NEW VACCINE FORMULATIONS - 1

Abstract: The invention relates to new vaccines for use as immunotherapeutic agents in the treatment of cancer particularly but not limited to prostate cancer. Further the invention relates to alternative culture techniques which offer improved scale-up and processing benefits and significantly improve the activity of vaccines based on whole allogeneic cells or derivatives thereof. It further relates to a method of producing whole cell cancer vaccines which comprise culturing malignant cells under conditions which are not conventional static 2D culture techniques.

Description: New Vaccine Formulations - 1 Summary New vaccines for use as immunotherapeutic agents in the treatment of cancer are described. First generation whole cell vaccines relied on traditional methodologies in the culture and production of the autologous or aUogeneic cell based products, whereby autologous tumour cells or immortalised aUogeneic tumour cell lines were grown under static two dimensional (2D) culture conditions. We describe alternative culture techniques which offer improved scale-up and processing benefits and in addition significantly improve the activity of vaccines based on whole cells or derivatives thereof. We describe changes in the phenotype of well-characterized established cell lines which enhances their overall potential as tumour immunotherapeutic agents. Furthermore, vaccines produced by the various culture techniques described have additional advantages over single cell suspension vaccines in the rate and extent of presentation to the immune system which leads to enhanced performance in treating specified tumours. Tumour cells or immortalised tumour cell lines grown in 2D culture bear little or no resemblance phenotypically or histologically to how the said tumour cells might grow in vivo. Tumour cells in 2D culture take up a regular contact inhibited growth pattern where the ceils grow as a uniform sheet on the plastic culture flask. We have found that alternative culture methodologies that are amenable to large-scale production of tumour cell vaccines and their derivatives also have surprising effects on the immunogenicity and/or antigenicity of the tumour cell lines. Furthermore the tumour vaccines produced have additional prolonged release activities related to the morphology of the cell masses resulting from the culture conditions used. The invention relates to culture methods for large scale production of vaccines, phenotype modulation of cell lines, processing methods for the production of the vaccines, analytical techniques to evaluate phenotype adjustment, extraction techniques to produce sub-cellular fractions of tumour cell lines and new compositions which display increased immunogenicity in standard models of tumour prevention and therapy. Background to the Invention It is known in the art that cancerous cells contain numerous mutations, qualitative and quantitative, spatial and temporal, relative to their normal, non- cancerous counterparts and that at certain periods during tumour growth and spread a proportion of the cancerous cells are capable of being recognised by the host's immune system as abnormal. Cancer cells express certain tumour- specific antigens (TSA) and tumour-associated antigens (TAA) that may serve as markers or targets for the immune system. This has led to numerous research efforts world-wide to develop immunotherapies that harness the power of the immune system and direct it to attack the cancerous cells, with the aim of eliminating such aberrant cells at least to a level that is not life- threatening (reviewed in Maraveyas, A. & Dalgleish, A.G. 1977 Active immunotherapy for solid tumours in vaccine design in The Role of Cytokine Networks, Ed. Grego adis et al., Plenum Press, New York, pages 129-145; Morton, D.L. and Ravindranath, M.H. 1996 Current concepts concerning melanoma vaccines in Tumor Immunology - Immunotherapy and Cancer Vaccines, ed. Dalgleish, A.G. and Browning, M., Cambridge University Press, pages 241-268. See also other papers in these publications for further detail). Tumours have the remarkable ability to counteract the immune system in a variety of ways which may include: down-regulation of the expression of potential target proteins; mutation of potential target proteins; down-regulation of surface expression of receptors and other proteins; down-regulation of MHC class I and II expression thereby disallowing direct presentation of TAA or TSA peptides; down-regulation of co-stimulatory molecules leading to incomplete stimulation of T-cells leading to anergy; shedding of selective, non representative membrane portions to act as decoy to the immune system; shedding of selective membrane portions to anergise the immune system; secretion of inhibitory molecules; induction of T-cell death; and many other ways. What is clear is that the phenotypic and genetic heterogeneity and plasticity of tumours in the body will have to be matched to a degree by immunotherapeutic strategies which similarly embody heterogeneity or polyvalency. The use of whole cancer cells, or crude derivatives thereof, as cancer immunotherapies can be viewed as analogous to the use of whole inactivated or attenuated viruses as vaccines against viral disease. The potential advantages are: (a) whole cells contain a broad range of antigens, providing an antigenic profile of sufficient heterogeneity to match that of the lesions as described above; (b) being multivalent (i.e. containing multiple antigens), the risk of immunological escape is reduced (the probability of cancer cells 'losing' through mutation all of these antigens is remote); and (c) cell-based vaccines include TSAs and TAAs that have yet to be identified as such; it is possible if not likely that currently unidentified antigens may be clinically more relevant than the relatively small number of TSAs/TAAs that are known. Cell-based vaccines fall into two categories. The first, based on autologous cells, involves the removal of a biopsy from a patient, cultivating tumour cells in vitro, modifying the cells through transfection and/or other means, irradiating the cells to render them replication-incompetent and then injecting the cells back into the same patient as a vaccine. Although this approach enjoyed considerable attention over the past decade, it has been increasingly apparent that this individually-tailored therapy is inherently impractical for several reasons. The approach is time consuming (often the lead-time for producing clinical doses of vaccine exceeds the patients life expectancy), expensive and, as a 'bespoke' product, it is not possible to specify a standardised product (only the procedure, not the product, can be standardised and hence optimised and quality controlled). Furthermore, the tumour biopsy used to prepare the autologous vaccine will have certain growth characteristics, interactions and communication with surrounding tissue that makes it somewhat unique. This alludes to a potentially significant disadvantage to the use of autologous cells for immunotherapy: a biopsy which provides the initial cells represents an immunological snapshot of the tumour, in that environment, at that point in time, and this may be inadequate as a phenotypic representation over time for the purpose of a vaccine with sustained activity that can be given over the entire course of the disease. The second type of cell-based vaccine describes the use of aUogeneic cells which are genetically (and hence immunologically) mismatched to the patients. AUogeneic cells benefit from the same advantages of multivalency as autologous cells. In addition, as aUogeneic cell vaccines can be based on immortalised cell lines which can be cultivated indefinitely in vitro, this approach does not suffer the lead-time and cost disadvantages of autologous approaches. Similarly the aUogeneic approach offers the opportunity to use combinations of cells types which may match or anticipate the disease profile of an individual in terms of stage of the disease, the location of the lesion and potential resistance to other therapies. Previous human clinical studies utilising whole cell based approaches have relied upon growth and production of the cells in traditional 2D culture systems - cells grown in static plastic flasks within incubators, with or without CO 2 atmospheric supplementation (termed "2D" culture herein). The cells grow in two dimensions only as a flat monolayer or sheet of cells adherent to the plastic or glass vessel. Cell growth usually becomes contact inhibited at confluence and attempts to grow the cells beyond this point usually results in the loss of the culture. It is generally not possible to control the healthiness of the cells in 2D static culture to any degree, since progression to apoptosis inevitably leads to ceils leaving the substratum and lysing in the media. Furthermore protracted culture under anoxia or media starvation, conditions which favour selection of aggressive cell phenotypes, is generally not possible in 2D culture without significant efforts aimed at growing out new cell variants that may have lost the relevant phenotype. The growth of cells in 2D culture relies on the serial passage of cells, wherein the cells are repeatedly removed from the substratum and subjected to a harsh cycle of centrifugation, washing and manipulation in suspension. The cells are grown and then harvested by mechanical means or treatment with trypsin to remove the cells from the substratum. The effect of trypsin, other proteolytic enzymes or other agents used for the purpose is to break attachment of the cells to the flask substrate. Proteolytic treatment inevitably removes some protein molecules from the surface of the cells, which may result in loss of potential epitopes that enable the recipient to raise an humoral or cell based immune response to the tumour. For example, proteolysis may remove MHC-I molecules that could present peptides in a direct manner to patient T cells, or adhesion molecules that offer co-stimulatory activity to direct presentation. Alternatively proteolysis may simply remove aberrantly expressed TAA or TSA which when processed through a cross-priming or other route could also contribute to an anti-tumour response. Furthermore, there is relatively little opportunity to manipulate the cell phenotype since, in order to attain the quantity of ceils required, a number of passages under standard growth conditions are required and attempts to drive the cells into a stressed or apoptotic or necrotic or otherwise altered phenotype prior to harvest results in significant losses of fragile cells at harvest undermining the feasibility of producing vaccine at a practical scale. This invention describes culture techniques not previously utilized in the production of vaccines for cancer immunotherapy that in one form do not require the removal of cells from a substrate In another embodiment this invention discloses culture techniques which elicit a modified phenotypic expression of proteins and/or peptides which, in some cases, are detectable even after trypsinization of the cells, and which alters the immunogenicity of the cells being cultured Description of the Invention An alternative to the 2D growth technique is the use of roller bottle culture where adherent cells are transiently immersed in media as the bottles rotate A methodology described herein to grow ceils in roller bottles has unexpectedly yielded excellent biomass production in conjunction with an increase of a set of immunogenic peptides which resulted from growth under these conditions. This type of growth technique has not generally been applied in the field of tumour cell vaccine production The technique requires the use of trypsin or mechanical techniques to remove the ceils from the bottles, however the recoveries are generally very good More importantly the cells whilst attached to a substratum receive gas and nutrients in ways which are different to normal static 2D culture which result in retained phenotypic changes even after trypsin treatment Comparison of peptide extracts from 2D grown cells in static flasks with the same cells grown in roller bottles has shown that whilst there are many common peptides expressed under both growth conditions, there are both higher levels of peptides produced in the roller grown cells and a spectrum of new peptides are uniquely produced by cells grown using roller technology In a further embodiment of this invention cells grown under roller bottle conditions were tested in vaccination models of tumour prevention and shown to be immunogenic and perform comparably with cells grown under normal 2D culture conditions A separate embodiment of this invention is the use of peptide extracts and other sub-ceiiular fractions derived from cells grown under non-standard conditions This includes cells grown on roller bottles, on microcarπers, on polyester fibre discs, 3D culture on non adherent flasks and 3D culture under microgravity conditions. The sub-cellular fractions or peptide fractions derived from the cells offer the opportunity to significantly increase the active principle of a given vaccine. This aspect of the invention also allows for further fractionation of the sub-cellular fractions or peptides to select the active principles and to simplify the vaccine composition if so required. A further advance in culture techniques for vaccine production is the use of micro-carrier culture whereby adherent cell lines are grown on small particles held in suspension by various means of agitation. The advantage of this growth method is that a significant increase of surface area can be achieved for a similar sized culture vessel. For instance a 500 ml bioreactor with a stirred bed of 1x10 6 microbeads/ml of 50μm in diameter will have the same surface area of 22-25 T175 flasks normally used for 2D culture. The resulting biomass can be removed from the beads by trypsinization or injected whilst adherent to the bead. If the bead itself were to be injected then this should be biocompatible and preferably biodegradable. A different methodology for the growth of the tumour cells relies on immobilization of cells on an inert matrix comprised of non-woven polyester discs which may be suspended in a bioreactor providing a very large surface area within a relatively small volume of media. This growth matrix results in excellent scale-up potential due to the significant enhancement of surface area over which the cells can grow. The bioreactor allows for very close control of nutrient utilization and oxygen consumption giving far greater control of growth properties. The morphology of the cells grown under these conditions is very different to 2D cultures; the cells develop extended membrane processes which grow out along the fibres of the disc. As a result of growth in this orientation the cells take on new phenotypic expression of immunologically significant molecules which are beneficial to direct and/or indirect antigen presentation following vaccination. Most significantly using this growth methodology we have unexpectedly shifted the phenotypic expression of the cells from being effectively MHC-I negative to the whole population becoming MHC-I positive, albeit at low levels. A further aspect of this invention is the manipulation of cell growth in static 2D flask culture, whereby cell adherence to the substratum is prevented by coating the flask with alginates or other similar hydrocolloid. This methodology is commonly used in the production of anchorage independent clones but hitherto has not been used in the production of vaccines or immunotherapeutic agents. Adaption of the cells to anchorage independent growth leads to the production of a spheroid or cell aggregate morphology which is phenotypically closer to in-vivo growth. The most advanced culture technique embodied herein is the use of a 3D microgravity culture which is similar to growth of cells on microcarriers in that normally adherent cells are held in suspension, though in the case of 3D microgravity this is not necessarily achieved on microcarriers and, furthermore, the rotation of the vessel is perpendicular to gravity. The microgravity culture technique as applied in this invention uses a revolving chamber filled with media which rotates horizontally. The seeded cells are prevented from adhering to the chamber walls by the speed of rotation and thus the cells are constantly in a state of "free fall" which resembles microgravity conditions achieved in space. We have found that it is possible to achieve the same biomass production in one 50 ml 3D microgravity vessel as is possible in 30 T175 flasks. By utilising a 500 ml 3D microgravity vessel we can achieve the same vaccine production of mammalian tumour cell lines as we can obtain by culturing approximately 300 T175 flasks or 30 1 ,700 cm 2 roller bottles. Under such microgravity conditions cells take on new and unexpected growth properties such as the propensity to form masses of cells or spheroids even without the aid of microcarriers. A further aspect of this invention is a direct process of formulation of cells harvested from 3D microgravity culture into vaccine, without the need to treat the cells with trypsin. This process therefore has the advantage of significantly increasing the yield of cells following harvest. Whereas for traditionally grown 2D cultures significant losses are incurred during the trypsinization, neutralization, washing and formulation, cells harvested as spheroids are significantly more robust to handle and wash, thereby leading to enhanced recoveries. We also disclose an enhanced phenotype of a cancer vaccine resulting from growth as a spheroid in 3D-microgravity culture. Cells grown under 2D culture bear little resemblance to tumour cells in vivo. The monolayers of 2D grown cells have a uniform, cobblestone appearance, become contact inhibited, grow under conditions of optimal gas and nutrient supply and adhere directly to the plastic or glass vessel in which they are being grown. We have found that cells grown in 3D microgravity conditions grow in a way much more akin to tumour cell growth in vivo; cells seeded directly into the microgravity chamber form spheroids which have an outer layer supplied optimally with gas and nutrients, possess an inner layer which is somewhat gas and nutrient deprived and slightly apoptotic, are adherent not to plastic or glass but to other cells and any extra-cellular matrix secreted by those cells, and which maintain a micro environment within the spheroid which may retain growth factors and other para and autocrine secretions which would otherwise be diluted out by the media. The net result of the microgravity growth of the tumour cells is that the spheroids grow with an heterogeneous phenotype similar in many ways to tumour cells in vivo. This type of phenotype is advantageous for a cell line grown for use as a tumour vaccine to elicit a response against an existing tumour. A particular aspect of the invention is counterintuitive in the field of tumour immunotherapy. Currently held belief is that a uniform cell population and single cell suspension is the desired form of a tumour vaccine. We have found that contrary to expectation, heterogeneous multicellular spheroids perform as excellent vaccines in immunisation protocols. The spheroids are heterogeneous with respect to their exposure to both media and oxygen which impacts on their metabolic status. Furthermore 3D culture allows far greater control of the apoptotic index of the cells, in that cells can be harvested in a form suitable for vaccination at almost every point from an healthy to a fully apoptotic population. In addition, cells in 3D culture can be held in stressed conditions for significant periods of time which cannot simply be achieved in traditional 2D culture. Cells held at confluency in 2D culture very quickly loose viability. Analytical techniques such as immunohistochemistry, flow cytometry, 2D electrophoresis, crossed immuno-electrophoresis, Western blotting, DNA hybridisation, fluorescent in situ hybridisation and other techniques has revealed significant shifts in phenotype of commonly used cell lines which result in significantly enhanced immunogenicity. A further embodiment of this invention is the use of 3D-microgravity culture to enhance the phenotype of existing cell lines that hitherto have not been grown in 3D culture. A further embodiment of the invention results from the use of spheroids grown in 3D-microgravity culture as a tumour vaccine or immunotherapeutic. Single aUogeneic cells when injected may be phagocytosed by dendritic cells (DC) or macrophages (M and processed for presentation to naive and memory T cells. Whilst DC and M have remarkable capacity to engulf single cells and many single cells there is a size limitation beyond which the DC and M cannot engulf a particle or cell. Spheroids may be grown to visible size in the 3D-microgravity culture where each spheroid is a millimeter or more in size. The spheroids offer an advantage over single cells in use as a vaccine since they represent a slow release formulation whereby concerted action by DC and M over several days is required to resolve the cell mass of the spheroid at the site of injection thereby leading to prolonged presentation of the vaccine components which is known for peptide and protein antigens to be a favoured means of enhancing the immune response. A further embodiment of this patent is the production and formulation of spheroids with a range of sizes to encompass differing size bands to effectively control the rate of presentation to the immune system. We also anticipate a process to separate and blend spheroids to give precisely controlled compositions of cell masses in the range from 5um-5mm utilising standard differential centrifugation, gradient centrifugation, and elutriation techniques. Description of the Drawings Figure 1 SELDI mass spec analysis for peptides extracted from cells grown in flasks and bottles Figure 2 Flow cytometry analysis of K1735 and B16 cells grown in 2D flask and polyester fibre disks Figure 3 Cell morphology of PNT2-C2, NIH 1542 and K1735 grown in microgravity culture Figure 4 Comparative analysis of apoptosis in PNT2-C2, NIH 1542 and K1735 cells grown in 2D and 3D culture Figure 5 Survival curves for rat protection experiment Examples Mammalian Tumour Cells Grown in Roller Bottles Retain Altered Phenotype Mouse melanoma cell lines K1735 and B16F10 were grown in DMEM in T175, 2D culture and also in roller bottles. Cells were seeded into the 2D or 3D vessels at 0.5-2 x10 6 or 0.5-2.0 x 10 7 cells, respectively. Growth was allowed to proceed until near confluency had been reached. For both cell lines a similar number of cells was achieved compared to growth in roller bottles as opposed to growth in 2D flasks (Table 1 ). More significantly even after trypsinisation there were clear phenotypic differences between the cells grown in 2D flasks and roller bottle grown cells. Analysis of small immunogenic peptides extracted by the method of Nair et al (1997 Eur J Immunol 27, p589-597) was performed using Surface Enhanced Laser Desorption lonisation (SELDI) mass spectrometry Immunogenic peptides extracted from the cells provided a unique fingerprint to that cell line. Whilst there were some common peptides between K1735 and B16/F10 there were also clearly unique peptides to each cell line (Figure 1 ) Furthermore there were higher levels of peptides extracted for each of the roller bottle grown cultures than the corresponding flask grown culture In addition there was a greater degree of complexity in peptides extracts from the roller bottles for each of the cell lines Mammalian Tumor Cell Lines Grown in Roller Bottles Retain Immunogenicity K1735 murine melanoma cells were grown in T175, 2D culture and also in roller bottles in similar media The ceils were harvested just prior to confluency and used in a standard model of aUogeneic tumour protection in the B16F10 tumour model Groups of 5, C57/B6 mice were immunised with 1x10 6 K1735 cells irradiated at 150Gy harvested from 2D flasks or roller bottles, at T-10 days The mice were then challenged on Day 0 with 5x10 5 live B16F10 cells and then monitored for tumour growth Both groups of mice showed a level of protection against B16F10 challenge over and above the PBS sham injected animals Growth of Mammalian Tumour Cells on Immobilised Matrix It has proven possible to grow B16F10 murine melanoma cells, DU145, LnCap and PC3 human prostate tumour lines and MatLyLu and PAIII rat prostate tumour cells on non-woven polyester discs ( Fibra-Cel™ New Brunswick Scientific) which may be suspended in bioreactors (Spinner Basket™ New Brunswick Scientific) Cells were seeded at 1x10 4 - 1x10 6 onto each disc and allowed to grow in culture media for five days before being stained and counted Cells grown on the discs were trypsinised and then stained for a variety of surface markers For the B16F10 cell line we have found a variable phenotype shift for MHC-I expression such that growth on the discs promotes enhanced MHC-I expression over and above cells grown in normal 2D culture, (Figure 2) Growth of Mammalian Tumour Cells in 3D Microgravity Culture Murine K1735 cells, PNT2-C2 normal human prostate epithelial cells and NIH 1542 prostate tumour cells were seeded into three 50ml Synthecon cassettes at 1x10 7 cells and placed on a microgravity apparatus rotating initially at 12 revolutions per minute After 10 days growth with intermittent replenishment of media and growth factors, the cassettes were harvested and analysed for cell growth, morphology and biomass increment All of the cell lines showed significant biomass increment as depicted (Table 3) and also showed formation of spheroid structures with slightly different morphology dependent on the cell line (Figure 3) It was also possible to utilise the microgravity apparatus in a semi continuous batch production mode whereby cell biomass was extracted after 7-10 days and some 5-10 ml of the remaining biomass was used to seed the next 45ml of fresh media and thereafter allowed to grow for a further period of 7-10 days in which time the biomass re-grew to similar levels found in the primary microgravity culture Production of Whole Cell AUogeneic Vaccine by 3D Culture Techniques A mammalian cell line was seeded at between 1x10 - 1x10 8 cells in a 50ml rotating microgravity cassette (Synthecon) in appropriate media. The cells were allowed to grow for a period of 7-14 days with intermediate media and growth factor replenishment. At harvest the cassette was emptied into a centrifuge vessel and lightly centrifuged to pellet the biomass. The visible and invisible spheroids were washed three times in PBS or other inert media, irradiated at 50-300 Gy and then formulated in appropriate freezing mixture and dispensed on the basis of either cell number determined by trypsinisation and counting or on a protein basis. No trypsinisation was required to remove the vaccine cells from the culture vessel thereby retaining valuable TAA and TSA which will elicit an anti-tumour response in the recipient mammal. Furthermore the spheroids are significantly easier to harvest as a result of their size, requiring less aggressive centrifugation than is normally required for individual cells which leads to significant enhancement of the recovered cell mass. Control of Apoptotic Index of Cells Grown in 3D Culture. Direct comparison of ceils grown in 2D and 3D culture was made following trypsinisation of the respective cultures. Analysis was performed by flow cytometry to determine the level of apoptosis in the cells harvested from both type of culture. A significant difference between 2D and 3D culture grown cells was observed for all of the cell lines grown, namely the murine K1735, human PNT2-C2 and human NIH 1542. Analysis of the viability of the cells grown either in 2D or 3D culture revealed similar viability in all cases as determined by trypan blue exclusion. Analysis of apoptosis revealed there to be no significant differences between cells grown by 2D and 3D methodologies at early stages of spheroid development. At later time points in the growth of the spheroids flow cytometry analysis of cells following annexin staining revealed whole population shifts towards annexin positive staining for cells grown in 3D culture, whereas propidium iodide staining revealed little or no change for cells grown either in 2D or 3D culture (Figure 4). This suggests that cells grown in 3D culture are slightly apoptotic hence they stain positive with annexin, but are no more late stage apoptotic than 2D grown cells, manifest by similar levels of propidium iodide staining. Previous attempts to drive 2D cultures into a more apoptotic phenotype by holding trypsinised adherent cells in suspension, or irradiation of trypsinised cells has failed to show such a significant shift towards apoptosis as achieved in this experiment. It is doubtful whether a 2D culture could be driven into apoptosis and then subsequently be harvested without incurring significant losses of the inevitably fragile cells. The embodiment herein allows for the majority of the cells in the harvested material to express an apoptotic index of a predetermined level dependent on the time of harvesting, which using traditional 2D culture could not be achieved. It is well known in the literature that phosphatidyl serine exposure on the outer membrane in cells (the basis for annexin staining) is an early apoptotic event and furthermore a significant signal for phagocytosis by macrophages and dendritic cells. Hence cells grown under 3D culture can be pushed into an early apoptotic phenotype, recovered from harvest and have a significant targeting signal for the attraction and uptake by antigen presenting cells. Since most tumour cell lines have low MHC-I expression and similarly low co- stimulatory molecule expression the direct route of T-cell activation is unlikely. Therefore to illicit an CD8+ anti-tumour response it is likely that aUogeneic vaccine cells will need to be processed by an antigen presenting cell and thus the targeting function of PS externalisation on cells grown in 3D microgravity conditions will prove advantageous in the production of cancer immunotherapeutics. Production of a Whole Cell AUogeneic Vaccine by 3D Microgravity Culture Techniques A whole cell aUogeneic vaccine was produced by 3D microgravity culture techniques for evaluation in either a rat therapy or rat potection model. PA3 Clone 1 was grown in 3D microgravity culture as follows. The microgravity cassettes were filled with 35 ml culture medium (RPMI 1640 10% FCS/2mM Glutamine) through the screw cap, to this 6 x 10 6 cells suspended 6 ml of culture medium was added. The cassette was then topped up to full (approx. 50ml) with -10 ml culture medium and the screw cap replaced. A 20ml syringe fitted with a 20μm filter attached was screwed onto the outlet valve of the cassette. A second 20ml syringe fitted with a 20μm was filled with culture medium and screwed onto the inlet valve of the cassette. Both valves on the cassette were opened and pressure applied to the inlet syringe to force any remaining air bubbles from within the cassette. Once all the air bubbles had been removed both valves were closed and any remaining culture medium was aspirated off and the valves sealed with stoppers. The cassettes were attached to the microgravity apparatus and rotated at 16 rpm at 37°C/5% C0 2 . Cultures were allowed to continue for 3-4 days when they were harvested by unscrewing the cap on the microgravityand pouring the contents into a 50ml tube. The cell suspension was washed three times in PBS with centrifugation, and stored on ice, prior to irradiation. The harvested spheroid suspension was irradiated as follows. Tubes containing cell suspensions were irradiated with 6 x 25 Gy (Total: 150 Gy). A ceil count is then performed on each sample using a haemocytometer. Briefly 10μl of each cell suspension in mixed with 90μl of tryphan blue and approx. 10μl was loaded under the coverslip of the haemocytometer. A cell count was performed using a x20 objective lens. Each cell suspension was then centrifuged at 18,000 rpm for 3 minutes. The resultant pellet was re-suspended into vaccine freezing medium (RPMI 1640/8%FCS/8%DMSO) at 5 x 10 6 cells per ml of freezing medium. Suspensions were then aliquoted into cryovials at 1.2ml per vial; vials were then placed into a -80°C freezer overnight prior to storage in liquid nitrogen. The aliquoted products were then used in vaccination studies in either protection experiments or therapy experiments utilising Copenhagen rats challenged with MatLyLu tumours. Comparison of 2D with 3D Grown cells as AUogeneic Vaccines in Rats PA3 Clone 1 rat prostate tumour cells were grown in either 2D or 3D microgravity culture for comparison as an aUogeneic vaccine in Copenhagen rats. PA3 Clone 1 was grown in 3D microgravity culture and produced for vaccine as described in the previous section. PA3 Clone 1 was grown in 2D as follows. 1 x 10 6 cells were seeded into a T175 tissue culture flask in 35 ml of RPMI 1640 culture medium supplemented with 10% FCS/2 mM Glutamine. Cells were allowed to grow until confluent under conditions of 37°C/5% C0 2 . Confluent cells were harvested by washing the monolayer with 10ml Hank's buffered salt solution, followed by 5ml trypsin/EDTA solution to remove cells from the flask. Once all cells had lifted away from the flask surface the trypsin reaction was stopped by the addition of 5ml FCS. The 10ml of cell suspension was collected in a 50ml tube, centrifuged at 18,000 rpm for 3 minutes. The resultant pellet was re-suspended into 10ml PBS and stored on ice, prior to irradiation The harvested cell suspension was irradiated as follows. Tubes containing cell suspensions were irradiated with 6 x 25 Gy (Total: 150 Gy). A cell count is then performed on each sample using a haemocytometer. Briefly 10μl of each cell suspension in mixed with 90μl of tryphan blue and approx 10μl was loaded under the coverslip of the haemocytometer A cell count was performed using a x20 objective lens Each cell suspension was then centrifuged at 18,000 rpm for 3 minutes The resultant pellet was re-suspended into vaccine freezing medium (RPMI 1640/8%FCS/8%DMSO) at 5 x 10 6 cells per ml of freezing medium Suspensions were then aliquoted into cryovials at 1 2ml per vial, vials were then placed into a -80°C freezer overnight prior to storage in liquid nitrogen The appropriate vials were removed from storage in liquid nitrogen, thawed and 0 2 ml from the vial was injected into shaved right hind flank of a Copenhagen rat 1 vial is sufficient to inject 5 animals (1 experimental group) See below for injection regime for protection and therapy The challenge material used for these experiments is the cell line Mat-Ly-Lu (Wild Type) 1 x 10 6 cells was seeded into a T175 tissue culture flask in 35 ml of RPMI 1640 culture medium supplemented with 10% FCS/2 mM Glutamine Cells were allowed to grow until confluent under conditions of 37°C/5% C0 2 Confluent cells were harvested by washing the monolayer with 10ml Hank's buffered salt solution, followed by 5ml trypsin/EDTA solution to remove cells from the flask Once all cells had lifted away from the flask surface the trypsin reaction was stopped by the addition of 5ml FCS The 10ml of cell suspension was collected in a 50ml tube, centrifuged at 18,000 rpm for 3 minutes The resultant pellet was re-suspended into 10ml PBS and stored on ice A cell count was performed as detailed above and cell suspension adjusted to 5 x 105 per ml and aliquoted into cryovials at 1 2 ml per vial Vials were then held on ice prior to injection 0 2 ml from a vial of Mat-Ly-Lu was injected into shaved left hind flank of a Copenhagen rat 1 vial is sufficient to inject 5 animals (1 experimental group) See below for injection regime for protection and therapy Protection Model Tumour dimensions were collected using calipers, two measurements- longitudinal and transverse were taken for each tumor. These values were multiplied together to give an indication of tumor size. A kill size of 25 mm was set such that any tumor reaching this size in one of the two measurable dimension was culled by exposure to a rising concentration of C0 2 and cervical dislocation. Kill curves were then generated. Results Animals grown with the 2D and 3D grown cells showed a survival benefit over and above the vehicle control injected animals. The 3D grown vaccine showed superior results to the 2D grown material confirming the advantage of 3D growth in terms of scale up, yield, process and efficacy. Tables Table 1 Cell Numbers per cm 2 for Flask and Roller Bottle Grown Cells Table 2 Cell Number Increment for PNT2-C2. NIH1542 and K1735 Grown in Microgravity Culture LEGEND TO FIGURES Figure 1 a. K1735 flask grown b. K1735 Roller bottle c. B16F10 Flask grown d. B16F10 Roller bottle Figure 2 rue 050399 008 Sacple ID g) 6/f]Q G ted Events 5000 PA r am πaικ f l,o High .Gated GH----n CV Peak Viluc COHTROL ΠTC 0 0 1023 L00 00 2 11 61 2 31, 1 1 WO 1023 6 3? 6 66 21 11 -.4 , b 002! r.\e 050B9Θ 009 Sample TD 6-6/FlO Gated Cvent:- 5000 Paraα name M Low H-gh .Gated GHc-n CV Pe»k, alue H2 Kb riTC O 0, 1023 100 00 - OS 55 16 113, 1 1 HO 1023 19 38 ■ 81 2 ") .9 19, 6 09^56 File 050898 01) Sample ID B (,/T 10 D15HS Gated Event ' s 5000 Paraα- name H l-ou H-gh ' .Gated CHe-n CV Pejk.V- lue HZ-X1 . πTC 0 0, 1023 100 00 θ lb 2. IS 64, 8 0S8.2 1 170 1023 91 92 9 52 20 02 61, 8 05B42

Claims: Claims 1. A method for producing a whole cell cancer vaccine which comprises: culturing malignant cells under conditions which are not conventional static 2D culture conditions; harvesting the malignant cells; and adding the harvested malignant ceils to a pharmaceutically acceptable carrier, excipient or diluent. 2. A method according to claim 1 wherein the malignant cells are cultured under non-adherent culture conditions. 3. A method according to claim 2 wherein the malignant cells are cultured in non adherent culture vessels. 4. A method according to claim 1 wherein the malignant cells are cultured under adherent non-static culture conditions. 5. A method according to claim 4 wherein the malignant cells are cultured in roller culture bottles. 6. A method according to claim 1 wherein the malignant cells are cultured under microgravity. 7. A method according to claim 6 wherein the malignant cells form spheroids under microgravity and the spheroids are harvested and added to the pharmaceutically acceptable carrier, excipient or diluent. 8. A method according to claim 1 wherein the malignant cells are cultured on microcarriers suspended in culture medium. 9. A method according to claim 8 wherein the microcarriers are beads or non woven fibre discs. 10. A method for producing a whole cell cancer vaccine which comprises culturing malignant cells under two or more of the following culture conditions: non-adherent; adherent non-static; microgravity; on microcarriers suspended in culture medium. 11. A method according to any of claims 1 to 10 wherein culturing the malignant cells alters their immunogenicity. 12. A method according to claim 11 wherein the immunogenicity of the malignant cells is altered in comparison with equivalent malignant cells cultured under conventional static 2D conditions. 13. A method according to any of claims 1 to 12 wherein the malignant cells are derived from a tumour cell line. 14. A method according to any of claims 1 to 13 further comprising gamma irradiating the malignant cells. 15. A whole cell cancer vaccine produced according to any of claims 1 to 14. 16. A whole cell cancer vaccine having: an immunogenic component which comprises a malignant cell having an altered immunogenicity; and a pharmaceutically acceptable carrier, excipient or diluent. 17. A whole cell cancer vaccine according to claim 16 wherein the malignant cell was cultured under conditions which were not conventional static 2D culture conditions and wherein the immunogenicity of the malignant cell was altered in comparison with the immunogenicity of an equivalent malignant cell grown under conventional static 2D culture conditions. 18. A whole cell cancer vaccine having: an immunogenic component which comprises a spheroid of malignant cells; and a pharmaceutically acceptable carrier, excipient or diluent. 19. A whole cell cancer vaccine according to claim 18 wherein one or more of the malignant cells of the spheroid has an altered immunogenicity. 20. A whole cell cancer vaccine according to claim 19 wherein the malignant cells were cultured under microgravity and the or each malignant cell has an altered immunogenicity compared to equivalent malignant cells grown under conventional 2D static culture conditions. 21. A whole cell cancer vaccine according to any of claims 15 to 20 further comprising an adjuvant. 22. A method for producing a whole cell cancer vaccine according to any of claims 1 to 14 wherein the malignant cells are prostate cells. 23. A whole cell cancer vaccine according to any of claims 15 to 21 wherein the cancer to be treated is prostate cancer.

Inventors: CHANA HAJ, SMITH PETER, STEVENSON DARREN, SUTTON ANDREW, CHANA, HAJ, SMITH, PETER, STEVENSON, DARREN, SUTTON, ANDREW
Applicants: CHANA HAJ, ONYVAX LTD, SMITH PETER, STEVENSON DARREN, SUTTON ANDREW, CHANA, HAJ, ONYVAX LIMITED, SMITH, PETER, STEVENSON, DARREN, SUTTON, ANDREW
Application year: 2000
Main classification: A61K39
IPC codes: A61K35, A61K39, A61P35, C12N5