FIELD OF THE INVENTION The present invention relates to a vaccine for the treatment and/or prevention of cancer. In particular, but not exclusively, the invention relates to a hybrid cell vaccine for the treatment of cancer. Further aspects of the invention relate to hybrid cells for use in such vaccines, to methods of producing such vaccines and cells, and to additional uses for such cells.

BACKGROUND TO THE INVENTION A number of approaches exist to the treatment and prevention of cancers. One such approach is the stimulation of the patient's own immune system to attack cancerous cells. Such stimulation may be achieved by means of cellular vaccines; that is, by the administration of cells to a patient which will stimulate the immune system such that it recognises and attacks cancerous cells. This may be achieved through the use of cells which express similar or identical antigens to in vivo cancer cells. One promising approach is the use of hybrid cell vaccines, with the cells being derived from a fusion of a professional antigen presenting cell, such as dendritic cells, with a tumour cell. It is believed that the fused cell presents antigens derived from the tumour cell to the patient's immune system, thereby stimulating recognition and attack of the in vivo tumour. The aim of these approaches is essentially to make the tumour cell resemble professional antigen presenting cells in the induction of the primary tumour specific T~cell responses. Hybrid cell vaccines are generally derived from antigen presenting cells fused with tumour cell lines. However, during creation and growth of a tumour cell line, there is a strong possibility that the cell line may have undergone some mutation or modification which may disrupt or alter the antigens presented by the tumour cell line, so reducing the effectiveness of the vaccine. This unpredictable variation in vaccine composition is undesirable. 2 Further undesirable variation in vaccine composition arises from the use of dendritic cells as the antigen presenting cell Such cells may be difficult to maintain in culture after fusion, and may be difficult to characterise, both of which features could reduce the efficacy of vaccines derived therefrom. Further, it is believed that fusions of dendritic cells may be unstable, and that vaccines derived from such fusions may include non-fused dendritic cells, or cells simply transformed with tumour antigens. This unpredictability could lead to a reduction in vaccine efficacy or use on patients for whom it is not appropriate. There is a need for an alternative source of hybrid cell vaccines.

SUMMARY OF THE INVENTION In accordance with a first aspect of the present invention, there is provided a method of producing a vaccine for use in the treatment and/or prevention of cancer, the method comprising: obtaining at least one tumour cell ex vivo, and fusing the tumour cell with at least one lymphoblastoid cell, to provide at least one hybrid cell. The present invention therefore allows for a cellular vaccine to be generated from ex vivo tumour cells. By ex vivo is meant tumour cells obtained directly from a patient, rather than cells which have been cultured to provide an in vitro cell line; such cells may also be known as primary cells. This has a number of advantages over vaccines derived from tumour cell lines. For example, ex vivo cells are less likely to have undergone modification or mutation (other than those leading to the cancerous condition) than tumour cell lines. The use of ex vivo cells also permits the generation of fusion cell lines derived from a specific tumour type, without being limited to those tumour types from which cell lines have previously been derived. This allows for a wider range of possible vaccine targets than with prior art methods. The use of lymphoblastoid cells as antigen presenting cells has a number of advantages over the use of dendritic cells; for example, stability of fusions and ease of post-fusion cultivation, cloning, and characterisation. This allows for a greater consistency in vaccine composition, and for more stable vaccines. Preferably the lymphoblastoid cell is a B lymphoblastoid cell. The lymphoblastoid cell is preferably activated; activation increases the expression of a range of molecules, such as MHC and costϊmulatory ligands such as CD80 and CD86 which enhance the cell's function as a professional antigen presenting cell and improve immunogenicity. The lymphoblastoid cell may conveniently be activated with a virus, preferably with Epstein-Ban- Virus. The method may also comprise the step of generating activated lymphoblastoid cells. Suitable protocols for generating such activated cells are known in the art. It is possible that the EBV latency of the lymphoblastoid cell may also affect the immunogenicity of the hybrid cell; for this reason, it is preferred that EBV latency type III lymphoblastoid cells (Kerr et al. Virology 1992; 187: 189-201) are used, since we believe these are more immunogenic than other latency types. However, other latency types may be used if desired. An alternative method of activation which may be used is activation by CD40 ligation. Fusion of cells may be achieved by any convenient method. Polyethylene glycol (PEG), or PEG and DMSO (dimethyl sulphoxide) is preferred, but any suitable method may be used, for example electrofusion, virally-derived fusogenic peptides, and so forth. The skilled person will be aware of appropriate methods which may be used. The vaccine may be autologous (that is, both tumour cells and antigen presenting cells are derived from the individual on whom the vaccine is to be used), semi-autologous (one type of cell, preferably the tumour cell, is derived from the individual on whom the vaccine is to be used), or allogeneic (neither cell type is derived from the individual on whom the vaccine is to be used). Allogeneic and semi- autologous vaccines, with antigen presenting cells derived from a different individual from that on whom the vaccine is to be used, can in certain circumstances have an adjuvant effect of allogeneic MHC expression obtained from the antigen presenting cells* This may improve the performance of such vaccines. The method may further comprise the step of selecting hybrid cells. Selection may be achieved by any convenient method; for example, using selectable markers introduced into the antigen presenting cells, double-dye selection, chemical resistance or sensitivity of hybrid cells, and the like. The method may further comprise the step of culturing hybrid cells. Hybrid cells may be propagated in culture, and may conveniently be maintained under the same selection means as used to select hybrid cells. Hybrid cells may be cultured clonally, such that a cell line is obtained which is derived from a single hybrid cell. This allows for uniformity of cells within a single line, and for accurate characterisation of cells within the vaccine. The hybrid cells, and / or the lymphoblastoid cells, may be further modified. Modifications may include transfection of cytokine genes (e.g., GM-CSF), genes to further enhance immunogenicity (for example, other costimulalory molecules not expressed), or specific tumour antigens; or may include knocking out particular genes, for example immunoglobulin genes of the parent lymphoblastoid cell so that only the tumour idiotype is expressed. The method may further comprise the step of combining hybrid cells derived from two or more different hybrid cell clones. This allows for the possibility of some variation in cell lines within the vaccine, which may improve performance by for example allowing complementation of different characteristics of different cell lines. Alternatively, or in addition, the method may comprise the step of combining hybrid cells derived from two or more different fusion events. In some embodiments of the invention, however, it may be preferred to produce the vaccine from a single cell line clone. The tumour cell may be obtained from any type of malignancy. Hematological malignancies are preferred; for example, leukaemias, lymphomas, and the like; although solid tumours may be used in certain embodiments. The method may further comprise the step of characterising the hybrid cells; the self-propagating nature of hybrids derived from lymphoblastoid cells, plus the potential for cloning, means that the hybrid cells lend themselves to accurate characterisation, which is of benefit to the targeted use of the vaccine on particular tumour types. Hybrid cells may be characterised by genetic / genomic, proteomic, and other means. For example, hybrid cells may be characterised to determine cell surface and intracellular protein expression (for example, cell surface molecules, T cell costimitlatory ligand molecules); HLA (antigen presenting molecules) by genetic or protein expression methods; expression of known tumour antigens (e.g. MAGE family, fusion proteins from gene translocations e.g. bcr/abl, immunoglobulin idiotype, etc); characterisation of antigen processing pathways; responses to cytokines and other biochemical / biological stimuli; ability of hybrid cells to stimulate T cell responses in vitro, using either allogeneic or autologous T cells as responder cells(methods include lymphocyte proliferation, cytokine synthesis and release, cellular cytotoxicity, Elispot assays, etc). The invention further provides the use of a hybrid cell derived from an ex vivo tumour cell fused with a lymphoblastoid cell in the preparation of a medicament for the treatment of cancer. A further aspect of the present invention provides a method of treating cancer, the method comprising administering a hybrid cell derived from an ex vivo tumour cell fused with a lymphoblastoid cell to a patient in need of such treatment The method may further comprise obtaining at least one tumour cell ex vivo, and fusing the tumour cell with at least one lymphoblastoid cell, to provide at least one hybrid cell. The tumour cell may be obtained from the patient in need of such treatment, or the cell may be obtained from a different patient. The present invention also provides a method of treating ex vivo tumour cells, the method comprising fusing the tumour cell with at least one lymphoblastoid cell, to provide at least one hybrid cell. The method may also comprise the step of removing the tumour cell from a patient. The hybrid cell may be returned to the same patient after fusion. Alternatively, the hybrid cell may be returned to a different patient. According to a further aspect of the present invention, there is provided a hybrid cell derived from an ex vivo tumour cell fused with a lymphoblastoid cell, for use as a vaccine. The invention also provides a hybrid cell derived from an ex- vivo tumour cell fused with a lymphoblastoid cell. Cells of the present invention may be useful for purposes other than as vaccines and treatments. Other potential uses include stimulation of tumour-specific immune responses in vitro, induction and expansion of tumour- specific effector cells (or other potential immune mediators) in vitro for adoptive transfer; tools for identifying novel tumour antigens; study of mechanisms involved in antigen-specific immune responses and / or oncogenesis. A still further aspect of the present invention provides a method for generating hybrid cells, the method comprising obtaining at least one ex vivo tumour cell from a subject, and fusing the tumour cell with at least one lymphoblastoid cell.

BRIEF DESCRIPTION OF THE DRAWINGS These and other aspects of the present invention will now be described by way of example only and with reference to the accompanying drawings, in which; Figure 1 shows a cell surface antigen expression phenotype for a particular hybrid cell type in accordance with an embodiment of the present invention; and Figure 2 and 3 show stimulation of T cell proliferation in vitro by hybrid cells in accordance with an embodiment of the present invention. DETAILED DESCRIPTION OF THE DRAWINGS

Materials and Methods Experimental protocols and materials were generally as described in Dimnion et al (Immunology. 1999 Dec;98(4):541-50), to which the skilled reader is referred for further details. Briefly, the EBV B-LCL cell line HMy2 was used to generate APC / tumour cell hybrids by cell fusion with tumour cells in the presence of PEG / DMSO in accordance with established techniques. Haernatological tumour cells were obtained ex vivo from patients using conventional techniques. The HMy2 celϊ line is resistant to ouabain, but sensitive to hypoxaπthine, aminopterin and thymidine (HAT), allowing for chemical selection of hybrid cells in vitro. Hybrid cell lines were analysed for the expression of cell surface markers by monoclonal antibody (mAb) staining of viable cells using immunofluorescence (IF) techniques, followed by flow cytometric analysis using a FACSCalibur flow cytonieter (Becton Dickinson Ltd, Cowley, UK). Stimulation of proliferation of allogeneic (normal healthy donor) or autologous (patient from whom the tumour cells were derived) T cells was performed by co-culture (5 days, 37°, 5%CO2) of peripheral blood mononuclear cells or separated T cells with hybrid cells, parent EBV B-LCL or tumour cells, and was estimated by pulsing cultures with [3H]thyrnidine and harvested after incubation (overnight, 37°, 5% CO2) with a Tomtec cell harvester onto glass fibre filters. Incorporated radioactivity was measured (as counts per minute [c.p.m.]) in a Wallac 1450 Microbeta liquid scintillation counter.

Results

A number of hybrids were made using EBV B-LCL and ex vivo haematological tumour cells. The number of cell lines derived from various types of tumour was as follows. B-CLL (B cell chronic lymphocytic leukaemia) 5 c-ALL (common acute lymphoblastic leukaemia) 1 Follicular lymphoma 1 Mantle cell lymphoma 2 Multiple myeloma 5 Acute myeloid leukaemia 2 Analysis of the expression of cell surface markers was carried out as described. Figure 1 shows a typical pheπotype of one B-CLL hybrid (identified as hybrid MD049). Markers which were studied include CDS, CDl 9, CD40, CDl Ia, CD50, CD54, CD58, CDSO5 CD86, anti-HLA-ABC, and anti-HLA-DR. The expression profile indicates that the hybrid cell (shaded plots) presents a largely identical profile to the parent EBV B- LCL cell (the lighter of the two line plots; the darker line plot refers to the parent tumour cells). Similar results were obtained for other hybrids. The stimulation of T cell proliferation in vitro by the hybrids was then studied. Figures 2 and 3 show, respectively, stimulation of allogeneic and autologous T cell proliferation in response to the indicated cells. It can be seen that the hybrid cells (striped bars; HMy2 - SB040) stimulate strong T cell responses in vitro, similar to levels stimulated by the parent EBV B-LCL (solid bars; HMy2), and significantly greater than the equivalent responses to tumour cells alone (shaded bars; SB040).

Discussion The present inventor has performed a range of experiments to demonstrate the feasibility of the use of hybrid cells derived from ex vivo tumour cells as cellular vaccines. Stable, self-propagating hybrid cell lines have been generated by fusion of EBV B-LCL with a range of haematological malignancies (including chronic lymphocytic leukaemia, πon-Hodgkin's lymphomas, acute lymphoblastic leukaemia, multiple myeloma, acute myeloid leukaemia). We have demonstrated that these express both class I and class II MHC, and a range of costimulatory and other surface molecules typical of professional antigen presenting cells, including CD80 and CD86 (where parent tumour cells lacked such expression). We have further demonstrated that these stimulate strong T cell proliferative responses in vitro (where unmodified tumour cells stimulated little or no response), where T cells derived from either normal healthy donor (allogeneic responses) or patient from whom the tumour cells were derived (autologous responses). Such responses are important for potential vaccine responses in vivo, to know that patients' immune cells are able to respond to the hybrid cells. Iii addition to these results from ex vivo tumour cells, we have also obtained preliminary results from human tumour cell lines derived from solid tumours, such as melanomas and cervical cancers. We have demonstrated the feasibility of generating stable hybrid cell lines using tumour cell lines derived from solid tumours (melanoma, cervical cancer). We have demonstrated that these hybrids stimulate significantly stronger allogeneic T cell responses in vitro than the parent tumour cell lines - also applies to T cell subpopulations CD4+, CD8+, naϊve (CD45RA+) and memory (CD45RO+). We have demonstrated that the enhanced T cell responses were dependent (at least in part) on expression of CD80 and CD86 by the hybrid cells. Further, we have demonstrated the ability of hybrid cells to stimulate cytotoxic T cell responses in vitro, as well as showing highly efficient antigen processing and presentation by MHC class I to antigen-specific cytotoxic T cell clones. We have further demonstrated expression of relevant tumour associated antigens by hybrid cells, showing a similar antigen expression to tumour cells from which the hybrids were derived; and we have demonstrated co-expression of HLA molecules derived from both parent cell lines (tumour cells and EBV B-LCL). The generation and production of hybrid cells from ex vivo tumour cells has a number of advantages for vaccine production over conventional techniques based on cell lines. These advantages include: o Expression of a range of relevant tumour antigens by the hybrid vaccine cells for the tumour type being treated; o Processing and presentation of these antigens to the immune system of the recipient of the vaccine o Stimulation of the recipient's immune system though the expression of appropriate T cell costimulatory ligaπd molecules by the vaccine cells o Induction of tumour-specific immune responses o Adjuvant effect of allogeneic MHC expression (when used in allogeneic and semi-autologous settings) o Potentially of use in a broad range of cancer types, although each hybrid cell cancer vaccine would be expected to be specific for that cancer type o Vaccines could be used as monotherapies in the treatment of cancer, or in combination with other forms of cancer treatment o Ease of culture and cloning of hybrid cells compared with hybrids produced with other antigen presenting cells o Stability of hybrids based on Iymphoblastoid cells The key advantage of the use of ex vivo cells is that such cells are less likely to have undergone additional mutations or modifications in vitro compared with cell lines, and so are more likely to provoke suitable specific immune responses.