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WO2010085845A1 - Cancer therapy and/or diagnosis - Google Patents

Cancer therapy and/or diagnosis Download PDF

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Publication number
WO2010085845A1
WO2010085845A1 PCT/AU2010/000081 AU2010000081W WO2010085845A1 WO 2010085845 A1 WO2010085845 A1 WO 2010085845A1 AU 2010000081 W AU2010000081 W AU 2010000081W WO 2010085845 A1 WO2010085845 A1 WO 2010085845A1
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breast cancer
metastatic breast
cells
brain
receptor
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PCT/AU2010/000081
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French (fr)
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Leonard Medeiros Da Silva
Sunil R. Lakhani
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The University Of Queensland
Ludwig Institute For Cancer Research Limited
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Publication of WO2010085845A1 publication Critical patent/WO2010085845A1/en

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    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
    • A—HUMAN NECESSITIES
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    • C07—ORGANIC CHEMISTRY
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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3015—Breast
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57415—Specifically defined cancers of breast
    • A—HUMAN NECESSITIES
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
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    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/475—Assays involving growth factors
    • G01N2333/4756—Neuregulins, i.e. p185erbB2 ligands, glial growth factor, heregulin, ARIA, neu differentiation factor
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/50—Determining the risk of developing a disease

Definitions

  • THIS invention relates to cancer therapy and/or diagnosis. More particularly, this invention relates to therapeutic targets and diagnostic markers for metastatic breast cancer and preferably metastatic breast cancer in the brain.
  • Breast cancer is one of the most common malignant neoplasias and a leading cause of female cancer mortality (1, 2). Among women with breast cancer in western countries, 30%-40% will develop metastatic disease (3).
  • the epidermal growth factor receptor family comprises four receptors, HERl, HER2, HER3 and HER4. Such receptors are triggered by several ligands. Upon activation, hetero or homo-dimerization occurs, followed by phosphorylation of specific tyrosine residues in the intracellular region. Such activation, leads to regulation of a variety of cellular processes including cell proliferation and survival. Much evidence suggests that the kinases Akt and Erk mediate a substantial part of the HER signaling (70). Basal-like tumours are a molecular subgroup of breast cancer (13, 14) that generally are high grade, negative for estrogen and progesterone receptors and do not overexpress HER2.
  • trastuzumab Herceptin®; Genentech Inc, South San Francisco, CA
  • trastuzumab treatment aims to block HER2 and its downstream pathways.
  • Other studies showed a trend for primary tumours of patients who developed brain metastases during disease progression to express markers related to the basal-like tumours (8, 16-18).
  • Palmieri et al (2007) Cancer Research, 67: 4190 to 4198 examined Her-2 expression profiles of breast cancer cells in the brain and demonstrated that Her-2 overexpression increases the outgrowth of metastatic tumour cells in the brain. Moreover, expression levels of Her-2, Her-3 and Her-4 proteins were examined in MDA-MB-231BR brain-seeking cell line and found to be comparable and at the lower limit of detection.
  • the invention is broadly directed to targets and methods for the diagnosis and/or treatment of metastatic outgrowth of breast cancer cells, particularly to the brain.
  • the invention is directed to targeting one or more members of the epidermal growth factor receptor family for treatment of metastatic outgrowth of breast cancer cells, particularly located in the brain, and more particularly in basal-like breast cancer tumours in the brain.
  • the invention provides a method of treating a metastatic breast cancer in a human, said method including the step of administering to an individual with an elevated level of Her-3 in one or more metastatic breast cancer cells of the brain, a therapeutic agent effective in inhibiting at least one ErbB receptor.
  • the method further includes the step of determining whether said individual has an elevated level of Her-3 in one or more metastatic breast cancer cells of the brain.
  • the invention provides a method of designing, engineering, screening or otherwise producing a therapeutic agent for treating a metastatic breast cancer, said method including the step of identifying a candidate agent that is suitable for use in treatment of a metastatic breast cancer, by the presence of an elevated expression level of Her-3 in one or more metastatic breast cancer cells of the brain.
  • the invention provides a therapeutic agent for treating a metastatic breast cancer designed, engineered, screened or otherwise produced according to a method of the second aspect.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutic agent for treating a metastatic breast cancer of the third aspect, together with a pharmaceutically-acceptable carrier, diluent or excipient.
  • the invention provides a method of treating a metastatic breast cancer, said method including the step of administering a therapeutic agent for treating a metastatic breast cancer according to the third aspect or a pharmaceutical composition according to the fourth aspect, to an individual with an elevated level of Her-3 in one or more metastatic breast cancer cells of the brain.
  • the invention provides a method of determining whether a human with a metastatic breast cancer is potentially responsive to treatment with a therapeutic agent effective in inhibiting at least one ErbB receptor and/or a therapeutic agent for treating a metastatic breast cancer designed, engineered, screened or otherwise produced according the method of the second aspect, said method including the step of detecting an elevated level of Her-3 in one or more metastatic breast cancer cells of the brain.
  • the invention provides a method of determining whether a human is predisposed to a metastatic breast cancer or is suffering from a metastatic breast cancer, said method including the step of detecting an elevated level of Her-3 in one or more metastatic breast cancer cells of the brain.
  • the metastatic breast cancer has one or more cells with normal expression levels of Her- 2. More preferably, the metastatic breast cancer has one or more breast cancer cells with normal expression levels of Her-2.
  • the metastatic breast cancer is located in the brain.
  • the metastatic breast cancer is derived from one or more breast cancer cells.
  • the at least one ErbB receptor is selected from the group consisting Her-1, Her-2, Her-3, Her-4, and combinations thereof. More preferably, the at least one ErbB receptor is selected from Her-1 and Her-2.
  • the at least one ErbB receptor is Her-2.
  • the candidate agent inhibits at least one ErbB receptor.
  • the therapeutic agent and/or the candidate agent inhibits at least one ErbB receptor by directly inhibiting at least one ErbB receptor.
  • the therapeutic agent and/or the candidate agent inhibits at least one ErbB receptor by indirectly inhibiting at least one ErbB receptor.
  • the therapeutic agent and/or the candidate agent selectively inhibits at least one ErbB receptor.
  • the one or more cells with normal levels of Her-2 have levels of an estrogen receptor and a progesterone receptor which are below a threshold level of detection.
  • the therapeutic agent and/or the candidate agent is selected from the group consisting of an isolated protein, an isolated nucleic acid, a small-molecule compound, and combinations thereof.
  • the therapeutic agent or the candidate agent is an antibody, or a fragment thereof and even more preferably, a monoclonal antibody.
  • the therapeutic agent and/or the candidate agent is selected from the group consisting of trastuzumab, pertuzumab, scFvFRP5, CAB051, ertumaxomab, lapatinib, CI- 1033, HKI-272, AEE-788, BIBW-2992, TAKl 65, BMS-599626, matuzumab, cetuximab, panitumumab, gefitinib, erlotinib, ICR62, nimotuzumab, Ch806, L8A4, MDX-447, zalutumumab, IMC-11F8, AZD4769, PF299804, EGFR501, ZD6474, EKB-569, EXEL 7647, EXEL 0999, AZD8931, MP412, herstatin, pelitinib, CP724714, XL647, PD- 1694
  • the therapeutic agent and/or candidate agent is trastuzumab.
  • FIGURE 1 A- Grade III invasive ductal carcinoma; B- Same tumour as in “A” showing CK 14 expression; C- Primary breast carcinoma negative for CD44; D- Matched metastasis of "C” showing positivity for CD44.
  • FIGURE 2 A -Unsupervised hierarchical clustering of DASL gene expression data from 22 unmatched (black bar) and 15 matched primary and brain metastases (shades of grey bars). Thirteen out of 15 matched samples are clustering together.
  • B Heatmap and dendogram showing clustering of the samples based on the 20 genes differentially expressed between primary tumors (light grey bar) and brain metastases (dark grey bar).
  • FIGURE 3 Her-3 expression. Summary of RT-PCR (first bar at each point) and DASL assay (second bar at each point) for 12 matched samples - Positive values indicate increased fold change in metastases and negative values increased fold change in primary.
  • X-axis are 12 matched primary breast cancers and brain metastases;
  • Y-axis is ration of fold-change-Brain metastasis/Primary cancer
  • FIGURE 4 A-Brain metastasis; B- Nuclear expression of HER3 for the tumour shown in "A”; C- Negativity for pAKT in the matched primary of "B”; D- Positivity for ERK1/2 in the brain metastasis shown in "B”.
  • F Positivity for EGFR in the brain metastasis from a non- breast cancer.
  • FIGURE 5 Growth curves of the cell line SKBR-3 grown on its own (BC only; vertical line) and on co-culture with ReNcell CX neural cells (NC; circle).
  • the double-headed arrow highlights statistical significant (p ⁇ 0.05, ANOVA) between the curves above and below the blue arrow.
  • X-axis is time in hours while Y axis is EpCam positive particles per 250 ⁇ l of buffer.
  • FIGURE 6 MRI images from week 10 showing brain tumours in the left hemisphere from an animal of (A) the control group, (B) the trastuzumab treated group, (C) the anti-neuregulin 1 treated group. Average tumor size was smaller in B and C compared to control A (p ⁇ 0.05, unpaired T test).
  • D Relative levels of HER3 transcripts in the parental cell line and in the four groups of mice. *Highlights statistical significance (p ⁇ 0.05, unpaired T test) between the parental group and the other groups; Y-axis is HER3 expression relative to GAPDH and X-axis the subgroups of treatment.
  • FIGURE 7 Colony forming assay. Effects of traztuzumab and neuregulin on the number of colonies of MB-MD A-231 breast cancer cell line. Y -axis is average number of colonies and X-axis the subgroups of treatment.
  • FIGURE 8 Colony forming assay. Effects of traztuzumab and neuregulin on the size of colonies of MB-MD A-231 breast cancer cell line. Y -axis is average number of colonies and X-axis the subgroups of treatment.
  • the present invention is predicated, at least in part, on the finding that Her-3 is overexpressed in tumours located in the brain which result from metastatic outgrowth of breast cancer tumours that exhibit a basal-like phenotype. Furthermore, even in the absence of HER2 amplification/overexpression in such tumours, an anti ( ⁇ )-Her-2 monoclonal antibody is useful in reducing a tumour growth located in the brain resulting from metastatic breast cancer outgrowth in vitro and in vivo.
  • the invention provides methods of treatment and/or diagnosis of a metastatic breast cancer in a human with elevated levels of Her-3 in one or more metastatic breast cancer cells of the brain, hi preferred embodiments, the metastatic breast cancer is located in the brain.
  • the metastatic breast cancer is derived from one or more cells, and more preferably breast cancer cells, with normal expression levels of Her-2.
  • breast cancer is meant a malignant tumour of the breast tissue.
  • breast cancer forms in tissues of the breast, usually the ducts (tubes that carry milk to the nipple) and lobules (glands that make milk).
  • metastatic breast cancer is meant one or more primary breast cancer cells which have undergone metastases and spread to one or more parts of the body to form secondary tumours which are otherwise referred to as metastatic tumours.
  • a “metastatic breast cancer” may be derived from one or more cancer cells which originate from a breast cancer, or alternatively the “metastatic breast cancer " may derived from a non-breast cancer cell source.
  • one or more cells with normal expression levels ofHer- 2 is meant that said one or more cells are considered to have, exhibit or display a lack of expression or amplification of Her-2, and preferably either gene amplification from the HER-2 gene locus or a HER-2 amplicon or protein expression, when compared to one or more cells which overexpress Her-2.
  • the "one or more cells with normal expression levels of Her-2” have expression levels of Her-2 which are below a threshold level of detection and are thus considered not to be Her-2 positive.
  • a positive HER2 result may be considered as immunohistochemistry (IHC) staining of 3+ (uniform, intense membrane staining of >30% of invasive tumour cells), a fluorescent in situ hybridization (FISH) result of more than six HER2 gene copies per nucleus or a FISH ratio (HER2 gene signals to chromosome 17 signals) of more than 2.2.
  • a negative result for Her-2 may be an IHC staining value of 0 or 1+, a fluorescence in situ hybridization (FISH) result of less than 4.0 HER2 gene copies per nucleus, or FISH ratio of less than 1.8.
  • Her-2 expression levels may result from basal or normal expression of the Her-2 gene locus located on genomic DNA, and in particular, two chromosomal copies of the Her-2 gene locus or alternatively expression of Her-2 which results from one or more cancer cells that are polysomic.
  • one or more cells with normal expression levels of Her-2 are considered not amenable to conventional targeted therapies for breast cancer such as ⁇ -Her-2 monoclonal antibodies (eg. trastuzumab). It will be appreciated that the "one or more cells with normal expression levels of Her-2 " may originate from a variety of sources such as in vitro culture (such as cultured breast cancer cell lines), ex vivo or alternatively, in vivo. In preferred embodiments, the "one or more cells with normal expression levels of Her-2" are one or more cancer cells and more preferably, one or more breast cancer cells.
  • applicable detection or assay methods include immunohistochemistry, histopathology, genomic analysis (such as fluorescence in situ hybridization) and gene expression profiling (such as DNA microarray-based techniques), although without limitation thereto.
  • Detection methods include those and for example as described in Korsching et al J Clin Pathol. 2008 May;61(5):553-60; Kusi ⁇ skaet al Pol J Pathol.2005;56(3): 107-10; andMullan and Millikan, Cell MoI Life Sci. 2007 Dec;64(24):3219-32.
  • the one or more cells which exhibit a phenotype of normal Her-2 levels as defined hereinabove and have expression levels of an estrogen receptor and a progesterone receptor which are below a threshold level of detection are also known in the art as "triple-negative tumour ", " basal-like tumour " or “basal-like phenotype " and may be used interchangeably herein.
  • ErbB receptor is meant any member of the epidermal growth factor receptor family of receptor tyrosine kinase or Her receptors.
  • the ErbB (or otherwise referred to as Her receptors or epidermal growth factor receptors) family of receptor tyrosine kinases are important mediators of cell growth, differentiation and survival.
  • Her-1 is specific for epidermal growth factor and EGF related peptides including transforming growth factor-alpha, amphiregulin, and heparin-binding EGF-like growth factor.
  • EGF related peptides including transforming growth factor-alpha, amphiregulin, and heparin-binding EGF-like growth factor.
  • the members of the ErbB family and particularly Her-1, Her-2, Her-3 and Her-4 share extensive homology and are able to homodimerise and heterodimerise with other members of the family. Reference is made to Zhang et al (2007) J. Clin. Invest. 117:2051-2058, which provides a review of ErbB receptor biology.
  • Her-3 refers to a Her-3 protein, and preferably a human Her-3 protein, as for example described in US Patent No. 5,480,968; Plowman et al, Proc Natl Acad Sci U S A. 1990 Jul;87(13):4905-9 and Kraus et al Proc Natl Acad Sci U S A. 1989 Dec;86(23):9193-7, which are incorporated herein by reference.
  • Her-3 may also referred to as “ErbB3 ", "Her-3 receptor” and “ErbB3 receptor” and other terms as are known in the art such as “LCCS2 ", “c- erbB3 “, “erbB3-S”, “MDA-BF-I “, “MGC88033 “, “c-erbB-3 “, “p!80-ErbB3 “, “p45-sErbB3 “ and “p85-sErbB3 " and may be used interchangeably herein.
  • Her-2 refers to a Her-2 protein, and preferably a human Her-2 protein, as for example described in King et al 1985 Science, Sep 6;229(4717):974-6.
  • ffer-2 may also be referred to as "ErbB2", “neu”, “Her-2 receptor “, “ErbB2 receptor “ and other terms as are known in the art such as “ «g/ ", “TKR1 “, “CD34O”, “HER-2/neu” and “pJSS ⁇ 2 ⁇ ”” and may be used interchangeably herein.
  • Her-1 refers to a Her-1 protein, and preferably a human Her-1 protein which is the mature, tyrosine kinase cell surface receptor, as for example described in Lin et al (1984) Science 224: 843 - 848 and Ullrich et al, (1984) Nature, 309:418- ⁇ 25.
  • Her-1 is also referred to "epidermal growth factor receptor (EGFR) ", "ErbBl”, “c-erbB-1 “, “ErbB” and “PIG61 " and may be used interchangeably hereia
  • EGFR epidermal growth factor receptor
  • ErbBl epidermal growth factor receptor
  • c-erbB-1 epidermal growth factor receptor
  • ErbB ErbBl
  • PEG61 epidermal growth factor receptor
  • Her-4 refers to a Her-4 protein, and preferably a human Her-4 protein, as for example described in Plowman et ⁇ l, Proc Natl Acad Sci U S A. 1993 Mar l;90(5):l 746-50.
  • Her-4" may also be referred to as “MGCl 38404", “pl8(T bB4 “, “tyro2” and “ErbB4 " and may be used interchangeably herein.
  • General aspects of the present invention contemplate detecting, determining or otherwise measuring an elevated level of Her-3 in one or more metastatic breast cancer cells of the brain, either as part of diagnostic methods or in methods of treatment of humans (preferably as a selection step), according to the present invention.
  • the present invention contemplates methods of treating a metastatic breast cancer in a human by administering to an individual with an elevated level of Her-3 in one or more metastatic breast cancer cells of the brain, a therapeutic agent designed, screened, engineered or otherwise produced according to methods of the present invention as described herein.
  • the present invention contemplates methods of treating one or more breast cancer cells with normal expression of Her-2 in a human which includes the step of administering to an individual with an elevated level of Her-3, a therapeutic agent effective in inhibiting at least one ErbB receptor.
  • the invention relates to methods of diagnosis of a metastatic breast cancer, preferably located in the brain, derived from one or more breast cancer cells with normal expression levels of Her-2. Such methods may assist in determining whether a human suffers from said metastatic breast cancer, is predisposed to said metastatic breast cancer and/or is more or less responsive to therapies directed to metastatic breast cancer and in particular metastatic breast cancer located in the brain.
  • predisposed and “predisposition” are used in the context of a probability that an individual may display clinical symptoms of a metastatic breast cancer, preferably located in the brain, derived from one or more breast cancer cells with normal expression levels of Her-2, or that any existing, manifest clinical symptoms of said metastatic breast cancer are the result of an underlying biochemical cause.
  • diagnostic methods of the invention may be used alone or combined with other forms of molecular and/or clinical diagnosis to improve the accuracy of diagnosis.
  • an elevated level ofHer-3 in one or more metastatic breast cancer cells of the brain is meant an amplified, overexpressed, up-regulated, enhanced or otherwise increased amount of Her-3 when compared to breast cancer cells which have not metastasized to the brain.
  • Her-3 per se may be assayed or measured to determine an elevated level of Her-3.
  • the present invention also contemplates detecting or assaying alternative forms of Her-3, such as post- translationally modified Her-3 (for example, phosphorylated Her-3) or variants such as splice variants.
  • molecules associated with Her-3 or its function such as downstream signalling proteins of the mitogen- activated protein kinase pathway which Her-3 activates, may also be assayed.
  • a ligand of Her-3 such as, but not limited to, neuregulin-1 may also be assayed in order to determine whether there is an elevated level of Her-3 in one or more metastatic breast cancer cells of the brain.
  • the invention contemplates protein-based detection techniques and in particular for detection of Her-3, or a fragment thereof.
  • protein is also meant “polypeptide ", “peptide “ or fragments thereof, referring to an amino acid polymer, comprising natural and/or non-natural amino acids, including L- and D-isomeric forms as are well understood in the art.
  • polypeptide a polypeptide
  • peptide or fragments thereof, referring to an amino acid polymer, comprising natural and/or non-natural amino acids, including L- and D-isomeric forms as are well understood in the art.
  • Her-3 or any other ErbB receptor or ErbB receptor ligand may be referred to as either a protein or polypeptide.
  • the protein or polypeptide is an isolated protein or an isolated polypeptide.
  • a “peptide” is a protein having no more than fifty (50) amino acids.
  • a “polypeptide” is a protein having fifty (50) or more amino acids.
  • Proteins and peptides may be useful in native, chemical synthetic or recombinant synthetic form.
  • isolated material that has been removed from its natural state or otherwise been subjected to human manipulation. Isolated material may be substantially or essentially free from components that normally accompany it in its natural state, or may be manipulated so as to be in an artificial state together with components that normally accompany it in its natural state. Isolated material may be in native, chemical synthetic or recombinant form.
  • a “fragment” is a segment, domain, portion or region of an ErbB receptor, and in particular embodiments Her-3, which constitutes less than 100% of the full- length protein.
  • a fragment preferably comprises less than 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 60%, 50%, 40%, 30%, 20% or as little as even 10%, 5% or 3% of the entire protein.
  • a protein fragment may comprise, for example, at least 5, 10, 20, 30, 40, 5060, 70, 80, 90, 100, 120, 140, 150, 200, 250, 300, 350, 400, 450 or 500 contiguous amino acids of an ErbB receptor, and in particular Her-3.
  • a fragment is a "biologically-active fragment ".
  • biologically-active fragment is meant a segment, portion or fragment of a biological active molecule which has at least about 0.1%, preferably at least about 10%, more preferably at least about 25% and even more preferably at least 50% of the activity of the molecule and even more preferably at least 70%, 80% or 90% of the biological activity of the entire or full length protein.
  • fluorescence activated cell sorting (FACS) analysis using labelled antibodies is readily amenable to quantitative measurement of cell surface expression of proteins and in particular receptor proteins such as Her-3.
  • Immunofluorscence and other fluorescence microscopy methods can also be used to stain metastatic breast cancer outgrowth in the brain to detect levels of Her-3 as well as other conventional immunohistochemistry techniques.
  • relative protein expression levels may be determined by other protein-based methods which include immunoassays, for example ELISA and immunoblotting (such as western blotting using ⁇ -Her-3 antibodies) to detect relative expression levels of Her-3.
  • the invention also contemplates techniques which detect nucleic acid-protein interactions such as, but not limited to, northwestern blotting and electrophoretic mobility shift assay
  • the invention further contemplates use of protein-based microarray technology, preferably to determine the expression pattern profile of metastatic breast cancer in order to analyse whether Her-3 expression is up-regulated or overexpressed in patients with metastatic breast cancer, preferably a metastatic breast cancer located in the brain.
  • Proteomic pattern analysis provides an alternative diagnostic method which is particularly useful for global expression pattern analysis of proteins.
  • Methods of cancer diagnosis using proteomic patterns are provided in Conrads et al Expert Rev MoI Diagn. 2003 Jul;3(4):411-20 and is incorporated herein by reference.
  • a plurality of proteins may be used in a protein library displayed in a number of ways, e.g., in phage display or cell display systems or by two-dimensional gel electrophoresis, or more specifically, differential two- dimensional gel electrophoresis (2D-DIGE).
  • 2D-DIGE differential two- dimensional gel electrophoresis
  • proteomic or “protein profiling” methods, such as described in Chapters 3.9.1 and 22 of CURRENT PROTOCOLS IN PROTEIN SCIENCE Eds. Coligan et al., John Wiley & Sons NY USA (1996-2002).
  • the protein array comprises a substrate to which is immobilized, impregnated, bound or otherwise coupled ErbB protein, or a fragment thereof.
  • the substrate may be a chemically-derivatized aluminium chip, a synthetic membrane such as PVDF or nitrocellulose, a glass slide or microtiter plates.
  • Detection of substrate-bound proteins may be performed using mass spectrometry, ELISA, immunohistochemistry, fluorescence microscopy or by colorimetric detection but is not limited thereto.
  • the invention provides methods to detect elevated levels of Her-3 indirectly by measurement of Her-3 activity and/or function.
  • nucleic acid detection techniques as are well known in the art to measure levels of a nucleic acid encoding a Her-3 in order to ascertain expression levels of Her-3.
  • any nucleic acid sequence detection technique may be applicable, such as nucleic acid sequencing, northern hybridization, nucleic acid sequence amplification, nucleic acid arrays and methods that detect melting temperature differences to identify whether a nucleic acid harbours one or more polymorphisms compared to a "wild type" reference nucleic acid.
  • nucleic acid designates single or double stranded mRNA, RNA, cRNA and DNA, said DNA inclusive of cDNA and genomic DNA.
  • a nucleic acid may be native or recombinant and may comprise one or more artificial nucleotides, e.g. nucleotides not normally found in nature.
  • RNA includes single- stranded and double-stranded unprocessed RNA, mRNA, siRNA, miRNA, RNAi and tRNA.
  • Nucleic acid also encompasses modified purines (for example, inosine, methylinosine and methyladenosine) and modified pyrimidines (thiouridine and methylcytosine).
  • isolated nucleic acid refers to a nucleic acid subjected to in vitro manipulation into a form not normally found in nature. Isolated nucleic acid includes both native and recombinant (non-native) nucleic acids. For example, a nucleic acid may be isolated from human.
  • mRNA RNA
  • RNA RNA
  • transcript are used interchangeably when referring to a transcribed copy of a transcribable nucleic acid.
  • the present invention also contemplates detection of variant nucleic acids.
  • Variants include within their scope naturally-occurring variants such as allelic variants, orthologs and homologs and mutants, for example.
  • mutant refers herein generally to encompass conservative or non-conservative amino acid substitutions, deletions and/or insertions present in a protein, or fragment thereof or introduced into an isolated protein or fragment thereof.
  • nucleic acid detection techniques may include detection of variants such as alternate transcriptional splice variants encoding different isoforms of Her-3.
  • variants such as alternate transcriptional splice variants encoding different isoforms of Her-3.
  • one particular Her-3 isoform represents a longer mRNA transcript and thereby encodes a longer isoform.
  • Suitable nucleic acid amplification techniques are well known to the skilled addressee, and include polymerase chain reaction (PCR) as for example described in Ausubel et al. ⁇ supra) which is incorporated herein by reference; strand displacement amplification (SDA) as for example described in U.S. Patent No 5,422,252 which is incorporated herein by reference; rolling circle replication (RCR) as for example described in Liu et al, (1996, J. Am. Chem. Soc. 118:1587-1594 and International application WO 92/01813) and Lizardi et al.
  • PCR polymerase chain reaction
  • SDA strand displacement amplification
  • RCR rolling circle replication
  • nucleic acid sequence-based amplification as for example described by Sooknanan et al., (1994, Biotechniques 17:1077-1080) which is incorporated herein by reference; and Q- ⁇ replicase amplification as for example described by Tyagi et al., (1996, Proc. Natl. Acad. ScL USA 93:5395-5400) which is incorporated herein by reference.
  • nucleic acid sequence amplification techniques are not presented as an exhaustive list of techniques. Persons skilled in the art will be well aware of a variety of other applicable techniques as well as variations and modifications to the techniques described herein.
  • an "amplification product” is a nucleic acid generated by a nucleic acid sequence amplification technique as hereinbefore described. Detection of amplification products may be achieved by detection of a probe hybridized to an amplification product, by direct representationation of amplification products by way of agarose gel electrophoresis, nucleotide sequencing of amplification products or by detection of fluorescently-labelled amplification products.
  • a “probe” is a single- or double-stranded oligonucleotide or polynucleotide, one and/or the other strand of which is capable of hybridizing to another nucleic acid, to thereby form a "hybrid" nucleic acid.
  • Probes and/or primers of the invention may be labelled, for example, with biotin or digoxigenin, with fluorochromes or donor fluorophores such as FITC, TRITC, Texas Red, TET, FAM6, HEX, ROX or Oregon Green, acceptor fluorophores such as LC-Red640, enzymes such as horseradish peroxidase (HRP) or alkaline phosphatase (AP) or with radionuclides such as 125 1, 32 P, 33 P or 35 S to assist detection of amplification products by techniques as are well known in the art.
  • fluorochromes or donor fluorophores such as FITC, TRITC, Texas Red, TET, FAM6, HEX, ROX or Oregon Green
  • acceptor fluorophores such as LC-Red640
  • enzymes such as horseradish peroxidase (HRP) or alkaline phosphatase (AP) or with radionuclides such as 125 1, 32 P, 33 P
  • an "oligonucleotide” is a single- or double-stranded nucleic acid having no more than one hundred (100) nucleotides (bases) or nucleotide pairs (base pairs).
  • a "polynucleotide '1' ' has more than one hundred (100) nucleotides or nucleotide pairs.
  • an oligonucleotide of the invention may be in the form of a primer.
  • a “primer” is a single-stranded oligonucleotide which is capable of hybridizing to a nucleic acid "template” and being extended in a template- dependent fashion by the action of a suitable DNA polymerase such as Taq polymerase, RNA-dependent DNA polymerase or SequenaseTM.
  • a primer may have at least twelve, fifteen, twenty, twenty-five, thirty, thirty five, forty or fifty contiguous nucleotide bases.
  • “hybridization” ', “hybridize” and “hybridizing” refers to formation of a hybrid nucleic acid through base-pairing between complementary or at least partially complementary nucleotide sequences under defined conditions, as is well known in the art. Normal base-pairing occurs through formation of hydrogen bonds between complementary A and T or U bases, and between G and C bases. It will also be appreciated that base-pairing may occur between various derivatives of purines (G and A) and pyrimidines (C, T and U).
  • Purine derivatives include inosine, methylinosine and methyladenosines.
  • Pyrimidine derivatives include sulfur- containing pyrimidines such as thiouridine and methylated pyrimidines such as methylcytosine.
  • anneal and “annealing” are used in the context of primer hybridization to a nucleic acid template for a subsequent primer extension reaction, such as occurs during nucleic acid sequence amplification or nucleotide sequencing, as for example described in Chapter 15 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds Ausubel et al. (John Wiley & Sons NY 1995- 1999).
  • detection may be performed by melting curve analysis using probes incorporating fluorescent labels that hybridize to amplification products in a sequence amplification reaction.
  • probes incorporating fluorescent labels that hybridize to amplification products in a sequence amplification reaction.
  • Fluorescent Resonance Energy Transfer (FRET) probes to hybridize with amplification products in "real time” as amplification products are produced with each cycle of amplification.
  • the invention contemplates use of melting curve analysis whereby nucleic acid-intercalating dyes such as ethidium bromide (EtBr) or SYBR Green I bind amplification products and fluorescence emission by the intercalated complexes is detected.
  • nucleic acid-intercalating dyes such as ethidium bromide (EtBr) or SYBR Green I bind amplification products and fluorescence emission by the intercalated complexes is detected.
  • the invention contemplates in particular embodiments detection of variant nucleic acid with polymorphisms. It will also be well understood by the skilled person that detection of nucleotide sequence polymorphisms may be performed using any of a variety of techniques such as PCR-RFLP analysis, fluorescence-based melt curve analysis, SSCP analysis, denaturing gradient gel electrophoresis (DGGE) or direct sequencing of amplification products.
  • PCR-RFLP analysis fluorescence-based melt curve analysis
  • SSCP analysis SSCP analysis
  • denaturing gradient gel electrophoresis (DGGE) denaturing gradient gel electrophoresis
  • Measurement of relative levels of Her-3 nucleic acid levels compared to an expressed level of a reference nucleic acid may be conveniently performed using a nucleic acid array.
  • Nucleic acid array technology has become well known in the art and examples of methods applicable to array technology are provided in Chapter 22 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel etal. (John Wiley & Sons NY USA 1995-2001).
  • An array can be generated by various methods, e.g., by photolithographic methods (see, e.g., U.S. Patent Nos. 5,143,854; 5,510,270; and 5,527,681), mechanical methods ⁇ e.g., directed-flow methods as described in U.S. Patent No.
  • Affymetrix nucleic acid array systems such as described in United States Patent 5,858,659 and United States Patent 6,300,063 which provide specific teaching in relation to nucleic acid array-based detection of disease-related polymorphisms but may provide useful methodology applicable to the present invention.
  • nucleic acid arrays are useful with a variety of different techniques such as primer extension, allele-specific hybridization, allele-specific ligation and allele-specific cleavage of a flap probe, as for example described in Kwok, 2000, supra, Pastinen et al, 2000, Genome Res. 10 1031.
  • Nucleic acid or gene expression levels may also be measured by a variety of other gene expression profiling methods as are known in the art such as, but not limited to, the DASL assay (cDNA-mediated annealing, selection extension and ligation) as described hereinafter.
  • DASL assay cDNA-mediated annealing, selection extension and ligation
  • In situ hybridisation techniques are also particularly useful for the detection of expression levels and includes chromogenic in situ hybridisation and fluorescence in situ hybridisation as are known in the art. Reference is made to Chapter 14 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al. (John Wiley & Sons NY USA 1995-2008) for non-limiting examples of applicable in situ hybridisation technology.
  • PCR amplification is not linear and hence end point analysis does not always allow for the accurate determination of nucleic acid expression levels.
  • Real-time PCR analysis provides a high throughput means of measuring gene expression levels. It uses specific primers, and fluorescence detection to measure the amount of product after each cycle. Hydridization probes utilise either quencher dyes or fluorescence directly to generate a signal. This method may be used to validate and quantify nucleic acid expression differences in cells and/or tissues obtained from metastatic breast cancer sufferers compared to cells and/or tissues obtained from non- sufferers or alternatively, comparison of the expression levels between the primary tumour and the metastases.
  • the metastatic breast cancer is located in the brain of a sufferer.
  • samples for diagnostic or analysis may be isolated from any cell and/or tissue source, inclusive of neural tissue, although more easily obtained cells and tissues such as blood, skin and the like may also be advantageous.
  • the invention contemplates particular embodiments of such methods which utilise the aforementioned techniques either alone or in combination.
  • the invention relates to methods of treatment of a human which includes the step of administering to an individual with an elevated level of Her-3 in one or more metastatic breast cancer cells of the brain, a therapeutic agent effective in inhibiting at least one ErbB receptor.
  • suitable therapeutic agents of the invention may be peptides, proteins such as antibodies or other organic molecules, preferably small organic molecules, with a desired biological activity and half-life.
  • the invention relates to methods of treatment of a human which includes the step of administering to an individual with an elevated level of Her- 3 in one or more metastatic breast cancer cells of the brain, a therapeutic agent effective in directly inhibiting at least one ErbB receptor.
  • the invention relates to methods of treatment of a human which includes the step of administering to an individual with an elevated level of Her-3 in one or more metastatic breast cancer cells of the brain, a therapeutic agent effective in indirectly inhibiting at least one ErbB receptor.
  • suitable indirect inhibitors include molecules which disrupt an interaction between at least one ErbB receptor and its respective ligand such as an antibody directed to an ErbB ligand.
  • inhibitor is meant a therapeutic agent and/or a candidate agent which interferes with, inhibits, blocks or hinders at least one ErbB receptor, either by a direct or an indirect mechanism.
  • the inhibitory effect may be of a function or functional activity of at least one ErbB receptor, such as inhibition of tyrosine kinase activity of said ErbB receptor.
  • an inhibitor may block dimerisation between two different types of
  • ErbB receptors which in turn may block signaling by other ErbB family receptors.
  • the therapeutic agent is an antagonist, hi yet other embodiments, the therapeutic agent is an agonist. In other particular embodiments, the therapeutic agent inhibits or disrupts the heterodimeric interaction between different ErbB receptors such as, but not limited to, Her-2 and Her-3.
  • the therapeutic agent is a direct inhibitor of at least one ErbB receptor which binds or interacts directly with at least one ErbB receptor.
  • a non-limiting example of an direct inhibitor is an antibody which binds to or has been raised against the at least one ErbB receptor or a fragment thereof, although without limitation thereto.
  • an direct inhibitor is a small-molecule which binds to at least one ErbB receptor or a fragment thereof.
  • the therapeutic agent is an indirect inhibitor of at least one ErbB receptor which does not bind to or interact with directly with the at least one ErbB receptor but inhibit the activity or function of the at least one ErbB receptor.
  • indirect inhibitors target or are directed to a modulator of at least one ErbB receptor.
  • an indirect inhibitor of at least one ErbB receptor targets or is directed to one or more ligands of at least one ErbB receptor.
  • Non-limiting examples of suitable ligands of ErbB receptors which may be a target for an indirect inhibitor include epidermal growth factor (EGF), transforming growth factor (TGF)-alpha, amphiregulin, betacellulin, heparin-binding EGF, epiregulin and members of the neuregulin family such as neuregulin 1, 2, 3 and 4, although without limitation thereto.
  • EGF epidermal growth factor
  • TGF transforming growth factor
  • amphiregulin betacellulin
  • betacellulin betacellulin
  • heparin-binding EGF epiregulin
  • members of the neuregulin family such as neuregulin 1, 2, 3 and 4, although without limitation thereto.
  • an indirect inhibitor of at least one ErbB receptor targets or is directed to a modulator, and in particular an activator, of ligands of ErbB receptors.
  • a modulator of the ErbB receptor ligand may be an enzyme which cleaves an ErbB receptor ligand from the cell surface, which in turn activates the ErbB receptor ligand.
  • a non-limiting example of a suitable enzyme are any one of the members of the ADAM (A Disintegrin And Metalloprotease) family such as ADAMlO, ADAM12, ADAMl 7 and ADAM 19, although without limitation thereto.
  • the therapeutic agent selectively inhibits at least one ErbB receptor.
  • “selective” or “selectively” is meant a therapeutic agent that primarily affects the function of an ErbB receptor but may also have an effect upon other ErbB receptors. Therefore in one embodiment, “selective " or “selectively” encompasses a situation where at least 50%, 55%, 60%, 65%, preferably at least 70%, 75%, 80%, 85% and more preferably 90%, 95%, 96%, 98%, 99% and 100% of the inhibitory activity of a therapeutic agent can be attributable to a single ErbB receptor.
  • “selective " or “selectively” includes a therapeutic agent that can bind to and inhibit an ErbB receptor.
  • a non-limiting example of such a therapeutic agent or candidate agent is a suitable binding and/interaction partner, such as a protein.
  • the protein is an antibody.
  • “selective” or “selectively” includes a therapeutic agent that specifically regulates an ErbB receptor such as, but not limited to, nucleic acid molecules.
  • “selective " or “selectively” includes a therapeutic agent that specifically regulates post-translational forms of an ErbB receptor.
  • the methods of the present invention contemplate a therapeutic agent that is an antibody, or an antibody fragment, which binds and/or has been raised against at least one ErbB receptor or alternatively, an antibody (or an antibody fragment) which binds and/or has been raised against a ligand of at least one ErbB receptor. It is envisaged that both polyclonal and monoclonal antibodies directed to either the entire protein or a biologically-active fragment thereof are suitable therapeutic agents.
  • the therapeutic agent is an ⁇ -Her-3 antibody.
  • the therapeutic agent is an ⁇ -Her-2 antibody.
  • ⁇ -Her2 antibodies see, for example, United States Patent No 6,165,464; 5,677,171; and 6,054,561.
  • the ⁇ -Her-2 antibody is trastuzumab which is sold under the name Herceptin® by Genentech.
  • the therapeutic agent is an ⁇ -Her-1 antibody.
  • Suitable non-limiting examples of ⁇ -Her-1 antibodies are provided in US Patent Nos. 6,235,883 and 5,844,093 and International Publication No. WO/2007/058823.
  • the therapeutic agent is an ⁇ -Her-4 antibody.
  • antibodies may be monoclonal or polyclonal, obtained for example by immunizing a suitable production animal ⁇ e.g. a mouse, rat, rabbit, sheep, chicken or goat). Serum or spleen cells may be then isolated from the immunized animal according to whether polyclonal or monoclonal antibodies are required.
  • Monoclonal antibodies may be produced by standard methods such as described in CURRENT PROTOCOLS IN IMMUNOLOGY (Eds. Coligan et al. John Wiley & Sons. 1995-2000) and Harlow, E. & Lane, D. Antibodies: A Laboratory Manual (Cold Spring Harbour, Cold Spring Harbour Laboratory, 1988). Such methods generally involve obtaining antibody-producing cells, such as spleen cells, from an animal immunized as described above, and fusing spleen cells with an immortalized fusion partner cell.
  • Recombinant antibodies are also contemplated. Selection of appropriate recombinant antibodies can be achieved by any of a number of methods including phage display, microarray or ribosome display, such as discussed in Hoogenboom, 2005, Nature Biotechnol. 23 1105, by way of example only.
  • antibody fragments such as Fab, F(ab)2, Fv, scFV and Fc fragments as well understood in the art.
  • antibodies may be conjugated with labels including but not limited to a chromogen, a catalyst, an enzyme, a fluorophore, a chemiluminescent molecule, biotin and/or a radioisotope.
  • labels including but not limited to a chromogen, a catalyst, an enzyme, a fluorophore, a chemiluminescent molecule, biotin and/or a radioisotope.
  • antibody or “immunoglobulin”, as used interchangeably herein, includes whole antibodies and any antigen binding fragment or single chains thereof.
  • antibody or “immunoglobulin” includes any antigen-binding protein product of the immunoglobulin gene complex, including immunoglobulin isotypes IgA, IgD, IgM, IgG and IgE and antigen-binding fragments thereof. Examples of antigen-binding fragments include, but are not limited to, Fab, F(ab) 2 , Fv, scFv, etc. It will be appreciated by a person of skill in the art that antibodies employed for therapeutic applications in humans must have specific properties which make these antibodies suitable for use in humans.
  • therapeutic antibodies are "humanised” , wherein the antibody or at least one chain thereof, typically comprises a variable framework region substantially from a human antibody and the complementary determining regions substantially from an antibody derived from a non-human (such as, but not limited to, rodent antibodies and shark antibodies).
  • Humanised antibodies are particularly advantageous for medical applications due to the decrease likelihood of eliciting a foreign body immune reaction.
  • compositions and treatment methods may utilize nucleic acid constructs (including but not limited to inhibitory RNA constructs) for treatment of a metastatic breast cancer derived from one or more breast cancer cells with normal expression levels of Her-2.
  • the metastatic breast cancer is located in the brain.
  • a nucleic acid construct may be any recombinant nucleic acid that facilitates delivery, expression, propagation or manipulation of a desired nucleic acid component of the construct.
  • a construct may be a plasmid, a cosmid, a modified virus or containing virus-derived elements, an artificial chromosome, a phagemid, an anti-sense oligonucleotide, RNA (e.g. siRNA or shRNA) or the like.
  • RNA e.g. siRNA or shRNA
  • virus-derived expression constructs suitable for human delivery include constructs comprising adenovirus-, adeno-associated virus-, lentivirus-, flavivirus- and/or vaccinia virus-derived elements.
  • RNA-based methods may be employed for inhibition of at least one ErbB receptor.
  • RNA interference and in particular (but not limited thereto) siRNA and shRNA, provides an attractive method for silencing of potential therapeutic gene targets by sequence-specific cleavage of cognate mRNA. Takeshita and Ochiya (Cancer Sci, 2006, 97: 689-696) provides numerous examples of the therapeutic potential of RNA interference against cancer and is incorporated herein by reference.
  • gene is used herein to describe a discrete nucleic acid locus, unit or region within a genome that may comprise one or more of introns, exons, splice sites, open reading frames and 5' and/or 3' non-coding regulatory sequences such as a promoter and/or a polyadenylation sequence.
  • the invention contemplates a genetic construct which comprises one or more nucleotide sequences capable of directing synthesis of an RNA molecule, said nucleotide sequence selected from the list comprising:- (i) a nucleotide sequence transcribable to an RNA molecule comprising an RNA sequence which is substantially homologous to an RNA sequence encoded by a nucleotide sequence of interest;
  • nucleotide sequence of (i) represents an inverted repeat of the nucleotide sequence of (i), separated by a spacer sequence
  • the nucleotide sequence comprises an inverted repeat separated by a non-intron spacer sequence
  • the presence of the non-intron spacer sequence facilitates the formation of a stem-loop structure by virtue of the binding of the inverted repeat sequences to each other.
  • the presence of the non-intron spacer sequence causes the transcribed RNA sequence (also referred to herein as a
  • transcript so formed to remain substantially in one piece, in a form that may be referred to herein as a "hairpin”.
  • the nucleotide sequence comprises an inverted repeat wherein the spacer sequence comprises an intron sequence
  • the presence of intron/exon splice junction sequences on either side of the intron sequence facilitates the removal of what would otherwise form into a loop structure.
  • the resulting transcript comprises a double-stranded RNA
  • RNA RNA
  • the RNA molecules may comprise a single hairpin or multiple hairpins including "bulges" of single-stranded DNA occurring in regions of double-stranded DNA sequences.
  • the RNA molecule may directed to a single target or alternatively, a plurality of targets.
  • the RNA molecule encodes at least one ErbB receptor selected from the group consisting of Her- 1, Her-2, Her-3 and Her-4.
  • the RNA molecule encodes a ligand to at least one ErbB receptor.
  • inhibitory RNA constructs include siRNA or shRNA construct that down-regulate expression of at least one ErbB receptor and more preferably at least one ErbB receptor selected from group consisting of Her-1 , Her-2, Her-3 and Her-4.
  • Non-limiting examples of suitable siRNA molecules, and in particular ErbB receptor-specific siRNA, according to the present invention are provided in Sergina et al Nature. 2007445(7126):437-41 and Sithanandam et al Cancer Gene Ther.2008 15(7):413-48.
  • the present invention also contemplates peptide mimetics as therapeutic agents, and more particularly antibody-like peptidomimetics such as described in Zhang et al (2007) J. Clin. Invest. 117:2051-2058 in relation to ErbB receptors, although without limitation thereto.
  • Particular embodiments contemplate use of small-molecule inhibitors which directly or indirectly inhibit one or a plurality of ErbB receptors. Such inhibitors may be reversible or irreversible. It is also envisage that such inhibitors are selective against a particular ErbB receptor protein or in other embodiments, inhibit more than one ErbB receptor protein.
  • a non-limiting example of a multi-target inhibitor is BIBW-2992, which inhibits Her-1 and Her-2.
  • small-molecule inhibitors may inhibit a particular function of the at least one ErbB receptor and in preferred embodiments, is a tyrosine kinase inhibitor.
  • a suitable small-molecule inhibitors includes derivatives of quinazolines and more particularly, 4-anilinoquinazoline-based inhibitors such as those inhibitors described in United States Patent No's 6391874 and 6713485 and International Publication No. WO/1999/035146.
  • Non-limiting examples of selective inhibitors include N-(3-chloro-4- fiuorophenyl)-7-methoxy-6-(3-morpholin-4-ylpropoxy)quinazolin-4-amine (otherwise known as "gefitinib” which is sold under the name IressaTM by Astra Zeneca; PubChem Substance Compound ID: 123631) and N-(3 -ethynylphenyl)-6,7- bis(2-methoxyethoxy)quinazolin-4-amine (otherwise known as "erlotinib” which is sold under the name Tarceva® by Genentech; PubChem Substance Compound ID: 176870).
  • suitable irreversible inhibitors include CI- 1033,
  • EKB-569 (PubChem Substance Compound ID: 6445562) and HKI-272 as provided in Zhang et al (2007) J. Clin. Invest. 117:2051-2058.
  • a therapeutic agent of the invention is a small- molecule inhibitor of Her-2.
  • a small- molecule inhibitor of Her-2 Reference is made to Spector et al (2007) Breast Cancer Research 9:205 which provides non-limiting examples of suitable small molecule Her-2 inhibitors and is incorporated herein by reference.
  • the invention relates to methods of designing, engineering, screening or otherwise producing a therapeutic agent for treating a metastatic breast cancer derived from one or more metastatic breast cancer cells with normal expression levels of Her-2, which includes the step of identifying a candidate agent that is suitable for use in treatment of a metastatic breast cancer derived from one or more breast cancer cells with normal expression levels of Her-2, by the presence of an elevated expression level of Her-3 in one or more metastatic breast cancer cells of the brain.
  • suitable therapeutic agents and/or candidate agents of the invention may be peptides, proteins such as antibodies or other organic molecules, preferably small organic molecules, with a desired biological activity and half-life.
  • a therapeutic agent for treating a metastatic breast cancer derived from one or more metastatic breast cancer cells with normal expression levels of Her-2 designed, screened, engineered or other produced according to the present invention inhibits at least one ErbB receptor. More preferably, said therapeutic agent selectively inhibits at least one ErbB receptor.
  • the therapeutic agents of the invention may be identified by any number of methods.
  • suitable therapeutic agents may be identified by way of screening libraries of molecules such as synthetic chemical libraries, including combinatorial libraries, by methods such as described in Nestler & Liu, 1998, Comb. Chem. High Throughput Screen. 1 113 and Kirkpatrick et al, 1999, Comb. Chem. High Throughput Screen 2 211.
  • libraries of naturally-occurring molecules may be screened by methodology such as reviewed in KoIb, 1998, Prog. Drug. Res. 51 185.
  • Screening methods of the present invention also contemplate use of in vitro assays which mimic and/or replicate the natural biological environment of a metastatic breast cancer, in order to test the potential therapeutic efficacy of one or more candidate agents.
  • Particularly preferred embodiments contemplate an in vitro assay which mimics a metastatic breast cancer located in the brain, wherein differentiated neural cells are co-cultured with a breast cancer cell line, with the result being a cell-based matrix derived from the co-culture neural cells and breast cancer cells.
  • Differentiated neural cells may be derived from differentiation of neural progenitor cells (and preferably neural stem cells) such as, but not limited to ReNcell CX cell line (Chemicon International).
  • suitable breast cancer cells for use in the aforementioned in vitro assays include SKBR-3 and basal-like breast cancer cell lines such as, but not limited to, MDA-MB-231.
  • More rational approaches to designing therapeutic agents of the present invention may employ X-ray crystallography, NMR spectroscopy, computer assisted screening of structural databases, computer-assisted modelling, or more traditional biophysical techniques which detect molecular binding interactions, as are well known in the art.
  • suitable therapeutic agents may be in the form of a binding partner and as such, identified by interaction assays such as yeast two-hybrid approaches and the like, but without limitation thereto.
  • Two- hybrid screening methods are provided in Chapter 20 of CURRENT PROTOCOLS IN PROTEIN SCIENCE Eds. Coligan et al, (John Wiley & Sons, 1997) which is incorporated herein by reference.
  • the present invention also contemplates particular embodiments where the therapeutic agents of the invention may be used in combination with other therapeutic agents such as chemotherapeutic agents including cytotoxic drugs (for example capecitabine, doxorubicin (including its liposomal formulation), fluorouracil, cisplatin, gemcitabine, the taxanes paclitaxel and docetaxel, and vinorelbine) and hormone therapies (such as endocrine therapies) for individuals that are estrogen or progesterone positive and in particular modulators of a hormone receptor such as estrogen and/or progesterone (eg: tamoxifen, luteinising hormone releasing hormone analogues and aromatase inhibitors).
  • chemotherapeutic agents including cytotoxic drugs (for example capecitabine, doxorubicin (including its liposomal formulation), fluorouracil, cisplatin, gemcitabine, the taxanes paclitaxel and docetaxel, and vinorelbine) and hormone therapies (such
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutic agent effective for treatment of a metastatic breast cancer, preferably located in the brain, derived from one or more breast cancer cells with normal expression levels of Her-2 designed, screened, engineered or otherwise produced according as hereinbefore described, together with a pharmaceutically acceptable carrier, diluent, or excipient.
  • the invention provides a method of treating a metastatic breast cancer derived from one or more breast cancer cells with normal expression levels of Her-2 in a human including the step of administering to an individual with an elevated level of Her-3 in one or more metastatic breast cancer cells of the brain, a therapeutic agent effective for treatment of said metastatic breast cancer designed, screened, engineered or otherwise produced as hereinbefore described.
  • the metastatic breast cancer is located in the brain.
  • the therapeutic agents of the present invention may be in the form of a small organic molecule, peptide or the like formulated with a pharmaceutically-acceptable carrier, diluent or excipient suitable for oral administration, as a transdermal patch or other noninvasive route of admim ' stration.
  • pharmaceutically-acceptable carrier diluent or excipient
  • a solid or liquid filler diluent or encapsulating substance that may be safely used in systemic administration.
  • a variety of carriers well known in the art may be used.
  • These carriers may be selected from a group including sugars, starches, cellulose and its derivatives, malt, gelatine, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline and salts such as mineral acid salts including hydrochlorides, bromides and sulfates, organic acids such as acetates, propionates and malonates and pyrogen-free water.
  • any safe route of administration may be employed for providing a patient with the composition of the invention.
  • oral, rectal, parenteral, sublingual, buccal, intravenous, intra-articular, intra-muscular, intra-dermal, subcutaneous, intracranial, inhalational, intraocular, intraperitoneal, intracerebroventricular and transdermal administration may be employed.
  • Dosage forms include tablets, dispersions, suspensions, injections, solutions, syrups, troches, capsules, suppositories, aerosols, transdermal patches and the like. These dosage forms may also include injecting or implanting controlled releasing devices designed specifically for this purpose or other forms of implants modified to act additionally in this fashion.
  • Controlled release of the therapeutic agent may be effected by coating the same, for example, with hydrophobic polymers including acrylic resins, waxes, higher aliphatic alcohols, polylactic and polyglycolic acids and certain cellulose derivatives such as hydroxypropylmethyl cellulose.
  • the controlled release may be effected by using other polymer matrices, liposomes and/or microspheres.
  • compositions of the present invention suitable for oral or parenteral administration may be presented as discrete units such as capsules, sachets or tablets each containing a pre-determined amount of one or more therapeutic agents of the invention, as a powder or granules or as a solution or a suspension in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion or a water-in-oil liquid emulsion.
  • Such compositions may be prepared by any of the methods of pharmacy but all methods include the step of bringing into association one or more agents as described above with the carrier which constitutes one or more necessary ingredients.
  • the compositions are prepared by uniformly and intimately admixing the agents of the invention with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation.
  • compositions may be administered in a manner compatible with the dosage formulation, and in such amount as is pharmaceutically-effective.
  • the dose administered to a patient should be sufficient to effect a beneficial response in a patient over an appropriate period of time.
  • the quantity of agent(s) to be administered may depend on the subject to be treated inclusive of the age, sex, weight and general health condition thereof, factors that will depend on the judgement of the practitioner.
  • Metastatic breast cancer to the brain implications of the local microenvironment for colonization and treatment
  • the present inventors used a set of 29 matched pairs of human primary breast cancer and brain metastases, 22 unmatched brain metastases of breast cancer and a second set for validation comprised of 10 matched pairs of primary breast cancer and brain metastases and 11 non-breast metastatic brain cancers to explore the metastatic breast cancer colonization of the brain.
  • a combination of morphology, immunohistochemistry, differential gene expression, co- culture of human neural cells and breast cancer cell lines and in vivo brain injections of tumour cells in mice (treated with trastuzumab and neutralizing anti-neuregulin antibody) was used in order to elucidate possible mechanisms that could enhance the colonization of the brain parenchyma by tumour cells.
  • the present inventors provide in vitro and in vivo evidence that suggests that the inhibition of HER family receptors or the neutralization of local secreted growth factors like neuregulin could play a role in preventing tumour progression and colonization in the brain microenvironment and also may shed light on the paradigm that basal-like tumours, for not overexpressing HER2, cannot have response to trastuzumab.
  • Material and Methods Clinical samples:
  • Formalin fixed-paraffin embedded (FFPE) tumour blocks were retrieved from the archives of the Pathology Queensland: The Royal Brisbane & Women's Hospital, Brisbane, Australia - Medical Faculty of Charles University in Plzen, Czech Republic - Instituto Nacional do Cancer, Brazil - Laborat ⁇ rio Salomao e Zoppi, Brazil and Institute of Clinical Pathology and Medical Research, Westmead Hospital, University of Sydney, Australia.
  • An initial set of 29 matched pairs of primary breast cancer and their brain metastases and 22 unmatched brain metastases of breast cancer was available.
  • a second set of samples comprised of 10 matched pairs of primary breast cancer and their brain metastases and 11 non-breast metastatic brain cancers was also available.
  • Haematoxylin and eosin slides were dotted by one pathologist in order to take cores to the recipient block. Both arrays were built using the tissue arrayer, model MTAI (Beecher Instruments, Inc, Sun Prairie, WI 53590 USA). The tissue cores had 0,6 mm in diameter and were 1 ,0mm away from each other. For the second set of samples whole sections were used for haematoxylin & eosin staining. Another TMA was built for the second set of samples following the same descriptions as for the initial set.
  • RNA expression profiling was performed using the DASL assay (cDNA- mediated annealing, selection extension and ligation, Illumina Inc., California, USA) interrogating the DASL Cancer Panel which contained 512 cancer related genes (49, 50).
  • the above-mentioned first and second sets of samples were analyzed in two separated experiments.
  • the RNA derived from the animal experiment was analyzed in conjunction with the second set of samples. All protocols were as specified by Illumina Inc. Briefly, RNA (250ng) was converted to cDNA through a reverse transcription reaction with biotinylated oligo-d(T)i 8 and random nonamers.
  • Oligonucleotides (three unique pairs for each of the 512 genes) were annealed to the biotinylated cDNA, the duplexes were bound to streptavidin conjugated paramagnetic particles to remove non-hybridized oligos. The annealed oligos were then extended and ligated (to incorporate an address sequence and primer site) to generate amplifiable products. These products were subjected to PCR amplification using fluorescently labeled (Cy3 and Cy5) primers. The labeled PCR products were hybridized to a Sentrix array or Beadchip.
  • cDNA Complementary DNA
  • cDNA Complementary DNA
  • cDNA was synthesized from 240ng of total RNA using random hexamers and the SuperscriptTM III Reverse Transcriptase kit (Invitrogen, Carsbald, CA, USA).
  • TaqMan One-Step UniversalMaster Mix (Applied Biosystems) was used for all reactions.
  • TaqMan reaction was done in a standard 96- well plate format with ABI 7500 OneStepPlus PCR system.
  • raw deltaCt dCt
  • RPLl 3 a endogenous control gene
  • the normalized dCt was then calibrated to a cell line pool reference (MCF-7, SKBR-3 and MDA-MB-231) to generate a ddCt.
  • MCF-7 cell line pool reference
  • SKBR-3 cell line SKBR-3
  • MDA-MB-2311 cell line pool reference
  • the ddCt was converted to fold change (2 "ddCt ) relative to the reference allowing comparison between samples.
  • MCF-7, MDA-MB-231 and SKBR-3 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) plus 10% heat-inactivated fetal bovine serum (FBS), 2 mmol/L glutamine, and 1 % penicillin G-streptomycin solution.
  • DMEM Dulbecco's Modified Eagle's Medium
  • FBS heat-inactivated fetal bovine serum
  • FBS heat-inactivated fetal bovine serum
  • penicillin G-streptomycin solution CHEMICON International, Inc. Catalogue SCC007
  • ReNcell CX CHEMICON International, Inc. Catalogue SCC007 is an immortalized human neural progenitor cell line with the ability to differentiate into neurons and glial cells.
  • Resuspended ReNcells were allowed to grow in a total volume of 5 mL of serum-free DMEM/F12 (NSA) medium containing freshly added 20 ng/ml rhEGF (R&D Systems), 10 ng/ml rhFGF (R&D Systems), 4 ug/ml heparin, 10% proliferation supplement (NeuroCult®, Stem Cell Technologies Inc.), 2% BSA (Sigma). After formation of neurospheres, the cells were differentiated into neurons and glial cells in order to create a neural cell layer following the replacement of the NSA media by DMEM/F12 with 10% FBS, 2 mmol/L glutamine, and 1% penicillin G-streptomycin solution.
  • NSA serum-free DMEM/F12
  • Co-culture was established after plating in 24 wells dishes 15,000 cells in triplicates of the MCF-7 and SKBR-3 cancer cell lines with 10,000 differentiated human neural cells. Triplicates of the cell lines without neural cells were cultured for comparison of the growth and the co-culture was also performed in the presence of trastuzumab 20ng/ ⁇ l, lapatinib 5nmol/mL, anti-neuregulin antibody 50 ⁇ g/mL and a combination of trastuzumab plus lapatinib. A growth curve was generated after counting the cells in a flow cytometer. The cells were washed once with phosphate- buffered saline (PBS) and then harvested with 0.05% trypsin/0.025% EDTA.
  • PBS phosphate- buffered saline
  • Detached cells were washed with PBS. Combinations of fluorochrome-conjugated monoclonal antibodies against human CD56 and Epcam (BD Biosciences,San Diego, CA, USA) were used to distinguish between neural and epithelial cells, were added to the cell suspension at concentrations recommended by the manufacturer and incubated at 4 0 C in the dark for 30 minutes. The labeled cells were washed with FACS buffer (PBS supplemented with 1%BSA) and then analyzed on a LSRII (BD Biosciences). Comparisons between cell lines growth curves were done using one way ANOVA. This experiment was repeated twice. Soft agar colonization assay
  • MDA-MB-231 cells Two hundred thousand MDA-MB-231 cells were cultured in a six well plate containing 1 ml of RPMI-10% FBS, with the addition of agarose (final 0.5% ,w/v), over a 1 ml layer of bottom agar (final 0.35%, w/v).
  • Cells were cultured with the addition of trastuzumab (20ng/ ⁇ l), recombinat beta-1-neuregulin (50ng/ml) and combination of recombinant neuregulin and trastuzumab. Cells without the addition of traztuzumab and neuregulin were used for control. Trastuzumab and neuregulin were replenished every four days. Three replicates per combination were used.
  • the research complied with national legislation and was authorized by the local animal ethical committees.
  • the animals were divided into four groups; neuregulin-group which had anti-neuregulin antibody mixed with the cells at the time of injection, trastuzumab-group which was treated with intraperitoneal trastuzumab on the day of the injection of the breast cancer cells in the brain, one control group not treated and a fourth group designed to be treated once tumour was detected.
  • Each group was comprised of three animals.
  • the mice were inoculated directly in the right brain hemisphere sub ventricular zone with 200,000 MB-MD A-231 cells.
  • the concentrations of the anti-neuregulin antibody and trastuzumab were 50 ⁇ g/mL and 30mg/Kg respectively.
  • mice were weighted before injections. Statistical significance of the differences across all four groups was analyzed by t-Student test. Cells were allowed to grow in the brain for approximately 10 weeks. At this point, the mice in the neuregulin, trastuzumab and controls group were sacrificed and the treatment of the fourth group started. Magnetic resonance imaging of the mice and measurement of the tumours:
  • MRI scans were performed using Bruker 16.4T vertical wide-bore scanner, equipped with a micro-mouse AHS probe and a head coil (20 mm SAW linear coil, M2M imaging).
  • the animal was kept under anesthesia with isoflurane (1% at flow rate of 0.5-0.7L/min), its respiration rate was monitored using the BIOTRJG, and the animal body temperature is maintained at 30 degree Celsius.
  • the animal heads are scanned at 78 x 78 micron in-plane resolution and 1 mm slice thickness.
  • Images were processed and the tumour sizes were measured by manual ROI segmentation using the software Paravision 4.0. Final sizes were reconfirmed in the post-mortem examination when haematoxilin & eosin stained sections were cut at 4 ⁇ m and scanned using a scanscope® (Aperio Technologies, Vista, CA, USA). Sizes were achieved after analysing the images with Image scope (Aperio Technologies, Vista, CA, USA). Results Clinical features and morphological-immunohistochemical profiling of primary and metastatic breast cancer to the brain:
  • the one which was not grade III was a mucinous carcinoma, hi the second set of samples, 10 matched pairs of primary breast cancer and their brain metastases were also grade III invasive ductal carcinomas not-otherwise-specified and the 11 non-breast metastatic brain cancers were comprised of metastases from one melanoma, six lung adenocarcinomas, one prostate carcinoma and two kidney carcinomas. Table 2 shows the summary of the results for all of the antibodies applied.
  • FIG. 1 shows an example of a high grade tumour, positive for CK 14 and the matched pair immunohistochemistry staining for CD44.
  • RNA and stringent filtering of the DASL data yielded gene expression profiling data on 37/61 brain metastases from breast cancer (15/39 from matched pairs and 22/22 from unmatched metastases) and 15 matched primaries.
  • HER3, EGFR, HER2, HER4 and HIFl-alfa expression was assessed using quantitative RT-PCR in 12 matched breast/brain samples for which DASL data and RNA were available. Similar to the DASL data, ten cases showed increased fold change by RT-PCR of HER3 gene expression relative to their matched primaries ranging from 1.12 to 5.8 and with an average of 2.4. Immunohistochemistry for HER3 was similar, showing positivity in 11/37 (29.7%) of the primary tumors, 22/37 (59%) of the matched metastases and 13/21 (62%) of the unmatched brain metastases.
  • phosphorylated HER3 confirmed more frequent activation in the brain metastases, with positivity in 15/37 (40%) of the primary tumors, 24/37 (64%) of the matched metastases and 18/21 (85%) of the unmatched brain metastases.
  • ReNcell CX CHEMICON International, Inc. Catalogue SCC007
  • ReNcell CX CHEMICON International, Inc. Catalogue SCC007
  • EpCam epithelial phenotype
  • CD56 neuronal phenotype
  • resistance to the trastuzumab was seen after day 3 of the cell culture and surprisingly the same pattern of growth was observed for the anti-neuregulin antibody. As described previously, resistance could be due to activation of other member of the HER family of receptors.
  • the group of cells treated with neuregulin had an average colony size of 29.8 ⁇ m, the control 27.3 ⁇ m, the trastuzumab group 21.3 ⁇ m and the neuregulin + traztuzumab 18.7 ⁇ m.
  • the summary of results is shown in Figures 7 and 8. Intracerebral injections of a basal-like breast cancer cell line (MDA-MB-231) and treatment with trastuzumab and anti-neuregulin antibody and differential gene expression
  • the present inventors aimed to investigate if trastuzumab would have any effect on the cells in the brain microenvironment and also if the blocking of the local factor growth neuregulin with an anti-neuregulin antibody would interfere in the rate of growth of the injected cells.
  • the tumour sizes are available in Table 3.
  • breast cancer is one of the most common cancers and a leading cause of mortality in women (1, 2). Because there are few long-term survivors among patients with systemic metastatic breast cancer, the main objectives of therapy are to increase the time lapse before progressive disease is established and to improve symptom relief without increasing toxicity and comprising quality of life. As systemic therapy of metastatic breast cancer improves, central nervous system involvement is becoming a more widespread problem. (5) It is predicted that brain metastases will become increasingly prevalent as greater control over systemic metastases is achieved, particularly with regard to HER2 -positive tumours (6).
  • Brain metastases from breast cancer may become a serious problem that oncologists may encounter frequently (10) and it is considered one of the most feared complications of cancer because even small tumours may cause incapacitating neurologic symptoms (4).
  • autopsies series have reported brain metastases in 6,7%-36% of selected patients (57, 58).
  • the median age of presentation of the brain metastases in the group of patients where follow-up data was available was 48.5 years old, most of the tumours showed negativity for estrogens and progesterone receptors and around 23% showed overexpression of HER2, confirmed by CISH.
  • the percentage of HER2 overexpression tumours was in accordance with previous studies (25, 26).
  • CD44 + CD24 cells have been shown to express higher levels of preinvasive genes and have highly invasive properties (29). In addition, this phenotype is also believed to enrich for cells with stem cell properties (27).
  • Hyaluronic acid stimulates CD44s-associated pi 85 (HER2) tyrosine kinase activity, leading to an increase in the ovarian carcinoma cell growth (35).
  • the HER/erbb family of tyrosine kinase receptors consists of four members:
  • HERl, HER2, HER3, and HER4 (38).
  • HERl, HER2, and HER3 have been shown to contribute to aggressive tumour formation (61), on the other hand HER4 signaling may impair cellular proliferation of human breast cells and promote differentiation (62).
  • the differential gene expression between the primary tumours and the brain metastases showed that, in the first set of samples, HER3 was differentially expressed with statistical significance and in the second set a twofold up-regulation was detected. This differential expression was confirmed by RT-PCR and immunohistochemistry.
  • the samples stained with antibody against phosphorylated HER3 showed nuclear positivity.
  • ErbB receptors have been reported to translocate to the nucleus.
  • HERl , HER2, HER3 and HER4 may translocate to the nucleus as full-length receptors (HERl -3) or as a proteolytically derived intracellular domain (HER4) (52-55, 63).
  • induced HER2 overexpression has been shown to enhance the outgrowth of a specialized brain-seeking breast cancer cell line in animal model (65).
  • reactive glia was shown to enhance growth of this same cell line (66).
  • the 231 -BrI cell line which was derived from a brain metastases in a nude mouse, showed increased adhesion to astrocytes and enhanced growth in vitro in the presence of an astrocyte conditioneted media when compared to the parental MD A-MB-231 and the lung metastasis-derived variant 231- Lung2 (67).
  • the ligand for HER3 is neuregulin-1, a transmembrane epidermal growth factor-like molecule which is abundant in the brain microenvironment (44, 64).
  • neuregulin-1 a transmembrane epidermal growth factor-like molecule which is abundant in the brain microenvironment (44, 64).
  • trastuzumab and lapatinib reduced the growth rate but drug resistance was seen from day 3 to 4 (42, 68).
  • the same result was seen when the neutralizing anti-neuregulin antibody was added suggesting that the neuregulin of the neural cells could also be playing a role in the growth enhancement.
  • the combination of trastuzumab and lapatinib overcame the resistance from day 3 to 4, suggesting that the resistance could be via activation of the other members of the HER family of receptors as indicated previously (42, 43).
  • the mechanisms of action of lapatinib and trastuzumab are different and their combination was shown to lead to synergism (69).
  • trastuzumab The mechanism of action of trastuzumab is distinct from tyrosine kinase inhibitors such as gefitinib or erlotinib, which bind competitively to the intracellular adenosine triphosphate binding site of erbb receptors (70, 71) and which is the same site of action for lapatinib. (72)
  • the MDA-MB-231 cell line has low levels of HER2 and HER3
  • the treatment of the mice with trastuzumab and anti-neuregulin antibody resulted in smaller tumours when compared to the control group.
  • This may reflect a potential for some tumours with low expression of HER2 to be responsive to trastuzumab and also suggests that the brain local environment may be playing a role in the enhancement of growth in vivo.
  • the colony forming assay supports this view since less and smaller colonies were detected in the group treated with traztuzumab compared to the control group and the group treated with neuregulin. These values were statistical significant.
  • Pertuzumab is a recombinant humanized monoclonal antibody and binds to extracellular domain II of the HER-2 receptor and blocks its ability to dimerize with other HER receptors.
  • the pertuzumab binding site within domain II does not overlap with the epitope on HER-2 (76) that is recognized by trastuzumab.
  • this monoclonal antibody could play a major role in cases where a tumour is also dependent on other members of the HER family of receptors.
  • the present study provides evidence that local factor neuregulin may enhance tumour growth in the brain microenvironment whether in vitro or in vivo and shed some light on the paradigm that HER2 negative cannot have response to trastuzumab.
  • HER2 negative cannot have response to trastuzumab.
  • trastuzumab or combinations of HER family receptors inhibitors.
  • tumours are dependent on members of the erbB family receptors that are fueled by local factors, autocrine loops or even autophophorylated.
  • HER-family of receptors in plasmids (pLHCX-EGFR; hygromycin resistant, pLPCX-HER2; puromycin resistant and pLNCX2-HER3; G418 resistant).
  • the transduction will be performed using an amphotropic producer cell line such as Phoenix amphotropic cells.
  • an amphotropic producer cell line such as Phoenix amphotropic cells.
  • the medium will be collected filtered and used for infecting the breast cancer cells.
  • Luminal and basal-like breast cancer cell lines (with and without the insertion of the HER-family of receptor genes) will be co-cultured with adult CNS cells in order to mimicry an artificial brain matrix.
  • the epithelial luminal and basal-like breast cancer cell lines will be place onto a matrix of adult CNS cells.
  • the epithelial and CNS cells will be differentially labelled with epithelial and neuronal florescent antibodies so that number of viable cells can be assessed by this device.
  • Human fibroblasts and cells which are not in co-culture will be used as controls. Growth curve for each experiment will be then generated and the influence of the co-culture conditions analysed.
  • EpCAM and CD56 for differential fluorescent labelling the epithelial and neuronal cells respectively.
  • Neurosphere assay 2 ⁇ is a serum-free culture system whereby the isolation and propagation of CNS-derived stem cells is feasible.
  • Adult precursors are dissociated and plated in a liquid growth medium that contains the stem-cell mitogens epidermal growth factor and/or fibroblast growth factor. Because of the lack of serum and the low plating density, most cells die, except those that divide in response to the stem- cell mitogens.
  • the growth-factor responsive cells proliferate to form floating clusters of cells that are referred to as neurospheres. These can be further dissociated into a single-cell suspension and then re-plated in fresh medium to produce secondary neurospheres. After removal of mitogenic factors, the progeny of the proliferating precursors can be differentiated into neurons, astrocytes and oligodendrocytes, which are the three primary cell types that are found in the adult human CNS. This will be our source of adult CNS cells to create the artificial brain matrix.
  • modulators of the HER-family of receptor into the 96-well plates will be performed.
  • modulators will comprise drugs currently used in clinical practice to target the HER-family of receptor, for example trastuzumab and lapatinib but not limited thereto.
  • Non-limiting examples of other drugs currently used in clinical practice to target the HER-family of receptor are given in this text, particularly at Table 5.
  • the wells will have quadruplicates samples and the statistical difference between the growth curves will be established using one-way Analysis of variance.
  • the growth curves will use time points at 12, 36, 60 and 84 hours and the drugs will be titrated.
  • RNA interference RNA interference
  • the above mentioned experiment will be also performed with the use of RNAi targeting the HER-family of receptor and also using an antibody targeting the local growth factor neuregulin 1 , the ligand of HER3 (the adult CNS cells express this molecule).
  • RNAi targeting HER-family of receptors and scrambles for negative controls are available for purchasing from several companies. We expect this second experimental phase to mirror at some extend the findings of the experimental phase where drugs were used.
  • the BD FACSArrayTM bioanalyzer from BD Biosciences is capable fast and sensitive high-content measurements of cells and proteins in cell biology, supporting analysis of apoptosis, cytokine and chemokine profiling, and phosphorylation of key signal transduction proteins.
  • This flow cytometer rapidly detects and quantifies concentrations of secreted proteins, proteins in cell lysates, and cell-associated proteins using small sample volumes directly from a 96-well microtiter plate. This device will be used for the above mentioned experiments.
  • the same co-culture system will also be used to assess activation of molecules which are downstream of the HER-family of receptors.
  • the molecules targeted will be the ones assessed in AIM 1(RAS, SOS, RAFl, ERK1/2, MEKland ELK-I for the MAPK pathway and p21, p27, MDM2, BAD, CASP9 and mTOR for the AKT pathway).
  • the BD FACSArrayTM bioanalyzer will again be used.
  • We expect that the results under non-co-culture conditions should mirror the results of the baseline assessment with western blots across the cell lines.
  • This experimental phase will also show the changes the will occur in the AKT and MAPK related molecules under co-culture conditions, allowing a better understanding of the behaviour of breast cancer cells in a brain environment.
  • apoptotic indexes can also be generated by the BD FACSArrayTM bioanalyzer using markers such as annexine and caspases.
  • 35.Bourguignon LY Zhu H, Chu A, IidaN, Zhang L, Hung MC. Interaction between the adhesion receptor, CD44, and the oncogene product, pl85HER2, promotes human ovarian tumour cell activation. J Biol Chem 1997;272(44):27913-8. 36.Bourguignon LY, Zhu H, Zhou B, Diedrich F, Singleton PA, Hung MC.
  • Hyaluronan promotes CD44v3-Vav2 interaction with Grb2-pl 85(HER2) and induces Racl and Ras signaling during ovarian tumour cell migration and growth. J Biol
  • ErbB-2/HER2 oncoprotein of human carcinomas may function solely as a shared coreceptor for multiple stroma-derived growth factors.
  • MET amplification leads to gefitinib resistance in lung cancer by activating ERBB3 signaling. Science 2007;316(5827):1039-43.
  • c-erbB-3 a nuclear protein in mammary epithelial cells. J Cell Biol 2002;157(6):929-39.
  • Her-2 overexpression increases the metastatic outgrowth of breast cancer cells in the brain.
  • Reactive glia are recruited by highly proliferative brain metastases of breast cancer and promote tumour cell colonization. Clin Exp Metastasis 2008.
  • N number of tumour cores showing positivity
  • T total number of cores assessable for the antibody specified
  • % percentage of cores showing positivity
  • NP not performed
  • Control Group not Tumour size- Days to death Average of days to treated mm 3 death
  • mice treated at Day 84 MRI Tumour Size Tumour Size at Days to death after with Trastuzumab at day of autopsy-mm 3 treatment injection-mm 3
  • Table 5 A sample of ErbB inhibitors which are either used for treatment or in pre-clinical and clinical trials

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Abstract

A method of treating a treating a metastatic breast cancer in a human is provided which includes the step of administering to an individual with an elevated level of Her-3 in one or more metastatic breast cancer cells of the brain, a therapeutic agent effective in inhibiting at least one ErbB receptor. Also provided are methods of identifying a therapeutic agent for treating a metastatic breast as well as methods of diagnosing a metastatic breast cancer which utilise detecting elevated levels of Her-3.

Description

TITLE
CANCER THERAPY AND/OR DIAGNOSIS
FIELD OF THE INVENTION
THIS invention relates to cancer therapy and/or diagnosis. More particularly, this invention relates to therapeutic targets and diagnostic markers for metastatic breast cancer and preferably metastatic breast cancer in the brain.
BACKGROUND TO THE INVENTION
Breast cancer is one of the most common malignant neoplasias and a leading cause of female cancer mortality (1, 2). Among women with breast cancer in western countries, 30%-40% will develop metastatic disease (3).
The natural history of metastatic breast cancer to the brain demonstrates that symptomatic brain metastases develop in 10% to 20% of patients with metastatic breast cancer and most of these patients will have a mean survival of 6 months following the diagnosis of the brain disease (4-6). Some studies have found associations between younger age, p53 positive tumours (7) and enrichment for estrogen receptor negative and epidermal growth factor receptor positive cancers (8) and the development of brain metastases.
Presently, the epidermal growth factor receptor family comprises four receptors, HERl, HER2, HER3 and HER4. Such receptors are triggered by several ligands. Upon activation, hetero or homo-dimerization occurs, followed by phosphorylation of specific tyrosine residues in the intracellular region. Such activation, leads to regulation of a variety of cellular processes including cell proliferation and survival. Much evidence suggests that the kinases Akt and Erk mediate a substantial part of the HER signaling (70). Basal-like tumours are a molecular subgroup of breast cancer (13, 14) that generally are high grade, negative for estrogen and progesterone receptors and do not overexpress HER2. Hence, these tumours are believed to be unlikely to respond to trastuzumab-based therapy and no other targeted therapy for this group is currently available, although clinical trials are ongoing (15). Trastuzumab (Herceptin®; Genentech Inc, South San Francisco, CA) treatment aims to block HER2 and its downstream pathways. Other studies showed a trend for primary tumours of patients who developed brain metastases during disease progression to express markers related to the basal-like tumours (8, 16-18).
Palmieri et al (2007) Cancer Research, 67: 4190 to 4198 examined Her-2 expression profiles of breast cancer cells in the brain and demonstrated that Her-2 overexpression increases the outgrowth of metastatic tumour cells in the brain. Moreover, expression levels of Her-2, Her-3 and Her-4 proteins were examined in MDA-MB-231BR brain-seeking cell line and found to be comparable and at the lower limit of detection.
SUMMARY OF THE INVENTION The invention is broadly directed to targets and methods for the diagnosis and/or treatment of metastatic outgrowth of breast cancer cells, particularly to the brain.
In another broad form, the invention is directed to targeting one or more members of the epidermal growth factor receptor family for treatment of metastatic outgrowth of breast cancer cells, particularly located in the brain, and more particularly in basal-like breast cancer tumours in the brain.
In a first aspect, the invention provides a method of treating a metastatic breast cancer in a human, said method including the step of administering to an individual with an elevated level of Her-3 in one or more metastatic breast cancer cells of the brain, a therapeutic agent effective in inhibiting at least one ErbB receptor.
In preferred embodiments, the method further includes the step of determining whether said individual has an elevated level of Her-3 in one or more metastatic breast cancer cells of the brain. In a second aspect, the invention provides a method of designing, engineering, screening or otherwise producing a therapeutic agent for treating a metastatic breast cancer, said method including the step of identifying a candidate agent that is suitable for use in treatment of a metastatic breast cancer, by the presence of an elevated expression level of Her-3 in one or more metastatic breast cancer cells of the brain.
In a third aspect, the invention provides a therapeutic agent for treating a metastatic breast cancer designed, engineered, screened or otherwise produced according to a method of the second aspect.
In a fourth aspect, the invention provides a pharmaceutical composition comprising a therapeutic agent for treating a metastatic breast cancer of the third aspect, together with a pharmaceutically-acceptable carrier, diluent or excipient.
In a fifth aspect, the invention provides a method of treating a metastatic breast cancer, said method including the step of administering a therapeutic agent for treating a metastatic breast cancer according to the third aspect or a pharmaceutical composition according to the fourth aspect, to an individual with an elevated level of Her-3 in one or more metastatic breast cancer cells of the brain.
In a sixth aspect, the invention provides a method of determining whether a human with a metastatic breast cancer is potentially responsive to treatment with a therapeutic agent effective in inhibiting at least one ErbB receptor and/or a therapeutic agent for treating a metastatic breast cancer designed, engineered, screened or otherwise produced according the method of the second aspect, said method including the step of detecting an elevated level of Her-3 in one or more metastatic breast cancer cells of the brain.
In a seventh aspect, the invention provides a method of determining whether a human is predisposed to a metastatic breast cancer or is suffering from a metastatic breast cancer, said method including the step of detecting an elevated level of Her-3 in one or more metastatic breast cancer cells of the brain.
In preferred embodiments of any one of the aforementioned aspects, the metastatic breast cancer has one or more cells with normal expression levels of Her- 2. More preferably, the metastatic breast cancer has one or more breast cancer cells with normal expression levels of Her-2.
Preferably, the metastatic breast cancer is located in the brain. In preferred embodiments, the metastatic breast cancer is derived from one or more breast cancer cells. Preferably, the at least one ErbB receptor is selected from the group consisting Her-1, Her-2, Her-3, Her-4, and combinations thereof. More preferably, the at least one ErbB receptor is selected from Her-1 and Her-2.
Even more preferably, the at least one ErbB receptor is Her-2.
In preferred embodiments, the candidate agent inhibits at least one ErbB receptor.
In preferred embodiments, the therapeutic agent and/or the candidate agent inhibits at least one ErbB receptor by directly inhibiting at least one ErbB receptor. hi other preferred embodiments, the therapeutic agent and/or the candidate agent inhibits at least one ErbB receptor by indirectly inhibiting at least one ErbB receptor.
According to any of the aforementioned aspects, the therapeutic agent and/or the candidate agent selectively inhibits at least one ErbB receptor.
In preferred embodiments, the one or more cells with normal levels of Her-2 have levels of an estrogen receptor and a progesterone receptor which are below a threshold level of detection.
Preferably, the therapeutic agent and/or the candidate agent is selected from the group consisting of an isolated protein, an isolated nucleic acid, a small-molecule compound, and combinations thereof.
More preferably, the therapeutic agent or the candidate agent is an antibody, or a fragment thereof and even more preferably, a monoclonal antibody.
According to preferred embodiments of the aforementioned aspects, the therapeutic agent and/or the candidate agent is selected from the group consisting of trastuzumab, pertuzumab, scFvFRP5, CAB051, ertumaxomab, lapatinib, CI- 1033, HKI-272, AEE-788, BIBW-2992, TAKl 65, BMS-599626, matuzumab, cetuximab, panitumumab, gefitinib, erlotinib, ICR62, nimotuzumab, Ch806, L8A4, MDX-447, zalutumumab, IMC-11F8, AZD4769, PF299804, EGFR501, ZD6474, EKB-569, EXEL 7647, EXEL 0999, AZD8931, MP412, herstatin, pelitinib, CP724714, XL647, PD- 169414, and combinations thereof.
Even more preferably, the therapeutic agent and/or candidate agent is trastuzumab.
Throughout this specification, unless the context requires otherwise, the words "comprise", "comprises" and "comprising" will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.
BRIEF DESCRIPTION OF FIGURES In order that the invention may be readily understood and put into practical effect, preferred embodiments will now be described by way of example with reference to the accompanying:
FIGURE 1 - A- Grade III invasive ductal carcinoma; B- Same tumour as in "A" showing CK 14 expression; C- Primary breast carcinoma negative for CD44; D- Matched metastasis of "C" showing positivity for CD44.
FIGURE 2 - A -Unsupervised hierarchical clustering of DASL gene expression data from 22 unmatched (black bar) and 15 matched primary and brain metastases (shades of grey bars). Thirteen out of 15 matched samples are clustering together. B - Heatmap and dendogram showing clustering of the samples based on the 20 genes differentially expressed between primary tumors (light grey bar) and brain metastases (dark grey bar).
FIGURE 3 - Her-3 expression. Summary of RT-PCR (first bar at each point) and DASL assay (second bar at each point) for 12 matched samples - Positive values indicate increased fold change in metastases and negative values increased fold change in primary. X-axis are 12 matched primary breast cancers and brain metastases; Y-axis is ration of fold-change-Brain metastasis/Primary cancer FIGURE 4 - A-Brain metastasis; B- Nuclear expression of HER3 for the tumour shown in "A"; C- Negativity for pAKT in the matched primary of "B"; D- Positivity for ERK1/2 in the brain metastasis shown in "B". E - Positivity for ERK5 in the brain metastasis shown in "B"; F - Positivity for EGFR in the brain metastasis from a non- breast cancer.
FIGURE 5 - Growth curves of the cell line SKBR-3 grown on its own (BC only; vertical line) and on co-culture with ReNcell CX neural cells (NC; circle). Treatment with trastuzumab (square), anti-neuregulin 1 antibody (Neu-Ab; diamond), lapatinib (asterisk) and their combinations (trastuzumab plus Neu-Ab is triangle; lapatinib plus trastuzumab is cross ) was also added to the co-culture system. The double-headed arrow highlights statistical significant (p<0.05, ANOVA) between the curves above and below the blue arrow. X-axis is time in hours while Y axis is EpCam positive particles per 250μl of buffer.
FIGURE 6 - MRI images from week 10 showing brain tumours in the left hemisphere from an animal of (A) the control group, (B) the trastuzumab treated group, (C) the anti-neuregulin 1 treated group. Average tumor size was smaller in B and C compared to control A (p<0.05, unpaired T test). D - Relative levels of HER3 transcripts in the parental cell line and in the four groups of mice. *Highlights statistical significance (p<0.05, unpaired T test) between the parental group and the other groups; Y-axis is HER3 expression relative to GAPDH and X-axis the subgroups of treatment.
FIGURE 7 - Colony forming assay. Effects of traztuzumab and neuregulin on the number of colonies of MB-MD A-231 breast cancer cell line. Y -axis is average number of colonies and X-axis the subgroups of treatment. FIGURE 8 - Colony forming assay. Effects of traztuzumab and neuregulin on the size of colonies of MB-MD A-231 breast cancer cell line. Y -axis is average number of colonies and X-axis the subgroups of treatment.
DETAILED DESCRIPTION OF THE INVENTION The present invention is predicated, at least in part, on the finding that Her-3 is overexpressed in tumours located in the brain which result from metastatic outgrowth of breast cancer tumours that exhibit a basal-like phenotype. Furthermore, even in the absence of HER2 amplification/overexpression in such tumours, an anti (α)-Her-2 monoclonal antibody is useful in reducing a tumour growth located in the brain resulting from metastatic breast cancer outgrowth in vitro and in vivo. Moreover, the association of Her-3 with other members of the ErbB receptor family suggests that therapies directed to members of the ErbB receptor family could be useful for treatment of a subset of metastatic breast cancers in the brain which normally do not respond well to, at least, α-Her-2 therapies such as trastuzumab.
In general aspects, the invention provides methods of treatment and/or diagnosis of a metastatic breast cancer in a human with elevated levels of Her-3 in one or more metastatic breast cancer cells of the brain, hi preferred embodiments, the metastatic breast cancer is located in the brain. In other preferred embodiments, the metastatic breast cancer is derived from one or more cells, and more preferably breast cancer cells, with normal expression levels of Her-2.
By "breast cancer" is meant a malignant tumour of the breast tissue. Typically, breast cancer forms in tissues of the breast, usually the ducts (tubes that carry milk to the nipple) and lobules (glands that make milk).
By "metastatic breast cancer " is meant one or more primary breast cancer cells which have undergone metastases and spread to one or more parts of the body to form secondary tumours which are otherwise referred to as metastatic tumours. A "metastatic breast cancer " may be derived from one or more cancer cells which originate from a breast cancer, or alternatively the "metastatic breast cancer " may derived from a non-breast cancer cell source.
As used herein, by "one or more cells with normal expression levels ofHer- 2 " is meant that said one or more cells are considered to have, exhibit or display a lack of expression or amplification of Her-2, and preferably either gene amplification from the HER-2 gene locus or a HER-2 amplicon or protein expression, when compared to one or more cells which overexpress Her-2. In particular embodiments, the "one or more cells with normal expression levels of Her-2" have expression levels of Her-2 which are below a threshold level of detection and are thus considered not to be Her-2 positive. Although not wishing to be bound by any particular theory, a positive HER2 result may be considered as immunohistochemistry (IHC) staining of 3+ (uniform, intense membrane staining of >30% of invasive tumour cells), a fluorescent in situ hybridization (FISH) result of more than six HER2 gene copies per nucleus or a FISH ratio (HER2 gene signals to chromosome 17 signals) of more than 2.2. By way of example, a negative result for Her-2 may be an IHC staining value of 0 or 1+, a fluorescence in situ hybridization (FISH) result of less than 4.0 HER2 gene copies per nucleus, or FISH ratio of less than 1.8. Reference is made to Wolff et al (2007) J Clin Oncol 25: 118-145 which provides non-limiting examples of assays and criteria for use of Her-2 as a marker in breast cancer and is incorporated herein by reference. In light of the foregoing it will be appreciated that normal Her-2 expression levels may result from basal or normal expression of the Her-2 gene locus located on genomic DNA, and in particular, two chromosomal copies of the Her-2 gene locus or alternatively expression of Her-2 which results from one or more cancer cells that are polysomic.
Although not wishing to be bound by any particular theory, "one or more cells with normal expression levels of Her-2 " are considered not amenable to conventional targeted therapies for breast cancer such as α-Her-2 monoclonal antibodies (eg. trastuzumab). It will be appreciated that the "one or more cells with normal expression levels of Her-2 " may originate from a variety of sources such as in vitro culture (such as cultured breast cancer cell lines), ex vivo or alternatively, in vivo. In preferred embodiments, the "one or more cells with normal expression levels of Her-2" are one or more cancer cells and more preferably, one or more breast cancer cells.
In determining whether "one or more cells with normal expression levels of Her-2 " have Her-2 expression levels that are below the threshold level of detection, applicable detection or assay methods include immunohistochemistry, histopathology, genomic analysis (such as fluorescence in situ hybridization) and gene expression profiling (such as DNA microarray-based techniques), although without limitation thereto. Reference is made to Sorlie et al Proc Natl Acad Sci USA 2001;98( 19): 10869-74; and Perou et al Nature 2000;406(6797):747-52 which provide non-limiting examples of suitable molecular methods, and in particular gene expression profiling methods, to determine or evaluate various markers in breast cancer whilst LaI et al Am J Clin Pathol 2004, 121:631 -636 provides non-limiting examples of Her-2 testing in breast cancer using immunohistochemical analysis and fluorescence in situ hybridization. In particular embodiments, the "one or more cells with normal expression levels of Her-2 " may have also have expression levels of an estrogen receptor and progesterone receptor which are below a threshold level of detection. Detection methods include those and for example as described in Korsching et al J Clin Pathol. 2008 May;61(5):553-60; Kusiήskaet al Pol J Pathol.2005;56(3): 107-10; andMullan and Millikan, Cell MoI Life Sci. 2007 Dec;64(24):3219-32.
The one or more cells which exhibit a phenotype of normal Her-2 levels as defined hereinabove and have expression levels of an estrogen receptor and a progesterone receptor which are below a threshold level of detection are also known in the art as "triple-negative tumour ", " basal-like tumour " or "basal-like phenotype " and may be used interchangeably herein. In the context of the present invention, by "ErbB receptor " is meant any member of the epidermal growth factor receptor family of receptor tyrosine kinase or Her receptors. The ErbB (or otherwise referred to as Her receptors or epidermal growth factor receptors) family of receptor tyrosine kinases are important mediators of cell growth, differentiation and survival. Her-1 is specific for epidermal growth factor and EGF related peptides including transforming growth factor-alpha, amphiregulin, and heparin-binding EGF-like growth factor. The members of the ErbB family and particularly Her-1, Her-2, Her-3 and Her-4 share extensive homology and are able to homodimerise and heterodimerise with other members of the family. Reference is made to Zhang et al (2007) J. Clin. Invest. 117:2051-2058, which provides a review of ErbB receptor biology.
The term "Her-3 " as used herein refers to a Her-3 protein, and preferably a human Her-3 protein, as for example described in US Patent No. 5,480,968; Plowman et al, Proc Natl Acad Sci U S A. 1990 Jul;87(13):4905-9 and Kraus et al Proc Natl Acad Sci U S A. 1989 Dec;86(23):9193-7, which are incorporated herein by reference. "Her-3 " may also referred to as "ErbB3 ", "Her-3 receptor" and "ErbB3 receptor " and other terms as are known in the art such as "LCCS2 ", "c- erbB3 ", "erbB3-S", "MDA-BF-I ", "MGC88033 ", "c-erbB-3 ", "p!80-ErbB3 ", "p45-sErbB3 " and "p85-sErbB3 " and may be used interchangeably herein.
The term "Her-2 " as used herein refers to a Her-2 protein, and preferably a human Her-2 protein, as for example described in King et al 1985 Science, Sep 6;229(4717):974-6. "ffer-2 " may also be referred to as "ErbB2", "neu", "Her-2 receptor ", "ErbB2 receptor " and other terms as are known in the art such as "«g/ ", "TKR1 ", "CD34O", "HER-2/neu" and "pJSS^2^"" and may be used interchangeably herein. The term "Her-1 " as used herein refers to a Her-1 protein, and preferably a human Her-1 protein which is the mature, tyrosine kinase cell surface receptor, as for example described in Lin et al (1984) Science 224: 843 - 848 and Ullrich et al, (1984) Nature, 309:418-^25. "Her-1 " is also referred to "epidermal growth factor receptor (EGFR) ", "ErbBl ", "c-erbB-1 ", "ErbB" and "PIG61 " and may be used interchangeably hereia The term "Her-4 "as used herein refers to a Her-4 protein, and preferably a human Her-4 protein, as for example described in Plowman et αl, Proc Natl Acad Sci U S A. 1993 Mar l;90(5):l 746-50. "Her-4" may also be referred to as "MGCl 38404", "pl8(TbB4", "tyro2" and "ErbB4 " and may be used interchangeably herein. General aspects of the present invention contemplate detecting, determining or otherwise measuring an elevated level of Her-3 in one or more metastatic breast cancer cells of the brain, either as part of diagnostic methods or in methods of treatment of humans (preferably as a selection step), according to the present invention. In certain broad aspects, the present invention contemplates methods of treating a metastatic breast cancer in a human by administering to an individual with an elevated level of Her-3 in one or more metastatic breast cancer cells of the brain, a therapeutic agent designed, screened, engineered or otherwise produced according to methods of the present invention as described herein. In other general aspects, the present invention contemplates methods of treating one or more breast cancer cells with normal expression of Her-2 in a human which includes the step of administering to an individual with an elevated level of Her-3, a therapeutic agent effective in inhibiting at least one ErbB receptor.
In other aspects, the invention relates to methods of diagnosis of a metastatic breast cancer, preferably located in the brain, derived from one or more breast cancer cells with normal expression levels of Her-2. Such methods may assist in determining whether a human suffers from said metastatic breast cancer, is predisposed to said metastatic breast cancer and/or is more or less responsive to therapies directed to metastatic breast cancer and in particular metastatic breast cancer located in the brain.
Thus "predisposed" and "predisposition" are used in the context of a probability that an individual may display clinical symptoms of a metastatic breast cancer, preferably located in the brain, derived from one or more breast cancer cells with normal expression levels of Her-2, or that any existing, manifest clinical symptoms of said metastatic breast cancer are the result of an underlying biochemical cause.
It will also be appreciated that the diagnostic methods of the invention may be used alone or combined with other forms of molecular and/or clinical diagnosis to improve the accuracy of diagnosis.
In the context of the present invention, by "an elevated level ofHer-3 in one or more metastatic breast cancer cells of the brain " is meant an amplified, overexpressed, up-regulated, enhanced or otherwise increased amount of Her-3 when compared to breast cancer cells which have not metastasized to the brain.
It will be appreciated that in particular embodiments, Her-3 per se may be assayed or measured to determine an elevated level of Her-3. The present invention also contemplates detecting or assaying alternative forms of Her-3, such as post- translationally modified Her-3 (for example, phosphorylated Her-3) or variants such as splice variants.
It is also contemplated that in other embodiments that molecules associated with Her-3 or its function, such as downstream signalling proteins of the mitogen- activated protein kinase pathway which Her-3 activates, may also be assayed. In particularly preferred embodiments, a ligand of Her-3 such as, but not limited to, neuregulin-1 may also be assayed in order to determine whether there is an elevated level of Her-3 in one or more metastatic breast cancer cells of the brain.
In light of the foregoing, it will be appreciated that a variety of methods as are known in the art may be utilised to determine an expression level of Her-3 in one or more metastatic breast cancer cells of the brain in methods of the present invention.
In particular embodiments, the invention contemplates protein-based detection techniques and in particular for detection of Her-3, or a fragment thereof.
By "protein" is also meant "polypeptide ", "peptide " or fragments thereof, referring to an amino acid polymer, comprising natural and/or non-natural amino acids, including L- and D-isomeric forms as are well understood in the art. For example, Her-3 or any other ErbB receptor or ErbB receptor ligand may be referred to as either a protein or polypeptide. In particular embodiments, the protein or polypeptide is an isolated protein or an isolated polypeptide.
A "peptide" is a protein having no more than fifty (50) amino acids. A "polypeptide" is a protein having fifty (50) or more amino acids.
Proteins and peptides may be useful in native, chemical synthetic or recombinant synthetic form.
For the purposes of this invention, by "isolated" is meant material that has been removed from its natural state or otherwise been subjected to human manipulation. Isolated material may be substantially or essentially free from components that normally accompany it in its natural state, or may be manipulated so as to be in an artificial state together with components that normally accompany it in its natural state. Isolated material may be in native, chemical synthetic or recombinant form. A "fragment" is a segment, domain, portion or region of an ErbB receptor, and in particular embodiments Her-3, which constitutes less than 100% of the full- length protein.
A fragment preferably comprises less than 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 60%, 50%, 40%, 30%, 20% or as little as even 10%, 5% or 3% of the entire protein.
In particular aspects, a protein fragment may comprise, for example, at least 5, 10, 20, 30, 40, 5060, 70, 80, 90, 100, 120, 140, 150, 200, 250, 300, 350, 400, 450 or 500 contiguous amino acids of an ErbB receptor, and in particular Her-3.
In particular embodiments, a fragment is a "biologically-active fragment ". By "biologically-active fragment" is meant a segment, portion or fragment of a biological active molecule which has at least about 0.1%, preferably at least about 10%, more preferably at least about 25% and even more preferably at least 50% of the activity of the molecule and even more preferably at least 70%, 80% or 90% of the biological activity of the entire or full length protein. By way of example only, fluorescence activated cell sorting (FACS) analysis using labelled antibodies is readily amenable to quantitative measurement of cell surface expression of proteins and in particular receptor proteins such as Her-3. Immunofluorscence and other fluorescence microscopy methods can also be used to stain metastatic breast cancer outgrowth in the brain to detect levels of Her-3 as well as other conventional immunohistochemistry techniques. Alternatively, relative protein expression levels may be determined by other protein-based methods which include immunoassays, for example ELISA and immunoblotting (such as western blotting using α-Her-3 antibodies) to detect relative expression levels of Her-3.
The invention also contemplates techniques which detect nucleic acid-protein interactions such as, but not limited to, northwestern blotting and electrophoretic mobility shift assay
The invention further contemplates use of protein-based microarray technology, preferably to determine the expression pattern profile of metastatic breast cancer in order to analyse whether Her-3 expression is up-regulated or overexpressed in patients with metastatic breast cancer, preferably a metastatic breast cancer located in the brain.
Proteomic pattern analysis provides an alternative diagnostic method which is particularly useful for global expression pattern analysis of proteins. Methods of cancer diagnosis using proteomic patterns are provided in Conrads et al Expert Rev MoI Diagn. 2003 Jul;3(4):411-20 and is incorporated herein by reference.
In particular embodiments, a plurality of proteins may be used in a protein library displayed in a number of ways, e.g., in phage display or cell display systems or by two-dimensional gel electrophoresis, or more specifically, differential two- dimensional gel electrophoresis (2D-DIGE). These particular embodiments may generally be referred to as "proteomic" or "protein profiling" methods, such as described in Chapters 3.9.1 and 22 of CURRENT PROTOCOLS IN PROTEIN SCIENCE Eds. Coligan et al., John Wiley & Sons NY USA (1996-2002).
In embodiments relating to protein arrays, preferably Her-3 is located at an identifiable address on the array. Preferably, the protein array comprises a substrate to which is immobilized, impregnated, bound or otherwise coupled ErbB protein, or a fragment thereof. The substrate may be a chemically-derivatized aluminium chip, a synthetic membrane such as PVDF or nitrocellulose, a glass slide or microtiter plates.
Detection of substrate-bound proteins may be performed using mass spectrometry, ELISA, immunohistochemistry, fluorescence microscopy or by colorimetric detection but is not limited thereto.
In alternative or additional embodiments, the invention provides methods to detect elevated levels of Her-3 indirectly by measurement of Her-3 activity and/or function.
A person of skill in the art will appreciate that the methods of the invention, and in particular the diagnostic methods of the invention, could employ nucleic acid detection techniques as are well known in the art to measure levels of a nucleic acid encoding a Her-3 in order to ascertain expression levels of Her-3. hi principle, any nucleic acid sequence detection technique may be applicable, such as nucleic acid sequencing, northern hybridization, nucleic acid sequence amplification, nucleic acid arrays and methods that detect melting temperature differences to identify whether a nucleic acid harbours one or more polymorphisms compared to a "wild type" reference nucleic acid.
The term "nucleic acid" as used herein designates single or double stranded mRNA, RNA, cRNA and DNA, said DNA inclusive of cDNA and genomic DNA. A nucleic acid may be native or recombinant and may comprise one or more artificial nucleotides, e.g. nucleotides not normally found in nature. RNA includes single- stranded and double-stranded unprocessed RNA, mRNA, siRNA, miRNA, RNAi and tRNA. Nucleic acid also encompasses modified purines (for example, inosine, methylinosine and methyladenosine) and modified pyrimidines (thiouridine and methylcytosine).
The term "isolated nucleic acid" as used herein refers to a nucleic acid subjected to in vitro manipulation into a form not normally found in nature. Isolated nucleic acid includes both native and recombinant (non-native) nucleic acids. For example, a nucleic acid may be isolated from human. The terms "mRNA", "RNA" and "transcript" are used interchangeably when referring to a transcribed copy of a transcribable nucleic acid. The present invention also contemplates detection of variant nucleic acids.
"Variants" include within their scope naturally-occurring variants such as allelic variants, orthologs and homologs and mutants, for example.
The terms "mutant", "mutation" and "mutated" are used herein generally to encompass conservative or non-conservative amino acid substitutions, deletions and/or insertions present in a protein, or fragment thereof or introduced into an isolated protein or fragment thereof.
In particular embodiments, nucleic acid detection techniques may include detection of variants such as alternate transcriptional splice variants encoding different isoforms of Her-3. By way of example only, one particular Her-3 isoform represents a longer mRNA transcript and thereby encodes a longer isoform.
Suitable nucleic acid amplification techniques are well known to the skilled addressee, and include polymerase chain reaction (PCR) as for example described in Ausubel et al. {supra) which is incorporated herein by reference; strand displacement amplification (SDA) as for example described in U.S. Patent No 5,422,252 which is incorporated herein by reference; rolling circle replication (RCR) as for example described in Liu et al, (1996, J. Am. Chem. Soc. 118:1587-1594 and International application WO 92/01813) and Lizardi et al. (International Application WO 97/19193) which are incorporated herein by reference; nucleic acid sequence-based amplification (NASBA) as for example described by Sooknanan et al., (1994, Biotechniques 17:1077-1080) which is incorporated herein by reference; and Q-β replicase amplification as for example described by Tyagi et al., (1996, Proc. Natl. Acad. ScL USA 93:5395-5400) which is incorporated herein by reference.
The abovementioned are examples of nucleic acid sequence amplification techniques but are not presented as an exhaustive list of techniques. Persons skilled in the art will be well aware of a variety of other applicable techniques as well as variations and modifications to the techniques described herein.
As used herein, an "amplification product" is a nucleic acid generated by a nucleic acid sequence amplification technique as hereinbefore described. Detection of amplification products may be achieved by detection of a probe hybridized to an amplification product, by direct vizualization of amplification products by way of agarose gel electrophoresis, nucleotide sequencing of amplification products or by detection of fluorescently-labelled amplification products.
As used herein, a "probe" is a single- or double-stranded oligonucleotide or polynucleotide, one and/or the other strand of which is capable of hybridizing to another nucleic acid, to thereby form a "hybrid" nucleic acid.
Probes and/or primers of the invention may be labelled, for example, with biotin or digoxigenin, with fluorochromes or donor fluorophores such as FITC, TRITC, Texas Red, TET, FAM6, HEX, ROX or Oregon Green, acceptor fluorophores such as LC-Red640, enzymes such as horseradish peroxidase (HRP) or alkaline phosphatase (AP) or with radionuclides such as 1251, 32P, 33P or 35S to assist detection of amplification products by techniques as are well known in the art.
As used herein, an "oligonucleotide" is a single- or double-stranded nucleic acid having no more than one hundred (100) nucleotides (bases) or nucleotide pairs (base pairs). A "polynucleotide'1'' has more than one hundred (100) nucleotides or nucleotide pairs.
In the particular context of nucleic acid sequence amplification, an oligonucleotide of the invention may be in the form of a primer.
As used herein, a "primer" is a single-stranded oligonucleotide which is capable of hybridizing to a nucleic acid "template" and being extended in a template- dependent fashion by the action of a suitable DNA polymerase such as Taq polymerase, RNA-dependent DNA polymerase or Sequenase™.
Typically, a primer may have at least twelve, fifteen, twenty, twenty-five, thirty, thirty five, forty or fifty contiguous nucleotide bases. As used herein, "hybridization" ', "hybridize" and "hybridizing" refers to formation of a hybrid nucleic acid through base-pairing between complementary or at least partially complementary nucleotide sequences under defined conditions, as is well known in the art. Normal base-pairing occurs through formation of hydrogen bonds between complementary A and T or U bases, and between G and C bases. It will also be appreciated that base-pairing may occur between various derivatives of purines (G and A) and pyrimidines (C, T and U). Purine derivatives include inosine, methylinosine and methyladenosines. Pyrimidine derivatives include sulfur- containing pyrimidines such as thiouridine and methylated pyrimidines such as methylcytosine. For a detailed discussion of the factors that generally affect nucleic acid hybridization, the skilled addressee is directed to Chapter 2 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, supra.
More specifically, the terms "anneal" and "annealing" are used in the context of primer hybridization to a nucleic acid template for a subsequent primer extension reaction, such as occurs during nucleic acid sequence amplification or nucleotide sequencing, as for example described in Chapter 15 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds Ausubel et al. (John Wiley & Sons NY 1995- 1999).
In another embodiment, detection may be performed by melting curve analysis using probes incorporating fluorescent labels that hybridize to amplification products in a sequence amplification reaction. A particular example is the use of
Fluorescent Resonance Energy Transfer (FRET) probes to hybridize with amplification products in "real time" as amplification products are produced with each cycle of amplification.
In yet another embodiment, the invention contemplates use of melting curve analysis whereby nucleic acid-intercalating dyes such as ethidium bromide (EtBr) or SYBR Green I bind amplification products and fluorescence emission by the intercalated complexes is detected.
In light of the foregoing, it will be appreciated that the invention contemplates in particular embodiments detection of variant nucleic acid with polymorphisms. It will also be well understood by the skilled person that detection of nucleotide sequence polymorphisms may be performed using any of a variety of techniques such as PCR-RFLP analysis, fluorescence-based melt curve analysis, SSCP analysis, denaturing gradient gel electrophoresis (DGGE) or direct sequencing of amplification products.
Measurement of relative levels of Her-3 nucleic acid levels compared to an expressed level of a reference nucleic acid may be conveniently performed using a nucleic acid array.
Nucleic acid array technology has become well known in the art and examples of methods applicable to array technology are provided in Chapter 22 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel etal. (John Wiley & Sons NY USA 1995-2001).
An array can be generated by various methods, e.g., by photolithographic methods (see, e.g., U.S. Patent Nos. 5,143,854; 5,510,270; and 5,527,681), mechanical methods {e.g., directed-flow methods as described in U.S. Patent No.
5,384,261), pin-based methods {e.g., as described in U.S. Pat. No. 5,288,514), and bead-based techniques {e.g., as described in PCT US/93/04145).
Reference is also made to Affymetrix nucleic acid array systems such as described in United States Patent 5,858,659 and United States Patent 6,300,063 which provide specific teaching in relation to nucleic acid array-based detection of disease-related polymorphisms but may provide useful methodology applicable to the present invention.
A particular advantage of nucleic acid arrays is that they are useful with a variety of different techniques such as primer extension, allele-specific hybridization, allele-specific ligation and allele-specific cleavage of a flap probe, as for example described in Kwok, 2000, supra, Pastinen et al, 2000, Genome Res. 10 1031.
Nucleic acid or gene expression levels may also be measured by a variety of other gene expression profiling methods as are known in the art such as, but not limited to, the DASL assay (cDNA-mediated annealing, selection extension and ligation) as described hereinafter.
In situ hybridisation techniques are also particularly useful for the detection of expression levels and includes chromogenic in situ hybridisation and fluorescence in situ hybridisation as are known in the art. Reference is made to Chapter 14 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al. (John Wiley & Sons NY USA 1995-2008) for non-limiting examples of applicable in situ hybridisation technology.
In another particular form of this embodiment, quantitative or semiquantitative PCR using primers corresponding to Her3 -encoding nucleic acids may be used to quantify relative expression levels Her-3 nucleic acid to thereby determine whether an individual is predisposed to or suffering from breast cancer. PCR amplification is not linear and hence end point analysis does not always allow for the accurate determination of nucleic acid expression levels.
Real-time PCR analysis provides a high throughput means of measuring gene expression levels. It uses specific primers, and fluorescence detection to measure the amount of product after each cycle. Hydridization probes utilise either quencher dyes or fluorescence directly to generate a signal. This method may be used to validate and quantify nucleic acid expression differences in cells and/or tissues obtained from metastatic breast cancer sufferers compared to cells and/or tissues obtained from non- sufferers or alternatively, comparison of the expression levels between the primary tumour and the metastases. In preferred embodiments, the metastatic breast cancer is located in the brain of a sufferer.
With regard to the above and any method of the present invention, samples for diagnostic or analysis may be isolated from any cell and/or tissue source, inclusive of neural tissue, although more easily obtained cells and tissues such as blood, skin and the like may also be advantageous.
For the purposes of diagnostic methods or selection methods as described herein, the invention contemplates particular embodiments of such methods which utilise the aforementioned techniques either alone or in combination.
It will be appreciated that the aforementioned techniques for detection of either nucleic acid or protein expression levels of Her-3 are also applicable for detection of expression levels of other ErbB receptors, and in particular Her-2, in methods of the present invention, as may be required.
In further aspects, the invention relates to methods of treatment of a human which includes the step of administering to an individual with an elevated level of Her-3 in one or more metastatic breast cancer cells of the brain, a therapeutic agent effective in inhibiting at least one ErbB receptor.
Accordingly, suitable therapeutic agents of the invention may be peptides, proteins such as antibodies or other organic molecules, preferably small organic molecules, with a desired biological activity and half-life. In particular embodiments, the invention relates to methods of treatment of a human which includes the step of administering to an individual with an elevated level of Her- 3 in one or more metastatic breast cancer cells of the brain, a therapeutic agent effective in directly inhibiting at least one ErbB receptor.
In other particular embodiments, the invention relates to methods of treatment of a human which includes the step of administering to an individual with an elevated level of Her-3 in one or more metastatic breast cancer cells of the brain, a therapeutic agent effective in indirectly inhibiting at least one ErbB receptor. Non-limiting examples of suitable indirect inhibitors include molecules which disrupt an interaction between at least one ErbB receptor and its respective ligand such as an antibody directed to an ErbB ligand. In the context of the present invention, by "inhibit", "inhibition",
"inhibited", "inhibitory" or "inhibitor" is meant a therapeutic agent and/or a candidate agent which interferes with, inhibits, blocks or hinders at least one ErbB receptor, either by a direct or an indirect mechanism. In particular embodiments, the inhibitory effect may be of a function or functional activity of at least one ErbB receptor, such as inhibition of tyrosine kinase activity of said ErbB receptor. In other embodiments, an inhibitor may block dimerisation between two different types of
ErbB receptors, which in turn may block signaling by other ErbB family receptors.
In particular embodiments, the therapeutic agent is an antagonist, hi yet other embodiments, the therapeutic agent is an agonist. In other particular embodiments, the therapeutic agent inhibits or disrupts the heterodimeric interaction between different ErbB receptors such as, but not limited to, Her-2 and Her-3.
According to certain embodiments, the therapeutic agent is a direct inhibitor of at least one ErbB receptor which binds or interacts directly with at least one ErbB receptor. A non-limiting example of an direct inhibitor is an antibody which binds to or has been raised against the at least one ErbB receptor or a fragment thereof, although without limitation thereto. In other non-limiting embodiments, an direct inhibitor is a small-molecule which binds to at least one ErbB receptor or a fragment thereof.
According to other embodiments, the therapeutic agent is an indirect inhibitor of at least one ErbB receptor which does not bind to or interact with directly with the at least one ErbB receptor but inhibit the activity or function of the at least one ErbB receptor. In particular, indirect inhibitors target or are directed to a modulator of at least one ErbB receptor. In particular embodiments, an indirect inhibitor of at least one ErbB receptor targets or is directed to one or more ligands of at least one ErbB receptor. Non-limiting examples of suitable ligands of ErbB receptors which may be a target for an indirect inhibitor include epidermal growth factor (EGF), transforming growth factor (TGF)-alpha, amphiregulin, betacellulin, heparin-binding EGF, epiregulin and members of the neuregulin family such as neuregulin 1, 2, 3 and 4, although without limitation thereto.
In other preferred embodiments, an indirect inhibitor of at least one ErbB receptor targets or is directed to a modulator, and in particular an activator, of ligands of ErbB receptors. According to these embodiments, a modulator of the ErbB receptor ligand may be an enzyme which cleaves an ErbB receptor ligand from the cell surface, which in turn activates the ErbB receptor ligand. A non-limiting example of a suitable enzyme are any one of the members of the ADAM (A Disintegrin And Metalloprotease) family such as ADAMlO, ADAM12, ADAMl 7 and ADAM 19, although without limitation thereto.
In particular preferred embodiments, the therapeutic agent selectively inhibits at least one ErbB receptor.
By "selective " or "selectively " is meant a therapeutic agent that primarily affects the function of an ErbB receptor but may also have an effect upon other ErbB receptors. Therefore in one embodiment, "selective " or "selectively" encompasses a situation where at least 50%, 55%, 60%, 65%, preferably at least 70%, 75%, 80%, 85% and more preferably 90%, 95%, 96%, 98%, 99% and 100% of the inhibitory activity of a therapeutic agent can be attributable to a single ErbB receptor. hi another embodiment, "selective " or "selectively" includes a therapeutic agent that can bind to and inhibit an ErbB receptor. A non-limiting example of such a therapeutic agent or candidate agent is a suitable binding and/interaction partner, such as a protein. In particular embodiments, the protein is an antibody.
In yet another embodiment, "selective" or "selectively" includes a therapeutic agent that specifically regulates an ErbB receptor such as, but not limited to, nucleic acid molecules. In yet a further embodiment, "selective " or "selectively" includes a therapeutic agent that specifically regulates post-translational forms of an ErbB receptor.
In preferred embodiments, the methods of the present invention contemplate a therapeutic agent that is an antibody, or an antibody fragment, which binds and/or has been raised against at least one ErbB receptor or alternatively, an antibody (or an antibody fragment) which binds and/or has been raised against a ligand of at least one ErbB receptor. It is envisaged that both polyclonal and monoclonal antibodies directed to either the entire protein or a biologically-active fragment thereof are suitable therapeutic agents.
In preferred embodiments, the therapeutic agent is an α-Her-3 antibody. Reference is made to United States Patent Nos 5,480,968 and 5,968,511 and International Publication No.2008/100624 which provides non-limiting examples of suitable α-Her-3 antibodies. In other preferred embodiments, the therapeutic agent is an α-Her-2 antibody.
For non-limiting examples of α-Her2 antibodies, see, for example, United States Patent No 6,165,464; 5,677,171; and 6,054,561. In particularly preferred embodiments the α-Her-2 antibody is trastuzumab which is sold under the name Herceptin® by Genentech. In yet other preferred embodiments, the therapeutic agent is an α-Her-1 antibody. Suitable non-limiting examples of α-Her-1 antibodies are provided in US Patent Nos. 6,235,883 and 5,844,093 and International Publication No. WO/2007/058823.
In further preferred embodiments, the therapeutic agent is an α-Her-4 antibody. Reference is made to United States Patent Nos. 5,811 ,098 and 7,332,579 which provide non-limiting examples of suitable α-Her-4 antibodies.
As mentioned above, antibodies may be monoclonal or polyclonal, obtained for example by immunizing a suitable production animal {e.g. a mouse, rat, rabbit, sheep, chicken or goat). Serum or spleen cells may be then isolated from the immunized animal according to whether polyclonal or monoclonal antibodies are required. Monoclonal antibodies may be produced by standard methods such as described in CURRENT PROTOCOLS IN IMMUNOLOGY (Eds. Coligan et al. John Wiley & Sons. 1995-2000) and Harlow, E. & Lane, D. Antibodies: A Laboratory Manual (Cold Spring Harbour, Cold Spring Harbour Laboratory, 1988). Such methods generally involve obtaining antibody-producing cells, such as spleen cells, from an animal immunized as described above, and fusing spleen cells with an immortalized fusion partner cell.
Recombinant antibodies are also contemplated. Selection of appropriate recombinant antibodies can be achieved by any of a number of methods including phage display, microarray or ribosome display, such as discussed in Hoogenboom, 2005, Nature Biotechnol. 23 1105, by way of example only.
Also contemplated are antibody fragments such as Fab, F(ab)2, Fv, scFV and Fc fragments as well understood in the art.
As is also well understood in the art, in order to assist detection of antibody- antigen complexes, antibodies may be conjugated with labels including but not limited to a chromogen, a catalyst, an enzyme, a fluorophore, a chemiluminescent molecule, biotin and/or a radioisotope.
As used herein, the terms "antibody" or "immunoglobulin", as used interchangeably herein, includes whole antibodies and any antigen binding fragment or single chains thereof. The terms "antibody" or "immunoglobulin" includes any antigen-binding protein product of the immunoglobulin gene complex, including immunoglobulin isotypes IgA, IgD, IgM, IgG and IgE and antigen-binding fragments thereof. Examples of antigen-binding fragments include, but are not limited to, Fab, F(ab)2, Fv, scFv, etc. It will be appreciated by a person of skill in the art that antibodies employed for therapeutic applications in humans must have specific properties which make these antibodies suitable for use in humans. Typically, therapeutic antibodies are "humanised" , wherein the antibody or at least one chain thereof, typically comprises a variable framework region substantially from a human antibody and the complementary determining regions substantially from an antibody derived from a non-human (such as, but not limited to, rodent antibodies and shark antibodies). Humanised antibodies are particularly advantageous for medical applications due to the decrease likelihood of eliciting a foreign body immune reaction.
Di other embodiments, pharmaceutical compositions and treatment methods may utilize nucleic acid constructs (including but not limited to inhibitory RNA constructs) for treatment of a metastatic breast cancer derived from one or more breast cancer cells with normal expression levels of Her-2. In preferred embodiments, the metastatic breast cancer is located in the brain.
Generally, a nucleic acid construct may be any recombinant nucleic acid that facilitates delivery, expression, propagation or manipulation of a desired nucleic acid component of the construct.
By way of example only, a construct may be a plasmid, a cosmid, a modified virus or containing virus-derived elements, an artificial chromosome, a phagemid, an anti-sense oligonucleotide, RNA (e.g. siRNA or shRNA) or the like.
Particular virus-derived expression constructs suitable for human delivery include constructs comprising adenovirus-, adeno-associated virus-, lentivirus-, flavivirus- and/or vaccinia virus-derived elements. hi light of the foregoing it will be appreciated that RNA-based methods may be employed for inhibition of at least one ErbB receptor. RNA interference, and in particular (but not limited thereto) siRNA and shRNA, provides an attractive method for silencing of potential therapeutic gene targets by sequence-specific cleavage of cognate mRNA. Takeshita and Ochiya (Cancer Sci, 2006, 97: 689-696) provides numerous examples of the therapeutic potential of RNA interference against cancer and is incorporated herein by reference.
The term "gene" is used herein to describe a discrete nucleic acid locus, unit or region within a genome that may comprise one or more of introns, exons, splice sites, open reading frames and 5' and/or 3' non-coding regulatory sequences such as a promoter and/or a polyadenylation sequence.
Therefore a person of skill in the art will readily appreciate that the invention contemplates a genetic construct which comprises one or more nucleotide sequences capable of directing synthesis of an RNA molecule, said nucleotide sequence selected from the list comprising:- (i) a nucleotide sequence transcribable to an RNA molecule comprising an RNA sequence which is substantially homologous to an RNA sequence encoded by a nucleotide sequence of interest;
(ii) a reverse complement of the nucleotide sequence of (i); (iii) a combination of the nucleotide sequences of (i) and (ii),
(iv) multiple copies of nucleotide sequences of (i), (ii) or (iii), optionally separated by a spacer sequence;
(v) a combination of the nucleotide sequences of (i) and (ii), wherein the nucleotide sequence of (ii) represents an inverted repeat of the nucleotide sequence of (i), separated by a spacer sequence; and
(vi) a combination as described in (v), wherein the spacer sequence comprises an intron sequence spliceable from said combination.
Where the nucleotide sequence comprises an inverted repeat separated by a non-intron spacer sequence, upon transcription, the presence of the non-intron spacer sequence facilitates the formation of a stem-loop structure by virtue of the binding of the inverted repeat sequences to each other. The presence of the non-intron spacer sequence causes the transcribed RNA sequence (also referred to herein as a
"transcript") so formed to remain substantially in one piece, in a form that may be referred to herein as a "hairpin". Alternatively, where the nucleotide sequence comprises an inverted repeat wherein the spacer sequence comprises an intron sequence, upon transcription, the presence of intron/exon splice junction sequences on either side of the intron sequence facilitates the removal of what would otherwise form into a loop structure. The resulting transcript comprises a double-stranded RNA
(dsRNA) molecule, optionally with overhanging 3' sequences at one or both ends. Such a dsRNA transcript is referred to herein as a "perfect hairpin". The RNA molecules may comprise a single hairpin or multiple hairpins including "bulges" of single-stranded DNA occurring in regions of double-stranded DNA sequences.
Depending upon the application, the RNA molecule may directed to a single target or alternatively, a plurality of targets. In particular embodiments, the RNA molecule encodes at least one ErbB receptor selected from the group consisting of Her- 1, Her-2, Her-3 and Her-4. In other particular embodiments, the RNA molecule encodes a ligand to at least one ErbB receptor.
In particular embodiments, inhibitory RNA constructs include siRNA or shRNA construct that down-regulate expression of at least one ErbB receptor and more preferably at least one ErbB receptor selected from group consisting of Her-1 , Her-2, Her-3 and Her-4.
Non-limiting examples of suitable siRNA molecules, and in particular ErbB receptor-specific siRNA, according to the present invention are provided in Sergina et al Nature. 2007445(7126):437-41 and Sithanandam et al Cancer Gene Ther.2008 15(7):413-48.
The present invention also contemplates peptide mimetics as therapeutic agents, and more particularly antibody-like peptidomimetics such as described in Zhang et al (2007) J. Clin. Invest. 117:2051-2058 in relation to ErbB receptors, although without limitation thereto. Particular embodiments contemplate use of small-molecule inhibitors which directly or indirectly inhibit one or a plurality of ErbB receptors. Such inhibitors may be reversible or irreversible. It is also envisage that such inhibitors are selective against a particular ErbB receptor protein or in other embodiments, inhibit more than one ErbB receptor protein. A non-limiting example of a multi-target inhibitor is BIBW-2992, which inhibits Her-1 and Her-2. Spector et al (2007) Breast Cancer Research 9:205 provides other examples of multi-target inhibitors. It is also appreciated that according to these embodiments that small-molecule inhibitors may inhibit a particular function of the at least one ErbB receptor and in preferred embodiments, is a tyrosine kinase inhibitor. Non-limiting examples of a suitable small-molecule inhibitors includes derivatives of quinazolines and more particularly, 4-anilinoquinazoline-based inhibitors such as those inhibitors described in United States Patent No's 6391874 and 6713485 and International Publication No. WO/1999/035146. Reference is also made to N-[3-chloro-4-[(3-fluorophenyl)methoxy]phenyl]-6-[5-[(2- methylsulfonylethylamino)methyl]furan-2-yl]quinazolin-4-amine (otherwise known as "lapatinib" and sold under the name Tykerb® by GlaxoSmithKline; PubChem Substance Compound ID: 208908) which is dual inhibitor of Her- 1 and Her-2 and provides a further non-limiting example of a suitable small molecule inhibitor of ErbB inhibitors. Non-limiting examples of selective inhibitors include N-(3-chloro-4- fiuorophenyl)-7-methoxy-6-(3-morpholin-4-ylpropoxy)quinazolin-4-amine (otherwise known as "gefitinib" which is sold under the name Iressa™ by Astra Zeneca; PubChem Substance Compound ID: 123631) and N-(3 -ethynylphenyl)-6,7- bis(2-methoxyethoxy)quinazolin-4-amine (otherwise known as "erlotinib" which is sold under the name Tarceva® by Genentech; PubChem Substance Compound ID: 176870). Non-limiting examples of suitable irreversible inhibitors include CI- 1033,
EKB-569 (PubChem Substance Compound ID: 6445562) and HKI-272 as provided in Zhang et al (2007) J. Clin. Invest. 117:2051-2058.
In preferred embodiments, a therapeutic agent of the invention is a small- molecule inhibitor of Her-2. Reference is made to Spector et al (2007) Breast Cancer Research 9:205 which provides non-limiting examples of suitable small molecule Her-2 inhibitors and is incorporated herein by reference.
Reference is made to Table 5 which provides non-limiting examples of suitable ErbB inhibitors which may be applied or used according to preferred embodiments of the present invention. In further aspects, the invention relates to methods of designing, engineering, screening or otherwise producing a therapeutic agent for treating a metastatic breast cancer derived from one or more metastatic breast cancer cells with normal expression levels of Her-2, which includes the step of identifying a candidate agent that is suitable for use in treatment of a metastatic breast cancer derived from one or more breast cancer cells with normal expression levels of Her-2, by the presence of an elevated expression level of Her-3 in one or more metastatic breast cancer cells of the brain.
Accordingly, suitable therapeutic agents and/or candidate agents of the invention may be peptides, proteins such as antibodies or other organic molecules, preferably small organic molecules, with a desired biological activity and half-life. In preferred embodiments, a therapeutic agent for treating a metastatic breast cancer derived from one or more metastatic breast cancer cells with normal expression levels of Her-2 designed, screened, engineered or other produced according to the present invention inhibits at least one ErbB receptor. More preferably, said therapeutic agent selectively inhibits at least one ErbB receptor. Persons skilled in the art will be aware that the therapeutic agents of the invention may be identified by any number of methods.
In one embodiment, suitable therapeutic agents may be identified by way of screening libraries of molecules such as synthetic chemical libraries, including combinatorial libraries, by methods such as described in Nestler & Liu, 1998, Comb. Chem. High Throughput Screen. 1 113 and Kirkpatrick et al, 1999, Comb. Chem. High Throughput Screen 2 211.
It is also contemplated that libraries of naturally-occurring molecules may be screened by methodology such as reviewed in KoIb, 1998, Prog. Drug. Res. 51 185.
Screening methods of the present invention also contemplate use of in vitro assays which mimic and/or replicate the natural biological environment of a metastatic breast cancer, in order to test the potential therapeutic efficacy of one or more candidate agents. Particularly preferred embodiments contemplate an in vitro assay which mimics a metastatic breast cancer located in the brain, wherein differentiated neural cells are co-cultured with a breast cancer cell line, with the result being a cell-based matrix derived from the co-culture neural cells and breast cancer cells. Differentiated neural cells may be derived from differentiation of neural progenitor cells (and preferably neural stem cells) such as, but not limited to ReNcell CX cell line (Chemicon International). Examples of suitable breast cancer cells for use in the aforementioned in vitro assays include SKBR-3 and basal-like breast cancer cell lines such as, but not limited to, MDA-MB-231.
More rational approaches to designing therapeutic agents of the present invention may employ X-ray crystallography, NMR spectroscopy, computer assisted screening of structural databases, computer-assisted modelling, or more traditional biophysical techniques which detect molecular binding interactions, as are well known in the art.
A review of structural bioinformatics approaches to drug discovery is provided in Fauman et al, 2003, Meth. Biochem. Anal. 44: 477.
Computer-assisted structural database searching and bioinformatic approaches are becoming increasingly utilized as a procedure for identifying and/or engineering agonists and antagonist molecules. Examples of database searching methods may be found in United States Patent No. 5,752,019 and International Publication WO 97/41526 (directed to identifying EPO mimetics) and United States Patents 7,158,891 and 5,680,331 which are directed to more general computational approaches to protein modelling and structural mimicry of protein activity.
Generally, other applicable methods include any of a variety of biophysical techniques which identify molecular interactions. Methods applicable to potentially useful techniques such as competitive radioligand binding assays, electrophysiology, analytical ultracentrifugation, microcalorimetry, surface plasmon resonance and optical biosensor-based methods are provided in Chapter 20 of CURRENT PROTOCOLS IN PROTEIN SCIENCE Eds. Coligan et al, (John Wiley & Sons, 1997) which is incorporated herein by reference.
A person skilled in the art will appreciate that suitable therapeutic agents may be in the form of a binding partner and as such, identified by interaction assays such as yeast two-hybrid approaches and the like, but without limitation thereto. Two- hybrid screening methods are provided in Chapter 20 of CURRENT PROTOCOLS IN PROTEIN SCIENCE Eds. Coligan et al, (John Wiley & Sons, 1997) which is incorporated herein by reference.
For the purpose of methods of treatment described herein, the present invention also contemplates particular embodiments where the therapeutic agents of the invention may be used in combination with other therapeutic agents such as chemotherapeutic agents including cytotoxic drugs (for example capecitabine, doxorubicin (including its liposomal formulation), fluorouracil, cisplatin, gemcitabine, the taxanes paclitaxel and docetaxel, and vinorelbine) and hormone therapies (such as endocrine therapies) for individuals that are estrogen or progesterone positive and in particular modulators of a hormone receptor such as estrogen and/or progesterone (eg: tamoxifen, luteinising hormone releasing hormone analogues and aromatase inhibitors). In light of the foregoing, it will be appreciated that the invention provides a pharmaceutical composition comprising a therapeutic agent effective for treatment of a metastatic breast cancer, preferably located in the brain, derived from one or more breast cancer cells with normal expression levels of Her-2 designed, screened, engineered or otherwise produced according as hereinbefore described, together with a pharmaceutically acceptable carrier, diluent, or excipient.
In other aspects, the invention provides a method of treating a metastatic breast cancer derived from one or more breast cancer cells with normal expression levels of Her-2 in a human including the step of administering to an individual with an elevated level of Her-3 in one or more metastatic breast cancer cells of the brain, a therapeutic agent effective for treatment of said metastatic breast cancer designed, screened, engineered or otherwise produced as hereinbefore described. According to these aspects, the metastatic breast cancer is located in the brain.
In light of the foregoing, it will be appreciated that preferably the therapeutic agents of the present invention may be in the form of a small organic molecule, peptide or the like formulated with a pharmaceutically-acceptable carrier, diluent or excipient suitable for oral administration, as a transdermal patch or other noninvasive route of admim'stration.
By "pharmaceutically-acceptable carrier, diluent or excipient" is meant a solid or liquid filler, diluent or encapsulating substance that may be safely used in systemic administration. Depending upon the particular route of administration, a variety of carriers, well known in the art may be used. These carriers may be selected from a group including sugars, starches, cellulose and its derivatives, malt, gelatine, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline and salts such as mineral acid salts including hydrochlorides, bromides and sulfates, organic acids such as acetates, propionates and malonates and pyrogen-free water.
A useful reference describing pharmaceutically acceptable carriers, diluents and excipients is Remington's Pharmaceutical Sciences (Mack Publishing Co. N.J. USA, 1991) which is incorporated herein by reference.
Any safe route of administration may be employed for providing a patient with the composition of the invention. For example, oral, rectal, parenteral, sublingual, buccal, intravenous, intra-articular, intra-muscular, intra-dermal, subcutaneous, intracranial, inhalational, intraocular, intraperitoneal, intracerebroventricular and transdermal administration may be employed. Dosage forms include tablets, dispersions, suspensions, injections, solutions, syrups, troches, capsules, suppositories, aerosols, transdermal patches and the like. These dosage forms may also include injecting or implanting controlled releasing devices designed specifically for this purpose or other forms of implants modified to act additionally in this fashion. Controlled release of the therapeutic agent may be effected by coating the same, for example, with hydrophobic polymers including acrylic resins, waxes, higher aliphatic alcohols, polylactic and polyglycolic acids and certain cellulose derivatives such as hydroxypropylmethyl cellulose. In addition, the controlled release may be effected by using other polymer matrices, liposomes and/or microspheres. Pharmaceutical compositions of the present invention suitable for oral or parenteral administration may be presented as discrete units such as capsules, sachets or tablets each containing a pre-determined amount of one or more therapeutic agents of the invention, as a powder or granules or as a solution or a suspension in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion or a water-in-oil liquid emulsion. Such compositions may be prepared by any of the methods of pharmacy but all methods include the step of bringing into association one or more agents as described above with the carrier which constitutes one or more necessary ingredients. In general, the compositions are prepared by uniformly and intimately admixing the agents of the invention with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation.
The above compositions may be administered in a manner compatible with the dosage formulation, and in such amount as is pharmaceutically-effective. The dose administered to a patient, in the context of the present invention, should be sufficient to effect a beneficial response in a patient over an appropriate period of time. The quantity of agent(s) to be administered may depend on the subject to be treated inclusive of the age, sex, weight and general health condition thereof, factors that will depend on the judgement of the practitioner.
So that the invention may be readily understood and put into practical effect, the following non-limiting Examples are provided EXAMPLES
EXAMPLE 1
Metastatic breast cancer to the brain: implications of the local microenvironment for colonization and treatment
In the present study, the present inventors used a set of 29 matched pairs of human primary breast cancer and brain metastases, 22 unmatched brain metastases of breast cancer and a second set for validation comprised of 10 matched pairs of primary breast cancer and brain metastases and 11 non-breast metastatic brain cancers to explore the metastatic breast cancer colonization of the brain. A combination of morphology, immunohistochemistry, differential gene expression, co- culture of human neural cells and breast cancer cell lines and in vivo brain injections of tumour cells in mice (treated with trastuzumab and neutralizing anti-neuregulin antibody) was used in order to elucidate possible mechanisms that could enhance the colonization of the brain parenchyma by tumour cells.
Herein, the present inventors provide in vitro and in vivo evidence that suggests that the inhibition of HER family receptors or the neutralization of local secreted growth factors like neuregulin could play a role in preventing tumour progression and colonization in the brain microenvironment and also may shed light on the paradigm that basal-like tumours, for not overexpressing HER2, cannot have response to trastuzumab. Material and Methods: Clinical samples:
Formalin fixed-paraffin embedded (FFPE) tumour blocks were retrieved from the archives of the Pathology Queensland: The Royal Brisbane & Women's Hospital, Brisbane, Australia - Medical Faculty of Charles University in Plzen, Czech Republic - Instituto Nacional do Cancer, Brazil - Laboratόrio Salomao e Zoppi, Brazil and Institute of Clinical Pathology and Medical Research, Westmead Hospital, University of Sydney, Australia. An initial set of 29 matched pairs of primary breast cancer and their brain metastases and 22 unmatched brain metastases of breast cancer was available. A second set of samples comprised of 10 matched pairs of primary breast cancer and their brain metastases and 11 non-breast metastatic brain cancers was also available. Clinical data regarding the timeframe from the day of the primary diagnosis to the day of the removal of the brain metastases and patient age was obtained for the initial set of samples. The study was approved by the local research ethics committees. Haematoxylin & eosin sections were reviewed by two pathologists (LDS, SRL) to confirm diagnosis, assess morphology and grade the tumours according to the World Health Organization criteria (48). For the initial set of samples two tissue arrays containing replicates of primary female breast cancer and their metastases to the brain were built. Both tissue microarrays were comprised of 51 cores of metastases from female breast cancer to the brain and 29 cores of primary female breast cancer. Haematoxylin and eosin slides were dotted by one pathologist in order to take cores to the recipient block. Both arrays were built using the tissue arrayer, model MTAI (Beecher Instruments, Inc, Sun Prairie, WI 53590 USA). The tissue cores had 0,6 mm in diameter and were 1 ,0mm away from each other. For the second set of samples whole sections were used for haematoxylin & eosin staining. Another TMA was built for the second set of samples following the same descriptions as for the initial set.
Immunohistochemistry and Chromogenic in Situ hybridization:
For the immunohistochemistry assay, sections of the paraffin blocks of the tissue arrays and whole sections of the second set of samples were cut to generate 4μm sections that were mounted on silane-coated slides. Immunohistochemistry was performed using the Envision® dual link system (Dakocytomation, Denmark) according to the manufacturer's recommendations. Table 1 shows all antibodies used, along with the antibody clone and dilution. Antigenic retrieval for all antibodies (except smooth muscle actin (SMA) which did not require any antigen retrieval) required two minutes pressure cooking (1050C) in EDTA (pH8.0) buffer. Positive and negative controls were included in all runs and all slides were analysed by at least two pathologists (LDS, PP or SRL) under a double headed optical light microscope. Cellular localization, intensity and percentage/number of positive and negative tumours for each antibody were recorded on primary tumours and brain metastases. Intensity was based on a three tier-system - weak, moderate, strong. Samples were considered positive if more than 10% of cells were stained. HER2 staining and scoring was according to the Herceptest® (Dakocytomation, Denmark) protocol. Chromogenic in situ hybridization was performed following instructions of the Zymed Spot-Light®HER2 CISH™ Kit (Zymed, California, USA) on the first set of samples. Briefly, the paraffin embedded sections were dewaxed and subjected to heat and enzyme digestion treatments followed by denaturation and hybridization to labelled nucleic acid probes. Immunodetection was performed and 3, 3'- Diaminobenzidine tetrachloride was used for visualization. Slides were counterstained with haematoxylin. One slide with a HER2 amplified - formalin fixed cell line was included as a positive control. Signals were counted under a brightfield microscope and results were classified into diploid, polysomy, low amplification and high amplification according to the manufacture's recommendation. Gene expression profiling:
Gene expression profiling was performed using the DASL assay (cDNA- mediated annealing, selection extension and ligation, Illumina Inc., California, USA) interrogating the DASL Cancer Panel which contained 512 cancer related genes (49, 50). The above-mentioned first and second sets of samples were analyzed in two separated experiments. The RNA derived from the animal experiment was analyzed in conjunction with the second set of samples. All protocols were as specified by Illumina Inc. Briefly, RNA (250ng) was converted to cDNA through a reverse transcription reaction with biotinylated oligo-d(T)i8 and random nonamers. Oligonucleotides (three unique pairs for each of the 512 genes) were annealed to the biotinylated cDNA, the duplexes were bound to streptavidin conjugated paramagnetic particles to remove non-hybridized oligos. The annealed oligos were then extended and ligated (to incorporate an address sequence and primer site) to generate amplifiable products. These products were subjected to PCR amplification using fluorescently labeled (Cy3 and Cy5) primers. The labeled PCR products were hybridized to a Sentrix array or Beadchip. Following hybridization, the arrays were scanned with a BeadArray Reader (Illumina) and data was extracted using BeadStudio software (Illumina). Samples exhibiting a median hybridization intensity across all probes of <600 (background corrected) were excluded from analysis. Probes with a BeadStudio detection score of <0.99 were also excluded. Array data transformation and normalization (per chip normalized to 50th percentile and per gene normalized to median) was done in Genespring® version 7.0 software (Agilent Technologies, Santa Clara, USA). Hierarchical clustering was performed using: Similarity measure; Pearson Correlation and clustering algorithm; Average Linkage. Genes with no data in half the starting conditions were discarded. Genes differentially expressed between primary tumours and brain metastases were identified using analysis of variance (ANOVA, p<0.01) or parametric test, variances not assumed equal (Welch t-test, p < 0.005). Up and down regulation was considered for normalized data values that were greater or less than a factor of 2 fold for the conditions compared. RNA extraction and Real-Time RT-PCR
Three 5μm thick FFPE sections of clinical samples were cut and de-waxed in two lots of xylene and two lots of 100% ethanol, 10 minutes each. RNA was extracted using the High Pure RNA Paraffin Kit® (Roche Applied Science, Mannheim, Germany) according to the manufacturer's protocol. RNA was quantified using the NanoDrop® ND- 1000 (NanoDrop Technologies, DE, USA). Relative expression levels of HER3, HER2, EGFR, HER4, HIF 1-alfa and CCNH was assessed using RT-PCR. Complementary DNA (cDNA) was synthesized from 240ng of total RNA using random hexamers and the Superscript™ III Reverse Transcriptase kit (Invitrogen, Carsbald, CA, USA). TaqMan One-Step UniversalMaster Mix (Applied Biosystems) was used for all reactions. TaqMan reaction was done in a standard 96- well plate format with ABI 7500 OneStepPlus PCR system. For data analysis, raw deltaCt (dCt) was first normalized to an endogenous control gene (RPLl 3 a) for each sample to generate normalized dCt. The normalized dCt was then calibrated to a cell line pool reference (MCF-7, SKBR-3 and MDA-MB-231) to generate a ddCt. In the final step of data analysis, the ddCt was converted to fold change (2"ddCt) relative to the reference allowing comparison between samples. Cell lines, co-culture and flow cytometry assays:
MCF-7, MDA-MB-231 and SKBR-3 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) plus 10% heat-inactivated fetal bovine serum (FBS), 2 mmol/L glutamine, and 1 % penicillin G-streptomycin solution. ReNcell CX (CHEMICON International, Inc. Catalogue SCC007) is an immortalized human neural progenitor cell line with the ability to differentiate into neurons and glial cells. Resuspended ReNcells were allowed to grow in a total volume of 5 mL of serum-free DMEM/F12 (NSA) medium containing freshly added 20 ng/ml rhEGF (R&D Systems), 10 ng/ml rhFGF (R&D Systems), 4 ug/ml heparin, 10% proliferation supplement (NeuroCult®, Stem Cell Technologies Inc.), 2% BSA (Sigma). After formation of neurospheres, the cells were differentiated into neurons and glial cells in order to create a neural cell layer following the replacement of the NSA media by DMEM/F12 with 10% FBS, 2 mmol/L glutamine, and 1% penicillin G-streptomycin solution. Co-culture was established after plating in 24 wells dishes 15,000 cells in triplicates of the MCF-7 and SKBR-3 cancer cell lines with 10,000 differentiated human neural cells. Triplicates of the cell lines without neural cells were cultured for comparison of the growth and the co-culture was also performed in the presence of trastuzumab 20ng/μl, lapatinib 5nmol/mL, anti-neuregulin antibody 50μg/mL and a combination of trastuzumab plus lapatinib. A growth curve was generated after counting the cells in a flow cytometer. The cells were washed once with phosphate- buffered saline (PBS) and then harvested with 0.05% trypsin/0.025% EDTA. Detached cells were washed with PBS. Combinations of fluorochrome-conjugated monoclonal antibodies against human CD56 and Epcam (BD Biosciences,San Diego, CA, USA) were used to distinguish between neural and epithelial cells, were added to the cell suspension at concentrations recommended by the manufacturer and incubated at 40C in the dark for 30 minutes. The labeled cells were washed with FACS buffer (PBS supplemented with 1%BSA) and then analyzed on a LSRII (BD Biosciences). Comparisons between cell lines growth curves were done using one way ANOVA. This experiment was repeated twice. Soft agar colonization assay
Two hundred thousand MDA-MB-231 cells were cultured in a six well plate containing 1 ml of RPMI-10% FBS, with the addition of agarose (final 0.5% ,w/v), over a 1 ml layer of bottom agar (final 0.35%, w/v). Cells were cultured with the addition of trastuzumab (20ng/μl), recombinat beta-1-neuregulin (50ng/ml) and combination of recombinant neuregulin and trastuzumab. Cells without the addition of traztuzumab and neuregulin were used for control. Trastuzumab and neuregulin were replenished every four days. Three replicates per combination were used. After 20 days in culture, colonies were counted and measured using a CKX41 Olympus inverted microscope. Images were captured using QCapture Pro 6.0 (Qimaging, Canada). Results are representative of three independent wells. A total of 30 colonies (only size bigger than 1 Oμm were counted) were measured per well and the average number of colonies was derived from twenty 0,196 mm2 random optical fields per well. One way ANOVA was used to determine statistical significance between experiments. Intracerebral injections of breast cancer cell line in mice and drug treatment: Severe combined immunodeficiency (SCID)-beige female mice (6 weeks old) were obtained from a colony housed by the University of Queensland Animal House and were maintained according to the University of Queensland Animal House guidelines. The research complied with national legislation and was authorized by the local animal ethical committees. The animals were divided into four groups; neuregulin-group which had anti-neuregulin antibody mixed with the cells at the time of injection, trastuzumab-group which was treated with intraperitoneal trastuzumab on the day of the injection of the breast cancer cells in the brain, one control group not treated and a fourth group designed to be treated once tumour was detected. Each group was comprised of three animals. The mice were inoculated directly in the right brain hemisphere sub ventricular zone with 200,000 MB-MD A-231 cells. The concentrations of the anti-neuregulin antibody and trastuzumab were 50μg/mL and 30mg/Kg respectively. Mice were weighted before injections. Statistical significance of the differences across all four groups was analyzed by t-Student test. Cells were allowed to grow in the brain for approximately 10 weeks. At this point, the mice in the neuregulin, trastuzumab and controls group were sacrificed and the treatment of the fourth group started. Magnetic resonance imaging of the mice and measurement of the tumours:
The tumour progression was followed up using MRI at weeks 4, 6 and 10. MRI scans were performed using Bruker 16.4T vertical wide-bore scanner, equipped with a micro-mouse AHS probe and a head coil (20 mm SAW linear coil, M2M imaging). The animal was kept under anesthesia with isoflurane (1% at flow rate of 0.5-0.7L/min), its respiration rate was monitored using the BIOTRJG, and the animal body temperature is maintained at 30 degree Celsius. The animal heads are scanned at 78 x 78 micron in-plane resolution and 1 mm slice thickness. The differentiation of the tumour and normal tissues were achieved using 2D Tl-multislice spin echo sequence with TR/TE=600/9 ms, NEX = 8; and 2D diffusion- weighted imaging with TR/TE = 2400/21.6 ms, diffusion encoding was applied in the read direction, with the parameters d/D = 2/14ms, and b = 1200 s/mm2, with 50 kHz bandwidth. Images were processed and the tumour sizes were measured by manual ROI segmentation using the software Paravision 4.0. Final sizes were reconfirmed in the post-mortem examination when haematoxilin & eosin stained sections were cut at 4 μm and scanned using a scanscope® (Aperio Technologies, Vista, CA, USA). Sizes were achieved after analysing the images with Image scope (Aperio Technologies, Vista, CA, USA). Results Clinical features and morphological-immunohistochemical profiling of primary and metastatic breast cancer to the brain:
Clinical data regarding age and time of relapse of the disease in the brain was available for 29 matched pairs of primaries and brain metastases. The median age was 48.5 years old at the initial diagnosis and the median time for the development of the brain metastasis was 3.5 years. All but one of the initial set of 29 matched pairs of primary breast cancer and their brain metastases and 22 unmatched brain metastases of breast cancer were grade III invasive ductal carcinomas not-otherwise- specified (48). The one which was not grade III was a mucinous carcinoma, hi the second set of samples, 10 matched pairs of primary breast cancer and their brain metastases were also grade III invasive ductal carcinomas not-otherwise-specified and the 11 non-breast metastatic brain cancers were comprised of metastases from one melanoma, six lung adenocarcinomas, one prostate carcinoma and two kidney carcinomas. Table 2 shows the summary of the results for all of the antibodies applied. Noteworthy, 68% of the metastases and 61% of the primary tumours of the initial set of samples displayed positivity for at least one of the basal-like markers (CK14, CK5/6, p63, SlOO and SMA) confirming an enrichment of metastatic breast cancer to brain by tumours with the basal-like phenotype. 37% and 30% of the matched pairs brain metastases showed positivity for ER and PR respectively. The unmatched brain metastasis had 15% of positivity for both receptors. Regarding HER2 status, 24% of the primaries and metastases demonstrated overexpression of the protein and the same percentage of amplification of the gene was confirmed by CISH. Interestingly, the percentage of CD44 positive tumours was higher amongst the metastases (56%) in comparison to the primaries (30%). Six matched brain metastases and 3 unmatched brain metastases displayed positivity for EGFR, in contrast to the non-brain metastases where 9 out of 11 tumours were positive. Figure 1 shows an example of a high grade tumour, positive for CK 14 and the matched pair immunohistochemistry staining for CD44.
Gene expression profiling of human primary and metastatic breast cancer to the brain
The availability of good quality RNA and stringent filtering of the DASL data yielded gene expression profiling data on 37/61 brain metastases from breast cancer (15/39 from matched pairs and 22/22 from unmatched metastases) and 15 matched primaries. Unsupervised analysis highlighted a strong similarity between primary tumors and their matched metastases (Figure 2A). Only 20 genes were, differentially expressed genes between the matched primaries and metastases (Figure 2B). This may be a consequence of the overall strong similarity between primaries and metastases coupled with the sample size (n=30) and number of genes analyzed (n=512 cancer genes in DASL panel). The comparison between primaries and all metastases (matched and unmatched) identified 27 statistically significant differentially expressed genes. All twenty genes identified in the matched pair analysis were part of this 27-gene set. Among this 20-gene set, were HER3 and one of its downstream target molecules GRB 2, hypoxia related molecule HIFl-alfa, MAPKinase cascade related protein CREBBP, cell cycle regulator RBl and proliferation related genes CCNH, CDK7 and CDC25B. Since the brain is rich in neuregulin 1 and this is a ligand for HER3, we hypothesized that the neuregulin- HER3 activation was important in allowing breast cancer cells to colonize the brain. HER family receptors and downstream molecules expression
HER3, EGFR, HER2, HER4 and HIFl-alfa expression was assessed using quantitative RT-PCR in 12 matched breast/brain samples for which DASL data and RNA were available. Similar to the DASL data, ten cases showed increased fold change by RT-PCR of HER3 gene expression relative to their matched primaries ranging from 1.12 to 5.8 and with an average of 2.4. Immunohistochemistry for HER3 was similar, showing positivity in 11/37 (29.7%) of the primary tumors, 22/37 (59%) of the matched metastases and 13/21 (62%) of the unmatched brain metastases. In agreement, phosphorylated HER3 confirmed more frequent activation in the brain metastases, with positivity in 15/37 (40%) of the primary tumors, 24/37 (64%) of the matched metastases and 18/21 (85%) of the unmatched brain metastases.
Immunohistochemistry for GRB2, HIFl-alfa (p value check , remove if not significant) and phosphorylated ERK1/2, JNK1/2, ERK5 and p38 also demonstrated increased activation in the metastases compared to the primary tumors. In contrast, phosphorylated AKT was equally high in both the primaries and metastases. Interestingly, the non-breast derived brain metastases showed similarly high activation of the MAPK pathway together with over-expression (3+ stain) of EGFR (in 9/11 (81%) metastases (a prostate and one colon carcinoma did not) but in the absence of HER3 activation. Co-culture of MCF-7 and SKBR-3 cell lines and human differentiated neural cells
In order to investigate if the neuronal cells would enhance the growth of breast cancer cell lines, we co-cultured MCF-7 and SKBR-3 cell lines with ReNcell CX (CHEMICON International, Inc. Catalogue SCC007). ReNcell CX cell line was grown as neurosphere and differentiated into adult type neuronal cells. The cells were co-cultured and the number of cell was counted using flow cytometry and antibodies to discriminate between epithelial phenotype (EpCam) and neuronal phenotype (CD56) every 24 hours for four days. The total number of cells in the wells was generated in triplicates so that a growth curve was created. The results revealed an enhancement of growth in the group of cells which was co-cultured when compared to the group where neuronal cells were not added to the plate. In parallel, the same co-culture experiment was performed with addition of trastuzumab and a neutralizing anti-neuregulin antibody to the cell culture media. This aimed to test if neuregulin could be playing a role in the growth enhancement. The results showed that both the trastuzumab and the anti-neuregulin antibody reduced the rate of growth of these cell lines. However, resistance to the trastuzumab was seen after day 3 of the cell culture and surprisingly the same pattern of growth was observed for the anti-neuregulin antibody. As described previously, resistance could be due to activation of other member of the HER family of receptors. Therefore, the experiment was repeated with addition of lapatinib plus trastuzumab and lapatinib and anti-neuregulin antibody to another group two groups of co-culture cells. This overcame the resistance found on day 3. Figure 5 shows the growth curves summary for this experiment. Soft agar colonization assay
The colony forming assay demonstrated that the average number of colonies was higher among the control group compared to the group treated with traztuzumab (p = 0.049) . When compared to the group of cells treated with trastuzumab and neuregulin, the number of colonies in the control group was still higher although not statistically significant ( p = 0.275). The use neuregulin showed enhancement of cell growth in the group treated with neuregulin only but this did not reach statistical significance ( p = 0.184) when compared to the control group. In terms of size, the group of cells treated with neuregulin had an average colony size of 29.8 μm, the control 27.3 μm, the trastuzumab group 21.3 μm and the neuregulin + traztuzumab 18.7 μm. The summary of results is shown in Figures 7 and 8. Intracerebral injections of a basal-like breast cancer cell line (MDA-MB-231) and treatment with trastuzumab and anti-neuregulin antibody and differential gene expression
The present inventors aimed to investigate if trastuzumab would have any effect on the cells in the brain microenvironment and also if the blocking of the local factor growth neuregulin with an anti-neuregulin antibody would interfere in the rate of growth of the injected cells. Four groups of mice, each containing 3 animals, were used. Remarkably, the groups of mice that were treated on the day of injection with trastuzumab and anti-neuregulin antibody had smaller average tumours sizes compared to the control group (p <0.01). The difference in size for the control group and the group of mice treated after detection of the tumour masses by MRI was not statistically significant. The tumour sizes are available in Table 3. Differential gene expression by ANOVA of the parental cells and the cells injected in the brain of the mice revealed several genes expressed (Table 4). Interestingly, HER3 and CD44 were some of such genes. RT-PCR confirmed higher levels of HER3 compared to the parental cells and immunohistochemistry showed higher percentage of positive cell among the cells injected in the brain compared to the parental. Immunohistochemistry also revealed higher percentage of CD44 positive cells among the injected cells compared to the parental. Figure 6 illustrates MRI images at week 10, patterns of expression of CD44 and HER3 in the parental cell line and in the cells that colonized the brain as well as the RT-PCR for HER3 in the parental cell line and the injected cells. Discussion Breast cancer is one of the most common cancers and a leading cause of mortality in women (1, 2). Because there are few long-term survivors among patients with systemic metastatic breast cancer, the main objectives of therapy are to increase the time lapse before progressive disease is established and to improve symptom relief without increasing toxicity and comprising quality of life. As systemic therapy of metastatic breast cancer improves, central nervous system involvement is becoming a more widespread problem. (5) It is predicted that brain metastases will become increasingly prevalent as greater control over systemic metastases is achieved, particularly with regard to HER2 -positive tumours (6). Brain metastases from breast cancer may become a serious problem that oncologists may encounter frequently (10) and it is considered one of the most feared complications of cancer because even small tumours may cause incapacitating neurologic symptoms (4). Over the last five decades, autopsies series have reported brain metastases in 6,7%-36% of selected patients (57, 58).
In the present study, the median age of presentation of the brain metastases in the group of patients where follow-up data was available was 48.5 years old, most of the tumours showed negativity for estrogens and progesterone receptors and around 23% showed overexpression of HER2, confirmed by CISH. The percentage of HER2 overexpression tumours was in accordance with previous studies (25, 26). We assessed the expression of basal-like tumour markers such as CK14, CKl 7, CK5/6, SMA and p63 and found that 68% of the metastases and 61 % of the primary tumours of the initial set had expression of at least one of these markers. This is consistent with other studies where patients with expression of basal-like and negativity for estrogens and progesterone receptors had a trend to develop brain metastases (8, 16- 18, 59).
Interestingly, the percentage of CD44+CD24' positive tumours was higher among the brain metastases compared to the primary tumours. This is noteworthy because CD44+CD24" cells have been shown to express higher levels of preinvasive genes and have highly invasive properties (29). In addition, this phenotype is also believed to enrich for cells with stem cell properties (27). Hyaluronic acid stimulates CD44s-associated pi 85 (HER2) tyrosine kinase activity, leading to an increase in the ovarian carcinoma cell growth (35). The brain matrix and is rich in hyaluronan (60). Interaction between CD44 and hyaluronan has been shown to provide resistance to shear under physiologic conditions and to support the initial steps of lymphocyte extravasation (34). Hence, it is logical to hypothesize that CD44 could be implicated in the homing and local facilitation of growth of breast cancer cell in the brain. The HER/erbb family of tyrosine kinase receptors consists of four members:
HERl, HER2, HER3, and HER4 (38). On one hand HERl, HER2, and HER3 have been shown to contribute to aggressive tumour formation (61), on the other hand HER4 signaling may impair cellular proliferation of human breast cells and promote differentiation (62). The differential gene expression between the primary tumours and the brain metastases showed that, in the first set of samples, HER3 was differentially expressed with statistical significance and in the second set a twofold up-regulation was detected. This differential expression was confirmed by RT-PCR and immunohistochemistry. Interestingly, the samples stained with antibody against phosphorylated HER3 showed nuclear positivity. ErbB receptors have been reported to translocate to the nucleus. HERl , HER2, HER3 and HER4, may translocate to the nucleus as full-length receptors (HERl -3) or as a proteolytically derived intracellular domain (HER4) (52-55, 63).
In the brain microenvironment, induced HER2 overexpression has been shown to enhance the outgrowth of a specialized brain-seeking breast cancer cell line in animal model (65). In addition, reactive glia was shown to enhance growth of this same cell line (66). Furthermore, the 231 -BrI cell line, which was derived from a brain metastases in a nude mouse, showed increased adhesion to astrocytes and enhanced growth in vitro in the presence of an astrocyte conditioneted media when compared to the parental MD A-MB-231 and the lung metastasis-derived variant 231- Lung2 (67). The ligand for HER3 is neuregulin-1, a transmembrane epidermal growth factor-like molecule which is abundant in the brain microenvironment (44, 64). We aimed to test if the growth of the of breast cancer cell lines (MCF-7 and SKBR-3) would be enhanced when co-cultured with adult neural cells and to verify what growth response would be seen when trastuzumab, lapatinib and a neutralizing anti-neuregulin antibody were added to the cell co-culture media. The cells were counted using flow cytometry and the results demonstrated an enhancement of growth compared to the breast cancer cell lines which were not co-cultured. In keeping with what has been shown before, trastuzumab and lapatinib reduced the growth rate but drug resistance was seen from day 3 to 4 (42, 68). The same result was seen when the neutralizing anti-neuregulin antibody was added suggesting that the neuregulin of the neural cells could also be playing a role in the growth enhancement. The combination of trastuzumab and lapatinib overcame the resistance from day 3 to 4, suggesting that the resistance could be via activation of the other members of the HER family of receptors as indicated previously (42, 43). The mechanisms of action of lapatinib and trastuzumab are different and their combination was shown to lead to synergism (69). The mechanism of action of trastuzumab is distinct from tyrosine kinase inhibitors such as gefitinib or erlotinib, which bind competitively to the intracellular adenosine triphosphate binding site of erbb receptors (70, 71) and which is the same site of action for lapatinib. (72)
Despite the fact that the MDA-MB-231 cell line has low levels of HER2 and HER3, the treatment of the mice with trastuzumab and anti-neuregulin antibody resulted in smaller tumours when compared to the control group. This may reflect a potential for some tumours with low expression of HER2 to be responsive to trastuzumab and also suggests that the brain local environment may be playing a role in the enhancement of growth in vivo. The colony forming assay supports this view since less and smaller colonies were detected in the group treated with traztuzumab compared to the control group and the group treated with neuregulin. These values were statistical significant. Furthermore, the combination of neuregulin and traztuzumab resulted in a difference not statistical significant when compared to the control group, suggesting that neuregulin was interfering in the action of trastuzumab on the cells. Recently, it has been shown that subgroups of HER2 negative patients can benefit of the treatment with trastuzumab. One of these studies was a retrospective analysis of the phase III Cancer and Leukemia Group B (CALGB) 9840 trial and it demonstrated that HER2-negative metastatic breast cancer patients with multiple copies of the chromosome carrying HER2 had significantly better response rates (63% vs. 26%, P = .048) when they were treated with trastuzumab in addition to paclitaxel (73). The second study showed that a small group of HER2-negative patients in the phase III National Surgical Adjuvant Breast and Bowel Project (NSABP) B-31 trial had significantly better disease-free survival with a relative risk of 0.40 (P = .026) when given trastuzumab after completing treatment for early breast cancer (74). Furthermore, another study suggested that the spectrum of patients who may benefit from trastuzumab-based therapies could be expanded to include patients with metastatic breast cancer without HER-2 amplification but who express transmembrane neuregulin, the ligand of HER3 (75). Pertuzumab, is a recombinant humanized monoclonal antibody and binds to extracellular domain II of the HER-2 receptor and blocks its ability to dimerize with other HER receptors. The pertuzumab binding site within domain II does not overlap with the epitope on HER-2 (76) that is recognized by trastuzumab. Hence, this monoclonal antibody could play a major role in cases where a tumour is also dependent on other members of the HER family of receptors.
In conclusion, the present study provides evidence that local factor neuregulin may enhance tumour growth in the brain microenvironment whether in vitro or in vivo and shed some light on the paradigm that HER2 negative cannot have response to trastuzumab. We suggest that there might be cohorts of HER-negative patients that may benefit of trastuzumab or combinations of HER family receptors inhibitors. Specially, in the cases where tumours are dependent on members of the erbB family receptors that are fueled by local factors, autocrine loops or even autophophorylated.
Example 2
We have already replicated the 3 members of the HER-family of receptors in plasmids (pLHCX-EGFR; hygromycin resistant, pLPCX-HER2; puromycin resistant and pLNCX2-HER3; G418 resistant). The transduction will be performed using an amphotropic producer cell line such as Phoenix amphotropic cells. Within 72 hours the medium will be collected filtered and used for infecting the breast cancer cells. Luminal and basal-like breast cancer cell lines (with and without the insertion of the HER-family of receptor genes) will be co-cultured with adult CNS cells in order to mimicry an artificial brain matrix. We will utilize 96-well plates that can be analysed by the BD FACSArray™ bioanalyzer from BD Biosciences. The epithelial luminal and basal-like breast cancer cell lines will be place onto a matrix of adult CNS cells. The epithelial and CNS cells will be differentially labelled with epithelial and neuronal florescent antibodies so that number of viable cells can be assessed by this device. Human fibroblasts and cells which are not in co-culture will be used as controls. Growth curve for each experiment will be then generated and the influence of the co-culture conditions analysed. We are currently using EpCAM and CD56 for differential fluorescent labelling the epithelial and neuronal cells respectively. A baseline characterization of the expression of these two markers in the chosen cell lines will necessary as to whether they are reliable in distinguishing the epithelial and neuronal cells. This will be done using equal number of both types of cells in the flow cytometer with an expectation of obtaining even figures at the end. Neurosphere assay 2β is a serum-free culture system whereby the isolation and propagation of CNS-derived stem cells is feasible. Adult precursors are dissociated and plated in a liquid growth medium that contains the stem-cell mitogens epidermal growth factor and/or fibroblast growth factor. Because of the lack of serum and the low plating density, most cells die, except those that divide in response to the stem- cell mitogens. The growth-factor responsive cells proliferate to form floating clusters of cells that are referred to as neurospheres. These can be further dissociated into a single-cell suspension and then re-plated in fresh medium to produce secondary neurospheres. After removal of mitogenic factors, the progeny of the proliferating precursors can be differentiated into neurons, astrocytes and oligodendrocytes, which are the three primary cell types that are found in the adult human CNS. This will be our source of adult CNS cells to create the artificial brain matrix.
Concurrently, the addition of modulators of the HER-family of receptor into the 96-well plates will be performed. These modulators will comprise drugs currently used in clinical practice to target the HER-family of receptor, for example trastuzumab and lapatinib but not limited thereto. Non-limiting examples of other drugs currently used in clinical practice to target the HER-family of receptor are given in this text, particularly at Table 5. The wells will have quadruplicates samples and the statistical difference between the growth curves will be established using one-way Analysis of variance. The growth curves will use time points at 12, 36, 60 and 84 hours and the drugs will be titrated.
The selective and robust effect of RNA interference (RNAi) on gene expression makes it a valuable research tool, both in cell culture and in living organisms because synthetic dsRNA introduced into cells can induce suppression of specific genes of interest. So, the above mentioned experiment will be also performed with the use of RNAi targeting the HER-family of receptor and also using an antibody targeting the local growth factor neuregulin 1 , the ligand of HER3 (the adult CNS cells express this molecule). RNAi targeting HER-family of receptors and scrambles for negative controls are available for purchasing from several companies. We expect this second experimental phase to mirror at some extend the findings of the experimental phase where drugs were used. The BD FACSArray™ bioanalyzer from BD Biosciences is capable fast and sensitive high-content measurements of cells and proteins in cell biology, supporting analysis of apoptosis, cytokine and chemokine profiling, and phosphorylation of key signal transduction proteins. This flow cytometer rapidly detects and quantifies concentrations of secreted proteins, proteins in cell lysates, and cell-associated proteins using small sample volumes directly from a 96-well microtiter plate. This device will be used for the above mentioned experiments.
The same co-culture system will also be used to assess activation of molecules which are downstream of the HER-family of receptors. The molecules targeted will be the ones assessed in AIM 1(RAS, SOS, RAFl, ERK1/2, MEKland ELK-I for the MAPK pathway and p21, p27, MDM2, BAD, CASP9 and mTOR for the AKT pathway). The BD FACSArray™ bioanalyzer will again be used. We expect that the results under non-co-culture conditions should mirror the results of the baseline assessment with western blots across the cell lines. This experimental phase will also show the changes the will occur in the AKT and MAPK related molecules under co-culture conditions, allowing a better understanding of the behaviour of breast cancer cells in a brain environment. In addition the assessment of apoptotic indexes can also be generated by the BD FACSArray™ bioanalyzer using markers such as annexine and caspases. Throughout the specification the aim has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. It will therefore be appreciated by those of skill in the art that, in light of the instant disclosure, various modifications and changes can be made in the particular embodiments exemplified without departing from the scope of the present invention.
All computer programs, algorithms, patent and scientific literature referred to herein is incorporated herein by reference.
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TABLES
Table 1 - Details of Antibodies Used in lmmunohistochemistry
Figure imgf000058_0001
Table 2 - Antibodies positivity across the samples
Figure imgf000059_0001
Legend: N = number of tumour cores showing positivity; T = total number of cores assessable for the antibody specified; % = percentage of cores showing positivity; NP = not performed
Table 3 - Summary of brain injections experiment
Control Group - not Tumour size- Days to death Average of days to treated mm3 death
Animal 1 544 80
Animal 2 250 77
Animal 3 187 77 74
Average tumour size mm3 327
Mice treated on the day Tumour size- Days to death Average of days to of injection of tumour mm3 death cells with Trastuzumab
Animal 1 78 77
Animal 2 65 90
Animal 3 74 95 88
Average tumour size
72 mm3
Mice treated on the day Tumour size- Days to death Average of days to of injection of tumour mm3 death cells with anti- neuregulin antibody
Animal 1 99 77
Animal 2 100 90
Animal 3 64 95 88
Average tumour size
87 mm3
Mice treated at Day 84 MRI Tumour Size Tumour Size at Days to death after with Trastuzumab at day of autopsy-mm3 treatment injection-mm3
Animal 1 144 142 0
Animal 2 193 199 11
Animal 3 239 250 14
Average tumour size
210 197 mm3 Table 4 - Genes differentially expressed between parental and tumour cells injected in the brain o
Figure imgf000061_0001
OS
Figure imgf000062_0001
Table 5 A sample of ErbB inhibitors which are either used for treatment or in pre-clinical and clinical trials
ON
Figure imgf000063_0001
Os
Figure imgf000064_0001
Table 5 continued

Claims

1. A method of treating a metastatic breast cancer in a human, said method including the step of administering to an individual with an elevated level of Her-3 in one or more metastatic breast cancer cells of the brain, a therapeutic agent effective in inhibiting at least one ErbB receptor.
2. The method of Claim 1 , wherein the metastatic breast cancer has one or more cells with normal expression levels of Her-2.
3. The method according to any one of the preceding claims, wherein the metastatic breast cancer has one or more breast cancer cells with normal expression levels of Her-2.
4. The method according to any one of the preceding claims, wherein the metastatic breast cancer is located in the brain.
5. The method according to any one of the preceding claims, wherein the at least one ErbB receptor is selected from the group consisting of Her-1 , Her-2, Her-3, Her- 4, and combinations thereof.
6. The method according to any one of the preceding claims, wherein the at least one ErbB receptor is selected from the group consisting of Her-1, Her-2 and Her-3.
7. The method according to any one of the preceding claims, wherein the at least one ErbB receptor is Her-2.
8. The method according to any one of the preceding claims, wherein the therapeutic agent directly inhibits at least one ErbB receptor or indirectly inhibits at least one ErbB receptor.
9. The method according to any one of the preceding claims, wherein the therapeutic agent selectively inhibits at least one ErbB receptor.
10. The method according to any one of the preceding claims, wherein the therapeutic agent is selected from the group consisting of an isolated protein or a fragment thereof, an isolated nucleic acid or a fragment thereof, a small-molecule compound, and combinations thereof.
11. The method according to any one of the preceding claims, wherein the therapeutic agent is an antibody, or a fragment thereof.
12. The method according to any one of the preceding claims, wherein the antibody is a monoclonal antibody.
13. The method according to any one of the preceding claims, wherein the antibody binds to and/or has been raised against Her-2.
14. The method according to any one of the preceding claims, wherein the therapeutic agent is selected from the group consisting of trastuzumab, pertuzumab, scFvFRP5, CAB051, ertumaxomab, lapatinib, CI- 1033, HKI-272, AEE-788, BIBW- 2992, TAKl 65, BMS-599626, matuzumab, cetuximab, panitumumab, gefitinib, erlotinib, ICR62, nimotuzumab, Ch806, L8A4, MDX-447, zalutumumab, IMC-11F8, AZD4769, PF299804, EGFR501, ZD6474, EKB-569, EXEL 7647, EXEL 0999, AZD8931, MP412, herstatin, pelitinib, CP724714, XL647, PD- 169414, and combinations thereof.
15. The method according to any one of the preceding claims, wherein the therapeutic agent is trastuzumab.
16. The method according to any one of the preceding claims, which further includes the step of administering to an individual, one or more therapeutic agents selected from the group consisting of a chemotherapeutic agent and a modulator of a hormone receptor.
17. The method according to any one of the preceding claims, which further includes the step of determining whether said individual has an elevated level of Her- 3 in one or more metastatic breast cancer cells of the brain.
18. A method of designing, engineering, screening or otherwise producing a therapeutic agent for treating a metastatic breast cancer, said method including the step of identifying a candidate agent that is suitable for use in treatment of a metastatic breast cancer by the presence of an elevated expression level of Her-3 in one or more metastatic breast cancer cells of the brain.
19. The method of Claim 18, wherein the candidate agent inhibits at least one ErbB receptor.
20. The method of Claim 18 or Claim 19, wherein the candidate agent directly inhibits at least one ErbB receptor or indirectly inhibits at least one ErbB receptor.
21. The method according to any one of Claims 18 to Claim 20, wherein the candidate agent selectively inhibits at least one ErbB receptor.
22. The method according to any one of Claims 18 to 21 , wherein the at least one ErbB receptor is selected from the group consisting of Her- 1, Her-2, Her-3, Her-4, and combinations thereof.
23. The method according to any one of Claims 18 to 22, wherein the at least one ErbB receptor is selected from the group consisting of Her- 1, Her-2 and Her-3.
24. The method according to any one of Claims 18 to 23, wherein the at least one ErbB receptor is Her-2.
25. The method according to any one of Claims 18 to 24, wherein the candidate agent is selected from the group consisting of an isolated protein or a fragment thereof, an isolated nucleic acid or a fragment thereof, a small-molecule compound, and combinations thereof.
26. The method according to any of Claims 18 to 25, wherein the candidate agent is an antibody, or a fragment thereof.
27. The method according to any one of Claims 18 to 26, wherein the antibody is a monoclonal antibody.
28. The method according to any one of Claims 18 to 27, wherein the antibody binds to and/or has been raised against Her-2.
29. The method according to any one of Claims 18 to 28, wherein the metastatic breast cancer has one or more cells with normal expression levels of Her-2.
30. The method according to any one of Claims 18 to 29, wherein the metastatic breast cancer has one or more breast cancer cells with normal expression levels of Her-2.
31. The method according to any one of Claims 18 to 30, wherein the one or more cells with normal levels of Her-2 have levels of an estrogen receptor and a progesterone receptor which are below a threshold level of detection.
32. The method according to any one of Claims 18 to 31 , wherein the metastatic breast cancer is located in the brain.
33. A therapeutic agent for treating a metastatic breast cancer designed, engineered, screened or otherwise produced according to a method of any one of Claims 18 to 32.
34. A pharmaceutical composition comprising a therapeutic agent for treating a metastatic breast cancer according to Claim 33, together with a pharmaceutically- acceptable carrier, diluent or excipient.
35. The pharmaceutical composition of Claim 34, which further comprises one or more therapeutic agents selected from the group consisting of a chemotherapeutic agent and a modulator of a hormone receptor.
36. A method of treating a metastatic breast cancer, said method including the step of administering a therapeutic agent for treating a metastatic breast cancer according to Claim 33 or a pharmaceutical composition according to Claim 34 or Claim 35, to an individual with an elevated level of Her-3 in one or more metastatic breast cancer cells of the brain.
37. The method according to Claim 36, wherein the metastatic breast cancer has one or more cells with normal expression levels of Her-2.
38. The method according to Claim 36 or Claim 37, wherein the metastatic breast cancer has one or more breast cancer cells with normal expression levels of Her-2.
39. The method according to any one of Claims 36 to 38, wherein the one or more cells with normal levels of Her-2 have levels of an estrogen receptor and a progesterone receptor which are below a threshold level of detection.
40. The method according to any one of Claims 36 to 39, wherein the metastatic breast cancer is located in the brain.
41. A method of determining whether a human with a metastatic breast cancer is potentially responsive to treatment with a therapeutic agent effective in inhibiting at least one ErbB receptor and/or a therapeutic agent for treating a metastatic breast cancer designed, engineered, screened or otherwise produced according to any one of Claims 18 to 32, said method including the step of detecting elevated levels of Her-3 in one or more metastatic breast cancer cells of the brain.
42. The method of Claim 41, wherein the at least one ErbB receptor is selected from the group consisting Her-1, Her-2, Her-3, Her-4, and combinations thereof.
43. The method according to Claim 41 or Claim 42, wherein the at least one ErbB receptor is selected from the group consisting of Her-1, Her-2 and Her-3.
44. The method according to any one of Claims 41 to 43, wherein the at least one ErbB receptor is Her-2.
45. The method according to any one of Claims 41 to 44, wherein the metastatic breast cancer has one or more cells with normal expression levels of Her-2.
46. The method according to any one of Claims 41 to 45, wherein the metastatic breast cancer has one or more breast cancer cells with normal expression levels of Her-2.
47. The method according to any one of Claims 41 to 46, wherein the one or more cells with normal levels of Her-2 have levels of an estrogen receptor and a progesterone receptor which are below a threshold level of detection.
48. The method according to any one of Claims 41 to 47, wherein the metastatic breast cancer is located in the brain.
49. A method of determining whether a human is predisposed to a metastatic breast cancer or is suffering from a metastatic breast cancer, said method including the step of detecting an elevated level of Her-3 in one or more metastatic breast cancer cells of the brain.
50. The method according to Claim 49, wherein the metastatic breast cancer has one or more cells with normal expression levels of Her-2.
51. The method according to Claim 49 or Claim 50, wherein the metastatic breast cancer has one or more breast cancer cells with normal expression levels of Her-2.
52. The method according to any one of Claims 49 to 51, wherein the one or more cells with normal levels of Her-2 have levels of an estrogen receptor and a progesterone receptor which are below a threshold level of detection.
53. The method according to any one of Claims 49 to 52, wherein the metastatic breast cancer is located in the brain.
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