ES2422884T3 - ErbB surface receptor complexes as biomarkers - Google Patents

Abstract

A method of determining the cancer status of a patient suffering from a cancer characterized by an aberrant expression of one or more ErbB cell surface receptor complexes, the method comprising the following steps: directly measuring in a patient sample an amount of one or more ErbB cell surface receptor complexes; comparing the amount of one or more ErbB cell surface receptor complexes to the corresponding amount of one or more ErbB cell surface receptor complexes in a reference sample; and correlating differences in the amount of one or more ErbB cell surface receptor complexes in the patient sample and the respective corresponding amount of one or more ErbB cell surface receptor complexes in the reference sample to determine the cancer status of the patient, wherein the ErbB cell surface receptor complex(es) are selected from the group consisting of Her1-Her1 homodimers, Her2-Her2 homodimers, Her1-Her3 receptor dimers, Her1-Her4 receptor dimers, Her2-Her4 receptor dimers, Her3-Her4 receptor homodimers, Her4-Her4 receptor homodimers, Her1-PI3K complexes, Her2-PI3K complexes, Her3-PI3K complexes, Her1-SHC complexes, Her2-SHC complexes, Her3-SHC complexes, Her3-SHC dimers Her1-IGF-IR receptor, Her2-IGF-IR receptor dimers, Her3-IGF-IR receptor dimers, Her1-PDGFR receptor dimers, Her2-PDGFR receptor dimers, Her3-PDGFR receptor dimers, receptor dimers p95Her2-Her3 ptor, p95Her2-Her2 receptor dimers, p95Her2-Her1 receptor dimers, EGFRvlll-Her1 receptor dimers, EGFRvlll-Her2 receptor dimers, and EGFRvlll-Her3 receptor dimers; wherein the ErbB cell surface receptor complex(es) are measured by the following steps: providing for each one or more ErbB cell surface receptor complexes a pair of reagents comprising a cleavage probe having a cleavage-inducing moiety with effective proximity, and one or more binding compounds each having one or more molecular tags attached through a cleavable bond, the molecular tags of the different binding compounds having different release characteristics; mixing the cleavage probe and the binding compound(s) for the one or more ErbB cell surface receptor complex(es) with the patient sample such that the cleavage probe and the one or more binding compound(s) bind specifically to their respective ErbB cell surface receptor complexes and that the cleavable bonds of one or more binding compounds are within effective proximity of the cleavage-inducing moiety for the molecular tags to be released; and separating and identifying the released molecular tags to determine the presence or absence or quantity of one or more ErbB cell surface receptor complexes in said patient's sample.

Description

ErbB surface receptor complexes as biomarkers

Field of the Invention

The present invention relates, in general terms, to biomarkers, and more specifically, to the use as biomarkers of ErbB cell surface receptor complexes, such as dimers and oligomers.

Background of the invention

A biomarker is a characteristic that is measured and evaluated objectively as an indicator of normal biological processes, pathogenic processes or pharmacological responses to a therapeutic intervention, Atkinson et al, Clin. Pharmacol Ther., 69: 89-95 (2001). Biomarkers vary greatly in their nature, ease of measurement and correlation with physiological states of interest, for example Frank et al, Nature Reviews Drug Discovery, 2: 566-580 (2003). In general, it is believed that the development of new validated biomarkers will lead both to a significant reduction in drug and healthcare development costs and to significant improvements in the treatment of various diseases and conditions. Therefore, a great effort has been devoted to the use of new technologies to find new classes of biomarkers, for example Petricoin et al, Nature Reviews Drug Discovery, 1: 683-695 (2002); Sidransky, Nature Reviews Cancer, 2: 210-219 (2002).

The interactions of the cell surface membrane components perform fundamental functions to transmit extracellular signals to a cell, both in normal physiology and in pathological states. In particular, many types of cell surface receptors undergo dimerization, oligomerization or clustering in relation to the transduction of an extracellular signal or event, for example ligand-receptor binding, in a cellular response, such as proliferation, augmentation. or decreased genetic expression or the like, for example George et al, Nature Reviews Drug Discovery, 1: 808-820 (2002); Mellado et al, Ann. Rev. Immunol., 19: 397-421 (2001); Schlessinger, Cell, 103: 211-225 (2000); Yarden, Eur. J. Cancer, 37: S3-S8 (2001). The role of these signal transduction events in diseases, such as cancer, has been the subject of intensive research and has led to the development of several new drugs and candidate drugs, for example Herbst and Shin, Cancer, 94: 1593-1611 (2002); Yarden and Sliwkowski, Nature Reviews Molecular Cell Biology, 2: 127-137 (2001); McCormick, Trends in Cell Biology, 9: 53-56 (1999); Blume-Jensen and Hunter, Nature, 411: 355-365 (2001).

Expression levels of individual cell surface receptors have been successfully employed as biomarkers, for example Slamon et al, US Patent 4,968,603 (Her2 expression). However, the level of expression of the individual receptor alone is not always a reliable indicator of a pathological condition or condition, for example Chow et al, Clin. Cancer Res., 7: 1957-1962 (2001) (EGFR, or Her1, expression). Despite the important role played by the dimerization of the receptor in cellular and pathological processes, the expression of the receptor dimer has not been used as a biomarker, partly due to the discomfort and lack of sensitivity of current measurement technologies and the inability or infeasibility of the use of these technologies to perform measurements on patient samples, which may be fixed in formalin and / or present an amount too small for the analysis, for example Price et al, Methods in Molecular Biology, 218: 255- 267 (2003); Stagljar, Science STKE 2003, pe56 (2003); Koll et al, International Patent Publication WO 2004/008099; Golemis, editor, Protein-Protein Interactions (Cold Spring Harbor Laboratory Press, New York, 2002); Sorkin et al, Curr. Biol., 10: 1395-1398 (2000); McVey et al, J. Biol. Chem., 17: 14092-14099 (2001); Salim et al, J. Biol. Chem.,

277: 15482-15485 (2002); Angers et al, Annu. Rev. Pharmacol. Toxicol., 42: 409-435 (2002); Szollosi et al, Reviews in Molecular Biotechnology, 82: 251-266 (2002); Matko et al, Meth. in Enzymol., 278: 444-462 (1997); Reed-Gitomer, U.S. Patent 5,192,660.

In view of the above, the availability of a new class of biomarkers in patient samples based on the presence, absence and / or profile or ratios of cell surface receptor dimers or complexes involved in key intracellular processes, such as transduction of the signal, they would mean an advance in the medical field, by providing a new tool for diagnosis, prognosis, patient stratification and drug development.

US-A-6001583 discloses the usefulness of Her2-GRB7 complexes as a cancer marker.

Gilbertson et al., CANCER RESEARCH, AMERICAN ASSOCIATION FOR CANCER RESEARCH, US, vol. 57, no. 15, August 1, 1997 (1997-08-01), pages 3272-3280 discloses the association of survival of patients suffering from childhood medulloblastoma and the coexpression of Her 2 and Her 4.

Bazin H et al., JOURNAL OF BIOTECHNOLOGY, vol. 82, no. 3, January 1, 2002, pages 233-250 discloses the Her receptor complexes and their potential use as a marker in conjunction with cancer.

WO 2004 08099 which is relevant under art. 54 (3) of the CPE only, refers to the detection of Her 1 / Her 2 and / or Her 2 / Her 3 protein complexes to determine the degree of response of a cancer.

Summary of the Invention

The invention relates to a method mentioned in claim 1. The disclosure provides biomarkers comprising ErbB receptor complexes on cell surface membranes of tissue samples.

or cells of a patient, particularly samples preserved by conventional procedures, such as freezing or fixation. In one aspect, the disclosure includes a method for determining the status of a healthy disease or condition, correlating this condition with the amounts of one or more ErbB receptor complexes in the cell surface membranes of a tissue sample or cells of a individual. In another aspect, the disclosure includes a method for determining a cancer state in a sample of an individual, correlating measurements of the amounts of one or more complexes of ErbB surface receptors in the sample with said state. The invention also provides a method for predicting the effectiveness of drugs acting on the ErbB dimer, for example, in the treatment of cancer, by relating the amounts and types of ErbB dimers that respond to drugs with efficacy or probability of patient response.

In one aspect, the invention allows to determine a pathological state of a patient suffering from a disease characterized by an aberrant expression of one or more complexes of ErbB cell surface receptors, through the following steps: (i) measurement of a quantity of one or more complexes of ErbB cell surface receptors in a sample of a patient, as mentioned in claim 1 (ii) comparing this amount with its corresponding amount in a reference sample; and (iii) correlation of the differences in the quantities of the patient sample with the corresponding quantities of the reference sample for the pathological state of the patient. A sample of a patient can be fixed or frozen; however, preferably, a sample of a patient is fixed using conventional protocols.

In a specific aspect, the disclosure provides a method for determining from the measurements in patient samples, especially fixed samples, the pathological state of a patient suffering from cancer, where said measurement is made of the types and / or amounts of ErbB receptor complexes, which are also referred to herein as "Her receptor complexes." These receptor complexes include, by way of example only, one or more Her1-Her1 homodimers, Her2-Her2 homodimers, Her1-Her2 receptor dimers, Her2-Her3 receptor dimers, Her1-Her3 receptor dimers, receptor dimers Her2-Her4, Her1-PI3K complexes, Her2-PI3K complexes, Her3-PI3K complexes, Her1-SHC complexes, Her2-SHC complexes, Her3-SHC complexes, Her1-IGF-IR receptor dimers, dimers of Her2-IGF-1R receptor, Her3-IGF-1R receptor dimers, Her1-PDGFR receptor dimers, Her2-PDGFR receptor dimers, Her3-PDGFR receptor dimers, p95Her2-Her3 receptor dimers, p95Her2- receptor dimers Her2, p95Her2-Her1 receptor dimers, EGFRvIII-Her1 receptor dimers, EGFRvIII-Her2 receptor dimers, and EGFRvIII-Her3 receptor dimers. In other embodiments, these Her receptor complexes are selected from the group consisting of Her1-Her2 receptor dimers and Her2-Her3 receptor dimers; or from the group consisting of Her1-Her2 receptor dimers, Her2-Her3 receptor dimers, and Her1-Her3 receptor dimers. In another embodiment, the disclosure includes the measurement of complexes comprising a Her receptor in an intracellular adapter molecule, in particular intracellular adapter molecules that complex with a Her receptor in response to phosphorylation of said receptor. Some examples of Her receptor complexes and intracellular adapter molecules include the complexes selected from the group consisting of Her1-PDK complexes, Her2-PI3K complexes, Her3-PI3K complexes, Her1-SHC complexes, Her2-SHC complexes, and Her3-SHC complexes. The disclosure also includes the association of receptor heterodimers comprising a Her receptor and another tyrosine kinase receptor with a pathological state. Some examples of Her receptor and other tyrosine kinase receptor complexes include receptor complexes selected from the group consisting of Her1-IGF-IR receptor dimers, Her2-IGF-1R receptor dimers, Her3-IGF-1R receptor dimers, Her1-PDGFR receptor dimers, Her2-PDGFR receptor dimers, and Her3-PDGFR receptor dimers. The disclosure also includes the association of receptor dimers comprising a full-length Her receptor and a truncated Her receptor with a pathological state. Some examples of full-length Her receptor receptors and truncated Her receptors include receptor complexes selected from the group consisting of p95Her2-Her3 receptor dimers, EGFRvIII-Her1 receptor dimers, EGFRvIII-Her2 receptor dimers, and EGFRvIII receptor dimers -Her3. In another aspect, this method to determine the pathological state includes the determination of the effectiveness of the drugs acting on the dimers, or the degree of response of a patient to them, for the treatment of cancer, the drug acting on the dimer on the Her receptor complexes, as described above.

In another aspect, the disclosure includes improved determinations of a pathological state by measuring the expression of Her1-Her3 receptor complexes in a sample of a patient, as well as the expression of Her1-Her2 and Her2-Her3 receptor complexes.

In another aspect, the invention provides a method for determining a cancer status in a patient, by determining the amounts of one or more ErbB cell surface membrane receptor dimers or relative amounts of a plurality of membrane receptor dimers. of the cell surface in a sample of tissue or cells of said patient. These dimers are measured using at least two reagents, referred to herein as reagent pairs, which are specific for different members of each dimer: a reagent, referred to in the present cleavage probe, has a cleavage inducing fraction that can be induced to Clivate susceptible links within its immediate proximity; and the other reagent, referred to in the present binding compound, has one or more molecular tags linked by bonds that can be cleaved by the cleavage-inducing fraction. According to the embodiment, whenever these different members form a dimer, the clivable bonds are brought to the proximity of effective cleavage of the cleavage-inducing fraction, so that the molecular tags are released. The released molecular tags are then separated from the reaction mixture and quantified to provide a measure of the formation of dimers.

In another aspect of the disclosure, ErbB receptor dimers in a patient sample are measured radiometrically; that is, the amount of an ErbB dimer is provided as a ratio of a measure of a compound present in the dimer to measure the total amount of the other dimer component, whether it is present in the dimer or in monomeric form. In one embodiment, typical measurements include peak height or peak surface of the peaks in an electropherogram that are correlated with specific molecular labels.

In a specific embodiment of this aspect, the invention provides a method for determining a cancer status in a patient, simultaneously determining the amounts of a plurality of Her receptor dimers in a fixed tissue sample of the patient. These dimers can be measured using at least two reagents that are specific to different members of each dimer: a reagent, referred to in the present cleavage probe, has a cleavage inducing fraction that can be induced to clivate susceptible bonds within its immediate proximity. ; and the other reagent, referred to in the present binding compound, has one or more molecular tags linked by bonds that can be cleaved by the cleavage-inducing fraction. According to the embodiment, whenever dimers of the Her receptor are formed, the clivable bonds of the binding compounds are brought to the proximity of effective cleavage of the cleavage-inducing fraction, so that the molecular tags are released. The molecular tags are then separated from the reaction mixture and quantified to provide a measure of the Her receptor dimer populations. In another embodiment of this aspect, the relative amounts of a plurality of Her receptor dimers are measured and related to a cancer state in a patient. Some examples of cancer include, by way of example only, breast cancer, ovarian cancer and prostate cancer. Some examples of Her receptor dimer of the disclosure include, by way of example, Her1-Her2 receptor dimers, Her1-Her3 receptor dimers, Her2-Her3 receptor dimers, and Her2-Her4 receptor dimers, as listed above. .

The present disclosure provides biomarkers comprising measurements of the amounts of ErbB receptor complexes in patient samples. In particular, profiles of populations of ErbB receptor complexes can be correlated with the pathological status of a patient and, in some embodiments, with the prognosis, the efficacy of drugs acting against ErbB dimers and the likelihood of response. from the patient to the therapy. According to the invention, the deficiencies existing in the field are overcome by allowing direct measurement of ErbB receptor complexes in patient samples, without the need to cultivate or otherwise process tissue or cell samples by methodologies, such as xenograft. , which increase the cost and the work, besides introducing sources of noise and potential artifacts in the final readings of the essay. The present invention also provides a secondary measurement for intracellular receptor phosphorylation or other modifications that are easily destroyed in sample preparation procedures. These secondary measurements are based on the measurement of complexes, such as receptor complexes with PI3K or SHC, and the like, which depend on the previous modifications to their formation and which are less affected by the sample preparation procedures.

Brief description of the illustrations

Figures 1A-1F illustrate by diagrams the use of releasable molecular labels to measure populations of receptor dimers.

Figures 1G-1H illustrate by diagrams the use of releasable molecular tags to measure cell surface receptor complexes in fixed tissue samples.

Figures 2A-2E illustrate by diagrams an embodiment of the method of the invention to profile the relative amounts of dimers of a plurality of receptor types.

Figures 3A-3D illustrate by diagrams methods for attaching molecular tags to antibodies.

Figures 4A-4E illustrate data from SKBR-3 and BT-20 cell lysate assays for receptor heterodimers using a method of the invention.

Figures 5A-5C illustrate test data for receptor heterodimers on human tumor and normal breast tissue samples, using a method of the invention.

Figures 6A and 6B illustrate test data of the invention to detect homodimers and phosphorylation of Her 1 in lysates of BT-20 cells.

Figure 7 shows data from assays of the invention showing populations of Her2 homodimers on MCF-7 and SKBR-3 cell lines.

Figures 8A-8B show data from assays of the invention that detect Her1 and Her3 heterodimers on cells, in response to increased heregulin concentrations (HRG).

Figures 9A and 9B show data on the increases in the amounts of Her1-Her3 heterodimers on 22Rvl and A549 cells, respectively, with the increase in epidermal growth factor (EGF) concentrations.

Figures 1 OA-1OC show data on the expression of IGF-1R heterodimers and various Her receptors in frozen samples of human breast tissue.

Figures 11 A-11D illustrate the trial design and experimental results for detecting a Her3-PI3 kinase receptor activation complex.

Figures 12A-12D illustrate the assay design and experimental results for detecting a Her3 / Shc adapter-receptor complex.

Fig. 13 shows data for a correlation between the expression of Her2-Her3 heterodimers and PI3K // Her3 complexes in tumor cells.

Fig. 14A-14B show measurements of populations of Her1-Her2 and Her2-Her3 receptor dimers obtained from normal breast tissue samples and tumor breast tissue samples.

Fig. 15A-15G show measurements of homodimers of Her1-Her1 and Her2-Her2 and heterodimers of Her1-Her2 and Her2-Her3 in fixed pearl sections of cancer cell lines.

Definitions

"Antibody" means an immunoglobulin that specifically binds to a specific polar and spatial organization of another molecule and, therefore, is defined as complementary to it. The antibody can be monoclonal or polyclonal and can be prepared by techniques that are well known in the field, such as immunization of a host and serum collection (polyclonal) or by preparing continuous hybrid cell lines and collecting secreted (monoclonal) protein. or by cloning and expressing nucleotide sequences or mutagenized versions thereof that encode at least the amino acid sequences necessary for the specific binding of natural antibodies. The antibodies may include a complete immunoglobulin or a fragment thereof, including these immunoglobulins 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. On the other hand, the aggregates, polymers and conjugates of immunoglobulins or their fragments can be used, when appropriate, provided that the binding affinity to a particular polypeptide is maintained. The guidelines for the production and selection of antibodies used in immunoassays, including assays that employ the releasable molecular tag (as described below) can be found in easily accessible texts and manuals, for example Harlow and Lane, Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, New York, 1988); Howard and Bethell, Basic Methods in Antibody Production and Characterization (CRC Press, 2001); Wild, editor, The Immunoassay Handbook (Stockton Press, New York, 1994), and the like.

"Antibody binding composition" means a molecule or a complex of molecules comprising one or more antibodies, or fragments thereof, and obtaining its binding specificity for said antibody or antibody fragment. Antibody binding compositions include, by way of example only, (i) pairs of antibodies in which a first antibody specifically binds to a target molecule and a second antibody specifically binds to a constant region of the first antibody; a biotinylated antibody that specifically binds to a target molecule and a streptavidin protein, this protein being derivatized with fractions such as molecular tags or photosensitizers, or the like, through a biotin fraction; (ii) antibodies specific for a target molecule and conjugated with a polymer, such as dextran, which, in turn, is derivatized with fractions such as molecular tags or photosensitizers, either directly by covalent bonds or indirectly by streptavidin-biotin junctions; (iii) antibodies specific for a target molecule and conjugated with a bead, or microbead, or other solid phase support, which, in turn, is derivatized, directly or indirectly, with fractions such as molecular tags or photosensitizers, or polymers that They contain the latter.

"Antigenic determinant" or "epitope" means a site on the surface of a molecule, usually a protein, to which a single antibody molecule binds; In general, a protein has several or multiple different antigenic determinants and reacts with antibodies of numerous different specificities. A preferable antigenic determinant is a phosphorylation site of a protein.

"Binding fraction" means any molecule to which molecular tags can be directly or indirectly bound and that is capable of specifically binding to an analyte. Binding fractions include, by way of example only, antibodies, antibody binding compositions, peptides, proteins, nucleic acids and organic molecules having a molecular weight of up to 1,000 daltons and which are composed of atoms selected from the group consisting of hydrogen, carbon, oxygen, nitrogen, sulfur and phosphorus. Preferably, the binding fractions are antibodies or antibody binding compositions.

"Cancer" and "cancer" describe or refer to the physiological condition in mammals that is typically characterized by deregulated cell growth. Some examples of cancers include, by way of example only, a carcinoma, lymphoma, blastoma, sarcoma and leukemia. Some more concrete examples of these cancers include squamous cell cancer, small cell lung cancer, 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, Vulvar cancer, thyroid cancer, liver carcinoma and various types of cancers that affect the head and neck.

For the purposes of the present, "complex" means a concurrence or set of molecules in direct or indirect contact with each other. In one aspect, "contact" or, more specifically, "direct contact" in relation to a complex of molecules, or in relation to specificity or specific binding, means two or more molecules that are close enough so that the Attractive non-covalent interactions, such as Van der Waal forces, hydrogen bonding, ionic and hydrophobic interactions, and the like, dominate the interaction of molecules. In this aspect, a complex of molecules is stable in the sense that, under test conditions, the complex is thermodynamically more favorable than a state that does not form a set or complex of its component molecules. For the purposes of the present, "complex" usually refers to a stable set of two or more proteins and is equivalently called "protein-protein complex." More typically, a "complex" refers to a stable set of two proteins.

With respect to cell surface membrane receptors, "dimer" means a complex of two or more membrane bound receptor proteins that may be the same or different. The identical receptor dimers are called "homodimers" and the different receptor dimers are called "heterodimers." Typically, dimers consist of two receptors in contact with each other. The dimers can be created on 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 they can also be created by active processes, such such as ligand-induced dimerization, covalent bonds, interaction with intracellular components or the like, for example Schlessinger, Cell, 103: 211-225 (2000). For the purposes of the present, it will be understood that the term "dimer" refers to the "dimer of the cell surface membrane receptor", unless the context implies otherwise.

"Pathological status" includes, by way of example only, the following characteristics: probability of contracting a disease, presence or absence of a disease, prognosis of the severity of a disease and probability of a patient responding to treatment with a specific therapeutic agent that It acts through a receptor complex. With respect to cancer, the "pathological state" also includes the detection of precancerous or cancerous cells or tissues, the selection of patients that are likely to respond to treatment with a therapeutic agent that acts through one or more receptor complexes, such as one or more receptor dimers, and the effects of improved treatment with these therapeutic agents. In one aspect, pathological state, with respect to the Her receptor complexes, means the probability that a cancer patient responds to treatment with a drug acting on the Her or ErbB dimer. Preferably, this cancer patient is a patient with breast or ovarian cancer and these drugs acting on Her dimer include Omnitarg ™ (2C4), Herceptin, ZD-1839 (Iressa), and OSI-774 (Tarceva).

"ErbB receptor" or "Her receptor" is a tyrosine kinase protein receptor that belongs to the ErbB receptor family and includes the EGFR ("Her1"), ErbB2 ("Her2"), ErbB3 ("Her3 ") And ErbB4 (" Her4 "). ErbB receptor generally comprises an extracellular domain, which can bind to an ErbB ligand; a lipophilic transmembrane domain; a conserved intracellular tyrosine kinase domain; and a signaling domain. carboxy-terminal that stores several tyrosine residues that can be phosphorylated.The ErbB receptor can be a native sequence ErbB receptor or a variant of an amino acid sequence thereof Preferably, the ErbB receptor is a human native sequence ErbB receptor. In one aspect, the ErbB receptor includes truncated versions of the Her receptors, including, but not limited to, EGFRvIII and p95Her2, disclosed in Chu et al, Biochem. J., 324: 855-861 (1997); Xia et al, Oncogene, 23: 646-653 (20 04); and the like.

The terms "ErbB1", "epidermal growth factor receptor" and "EGFR" and "Her1" are used interchangeably herein and refer to the native sequence EGFR disclosed, for example, in Carpenter et al. Ann. Rev. Biochem. 56: 881-914 (1987), including variants thereof (such as mutant EGFR by deletion of Humphrey et al. PNAS (USA) 87: 4207-4211 (1990)). ErbB1 refers to the gene that encodes the EGFR protein product. Examples of antibodies that bind to EGFR include MAb 579 (ATCC CRL RB 8506), MAb 455 (ATCC CRL HB8507), MAb 225 (ATCC CRL 8508), MAb 528 (ATCC CRL 8509) (see, U.S. Patent No. 4,943 .533, Mendelsohn et al.) And variants thereof, such as 225 chimerized (C225) and 225 human reformed (H225) (see, WO 96/40210, Imclone Systems Inc.).

"Her2", "ErbB2" and "c-Erb-B2" are used interchangeably. Unless otherwise indicated, the terms "ErbB2", "c-Erb-B2" and "Her2" used herein refer to the human protein. The human ErbB2 gene and the ErbB2 protein are described, for example, in Semba et al., PNAS (USA) 82: 6497-650 (1985) and Yamamoto et al. Nature 319: 230-234 (1986) (Genbank accession number X03363). Some examples of antibodies that specifically bind to Her2 are disclosed in US Patents 5,677,171; 5,772,997; Fendly et al, Cancer Res., 50: 1550-1558 (1990); and the like

"ErbB3" and "Her3" refers to the receptor polypeptide disclosed, for example, in US Pat. Nos.

5,183,884 and 5,480,968, as well as in Kraus et al. PNAS (USA) 86: 9193-9197 (1989), including variants thereof. Some examples of antibodies that bind to Her3 are described in US Patent No. 5,968,511, for example antibody 8B8 (ATCC HB 12070).

The terms "ErbB4" and "Her4" used herein refer to the receptor polypeptide disclosed, for example, in EP Patent Application No. 599,274; Plowman et al., Proc. Natl Acad. Sci. USA, 90: 1746-1750 (1993); and Plowman et al., Nature, 366: 473-475 (1993), including variants thereof, such as the Her4 isoforms disclosed in WO 99/19488.

"Type 1 insulin-like growth factor receptor" or "IGF-1R" means a human tyrosine kinase receptor substantially identical to those disclosed in Ullrich et al, EMBO J., 5: 2503-2512 (1986) or Steele -Perkins et al, J. Biol. Chem., 263: 11486-11492 (1988).

"Isolated" in relation to a polypeptide or protein means substantially separated from the components of its natural environment. Preferably, an isolated protein or polypeptide is a composition that is composed of at least eighty percent by weight of the protein or polypeptide identified by the sequence compared to the components of its natural environment; more preferably, this composition is composed of at least ninety-five percent by weight of the protein or polypeptide identified by the sequence compared to the components of its natural environment; and even more preferably, said composition is composed of at least ninety-nine percent by weight of the protein or polypeptide identified by the sequence compared to the components of its natural environment. More preferably, an isolated protein or polypeptide is a homogeneous composition that can be resolved as a single point after conventional separation by two-dimensional gel electrophoresis based on molecular weight and isoelectric point. The protocols for this analysis using conventional two-dimensional gel electrophoresis are well known to people normally qualified in the field, for example Hames and Rickwood, Editors, Gel Electrophoresis of Proteins: A Practical Approach (TRL Press, Oxford, 1981); Scopes, Protein Purification (Springer-Verlag, New York, 1982); Rabilloud, Editor, Proteome Research: Two-Dimensional Gel Electrophoresis and Identification Methods (Springer-Verlag, Berlin, 2000).

"Kit" refers to any administration system for administering materials or reagents to carry out a method of the invention. In the context of reaction assays, these delivery systems include systems that allow the storage, transport or administration of reaction reagents (eg, probes, enzymes, etc. in appropriate containers) and / or complementary materials. (for example, buffer solutions, written instructions for carrying out the test, etc.) from one place to another. For example, the kits include one or more containers (eg, boxes) containing the corresponding reaction reagents and / or complementary materials. These contents can be administered in the intended container together or separately. For example, a first container may contain an enzyme for use in an assay, while a second container contains the probes.

"Identical percentage", or a similar term, used with respect to the comparison of a reference sequence with another sequence (ie a "candidate" sequence), means that in an optimal alignment between the two sequences, the candidate sequence is identical to the reference sequence in a series of subunit positions equivalent to the indicated percentage, the nucleotide subunits being for polynucleotide or amino acid comparisons for polypeptide comparisons. For the purposes of the present, an "optimal alignment" of the sequences being compared is one that maximizes the coincidences between subunits and minimizes the number of gaps used to construct an alignment. Identity percentages can be determined with commercially available algorithm applications, described by Needleman and Wunsch, J. Mol. Biol., 48: 443-453 (1970) ("GAP" program of Wisconsin Sequence Analysis Package, Genetics Computer Group, Madison, WT). Other software packages in the field for building alignments and calculating the percentage of identity or performing other similarity measurements include the "BestFit" program, based on the Smith and Waterman algorithm, Advances in Applied Mathematics, 2: 482-489 ( 1981) (Wisconsin Sequence Analysis Package, Genetics Computer Group, Madison, WI). In other words, for example, to obtain a polypeptide having an amino acid sequence at least 95 percent identical to a reference amino acid sequence, up to five percent of the amino acid residues of the reference sequence can be subjected by deletion or substituted by another amino acid, or a number of amino acids of up to five percent of the total amino acid residues of the reference sequence can be inserted into the reference sequence. These alterations of the reference sequence may occur at the amino-terminal or carboxy-terminal positions of the reference amino acid sequence or anywhere between said terminal positions, sandwiched either individually between residues of the reference sequence or at one or more contiguous groups of the reference sequence. It is understood that to make comparisons with reference sequences of the invention, the candidate sequence may be a component or segment of a longer polynucleotide or polypeptide and that these comparisons oriented to compute the percentage of identity will be made with respect to the relevant segment or component .

"Phosphatidylinositol 3-kinase protein" or, equivalently, a "PI3K protein" means a human intracellular protein from the set of human proteins described under NCBI accession numbers NP_852664, NP_S52556, and NP_852665, as well as proteins that have substantially amino acid sequences identical to them.

"Platelet-derived growth factor receptor" or "PDGFR" means a human tyrosine kinase protein receptor that is substantially identical to PDGFRa or PDGFR, or variants thereof, described in Heldin et al., Physiological Reviews, 79: 1283 -1316 (1999). In one aspect, the invention includes the determination of cancer states, precancerous conditions, fibrotic or sclera conditions, measuring one or more dimers of the following group: PDGFRa homodimers, PDGFR� homodimers, and PDGFRa-PDGFR� heterodimers. In particular, fibrotic conditions include pulmonary or renal fibrosis and sclera conditions include atherosclerosis. Cancers include, by way of example only, breast cancer, colorectal carcinoma, glioblastoma and ovarian carcinoma. It is understood that the reference to "PDGFR" only means "PDGFRa"

or "PDGFR�".

"Polypeptide" refers to a class of compounds consisting of chemically linked amino acid residues by amide bonds with water removal between the carboxy group of one amino acid and the amino group of the other amino acid. A polypeptide is a polymer of amino acid residues, which can contain a large number of these residues. Peptides are similar to polypeptides, except that they are generally made up of a smaller number of amino acids.

Sometimes the peptides are called oligopeptides. There is no precise distinction between polypeptides and peptides. For convenience, in the present disclosure and in the claims, the term "polypeptide" will be used to refer generally to peptides and polypeptides. The amino acid residues can be natural or synthetic.

"Protein" refers to a polypeptide, usually synthesized by a biological cell, folded into a defined three-dimensional structure. In general, proteins have from about 5,000 to about 5,000,000 or more molecular weight, more usually from about 5,000 to about 1,000,000 molecular weight, and may include posttranslational modifications, such as acetylation, acylation, ADP-ribosylation, amidation, covalent bond of flavin, covalent bond of a heme fraction, covalent bond of a nucleotide or nucleotide derivative, covalent bond of a lipid or lipid derivative, covalent bond of phosphotidylinositol, cross bond, cyclisation, disulfide bond formation, farnesylation, dimethylation, covalent cross-linking formation, cystine formation, pyroglutamate formation, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myrisylation, oxidation, phosphorylation, pyrilage, racemization, selenization, sulfonation and ubiquitination, for example Wold, F., Post-translational Protein Modifications : Perspectives and Prospects, p. 1-12 in Post-translational Covalent Modification of Proteins, BC Johnson, Ed., Academic Press, New York, 1983. Proteins include, by way of illustration and without limitation, cytokines or interleukins, enzymes such as, for example, kinases, proteases, galactosidases, etc., protamines, histones, albumins, immunoglobulins, scleroproteins, phosphoproteins, mucoproteins, chromoproteins, lipoproteins, nucleoproteins, glycoproteins, T cell receptors, proteoglycans and the like.

"Reference sample" means one or more samples of tissue, xenograft or cells that are representative of a normal or non-pathological state with which measurements made with patient samples are compared to determine if a receptor complex is present in excess or It is present in a limited amount in the patient sample. The nature of the reference sample is a matter of design selection for a specific trial and can be obtained or determined from the patient's normal tissue or from tissues of a population of healthy individuals. Preferably, the values relative to amounts of receptor complexes in the reference samples are obtained under experimental conditions essentially identical to the corresponding values of the samples of the patients to be tested. The reference samples can be of the same type of tissue as the patient sample or they can be of different types of tissues and the population from which the reference samples are obtained can be selected based on the characteristics that coincide with those of the patient. , such as age, sex, race, etc. Typically, in the assays of the invention, the amounts of receptor complexes of the patient samples are compared with the corresponding reference sample values that have been previously tabulated and are provided as mean ranges, average values with standard deviations or similar representations. . "Receptor complex" means a complex comprising at least one cell surface membrane receptor. The receptor complexes may include a dimer of cell surface membrane receptors or one or more intracellular proteins, such as adapter proteins, that form bonds in the various signaling pathways. Some examples of intracellular proteins that may be part of a receptor complex include, but are not limited to, PI3K proteins, Grb2 proteins, Grb7 proteins, She proteins, and Sos proteins, Src proteins, Cb1 proteins, PLC proteins and Shp2 proteins, GAP proteins, Nek proteins, Vav proteins, and Crk proteins. In one aspect, receptor complexes include PI3K or Shc proteins.

"Tyrosine kinase receptor" or "RTK" means a human receptor of a protein that exhibits intracellular kinase activity and selected from the RTK protein family described in Schlessinger, Cell, 103: 211-225 (2000); and Blume-Jensen and Hunter (cited above). "Tyrosine kinase receptor dimer" means a complex on a cell surface membrane comprising two protein tyrosine kinase receptors. In some aspects, a tyrosine kinase receptor dimer may comprise two covalently linked tyrosine kinase protein receptors. Some examples of RTK dimers are shown in Table I. RTK dimers of particular interest are Her receptor dimers and VEGFR dimers.

"Sample" or "tissue sample" or "patient sample" or "patient tissue or cell sample" means a collection of similar cells obtained from a tissue of a subject or patient. The source of the tissue sample may be tissue, such as a sample of a tissue or organ or a fresh, frozen and / or preserved biopsy or aspirate; blood or any component of the blood; body fluids, such as cerebrospinal fluid, amniotic fluid, peritoneal fluid or interstitial fluid; or cells of any point in the gestation or development of the subject. The tissue sample may contain compounds that are not naturally intermingled with the tissue in nature, such as preservatives, anticoagulants, buffer solutions, fixatives, nutrients, antibiotics or the like. In one aspect of the invention, tissue samples or patient samples are fixed, particularly conventional samples embedded in paraffin and fixed with formalin. These samples are typically employed in an assay for receptor complexes in the form of thin sections, for example 3-10 µm thick, of fixed tissue mounted on a microscope slide or equivalent surface. These samples are also typically subjected to a conventional rehydration procedure and, optimally, to an antigen recovery procedure as part of the test measurements or as preliminary to them.

"Separation profile" in relation to the separation of molecular labels means a diagram, a graph, a curve, a bar graph or other representation of the signal strength data versus a parameter related to molecular labels, such as retention time, mass or the like, which provides a reading or measurement of the number of molecular labels of each type produced in an assay. A separation profile can be an electropherogram, a chromatogram, an electrochromatogram, a mass spectrogram or a similar graphical representation of the data, depending on the separation technique employed. A "peak" or a "band" or a "zone" in relation to a separation profile means a region in which a separate compound is concentrated. Multiple separation profiles may exist for a single assay if, for example, different molecular tags have different fluorescent tags with different emission spectra and the data is collected and recorded at multiple wavelengths. In one aspect, the released molecular tags are separated by differences in electrophoretic mobility to form an electropherogram in which the different molecular tags correspond to different peaks in the electropherogram. A measure of the distinction or absence of overlapping of adjacent peaks in an electropherogram is the "electrophoretic resolution", which can be taken as the distance between maximum adjacent peaks divided by four times the greater of the two standard deviations of the peaks. Preferably, the adjacent peaks have a resolution of at least 1.0, more preferably of at least 1.5 and more preferably of at least 2.0. In a particular separation and detection system, the desired resolution can be obtained by selecting a plurality of molecular tags whose members have electrophoretic mobilities that differ at least in a resolutory amount from a peak, this amount depending on several factors well known to people qualified in the field, including the signal detection system, the nature of the fluorescent fractions, the diffusion coefficients of the labels, the presence or absence of screening matrices, the nature of the electrophoretic apparatus, for example the presence or absence of channels, the length of the separation channels and the like. Electropherograms can be analyzed to associate characteristics in the data with presence, absence

or the amounts of molecular labels, using analysis programs such as that disclosed in Williams et al, US Patent Publication 2003/0170734 A1.

"SHC" (meaning "Src 2 homology / related to alpha collagen") means any of a family of adapter proteins (66, 52 and 46 kDalton) in RTK signaling pathways substantially identical to those described in Pelicci et al, Cell, 70: 93-104 (1992). In one aspect, SHC means the human versions of these adapter proteins.

"Signaling pathway" or "signal transduction pathway" means a series of molecular events that usually begin with the interaction of the cell surface receptor with an extracellular ligand or with the binding of an intracellular molecule with a phosphorylated site of a receptor cell surface that triggers a series of molecular interactions, where the series of molecular interactions leads to the regulation of genetic expression in the nucleus of a cell. "Ras-MAPK pathway" means a signaling pathway that includes phosphorylation of a MAPK protein after the formation of a Ras-GTP complex. "Via PI3K-Akt" means a signaling pathway that includes phosphorylation of an Akt protein by a PI3K protein.

"Specific" or "specificity", in relation to the binding of one molecule to another, as a binding compound, or a probe, for a complex or target analyte, means the recognition, contact and formation of a stable complex between the probe and the objective, together with the recognition, contact or formation of substantially smaller complex of the probe with other molecules. In a "specific" aspect, in relation to the union of a first molecule to a second molecule it means that, to the extent that the first molecule recognizes and forms a complex with other molecules in a reaction or sample, it forms the largest number of complexes with the second molecule. In one aspect, this largest number is at least fifty percent of all these complexes formed by the first molecule. In general, the molecules involved in a specific binding event have areas on their surfaces or in cavities that give rise to specific recognition between the molecules that bind together. Some examples of specific binding include antibody-antigen interactions, enzyme-substrate interactions, duplex or triplex formation between polynucleotides and / or oligonucleotides, receptor-ligand interactions and the like.

"Spectrally resolvable" in relation to a plurality of fluorescent labels means that the fluorescent emission bands of the labels are sufficiently different, that is not sufficiently overlapping, that the molecular labels to which the respective labels are attached can be distinguished on the basis of the fluorescent signal generated by the respective tags by standard photodetection systems, for example using a system of bandpass filters and photomultiplier tubes or the like, as exemplified by the systems described in US Patents 4,230,558; 4,811,218, or the like, or in Wheeless et al, p. 21-76, in Flow Cytometry: Instrumentation and Data Analysis (Academic Press, New York, 1985).

"Substantially identical" in relation to the proteins or amino acid sequences of the proteins of a family of related proteins to be compared means well that one protein has an amino acid sequence that is at least fifty percent identical to the other. protein or that a protein is an isoform or binding variant of the same gene as the other protein. In one aspect, substantially identical means a protein, or an amino acid sequence thereof, that is at least eighty percent identical to the other protein, or to the amino acid sequence thereof.

Detailed description of the invention

The disclosure provides a method for using ErbB cell surface receptor complexes as biomarkers for a pathological state or other physiological conditions in a biological organism, particularly a cancer state in a human being. In one aspect, ErbB receptor complexes are measured directly in patient samples; that is, the measurements are made without culture, without formation of xenografts or without using similar techniques, which involve increased costs and labor and that could introduce artifacts and / or noise into the measurement process. In a particular aspect of the invention, measurements of one or more receptor complexes are made directly on tissue lysates of frozen patient samples or on sections of fixed patient samples. In a preferable embodiment, one or more ErbB receptor complexes are measured in sections of paraffin embedded and formalin fixed samples (FFPE).

In another aspect, the disclosure provides an indirect measurement of ErbB receptor phosphorylation through the measurement of complexes that depend on these posttranslational modifications for their formation.

In one aspect, a plurality of ErbB receptor complexes, such as receptor dimers, are measured simultaneously in the same reaction mixture of the assay. Preferably, these complexes are measured using binding compounds that have one or more bound releasable molecular tags, so that after binding to a protein in a complex, the molecular tags can be released and separated from the reaction, the assay or the mixture for detection and / or quantification.

In one aspect, the invention provides a method for determining a pathological state of a patient comprising the following steps: measuring an amount of one or more ErbB receptor dimers in a sample of a patient; comparison of this quantity with its corresponding quantity in a reference sample; and correlation of the differences in the amounts of the patient sample with the corresponding amounts of the reference sample to determine the presence or severity of a patient's pathological condition. In a preferable embodiment, the measurement step comprises the following steps: (i) providing one or more specific binding compounds for a protein of one or more receptor dimers, so that each binding compound has one or more molecular tags attached by means of a clivable bond and that each molecular tag or labels attached to different binding compounds have different separation characteristics, so that after separation the molecular labels of the different binding compounds form different peaks in a separation profile; (ii) combination of the binding compounds and the complex or complexes, so that the binding compounds specifically bind to their respective receptor dimers to form detectable complexes; (iii) cleavage of the clivable bond of each binding compound forming detectable complexes, and (iv) separation and identification of the molecular tags released to determine the presence or absence or the amount of one or more receptor dimers.

In another aspect, the step of measuring the amounts of one or more types of ErbB receptor dimers comprises the following steps: (i) providing for each type or types of receptor dimers a specific cleavage probe for a first receptor in each dimer or dimers of the receptor, each cleavage probe having an induction fraction of the cleavage with an effective proximity; (ii) provide one or more specific binding compounds for a second receptor of each dimer or dimers of the receptor, such that each binding compound has one or more molecular tags attached through a clivable bond and that the molecular tag or labels attached to the different binding compounds have different separation characteristics, so that after separation the molecular labels of the different binding compounds form different peaks in a separation profile; (iii) combination of the cleavage probes, the binding compounds and the type or types of receptor dimers, so that the cleavage probes specifically bind to the second receptors of the receptor dimers and that the clivable bonds of the receptor binding compounds are within the effective proximity of the cleavage-inducing fractions of the cleavage probes, so that the molecular tags are released; Y

(iv) separation and identification of the molecular labels released to determine the presence or absence or the amount of one or more types of receptor dimers. Preferably, the receptor dimers and the first and second receptors are selected from the receptor dimers listed in Table I.

In another aspect of the disclosure, a biological sample, comprising a heterogeneous population of cells suspected of containing the rare cell of interest, is obtained from a patient. A sample is then prepared by mixing the biological sample with magnetic particles that bind a specifically reactive biospecific ligand with an antigen on the rare cell that is different or is not found in blood cells (referred to herein as "capture antigen" ), so that other components can be removed substantially from the sample. The sample is subjected to a magnetic field that is effective in separating cells labeled with magnetic particles, including rare cells of interest, if one is present in the sample. The population of cells isolated in this way is subsequently analyzed, using molecular labels conjugated to specific binding fractions for biomarkers, in order to determine the presence and / or the number of rare cells. In a preferable embodiment, the magnetic particles employed in this method are colloidal magnetic nanoparticles. Preferably, these rare cell populations are circulating epithelial cells, which can be isolated from the blood of a patient using epithelial-specific capture antigens, for example as disclosed in Hayes et al, International J. of Oncology, 21: 11111117 (2002); Soria et al, Clinical Cancer Research, 5: 971-975 (1999); Ady et al, British J. Cancer, 90: 443-448 (2004); which are incorporated herein by reference. In particular, the BerEP4 monoclonal antibody (Dynal A.S., Oslo, Norway) can be used to capture human epithelial cells with magnetic particles.

In another aspect, the disclosure provides a method for determining a cancer state of a patient comprising the following steps: (i) immunomagnetically isolating the sample from a patient comprising circulating epithelial cells, contacting a blood sample from a patient with one or more antibody compositions, each antibody composition being specific for a capture antigen and being bound to a magnetic particle; (ii) measurement of an amount of one or more ErbB receptor complexes in the patient sample; comparison of each of these quantities with their corresponding quantity in a reference sample; and correlation of differences in the amounts of the patient sample and the corresponding corresponding amounts in the reference sample to determine the presence or severity of a cancer condition in the patient. In a preferable embodiment, the measurement step comprises the following steps: (i)

providing one or more specific binding compounds for a protein of one or more ErbB receptor complexes, so that each binding compound has one or more molecular tags linked by a clivable bond and that each molecular tag or labels attached to different compounds of binding have different separation characteristics, so that after separation the molecular labels of the different binding compounds form different peaks in a separation profile; (ii) combination of the binding compounds and the ErbB receptor complexes or complexes, so that the binding compounds specifically bind to their respective ErbB receptor complexes or complexes to form detectable complexes; (iii) cleavage of the clivable bond of each binding compound forming detectable complexes, and (iv) separation and identification of the molecular tags released to determine the presence or absence or the amount of one or more ErbB receptor complexes.

In another aspect, the step of measuring the amounts of one or more ErbB receptor complexes comprises the following steps: (i) providing for each ErbB receptor complex or complexes a specific cleavage probe for a first protein in each complex or complexes of the ErbB receptor, each cleavage probe having an induction fraction of the cleavage with an effective proximity; (ii) provide one or more specific binding compounds for a second protein of each ErbB receptor complex or complexes, such that each binding compound has one or more molecular tags attached through a clivable bond and that the tag or tags molecular bonds attached to the different binding compounds have different separation characteristics, so that after separation the molecular labels of the different binding compounds form different peaks in a separation profile; (iii) combination of the cleavage probes, the binding compounds and the complex or complexes, so that the cleavage probes specifically bind to the first proteins of the ErbB receptor complexes and that the binding compounds specifically bind to the second proteins of the ErbB receptor complexes and the clivable bonds of the binding compounds are within the effective proximity of the cleavage inducing fractions of the cleavage probes, so that the molecular tags are released; and (iv) separation and identification of the molecular tags released to determine the presence or absence or the amount of one or more ErbB receptor complexes.

Examples of biomarkers of receptor dimers and drugs acting on dimers

The biomarkers of the disclosure include dimers and oligomers of the following receptors.

Table 1

Examples of cell surface membrane receptor complexes

 Dimer dimer

Her1-Her1

IGF-1R-Her1 heterodimer

Her1-Her2

IGF-1R-Her3 heterodimer

Her1-Her3

Her1-PDGFR heterodimers

Her1-Her4

Her3-PDGFR heterodimers

Her2-Her2

Her2-PI3K

Her2-Her3

Her1-SHC

Her2-Her4

Her3-SHC

Her3-Her4

Her2-SHC

Her4-Her4

Her3-PI3K

Her2-PDGFR heterodimers

Her1-PI3K

IGF-1R-Her2 heterodimer

The mechanisms of action of many drugs that are used or that are in the development phase require the inhibition of one or more functions of ErbB receptor dimers, such as the association of component receptors in a dimer structure, or a function, such as enzymatic activity, for example kinase activity or autophosphorylation, which depends on dimerization. These drugs are referred to herein as "drugs acting on dimers" or "drugs acting on dimers of ErbB". The number, type, formation and / or dissociation of the receptor dimers in the cells of a patient being treated, or whose treatment is contemplated, influence the effectiveness or suitability of the use of a certain drug that acts on the dimer of ErbB The following ErbB receptor dimers are biomarkers related to the indicated drugs. In one aspect, the invention provides biomarkers to control the effect on the pathological state of a drug acting on the ErbB dimer.

Table II

Drugs associated with dimers of cell surface membranes

Her1-Her1, Her1-Her2, Her1-Her3, Her4, Her1-IGF-IR, Her2-IGF-1R

 Her1- Cetuximab (Erbitux), Trastuzumab (Herceptin), h-R3 (TheraCIM), ABX-EGF, MDX-447, ZD-1839 (Iressa), OSI-774 (Tarceva), PKI 166, GW572016, CI-1033, EKB- 569, EMD 72000

Her2-Her1, Her2-Her3, Her2-Her2, Her4

 Her2 4D4 Mab, Trastuzumab (Herceptin), 2C4, GW572016

The following references describe the drugs that act on dimers listed in Table II: Traxler, Expert 10 Opin. Ther. Targets, 7: 215-234 (2002); Baselga, editor, Oncology Biotherapeutics, 2: 1-36 (2002); Nam et al, Current Drug Targets, 4: 159-179 (2003); Seymour, Current Drug Targets, 2: 117-133 (2001); and the like

Table III

Receiver complexes associated with PI3K

Dimer dimer

Herl-herl

IGF-IR-Herl heterodimer

Herl-Her2

Her4-Her4

Herl-Her3

Her3-Her4

Herl-Her4

Her2-Her4

Her2-Her2

Her2-Her3

Heterodimer IGF-1R-Her2 Heterodimers Her3-PDGFR

Heterodimer IGF-1R-Her3 Heterodimers Her1-PDGFR

Her2-PDGFR heterodimers

Her2-XX-PI3K *

* "XX" refers to any Her2 receptor with which it is capable of forming a dimer.

15 Sample preparation

Samples containing molecular complexes can come from a wide variety of sources for use with the present invention, in order to relate the populations of receptor complexes with the pathological state or the healthy state, including cell cultures, animal or plant tissues , patient biopsies

or similar. Preferably, the samples are samples from human patients. The samples are prepared for

20 tests of the invention using conventional techniques, which may depend on the source from which a sample is obtained.

A. Solid tissue samples. For biopsies and medical samples, guidance is provided in the following references: Bancroft JD & Stevens A, eds. Theory and Practice of Histological Techniques (Churchill Livingstone, Edinburgh, 1977); Pearse, Histochemistry. Theory and applied. 4th ed. (Churchill Livingstone, Edinburgh, 1980).

25 Within the scope of the cancerous disease state, some examples of tissue samples from patients that can be used include, by way of example only, breast, prostate, ovary, colon, lung, endometrium, stomach, salivary gland or pancreas. The tissue sample can be obtained by various procedures, including, but not limited to, surgical excision, aspiration or biopsy. The tissue can be fresh or frozen. In one embodiment, tests of the invention are performed on tissue samples that have been fixed and embedded in paraffin or the like; therefore, in these embodiments a dewaxing step is performed. A tissue sample can be fixed (ie preserved) by a conventional methodology [see, for example, "Manual of Histological Staining Method of the Armed Forces Institute of Pathology", 3rd edition (1960) Read

G. Luna, HT (ASCP) Editor, The Blakston Division McGraw-Hill Book Company, New York; The Armed Forces Institute of Pathology Advanced Laboratory Methods in Histology and Pathology (1994) Ulreka V. Mikel, Editor, Armed Forces Institute of Pathology, American Registry of Pathology, Washington, D.C. A qualified person in the field will appreciate that the choice of a fixative is determined by the purpose for which the tissue is to be histologically stained or otherwise analyzed. A person qualified in the field will also appreciate that the length of the fixation depends on the size of the tissue sample and the fixative used. By way of example, the neutral formalin buffer solution, Bouin or paraformaldehyde, can be used to fix a tissue sample.

Usually, a tissue sample is fixed first and subsequently dehydrated through an ascending series of alcohols, infiltrated and embedded in paraffin or other sectioning means, so that the tissue sample can be sectioned. Alternatively, the tissue can be sectioned and the sections obtained fixed. As an example, the tissue sample can be embedded and processed in paraffin by a conventional methodology (see, for example, "Manual of Histological Staining Method of the Armed Forces Institute of Pathology", above). Some examples of paraffin that can be used include, by way of example, Paraplast, Broiloid and Tissuemay. Once the tissue sample is embedded, the sample can be sectioned with a microtome or the like (see, for example, "Manual of Histological Staining Method of the Armed Forces Institute of Pathology", above). As an example for this procedure, the sections may have a thickness in a range of about three microns to about 12 microns and, preferably, a thickness in a range of about five microns to about 10 microns. In one aspect, a section can have an area of about 10 mm2 to about 1 cm2. Once cut, the sections can be attached to slides using various standard methods. Examples of slide adhesives include, by way of example only, silane, gelatin, poly-L-lysine and the like. By way of example, the paraffin embedded sections can be attached to positively charged slides and / or poly-L-lysine coated slides.

If paraffin has been used as a material for embedding, the tissue sections are generally deparaffinized and rehydrated with water. Tissue sections can be deparaffinized by various conventional standard methodologies. For example, xylenes and a gradually descending series of alcohols can be used (see, for example, "Manual of Histological Staining Method of the Armed Forces Institute of Pathology", above). Alternatively, commercially available non-organic dewaxing agents, such as Hemo-De® (CMS, Houston, Tex.), Can be employed.

For tissue culture cells, fresh tissues or similar sources of mammals, samples can be prepared by conventional cell lysis techniques (for example, 0.14 M NaC1, 1.5 mM MgC12, 10 mM Tris-CI (pH 8.6), 0.5% Nonidet P-40, and protease and / or phosphatase inhibitors, when necessary). For fresh mammalian tissues, sample preparation may also include a tissue disaggregation step, such as crushing, chopping, crushing, sonication or the like.

B. Magnetic isolation of cells. In some applications, such as the measurement of dimers on rare metastatic cells of a patient's blood, an enrichment step can be performed before carrying out an assay to detect the dimer populations of the surface receptor. Immunomagnetic enrichment or isolation can be performed using various techniques and materials known in the field, disclosed in the following representative references incorporated herein as references: Terstappen et al, US Patent 6,365,362; Terstappen et al, US Patent 5,646,001; Rohr et al, US Patent 5,998,224; Kausch et al, U.S. Patent 5,665,582; Kresse et al, US Patent 6,048,515; Kausch et al, U.S. Patent 5,508,164; Miltenyi et al, US Patent 5,691,208; Molday, US Patent 4,452,773; Kronick, U.S. Patent 4,375,407; Radbruch et al, chapter 23, in Methods in Cell Biology, Vol, 42 (Academic Press, New York, 1994); Uhlen et al, Advances in Biomagnetic Separation (Eaton Publishing, Natick, 1994); Safarik et al, J. Chromatography B, 722: 33-53 (1999); Miltenyi et al, Cytometry, 11: 231238 (1990); Nakamura et al, Biotechnol. Prog., 17: 1145-1155 (2001); Moreno et al, Urology, 58: 386-392 (2001); Racila et al, Proc. Natl Acad. Sci., 95: 4589-4594 (1998); Zigeuner et al, J. Urology, 169: 701-705 (2003); Ghossein et al, Seminars in Surgical Oncology, 20: 304-311 (2001).

The preferable magnetic particles for use in the embodiment of the present invention are particles that behave like colloids. These particles are characterized by their submicron particle size, which is generally less than about 200 nanometers (nm) (0.20 microns) and their stability for gravitational separation of the solution for prolonged periods of time. In addition to many other advantages, this size range makes them basically invisible to analytical techniques commonly used for cell analysis. Particles within a range of 90-150 nm and having between 70-90% magnetic mass are contemplated for use in the present invention. Suitable magnetic particles consist of a crystalline core of a superparamagnetic material surrounded by molecules that are attached, for example physically absorbed or covalently bound, to the magnetic core and that confer stabilizing colloidal properties. The coating material should preferably be applied in an amount effective to avoid non-specific interactions between the biological macromolecules found in the sample and the magnetic cores. These biological macromolecules can include sialic acid residues on the surface of cells, lectins, gliproteins and other non-target components of the membrane. On the other hand, the material should contain as much mass / magnetic nanoparticles as possible. The size of the magnetic crystals that comprise the core is small enough that they do not contain a complete magnetic domain. The size of the nanoparticles is small enough that Brownian's movement exceeds its magnetic moment. As a consequence, the alignment of the north pole, the south pole and the subsequent attraction / mutual repulsion of these colloidal magnetic particles does not seem to occur even in moderately strong magnetic fields, which contributes to the stability of their solution. Finally, the magnetic particles should be separable in external field separators with a high magnetic gradient. This feature facilitates the handling of the samples and provides economic advantages over the more complicated internal gradient columns loaded with ferromagnetic beads or steel wool. Magnetic particles having the properties described above can be prepared by modifying base materials described in U.S. Patent Nos. 4,795,698, 5,597,531 and 5,698,271, which are incorporated herein by reference.

Assays using releasable molecular labels

The measurement of dimer populations using releasable molecular tags offers numerous advantages, including (1) the separation of molecular tags released from a test mixture provides a markedly reduced background noise and a significant increase in sensitivity; and (2) the use of specially designed molecular tags to facilitate separation and detection offers a practical multiplexing capability, so that multiple components of the receptor complex can be easily measured simultaneously in the same assay. The tests using these labels can take various forms and are disclosed in the following references: Singh et al, US Patent 6,627,400; US Patent Publications Singh et al, 2002/0013126; and 2003/0170915, and Williams et al, 2002/0146726; and Chan-Hui et al, International Patent Publication WO 2004/011900, all of which are incorporated herein by reference. For example, a wide variety of separation techniques can be employed that can distinguish molecules based on one or more physical, chemical or optical differences between the molecules that are being separated, including, by way of example only, electrophoretic mobility, weight molecular, shape, solubility, pKa, hydrophobicity, charge, charge / mass ratio, polarity or the like. In one aspect, molecular tags in a plurality or set differ in the characteristics of electrophoretic mobility and optical detection, and are separated by electrophoresis. In another aspect, molecular tags in a plurality or assembly may differ in molecular weight, shape, solubility, pKa, hydrophobicity, charge, polarity, and are separated by reverse phase or normal phase HPLC, Ion exchange HPLC, capillary electrochromatography, mass spectroscopy, gas phase chromatography or a similar technique.

Molecular tag sets are provided that are separated into different bands or peaks by a separation technique after they have been released from the binding compounds. The identification and quantification of these peaks provides a measure or profile of the types and amounts of receptor dimers. The molecular labels within a set can be chemically diverse; however, usually and for reasons of convenience, molecular tag sets are chemically related. For example, they can all be peptides, or they can be composed of different combinations of the same basic elements or monomers, or they can be synthesized using the same basic support with different substitute groups to impart different separation characteristics, as described in more detail below. . The number of molecular tags in a plurality may vary depending on several factors, including, the mode of separation employed, the marks used in the molecular tags for detection, the sensitivity of the binding fractions, the efficiency with the that clivable links are cloned, etc. In one aspect, the number of molecular tags in a plurality to measure populations of receptor dimers is in a range of 2 to 10. In other aspects, the size of the plurality can be in a range of 2 to 8, 2 to 6, 2 to 4, or 2 to 3.

Receptor dimers can be detected in assays that have homogeneous or non-homogeneous formats, that is, heterogeneous formats. In a homogeneous format, no step is required to separate binding compounds specifically bound to target complexes from unbound binding compounds. In a preferable embodiment, the homogeneous formats employ pairs of reagents comprising (i) one or more binding compounds with releasable molecular tags and (ii) at least one cleavage probe that is capable of generating an active species that reacts with molecular tags and releases them within an effective proximity of the cleavage probe.

Receiver dimers can also be detected by assays that use a heterogeneous format. Typically heterogeneous techniques involve a separation step, where intracellular complexes having specifically bound binding compounds are separated from unbound binding compounds and, optionally, other sample components, such as proteins, membrane fragments and the like. . The separation can be achieved in various ways, such as using a reagent bound to a solid support that distinguishes between the binding compounds bound to the complex and unbound. The solid support can be the wall of a container, for example the well of a microtiter plate, a capillary, a plate, a slide, beads, including magnetic beads, liposomes or the like. The main characteristics of the solid support are that (1) allows segregation of bound and unbound binding compounds, and (2) does not interfere in the formation of the binding complex or in the other operations for the determination of receptor dimers . Usually, in fixed samples, unbound binding compounds are simply removed by washing.

With the detection using molecular labels in a heterogeneous format, after washing, a sample can be combined with a solvent in which the molecular labels are to be released. Depending on the nature of the clivable bond and the cleavage method, the solvent may include any additional reagents for cleavage. When reagents are not necessary for cleavage, the solvent may conveniently be a separation buffer solution, for example an electrophoretic separation medium. For example, when the cleavage link is photolilable or clivable through an active species generated by a photosensitizer, light of the appropriate band length can be irradiated on the medium to release the molecular labels in the buffer solution.

In any of the formats, if the conditions of the test reaction interfere with the separation technique employed, it may be necessary to remove or change the buffer solution of the test reaction before cleavage and separation of the molecular labels. For example, in some embodiments, the test conditions include salt concentrations (eg, necessary for specific binding) that degrade the separation performance when the molecular labels are separated on the basis of electrophoretic mobility. In these embodiments, an assay buffer solution is replaced by a separation buffer medium or solution, prior to the release and separation of the molecular labels.

Assays using releasable molecular tags and cleavage probes can be performed in multiple different configurations and formats, depending on the complexes that are detected or measured. Based on the present disclosure, a qualified person in the field will choose the appropriate design to select the quantities and specificities of cleavage probes and specific binding compounds.

In one aspect of the invention, the use of releasable molecular labels to measure dimers of cell surface membranes is shown in diagram form in Figs. 1A and 1B. Binding compounds (100) having molecular tags "mT1" and "mT2" and cleavage probe (102) with a "PS" photosensitizer are combined with biological cells (104). The binding compounds that have the molecular tag "mT1" are specific for the cell surface receptors R1 (106) and the binding compounds that have the molecular tag "mT2" are specific for the cell surface receptors R2 (108). The cell surface receptors R1 and R2 are present as monomers, for example (106) and (108), and as dimers (110) in the cell surface membrane (112). These test components are then incubated in a suitable binding buffer solution to allow the formation (114) of stable complexes between the binding compounds and their respective target receptors and between the cleavage probe and its target receptor. As illustrated, preferably both the binding compounds and the cleavage probes comprise an antibody binding composition, which allows the molecular tags and the cleavage-inducing fraction to be specifically directed to the membrane components. In one aspect, these antibody binding compositions are monoclonal antibodies. In these embodiments, the binding buffer solutions may comprise buffer solutions employed in conventional ELISA techniques or the like. After joining the compounds and cleavage probes for the stable compounds (116), the test mixture is illuminated (118) to induce the photosensitizers (120) to generate singlet oxygen. The singlet oxygen reacts rapidly with the components of the assay mixture, so that its effective proximity (122) to dodge the clivable bonds of the molecular tags is spatially limited so that only the molecular tags that are within proximity effective be released (124). As illustrated, the only molecular labels released are those of the binding compounds that form stable complexes with the dimers of R1-R2 and a cleavage probe. The released molecular labels (126) are removed from the test mixture and separated (128) according to a separation characteristic, so that a distinct peak (130) is formed in a separation profile (132). According to the invention, this withdrawal and separation can be the same step. Optionally, prior to illumination, the binding buffer solution can be removed and replaced with a buffer solution more suitable for separation, that is, a separation buffer solution. For example, binding buffer solutions typically have salt concentrations that can degrade the performance of some separation techniques, such as capillary electrophoresis, to separate molecular tags at different peaks. In one embodiment, this change of buffer solutions can be performed by membrane filtration.

An embodiment illustrating the ratiometric measurement of heterodimers is illustrated in Fig. 1C, in which an additional binding compound is used to give a measure of the total amount of protein (1104) in a sample. Reagents (1122) of the invention comprise (i) cleavage probes (1108), a first binding compound (1106) and a second binding compound (1107), wherein the first binding compound (1106) is specific for the Protein (1102) and a second binding compound (1107) is specific for protein (1104) to an antigenic determinant other than that for which the cleavage probe (1108) is specific. After reagent binding, the cleavage probe (1108) is activated to produce active species that shed the clivable bonds of the molecular labels within the effective proximity of the photosensitizer. In this embodiment, the molecular tags are released from the monomers of the protein (1104) that have both reagents (1107) and (1108) attached and from the heterodimers that have the reagent (1108) attached and some of the reagents or both ( 1106) and (1107) united. The released molecular tags (1123) are separated and the peaks (1118 and 1124) of a separation profile (1126) are correlated with the amounts of the released molecular tags. In this embodiment, the relative heights of the peaks, or areas, may reflect (i) the differences in the affinity of the first and second binding compounds with their respective antigenic determinants, and / or (ii) the presence or absence of the antigenic determinant for which the binding compound is specific. The second situation is important as long as a binding compound is used to control the posttranslational state of a protein, for example the phosphorylation state.

Homodimers can be measured as illustrated in Fig. 1D. As noted above, an assay may comprise three reagents (1128): cleavage probes (1134), a first binding compound (1130) and a second binding compound (1132). The first binding compound (1130) and the cleavage probe (1134) are structured to be specific for the same antigenic determinant (1135) of the protein (1138) that exists (1140) in a sample as homodimer (1136) or as monomer (1138). Once the 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 to 1150) are formed in the test mixture. Since the cleavage probe (1134) and the binding compound (1130) are specific for the same antigenic determinant (1135), four different combinations (1144 to 1150) of reagents can complex with homodimers. Of the complexes in the test sample, only those (1143) with both a cleavage probe (1134) and at least one binding compound will provide released molecular tags (1151) for separation and detection (1154). In this embodiment, the peak size (1153) is proportional to the amount of homodimer in the test mixture, while the peak size (1152) is proportional to the total amount of protein (1138) in the test sample, both in monomeric form (1142) and in homodimeric form (1146 and 1148). Fig. 1E illustrates analogous measurements for cell surface receptors that form heterodimers in the cell surface membrane (1161). A person qualified in the field would understand that dimers can be measured in cell or tissue lysates, or in fixed samples whose membranes have been permeabilized or removed through the fixation process. In these cases, the binding compounds may be specific for extracellular or intracellular domains of cell surface membrane receptors. As illustrated in Figs. 1E and 1F, releasable molecular tags can also be used for the simultaneous detection or measurement of multiple intracellular dimers and complexes in a cell sample. The cells (160), which may be from a sample of in vitro cultures or a sample of patient tissue, are lysed (172) to obtain accessible molecular complexes associated with the cell membrane and / or posttranslational modification sites, such as phosphorylation sites, within the cytoplasmic domains of membrane molecules. Subsequently, the resulting lysate (174) is combined with test reagents (176) that include multiple cleavage probes (175) and multiple binding compounds (177). Assay conditions (178) are selected which allow the reagents (176) to specifically bind their respective target, so that upon activation the clivable bonds within the effective proximity (180) of the cleavage inducing fractions are cloned and the molecular labels released (182). As noted above, after cleavage, the released molecular tags are separated (184) and identified in a separation profile (186), such as an electropherogram, and based on the number and amounts of molecular tags measured, a profile of selected molecular complexes in the sample cells.

Fig. 1G and 1H illustrate an embodiment of the invention for measuring receptor complexes in fixed or frozen tissue samples. The fixed tissue sample (1000), for example a sample embedded in paraffin and fixed with formalin, is cut to provide a section (1004) using a microtome or similar instrument and after placing it on a surface (1006), which can Being the microscope slide, it is deparaffinized and rehydrated for the application of the test reagents. The magnification (1007) shows the portion (1008) of the section (1004) on the portion (1014) of a microscope slide (1006). The molecules of the receptor dimer (1018) are illustrated as embedded in the residues of the membrane structure (1016) of the fixed sample. In accordance with this aspect of the invention, the cleavage probe and the binding components are incubated with the fixed sample so that they bind to their target molecules. For example, cleavage probes (1012) (illustrated in the figure as an antibody having a photosensitizer ("PS") attached) and a first binding compound (1010) (illustrated as an antibody having a molecular tag "mT1 »Bound) specifically bind to the receptor (1011) common to all dimers shown, a second binding compound (1017) (with« mT2 ») specifically binds to the receptor (1015), and a third binding compound (1019) (with "mT3") specifically binds to the receptor (1013). After washing to remove the binding compounds and the cleavage probe that are not specifically bound to their respective target molecules, buffer solution (1024) (called "illumination buffer solution" in the Figure) is added. For convenience, the buffer solution (1024) may be contained in section (1004) or a portion thereof, creating a hydrophobic barrier on the slide (1006), for example with a wax marker. After the illumination of the photosensitizers and the release of the molecular tags (1026), the buffer solution (1024) that now contains the released molecular tags (1025) is transferred to a separation device, such as a capillary electrophoresis instrument, to the separation (1028) and identification of the molecular labels released, for example, in an electropherogram (1030).

Measurements made directly on tissue samples, particularly as illustrated in Fig. 1G and 1H, can be normalized including measurements on cell or tissue targets that are representative of the total number of cells in the sample and / or the amounts of certain subtypes of cells in the sample. These tissue targets are referred to herein as "tissue indicators." Additional measurement may be preferable, or even necessary, due to cellular and tissue heterogeneity of patient samples, in particular tumor samples, which may comprise substantial fractions of normal cells. For example, in Fig. 1H, the values for the total amount of the receptor (1011) can be provided as a ratio of the following two measurements: the peak area (1032) of the molecular label ("mT1") and the area of a peak corresponding to a molecular tag correlated with a cellular or tissue component common to all cells in the sample, for example tubulin or the like. In some cases, when all the cells in the sample are epithelial cells, cytokeratin can be used. Accordingly, detection methods based on releasable molecular tags may include an additional step consisting of providing a binding compound (with a different molecular tag) specific for a normalization protein, such as tubulin.

Figures 2A-2E illustrate another embodiment of the invention for preparing a dimerization profile between a plurality of receptor types. Figure 2A explains the basic steps of this essay. Cell membranes

(200) to be tested for the presence of cell surface receptor dimers are combined with sets of binding compounds (202) and (204) and a cleavage probe (206). The membrane fractions (200) contain three different types of monomer receptor molecules ("1", "2" and "3") in their cell membrane, which are associated to form three different heterodimers: 1-2, 1 -3, and 2-3. Three antibody reagents (202) and (204) are combined with the membrane fraction (200), each of the antibody reagents having specificity for one of the three receptor molecules, where the antibody (206) is specific for the receptor molecule 1, the antibody (204) is specific for the receptor molecule 2 and the antibody (202) is specific for the receptor molecule 3. The antibody for the first receptor molecule is covalently bound with a photosensitizing molecule, labeled with PS. Antibodies to the second and third receptor molecules are linked to two different tags, labeled with T2 and T3, respectively, through a cleavage linkage through an active species generated by the photosensitizer fraction.

After mixing, the antibodies are allowed to bind (208) to the molecules on the surface of the membranes. The photosensitizer is activated (210), bypassing the link between the tags and the antibodies that are at an actionable distance from a sensitizing molecule, thereby releasing tags in the assay medium. The reaction material is separated (212) subsequently, for example by capillary electrophoresis, as illustrated. As shown in the lower part of Figure 2A, the T2 and T3 tags are released and separation by electrophoresis will reveal two bands corresponding to these tags. Since 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 test.

As shown in Fig. 2A, only two of the three different heterodimers that are present in the cell membrane will bind both a photosensitizer-containing antibody and a label-containing antibody, so only these two species would result in tags released. However, multiple experiments are required to measure the relative amounts of the different dimers. Fig. 2B provides a table that collects five different test combinations. The illustrative results for each test composition are found in Fig. 2C. Test I represents the results of the complete test, as described in Figure 2A. In Test II, the specific antibody for receptor molecule 1, which is bound to the photosensitizer, is suppressed. This test does not produce any signal, which indicates that the T2 and T3 signals obtained in Test I need the photosensitizer reagent. Also, Test V shows that the label signals need the presence of membranes. Tests III and IV show that each of the labeled reagents does not need the presence of the other to be clivated. These results, when analyzed together, allow conclusions to be drawn regarding the presence and composition of the receptor heterodimers present in the membrane, shown in Figure 2C, that is, that both heterodimer 1-2 and 1- 3 are present. On the other hand, the relative signal intensities of each tag allow estimating the relative abundance of each of the heterodimers.

However, a conclusion regarding the existing heterodimer 2-3 cannot be drawn with the combination of reagents used in this assay. No signal representing this complex will be obtained, regardless of whether it is present or not, because it will not have a photosensitizer reagent attached to it. In order to draw conclusions about each possible dimeric combination of the three monomers, it is necessary to use a fourth reagent that can be located in all possible oligomers that comprise monomers 1, 2 and / or 3, or join the three binding agents used in this experiment with labels and sensitizing molecules in different combinations. This last strategy is illustrated in Figures 2D and 2E. Three possible combinations of label distribution and photosensitizers among the three antibody reagents are listed in the table on the left of Figure 2D. The first combination comprises a photosensitizer bound to the specific antibody for monomer number 1 and is the same combination used in the illustration of Figure 2A-2C, and has the same population of dimers as in Figure 2C. The second combination comprises a photosensitizer bound to the specific antibody for monomer number 2 and the population profile produces the same number for heterodimer 1-2, plus a value for heterodimer 2-3. The third combination comprises a photosensitizer bound to the specific antibody for monomer number 3 and the population profile produces the same number for heterodimer 1-3 and 2-3 as that obtained from the first two combinations. These results can be combined to produce the profile of the total heterodimer population collected in Figure 2E.

A preferable embodiment for measuring the relative amounts of receptor dimers containing a common component receptor is illustrated in Fig. 2F. In the design of this assay, two different receptor dimers ("1-2"

(240) and "2-3" (250)), each having a common component "2", can be measured radiometrically with respect to the common component. An assay design for measuring the receptor heterodimer (240) comprising the "1" receptor (222) and the "2" receiver (220) and the receptor heterodimer (250) comprising the "2" receptor is shown ( 220) and receiver "3" (224). A main feature of this embodiment is that the cleavage probe

(227)

 it can be made specific for the common receptor of the heterodimer pair. The binding compound (228) specific for the "2" receptor provides a signal (234) related to the total amount of the "2" receptor in the assay, while the binding compound (226) specific for the "1" receptor and the binding compound (230) specific for the receiver "3" provide signals (232 and 236, respectively) related only to the amount of the receiver "1" and the receiver "3" present as heterodimers with the receiver "2", respectively. The design of Fig. 2F can be generalized to more than two receptor complexes containing a common component, simply by adding specific binding compounds to the components of the additional complexes.

TO.

 Binding compounds

As mentioned above, mixtures containing pluralities of different binding compounds may be provided, where each different binding compound has one or more molecular tags attached through cleavable bonds. The nature of the binding compound, the clivable bond and the molecular tag can vary greatly. 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 molecule or protein or to a stable complex formation with an analyte of interest, such as a protein complex. In one aspect, a binding compound, which can be represented by the following formula, comprises one or more molecular tags attached to a binding fraction.

B- (L-E) k

where B is the junction fraction; L is a clivable link; and E is a molecular tag. In homogeneous assays, the clivable bond L can be an oxidation labile bond and, more preferably, is a bond that can be cloned by singlet oxygen. The fraction "- (L-E) k" indicates that a single binding compound can have multiple molecular tags linked by clivable bonds. In one aspect, k is an integer greater than or equal to one, although in other embodiments, k may be greater than several hundred, for example 100 to 500, ok it may be several hundred to several thousand, for example 500 to 5,000 Typically, each of the pluralities of different types of binding compounds have a different molecular tag, E. Clivable bonds, for example oxidation labile bonds, and molecular tags E are attached to B by conventional chemical processes.

Preferably, B is an antibody binding composition that specifically binds to a target, as a predetermined antigenic determinant of a target protein, as a cell surface receptor. These compositions can be easily formed from a wide variety of commercially available antibodies, both monoclonal and polyclonal, specific for the proteins of interest. In particular, antibodies specific for epidermal growth factor receptors are disclosed in the following patents, which are incorporated herein by reference: 5,677,171; 5,772,997; 5,968,511; 5,480,968;

5,811,098. US Patent 6,488,390, incorporated herein by reference, discloses antibodies specific for a receptor bound to protein G, CCR4. US Patent 5,599,681, incorporated herein by reference, discloses antibodies specific for protein phosphorylation sites. Commercial distributors such as Cell Signaling Technology (Beverly, MA), Biosource International (Camarillo, CA), and Upstate (Charlottesville, VA) also provide specific monoclonal and polyclonal antibodies to many receptors.

The clivable link L can be virtually any chemical link group that can be cleaved under conditions that do not degrade the structure or affect the detection characteristics of the released molecular tag E. Whenever a cleavage probe is used in a format of In a homogeneous test, the cleavage link L is cloned by means of a cleavage agent generated by the cleavage probe that acts at a short distance, so that only the cleavage of the cleavable bonds is performed in the immediate proximity of the cleavage probe. Typically this agent must be activated by performing a physical or chemical variation in the reaction mixture, so that the agent produces an ephemeral active species that diffuses into a clivable bond to effect cleavage. In a homogeneous format, the cleavage agent preferably binds to a binding fraction, such as an antibody, directed before activation of the cleavage agent to a particular site in the vicinity of a binding compound with releasable molecular tags. In these embodiments, the cleavage agent is referred to herein as the "cleavage-inducing fraction", which is discussed in more detail below.

In a non-homogeneous format, since specifically bound binding compounds are separated from unbound binding compounds, a broader selection of cleavable bonds and cleavage agents can be employed. Clivable bonds can not only include bonds that are labile to the reaction with an active species that acts locally, such as hydrogen peroxide, singlet oxygen and the like, but also bonds that are labile to agents that operate throughout a mixture of reaction, such as labile bonds to the bases, photoclivable bonds, clivable bonds by reduction, oxidated clivated bonds, acid labile bonds, specific protease cleavable peptide bonds and the like. References describing many of these links include Greene and Wuts, Protective Groups in Organic Synthesis, Second Edition (John Wiley & Sons, New York, 1991); Hermanson, Bioconjugate Techniques (Academic Press, New York, 1996); and Still et al, U.S. Patent 5,565,324.

In one aspect, the commercially available clivable reagent systems can be used with the invention. For example, a disulfide bond can be introduced between an antibody-binding composition and a molecular tag, using a heterofunctional agent such as N-succinimidyl 3- (2-pyridyldithio) propionate (SPDP), succinimidyloxycarbonyl-a-methyl-a; - (2-Pyridyldithium) toluene (SMPT), or the like, offered by commercial distributors such as Pierce Chemical Company (Rockford, EL). The disulfide bonds introduced by these bonds can be broken by treatment with a reducing agent, such as dithiothreitol (DTT), dithioerythritol (DTE), 2-mercaptoethanol, sodium borohydride or the like. Typical concentrations of reducing agents for cleavage of disulfide bonds are in the range of 10 to 100 mM. An oxidation labile linkage can be introduced between an antibody binding composition and a molecular tag, using the cross-linking reagent of a homobifunctional NHS ester, disuccinimidyl tartarate (DST) (distributed by Pierce) containing susceptible cis diols upon cleavage with sodium periodate (for example 15 mM of periodate at a physiological pH for four hours). The linkages containing esterified separator components can be cleaved with strong nucleophilic agents, such as hydroxylamine, for example 0.1 N hydroxylamine, pH 8.5, for 36 hours at 37 ° C. These separators can be introduced by a homobifunctional cross linking agent such as ethylene bis (succinimidylsuccinate) (EGS) glycol marketed by Pierce (Rockford, IL). A labile bond can be introduced to the bases with a sulfone group. Homobifunctional cross-linking agents that can be used to introduce sulfone groups into a clivable bond include bis [2 (succinimidyloxycarbonyloxy) ethyl] sulfone (BSOCOES), and 4,4-difluoro-3,3-dinitrophenylsulfone (DFDNPS) . Some examples of basic conditions for cleavage include 0.1 M sodium phosphate, adjusted to pH 11.6 by the addition of Tris base, containing 6 M urea, 0.1% SDS, and 2 mM DTT, with incubation at 37 ° C for two hours. The photoclivable links include those disclosed in Rothschild et al, U.S. Patent No.

5,986,076.

When L is oxidation labile, L can be a trioether or its selenium analog; or an olefin, which contains double carbon-carbon bonds, where cleavage of a double bond with an oxo group releases the molecular tag E. Some examples of oxidation labile bonds are disclosed in Singh et al, US Patent No. 6,627. 400; and in the US Patent Publications Singh et al, 2002/0013126; and 2003/0170915, and in Willner et al, US Patent No. 5,622,929, all of which are incorporated herein by reference.

The molecular tag E of the present invention may comprise an electrophonic tag, as described in the following references, when the separation of the pluralities of molecular tags is performed by gas chromatography or mass spectrometry: Zhang et al, Bioconjugate Chem., 13: 1002-1012 (2002); Giese, Anal. Chem., 2: 165-168 (19S3); and U.S. Patent Nos. 4,650,750; 5,360,819; 5,516,931; 5,602,273; and the like

The molecular tag E is preferably a water soluble organic compound that is stable with respect to active species, especially singlet oxygen, and that includes a detection group or indicator. On the other hand, E can be very variable in terms of size and structure. In one aspect, E has a molecular weight in the range of about 50 to about 2500 daltons, more preferably about 50 to about 1500 daltons. Preferable structures of E are described in more detail below. E may comprise a detection group to generate an electrochemical, fluorescent or chromogenic signal. In embodiments that employ mass detection, E may not have a separate fraction for the purpose of detection. 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. In one aspect, the characteristic of chromatographic or electrophoretic separation is the retention time in a set of separation conditions standard in the art, for example voltage, column pressure, column type, mobile phase, electrophoretic separation medium and the like. . In another aspect, the optical property is a fluorescence property, such as the emission spectrum, the duration of the fluorescence, the intensity of the fluorescence at a certain wavelength or band of wavelengths and the like. Preferably, the fluorescence property is the intensity of the fluorescence. For example, each molecular tag of a plurality may have the same fluorescent emission properties, but each will differ from the others by virtue of a unique retention time. On the other hand, two or more molecular tags of a plurality may have identical retention or migration times, but will have unique fluorescent properties, for example spectrally resolvable emission spectra, so that all members of the plurality can be distinguished by combination of molecular separation with fluorescence measurement.

Preferably, the released molecular tags are detected by electrophoretic separation and fluorescence of a detection group. In these embodiments, molecular tags that have substantially identical fluorescence properties have different electrophoretic mobilities, such that different spikes form in an electropherogram under separation conditions. Preferably, the pluralities of molecular tags of the invention are separated by conventional capillary electrophoresis apparatus, either in the presence or absence of a conventional screening matrix. Some examples of capillary electrophoresis appliances include Applied Biosystems (Foster City, CA) models 310, 3100 and 3700; Beckman (Fullerton, CA) model P / ACE MDQ; Amersham Biosciences (Sunnyvale, CA) MegaBACE 1000 or 4000; the SpectruMedix genetic analysis system; and the like Electrophoretic mobility is proportional to q / M2 / 3, where q is the charge on the molecule and M is the mass of the molecule. Preferably, the difference in mobility in the conditions of the determination between the nearest electrophoretic tags will be at least 0.001, usually 0.002, more usually at least 0.01, and even about 0.02 or more.

Preferably, in this conventional apparatus, the electrophoretic mobilities of the molecular tags of a plurality differ by at least one percent and, more preferably, at least by a percentage ranging from 1-10 percent. Molecular labels are identified and quantified by analyzing a separation profile or, more specifically, an electropherogram and these values are correlated with the amounts and types of receptor dimers present in a sample. For example, during or after an electrophoretic separation, molecular tags are detected or identified by recording the fluorescence signals and migration times (or migration distances) of the separated compounds, or by making a graph of the relative fluorescence and the order of migration of molecular labels (for example, as an electropherogram). Preferably, the presence, absence and / or amounts of molecular labels are measured using one or more standards disclosed by Williams et al, US Patent Publication 2003 / 0170734A1, which is incorporated herein by reference.

The pluralities of molecular labels may also be designed for separation by chromatography, based on one or more physical characteristics, including, but not limited to, molecular weight, shape, solubility, pKa, hydrophobicity, charge, polarity or similar, for example, as disclosed in US Patent Publication 2003/0235832, which is incorporated herein by reference. A chromatographic separation technique is selected based on parameters such as the type of column, the solid phase, the mobile phase, etc. and then a plurality of molecular labels is selected that can be separated to form different peaks or bands in a single operation. Several factors determine the HPLC technique selected for use in the invention, including the number of molecular tags to be detected (ie, the size of the plurality), the estimated amounts of each molecular tag that will be generated in the tests, the availability and ease of synthesis of molecular labels that are candidates for the set that will be used in multiplexed tests, the detection modality used and the availability, strength, cost and ease of handling of HPLC instrumentation, columns and solvents. In general, appropriate columns and techniques are preferred for analyzing limited quantities of sample and providing maximum resolution separations. Guidance for making these selections can be found in the literature, for example in Snyder et al, Practical HPLC Method Development, (John Wiley & Sons, New York, 1988); Millner, "High Resolution Chromatography: A Practical Approach", Oxford University Press, New York (1999), Chi-San Wu, "Column Handbook for Size Exclusion Chromatography", Academic Press, San Diego (1999), and Oliver, "HPLC of Macromolecules: A Practical Approach, Oxford University Press », Oxford, England (1989). In particular, there are procedures available for the systematic development and optimization of certain conditions of chromatographic separations, such as column type, solid phase and the like, for example Haber et al, J. Chromatogr. Sci., 38: 386-392 (2000); Outinen et al, Eur. J. Pharm. Sci., 6: 197-205 (1998); Lewis et al, J. Chromatogr., 592: 183-195 and 197-208 (1992); etc. An example of an HPLC instrumentation system suitable for use with the present invention is the Agilent 1100 Series HPLC system (Agilent Technologies, Palo Alto, CA).

In one aspect, the molecular tag E is (M, D), where M is a mobility modifying fraction and D is a detection fraction. The notation "(M, D)" is used to indicate that the order of fractions M and D may be such that any of the fractions may be adjacent to the cleavage link L. That is, "BL- (M, D) »Designates the binding compound in either of two ways:« BLMD »or« BLDM ».

The detection fraction D may be a fluorescent dye or tag, a chromogenic dye or tag, an electrochemical tag, etc. Preferably D is a fluorescent dye. Some examples of fluorescent dyes for use with the invention include water-soluble rhodamine dyes, fluoresceins, 4,7-dichlorofluoresceins, benzoxanthene dyes and energy transfer dyes, disclosed in the following references: Handbook of Molecular Probes and Research Reagents , 8th ed., (Molecular Probes, Eugene, 2002); Lee et al, U.S. Patent 6,191,278; Lee et al, U.S. Patent 6,372,907; Menchen et al, US Patent 6,096,723; Lee et al, U.S. Patent 5,945,526; Lee et al, Nucleic Acids Research, 25: 2816-2822 (1997); Hobb, Jr., U.S. Patent 4,997,928; Khanna et al, U.S. Patent 4,318,846; etc. Preferably, D is a fluorescein or a fluorescein derivative.

In an embodiment illustrated in Fig. 3A, the binding compounds comprise a biotinylated antibody (300) as the binding fraction. Molecular labels are attached to the junction fraction (300) by means of a bridge

(306) streptavidin or avidin. Preferably, in practice, the binding fraction (300) is first reacted with a target complex, after which streptavidin or avidin (304) is added to form an antibody-biotin-avidin complex (305). To these complexes (305) are added (308) biotinylated molecular tags (310) to form binding compound (312).

In another embodiment more illustrated in Fig. 3B, the binding compounds comprise an antibody (314) derivatized with a multifunctional fraction (316) containing multiple functional groups (318) that are precursors of the molecular labels that have been reacted (320) to obtain a final binding compound that has multiple molecular tags (322) attached. Some examples of multifunctional fractions include aminodextran and similar materials.

Once each of the binding compounds is derivatized separately by a different molecular tag, it is grouped with other binding compounds to form a plurality of binding compounds. Typically, each class of different binding compound is present in a composition in the same proportion; however, the proportions can be varied depending on the design choice, so that a binding compound or a subset of concrete binding compounds are present in greater or lesser proportion, depending on the suitability or the requirements for a given embodiment. or essay. Factors that may affect this design choice include, by way of example only, the affinity of the antibody and its avidity for a specific target, the relative prevalence of a target, the fluorescent characteristics of a detection fraction of a molecular tag, etc. .

B. Active species that produce the cleavage induction fraction

A cleavage-inducing fraction, or a cleavage agent, is a group that produces an active species capable of cloning a clivable bond, preferably by oxidation. Preferably, the active species is a chemical species that has an ephemeral activity, so that its cleavage-inducing effects occur only in the vicinity of the place where it was generated. Either the active species is inherently ephemeral, so as not to generate a significant background noise beyond the proximity of its creation, or a scrubber is used that efficiently purifies the active species, so that they are not available to react with Clivable links beyond a short distance from where they were generated. Some examples of active species include singlet oxygen, hydrogen peroxide, NADH and hydroxyl radicals, phenoxy radical, superoxide, etc. Some examples of neutralizers for active species that cause oxidation include polyenes, carotenoids, vitamin E, vitamin C, N-conjugates amino acid-pyrrole tyrosine, histidine, and glutathione, and the like, for example Beutner et al, Meth. Enzymol., 319: 226-241 (2000).

An important consideration in designing assays that employ a clivating inducer fraction and a clivable link is that they cannot be so far apart from each other, when they bind to a receptor complex, that the active species generated by the cleavage inducing fraction cannot clivate efficiently the cleavage link. In one aspect, the clivable bonds are preferably less than 1000 nm, and preferably 20-200 nm, of an inducing fraction of the linked cleavage. More preferably, for the inductive fractions of photosensitizer cleavage that generate singlet oxygen, the clivable bonds are located about 20-100 nm from a photosensitizer in a receptor complex. The range in which a cleavage-inducing fraction can effectively clivate a clivable bond (that is, clivar enough molecular tag to generate a detectable signal) is referred to herein as "effective proximity." An expert in the field recognizes that the effective proximity of a specific sensitizer may depend on the details of a particular test design and may be determined.

or be modified by routine experimentation.

A sensitizer is a compound that can be induced to generate a reactive intermediate, or a species, usually singlet oxygen. Preferably, a sensitizer used in accordance with the invention is a photosensitizer. Other sensitizers included in the scope of the invention are compounds that, upon excitation by heat, light, ionizing radiation or chemical activation, will release a singlet oxygen molecule. The best known members of this class of compounds include endoperoxides, such as 1,4biscarboxyethyl-1,4-naphthalene endoperoxide, 9,10-diphenylantracen-9,10-endoperoxide and 5,6,11,12-tetraphenyl naphthalene 5, 12-endoperoxide. The heating or direct absorption of light by these compounds releases singlet oxygen. Other sensitizers are disclosed in the following references: Di Mascio et al, FEBS Lett., 355: 287 (1994) (peroxidases and oxygenases); Kanofsky, J.Biol. Chem. 258: 5991-5993 (1983) (lactoperoxidase); Pierlot et al, Meth. Enzymol., 319: 3-20 (2000) (thermal lysis of endoperoxides); etc. The binding of a binding agent to the cleavage-inducing fraction can be direct or indirect, covalent or non-covalent and can be performed by well known techniques, usually available in the literature. See, for example, "Immobilized Enzymes," Ichiro Chibata, Halsted Press, New York (1978); Cuatrecasas, J. Biol. Chem., 245: 3059 (1970).

As mentioned above, the preferable cleavage fraction in accordance with the present invention is a photosensitizer that produces singlet oxygen. For the purposes of the present, a "photosensitizer" is a molecule that absorbs light and that when activated by light converts molecular oxygen into singlet oxygen. The photosensitizers can be linked directly or indirectly, through covalent or non-covalent bonds, to the binding agent of a specific class reagent. There are guidelines for making these compositions, particularly for antibodies as binding agents, available in the literature, such as, for example, in the fields of photodynamic therapy, immunodiagnostic, etc. Some examples of references are the following: Ullman, et al, Proc. Natl Acad. Sci. USA 91, 5426-5430 (1994); Strong et al, Ann. New York Acad. Sci., 745: 297-320 (1994); Yarmush et al, Crit. Rev. Therapeutic Drug Carrier Syst, 10: 197-252 (1993); Pease et al, U.S. Patent 5,709,994; Ullman et al, U.S. Patent 5,340,716; Ullman et al, US Patent 6,251,581; McCapra, U.S. Patent 5,516,636; etc.

There is a wide variety of light sources available to photoactivate photosensitizers that generate singlet oxygen. Both monochromatic and polychromatic sources can be used, as long as the source is intense enough to produce enough singlet oxygen in a practical period of time. The length of the irrigation depends on the nature of the photosensitizer, the nature of the clivable bond, the power of the irradiation source and its distance from the sample, etc. In general, the period for irradiation can be from less than about a microsecond to about 10 minutes, usually in an approximate range of one millisecond to about 60 seconds. The intensity and length of the irradiation should be sufficient to excite at least about 0.1% of the photosensitizer molecules, usually at least 30% of the photosensitizer molecules and preferably almost all photosensitizer molecules. Some examples of light sources include, but are not limited to, lasers, such as helium-neon lasers, argon lasers, YAG lasers, He / Cd lasers and ruby lasers; photodiodes; mercury, sodium and xenon vapor lamps; incandescent lamps, such as tungsten and tungsten / halogen lamps; flash lamps; etc. As an example, a photoactivation device disclosed in Bjomson et al, International Patent Publication WO 03/051669 is used. Briefly said, the photoactivation device is a beam of light emitting diodes (LEDs) mounted in a housing that allows simultaneous illumination of all the wells in a 96-well plate. An LED suitable for use in the present invention is a high power GaAIAs IR emitter, such as the OD-880W model manufactured by OPTO DIODE CORP. (Newbury Park, CA).

Some examples of photosensitizers that can be used in the present invention are those that have the aforementioned properties and are included in the following references: Singh and Ullman, US Patent 5,536,834; Li et al, US Patent 5,763,602; Martin et al, Methods Enzymol., 186: 635-645 (1990); Yarmush et al, Crit. Rev. Therapeutic Drug Carrier Syst, 10: 197-252 (1993); Pease et al, U.S. Patent 5,709,994; Ullman et al, U.S. Patent 5,340,716; Ullman et al, US Patent 6,251,581; McCapra, U.S. Patent 5,516,636; Thetford, European Patent Publication 0484027; Sessler et al, SPIE, 1426: 318-329 (1991); Magda et al, U.S. Patent 5,565,552; Roelant, US Patent 6,001,673; etc.

As in the case of sensitizers, in certain embodiments a photosensitizer may be associated with a solid phase support, being connected by a covalent or non-covalent bond to the surface of the support or incorporated into the body of the support. In general, the photosensitizer is associated with the support in an amount necessary to achieve the necessary amount of singlet oxygen. Generally, the amount of photosensitizer is determined empirically.

In one embodiment, a photosensitizer is incorporated into a latex particle to form photosensitizing beads, for example as disclosed in Pease et al., U.S. Patent 5,709,994; Pollner, US Patent 6,346,384; and Pease et al, PCT Publication WO 01/84157. Alternatively, the photosensitizer beads can be prepared by bonding a photosensitizer, such as rose flare, to 0.5 micron latex beads, by chloromethyl groups on the latex to provide an ester bonding group, such as described in J. Amer. Chem. Soc, 97: 3741 (1975). The use of photosensitizing beads is illustrated in Fig. 3C. As described in Fig. 1C for the detection of heteroduplex, complexes (330) are formed after combining reagents (1122) with a sample. This reaction can be carried out, for example, in a conventional 96 or 384 well microtiter plate, or the like, having a filter membrane that forms a wall, for example, in the lower part of the wells, which allows removal Reagents by applying vacuum. This allows the buffer solutions to be changed comfortably, if the buffer solution necessary for the specific binding of binding compounds is different from that necessary for the generation of singlet oxygen or for separation. For example, in the case of antibody-based binding compounds, a buffer solution with a high salt content is needed. If the electrophoretic separation of the released labels is used, then a better result is achieved by changing the buffer solution to one with a lower salt concentration for electrophoresis. In this embodiment, instead of attaching a photosensitizer directly to a binding compound, such as an antibody, a cleavage probe comprises two components: antibody (332) derivatized with a capture fraction, such as biotin (indicated in Fig. 3C as "Bio") and a photosensitizing pearl (338) whose surface is derivatized with an agent (334) that specifically binds to the capture fraction, such as avidin or streptavidin. The complexes (330) are then captured (335) by the photosensitizing beads through the capture fraction, such as biotin (336). Preferably, if the pore diameter of the filter membrane is selected so that the sensitizing beads (338) cannot pass, then the change of buffer solution also serves to remove unbound binding compounds, which results in a signal improved Once an appropriate buffer solution for separation has been added, if necessary, the photosensitizing beads (338) are illuminated, in order to generate singlet oxygen (342) and release the molecular tags (344). These released molecular tags (346) are subsequently separated to form the separation profile (352) and the dimers are quantified radiometrically from the peaks (348) and (350). Photosensitizing pearls can be used in homogeneous and heterogeneous assay formats.

Preferably, when analytes, such as cell surface receptors, or antigens are to be detected in a fixed sample, the cleavage probe may comprise a primary haptenized antibody and a secondary anti-hapten binding protein derivatized with multiple photosensitizing molecules. A preferable primary haptenized antibody is a biotinylated antibody and a preferable secondary secondary hapten binding protein can be an anti-biotin or streptavidin antibody. Other combinations of these primary and secondary assets are well known in the field, for example Haugland, Handbook of Fluorescent Probes and Research Reagents, 9th edition (Molecular Probes, Eugene, OR, 2002). An example of a combination of these reagents is illustrated in Fig. 3E. The binding compounds (366 and 368) having releasable labels ("mT1" and "mT2" in the Figure) and the primary antibody (368) biotin derivatized (369) are specifically bound to different epitopes of the receptor dimer (362 ) in the membrane (360). Biotin-specific binding protein (370), for example, streptavidin, is bound to biotin (369) carrying multiple photosensitizers (372) in close proximity to binding compounds (366 and 368). The biotin-specific binding protein (370) can also be an anti-biotin antibody and the photosensitizers can be linked through a free amino group in the protein, by conventional chemical coupling processes, as in Hermanson (cited above). An example of a photosensitizer for this use is an NHS methylene blue ester prepared as disclosed in Shimadzu et al, European Patent Publication 0510688.

Test conditions

The following general discussion of specific methods and conditions and materials are offered for illustrative purposes only and without limitation. An expert in the field will understand how the methods described herein can be adapted to other applications, particularly with the use of different samples, cell types and target complexes.

To perform the methods of the invention, a combination of the test components, including the sample to be tested, the binding compounds and, optionally, the cleavage probe is performed. Usually, the test components can be combined in any order. However, in certain applications the order in which they are added may be important. For example, it can be tried to control the competitive union, as in a quantitative trial. Or you can try to control the stability of a complex formed. In these applications the reactions can be carried out in phases and may require incubations to be able to achieve the complete mixture or to initiate the cleavage reaction.

Generally, the amounts of each reagent are determined empirically. The amount of sample used in an assay shall be determined by the expected number of target complexes present and by the separation and detection means used to control the assay signal. In general, the amounts of the binding compounds and the cleavage probe are provided in molar excess with respect to the expected amount of the target molecules in the sample, usually at a molar excess of at least 1.5, more preferably at a excess of 10 or higher. In specific applications, the concentration employed may be higher or lower, depending on the affinity of the binding agents and the expected number of target molecules present in a single cell. When it is desired to determine the effect of a chemical compound on a formation of oligomeric cell surface complexes, the compound can be added to the cells before adding the probes, simultaneously or after adding them, depending on the effect that is pretend to control

The assay mixture is combined and incubated under conditions that allow the binding of the probes to the cell surface molecules, usually in an aqueous medium, usually at a physiological pH (comparable to the pH at which the cells are grown), maintained by a buffer solution at a concentration in a range of about 10 to 200 mM. Conventional buffer solutions can be used, in addition to other necessary conventional additives, such as salts, culture medium, stabilizers, etc. Physiological and constant temperatures are used normally. Typically, incubation temperatures range from about 4 ° C to 70 ° C, usually between about 15 ° C to 45 ° C, more usually from 25 ° C to 37 ° C. After preparation of the assay mixture and incubation to allow the probes to bind to cell surface molecules, the mixture is treated to activate the cleavage agent to cleanse the labels of the binding compounds that are within the effective proximity of the cleavage agent, releasing the corresponding label from the cell surface in the solution. The nature of this treatment will depend on the mechanism of action of the cleavage agent. For example, when a photosensitizer is used as a cleavage agent, activation of the cleavage will comprise irradiation of the mixture at the wavelength of the light appropriate to the particular sensitizer employed.

After cleavage, the probe is analyzed to determine the identity of the labels that have been released. When an assay using a plurality of binding compounds is used, the separation of the released tags will generally precede their detection. The methods for both separation and detection are determined during the design of the labels for the assay. A preferable mode of separation employs electrophoresis, in which the various tags are separated based on known differences in their electrophoretic mobilities.

As mentioned above, in some embodiments, if the conditions of the test reaction may interfere with the separation technique employed, it may be necessary to remove or change the buffer solution of the test reaction before cleavage and label separation. molecular. For example, test conditions may include salt concentrations (for example, necessary for specific binding) that degrade separation performance when molecular tags are separated based on electrophoretic mobility. In this way, buffer solutions with a high salt content can be removed, for example before the cleavage of the molecular labels, and replaced by other buffer solutions suitable for electrophoretic separation through filtration, aspiration, dilution or other means. .

Examples

Any example that does not fall within the scope of the invention is offered exclusively for the purpose of comparison.

Sources of materials used in the examples

Specific antibodies to Her receptors, adapter molecules and standardization standards are obtained from commercial distributors, including Labvision, Cell Signaling Technology, and BD Biosciences. All cell lines were purchased from ATCC. All frozen human tissue samples were instantly purchased from William Bainbridge Genome Foundation (Seattle, WA) or Bio Research Support (Boca Raton, FL) were approved by the provider's Institutional Research Council (IRB).

The molecular-antibody tag conjugates employed below are formed by reacting NHS esters of the molecular tag with a free amine of the indicated antibody using conventional procedures. Molecular labels, identified below by their designation "Pro_N" are disclosed in the following references: Singh et al, US Patent Publications 2003/017915 and 2002/0013126, which are incorporated herein by reference. Briefly said, the following binding compounds are monoclonal antibody-molecular tag conjugates formed by reacting an NHS ester of a molecular tag with free amines of the antibodies in a conventional reaction.

Example 1

Analysis of cell lysates for Her-2 heterodimerization and receptor phosphorylation

In this example, Her1-Her2 and Her2-Her3 heterodimers and phosphorylation states are measured in cell lysates of various cell lines after treatment with various concentrations of epidermal growth factor (EGF) and heregulin (HRG). Measurements are made using three binding compounds and a cleavage probe, as described below.

Sample preparation:

one.

Culture of a serum-deprived breast cancer cell line overnight, before use.

2.

Stimulation of cell lines with EGF and / or HRG in culture media for 10 minutes at 37 ° C. Some examples of doses of EGF / HRG are 0, 0.032, 0.16, 0.8, 4, 20, 100 nM for all cell lines (for example, MCF-7, T47D, SKBR-3) except BT20 in the maximum dose is increased up to 500 nM because saturation is not achieved with 100 nM EGF.

3.

Aspiration of the culture media, transfer to ice and addition of the lysis buffer solution for lysing the cells in situ.

Four.

Scraping and transfer of the lysate to the microfuge tube. Incubation on ice for 30 min. Microfuge at 14,000 rpm, 4 ° C, for 10 min. (Spinning is optional.)

5.

Collection of supernatants as lysates and aliquots for storage at -80 ° C until use.

Test.

Assay design: As illustrated in diagram form in Fig. 4A, Her2-Her3 heterodimers (900) are ratiometrically quantified based on the binding of the cleavage probe (902) and the binding compounds (904) , (906), and (908). A photosensitizer indicated by "PS" binds to the cleavage probe (902) through an avidin-biotin bond and the binding compounds (904), (906), and (908) are labeled with the molecular labels Prol4 , Pro10, and Pro 11, respectively. The binding compound (904) is specific for a phosphorylation site in Her3.

The total volume of the test is 40 ul. The volume of the lysate is adjusted to 30 ul with the lysis buffer solution. The antibodies are diluted in the lysis buffer solution up to 10ul. Typically between ~ 5000 and 15000 equivalents are used in lysate cells per reaction. The detection limit is ~ 1000 equivalents in lysate cells.

Procedure: The final concentrations of the binding compounds before mixing (i.e. molecular label or biotin-antibody conjugates) in the reaction:

Pro4_anti-Her-2: 0.1 ug / ml

Pro10_anti-Her-1: 0.05-0.1 ug / ml

Pro11_anti-Her-3: 0.1 ug / ml

Pro2_anti-phospho-Tir: 0.1 ug / ml

Biotin_anti-Her-2: 1-2 ug / ml

one.

To test 96 wells, add 10 ul of antibody mixture to 30 ul of lysate and incubate for one hour at RT.

2.

Add 2 ul of streptavidin derivatized cleavage probe (final 2 ug / well) to test the well and incubate for 45 minutes.

3.

Add 150 ul of PBS with 1% BSA to the 96-well filter plate (Millipore MAGVN2250) and incubate for one hour at RT to block.

Four.

Empty the filter plate by vacuum suction. Transfer the test reactions to the filter plate and apply vacuum to empty.

5.

Add 200 ul of wash buffer solution and apply vacuum to empty. Repeat once.

6.

Add 200 ul of lighting buffer solution and apply vacuum to empty. Repeat once.

7.

Add 30 ul of lighting buffer solution and illuminate for 20 min.

8.

Transfer 10 ul of each reaction to the EC test plate for analysis, using an ABI13100 CE instrument with a 22 cm capillary (injection conditions: 5 kV, 75 sec, 30 ° C; conditions of

operation: 600 sec, 30 ° C). The assay buffer solutions are as follows:

5 Lysis buffer solution (freshly made and stored on ice) Final ul Reserva 1% Triton X-100 1000 10% 20 mM Tris-HCI (pH 7.5) 200 1 M 100 mMNaCl 200 5M 50 mM NaF 500 1 M 50 mM Na beta-glycerophosphate 1000 0.5 M 1 mM Na3V04 100 0.1 M 5 mM EDTA 100 0.5 M 10 ug / ml pepstatin 100 1 mg / ml 1 tablet (per 10 ml) of Roche Complete protease inhibitor N / AN /TO

(# 1836170) Water 6500 N / A 10 ml Total

Wash buffer solution (stored at 4 ° C) Final ml Reserve 1% NP-40 50 10% 1xPBS 50 10x 150 mM NaCl 15 5M 5 mM water with EDTA 5 0.5 M Water 380 N / A

500 ml Total

Lighting buffer solution:

Final ul Reserve

0.005x PBS 50 1x

CE std 3 100x 10 mM Tris-HCl (pH 8.0) 0.1M 10pMA160 1nM 10pMA315 1nM

10pMHABA 1nM

 Water 10,000 N / A

10 ml Total

Analysis of data:

one.

Normalize the signal of the relative fluorescence units (RFU) of each molecular tag against the EC A315 reference standard (a fluorescein derivatized deoxyadenosine monophosphate having a known peak position with respect to the molecular tags of the test after electrophoretic separation) .

2.

Subtract the RFU from the "non-lysed" background control of the corresponding molecular label signals.

3.

Report heterodimerization for Her-1 or Her-3 as the corresponding RFU ratiometric for Pro4_anti-Her-2 RFU from the test wells using biotin-anti-Her-2.

Four.

Report phosphorylation of the Her-1,2,3 receptor as RFU of the ratiometric of Pro2_PT100 anti-phospho-Tir for RFU of Pro4_anti-Her-2 from the test wells using biotin-anti-Her-2.

The test results are illustrated in Fig. 4B-4H. Fig. 4B shows the amount of increases in Her1-Her2 heterodimers in MCF-7 cells with an increase in EGF concentration, while the amount of the same dimer basically shows no variation with an increase in HRG concentration. . Fig. 4 shows the opposite result for Her2-Her3 heterodimers. That is, the amount of Her2-Her3 heterodimers increases in MCF-7 cells with an increase in HRG concentrations, while the amount of the same dimer basically shows no change with an increase in EGF concentration.

Fig. 4D and 4E show the amount of increases in Her1-Her2 heterodimers in SKBR-3 cells and BT-20 cells, respectively, with an increase in EGF concentrations.

Example 2 Analysis of tissue lysates for Her2 heterodimerization and receptor phosphorylation

In this example, the heterodimers of Her1-Her2 and Her2-Her3 and phosphorylation states are measured in tissue lysates of human breast cancer samples.

Sample preparation:

one.

Instantly frozen tissues are mechanically altered in a frozen state by cutting.

2.

Transfer tissues to a microfuge tube and add 3x volumes of lysis buffer tissue (from Appendix I). Next, mix with vortex shaker to disperse the tissues in the buffer solution.

3.

Incubate on ice for 30 min., Stirring with vortex to mix.

Four.

Centrifuge at 14,000 rpm, 4 ° C, for 20 min.

5.

Collect supernatants and lysates and determine the total protein concentration with a BCA (Pierce) assay using a small aliquot.

6.

Divide the rest into aliquots for storage at -80 ° C until use.

Trial Design:

one.

The total volume of the test is 40 ul.

2.

Lysates are tested in titration series of 40, 20, 10, 5, 2.5, 1.25, 0.63, 0.31 ug of total equivalents and the volume is adjusted to 30 ul with the lysis buffer solution . Titration series data confirm the specificity of dimerization or phosphorylation signals.

3.

A universal antibody mixture comprising all eTag-antibodies diluted in lysis buffer solution is used at the following concentrations.

Four.

Individual biotin-antibody is added for each receptor separately to the reactions.

5.

Three eTag assays are performed with each tissue lysate, each using a different biotin-antibody, corresponding to the dimerization of the specific receptor to be measured.

6.

The level of expression of each receptor is determined from the different assays that contain specific receptor biotinantibody.

7.

The dimerization and phosphorylation signals are determined ratiometrically only in the test containing

Biotin-anti-Her-2.

Assay Controls: The MCF-10A and MCF-7 cell lines are used as negative qualitative controls and

positive, respectively. Cell lines are stimulated or not stimulated with 100 nM EGF or 100 nM

of HRG. The lysis buffer solution is included as background control when tissue samples are replaced.

Final concentrations of antibodies before mixing in the reactions:

Universal antibody mixture: Pro4_anti-Her-2: 0.1 ug / ml Prol0_anti-Her-1: 0.05 ug / ml Pro11_anti-Her-3: 0.1 ug / ml Pro2_anti-phospho-Tir: 0.01 ug / ml

Individual biotin antibody: Biotin_anti-Her-1: 2 ug / ml Biotin_anti-Her-2: 2 ug / ml Biotin_anti-Her-3: 2 ug / ml

Process:

one.

Prepare the antibody reaction mixture, adding biotin antibody to the universal antibody mixture.

2.

To test 96 wells, add 10 ul of universal reaction mixture to 30 ul of lysate and incubate for one hour at RT.

3.

Add 2 ul of streptavidin derivatized cleavage probe (final 2 ug / well) to test the well and incubate for 45 minutes.

Four.

Add 150 ul of PBS with 1% BSA to the 96-well filter plate (Millipore MAGVN2250) and incubate for one hour at RT to block.

5.

Empty the filter plate by vacuum suction. Transfer the test reactions to the filter plate and apply vacuum to empty.

6.

Add 200 ul of wash buffer solution and apply vacuum to empty. Repeat once.

7.

Add 200 ul of lighting buffer solution and apply vacuum to empty. Repeat once.

8.

Add 30 ul of lighting buffer solution and illuminate for 20 min.

9.

Transfer 10 ul of each reaction to the EC test plate for analysis, using an ABI3100 capillary electrophoresis instrument with a 22 cm capillary (injection conditions: 5 kV, 75 sec, 30 ° C; operating conditions: 600 sec, 30 ° C).

Analysis of data:

one.

Normalize the RFU signal of each molecular tag against the CE A315 reference standard.

2.

Determine the RFU cut-off values (each for dimerization or phosphorylation) below which the ratios are not calculated, because the signals are too low to be reliable. Below the cut-off values, the RFU signals cannot be titrated in the series of dilutions of lysate tested. Values can be determined with a broad set of normal tissues in which dimerization and phosphorylation signals are expected to be absent or kept at minimum levels. These values also represent the basal level of dimerization or phosphorylation on normal tissues with which the tumor tissues will be compared.

3.

For the minority of normal tissues, if present, with RFU values above the cutoff level, determine the level of individual RFU and the ratiometric readings of the Her-1 or Her-3 heterodimerization or phosphorylation peaks detected. These samples represent atypical cases that should be used as corresponding donor controls for the respective tumor tissue samples during scoring.

Four.

For all tumor samples showing RFU signals susceptible to titration, use the lowest signal of each Her-1, Her-2, Her-3, or phosphorylation of the tissue lysate titration series as background. Subtract this background from the molecular label signals of high dose lysates (eg, 40 ug) to produce the specific RFU signals. If there is no response of the dose of the signal in the titration series, all signals (which are usually very low) are considered background, without any specific signal being used for ratiometric analysis.

5.

Report heterodimerization for Her-1 or Her-3 as the corresponding specific RFU ratiometric for Pro4_anti-Her-2 specific RFUs. If no specific RFU is obtained, dimerization is negative.

6.

Report the phosphorylation of the receptor for Her-1,2,3 as the ratiometric specific RFUs of Pro2_anti-phospho-Tir for the specific RFUs of Pro4_anti-Her-2. If no specific RFU is obtained, the phosphorylation is negative.

In Fig. 5A-5C, the data shown are representative of multiple breast tissue samples from patients tested with trials of the invention. The clinical status of Her-2 immunohistochemistry (DAKO Herceptest) of 9 out of 10 tumor samples was negative, indicating an undetectable Her-2 staining, or a staining of less than 10% of the tumor cells, or an imperceptible or barely perceptible staining on part of the cell membrane of more than 10% of the tumor cells. The assays of the invention determined the expression of Her-1, Her-2 and Her-3 in both normal and tumor tissues. Heterodimerization of Her1 and Her2 and Her2 and Her3 was detected only in tumor tissue, but not in any normal tissue.

Example 3

Analysis of cell lysates for homodimerization of Her1 and Her2 and receptor phosphorylation

Sample preparation was basically performed as described in Example 2. Homodimerization of Her1 was induced by treating cell lines with EGF or TGFa. For homodimerization of Her2 that does not have a ligand, non-stimulated SKBR-3 or MDA-MD-453 cells that overexpress Her2 are compared to non-stimulated MCF-7 cells that express a low level of Her2.

Assay design: A monoclonal antibody specific for the receptor is conjugated separately with a molecular tag or with biotin (which is subsequently attached to a photosensitizer via an avidin bridge), so that the cleavage probe and binding compound compete for joining the same epitope in this example. Another binding compound consisting of a second antibody that recognizes an overlapping epitope at the receptor is used, so that a ratiometric signal can be generated as a measure of homodimerization. The signal obtained from the second antibody also provides a measure of the total amount of receptor in a sample. The total amount of receptor is determined in a separate test well. Phosphorylation of the receptor can be quantified together with homodimerization or with the total amount of the receptor.

Procedure: The test volume is 40 ul and the general procedure is similar to that in Example 2. Two test wells, A and B, are prepared for each sample, in order to quantify the homodimerization and the total amount of the receptor separately. .

For the quantification of Her1-Her1 homodimers:

Final concentrations in the antibody mixture in test well A:

Prol2_anti-Her-l: 0.05-0.1 ug / ml

Biotin_anti-Her-1: 1-2 ug / ml

Final concentrations in the antibody mixture in test well B: Pro10_anti-Her-1: 0.05-0.1 ug / ml Pro2_anti-phospho-Tir: 0.1 ug / ml Biotin_anti-Her-l: 1- 2 ug / ml

For the quantification of Her2-Her2 homodimers: Final concentrations in the antibody mixture in test well A:

Pro4_anti-Her-1: 0.05-0.1 ug / ml

Biotin_anti-Her-1: 1-2 ug / ml

Final concentrations in the antibody mixture in test well B:

Pro4_anti-Her-1: 0.05-0.1 ug / ml Pro2_anti-phospho-Tir: 0.1 ug / ml Biotin_anti-Her-1: 1-2 ug / ml

Analysis of data:

one.

Normalize the RFU signal of each molecular tag against the CE A315 reference standard.

2.

Subtract the RFU from the "non-lysed" background control of the corresponding molecular label signals.

3.

Report homodimerization for Her-1 or Her-2 as the corresponding standardized RFUs of test well A as the ratiometric for standardized RFUs of the total amount of the receptor of the corresponding test well B.

Four.

Report phosphorylation of the receptor for the homodimer of Her-1 or Her-2 as the standardized RFUs of Pro2_PT100 anti-phospho-Tir of test well B as the ratiometric for standardized RFUs of the total amount of receptor of the same test well B.

The test results are illustrated in Fig. 6A-6B and Fig. 7. Fig. 6A shows that the amount of Her1-Her1 homodimers in BT-20 cells increases with a higher concentration of EGF. Fig. 6B shows that the amount of phosphorylation of Her1 in BT-20 cells increases with a higher concentration of EGF. The detection of Her2-Her2 homodimers was demonstrated by comparing signals from SKBR-3 cells expressing Her2 with signals from MCF-7 cells expressing a reduced level of Her2 on the cell surface. As shown in the diagrams of Fig. 7, no signal of the Her2-Her2 homodimer capable of specific titration was detected in the MCF-7 cells, while the signals of the Her2-Her2 homodimer in SKBR-3 cells they were clearly above the signals of the MCF-7 cells.

Example 4 Analysis of cell lysates for heterodimerization of Her1-Her3 and receptor phosphorylation

Samples are prepared as follows:

one.

Culture of a serum-deprived breast cancer cell line overnight, before use.

2.

Stimulation of cell lines with HRG in culture media for 10 minutes at 37 ° C. Some examples of doses of HRG are 0.032, 0.16, 0.8, 4, 20, 100 nM for T47D cells.

3.

Aspiration of the culture media, transfer to ice and addition of the lysis buffer solution for lysing the cells in situ.

Four.

Scraping and transfer of the lysate to the microfuge tube. Incubation on ice for 30 min. Microfuge to

14,000 rpm, 4 ° C, for 10 min. (Spinning is optional.)

5.

Collection of supernatants as lysates and aliquot division for storage at -80ºC up to

the utilization. Test design: The total volume of the test is 40 ul. The volume of the lysate is adjusted to 30 ul with the lysis buffer solution. The antibodies are diluted in the lysis buffer solution to 5 ul. Typically between ~ 5000 and 50,000 equivalents are used in lysate cells per reaction. Final concentrations of antibodies before mixing in the reaction:

Pro10_anti-Her-1: 0.05-0.1 ug / ml Proll_anti-Her-3: 0.1 ug / ml Pro2_anti-phospho-Tir: 0.1 ug / ml Biotin_anti-Her-3: 1-2 ug / ml

one.

To test 96 wells, add 5 ul of antibody mixture to 30 ul of lysate and incubate for one hour in RT.

2.

Add 5 ul of streptavidin derivatized molecular scissors (final 4 ug / well) to test the well and incubate for 45 minutes.

3.

Add 150 ul of PBS with 1% BSA to the 96-well filter plate (Millipore MAGVN2250) and incubate for one hour in RT to block.

Four.

Empty the filter plate by vacuum suction. Transfer the test reactions to the filter plate and apply vacuum to empty.

5.

Add 200 ul of wash buffer solution and apply vacuum to empty. Repeat once.

6.

Add 200 ul of lighting buffer solution and apply vacuum to empty. Repeat once.

7.

Add 30 ul of lighting buffer solution and illuminate for 20 min.

8.

Transfer 10 ul of each reaction to the EC test plate for analysis, using an ABI3100 capillary electrophoresis instrument with a 22 cm capillary (injection conditions: 5 kV, 425 sec, 30 ° C; operating conditions: 600 sec, 30 ° C).

Analysis of data:

one.

Normalize the RFU signal of each eTag indicator against the CE A315 reference standard.

2.

Subtract the background control RFUs "not lysed" from the corresponding eTag indicator signals.

3.

Report heterodimerization as the ratiometric RFU of Pro 10 derived from Her-1 versus RFUs of Prol 1 from anti-Her-3.

Four.

Report phosphorylation of the Her-1/3 receptor as RFU of the ratiometric of Pro2_PT100 anti-phospho-Tir for RFU of Pro 1 l_anti-Her-3 of the test wells using biotin-anti-Her-3.

The test results are illustrated in Fig. 8A and SB. The data shows that both Her-Her3 heterodimerization and dimer phosphorylation increase with higher concentrations of HRG.

Example 5

Increased expression of the Her-Her2 receptor dimer in cancer cell lines in response to increased epidermal growth factor

In this example, Her1-Her3 heterodimers are measured in cell lysates of the 22Rvl and A549 cancer cell lines after treatment with various concentrations of epidermal growth factor (EGF). Measurements are made using three binding compounds and a cleavage probe, as described below.

Sample preparation:

one.

Culture of a serum-deprived breast cancer cell line overnight, before use.

2.

Stimulation of cell lines with EGF in culture media for 10 minutes at 37 ° C. Some examples of doses of EGF applied to both cell lines ranged from 0-100 nM.

3.

Aspiration of the culture media, transfer to ice and addition of the lysis buffer solution for lysing the cells in situ.

Four.

Scraping and transfer of the lysate to the microfuge tube. Incubation on ice for 30 min. Microfuge to

14,000 rpm, 4 ° C, for 10 min. (Centrifugation is optional.) Determination of protein concentration. 5. Collection of supernatants as lysates and division into aliquots for storage at 80 ° C until use.

The test design is basically the same as that illustrated in Fig. 4A, with the following exceptions: the binding compounds (904), (906) and (908) are labeled with the molecular labels Prol0, Prol 1 and Pro 2 respectively. The total volume of the test is 40 ul. The volume of the lysate is adjusted to 30 ul with the lysis buffer solution. The antibodies are diluted in the lysis buffer solution to 5 ul. Typically between ~ 5000 and 15,000 equivalents are used in lysate cells per reaction. The detection limit is ~ 1000 equivalents in lysate cells. Procedure: The final concentrations of the binding compounds before mixing (i.e. molecular label or biotin-antibody conjugates) in the reaction:

Pro10_anti-Her-1: 0.05-0.1 ug / ml

Proll_anti-Her-3: 0.1 ug / ml

Pro2_anti-phospho-Tir: 0.1 to 0.2 ug / ml

Biotin_anti-Her-3: 1-2 ug / ml

one.

To test 96 wells, add 5 ul of antibody mixture to 30 ul of lysate and incubate for one hour in RT.

2.

Add 5 ul of streptavidin derivatized cleavage probe (final 4 ug / well) to test the well and incubate for 45 minutes.

3.

Add 150 ul of PBS with 1% BSA to the 96-well filter plate (Millipore MAGVN2250) and incubate for one hour in RT to block.

Four.

Empty the filter plate by vacuum suction. Transfer the test reactions to the filter plate and apply vacuum to empty.

5.

Add 200 ul of wash buffer solution and apply vacuum to empty. Repeat once.

6.

Add 200 ul of lighting buffer solution and apply vacuum to empty. Repeat once.

7.

Add 30 ul of lighting buffer solution and illuminate for 20 min.

8.

Transfer 10 ul of each reaction to the EC test plate for analysis, using an AB13100 CE instrument with a 22 cm capillary (injection conditions: 5 kV, 70 sec, 30 ° C; operating conditions: 425 sec, 30 ° C).

 The test buffer solutions are as follows: Lysis buffer solution (freshly made and stored on ice) Final ul Reserve 1% Triton X-l00 1000 10%

20 mM Tris-HCl (pH 7.5) 500 1 M 100 mM NaCl 200 5M 50 mM NaF 500 1M 50 mM Na beta-glycerophosphate 500 1.0M 1 mM Na3VO4 100 0.1 M 5 mM EDTA 100 0.5 M 10 ug / ml pepstatin 100 1 mg / ml 1 tablet (per 10 ml) of Roche Complete protease inhibitor N / AN / A

(# 1836170) Water 7 ml N / A 10 ml Total

Wash buffer solution (stored at 4 ° C): 0.5% Triton X100 in lx PBS. Lighting buffer solution: Final ul Reserva 0.005x PBS 50 lx CE std 1 (A27, ACLARA Biosciences, Inc., Mountain View, CA) 4 5000x CE std 2 (fluorescein) 4 5000x Water 9942 N / A

10 ml Total

Analysis of data:

one.

Normalize the signal of relative fluorescence units (RFU) of each molecular tag against reference standard 2.

2.

Subtract the RFU from the "non-lysed" background control of the corresponding molecular label signals.

3.

Report heterodimerization for Her-1 as the corresponding RFU ratiometric for Prol l_anti-Her-3 RFU from the test wells using biotin-anti-Her-3.

Four.

Report the phosphorylation of the receptor for Her-1,2,3 as RFU of the ratiometric of Pro2_PT100 anti-phospho-Tir for RFU of Prol l_anti-Her-3 of the test wells using biotin-anti-Her-3 shown).

Figures 9A and 9B show the increases in the amounts of Her1-Her3 heterodimers on 22Rvl and A549 cells, respectively, with the increase in EGF concentrations.

Example 6 Emergence of IGF-1R heterodimers with Her1, Her2, and Her3 in the lysates of tumor breast tissue

In this example, cells from 12 different human tumor breast tissues were tested for the presence of Her1-IGF-IR, Her2-IGF-1R, and Her3-IGF-1R dimers, using basically the same assays as those illustrated in Fig. 4A. Sample preparation was performed as follows:

one.

Instantly frozen tissues are mechanically altered in a frozen state by cutting.

2.

Transfer tissues to a microfuge tube and add 3x volumes of lysis buffer tissue. Next, mix with vortex shaker to disperse the tissues in the buffer solution.

3.

Incubate on ice for 30 min., Stirring with vortex to mix.

Four.

Centrifuge at 14,000 rpm, 4 ° C, for 20 min.

5.

Collect supernatants and lysates and determine the total protein concentration with a BCA (Pierce) assay using a small aliquot.

6.

Divide the rest into aliquots for storage at -80 ° C until use.

The trial was prepared as follows:

one.

 The total volume of the test is 40 ul.

2.

Lysates are tested in titration series of 40, 20, 10, 5, 2.5, 1.25, 0.63, 0.31 ug of total equivalents and the volume is adjusted to 30 ul with the lysis buffer solution . The data of the titration series confirm the specificity of the dimerization.

3.

A mixture of universal antibody comprising all binding compounds and biotin antibody diluted in a lysis buffer solution is used, at the concentrations indicated below.

Final antibody concentrations before mixing in the reactions: Pro10_anti-Her-2: 0.1 ug / ml Pro14_anti-Her-1: 0.1 ug / ml Prol 1_ anti-Her-3: 0.1 ug / ml Pro7_anti- IGF-lR: 0.1 ug / ml Pro2_anti-phospho-Tir: 0.2 ug / ml Biotin_anti-Her-2: 2 ug / ml

Process:

one.

To test 96 wells, add 5 ul of universal reaction mixture to 30 ul of lysate and incubate for one hour in RT.

2.

Add 5 ul of streptavidin derivatized molecular scissors, ie the cleavage probe (final 4 ug / well), to test the well and incubate for 45 minutes.

3.

Add 150 ul of PBS with 1% BSA to the 96-well filter plate (Millipore MAGVN2250) and incubate for one hour in RT to block.

Four.

Empty the filter plate by vacuum suction. Transfer the test reactions to the filter plate and apply vacuum to empty.

5.

Add 200 ul of wash buffer solution and apply vacuum to empty. Repeat once.

6.

Add 200 ul of lighting buffer solution and apply vacuum to empty. Repeat once.

7.

Add 30 ul of lighting buffer solution and illuminate for 20 min.

8.

Transfer 10 ul of each reaction to the EC test plate for analysis, using: (i) EC equipment: ABI3100, 22 cm capillary, (ii) EC injection conditions: 5 kV, 70 sec, 30 ° C, and (iii) EC operating conditions: 425 sec, 30 ° C

Analysis of data:

one.

Normalize the RFU signal of each molecular tag against the CE 1 reference standard.

2.

Look for signals that can be titrated for each molecular tag. It is understood that signals that are not susceptible to titration are non-specific signals and are not used for the interpretation of the data. A cut-off value is determined based on the values of a broad set of normal tissues in which dimerization signals are expected to be absent or kept at minimum levels. These values also represent the basal level of dimerization on normal tissues with which tumor tissues are compared.

3. Heterodimerization for IGF-1R with Her-1 or Her-2 or Her-3 is reported as the corresponding specific RFUs.

Two of the 12 breast tumors tested expressed heterodimers of Her1-IGF-1R, Her2-IGF-1R, and Her3-IGF-1R, as shown in Fig. 10A-C. The lines in the graph of each Figure show the trend between the amount of heterodimer of the measured receptor and the amount of lysate tested for the two breast tumor samples that obtained positive results for the indicated heterodimers.

Example 7 P13K / Her-3 receptor activation complex

In this example, the assays were designed as shown in Figs. 11A and 11C to measure a receptor complex comprising Her2, Her3, and PI3K in the breast cancer cell line, MCF-7. The binding compound (1106) having a first molecular tag ("mT1" in the Figure and "eTag1" below) is specific for the extracellular domain of the Her3 receptor (1102); the binding compound (1110) having a second molecular tag ("mT2" in the Figure and "eTag2" below) is specific for component p185 (1111) of the PI3K protein (1100); and the cleavage probe (1108) having a attached photosensitizer is specific for the intracellular domain of the Her3 receptor (1102), where "H2" indicates a Her2 receptor (1104), "H3" indicates a Her3 receptor (1102), " p85 »and« p110 »are components of PI3-kinase (1100), which binds to a phosphorylation site of H3 (denoted by« P ») through its p85 fraction. The two test designs are similar, except that in the design of Fig. 11A the cleavage probe is specific for the Her3 receptor and in the design of Fig. 11C the cleavage probe is specific for the p85 component of PI3- kinase The tests were performed as follows:

Sample preparation:

one.

 Culture of a serum-deprived breast cancer cell line overnight, before use.

2.

 Stimulation of cell lines with HRG in culture media for 10 minutes at 37 ° C. Some examples of doses of HRG are 0.032, 0.16, 0.8, 4, 20, 100 nM for MCF-7 cells.

3.

Aspiration of the culture media, transfer to ice and addition of the lysis buffer solution (described above) for lysing the cells in situ.

Four.

 Scraping and transfer of the lysate to the microfuge tube. Incubation on ice for 30 min. Microfuge at 14,000 rpm, 4 ° C, for 10 min.

5.

 Collection of supernatants as lysates and aliquots for storage at -80 ° C until use.

Lysis buffer solution (freshly made and stored on ice):

 Final ul Reserva 1% Triton Xl 00 1000 10% 20 mM Tris-HCl (pH 7.5) 200 1 M 100 mMNaCl 200 5M 50 mM NaF 500 1M 50 mM Na beta-glycerophosphate 1000 0.5 M 1 mM Na3VO4 100 0.1 M 5 mM EDTA 100 0.5 M 10 ug / ml pepstatin 100 1 mg / ml 1 tablet (per 10 ml) of Roche Complete protease inhibitor N / AN / A

(# 1836170) Water 6500 N / A 10 ml Total

Assay Design: The formation of the receptor complex is quantified ratiometrically based on the schemes illustrated in each Figure. That is, the reading of the tests are the ratios of the molecular label peaks, eTag2 / eTagl.

The total volume of the test is 40 ul. The volume of the lysate is adjusted to 10 ul with the lysis buffer solution. The antibodies are diluted in the lysis buffer solution up to 20 ul. Typically between ~ 5000 and

500,000 equivalents in lysate cells per reaction. Procedure: Operational concentrations of the premixed antibodies before adding them to the reaction: To

the Her-3 / PI3K complex with cleavage probe in Her-3 (the design of Fig. 11 A) eTagl_anti-Her-3 at 10 nM (eTagl was Pro14 in this test) eTag2_anti-PI3K at 10 nM (eTag2 was Pro1 in this test) Biotin_anti-Her-3 at 20 nM Universal Standard US-1 at 700 nM

[The Universal Standard US-1 is biotin-conjugated BSA and the Pro8 molecular tag, which is used to normalize the amount of streptavidin photosensitizer beads in one assay]. Molecular tags bound directly to the antibodies, reacting an NHS ester of a molecular tag precursor with free amines of the antibodies, using conventional techniques, see for example Hermanson (cited above).

For the Her-3 / PI3K complex with PI3K cleavage probe (the design of Fig. 11 C): eTagl_anti-PI3K at 10 nM (eTagl was Prol in this assay) eTag2_anti-Her-3 at 10 nM (eTag2 was Pro 14 in this test) Biotin anti-PI3K at 20 nM

Universal Standard US-1 at 700 nM

9.

 To test a 96-well filter plate (Millipore MAGVN2250), add 20 ul of antibody mixture to 10 ul of lysate and incubate for one hour at 4 ° C.

10.

 Add 10 ul of streptavidin derivatized cleavage probe (final 4 ug / well) to test the well and incubate for 40 minutes.

eleven.

 Add 200 ul of wash buffer solution and apply vacuum to empty.

12.

 Add 30 ul of lighting buffer solution and illuminate.

13.

 Transfer 10 ul of each reaction to the EC test plate for analysis. Analysis of data:

one.

Normalize the signal of relative fluorescence units (RFU) of each molecular label against that of the internal Universal Standard US-1.

2.

Subtract the background control RFUs "not lysed" from the corresponding standardized eTag indicator signals.

3.

Report the formation of the receptor complex as the ratiometric of the normalized eTag2 / eTagI signal (as shown in Fig. 1 IB and 1 ID).

Example 8 Interaction of the Shc / Her-3 adapter-receiver

In this example, the tests were designed as shown in Figs. 12A and 12C. In Fig. 12A, the Her2 receptor (1200) and the Her3 receptor (1202) form a dimer in the cell surface membrane (1204) and each receptor is represented as having phosphorylated sites (1209 and 1210). Proteins (1206 and 1208) bind to phosphorylation sites (1210) and (1209), respectively. A first binding compound (1214) and the cleavage probe (1216) are specific for different antigenic determinants of the extracellular domain of the Her 2 receptor (1200). A second binding compound (1212) is specific for Shc proteins (1206 and 1208). The test designs of Fig. 12A and 12C are similar, except that in the design of Fig. 12A the cleavage probe is specific for the Her2 receptor and in the design of Fig. 12C the cleavage probe is specific for the Her3 receiver. Therefore, in the first case, the total Her2 receptor is measured, while in the second case the total Her3 receptor is measured. The tests were performed as follows: sample preparation was performed as before (Example 7).

Assay Design: The formation of the receptor complex is quantified ratiometrically based on the schemes illustrated in each Figure. That is, in Fig. 12B and 12D the reading of the tests are the ratios of the mT2 / mTi peaks as a function of the concentration of HRG.

The total volume of the test is 40 ul. The volume of the lysate is adjusted to 10 ul with the lysis buffer solution. The antibodies are diluted in the lysis buffer solution up to 20 ul. Typically they are used between ~ 5000 and

500,000 equivalents in lysate cells per reaction. Procedure: Operational concentrations of the premixed antibodies before adding them to the reaction: For the Her-3 / Shc complex with cleavage probe in Her-3 (the design of Fig. 12B)

eTag1_anti-Her-3 at 10 nM (eTagl was Prol4 in this test)

eTag2_anti-Shc at 10 nM (eTag2 was Prol2 in this test)

eTag3_anti-phospho-Tir at 10 nM (eTag3 was Pro2 in this test)

Biotin_anti-Her-3 at 20 nM

Universal Standard US-1 at 700 nM For the Her-2 / Shc complex with the Her-2 cleavage probe (the design in Fig. 12A):

eTagl_anti-Her-2 at 10 nM (eTagl was Prol4 in this test)

eTag2_anti-Shc at 10 nM (eTag2 was Pro 12 in this test)

eTag3_anti-phospho-Tir at 10 nM (eTag3 was Pro2 in this test)

Biotin_anti-Her-2 at 20 nM

Universal Standard US-1 at 700 nM

one.

To test a 96-well filter plate (Millipore MAGVN2250), add 20 ul of antibody mixture to 10 ul of lysate and incubate for one hour at 4 ° C.

2.

Add 10 ul of streptavidin derivatized cleavage probe (final 4 ug / well) to test the well and incubate for 40 minutes.

3.

Add 200 ul of wash buffer solution and apply vacuum to empty.

Four.

Add 30 ul of lighting buffer solution and illuminate.

5.

Transfer 10 ul of each reaction to the EC test plate for analysis.

Analysis of data:

one.

Normalize the signal of relative fluorescence units (RFU) of each molecular label against that of the internal Universal Standard US-1.

2.

Subtract the RFU from the "non-lysed" background control of the corresponding signals for the standardized molecular labels.

3.

Report the formation of the receptor complex as the ratiometric of the standardized mT2 / mT1 signals (as shown in Figs. 12B and 12D) and the phosphorylation of the receptor (data not shown) as mT3 / mT1 signals.

Example 9

Correlation between Her2-Her3 heterodimer measurements and Her3-PI3K complex measurements in breast tumor samples

In this example, human breast tumor samples were tested separately, using the methods described above to determine the amounts of Her2-Her3 heterodimers and the amounts of the Her3-PI3K complex. Fig. 13 illustrates the data obtained from these tests, which show that the two measurements are correlated.

Example 10

Expression of Her1-Her2 and Her2-Her3 heterodimers in breast tumor lysates and normal tissue lysates

Normal tissue samples and frozen human breast tumor tissue samples were obtained from the William Bainbridge Genomic Foundation (Bainbridge Island, WA). Assays with a format such as that shown in Fig. 3E were performed with 32 samples of tumor tissue and 30 samples of normal tissue. Tumor tissues were composed of a mixture of tumor and normal cells that ranged between 25 and more than 90 percent, according to the pathological data supplied with the tissues by the distributor. Samples were prepared and assays were performed basically as described for Examples 2 and 6. The data is reported as the intensity or peak area of the separated molecular tag released from the binding compound specifically bound to the receptor opposite to the cleavage probe, ie the molecular tag corresponding to "mT1" in Fig. 3E. No attempt was made to normalize the signals generated based on the percentage of tumor cells in the sample.

The data of these measurements are shown in Fig. 14A (Her1-Her2 heterodimer measurements) and in Fig. 14B (Her2-Her3 heterodimer measurements), where the white squares (0) indicate measurements on tumor tissues and black diamonds (+) indicate measurements on normal tissues. The data shows that tumor cells in important fractions of tumor tissue samples express large amounts of Her1-Her2 heterodimers and Her2-Her3 heterodimers compared to those expressed in normal tissue sample cells.

Example 11

Measurement of receptor dimers in tissue samples embedded in paraffin and fixed with formalin

In this example, fixed tissue models made of pelletized cell lines were tested for the presence of Her receptor dimers. The assay design for the heterodimers was basically the same as that described in Fig. 4A, with the exceptions indicated below. That is, four components are used: (i) a cleavage probe comprising a biotinylated monoclonal antibody conjugated to a cleavage-inducing fraction (in this example, a streptavidin derivatized from the photosensitizer, as illustrated in Fig. 3E) and specific for one of the dimer receptors, (ii) a derivatized monoclonal antibody with a first molecular tag and specific for the same receptor as the cleavage probe, (iii) a derivatized monoclonal antibody with a second molecular tag and specific for the receptor opposite to that for which the cleavage probe is specific, and (iv) a derivatized monoclonal antibody with a third molecular tag and specific for an intracellular phosphorylated tyrosine. The test design for homodimers was basically the same as that described in Fig. 1D, with the exceptions indicated below.

In each case, the fixed tissue models were prepared as follows: the cells grown in culture plates were stimulated with EGF or HRG, as described in the previous examples. They were then washed and removed by scraping. The removed cells were centrifuged to form a pearl, after which formalin was added and the mixture was incubated until the next day at 4 ° C. The fixed pearl was embedded in paraffin, using a Miles Tissue Tek HI Embedding Center, after which 10 um tissue sections of the pearl were cut using a microtome (Leica model 2145). Tissue sections were placed on microscope positively charged glass slides (usually multiple sections of tissue per slide) and baked for one hour at 60 ° C.

The tissue sections of the slides were tested as follows: The tissue sections of a slide were dewaxed with the EZ-Dewax reagent (Biogenex, San Ramon, CA) following the protocol recommended by the manufacturer. Briefly said, 500 µL of EZ-Dewax was added to each tissue section and the sections were incubated at RT for five minutes, after which they were washed with 70% EtOH. This step was repeated and the slide was finally rinsed with deionized water, after which the slide was incubated in RT water for 20 min. The slide was then immersed in a 1X Antigen Retrieval solution (Biogenesis, Brentwood, NH) at pH 10, after which it was heated for 15 minutes in a microwave oven (5 min at maximum power, followed by 10 minutes at low power ). After cooling in RT (about 45 minutes), the slide was immersed in a water bath for 5 minutes and then dried. The tissue sections on the dry slide were surrounded with a hydrophobic wax marker to create regions capable of containing reagents placed on the tissue sections (as illustrated in Fig. 1H-1I), after which it was washed the slide three times in IX Perm / Wash (BD Biosciences). 50-100 µL of blocking buffer solution was added to each section and the slide was placed in a humidified covered box containing deionized water for two hours at 4 ° C. Next, the blocking buffer solution of each section was removed by suction. (The blocking buffer solution is IX Perm / Wash with protease inhibitors (Roche), phosphatase inhibitors (sodium fluoride, sodium vanadate, (3-glycerol phosphate), and 10% mouse serum). each 40-50 μL section of the antibody mixture containing binding compounds and the cleavage probe (5 μg / mL each, except for biotin-Ab5 (anti-Her1) at 10 μg / mL in the Her1 assay -Her2), and the slide was placed in a humidified box until the next day at 4 ° C. The sections were then washed three times with 100 μL Perm / Wash containing phosphatase and protease inhibitors, after which 50 μL of photosensitizer in solution IX Perm / Wash (containing phosphatase and protease inhibitors) The slide was then incubated for an hour or hour and a half at 4 ° C in the dark in a humidified box, after which the photosensitizer was removed by suction, keeping the slide in the dark ity While remaining in the dark, the slide was then immersed in .01X PBS and incubated on ice for one hour. The slide was removed from the PBS, dried and 40-50 μL of 0.0IX PBS with 2 pM fluorescein was added to each section, after which it was illuminated with a high-power laser diode (GaAIAs JJR emitter, model OD- 880W, OPTO DIODE CORF, Newbury Park, CA) for one hour. Fluorescein acts as a standard to help correlate the peaks of an electropherogram with molecular tags. After illumination, the solution covering each section of tissue was mixed by gently shaking and transferred to an EC plate for analysis in an Applied Biosystems capillary electrophoresis instrument (Foster City, CA) model 3100.

Fig. 15A shows the data of the analysis of the homodimers of Her1-Her1 and the phosphorylation of the receptor in sections of the fixed beads of the breast adenocarcinoma cell line, MDA-MB-468 (ATCC accession number: HTB- 132), prepared from unstimulated cells or cells stimulated with 100 μM EGF. The biotinylated anti-Her1 monoclonal antibody (Labvision) at 2 μg / mL was used as the primary antibody of the cleavage probe (for the cleavage of methylene blue derivatized streptavidin (previously described) was bound through biotin). The anti-Her1 monoclonal antibody derivatized from Pro10 (Labvision) at 2 ug / mL was used to measure homodimerized Her1. The anti-Her1 monoclonal antibody derivatized from Pro1 (Labvision) at 0.8 ug / mL was used to measure total Her1. The unlabeled antibody Ab-5 was also included in the reactions at 3.2 μg / mL. Pro2 derivatized monoclonal antibody (anti-phosphorylated-Tir, Cell Signaling) at 0.5 μg / mL was used to measure intracellular phosphorylation. The data of the fixed tissue measurements confirm and are consistent with the measurements on cell lysates, which show increases in the expression of the Her-Her1 homodimer and intracellular phosphorylation due to EGF stimulation.

Fig. 15B shows the data of the Her2-Her2 homodimer analysis and the receptor phosphorylation in fixed pearl sections of the MCF-7 and SKBR-3 breast cancer cell lines. All monoclonal antibodies used as cleavage probes or binding compounds were used at concentrations of 5 μg / mL. In order to generate a better cleavage, two cleavage probes were used in this assay: one directed towards an extracellular Her2 antigenic determinant and the other directed towards an Her2 intracellular antigenic determinant. The fixed tissue measurement data confirm that SKBR3 cells express higher levels of Her2-Her2 homodimers than MCF-7 cells.

Fig. 15C shows the analysis data of Her1-Her2 heterodimers and receptor phosphorylation in sections of the fixed beads of the breast adenocarcinoma cell line, MCF-7, prepared from unstimulated cells or of cells stimulated with 40 nM of EGF. Two cleavage probes were used: one comprising the anti-Her1 monoclonal antibody (at 5 μg / mL) and the other comprising the monoclonal antibody (at 10 μg / mL) (both from Lab vision), in order to increase the release rate of molecular tags. The data shows that increases in Her1-Her2 heterodimer expression caused by EGF stimulation are detected in the fixed tissue.

Fig. 15D shows the data of the analysis of Her1-Her2 heterodimers and receptor phosphorylation in sections of the fixed pearls of the breast adenocarcinoma cell line, 22Rv1, prepared from unstimulated cells or cells stimulated with 100 nM of EGF. Once again, measurements on fixed tissues demonstrate an upward regulation of Her1-Her2 dimers and phosphorylation of the Her receptor, in response to EGF treatment.

Fig. 15E shows data from the analysis of Her2-Her3 heterodimers and receptor phosphorylation in sections of fixed beads of the breast adenocarcinoma cell line, MCF-7, prepared from

5 cells not stimulated or cells stimulated with 40 μM HRG. In this example, the binding reactions and cleavage reactions took place in tubes containing sections, instead of microscope slides. For the rest, the protocol was basically the same as that used for the detection of Her1-Her2 dimers. The data shows that increases in Her2-Her3 heterodimer expression caused by stimulation with HRG are detected in the fixed tissue.

10 Figure 15F shows the analysis data of Her2-Her3 heterodimers and PI3K-Her3 dimers in fixed bead sections of MCF-7 cells, both unstimulated and stimulated with 40 μM HRG. The test design for PI3K-Her3 was basically like the one described in Fig. 11A. The previous fixation protocol was followed in both cases, except that none of the samples was treated with reagents for antigen recovery. The data shows that Her2-Her3 dimers increased with treatment with

15 HRG, but that the amount of PI3K-Her3 dimer remained basically unchanged.

Fig 15G shows the data of the analysis of total PI3K, the dimer of Her2-Her3 total, and Her3 total, all of them with respect to the amount of tubulin. Tubulin was measured in a conventional sandwich test, using a cleavage probe and a binding compound with a molecular tag. Tubulin was measured to test procedures to normalize the measurement of the dimer with respect to a target representative of the number of

20 total cells of a sample, which may be necessary for measurements on samples with heterogeneous cell types. The data show that the ratios of PI3K-Her3 and Her2-Her3 with respect to tubulin are qualitatively the same as in measurements made directly on PI3K-Her3 and Her2-Her3.

Claims (13)

1. A method for determining the cancer status of a patient suffering from cancer characterized by aberrant expression of one or more ErbB cell surface receptor complexes, the method comprising the following steps: measure directly in a sample of the patient an amount of one or more complexes of ErbB cell surface receptors; comparing the amount of one or more ErbB cell surface receptor complexes with the corresponding amount of one or more ErbB cell surface receptor complexes in a reference sample; Y correlate differences in the amount of one or more ErbB cell surface receptor complexes of the patient sample and the corresponding corresponding amount of one or more ErbB cell surface receptor complexes of the reference sample to determine the cancer status of the patient. patient, where the ErbB cell surface receptor complexes or complexes are selected from the group consisting of Her1-Her1 homodimers, Her2-Her2 homodimers, Her1-Her3 receptor dimers, Her1-Her4 receptor dimers, Her2-Her4 receptor dimers , Her3-Her4 receptor dimers, Her4-Her4 receptor homodimers, Her1-PI3K complexes, Her2-PI3K complexes, Her3-PI3K complexes, Her1-SHC complexes, Her2-SHC complexes, Her3-SHC complexes , Her1-IGF-IR receptor dimers, Her2-IGF-IR receptor dimers, Her3-IGF-IR receptor dimers, Her1-PDGFR receptor dimers, Her2-PDGFR receptor dimers, Her3-PDGFR receptor dimers, dimers of the p95Her2-Her3 receptor, dimers of the p95Her2-Her2 receptor, dimers of the p95Her2-Her1 receptor, dimers of the EGFRvlll-Her1 receptor, dimers of the EGFRvlll-Her2 receptor, and dimers of the EGFRvlll-Her3 receptor; wherein the ErbB cell surface receptor complex or complexes are measured through the following steps: provide for each or more complexes of ErbB cell surface receptors a pair of reagents comprising a cleavage probe having an induction fraction of the cleavage with an effective proximity, and one or more binding compounds having, each of them, one or more molecular tags attached through a clivable bond, the molecular tags of the different binding compounds having distinct separation characteristics; mixing the cleavage probe and the compound or binding compounds for the one or more ErbB cell surface receptor complexes with the patient sample, such that the cleavage probe and the one or more binding compounds specifically bind to their respective ErbB cell surface receptor complexes and that the clivable bonds of one or more binding compounds are within the effective proximity of the cleavage inducing fraction so that the molecular tags are released; Y separating and identifying the molecular tags released to determine the presence or absence or the amount of the one or more complexes of ErbB cell surface receptors in the sample of said patient.

2.

 The method according to claim 1, wherein the patient's cancer status is the degree of response of said patient to treatment with a drug acting on the ErbB dimer.

3.

 The method of claim 2, wherein the cancer is selected from the group consisting of breast cancer, ovarian cancer, prostate cancer and colorectal cancer.

Four.

 The method of claim 1, wherein the one or more ErbB cell surface receptor complexes are one or more heterodimers that include a PDGF receptor.

5.

 The method of claim 4, wherein the one or more ErbB cell surface receptor complexes are selected from the group consisting of Her1-PDGFR receptor dimers, Her2-PDGFR receptor dimers, and Her3-PDGFR receptor dimers.

6.

The method of claim 1 or 5, wherein said patient sample is a fixed tissue sample or a frozen tissue sample.

7.

The method of claim 4, 5 or 6, wherein the cancer is selected from the group consisting of breast cancer, ovarian cancer and glioblastoma.

8.

The method of claim 1, wherein the patient sample is a fixed tissue sample and wherein the one or more ErbB cell surface receptor complexes are Her1-Her1, Her1-Her3, Her1-Her4, Her2-Her2, Her3-Her4, or Her4-Her4.

9.

The method of claim 1, wherein the patient sample is a fixed tissue sample and wherein the one or more ErbB cell surface receptor complexes are p95Her2-Her1, p95Her2-Her2, or p95Her2-Her3.

10.

The method of claim 1, wherein the one or more ErbB cell surface receptor complexes are Her1-Her1, Her2-Her2 or Her1-Her3.

The method of claim 1, wherein the one or more ErbB cell surface receptor complexes are Her1-IGF-1R, Her2-IGF-1R or Her3-IGF-1R. 12. The method of claim 1, wherein the one or more ErbB cell surface receptor complexes are Her3-PI3K. 13. The method of claim 1, wherein the one or more ErbB cell surface receptor complexes are Her2-SHC or Her3-SHC. 14. The method of any one of the preceding claims wherein the determination of the patient's cancer status includes the prediction of the patient's effectiveness with respect to treatment with a drug acting on the ErbB dimer.