise irradiation of the mixture at the wavelength of light appropriate to the particular sensitizer used.
  • the sample can then be analyzed to determine the identity of tags that have been released.
  • separation of the released tags will generally precede their detection.
  • the methods for both separation and detection are determined in the process of designing the tags for the assay.
  • a preferred mode of separation employs electrophoresis, in which the various tags are separated based on known differences in their electrophoretic mobilities.
  • assay reaction conditions may interfere with the separation technique employed, it may be necessary to remove, or exchange, the assay reaction buffer prior to cleavage and separation of the molecular tags.
  • assay conditions may include salt concentrations (e.g. required for specific binding) that degrade separation performance when molecular tags are separated on the basis of electrophoretic mobility.
  • salt concentrations e.g. required for specific binding
  • such high salt buffers may be removed, e.g., prior to cleavage of molecular tags, and replaced with another buffer suitable for electrophoretic separation through filtration, aspiration, dilution, or other means.
  • the invention further provides methods of treating a subject with cancer.
  • the methods comprise determining that the subject has a cancer comprising a cancer cell that is likely to respond to treatment with a Her2- acting according to a method of the invention, and administering an effective amount of a Her2-acting agent to the subject.
  • the Her2-acting agent is trastuzumab.
  • the methods comprise determining that a subject has a cancer comprising a cancer cell that is likely to respond to treatment with a Her2-acting agent according to a method of the invention, then advising a medical professional of the treatment option of administering to the subject an effective amount of a Her2-acting agent.
  • the Her2-acting agent is trastuzumab.
  • the methods comprise determining that a subject is has cancer comprising a cancer cell that is likely to respond to treatment with a Her2-acting agent according to a method of the invention, then advising a medical professional to treat the subject with an effective amount of a Her2-acting agent.
  • the Her2-acting agent is trastuzumab.
  • the methods comprise determining that a subject has a pre-cancerous condition that is likely to respond to treatment with a Her2 -acting according to a method of the invention, and administering an effective amount of a Her2- acting agent to the subject.
  • the Her2-acting agent is trastuzumab.
  • the methods comprise determining that a subject has a pre-cancerous condition that is likely to respond to treatment with a Her2 -acting according to a method of the invention, then advising a medical professional of the treatment option of administering to the subject an effective amount of a Her2-acting agent.
  • the Her2-acting agent is trastuzumab.
  • the methods comprise determining that a subject has a pre-cancerous condition that is likely to respond to treatment with a Her2-acting agent according to a method of the invention, then advising a medical professional to treat the subject with an effective amount of a Her2-acting agent.
  • the Her2-acting agent is trastuzumab.
  • the methods comprise determining that a subject has a cancer or pre-cancerous condition that is likely to respond to treatment with a Her2- acting agent according to a method of the invention at a first time, then determining that the subject remains with a cancer or pre-cancerous condition that is likely to respond to treatment with a Her2-acting agent according to a method of the invention at a later second time.
  • the methods comprise determining that a subject has a cancer or pre-cancerous condition that is likely to respond to treatment with a Her2- acting agent according to a method of the invention at a first time, then determining that the subject remains with a cancer or pre-cancerous condition that is not likely to respond to treatment with a Her2-acting agent according to a method of the invention at a later second time.
  • the subject has locally advanced or metastatic breast cancer.
  • the cancer has failed to respond to platinum- based chemotherapy.
  • the cancer has failed to respond to docetaxel.
  • about 250 mg trastuzumab is administered.
  • about 500 mg trastuzumab is administered.
  • between about 10 mg and about 500 mg trastuzumab is administered.
  • an amount of trastuzumab effective to treat the subject is administered to the subject.
  • the subject is administered a combination therapy that includes trastuzumab.
  • the combination therapy can include trastuzumab in combination with one or more of any anti-cancer therapeutic agent known to one of skill in the art without limitation.
  • the anti-cancer therapeutic agent has a different mechanism of action from trastuzumab.
  • the anti-cancer therapeutic agent can be an anti-metabolite (e.g., 5-flourouricil (5-FU) 5 methotrexate (MTX), fludarabine, etc.), an anti-microtubule agent (e.g., vincristine; vinblastine; taxanes such as paclitaxel and docetaxel; etc.), an alkylating agent (e.g., cyclophosphamide, melphalan, bischloroethylnitrosurea, etc.), platinum agents (e.g., cisplatin, carboplatin, oxaliplatin, JM-216, CI-973, etc.), anthracyclines (e.g., doxorubicin, daunorubicin, etc.), antibiotic agents (e.g., mitomycin-C, actinomycin D, etc.), topoisomerase inhibitors (e.g., etoposide, camptothecins, etc.
  • anti-cancer therapeutic agents include abarelix, aldesleukin, alemtuzumab, alitretinoin, allopurinol, altretamine, amifostine, anastrozole, arsenic trioxide, asparaginase, BCG live, bevaceizumab, bexarotene, bleomycin, bortezomib, busulfan, calusterone, camptothecin, capecitabine, carboplatin, carmustine, celecoxib, cetuximab, chlorambucil, cinacalcet, cisplatin, cladribine, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, darbepoetin alfa, daunorubicin, denileukin diftitox, dexrazoxane, docetaxel, doxorubicin,
  • the invention provides several additional embodiments of the invention.
  • the invention provides a computer- implemented method for determining whether a cancer cell is likely to respond to treatment with a Her2-acting agent.
  • Such methods generally comprise performing a method of the invention with a computer system adapted to perform the method of the invention. Such adaptation is well within the skill of those in the art.
  • the methods comprise calculating a score of a cancer or a probability that the cancer will respond to treatment with an Her2-acting agent using a formula of the invention with a computer.
  • the method further comprises the step of displaying the score of the cancer or the probability of responding to treatment with a Her2-acting agent on a computer display.
  • the method further comprises the step of printing the score of the cancer or the probability of responding to treatment with a Her2-acting agent onto a tangible medium, such as, for example, paper.
  • the invention provides a display that indicates that a cancer or cancer cell is likely to respond to treatment with a Her2-acting agent.
  • the display is a computer display.
  • the likelihood of the cancer or cancer cell to respond to such treatment is determined according to a method or formula of the invention.
  • the invention provides a paper document that indicates that a cancer or cancer cell is likely to respond to treatment with a Her2-acting agent.
  • the paper document is a printed document, In certain embodiments, the printed document is a computer print-out.
  • the likelihood of the cancer or cancer cell to respond to such treatment is determined according to a method or formula of the invention.
  • the invention provides a computer-readable memory that comprises data indicating that a cancer or cancer cell is likely to respond to treatment with a Her2 -acting agent.
  • the computer-readable memory is a random-access memory.
  • the computer-readable memory is a fixed disk.
  • the computer-readable memory is a floppy disk.
  • the computer-readable memory is a portable memory device, such as, e.g., a USB key or an iPodTM.
  • the likelihood of the cancer or cancer cell to respond to such treatment is determined according to a method of the invention.
  • the invention provides a computer-readable memory that comprises data comprising a number of biomarkers on a cancer or cancer cell that are associated with responsiveness to trastuzumab therapy as described herein and computer-readable instructions for determining the score of the cancer or cancer cell or probability that the cancer or cancer cell will respond to trastuzumab therapy.
  • the computer-readable memory is a random-access memory.
  • the computer-readable memory is a fixed disk.
  • the computer-readable memory is a floppy disk.
  • the computer- readable memory is a portable memory device, such as, e.g., a USB key or an iPodTM.
  • the likelihood of the cancer or cancer cell to respond to such treatment is determined according to a method or formula of the invention.
  • kits that are useful in determining whether a cancer or cancer cell is likely to respond to treatment with a Her2- acting agent.
  • the kits of the present invention comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195 or 200 or more binding compounds that can be used to detect and/or quantify one or more biomarkers correlated with responsiveness to treatment with a Her2-acting agent.
  • kits of the present invention comprise at least 2, but as many as several hundred or more such binding compounds.
  • the kit may also comprise one or more cleaving probes for use in a method of the invention.
  • the kit may also comprise at least one internal standard to be used in generating the biomarker profiles of the present invention.
  • the internal standard or standards can be any of the classes of compounds described above.
  • kits of the present invention may also include reagents such as buffers, or other reagents that can be used in detecting the biomarker(s) associated with responsiveness to treatment with a Her2-acting agent.
  • reagents such as buffers, or other reagents that can be used in detecting the biomarker(s) associated with responsiveness to treatment with a Her2-acting agent.
  • Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents such as sugars, sodium chloride, and the like.
  • kits of the invention may further comprise a computer program product for use in conjunction with a computer system, wherein the computer program product comprises a computer readable storage medium and a computer program mechanism embedded therein.
  • the computer program mechanism comprises instructions for evaluating whether a plurality of features in a biomarker profile of a cancer or cancer cell satisfies a first value set. Satisfying the first value set predicts that . the cancer or cancer cell is likely to respond to treatment with a Herl acting agent.
  • the plurality of features corresponds to the presence and/or amount of expression of Herl -Herl dimers, Herl-Her2 dimers, Herl-Her3 dimers, Her2-Her3 dimers, Herl phosphorylation, Her2 phosphorylation, Her3 phosphorylation, or any other biomarker described herein as correlated with responsiveness or non-responsiveness to treatment with a Her2-acting agent.
  • the computer program product further comprises instructions for evaluating whether the plurality of features in the biomarker profile of the test subject satisfies a second value set. Satisfying the second value set predicts that the cancer or cancer cell is not likely to respond to treatment with a Herl acting agent.
  • kits of the present invention comprise a computer having a central processing unit and a memory coupled to the central processing unit.
  • the memory stores instructions for evaluating whether a plurality of features in a biomarker profile of a cancer or cancer cell satisfies a first value set.
  • the plurality of features corresponds the presence and/or amount of expression of Herl -Herl dimers, Herl-Her2 dimers, Herl-Her3 dimers, Her2-Her3 dimers, Herl phosphorylation, Her2 phosphorylation, Her3 phosphorylation, or any other biomarker described herein as correlated with responsiveness or non-responsiveness to treatment with a Her2-acting agent.
  • Figure 9 details an exemplary system that supports the functionality described above.
  • the system is preferably a computer system 10 having:
  • a central processing unit 22 a main non-volatile storage unit 14, for example, a hard disk drive, for storing software and data, the storage unit 14 controlled by storage controller 12; a system memory 36, preferably high speed random-access memory (RAM), for storing system control programs, data, and application programs, comprising programs and data loaded from non-volatile storage unit 14; system memory 36 may also include read-only memory (ROM); a user interface 32, comprising one or more input devices (e.g., keyboard 28) and a display 26 or other output device; a network interface card 20 for connecting to any wired or wireless communication network 34 (e.g., a wide area network such as the Internet); an internal bus 30 for interconnecting the aforementioned elements of the system; and a power source 24 to power the aforementioned elements.
  • ROM read-only memory
  • a user interface 32 comprising one or more input devices (e.g., keyboard 28) and a display 26 or other output device
  • a network interface card 20 for connecting to any wired or wireless
  • system memory 36 includes:
  • a file system 42 for controlling access to the various files and data structures used by the present invention; a training data set 44 for use in construction one or more decision rules in accordance with the present invention; a data analysis algorithm module 54 for processing training data and constructing decision rules; one or more decision rules 56; a biomarker profile evaluation module 60 for determining whether a plurality of features in a biomarker profile of a cancer or cancer cell satisfies a first value set; a test subject biomarker profile 62 comprising biomarkers 64 and, for each such biomarkers, features 66; and a database 68 of select biomarkers of the present invention as described herein.
  • Training data set 46 comprises data for a plurality of subjects 46. For each subject 46 there is a subject identifier 48 and a plurality of biomarkers 50. For each biomarker 50, there is at least one feature 52. Although not shown in Figure 35, for each feature 52, there is a feature value. For each decision rule 56 constructed using data analysis algorithms, there is at least one decision rule value set 58.
  • computer 10 comprises software program modules and data structures.
  • the data structures stored in computer 10 include training data set 44, decision rules 56, test subject biomarker profile 62, and biomarker database 68.
  • Each of these data structures can comprise any form of data storage system including, but not limited to, a flat ASCII or binary file, an Excel spreadsheet, a relational database (SQL), or an on-line analytical processing (OLAP) database (MDX and/or variants thereof).
  • data structures are each in the form of one or more databases that include hierarchical structure (e.g., a star schema).
  • such data structures are each in the form of databases that do not have explicit hierarchy (e.g., dimension tables that are not hierarchically arranged).
  • each of the data structures stored or accessible to system 10 are single data structures.
  • such data structures in fact comprise a plurality of data structures (e.g., databases, files, archives) that may or may not all be hosted by the same computer 10.
  • training data set 44 comprises a plurality of Excel spreadsheets that are stored either on computer 10 and/or on computers that are addressable by computer 10 across wide area network 34.
  • training data set 44 comprises a database that is either stored on computer 10 or is distributed across one or more computers that are addressable by computer 10 across wide area network 34,
  • biomarker profile evaluation module 60 and/or other modules can reside on a client computer that is in communication with computer 10 via network 34.
  • biomarker profile evaluation module 60 can be an interactive web page
  • training data set 44, decision rules 56, and/or biomarker database 68 illustrated in Figure 9 are on a single computer (computer 10) and in other embodiments one or more of such data structures and module are hosted by one or more remote computers (not shown). Any arrangement of the data structures and software modules illustrated in Figure 9 on one or more computers is within the scope of the present invention so long as these data structures and software modules are addressable with respect to each other across network 34 or by other electronic means. Thus, the present invention fully encompasses a broad array of computer systems. 6.
  • Antibodies that specifically bind Her receptors, adaptor molecules, and normalization standards were obtained from commercial vendors, including Labvision, Cell Signaling Technology, and BD Biosciences. All cell lines were purchased from ATCC (Manassas, VA).
  • NHS esters of the molecular tag with a free amine on the indicated antibody using conventional procedures.
  • Molecular tags are disclosed in U.S. Published Application Nos. 2003/017915 and 2002/0013126, which are each incorporated by reference. Briefly, binding compounds below are molecular tag-monoclonal antibody conjugates formed by reacting an NHS ester of a molecular tag with free amines of the antibodies in a conventional reaction.
  • Samples were prepared according to the following protocol. For double- coated formalin-fixed paraffin embedded (FFPE slides) tissue samples, the FFPE samples were incubated in solvent baths filled with xylene at 55 0 C for 20 minutes. The slides were then transferred to fresh xylene jars and incubated for 10 min at 55°C. The slides were then treated as regular FFPE slides. Antigen retrieval was performed either by boiling the slides in citrate buffer (pH 6.4) in a microwave for 10 minutes or by protease digestion of the tissue section using Protease 1 from Ventana Medical Systems (Tucson, AZ) following their protocol. This treatment was followed by pre-blocking of the tissue with 100 ⁇ blocking buffer containing 2% mouse serum, protease and phosphatase inhibitors for 1 hour at room temperature.
  • citrate buffer pH 6.4
  • Each antibody used in the eTag assays was conjugated to a unique species of eTag reporter that is distinguishable from other eTag reporters used in the assay following capillary electrophoresis, or to a molecular scissors molecule using standard conjugation chemistry.
  • General methods for constructing and using such reagents to detect receptor monomoers, homodimers, heterodimers, and phosphorylated and unphosphorylated proteins and are described in U.S. Patent Nos. 6,627,400, 6,630,296, 6,649,351, 6,673,550, and 6,818,399 and in U.S. Patent Application Publication Nos. 2003/0013126, 20030040016, 20030170734, 2004/0265858, 2004/0063114, 2004/0126818, and 2005/0089940.
  • eTag-labeled and biotinylated antibodies were added to the processed sections in blocking buffer (100 ⁇ l per section) for 14 h at 4 0 C.
  • Receptor phosphorylation was determined by analyzing the eTags released in a standard eTag proximity assay using eTag-labeled anti-phospho tyrosine antibody and biotinylated anti-receptor specific antibody.
  • eTag-labeled antibody specific to one member of the dimer was used, in conjunction with a biotin-labeled antibody specific to the second member.
  • a single specific antibody to the receptor, labeled separately with eTag or biotin was used in the assay.
  • total PTEN was determined by analyzing the eTags relased in a standard eTag proximity assay using eTag labeled with PTEN antibodies and biotinylated PTEN antibodies. See Figures IA- IF.
  • Labeled antibodies were used at a final concentration of 1 ⁇ g/ml in the blocking buffer. After incubation with appropriate antibodies, the slides were rinsed, and the sections incubated with streptavidin-labeled scissors molecules (2.5 ⁇ g per section) for 1 h at room temperature. The slides were rinsed and 60 ⁇ illumination buffer (containing 2 pM fluorescein, and CE markers A 160 and A315) was added to the sections, followed by illumination with -700 nm LED light for 1 h, with samples maintained at 4 0 C. The released eTag molecules in the illumination buffer were analyzed by capillary electrophoresis (using ABI 3100 genetic analyzer, Applied Biosystems, Foster City, CA). Fluorescein was used as an internal marker for all CE runs for injection control.
  • eTag InformerTM software Mongram Biosciences, Inc.; South San Francisco, CA
  • Cytokeratin was used as an internal control and all peak areas obtained were further normalized for the epithelial cell content to the cytokeratin peak area readout to account for variation in the biological samples.
  • hemotoxylin and eosin staining was performed on a slide from each patient to determine tumor percent for each sample.
  • This example provides methods and results for experiments that correlate expression levels of particular ErbB receptor dimers with responsiveness to therapy with an exemplary Her2-acting agent, trastuzumab.
  • this example describes correlations between amounts of Herl-Her2 dimer expression, Herl-Her3 dimer expression, Her2-Her3 dimer expression, Her2-Her2 dimer expression, and/or PTEN expression, and responsiveness to trastuzumab therapy, [Oil 1] Although 32 patient samples were analyzed by eTag assays as described above, one of these samples was excluded from data analysis due to low tumor content (percent tumor ⁇ 1%), One additional sample was excluded from the statistical analysis since no treatment data was available for this patient. Thus, this training set comprised 30 individual samples.
  • the tumor percentage of each sample was also measured using the immunohistochemistry (IHC) or hematoxylin and eosin (H&E) image method and used to calculate the number of cancer cells present in each sample and the ratio of cancer cells to normal cells in the sample. Briefly, samples were scored for tumor cell percentage relative to normal cell based on cell morphology, differentiation patterns, and grade and invasiveness of the cancer based on standard histological techniques. During measurements of dimer expression, molecular tags that recognize cytokeratin and/or tubulin were used to estimate the total number of cells, both normal and cancerous, present in the sample. The total number of cells was then multiplied by the proportion of cancer cells in the sample to identify the number of cancer cells in the sample, which were then used to normalize the numbers of dimers expressed per cell.
  • IHC immunohistochemistry
  • H&E hematoxylin and eosin
  • III and IV each defines a score method in which levels of heterodimers (and in the case of Formula IV, one homodimer) along with PTEN are utilized to derive a score and make a prediction. Based on these algorithms, patients were predicted to be either responsive (1) or non-responsive (0) to trastuzumab treatment.
  • Sensitivity (Sn) and Specificity (Sp) were determined for each method.
  • Sn defines the probability that the test can correctly predict response to trastuzumab (a "1” by these methods)
  • Sp defines the probability that the test can correctly predict non-response to Herceptin (a "0” by these methods).
  • Positive Predictive Value (PPV) defines the probability that a patient responds to the drug (is a responder) given a positive test result (a "1” by these methods)
  • Negative Predictive Value (NPV) defines the probability that a patient does not respond to the drug (is a non-responder) given a negative test result (a "0” by these methods).
  • Tables 1 and 2 below. As discussed below, the patients were split into two groups: Group 1, including patients treated with trastuzumab monotherapy, and Group 2, including patients treated with combination therapy including trastuzumab. Results from Group 1 are presented in Table 3, while results from Group 2 are presented in Table 4.
  • Her2 phosphorylation levels often varied significantly from the Her2 total protein levels. For example, Patient 15 showed moderate levels of total Her2, but high phosphorylation levels of this receptor. In contrast, Patient 20 had high Her2 levels but no detectable phosphorylation. Total expression of Her3 varied significantly between patients. See Tables 3 and 4, above, and Figure 12. The Herl/Her3 and Her2/Her3 heterodirner levels (Tables 3 and 4, Figure 12) showed no correlation with the total receptor expression levels of either Her3 or Her2. In some patients neither of these dimers were observed even when some level of Her3 protein was present, e.g., Patient 9 ⁇ See Figure 12). All the parameters measured by eTag assays as described above were tested as negative or positive predictors of response in our statistical analysis.
  • Herl/Her2 dimer, PTEN and Her2/Her3 dimer were used in the analyses discussed below. These parameters were chosen because they exhibited the broadest distribution within the class of patients including responders and nonresponders, so they seemed likely candidates for discriminating between responders and non-resporiders. A representative distribution of these parameters is presented in Figure 10. In general, increased Her3-containing heterodimer levels negatively correlated with responsiveness to trastuzumab thereapy, while elevated PTEN concentrations, which is a phosphatase known to de-phosphorylate PIP 35 reverses the actions OfPI 3 kinase and therefore inhibits the Akt signaling pathway, positively correlated with patient response ,
  • the patients were divided into two groups based on their treatment regimen.
  • Group 1 included patients who had trastuzumab monotherapy at some point during their treatment, while Group 2 included patients who had received trastuzumab in combination with chemotherapy.
  • Each of these groups of patients was further divided into two groups based on their responsiveness to treatment: those that showed a clinical response ("R” including the clinically determined NC (No Change, which is equivalent to Stable Disease or SD), PR (Partial Response), CR (Complete Response)) and those that showed no response ("NR" including PD (Progressive Disease)).
  • R including the clinically determined NC (No Change, which is equivalent to Stable Disease or SD)
  • PR Partial Response
  • CR Complete Response
  • NR including PD (Progressive Disease)
  • a prediction of likely response for the patients to trastuzumab therapy was either 1 for responders or 0 for non-responders.
  • Formula I was used to predict a patient's response to trastuzumab therapy.
  • a patient was predicted to respond to treatment with trastuzumab if the sum of Herl/Her2 and Herl/Her3 heterodimers detected was less than or equal to 0.
  • NR non-responders
  • NPV negative predictive value
  • Formula I was applied to patient Group 2, including all patients that received chemotherapy in combination with trastuzurnab.
  • 11 patients were designated as responders and five as non-responders (Table 7). All 11 responders (R) and five non- responders (NR) were correctly identified, therefore the PPV and NPV are both 100% (11/11) and (5/5). The Sn and the Sp are also both 100%, and the p-value for this analysis is 0.0002.
  • Figure 13 shows a Kaplan-Meier curve comparing surivial of patients predicted to respond to trastuzumab therapy versus patients predicted not to respond to such therapy. As shown in Figure 13, the assortment of such patients by Formula I was statistically significant (p-value of 0.0106 by logrank). Further, the Kaplan-Meier curve constructed by applying Formula I to the patient cohort is very similar to a curve plotting CR/PR/SD patients versus PD patients. See Figure 14. [0126] Finally, results from applying Formula I to combined Groups 1 and 2 are shown in Table 8, below:
  • Formula II is defined as follows:
  • each of the dimers is expressed as a number of dimers per cell and PTEN is expressed as the amount of RFUs normalized against cytokeratin as described above. If the first aspect of Formula II gives a number less than or equal to about 2.091, the patient is predicted to respond to trastuzumab therapy. Further, even if the first aspect of Formula II yields a score greater than about 2.091, the patient would still be predicted to respond to trastuzumab therapy if the second aspect of Formula H yields a value greater than about 2.779.
  • Table 9 the patients of Groups 1 and 2 were combined, Table 10 presents Group 1 alone, and Table 11 presents Group 2 alone.
  • Formula IH was used to predict patient response to trastuzumab.
  • Formula 111 defines a method of scoring whether a patient is predicted to respond to treatment with trastuzumab based upon the number of Herl/Her2, Herl/Her3, and Her2/Her3 heterodimers detected and the normalized amount of PTEN. Specifically, a score of 0 was assigned to a patient if any of each of Herl/Her2 dimers per cell, Herl/Her3 dimers per cell, or Her2/Her3 dimers per cell were detected in the patient's sample. If no heterodimer was detected, the sample was assigned a value of 1.
  • Formula IV was used to predict patient response to trastuzumab.
  • Formula IV defines a method of scoring whether a patient is predicted to respond to treatment with trastuzumab based upon the number of Herl/Her2, Herl/Her3, and Her2/Her3 heterodimers detected and the normalized amount of PTEN. Specifically, a score of 0 was assigned to a patient if any of each of Herl/Her2 dimers per cell, Herl/Her3 dimers per cell, or Her2/Her3 dimers per cell were detected in the patient's sample. If no heterodirner was detected, the sample was assigned a value of 1.
  • Formula V was used to predict patient response to trastuzumab.
  • a patient was predicted to respond to treatment with trastuzumab if the sum of Herl/Her2, Herl/Her3, and Her2/Her3 heterodimers detected was less than or equal to 2700 dimers, or the normalized amount of PTEN detected was greater than 600.
  • Formula V was applied to patient Group I. As shown in Table 11, below, Herl/Her3, Herl/Her2 and Her2/Her3 heterodimers along with PTEN were used to predict patient response to Herceptin. In Group I, Formula V predicted nine patients to be responders (1) and six of them showed a clinical response (R), thus the PPV was 66% (6/9). Of the five patients we predicted to be non-responders (0), none showed a clinical response, thus the NPV was 100% (5/5).
  • a score of 1 was assigned for patients exhibiting an amount of PTEN expression corresponding to more than about about 93252 unnormalized RFUs, while patients with less PTEN were assigned a score of 0.
  • the scores for each of these four variables were summed, and patients with a sum equal to or greater than 2 were identified as responders, while patients with a sum less than 2 were scored as non-responders.
  • Her2/Her3 dimers per cell less than 2700 were scored as responders.
  • patients with an amount of Herl/Her2 dimers per cell + Her2/Her3 dimers per cell less than 1730 or an amount of PTEN corresponding to more than about 80000 unno ⁇ nalized RFUs were scored as responders.
  • patients with an amount of Herl/Her3 dimers per cell + Her2/Her3 dimers per cell less than about 2700 were scored as responders.
  • In Formula XII patients with an amount of Herl/Her3 dimers per cell + Her2/Her3 dimers per cell less than about 1267 or an amount of PTEN corresponding to more than about 80000 unnormalized RFUs were scored as responders.
  • Example 3 Application of Formulae I-IV to a Naive Dataset
  • Formulae I-IV determined as described above, were applied to a naive dataset to test the predictive power of the algorithms. Briefly, the dimer and PTEN concentrations of the samples in the na ⁇ ve dataset were determined according to Example 1, then the formulae determined in Example 2 were used to predict whether the patients were predicted to respond to trastuzumab therapy. The results of the analysis are presented in Table 15, below.

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Abstract

Dans certains aspects, la présente invention a trait à des procédés et des compositions pour déterminer si une cellule cancéreuse est susceptible de réagir à un traitement avec un agent activité Her2. De préférence, les procédés de l'invention sont mis en oeuvre à l'aide d'ensembles de composés de liaison ayant des étiquettes moléculaires libérables qui sont spécifiques pour de constituants multiples d'un ou de plusieurs types de dimères. Suite à la liaison, les étiquettes moléculaires sont libérées et séparées du mélange de dosage pour analyse.
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US9487588B2 (en) 2007-02-16 2016-11-08 Merrimack Pharmaceuticals, Inc. Antibodies against the ectodomain of ERBB3 and uses thereof
US7846440B2 (en) 2007-02-16 2010-12-07 Merrimack Pharmaceuticals, Inc. Antibodies against ErbB3 and uses thereof
US8691225B2 (en) 2007-02-16 2014-04-08 Merrimack Pharmaceuticals, Inc. Antibodies against the ectodomain of ErbB3 and uses thereof
US8961966B2 (en) 2007-02-16 2015-02-24 Merrimack Pharmaceuticals, Inc. Antibodies against ERBB3 and uses thereof
EP2235536A4 (fr) * 2007-12-20 2011-05-04 Lab Corp America Holdings Procédés de diagnostic du her-2
US10416162B2 (en) 2007-12-20 2019-09-17 Monogram Biosciences, Inc. Her2 diagnostic methods
EP2235536A1 (fr) * 2007-12-20 2010-10-06 Laboratory Corporation of America Holdings Procédés de diagnostic du her-2
US20110189173A1 (en) * 2008-07-08 2011-08-04 George Mason Intellectual Properties, Inc. PHOSPHORYLATED C-ErbB2 AS A SUPERIOR PREDICTIVE THERANOSTIC MARKER FOR THE DIAGNOSIS AND TREATMENT OF CANCER
US9086414B2 (en) * 2008-07-08 2015-07-21 George Mason Research Foundation, Inc. Phosphorylated C-ErbB2 as a superior predictive theranostic marker for the diagnosis and treatment of cancer
US8623592B2 (en) 2008-08-15 2014-01-07 Merrimack Pharmaceuticals, Inc. Methods and systems for predicting response of cells to a therapeutic agent
US10273308B2 (en) 2008-12-01 2019-04-30 Laboratory Corporation Of America Holdings Methods of producing antibodies specific for p95
US9081019B2 (en) 2008-12-01 2015-07-14 Laboratory Corporation Of America Holdings Methods and assays for measuring p95 and/or p95 complexes in a sample and antibodies specific for p95
EP2387711A4 (fr) * 2009-01-15 2012-07-18 Lab Corp America Holdings Procédés permettant de déterminer la réponse d'un patient par mesure de her-3
US9766242B2 (en) 2009-01-15 2017-09-19 Laboratory Corporation Of America Holdings Methods of determining patient response by measurement of HER-3 and P95
EP2387711A1 (fr) * 2009-01-15 2011-11-23 Laboratory Corporation of America Holdings Procédés permettant de déterminer la réponse d'un patient par mesure de her-3
US10775382B2 (en) 2009-01-15 2020-09-15 Laboratory Corporation Of America Holdings Methods of determining patient response by measurement of HER-3
US9518130B2 (en) 2010-03-11 2016-12-13 Merrimack Pharmaceuticals, Inc. Use of ERBB3 inhibitors in the treatment of triple negative and basal-like breast cancers
US8895001B2 (en) 2010-03-11 2014-11-25 Merrimack Pharmaceuticals, Inc. Use of ErbB3 inhibitors in the treatment of triple negative and basal-like breast cancers
US9688761B2 (en) 2013-12-27 2017-06-27 Merrimack Pharmaceuticals, Inc. Biomarker profiles for predicting outcomes of cancer therapy with ERBB3 inhibitors and/or chemotherapies
US10273304B2 (en) 2013-12-27 2019-04-30 Merrimack Pharmaceuticals, Inc. Biomarker profiles for predicting outcomes of cancer therapy with ERBB3 inhibitors and/or chemotherapies
US10184006B2 (en) 2015-06-04 2019-01-22 Merrimack Pharmaceuticals, Inc. Biomarkers for predicting outcomes of cancer therapy with ErbB3 inhibitors

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