WO2003062829A2

WO2003062829A2 - Identifying chemotherapeutic compounds

Info

Publication number: WO2003062829A2
Application number: PCT/GB2003/000185
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: WO2003062829A3; AU2003205816A1; GB0200970D0

Abstract

A method of identifying chemotherapeutic activity in a compound other than etoposide, or combination of compounds, comprises: i) treating a cell in which an anti-apoptotic protein is over-expressed with the compound, or combination of compounds, to be assessed for chemotherapeutic activity; and ii) assaying for a change in the conformation of Bak expressed in the cell. The presence of a change in the conformation of Bak indicates that the compound, or combination of compounds, has chemotherapeutic activity, and the absence of a change in the conformation of Bak indicates that the compound, or combination of compounds, does not have chemotherapeutic activity.

Description

IDENTIFYING CHEMOTHERAPEUTIC COMPOUNDS.

The present invention relates to a method of identifying chemotherapeutic activity in a compound or combination of compounds.

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.

Whilst there are many known chemotherapeutic agents there is a need to identify new chemotherapeutic agents that may be used in the treatment of cancers. New agents are required both in an attempt to identify agents that may have an effect on cancers for which no chemotherapeutic agents are currently known, and also to provide suitable agents for cancerous cells that have developed resistance to known chemotherapy agents.

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.

We have also found that cells exposed to an agent capable of inducing apoptosis also exhibit a change in the conformation of the Bcl-2 homology 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. Although chemotherapeutic agents exert much of their effect through apoptosis not all compounds that are capable of inducing apoptosis are suitable for use in chemotherapy. One way in which apoptosis-inducing compounds may be classified is by the organelles that they effect in order to cause apoptosis. For a compound to be capable of use as a chemotherapeutic agent it must be capable of inducing apoptosis through a mechanism that involves perturbation of the nucleus. Methods of inducing apoptosis that achieve their effect through other pathways, including methods such as serum starvation or exposure to raised temperatures, transcriptional regulation, kinase inhibition and exposure to Fas ligand are not capable of use as chemotherapeutic substances.

The paper of Griffiths et al. illustrates that apoptosis-inducing agents that act through nuclear perturbation and apoptosis-inducing agents that act through different mechanisms are both able to induce the 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 amino acid residue sequence: (Sequence ID No. 1)

MASGQGPGPP RQECGEPALP SASEEQVAQD TEENFRSYVF YRHQQEQEAE GNAAPADPEM NTLPLQPSST MGQNGRQLAI IGDDIΝRRYD SEFQTMLQHL QPTAEΝAYEY FTKIATSLFE SGTΝWGRNNA LLGFGYRLAL HNYQHGLTGF LGQNTRFNND FMLHHCIARW IAQRGGWNAA LΝLGΝGPILΝ NLVNLGNNLL GQFNNRRFFK S

Specifically, data published in the paper show that etoposide, a known chemotherapeutic agent, and staurosporine, which causes cell death through non-nuclear mechanisms, both cause the ΝH₂ terminal of Bak to alter its conformation. The paper also illustrates that in cells over-expressing the anti-apoptotic protein Bcl-2 etoposide treatment induces a change in the conformation of Bak protein. These data indicate that the ability of a compound to cause a conformational change of Bak in a cell exposed to the compound does not provide any information about the way in which the compound causes apoptosis, and so does not provide an indication as to the suitability of the compound for use as a chemotherapeutic agent. It is an object of the present invention to provide a novel method of identifying chemotherapeutic activity in a compound or combination of compounds.

According to a first aspect of the present invention, there is provided a method of identifying chemotherapeutic activity in a compound other than etoposide, or combination of compounds, comprising the steps of: i) treating a cell in which an anti-apoptotic protein is over-expressed with the compound, or combination of compounds, to be assessed for chemotherapeutic activity; and ii) assaying for a change in the conformation of Bak expressed in the cell whereby the presence of a change in the conformation of Bak indicates that the compound, or combination of compounds, has chemotherapeutic activity, and the absence of a change in the conformation of Bak indicates that the compound, or combination of compounds, does not have chemotherapeutic activity.

According to a second aspect of the present invention, there is provided a method of screening compounds, or combinations of compounds, to identify chemotherapeutic activity, the method comprising for each compound or combination of compounds to be screened the steps of: i) treating a cell in which an anti-apoptotic protein is over-expressed with the compound, or combination of compounds, to be assessed for chemotherapeutic activity; and ii) assaying for a change in the conformation of Bak expressed in the cell whereby the screen indicates that compounds or combinations of compounds producing a conformation change in Bak have chemotherapeutic activity and that compounds or combinations of compounds that do not produce a conformation change in Bak do not have chemotherapeutic activity.

Compounds to be screened will generally be known compounds for which it is wished to determine whether they possess chemotherapeutic activity. We have found that in cells transfected such that they over-express an anti-apoptotic protein the conformational change in the pro-apoptotic protein Bak is abrogated in response to all compounds other than those that cause cell death through mechanisms involving nuclear perturbation.

We have also found that chemotherapeutic compounds that have a greater specificity for cancerous cells than for non-cancerous cells can be identified by their ability to induce an increased conformational change of Bak in cells over-expressing an anti-apoptotic protein as opposed to that change induced in cells in which the anti-apoptotic protein is not over- expressed. The ability to identify such compounds is highly desirable. The specificity of the compounds means that they are likely to be very effective in cancer treatment, but with less of the side-effects caused by damage to non-cancerous cells. Accordingly there is provided a method according to the first or second aspects of the invention, further comprising: iii) comparing the results of step ii) with the change in conformation of Bak induced by the compound, or combination of compounds, in cells in which the anti- apoptotic protein is not over-expressed; whereby the method indicates that compounds, or combinations of compounds, which induce an increased conformational change in Bak of cells in which an anti-apoptotic protein is over-expressed, as compared to cells in which the anti-apoptotic protein is not over-expressed, have chemotherapeutic activity with a greater specificity for cancerous rather than non-cancerous cells.

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. 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. Alternatively the conformational change in Bak may be a change in the conformation of the Bcl-2 homology 1 (BHl) 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 anti-apoptotic protein over-expressed by the cells to be treated may be a member of the Bcl-2 family. It may, for instance, be Bcl-2 or BC1-X . The anti-apoptotic protein may be any homologue of Bcl-2 or of BC1-X_L which has anti-apoptotic activity.

The over-expression of the anti-apoptotic protein may be induced in a number of ways. For instance the cell may be induced to over-express the anti-apoptotic protein by incorporation in the cell of additional genetic material coding for the anti-apoptotic protein. Such genetic material may be incorporated in the cell under the control of a suitable promoter by means of a plasmid or other such suitable vector. The over- expression of the anti-apoptotic protein may, for instance, be induced by the incorporation in the cell of a pcDNA3.1 vector used in combination with a CMV (cytomegalovirus) promoter.

Alternatively the over-expression of anti-apoptotic proteins may be induced by other methods, such as treatment of cells to be used with compounds that induce a suitable anti- apoptotic protein's expression. In an alternative method cell lines which constitutively express elevated levels of anti-apoptotic proteins may be selected for use according to the invention.

Generally the degree of over-expression of the chosen anti-apoptotic protein need only be in the region of a 10% increase over normal cellular levels of expression. Preferably, however the degree of over-expression of the anti-apoptotic protein may be a 40% or greater increase over normal cellular levels of expression.

The human form of Bcl-2 has the amino acid residue sequence:

(Sequence ID No. 2)

MAHAGRTGYD NREIVMKYIH YKLSQRGYEW DAGDVGAAPP GAAPAPGIFS

SQPGHTPHPA ASRDPVARTS PLQTPAAPGA AAGPALSPVP PVVHLTLRQA GDDFSRRYRR DFAEMSSQLH LTPFTARGRF ATNNEELFRD GVΝWGRTNAF FEFGGNMCNE SNΝREMSPLN DΝIALWMTEY LΝRHLHTWIQ DΝGGWDAFNE LYGPSMRPLF DFSWLSLKTL LSLALVGACI TLGAYLGHK

The human form of Bel X_L has the amino acid residue sequence:

(Sequence ID Νo.3)

MSQSNRELNN DFLSYKLSQK GYSWSQFSDN EENRTEAPEG TESEMETPSA

INGNPSWHLA DSPAVNGATG HSSSLDAREV MAAVKQALRE AGDEFELRYR

RAFSDLTSQL HITPGTAYQS FEQWNELFR DGVNWGRTNA FFSFGGALCN

ESNDKEMQNL NSRIAAWMAT YLΝDHLEPWI QEΝGGWDTFN ELYGΝΝAAAE

SRKGQERFΝR FLTGMTNAGN NLLGSLFSRK

The reference to a change in the conformation of Bak encompasses any change in the conformation of Bak expressed in the treated cell from the conformation of the protein present in the anti-apoptotic protein over-expressing cells before treatment. The ability of a compound, or combination of compounds, to bring about such a change in cells transfected to over-express an anti-apoptotic protein is an indicator that the compound, or combination of compounds, in question is capable of inducing cell death through perturbation of the nucleus. Thus such compounds demonstrate potential chemotherapeutic activity.

The change in the conformation of Bak occurs shortly after administration of the compound or combination of compounds to be tested, often within eight hours of treatment, in some cases as shortly as four hours after treatment. The method is therefore particularly suited to produce rapid results, and so may form the basis of a high throughput screen.

It may be preferred to treat the cells with a combination of compounds to be tested. Such combinations may include combinations of known or putative 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 to screen for such compounds by adding a further step in which the anti-apoptotic protein over-expressing cells are first exposed to a potential enhancer of chemotherapeutic activity before being treated with a known or putative chemotherapeutic agent.

The invention may further be used to determine the point in the cell cycle at which a chemotherapeutic agent has its effect. It is advantageous to select chemotherapeutic agents that have their effect at "checkpoints" in mitosis as such agents have less damaging effect on a patient's non-cancerous cells since such cells have a relatively slower rate of mitosis. The accumulation of DNA in cells enables the cell cycle status of the cells to be determined. Therefore the invention may preferably be effected in the presence of a substance capable of labelling DNA, such as propidium iodide. Thus cells treated with the compound, or combination of compounds, to be tested for chemotherapeutic activity may be assayed for a conformational change in Bak, those cells in which such a change is detected are selected, and the DNA content of these selected cells determined from their labelling. The DNA content of the cells may then be used to ascertain during which phase of the mitotic cell cycle the tested compound causes commitment of the cells to apoptosis.

Cells which are suitable to be induced to over-express anti-apoptotic proteins and be used to effect the invention include human cell lines such as CEM cells. However, as it is currently believed that all naturally occurring cells express Bak, it is anticipated that cells derived from any source tissue, including primary cell lines, may be induced to over- express a suitable anti-apoptotic protein and be used in accordance with the invention.

Certain chemotherapeutic compounds are known to have tissue specific effects, therefore the invention may be effected in cells derived from selected tissue types in order to assess the effectiveness of known or putative chemotherapeutic compounds in those tissues. For instance the invention may be practised in cells derived from tissues affected by particular forms of cancer in order to assess the effectiveness of known or putative chemotherapeutic compounds in treating those cancers.

Assaying Bak protein in the cells for a conformational change may be achieved by any suitable method known in the prior art. For instance the assay may be effected using an antibody that binds specifically to the form of Bak in which the NH₂ terminal has undergone a conformational change. An example of an antibody that is capable of binding to Bak that has undergone a conformational change at the NH₂ terminal 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 a 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.

Alternatively the assay may be effected using an agent that specifically binds to Bak that has undergone a conformational change within the BHl domain. An example of such an agent is the antibody identified as Ab-3 in Griffiths et al.. Ab-3 may be a purified IgG2a antibody that is the product of the TC-98 clone hybridoma cell line. The immunogen used in the generation of this antibody was the BHl 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 maybe 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 culturing the anti-apoptotic protein over-expressing cells in a suitable medium, for instance in the case of suitably transfected CEM cells Optimem-1 medium (Gibco BRL), to which the compound or combination of compounds to be tested has been added. Suitable concentrations of the compound, or combination, and suitable incubation times may be readily determined through dose response curves.

Once the cells have been exposed to the compound, or combination, to be tested they may then be fixed, for instance in acetone, and permeabilised, if necessary through treatment with a detergent such as Triton X-100. In the case of an indirect localisation protocol using a specific antibody (as primary antibody) the cells may then be incubated for a suitable time, such as one hour, with antibody diluted to a suitable concentration, for instance 1:100 dilution in phosphate buffered saline (PBS). Excess antibody may then be removed by means of multiple washes in PBS. The cells, containing the Bak-primary antibody complex may then be incubated in a dilute solution of a further labelled antibody (secondary antibody) capable of specific binding to the primary antibody. In the case of a secondary antibody labelled with a fluorophore, such as FITC, the cells may then be washed in PBS ,to remove excess secondary antibody, and mounted in a suitable medium such as gelvatol. Presence of labelled Bak may then be determined by use of immunofluorescence microscopy using light with a suitable excitation spectrum.

Such a protocol may be readily altered to accommodate dispersed cells, for example for FACS analysis, or different labelling protocols, such as immuno-peroxidase labelling or the like. Suitable variations will be apparent to those skilled in the art.

The invention will now be described, by way of example only, with reference to the accompanying experimental data and accompanying Figures 1 to 10 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 the NH terminal 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 the NH₂ terminal 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).

Cells over-expressing anti-apoptotic proteins do not exhibit a change in the conformation of the NH? terminal of Bak when exposed to apoptotic agents that do not cause nuclear perturbation.

Figures 3 and 4 show the results of fluorescence activated cell sorting (FACS) analysis of the conformational change in the NH₂ terminal of Bak (determined by labelling with antibody Ab-1) in cells treated with the apoptosis-inducing agents staurosporine and cyclohexamide respectively. Staurosporine and cyclohexamide both achieve their effects through inhibition of protein kinase activity, and not through nuclear perturbation.

Turning first to Figure 3, in staurosporine-treated cells transfected to over-express the anti-apoptotic proteins Bcl-2 or BC1-X (designated Bcl-2 or BC1-X respectively) it can be seen that the level of labelling by Ab-1 does not differ from that seen in the FACS controls. This indicates that staurosporine does not induce a change in the conformation of the NH₂ terminal of Bak. In contrast cells transfected with a vector control (designated Neo) exhibit levels of labelling by Ab-1 that are greatly increased over the level of the FACS controls. This indicates that staurosporine is able to cause commitment to apoptosis, and hence a change in the NH₂ conformation of Bak, in these cells.

Similarly, in Figure 4, it can be seen that vector control transfected cells (Neo) treated with cyclohexamide exhibit an increased level of Ab-1 labelling, indicating that cyclohexamide is able to induce a conformational change in the NH₂ terminal of Bak, and hence the cells' commitment to apoptosis. In contrast, identically treated cells in which Bcl-2 is over-expressed exhibit no increase in Ab-1 labelling. Thus it can be seen that the ability of apoptotic agents that do not cause nuclear perturbation to bring about a conformational change in the NH₂ terminal of' Bak is negated by cellular over-expression of anti-apoptotic proteins.

Cells over-expressing anti-apoptotic proteins exhibit a change in the conformation of the NH? terminal of Bak when exposed to apoptotic agents that cause nuclear perturbation. Figures 5 and 6 illustrate the results of FACS analysis of the conformational change in the NH₂ terminal of Bak (determined by labelling with Ab-1) in Neo and Bcl-2 over- expressing cells. Figures 4 and 5 show the response to treatment with the chemotherapeutic compounds oxaliplatin and taxol respectively, both of which cause apoptosis through nuclear perturbation.

Figure 5 shows that four hours after treatment with oxaliplatin, Neo cells show no increase in Ab-1 labelling indicative of a change in the conformation of the NH₂ terminal of Bak. In contrast cells over-expressing Bcl-2 exhibit elevated Ab-1 labelling at this time-point, characteristic of a conformational change in the NH2 terminal of Bak, which is in turn indicative of cellular commitment to apoptosis.

At six hours after treatment with oxaliplatin Neo cells exhibit increased levels of Ab-1 labelling compared to controls indicating that a conformational change in the NH terminal of Bak, and hence commitment to apoptosis, has occurred. Bcl-2 over- expressing cells from this time-point maintain the raised levels of Ab-1 labelling observed at four hours after treatment.

In Figure 6 it can be seen that taxol treatment of both Neo and Bcl-2 over-expressing cells causes increased Ab-1 labelling compared to controls, indicating that a conformational change in the NH₂ terminal of Bak has taken place in both populations of treated cells.

Thus it can be seen that in both control-transfected cells and cells over-expressing anti- apoptotic proteins treatment with apoptotic agents that cause nuclear perturbation induces detectable changes in the conformation of the NH₂ terminal of Bak. Assaying for chemotherapeutic activity by analysis of induction of Bak NH2 terminal conformational change in control cells and anti-apoptotic protein over-expressing cells. The results reported above illustrate that the ability of a compound to induce apoptosis through nuclear perturbation, and hence to potentially act as a chemotherapeutic agent, is indicated by an ability to cause a conformational change in the NH2 terminal of Bak in both control and anti-apoptotic protein over-expressing cells.

Figure 7 illustrates the results of comparison of levels of Ab-1 labelling (indicative of Bak NH2 terminal conformational change) in both transfection-control cells and anti-apoptotic protein over-expressing cells treated with a range of apoptosis-inducing factors. The results of Ab-1 labelling are recorded using a scale as follows:

- indicates Ab-1 labelling no greater than relevant controls

+ indicates Ab-1 labelling increased compared to controls

++ indicates Ab-1 labelling much increased compared to controls

Agents that cause a greater degree of labelling in transfection-control cells as opposed to anti-apoptotic protein over-expressing cells induce apoptosis through methods other than nuclear perturbation. Such agents are not suitable as candidate chemotherapeutic agents.

Agents that cause an increase in the labelling of both transfection-control cells and anti- apoptotic protein over-expressing cells induce apoptosis through nuclear perturbation. Such agents therefore represent potential chemotherapeutic agents.

Agents that cause a greater degree of labelling in anti-apoptotic protein over-expressing cells than in transfection-control cells represent potential chemotherapeutic agents exhibiting a greater specificity for cancerous cells than for non-cancerous cells. This relative difference in specificity of action may, at least in part, explain the improved chemotherapeutic effectiveness of agents such as oxaliplatin and taxol.

The results generated by the assay and reported in Table 1 are consistent with previously reported activities of the agents tested. Agents inducing apoptosis through nuclear perturbation can still cause death of cells over- expressing anti-apoptotic proteins (although morphological changes associated with apoptosis are delayed).

Figure 8 shows the results of measurement of apoptosis (based on assessment of nuclear morphology of Hoechst 33258 labelled cells) in Neo, Bcl-2 and BC1-X_L cells in response to treatment with the chemotherapeutic agent cisplatin at a concentration of 25μM. Cisplatin induces apoptosis through nuclear perturbation.

It can be seen that while cisplatin is able to induce apoptosis in all three cell types the onset of apoptosis is very much delayed in the cells over expressing anti-apoptotic proteins (Bcl-2 and BC1-X_L cells) as compared to the cells transfected with the vector control.

Change in the conformation of the NH? terminal of Bak in cells over-expressing anti- apoptotic proteins can be detected before apoptosis.

Figure 9 shows the results of assessment of conformational change of the NH₂ terminal of cellular Bak of Neo, Bcl-2 and BC1-X cells in response to treatment with cisplatin at 25μM. It can be seen that labelling by antibody Ab-1 (indicating NH terminal conformational change) increases in all cell types at approximately the same rate.

Comparison of the results shown in Figure 8 with those shown in Figure 9 indicates that the change in the conformation of Bak can be detected earlier than can apoptosis in cells over-expressing anti-apoptotic proteins.

Determination of phase of cell cycle effected by chemotherapeutic agents. Figure 10 shows DNA accumulation (as assessed by propidium iodide labelling) in cells labelling positively for antibody Ab-1 (indicating commitment to apoptosis) after treatment with a number of different compounds able to induce apoptosis.

Analysis of DNA accumulation allows analysis of the cell cycle state of the cells that have undergone commitment to apoptosis. Thus it can be seen that cells treated with etoposide have a characteristic profile of DNA accumulation indicating that etoposide causes commitment to apoptosis during the S and G2M phases of the cell cycle. In contrast Taxol can be seen to effect cells in the M phase, whilst staurosporine and heatshock effect cells throughout the cell cycle.

Claims

CLAIMS.

1. A method of identifying chemotherapeutic activity in a compound other than etoposide, or combination of compounds, comprising the steps of: i) treating a cell in which an anti-apoptotic protein is over-expressed with the compound, or combination of compounds, to be assessed for chemotherapeutic activity; and ii) assaying for a change in the conformation of Bak expressed in the cell whereby the presence of a change in the conformation of Bak indicates that the compound, or combination of compounds, has chemotherapeutic activity, and the absence of a change in the conformation of Bak indicates that the compound, or combination of compounds, does not have chemotherapeutic activity.

2. A method of screening compounds, or combinations of compounds, to identify chemotherapeutic activity, the method comprising for each compound or combination of compounds to be screened the steps of: i) treating a cell in which an anti-apoptotic protein is over-expressed with the compound, or combination of compounds, to be assessed for chemotherapeutic activity; and ii) assaying for a change in the conformation of Bak expressed in the cell whereby the screen indicates that compounds or combinations of compounds producing a conformation change in Bak have chemotherapeutic activity and that compounds or combinations of compounds that do not produce a conformation change in Bak do not have chemotherapeutic activity.

3. A method according to claim 1 or claim 2, further comprising: iii) comparing the results of step ii) with the change in conformation of Bak induced by the compound, or combination of compounds, in cells in which the anti- apoptotic protein is not over-expressed; whereby the method indicates that compounds, or combinations of compounds, which induce an increased conformational change in Bak of cells in which an anti-apoptotic protein is over-expressed, as compared to cells in which the anti-apoptotic protein is not over-expressed, have chemotherapeutic activity with a greater specificity for cancerous rather than non-cancerous cells.

4. A method according to any preceding claim, wherein the anti-apoptotic protein is Bcl-2.

5. A method according to any preceding claim, wherein the anti-apoptotic protein is Bcl-X_L.

6. A method according to any preceding claim, wherein the over-expression of the anti-apoptotic protein is induced by incorporation in the cell of a pcDNA3.1 vector encoding the anti-apoptotic protein under the control of a CMV (cytomegalovirus) promoter.

7. A method according to any preceding claim, wherein the change in the conformation of Bak is a change at the NH₂ terminal of Bak.

8. A method according to any preceding claim, wherein the change in the conformation of Bak is a change in the BHl region of Bak.

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

10. A method according to claim 9, wherein the specific binding partner is an antibody.

11. A method according to any of claims 1 to 6, wherein the change in the conformation of Bak 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.

12. A method according to any of claims 1 to 6, wherein the change in the conformation of Bak is a change at the BHl 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.

13. A method according to claim 9, wherein the specific binding partner is an antibody fragment.

14. A method according to claim 13, wherein the specific binding partner is an antibody fragment expressed intracellularly.

15. A method of identifying chemotherapeutic activity in a compound, or combination of compounds according to any of claims 9 to 14, wherein the specific binding partner additionally comprises a reporter moiety.

16. A method according to claim 15, wherein the reporter moiety is a fluorophore.

17. A method according to claim 15, wherein the reporter moiety is an enzyme capable of catalysing a chromogenic reaction of a suitable substrate.

18. A method according to any of claims 9 to 14, wherein the presence of the specific binding partner is detected by an indirect labelling technique.

19. A method according to any of claims 10 to 14, wherein the presence of the specific binding partner is detected by an enzyme linked immunosorbent assay (ELISA).

20. A method according to any preceding claim, wherein the chemotherapeutic activity of a combination of compounds is compared with the chemotherapeutic activity of the compounds comprising the combination.

21. A method according to any of claims 1 to 19, wherein the combination of compounds to be tested includes one known chemotherapeutic compound and an adjuvant, or potential adjuvant, of the chemotherapeutic compound.

22. A method according to any preceding claim, wherein the assay for the change in the conformation of Bak is conducted within 8 hours of the treatment of the cells with the compound or combination of compounds.

23. A method according to claim 22, wherein the assay for the change in the conformation of Bak is conducted within 4 hours of the treatment of the cells with the compound or combination of compounds.

24. A kit for identifying chemotherapeutic activity in a compound, or combination of compounds, comprising a cell engineered to over-express an anti-apoptotic protein and an agent for detecting a change in the conformation of the NH₂ terminal of Bak protein in the cell.

25. A kit according to claim 24, wherein the anti-apoptotic protein is Bcl-2.

26. A kit according to claim 24, wherein the anti-apoptotic protein is BC1-X_L.

27. A kit according to any of claims 24 to 26, wherein the agent is an antibody.

28. A kit according to any of claims 24 to 26, wherein the agent is an antibody fragment.

29. A kit according to claim 28, wherein the agent is an intracellularly expressed antibody fragment (intrabody).