WO2003062828A2

WO2003062828A2 - Determining chemotherapeutic effectiveness

Info

Publication number: WO2003062828A2
Application number: PCT/GB2003/000162
Authority: WO
Inventors: Gareth Joseph Griffiths
Original assignee: The Victoria University Of Manchester
Priority date: 2002-01-17
Filing date: 2003-01-17
Publication date: 2003-07-31
Also published as: WO2003062828A3; AU2003202032A1; GB0200971D0

Abstract

A method of determining the potential effectiveness of a chemotherapeutic compound, or combination of compounds, for treating cancerous cells in a human or animal patient comprises the steps of: i) exposing a sample of the cancerous cells taken from the patient to a chemotherapeutic compound, or combination of compounds the effectiveness of which is to be determined; and ii) assaying for a conformational change in the Bak protein of the cells. The change in the conformation of Bak represents a very early indicator of cellular commitment to apoptosis, and the ability of a compound, or combination of compounds, to cause sucks a change in cancerous cells provides an indication that the compound, or combination, will be effective in chernotherapeutic treatment of the cells.

Description

DETERMINING CHEMOTHERAPEUTIC EFFECTIVENESS.

The present invention relates to a method of determining the potential effectiveness of a chemotherapeutic agent for treating cancerous cells in a human or animal patient.

Cancer represents the second highest cause of mortality in most developed countries after heart disease. It is estimated that one in three Americans presently alive will ultimately develop cancer. Many different treatments for cancer are currently known, although none are universally effective. Amongst the most commonly used treatments are surgical procedures, radiotherapy and chemotherapy.

Chemotherapy may have many purposes. It may be given in the treatment of cancer, to reduce the size of tumours, to prevent a tumour spreading or to kill secondary tumours formed by metastasis.

Many different chemotherapeutic agents are known and are used either individually or in combination to provide defined chemotherapy regimes.

Currently the chemotherapy regime that will be used to treat a patient's tumour is chosen on the basis of the histology of the tumour and the extent of the disease. On the basis of this information the regime will be chosen from a number of standardised regimes. Since the regimes are not selected on the basis of the sensitivity of the patient's tumour to the specific drugs of the regime many patients receive as part of their treatment one or more drugs to which their tumour is resistant. - These drugs are therefore inactive against the patient's tumour, though their toxicities limit the administration of other, more effective, agents to the patient.

One mechanism by which chemotherapeutic agents achieve their effect is through causing cancerous cells to undergo apoptosis. There are a number of different steps involved in the process of cell death through apoptosis including commitment of the cell to the process and the cell death, or execution, phase itself. There is growing evidence that these steps are separate and that cells exhibit different markers during the different phases. Therefore, when assessing a chemotherapeutic compound's ability to induce apoptosis, it may be desirable to assay for markers of commitment to apoptosis, , as opposed to markers indicating that apoptosis is in progress. Thus one may ascertain, at the earliest possible time, that an agent is indeed capable of causing the death of cells. It is particularly beneficial to assess a compound's ability to cause commitment to apoptosis since different compounds may take very different lengths of time between committing a cell to die and bringing about cell death;

Research has been conducted to identify cellular markers that indicate a commitment to apoptosis. One recently identified example of such a marker is a conformational change observed at the NH₂ terminal of the pro-apoptotic protein Bak (Griffiths et al, 1999 J. Cell Biol. March, 8, 144:5, pages 903-914). This change correlates strongly with loss of clonogenicity by cells which is known to be an indicator of commitment to apoptosis. This is shown by experiments conducted in CEM T-cells exposed to dexamethasone. It is known that CEM T-cells exposed to dexamethasone for a period longer than 32 hours undergo commitment to apoptosis, and thus lose clonogenicity. If the period of exposure is less than 32 hours the cell retain their ability to produce clones, which shows that the cells have not committed to apoptosis. Our unpublished data shows that in these cases the proportion of cells losing clonogenicity corresponds to the proportion of cells exhibiting changes in the conformation of Bak.

Our unpublished data shows that the conformational change in the NH₂ terminal of Bak is conserved between all cell types studied thus far, including primary cells and a number of different cell lines such as leukaemic cell lines. Furthermore the altered conformation is induced in response to a number of agents that are able to induce apoptosis through different pathways. The change in the conformation of Bak represents a very early indicator of cellular commitment to apoptosis, that precedes caspase activation, nuclear condensation and cellular blebbing.

The paper of Griffiths et al. illustrates that apoptosis-inducing agents are able to induce a change in the conformation of the NH₂ terminal of Bak, which is a cellularly expressed pro-apoptotic protein the human form of which has the a ino acid residue sequence: (Sequence ID No. 1)

MASGQGPGPP RQECGEPALP SASEEQNAQD TEENFRSYNF YRHQQEQEAE GNAAPADPEM NTLPLQPSST MGQNGRQLAI IGDDLΝRRYD SEFQTMLQHL QPTAEΝAYEY FTKIATSLFE SGLΝWGRNNA LLGFGYRLAL HNYQHGLTGF LGQVTRFNVD FMLHHCIARW IAQRGGWVAA LΝLGΝGPILΝ VLVVLGVVLL GQFVVRRFFK S

Currently, to determine whether a chosen chemotherapeutic agent is having an effect upon the cancerous cells of a patient, it is necessary to measure the level of cell death caused in a sample of the cells. The techniques that are used to achieve this in clinical practice are based mainly on histological assays. These involve the use of dyes to measure membrane integrity (a property lost during death), the DΝA and chromatin status of the cells (DΝA and chromatin become condensed as cells undergo apoptosis), and other features such as the exposure of phosphatidyl serine (which binds annexin V) on the outer membrane of the cell. All of these methods suffer from the fact that cell death is temporally a very late event when assessing the effectiveness of drug treatment. Thus the study of cells dividing and dying within a tumour can give a very false picture of the effectiveness of a chemotherapeutic regime. The rate of death varies dramatically in response to different chemotherapeutic agents, and may vary between hours and days. A major drawback of these assays is therefore their inability to predict the effectiveness of certain chemotherapy drugs. More accurate is the measurement of cellular commitment to apoptosis caused by the chosen agents. This may be accurately measured using clonogenic assays, whereby two populations are exposed for the same length of time to a given drug and then individual cells plated into microplate wells. The ability of cell to form colonies from these individual cells is a much more accurate method of predicting the effectiveness of a drug upon the survival of cancerous cells. Unfortunately such assays are technically demanding, and the cultured cells take time to form colonies of clones.

It is an object of the present invention to obviate or mitigate the abovementioned disadvantages of the prior art. According to the present invention there is provided a method of determining the potential effectiveness of a chemotherapeutic compound, or combination of compounds, for treating cancerous cells in a human or animal patient comprising the following steps: i) exposing a sample of the cancerous cells taken from the patient to a chemotherapeutic compound, or combination of compounds, the effectiveness of which is to be determined; and ii) assaying for a conformational change in the Bak protein of the cells.

The effectiveness of a chemotherapeutic agent, or combination of agents, for treating cancerous cells in a human or animal patient may for the purposes of this invention be considered to be represented by the ability of the chemotherapeutic compound, or combination of compounds, to induce a conformational change in Bak expressed within the cancerous cells. Such a change in the conformation of Bak may include any change in the conformation of Bak caused by the treatment of the cells with the compound, or combination of compounds, to be tested.

The conformational change in Bak may be a change at NH₂ terminal of the protein. We have found that the conformational change in the NH₂ terminal of Bak is conserved between all cell types studied thus far, including primary cells and a number of different cell lines such as leukaemic cell lines. Furthermore the altered conformation is induced in response to a. number of agents that are able to induce apoptosis through different pathways. The change in the conformation of Bak represents a very early indicator of cellular commitment to apoptosis, that precedes caspase activation, nuclear condensation and cellular blebbing.

We have further found that cells exposed to an agent capable of inducing apoptosis also exhibit a change in the conformation of the Bcl-2 homo logy 1 (BH1) region of Bak. This change occurs later than the change in the NH₂ region, at a point closer the death of the cell via apoptotic mechanisms.

The cells to be tested may be primary cell cultures, that is to say cells taken from the cancerous tissue source and their progeny, grown in culture, but exposed to the chemotherapeutic compound, or combination of compounds, to be tested before their subdivision and transfer to a subculture.

Alternatively it may be preferred that the cells to be tested be cells taken from the cancerous tissue source and subcultured prior to their being exposed to the chemotherapeutic compound, or combination of compounds, to be tested. Such an approach may be advantageous when only possible to obtain a small number of cells from the patient, allowing the number of cells available for the assay to be expanded.

The cancerous cells to be assayed may be obtained in a number of ways. In the case of cells from a solid tumour it may be preferred to take a biopsy sample from the tumour to provide the cells.^' In the case of, for example, a cancer effecting cells of the circulation it may be advantageous to acquire the cancerous cells by means of a blood sample.

It may be preferred that the cancerous cells be dispersed from the tissue of the biopsy, for example by explant culture or by enzymatic dispersion. Such an approach may facilitate the exposure of the cancerous cells to the chemotherapeutic agent. Dispersed cells may also be more readily assayed, by for example labelling with a fluorophore-coηjugated antibody followed by fluorescent activated cell sorter (FACS) analysis, to cancerous cells immersed in the medium.

The method of the invention may generally be applied to use of a plurality of different chemotherapeutic agents, or regimes of agents, to determine which of the tested agents or regimes is likely to be most effective. A plurality of regimes to be tested may comprise a number of different combinations of agents, or a number of combinations of the same agents in different proportions or at different concentrations.

The method may readily be applied to at least 5 different chemotherapeutic agents or regimes of agents. The method may further be applied to at least 10 different chemotherapeutic agents or regimes of agents. It may be preferred that the method be applied to at least 20 different chemotherapeutic agents or regimes of agents. It may be preferred to treat the cancerous cells with a combination of compounds to be tested. Such combinations may include combinations of known chemotherapeutic agents. Alternatively compounds with chemotherapeutic activity may be combined with other compounds potentially able to increase the chemotherapeutic agent's efficacy. Known examples of such compounds include the active folate leucovorin, which does not have chemotherapeutic activity itself, but is known to increase the chemotherapeutic activity of fluorouracil.

In order to test for additive or synergistic effects produced by combinations of compounds to be tested the effect of the combination may be compared with the effect achieved by use of the individual compounds that comprise the combination.

It is known that in some cases cells may lose sensitivity to chemotherapeutic agents. For example the loss of mismatch repair function by cells may cause them to lose sensitivity to chemotherapeutic compounds such as cisplatin or doxorubicin, since the cells are unable to detect the DNA damage caused by the compound, thereby preventing chemotherapeutic function of these agents. However treatment of such insensitive cells with certain compounds that do not themselves have chemotherapeutic activity causes the cells to regain sensitivity to the chemotherapeutic agents. A known example of such an enhancer of chemotherapeutic activity is 5-azacytidine, which is able to restore the chemotherapeutic action of cisplatin in the treatment of otherwise resistant cells. The invention may be used determine the chemotherapeutic effect of combinations comprising such compounds by adding a further step in which the cancerous cells are first exposed to a potential enhancer of chemotherapeutic activity before being treated with a known chemotherapeutic agent.

Assaying Bak protein in the cells for a conformational change may be achieved by any suitable method known in the prior art. For instance it may be wished to use an antibody that that binds specifically to Bak the NH₂ terminal of which has undergone a conformational change. An example of an antibody is that is capable of binding to Bak that has undergone a conformational change indicative of a cellular commitment to apoptosis, but does not bind to Bak in its usual conformation, is the Ab-1 antibody referred to in Griffiths et al. Ab-1 may be purified IgG2a monoclonal antibody that is the product of the TC-100 clone hybridoma cell line. It is commercially available from Calbiochem and Oncogene (Catalogue number AM03). The immunogen used in production of this antibody was human recombinant Bak from which the transmembrane and C-terminal portions had been deleted. The antibody recognises an epitope located in the N-terminal region of Bak, between the first and fifty-second amino acid residues.

A further example of an antibody able to bind specifically to Bak the NH₂ terminal of which has undergone a conformational change is Ab-2 referred to in Griffiths et al.. Ab-2 may be a purified IgG2a monoclonal antibody that is the product of the TC-102 clone hybridoma cell line. It is commercially available from Calbiochem and Oncogene (Catalogue number AM04). The immunogen used in production of this antibody was human recombinant Bak from which the transmembrane and C-terminal portions had been deleted. The antibody recognises an epitope located in the N-terminal region of Bak, between the first and fifty-second amino acid residues.

As an alternative it may be preferred to assay for Bak protein within the cancerous cells that has undergone a conformational • change within the BH1 domain. Such a conformational change may be detected using an antibody that binds specifically to Bak in which the conformation of the BH1 domain is altered. An example of such an antibody is Ab-3 identified in Griffiths et al.. Ab-3 may be a purified IgG2a monoclonal antibody that is the product of the TC-98 clone hybridoma cell line. The immunogen used in production of this antibody was the BH1 domain of human recombinant Bak protein.

As an alternative to the use of a whole antibody it may be preferred to use an antibody fragment having the same binding specificities. It may further be wished that the cell express the antibody fragment intra-cellularly as an "intrabody". Suitable methods by which a chosen antibody may be adapted are well known in the art.

The change in the conformation of the NH₂ terminal of Bak may alternatively be investigated using a protein, other than an antibody or antibody fragment, or other such binding partner that is capable of binding specifically to Bak that has undergone a conformational change. Such a specific binding partner may be naturally occurring, or may be artificially produced. For example the paper by Griffiths et al. reveals that the binding of Bak to other intracellular proteins is altered upon the change in the conformation of Bak's NH₂ terminal. Proteins that bind only to Bak once the conformation of its NH₂ terminal has changed may be used as specific binding partners for use in the invention, or may be further engineered to produce improved binding partners.

It may be desired to use a specific binding partner, including antibodies or antibody fragments, that additionally comprises a reporter moiety to allow direct labelling of the conformationally-altered Bak. Such a reporter moiety may, for example, comprise a fluorophore (such as fluorescein isothiocyanate or the like) or an enzyme (such as horseradish peroxidase) capable of catalysing a chromogenic reaction of a suitable substrate (such as diaminobenzidine).

It may alternatively be preferred to use an indirect labelling technique to detect the presence of Bak that has undergone a conformational change.

Suitable examples of such direct or indirect labelling techniques are well known. For instance when using immunological methods immunocytochemistry, such as immunofluorescence or immunoperoxidase labelling, may be used. Such techniques are suited to both direct and indirect labelling. In the case of indirect labelling a protocol allowing amplification of the reporter signal generated, for instance amplification through use of avidin/biotin complexes or "primary" and "secondary" antibodies may be used.

An alternative approach to the use of immunological methods to assay for the presence of Bak which has undergone a conformational change at the NH₂ terminal is to use an enzyme linked immunosorbent assay (ELISA) using a suitable antibody. Such immunological techniques may readily be adapted to use specific binding partners other than antibodies. The invention may be effected by taking a selection of cells from, for example, a tissue biopsy. The biopsy may be divided into smaller samples and the samples transferred to individual wells of a multiwell plate. The sample of cells within each different well may then be exposed to a number of different chemotherapeutic compounds, such that the cells in each well are exposed to a different chemotherapeutic compound. The chemotherapeutic compounds to be tested may, for instance be administered to the cells by means of adding the compounds to be tested to cell culture medium incubating the cells.

Once the cells of the biopsy samples have been exposed to the chemotherapeutic compounds to be tested they may then be removed and the samples processed for cryotomy. The samples may, for example, be mounted in a suitable medium and frozen, before being cut into tissue sections on a cryostat. The resultant tissue sections may then be mounted on microscope slides and used for immuno-labelling according to well established protocols.

A suitable immuno-labelling protocol may, for example, comprise incubating the tissue sections in a dilute solution of a specific antibody, such as Ab-1 described above, directly conjugated with a fluorophore such as fluorescein isothiocyanate (FITC). The presence of cells in which Bak has undergone a conformational change may then be determined by fluorescence microscopy.

The invention will now be described, by way of example only, with reference to the accompanying experimental data and accompanying Figures 1 to 3 of the drawings which illustrate the experimental data.

EXPERIMENTAL DATA.

Change in the conformation of Bak is conserved between cell types.

The change in the conformation of Bak, indicating cellular commitment to apoptosis, in response to treatment with apoptosis-inducing agents occurs in all cell types thus far tested. Figure 1 shows labelling of the cell nucleus and specific labelling of Bak which has undergone a conformational change (lighter labelling around the periphery of the nucleus) in a range of cell types and lines treated with staurosporine, at a concentration of 250nM, for 6 hours to induce apoptosis. It can thus be seen that induction of apoptosis is associated with a change in the conformation of the NH₂ terminal of Bak in both primary cell cultures and cell lines (including examples of precursor cell lines, differentiated cell lines and transformed cell lines).

Change in the conformation of Bak corresponds with loss of clonogenicity. Loss of clonogenicity by cells is known to indicate cellular commitment to apoptosis. The results shown in Figure 2 illustrate that in cells exposed to the apoptosis inducing compound dexamethasone the rate of loss of clonogenicity by the cells corresponds with the rate of the increase in the expression of the conformational change in the NH₂ terminal of Bak (as determined by labelling with antibody Ab-1).

Temporal difference between different conformational changes in Bak in response to apoptotic stimuli.

Figure 3 compares labelling with Ab-1 (indicative of a conformational change at the NH terminal of Bak) and labelling with Ab-3 (which indicates a conformational change within the BH1 region of Bak).

It can be seen, from the fluorescence activated cell sorting (FACS) data shown on the left hand side of the Figure and the graph on the right hand side of the figure, that cells induced to undergo apoptosis by treatment with lOμm etoposide label positively for Ab-1 earlier than for Ab-3. This indicates that the conformational change in the NH₂ terminal of Bak occurs earlier in the process of apoptotic cell death than the change in the BH1 region of Bak.

Claims

CLAIMS.

1. A method of determining the potential effectiveness of a chemotherapeutic compound, or combination of compounds, for treating cancerous cells in a human or animal patient comprising the following steps: i) exposing a sample of the cancerous cells taken from the patient to a chemotherapeutic compound, or combination of compounds the effectiveness of which is to be determined; and ii) assaying for a conformational change in the Bak protein of the cells.

2. A method according to claim 1, wherein the conformational change is a change at the NH₂ terminal of Bak.

3. A method according to claim 1, wherein the conformational change is a change in the BH1 domain of Bak.

4. A method according to any preceding claim, wherein the assay for the change in the conformation of of Bak is conducted by means of a specific binding partner for Bak that has undergone a conformational change.

5. A method according to claim 4, wherein the specific binding partner is an antibody.

6. A method according to claim 1, wherein the conformational change is a change at the NH2 terminal of Bak that is detected by a specific antibody binding partner for Bak that has undergone a conformational change, the antibody being Ab-1 a purified IgG2a monoclonal antibody that is the product of the TC-100 clone hybridoma cell line.

7. A method according to claim 1, wherein the conformational change is a change at the NH2 terminal of Bak that is detected by a specific antibody binding partner for Bak that has undergone a conformational change, the antibody being Ab-3 a purified IgG2a monoclonal antibody that is the product of the TC-98 clone hybridoma cell line.

8. A method according to claim 4, wherein the specific binding partner is an antibody fragment.

9. A method according to claim 4, wherein the specific binding partner is protein other than an antibody or antibody fragment.

10. A method according to any one of claims 4 to 9, wherein the specific binding partner additionally comprises a reporter moiety.

11. A method according to claim 10, wherein the reporter moiety is a fluorophore.

12. A method according to claim 10, wherein the reporter moiety is an enzyme capable of catalysing a chromogenic reaction of a suitable substrate.

13. A method according to any of claims 4 to 12, wherein the presence of the specific binding partner is detected by an indirect labelling technique.

14. A method according to any one of claims 4 to 13, wherein the presence of the specific binding partner is detected by an enzyme linked immunosorbent assay (ELISA).

15. A method according to any preceding claim, wherein the cancerous cells exposed to the chemotherapeutic compound or combination of compounds are primary cells.

16. A method according to any preceding claim, wherein the cancerous cells are cultured prior to their exposure to the chemotherapeutic compound or combination of compounds.

17. A method according to any preceding claim, wherein the cancerous cell are dispersed prior to their exposure to the chemotherapeutic compound or combination of compounds.