METHOD FOR PREDICTING THE RESPONSE TO A TREATMENT

Abstract

to a method of predicting the response to a treatment with a HER inhibitor in a patient comprising the steps of assessing a biomarker or a combination of biomarkers selected from the group consisting of amphiregulin, an epidermal growth factor, a transforming growth factor alpha, and a HER2 biomarker in a biological sample from the patient and predicting the response to the treatment with the HER inhibitor in the patient by evaluating the results of the first step. Further uses and methods wherein these markers are used are disclosed.

Description

PRIORITY TO RELATED APPLICATIONS

[0001]This application is a continuation of U.S. application Ser. No. 11/438,033, filed May 19, 2006, now pending; which claims the benefit of European Application No. 05017663.5, filed Aug. 12, 2005. The entire contents of the above-identified applications are hereby incorporated by reference.

FIELD OF THE INVENTION

[0002]The invention is related to a method of predicting the response to a treatment with a HER inhibitor, preferably a HER dimerization inhibitor, in a patient comprising the steps of assessing a marker gene or a combination of marker genes selected from the group consisting of an epidermal growth factor, a transforming growth factor alpha and a HER2 marker gene or a combination of marker genes comprising an amphiregulin marker gene and a marker gene selected from an epidermal growth factor, a transforming growth factor alpha and a HER2 marker gene in a biological sample from the patient and predicting the response to the treatment with the HER inhibitor in the patient by evaluating the results of the first step. Further uses and methods wherein these markers are used are disclosed.

BACKGROUND OF THE INVENTION

[0003]The human epidermal growth factor receptor (ErbB or HER) family comprises four members (HER1-4) that, through the activation of a complex signal cascade, are important mediators of cell growth, survival and differentiation. At least 11 different gene products from the epidermal growth factor (EGF) superfamily bind to three of these receptors, EGFR (also called ErbB1 or HER1), HER3 (ErbB3) and HER4 (ErbB4). Although no ligand has been identified that binds and activates HER2 (ErbB2 or neu), the prevailing understanding is that HER2 is a co-receptor that acts in concert with other HER receptors to amplify and in some cases initiate receptor-ligand signaling. Dimerization with the same receptor type (homodimerization) or another member of the HER family (heterodimerization) is essential for their activity. HER2 is the preferred dimerization partner for other HER family members. The role of the HER family in many epithelial tumor types is well documented and has led to the rational development of novel cancer agents directed specifically to HER receptors. The recombinant humanized anti-HER2 monoclonal antibody (MAb) trastuzumab is a standard of care in patients with HER2-positive metastatic breast cancer (MBC). Overexpression/amplification of the HER2 protein/gene, which occurs in 20-30% of breast cancer cases, is a prerequisite for treatment with trastuzumab.

[0004]Pertuzumab (Omnitarg®; formerly 2C4) is the first of a new class of agents known as HER dimerization inhibitors (HDIs). Pertuzumab binds to HER2 at its dimerization domain, thereby inhibiting its ability to form active dimer receptor complexes and thus blocking the downstream signal cascade that ultimately results in cell growth and division. Pertuzumab is a fully humanized recombinant monoclonal antibody directed against the extracellular domain of HER2. Binding of Pertuzumab to the HER2 on human epithelial cells prevents HER2 from forming complexes with other members of the HER family (including EGFR, HER3, HER4) and probably also HER2 homodimerization. By blocking complex formation, Pertuzumab prevents the growth-stimulatory effects and cell survival signals activated by ligands of HER1, HER3 and HER4 (e.g. EGF, TGFα, amphiregulin, and the heregulins). Other names for Pertuzumab are 2C4 or Pertuzumab. Pertuzumab is a fully humanized recombinant monoclonal antibody based on the human IgG1(κ) framework sequences. The structure of Pertuzumab consists of two heavy chains (449 residues) and two light chains (214 residues). Compared to Trastuzumab (Herceptin®), Pertuzumab has 12 amino acid differences in the light chain and 29 amino acid differences in the IgG1 heavy chain. WO 2004/092353 and WO 2004/091384 present investigations that the formation of heterodimers of HER2 with other receptors should be linked to the effectiveness or suitability of Pertuzumab.

[0005]Zabrecky, J. R. et al., J. Biol. Chem. 266 (1991) 1716-1720 disclose that the release of the extracellular domain of HER2 may have implications in oncogenesis and its detection could be useful as a cancer diagnostic. Colomer, R. et al., Clin. Cancer Res. 6 (2000) 2356-2362 disclose circulating HER2 extracellular domain and resistance to chemotherapy in advanced breast cancer. The prognostic and predictive values of the extracellular domain of HER2 is reviewed by Hait, W. N., Clin. Cancer Res. 7 (2001) 2601-2604.

SUMMARY OF THE INVENTION

[0006]There is still a need to provide further methods for determining the progression of disease in a cancer patient treated with a HER dimerization inhibitor.

[0007]Therefore, in an embodiment of the invention, a method of predicting the response to a treatment with a HER inhibitor, preferably a HER dimerization inhibitor, in a patient is provided comprising the steps of: [0008](a) determining the expression level or amount of one or more biomarker in a biological sample from a patient wherein the biomarker or biomarkers are selected from the group consisting of: [0009](1) transforming growth factor alpha; [0010](2) HER2; [0011](3) amphiregulin; and [0012](4) epidermal growth factor; [0013](b) determining whether the expression level or amount assessed in step (a) is above or below a certain quantity that is associated with an increased or decreased clinical benefit to a patient; and [0014](c) predicting the response to the treatment with the HER inhibitor in the patient by evaluating the results of step (b).

[0015]In another embodiment of the invention, a probe that hybridizes with the polynucleotides of the above biomarkers under stringent conditions or an antibody that binds to the proteins of the above biomarkers is used for predicting the response to treatment with a HER inhibitor in a patient or used for selecting a composition for inhibiting the progression of disease in a patient.

[0016]In still another embodiment of the invention, a kit is provided comprising a probe that anneals with a biomarker polynucleotide under stringent conditions or an antibody that binds to the biomarker protein.

[0017]In still another embodiment of the invention, a method of selecting a composition for inhibiting the progression of disease in a patient is provided, the method comprising: [0018](a) separately exposing aliquots of a biological sample from a cancer patient in the presence of a plurality of test compositions; [0019](b) comparing the level of expression of one or more biomarkers selected from the group consisting of amphiregulin, epidermal growth factor, transforming growth factor alpha and HER2 in the aliquots of the biological sample contacted with the test compositions and the level of expression of such biomarkers in an aliquot of the biological sample not contacted with the test compositions; and [0020](c) selecting one of the test compositions which alters the level of expression of a particular biomarker or biomarkers in the aliquot of the biological sample contacted with the test composition and the level of expression of the corresponding biomarker or biomarkers in the aliquot of the biological sample not contacted with the test composition is an indication for the selection of the test composition.

[0021]In yet another embodiment of the invention, a method of identifying a candidate agent is provided said method comprising: [0022](a) contacting an aliquot of a biological sample from a cancer patient with the candidate agent and determining the level of expression of one or more biomarkers selected from the group consisting of amphiregulin, epidermal growth factor, transforming growth factor alpha and HER2 in the aliquot; [0023](b) determining the level of expression of a corresponding biomarker or of a corresponding combination of biomarkers in an aliquot of the biological sample not contacted with the candidate agent; [0024](c) observing the effect of the candidate agent by comparing the level of expression of the biomarker or biomarkers in the aliquot of the biological sample contacted with the candidate agent and the level of expression of the corresponding biomarker or biomarkers in the aliquot of the biological sample not contacted with the candidate agent; and [0025](d) identifying said agent from said observed effect, wherein an at least 10% difference between the level of expression of the biomarker or biomarkers in the aliquot of the biological sample contacted with the candidate agent and the level of expression of the corresponding biomarker or biomarkers in the aliquot of the biological sample not contacted with the candidate agent is an indication of an effect of the candidate agent.

[0026]In yet another embodiment, a candidate agent identified by the method according to the invention or a pharmaceutical preparation comprising an agent according to the invention is provided.

[0027]In yet another embodiment of the invention, an agent according to the invention is provided for the preparation of a composition for the treatment of cancer.

[0028]In still another embodiment of the invention, a method of producing a drug is provided comprising: [0029](i) synthesizing the candidate agent identified as described above or an analog or derivative thereof in an amount sufficient to provide said drug in a therapeutically effective amount to a subject; and/or [0030](ii) combining the drug candidate or the candidate agent identified as described above or an analog or derivative thereof with a pharmaceutically acceptable carrier.

[0031]In yet another embodiment of the invention, a biomarker protein or a biomarker polynucleotide selected from the group consisting of an amphiregulin biomarker, and epidermal growth factor biomarker, a transforming growth factor alpha biomarker and a HER2 biomarker protein or polynucleotide is used for deriving a candidate agent or for selecting a composition for inhibiting the progression of a disease in a patient.

[0032]In another embodiment of the invention, a HER inhibitor, preferably a HER dimerization inhibitor, is used for the manufacture of a medicament for treating a human cancer patient characterized in that said treating or treatment includes assessing in a biological sample from the patient: one or more biomarkers selected from the group consisting of amphiregulin biomarker, epidermal growth factor biomarker, transforming growth factor alpha biomarker, and HER2 biomarker. In a particular embodiment, one or more biomarkers are assessed wherein the biomarkers are selected from the group consisting of epidermal growth factor, transforming growth factor alpha, and HER2. In another particular embodiment, a transforming growth factor alpha biomarker is assessed in combination with one or more biomarkers selected from the group consisting of epidermal growth factor, amphiregulin, and HER2. In another particular embodiment, a HER2 biomarker is assessed in combination with one or more biomarkers selected from the group consisting of epidermal growth factor, transforming growth factor alpha, and amphiregulin.

[0033]In another particular embodiment, a epidermal growth factor biomarker is assessed in combination with one or more biomarkers selected from the group consisting of amphiregulin, transforming growth factor alpha, and HER2.

[0034]In another particular embodiment, an amphiregulin biomarker is assessed in combination with one or more biomarkers selected from the group consisting of epidermal growth factor, transforming growth factor alpha, and HER2.

BRIEF DESCRIPTION OF THE FIGURES

[0035]FIG. 1: Scatterplot TGF-alpha logarithmic transformation versus categorized clinical benefit

[0036]FIG. 2: Scatterplot Amphiregulin logarithmic transformation versus categorized clinical benefit

[0037]FIG. 3: Ordinal clinical benefit TGF-alpha

[0038]FIG. 4: Ordinal clinical benefit Amphiregulin

[0039]FIG. 5: Ordinal clinical benefit EGF

[0040]FIG. 6: Ordinal clinical benefit HER2-ECD

[0041]FIG. 7: Overview exploratory cut-points and log-rank p-values for TTP and TTD for Amphiregulin, EGF, TGF-alpha, HER2-ECD

[0042]FIG. 8: TGF-alpha Kaplan Meier plot for time to progression/death based on exploratory single marker cut-point

[0043]FIG. 9: TGF-alpha Kaplan Meier plot for time to death based on exploratory single marker cut-point

[0044]FIG. 10: Amphiregulin Kaplan Meier plot for time to progression/death based on exploratory single marker cut-point

[0045]FIG. 11: Amphiregulin Kaplan Meier plot for time to death based on exploratory single marker cut-point

[0046]FIG. 12: EGF Kaplan Meier plot for time to progression/death based on exploratory single marker cut-point

[0047]FIG. 13: EGF Kaplan Meier plot for time to death based on exploratory single marker cut-point

[0048]FIG. 14: HER2-ECD Kaplan Meier plot for time to progression/death based on exploratory single marker cut-point

[0049]FIG. 15: HER2-ECD Kaplan Meier plot for time to death based on exploratory single marker cut-point

[0050]FIG. 16: As example for a combination score, further improving the separation between the greater clinical benefit/lesser clinical benefit groups in TTP: Ordinal clinical benefit HER2-ECD TGF alpha combination

[0051]FIG. 17: Overview exploratory cut-points and log-rank p-values for TTP and TTD for a combination of TGF-alpha and HER2-ECD

[0052]FIG. 18: HER2-ECD/TGF-alpha Kaplan Meier plot for time to progression/death based on exploratory combination marker cut-point

[0053]FIG. 19: HER2-ECD/TGF-alpha Kaplan Meier plot for time to death based on exploratory combination marker cut-point

DETAILED DESCRIPTION OF THE INVENTION

[0054]The articles "a" and "an" are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0055]The term "biological sample" shall generally mean any biological sample obtained from an individual, body fluid, cell line, tissue culture, or other source. Body fluids are e.g. lymph, sera, plasma, urine, semen, synovial fluid and spinal fluid. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art. If the term "sample" is used alone, it shall still mean that the "sample" is a "biological sample", i.e. the terms are used interchangeably.

[0056]The term "response of a patient to treatment with a HER inhibitor" or "response of a patient to treatment with a HER dimerization inhibitor" refers to the clinical benefit imparted to a patient suffering from a disease or condition (such as cancer) from or as a result of the treatment with the HER inhibitor (e.g., a HER dimerization inhibitor). A clinical benefit includes a complete remission, a partial remission, a stable disease (without progression), progression-free survival, disease free survival, improvement in the time-to-progression (of the disease), improvement in the time-to-death, or improvement in the overall survival time of the patient from or as a result of the treatment with the HER dimerization inhibitor. There are criteria for determining a response to therapy and those criteria allow comparisons of the efficacy to alternative treatments (Slapak and Kufe, Principles of Cancer Therapy, in Harrisons's Principles of Internal Medicine, 13th edition, eds. Isselbacher et al., McGraw-Hill, Inc., 1994). For example, a complete response or complete remission of cancer is the disappearance of all detectable malignant disease. A partial response or partial remission of cancer may be, for example, an approximately 50 percent decrease in the product of the greatest perpendicular diameters of one or more lesions or where there is not an increase in the size of any lesion or the appearance of new lesions.

[0057]As used herein, the term "progression of cancer" includes and may refer to metastasis; a recurrence of cancer, or an at least approximately 25 percent increase in the product of the greatest perpendicular diameter of one lesion or the appearance of new lesions. The progression of cancer, preferably breast cancer, is "inhibited" if recurrence or metastasis of the cancer is reduced, slowed, delayed, or prevented.

[0058]As used herein, the term "Time To Progression/death" (also referred to as "TPP") or Progression-Free Survival (also referred to as "PFS") refers to a clinical endpoint frequently used in oncology trials (that includes but is not limited to clinical trials with reference to the present invention). The measurement for each patient equals the time elapsed from onset of the treatment of a patient in a trial (as defined in the protocol [i.e, see the examples infra]) until the detection of a malignancy progression (as defined in the protocol) or the occurrence of any fatality (whatever is first). If the observation of the patient was stopped (e.g. at study end) after a period and no event was observed, then this observation time t is called "censored."

[0059]As used herein, the term "Time To Death" (also referred to as "TTD") or "Overall Survival" (also referred to as "OS") refers to a clinical endpoint frequently used in oncology trials (that includes but is not limited to clinical trials with reference to the present invention). The measurement for each patient equals the time elapsed from onset of the treatment of a patient in a trial (as defined in the protocol [i.e., see the examples infra]) until the occurrence of any fatality. If the observation of the patient is stopped (e.g. at study end) after a period t and the patient survived to this time, then this observation time t is called "censored."

[0060]As used herein, the term "covariate" refers to certain variables or information relating to a patient. The clinical endpoints are frequently considered in regression models, where the endpoint represent the dependent variable and the biomarkers represent the main or target independent variables (regressors). If additional variables from the clinical data pool are considered these are denoted as (clinical) covariates. The term "clinical covariate" here is used to describe all clinical information about the patient, which are in general available at baseline. These clinical covariates comprise demographic information like sex, age etc., other anamnestic information, concomitant diseases, concomitant therapies, result of physical examinations, common laboratory parameters obtained, known properties of the target tumor, information quantifying the extent of malignant disease, clinical performance scores like ECOG or Karnofsky index, clinical disease staging, timing and result of pretreatments and disease history as well as all similar information, which may be associated with the clinical prognosis.

[0061]As used herein, the term "raw analysis" or "unadjusted analysis" refers to regression analyses, where over the considered biomarkers no additional clinical covariates were used in the regression model, neither as independent factors nor as stratifying covariate.

[0062]As used herein, the term "adjusted by covariates" refers to regression analyses, where over the considered biomarkers additional clinical covariates were used in the regression model, either as independent factors or as stratifying covariate.

[0063]As used herein, the term "univariate" refers to regression models or graphical approaches where as independent variable only one of the target biomarkers is part of the model. These univariate models can be considered with and without additional clinical covariates.

[0064]As used herein, the term "multivariate" refers to regression models or graphical approaches where as independent variables more than one of the target biomarkers are part of the model.

[0065]These multivariate models can be considered with and without additional clinical covariates.

[0066]"Nucleotides" are "nucleosides" that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those "nucleosides" that include a pentofuranosyl sugar, the phosphate group can be linked to either the 2', 3' or 5' hydroxyl moiety of the sugar. A "nucleotide" is the "monomeric unit" of an "oligonucleotide", more generally denoted herein as an "oligomeric compound", or a "polynucleotide", more generally denoted as a "polymeric compound". Another general expression therefor is desoxyribonucleic acid (DNA) and ribonucleic acid (RNA). As used herein the term "polynucleotide" is synonymous with "nucleic acid."

[0067]As used herein, the term "probe" refers to synthetically or biologically produced nucleic acids (DNA or RNA) which, by design or selection, contain specific nucleotide sequences that allow them to hybridize under defined predetermined stringencies specifically (i.e., preferentially) to "nucleic acids". A "probe" can be identified as a "capture probe" meaning that it "captures" the nucleic acid so that it can be separated from undesirable materials which might obscure its detection. Once separation is accomplished, detection of the captured "target nucleic acid" can be achieved using a suitable procedure. "Capture probes" are often already attached to a solid phase. According to the present invention, the term hybridization under "stringent conditions" is given the same meaning as in Sambrook et al. (Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), paragraph 1.101-1.104). Preferably, a "stringent hybridization" is the case when a hybridization signal is still detectable after washing for 1 h with 1×SSC and 0.1% SDS at 50° C., preferably at 55° C., more preferably at 62° C., and most preferably at 68° C., and more preferably for 1 hour with 0.2×SSC and 0.1% SDS at 50°., preferably at 55° C., more preferably at 62°, and most preferably at 68° C. The composition of the SSC buffer is described in Sambrook et al. (Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989)).

[0068]As used herein, a "transcribed polynucleotide" is a polynucleotide (e.g an RNA, a cDNA, or an analog of one of an RNA or cDNA) which is complementary to or homologous with all or a portion of a mature RNA made by transcription of a gene, such as the marker gene of the invention, and normal post-transcriptional processing (e.g. splicing), if any, of the transcript. The term "cDNA" is an abbreviation for complementary DNA, the single-stranded or double-stranded DNA copy of a mRNA. The term "mRNA" is an abbreviation for messenger RNA- the RNA that serves as a template for protein synthesis.

[0069]As used herein, the term "marker gene" or "biomarker gene" is meant to include a gene which is useful according to this invention for determining the progression of cancer in a patient, particularly in a breast cancer patient.

[0070]As used herein, the term "marker polynucleotide" or "biomarker polynucleotide" is meant to include a nucleotide transcript (hnRNA or mRNA) encoded by a marker gene according to the invention, or cDNA derived from the nucleotide transcript, or a segment of said transcript or cDNA.

[0071]As used herein, the term "marker protein," "marker polypeptide," "biomarker protein," or "biomarker polypeptide" is meant to include a protein or polypeptide encoded by a marker gene according to the invention or to a fragment thereof.

[0072]As used herein, the term "marker" and "biomarker" are used interchangeably and refer to a marker gene, marker polynucleotide, or marker protein as defined above.

[0073]As used herein, the term "gene product" refers to a marker polynucleotide or marker protein encoded by a marker gene.

[0074]The expression of a marker gene "significantly" differs from the level of expression of the marker gene in a reference sample if the level of expression of the marker gene in a sample from the patient differs from the level in a sample from the reference subject by an amount greater than the standard error of the assay employed to assess expression, and preferably at least 10%, and more preferably 25%, 50%, 75%, 100%, 125%, 150%, 175%, 200%, 300%, 400%, 500% or 1,000% of that amount. Alternatively, expression of the marker gene in the patient can be considered "significantly" lower than the level of expression in a control subject if the level of expression in a sample from the patient is lower than the level in a sample from the control subject by an amount greater than the standard error of the assay employed to assess expression, and preferably at least 10%, and more preferably 25%, 50%, 75%, 100%, 125%, 150%, 175%, 200%, 300%, 400%, 500% or 1,000% that amount.

[0075]A marker polynucleotide or a marker protein "corresponds to" another marker polynucleotide or marker protein if it is related thereto, and in preferred embodiments is identical thereto.

[0076]The terms "level of expression" or "expression level" are used interchangeably and generally refer to the amount of a polynucleotide or an amino acid product or protein in a biological sample. "Expression" generally refers to the process by which gene encoded information is converted into the structures present and operating in the cell. Therefore, according to the invention "expression" of a gene may refer to transcription into a polynucleotide, translation into a protein or even posttranslational modification of the protein. Fragments of the transcribed polynucleotide, the translated protein or the postranslationally modified protein shall also be regarded as expressed whether they originate from a transcript generated by alternative splicing, a degraded transcript or from a posttranslational processing of the protein, e.g. by proteolysis. "Expressed genes" include those that are transcribed into a polynucleotide as mRNA and then translated into a protein; and also include expressed genes that are transcribed into RNA but not translated into a protein (for example, transfer and ribosomal RNAs).

[0077]The term "overexpression" or "increased expression" refers to an upward deviation in levels of expression as compared to the baseline expression level in a sample used as a control.

[0078]The term "underexpression" or "decreased expression" refers to a downward deviation in levels of expression as compared to the baseline expression level in a sample used as a control.

[0079]The term "amphiregulin" relates to a gene that encodes a protein and to the protein itself that is a member of the epidermal growth factor family. It is an autocrine growth factor as well as a mitogen for astrocytes, Schwann cells, and fibroblasts. It is related to epidermal growth factor (EGF) and transforming growth factor alpha (TGF-alpha). This protein interacts with the EGF/TGF-alpha receptor to promote the growth of normal epithelial cells and inhibits the growth of certain aggressive carcinoma cell lines. According to the invention, the amino acid sequence of amphiregulin is the amino acid sequence according to SEQ ID NO: 1. According to the invention, the nucleic acid sequence of the "amphiregulin" cDNA is the nucleic acid sequence according to SEQ ID NO: 5 which is accessible at GenBank with the accession number NM_--001657.

[0080]The term "transforming growth factor alpha" relates to a gene that encodes a protein and to the protein itself that is a member of the family of transforming growth factors (TGFs). These are biologically active polypeptides that reversibly confer the transformed phenotype on cultured cells. "Transforming growth factor-alpha" shows about 40% sequence homology with epidermal growth factor and competes with EGF for binding to the EGF receptor, stimulating its phosphorylation and producing a mitogenic response. According to the invention, the amino acid sequence of "Transforming growth factor-alpha" is the amino acid sequence according to SEQ ID NO: 3. According to the invention, the nucleic acid sequence of the "transforming growth factor-alpha" cDNA is the nucleic acid sequence according to SEQ ID NO: 7 which is accessible at GenBank with the accession number NM_--003236.

[0081]The term "epidermal growth factor" relates to a gene that encodes a protein and to the protein itself that is a member of the family of growth factors. "Epidermal growth factor (EGF)" has a profound effect on the differentiation of specific cells in vivo and is a potent mitogenic factor for a variety of cultured cells of both ectodermal and mesodermal origin. The EGF precursor is believed to exist as a membrane-bound molecule which is proteolytically cleaved to generate the 53-amino acid peptide hormone that stimulates cells to divide. According to the invention, the amino acid sequence of "Epidermal growth factor" is the amino acid sequence according to SEQ ID NO: 2. According to the invention, the nucleic acid sequence of the "Epidermal growth factor (EGF)" cDNA is the nucleic acid sequence according to SEQ ID NO: 6 which is accessible at GenBank with the accession number NM_--001963. The "Epidermal Growth Factor Receptor" abbreviated as EGFR, a 170-kD glycoprotein, is composed of an N-terminus extracellular domain, a hydrophobic transmembrane domain, and a C-terminus intracellular region containing the kinase domain. The mRNA has different variants translated into different receptor proteins. According to the invention, the amino acid sequence of the "Epidermal growth factor receptor" is the amino acid sequence according to SEQ ID NO: 11 (transcript variant 1; GenBank accession number NM_--005228), SEQ ID NO: 12 (transcript variant 2; GenBank accession number NM_--201282), SEQ ID NO: 13 (transcript variant 3; GenBank accession number NM_--201283), or SEQ ID NO: 14 (transcript variant 4; GenBank accession number NM_--201284). EGFR, encoded by the erbB1 gene, has been causally implicated in human malignancy. In particular, increased expression of EGFR has been observed in breast, bladder, lung, head, neck and stomach cancer as well as glioblastomas. EGFR ligandinduced dimerization activates the intrinsic RTK domain (an Src homology domain 1, SH1), resulting in autophosphorylation on six specific EGFR tyrosine residues in the noncatalytic tail of the cytoplasmic domain. The cellular effects of EGFR activation in a cancer cell include increased proliferation, promotion of cell motility, adhesion, invasion, angiogenesis, and enhanced cell survival by inhibition of apoptosis. Activated EGFR induces tumor cell proliferation through stimulation of the mitogen-activated protein kinase (MAPK) cascade.

[0082]The terms "human neu", "c-erbB-2", "erbB2", "erbB-2", "HER-2/neu", "HER-2" and "HER2" are used interchangeably herein. These terms relate to a gene that encodes a protein and to the protein itself that is a member of the family of the epidermal growth factor (EGF) receptor family of receptor tyrosine kinases. This protein has no ligand binding domain of its own and therefore cannot bind growth factors. However, it does bind tightly to other ligand-bound EGF receptor family members to form a heterodimer, stabilizing ligand binding and enhancing kinase-mediated activation of downstream signalling pathways, such as those involving mitogen-activated protein kinase and phosphatidylinositol-3 kinase. Allelic variations at amino acid positions 654 and 655 of isoform a (positions 624 and 625 of isoform b) have been reported, with the most common allele, Ile654/Ile655 being preferred according to the invention. Amplification and/or overexpression of this gene has been reported in numerous cancers, including breast and ovarian tumors. Alternative splicing results in several additional transcript variants, some encoding different isoforms and others that have not been fully characterized. According to the invention, the amino acid sequence of HER2 is the amino acid sequence according to SEQ ID NO: 4. According to the invention, the nucleic acid sequence of the "HER2" cDNA is the nucleic acid sequence according to SEQ ID NO: 8 which is accessible at GenBank with the accession number NM_--004448.2.

[0083]The "extracellular domain of HER2" or "shed extracellular domain of HER2" or "HER2-ECD" is a glycoprotein of between 97 and 115 kDa which corresponds substantially to the extracellular domain of the human HER2 gene product. It can be referred to as p105 (Zabrecky, J. R. et al., J. Biol. Chem. 266 (1991) 1716-1720; U.S. Pat. No. 5,401,638; U.S. Pat. No. 5,604,107). The quantitation and detection of the extracellular domain of HER2 is described in U.S. Pat. No. 5,401,638 and U.S. Pat. No. 5,604,107.

[0084]The term "HER3" stands for another member of the epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases. This membrane-bound protein has not an active kinase domain. The protein can bind ligands but not transmit a signal into the cell. It forms heterodimers with other EGF receptor family members which do have kinase activity which leads to cell proliferation or differentiation. Amplification of this gene and/or overexpression of its protein is found in numerous cancers. According to the invention, the amino acid sequence of the "HER3" cDNA is the amino acid sequence according to SEQ ID NO: 9 which is accessible at GenBank from the translation of the nucleic acid sequence of HER3 with the accession number NM_--001005915. According to the invention, the nucleic acid sequence of the "HER3" cDNA is the nucleic acid sequence according to SEQ ID NO: 10 which is accessible at GenBank with the accession number NM_--001005915.

[0085]The term "antibody" herein is used in the broadest sense and specifically covers intact monoclonal antibodies, polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments, so long as they exhibit the desired biological activity of an antibody.

[0086]The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler, G. et al., Nature 256 (1975) 495-497, or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). "Antibody fragments" comprise a portion of an intact antibody.

[0087]An antibody "which binds" an antigen of interest according to the invention is one capable of binding that antigen with sufficient affinity such that the antibody is useful in detecting the presence of the antigen. One antibody according to the invention binds human HER2 and does not (significantly) cross-react with other proteins. In such embodiments, the extent of binding of the antibody to other proteins will be less than 10% as determined by fluorescence activated cell sorting (FACS) analysis or radioimmunoprecipitation (RIA).

[0088]Dimerization--the pairing of receptors--is essential to the signaling activity of all HER receptors. According to the invention, the term "HER dimerization inhibitor" or preferably "HER2 heterodimerization inhibitor" refers to a therapeutic agent that binds to HER2 and inhibits HER2 heterodimerization. These are preferably antibodies, preferably monoclonal antibodies, more preferably humanized antibodies that bind to HER2 and inhibit HER2 heterodimerization. Examples of antibodies that bind HER2 include 4D5, 7C2, 7F3 or 2C4 as well as humanized variants thereof, including huMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 and huMAb4D5-8 as described in Table 3 of U.S. Pat. No. 5,821,337; and humanized 2C4 mutant numbers 560, 561, 562, 568, 569, 570, 571, 574, or 56869 as described in WO 01/00245. 7C2 and 7F3 and humanized variants thereof are described in WO 98/17797. The term "HER dimerization inhibitor" or "HER2 heterodimerization inhibitor" shall not apply to Trastuzumab monoclonal antibodies commercially available as "Herceptin®" as the mechanism of action is different and as Trastuzumab does not inhibit HER dimerization.

[0089]Preferred throughout the application is the "antibody 2C4", in particular the humanized variant thereof (WO 01/00245; produced by the hybridoma cell line deposited with the American Type Culture Collection, Manassass, Va., USA under ATCC HB-12697), which binds to a region in the extracellular domain of HER2 (e.g., any one or more residues in the region from about residue 22 to about residue 584 of HER2, inclusive). The "epitope 2C4" is the region in the extracellular domain of ErbB2 to which the antibody 2C4 binds. The expression "monoclonal antibody 2C4" refers to an antibody that has antigen binding residues of, or derived from, the murine 2C4 antibody of the Examples in WO 01/00245. For example, the monoclonal antibody 2C4 may be murine monoclonal antibody 2C4 or a variant thereof, such as humanized antibody 2C4, possessing antigen binding amino acid residues of murine monoclonal antibody 2C4. Examples of humanized 2C4 antibodies are provided in Example 3 of WO 01/00245. Unless indicated otherwise, the expression "rhuMAb 2C4" when used herein refers to an antibody comprising the variable light (VL) and variable heavy (VH) sequences of SEQ ID Nos. 3 and 4 of WO 01/00245, respectively, fused to human light and heavy IgG1 (non-A allotype) constant region sequences optionally expressed by a Chinese Hamster Ovary (CHO) cell. Preferred embodiments of WO 01/00245 are preferred herein as well. The humanized antibody 2C4 is also called Pertuzumab.

[0090]A "kit" is any manufacture (e.g a package or container) comprising at least one reagent, e.g a probe, for specifically detecting a marker gene or protein of the invention. The manufacture is preferably promoted, distributed, or sold as a unit for performing the methods of the present invention.

[0091]The verbs "determine" and "assess" shall have the same meaning and are used interchangeably throughout the application.

[0092]Conventional techniques of molecular biology and nucleic acid chemistry, which are within the skill of the art, are explained in the literature. See, for example, Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; Gait, M. J. (ed.), Oligonucleotide synthesis--a practical approach, IRL Press Limited, 1984; Hames, B. D. and Higgins, S. J. (eds.), Nucleic acid hybridisation--a practical approach, IRL Press Limited, 1985; and a series, Methods in Enzymology, Academic Press, Inc., all of which are incorporated herein by reference. All patents, patent applications, and publications mentioned herein, both supra and infra, are hereby incorporated by reference in their entirety.

[0093]As used herein, the general form of a prediction rule consists in the specification of a function of one or multiple biomarkers potentially including clinical covariates to predict response or non-response, or more generally, predict benefit or lack of benefit in terms of suitably defined clinical endpoints.

[0094]The simplest form of a prediction rule consists of an univariate model without covariates, where the prediction is determined by means of a cutoff or threshold. This can be phrased in terms of the Heaviside function for a specific cutoff c and a biomarker measurement x, where the binary prediction A or B is to be made, then

If H(x-c)=0 then predict A.

If H(x-c)=1 then predict B.

[0095]This is the simplest way of using univariate biomarker measurements in prediction rules. If such a simple rule is sufficient, it allows for a simple identification of the direction of the effect, i.e. whether high or low expression levels are beneficial for the patient.

[0096]The situation can be more complicated if clinical covariates need to be considered and/or if multiple biomarkers are used in multivariate prediction rules. In order to illustrate the issues here are two hypothetical examples:

Covariate Adjustment (Hypothetical Example):

[0097]For a biomarker X it is found in a clinical trial population that high expression levels are associated with a worse prognosis (univariate analysis). A closer analysis shows that there are two tumor types in the population, one of which possess a worse prognosis than the other one and at the same time the biomarker expression for this tumor group is generally higher. An adjusted covariate analysis reveals that for each of the tumor types the relation of clinical benefit and prognosis is reversed, i.e. within the tumor types, lower expression levels are associated with better prognosis. The overall opposite effect was masked by the covariate tumor type--and the covariate adjusted analysis as part of the prediction rule reversed the direction.

Multivariate Prediction (Hypothetical Example):

[0098]For a biomarker X it is found in a clinical trial population that high expression levels are slightly associated with a worse prognosis (univariate analysis). For a second biomarker Y a similar observation was made by univariate analysis. The combination of X and Y revealed that a good prognosis is seen if both biomarkers are low. This makes the rule to predict benefit if both biomarkers are below some cutoffs (AND- connection of a Heaviside prediction function). For the combination rule there is no longer a simple rule phraseable in an univariate sense. E.g. having low expression levels in X will not automatically predict a better prognosis.

[0099]These simple examples show that prediction rules with and without covariates cannot be judged on the univariate level of each biomarker. The combination of multiple biomarkers plus a potential adjustment by covariates does not allow to assign simple relationships towards single biomarkers.

[0100]In one embodiment of the invention, a method of predicting the response to a treatment with a HER inhibitor, preferably a HER dimerization inhibitor, in a patient comprises the steps of: [0101](a) determining the expression level or amount of one or more biomarkers in a biological sample from a patient wherein the biomarker or biomarkers are selected from the group consisting of: [0102](1) transforming growth factor alpha; [0103](2) HER2; [0104](3) amphiregulin; and [0105](4) epidermal growth factor; [0106](b) determining whether the expression level or amount assessed in step (a) is above or below a certain quantity that is associated with an increased or decreased clinical benefit to a patient; and [0107](c) predicting the response to the treatment with the HER inhibitor in the patient by evaluating the results of step (b).

[0108]In a more particular embodiment of the above method, the expression level of the transforming growth factor alpha biomarker is determined in combination with one or more biomarkers selected from the group consisting of epidermal growth factor, amphiregulin, and HER2. In another more particular embodiment of the above method, the expression level of the HER2 biomarker is determined in combination with one or more biomarkers selected from the group consisting of epidermal growth factor, transforming growth factor alpha, and amphiregulin. In another more particular embodiment of the above method, the expression level of the epidermal growth factor biomarker is determined in combination with one or more biomarkers selected from the group consisting of amphiregulin, transforming growth factor alpha, and HER2. In another more particular embodiment of the above method, an amphiregulin biomarker is assessed in combination with one or more biomarkers selected from the group consisting of epidermal growth factor, transforming growth factor alpha, and HER2.

[0109]The "quantity that is associated with an increased or decreased clinical benefit to a patient" of the above method is preferably a value expressed in mass/volume for blood serum or blood plasma or mass/mass for tumor tissue. It can be measured by methods known to the expert skilled in the art and also disclosed by this invention. If the expression level or amount determined in step (a) is above or below a certain quantity or value, the response to the treatment can be determined.

[0110]With respect to the quantity in blood serum for the transforming growth factor alpha marker protein, a range between 2.0-10.0 pg/ml, preferably a range between 2.0-5.0 pg/ml, and more preferably about 3.5 pg/ml may be favorable for progression free survival and overall survival when treatment with a HER inhibitor is considered. See FIG. 7. Thus, in a preferred embodiment, the quantity of transforming growth factor alpha marker protein in the blood serum of a patient is within one of the foregoing ranges for predicting a good response to treatment with a HER inhibitor in the patient.

[0111]With respect to the quantity in blood serum for the HER2 marker protein (preferably the soluble HER2 extracellular domain (HER2-ECD)), a range between 12-22 ng/ml, preferably about 18 ng/ml, may be favorable for progression free survival and overall survival when treatment with a HER inhibitor is considered. See FIG. 7. Thus, in a preferred embodiment, the quantity of HER2 marker protein in the blood serum of a patient is within the foregoing range for predicting a good response to treatment with a HER inhibitor in the patient.

[0112]With respect to the quantity in blood serum for the epidermal growth factor marker protein, a range between 100-250 pg/ml, preferably about 150 pg/ml, may be favorable for progression free survival and overall survival when treatment with a HER inhibitor is considered. See FIG. 7. Thus, in a preferred embodiment, the quantity of epidermal growth factor marker protein in the blood serum of a patient is within the foregoing range for predicting a good response to treatment with a HER inhibitor in the patient.

[0113]With respect to the quantity in blood serum for the amphiregulin marker protein, a range between 6-15 pg/ml, preferably about 12 pg/ml, may be favorable for progression free survival and overall survival when treatment with a HER inhibitor is considered. See FIG. 7. Thus, in a preferred embodiment, the quantity of amphiregulin marker protein in the blood serum of a patient is within the foregoing range for predicting a good response to treatment with a HER inhibitor in the patient.

[0114]Since the marker genes, in particular in serum, may be used in multiple-marker prediction models potentially including other clinical covariates, the direction of a beneficial effect of a single marker gene within such models cannot be determined in a simple way, and may contradict the direction found in univariate analyses, i.e. the situation as described for the single marker gene.

[0115]More preferably, in the method according to the invention, the quantity or value (below or above which is associated with an increased or decreased clinical benefit) is determined by: [0116](1) determining the expression level or amount of a biomarker or combination of biomarkers in a plurality of biological samples from patients before treatment with the HER inhibitor, [0117](2) treating the patients with the HER inhibitor, [0118](3) determining the clinical benefit of each patient; and [0119](4) correlating the clinical benefit of the patients treated with the HER inhibitor to the expression level or amount of the biomarker or combination of biomarkers.

[0120]The "quantity" is preferably a value expressed in mass/volume for blood serum or blood plasma or mass/mass for tumor tissue.

[0121]The present invention also considers mutants or variants of the marker genes according to the present invention and used in the methods according to the invention. In those mutants or variants the native sequence of the marker gene is changed by substitutions, deletions or insertions. "Native sequence" refers to an amino acid or nucleic acid sequence which is identical to a wild-type or native form of a marker gene or protein.

[0122]The present invention also considers mutants or variants of the proteins according to the present invention and used in the methods according to the invention. "Mutant amino acid sequence," "mutant protein" or "mutant polypeptide" refers to a polypeptide having an amino acid sequence which varies from a native sequence or is encoded by a nucleotide sequence intentionally made variant from a native sequence. "Mutant protein," "variant protein" or "mutein" means a protein comprising a mutant amino acid sequence and includes polypeptides which differ from the amino acid sequence of the native protein according to the invention due to amino acid deletions, substitutions, or both.

[0123]The present invention also considers a method of predicting the response to a treatment with a combination of a HER inhibitor and another substance or agent as a chemotherapeutic agent or a therapeutic antibody used for treating cancer. The chemotherapeutic agent may be e.g. gemcitabine (Gemzar®; chemical name: 2',2'-difluorodeoxycytidine (dFdC)), carboplatin (diammine-(cyclobutane-1,1-dicarboxylato (2-)-O,O')-platinum), or paclitaxel (Taxol®, chemical name: β-(benzoylamino)-α-hydroxy-,6,12b-bis(acetyloxy)-12-(benzoylox- y)-2a,3,4,4a,5,6,9,10,11,12,12a,12b-dodecahydro-4,11-dihydroxy-4a,8,13,13-- tetramethyl-5-oxo-7,11-methano-1H-cyclodeca(3,4)benz(1,2-b)oxet-9-yl ester,(2aR-(2a-α,4-β,4a-β,6-β, 9-α(α-R*,β-S*),11-α,12-α,12a-α, 2b-α))-benzenepropanoic acid); or transtuzumab; or erlotinib.

[0124]In a preferred embodiment of the invention, the biological sample is blood serum, blood plasma or tumor tissue. Tumor tissue may be formalin-fixed paraffin embedded tumor tissue or fresh frozen tumor tissue.

[0125]In another preferred embodiment of the invention, the HER dimerization inhibitor inhibits heterodimerization of HER2 with EGFR or HER3, or HER4. Preferably, the HER dimerization inhibitor is an antibody, preferably the antibody 2C4. Preferred throughout the application is the "antibody 2C4", in particular the humanized variant thereof (WO 01/00245; produced by the hybridoma cell line deposited with the American Type Culture Collection, Manassass, Va., USA under ATCC HB-12697), which binds to a region in the extracellular domain of HER2 (e.g., any one or more residues in the region from about residue 22 to about residue 584 of HER2, inclusive). Examples of humanized 2C4 antibodies are provided in Example 3 of WO 01/00245. The humanized antibody 2C4 is also called Pertuzumab.

[0126]In still another preferred embodiment of the invention, the patient is a cancer patient, preferably a breast cancer, ovarian cancer, lung cancer or prostate cancer patient. The breast cancer patient is preferably a metastatic breast cancer patient or a HER2 low expressing breast or metastatic breast cancer patient, or a HER2 high expressing breast or metastatic breast cancer patient. The ovarian cancer patient is preferably a metastatic ovarian cancer patient. The lung cancer patient is preferably a non-small cell lung cancer (NSCLC) patient.

[0127]It is preferred that two, three or all four marker genes, marker polynucleotides or marker proteins are used in combination, i.e. used in all disclosed embodiments of the invention or methods, uses or kits according to the invention. The following are preferred combinations of biomarkers in which the level of expression or amounts are determined in accordance with the invention:

[0128]In one particular embodiment, a transforming growth factor alpha biomarker is assessed in combination with one or more biomarkers selected from the group consisting of epidermal growth factor, amphiregulin, and HER2. In another particular embodiment, a HER2 biomarker is assessed in combination with one or more biomarkers selected from the group consisting of epidermal growth factor, transforming growth factor alpha, and amphiregulin.

[0129]In another particular embodiment, a epidermal growth factor biomarker is assessed in combination with one or more biomarkers selected from the group consisting of amphiregulin, transforming growth factor alpha, and HER2. In another particular embodiment, an amphiregulin biomarker is assessed in combination with one or more biomarkers selected from the group consisting of epidermal growth factor, transforming growth factor alpha, and HER2.

[0130]In a particularly preferred embodiment of the invention, the combination of biomarkers consists of: [0131]the transforming growth factor alpha and the HER2 biomarkers, or [0132]the transforming growth factor alpha and the EGF biomarkers, or [0133]the amphiregulin, the epidermal growth factor, the transforming growth factor alpha and the HER2 biomarkers,

[0134]In a preferred embodiment of the invention, the level of expression of the marker gene or the combination of marker genes in the sample is assessed by detecting the level of expression of a marker protein or a fragment thereof or a combination of marker proteins or fragments thereof encoded by the marker gene or the combination of marker genes. Preferably, the level of expression of the marker protein or the fragment thereof or the combination of marker proteins or the fragments thereof is detected using a reagent which specifically binds with the marker protein or the fragment thereof or the combination of marker proteins or the fragments thereof. Preferably, the reagent is selected from the group consisting of an antibody, a fragment of an antibody, and an antibody derivative.

[0135]There are many different types of immunoassays which may be used in the method of the present invention, e.g. enzyme linked immunoabsorbent assay (ELISA), fluorescent immunosorbent assay (FIA), chemical linked immunosorbent assay (CLIA), radioimmuno assay (RIA), and immunoblotting. For a review of the different immunoassays which may be used, see: Lottspeich and Zorbas (eds.), Bioanalytik, 1^st edition 1998, Spektrum Akademischer Verlag, Heidelberg, Berlin, Germany. Therefore, in yet another preferred embodiment of the invention, the level of expression is determined using a method selected from the group consisting of proteomics, flow cytometry, immunocytochemistry, immunohistochemistry, enzyme-linked immunosorbent assay, multi-channel enzyme-linked immunosorbent assay, and variations of these methods. Therefore more preferably, the level of expression is determined using a method selected from the group consisting of proteomics, flow cytometry, immunocytochemistry, immunohistochemistry, enzyme-linked immunosorbent assay, multi-channel enzyme-linked immunosorbent assay, and variations of these methods.

[0136]In another preferred embodiment of the invention, the fragment of the marker protein is the extracellular domain of the HER2 marker protein (HER2-ECD). Preferably, the extracellular domain of the HER2 marker protein has a molecular mass of approximately 105,000 Dalton. "Dalton" stands for a mass unit that is equal to the weight of a hydrogen atom, or 1.657×10^-24 grams.

[0137]In another preferred embodiment of the invention [0138]the amino acid sequence of the amphiregulin marker protein is the amino acid sequence SEQ ID NO: 1, [0139]the amino acid sequence of the epidermal growth factor marker protein is the amino acid sequence SEQ ID NO: 2, [0140]the amino acid sequence of the transforming growth factor alpha marker protein is the amino acid sequence SEQ ID NO: 3, or [0141]the amino acid sequence of the HER2 marker protein is the amino acid sequence SEQ ID NO: 4.

[0142]In another preferred embodiment of the invention, the quantity in blood serum for [0143]the transforming growth factor alpha marker protein is between 2.0 to 10.0 pg/ml, preferably about 3.5 pg/ml, [0144]the epidermal growth factor marker protein is between 100 to 250 pg/ml, preferably about 150 pg/ml, or pg/ml. [0145]the amphiregulin marker protein is between 6 to 15 pg/ml, preferably about 12 the HER2 marker protein is between 12 to 22 ng/ml, preferably about 18 ng/ml.

[0146]In still another preferred embodiment of the invention, the "quantity" in blood serum for the extracellular domain of the HER2 marker protein is between 12 to 22 ng/ml, preferably about 18 ng/ml.

[0147]In yet another preferred embodiment of the invention, the level of expression of the marker gene or the combination of marker genes in the biological sample is assessed by detecting the level of expression of a transcribed marker polynucleotide encoded by the marker gene or a fragment of the transcribed marker polynucleotide or of transcribed marker polynucleotides encoded by the combination of marker genes or fragments of the transcribed marker polynucleotide. Preferably, the transcribed marker polynucleotide is a cDNA, mRNA or hnRNA or wherein the transcribed marker polynucleotides are cDNA, mRNA or hnRNA.

[0148]Preferably, the step of detecting further comprises amplifying the transcribed polynucleotide. The amplification is performed preferably with the polymerase chain reaction which specifically amplifies nucleic acids to detectable amounts. Other possible amplification reactions are the Ligase Chain Reaction (LCR; Wu D. Y. and Wallace R. B., Genomics 4 (1989) 560-569; and Barany F., Proc. Natl. Acad. Sci. USA 88 (1991)189-193); Polymerase Ligase Chain Reaction (Barany F., PCR Methods and Applic. 1 (1991) 5-16); Gap-LCR (WO 90/01069); Repair Chain Reaction (EP 0439182 A2), 3SR (Kwoh, D. Y. et al., Proc. Natl. Acad. Sci. USA 86 (1989) 1173-1177; Guatelli, J. C. et al., Proc. Natl. Acad. Sci. USA 87 (1990) 1874-1878; WO 92/08808), and NASBA (U.S. Pat. No. 5,130,238). Further, there are strand displacement amplification (SDA), transcription mediated amplification (TMA), and Q(3-amplification (for a review see e.g. Whelen, A. C. and Persing, D. H., Annu. Rev. Microbiol. 50 (1996) 349-373; Abramson, R. D. and Myers T. W., Curr. Opin. Biotechnol. 4 (1993) 41-47). More preferably, the step of detecting is using the method of quantitative reverse transcriptase polymerase chain reaction.

[0149]Other suitable polynucleotide detection methods are known to the expert in the field and are described in standard textbooks as Sambrook J. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; and Ausubel, F. et al., Current Protocols in Molecular Biology, 1987, J. Wiley and Sons, NY. There may be also further purification steps before the polynucleotide detection step is carried out as e.g. a precipitation step. The detection methods may include but are not limited to the binding or intercalating of specific dyes as ethidiumbromide which intercalates into the double-stranded polynucleotides and changes their fluorescence thereafter. The purified polynucleotide may also be separated by electrophoretic methods optionally after a restriction digest and visualized thereafter. There are also probe-based assays which exploit the oligonucleotide hybridisation to specific sequences and subsequent detection of the hybrid. It is also possible to sequence the DNA after further steps known to the expert in the field. The preferred template-dependent DNA polymerase is Taq polymerase.

[0150]In yet another preferred embodiment of the invention, the level of expression of the marker gene is assessed by detecting the presence of the transcribed marker polynucleotide or the fragment thereof in a sample with a probe which anneals with the transcribed marker polynucleotide or the fragment thereof under stringent hybridization conditions or the level of expression of the combination of the marker genes in the samples is assessed by detecting the presence of transcribed marker polynucleotides or the fragments thereof in a sample with probes which anneal with the transcribed marker polynucleotides or the fragments thereof under stringent hybridization conditions. This method may be performed in a homogeneous assay system. An example for a "homogeneous" assay system is the TagMan® system that has been detailed in U.S. Pat. No. 5,210,015, U.S. Pat. No. 5,804,375 and U.S. Pat. No. 5,487,972. Briefly, the method is based on a double-labelled probe and the 5'-3' exonuclease activity of Taq DNA polymerase. The probe is complementary to the target sequence to be amplified by the PCR process and is located between the two PCR primers during each polymerisation cycle step. The probe has two fluorescent labels attached to it. One is a reporter dye, such as 6-carboxyfluorescein (FAM), which has its emission spectra quenched by energy transfer due to the spatial proximity of a second fluorescent dye, 6-carboxy-tetramethyl-rhodamine (TAMRA). In the course of each amplification cycle, the Taq DNA polymerase in the process of elongating a primed DNA strand displaces and degrades the annealed probe, the latter due to the intrinsic 5'-3' exonuclease activity of the polymerase. The mechanism also frees the reporter dye from the quenching activity of TAMRA. As a consequence, the fluorescent activity increases with an increase in cleavage of the probe, which is proportional to the amount of PCR product formed. Accordingly, an amplified target sequence is measured by detecting the intensity of released fluorescence label. Another example for "homogeneous" assay systems are provided by the formats used in the LightCycler® instrument (see e.g. U.S. Pat. No. 6,174,670), some of them sometimes called "kissing probe" formats. Again, the principle is based on two interacting dyes which, however, are characterized in that the emission wavelength of a donor-dye excites an acceptor-dye by fluorescence resonance energy transfer. The COBAS® AmpliPrep instrument (Roche Diagnostics GmbH, D-68305 Mannheim, Germany) was recently introduced to expand automation by isolating target sequences using biotinylated sequence-specific capture probes along with streptavidin-coated magnetic particles (Jungkind, D., J. Clin. Virol. 20 (2001) 1-6; Stelzl, E. et al., J. Clin. Microbiol. 40 (2002) 1447-1450). It has lately been joined by an additional versatile tool, the Total Nucleic Acid Isolation (TNAI) Kit (Roche Diagnostics). This laboratory-use reagent allows the generic, not sequence-specific isolation of all nucleic acids from plasma and serum on the COBAS® AmpliPrep instrument based essentially on the method developed by Boom, R. et al., J. Clin. Microbiol. 28 (1990) 495-503.

[0151]In another preferred embodiment of the invention, the nucleic acid sequence of the amphiregulin marker polynucleotide is the nucleic acid sequence SEQ ID NO: 5, the nucleic acid sequence of the epidermal growth factor marker polynucleotide is the nucleic acid sequence SEQ ID NO: 6, the nucleic acid sequence of the transforming growth factor alpha marker polynucleotide is the nucleic acid sequence SEQ ID NO: 7, or the nucleic acid sequence of the HER2 marker polynucleotide is the nucleic acid sequence SEQ ID NO: 8.

[0152]In another embodiment of the invention, a probe that hybridizes with the epidermal growth factor, transforming growth factor alpha or HER2 marker polynucleotide under stringent conditions or an antibody that binds to the epidermal growth factor, transforming growth factor alpha or HER2 marker protein is used for predicting the response to treatment with a HER inhibitor in a patient or a probe that hybridizes with the amphiregulin, epidermal growth factor, transforming growth factor alpha or HER2 marker polynucleotide under stringent conditions or an antibody that binds to the amphiregulin, epidermal growth factor, transforming growth factor alpha or HER2 marker protein is used for selecting a composition for inhibiting the progression of disease in a patient. The disease is preferably cancer and the patient is preferably a cancer patient as disclosed above.

[0153]In another embodiment of the invention, a kit comprising a probe that anneals with the amphiregulin, epidermal growth factor, transforming growth factor alpha or HER2 marker polynucleotide under stringent conditions or an antibody that binds to the amphiregulin, epidermal growth factor, transforming growth factor alpha or HER2 marker protein is provided. Such kits known in the art further comprise plastics ware which can be used during the amplification procedure as e.g. microtitre plates in the 96 or 384 well format or just ordinary reaction tubes manufactured e.g. by Eppendorf, Hamburg, Germany and all other reagents for carrying out the method according to the invention, preferably an immunoassay, e.g. enzyme linked immunoabsorbent assay (ELISA), fluorescent immunosorbent assay (FIA), chemical linked immunosorbent assay (CLIA), radioimmuno assay (RIA), and immunoblotting. For a review of the different immunoassays and reagents which may be used, see: Lottspeich and Zorbas (eds.), Bioanalytik, 1^st edition, 1998, Spektrum Akademischer Verlag, Heidelberg, Berlin, Germany. Preferably combinations of the probes or antibodies to the various marker polynucleotides or marker proteins are provided in the form of kit as the preferred combinations of the marker polynucleotides or marker proteins as disclosed above.

[0154]In another embodiment of the invention, a method of selecting a composition for inhibiting the progression of disease in a patient is provided, the method comprising: [0155](a) separately exposing aliquots of a biological sample from a cancer patient in the presence of a plurality of test compositions; [0156](b) comparing the level of expression of one or more biomarkers selected from the group consisting of amphiregulin, epidermal growth factor, transforming growth factor alpha and HER2 in the aliquots of the biological sample contacted with the test compositions and the level of expression of such biomarkers in an aliquot of the biological sample not contacted with the test compositions; and [0157](c) selecting one of the test compositions which alters the level of expression of the biomarker or biomarkers in the aliquot containing that test composition relative to the aliquot not contacted with the test composition wherein an at least 10% difference between the level of expression of the biomarker or biomarkers in the aliquot of the biological sample contacted with the test composition and the level of expression of the corresponding biomarker or biomarkers in the aliquot of the biological sample not contacted with the test composition is an indication for the selection of the test composition. The disease is preferably cancer and the patient is preferably a cancer patient as disclosed above.

[0158]In another embodiment of the invention, a method of selecting a composition for inhibiting the progression of disease in a patient is provided, the method comprising: [0159](a) separately exposing aliquots of a biological sample from a cancer patient in the presence of a plurality of test compositions; [0160](b) comparing the level of expression of one or more biomarkers selected from the group consisting of the amphiregulin, epidermal growth factor, transforming growth factor alpha and HER2 in the aliquots of the biological sample contacted with the test compositions and the level of expression of such biomarkers in an aliquot of the biological sample not contacted with the test compositions; and [0161](c) selecting one of the test compositions which alters the level of expression of the biomarker or biomarkers in the aliquot containing that test composition relative to the aliquot not contacted with the test composition wherein an at least 10% difference between the level of expression of the biomarker or biomarkers in the aliquot of the biological sample contacted with the test composition and the level of expression of the corresponding biomarker or biomarkers in the aliquot of the biological sample not contacted with the test composition is an indication for the selection of the test composition. The disease is preferably cancer and the patient is preferably a cancer patient as disclosed above.

[0162]The expression of a marker gene "significantly" differs from the level of expression of the marker gene in a reference sample if the level of expression of the marker gene in a sample from the patient differs from the level in a sample from the reference subject by an amount greater than the standard error of the assay employed to assess expression, and preferably at least 10%, and more preferably 25%, 50%, 75%, 100%, 125%, 150%, 175%, 200%, 300%, 400%, 500% or 1,000% of that amount. Alternatively, expression of the marker gene in the patient can be considered "significantly" lower than the level of expression in a reference subject if the level of expression in a sample from the patient is lower than the level in a sample from the reference subject by an amount greater than the standard error of the assay employed to assess expression, and preferably at least 10%, and more preferably 25%, 50%, 75%, 100%, 125%, 150%, 175%, 200%, 300%, 400%, 500% or 1,000% that amount. The difference of the level of expression be up to 10,000 or 50,000%. The difference of the level of expression is preferably between 10% to 10,000%, more preferably 25% to 10,000%, 50% to 10,000%, 100% to 10,000%, even more preferably 25% to 5,000%, 50% to 5,000%, 100% to 5,000%.

[0163]In another embodiment of the invention, a method of identifying a candidate agent is provided said method comprising: [0164](a) contacting an aliquot of a biological sample from a cancer patient with the candidate agent and determining the level of expression of one or more biomarkers selected from the group consisting of amphiregulin, epidermal growth factor, transforming growth factor alpha and HER2 in the aliquot; [0165](b) determining the level of expression of a corresponding biomarker or biomarkers in an aliquot of the biological sample not contacted with the candidate agent; [0166](c) observing the effect of the candidate agent by comparing the level of expression of the biomarker or biomarkers in the aliquot of the biological sample contacted with the candidate agent and the level of expression of the corresponding biomarker or biomarkers in the aliquot of the biological sample not contacted with the candidate agent; and [0167](d) identifying said agent from said observed effect, wherein an at least 10% difference between the level of expression of the biomarker gene or combination of biomarker genes in the aliquot of the biological sample contacted with the candidate agent and the level of expression of the corresponding biomarker gene or combination of biomarker genes in the aliquot of the biological sample not contacted with the candidate agent is an indication of an effect of the candidate agent.

[0168]In still another embodiment of the invention, a method of identifying a candidate agent is provided said method comprising: [0169](a) contacting an aliquot of a biological sample from a cancer patient with the candidate agent and determining the level of expression in the aliquot of: [0170](1) a biomarker or a combination of biomarkers selected from the group consisting of epidermal growth factor, transforming growth factor alpha and HER2 or; [0171](2) a combination of biomarkers comprising amphiregulin and one or more biomarkers selected from the group consisting of an epidermal growth factor, a transforming growth factor alpha, and HER2, [0172](b) determining the level of expression of a corresponding biomarker or biomarkers in an aliquot of the biological sample not contacted with the candidate agent, [0173](c) observing the effect of the candidate agent by comparing the level of expression of the biomarker or biomarkers in the aliquot of the biological sample contacted with the candidate agent and the level of expression of the corresponding biomarker or biomarkers in the aliquot of the biological sample not contacted with the candidate agent, [0174](d) identifying said agent from said observed effect, wherein an at least 10% difference between the level of expression of the biomarker or biomarkers in the aliquot of the biological sample contacted with the candidate agent and the level of expression of the corresponding biomarker or biomarkers in the aliquot of the biological sample not contacted with the candidate agent is an indication of an effect of the candidate agent.

[0175]Preferably, the candidate agent is a candidate inhibitory agent. Preferably, said candidate agent is a candidate enhancing agent.

[0176]In another embodiment of the invention, a candidate agent derived by the method according to the invention is provided.

[0177]In another embodiment of the invention, a pharmaceutical preparation comprising an agent according to the invention is provided.

[0178]In yet another embodiment of the invention, an agent according to the invention is used for the preparation of a composition for the treatment of cancer. Preferred forms of cancer are disclosed above.

[0179]In another preferred embodiment of the invention, a method of producing a drug comprising the steps of the method according to the invention and [0180](i) synthesizing the candidate agent identified in step (c) above or an analog or derivative thereof in an amount sufficient to provide said drug in a therapeutically effective amount to a subject; and/or [0181](ii) combining the drug candidate the candidate agent identified in step (c) above or an analog or derivative thereof with a pharmaceutically acceptable carrier.

[0182]In another embodiment of the invention, a marker protein or a marker polynucleotide selected from the group consisting of a amphiregulin, epidermal growth factor, transforming growth factor alpha and HER2 marker protein or marker polynucleotide is used for identifying a candidate agent or for selecting a composition for inhibiting the progression of a disease in a patient. The disease is preferably cancer and the patient is preferably a cancer patient as disclosed above.

[0183]In another embodiment of the invention, a HER inhibitor is used for the manufacture of a pharmaceutical composition for treating a human cancer patient characterized in that said treating or treatment includes assessing in a biological sample from the patient [0184](a) a marker gene or a combination of marker genes selected from the group consisting of an epidermal growth factor, a transforming growth factor alpha and a HER2 marker gene or; [0185](b) a combination of marker genes comprising an amphiregulin marker gene and a marker gene selected from the group consisting of an epidermal growth factor, a transforming growth factor alpha and a HER2 marker gene.

[0186]The manufacture of a pharmaceutical composition for treating a human cancer patient and particularly the formulation is described in WO 01/00245, incorporated herein by reference, particularly for the antibody 2C4.

[0187]In an preferred embodiment of the invention, in the use of the HER dimerization inhibitor for the manufacture of a pharmaceutical composition for treating a human cancer patient, the treatment includes assessing the marker gene or the combination of marker genes at least one time or repeatedly during treatment. Preferably, the level of expression of the marker gene or the level of expression of the combination of marker genes is assessed. Preferably, the HER inhibitor is an antibody, preferably the antibody 2C4. Preferably, the patient is a breast cancer, ovarian cancer, lung cancer or prostate cancer patient.

[0188]In all embodiments of the invention, combinations of the marker genes, marker polynucleotides or marker proteins are used as disclosed above. In all embodiments of the invention, preferred values for the difference of the level of expression determined in the respective steps are also as disclosed above.

[0189]The following examples, sequence listing and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention.

EXAMPLES

Statistical Methods

[0190]The statistical tasks comprise the following steps:

1. Pre-selection of candidate biomarkers2. Pre-selection of relevant clinical prognostic covariates3. Selection of biomarker prediction functions at an univariate level4. Selection of biomarker prediction functions including clinical covariates at an univariate level5. Selection of biomarker prediction functions at a multivariate level6. Selection of biomarker prediction functions including clinical covariates at a multivariate levelThe following text details the different steps:

[0191]Ad1: Pre-selection of candidate biomarkers: The statistical pre-selection of candidate biomarkers is oriented towards the strength of association with measures of clinical benefit. For this purpose the different clinical endpoints may be transformed in derived surrogate scores, as e.g. an ordinal assignment of the degree of clinical benefit or morbidity scores regarding TTP or TTD which avoid censored observations. These surrogate transformed measures can be easily used for simple correlation analysis, e.g. by the non-parametric Spearman rank correlation approach. An alternative here is to use the biomarker measurements as metric covariates in Time-to-event regression models, as e.g. Cox proportional hazard regression. Depending on the statistical distribution of the biomarker values this step may require some pre-processing, as e.g. variance stabilizing transformations and the use of suitable scales or, alternatively, a standardization step like e.g. using percentiles instead of raw measurements. A further approach is inspection of bivariate scatter plots, e.g. by displaying the scatter of (x-axis=biomarker value, y-axis=measure of clinical benefit) on a single patient basis. Here also some non-parametric regression line as e.g. achieved by smoothing splines can be useful to visualize the association of biomarker and clinical benefit.

[0192]The goal of these different approaches is the pre-selection of biomarker candidates, which show some association with clinical benefit in at least one of the benefit measures employed, while results for other measures are not contradictory. When there are available control groups, then differences in association of biomarkers with clinical benefit in the different arms could be a sign of differential prediction which makes the biomarker eligible for further consideration.

[0193]Ad2: Pre-selection of relevant clinical prognostic covariates: The term "clinical covariate" here is used to describe all other information about the patient, which are in general available at baseline. These clinical covariates comprise demographic information like sex, age etc., other anamnestic information, concomitant diseases, concomitant therapies, result of physical examinations, common laboratory parameters obtained, known properties of the target tumor, information quantifying the extent of malignant disease, clinical performance scores like ECOG or Karnofsky index, clinical disease staging, timing and result of pretreatments and disease history as well as all similar information, which may be associated with the clinical prognosis. The statistical pre-selection of clinical covariates parallels the approaches for pre-selecting biomarkers and is as well oriented towards the strength of association with measures of clinical benefit. So in principle the same methods apply as considered under 1. In addition to statistical criteria, also criteria from clinical experience and theoretical knowledge may apply to pre-select relevant clinical covariates.

[0194]The prognosis by clinical covariates could interact with the prognosis of the biomarkers. They will be considered for refined prediction rules if necessary.

[0195]Ad3: Selection of biomarker prediction functions at an univariate level: The term "prediction function" will be used in a general sense to mean a numerical function of a biomarker measurement which results in a number which is scaled to imply the target prediction.

[0196]A simple example is the choice of the Heaviside function for a specific cutoff c and a biomarker measurement x, where the binary prediction A or B is to be made, then

If H(x-c)=0 then predict A.

If H(x-c)=1 then predict B.

[0197]This is probably the most common way of using univariate biomarker measurements in prediction rules. The definition of a prediction function usually recurs to an existing training data set which can be used to explore the prediction possibilities. In order to achieve a suitable cutoff c from the training set different routes can be taken. First the scatterplot with smoothing spline mentioned under 1 can be used to define the cutoff. Alternatively some percentile of the distribution could be chosen, e.g. the median or a quartile. Cutoffs can also be systematically extracted by investigating all possible cutoffs according to their prediction potential with regard to the measures of clinical benefit. Then these results can be plotted to allow for an either manual selection or to employ some search algorithm for optimality. This was realized based on the endpoints TTP and TTD using a Cox model, where at each test cutoff the biomarker was used as a binary covariate. Prediction criteria were the resulting Hazard ratios. Then the results for TTP and TTD can be considered together in order to chose a cutoff which shows prediction in line with both endpoints

[0198]Another uncommon approach for choosing a prediction function can be based on a fixed parameter Cox regression model obtained from the training set with biomarker values (possibly transformed) as covariate. Then the prediction could simply depend on whether the computed Hazard ratio is smaller or greater than 1.

[0199]A further possibility is to base the decision on some likelihood ratio (or monotonic transform of it), where the target probability densities were pre-determined in the training set for separation of the prediction states. Then the biomarker would be plugged into some function of the density ratios.

[0200]Ad4: Selection of biomarker prediction functions including clinical covariates at an univariate level: Univariate here refers to using only one biomarker--with regard to clinical covariates this can be a multivariate model. This approach parallels the search without clinical covariates, only that the methods should allow for incorporating the relevant covariate information. The scatterplot method of choosing a cutoff allows only a limited use of covariates, e.g. a binary covariate could be color coded within the plot. If the analysis relies on some regression approach then the use of covariates (also many of them at a time) is usually facilitated. The cutoff search based on the Cox model described under 3, allows for an easy incorporation of covariates and thereby leads to a covariate adjusted univariate cutoff search. The adjustment by covariates may be done as covariates in the model or via the inclusion in a stratified analysis.

[0201]Also the other choices of prediction functions allow for the incorporation of covariates.

[0202]This is straightforward for the Cox model choice as prediction function. There is the option to estimate the influence of covariates on an interaction level, which means that e.g. for different age groups different Hazard ratios apply.

[0203]For the likelihood ratio type of prediction functions, the prediction densities must be estimated including covariates. Here the methodology of multivariate pattern recognition can be used or the biomarker values can be adjusted by multiple regression on the covariates (prior to density estimation).

[0204]The CART technology (Classification And Regression Trees; Breiman L., Friedman J. H., Olshen R. A., Stone C. J., Chapman & Hall (Wadsworth, Inc.), New York, 1984) can be used for a biomarker (raw measurement level) plus clinical covariates employing a clinical benefit measure as response. This way cutoffs are searched and a decision tree type of functions will be found involving the covariates for prediction. The cutoffs and algorithms chosen by CART are frequently close to optimal and may be combined and unified by considering different clinical benefit measures.

[0205]Ad5: Selection of biomarker prediction functions at a multivariate level: When there are several biomarker candidates which maintain their prediction potential within the different univariate prediction function choices, then a further improvement may be achieved by combinations of biomarkers, i.e. considering multivariate prediction functions.

[0206]Based on the simple Heaviside function model combinations of biomarkers may be evaluated, e.g. by considering bivariate scatterplots of biomarker values where optimal cutoffs are indicated. Then a combination of biomarkers can be achieved by combining different Heaviside function by the logical AND and OR operators in order to achieve an improved prediction.

[0207]The CART technology (Classification And Regression Trees) can be used for multiple biomarkers (raw measurement level) and a clinical benefit measure as response, in order to achieve cutoffs for biomarkers and decision tree type of functions for prediction. The cutoffs and algorithms chosen by CART are frequently close to optimal and may be combined and unified by considering different clinical benefit measures.

[0208]The Cox-regression can be employed on different levels. A first way is to incorporate the multiple biomarkers in a binary way (i.e. based on Heaviside functions with some cutoffs). The other option is to employ biomarkers in a metric way (after suitable transformations), or a mixture of the binary and metric approach. The evolving multivariate prediction function is of the Cox type as described under 3.

[0209]The multivariate likelihood ratio approach is difficult to realize but presents as well as an option for multivariate prediction functions.

[0210]Ad6: Selection of biomarker prediction functions including clinical covariates at a multivariate level: When there are relevant clinical covariates then a further improvement may be achieved by combining multiple biomarkers with multiple clinical covariates. The different prediction function choices will be evaluated with respect to the possibilities to include clinical covariates.

[0211]Based on the simple logical combinations of Heaviside functions for the biomarkers, further covariates may be included to the prediction function based on logistic regression model obtained in the training set.

[0212]The CART technology and the evolving decision trees can be easily used with additional covariates, which would include these in the prediction algorithm.

[0213]All prediction functions based on the Cox-regression can use further clinical covariates. There is the option to estimate the influence of covariates on an interaction level, which means that e.g. for different age groups different Hazard ratios apply.

[0214]The multivariate likelihood ratio approach is not directly extendible to the use of additional covariates.

Example 1

Baseline Blood Sera from HER2 Low Expressing Metastatic Breast Cancer Patients Treated with Pertuzumab were Assessed for Levels of HER Ligands and Shedded HER2 (HER2 ECD), as Described Below

[0215]Kits used for assessment of the serum biomarkers:

TABLE-US-00001 Marker Assay Distribution HER2-ECD Bayer HER-2/neu ELISA, DakoCytomation N.V./S.A., Cat. #: EL501 Interleuvenlaan 12B, B-3001 Heverlee Amphiregulin DuoSet ELISA Development R&D Systems Ltd., 19 Barton System Human Amphiregulin, Lane, Abingdon OX14 3NB, UK Cat. #: DY262 EGF Quantikine human EGF R&D Systems Ltd., 19 Barton ELISA kit, Cat. #: DEG00 Lane, Abingdon OX14 3NB, UK TGF-alpha Quantikine ® Human TGF-alpha R&D Systems Ltd., 19 Barton Immunoassay, Cat. #: DTGA00 Lane, Abingdon OX14 3NB, UK

Protocols:

HER2-ECD:

[0216]HER2-ECD ELISA was performed according to the recommendations of the manufacturer.

[0217]Amphiregulin: [0218]Prepare all reagents (provided with the kit), standard dilutions (provided with the kit) and samples

[0219]Provide EvenCoat Goat Anti-mouse IgG microplate strips (R&D, Cat. # CP002; not provided with the kit) in the frame. The frame is now termed ELISA plate.

[0220]Determine of the required number of wells (number of standard dilutions+number of samples).

[0221]Determine the plate layout.

[0222]Add 100 μl diluted capture antibody (provided with the kit; 1:180 in PBS) to each well.

[0223]Incubate at r.t. for 1 hour.

[0224]Aspirate each well and wash, repeating the process three times for a total of four washes. Wash by filling each well with 400 μl Wash buffer (not provided with the kit; 0.05% Tween-20 in PBS was used), using a manifold dispenser, and subsequent aspiration. After the last wash, remove any remaining Wash buffer by aspirating. Invert the plate and blot it against clean paper towels.

[0225]Add 100 μl standard dilution or diluted sample (see below) per well. Change tip after every pipetting step.

[0226]Cover plate with the adhesive strip (provided with the kit).

[0227]Incubate for 2 hours at r.t. on a rocking platform.

[0228]Repeat the aspiration/wash as described previously.

[0229]Aspirated samples and wash solutions are treated with laboratory disinfectant.

[0230]Add 100 μl Detection Antibody (provided with the kit) diluted 1:180 in Reagent diluent (not provided with the kit; 1% BSA (Roth; Albumin Fraction V, Cat. # T844.2) in PBS was used) per well

[0231]Incubate for 2 hours at r.t.

[0232]Repeat the aspiration/wash as described previously.

[0233]Add 100 μl working dilution of the Streptavidin-HRP to each well (provided with the kit; 1:200 dilution in Reagent diluent). Cover with a new adhesive strip.

[0234]Incubate for 20 min at r.t.

[0235]Repeat the aspiration/wash as described previously.

[0236]Add 100 μl Substrate Solution (R&D, Cat. # DY999; not provided with the kit) to each well.

[0237]Incubate for 20 min at r.t. Protect from light. Add 50 μl Stop Solution (1.5 M H2504 (Schwefelsaure reinst, Merck, Cat. # 713); not provided with the kit) to each well. Mix carefully.

[0238]Determine the optical density of each well immediately, using a microplate reader set to 450 nm.

Amphiregulin Standard Curve:

[0239]A 40 ng/ml amphiregulin stock solution was prepared in 1% BSA in PBS, aliquotted and stored at -80° C. Amphiregulin solutions in 20% BSA in PBS were not stable beyond 2 weeks and were therefore not used. From the aliquotted amphiregulin stock solution, the amphiregulin standard curve was prepared freshly in 20% BSA in PBS prior to each experiment. The highest concentration was 1000 pg/ml (1:40 dilution of the amphiregulin stock solution). The standards provided with the ELISA kit produced a linear standard curve. Excel-based analysis of the curves allowed the determination of curve equations for every ELISA.

Amphiregulin Samples:

[0240]When samples were diluted 1:1 in Reagent Diluent, all samples were within the linear range of the ELISA. Each sample was measured in duplicates. Dependent on the quality of the data, and on sufficient amounts of serum, determinations were repeated in subsequent experiments if necessary.

[0241]EGF:

[0242]Prepare all reagents (provided with the kit), standard dilutions (provided with the kit) and samples

[0243]Remove excess antibody-coated microtiter plate strips (provided with the kit) from the frame. The frame is now termed ELISA plate.

[0244]Determine of the required number of wells: (Number of standard dilutions+number of samples)×2

[0245]Determine the plate layout.

[0246]Add 50 μl Assay Diluent RD1 (provided with the kit) to each well

[0247]Add 200 μl standard dilution or diluted sample (e.g. 1:20 in Calibrator Diluent RD6H) per well. Change tip after every pipetting step.

[0248]Cover plate with the adhesive strip (provided with the kit).

[0249]Incubate for 2 hours at r.t. on a rocking platform.

[0250]Aspirate each well and wash, repeating the process three times for a total of four washes. Wash by filling each well with 400 μl Wash Buffer (provided with the kit), using a manifold dispenser, and subsequent aspiration. After the last wash, remove any remaining Wash buffer by aspirating. Invert the plate and blot it against clean paper towels.

[0251]Aspirated samples and wash solutions are treated with laboratory disinfectant. Add 200 μl of Conjugate (provided with the kit) to each well. Cover with a new adhesive strip.

[0252]Incubate for 2 hours at r.t.

[0253]Repeat the aspiration/wash as described previously.

[0254]Add 200 μl Substrate Solution (provided with the kit) to each well.

[0255]Incubate for 20 min at r.t. Protect from light.

[0256]Add 50 μl Stop Solution (provided with the kit) to each well. Mix carefully.

[0257]Determine the optical density of each well within 30 minutes, using a microplate reader set to 450 nm.

EGF Standard Curve:

[0258]The standards provided with the ELISA kit produced a linear standard curve. Also very small concentrations showed detectable results.

[0259]EGF samples:

[0260]A total of four assays with the samples was performed. Each sample was measured 2-5 times, the number of determinations being dependent on the quality of the results (mean+/-SD) and the availability of sufficient amounts of serum. When samples were diluted 1:20 in Calibrator Diluent RD6H, all samples were within the linear range of the ELISA.

TGF-Alpha:

[0261]Prepare all reagents (provided with the kit), standard dilutions (provided with the kit) and samples

[0262]Remove excess antibody-coated microtiter plate strips (provided with the kit) from the frame. The frame is now termed ELISA plate.

[0263]Determine of the required number of wells: (Number of standard dilutions+number of samples)×2

[0264]Determine the plate layout.

[0265]Add 100 μl Assay Diluent RD1W (provided with the kit) to each well

[0266]Add 50 μl standard dilution or sample per well. Change tip after every pipetting step.

[0267]Cover plate with the adhesive strip (provided with the kit).

[0268]Incubate for 2 hours at r.t. on a rocking platform.

[0269]Aspirate each well and wash, repeating the process three times for a total of four washes. Wash by filling each well with 400 μl Wash Buffer (provided with the kit), using a manifold dispenser, and subsequent aspiration. After the last wash, remove any remaining Wash buffer by aspirating. Invert the plate and blot it against clean paper towels.

[0270]Aspirated samples and wash solutions are treated with laboratory disinfectant.

[0271]Add 200 μl of TGF-alpha Cojugate (provided with the kit) to each well. Cover with a new adhesive strip.

[0272]Incubate for 2 hours at r.t.

[0273]Repeat the aspiration/wash as described previously.

[0274]Add 200 μl Substrate Solution (provided with the kit) to each well.

[0275]Incubate for 30 min at r.t. Protect from light.

[0276]Add 50 μl Stop Solution (provided with the kit) to each well. Mix carefully.

[0277]Determine the optical density of each well within 30 minutes, using a microplate reader set to 450 nm.

TGF-Alpha Standard Curve:

[0278]The standards provided with the ELISA kit produced a linear standard curve. Also very small concentrations showed detectable results.

TGF-Alpha Samples:

[0279]A total of four assays with the samples was performed. Samples were measured in 2-4 independent assays.

[0280]The serum data was analyzed to identify factors the baseline serum levels of which would be associated with response to the Pertuzumab treatment. For all factors a skewed pattern of the distribution (mean, standard deviation, median, minimum, maximum) was observed. A monotonic transform was used to reduce the skewness based on the logarithm: Log(x+1). In a univariate analysis, it was explored whether suitable cut-points for the factors could be defined which would relate to the probability of response (in this example defined as clinical benefit). Here, patients with clinical benefit were defined as those who achieved a partial response (PR) or maintained stable disease for at least 6 months. Scatterplots of the factors versus the response categories were investigated. FIG. 1 and FIG. 2 show a plotting of the clinical response categories versus the logarithmic transformation of the serum levels of TGF-alpha and amphiregulin, respectively, to exemplify the approach.

[0281]Based on the scatterplots, cut-points were selected for the factors to define groups of patients, who have experienced greater clinical benefit. FIG. 3 (TGF-alpha), FIG. 4 (Amphiregulin), FIG. 5 (EGF), and FIG. 6 (HER2-ECD) show the clinical benefit in relation to the different factor groupings based on the exploratory cut-points calculated to the original factor units. The cut-points separate out some of the patients without clinical benefit, and hence, elevate the response rate for the group with greater clinical benefit.

Example 2

[0282]In this example the exploratory cut-points from Example 1 were used to assess the univariate effect of the factor groupings on different measures of the clinical benefit of the Pertuzumab treatment, using time to progression/or death (TTP) and time to death (TTD) as alternative clinical endpoints. Significant effects were observed for TGF-alpha, Amphiregulin, EGF and HER2-ECD in Kaplan-Meier estimates and log-rank tests for TTP and/or TTD, as shown in an overview in FIG. 7.

[0283]The Kaplan-Meier plots displaying the hazard ratio are given for TTP and TTD (highest number of events observed) in FIG. 8 and FIG. 9 (TGF-alpha), 10 and 11 (Amphiregulin), 12 and 13 (EGF), and 14 and 15 (HER2-ECD), showing the pronounced effect of a grouping based on these factors on the clinical outcome of the patients treated with Pertuzumab.

Example 3

[0284]In this example multivariate approaches were used to identify combinations of factors that would further improve the identification of patients with greater benefit from the Pertuzumab treatment. Results, as derived from a CART approach (Classification And Regression Trees), are reflected. The CART classification approach made it necessary to specify as the benefit group all values in clinical benefit above of 0. As variables serum levels of HER2-ECD, TGF-alpha, Amphiregulin, and EGF were employed. A combination of serum HER2-ECD and serum TGF-alpha levels were selected to give best results. From the CART results optimized cut-points for a combination of serum HER2-ECD and serum TGF-alpha levels were derived, resulting in a rule for exploratory categorization of clinical benefit in the study population--a combination of low serum HER2-ECD values and low serum TGF-alpha values capturing 2/2 PR and 2/3 SD>6 months in the study population and excluding a reasonable number of fast progressing patients. FIG. 16 shows the clinical benefit in relation to the TGF-alpha/HER2-ECD combination groupings based on the exploratory combination cut-point. FIG. 17 summarizes the effect of a combination of TGF-alpha and HER2-ECD on TTP and TTD. The Kaplan-Meier estimates and the hazard ratios given in FIG. 18 (TTP) and FIG. 19 (TTD) demonstrate the significant effect of the grouping based on a combination of these factors for on the clinical outcome of the patients treated with Pertuzumab.

[0285]Unless stated to the contrary, all compounds in the examples were prepared and characterized as described. All ranges recited herein encompass all combinations and subcombinations included within that range limit. All patents and publications cited herein are hereby incorporated by reference in their entirety for any purpose.

Sequence CWU 1

141252PRTHomo sapiens 1Met Arg Ala Pro Leu Leu Pro Pro Ala Pro Val Val Leu Ser Leu Leu1 5 10 15Ile Leu Gly Ser Gly His Tyr Ala Ala Gly Leu Asp Leu Asn Asp Thr 20 25 30Tyr Ser Gly Lys Arg Glu Pro Phe Ser Gly Asp His Ser Ala Asp Gly 35 40 45Phe Glu Val Thr Ser Arg Ser Glu Met Ser Ser Gly Ser Glu Ile Ser 50 55 60Pro Val Ser Glu Met Pro Ser Ser Ser Glu Pro Ser Ser Gly Ala Asp65 70 75 80Tyr Asp Tyr Ser Glu Glu Tyr Asp Asn Glu Pro Gln Ile Pro Gly Tyr 85 90 95Ile Val Asp Asp Ser Val Arg Val Glu Gln Val Val Lys Pro Pro Gln 100 105 110Asn Lys Thr Glu Ser Glu Asn Thr Ser Asp Lys Pro Lys Arg Lys Lys 115 120 125Lys Gly Gly Lys Asn Gly Lys Asn Arg Arg Asn Arg Lys Lys Lys Asn 130 135 140Pro Cys Asn Ala Glu Phe Gln Asn Phe Cys Ile His Gly Glu Cys Lys145 150 155 160Tyr Ile Glu His Leu Glu Ala Val Thr Cys Lys Cys Gln Gln Glu Tyr 165 170 175Phe Gly Glu Arg Cys Gly Glu Lys Ser Met Lys Thr His Ser Met Ile 180 185 190Asp Ser Ser Leu Ser Lys Ile Ala Leu Ala Ala Ile Ala Ala Phe Met 195 200 205Ser Ala Val Ile Leu Thr Ala Val Ala Val Ile Thr Val Gln Leu Arg 210 215 220Arg Gln Tyr Val Arg Lys Tyr Glu Gly Glu Ala Glu Glu Arg Lys Lys225 230 235 240Leu Arg Gln Glu Asn Gly Asn Val His Ala Ile Ala 245 25021207PRTHomo sapiens 2Met Leu Leu Thr Leu Ile Ile Leu Leu Pro Val Val Ser Lys Phe Ser1 5 10 15Phe Val Ser Leu Ser Ala Pro Gln His Trp Ser Cys Pro Glu Gly Thr 20 25 30Leu Ala Gly Asn Gly Asn Ser Thr Cys Val Gly Pro Ala Pro Phe Leu 35 40 45Ile Phe Ser His Gly Asn Ser Ile Phe Arg Ile Asp Thr Glu Gly Thr 50 55 60Asn Tyr Glu Gln Leu Val Val Asp Ala Gly Val Ser Val Ile Met Asp65 70 75 80Phe His Tyr Asn Glu Lys Arg Ile Tyr Trp Val Asp Leu Glu Arg Gln 85 90 95Leu Leu Gln Arg Val Phe Leu Asn Gly Ser Arg Gln Glu Arg Val Cys 100 105 110Asn Ile Glu Lys Asn Val Ser Gly Met Ala Ile Asn Trp Ile Asn Glu 115 120 125Glu Val Ile Trp Ser Asn Gln Gln Glu Gly Ile Ile Thr Val Thr Asp 130 135 140Met Lys Gly Asn Asn Ser His Ile Leu Leu Ser Ala Leu Lys Tyr Pro145 150 155 160Ala Asn Val Ala Val Asp Pro Val Glu Arg Phe Ile Phe Trp Ser Ser 165 170 175Glu Val Ala Gly Ser Leu Tyr Arg Ala Asp Leu Asp Gly Val Gly Val 180 185 190Lys Ala Leu Leu Glu Thr Ser Glu Lys Ile Thr Ala Val Ser Leu Asp 195 200 205Val Leu Asp Lys Arg Leu Phe Trp Ile Gln Tyr Asn Arg Glu Gly Ser 210 215 220Asn Ser Leu Ile Cys Ser Cys Asp Tyr Asp Gly Gly Ser Val His Ile225 230 235 240Ser Lys His Pro Thr Gln His Asn Leu Phe Ala Met Ser Leu Phe Gly 245 250 255Asp Arg Ile Phe Tyr Ser Thr Trp Lys Met Lys Thr Ile Trp Ile Ala 260 265 270Asn Lys His Thr Gly Lys Asp Met Val Arg Ile Asn Leu His Ser Ser 275 280 285Phe Val Pro Leu Gly Glu Leu Lys Val Val His Pro Leu Ala Gln Pro 290 295 300Lys Ala Glu Asp Asp Thr Trp Glu Pro Glu Gln Lys Leu Cys Lys Leu305 310 315 320Arg Lys Gly Asn Cys Ser Ser Thr Val Cys Gly Gln Asp Leu Gln Ser 325 330 335His Leu Cys Met Cys Ala Glu Gly Tyr Ala Leu Ser Arg Asp Arg Lys 340 345 350Tyr Cys Glu Asp Val Asn Glu Cys Ala Phe Trp Asn His Gly Cys Thr 355 360 365Leu Gly Cys Lys Asn Thr Pro Gly Ser Tyr Tyr Cys Thr Cys Pro Val 370 375 380Gly Phe Val Leu Leu Pro Asp Gly Lys Arg Cys His Gln Leu Val Ser385 390 395 400Cys Pro Arg Asn Val Ser Glu Cys Ser His Asp Cys Val Leu Thr Ser 405 410 415Glu Gly Pro Leu Cys Phe Cys Pro Glu Gly Ser Val Leu Glu Arg Asp 420 425 430Gly Lys Thr Cys Ser Gly Cys Ser Ser Pro Asp Asn Gly Gly Cys Ser 435 440 445Gln Leu Cys Val Pro Leu Ser Pro Val Ser Trp Glu Cys Asp Cys Phe 450 455 460Pro Gly Tyr Asp Leu Gln Leu Asp Glu Lys Ser Cys Ala Ala Ser Gly465 470 475 480Pro Gln Pro Phe Leu Leu Phe Ala Asn Ser Gln Asp Ile Arg His Met 485 490 495His Phe Asp Gly Thr Asp Tyr Gly Thr Leu Leu Ser Gln Gln Met Gly 500 505 510Met Val Tyr Ala Leu Asp His Asp Pro Val Glu Asn Lys Ile Tyr Phe 515 520 525Ala His Thr Ala Leu Lys Trp Ile Glu Arg Ala Asn Met Asp Gly Ser 530 535 540Gln Arg Glu Arg Leu Ile Glu Glu Gly Val Asp Val Pro Glu Gly Leu545 550 555 560Ala Val Asp Trp Ile Gly Arg Arg Phe Tyr Trp Thr Asp Arg Gly Lys 565 570 575Ser Leu Ile Gly Arg Ser Asp Leu Asn Gly Lys Arg Ser Lys Ile Ile 580 585 590Thr Lys Glu Asn Ile Ser Gln Pro Arg Gly Ile Ala Val His Pro Met 595 600 605Ala Lys Arg Leu Phe Trp Thr Asp Thr Gly Ile Asn Pro Arg Ile Glu 610 615 620Ser Ser Ser Leu Gln Gly Leu Gly Arg Leu Val Ile Ala Ser Ser Asp625 630 635 640Leu Ile Trp Pro Ser Gly Ile Thr Ile Asp Phe Leu Thr Asp Lys Leu 645 650 655Tyr Trp Cys Asp Ala Lys Gln Ser Val Ile Glu Met Ala Asn Leu Asp 660 665 670Gly Ser Lys Arg Arg Arg Leu Thr Gln Asn Asp Val Gly His Pro Phe 675 680 685Ala Val Ala Val Phe Glu Asp Tyr Val Trp Phe Ser Asp Trp Ala Met 690 695 700Pro Ser Val Ile Arg Val Asn Lys Arg Thr Gly Lys Asp Arg Val Arg705 710 715 720Leu Gln Gly Ser Met Leu Lys Pro Ser Ser Leu Val Val Val His Pro 725 730 735Leu Ala Lys Pro Gly Ala Asp Pro Cys Leu Tyr Gln Asn Gly Gly Cys 740 745 750Glu His Ile Cys Lys Lys Arg Leu Gly Thr Ala Trp Cys Ser Cys Arg 755 760 765Glu Gly Phe Met Lys Ala Ser Asp Gly Lys Thr Cys Leu Ala Leu Asp 770 775 780Gly His Gln Leu Leu Ala Gly Gly Glu Val Asp Leu Lys Asn Gln Val785 790 795 800Thr Pro Leu Asp Ile Leu Ser Lys Thr Arg Val Ser Glu Asp Asn Ile 805 810 815Thr Glu Ser Gln His Met Leu Val Ala Glu Ile Met Val Ser Asp Gln 820 825 830Asp Asp Cys Ala Pro Val Gly Cys Ser Met Tyr Ala Arg Cys Ile Ser 835 840 845Glu Gly Glu Asp Ala Thr Cys Gln Cys Leu Lys Gly Phe Ala Gly Asp 850 855 860Gly Lys Leu Cys Ser Asp Ile Asp Glu Cys Glu Met Gly Val Pro Val865 870 875 880Cys Pro Pro Ala Ser Ser Lys Cys Ile Asn Thr Glu Gly Gly Tyr Val 885 890 895Cys Arg Cys Ser Glu Gly Tyr Gln Gly Asp Gly Ile His Cys Leu Asp 900 905 910Ile Asp Glu Cys Gln Leu Gly Val His Ser Cys Gly Glu Asn Ala Ser 915 920 925Cys Thr Asn Thr Glu Gly Gly Tyr Thr Cys Met Cys Ala Gly Arg Leu 930 935 940Ser Glu Pro Gly Leu Ile Cys Pro Asp Ser Thr Pro Pro Pro His Leu945 950 955 960Arg Glu Asp Asp His His Tyr Ser Val Arg Asn Ser Asp Ser Glu Cys 965 970 975Pro Leu Ser His Asp Gly Tyr Cys Leu His Asp Gly Val Cys Met Tyr 980 985 990Ile Glu Ala Leu Asp Lys Tyr Ala Cys Asn Cys Val Val Gly Tyr Ile 995 1000 1005Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys Trp Trp Glu Leu Arg 1010 1015 1020His Ala Gly His Gly Gln Gln Gln Lys Val Ile Val Val Ala Val 1025 1030 1035Cys Val Val Val Leu Val Met Leu Leu Leu Leu Ser Leu Trp Gly 1040 1045 1050Ala His Tyr Tyr Arg Thr Gln Lys Leu Leu Ser Lys Asn Pro Lys 1055 1060 1065Asn Pro Tyr Glu Glu Ser Ser Arg Asp Val Arg Ser Arg Arg Pro 1070 1075 1080Ala Asp Thr Glu Asp Gly Met Ser Ser Cys Pro Gln Pro Trp Phe 1085 1090 1095Val Val Ile Lys Glu His Gln Asp Leu Lys Asn Gly Gly Gln Pro 1100 1105 1110Val Ala Gly Glu Asp Gly Gln Ala Ala Asp Gly Ser Met Gln Pro 1115 1120 1125Thr Ser Trp Arg Gln Glu Pro Gln Leu Cys Gly Met Gly Thr Glu 1130 1135 1140Gln Gly Cys Trp Ile Pro Val Ser Ser Asp Lys Gly Ser Cys Pro 1145 1150 1155Gln Val Met Glu Arg Ser Phe His Met Pro Ser Tyr Gly Thr Gln 1160 1165 1170Thr Leu Glu Gly Gly Val Glu Lys Pro His Ser Leu Leu Ser Ala 1175 1180 1185Asn Pro Leu Trp Gln Gln Arg Ala Leu Asp Pro Pro His Gln Met 1190 1195 1200Glu Leu Thr Gln 12053160PRTHomo sapiens 3Met Val Pro Ser Ala Gly Gln Leu Ala Leu Phe Ala Leu Gly Ile Val1 5 10 15Leu Ala Ala Cys Gln Ala Leu Glu Asn Ser Thr Ser Pro Leu Ser Ala 20 25 30Asp Pro Pro Val Ala Ala Ala Val Val Ser His Phe Asn Asp Cys Pro 35 40 45Asp Ser His Thr Gln Phe Cys Phe His Gly Thr Cys Arg Phe Leu Val 50 55 60Gln Glu Asp Lys Pro Ala Cys Val Cys His Ser Gly Tyr Val Gly Ala65 70 75 80Arg Cys Glu His Ala Asp Leu Leu Ala Val Val Ala Ala Ser Gln Lys 85 90 95Lys Gln Ala Ile Thr Ala Leu Val Val Val Ser Ile Val Ala Leu Ala 100 105 110Val Leu Ile Ile Thr Cys Val Leu Ile His Cys Cys Gln Val Arg Lys 115 120 125His Cys Glu Trp Cys Arg Ala Leu Ile Cys Arg His Glu Lys Pro Ser 130 135 140Ala Leu Leu Lys Gly Arg Thr Ala Cys Cys His Ser Glu Thr Val Val145 150 155 16041255PRTHomo sapiens 4Met Glu Leu Ala Ala Leu Cys Arg Trp Gly Leu Leu Leu Ala Leu Leu1 5 10 15Pro Pro Gly Ala Ala Ser Thr Gln Val Cys Thr Gly Thr Asp Met Lys 20 25 30Leu Arg Leu Pro Ala Ser Pro Glu Thr His Leu Asp Met Leu Arg His 35 40 45Leu Tyr Gln Gly Cys Gln Val Val Gln Gly Asn Leu Glu Leu Thr Tyr 50 55 60Leu Pro Thr Asn Ala Ser Leu Ser Phe Leu Gln Asp Ile Gln Glu Val65 70 75 80Gln Gly Tyr Val Leu Ile Ala His Asn Gln Val Arg Gln Val Pro Leu 85 90 95Gln Arg Leu Arg Ile Val Arg Gly Thr Gln Leu Phe Glu Asp Asn Tyr 100 105 110Ala Leu Ala Val Leu Asp Asn Gly Asp Pro Leu Asn Asn Thr Thr Pro 115 120 125Val Thr Gly Ala Ser Pro Gly Gly Leu Arg Glu Leu Gln Leu Arg Ser 130 135 140Leu Thr Glu Ile Leu Lys Gly Gly Val Leu Ile Gln Arg Asn Pro Gln145 150 155 160Leu Cys Tyr Gln Asp Thr Ile Leu Trp Lys Asp Ile Phe His Lys Asn 165 170 175Asn Gln Leu Ala Leu Thr Leu Ile Asp Thr Asn Arg Ser Arg Ala Cys 180 185 190His Pro Cys Ser Pro Met Cys Lys Gly Ser Arg Cys Trp Gly Glu Ser 195 200 205Ser Glu Asp Cys Gln Ser Leu Thr Arg Thr Val Cys Ala Gly Gly Cys 210 215 220Ala Arg Cys Lys Gly Pro Leu Pro Thr Asp Cys Cys His Glu Gln Cys225 230 235 240Ala Ala Gly Cys Thr Gly Pro Lys His Ser Asp Cys Leu Ala Cys Leu 245 250 255His Phe Asn His Ser Gly Ile Cys Glu Leu His Cys Pro Ala Leu Val 260 265 270Thr Tyr Asn Thr Asp Thr Phe Glu Ser Met Pro Asn Pro Glu Gly Arg 275 280 285Tyr Thr Phe Gly Ala Ser Cys Val Thr Ala Cys Pro Tyr Asn Tyr Leu 290 295 300Ser Thr Asp Val Gly Ser Cys Thr Leu Val Cys Pro Leu His Asn Gln305 310 315 320Glu Val Thr Ala Glu Asp Gly Thr Gln Arg Cys Glu Lys Cys Ser Lys 325 330 335Pro Cys Ala Arg Val Cys Tyr Gly Leu Gly Met Glu His Leu Arg Glu 340 345 350Val Arg Ala Val Thr Ser Ala Asn Ile Gln Glu Phe Ala Gly Cys Lys 355 360 365Lys Ile Phe Gly Ser Leu Ala Phe Leu Pro Glu Ser Phe Asp Gly Asp 370 375 380Pro Ala Ser Asn Thr Ala Pro Leu Gln Pro Glu Gln Leu Gln Val Phe385 390 395 400Glu Thr Leu Glu Glu Ile Thr Gly Tyr Leu Tyr Ile Ser Ala Trp Pro 405 410 415Asp Ser Leu Pro Asp Leu Ser Val Phe Gln Asn Leu Gln Val Ile Arg 420 425 430Gly Arg Ile Leu His Asn Gly Ala Tyr Ser Leu Thr Leu Gln Gly Leu 435 440 445Gly Ile Ser Trp Leu Gly Leu Arg Ser Leu Arg Glu Leu Gly Ser Gly 450 455 460Leu Ala Leu Ile His His Asn Thr His Leu Cys Phe Val His Thr Val465 470 475 480Pro Trp Asp Gln Leu Phe Arg Asn Pro His Gln Ala Leu Leu His Thr 485 490 495Ala Asn Arg Pro Glu Asp Glu Cys Val Gly Glu Gly Leu Ala Cys His 500 505 510Gln Leu Cys Ala Arg Gly His Cys Trp Gly Pro Gly Pro Thr Gln Cys 515 520 525Val Asn Cys Ser Gln Phe Leu Arg Gly Gln Glu Cys Val Glu Glu Cys 530 535 540Arg Val Leu Gln Gly Leu Pro Arg Glu Tyr Val Asn Ala Arg His Cys545 550 555 560Leu Pro Cys His Pro Glu Cys Gln Pro Gln Asn Gly Ser Val Thr Cys 565 570 575Phe Gly Pro Glu Ala Asp Gln Cys Val Ala Cys Ala His Tyr Lys Asp 580 585 590Pro Pro Phe Cys Val Ala Arg Cys Pro Ser Gly Val Lys Pro Asp Leu 595 600 605Ser Tyr Met Pro Ile Trp Lys Phe Pro Asp Glu Glu Gly Ala Cys Gln 610 615 620Pro Cys Pro Ile Asn Cys Thr His Ser Cys Val Asp Leu Asp Asp Lys625 630 635 640Gly Cys Pro Ala Glu Gln Arg Ala Ser Pro Leu Thr Ser Ile Ile Ser 645 650 655Ala Val Val Gly Ile Leu Leu Val Val Val Leu Gly Val Val Phe Gly 660 665 670Ile Leu Ile Lys Arg Arg Gln Gln Lys Ile Arg Lys Tyr Thr Met Arg 675 680 685Arg Leu Leu Gln Glu Thr Glu Leu Val Glu Pro Leu Thr Pro Ser Gly 690 695 700Ala Met Pro Asn Gln Ala Gln Met Arg Ile Leu Lys Glu Thr Glu Leu705 710 715 720Arg Lys Val Lys Val Leu Gly Ser Gly Ala Phe Gly Thr Val Tyr Lys 725 730 735Gly Ile Trp Ile Pro Asp Gly Glu Asn Val Lys Ile Pro Val Ala Ile 740 745 750Lys Val Leu Arg Glu Asn Thr Ser Pro Lys Ala Asn Lys Glu Ile Leu 755 760 765Asp Glu Ala Tyr Val Met Ala Gly Val Gly Ser Pro Tyr Val Ser Arg 770 775 780Leu Leu Gly Ile Cys Leu Thr Ser Thr Val Gln Leu Val Thr Gln Leu785 790 795 800Met Pro Tyr Gly Cys Leu Leu Asp His Val Arg Glu Asn Arg Gly Arg 805 810 815Leu Gly Ser Gln Asp Leu Leu Asn Trp Cys Met Gln Ile Ala Lys Gly 820 825 830Met Ser Tyr Leu Glu Asp Val Arg Leu Val His Arg Asp Leu Ala Ala 835

840 845Arg Asn Val Leu Val Lys Ser Pro Asn His Val Lys Ile Thr Asp Phe 850 855 860Gly Leu Ala Arg Leu Leu Asp Ile Asp Glu Thr Glu Tyr His Ala Asp865 870 875 880Gly Gly Lys Val Pro Ile Lys Trp Met Ala Leu Glu Ser Ile Leu Arg 885 890 895Arg Arg Phe Thr His Gln Ser Asp Val Trp Ser Tyr Gly Val Thr Val 900 905 910Trp Glu Leu Met Thr Phe Gly Ala Lys Pro Tyr Asp Gly Ile Pro Ala 915 920 925Arg Glu Ile Pro Asp Leu Leu Glu Lys Gly Glu Arg Leu Pro Gln Pro 930 935 940Pro Ile Cys Thr Ile Asp Val Tyr Met Ile Met Val Lys Cys Trp Met945 950 955 960Ile Asp Ser Glu Cys Arg Pro Arg Phe Arg Glu Leu Val Ser Glu Phe 965 970 975Ser Arg Met Ala Arg Asp Pro Gln Arg Phe Val Val Ile Gln Asn Glu 980 985 990Asp Leu Gly Pro Ala Ser Pro Leu Asp Ser Thr Phe Tyr Arg Ser Leu 995 1000 1005Leu Glu Asp Asp Asp Met Gly Asp Leu Val Asp Ala Glu Glu Tyr 1010 1015 1020Leu Val Pro Gln Gln Gly Phe Phe Cys Pro Asp Pro Ala Pro Gly 1025 1030 1035Ala Gly Gly Met Val His His Arg His Arg Ser Ser Ser Thr Arg 1040 1045 1050Ser Gly Gly Gly Asp Leu Thr Leu Gly Leu Glu Pro Ser Glu Glu 1055 1060 1065Glu Ala Pro Arg Ser Pro Leu Ala Pro Ser Glu Gly Ala Gly Ser 1070 1075 1080Asp Val Phe Asp Gly Asp Leu Gly Met Gly Ala Ala Lys Gly Leu 1085 1090 1095Gln Ser Leu Pro Thr His Asp Pro Ser Pro Leu Gln Arg Tyr Ser 1100 1105 1110Glu Asp Pro Thr Val Pro Leu Pro Ser Glu Thr Asp Gly Tyr Val 1115 1120 1125Ala Pro Leu Thr Cys Ser Pro Gln Pro Glu Tyr Val Asn Gln Pro 1130 1135 1140Asp Val Arg Pro Gln Pro Pro Ser Pro Arg Glu Gly Pro Leu Pro 1145 1150 1155Ala Ala Arg Pro Ala Gly Ala Thr Leu Glu Arg Pro Lys Thr Leu 1160 1165 1170Ser Pro Gly Lys Asn Gly Val Val Lys Asp Val Phe Ala Phe Gly 1175 1180 1185Gly Ala Val Glu Asn Pro Glu Tyr Leu Thr Pro Gln Gly Gly Ala 1190 1195 1200Ala Pro Gln Pro His Pro Pro Pro Ala Phe Ser Pro Ala Phe Asp 1205 1210 1215Asn Leu Tyr Tyr Trp Asp Gln Asp Pro Pro Glu Arg Gly Ala Pro 1220 1225 1230Pro Ser Thr Phe Lys Gly Thr Pro Thr Ala Glu Asn Pro Glu Tyr 1235 1240 1245Leu Gly Leu Asp Val Pro Val 1250 125551270DNAHomo sapiens 5agacgttcgc acacctgggt gccagcgccc cagaggtccc gggacagccc gaggcgccgc 60gcccgccgcc ccgagctccc caagccttcg agagcggcgc acactcccgg tctccactcg 120ctcttccaac acccgctcgt tttggcggca gctcgtgtcc cagagaccga gttgccccag 180agaccgagac gccgccgctg cgaaggacca atgagagccc cgctgctacc gccggcgccg 240gtggtgctgt cgctcttgat actcggctca ggccattatg ctgctggatt ggacctcaat 300gacacctact ctgggaagcg tgaaccattt tctggggacc acagtgctga tggatttgag 360gttacctcaa gaagtgagat gtcttcaggg agtgagattt cccctgtgag tgaaatgcct 420tctagtagtg aaccgtcctc gggagccgac tatgactact cagaagagta tgataacgaa 480ccacaaatac ctggctatat tgtcgatgat tcagtcagag ttgaacaggt agttaagccc 540ccccaaaaca agacggaaag tgaaaatact tcagataaac ccaaaagaaa gaaaaaggga 600ggcaaaaatg gaaaaaatag aagaaacaga aagaagaaaa atccatgtaa tgcagaattt 660caaaatttct gcattcacgg agaatgcaaa tatatagagc acctggaagc agtaacatgc 720aaatgtcagc aagaatattt cggtgaacgg tgtggggaaa agtccatgaa aactcacagc 780atgattgaca gtagtttatc aaaaattgca ttagcagcca tagctgcctt tatgtctgct 840gtgatcctca cagctgttgc tgttattaca gtccagctta gaagacaata cgtcaggaaa 900tatgaaggag aagctgagga acgaaagaaa cttcgacaag agaatggaaa tgtacatgct 960atagcataac tgaagataaa attacaggat atcacattgg agtcactgcc aagtcatagc 1020cataaatgat gagtcggtcc tctttccagt ggatcataag acaatggacc ctttttgtta 1080tgatggtttt aaactttcaa ttgtcacttt ttatgctatt tctgtatata aaggtgcacg 1140aaggtaaaaa gtattttttc aagttgtaaa taatttattt aatatttaat ggaagtgtat 1200ttattttaca gctcattaaa cttttttaac caaacagaaa aaaaaaaaaa aaaaaaaaaa 1260aaaaaaaaaa 127064877DNAHomo sapiens 6actgttggga gaggaatcgt atctccatat ttcttctttc agccccaatc caagggttgt 60agctggaact ttccatcagt tcttcctttc tttttcctct ctaagccttt gccttgctct 120gtcacagtga agtcagccag agcagggctg ttaaactctg tgaaatttgt cataagggtg 180tcaggtattt cttactggct tccaaagaaa catagataaa gaaatctttc ctgtggcttc 240ccttggcagg ctgcattcag aaggtctctc agttgaagaa agagcttgga ggacaacagc 300acaacaggag agtaaaagat gccccagggc tgaggcctcc gctcaggcag ccgcatctgg 360ggtcaatcat actcaccttg cccgggccat gctccagcaa aatcaagctg ttttcttttg 420aaagttcaaa ctcatcaaga ttatgctgct cactcttatc attctgttgc cagtagtttc 480aaaatttagt tttgttagtc tctcagcacc gcagcactgg agctgtcctg aaggtactct 540cgcaggaaat gggaattcta cttgtgtggg tcctgcaccc ttcttaattt tctcccatgg 600aaatagtatc tttaggattg acacagaagg aaccaattat gagcaattgg tggtggatgc 660tggtgtctca gtgatcatgg attttcatta taatgagaaa agaatctatt gggtggattt 720agaaagacaa cttttgcaaa gagtttttct gaatgggtca aggcaagaga gagtatgtaa 780tatagagaaa aatgtttctg gaatggcaat aaattggata aatgaagaag ttatttggtc 840aaatcaacag gaaggaatca ttacagtaac agatatgaaa ggaaataatt cccacattct 900tttaagtgct ttaaaatatc ctgcaaatgt agcagttgat ccagtagaaa ggtttatatt 960ttggtcttca gaggtggctg gaagccttta tagagcagat ctcgatggtg tgggagtgaa 1020ggctctgttg gagacatcag agaaaataac agctgtgtca ttggatgtgc ttgataagcg 1080gctgttttgg attcagtaca acagagaagg aagcaattct cttatttgct cctgtgatta 1140tgatggaggt tctgtccaca ttagtaaaca tccaacacag cataatttgt ttgcaatgtc 1200cctttttggt gaccgtatct tctattcaac atggaaaatg aagacaattt ggatagccaa 1260caaacacact ggaaaggaca tggttagaat taacctccat tcatcatttg taccacttgg 1320tgaactgaaa gtagtgcatc cacttgcaca acccaaggca gaagatgaca cttgggagcc 1380tgagcagaaa ctttgcaaat tgaggaaagg aaactgcagc agcactgtgt gtgggcaaga 1440cctccagtca cacttgtgca tgtgtgcaga gggatacgcc ctaagtcgag accggaagta 1500ctgtgaagat gttaatgaat gtgctttttg gaatcatggc tgtactcttg ggtgtaaaaa 1560cacccctgga tcctattact gcacgtgccc tgtaggattt gttctgcttc ctgatgggaa 1620acgatgtcat caacttgttt cctgtccacg caatgtgtct gaatgcagcc atgactgtgt 1680tctgacatca gaaggtccct tatgtttctg tcctgaaggc tcagtgcttg agagagatgg 1740gaaaacatgt agcggttgtt cctcacccga taatggtgga tgtagccagc tctgcgttcc 1800tcttagccca gtatcctggg aatgtgattg ctttcctggg tatgacctac aactggatga 1860aaaaagctgt gcagcttcag gaccacaacc atttttgctg tttgccaatt ctcaagatat 1920tcgacacatg cattttgatg gaacagacta tggaactctg ctcagccagc agatgggaat 1980ggtttatgcc ctagatcatg accctgtgga aaataagata tactttgccc atacagccct 2040gaagtggata gagagagcta atatggatgg ttcccagcga gaaaggctta ttgaggaagg 2100agtagatgtg ccagaaggtc ttgctgtgga ctggattggc cgtagattct attggacaga 2160cagagggaaa tctctgattg gaaggagtga tttaaatggg aaacgttcca aaataatcac 2220taaggagaac atctctcaac cacgaggaat tgctgttcat ccaatggcca agagattatt 2280ctggactgat acagggatta atccacgaat tgaaagttct tccctccaag gccttggccg 2340tctggttata gccagctctg atctaatctg gcccagtgga ataacgattg acttcttaac 2400tgacaagttg tactggtgcg atgccaagca gtctgtgatt gaaatggcca atctggatgg 2460ttcaaaacgc cgaagactta cccagaatga tgtaggtcac ccatttgctg tagcagtgtt 2520tgaggattat gtgtggttct cagattgggc tatgccatca gtaataagag taaacaagag 2580gactggcaaa gatagagtac gtctccaagg cagcatgctg aagccctcat cactggttgt 2640ggttcatcca ttggcaaaac caggagcaga tccctgctta tatcaaaacg gaggctgtga 2700acatatttgc aaaaagaggc ttggaactgc ttggtgttcg tgtcgtgaag gttttatgaa 2760agcctcagat gggaaaacgt gtctggctct ggatggtcat cagctgttgg caggtggtga 2820agttgatcta aagaaccaag taacaccatt ggacatcttg tccaagacta gagtgtcaga 2880agataacatt acagaatctc aacacatgct agtggctgaa atcatggtgt cagatcaaga 2940tgactgtgct cctgtgggat gcagcatgta tgctcggtgt atttcagagg gagaggatgc 3000cacatgtcag tgtttgaaag gatttgctgg ggatggaaaa ctatgttctg atatagatga 3060atgtgagatg ggtgtcccag tgtgcccccc tgcctcctcc aagtgcatca acaccgaagg 3120tggttatgtc tgccggtgct cagaaggcta ccaaggagat gggattcact gtcttgatat 3180tgatgagtgc caactggggg tgcacagctg tggagagaat gccagctgca caaatacaga 3240gggaggctat acctgcatgt gtgctggacg cctgtctgaa ccaggactga tttgccctga 3300ctctactcca ccccctcacc tcagggaaga tgaccaccac tattccgtaa gaaatagtga 3360ctctgaatgt cccctgtccc acgatgggta ctgcctccat gatggtgtgt gcatgtatat 3420tgaagcattg gacaagtatg catgcaactg tgttgttggc tacatcgggg agcgatgtca 3480gtaccgagac ctgaagtggt gggaactgcg ccacgctggc cacgggcagc agcagaaggt 3540catcgtggtg gctgtctgcg tggtggtgct tgtcatgctg ctcctcctga gcctgtgggg 3600ggcccactac tacaggactc agaagctgct atcgaaaaac ccaaagaatc cttatgagga 3660gtcgagcaga gatgtgagga gtcgcaggcc tgctgacact gaggatggga tgtcctcttg 3720ccctcaacct tggtttgtgg ttataaaaga acaccaagac ctcaagaatg ggggtcaacc 3780agtggctggt gaggatggcc aggcagcaga tgggtcaatg caaccaactt catggaggca 3840ggagccccag ttatgtggaa tgggcacaga gcaaggctgc tggattccag tatccagtga 3900taagggctcc tgtccccagg taatggagcg aagctttcat atgccctcct atgggacaca 3960gacccttgaa gggggtgtcg agaagcccca ttctctccta tcagctaacc cattatggca 4020acaaagggcc ctggacccac cacaccaaat ggagctgact cagtgaaaac tggaattaaa 4080aggaaagtca agaagaatga actatgtcga tgcacagtat cttttctttc aaaagtagag 4140caaaactata ggttttggtt ccacaatctc tacgactaat cacctactca atgcctggag 4200acagatacgt agttgtgctt ttgtttgctc ttttaagcag tctcactgca gtcttatttc 4260caagtaagag tactgggaga atcactaggt aacttattag aaacccaaat tgggacaaca 4320gtgctttgta aattgtgttg tcttcagcag tcaatacaaa tagatttttg tttttgttgt 4380tcctgcagcc ccagaagaaa ttaggggtta aagcagacag tcacactggt ttggtcagtt 4440acaaagtaat ttctttgatc tggacagaac atttatatca gtttcatgaa atgattggaa 4500tattacaata ccgttaagat acagtgtagg catttaactc ctcattggcg tggtccatgc 4560tgatgatttt gccaaaatga gttgtgatga atcaatgaaa aatgtaattt agaaactgat 4620ttcttcagaa ttagatggcc ttatttttta aaatatttga atgaaaacat tttattttta 4680aaatattaca caggaggcct tcggagtttc ttagtcatta ctgtcctttt cccctacaga 4740attttccctc ttggtgtgat tgcacagaat ttgtatgtat tttcagttac aagattgtaa 4800gtaaattgcc tgatttgttt tcattataga caacgatgaa tttcttctaa ttatttaaat 4860aaaatcacca aaaacat 487774119DNAHomo sapiens 7ctggagagcc tgctgcccgc ccgcccgtaa aatggtcccc tcggctggac agctcgccct 60gttcgctctg ggtattgtgt tggctgcgtg ccaggccttg gagaacagca cgtccccgct 120gagtgcagac ccgcccgtgg ctgcagcagt ggtgtcccat tttaatgact gcccagattc 180ccacactcag ttctgcttcc atggaacctg caggtttttg gtgcaggagg acaagccagc 240atgtgtctgc cattctgggt acgttggtgc acgctgtgag catgcggacc tcctggccgt 300ggtggctgcc agccagaaga agcaggccat caccgccttg gtggtggtct ccatcgtggc 360cctggctgtc cttatcatca catgtgtgct gatacactgc tgccaggtcc gaaaacactg 420tgagtggtgc cgggccctca tctgccggca cgagaagccc agcgccctcc tgaagggaag 480aaccgcttgc tgccactcag aaacagtggt ctgaagagcc cagaggagga gtttggccag 540gtggactgtg gcagatcaat aaagaaaggc ttcttcagga cagcactgcc agagatgcct 600gggtgtgcca cagaccttcc tacttggcct gtaatcacct gtgcagcctt ttgtgggcct 660tcaaaactct gtcaagaact ccgtctgctt ggggttattc agtgtgacct agagaagaaa 720tcagcggacc acgatttcaa gacttgttaa aaaagaactg caaagagacg gactcctgtt 780cacctaggtg aggtgtgtgc agcagttggt gtctgagtcc acatgtgtgc agttgtcttc 840tgccagccat ggattccagg ctatatattt ctttttaatg ggccacctcc ccacaacaga 900attctgccca acacaggaga tttctatagt tattgttttc tgtcatttgc ctactgggga 960agaaagtgaa ggaggggaaa ctgtttaata tcacatgaag accctagctt taagagaagc 1020tgtatcctct aaccacgaga ctctcaacca gcccaacatc ttccatggac acatgacatt 1080gaagaccatc ccaagctatc gccacccttg gagatgatgt cttatttatt agatggataa 1140tggttttatt tttaatctct taagtcaatg taaaaagtat aaaacccctt cagacttcta 1200cattaatgat gtatgtgttg ctgactgaaa agctatactg attagaaatg tctggcctct 1260tcaagacagc taaggcttgg gaaaagtctt ccagggtgcg gagatggaac cagaggctgg 1320gttactggta ggaataaagg taggggttca gaaatggtgc cattgaagcc acaaagccgg 1380taaatgcctc aatacgttct gggagaaaac ttagcaaatc catcagcagg gatctgtccc 1440ctctgttggg gagagaggaa gagtgtgtgt gtctacacag gataaaccca atacatattg 1500tactgctcag tgattaaatg ggttcacttc ctcgtgagcc ctcggtaagt atgtttagaa 1560atagaacatt agccacgagc cataggcatt tcaggccaaa tccatgaaag ggggaccagt 1620catttatttt ccattttgtt gcttggttgg tttgttgctt tatttttaaa aggagaagtt 1680taactttgct atttattttc gagcactagg aaaactattc cagtaatttt tttttcctca 1740tttccattca ggatgccggc tttattaaca aaaactctaa caagtcacct ccactatgtg 1800ggtcttcctt tcccctcaag agaaggagca attgttcccc tgacatctgg gtccatctga 1860cccatggggc ctgcctgtga gaaacagtgg gtcccttcaa atacatagtg gatagctcat 1920ccctaggaat tttcattaaa atttggaaac agagtaatga agaaataata tataaactcc 1980ttatgtgagg aaatgctact aatatctgaa aagtgaaaga tttctatgta ttaactctta 2040agtgcaccta gcttattaca tcgtgaaagg tacatttaaa atatgttaaa ttggcttgaa 2100attttcagag aattttgtct tcccctaatt cttcttcctt ggtctggaag aacaatttct 2160atgaattttc tctttatttt ttttttataa ttcagacaat tctatgaccc gtgtcttcat 2220ttttggcact cttatttaac aatgccacac ctgaagcact tggatctgtt cagagctgac 2280cccctagcaa cgtagttgac acagctccag gtttttaaat tactaaaata agttcaagtt 2340tacatccctt gggccagata tgtgggttga ggcttgactg tagcatcctg cttagagacc 2400aatcaatgga cactggtttt tagacctcta tcaatcagta gttagcatcc aagagacttt 2460gcagaggcgt aggaatgagg ctggacagat ggcggaacga gaggttccct gcgaagactt 2520gagatttagt gtctgtgaat gttctagttc ctaggtccag caagtcacac ctgccagtgc 2580cctcatcctt atgcctgtaa cacacatgca gtgagaggcc tcacatatac gcctccctag 2640aagtgccttc caagtcagtc ctttggaaac cagcaggtct gaaaaagagg ctgcatcaat 2700gcaagcctgg ttggaccatt gtccatgcct caggatagaa cagcctggct tatttgggga 2760tttttcttct agaaatcaaa tgactgataa gcattggctc cctctgccat ttaatggcaa 2820tggtagtctt tggttagctg caaaaatact ccatttcaag ttaaaaatgc atcttctaat 2880ccatctctgc aagctccctg tgtttccttg ccctttagaa aatgaattgt tcactacaat 2940tagagaatca tttaacatcc tgacctggta agctgccaca cacctggcag tggggagcat 3000cgctgtttcc aatggctcag gagacaatga aaagccccca tttaaaaaaa taacaaacat 3060tttttaaaag gcctccaata ctcttatgga gcctggattt ttcccactgc tctacaggct 3120gtgacttttt ttaagcatcc tgacaggaaa tgttttcttc tacatggaaa gatagacagc 3180agccaaccct gatctggaag acagggcccc ggctggacac acgtggaacc aagccaggga 3240tgggctggcc attgtgtccc cgcaggagag atgggcagaa tggccctaga gttcttttcc 3300ctgagaaagg agaaaaagat gggattgcca ctcacccacc cacactggta agggaggaga 3360atttgtgctt ctggagcttc tcaagggatt gtgttttgca ggtacagaaa actgcctgtt 3420atcttcaagc caggttttcg agggcacatg ggtcaccagt tgctttttca gtcaatttgg 3480ccgggatgga ctaatgaggc tctaacactg ctcaggagac ccctgccctc tagttggttc 3540tgggctttga tctcttccaa cctgcccagt cacagaagga ggaatgactc aaatgcccaa 3600aaccaagaac acattgcaga agtaagacaa acatgtatat ttttaaatgt tctaacataa 3660gacctgttct ctctagccat tgatttacca ggctttctga aagatctagt ggttcacaca 3720gagagagaga gagtactgaa aaagcaactc ctcttcttag tcttaataat ttactaaaat 3780ggtcaacttt tcattatctt tattataata aacctgatgc ttttttttag aactccttac 3840tctgatgtct gtatatgttg cactgaaaag gttaatattt aatgttttaa tttattttgt 3900gtggtaagtt aattttgatt tctgtaatgt gttaatgtga ttagcagtta ttttccttaa 3960tatctgaatt atacttaaag agtagtgagc aatataagac gcaattgtgt ttttcagtaa 4020tgtgcattgt tattgagttg tactgtacct tatttggaag gatgaaggaa tgaacctttt 4080tttcctaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaa 411984624DNAHomo sapiens 8ggaggaggtg gaggaggagg gctgcttgag gaagtataag aatgaagttg tgaagctgag 60attcccctcc attgggaccg gagaaaccag gggagccccc cgggcagccg cgcgcccctt 120cccacggggc cctttactgc gccgcgcgcc cggcccccac ccctcgcagc accccgcgcc 180ccgcgccctc ccagccgggt ccagccggag ccatggggcc ggagccgcag tgagcaccat 240ggagctggcg gccttgtgcc gctgggggct cctcctcgcc ctcttgcccc ccggagccgc 300gagcacccaa gtgtgcaccg gcacagacat gaagctgcgg ctccctgcca gtcccgagac 360ccacctggac atgctccgcc acctctacca gggctgccag gtggtgcagg gaaacctgga 420actcacctac ctgcccacca atgccagcct gtccttcctg caggatatcc aggaggtgca 480gggctacgtg ctcatcgctc acaaccaagt gaggcaggtc ccactgcaga ggctgcggat 540tgtgcgaggc acccagctct ttgaggacaa ctatgccctg gccgtgctag acaatggaga 600cccgctgaac aataccaccc ctgtcacagg ggcctcccca ggaggcctgc gggagctgca 660gcttcgaagc ctcacagaga tcttgaaagg aggggtcttg atccagcgga acccccagct 720ctgctaccag gacacgattt tgtggaagga catcttccac aagaacaacc agctggctct 780cacactgata gacaccaacc gctctcgggc ctgccacccc tgttctccga tgtgtaaggg 840ctcccgctgc tggggagaga gttctgagga ttgtcagagc ctgacgcgca ctgtctgtgc 900cggtggctgt gcccgctgca aggggccact gcccactgac tgctgccatg agcagtgtgc 960tgccggctgc acgggcccca agcactctga ctgcctggcc tgcctccact tcaaccacag 1020tggcatctgt gagctgcact gcccagccct ggtcacctac aacacagaca cgtttgagtc 1080catgcccaat cccgagggcc ggtatacatt cggcgccagc tgtgtgactg cctgtcccta 1140caactacctt tctacggacg tgggatcctg caccctcgtc tgccccctgc acaaccaaga 1200ggtgacagca gaggatggaa cacagcggtg tgagaagtgc agcaagccct gtgcccgagt 1260gtgctatggt ctgggcatgg agcacttgcg agaggtgagg gcagttacca gtgccaatat 1320ccaggagttt gctggctgca agaagatctt tgggagcctg gcatttctgc cggagagctt 1380tgatggggac ccagcctcca acactgcccc gctccagcca gagcagctcc aagtgtttga 1440gactctggaa gagatcacag gttacctata catctcagca tggccggaca gcctgcctga 1500cctcagcgtc ttccagaacc tgcaagtaat ccggggacga attctgcaca atggcgccta 1560ctcgctgacc ctgcaagggc tgggcatcag ctggctgggg ctgcgctcac tgagggaact 1620gggcagtgga ctggccctca tccaccataa cacccacctc tgcttcgtgc acacggtgcc 1680ctgggaccag ctctttcgga acccgcacca agctctgctc cacactgcca accggccaga 1740ggacgagtgt gtgggcgagg gcctggcctg ccaccagctg tgcgcccgag ggcactgctg 1800gggtccaggg cccacccagt gtgtcaactg cagccagttc cttcggggcc aggagtgcgt 1860ggaggaatgc cgagtactgc aggggctccc cagggagtat gtgaatgcca ggcactgttt 1920gccgtgccac cctgagtgtc agccccagaa tggctcagtg acctgttttg gaccggaggc 1980tgaccagtgt gtggcctgtg cccactataa ggaccctccc ttctgcgtgg cccgctgccc 2040cagcggtgtg aaacctgacc tctcctacat gcccatctgg

aagtttccag atgaggaggg 2100cgcatgccag ccttgcccca tcaactgcac ccactcctgt gtggacctgg atgacaaggg 2160ctgccccgcc gagcagagag ccagccctct gacgtccatc atctctgcgg tggttggcat 2220tctgctggtc gtggtcttgg gggtggtctt tgggatcctc atcaagcgac ggcagcagaa 2280gatccggaag tacacgatgc ggagactgct gcaggaaacg gagctggtgg agccgctgac 2340acctagcgga gcgatgccca accaggcgca gatgcggatc ctgaaagaga cggagctgag 2400gaaggtgaag gtgcttggat ctggcgcttt tggcacagtc tacaagggca tctggatccc 2460tgatggggag aatgtgaaaa ttccagtggc catcaaagtg ttgagggaaa acacatcccc 2520caaagccaac aaagaaatct tagacgaagc atacgtgatg gctggtgtgg gctccccata 2580tgtctcccgc cttctgggca tctgcctgac atccacggtg cagctggtga cacagcttat 2640gccctatggc tgcctcttag accatgtccg ggaaaaccgc ggacgcctgg gctcccagga 2700cctgctgaac tggtgtatgc agattgccaa ggggatgagc tacctggagg atgtgcggct 2760cgtacacagg gacttggccg ctcggaacgt gctggtcaag agtcccaacc atgtcaaaat 2820tacagacttc gggctggctc ggctgctgga cattgacgag acagagtacc atgcagatgg 2880gggcaaggtg cccatcaagt ggatggcgct ggagtccatt ctccgccggc ggttcaccca 2940ccagagtgat gtgtggagtt atggtgtgac tgtgtgggag ctgatgactt ttggggccaa 3000accttacgat gggatcccag cccgggagat ccctgacctg ctggaaaagg gggagcggct 3060gccccagccc cccatctgca ccattgatgt ctacatgatc atggtcaaat gttggatgat 3120tgactctgaa tgtcggccaa gattccggga gttggtgtct gaattctccc gcatggccag 3180ggacccccag cgctttgtgg tcatccagaa tgaggacttg ggcccagcca gtcccttgga 3240cagcaccttc taccgctcac tgctggagga cgatgacatg ggggacctgg tggatgctga 3300ggagtatctg gtaccccagc agggcttctt ctgtccagac cctgccccgg gcgctggggg 3360catggtccac cacaggcacc gcagctcatc taccaggagt ggcggtgggg acctgacact 3420agggctggag ccctctgaag aggaggcccc caggtctcca ctggcaccct ccgaaggggc 3480tggctccgat gtatttgatg gtgacctggg aatgggggca gccaaggggc tgcaaagcct 3540ccccacacat gaccccagcc ctctacagcg gtacagtgag gaccccacag tacccctgcc 3600ctctgagact gatggctacg ttgcccccct gacctgcagc ccccagcctg aatatgtgaa 3660ccagccagat gttcggcccc agcccccttc gccccgagag ggccctctgc ctgctgcccg 3720acctgctggt gccactctgg aaaggcccaa gactctctcc ccagggaaga atggggtcgt 3780caaagacgtt tttgcctttg ggggtgccgt ggagaacccc gagtacttga caccccaggg 3840aggagctgcc cctcagcccc accctcctcc tgccttcagc ccagccttcg acaacctcta 3900ttactgggac caggacccac cagagcgggg ggctccaccc agcaccttca aagggacacc 3960tacggcagag aacccagagt acctgggtct ggacgtgcca gtgtgaacca gaaggccaag 4020tccgcagaag ccctgatgtg tcctcaggga gcagggaagg cctgacttct gctggcatca 4080agaggtggga gggccctccg accacttcca ggggaacctg ccatgccagg aacctgtcct 4140aaggaacctt ccttcctgct tgagttccca gatggctgga aggggtccag cctcgttgga 4200agaggaacag cactggggag tctttgtgga ttctgaggcc ctgcccaatg agactctagg 4260gtccagtgga tgccacagcc cagcttggcc ctttccttcc agatcctggg tactgaaagc 4320cttagggaag ctggcctgag aggggaagcg gccctaaggg agtgtctaag aacaaaagcg 4380acccattcag agactgtccc tgaaacctag tactgccccc catgaggaag gaacagcaat 4440ggtgtcagta tccaggcttt gtacagagtg cttttctgtt tagtttttac tttttttgtt 4500ttgttttttt aaagatgaaa taaagaccca gggggagaat gggtgttgta tggggaggca 4560agtgtggggg gtccttctcc acacccactt tgtccatttg caaatatatt ttggaaaaca 4620gcta 46249183PRTHomo sapiens 9Met Arg Ala Asn Asp Ala Leu Gln Val Leu Gly Leu Leu Phe Ser Leu1 5 10 15Ala Arg Gly Ser Glu Val Gly Asn Ser Gln Ala Val Cys Pro Gly Thr 20 25 30Leu Asn Gly Leu Ser Val Thr Gly Asp Ala Glu Asn Gln Tyr Gln Thr 35 40 45Leu Tyr Lys Leu Tyr Glu Arg Cys Glu Val Val Met Gly Asn Leu Glu 50 55 60Ile Val Leu Thr Gly His Asn Ala Asp Leu Ser Phe Leu Gln Trp Ile65 70 75 80Arg Glu Val Thr Gly Tyr Val Leu Val Ala Met Asn Glu Phe Ser Thr 85 90 95Leu Pro Leu Pro Asn Leu Arg Val Val Arg Gly Thr Gln Val Tyr Asp 100 105 110Gly Lys Phe Ala Ile Phe Val Met Leu Asn Tyr Asn Thr Asn Ser Ser 115 120 125His Ala Leu Arg Gln Leu Arg Leu Thr Gln Leu Thr Gly Gln Phe Pro 130 135 140Met Val Pro Ser Gly Leu Thr Pro Gln Pro Ala Gln Asp Trp Tyr Leu145 150 155 160Leu Asp Asp Asp Pro Arg Leu Leu Thr Leu Ser Ala Ser Ser Lys Val 165 170 175Pro Val Thr Leu Ala Ala Val 180101050DNAHomo sapiens 10acacacacac acccctcccc tgccatccct ccccggactc cggctccggc tccgattgca 60atttgcaacc tccgctgccg tcgccgcagc agccaccaat tcgccagcgg ttcaggtggc 120tcttgcctcg atgtcctagc ctaggggccc ccgggccgga cttggctggg ctcccttcac 180cctctgcgga gtcatgaggg cgaacgacgc tctgcaggtg ctgggcttgc ttttcagcct 240ggcccggggc tccgaggtgg gcaactctca ggcagtgtgt cctgggactc tgaatggcct 300gagtgtgacc ggcgatgctg agaaccaata ccagacactg tacaagctct acgagaggtg 360tgaggtggtg atggggaacc ttgagattgt gctcacggga cacaatgccg acctctcctt 420cctgcagtgg attcgagaag tgacaggcta tgtcctcgtg gccatgaatg aattctctac 480tctaccattg cccaacctcc gcgtggtgcg agggacccag gtctacgatg ggaagtttgc 540catcttcgtc atgttgaact ataacaccaa ctccagccac gctctgcgcc agctccgctt 600gactcagctc accggtcagt tcccgatggt tccttctggc ctcacccctc agccagccca 660agactggtac ctccttgatg atgacccaag actgctcact ctaagtgcct cttccaaggt 720gcctgtcacc ttggccgctg tctaaaggtc cattgctccc taagcaatag agggccccca 780gtagggggag ctaggggcat ctgctccagg gaaaggaacc ctgtgtcctt gtggggctgg 840agtcagagct ggatctgtta accgtttttc taatttcaaa gtacagtgta ccggaggcca 900ggcctgatgg cttacacctg taatcccagc attttgggag gccaaggagg gcagatcact 960tgagatcagg agtttgagac cagcctggcc aacatggcga aaccctgtct ctactaaaaa 1020tacaaaaaaa taaaataaaa taaaaaatta 1050111210PRTHomo sapiens 11Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala1 5 10 15Ala Leu Cys Pro Ala Ser Arg Ala Leu Glu Glu Lys Lys Val Cys Gln 20 25 30Gly Thr Ser Asn Lys Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe 35 40 45Leu Ser Leu Gln Arg Met Phe Asn Asn Cys Glu Val Val Leu Gly Asn 50 55 60Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys65 70 75 80Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val 85 90 95Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Met Tyr 100 105 110Tyr Glu Asn Ser Tyr Ala Leu Ala Val Leu Ser Asn Tyr Asp Ala Asn 115 120 125Lys Thr Gly Leu Lys Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu 130 135 140His Gly Ala Val Arg Phe Ser Asn Asn Pro Ala Leu Cys Asn Val Glu145 150 155 160Ser Ile Gln Trp Arg Asp Ile Val Ser Ser Asp Phe Leu Ser Asn Met 165 170 175Ser Met Asp Phe Gln Asn His Leu Gly Ser Cys Gln Lys Cys Asp Pro 180 185 190Ser Cys Pro Asn Gly Ser Cys Trp Gly Ala Gly Glu Glu Asn Cys Gln 195 200 205Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser Gly Arg Cys Arg 210 215 220Gly Lys Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys225 230 235 240Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Arg Lys Phe Arg Asp 245 250 255Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro 260 265 270Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly 275 280 285Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His 290 295 300Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu305 310 315 320Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val 325 330 335Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn 340 345 350Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp 355 360 365Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr 370 375 380Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu385 390 395 400Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp 405 410 415Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln 420 425 430His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu 435 440 445Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser 450 455 460Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu465 470 475 480Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu 485 490 495Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro 500 505 510Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn 515 520 525Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly 530 535 540Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro545 550 555 560Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro 565 570 575Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val 580 585 590Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp 595 600 605Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His Pro Asn Cys 610 615 620Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro Thr Asn Gly625 630 635 640Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val Gly Ala Leu Leu Leu 645 650 655Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met Arg Arg Arg His 660 665 670Ile Val Arg Lys Arg Thr Leu Arg Arg Leu Leu Gln Glu Arg Glu Leu 675 680 685Val Glu Pro Leu Thr Pro Ser Gly Glu Ala Pro Asn Gln Ala Leu Leu 690 695 700Arg Ile Leu Lys Glu Thr Glu Phe Lys Lys Ile Lys Val Leu Gly Ser705 710 715 720Gly Ala Phe Gly Thr Val Tyr Lys Gly Leu Trp Ile Pro Glu Gly Glu 725 730 735Lys Val Lys Ile Pro Val Ala Ile Lys Glu Leu Arg Glu Ala Thr Ser 740 745 750Pro Lys Ala Asn Lys Glu Ile Leu Asp Glu Ala Tyr Val Met Ala Ser 755 760 765Val Asp Asn Pro His Val Cys Arg Leu Leu Gly Ile Cys Leu Thr Ser 770 775 780Thr Val Gln Leu Ile Thr Gln Leu Met Pro Phe Gly Cys Leu Leu Asp785 790 795 800Tyr Val Arg Glu His Lys Asp Asn Ile Gly Ser Gln Tyr Leu Leu Asn 805 810 815Trp Cys Val Gln Ile Ala Lys Gly Met Asn Tyr Leu Glu Asp Arg Arg 820 825 830Leu Val His Arg Asp Leu Ala Ala Arg Asn Val Leu Val Lys Thr Pro 835 840 845Gln His Val Lys Ile Thr Asp Phe Gly Leu Ala Lys Leu Leu Gly Ala 850 855 860Glu Glu Lys Glu Tyr His Ala Glu Gly Gly Lys Val Pro Ile Lys Trp865 870 875 880Met Ala Leu Glu Ser Ile Leu His Arg Ile Tyr Thr His Gln Ser Asp 885 890 895Val Trp Ser Tyr Gly Val Thr Val Trp Glu Leu Met Thr Phe Gly Ser 900 905 910Lys Pro Tyr Asp Gly Ile Pro Ala Ser Glu Ile Ser Ser Ile Leu Glu 915 920 925Lys Gly Glu Arg Leu Pro Gln Pro Pro Ile Cys Thr Ile Asp Val Tyr 930 935 940Met Ile Met Val Lys Cys Trp Met Ile Asp Ala Asp Ser Arg Pro Lys945 950 955 960Phe Arg Glu Leu Ile Ile Glu Phe Ser Lys Met Ala Arg Asp Pro Gln 965 970 975Arg Tyr Leu Val Ile Gln Gly Asp Glu Arg Met His Leu Pro Ser Pro 980 985 990Thr Asp Ser Asn Phe Tyr Arg Ala Leu Met Asp Glu Glu Asp Met Asp 995 1000 1005Asp Val Val Asp Ala Asp Glu Tyr Leu Ile Pro Gln Gln Gly Phe 1010 1015 1020Phe Ser Ser Pro Ser Thr Ser Arg Thr Pro Leu Leu Ser Ser Leu 1025 1030 1035Ser Ala Thr Ser Asn Asn Ser Thr Val Ala Cys Ile Asp Arg Asn 1040 1045 1050Gly Leu Gln Ser Cys Pro Ile Lys Glu Asp Ser Phe Leu Gln Arg 1055 1060 1065Tyr Ser Ser Asp Pro Thr Gly Ala Leu Thr Glu Asp Ser Ile Asp 1070 1075 1080Asp Thr Phe Leu Pro Val Pro Glu Tyr Ile Asn Gln Ser Val Pro 1085 1090 1095Lys Arg Pro Ala Gly Ser Val Gln Asn Pro Val Tyr His Asn Gln 1100 1105 1110Pro Leu Asn Pro Ala Pro Ser Arg Asp Pro His Tyr Gln Asp Pro 1115 1120 1125His Ser Thr Ala Val Gly Asn Pro Glu Tyr Leu Asn Thr Val Gln 1130 1135 1140Pro Thr Cys Val Asn Ser Thr Phe Asp Ser Pro Ala His Trp Ala 1145 1150 1155Gln Lys Gly Ser His Gln Ile Ser Leu Asp Asn Pro Asp Tyr Gln 1160 1165 1170Gln Asp Phe Phe Pro Lys Glu Ala Lys Pro Asn Gly Ile Phe Lys 1175 1180 1185Gly Ser Thr Ala Glu Asn Ala Glu Tyr Leu Arg Val Ala Pro Gln 1190 1195 1200Ser Ser Glu Phe Ile Gly Ala 1205 121012628PRTHomo sapiens 12Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala1 5 10 15Ala Leu Cys Pro Ala Ser Arg Ala Leu Glu Glu Lys Lys Val Cys Gln 20 25 30Gly Thr Ser Asn Lys Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe 35 40 45Leu Ser Leu Gln Arg Met Phe Asn Asn Cys Glu Val Val Leu Gly Asn 50 55 60Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys65 70 75 80Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val 85 90 95Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Met Tyr 100 105 110Tyr Glu Asn Ser Tyr Ala Leu Ala Val Leu Ser Asn Tyr Asp Ala Asn 115 120 125Lys Thr Gly Leu Lys Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu 130 135 140His Gly Ala Val Arg Phe Ser Asn Asn Pro Ala Leu Cys Asn Val Glu145 150 155 160Ser Ile Gln Trp Arg Asp Ile Val Ser Ser Asp Phe Leu Ser Asn Met 165 170 175Ser Met Asp Phe Gln Asn His Leu Gly Ser Cys Gln Lys Cys Asp Pro 180 185 190Ser Cys Pro Asn Gly Ser Cys Trp Gly Ala Gly Glu Glu Asn Cys Gln 195 200 205Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser Gly Arg Cys Arg 210 215 220Gly Lys Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys225 230 235 240Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Arg Lys Phe Arg Asp 245 250 255Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro 260 265 270Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly 275 280 285Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His 290 295 300Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu305 310 315 320Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val 325 330 335Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn 340 345 350Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp 355 360 365Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr 370 375 380Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu385 390 395 400Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp 405 410 415Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln 420 425 430His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu 435 440 445Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser 450

455 460Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu465 470 475 480Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu 485 490 495Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro 500 505 510Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn 515 520 525Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly 530 535 540Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro545 550 555 560Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro 565 570 575Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val 580 585 590Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp 595 600 605Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His Pro Asn Cys 610 615 620Thr Tyr Gly Ser62513405PRTHomo sapiens 13Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala1 5 10 15Ala Leu Cys Pro Ala Ser Arg Ala Leu Glu Glu Lys Lys Val Cys Gln 20 25 30Gly Thr Ser Asn Lys Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe 35 40 45Leu Ser Leu Gln Arg Met Phe Asn Asn Cys Glu Val Val Leu Gly Asn 50 55 60Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys65 70 75 80Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val 85 90 95Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Met Tyr 100 105 110Tyr Glu Asn Ser Tyr Ala Leu Ala Val Leu Ser Asn Tyr Asp Ala Asn 115 120 125Lys Thr Gly Leu Lys Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu 130 135 140His Gly Ala Val Arg Phe Ser Asn Asn Pro Ala Leu Cys Asn Val Glu145 150 155 160Ser Ile Gln Trp Arg Asp Ile Val Ser Ser Asp Phe Leu Ser Asn Met 165 170 175Ser Met Asp Phe Gln Asn His Leu Gly Ser Cys Gln Lys Cys Asp Pro 180 185 190Ser Cys Pro Asn Gly Ser Cys Trp Gly Ala Gly Glu Glu Asn Cys Gln 195 200 205Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser Gly Arg Cys Arg 210 215 220Gly Lys Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys225 230 235 240Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Arg Lys Phe Arg Asp 245 250 255Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro 260 265 270Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly 275 280 285Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His 290 295 300Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu305 310 315 320Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val 325 330 335Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn 340 345 350Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp 355 360 365Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr 370 375 380Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu385 390 395 400Ile Thr Gly Leu Ser 40514705PRTHomo sapiens 14 Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala1 5 10 15Ala Leu Cys Pro Ala Ser Arg Ala Leu Glu Glu Lys Lys Val Cys Gln 20 25 30 Gly Thr Ser Asn Lys Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe 35 40 45Leu Ser Leu Gln Arg Met Phe Asn Asn Cys Glu Val Val Leu Gly Asn 50 55 60Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys65 70 75 80Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val 85 90 95Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Met Tyr 100 105 110Tyr Glu Asn Ser Tyr Ala Leu Ala Val Leu Ser Asn Tyr Asp Ala Asn 115 120 125Lys Thr Gly Leu Lys Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu 130 135 140His Gly Ala Val Arg Phe Ser Asn Asn Pro Ala Leu Cys Asn Val Glu145 150 155 160Ser Ile Gln Trp Arg Asp Ile Val Ser Ser Asp Phe Leu Ser Asn Met 165 170 175Ser Met Asp Phe Gln Asn His Leu Gly Ser Cys Gln Lys Cys Asp Pro 180 185 190Ser Cys Pro Asn Gly Ser Cys Trp Gly Ala Gly Glu Glu Asn Cys Gln 195 200 205Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser Gly Arg Cys Arg 210 215 220Gly Lys Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys225 230 235 240Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Arg Lys Phe Arg Asp 245 250 255Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro 260 265 270Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly 275 280 285Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His 290 295 300Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu305 310 315 320Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val 325 330 335Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn 340 345 350Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp 355 360 365Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr 370 375 380Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu385 390 395 400Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp 405 410 415Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln 420 425 430His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu 435 440 445Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser 450 455 460Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu465 470 475 480Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu 485 490 495Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro 500 505 510Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn 515 520 525Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly 530 535 540Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro545 550 555 560Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro 565 570 575Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val 580 585 590Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp 595 600 605Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His Pro Asn Cys 610 615 620Thr Tyr Gly Pro Gly Asn Glu Ser Leu Lys Ala Met Leu Phe Cys Leu625 630 635 640Phe Lys Leu Ser Ser Cys Asn Gln Ser Asn Asp Gly Ser Val Ser His 645 650 655Gln Ser Gly Ser Pro Ala Ala Gln Glu Ser Cys Leu Gly Trp Ile Pro 660 665 670Ser Leu Leu Pro Ser Glu Phe Gln Leu Gly Trp Gly Gly Cys Ser His 675 680 685Leu His Ala Trp Pro Ser Ala Ser Val Ile Ile Thr Ala Ser Ser Cys 690 695 700His705

Claims

1. A method of predicting the response of a patient to treatment with a HER dimerization inhibitor comprising the steps of:(a) determining the expression level one or more biomarker proteins with one such biomarker protein being transforming growth factor alpha;(b) determining whether the expression level assessed in step (a) is above or below a certain quantity that is associated with an increased or decreased clinical benefit to a patient; and(c) predicting the response to the treatment with the HER inhibitor in the patient by evaluating the results of step (b).

2. A method according to claim 1 wherein, in step (a), in addition to the expression level of transforming growth factor alpha, the level of expression of one or more biomarker proteins, selected from the group consisting of epidermal growth factor, amphiregulin, and HER2, is also determined.

3. A method according to claim 2 wherein, in step (a), the level of expression of transforming growth factor alpha is determined in combination with the level of expression of: epidermal growth factor; HER2; or both amphiregulin and HER2.

4. A method according to claim 1 wherein the HER dimerization inhibitor inhibits heterodimerization of HER2 with EGFR or HER3.

5. A method according to claim 1 wherein the HER dimerization inhibitor is an antibody.

6. A method according to claim 1 wherein the HER dimerization inhibitor is the antibody 2C4.

7. A method according to claim 1 wherein the patient is a cancer patient.

8. A method according to claim 1 wherein the patient is a breast cancer, ovarian cancer, lung cancer or prostate cancer patient.

9. A method according to claim 1 wherein the level of expression of said biomarker protein or proteins is assessed by detecting the level of expression of fragments thereof.

10. A method according to claim 1 wherein the level of expression said biomarker or biomarkers is determined using a reagent which specifically binds with the biomarker protein or the fragment thereof or the combination of biomarker proteins or the fragments thereof.

11. A method according to claim 10 wherein said reagent is selected from the group consisting of an antibody, a fragment of an antibody, and an antibody derivative.

12. A method according to claim 1 wherein a biomarker protein is the extracellular domain of HER2.

13. A method according to claim 12 wherein said extracellular domain extracellular domain of HER2 has a molecular mass of approximately 105,000 Daltons.

14. A method according to claim 1 wherein the amino acid sequence of the transforming growth factor alpha biomarker protein is SEQ ID NO: 3

15. A method according to claim 2 wherein the amino acid sequence of the amphiregulin biomarker protein is SEQ ID NO: 1, the amino acid sequence of the epidermal growth factor biomarker protein is SEQ ID NO: 2, the amino acid sequence of the transforming growth factor alpha biomarker protein is SEQ ID NO: 3, and the amino acid sequence of the HER2 biomarker protein is SEQ ID NO: 4.

16. A method according to claim 1 wherein the amount of transforming growth factor alpha is determined in a biological sample of blood serum and the quantity that is associated with an increased clinical benefit to a patient is within the range between 2.0 to 10.0 pg/ml of transforming growth factor alpha.

17. A method according to claim 2 wherein the amounts of transforming growth factor alpha, amphiregulin, epidermal growth factor, and HER2 marker proteins are determined in a biological sample of blood serum and the quantities that are associated with an increased clinical benefit to a patient are within the range of between 2.0 to 10.0 pg/ml of transforming growth factor alpha, between 6 to 15 pg/ml of amphiregulin, between 100 to 250 pg/ml of epidermal growth factor, and between 12 to 22 ng/ml of HER2 marker proteins.

18. A method according to claim 2 wherein the amounts of transforming growth factor alpha, amphiregulin, epidermal growth factor, and HER2 marker proteins are determined in a biological sample of blood serum and the quantities that are associated with an increased clinical benefit to a patient are about 3.5 pg/ml of transforming growth factor alpha, about 12 pg/ml of amphiregulin, about 150 pg/ml of epidermal growth factor, and about 18 ng/ml of HER2 marker proteins.

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