HK1139711A

HK1139711A - Method for predicting the response to a treatment

Info

Publication number: HK1139711A
Application number: HK10106101.1A
Authority: HK
Inventors: 约阿希姆‧默克斯; 安德烈亚斯‧斯特劳斯; 格哈德‧楚格边尔
Original assignee: 霍夫曼-拉罗奇有限公司
Priority date: 2005-08-12
Filing date: 2010-06-21
Publication date: 2010-09-24

Description

Methods of predicting response to treatment

The application is a divisional application with the international application number of PCT/EP2006/004950, the international application date of 24/5/2006, the Chinese application number of 200680028951.3, the date of entering the Chinese country of 2008/2/4, and the invention name of the divisional application is a method for predicting response to treatment.

Technical Field

The present invention relates to a method of predicting response to treatment with a HER dimerization inhibitor in a patient, the method comprising the steps of: assessing a marker gene or combination of marker genes selected from the group consisting of epidermal growth factor, transforming growth factor alpha and HER2 marker genes, or a combination of marker genes, comprising an amphiregulin marker gene and a marker gene selected from the group consisting of epithelial growth factor, transforming growth factor alpha and HER2 marker genes, in a biological sample from the patient and predicting the response of treatment with a HER dimerization inhibitor in the patient by assessing the outcome of the first step. Other applications and methods in which these markers are applied are also disclosed.

Background

The human epidermal growth factor receptor (ErbB or HER) family comprises 4 members (HER1-4) which are important regulators of cell growth, survival and differentiation by activating a complex signaling cascade. At least 11 different gene products from the Epidermal Growth Factor (EGF) superfamily bind to three of these receptors, EGFR (also known as ErbB1 or HER1), HER3(ErbB3) and HER4(ErbB 4). Although no ligands have been identified that bind and activate HER2(ErbB2 or neu), it is generally understood that HER2 is a co-receptor that works 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 HER family member (heterodimerization) is important for their activity. HER2 is a 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 specific for HER receptors. Recombinant humanized anti-HER 2 monoclonal antibody (MAb) trastuzumab is the standard of care for patients with HER 2-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.

Pertuzumab(Omnitarg^TM(ii) a Formerly 2C4) is the first known HER dimerization inhibitionNovel classes of agents (HDIs). Pertuzumab binds to HER2 in its dimerization domain, thereby inhibiting its ability to form an active dimeric receptor complex and thus blocking the downstream signaling cascade that ultimately leads to cell growth and division. Pertuzumab is a fully humanized recombinant monoclonal antibody directed against the extracellular domain of HER 2. Binding of Pertuzumab to HER2 on human epithelial cells prevents HER2 from forming complexes with other members of the HER family (including EGFR, HER3, HER4) and possibly HER2 dimerization. By blocking complex formation, Pertuzumab prevents growth-stimulation and cell survival signals activated by ligands of HER1, HER3, and HER4 (e.g., EGF, TGF α, amphiregulin, and heregulin). Other names of Pertuzumab are 2C4 or Omnitarg^TM. Pertuzumab is a fully humanized recombinant monoclonal antibody based on the framework sequence of human IgG1(κ). The structure of Pertuzumab consists of two heavy chains (449 residues) and two light chains (214 residues). With trastuzumab) In contrast, 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 describe that the heterodimer formation of Her2 with other receptors should be linked to the potency and suitability of Pertuzumab.

Zabercky, J.R. et al, J.biol.chem. (J.Chem.Biol.266) (1991)1716-1720 disclose that the release of the extracellular domain of Her2 may be involved in neoplasia and that its detection can be used as a diagnosis of cancer. Colomer, R. et al, Clin.cancer Res. (clinical cancer research) 6(2000)2356-2362 disclose circulating Her2 extracellular domains and resistance to chemotherapy in advanced breast cancer. Hait, w.n., clin.cancer Res, (clinical cancer research) 7(2001)2601-2604 commented on the diagnostic and prognostic value of Her2 extracellular domain in general.

Summary of The Invention

There remains a need to provide further methods of determining disease progression in cancer patients treated with HER dimerization inhibitors.

Accordingly, in one embodiment of the invention, there is provided a method of predicting response to treatment with a HER dimerization inhibitor in a patient, the method comprising the steps of:

a) evaluation in biological samples from patients

-a marker gene or combination of marker genes selected from the group consisting of epidermal growth factor, transforming growth factor alpha and HER2 marker genes, or

A marker gene combination comprising an amphiregulin marker gene and a marker gene selected from the group consisting of epidermal growth factor, transforming growth factor alpha and HER2 marker genes, and

b) predicting the response of a treatment with a HER dimerization inhibitor in a patient by evaluating the results of step a).

In another embodiment of the invention, a probe that hybridizes under stringent conditions to an epidermal growth factor, transforming growth factor alpha, or HER 2-labeled polynucleotide, or an antibody that binds to an epidermal growth factor, transforming growth factor alpha, or HER 2-labeled protein is used to predict the response to treatment with a HER dimerization inhibitor in a patient, or a probe that hybridizes under stringent conditions to an amphiregulin, epidermal growth factor, transforming growth factor alpha, or HER 2-labeled polynucleotide, or an antibody that binds to an amphiregulin, epidermal growth factor, transforming growth factor alpha, or HER 2-labeled protein is used to select a composition that inhibits disease progression in a patient.

In another embodiment of the invention, a kit is provided comprising a probe that anneals to a polynucleotide labeled with amphiregulin, epidermal growth factor, transforming factor alpha or HER2, or an antibody that binds to a protein labeled with amphiregulin, epidermal growth factor, transforming growth factor alpha or HER2, under stringent conditions.

In another embodiment of the present invention, there is provided a method of selecting a composition that inhibits the development of a disease in a patient, the method comprising:

a) separately exposing aliquots comprising biological samples from cancer patients in the presence of a plurality of test compositions;

b) comparing the expression level of a marker gene or combination of marker genes selected from the group consisting of amphiregulin, epidermal growth factor, transforming growth factor alpha and HER2 marker genes in the aliquot of the biological sample contacted with the test composition with the expression level of a marker gene or combination of marker genes selected from the group consisting of amphiregulin, epidermal growth factor, transforming growth factor alpha and HER2 marker genes in the aliquot of the biological sample not contacted with the test composition;

c) selecting a test composition that alters the expression level of the marker gene or combination of marker genes in an aliquot comprising the test composition relative to an aliquot not contacted with the test composition, wherein the presence of at least a 10% difference between the expression level of the marker gene or combination of marker genes in an aliquot of the biological sample contacted with the test composition and the expression level of the corresponding marker gene or combination of marker genes in an aliquot of the biological sample not contacted with the test composition is indicative of selecting the test composition.

In another embodiment of the present invention, there is provided a method of obtaining a candidate agent, the method comprising:

a) contacting an aliquot of a biological sample from a cancer patient with a candidate agent and determining the expression level in the aliquot of a marker gene or combination of marker genes selected from the group consisting of amphiregulin, epidermal growth factor, transforming growth factor alpha and HER2 marker genes,

b) determining the expression level of the corresponding marker gene or the corresponding combination of marker genes in an aliquot of the biological sample not contacted with the candidate agent,

c) observing the effect of the candidate agent by comparing the expression level of the marker gene or combination of marker genes in an aliquot of the biological sample contacted with the candidate agent with the expression level of the corresponding marker gene or corresponding combination of marker genes in an aliquot of the biological sample not contacted with the candidate agent,

d) obtaining said agent by said observed effect, wherein a difference of at least 10% between the expression level of said marker gene or combination of marker genes in an aliquot of the biological sample contacted with said candidate agent and the expression level of the corresponding marker gene or combination of marker genes in an aliquot of the biological sample not contacted with said candidate agent is indicative for the effect of said candidate agent.

In another embodiment of the invention, a candidate agent obtained by the method according to the invention or a pharmaceutical preparation comprising an agent according to the invention is provided.

In another embodiment of the invention, there is provided a medicament according to the invention for the preparation of a composition for the treatment of cancer.

In another embodiment of the present invention, there is provided a method of producing a medicament comprising the steps of the method of the present invention and

i) synthesizing the candidate agent identified in step (c), or an analog or derivative thereof, in an amount sufficient to provide the drug in a therapeutically effective amount to the subject; and/or

ii) combining the drug candidate agent identified in step (c) or an analogue or derivative thereof with a pharmaceutically acceptable carrier.

In another embodiment of the invention, a marker protein or marker polynucleotide selected from the group consisting of amphiregulin, epidermal growth factor, transforming growth factor alpha and HER2 marker protein or marker polynucleotide is used to obtain a candidate agent or to select a composition for inhibiting the development of a disease in a patient.

In another embodiment of the invention, a HER dimerization inhibitor is used for the preparation of a medicament for the treatment of a human cancer patient, characterized in that the treatment or therapy comprises evaluating a biological sample from the patient.

-a combination of marker genes comprising an amphiregulin marker gene and a marker gene selected from the group consisting of epidermal growth factor, transforming growth factor alpha and HER2 marker genes.

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

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

The term "response to treatment with a HER dimerization inhibitor" or "patient response to treatment with a HER dimerization inhibitor" refers to the clinical benefit that a treatment with a HER dimerization inhibitor or as a result of treatment with a HER dimerization inhibitor is given to a patient with a disease or condition, such as cancer, clinical benefits include complete remission, partial remission, stable disease (no development), survival without progression, survival without disease, improvement in time to progression, improvement in time to death, or improvement in the overall survival of the patient from treatment with a HER dimerization inhibitor or as a result of treatment with a HER dimerization inhibitor. Principles of Cancer Therapy, in Harrisons's Principles of Internal Medicine, (Cancer treatment Principles of Internal Medicine Principles of Harrisons), 13 th 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. For example, a partial response or partial remission of cancer may be an approximately 50% reduction in the products of the largest perpendicular diameter of one or more lesions, or the absence of an increase or appearance of new lesions in the size of any lesion.

As used herein, the term "cancer progression" includes and may refer to metastasis, cancer recurrence, or at least about a 25% increase in the products of the largest vertical diameter of a lesion or the appearance of new lesions. The development of cancer, preferably breast cancer, is "inhibited" if the recurrence or metastasis of the cancer is reduced, slowed, delayed or prevented.

The term "time to progression/death (TTP)" is synonymous with "survival without Progression (PFS)". This describes the clinical endpoints commonly used in oncology trials. Here, the examination of each patient is equal to the time that elapses from the start of treatment of the test patient (as defined during the test) until the detection of malignant development (as defined in the test procedure) or any untoward attack (whether or not it is a first attack). If the patient is stopped from viewing after a period of time (e.g., at the end of the study) and no symptoms are observed, then this viewing time t is called "checked".

The term "Time To Death (TTD)" is synonymous with "Total survival (OS)". This describes the clinical endpoints commonly used in oncology trials. Here, the examination of each patient is equal to the time elapsed from the start of treatment (as defined in the method) of the test patient until any unfortunate episodes. If the patient is stopped from being observed after a period of time t (e.g. at the end of the study) and the patient survives by this time, this observation time t is called "checked".

The term "covariate" has the following meaning. Clinical endpoints are typically considered in regression models, where the endpoints represent dependent variables and biomarkers represent primary or target independent variables (regressors). If other variables from the clinical database are considered, these are denoted as (clinical) co-variables. The term "clinical co-variable" is used herein to describe all clinical information about a patient, which is generally available at baseline. These clinical covariates include demographic information such as gender, age, etc., other past condition information, concomitant diseases, concomitant treatments, physical examination results, general laboratory parameters obtained, known characteristics of the target tumor, information quantifying the extent of malignancy, clinical performance scores such as ECOG or Karnofsky indices, clinical disease segmentation, timing and outcome of pretreatment and disease history, and all similar information, which may be relevant to clinical prognosis.

The term "raw or unadjusted analysis" refers herein to regression analysis in which no other clinical co-variables are applied in the regression model, either as independent factors or as hierarchical co-variables, except for the biomarker under consideration.

The term "adjusted by covariate" refers to regression analysis in which other clinical covariates are used in the regression model, in addition to the considered biomarkers, either as independent factors or as hierarchical covariates.

The term "univariate" refers herein to a regression model or a mapping method in which only one target biomarker is part of the model as an independent variable. These univariate models can be considered, with and without other clinical co-variables.

The term "multivariate" herein refers to a regression model or a mapping method in which more than one target biomarker is part of the model as independent variables. These multivariate models can be considered, with and without other clinical co-variables.

"nucleotide" is a "nucleoside" further comprising a phosphate group covalently linked to the sugar moiety of the nucleoside for those "nucleosides" comprising a five-membered furanosyl sugar, the phosphate group may be attached to the 2 ', 3 ' or 5 ' hydroxyl moiety of the sugar "nucleotide" is a "monomeric unit" of an "oligonucleotide", more generally denoted herein as an "oligomeric compound", or a "polynucleotide", more generally denoted herein as a "polymeric compound", whereby the other general expressions are deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

The term "probe" refers to synthetic or biologically produced nucleic acids (DNA or RNA) that, by design or selection, comprise specific nucleotide sequences that allow them to hybridize specifically (i.e., preferentially) to the "nucleic acid" under a defined predetermined stringency. A "probe" can be identified as a "capture probe", meaning that it "captures" the nucleic acid so that it can be separated from unwanted substances that may obscure its detection. When separation is complete, detection of the captured "target nucleic acid" can be achieved using appropriate methods. The "capture probe" is typically already attached to a solid phase. According to the present invention, the term hybridization under "stringent conditions" gives the same meaning as in Sambrook et al (Molecular Cloning, laboratory Manual), Cold Spring Harbor laboratory Press (1989), paragraphs 1.101-1.104). Preferably, a "stringent hybridization" is a situation where a hybridization signal is still detected after 1 hour of washing with 1 XSSC and 0.1% SDS at 50 ℃, preferably at 55 ℃, more preferably at 62 ℃, and most preferably at 68 ℃, and more preferably after 1 hour of washing with 0.2 XSSC and 0.1% SDS at 50 ℃, preferably at 55 ℃, more preferably at 62 ℃, and most preferably at 68 ℃. The composition of the SSC buffer is described in Sambrook et al (Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989)).

A "transcribed polynucleotide" is a polynucleotide (e.g., RNA, cDNA, or an analog of one of the RNAs or cdnas) that is complementary or homologous to all or part of a mature RNA formed by transcription of a gene, such as transcription of a marker gene of the invention, and normal post-transcriptional processing (e.g., splicing) of the transcript, if any. The term "cDNA" is an abbreviation for complementary DNA, a single-stranded or double-stranded DNA copy of mRNA. The term "mRNA" is an abbreviation for RNA that is the template for protein synthesis.

The term "marker gene" is intended to include genes for determining the development of cancer in a patient, in particular in a breast cancer patient, according to the invention. It may also be called a cancer, preferably breast cancer marker gene. The term "labeled polynucleotide" is intended to include a nucleotide transcript (hnRNA or mRNA) encoded by a marker gene according to the invention, or a cDNA derived from said nucleotide transcript, or a fragment of said transcript or cDNA.

The term "marker protein" or "marker polypeptide" is intended to include a protein or polypeptide encoded by a marker gene according to the invention, or a polypeptide or protein fragment comprising said marker protein.

The term "gene product" is intended to include both marker polynucleotides and marker proteins encoded by the referenced genes.

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

A tagged polynucleotide or tagged protein "corresponds" to another tagged polynucleotide or tagged protein if it is related to it, particularly preferably it is identical to it.

The terms "level of expression" or "expression level" are used interchangeably and generally refer to the amount of polynucleotide or amino acid product or protein in a sample. "expression" generally refers to the process by which information encoded by a gene is converted into a structure that is present and operational in a cell, and thus the term "expression" of a gene according to the present invention includes transcription into a polynucleotide, translation into a protein, and even post-translational modification of a protein. Transcribed polynucleotides, translated proteins or fragments of post-translationally modified proteins should also be considered as expressed, regardless of whether they originate, for example, from transcripts produced by alternative splicing, degraded transcripts, or from post-translational processing of proteins, for example, by proteolytic processing. As used herein, "expressed genes" include those genes that are transcribed into a polynucleotide that is mRNA and then translated into protein. The term should also include those expressed genes that are transcribed into RNA but not translated into protein (e.g., transport and ribosomal RNAs). The terms "overexpression" and "underexpression" refer to an upward or downward deviation, respectively, in expression level when compared to the baseline expression level in a sample used as a control. Thus, "overexpression" is also "increased expression" and "low expression" is "decreased expression".

The term "amphiregulin" relates to a gene encoding a protein, and to the protein itself, which is a member of the epidermal growth factor family. It is an autocrine growth factor and a mitogen for astrocytes, neural membrane cells and fibroblasts. It relates 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 inhibit the growth of certain invasive cancer cell lines. According to the invention, the amino acid sequence of amphiregulin is according to SEQ ID NO: 1. According to the invention, the nucleic acid sequence of the "amphiregulin" cDNA is according to SEQ ID NO: 5, available in GenBank under accession number NM _ 001657.

The term "transforming growth factor alpha" relates to the gene encoding the protein, and to the protein itself, which is a member of the Transforming Growth Factor (TGFs) family. These are biologically active polypeptides that reversibly confer a transformed phenotype on cultured cells. "transforming growth factor-alpha" exhibits about 40% sequence identity to epidermal growth factor and competes with EFG 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 according to SEQ ID NO: 3. According to the invention, the nucleic acid sequence of the "transforming growth factor-alpha" cDNA is according to SEQ ID NO: 7, available in GenBank under accession number NM _ 003236.

The term "epidermal growth factor" relates to a gene encoding a protein, and to the protein itself, which is a member of the growth factor family. "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 ectodermal and mesodermal origin. It is believed that the EGF precursor exists as a membrane-bound molecule whose proteolytic cleavage produces a 53 amino acid peptide hormone that stimulates cell division. According to the invention, the amino acid sequence of the "epidermal growth factor" is according to SEQ ID NO: 2. According to the invention, the nucleic acid sequence of the "Epidermal Growth Factor (EGF)" cDNA is according to SEQ ID NO: 6, available in GenBank under accession number NM _ 001963. "epidermal growth factor receptor", abbreviated EGFR, is a 170-kD glycoprotein consisting of an N-terminal extracellular domain, a hydrophobic transmembrane domain, and a C-terminal intracellular domain containing a kinase domain. mRNA has different variants translated into different receptor proteins. According to the invention, the amino acid sequence of the "epidermal growth factor receptor" is according to SEQ ID NO: 11 (transcript variant 1; GenBank accession No. NM — 005228), SEQ ID NO: 12 (transcript variant 2; GenBank accession No. NM — 201282), SEQ ID NO: 13 (transcript variant 3; GenBank accession No. NM — 201283), or SEQ ID NO: 14 (transcript variant 4; GenBank accession No. NM-201284). EGFR, which is encoded by the erbB1 gene, has been causally involved in human malignancies.in particular, increased EGFR expression has been observed in breast, bladder, lung, head, neck and stomach cancers as well as glioblastomas. EGFR ligand-induced dimerization activates the intrinsic RTK domain (Src homeodomain 1, SH1), this results in autophosphorylation of the 6 specific EGFR tyrosine residues in the non-catalytic tail of the cytoplasmic domain the cellular effects of EGFR activation in cancer cells include increased proliferation, promotion of cellular motility, adhesion, invasion, angiogenesis, activated EGFR induces tumor cell proliferation by stimulating a mitogen-activated protein kinase (MAPK) cascade.

The terms "human neu", "c-erbB-2", "erbB 2", "erbB-2", "HER-2/neu", "HER 2" and "HER-2" are used interchangeably herein. The term "Her 2" relates to the gene encoding a protein that is a member of the Epidermal Growth Factor (EGF) receptor family of receptor tyrosine kinases, and to the protein itself. This protein has no ligand binding to its own domain and therefore cannot bind to growth factors. However, it does bind strongly to other ligand-bound EGF receptor family members to form heterodimers, which stabilize ligands that bind and enhance kinase-regulated activation of downstream signaling pathways, such as those including mitogen-activated protein kinases and phosphatidylinositol-3 kinases. Allelic variation at amino acid positions 654 and 655 of isoform a (positions 624 and 625 of isoform b) has 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 many cancers, including breast and ovarian tumors. Alternative splicing results in some other transcriptional variants, some encoding different isoforms, and others not yet fully characterized. According to the invention, the amino acid sequence of Her2 is according to SEQ ID NO: 4. According to the invention, the nucleic acid sequence of the "Her 2" cDNA is according to SEQ ID NO: 8, available in GenBank under accession number NM _ 004448.2.

The "extracellular domain of Her 2" or "excised extracellular domain of Her 2" (shedexcellular domain of Her2) is a glycoprotein of 97-115kDa, which essentially corresponds to the extracellular domain of the human Her2 gene product. It can be used as p105 (Zabercky, J.R., et al, J.biol.chem. (J.Chem. biol.) 266(1991) 1716-1720; US5,401,638; US5,604,107). Quantification and detection of the extracellular domain of Her2 is described in US5,401,638 and US5,604,107.

The term "Her 3" represents another member of the Epidermal Growth Factor Receptor (EGFR) family of receptor tyrosine kinases. Such membrane-bound proteins have no active kinase domain. The protein can bind to a ligand, but is unable to transmit a signal into the cell. It forms heterodimers with other EGF receptor family members, which do have kinase activity that leads to cell proliferation or differentiation. Amplification of this gene and/or overexpression of its protein is found in many cancers. According to the invention, the amino acid sequence of the "Her 3" cDNA is according to SEQ ID NO: 9, obtainable in GenBank from the translation of a nucleic acid sequence of Her3 with accession No. NM _ 001005915. According to the invention, the nucleic acid sequence of the "Her 3" cDNA is according to seq id NO: 10, available in GenBank under accession number NM _ 001005915.

The term "antibody" is used herein in the broadest sense and specifically covers intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments, provided that they exhibit the desired biological activity.

As used herein, the term "monoclonal antibody" 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. G.et al, Nature 256(1975)495-497, or may be prepared by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567).

An antibody that "binds" an antigen of interest according to the invention is one that binds to the antigen with sufficient affinity that the antibody is used to detect the presence of the antigen. The antibody according to the invention is an antibody which binds to human Her2 and which does not (significantly) cross-react with other proteins. In such embodiments, the degree 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).

Dimerization-the pairing of receptors-is essential for the signaling activity of all HER receptors. According to the present invention, the term "Her dimerization inhibitor" or preferably "Her 2 heterodimerization inhibitor" refers to a therapeutic agent that binds to Her2 and inhibits Her2 heterodimerization. These are preferably antibodiesPreferably a monoclonal antibody, more preferably a humanized antibody, which binds to Her2 and inhibits 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. patent No. 5,821,337; humanized 2C4 mutants described in WO01/00245 are numbered 560, 561, 562, 568, 569, 570, 571, 574, or 56869. 7C2 and 7F3 and humanized variants thereof are described in WO 98/17797. Since the mechanism of action is different and since this antibody does not inhibit Her dimerization, this term should not be used e.g. as Herceptin^TMThe name of (1) monoclonal antibody to trastuzumab (trastuzumab).

Preferred in this application is "antibody 2C 4", particularly humanized variants thereof (WO 01/00245; produced by hybridoma cell lines deposited with ATCC HB-12697 at the American type culture Collection of Manassass, Va., USA), which binds to a region in the extracellular domain of Her2 (e.g., at any one or more of the residues in the region from about residue 22 to about residue 584 of Her2, inclusive). "epitope 2C 4" is the region in the extracellular domain of ErbB2 to which antibody 2C4 binds. The expression "monoclonal antibody 2C 4" refers to an antibody having the antigen binding residues of the murine 2C4 antibody in the examples of WO01/00245 or derived from said murine 2C4 antibody. For example, monoclonal antibody 2C4 may be murine monoclonal antibody 2C4 or a variant thereof, such as humanized antibody 2C4, which has the antigen binding amino acid residues of murine monoclonal antibody 2C4. An example of a humanized 2C4 antibody is provided in example 3 of WO 01/00245. Unless otherwise indicated, the expression "rhuMAb 2C 4" as used herein refers to an antibody comprising the variable light chain (VL) and variable heavy chain (VH) sequences of WO01/00245 fused to human light chain and heavy chain IgG1 (non-a allotypes) constant region sequences, respectively, optionally expressed by Chinese Hamster Ovary (CHO) cells. Preferred embodiments of WO01/00245 are also preferred herein. Humanized antibody 2C4 is also known as pertuzumab (Omnit)arg^TM)。

A "kit" is any product (e.g., package or container) that includes at least one reagent, such as a probe, for specifically detecting a marker gene or protein of the invention. The product is preferably marketed, distributed or sold as a unit for carrying out the method of the invention.

The verbs "determine" and "evaluate" shall have the same meaning and are used interchangeably in this application.

Detailed Description

Conventional techniques of molecular biology and nucleic acid chemistry are explained in the literature and are within the skill of the art. See, for example, Sambrook, j, et al, Molecular Cloning: a Laboratory Manual (molecular cloning: A Laboratory Manual), Cold Spring Harbor Laboratory Press (Cold Spring Harbor Laboratory Press), Cold Spring Harbor Laboratory Press (Cold Spring Harbor), New York, 1989; gait, m.j. (ed.), Oligonucleotide Synthesis-a Practical Approach, IRL publishers ltd, 1984; hames, b.d., and Higgins, S.J, (eds.), Nucleic Acid hybridization-a Practical Approach, IRL publishers ltd, 1985; and series of books, Methods in enzymology, Academic Press, Inc. (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 one embodiment of the invention, a method of predicting the response of a treatment with a HER dimerization inhibitor in a patient comprises the steps of:

a) evaluation in biological samples from patients

A marker gene or combination of marker genes selected from the group consisting of epidermal growth factor, transforming growth factor alpha and HER2 marker genes, or

Preferably, in the method according to the invention, the step a) of assessing a marker gene or a combination of marker genes comprises

a1) Assessing the expression level of the marker gene or combination of marker genes,

a2) determining whether the expression level in step a1) is above or below a threshold.

The threshold value is preferably a value expressed in terms of mass/volume in relation to serum or plasma or in relation to mass/mass in relation to tumour tissue. It can be detected by methods known to the expert skilled in the art and disclosed by the present invention.

In a preferred embodiment of the invention, the response to the treatment can be determined if the threshold is higher or lower than the threshold. As for the use of the single marker gene according to the present invention, the case for patients with metastatic breast cancer is as follows. However, this may depend on the indication, but may be determined based on the content of the present invention. With respect to the transforming growth factor alpha marker gene alone, low expression levels are advantageous for survival without progression (death or time to progression) and overall survival (time to death) when considering Her dimerization inhibitor treatment for Her2 low expressing metastatic breast cancer patients, i.e. in these patients low expression levels of the transforming growth factor alpha marker gene predict a good response to treatment with Her dimerization inhibitor (see figure 7). This is the case for both the original and adjusted clinical covariates. Therefore, it is preferred that the expression level of the transforming growth factor alpha marker gene assessed in step a1) is below a threshold value to predict a good response to treatment with a Her dimerization inhibitor in Her2 low expressing metastatic breast cancer patients. This is also the case with Her2 marker gene alone in these patients, in particular with soluble Her2 extracellular domain, and with epidermal growth factor marker gene alone in these patients.

With respect to the amphiregulin marker gene alone, in the original analysis, low expression levels were favorable for survival without progression (death or time to progression) and overall survival (time to death) when Her dimerization inhibitor treatment was considered for Her2 low expressing metastatic breast cancer patients, i.e. in these patients low expression levels of the amphiregulin marker gene predicted good response to treatment with Her dimerization inhibitor. This is the case for both the original and adjusted clinical covariates. Analysis of the modulation of clinical covariates showed that high expression levels were favourable for progression free survival (death or time to progression) and overall survival (time to death) in these patients after treatment with HER dimerization inhibitors.

Since marker genes, particularly in serum, can be used in multi-marker predictive models potentially including other clinical co-variables, the trend of the beneficial effect of a single marker gene in such models cannot be determined in a simple manner and may be contrary to the trend found in univariate analysis, i.e. the situation described for the use of a single marker gene.

More preferably, in the method according to the invention, the threshold value is determined before step a1) of the method according to the invention by:

1) assessing the expression level of a marker gene or a combination of marker genes in a plurality of biological samples from a patient prior to treatment with a HER dimerization inhibitor,

2) treating said patient with a HER dimerization inhibitor,

3) correlating the response of the patient treated with the HER dimerization inhibitor with the expression level of said marker gene or combination of marker genes determined in step a), thereby determining a threshold value.

The threshold value is preferably a value expressed in relation to the mass/volume of serum or plasma or the mass/mass of tumor tissue.

Mutants or variants of the marker gene according to the invention and mutants or variants of the marker gene for use in the method according to the invention are also contemplated by the invention. In these mutants or variants, the natural sequence of the marker gene is changed by substitution, deletion or insertion. "native sequence" refers to the same amino acid or nucleic acid sequence as the wild-type or native form of the marker gene or protein.

Mutants or variants of the proteins according to the invention, and mutants or variants of the proteins for use in the methods according to the invention are also contemplated by the present invention. "mutant amino acid sequence", "mutein" or "mutant polypeptide" refers to a polypeptide having an amino acid sequence that is different from the native sequence or that is encoded by a nucleotide sequence from which a variant is intentionally made. "mutein", "variant protein" or "mutein" (mutein) "means a protein comprising a mutated amino acid sequence and includes polypeptides that differ from the amino acid sequence of a native protein according to the invention by amino acid deletions, substitutions, or both.

The invention also contemplates methods of predicting response to treatment with a combination of a HER dimerization inhibitor and another substance or agent that is a chemotherapeutic agent or a therapeutic antibody for treating cancer. The chemotherapeutic agent may be, for example, gemcitabine (Gemcitabine)Chemical name: 2 ', 2 ' -difluorodeoxycytidine (dFdC)), carboplatin (diamine- (cyclobutane-1, 1-dicarboxxylato (2-) -O, O ') -platinum), or paclitaxel ((dFdC)Chemical name: beta- (benzoylamino) -alpha-hydroxy-, 6, 12 b-bis (acetyloxy) -12- (benzene)Formyloxy) -2a, 3, 4, 4a, 5,6, 9, 10, 11, 12, 12a, 12 b-dodecahydro-4, 11-dihydroxy-4 a, 8, 13, 13-tetramethyl-5-oxo-7, 11-methylene-1H-aryldecano (3, 4) benz (1, 2-b) oxet-9-yl ester, (2aR- (2a- α, 4- β, 4a- β, 6- β, 9- β 0(α -R, β -S), 11- α, 12- α, 12a- α, 2b- α)) -benzenepropanoic acid).

In a preferred embodiment of the invention, the biological sample is serum, plasma or tumor tissue. The tumor tissue may be formalin-fixed paraffin-embedded tumor tissue or freshly frozen tumor tissue.

In another preferred embodiment of the invention said HER dimerization inhibitor inhibits heterodimerization of HER2 with EGFR or HER3 or HER 4. Preferably, the HER dimerization inhibitor is an antibody, preferably antibody 2C4. Throughout this application, preference is given to "antibody 2C 4", particularly humanized variants thereof (WO 01/00245; produced by a hybridoma cell line deposited with ATCC HB-12697 at the American type culture Collection of Manassass, Va., USA), which binds to a region in the extracellular domain of Her2 (e.g., at any one or more of the residues in the region from about residue 22 to about residue 584 of Her2, inclusive). An example of a humanized 2C4 antibody is provided in example 3 of WO 01/00245. Humanized antibody 2C4 also called Omnitarg^TMOr pertuzumab.

In another preferred embodiment of the invention said patient is a cancer patient, preferably a breast cancer, ovarian cancer, lung cancer or prostate cancer patient, said breast cancer patient is preferably a metastatic breast cancer patient or a HER2 low expressing breast cancer or a metastatic breast cancer patient, or a Her2 high expressing breast cancer or a metastatic breast cancer patient, said ovarian cancer patient is preferably a metastatic ovarian cancer patient.

Preferably, two, three or all four marker genes, marker polynucleotides or marker proteins are used in combination, i.e. in all disclosed embodiments of the invention or in the methods, uses or kits according to the invention. Preferably, the expression levels of:

-a combination of epidermal growth factor, transforming growth factor alpha or HER2 marker gene, marker protein or marker polynucleotide,

a marker gene combination comprising an amphiregulin marker gene and a marker gene selected from the group consisting of epidermal growth factor, transforming growth factor alpha and HER2 marker genes,

a combination of epidermal growth factor and transforming growth factor alpha or HER2 marker gene, marker protein or marker polynucleotide, or

-a combination of transforming growth factor alpha and a HER2 marker gene, protein or polynucleotide.

In a particularly preferred embodiment of the invention, the marker gene combination consists of:

transforming growth factor alpha and HER2 marker genes,

-transforming growth factor alpha and epidermal growth factor marker gene, or

Amphiregulin, epidermal growth factor, transforming growth factor alpha and HER2 marker genes.

In another particularly preferred embodiment of the invention, the marker gene combination consists of:

epidermal growth factor and HER2 marker gene,

amphiregulin and epidermal growth factor marker genes,

amphiregulin and transforming growth factor alpha marker genes,

amphiregulin and the HER2 marker gene,

amphiregulin, epidermal growth factor and transforming growth factor alpha marker genes,

amphiregulin, epidermal growth factor and HER2 marker gene,

amphiregulin, TGF-alpha and HER2 marker genes, or

Epidermal growth factor, transforming growth factor alpha and HER2 marker genes.

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

There are many different types of immunoassays that can be used in the methods of the invention, for example, enzyme-linked immunosorbent assays (ELISA), Fluorescent Immunoabsorbents Assays (FIA), Chemically Linked Immunoabsorbents Assays (CLIA), Radioimmunoassays (RIA), and immunoblots. For a review of the different immunoassays that can be used, see: lottspich and Zorbas (eds.), Bioanalytik, first edition 1998, Spektrum Akademischer Verlag, Heidelberg, Berlin, Germany in another preferred embodiment of the invention, therefore, the expression level is determined using a method selected from the group consisting of: thus, more preferably, the expression level is determined using a method selected from the group consisting of proteomics, flow cytometry, immunocytochemistry, immunohistochemistry, enzyme-linked immunosorbent assay, multichannel enzyme-linked immunosorbent assay, and variants of these methods.

In another preferred aspect of the present inventionIn embodiments, the fragment of the marker protein is the extracellular domain of Her2 marker protein. Preferably, the extracellular domain of the Her2 marker protein has a molecular weight of about 105.000 daltons. "Dalton" means the equivalent of a hydrogen atom weight or 1.657X 10^-24Mass units of grams.

In another preferred embodiment of the present invention,

-the amino acid sequence of the amphiregulin marker protein is the amino acid sequence SEQ ID NO: 1,

-the amino acid sequence of the epidermal growth factor marker protein is the amino acid sequence of SEQ ID NO: 2,

-the amino acid sequence of the transforming growth factor alpha marker protein is the amino acid sequence SEQ ID NO: 3, or

The amino acid sequence of the HER2 marker protein is the amino acid sequence SEQ ID NO: 4.

in another preferred embodiment of the invention, the threshold values in the serum for

A threshold value for the transforming growth factor alpha marker protein of 2.0-10.0, preferably 2.0-5.0pg/ml, more preferably about 3.5pg/ml,

a threshold value of 100-250pg/ml, preferably about 150pg/ml, or for the epidermal growth factor marker protein

The threshold for amphiregulin marker proteins is 6-15pg/ml, preferably about 12 pg/ml.

In another preferred embodiment of the invention, the threshold value for the extracellular domain of the Her2 marker protein in serum is 12-22ng/ml, preferably about 18 ng/ml.

In another preferred embodiment of the present invention, the expression level of a marker gene or a combination of marker genes in a biological sample is assessed by detecting the expression level of a transcribed marker polynucleotide or a fragment of said transcribed marker polynucleotide encoded by said marker gene or by detecting the expression level of a transcribed marker polynucleotide or a fragment of said transcribed marker polynucleotide encoded by a combination of marker genes. Preferably, the transcribed tagged polynucleotide is a cDNA, mRNA or hnRNA, or wherein the plurality of transcribed tagged polynucleotides is a cDNA, mRNA or hnRNA.

Preferably, the detecting step further comprises amplifying the transcribed polynucleotide. Amplification is preferably performed using the polymerase chain reaction which specifically amplifies the nucleic acid to a detectable amount. Other amplification reactions that may be used are the ligase chain reaction (LCR; Wu D.Y. and Wallace R.B., Genomics 4(1989) 560-; polymerase ligase chain reaction (Barany f., PCR Methods and applications) (PCR Methods and applications) 1(1991) 5-16); Gap-LCR (WO 90/01069); repair chain reactions (EP 0439182A 2), 3SR (Kwoh, D.Y. et al, Proc. Natl. Acad. Sci. USA (Proc. Natl. Acad. Sci. USA) 86(1989) 1173-. In addition, there are Strand Displacement Amplification (SDA), transcription-mediated amplification (TMA), and Q.beta. -amplification (for review see, e.g., Whelen, A.C. and Persing, D.H., Annu.Rev.Microbiol. (annual journal of microbial review) 50(1996) 349) 373; Abramson, R.D., and Myers T.W., curr.Opin.Biotechnol. (Current Biotech concept) 4(1993) 41-47). More preferably, the detecting step employs a method of quantitative reverse transcriptase polymerase chain reaction.

Other suitable polynucleotide detection methods are known to those skilled in the art and are described in standard texts such as Sambrook J. et al, Molecular Cloning: a Laboratory Manual (molecular cloning: A Laboratory Manual), Cold Spring Harbor Laboratory Press (Cold Spring Harbor Laboratory Press), Cold Spring Harbor Laboratory Press (Cold Spring Harbor), New York, 1989; and Ausubel, F. et al, Current protocols in Molecular Biology (modern methods in Molecular Biology), 1987, J.Wiley and Sons, New York. there may also be further purification steps, such as precipitation steps, prior to performing the polynucleotide detection step.

In another preferred embodiment of the present invention, the expression level of a marker gene is assessed by detecting the presence of the transcribed marker polynucleotide or fragment thereof in a sample using a probe that anneals to the transcribed marker polynucleotide or fragment thereof under stringent hybridization conditions, or the expression level of a marker gene combination in a sample is assessed by detecting the presence of the transcribed marker polynucleotide or fragment thereof in a sample using a probe that anneals to the transcribed marker polynucleotide or fragment thereof under stringent hybridization conditions. This method can be performed in a homogeneous detection system. An example of a "homologous" detection system isSystems described in US5,210,015, US5,804,375 and US5,487,972. Briefly, the method is based on the 5 '-3' exonuclease activity of a dual-labeled probe and Taq DNA polymerase. The probe is complementary to the target sequence amplified by the PCR method and is located between two PCR primers during each polymerization cycle step. The probe has two fluorescent labels attached thereto. One label is a reporter dye, such as 6-carboxyfluorescein (FAM), which quenches its emission spectrum by energy transfer due to spatial proximity to a second fluorescent dye, 6-carboxy-tetramethyl-rhodamine (TAMRA). Taq DNA polymerization during extension of primed DNA strands during each amplification cycleThe enzyme displaces and degrades the annealed probe, degradation due to the intrinsic 5 '-3' exonuclease activity of the polymerase. This mechanism also releases the reporter dye from the quenching activity of TAMRA. As a result, the fluorescence activity increases with increasing cleavage of the probe, which is proportional to the amount of PCR product formed. Thus, the intensity of the released fluorescent label is detected to determine the amplified target sequence. Another example of a "homologous" detection system is used inForms of devices (see, e.g., US6,174,670) are provided, some of which are sometimes referred to as "kissing probe" forms. Also, the principle is based on two interacting dyes, however, they are characterized in that the emission wavelength of the donor-dye excites the acceptor-dye by fluorescence resonance energy transfer. Recently introducedThe AmplPrep device (Roche Diagnostics GmbH), D-68305Mannheim, Germany) to expand automation by separating target sequences from streptavidin-coated magnetic particles using biotinylated sequence-specific capture probes (Jungkind, D., J.Clin.Virol. (J.Clin.Virol.) 20(2001) 1-6; Stelzl, E., et al, J.Clin.Microbiol. (J.Clin.Microbiol.Microbiol. (J.Microbiol.Cl.) 40(2002) 1447-. Recently it has added other common tools, Total Nucleic Acid Isolation (TNAI) Kit (Total Nucleic acid isolation (TNAI) Kit, Roche Diagnostics). Such laboratory-use reagents are allowed to be used inAll nucleic acids were generally, sequence-specifically isolated from plasma and serum on the AmpliPrep instrument, which was essentially based on the method developed by Boom, r, et al, j.clin.microbiol. (journal of clinical microbiology) 28(1990) 495-.

In another preferred embodiment of the invention, the nucleic acid sequence of the amphiregulin marker polynucleotide is the nucleic acid sequence of SEQ ID NO: 5,

-the nucleic acid sequence of the epidermal growth factor marker polynucleotide is the nucleic acid sequence of SEQ ID NO: 6,

-the nucleic acid sequence of the transforming growth factor alpha tagged polynucleotide is the nucleic acid sequence of SEQ id no: 7, or

-the nucleic acid sequence of the HER2 marker polynucleotide is the nucleic acid sequence of SEQ ID NO: 8.

in another embodiment of the invention, a probe that hybridizes under stringent conditions to an epidermal growth factor, transforming growth factor alpha, or HER 2-labeled polynucleotide, or an antibody that binds to an epidermal growth factor, transforming growth factor alpha, or HER 2-labeled protein, is used to predict response in a patient to treatment with a HER dimerization inhibitor, or a probe that hybridizes under stringent conditions to an amphiregulin, epidermal growth factor, transforming growth factor alpha, or HER 2-labeled polynucleotide, or an antibody that binds to an amphiregulin, epidermal growth factor, transforming growth factor alpha, or HER 2-labeled protein, is used to select a composition that inhibits the development of a disease in a patient.

In another embodiment of the invention, a kit is provided comprising a probe that anneals to a polynucleotide labeled with amphiregulin, epidermal growth factor, transforming growth factor alpha or HER2, or an antibody that binds to a protein labeled with amphiregulin, epidermal growth factor, transforming growth factor alpha or HER2, under stringent conditions. Such kits known in the art also comprise a plastic vessel, which can be used, for example, as a microtiter plate in a 96-well or 384-well format in the amplification step, or just as a common reaction tube, for example, a common reaction tube prepared by Eppendorf, Hamburg, germany, and also all other reagents for carrying out the method according to the invention, preferably an immunoassay, such as an enzyme-linked immunosorbent assay (ELISA), a Fluorescent Immunoadsorption Assay (FIA), a chemically coupled immunoadsorption assay (CLIA), a Radioimmunoassay (RIA), and an immunoblot. For a review of the different immunoassays and reagents that may be used, see: lottspich and Zorbas (eds.), Bioanalytik, first edition, 1998, Spektrum Akademischer Verlag, Heidelberg, Berlin, Germany. Preferably, the probe or antibody is provided in combination with various labeled polynucleotides or labeled proteins in a kit format, such as the preferred combinations of labeled polynucleotides or labeled proteins disclosed above.

In another embodiment of the present invention, there is provided a method of selecting a composition for inhibiting the progression of a disease in a patient, the method comprising:

a) separately exposing aliquots of a biological sample from a cancer patient in the presence of a plurality of test compositions;

b) comparing the expression level of a marker gene or combination of marker genes selected from the group consisting of amphiregulin, epidermal growth factor, transforming growth factor alpha and HER2 marker gene in the aliquot of biological sample contacted with the test composition with the expression level of a marker gene or combination of marker genes selected from the group consisting of amphiregulin, epidermal growth factor, transforming growth factor alpha and HER2 marker gene in the aliquot of biological sample not contacted with the test composition;

c) selecting a test composition that alters the expression level of said marker gene or said combination of marker genes in an aliquot comprising said test composition relative to an aliquot not contacted with the test composition, wherein the presence of a significant or at least 10% difference between the expression level of said marker gene or said combination of marker genes in an aliquot of the biological sample contacted with said test composition and the expression level of the corresponding marker gene or combination of marker genes in an aliquot of the biological sample not contacted with said test composition is indicative of selecting said test composition. The disease is preferably cancer and the patient is preferably a cancer patient as described above.

b) (ii) the expression level of each of the following in an aliquot of a biological sample contacted with the test composition

A marker gene or marker gene combination selected from the group consisting of epidermal growth factor, transforming growth factor alpha and HER2 marker gene, or

comparing the expression levels of the following in an aliquot of a biological sample not contacted with the test composition

-a marker gene combination comprising an amphiregulin marker gene and a marker gene selected from the group consisting of epidermal growth factor, transforming growth factor alpha and HER2 marker genes;

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

In another embodiment of the invention, there is provided a method of derivatizing (or identifying) a candidate agent, the method comprising:

a) contacting an aliquot of a biological sample from a cancer patient with a candidate agent, and determining the expression level in the aliquot of a marker gene or combination of marker genes selected from the group consisting of amphiregulin, epidermal growth factor, transforming growth factor alpha, and HER2 marker genes,

d) deriving (or identifying) the agent from the observed effect, wherein a difference of at least 10% between the expression level of the marker gene or combination of marker genes in an aliquot of the biological sample contacted with the candidate agent and the expression level of the corresponding marker gene or combination of marker genes in an aliquot of the biological sample not contacted with the candidate agent is indicative of the effect of the candidate agent.

In another embodiment of the present invention, there is provided a method of derivatizing a candidate agent, the method comprising:

a) contacting an aliquot of a biological sample from a cancer patient with a candidate agent, and determining the expression levels of

d) deriving said agent from said observed effect, wherein a difference of at least 10% between the expression level of said marker gene or combination of marker genes in an aliquot of the biological sample contacted with said candidate agent and the expression level of the corresponding marker gene or combination of marker genes in an aliquot of the biological sample not contacted with said candidate agent is indicative of the effect of said candidate agent.

Preferably, the candidate agent is a candidate inhibitor. Preferably, the candidate agent is a candidate enhancer.

In another embodiment of the invention, candidate agents derived by the method according to the invention are provided.

In another embodiment of the invention, there is provided a pharmaceutical formulation comprising an agent according to the invention.

In another embodiment of the invention, the medicament according to the invention is used for the preparation of a composition for the treatment of cancer. Preferred forms of cancer are as described above.

In another preferred embodiment of the present invention, the method for producing a medicament comprises the steps of the method according to the present invention and

ii) combining the drug candidate, i.e. the candidate agent identified in step (c) or an analogue or derivative thereof, with a pharmaceutically acceptable carrier.

In another embodiment of the invention, a marker protein or marker polynucleotide selected from the group consisting of amphiregulin, epidermal growth factor, transforming growth factor alpha and HER2 marker proteins or marker polynucleotides is used to derive a candidate agent or to select a composition for inhibiting the development of a disease in a patient. The disease is preferably cancer and the patient is preferably a cancer patient as disclosed above.

In another embodiment of the invention, a HER dimerization inhibitor is used for the preparation of a medicament for the treatment of a human cancer patient, characterized in that the treatment comprises assessing the following items in a biological sample from the patient:

-a marker gene combination comprising an amphiregulin marker gene and a marker gene selected from the group consisting of epidermal growth factor, transforming growth factor alpha and HER2 marker genes.

The preparation of a medicament for the treatment of human cancer patients and in particular the formulation is described in WO01/00245, which is incorporated herein by reference, in particular with respect to antibody 2C4.

In a preferred embodiment of the invention, in the use of the Her dimerization inhibitor for the preparation of a medicament for the treatment of a human cancer patient, the treatment comprises assessing the marker gene or marker gene combination at least once or repeatedly during the course of the treatment, preferably assessing the expression level of the marker gene or marker gene combination.

In all embodiments of the invention, a combination of marker genes, marker polynucleotides or marker proteins as disclosed above is used. In all embodiments of the invention, preferred values for the difference in expression level determined in each step are also as described above.

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 may be made in the proposed method without departing from the spirit of the invention.

Description of the drawings

FIG. 1: scatter plot of clinical benefit of TGF-alpha log-transformed relative categorisation

FIG. 2: scatter plot of clinical benefit of amphiregulin log-transformed relative classifications

FIG. 3: sequential (Ordinal) clinical benefit TGF-alpha

FIG. 4: sequential clinical benefit amphiregulin

FIG. 5: sequential clinical benefit EGF

FIG. 6: sequential clinical benefit HER2-ECD

FIG. 7: comprehensive investigative intercept and logarithmically arranged p-values for TTP and TTD for amphiregulin, EGF, TGF-alpha, HER2-ECD

FIG. 8: TGF-alpha Kaplanimeer plots for progression/death time based on a investigational single marker cut-off

FIG. 9: TGF-alpha Kaplan Meier plot for death time based on investigational single marker cut-points

FIG. 10: amphiregulin Kaplan Meier plot for progression/death time based on investigational single marker intercept

FIG. 11: amphiregulin KaplanMeier plot for death time based on investigational single marker intercept

FIG. 12: EGF Kaplanimeier plot for progression/death time based on investigational single marker cut-off

FIG. 13: EGF Kaplan Meier plot for death time based on investigational single marker cut-off

FIG. 14: HER 2-ECDCKaplan Meier plot for progression/death time based on investigational single marker cut-points

FIG. 15: HER2-ECD Kaplan message plot for death time based on investigational single-marker cut-points

FIG. 16: as an example of a combined score, in TTP: sequential clinical benefit HER2-ECD TGF-alpha combinations further improving spacing between groups of greater clinical benefit/lesser clinical benefit

FIG. 17: comprehensive investigative intercept and logarithmically arranged p-values for TTP and TTD for TGF-alpha and HER2-ECD combinations

FIG. 18: HER 2-ECD/TGF-alpha Kaplan Meier plot for progression/death time based on investigational combinatorial marker cut-points

FIG. 19: HER 2-ECD/TGF-alpha Kaplan Meier plot for death time based on investigational combinatorial marker cut-points

Examples

General review of the prediction rules

The general form of the prediction algorithm consists of a specification of a function of one or more biomarkers, potentially including a clinical co-variable predicting response or non-response, or more generally, a predicted benefit or lack of benefit with respect to a properly defined clinical endpoint.

This can be expressed in terms of the Haviscidide function (Heaviside function) for the detection χ for a particular cut-off c and biomarker, where a or B is predicted in binary, then

And predicting A if H (x-c) ═ 0.

And predicting B if H (x-c) ═ 1.

This is the simplest way to apply univariate biomarker detection in the prediction algorithm. If such a simple rule is sufficient, it allows a simple identification of the direction of action, i.e.whether a high or low expression level is advantageous for the patient.

This situation may be more complicated if clinical co-variables need to be considered and/or if multiple biomarkers are used in the multivariate prediction algorithm. To illustrate this situation, there are two hypothetical embodiments:

covariate adjustment (assuming example):

for biomarker X, high expression levels were found in the clinical trial population to correlate with worse prognosis (univariate analysis). Closer analysis indicates the presence of two tumor types in the population, one of which has a worse prognosis than the other, and at the same time the biomarker expression is generally higher for this tumor group. Adjusted covariate analysis showed that the relationship between clinical benefit and prognosis was reversed for each tumor type, i.e. lower expression levels within a tumor type correlate with better prognosis. This diametrically opposite effect is masked by the covariate tumor type-and the covariate adjustment analysis as part of the prediction algorithm reverses this trend.

Multivariate prediction (hypothetical example):

for biomarker X, high expression levels were found in the clinical trial population to be somewhat correlated with worse prognosis (univariate analysis). For the second biomarker Y, a similar observation was made by univariate analysis. The combination of X and Y indicates that if both biomarkers are low, a good prognosis is observed. This makes the rule predict benefit if both biomarkers are below certain cut-points (AND-links of the Hevesedel prediction function). For the combination law, there is no longer a simple law expressed in the univariate sense. For example, having a low expression level in X no longer automatically predicts a better prognosis.

These simple examples show that the prediction with or without covariates cannot be judged at the univariate level of each biomarker. The combination of multiple biomarkers plus potential adjustments by covariates does not allow for specifying simple relationships with respect to a single biomarker.

Statistical method

The statistical task comprises the following steps:

1. pre-selection of candidate biomarkers

2. Pre-selection of relevant clinical prognostic co-variables

3. Selection of biomarker prediction function at univariate level

4. Selection of biomarker prediction function comprising clinical covariates at univariate level

5. Selection of biomarker prediction function at multivariate level

6. Selection of biomarker prediction functions comprising clinical covariates at the multivariate level

The following details the different steps:

ad 1: pre-selecting candidate biomarkers: statistical pre-selection of candidate biomarkers is localized to the strength of correlation with clinical benefit detection. For this purpose, different clinical endpoints may be translated into derived surrogate scores, e.g., as a sequential designation of the degree of clinical benefit or morbidity score on TTP or TTD that avoids the examined observations. Detection of these alternative transitions can be readily used for simple correlation analysis, for example, by the parametric-free Spearman rank correlation method. Another approach is the examination of bivariate scattergrams, e.g. by showing scatter points on an individual patient basis (x-axis: biomarker values, y-axis: clinical benefit detection), for example, where there are also parametric-free regression lines obtained by smoothing splines, which can be used to visualize the correlation of biomarkers and clinical benefits.

The purpose of these different methods is to pre-select biomarker candidates that show some correlation with clinical benefit in at least one of the benefit tests used, while the results with respect to the other tests are not contradictory. When there is a control group available, then the difference in the correlation of the biomarker to clinical benefit in different parts is an indication of the different predictions that made the biomarker suitable for further consideration.

Ad 2: pre-selection of relevant clinical prognostic co-variables: the term "clinical co-variable" is used herein to describe all other information about a patient that is generally available at baseline. These clinical covariates include demographic information such as gender, age, etc., other past condition information, concomitant diseases, concomitant treatments, physical examination results, general laboratory parameters obtained, known characteristics of the target tumor, information quantifying the extent of malignancy, clinical performance scores such as ECOG or Karnofsky indices, clinical disease segmentation, timing and outcome of pretreatment and disease history, and all similar information, which may be relevant to clinical prognosis. Statistical pre-selection of clinical covariates is similar to the method of pre-selecting biomarkers and is also localized to the strength of correlation with clinical benefit detection. In principle, therefore, the same method is used as considered under 1. In addition to statistical criteria, criteria and theoretical knowledge from clinical experience can also be used to pre-select relevant clinical co-variables.

The prognosis by clinical co-variable interacts with the prognosis of the biomarker. They will be considered accurate predictions if necessary.

Ad 3: biomarker prediction function was chosen at univariate level: the term "predictive function" will be used in a generic sense to mean a numerical function of biomarker detection that results in numbers that suggest a target prediction proportionally.

A simple example is the selection of a Hevesiad function for a particular intercept point c and biomarker detection x, where a binary prediction A or B is made, then

Predict a if H (x-c) ═ 0.

Predict B if H (x-c) ═ 1.

This is probably the most common way to use univariate biomarker detection in the prediction algorithm. The definition of the prediction function typically repeats existing training data sets (training data sets) that can be used to study the likelihood of prediction. In order to obtain the appropriate intercept point c from the exercise settings, different approaches may be taken. First, a scatter plot with a smooth spline mentioned under 1 may be used to define the intercept. Alternatively, certain distribution percentages may be selected, for example, a median or quartile value. The intercept points can also be systematically extracted by studying all possible intercept points according to their predictive potential for clinical benefit detection. These results can then be plotted to allow manual selection or optimization using some search algorithm. This is achieved based on the end-points TTP and TTD using the Cox model, where at each detection intercept the biomarker is used as a binary co-variable. The prediction criterion is the proportion of risk obtained. The results for TTP and TTD can then be considered together to select a cutoff point that displays a prediction that fits both endpoints.

Another less common method to select the prediction function may be based on a fixed parameter Cox regression model using biomarker values (possibly transformed) as covariates obtained from exercise settings. The prediction may then only depend on whether the calculated risk ratio is less than 1 or greater than 1.

Another possibility is based on a determination of some probability ratio (or monotonic transformation thereof), where the target probability of possibility is predetermined in the exercise setting regarding the split prediction situation. The biomarkers are then inserted into some probability ratio function.

Ad 4: biomarker prediction functions containing clinical co-variables were selected at the univariate level: univariate here means that only one biomarker is used-this may be a multivariate model with respect to clinical co-variables. This approach is similar to a search without clinical covariates, except that the approach should allow for the incorporation of relevant covariate information. The scatter-point method of selecting the intercept point allows only limited application of the co-variable, e.g., the binary co-variable may be a color of the code within the plot. If this analysis relies on some regression method, then the application of co-variables (or more of them at the same time) is often facilitated. Intercept point searches based on the Cox model described under 3 allow for easy incorporation of covariates and thus result in univariate intercept point searches for covariate adjustments. The co-variable adjustment can be performed as co-variables in the model or by inclusion in the hierarchical analysis.

Other choices of the prediction function also allow for the incorporation of co-variables.

This is directly used for Cox model selection as a prediction function. There is an option to assess the impact of co-variables on the level of interaction, which means, for example, that different risk proportions are applied for different age groups.

For the probability ratio type of the prediction function, the prediction probability must include a covariate to evaluate. A multivariate pattern recognition approach can be applied here, or the biomarker values can be adjusted (before probability estimation) by multiple regression on the covariates.

The CART technique (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 biomarkers (raw assay levels) plus clinical co-variables that apply clinical benefit assays as responses. This method intercept point is searched and a decision tree type that contains a function of the covariate used for prediction will be found. The intercept points and algorithms selected by CART are generally near optimal and can be combined and unified by considering different clinical benefit tests.

Ad 5: selecting biomarker prediction function at multivariate level: when there are some biomarker candidates that retain their predictive potential in different univariate predictor function selections, then further improvement can be achieved by biomarker combination, i.e. considering multivariate predictor functions.

Based on a simple Hevesseld function model, biomarker combinations may be evaluated, for example, by considering a bivariate scatter plot in which the biomarker values for the optimal intercept points are specified. The combination of biomarkers can be achieved by combining different haversonie functions by reasonable AND OR operations to obtain improved predictions.

CART technology (classification and regression trees) can be used for a variety of biomarkers (raw detection levels) and clinical benefit tests in response to obtain a decision tree type of cutoff and prediction functions for the biomarkers. The intercept points and algorithms selected by CART are generally near optimal and can be combined and unified by considering different clinical benefit tests.

Cox-regression can be applied at different levels. The first way is to combine multiple biomarkers in a binary way (i.e., based on a haversian function with some intercept points). Other options apply the biomarkers in a metric manner (after appropriate conversion), or a combination of binary and metric manners. This extrapolating multivariable prediction function (evolution multivariate prediction function) is of the Cox type as described under 3.

This multivariate probability ratio method is difficult to implement, but is also described as the selection of a multivariate prediction function.

Ad 6: selecting biomarker prediction functions comprising clinical covariates at a multivariate level: when there are clinically covariates of interest, then further improvements can be achieved by combining multiple biomarkers with multiple clinical covariates. Different prediction function choices will be evaluated with respect to the likelihood of containing clinical covariates.

Based on a simple logical combination of the Hevessie functions on the biomarkers, other covariates can be included in the prediction function based on the logarithmic regression model obtained in the exercise setting.

The CART technique and the calculation decision tree can be easily applied with other co-variables, which would include those in the prediction algorithm.

All predictor functions based on Cox-regression can apply other clinical co-variables. There are options to assess the impact of co-variables on the level of interaction, which means, for example, that different risk proportions are applied for different age groups.

The multivariate probability ratio method does not directly extend to the application of other co-variables.

Example 1

Baseline sera from HER2 low expressing metastatic breast cancer patients treated with Pertuzumab were evaluated for HER ligand and depleted HER2(HER2ECD) levels as described below.

Kit for serum biomarker assessment

Marking	Detection of	Vendors
Marking	Detection of	Vendors	HER2-ECD	Bayer HER-2/neuELISA, Cat No.: EL501	DakoCytomation N.V./S.A.，Interleuvenlaan 12B，B-3001Heverlee
Amphiregulin	DuoSet ELISA development system human amphiregulin, catalog No.: DY262	R&D Systems Ltd.(R&System D limited), 19Barton Lane, Abingdon OX 143 NB, uk	HER2-ECD	Bayer HER-2/neuELISA, Cat No.: EL501	DakoCytomation N.V./S.A.，Interleuvenlaan 12B，B-3001Heverlee

The process comprises the following steps:

HER2-ECD：

the HER2-ECD ELISA was performed according to the supplier's recommendations.

Amphiregulin:

preparation of all reagents (supplied by kit), Standard dilutions (supplied by kit) and samples

Evencoat goat anti-mouse IgG microplate strips provided in frame (R & D, catalog number CP 002; not provided by kit). The frame is now called an ELISA plate.

Determining the number of wells required (number of standard dilutions + number of samples).

-determining a plate layout.

Add 100. mu.l of diluted capture antibody (supplied by kit; 1: 180 in PBS) to each well.

Incubation at room temperature for 1 hour.

Pipette each well and wash, repeat this step three times, for a total of 4 washes the wash is performed by filling each well with 400 μ Ι of wash buffer (not provided by the kit; using 0.05% tween-20 in PBS), using a manifold dispenser, and then pipette.

Add 100. mu.l of standard dilution or diluted sample per well (see below). The tip is replaced after each pipetting step.

Cover the plate with adhesive tape (provided by the kit).

Incubate at room temperature on a shaking platform for 2 hours.

-repeating the blowing/washing as described before.

-the blown up sample and the washing solution are treated with a laboratory disinfectant.

100. mu.l of detection antibody (provided by the kit) diluted 1: 180 in reagent diluent (not provided by the kit; using 1% BSA in PBS (Roth; albumin fragment V, cat. No. T844.2)) were added to each well.

Incubation at room temperature for 2 hours.

-repeating the blowing/washing as described before.

Add 100. mu.l of streptavidin-HRP working dilution (supplied by kit; 1: 200 dilution in reagent dilution) to each well. Covered with a new adhesive strip.

Incubation at room temperature for 20 min.

-repeating the blowing/washing as described before.

Adding 100. mu.l of substrate solution (R & D, catalog number DY 999; not provided by the kit) to each well

Incubation at room temperature for 20 min. And (4) avoiding light.

Add 50. mu.l stop solution (1.5M H2SO 4: (1.5) to each wellReinst, Merck, catalog number 713); not provided by the kit). Carefully mixed.

The optical density of each well was determined immediately using a microplate reader set at 450 nm.

Amphiregulin standard curve:

40ng/ml amphiregulin stock was prepared in 1% BSA in PBS, aliquoted and stored at-80 ℃. Solutions of amphiregulin in 20% BSA in PBS are unstable for more than two weeks and therefore cannot be used. A standard curve of amphiregulin in 20% BSA in PBS was freshly prepared from an aliquot of the amphiregulin stock prior to each experiment. The maximum concentration was 1000pg/ml (1: 40 dilution of amphiregulin stock). The standards provided by the ELISA kit generated a linear standard curve. The curve-based analysis of Excel allowed the determination of the curve equation for each ELISA.

Amphiregulin samples:

when samples were diluted 1: 1 in reagent diluent, all samples were within the linear range of the ELISA. Each sample was tested in duplicate. Depending on the quality of the data, and sufficient amount of serum, if desired. The assay was repeated in subsequent experiments.

EGF：

Removal of excess antibody-coated microtiter plate strips from the frame (provided by the kit). The frame is now called an ELISA plate.

-determining the number of holes needed: (number of standard dilutions + number of samples). times.2

-determining a plate layout.

To each well 50 μ l of test dilution RD1 (provided by the kit) was added.

Add 200. mu.l of standard dilution or diluted sample per well (e.g., 1: 20 in Calibrator Diluent RD 6H). The tip is replaced after each pipetting step.

Cover the plate with adhesive tape (provided by the kit).

Incubate at room temperature on a shaking platform for 2 hours.

-pipetting each well and washing, repeating this step three times for a total of 4 washes. Washing was performed by filling each well with 400 μ l of wash buffer (supplied from the kit), using a manifold dispenser, and then pipetting. After the final wash, any residual wash buffer was removed by pipetting. The plate was inverted and blotted dry with a clean paper towel.

Add 200 μ Ι of conjugate to each well (provided by the kit). Covered with a new adhesive strip.

Incubation at room temperature for 2 hours.

-repeating the blowing/washing as described before.

Add 200 μ l substrate solution to each well (provided by the kit).

Incubation at room temperature for 20 min. And (4) avoiding light.

Add 50. mu.l of stop solution (supplied by kit) to each well. Carefully mixed.

The optical density of each well was determined within 30 minutes using a microplate reader set at 450 nm.

EGF standard curve:

the standards provided by the ELISA kit generated a linear standard curve. In addition, very small concentrations showed detectable results.

EGF samples:

a total of 4 tests were performed using the samples. Each sample was tested 2-5 times, the number of tests depending on the quality of the results (mean +/-SD) and the availability of sufficient amount of serum. All samples were in the linear range of the ELISA when the samples were diluted 1: 20 in Calibrator diluent RD 6H.

TGF-α：

-determining a plate layout.

To each well 100 μ l of test dilution RD1W (provided by the kit) was added.

Add 50 μ l of standard dilution or sample per well. The tip is replaced after each pipetting step.

Cover the plate with adhesive tape (provided by the kit).

Incubate at room temperature on a shaking platform for 2 hours.

Add 200 μ l of TGF- α conjugate to each well (provided by the kit). Covered with a new adhesive strip.

Incubation at room temperature for 2 hours.

-repeating the blowing/washing as described before.

Add 200 μ l substrate solution to each well (provided by the kit).

Incubation at room temperature for 30 min. And (4) avoiding light.

Add 50. mu.l of stop solution (supplied by kit) to each well. Carefully mixed.

TGF- α standard curve:

TGF-alpha samples:

a total of 4 tests were performed using the samples. The samples were tested in 2-4 independent tests.

Serum data was analyzed to identify factors whose baseline serum levels correlated with the response of Pertuzumab therapy. For all factors, a skewed pattern of distribution (mean, standard deviation, median, minimum, maximum) was observed. Based on the logarithm: log (x +1), monotonic transition is used to reduce skew. In univariate analysis, the study was able to determine the appropriate cut-off point for the factor in relation to the likelihood of a response (defined as clinical benefit in this example). Herein, patients with clinical benefit are defined as those patients who have acquired a Partial Response (PR) or have remained stable for at least 6 months. Scatter plots of the factors versus response species were studied. Figures 1 and 2 show plots of log-conversion of clinical response species against serum levels of TGF-alpha and amphiregulin, respectively, to illustrate the methods.

Based on the scatter plot, an intercept point is selected for the factor to determine a group of patients experiencing greater clinical benefit. FIG. 3 (TGF-. alpha.), FIG. 4 (amphiregulin), FIG. 5(EGF), and FIG. 6(HER2-ECD) show clinical benefit relative to different factor groupings based on research intercept points calculated as original factor units. The cut-off separates some asymptomatic patients and, therefore, increases the response rate for the group with greater clinical benefit.

Example 2

In this example, the investigational intercept from example 1 was used to evaluate the univariate effect of the factorial grouping on different measures of clinical benefit of Pertuzumab treatment using time to progression/or death (TTP) and Time To Death (TTD) as alternative clinical endpoints. In the Kaplan-Meier estimation and log-rank assays for TTP and/or TTD, significant effects on TGF- α, amphiregulin, EGF and HER2-ECD were observed, as shown in the summary of FIG. 7.

Kaplan-Meier plots showing risk ratios are given in figures 8 and 9(TGF- α), 10 and 11 (amphiregulin), 12 and 13(EGF), and 14 and 15(HER2-ECD) for TTP and TTD (highest number of events observed), showing the significant effects of these factor groupings based on clinical outcome for patients treated with Pertuzumab.

Example 3

In this example, a multivariate approach was applied to identify combinations of factors that further improve the identification of patients with greater benefit from Pertuzumab treatment, reflecting the results deduced from the CART approach (classification and regression trees), the CART classification approach made it necessary to indicate all values in the clinical benefit of greater than 0 as benefit groups the serum levels of Her2-ECD, TGF- α, amphiregulin and EGF were used as variables the combination of serum Her2-ECD and serum TGF- α levels was chosen to give the best results the optimal cut-off for the combination of serum Her2-ECD and serum TGF- α levels was deduced from the CART results, resulting in the rule for the investigative classification of clinical benefit in the study population-the combination of low serum Her2-ECD values and low serum TGF- α values captured 2/2PR and 2/3SD in the study population for > 6 months, FIG. 16 shows clinical benefit relative to TGF- α/HER2-ECD combination groupings based on investigative combination intercept points FIG. 17 summarizes the effect of TGF- α and HER2-ECD combinations on TTP and TTD the Kaplan-Meier estimates and risk ratios given in FIG. 18(TTP) and FIG. 19(TTD) demonstrate a significant effect of groupings based on combinations of these factors on clinical outcomes of patients treated with Pertuzumab.

Sequence listing

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Leu Ala Leu Ile His His Asn Thr His Leu Cys Phe Val His Thr Val

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Leu Pro Cys His Pro Glu Cys Gln Pro Gln Asn Gly Ser Val Thr Cys

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Ser Tyr Met Pro Ile Trp Lys Phe Pro Asp Glu Glu Gly Ala Cys Gln

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Pro Cys Pro Ile Asn Cys Thr His Ser Cys Val Asp Leu Asp Asp Lys

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Gly Cys Pro Ala Glu Gln Arg Ala Ser Pro Leu Thr Ser Ile Ile Ser

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Ala Val Val Gly Ile Leu Leu Val Val Val Leu Gly Val Val Phe Gly

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Arg Leu Leu Gln Glu Thr Glu Leu Val Glu Pro Leu Thr Pro Ser Gly

690 695 700

Ala Met Pro Asn Gln Ala Gln Met Arg Ile Leu Lys Glu Thr Glu Leu

705 710 715 720

Arg Lys Val Lys Val Leu Gly Ser Gly Ala Phe Gly Thr Val Tyr Lys

725 730 735

Gly Ile Trp Ile Pro Asp Gly Glu Asn Val Lys Ile Pro Val Ala Ile

740 745 750

Lys Val Leu Arg Glu Asn Thr Ser Pro Lys Ala Asn Lys Glu Ile Leu

755 760 765

Asp Glu Ala Tyr Val Met Ala Gly Val Gly Ser Pro Tyr Val Ser Arg

770 775 780

Leu Leu Gly Ile Cys Leu Thr Ser Thr Val Gln Leu Val Thr Gln Leu

785 790 795 800

Met Pro Tyr Gly Cys Leu Leu Asp His Val Arg Glu Asn Arg Gly Arg

805 810 815

Leu Gly Ser Gln Asp Leu Leu Asn Trp Cys Met Gln Ile Ala Lys Gly

820 825 830

Met Ser Tyr Leu Glu Asp Val Arg Leu Val His Arg Asp Leu Ala Ala

835 840 845

Arg Asn Val Leu Val Lys Ser Pro Asn His Val Lys Ile Thr Asp Phe

850 855 860

Gly Leu Ala Arg Leu Leu Asp Ile Asp Glu Thr Glu Tyr His Ala Asp

865 870 875 880

Gly Gly Lys Val Pro Ile Lys Trp Met Ala Leu Glu Ser Ile Leu Arg

885 890 895

Arg Arg Phe Thr His Gln Ser Asp Val Trp Ser Tyr Gly Val Thr Val

900 905 910

Trp Glu Leu Met Thr Phe Gly Ala Lys Pro Tyr Asp Gly Ile Pro Ala

915 920 925

Arg Glu Ile Pro Asp Leu Leu Glu Lys Gly Glu Arg Leu Pro Gln Pro

930 935 940

Pro Ile Cys Thr Ile Asp Val Tyr Met Ile Met Val Lys Cys Trp Met

945 950 955 960

Ile Asp Ser Glu Cys Arg Pro Arg Phe Arg Glu Leu Val Ser Glu Phe

965 970 975

Ser Arg Met Ala Arg Asp Pro Gln Arg Phe Val Val Ile Gln Asn Glu

980 985 990

Asp Leu Gly Pro Ala Ser Pro Leu Asp Ser Thr Phe Tyr Arg Ser Leu

995 1000 1005

Leu Glu Asp Asp Asp Met Gly Asp Leu Val Asp Ala Glu Glu Tyr

1010 1015 1020

Leu Val Pro Gln Gln Gly Phe Phe Cys Pro Asp Pro Ala Pro Gly

1025 1030 1035

Ala Gly Gly Met Val His His Arg His Arg Ser Ser Ser Thr Arg

1040 1045 1050

Ser Gly Gly Gly Asp Leu Thr Leu Gly Leu Glu Pro Ser Glu Glu

1055 1060 1065

Glu Ala Pro Arg Ser Pro Leu Ala Pro Ser Glu Gly Ala Gly Ser

1070 1075 1080

Asp Val Phe Asp Gly Asp Leu Gly Met Gly Ala Ala Lys Gly Leu

1085 1090 1095

Gln Ser Leu Pro Thr His Asp Pro Ser Pro Leu Gln Arg Tyr Ser

1100 1105 1110

Glu Asp Pro Thr Val Pro Leu Pro Ser Glu Thr Asp Gly Tyr Val

1115 1120 1125

Ala Pro Leu Thr Cys Ser Pro Gln Pro Glu Tyr Val Asn Gln Pro

1130 1135 1140

Asp Val Arg Pro Gln Pro Pro Ser Pro Arg Glu Gly Pro Leu Pro

1145 1150 1155

Ala Ala Arg Pro Ala Gly Ala Thr Leu Glu Arg Pro Lys Thr Leu

1160 1165 1170

Ser Pro Gly Lys Asn Gly Val Val Lys Asp Val Phe Ala Phe Gly

1175 1180 1185

Gly Ala Val Glu Asn Pro Glu Tyr Leu Thr Pro Gln Gly Gly Ala

1190 1195 1200

Ala Pro Gln Pro His Pro Pro Pro Ala Phe Ser Pro Ala Phe Asp

1205 1210 1215

Asn Leu Tyr Tyr Trp Asp Gln Asp Pro Pro Glu Arg Gly Ala Pro

1220 1225 1230

Pro Ser Thr Phe Lys Gly Thr Pro Thr Ala Glu Asn Pro Glu Tyr

1235 1240 1245

Leu Gly Leu Asp Val Pro Val

1250 1255

<210>5

<211>1270

<212>DNA

<213> human

<400>5

agacgttcgc acacctgggt gccagcgccc cagaggtccc gggacagccc gaggcgccgc 60

gcccgccgcc ccgagctccc caagccttcg agagcggcgc acactcccgg tctccactcg 120

ctcttccaac acccgctcgt tttggcggca gctcgtgtcc cagagaccga gttgccccag 180

agaccgagac gccgccgctg cgaaggacca atgagagccc cgctgctacc gccggcgccg 240

gtggtgctgt cgctcttgat actcggctca ggccattatg ctgctggatt ggacctcaat 300

gacacctact ctgggaagcg tgaaccattt tctggggacc acagtgctga tggatttgag 360

gttacctcaa gaagtgagat gtcttcaggg agtgagattt cccctgtgag tgaaatgcct 420

tctagtagtg aaccgtcctc gggagccgac tatgactact cagaagagta tgataacgaa 480

ccacaaatac ctggctatat tgtcgatgat tcagtcagag ttgaacaggt agttaagccc 540

ccccaaaaca agacggaaag tgaaaatact tcagataaac ccaaaagaaa gaaaaaggga 600

ggcaaaaatg gaaaaaatag aagaaacaga aagaagaaaa atccatgtaa tgcagaattt 660

caaaatttct gcattcacgg agaatgcaaa tatatagagc acctggaagc agtaacatgc 720

aaatgtcagc aagaatattt cggtgaacgg tgtggggaaa agtccatgaa aactcacagc 780

atgattgaca gtagtttatc aaaaattgca ttagcagcca tagctgcctt tatgtctgct 840

gtgatcctca cagctgttgc tgttattaca gtccagctta gaagacaata cgtcaggaaa 900

tatgaaggag aagctgagga acgaaagaaa cttcgacaag agaatggaaa tgtacatgct 960

atagcataac tgaagataaa attacaggat atcacattgg agtcactgcc aagtcatagc 1020

cataaatgat gagtcggtcc tctttccagt ggatcataag acaatggacc ctttttgtta 1080

tgatggtttt aaactttcaa ttgtcacttt ttatgctatt tctgtatata aaggtgcacg 1140

aaggtaaaaa gtattttttc aagttgtaaa taatttattt aatatttaat ggaagtgtat 1200

ttattttaca gctcattaaa cttttttaac caaacagaaa aaaaaaaaaa aaaaaaaaaa 1260

aaaaaaaaaa 1270

<210>6

<211>4877

<212>DNA

<213> human

<400>6

actgttggga gaggaatcgt atctccatat ttcttctttc agccccaatc caagggttgt 60

agctggaact ttccatcagt tcttcctttc tttttcctct ctaagccttt gccttgctct 120

gtcacagtga agtcagccag agcagggctg ttaaactctg tgaaatttgt cataagggtg 180

tcaggtattt cttactggct tccaaagaaa catagataaa gaaatctttc ctgtggcttc 240

ccttggcagg ctgcattcag aaggtctctc agttgaagaa agagcttgga ggacaacagc 300

acaacaggag agtaaaagat gccccagggc tgaggcctcc gctcaggcag ccgcatctgg 360

ggtcaatcat actcaccttg cccgggccat gctccagcaa aatcaagctg ttttcttttg 420

aaagttcaaa ctcatcaaga ttatgctgct cactcttatc attctgttgc cagtagtttc 480

aaaatttagt tttgttagtc tctcagcacc gcagcactgg agctgtcctg aaggtactct 540

cgcaggaaat gggaattcta cttgtgtggg tcctgcaccc ttcttaattt tctcccatgg 600

aaatagtatc tttaggattg acacagaagg aaccaattat gagcaattgg tggtggatgc 660

tggtgtctca gtgatcatgg attttcatta taatgagaaa agaatctatt gggtggattt 720

agaaagacaa cttttgcaaa gagtttttct gaatgggtca aggcaagaga gagtatgtaa 780

tatagagaaa aatgtttctg gaatggcaat aaattggata aatgaagaag ttatttggtc 840

aaatcaacag gaaggaatca ttacagtaac agatatgaaa ggaaataatt cccacattct 900

tttaagtgct ttaaaatatc ctgcaaatgt agcagttgat ccagtagaaa ggtttatatt 950

ttggtcttca gaggtggctg gaagccttta tagagcagat ctcgatggtg tgggagtgaa 1020

ggctctgttg gagacatcag agaaaataac agctgtgtca ttggatgtgc ttgataagcg 1080

gctgttttgg attcagtaca acagagaagg aagcaattct cttatttgct cctgtgatta 1140

tgatggaggt tctgtccaca ttagtaaaca tccaacacag cataatttgt ttgcaatgtc 1200

cctttttggt gaccgtatct tctattcaac atggaaaatg aagacaattt ggatagccaa 1260

caaacacact ggaaaggaca tggttagaat taacctccat tcatcatttg taccacttgg 1320

tgaactgaaa gtagtgcatc cacttgcaca acccaaggca gaagatgaca cttgggagcc 1380

tgagcagaaa ctttgcaaat tgaggaaagg aaactgcagc agcactgtgt gtgggcaaga 1440

cctccagtca cacttgtgca tgtgtgcaga gggatacgcc ctaagtcgag accggaagta 1500

ctgtgaagat gttaatgaat gtgctttttg gaatcatggc tgtactcttg ggtgtaaaaa 1560

cacccctgga tcctattact gcacgtgccc tgtaggattt gttctgcttc ctgatgggaa 1620

acgatgtcat caacttgttt cctgtccacg caatgtgtct gaatgcagcc atgactgtgt 1680

tctgacatca gaaggtccct tatgtttctg tcctgaaggc tcagtgcttg agagagatgg 1740

gaaaacatgt agcggttgtt cctcacccga taatggtgga tgtagccagc tctgcgttcc 1800

tcttagccca gtatcctggg aatgtgattg ctttcctggg tatgacctac aactggatga 1860

aaaaagctgt gcagcttcag gaccacaacc atttttgctg tttgccaatt ctcaagatat 1920

tcgacacatg cattttgatg gaacagacta tggaactctg ctcagccagc agatgggaat 1980

ggtttatgcc ctagatcatg accctgtgga aaataagata tactttgccc atacagccct 2040

gaagtggata gagagagcta atatggatgg ttcccagcga gaaaggctta ttgaggaagg 2100

agtagatgtg ccagaaggtc ttgctgtgga ctggattggc cgtagattct attggacaga 2160

cagagggaaa tctctgattg gaaggagtga tttaaatggg aaacgttcca aaataatcac 2220

taaggagaac atctctcaac cacgaggaat tgctgttcat ccaatggcca agagattatt 2280

ctggactgat acagggatta atccacgaat tgaaagttct tccctccaag gccttggccg 2340

tctggttata gccagctctg atctaatctg gcccagtgga ataacgattg acttcttaac 2400

tgacaagttg tactggtgcg atgccaagca gtctgtgatt gaaatggcca atctggatgg 2460

ttcaaaacgc cgaagactta cccagaatga tgtaggtcac ccatttgctg tagcagtgtt 2520

tgaggattat gtgtggttct cagattgggc tatgccatca gtaataagag taaacaagag 2580

gactggcaaa gatagagtac gtctccaagg cagcatgctg aagccctcat cactggttgt 2640

ggttcatcca ttggcaaaac caggagcaga tccctgctta tatcaaaacg gaggctgtga 2700

acatatttgc aaaaagaggc ttggaactgc ttggtgttcg tgtcgtgaag gttttatgaa 2760

agcctcagat gggaaaacgt gtctggctct ggatggtcat cagctgttgg caggtggtga 2820

agttgatcta aagaaccaag taacaccatt ggacatcttg tccaagacta gagtgtcaga 2880

agataacatt acagaatctc aacacatgct agtggctgaa atcatggtgt cagatcaaga 2940

tgactgtgct cctgtgggat gcagcatgta tgctcggtgt atttcagagg gagaggatgc 3000

cacatgtcag tgtttgaaag gatttgctgg ggatggaaaa ctatgttctg atatagatga 3060

atgtgagatg ggtgtcccag tgtgcccccc tgcctcctcc aagtgcatca acaccgaagg 3120

tggttatgtc tgccggtgct cagaaggcta ccaaggagat gggattcact gtcttgatat 3180

tgatgagtgc caactggggg tgcacagctg tggagagaat gccagctgca caaatacaga 3240

gggaggctat acctgcatgt gtgctggacg cctgtctgaa ccaggactga tttgccctga 3300

ctctactcca ccccctcacc tcagggaaga tgaccaccac tattccgtaa gaaatagtga 3360

ctctgaatgt cccctgtccc acgatgggta ctgcctccat gatggtgtgt gcatgtatat 3420

tgaagcattg gacaagtatg catgcaactg tgttgttggc tacatcgggg agcgatgtca 3480

gtaccgagac ctgaagtggt gggaactgcg ccacgctggc cacgggcagc agcagaaggt 3540

catcgtggtg gctgtctgcg tggtggtgct tgtcatgctg ctcctcctga gcctgtgggg 3600

ggcccactac tacaggactc agaagctgct atcgaaaaac ccaaagaatc cttatgagga 3660

gtcgagcaga gatgtgagga gtcgcaggcc tgctgacact gaggatggga tgtcctcttg 3720

ccctcaacct tggtttgtgg ttataaaaga acaccaagac ctcaagaatg ggggtcaacc 3780

agtggctggt gaggatggcc aggcagcaga tgggtcaatg caaccaactt catggaggca 3840

ggagccccag ttatgtggaa tgggcacaga gcaaggctgc tggattccag tatccagtga 3900

taagggctcc tgtccccagg taatggagcg aagctttcat atgccctcct atgggacaca 3960

gacccttgaa gggggtgtcg agaagcccca ttctctccta tcagctaacc cattatggca 4020

acaaagggcc ctggacccac cacaccaaat ggagctgact cagtgaaaac tggaattaaa 4080

aggaaagtca agaagaatga actatgtcga tgcacagtat cttttctttc aaaagtagag 4140

caaaactata ggttttggtt ccacaatctc tacgactaat cacctactca atgcctggag 4200

acagatacgt agttgtgctt ttgtttgctc ttttaagcag tctcactgca gtcttatttc 4260

caagtaagag tactgggaga atcactaggt aacttattag aaacccaaat tgggacaaca 4320

gtgctttgta aattgtgttg tcttcagcag tcaatacaaa tagatttttg tttttgttgt 4380

tcctgcagcc ccagaagaaa ttaggggtta aagcagacag tcacactggt ttggtcagtt 4440

acaaagtaat ttctttgatc tggacagaac atttatatca gtttcatgaa atgattggaa 4500

tattacaata ccgttaagat acagtgtagg catttaactc ctcattggcg tggtccatgc 4560

tgatgatttt gccaaaatga gttgtgatga atcaatgaaa aatgtaattt agaaactgat 4620

ttcttcagaa ttagatggcc ttatttttta aaatatttga atgaaaacat tttattttta 4680

aaatattaca caggaggcct tcggagtttc ttagtcatta ctgtcctttt cccctacaga 4740

attttccctc ttggtgtgat tgcacagaat ttgtatgtat tttcagttac aagattgtaa 4800

gtaaattgcc tgatttgttt tcattataga caacgatgaa tttcttctaa ttatttaaat 4860

aaaatcacca aaaacat 4877

<210>7

<211>4119

<212>DNA

<213> human

<400>7

ctggagagcc tgctgcccgc ccgcccgtaa aatggtcccc tcggctggac agctcgccct 60

gttcgctctg ggtattgtgt tggctgcgtg ccaggccttg gagaacagca cgtccccgct 120

gagtgcagac ccgcccgtgg ctgcagcagt ggtgtcccat tttaatgact gcccagattc 180

ccacactcag ttctgcttcc atggaacctg caggtttttg gtgcaggagg acaagccagc 240

atgtgtctgc cattctgggt acgttggtgc acgctgtgag catgcggacc tcctggccgt 300

ggtggctgcc agccagaaga agcaggccat caccgccttg gtggtggtct ccatcgtggc 360

cctggctgtc cttatcatca catgtgtgct gatacactgc tgccaggtcc gaaaacactg 420

tgagtggtgc cgggccctca tctgccggca cgagaagccc agcgccctcc tgaagggaag 480

aaccgcttgc tgccactcag aaacagtggt ctgaagagcc cagaggagga gtttggccag 540

gtggactgtg gcagatcaat aaagaaaggc ttcttcagga cagcactgcc agagatgcct 600

gggtgtgcca cagaccttcc tacttggcct gtaatcacct gtgcagcctt ttgtgggcct 660

tcaaaactct gtcaagaact ccgtctgctt ggggttattc agtgtgacct agagaagaaa 720

tcagcggacc acgatttcaa gacttgttaa aaaagaactg caaagagacg gactcctgtt 780

cacctaggtg aggtgtgtgc agcagttggt gtctgagtcc acatgtgtgc agttgtcttc 840

tgccagccat ggattccagg ctatatattt ctttttaatg ggccacctcc ccacaacaga 900

attctgccca acacaggaga tttctatagt tattgttttc tgtcatttgc ctactgggga 960

agaaagtgaa ggaggggaaa ctgtttaata tcacatgaag accctagctt taagagaagc 1020

tgtatcctct aaccacgaga ctctcaacca gcccaacatc ttccatggac acatgacatt 1080

gaagaccatc ccaagctatc gccacccttg gagatgatgt cttatttatt agatggataa 1140

tggttttatt tttaatctct taagtcaatg taaaaagtat aaaacccctt cagacttcta 1200

cattaatgat gtatgtgttg ctgactgaaa agctatactg attagaaatg tctggcctct 1260

tcaagacagc taaggcttgg gaaaagtctt ccagggtgcg gagatggaac cagaggctgg 1320

gttactggta ggaataaagg taggggttca gaaatggtgc cattgaagcc acaaagccgg 1380

taaatgcctc aatacgttct gggagaaaac ttagcaaatc catcagcagg gatctgtccc 1440

ctctgttggg gagagaggaa gagtgtgtgt gtctacacag gataaaccca atacatattg 1500

tactgctcag tgattaaatg ggttcacttc ctcgtgagcc ctcggtaagt atgtttagaa 1560

atagaacatt agccacgagc cataggcatt tcaggccaaa tccatgaaag ggggaccagt 1620

catttatttt ccattttgtt gcttggttgg tttgttgctt tatttttaaa aggagaagtt 1680

taactttgct atttattttc gagcactagg aaaactattc cagtaatttt tttttcctca 1740

tttccattca ggatgccggc tttattaaca aaaactctaa caagtcacct ccactatgtg 1800

ggtcttcctt tcccctcaag agaaggagca attgttcccc tgacatctgg gtccatctga 1860

cccatggggc ctgcctgtga gaaacagtgg gtcccttcaa atacatagtg gatagctcat 1920

ccctaggaat tttcattaaa atttggaaac agagtaatga agaaataata tataaactcc 1980

ttatgtgagg aaatgctact aatatctgaa aagtgaaaga tttctatgta ttaactctta 2040

agtgcaccta gcttattaca tcgtgaaagg tacatttaaa atatgttaaa ttggcttgaa 2100

attttcagag aattttgtct tcccctaatt cttcttcctt ggtctggaag aacaatttct 2160

atgaattttc tctttatttt ttttttataa ttcagacaat tctatgaccc gtgtcttcat 2220

ttttggcact cttatttaac aatgccacac ctgaagcact tggatctgtt cagagctgac 2280

cccctagcaa cgtagttgac acagctccag gtttttaaat tactaaaata agttcaagtt 2340

tacatccctt gggccagata tgtgggttga ggcttgactg tagcatcctg cttagagacc 2400

aatcaatgga cactggtttt tagacctcta tcaatcagta gttagcatcc aagagacttt 2460

gcagaggcgt aggaatgagg ctggacagat ggcggaacga gaggttccct gcgaagactt 2520

gagatttagt gtctgtgaat gttctagttc ctaggtccag caagtcacac ctgccagtgc 2580

cctcatcctt atgcctgtaa cacacatgca gtgagaggcc tcacatatac gcctccctag 2640

aagtgccttc caagtcagtc ctttggaaac cagcaggtct gaaaaagagg ctgcatcaat 2700

gcaagcctgg ttggaccatt gtccatgcct caggatagaa cagcctggct tatttgggga 2760

tttttcttct agaaatcaaa tgactgataa gcattggctc cctctgccat ttaatggcaa 2820

tggtagtctt tggttagctg caaaaatact ccatttcaag ttaaaaatgc atcttctaat 2880

ccatctctgc aagctccctg tgtttccttg ccctttagaa aatgaattgt tcactacaat 2940

tagagaatca tttaacatcc tgacctggta agctgccaca cacctggcag tggggagcat 3000

cgctgtttcc aatggctcag gagacaatga aaagccccca tttaaaaaaa taacaaacat 3060

tttttaaaag gcctccaata ctcttatgga gcctggattt ttcccactgc tctacaggct 3120

gtgacttttt ttaagcatcc tgacaggaaa tgttttcttc tacatggaaa gatagacagc 3180

agccaaccct gatctggaag acagggcccc ggctggacac acgtggaacc aagccaggga 3240

tgggctggcc attgtgtccc cgcaggagag atgggcagaa tggccctaga gttcttttcc 3300

ctgagaaagg agaaaaagat gggattgcca ctcacccacc cacactggta agggaggaga 3360

atttgtgctt ctggagcttc tcaagggatt gtgttttgca ggtacagaaa actgcctgtt 3420

atcttcaagc caggttttcg agggcacatg ggtcaccagt tgctttttca gtcaatttgg 3480

ccgggatgga ctaatgaggc tctaacactg ctcaggagac ccctgccctc tagttggttc 3540

tgggctttga tctcttccaa cctgcccagt cacagaagga ggaatgactc aaatgcccaa 3600

aaccaagaac acattgcaga agtaagacaa acatgtatat ttttaaatgt tctaacataa 3660

gacctgttct ctctagccat tgatttacca ggctttctga aagatctagt ggttcacaca 3720

gagagagaga gagtactgaa aaagcaactc ctcttcttag tcttaataat ttactaaaat 3780

ggtcaacttt tcattatctt tattataata aacctgatgc ttttttttag aactccttac 3840

tctgatgtct gtatatgttg cactgaaaag gttaatattt aatgttttaa tttattttgt 3900

gtggtaagtt aattttgatt tctgtaatgt gttaatgtga ttagcagtta ttttccttaa 3960

tatctgaatt atacttaaag agtagtgagc aatataagac gcaattgtgt ttttcagtaa 4020

tgtgcattgt tattgagttg tactgtacct tatttggaag gatgaaggaa tgaacctttt 4080

tttcctaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaa 4119

<210>8

<211>4624

<212>DNA

<213> human

<400>8

ggaggaggtg gaggaggagg gctgcttgag gaagtataag aatgaagttg tgaagctgag 60

attcccctcc attgggaccg gagaaaccag gggagccccc cgggcagccg cgcgcccctt 120

cccacggggc cctttactgc gccgcgcgcc cggcccccac ccctcgcagc accccgcgcc 180

ccgcgccctc ccagccgggt ccagccggag ccatggggcc ggagccgcag tgagcaccat 240

ggagctggcg gccttgtgcc gctgggggct cctcctcgcc ctcttgcccc ccggagccgc 300

gagcacccaa gtgtgcaccg gcacagacat gaagctgcgg ctccctgcca gtcccgagac 360

ccacctggac atgctccgcc acctctacca gggctgccag gtggtgcagg gaaacctgga 420

actcacctac ctgcccacca atgccagcct gtccttcctg caggatatcc aggaggtgca 480

gggctacgtg ctcatcgctc acaaccaagt gaggcaggtc ccactgcaga ggctgcggat 540

tgtgcgaggc acccagctct ttgaggacaa ctatgccctg gccgtgctag acaatggaga 600

cccgctgaac aataccaccc ctgtcacagg ggcctcccca ggaggcctgc gggagctgca 660

gcttcgaagc ctcacagaga tcttgaaagg aggggtcttg atccagcgga acccccagct 720

ctgctaccag gacacgattt tgtggaagga catcttccac aagaacaacc agctggctct 780

cacactgata gacaccaacc gctctcgggc ctgccacccc tgttctccga tgtgtaaggg 840

ctcccgctgc tggggagaga gttctgagga ttgtcagagc ctgacgcgca ctgtctgtgc 900

cggtggctgt gcccgctgca aggggccact gcccactgac tgctgccatg agcagtgtgc 960

tgccggctgc acgggcccca agcactctga ctgcctggcc tgcctccact tcaaccacag 1020

tggcatctgt gagctgcact gcccagccct ggtcacctac aacacagaca cgtttgagtc 1080

catgcccaat cccgagggcc ggtatacatt cggcgccagc tgtgtgactg cctgtcccta 1140

caactacctt tctacggacg tgggatcctg caccctcgtc tgccccctgc acaaccaaga 1200

ggtgacagca gaggatggaa cacagcggtg tgagaagtgc agcaagccct gtgcccgagt 1260

gtgctatggt ctgggcatgg agcacttgcg agaggtgagg gcagttacca gtgccaatat 1320

ccaggagttt gctggctgca agaagatctt tgggagcctg gcatttctgc cggagagctt 1380

tgatggggac ccagcctcca acactgcccc gctccagcca gagcagctcc aagtgtttga 1440

gactctggaa gagatcacag gttacctata catctcagca tggccggaca gcctgcctga 1500

cctcagcgtc ttccagaacc tgcaagtaat ccggggacga attctgcaca atggcgccta 1560

ctcgctgacc ctgcaagggc tgggcatcag ctggctgggg ctgcgctcac tgagggaact 1620

gggcagtgga ctggccctca tccaccataa cacccacctc tgcttcgtgc acacggtgcc 1680

ctgggaccag ctctttcgga acccgcacca agctctgctc cacactgcca accggccaga 1740

ggacgagtgt gtgggcgagg gcctggcctg ccaccagctg tgcgcccgag ggcactgctg 1800

gggtccaggg cccacccagt gtgtcaactg cagccagttc cttcggggcc aggagtgcgt 1860

ggaggaatgc cgagtactgc aggggctccc cagggagtat gtgaatgcca ggcactgttt 1920

gccgtgccac cctgagtgtc agccccagaa tggctcagtg acctgttttg gaccggaggc 1980

tgaccagtgt gtggcctgtg cccactataa ggaccctccc ttctgcgtgg cccgctgccc 2040

cagcggtgtg aaacctgacc tctcctacat gcccatctgg aagtttccag atgaggaggg 2100

cgcatgccag ccttgcccca tcaactgcac ccactcctgt gtggacctgg atgacaaggg 2160

ctgccccgcc gagcagagag ccagccctct gacgtccatc atctctgcgg tggttggcat 2220

tctgctggtc gtggtcttgg gggtggtctt tgggatcctc atcaagcgac ggcagcagaa 2280

gatccggaag tacacgatgc ggagactgct gcaggaaacg gagctggtgg agccgctgac 2340

acctagcgga gcgatgccca accaggcgca gatgcggatc ctgaaagaga cggagctgag 2400

gaaggtgaag gtgcttggat ctggcgcttt tggcacagtc tacaagggca tctggatccc 2460

tgatggggag aatgtgaaaa ttccagtggc catcaaagtg ttgagggaaa acacatcccc 2520

caaagccaac aaagaaatct tagacgaagc atacgtgatg gctggtgtgg gctccccata 2580

tgtctcccgc cttctgggca tctgcctgac atccacggtg cagctggtga cacagcttat 2640

gccctatggc tgcctcttag accatgtccg ggaaaaccgc ggacgcctgg gctcccagga 2700

cctgctgaac tggtgtatgc agattgccaa ggggatgagc tacctggagg atgtgcggct 2760

cgtacacagg gacttggccg ctcggaacgt gctggtcaag agtcccaacc atgtcaaaat 2820

tacagacttc gggctggctc ggctgctgga cattgacgag acagagtacc atgcagatgg 2880

gggcaaggtg cccatcaagt ggatggcgct ggagtccatt ctccgccggc ggttcaccca 2940

ccagagtgat gtgtggagtt atggtgtgac tgtgtgggag ctgatgactt ttggggccaa 3000

accttacgat gggatcccag cccgggagat ccctgacctg ctggaaaagg gggagcggct 3060

gccccagccc cccatctgca ccattgatgt ctacatgatc atggtcaaat gttggatgat 3120

tgactctgaa tgtcggccaa gattccggga gttggtgtct gaattctccc gcatggccag 3180

ggacccccag cgctttgtgg tcatccagaa tgaggacttg ggcccagcca gtcccttgga 3240

cagcaccttc taccgctcac tgctggagga cgatgacatg ggggacctgg tggatgctga 3300

ggagtatctg gtaccccagc agggcttctt ctgtccagac cctgccccgg gcgctggggg 3360

catggtccac cacaggcacc gcagctcatc taccaggagt ggcggtgggg acctgacact 3420

agggctggag ccctctgaag aggaggcccc caggtctcca ctggcaccct ccgaaggggc 3480

tggctccgat gtatttgatg gtgacctggg aatgggggca gccaaggggc tgcaaagcct 3540

ccccacacat gaccccagcc ctctacagcg gtacagtgag gaccccacag tacccctgcc 3600

ctctgagact gatggctacg ttgcccccct gacctgcagc ccccagcctg aatatgtgaa 3660

ccagccagat gttcggcccc agcccccttc gccccgagag ggccctctgc ctgctgcccg 3720

acctgctggt gccactctgg aaaggcccaa gactctctcc ccagggaaga atggggtcgt 3780

caaagacgtt tttgcctttg ggggtgccgt ggagaacccc gagtacttga caccccaggg 3840

aggagctgcc cctcagcccc accctcctcc tgccttcagc ccagccttcg acaacctcta 3900

ttactgggac caggacccac cagagcgggg ggctccaccc agcaccttca aagggacacc 3960

tacggcagag aacccagagt acctgggtct ggacgtgcca gtgtgaacca gaaggccaag 4020

tccgcagaag ccctgatgtg tcctcaggga gcagggaagg cctgacttct gctggcatca 4080

agaggtggga gggccctccg accacttcca ggggaacctg ccatgccagg aacctgtcct 4140

aaggaacctt ccttcctgct tgagttccca gatggctgga aggggtccag cctcgttgga 4200

agaggaacag cactggggag tctttgtgga ttctgaggcc ctgcccaatg agactctagg 4260

gtccagtgga tgccacagcc cagcttggcc ctttccttcc agatcctggg tactgaaagc 4320

cttagggaag ctggcctgag aggggaagcg gccctaaggg agtgtctaag aacaaaagcg 4380

acccattcag agactgtccc tgaaacctag tactgccccc catgaggaag gaacagcaat 4440

ggtgtcagta tccaggcttt gtacagagtg cttttctgtt tagtttttac tttttttgtt 4500

ttgttttttt aaagatgaaa taaagaccca gggggagaat gggtgttgta tggggaggca 4560

agtgtggggg gtccttctcc acacccactt tgtccatttg caaatatatt ttggaaaaca 4620

gcta 4624

<210>9

<211>183

<212>PRT

<213> human

<400>9

Met Arg Ala Asn Asp Ala Leu Gln Val Leu Gly Leu Leu Phe Ser Leu

1 5 10 15

Ala Arg Gly Ser Glu Val Gly Asn Ser Gln Ala Val Cys Pro Gly Thr

20 25 30

Leu Asn Gly Leu Ser Val Thr Gly Asp Ala Glu Asn Gln Tyr Gln Thr

35 40 45

Leu Tyr Lys Leu Tyr Glu Arg Cys Glu Val Val Met Gly Asn Leu Glu

50 55 60

Ile Val Leu Thr Gly His Asn Ala Asp Leu Ser Phe Leu Gln Trp Ile

65 70 75 80

Arg Glu Val Thr Gly Tyr Val Leu Val Ala Met Asn Glu Phe Ser Thr

85 90 95

Leu Pro Leu Pro Asn Leu Arg Val Val Arg Gly Thr Gln Val Tyr Asp

100 105 110

Gly Lys Phe Ala Ile Phe Val Met Leu Asn Tyr Asn Thr Asn Ser Ser

115 120 125

His Ala Leu Arg Gln Leu Arg Leu Thr Gln Leu Thr Gly Gln Phe Pro

130 135 140

Met Val Pro Ser Gly Leu Thr Pro Gln Pro Ala Gln Asp Trp Tyr Leu

145 150 155 160

Leu Asp Asp Asp Pro Arg Leu Leu Thr Leu Ser Ala Ser Ser Lys Val

165 170 175

Pro Val Thr Leu Ala Ala Val

180

<210>10

<211>1050

<212>DNA

<213> human

<400>10

acacacacac acccctcccc tgccatccct ccccggactc cggctccggc tccgattgca 60

atttgcaacc tccgctgccg tcgccgcagc agccaccaat tcgccagcgg ttcaggtggc 120

tcttgcctcg atgtcctagc ctaggggccc ccgggccgga cttggctggg ctcccttcac 180

cctctgcgga gtcatgaggg cgaacgacgc tctgcaggtg ctgggcttgc ttttcagcct 240

ggcccggggc tccgaggtgg gcaactctca ggcagtgtgt cctgggactc tgaatggcct 300

gagtgtgacc ggcgatgctg agaaccaata ccagacactg tacaagctct acgagaggtg 360

tgaggtggtg atggggaacc ttgagattgt gctcacggga cacaatgccg acctctcctt 420

cctgcagtgg attcgagaag tgacaggcta tgtcctcgtg gccatgaatg aattctctac 480

tctaccattg cccaacctcc gcgtggtgcg agggacccag gtctacgatg ggaagtttgc 540

catcttcgtc atgttgaact ataacaccaa ctccagccac gctctgcgcc agctccgctt 600

gactcagctc accggtcagt tcccgatggt tccttctggc ctcacccctc agccagccca 660

agactggtac ctccttgatg atgacccaag actgctcact ctaagtgcct cttccaaggt 720

gcctgtcacc ttggccgctg tctaaaggtc cattgctccc taagcaatag agggccccca 780

gtagggggag ctaggggcat ctgctccagg gaaaggaacc ctgtgtcctt gtggggctgg 840

agtcagagct ggatctgtta accgtttttc taatttcaaa gtacagtgta ccggaggcca 900

ggcctgatgg cttacacctg taatcccagc attttgggag gccaaggagg gcagatcact 960

tgagatcagg agtttgagac cagcctggcc aacatggcga aaccctgtct ctactaaaaa 1020

tacaaaaaaa taaaataaaa taaaaaatta 1050

<210>11

<211>1210

<212>PRT

<213> human

<400>11

Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala

1 5 10 15

Ala 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 45

Leu Ser Leu Gln Arg Met Phe Asn Asn Cys Glu Val Val Leu Gly Asn

50 55 60

Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys

65 70 75 80

Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val

85 90 95

Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Met Tyr

100 105 110

Tyr Glu Asn Ser Tyr Ala Leu Ala Val Leu Ser Asn Tyr Asp Ala Asn

115 120 125

Lys Thr Gly Leu Lys Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu

130 135 140

His Gly Ala Val Arg Phe Ser Asn Asn Pro Ala Leu Cys Asn Val Glu

145 150 155 160

Ser Ile Gln Trp Arg Asp Ile Val Ser Ser Asp Phe Leu Ser Asn Met

165 170 175

Ser Met Asp Phe Gln Asn His Leu Gly Ser Cys Gln Lys Cys Asp Pro

180 185 190

Ser Cys Pro Asn Gly Ser Cys Trp Gly Ala Gly Glu Glu Asn Cys Gln

195 200 205

Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser Gly Arg Cys Arg

210 215 220

Gly Lys Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys

225 230 235 240

Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Arg Lys Phe Arg Asp

245 250 255

Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro

260 265 270

Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly

275 280 285

Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His

290 295 300

Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu

305 310 315 320

Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val

325 330 335

Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn

340 345 350

Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp

355 360 365

Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr

370 375 380

Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu

385 390 395 400

Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp

405 410 415

Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln

420 425 430

His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu

435 440 445

Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser

450 455 460

Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu

465 470 475 480

Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu

485 490 495

Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro

500 505 510

Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn

515 520 525

Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly

530 535 540

Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro

545 550 555 560

Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro

565 570 575

Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val

580 585 590

Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp

595 600 605

Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His Pro Asn Cys

610 615 620

Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro Thr Asn Gly

625 630 635 640

Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val Gly Ala Leu Leu Leu

645 650 655

Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met Arg Arg Arg His

660 665 670

Ile Val Arg Lys Arg Thr Leu Arg Arg Leu Leu Gln Glu Arg Glu Leu

675 680 685

Val Glu Pro Leu Thr Pro Ser Gly Glu Ala Pro Asn Gln Ala Leu Leu

690 695 700

Arg Ile Leu Lys Glu Thr Glu Phe Lys Lys Ile Lys Val Leu Gly Ser

705 710 715 720

Gly Ala Phe Gly Thr Val Tyr Lys Gly Leu Trp Ile Pro Glu Gly Glu

725 730 735

Lys Val Lys Ile Pro Val Ala Ile Lys Glu Leu Arg Glu Ala Thr Ser

740 745 750

Pro Lys Ala Asn Lys Glu Ile Leu Asp Glu Ala Tyr Val Met Ala Ser

755 760 765

Val Asp Asn Pro His Val Cys Arg Leu Leu Gly Ile Cys Leu Thr Ser

770 775 780

Thr Val Gln Leu Ile Thr Gln Leu Met Pro Phe Gly Cys Leu Leu Asp

785 790 795 800

Tyr Val Arg Glu His Lys Asp Asn Ile Gly Ser Gln Tyr Leu Leu Asn

805 810 815

Trp Cys Val Gln Ile Ala Lys Gly Met Asn Tyr Leu Glu Asp Arg Arg

820 825 830

Leu Val His Arg Asp Leu Ala Ala Arg Asn Val Leu Val Lys Thr Pro

835 840 845

Gln His Val Lys Ile Thr Asp Phe Gly Leu Ala Lys Leu Leu Gly Ala

850 855 860

Glu Glu Lys Glu Tyr His Ala Glu Gly Gly Lys Val Pro Ile Lys Trp

865 870 875 880

Met Ala Leu Glu Ser Ile Leu His Arg Ile Tyr Thr His Gln Ser Asp

885 890 895

Val Trp Ser Tyr Gly Val Thr Val Trp Glu Leu Met Thr Phe Gly Ser

900 905 910

Lys Pro Tyr Asp Gly Ile Pro Ala Ser Glu Ile Ser Ser Ile Leu Glu

915 920 925

Lys Gly Glu Arg Leu Pro Gln Pro Pro Ile Cys Thr Ile Asp Val Tyr

930 935 940

Met Ile Met Val Lys Cys Trp Met Ile Asp Ala Asp Ser Arg Pro Lys

945 950 955 960

Phe Arg Glu Leu Ile Ile Glu Phe Ser Lys Met Ala Arg Asp Pro Gln

965 970 975

Arg Tyr Leu Val Ile Gln Gly Asp Glu Arg Met His Leu Pro Ser Pro

980 985 990

Thr Asp Ser Asn Phe Tyr Arg Ala Leu Met Asp Glu Glu Asp Met Asp

995 1000 1005

Asp Val Val Asp Ala Asp Glu Tyr Leu Ile Pro Gln Gln Gly Phe

1010 1015 1020

Phe Ser Ser Pro Ser Thr Ser Arg Thr Pro Leu Leu Ser Ser Leu

1025 1030 1035

Ser Ala Thr Ser Asn Asn Ser Thr Val Ala Cys Ile Asp Arg Asn

1040 1045 1050

Gly Leu Gln Ser Cys Pro Ile Lys Glu Asp Ser Phe Leu Gln Arg

1055 1060 1065

Tyr Ser Ser Asp Pro Thr Gly Ala Leu Thr Glu Asp Ser Ile Asp

1070 1075 1080

Asp Thr Phe Leu Pro Val Pro Glu Tyr Ile Asn Gln Ser Val Pro

1085 1090 1095

Lys Arg Pro Ala Gly Ser Val Gln Asn Pro Val Tyr His Asn Gln

1100 1105 1110

Pro Leu Asn Pro Ala Pro Ser Arg Asp Pro His Tyr Gln Asp Pro

1115 1120 1125

His Ser Thr Ala Val Gly Asn Pro Glu Tyr Leu Asn Thr Val Gln

1130 1135 1140

Pro Thr Cys Val Asn Ser Thr Phe Asp Ser Pro Ala His Trp Ala

1145 1150 1155

Gln Lys Gly Ser His Gln Ile Ser Leu Asp Asn Pro Asp Tyr Gln

1160 1165 1170

Gln Asp Phe Phe Pro Lys Glu Ala Lys Pro Asn Gly Ile Phe Lys

1175 1180 1185

Gly Ser Thr Ala Glu Asn Ala Glu Tyr Leu Arg Val Ala Pro Gln

1190 1195 1200

Ser Ser Glu Phe Ile Gly Ala

1205 1210

<210>12

<211>628

<212>PRT

<213> human

<400>12

Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala

1 5 10 15

Ala 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 45

Leu Ser Leu Gln Arg Met Phe Asn Asn Cys Glu Val Val Leu Gly Asn

50 55 60

Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys

65 70 75 80

Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val

85 90 95

Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Met Tyr

100 105 110

Tyr Glu Asn Ser Tyr Ala Leu Ala Val Leu Ser Asn Tyr Asp Ala Asn

115 120 125

Lys Thr Gly Leu Lys Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu

130 135 140

His Gly Ala Val Arg Phe Ser Asn Asn Pro Ala Leu Cys Asn Val Glu

145 150 155 160

Ser Ile Gln Trp Arg Asp Ile Val Ser Ser Asp Phe Leu Ser Asn Met

165 170 175

Ser Met Asp Phe Gln Asn His Leu Gly Ser Cys Gln Lys Cys Asp Pro

180 185 190

Ser Cys Pro Asn Gly Ser Cys Trp Gly Ala Gly Glu Glu Asn Cys Gln

195 200 205

Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser Gly Arg Cys Arg

210 215 220

Gly Lys Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys

225 230 235 240

Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Arg Lys Phe Arg Asp

245 250 255

Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro

260 265 270

Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly

275 280 285

Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His

290 295 300

Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu

305 310 315 320

Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val

325 330 335

Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn

340 345 350

Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp

355 360 365

Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr

370 375 380

Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu

385 390 395 400

Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp

405 410 415

Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln

420 425 430

His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu

435 440 445

Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser

450 455 460

Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu

465 470 475 480

Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu

485 490 495

Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro

500 505 510

Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn

515 520 525

Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly

530 535 540

Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro

545 550 555 560

Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro

565 570 575

Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val

580 585 590

Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp

595 600 605

Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His Pro Asn Cys

610 615 620

Thr Tyr Gly Ser

625

<210>13

<211>405

<212>PRT

<213> human

<400>13

Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala

1 5 10 15

Ala 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 45

Leu Ser Leu Gln Arg Met Phe Asn Asn Cys Glu Val Val Leu Gly Asn

50 55 60

Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys

65 70 75 80

Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val

85 90 95

Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Met Tyr

100 105 110

Tyr Glu Asn Ser Tyr Ala Leu Ala Val Leu Ser Asn Tyr Asp Ala Asn

115 120 125

Lys Thr Gly Leu Lys Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu

130 135 140

His Gly Ala Val Arg Phe Ser Asn Asn Pro Ala Leu Cys Asn Val Glu

145 150 155 160

Ser Ile Gln Trp Arg Asp Ile Val Ser Ser Asp Phe Leu Ser Asn Met

165 170 175

Ser Met Asp Phe Gln Asn His Leu Gly Ser Cys Gln Lys Cys Asp Pro

180 185 190

Ser Cys Pro Asn Gly Ser Cys Trp Gly Ala Gly Glu Glu Asn Cys Gln

195 200 205

Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser Gly Arg Cys Arg

210 215 220

Gly Lys Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys

225 230 235 240

Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Arg Lys Phe Arg Asp

245 250 255

Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro

260 265 270

Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly

275 280 285

Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His

290 295 300

Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu

305 310 315 320

Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val

325 330 335

Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn

340 345 350

Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp

355 360 365

Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr

370 375 380

Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu

385 390 395 400

Ile Thr Gly Leu Ser

405

<210>14

<211>705

<212>PRT

<213> human

<400>14

Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala

1 5 10 15

Ala 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 45

Leu Ser Leu Gln Arg Met Phe Asn Asn Cys Glu Val Val Leu Gly Asn

50 55 60

Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys

65 70 75 80

Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val

85 90 95

Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Met Tyr

100 105 110

Tyr Glu Asn Ser Tyr Ala Leu Ala Val Leu Ser Asn Tyr Asp Ala Asn

115 120 125

Lys Thr Gly Leu Lys Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu

130 135 140

His Gly Ala Val Arg Phe Ser Asn Asn Pro Ala Leu Cys Asn Val Glu

145 150 155 160

Ser Ile Gln Trp Arg Asp Ile Val Ser Ser Asp Phe Leu Ser Asn Met

165 170 175

Ser Met Asp Phe Gln Asn His Leu Gly Ser Cys Gln Lys Cys Asp Pro

180 185 190

Ser Cys Pro Asn Gly Ser Cys Trp Gly Ala Gly Glu Glu Asn Cys Gln

195 200 205

Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser Gly Arg Cys Arg

210 215 220

Gly Lys Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys

225 230 235 240

Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Arg Lys Phe Arg Asp

245 250 255

Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro

260 265 270

Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly

275 280 285

Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His

290 295 300

Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu

305 310 315 320

Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val

325 330 335

Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn

340 345 350

Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp

355 360 365

Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr

370 375 380

Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu

385 390 395 400

Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp

405 410 415

Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln

420 425 430

His Gly Gln Phc Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu

435 440 445

Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser

450 455 460

Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu

465 470 475 480

Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu

485 490 495

Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro

500 505 510

Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn

515 520 525

Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly

530 535 540

Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro

545 550 555 560

Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro

565 570 575

Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val

580 585 590

Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp

595 600 605

Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His Pro Asn Cys

610 615 620

Thr Tyr Gly Pro Gly Asn Glu Ser Leu Lys Ala Met Leu Phe Cys Leu

625 630 635 640

Phe Lys Leu Ser Ser Cys Asn Gln Ser Asn Asp Gly Ser Val Ser His

645 650 655

Gln Ser Gly Ser Pro Ala Ala Gln Glu Ser Cys Leu Gly Trp Ile Pro

660 665 670

ser Leu Leu Pro Ser Glu Phe Gln Leu Gly Trp Gly Gly Cys Ser His

675 680 685

Leu His Ala Trp Pro Ser Ala Ser Val Ile Ile Thr Ala Ser Ser Cys

690 695 700

His

705

Claims

1. A method of predicting response to treatment with a HER dimerization inhibitor in a patient comprising the steps of

a) Evaluation in biological samples from patients

b) predicting the response in the patient to treatment with a HER dimerization inhibitor by evaluating the results of step a).

2. The method according to claim 1, wherein the step a) of assessing a marker gene or a combination of marker genes comprises

a2) determining whether the expression level assessed in step a1) is above or below a threshold.

3. Method according to claim 2, wherein said threshold value is determined before step a1) of the method of claim 2 by

2) treating said patient with a HER dimerization inhibitor,

4. A method according to any one of claims 1 to 3 wherein the biological sample is serum, plasma or tumour tissue.

5. The method according to any one of claims 1 to 4, wherein the HER dimerization inhibitor inhibits heterodimerization of HER2 with EGFR or HER 3.

6. The method according to claim 5, wherein the HER dimerization inhibitor is an antibody, preferably antibody 2C4.

7. The method according to any one of claims 1 to 6, wherein the patient is a cancer patient, preferably a breast, ovarian, lung or prostate cancer patient.

8. The method according to any of claims 1 to 7, wherein the combination of marker genes consists of:

-transforming growth factor alpha and HER2 marker genes, or

9. The method according to any one of claims 1 to 8, wherein the expression level of the marker gene or the combination of marker genes in the sample is assessed by detecting the expression level 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.

10. The method of claim 9, wherein the expression level of the marker protein or fragment thereof or combination of marker proteins or fragments thereof is detected using a reagent that specifically binds to the marker protein or fragment thereof or combination of marker proteins or fragments thereof.

11. The method of claim 10, wherein the agent is selected from the group consisting of an antibody, an antibody fragment, or a fragment of an antibody derivative.

12. A method according to any one of claims 9 to 11, wherein the expression level is determined using a method selected from the group consisting of: proteomics, flow cytometry, immunocytochemistry, immunohistochemistry, enzyme-linked immunosorbent assays, multichannel enzyme-linked immunosorbent assays, and variants of these methods.

13. A method according to any one of claims 9 to 12 wherein the fragment of the marker protein is the extracellular domain of the Her2 marker protein.

14. The method according to claim 13, wherein the extracellular domain of said Her2 marker protein has a molecular weight of about 105.000 daltons.

15. A method according to any one of claims 9 to 14

Wherein the amino acid sequence of the amphiregulin marker protein is the amino acid sequence SEQ ID NO: 1,

wherein the amino acid sequence of the epidermal growth factor marker protein is the amino acid sequence of SEQ ID NO: 2,

wherein the amino acid sequence of the transforming growth factor alpha marker protein is the amino acid sequence SEQ ID NO: 3, or

Wherein the amino acid sequence of the HER2 marker protein is the amino acid sequence of SEQ ID NO: 4.

16. a method according to any one of claims 9 to 15 wherein the threshold values in the serum for

A threshold value for the transforming growth factor alpha marker protein of 2.0-5.0pg/ml, preferably about 3.5pg/ml,

17. Method according to any one of claims 13 to 15, wherein the threshold value for the extracellular domain of the Her2 marker protein in serum is between 12 and 22ng/ml, preferably about 18 ng/ml.

18. The method according to any one of claims 1 to 8, wherein the expression level of the marker gene or the combination of marker genes in the biological sample is assessed by detecting the expression level of a transcribed marker polynucleotide or a fragment of said transcribed marker polynucleotide encoded by said marker gene or the expression level of a transcribed marker polynucleotide or a fragment of said transcribed marker polynucleotide encoded by the combination of marker genes.

19. The method of claim 18, wherein the transcribed tagged polynucleotide is a cDNA, mRNA, or hnRNA, or wherein the plurality of transcribed tagged polynucleotides is a cDNA, mRNA, or hnRNA.

20. A method according to any one of claims 18 to 19, wherein the detecting step further comprises amplifying the transcribed polynucleotide or polynucleotides. .

21. The method according to claim 20, wherein the detecting step is performed using a quantitative reverse transcriptase polymerase chain reaction method.

22. The method according to any one of claims 18 to 21,

-wherein the expression level of said marker gene is assessed by detecting the presence of said transcribed marker polynucleotide or fragment thereof in a sample using a probe that anneals to said transcribed marker polynucleotide or fragment thereof under stringent hybridization conditions, or

-wherein the level of expression of said marker gene combination in the sample is assessed by detecting the presence of said transcribed marker polynucleotide or fragment thereof in the sample using a probe that anneals to said transcribed marker polynucleotide or fragment thereof under stringent hybridization conditions.

23. A method according to any one of claims 18 to 22

Wherein the nucleic acid sequence of the amphiregulin-tagged polynucleotide is the nucleic acid sequence of seq id NO: 5,

wherein the nucleic acid sequence of the epidermal growth factor marker polynucleotide is the nucleic acid sequence of SEQ ID NO: 6,

wherein the nucleic acid sequence of the transforming growth factor alpha tagged polynucleotide is the nucleic acid sequence of SEQ ID NO: 7, or

Wherein the nucleic acid sequence of the HER2 marker polynucleotide is the nucleic acid sequence of SEQ id no: 8.

24. use of a probe that hybridizes under stringent conditions to a polynucleotide labeled with epidermal growth factor, transforming growth factor alpha, or HER2, or an antibody that binds to an epidermal growth factor, transforming growth factor alpha, or HER2 marker protein, for predicting a response in a patient to treatment with a HER dimerization inhibitor, or use of a probe that hybridizes under stringent conditions to a polynucleotide labeled with amphiregulin, epidermal growth factor, transforming growth factor alpha, or HER2, or an antibody that binds to an amphiregulin, epidermal growth factor, transforming growth factor alpha, or HER2 marker protein, for selecting a composition that inhibits disease progression in a patient.

25. A kit comprising a probe that anneals to an amphiregulin, epidermal growth factor, transforming growth factor alpha or HER2 labeled polynucleotide under stringent conditions, or an antibody that binds to an amphiregulin, epidermal growth factor, transforming growth factor alpha or HER2 labeled protein.

26. A method of selecting a composition that inhibits disease progression in a patient, the method comprising:

c) selecting a test composition that alters the expression level of said marker gene or said combination of marker genes in an aliquot comprising said test composition relative to an aliquot not contacted with the test composition, wherein the presence of at least a 10% difference between the expression level of said marker gene or said combination of marker genes in an aliquot of the biological sample contacted with said test composition and the expression level of the corresponding marker gene or combination of marker genes in an aliquot of the biological sample not contacted with said test composition is indicative of selecting said test composition.

27. A method of derivatizing a candidate agent, the method comprising:

28. The method according to claim 27, wherein the candidate agent is a candidate inhibitor.

29. The method according to claim 27, wherein said candidate agent is a candidate enhancer.

30. A candidate agent derived by a method according to any one of claims 27 to 29.

31. A pharmaceutical formulation comprising a medicament according to claim 30.

32. Use of a medicament according to claim 30 for the preparation of a composition for the treatment of cancer.

33. A method of producing a medicament comprising the steps of the method of any one of claims 27-29; and is

ii) combining the drug candidate, i.e., the candidate agent identified in step (c), or an analog or derivative thereof, with a pharmaceutically acceptable carrier.

34. Use of a marker protein or a marker polynucleotide selected from the group consisting of amphiregulin, epidermal growth factor, transforming growth factor alpha and HER2 marker proteins or marker polynucleotides for deriving a candidate agent or for selecting a composition for inhibiting the development of a disease in a patient.

Use of a HER dimerization inhibitor for the preparation of a medicament for the treatment of a human cancer patient, characterized in that the treatment comprises evaluating the following items in a biological sample from the patient

36. Use according to claim 35, wherein the treatment comprises assessing the marker gene or marker gene combination at least once or repeatedly during the course of treatment.

37. Use according to any of claims 35 to 36, wherein the expression level of the marker gene or the expression level of the combination of marker genes is assessed.

38. Use according to any one of claims 35 to 37, wherein the HER dimerization inhibitor is an antibody, preferably antibody 2C4.

39. Use according to any one of claims 35 to 38, wherein the patient is a breast, ovarian, lung or prostate cancer patient.