WO2007022588A1

WO2007022588A1 - Method for assessing a response to an antiproliferative agent

Info

Publication number: WO2007022588A1
Application number: PCT/AU2006/001230
Authority: WO
Inventors: Junya Kuroda; Hamsa Puthalakath; Mark Cragg; Philippe Bouillet; Andrew Roberts; Priscilla Kelly; David Huang; Andreas Strasser
Original assignee: Walter and Eliza Hall Institute of Medical Research
Current assignee: Walter and Eliza Hall Institute of Medical Research
Priority date: 2005-08-24
Filing date: 2006-08-24
Publication date: 2007-03-01
Anticipated expiration: 2008-02-24

Abstract

The present invention relates generally to a method of diagnosing, predicting and/or monitoring cellular responsiveness to antiproliferative agents. More particularly, the present invention relates to a method of diagnosing, predicting and/or monitoring reduced cellular responsiveness to antiproliferative agents by screening for modulation in the levels of functional Bim and/or Bad in the subject cell. The present invention further provides a method for predicting, diagnosing and/or monitoring the responsiveness of a condition characterised by unwanted cellular proliferation to treatment with an antiproliferative agent. Also provided are diagnostic agents useful for detecting functional Bim and/or Bad expression levels.

Description

METHOD FOR ASSESSING A RESPONSE TO AN ANTIPROLIFERATIVE AGENT

FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION

Bibliographic details of the publications referred to by author in this specification are collected alphabetically at the end of the description.

The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in Australia.

Malignant tumours, or cancers, grow in an uncontrolled manner, invade normal tissues, and often metastasize and grow at sites distant from the tissue of origin. In general, cancers are derived from one or only a few normal cells that have undergone a poorly understood process called malignant transformation. Cancers can arise from almost any tissue in the body. Those derived from epithelial cells, called carcinomas, are the most common kinds of cancers. Sarcomas are malignant tumours of mesenchymal tissues, arising from cells such as fibroblasts, muscle cells, and fat cells. Solid malignant tumours of lymphoid tissues are called lymphomas, and marrow and blood-borne malignant tumours of lymphocytes and other hematopoietic cells are called leukemias.

Cancer is one of the three leading causes of death in industrialised nations. As treatments for infectious diseases and the prevention of cardiovascular disease continues to improve, and the average life expectancy increases, cancer is likely to become the most common fatal disease in these countries. Therefore, successfully treating cancer requires that all the malignant cells be removed or destroyed without killing the patient. An ideal way to achieve this would be to induce an immune response against the tumour that would discriminate between the cells of the tumour and their normal cellular counterparts. However, immunological approaches to the treatment of cancer have been attempted for over a century with unsustainable results.

Accordingly, current methods of treating cancer continue to follow the long used protocol of surgical excision (if possible) followed by radiotherapy and/or chemotherapy, if necessary. The success rate of this rather crude form of treatment is extremely variable but generally decreases significantly as the tumour becomes more advanced and metastasises. Further, these treatments are associated with severe side effects including disfigurement and scarring from surgery (e.g. mastectomy or limb amputation), severe nausea and vomiting from chemotherapy, and most significantly, the damage to normal tissues such as the hair follicles, gut and bone marrow which is induced as a result of the relatively nonspecific targeting mechanism of the toxic drugs which form part of most cancer treatments.

Further, most anti-cancer treatments, which include cytotoxic chemotherapeutic agents, signal transduction inhibitors, radiotherapy, monoclonal antibodies and cytotoxic lymphocytes, kill cancer cells by apoptosis. Although tumours may contain a proportion of apoptotic cells and even areas of necrosis before anti-cancer treatment is given, an increased number of apoptotic cells and larger areas of necrosis are anticipated in tumours that respond to the anti-cancer treatment. However, when cytotoxic chemotherapeutic agents are used for the treatment of advanced cancer, the degree of cell kill and thus the response of the tumour to the first treatment is frequently difficult to assess.

Conventionally, patients receive a minimum of three cycles of chemotherapy before a clinical and radiological assessment of tumour response is made. Usually, only a minority of patients with advanced cancer responds to cytotoxic drugs and so patients may experience the side effects of treatment without obtaining benefit. Hence, there is an unmet medical need for a diagnostic method that would enable the rapid and reliable assessment or monitoring of a patient's likely responsiveness to a chemotherapeutic drug. Knowing whether a neoplastic cell is capable of responding or is developing resistance to responding to a given antiproliferative drug would spare the majority of patients from ineffective and potentially toxic treatment. Then, non-responding patients can be offered second line treatments or clinical trials of investigational agents.

In work leading up to the present invention it has been determined that antiproliferative agents such as imatinib function via Bim and Bad mediated signalling and that in leukaemia patients exhibiting imatinib resistance there has occurred a mutation in the leukaemic cells wherein the functionality of Bim and/or Bad is adversely affected, for example by virtue of loss of expression. Accordingly, these findings have now facilitated the development of assessment technology directed to diagnosing, prognosing and/or monitoring for cellular resistance to antiproliferative agents. This now provides a means of effectively managing patients with conditions characterised by unwanted proliferation and who are likely to require treatment based on the administration of antiproliferative agents such as chemotherapy drugs.

SUMMARY OF THE INVENTION

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

As used herein, the term "derived from" shall be taken to indicate that a particular integer or group of integers has originated from the species specified, but has not necessarily been obtained directly from the specified source. Further, as used herein the singular forms of "a", "and" and "the" include plural referents unless the context clearly dictates otherwise.

The subject specification contains nucleotide sequence information prepared using the programme Patentln Version 3.1, presented herein after the bibliography. Each nucleotide sequence is identified in the sequence listing by the numeric indicator <210> followed by the sequence identifier (eg. <210>l, <210>2, etc). The length, type of sequence (DNA, etc) and source organism for each nucleotide sequence is indicated by information provided in the numeric indicator fields <211>, <212> and <213>, respectively. Nucleotide sequences referred to in the specification are identified by the indicator SEQ ID NO: followed by the sequence identifier (eg. SEQ ID NO: 1 , SEQ ID NO:2, etc.). The sequence identifier referred to in the specification correlates to the information provided in numeric indicator field <400> in the sequence listing, which is followed by the sequence identifier (eg. <400>l, <400>2, etc). That is SEQ ID NO:1 as detailed in the specification correlates to the sequence indicated as <400>l in the sequence listing.

One aspect of the present invention is directed to a method for assessing the responsiveness of a cell to an antiproliferative agent, said method comprising screening for the level of functional Bim and/or Bad protein and/or gene expression in said cell wherein a decrease in the level of said protein and/or gene expression relative to normal levels is indicative of reduced responsiveness . Another aspect of the present invention therefore provides a method for assessing the responsiveness of a neoplastic cell to an antiproliferative agent, said method comprising screening for the level of functional Bim and/or Bad protein and/or gene expression in said cell wherein a decrease in the level of said protein and/or gene expression relative to normal levels is indicative of reduced responsiveness.

Yet another aspect of the present invention is directed to a method for assessing the responsiveness of a malignant neoplastic cell to an antiproliferative agent, said method comprising screening for the level of functional Bim and/or Bad protein and/or gene expression in said cell wherein a decrease in the level of said protein and/or gene expression relative to normal levels is indicative of reduced responsiveness.

Still another aspect of the present invention is directed to a method for assessing the responsiveness of a malignant neoplastic cell to an antiproliferative agent, which agent induces death, said method comprising screening for the level of functional Bim and/or Bad protein and/or gene expression in said cell wherein a decrease in the level of said protein and/or gene expression relative to normal levels is indicative of reduced responsiveness.

Yet still another aspect of the present invention provides a method for assessing the responsiveness of a malignant neoplastic cell to an antiproliferative agent, which agent induces apoptosis, said method comprising screening for the level of functional Bim and/or Bad protein and/or gene expression in said cell wherein a decrease in the level of said protein and/or gene expression relative to normal levels is indicative of reduced responsiveness.

Still yet another aspect of the present invention is directed to a method for assessing the responsiveness of a malignant neoplastic cell to an antiproliferative agent, which agent induces apoptosis, said method comprising screening for the level of functional Bim and/or Bad protein and/or gene expression in said cell wherein the absence of said protein and/or gene expression is indicative of unresponsiveness. In a related aspect the present invention is directed to a method for assessing the responsiveness to an antiproliferative agent of a cell in a mammal or in a biological cell derived from said mammal, said method comprising screening for the level of functional Bim and/or Bad protein and/or gene expression in said cell wherein a decrease in the level of said protein and/or gene expression relative to normal levels is indicative of reduced responsiveness.

Another further aspect of the present invention relates to a method for monitoring the responsiveness of a cellular population to an antiproliferative agent, said method comprising screening for modulation of the level of functional Bim and/or Bad protein and/or gene expression wherein a decrease in the level of said protein and/or gene expression in the subject cellular population relative to a previously obtained level or a normal level is indicative of the maintenance or worsening of responsiveness and an increase in said level is indicative of an improvement in said responsiveness.

Yet another aspect of the present invention is directed to a method for assessing the actual or likely responsiveness to an antiproliferative agent of a condition characterised by unwanted cellular proliferation, said method comprising screening for the level of functional Bim and/or Bad protein and/or gene expression wherein a decrease in the level of said protein and/or gene expression relative to normal levels is indicative of reduced responsiveness.

In yet another aspect, the present invention relates to a method for monitoring the responsiveness to an antiproliferative agent of a condition characterised by unwanted cellular proliferation, said method comprising screening for modulation of the level of functional Bim and/or Bad protein and/or gene expression wherein a decrease in the level of said protein and/or gene expression in the subject cellular population relative to a previously obtained level or a normal level is indicative of the maintenance or worsening of responsiveness and an increase in said level is indicative of an improvement in said responsiveness. Yet another aspect of the present invention is directed to assessing and/or monitoring the effectiveness of an antiproliferative treatment regime in a mammal, said method comprising screening for the level of functional Bim and/or Bad protein and/or gene expression by a cellular population of interest wherein a decrease in the level of said protein and/or gene expression relative to normal levels is indicative of reduced responsiveness.

Another aspect of the present invention provides a diagnostic kit comprising an agent for detecting Bim and/or Bad or a nucleic acid molecule encoding Bim and/or Bad and reagents useful for facilitating the detection by said agent.

The present invention further contemplates the use of an interactive molecule directed to Bim and/or Bad in the manufacture of a quantitative or semi-quantitative diagnostic kit to assess cellular responsiveness to an antiproliferative agent. The kit may come with instructions for use and may be automated or semi-automated or in a form which is compatible with an automated machine or software.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 Effect of Bcl-2 over-expression or dominant negative FADD on the proliferation inhibitory and pro-apoptotic effects of imatinib. K562 and BV173 human Ph¹+ leukemia- derived cells were treated with the indicated concentrations of imatinib for 48 h. (A) Cell proliferation was measured using the MTS assay. (B) Staining with PI followed by flow cytometric analysis was used to discriminate between live and dead cells (PI⁺). Results represent mean ±SD of three experiments. (C) Western blotting was performed to determine the levels of anti-apoptotic Bcl-2 family members before and after treatment with imatinib in K562 and BV 173 cells. (D) Expression of FLAG-tagged Bcl-2 or FADD- DN proteins in K562 transfectants was measured by immunofluorescent staining with anti- FLAG antibody and flow cytometric analysis (H; high, M; intermediate, L; low level of expression of the transgenic protein). (E, F) Parental K562 cells, K562/bcl-2 cells and K562/fadd.dn cells (high, medium and low expressors) were treated with various concentrations of imatinib for 48 h. (E) Cell proliferation was measured using the MTS assay. (F) Staining with PI followed by flow cytometric analysis was used to discriminate between live and dead cells (PI⁺). Results represent mean ±SD of three experiments.

Figure 2 Treatment with imatinib causes increased expression of Bim and Bim de- phosphorylation in Ph¹+ human leukemic cells. (A) K562 cells were treated for the indicated amount of time with 0.5 μM or 1 μM imatinib (B) BV173 cells were treated for the indicated amount of time with 1.5 μM or 3 μM imatinib. Western blotting was performed with an anti-Bim antibody and as a loading control with an antibody to HSP70 or β-actin. (C) K562 cells were treated for 3 or 6 h with 1 μM imatinib. Semi-quantitative PCR was performed to determine levels of bim mRNA. Semi-quantitative PCR for β-actin mRNA was used as a control. (D) K562 cells were left untreated or treated for 24 h with 1.5 μM imatinib. Bim proteins were purified on a protein G sepharose column coupled with anti-Bim antibodies and then analysed by 2-D gel electrophoresis.

Figure 3 RNAi -mediated knock-down protects K562 and BVl 73 cells against imatinib. (A) Western blot analysis using antibodies to Bim and β-actin (loading control) documents the levels of Bim expression in parental (ctl: control) and RNAi Bim knock-down subclones of K562 and BV173 cells. (B, C) Parental and RNAi Bim knock-down subclones of K562 and BVl 73 cells were treated for the indicated amount of time with 1.5 μM (B) or 3 μM (C) imatinib respectively. Cell viability was determined by staining with PI followed by blow cytometric analysis. (D) Parental and RNAi Bim knock-down subclones of K562 cells were treated for 24 or 48 h with 0 (DMSO carrier alone), 1.5 or 3 μM imatinib. Cells were then plated in agar to measure frequency of colony formation. Data represent mean±SD of three experiments indicating percentage of colony formation compared to untreated cells.

Figure 4 Effect of imatinib on expression of Bim, Bad and Bmf and phosphorylation of Bad in control and RNAi Bim knock-down K562 and BV173 cells. Parental as well as RNAi Bim knock-down K562 and BVl 73 cells were treated for the indicated periods of time with 1.5 μM or 3 μM imatinib, respectively. Western blotting was performed using antibodies to Bim, Bad, phosphorylated Bad, Bmf and β-actin (loading control).

Figure 5 Loss of Bim or Bad and Bcl-2 over-expression protects bcr-c-abl transformed fetal liver cells against imatinib-induced cell death. Control (wt), bim-/-, bad-/-, bim-/- bad-/- (double knock-out, DKO) and vav-bcl-2 transgenic fetal liver cells transformed with bcr-c-abl (3 independent lines for each genotype) were treated with vehicle (DMSO) or 1.5 or 3 μM DMSO. (A) Cell growth was scored after 1, 2, 4 and 7 days treatment with vehicle (DMSO) (line), 1.5 μM (larger dotted line) or 3 μM (smaller dotted line) imatinib. Data represent mean ±S.D. of three independent experiments. (B) Cell viability was assessed after 7 days of treatment with 1.5 μM or 3 μM imatinib by staining with tryptan blue dye and counting in a hemocytometer. Results represent mean ±S.D. of three independent experiments indicating percentage of viable cells normalized to percentage of viable cells in control (DMSO) treated cells.

Figure 6 Effect of imatinib on expression of Bim, Bad, Bmf, Bcl-2, Bcl-xL and Bax as well as phosphorylation of Bad in bcr-c-abl transformed wt, bim^'1' and bad^1" fetal liver cells. (A) Cells were treated for 0, 24 or 48 h with 3 μM imatinib. Western blotting was performed with antibodies specific to Bim, Bad (total), phosphorylated Bad, Bmf, Bcl-2, BCI-X_L, Bax or β-actin (loading control). (B) Cells were treated for 3, 6, 12 or 24 h with 5 μM imatinib. Semi-quantitative RT-PCR analysis was performed to determine the levels of bmf, puma and β-actin (loading control) mRNA

Figure 7 Bim expression is induced in primary Ph¹+ leukemia cells from good responders after ex vivo imatinib treatment. Cells were cultured for O₅ 12 or 24 h with 3 μM imatinib. Western blotting was performed with antibodies to Bim or β-actin. Clinical response and, where known, mutational status of bcr-abl are indicated.

Figure 8 Cell viability was quantified by flow cytometric analysis with PI/FITC- conjugated Annexin V counterstaining. K562, BV173 and vΛ.bcr-c-abt FLCs were treated for 48 h with the indicated concentration of imatinib. X-axis represents staining with FITC labelled Annexin V, and Y-axis represents staining with PI.

Figure 9 Western Blot analysis for Bim expression in MEG-Ol PhI+ leukemia cell line.

Figure 10 Western Blot analysis for Bim expression in K562 cells treated with imatinib with or without the broad spectrum caspase inhibitor ZVAD-fmk.

Figure 11 Effect of the expression of a control RNAi construct on K562 cells: (A)Bcr/Abl expression level, and (B) the response to imatinib were examined.

Figure 12 RT-PCR analysis for bcr/abl expression in bcr-c-abl transformed FLCs cells from wt, bim-/-, bad-/-, bim-/-bad-/-, and vav-bcl-2 transgenic embryos. DETAILED DESCRIPTION OF THE INVENTION

The present invention is predicated, in part, on the determination that the loss of Bim and/or Bad functionality, for example due to loss of expression, serves as a highly sensitive indicator of the reduced sensitivity of a cell to the actions of an antiproliferative agent. Accordingly, the detection of reduced levels of functional Bim and/or Bad relative to normal levels provides a convenient and precise mechanism for assessing potential or actual cellular responsiveness to antiproliferative agents, in particular in the context of predicting or monitoring the responsiveness of a neoplastic condition to chemotherapy.

Accordingly, one aspect of the present invention is directed to a method for assessing the responsiveness of a cell to an antiproliferative agent, said method comprising screening for the level of functional Bim and/or Bad protein and/or gene expression in said cell wherein a decrease in the level of said protein and/or gene expression relative to normal levels is indicative of reduced responsiveness.

Reference to "cell" should be understood as a reference to any normal or abnormal cell. The subject cells may have been freshly isolated from an individual (such as an individual who may be the subject of treatment) or they may have been sourced from a non-fresh source, such as from a culture (for example, where cell numbers were expanded and/or the cells were cultured so as to render them receptive to differentiative signals) or a frozen stock of cells (for example, an established cell line), which had been isolated at some earlier time point either from an individual or from another source. It should also be understood that the subject cells, prior to undergoing analysis, may have undergone some other form of treatment or manipulation, such as but not limited to enrichment or purification, modification of cell cycle status or the formation of a cell line. Accordingly, the subject cell may be a primary cell or a secondary cell. A primary cell is one which has been isolated from an individual. A secondary cell is one which, following its isolation, has undergone some form of in vitro manipulation such as the preparation of a cell line, prior to the application of the method of the invention. Preferably, the subject cell is an abnormal cell and even more preferably a neoplastic cell. The present invention therefore more particularly provides a method for assessing the responsiveness of a neoplastic cell to an antiproliferative agent, said method comprising screening for the level of functional Bim and/or Bad protein and/or gene expression in said cell wherein a decrease in the level of said protein and/or gene expression relative to normal levels is indicative of reduced responsiveness.

Reference to a "neoplastic cell" should be understood as a reference to a cell exhibiting abnormal growth. The term "growth" should be understood in its broadest sense and includes reference to proliferation.

The phrase "abnormal growth" in this context is intended as a reference to cell growth which, relative to normal cell growth, exhibits one or more of an increase in the rate of cell division, an increase in the number of cell divisions, a decrease in the length of the period of cell division, an increase in the frequency of periods of cell division or uncontrolled proliferation and evasion of apoptosis. Without limiting the present invention in any way, the common medical meaning of the term "neoplasia" refers to "new cell growth" that results as a loss of responsiveness to normal growth controls, eg. to neoplastic cell growth. Neoplasias include "tumours" which may be either benign, pre-malignant or malignant. The term "neoplasm" should be understood as a reference to a lesion, tumour or other encapsulated or unencapsulated mass or other form of growth which comprises neoplastic cells.

The common medical meaning of the term "neoplasia" refers to "new cell growth" that results as a loss of responsiveness to normal growth controls, e.g. to neoplastic cell growth. A "hyperplasia" refers to cells undergoing an abnormally high rate of growth. However, as used herein, the terms "neoplasia" and "hyperplasia" can be used interchangeably, referring generally to cells experiencing abnormal cell growth rates. Neoplasias and hyperplasias include "tumours" which may be either benign, pre-malignant or malignant. The term "neoplasm" should be understood as a reference to a lesion, tumour or other encapsulated or unencapsulated mass or other form of growth which comprises neoplastic cells.

As used herein, the terms "hyperproliferative" and "neoplastic" are used interchangeably and refer to those cells in an abnormal state or condition characterized by rapid proliferation or neoplasm. The terms are meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues or organs irrespective of histopathologic type or state of invasiveness. "Pathologic hyperproliferative" cells occur in disease states characterized by malignant tumour growth.

The term "neoplasm", in the context of the present invention should be understood to include reference to all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly or non-malignantly transformed cells, tissues or organs irrespective of histopathologic type or state of invasiveness.

The term "carcinoma" is recognised by those skilled in the art and refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostate carcinomas, endocrine system carcinomas and melanomas. Exemplary carcinomas include those forming from tissue of the breast. The term also includes carcinosarcomas, e.g. which include malignant tumours composed of carcinomatous and sarcomatous tissues. An "adenocarcinoma" refers to a carcinoma derived from glandular tissue or in which the tumour cells form recognisable glandular structures.

The neoplastic cells comprising the neoplasm may be any cell type, derived from any tissue, such as an epithelial or non-epithelial cell. Reference to the terms "malignant neoplasm" and "cancer" and "carcinoma" herein should be understood as interchangeable.

The term "neoplasm" should be understood as a reference to a lesion, tumour or other encapsulated or unencapsulated mass or other form of growth which comprises neoplastic cells. The neoplastic cells comprising the neoplasm may be any cell type, derived from any tissue, such as an epithelial or non-epithelial cell. Examples of neoplasms and neoplastic cells encompassed by the present invention include, but are not limited to central nervous system tumours, retinoblastoma, neuroblastoma and other paediatric tumours, head and neck cancers (e.g. squamous cell cancers), breast and prostate cancers, lung cancer (both small and non-small cell lung cancer), kidney cancers (e.g. renal cell adenocarcinoma), oesophagogastric cancers, hepatocellular carcinoma, pancreaticobiliary neoplasias (e.g. adenocarcinomas and islet cell tumours), colorectal cancer, cervical and anal cancers, uterine and other reproductive tract cancers, urinary tract cancers (e.g. of ureter and bladder), germ cell tumours (e.g. testicular germ cell tumours or ovarian germ cell tumours), ovarian cancer (e.g. ovarian epithelial cancers), carcinomas of unknown primary, human immunodeficiency associated malignancies (e.g. Kaposi's sarcoma), lymphomas, leukemias, malignant melanomas, sarcomas, endocrine tumours (e.g. of thyroid gland), mesothelioma and other pleural or peritoneal tumours, neuroendocrine tumours and carcinoid tumours.

The present invention is therefore preferably directed to a method for assessing the responsiveness of a malignant neoplastic cell to an antiproliferative agent, said method comprising screening for the level of functional Bim and/or Bad protein and/or gene expression in said cell wherein a decrease in the level of said protein and/or gene expression relative to normal levels is indicative of reduced responsiveness.

Reference to an "antiproliferative agent" should be understood as a reference to any agent which acts to reduce the rate or level of cellular proliferation. The subject agent may function by any suitable mechanism including, but not limited to, inducing apoptosis, modulating cellular microtubule structure (eg. promoting microtubule polymerisation), inhibiting tyrosine kinase mediated signalling, antagonising cell surface receptor binding (eg. EGFR & VEGFR inhibitors), modulating glucocorticoid receptor functioning, downregulating angiogenesis (eg. inhibiting VEGF functioning) or otherwise inducing cell death or reducing cellular proliferation events. Without limiting the present invention to any one theory or mode of action, and by way of exemplification, Gleevec (also called imatinib) is an example of a tyrosine kinase inhibitor. It works efficiently in the treatment of CML, cases of AML that have a bcr-abl chromosomal translocation and in certain (rare) cases of gastric lymphoma. Other tyrosine kinase inhibitors include gefitinib, used for treatment of certain lung cancers, while still others are being developed for the treatment of lymphomas, leukemias, colon cancer, brain tumours etc. It is understood that Bim is critical for tumour cell killing using these drugs. Glucocorticoids, such as dexamethasone, bind to the glucocorticoid receptor, which is a transcription factor, and thereby cause upregulation of certain genes and downregulation of others. Glucocorticoids are used (in combination with other chemotherapeutic drugs) in the treatment of certain lymphomas. Again, it has been shown that Bim is essential for killing by dexamethasone. In another example, Taxol promotes excessive polymerisation of microtubules. It is used in the treatment of a range of cancers (usually in combination with other chemotherapeutic drugs). Bim is critical for Taxol-induced killing of at least certain types of cells. Avastin and related drugs inhibit VEGF and/or its receptor

(VEFG-R), which promote growth of blood vessels (angiogenesis). These drugs are new and are being used or trialed in the treatment of advanced breast cancer, lung cancer, colon cancer and other cancers. Examples of antiproliferative agents which act to reduce the rate or level of cellular proliferation are shown in Table 1.

TABLE 1

Antiproliferative Agent Structure

Avastin (Bevacizumab)

Gefitinib

Dexamethasone

Taxol

Imatinib (Gleevec)

It should also be understood that the subject antiproliferative agent is one which mediates its functional objective via the Bim signalling pathway. This may be effected in either a direct or an indirect manner. For example, the agent may directly act on either Bim or upstream signalling molecules. Alternatively, the agent may act on some other aspect of cellular functioning which itself acts to modulate Bim mediated signalling.

Preferably, the present invention is directed to a method for assessing the responsiveness of a malignant neoplastic cell to an antiproliferative agent, which agent induces death, said method comprising screening for the level of functional Bim and/or Bad protein and/or gene expression in said cell wherein a decrease in the level of said protein and/or gene expression relative to normal levels is indicative of reduced responsiveness.

More preferably, said cell death is apoptosis.

According to this preferred embodiment, the present invention provides a method for assessing the responsiveness of a malignant neoplastic cell to an antiproliferative agent, which agent induces apoptosis, said method comprising screening for the level of functional Bim and/or Bad protein and/or gene expression in said cell wherein a decrease in the level of said protein and/or gene expression relative to normal levels is indicative of reduced responsiveness.

Reference to "induce" should be understood to include reference to the subject agent either directly or indirectly inducing apoptosis, as described above.

Reference to "Bim" and "Bad" should be understood as a reference to all forms of these molecules and to fragments, mutants or variants thereof. It should also be understood to include reference to any isoforms which may arise from alternative splicing of Bim or Bad mRNA or mutant or polymorphic forms of Bim or Bad. For example, there are two known, isoforms of human Bim, these being Bini_L and BUTIEL, and three known isoforms of murine Bim, these being Binis, BiniL and Bini_EL- Without limiting the present invention to any one theory or mode of action, Bim is known as a "BH3-only" protein since the only Bcl-2 homology region which it encompasses is BH3. It thereby forms a novel member of a Bcl-2 related BH3-only pro-apoptotic group which also comprises, for example, Bik/Nbk, Bid and Hrk. However, Bim is the only BH3-only protein for which splice variants exist, thereby resulting in the expression of a variety of isoforms. The BH3-only proteins share with each other and the Bcl-2 family at large only the 9-16 amino acid BH3 region and they are essential for initiation of apoptosis signalling [Huang, 2000]. BH3-only proteins are regulated by a range of transcriptional and post-translational mechanisms [Puthalakath et al. Cell Death Differ. 2002, 9(5):505-12] and experiments with gene knockout mice have shown that different members of this subgroup are required for the execution of different death stimuli [Huang, 2000]. For example, Bim is needed for cytokine withdrawal-induced apoptosis [Bouillet et al, 1999, Science, 286:1735-1738] and for killing of autoreactive lymphocytes [Bouillet et al, Nature 2002, 415(6874):922-6].

Reference to Bim and Bad being "functional" should be understood as a reference to the capacity of these protein molecules to perform their normal range of activities. To this end, changes to the level of functional Bim or Bad can be caused in two ways, as follows:

(i) changes to the concentration of Bim or Bad expression product may occur. For example intracellular concentrations of Bim or Bad may be either reduced or increased to abnormal levels, for example due to the existence of mutations in the Bim or Bad promoter which thereby alter its level of expression. Without limiting the present invention in any way, both under expression and overexpression of Bim have been shown to result in reduced or eliminated responsiveness to apoptosis inducing agents such as imatinib; or

(ii) changes to the activity of Bim or Bad may occur. This may or may not also involve changes to the concentration of Bim or Bad. The activity of these molecules may be modulated by any one of a number of mechanisms including, for example, amino acid sequence mutations or truncations which impact on the range or level of Bim or Bad activities. Accordingly, in this particular embodiment the level of functional Bim or Bad is altered without necessarily changing absolute intracellular concentrations of these molecules.

In a preferred embodiment the changes to Bim or Bad levels which one is screening for are changes to the absolute intracellular concentrations of these molecules.

As detailed hereinbefore, the present invention is predicated on the finding that reduced intracellular levels of functional Bim and/or Bad expression result in reduced responsiveness to the actions of antiproliferative agents. By "responsiveness" is meant the capacity of the subject cell to undergo a slowing or cessation of its proliferative activity. Accordingly, it should be understood that although the present invention is exemplified in terms of cells which produce no Bim and have been found to be completely resistant to the effects of apoptosis inducing agents such as imatinib, in other situations a cell may produce lower than normal levels of Bim and/or Bad, thereby correlating to a reduced level of responsiveness, but not necessarily the complete absence of any responsiveness. The method of the present invention is nevertheless very useful in this situation since determination of the existence of sub-optimal potential to respond to a particular antiproliferative agent may indicate that consideration should be given to alternative treatment options.

In a most preferred embodiment the present invention is directed to a method for assessing the responsiveness of a malignant neoplastic cell to an antiproliferative agent, which agent induces apoptosis, said method comprising screening for the level of functional Bim and/or Bad protein and/or gene expression in said cell wherein the absence of said protein and/or gene expression is indicative of unresponsiveness.

The method of the present invention is predicated on the correlation of functional Bim and/or Bad levels in a cellular population with normal levels of these molecules. The "normal level" is the level of Bim and/or Bad in a corresponding cellular sample which does not exhibit reduced responsiveness to the actions of an antiproliferative agent.

Accordingly, the present invention is directed to screening for changes to Bim and/or Bad levels relative either to a normal reference level (or normal reference level range) or to an earlier Bim and/or Bad level result determined from a particular in vitro biological sample or subject. A normal reference level is the Bim and/or Bad level from a relevant biological sample of an in vitro source or a sample from a subject or group of subjects who are not exhibiting reduced responsiveness. In a preferred embodiment, said normal reference level is the level determined from one or more subjects of a relevant cohort to that of the subject being screened by the method of the invention. By "relevant cohort" is meant a cohort characterised by one or more features which are also characteristic of the subject who is the subject of screening. These features include, but are not limited to, age, gender, ethnicity or health status, for example.

This reference level may be a discrete figure or may be a range of figures. The reference level may vary between individual classes of Bim and/or Bad molecules (such as the differentially spliced forms of Bim). The method of the invention should be understood to encompass all suitable forms of analysis such as the analysis of test results relative to a standard result which reflects individual or collective results.

It should be understood that the present method may be performed in the context of analysing the responsiveness of an in vitro cellular population, such as a cell line, or the responsiveness of a cellular population which has been harvested from a mammal. Although the present invention will likely have its most significant application in terms of assessing the neoplastic cells of a patient, it is also conceivable that one might analyse a cellular population, such as a cell line, for the purpose of analysing the phenomenon of the existence of resistance or development of resistance to the apoptotic effects of a chemotherapy agent.

To this end, in a related aspect the present invention is directed to a method for assessing the responsiveness to an antiproliferative agent of a cell in a mammal or in a biological sample derived from said mammal, said method comprising screening for the level of functional Bim and/or Bad protein and/or gene expression in said cell wherein a decrease in the level of said protein and/or gene expression relative to normal levels is indicative of reduced responsiveness.

Preferably, said cell is a neoplastic cell and even more preferably a malignant neoplastic cell.

Still more preferably, said antiproliferative agent induces cell death, in particular apoptosis. The term "mammal" as used herein includes humans, primates, livestock animals (eg. horses, cattle, sheep, pigs, donkeys), laboratory test animals (eg. mice, rats, guinea pigs), companion animals (eg. dogs, cats) and captive wild animal (eg. kangaroos, deer, foxes). Preferably, the mammal is a human or a laboratory test animal. Even more preferably, the mammal is a human.

In accordance with the present invention, one may screen for Bim expression levels and/or Bad expression levels. Without limiting the present invention to any one theory or mode of action, Bim is thought to function as the critical protein for facilitating the apoptotic effects of antiproliferative agents such as imatinib. However, as foreshadowed hereinbefore, variation can and does exist in terms of the level and extent of unresponsiveness across a neoplastic cell population. For example, and without limiting the present invention in any way, in the context of imatinib resistance it has been determined that there exists a level of approximately 70% unresponsiveness across cellular populations where Bim alone is downregulated, approximately 20% unresponsiveness across a cellular population where Bad alone is downregulated and approximately 100% unresponsiveness where both Bad and Bim are downregulated. Accordingly, in a most preferred embodiment one is screening for modulation, in particular downregulation, of both Bad and Bim levels. Nevertheless, it should also be understood that the present invention extends to screening for Bad or Bim alone, in addition to screening for one or both of these molecules together with any other molecule which is of interest in the context of a particular situation, for example Bmf.

The method of the present invention has application in relation to a broad range of situations including, but not limited to:

(i) screening cellular populations, such as neoplastic cell populations, prior to the commencement of chemotherapy in order to determine the level of chemoresponsiveness

(ii) monitoring neoplastic cell populations during chemotherapy in order to identify modulation of chemoresponsiveness

(iii) monitoring a patient during or after a therapeutic treatment regime, irrespective of whether that regime is chemotherapeutic or not, in order to identify the emergence of chemoresistant cellular populations, such as chemoresistant metastises

(iv) to screen for or monitor normal cellular populations in the context of the extent of their Bim and/or Bad expression levels and, thereby, assess the likely extent and nature of unwanted normal cell death during a selected antiproliferative treatment regime.

Accordingly, the method of the present invention can be applied to assess the actual responsiveness of a cell to an antiproliferative agent, that is, after the cell has been exposed to the agent. Alternatively, it can be applied to assess the likely or potential response which may occur if that cell was exposed to the agent.

To this end, although the preferred method is to detect a decrease in Bim and/or Bad levels in order to diagnose the onset or predisposition to the onset of resistance to the effects of an antiproliferative agent, the detection of an increase in the levels of these molecules may be desired under certain circumstances. For example, to monitor for a reversal of chemoresistance, such as in the circumstance where a patient exhibiting chemoresistance is subjected to a treatment regime directed to upregulating or inducing functional Bim and/or Bad expression or otherwise increasing the intracellular levels of these molecules in resistant cells.

This aspect of the present invention also enables one to monitor the progression of a condition characterised by unwanted cellular proliferation, such as a neoplastic condition. By "progression" is meant the ongoing nature of the condition in terms of the existence or emergence of resistance to treatment with an antiproliferative agent. To this end, it should be understood that although the primary application of this technology is likely to be in the context of neoplastic conditions, the present invention should not be limited in this way. For instance, applications of the present technology lie with other conditions characterised by some form of unwanted cellular proliferation, such as autoimmunity.

Accordingly, another aspect of the present invention relates to a method for monitoring the responsiveness of a cellular population to an antiproliferative agent, said method comprising screening for modulation of the level of functional Bim and/or Bad protein and/or gene expression wherein a decrease in the level of said protein and/or gene expression in the subject cellular population relative to a previously obtained level or a normal level is indicative of the maintenance or worsening of responsiveness and an increase in said level is indicative of an improvement in said responsiveness.

Preferably, said cellular population is a neoplastic population and more preferably a malignant neoplastic population.

Still more preferably, said antiproliferative agent induces cell death, in particular apoptosis.

Yet another aspect of the present invention is directed to a method for assessing the actual or likely responsiveness to an antiproliferative agent of a condition characterised by unwanted cellular proliferation to an antiproliferative agent, said method comprising screening for the level of functional Bim and/or Bad protein and/or gene expression wherein a decrease in the level of said protein and/or gene expression relative to normal levels is indicative of reduced responsiveness.

Preferably, said condition is a neoplastic condition and more preferably a malignant neoplastic condition.

In yet another aspect, the present invention relates to a method for monitoring the responsiveness to an antiproliferative agent of a condition characterised by unwanted cellular proliferation, said method comprising screening for modulation of the level of functional Bim and/or Bad protein and/or gene expression wherein a decrease in the level of said protein and/or gene expression in the subject cellular population relative to a previously obtained level or a normal level is indicative of the maintenance or worsening of responsiveness and an increase in said level is indicative of an improvement in said responsiveness.

Yet another aspect of the present invention is directed to assessing and/or monitoring the effectiveness of an antiproliferative treatment regime in a mammal, said method comprising screening for the level of functional Bim and/or Bad protein and/or gene expression by a cellular population of interest wherein a decrease in the level of said protein and/or gene expression relative to normal levels is indicative of reduced responsiveness.

As detailed hereinbefore, it should be understood that although the effectiveness of an antiproliferative treatment regime will generally be assessed in terms of the responsiveness of the unwanted cellular population to the antiproliferative agent, one may also seek to determine the level of responsiveness of normal cells in order to make an assessment of the nature and extent of likely cell death related side effects which may be experienced by the patient.

In the context of this aspect of the present invention, said antiproliferative treatment regime is preferably targeted to a neoplastic condition and still more preferably a malignant neoplastic condition. Still more preferably, said antiproliferative agent induces cell death, in particular apoptosis.

The present invention therefore provides means for assessing both the existence and extent of cellular populations which exhibit reduced or changed responsiveness to an antiproliferative agent.

In accordance with these preferred aspects of the invention, said neoplastic condition is characterised by a neoplasm of the central nervous system tumours, retinoblastoma, neuroblastoma and other paediatric tumours, head and neck cancers (e.g. squamous cell cancers), breast and prostate cancers, lung cancer (both small and non-small cell lung cancer), kidney cancers (e.g. renal cell adenocarcinoma), oesophagogastric cancers, hepatocellular carcinoma, pancreaticobiliary neoplasias (e.g. adenocarcinomas and islet cell tumours), colorectal cancer, cervical and anal cancers, uterine and other reproductive tract cancers, urinary tract cancers (e.g. of ureter and bladder), germ cell tumours (e.g. testicular germ cell tumours or ovarian germ cell tumours), ovarian cancer (e.g. ovarian epithelial cancers), carcinomas of unknown primary, human immunodeficiency associated malignancies (e.g. Kaposi's sarcoma), lymphomas, leukemias, malignant melanomas, sarcomas, endocrine tumours (e.g. of thyroid gland), mesothelioma and other pleural or peritoneal tumours, neuroendocrine tumours and carcinoid tumours.

Reference to a "biological sample" should be understood as a reference to any sample of biological material derived from an animal such as, but not limited to, cellular material, biofluids (eg. blood) which contain cellular material, faeces, tissue biopsy specimens or surgical specimens. The biological sample which is tested according to the method of the present invention may be tested directly or may require some form of treatment prior to testing. For example, a biopsy or surgical sample may require homogenisation prior to testing or it may require sectioning for in situ testing. Alternatively, the cell sample may require permeabilisation prior to testing. Further, to the extent that the biological sample is not in liquid form, (if such form is required for testing) it may require the addition of a reagent, such as a buffer, to mobilise the sample.

To the extent that the target molecule (such as a protein molecule) is present in a biological sample, the biological sample may be directly tested or else all or some of the nucleic acid material present in the biological sample may be isolated prior to testing (assuming that one is screening for mRNA levels, for example, or genomic DNA mutations, such as in promoters or other regulatory regions which may affect Bim or Bad expression). In yet another example, the sample may be partially purified or otherwise enriched prior to analysis. For example, to the extent that a biological sample comprises a very diverse cell population, it may be desirable to select out a sub-population of particular interest, such as enriching for the cell population of which the neoplastic cell forms part. It is within the scope of the present invention for the target cell population or molecules derived therefrom to be pretreated prior to testing, for example, inactivation of live virus or being run on a gel. It should also be understood that the biological sample may be freshly harvested or it may have been stored (for example by freezing) prior to testing or otherwise treated prior to testing (such as by undergoing culturing).

The choice of what type of sample is most suitable for testing in accordance with the method disclosed herein will be dependent on the nature of the situation, such as the nature of the condition being monitored.

Means for screening for changes in Bim and/or Bad levels in a subject, a biological sample derived therefrom or other form of cellular population (such as a cell line), can be achieved by any suitable method, which would be well known to the person of skill in the art. Briefly, one may seek to detect Bim and/or Bad protein in a biological sample. Anti-Bim or anti-Bad immunointeractive molecules may be used to identify the protein directly. In terms of in vivo analyses, anti-Bim and/or anti-Bad immunointeractive molecules could be coupled to medical imaging agents in order to visualise specific binding to the cellular population of interest, in particular following the administration of anti-cancer treatments. This requires, however, that the technique which is utilised facilitates the cellular uptake of the imaging agent. More specifically, these methods include, but are not limited to: (i) In vivo detection of Bim and/or Bad. Molecular Imaging may be used following administration of imaging probes or reagents capable of disclosing altered expression levels of the Bim and/or Bad mRNA or protein expression product in the biological sample.

Molecular imaging [Moore et άl., Nature Medicine, (5:351-355, 2000] is the in vivo imaging of molecular expression that correlates with the macro-features currently visualized using "classical" diagnostic imaging techniques such as X-Ray, computed tomography (CT), MRI, Positron Emission Tomography (PET) or endoscopy.

(ii) Analysis of mRNA expression by Fluorescent In Situ Hybridization (FISH) or other suitable technique, or in extracts from the dead cells by technologies such as Quantitative Reverse Transcriptase Polymerase Chain Reaction (QRTPCR) or

Flow cytometric qualification of competitive RT-PCR products [Wedemeyer et al, Clinical Chemistry 48:9 1398-1405, 2002] or array technologies.

For example, a labelled polynucleotide encoding Bim and/or Bad may be utilized as a probe in a Northern blot of an RNA extract obtained from a biological sample.

Preferably, a nucleic acid extract from the subject is utilized in concert with oligonucleotide primers corresponding to sense and antisense sequences of a polynucleotide encoding Bim and/or Bad, or flanking sequences thereof, in a nucleic acid amplification reaction such as RT PCR, real time PCR or SAGE. A variety of automated solid-phase detection techniques are also appropriate. For example, very large scale immobilized primer arrays (VLSIP S™) are used for the detection of nucleic acids as, for example, described by Fodor et al., 1991 and Kazal et al., 1996. The above genetic techniques are well known to persons skilled in the art.

For example, to detect Bim and/or Bad encoding RNA transcripts, RNA is isolated from a suitable cellular sample. RNA can be isolated by methods known in the art, e.g. using TRIZOL™ reagent (GIBCO-BRL/Life Technologies, Gaithersburg, Md.). Oligo-dT, or random-sequence oligonucleotides, as well as sequence-specific oligonucleotides can be employed as a primer in a reverse transcriptase reaction to prepare first-strand cDNAs from the isolated RNA. Resultant first-strand cDNAs are then amplified with sequence-specific oligonucleotides in PCR reactions to yield an amplified product.

"Polymerase chain reaction" or "PCR" refers to a procedure or technique in which amounts of a preselected fragment of nucleic acid, RNA and/or DNA, are amplified as described in U.S. Patent No. 4,683,195. Generally, sequence information from the ends of the region of interest or beyond is employed to design oligonucleotide primers. These primers will be identical or similar in sequence to opposite strands of the template to be amplified. PCR can be used to amplify specific RNA sequences and cDNA transcribed from total cellular RNA. See generally Mullis et ah, 1987; Erlich, 1989. Thus, amplification of specific nucleic acid sequences by PCR relies upon oligonucleotides or "primers" having conserved nucleotide sequences wherein the conserved sequences are deduced from alignments of related gene or protein sequences. For example, one primer is prepared which is predicted to anneal to the antisense strand and another primer prepared which is predicted to anneal to the sense strand of a cDNA molecule which encodes Bim and/or Bad.

To detect the amplified product, the reaction mixture is typically subjected to agarose gel electrophoresis or other convenient separation technique and the relative presence of Bim and/or Bad specific amplified nucleic acid detected. For example, the Bim and/or Bad amplified nucleic acid may be detected using Southern hybridization with a specific oligonucleotide probe or comparing its electrophoretic mobility with nucleic acid standards of known molecular weight. Isolation, purification and characterization of the amplified Bim and/or Bad nucleic acid may be accomplished by excising or eluting the fragment from the gel (for example, see references Lawn et ah, 1981; Goeddel et ah, 1980), cloning the amplifϊed product into a cloning site of a suitable vector, such as the pCRII vector (Invitrogen), sequencing the cloned insert and comparing the sequence to the known sequence of Bim and/or Bad. The relative amounts of Bim and/or Bad mRNA and cDNA can then be determined.

(iii) Measurement of altered Bim and/or Bad protein levels in a suitable biological sample, either qualitatively or quantitatively, for example by immunoassay, utilising immunointeractive molecules such as monoclonal antibodies.

In one example, one may seek to detect Bim and/or Bad-immunointeractive molecule complex formation. For example, an antibody according to the invention, having a reporter molecule associated therewith, may be utilized in immunoassays. Such immunoassays include but are not limited to radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs) and immunochromatographic techniques (ICTs), Western blotting which are well known to those of skill in the art. For example, reference may be made to "Current Protocols in Immunology", 1994 which discloses a variety of immunoassays which may be used in accordance with the present invention. Immunoassays may include competitive assays. It will be understood that the present invention encompasses qualitative and quantitative immunoassays.

Suitable immunoassay techniques are described, for example, in U.S. Patent Nos. 4,016,043, 4,424,279 and 4,018,653. These include both single-site and two-site assays of the non-competitive types, as well as the traditional competitive binding assays. These assays also include direct binding of a labelled antigen-binding molecule to a target antigen. The antigen in this case is Bim and/or Bad or a fragment thereof.

In one example, a two-site assay is used. A number of variations of these assays exist, all of which are intended to be encompassed by the present invention.

Briefly, in a typical forward assay, an unlabelled antigen-binding molecule such as an unlabelled antibody is immobilized on a solid substrate and the sample to be tested brought into contact with the bound molecule. After a suitable period of incubation, for a period of time sufficient to allow formation of an antibody-antigen complex, another antigen-binding molecule, suitably a second antibody specific to the antigen, labelled with a reporter molecule capable of producing a detectable signal is then added and incubated, allowing time sufficient for the formation of another complex of antibody-antigen-labelled antibody. Any unreacted material is washed away and the presence of the antigen is determined by observation of a signal produced by the reporter molecule. The results may be either qualitative, by simple observation of the visible signal, or may be quantitated by comparing with a control sample containing known amounts of antigen. Variations on the forward assay include a simultaneous assay, in which both sample and labelled antibody are added simultaneously to the bound antibody. These techniques are well known to those skilled in the art, including minor variations as will be readily apparent.

In the typical forward assay, a first antibody having specificity for the antigen or antigenic parts thereof is either covalently or passively bound to a solid surface. The solid surface is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene. The solid supports may be in the form of tubes, beads, discs of microplates, or any other surface suitable for conducting an immunoassay. The binding processes are well known in the art and generally consist of cross-linking covalently binding or physically adsorbing, the polymer-antibody complex is washed in preparation for the test sample. An aliquot of the sample to be tested is then added to the solid phase complex and incubated for a period of time sufficient and under suitable conditions to allow binding of any antigen present to the antibody. Following the incubation period, the antigen-antibody complex is washed and dried and incubated with a second antibody specific for a portion of the antigen. The second antibody has generally a reporter molecule associated therewith that is used to indicate the binding of the second antibody to the antigen.

The amount of labelled antibody that binds, as determined by the associated reporter molecule, is proportional to the amount of antigen bound to the immobilized first antibody.

An alternative method involves immobilizing the antigen in the biological sample and then exposing the immobilized antigen to specific antibody that may or may not be labelled with a reporter molecule. Depending on the amount of target and the strength of the reporter molecule signal, a bound antigen may be detectable by direct labelling with the antibody. Alternatively, a second labelled antibody, specific to the first antibody is exposed to the target-first antibody complex to form a target-first antibody-second antibody tertiary complex. The complex is detected by the signal emitted by the reporter molecule.

From the foregoing, it will be appreciated that the reporter molecule associated with the antigen-binding molecule may include the following :-

(a) direct attachment of the reporter molecule to the antibody;

(b) indirect attachment of the reporter molecule to the antibody; i.e., attachment of the reporter molecule to another assay reagent which subsequently binds to the antibody; and

(c) attachment to a subsequent reaction product of the antibody.

The reporter molecule may be selected from a group including a chromogen, a catalyst, an enzyme, a fluorochrome, a chemiluminescent molecule, a paramagnetic ion, a lanthanide ion such as Europium (Eu³⁴), a radioisotope including other nuclear tags and a direct visual label.

In the case of a direct visual label, use may be made of a colloidal metallic or non- metallic particle, a dye particle, an enzyme or a substrate, an organic polymer, a latex particle, a liposome, or other vesicle containing a signal producing substance and the like.

A large number of enzymes suitable for use as reporter molecules is disclosed in U.S. Patent Nos. U.S. 4,366,241, U.S. 4,843,000, and U.S. 4,849,338. Suitable enzymes useful in the present invention include alkaline phosphatase, horseradish peroxidase, luciferase, β-galactosidase, glucose oxidase, lysozyme, malate dehydrogenase and the like. The enzymes may be used alone or in combination with a second enzyme that is in solution.

Suitable fluorochromes include, but are not limited to, fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), R-Phycoerythrin (RPE), and Texas Red. Other exemplary fluorochromes include those discussed by Dower et al., International Publication No. WO 93/06121. Reference also may be made to the fluorochromes described in U.S. Patent Nos. 5,573,909 (Singer et al), 5,326,692

(Brinkley et al). Alternatively, reference may be made to the fluorochromes described in U.S. Patent Nos. 5,227,487, 5,274,113, 5,405,975, 5,433,896, 5,442,045, 5,451,663, 5,453,517, 5,459,276, 5,516,864, 5,648,270 and 5,723,218.

In the case of an enzyme immunoassay, an enzyme is conjugated to the second antibody, generally by means of glutaraldehyde or periodate. As will be readily recognised, however, a wide variety of different conjugation techniques exist which are readily available to the skilled artisan. The substrates to be used with the specific enzymes are generally chosen for the production of, upon hydrolysis by the corresponding enzyme, a detectable colour change. Examples of suitable enzymes include those described supra. It is also possible to employ fluorogenic substrates, which yield a fluorescent product rather than the chromogenic substrates noted above. In all cases, the enzyme-labelled antibody is added to the first antibody- antigen complex, allowed to bind, and then the excess reagent washed away. A solution containing the appropriate substrate is then added to the complex of antibody-antigen-antibody. The substrate will react with the enzyme linked to the second antibody, giving a qualitative visual signal, which may be further quantitated, usually spectrophotometrically, to give an indication of the amount of antigen which was present in the sample.

Alternately, fluorescent compounds, such as fluorescein, rhodamine and the lanthanide, europium (EU), may be chemically coupled to antibodies without altering their binding capacity. When activated by illumination with light of a particular wavelength, the fluorochrome-labelled antibody adsorbs the light energy, inducing a state to excitability in the molecule, followed by emission of the light at a characteristic colour visually detectable with a light microscope. The fluorescent- labelled antibody is allowed to bind to the first antibody-antigen complex. After washing off the unbound reagent, the remaining tertiary complex is then exposed to light of an appropriate wavelength. The fluorescence observed indicates the presence of the antigen of interest. Immunofluorometric assays (IFMA) are well established in the art and are particularly useful for the present method. However, other reporter molecules, such as radioisotope, chemiluminescent or bioluminescent molecules may also be employed.

(iv) Determining altered protein expression based on any suitable functional test, enzymatic test or immunological test in addition to those detailed in point (iii) above.

(vi) Nanotechnology-related techniques such as those outlined in Ferrari [Nature

Reviews Cancer 5:161-171, 2005] and Duncan [Nature Reviews Drug Discovery, 2:347-360, 2003].

Another aspect of the present invention provides a diagnostic kit comprising an agent for detecting Bim and/or Bad or a nucleic acid molecule encoding Bim and/or Bad and reagents useful for facilitating the detection by said agent. The agent may be an antibody or other suitable detection molecule. The present invention further contemplates the use of an interactive molecule directed to Bim and/or Bad in the manufacture of a quantitative or semi-quantitative diagnostic kit to assess cellular responsiveness to an antiproliferative agent. The kit may come with instructions for use and may be automated or semi-automated or in a form which is compatible with an automated machine or software.

The present invention is now described by reference to the following non-limiting examples.

EXAMPLE 1

PRO-APOPTOTIC BH3-ONLY PROTEIMS BIM AND BAD MEDIATE IMATINIB MESYLATE-INDUCED KILLING OF BCR/ABL-POSITIVE LEUKEMIC CELLS IN CULTURE AND IN PATIENTS

Materials and Methods

Cell Lines

K562 is an erythroleukemia, BVl 73 is a pre-B cell leukemia, and MEG-Ol is a megakaryoblastic leukemia cell lines, all derived from Ph¹+ CML patients in blast-crisis phase. Cells were maintained as suspension cultures in RPMI- 1640 with 10% or 15% heat-inactivated foetal calf serum (FCS) and 2mM L-glutamate.

Expression Constructs and Cell Transfection

Expression constructs for human Bcl-2, BCI-XL and a dominant-interfering mutant of FADD/M0RT1 (FADD-DN), all containing an N-terminal FLAG epitope tag and the puromycin resistance gene were described previously [Huang, 1999]. A Gene-Pulser (BioRad Laboratories, Hercules, CA) was used for electroporation and selection was performed with 5 μg/mL puromycin. Cells were single cell cloned by limiting dilution. FLAG-tagged proteins were detected by cytoplasmic immunofluorescence staining with anti-FLAG antibody (Sigma) and flow cytometric analysis in a FACScan (Becton Dickinson, Franklin Lakes, NJ).

RNA Interference (RNAi) for Targeting Bim Expression

Generation of the anti-Bim short hairpin RNA construct cloned into pSUPER vector with the neomycin-resistant gene was described elsewhere. We also generated a control construct in which the target sequence was scrambled. Electroporation was performed and positive clones were selected with 1 mg/mL Geneticin (Gibco BRL, Grand Island, NY). Cell lines were single cell cloned by limiting dilution.

Mice and Retroviral Infection of Fetal Liver Cells

All experiments with mice were performed according to the guidelines of the Melbourne Directorate Animal Ethics Committee. The bim '^~ [Bouillet et al, 1999, supra], bad'^' [Ranger et al, Proc Natl Acad Sci USA. 2003; 100:9324-9329] and vav-bcl-2 transgenic mice have all been described. These animals were either generated on a C57BL/6 background or had been backcrossed with C57BL/6 mice for >8 generations. The him ^1' bad'^' mice were produced by intercrossing Mm^"1" and bad^1' mice. Fetal liver cells (FLCs) were harvested from 14.5 day old embryos and pre-cultured in DMEM containing 20% FCS, 2mM L-glutamate, 50μM 2-mercaptoethanol and cytokines for 24h (50 ng/mL stem cell factor, SCF, 50 ng/mL thrombopoietin: TPO, 500 ng/mL fetal liver kinase-ligand, FIkL and 100 LVmL interleukin-6, IL-6; all gifts of Dr. W Alexander, Walter and Eliza Hall Institute). The pMPZen. bcr-c-abl expression vector has been described [Hariharan et al, Oncogene Res 1988;3:387-399] and was transfected to packaging Phoenix cells using FuGENEό (Roche, Basel, Switzerland). Pre-cultured FLCs were infected by co-cultivation on Retronectin (Takara, Kusatsu, Japan)-coated plates in virus containing medium supplemented with cytokines (see above). Infected cells expressing Bcr/Abl were selected by growth in the absence of cytokines.

Western Blotting

Protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS/PAGE) and then electroblotted onto a Hybond-nitrocellulose membrane (Amersham Biosciences, Upsula, Sweden). The membranes were saturated with 5% (wt/vol) non-fat dry milk in PBS with 0.1% (vol/vol) Tween 20 (Sigma). Antibodies against human Bcl-2 (Bcl-2-100), mouse Bcl-2 (clone 3Fl 1), Bcl-w (clone 13F9), BcI-X (Transduction Laboratory, Lexington, KY), Bim (clone 3C5 or polyclonal Ab from Stressgen, British Columbia, Canada), Bad (Stressgen), phospho-Bad (Ser¹¹²) (Cell Signaling Technologies, Beverly, MA), phospho-Bad (Ser¹³⁶) (Cell Signaling Technologies), Bax (Upstate, Lake Placid, NY), mouse Bmf (clone 12E10), Bmf (polyclonal Ab from Alexis, San Diego, CA), Heat Shock Protein 70 (Hsp70) (N6; a gift from Dr R Anderson, Peter MacCallum Cancer Institute, Melbourne), McI-I (Dako, Glostrup, Denmark) and β-actin (Sigma) were utilized. Detection was performed with horseradish peroxidase-conjugated secondary Abs (specific to rat, mouse, hamster or rabbit IgG) and enhanced chemiluminescence (Amersham Biosciences).

Two-Dimensional (2-D) Gel Electrophoresis

2-D protein electrophoresis was performed using the IPGphor isoelectric focusing (IEF) system (Amersham Pharmacia Biotech, Piscataway, NJ). Protein lysates were loaded onto IPG gels, rehydrated at 20⁰C for 12 h, and subjected to IEF for at least 12,000 volt x h. After the equilibration with SDS/PAGE buffer for 10 minutes at room temperature, the IPG gel was subjected to second-dimensional SDS/PAGE. Transfer, immunoblotting and visualization were performed as described above (Western blotting).

Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)

Total RNA was extracted using the Micro-to-Midi Total RNA Extraction Kit (Invitrogen, San Diego, CA), and 20 ng/μL total RNA was subjected to RT. 1 μL cDNA was subjected to PCR using primers for him, bcr/abl, bmf ox puma. Primers were designed as follows: murine bmf: forward (fwd) 5'-cgagtgttcaccctgga-3' (SEQ ID NO:1), reverse (rev) 5'- ccccttccctgttttcttgt -3' (SEQ ID NO:2), human puma: fwd 5'-gacctcaacgcacagta-3' (SEQ ID NO:3), rev 5'-ctaattgggctccatct-3' (SEQ ID NO:4), murine puma: fwd 5'- gcccagcagcacttagagtc-3' (SEQ ID NO:5), rev 5'-tgtcgatgctgctcttcttg-3' (SEQ ID NO:6). Primers for bim, bcr/abl and human όm/were utilized as described, β-actin was used as a control for the quality and abundance of RNA. CeIl Death Assays and MTS Assay

Cell death was assessed either by tryptan-blue staining and cell counting in a hemocytometer or by staining with propidium iodide (PI) and flow cytometric analysis. The MTS assay was performed with the cell proliferation assay kit (MBL, Nagoya, Japan) as described elsewhere [Kuroda et al, Blood, 2003; 102:2229-2235]. For clonogenic survival assays, K562 cells were seeded at 2.0 x 10⁵ cells/mL and treated with 1.5 or 3.0 μM imatinib (Novartis Pharma AG, Basel, Switzerland) for 24 or 48h. Cells were then washed three times in complete medium to remove imatinib and before plating on 0.3% soft agar containing 20% FCS and 30% DMEM. Clonogenic potency was examined by counting colony numbers after 8 days of culture.

Analysis of Primary Ph¹+ Leukemia Samples

Bone marrow (BM) samples were harvested from four Ph¹+ leukemia patients before and after treatment by imatinib with informed consent according to the Declaration of Helsinki. BM mononuclear cells were isolated by centrifugation with Ficoll-Hypaque, resuspended in RPMI1640 containing 10% FCS at 2.0 x 10⁵ cells/mL and exposed to 3.0 μM imatinib for 12 or 24h. Protein extraction and Western blotting were performed as described.

Results

Imatinib Activates the Bcl-2-Regulated Apoptotic Pathway in Ph¹+ Leukemia Cell Lines

The Ph¹+ human leukemia lines K562 and BVl 73 were chosen for initial studies on the effects of imatinib. Imatinib inhibited cell proliferation with similar efficiency in both cell lines (Figure IA), but BV 173 cells were less susceptible to imatinib-induced cell death than K562 cells (Figure IB, Figure 8). Western blot analysis showed clear differences in Bcl-2 expression between K562 and BVl 73 cells. Upon treatment with imatinib, Bcl-2 levels dropped significantly in K562 cells but were maintained or even increased slightly in BVl 73 cells (Figure 1C). Imatinib treatment caused no obvious changes in Bcl-x_L, McI- 1 or Bcl-w in either of the two cell lines (Figure 1C). These results indicate that the pro- apoptotic effects of imatinib may depend on cell context and suggest that sustained Bcl-2 expression may explain the relative resistance of BVl 73 cells.

To examine this further and to determine whether imatinib triggers apoptosis via the Bcl-2- regulated or the 'death receptor' pathway, multiple K562 subclones over-expressing different levels of Bcl-2 (K562/Bcl-2), Bcl-x_L, or a dominant inhibitor of FADD (FADD- DN), which blocks death receptor signalling (Figure ID and not shown). Bcl-2 and BCI-X_L protected K562 cells in a dose dependent manner from the pro-apoptotic effects of imatinib, whereas FADD-DN had no effect (Figures IE and F and data not shown). K562 cells are insensitive to FasL and TNF; so the function of FADD-DN in these cells could not be demonstrated, but expression of this vector at similar levels was previously shown to protect several human lymphoma lines from FasL [Huang, 1999]. These results demonstrate that imatinib kills Ph¹+ leukemia cells by activating the Bcl-2-regulated apoptotic pathway.

Imatinib Increases Bim Expression in Ph¹+ Leukemia Cell Lines

Since imatinib kills cells by the Bcl-2 regulated pathway and since BH3-only proteins are essential initiators in this pathway [Huang, 2000], the effects of imatinib on the levels of some of these proteins were examined. Treatment with imatinib induced a rapid and sustained increase in the levels of both BiniE_L and Bini_L proteins in K562 (Figure 2A), BV173 (Figure 2B) and MEG-01 PhI+ leukemia cells (Figure 9). When K562 cells were pre-treated with the pan-caspase inhibitor Z-VAD-fmk, imatinib treatment still caused a marked increase in BimEL and BimL (Figure 10). These results demonstrate that imatinib causes an increase in BimEL and BimL expression during the early phase of apoptosis by a mechanism that is independent of caspase activation. Imatinib Increases Bim Expression through both Transcriptional and Post-translational Mechanisms

To investigate the mechanisms of imatinib-induced Bim induction, semi-quantitative RT- PCR were performed. Treatment of K562 cells with 1.0 μM increased bim mRNA levels within 3 h, indicating its transcriptional upregulation (Figure 2C).

BiniEL from imatinib-treated K562 cells migrated more rapidly in SDS-PAGE than Biπi_EL from untreated cells (Figure 2A and Figure 10), indicating that BCR-ABL inactivation might cause a change in Bini_EL phosphorylation. Accordingly, 2-D gel electrophoresis and Western blotting were used to examine the phosphorylation status of Bim before and after imatinib treatment. Figure 2D shows that BiniEL proteins from imatinib-treated K562 cells migrated more closely to the anode than Bim_EL proteins from untreated cells, indicating the accumulation of lesser phosphorylated forms of Bim. Collectively, these results show that imatinib causes increased Bim expression through both transcriptional up-regulation and post-translational modification.

Bim Is the Critical but not the only BH3-Only Protein Mediating Imatinib-induced Killing of Ph + Leukemia Lines

To examine the role of Bim in imatinib-induced cell killing, multiple subclones of K562 and BVl 73 cells in which Bim levels were suppressed to different degrees by stable expression of an RNAi construct were generated (Figure 3A). Expression of a control RNAi construct had no effect on cell proliferation, Bcr/Abl expression levels, or the response to imatinib (Figure 11). In contrast, Bim knock-down clones (K562/shBim and BV173/shBim) were generally less susceptible to imatinib-induced killing than the parental cells (Figures 3B and C). Importantly, the degree of Bim suppression correlated with the extent of protection from imatinib-induced cell killing in both K562 and BV173 subclones (Figures. 3B and C). To examine the role of Bim in long-term survival and retention of proliferative capacity, parental K562 cells, Bim knock-down K562 subclone #18 and Bcl-2 over-expressing cells were treated for 24 or 48 h with imatinib, washed and surviving cells enumerated in agar colony assays. Bim knock-down significantly rescued clonogenic potency with K562/shBim#18 cells forming twice or >10 times as many colonies as parental K562 cells after 24 or 48 h of imatinib treatment (Figure 3D). Compared to K562/shBim#18 cells, Bcl-2 over-expression promoted significantly greater retention of clonogenic potential after 48 h of imatinib treatment.

This observation and the finding that many Bim knock-down cells eventually died after exposure to imatinib in short-term culture assays (Figures 3B and C) indicated that the residual amount of Bim in these cells might be sufficient for cell killing. Alternatively, additional pro-apoptotic molecules might contribute to imatinib-induced cell killing. It was found that Bim levels were increased after imatinib treatment even in K562/shBim#18 and BV173/shBim#4 clones, although Bim was almost undetectable in these clones in the basal state (Figure 4A). In addition, it was observed that Bad became de-phosphorylated and that Bmf was up-regulated in response to imatinib treatment in both K562 and BVl 73 cells, including the Bim-knock-down clones (Figure 4A). Semi-quantitative RT-PCR analysis showed that imatinib caused increased transcription of bmf but not puma in K562 cells. Collectively, these results demonstrate that Bim is critical for imatinib-induced apoptosis in Ph¹+ leukemia cell lines and they indicate that additional BH3-only proteins, particularly Bad and Bmf, may contribute to cell killing.

Combined Loss of Bim and Bad Protects Bcr/Abl-Transformed Mouse Fetal Liver Cells from Imatinib -Lnduced Apoptosis

To avoid the complication of incomplete loss of protein expression in RNAi vector transfected cells, advantage was taken of BH3-only gene knock-out mice. Fetal liver cells from control (wt), vav-bcl-2 transgenic, bim^"1', bad'^' and bim^^'bad^1" embryos (E14.5) were transformed with a bcr-c-abl retrovirus and the derived stably growing cell lines tested for their sensitivity to imatinib. Although all lines of all genotypes expressed bcr-c-abl mRNA and exhibited morphology and surface marker expression characteristic of myeloid progenitors (Figure 12), they varied somewhat in their rate of proliferation (Figure 5A). This has previously been reported for bcr-abl retrovirus infected wt cells and is probably due to differences in additional oncogenic mutations that these cells have [Elefanty et ah, EMBO J. 1990, 9(4): 1069-78]. To obtain representative results, growth inhibitory (Figure 5A) and cell death inducing effects (Figure 5B) of imatinib were tested on at least three independent clones from each genetic background. Imatinib inhibited proliferation and induced death in wt.bcr-c-abt cells in a time- and dose-dependent manner: The bim^''^'bcr- c-abt and to a somewhat lesser extent also the bad ^"bcr-c-άbt cells were more resistant to imatinib-induced cell death than their wt counterparts, showing on average 2- to 3-fold higher viability (Figure 5B). Many of the bcr-c-abl transformed bim^~'^~ and bad^1' cells did, however, eventually die after treatment with imatinib, indicating that these two BH3-only proteins might have overlapping function. Consistent with the notion that Bim and Bad are activated independently by imatinib, similar induction of Bim was found in wt and bad ^" bcr-c-abl transformed fetal liver cells, and similar loss of phosphorylation of Bad in wt and bim^'1' bcr-c-abl transformed cells (Figure 6A). In addition, imatinib caused similar amounts of Bmf upregulation in lines of all genotypes (Figure 6A), but it had no impact on the levels of puma mRNA levels (Figure 6B) or the levels of Bcl-2, Bcl-x_L and Bax protein expression (Figure 6A). Intriguingly, bcr-c-abl transformed bim ^'bad ^' fetal liver cells were significantly more resistant to imatinib-induced killing compared to the bim^'1', bad^1' and wt cells (Figure 5B), although their proliferation was still inhibited in a time- and dose- dependent manners (Figure 5A). Remarkably, less than 10% bcr-c-abl transformed bim^'^bad^1' FLCs were killed even after 7 days of treatment with 3.0 μM imatinib, a degree of resistance that was only recapitulated by Bcl-2 over-expression (Figure 5B). These results demonstrate that Bim and Bad together are essential initiators of imatinib-induced killing of bcr-c-abl transformed cells and they indicate that Bmf might play an ancillary role in this process.

Bim Induction in Primary Human Ph¹+ Leukemia Cells

Examination was made of whether Bim levels are increased upon imatinib treatment in primary human Ph¹+ leukemia cells and whether this might serve as an indicator of therapeutic outcome. Firstly, blood leukocytes from X freshly diagnosed CML patients which were all classified as good responders (sustained complete haematologic response) were studied. Blood cells were cultured for 12 h either in medium alone or in the presence of 3.0 μM imatinib, a drug concentration that is routinely achieved in the serum of treated patients. Western blotting results are shown in Figure 7A.

Next, paired BM samples were obtained from two Ph¹+ CML and two Ph¹+ ALL patients both before imatinib treatment and at relapse/disease progression after imatinib treatment. By cytogenetic studies >90% of BM cells were identified to be leukemic (data not shown). Imatinib was provided as the initial treatment for the two CML patients, while given as the alternative treatment for the two chemoresistant Ph¹+ ALL patients. The primary imatinib efficacy was assessed by peripheral blood and BM cell counts and systemic evaluation: good responders were defined as sustained complete haematologic response, complete BM response and no extramedullar^ involvement that lasted for at least 4 weeks [Ottmann et al, Blood, 2002; 100:1965-1971]. The causes of resistance to imatinib were assessed in two patients at relapse and were found to be an E255K mutation in Bcr-Abl in Case 3 and a T3151 mutation in Case 4, respectively. BM cells were cultured for 12 h in medium alone (control) or in the presence of 3.0 μM imatinib. As shown in Figure 7, imatinib caused an increase in Bini_EL in two leukemic samples (cases 1 and 2) from good responders but no Bim increase was seen in leukemic cells from initial (case 4) or acquired poor responders (cases 1-3 relapse).

Finally, bone marrow samples from de novo imatinib-resistant patients were investigated. Upon culture for 12 h in 3.0 μM imatinib, none of these leukemic cells showed induction of Bim expression (Figure 7C). These results show that Bim induction correlates with good response to imatinib in Ph¹+ human CML and ALL cases.

Resistance to Imatinib Caused by Bcl-2 Over-Expression or Loss of Bim and/or Bad Can Be Overcome by Co-Treatment with the BHS -Mimetic Compound ABT? '37

Acquired or de novo resistance to chemotherapeutic drugs is a significant problem in the treatment of CML and other cancers. Since loss of Bim (and/or Bad) or Bcl-2 over- expression renders PhI+ human leukemia cells and bcr-c-abl transformed mouse fetal liver cells resistant to imatinib, consideration was given as to whether ABT737 could re- sensitize these cells. Accordingly parental K562 cells and subclones over-expressing BcI- 2 or those with suppressed levels of Bim were treated for 48 h with either imatinib alone, ABT737 alone or with both and then measured their survival. Treatment with ABT737 alone had little effect on any of the K562 sublines. As described above, imatinib readily killed parental K562 cells but not the Bcl-2 over-expressing or Bim-deficient clones (Figure 8A). Co-treatment with ABT737 greatly enhanced imatinib-induced killing.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

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Claims

CLAIMS:

1. A method for assessing the responsiveness of a mammalian cell to an antiproliferative agent, said method comprising screening for the level of functional Bim and/or Bad protein and/or gene expression in said cell wherein a decrease in the level of said protein and/or gene expression relative to normal levels is indicative of reduced responsiveness.

2. A method for assessing the responsiveness to an antiproliferative agent of a condition in a mammal, which condition is characterised by unwanted cellular proliferation, said method comprising screening for the level of functional Bim and/or Bad protein and/or gene expression in said proliferating cells wherein a decrease in the level of said protein and/or gene expression relative to normal levels is indicative of reduced responsiveness.

3. A method for assessing the effectiveness of an antiproliferative treatment regime in a mammal, said method comprising screening for the level of functional Bim and/or Bad protein and/or gene expression by a cellular population of interest wherein a decrease in the level of said protein and/or gene expression relative to normal levels or a previously obtained level is indicative of reduced responsiveness and an increase in said level is indicative of an improvement in said responsiveness.

4. The method according to claim 1 or 2 or 3 wherein said method is directed to assessing either the actual or potential responsiveness of said cell or condition to said antiproliferative agent.

5. The method according to claim 4 wherein said method is directed to assessing the actual responsiveness of said cell to said antiproliferative agent and said method is performed after the exposure of said cell to said antiproliferative agent.

6. The method according to claim 4 wherein said method is directed to assessing the potential responsiveness of said cell to said antiproliferative agent and said method is performed prior to the exposure of said cell to said antiproliferative agent.

7. A method for monitoring the responsiveness of a cellular population to an antiproliferative agent, said method comprising screening for modulation of the level of functional Bim and/or Bad protein and/or gene expression wherein a decrease in the level of said protein and/or gene expression in the subject cellular population relative to a previously obtained level or a normal level is indicative of the maintenance or worsening of responsiveness and an increase in said level is indicative of an improvement in said responsiveness.

8. A method for monitoring the responsiveness to an antiproliferative agent of a condition in a mammal, which condition is characterised by unwanted cellular proliferation, said method comprising screening for modulation of the level of functional Bim and/or Bad protein and/or gene expression wherein a decrease in the level of said protein and/or gene expression in the subject cellular population relative to a previously obtained level or a normal level is indicative of the maintenance or worsening of responsiveness and an increase in said level is indicative of an improvement in said responsiveness.

9. A method for monitoring the effectiveness of an antiproliferative treatment regime in a mammal, said method comprising screening for the level of functional Bim and/or Bad protein and/or gene expression by a cellular population of interest wherein a decrease in the level of said protein and/or gene expression relative to normal levels or a previously obtained level is indicative of reduced responsiveness and an increase in said level is indicative of an improvement in said responsiveness.

10. The method according to any one of claims 1-9 wherein said assessment is based on screening only for Bim.

11. The method according to any one of claims 1-9 wherein said assessment is based on screening only for Bad.

12. The method according to any one of claims 1-9 wherein said assessment is based on screening for both Bim and Bad.

13. The method according to any one of claims 10-12 wherein said cell or cellular population is a neoplastic cell or neoplastic cellular population.

14. The method according to claim 13 wherein said neoplastic cell or cellular population is benign, pre-malignant or malignant.

15. The method according to claim 14 wherein said neoplastic cell or cellular population is malignant.

16. The method according to claim 15 wherein said malignant cell is from a central nervous system tumour, retinoblastoma, neuroblastoma, paediatric tumour, head or neck cancer (e.g. squamous cell cancer), breast or prostate cancer, lung cancer (both small and non-small cell lung cancer), kidney cancer (e.g. renal cell adenocarcinoma), oesophagogastric cancer, hepatocellular carcinoma, pancreaticobiliary neoplasia (e.g. adenocarcinomas and islet cell tumour), colorectal cancer, cervical or anal cancer, uterine or other reproductive tract cancers, urinary tract cancer (e.g. of ureter and bladder), germ cell tumour (e.g. testicular germ cell tumour or ovarian germ cell tumour), ovarian cancer (e.g. ovarian epithelial cancer), carcinoma of unknown primary, human immunodeficiency associated malignancy (e.g. Kaposi's sarcoma), lymphoma, leukemia, malignant melanoma, sarcoma, endocrine tumour (e.g. of thyroid gland), mesothelioma or other pleural or peritoneal tumours, neuroendocrine tumour or carcinoid tumour.

17. The method according to claim 14 or 15 wherein said responsiveness is a reduction in the rate or level of cellular proliferation.

18. The method according to claim 17 wherein said responsiveness is the induction of cell death.

19. The method according to claim 18 wherein said cell death is apoptosis.

20. The method according to claim 17 or 18 wherein said rate or level of cellular proliferation is reduced by inducing apoptosis, modulating cellular microtubule structure, inhibiting tyrosine kinase mediated signalling, antagonising cell surface receptor binding, modulating glucocorticoid receptor functioning or downregulating angiogenesis.

21. The method according to claim 20 wherein said antiproliferative agent is a tyrosine kinase inhibitor.

22. The method according to claim 21 wherein said tyrosine kinase inhibitor is Gleevec, or gefitinib.

23. The method according to claim 20 wherein said antiproliferative agent is a modulator of glucocorticoid functioning.

24. The method according to claim 23 wherein said modulator of glucocorticoid functioning is dexamethasone.

25. The method according to claim 20 wherein said antiproliferative agent is a modulator of cellular microtubule structure.

26. The method according to claim 25 wherein said modulator of microtubule structure is taxol.

27. The method according to claim 20 wherein said antiproliferative agent is a downregulator of angiogenesis.

28. The method according to claim 27 wherein said downregulator of angiogenesis inhibits VEGF functioning or VEGF receptor signalling.

29. The method according to claim 28 wherein said downregulator is Avastin.

30. The method according to any one of claims 1-29 wherein said cell is a human cell.

31. The method according to claim 30 wherein said screening is performed in vivo or in situ.

32. The method according to claim 30 wherein said screening is performed in vitro.

33. The method according to claim 32 wherein said screening is performed on a biological sample derived from said mammal.

34. The method according to claim 33 wherein said biological sample is cellular material, biofϊuid which contain cellular material (eg. blood), faeces, tissue biopsy specimen or surgical specimen.

35. The method according to claim 31 or 32 wherein said method comprises screening for Bim and/or Bad protein levels.

36. The method according to claim 35 wherein said protein level is detected using antibodies directed to Bim and/or Bad.

37. The method according to claim 31 or 32 wherein said method comprises screening for Bim and/or Bad RNA transcript levels.

38. The method according to claim 37 wherein said RNA is mRNA.

39. A diagnostic kit comprising an agent for detecting Bim and/or Bad or a nucleic acid molecule encoding Bim and/or Bad and reagents useful for facilitating the detection by said agent.

40. The use of an interactive molecule directed to Bim and/or Bad in the manufacture of a quantitative or semi-quantitative diagnostic kit to assess cellular responsiveness to an antiproliferative agent.