WO2000071155A2 - New vaccine formulations - 2 - Google Patents
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- WO2000071155A2 WO2000071155A2 PCT/GB2000/001918 GB0001918W WO0071155A2 WO 2000071155 A2 WO2000071155 A2 WO 2000071155A2 GB 0001918 W GB0001918 W GB 0001918W WO 0071155 A2 WO0071155 A2 WO 0071155A2
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2502/00—Coculture with; Conditioned medium produced by
- C12N2502/28—Vascular endothelial cells
Definitions
- 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 allogeneic cell based vaccines, whereby autologous tumour cells or immortalised allogeneic tumour cell lines were cultured in static two dimensional (2D) culture conditions
- This invention relates to a co-culture techniques which leads to significant enhancement of the phenotype of cell lines towards a more in vivo phenotype resulting in a vaccine reflecting a more realistic phenotypic representation of an in vivo tumour
- We describe alternative culture techniques which offer significant scale up and processing benefits and in addition significantly improve the activity of the whole-cell vaccine
- vaccines produced by the various co-culture techniques described have additional advantages over single cell suspension vaccines in the mode of presentation to the immune system which leads to enhanced performance in treating the disease
- cancerous cells contain numerous mutations, qualitative and quantitative, spatial and temporal, relative to their non-malignant, 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
- TSA tumour-specific antigens
- TAA tumour-associated antigens
- 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 atten
- 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
- the second type of cell-based vaccine describes the use of allogeneic cells which are be genetically (and hence immunologically) mismatched to the patients Allogeneic cells benefit from the same advantages of multivalency as autologous cells
- allogeneic 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
- the allogeneic 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.
- Hitherto described cell based cancer vaccines are based on i) populations of primary cultures of autologous tumour cells, n) single populations of allogeneic immortalised tumour cell lines, in) allogeneic immortalised cell lines as many as three or four in combination and iv) genetically modified fibroblasts in combination with tumour cells
- This invention describes culture methods for large scale production of vaccines, phenotype adjustment of established cell lines, co-culture techniques, specific compositions of immortalised tumour cell lines and immortalised non-malignant cells, compositions comprising immortalised tumour cells and autologous non-malignant cells, processing methods for the production of the vaccines, analytical techniques to evaluate phenotype adjustment and new compositions which display increased immunogenicity in standard models of tumour prevention and therapy
- this invention describes a new strategy, targeting the immune response not only to the tumour itself but also to the local tumour environment and the non-malignant cells which comprise that environment
- This embodiment describes the preparation of a mixed cell type vaccine, in which tumour cell lines are grown in intimate contact with a non-malignant component
- the growth of this mixed cell type vaccine can be achieved by a number of means, for example co-culture in 2D flasks, growth in transwell inserts where conditioned medium exerts its effect on the tumour line or growth in a 3D mixed culture system
- a mixed culture approach in that the literature reports that in many cases cell-cell contact is required to elicit reciprocal phenotypic changes in both tumour and non-tumour components (e g Fibroblast mediated differentiation in human breast carcinoma cells (MCF-7) grown as nodules in vitro, Int J Cancer , (1994), 56, p731-735, Regulation of collagenase-3 expression in human breast carcinoma is mediated by stromal-epithelial cell interactions, Cancer Research (1997), 57, p4882-4888, Co-culture of human breast adenocarcinoma MCF-7 cells and human dermal fibroblasts enhances the
- This invention describes the use of 3D microgravity culture to grow significant quantities of tumour cells and co-cultured cells for use as vaccines
- 3D microgravity culture to grow significant quantities of tumour cells and co-cultured cells for use as vaccines
- a further embodiment of this invention is the use of 3D co-culture to achieve a mixed cell type vaccine comprising an immortalised tumour cell line in combination with an immortalised or non-immortalised non-malignant cell line such as epithelial cells, stromal fibroblasts, bone stromal cells, endothelial cells, palisade cells or any other immortalised or non immortalised non-malignant cell type
- an immortalised or non-immortalised non-malignant cell line such as epithelial cells, stromal fibroblasts, bone stromal cells, endothelial cells, palisade cells or any other immortalised or non immortalised non-malignant cell type
- an immortalised neuroendocnne lineage which are thought to have a parac ⁇ ne function which may drive the progression of tumour development from hyperplasia to neoplastic to metastatic phenotypes
- Two immortalised neuroendocnne tumour lines are known in the literature, the prostatic derived lung metastatic neuroendocnne line C
- a further aspect of this invention is a direct process of formication of cells harvested from 3D microgravity culture 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 tend to be significantly more robust to handle and wash, thereby leading to enhanced recoveries
- a further embodiment of this invention is the use of mixed 3D microgravity culture incorporating non-mahgnant cell lines to enhance the phenotype of cell lines which hitherto have not been grown in mixed 3D culture
- a further embodiment of this invention is enhanced immunogenicity arising from the use of non-mahgnant cells in combination with tumour cell lines arising from direct presentation of peptides on the MHC-I molecules of the non-mahgnant cell line, particularly if the non-mahgnant cell line is matched or partially matched to the recipient
- the peptides may be released from the tumour cell lines during growth, processing and irradiation of the 3D cell mass, or may be released during initial interaction of macrophages and dendritic cells at the time of injection
- a separate embodiment of this invention is the use of peptide extracts and other sub-cellular fractions derived from cells and co-cultures of cells grown under non- standard conditions This includes cells grown on roller bottles, on microcar ⁇ ers, 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 component of a
- a further embodiment of this invention is the production, composition and use of a mixed autologous / allogeneic cell vaccine comprising autologous non-mahgnant cells taken from an intended recipient of the vaccine grown in culture with an allogeneic immortalised tumour cell line
- the autologous non-ma gnant cells have non-mahgnant levels of MHC-I and will thus have the capability of presenting in the context of autologous MHC-I peptides released from lysed, destroyed or apoptotic tumour cells with which they have been grown
- FIG. 1 PSA expression analysis (PCR) of prostate cell lines grown in co-culture with bone marrow stromal cells
- the murine melanoma cell line K1735 was grown with the murine fibroblast cell line 3T3 at a ratio of 1 1
- the 3T3 cells were seeded at 5 x 10 6 cells in a 50 ml cassette for a microgravity instrument (Synthecon) and allowed to grow for 2 days in DMEM 2% foetal calf serum, 10 mM glutamine
- a further inoculum of 5x10 6 cells of either K1735 was made into the 50 ml rotating cassette (Synthecon) and the culture inoculated for a further 6-9 days
- the resulting spheroids were harvested and washed by light centrifugation in DMEM at day 8-1 1 and taken for further analysis
- the human prostatic epithelial tumour line NIH1542 was grown with the non- malignant human prostatic epithelial line PNT2-C2 at a 1 1 ratio in a 3D microgravity culture vessel (Synthecon)
- the cell lines were co-inoculated at 5x10 6 cells each and cultured in KSFM for a period of 6-1 1 days until a significant biomass of spheroids had accumulated
- the cell mass was harvested and washed in serum free medium and then trypsinized to prepare the sample for analysis
- the human prostatic epithelial tumour line NIH1542 was grown with the human prostatic neuroendocrine epithelial line CRL-5813 at a 1 1 ratio in a 3D microgravity culture vessel (Synthecon)
- the cell lines were co-inoculated at 5x10 6 cells each and cultured in KSFM for a period of 6-1 1 days until a significant biomass of spheroids had accumulated )
- two separate 2D cultures were seeded and grown to near confluence
- the cells were trypsinized and the mixture of cells from 2D and 3D co-culture were analyzed for the level of apoptosis by annexin and propidium iodide staining and also for phenotypic expression of various markers
- Further 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 spect
- the murine melanoma cell line K1735 was grown with the murine fibroblast cell line 3T3 at a ratio of 1 1
- the 3T3 cells were seeded at 5 x 10 6 cells in a 50 ml cassette for a microgravity instrument (Synthecon) and allowed to grow for 2 days in DMEM 2% foetal calf serum, 10 mM glutamine
- a further inoculum of 5x10 6 cells of either K1735 was made into the 50 ml rotating cassette (Synthecon) and the culture inoculated for a further 6-9 days
- the cells were allowed to grow for a period of 7-14 days with intermediate media and growth factor replenishment
- the cassette was emptied into a centrifuge vessel and lightly centnfuged 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 either used directly in the protection experiment or, formulated in appropriate freezing mixture (10% DMSO in Hanks) and ahquoted on the basis of either cell number determined by trypsinization and counting or on a protein or nucleic acid basis No trypsinization 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
- 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
- mice Groups of 8 C57/B6 mice were immunized with 1-10 x10 6 K1735 / 3T3 spheroids and compared to 2D flask grown K1735 cells irradiated at 150 Gy, at T-10 days The mice were then challenged on Day 0 with 5x10 5 live B16F10 cells and then monitored for tumour growth PSA expression in prostate cell lines grown alone and in co-culture with normal bone marrow stromal cells
- Onycapl and Onycap 23 have been deposited under the Budapest Treaty at ECACC on 28 March 2000 under the Accession numbers 00032802 and 00032801 respectively
- RNA precipitated with isopropanol RNA pellets were washed with 75% ethanol, dried and re-suspended in TE buffer
- RNA sample was treated with Deoxy ⁇ bonuclease I (Life Technologies #18068-015) to ensure there was no contamination with genomic DNA Reactions were incubated for 15 minutes at room temperature then the DNase inactivated by the addition of 25mM EDTA and heating to 65°C for 10 minutes
- Reverse transcription was performed using the 1 st strand cDNA synthesis kit for RT-PCR (AMV) from Boehnnger Mannheim (#1 483 188) Reactions were incubated at 25°C for 10 minutes then at 42°C for 1 hour The AMV enzyme was denatured by heating to 99°C for 5 minutes then the reaction was cooled to 4°C
- PCR primers were designed from published sequences Verification of cDNA integrity was achieved by PCR amplification using GapdH housekeeping primers Amplification reactions with the external primer set were carried out using thermocycler program (1 ) The internal nested PCR set was run on program (2) below
- the PCR samples were run in pairs.
- the prostate cells alone then the same cells grown in co-culture with NHBMSC followed by NHBMSC alone then a positive and negative control Figure 1.
- PSA expression was not detected in 3 out of 4 prostate cells grown alone however, cDNA isolated from all of the cell lines grown in co-culture with NHBMSC produced a band at 200bp following 2 rounds of PCR amplification suggesting an up-regulation of PSA as a result of co-culture with bone marrow stromal cells.
- Cell line ONYCAP-23 were seeded at 1 x 10 6 cells onto confluent monolayers of NHBMSC cells in T175 culture flasks. Co-cultures were maintained for 2 days using keratinocyte growth medium supplemented with 5% foetal calf serum. Cells were harvested by scraping from the surface of the plastic and washed in Hanks balanced salt solution before RNA extraction.
- RNA precipitated with isopropanol RNA pellets were washed with 75% ethanol, dried and re-suspended in TE buffer
- RNA sample was treated with Deoxynbonuclease I (Life Technologies #18068-015) to ensure there was no contamination with genomic DNA Reactions were incubated for 15 minutes at room temperature then the DNase inactivated by the addition of 25mM EDTA and heating to 65°C for 10 minutes
- Reverse transcription was performed using the 1 st strand cDNA synthesis kit for RT-PCR (AMV) from Boehrmger Mannheim (#1 483 188) Reactions were incubated at 25°C for 10 minutes then at 42°C for 1 hour The AMV enzyme was denatured by heating to 99°C for 5 minutes then the reaction was cooled to 4°C
- PSA PCR primers were designed from published sequences tabulated below Verification of cDNA integrity was achieved by PCR amplification using GapdH housekeeping primers Amplification reactions with the external primer set were carried out using thermocycler program (1 ) The internal nested PCR set was run on program (2) below (1 ) 95°C 3 m ⁇ n (2) 95°C 3 mm 95°C 45 sec ] 95°C 45 sec ] 57°C 45 sec ⁇ 30 cycles 60°C 45 sec r 30 cycles 72°C 45 sec J 72°C 45 sec J 72°C 5 mm 72°C 5 mm
- thermocycler program was given below and were run on the thermocylcer
- PCR samples were run in pairs on agarose gels
- tumour elicited stroma shows aberrant expression of proteins in response to the tumour cells
- K1735 (P25) cells were grown in RPMI 1640 medium supplemented with 10% FCS and 2 mM L-glutamme, trypsmised, irradiated at 100 Gy and formulated into 10%DMSO in RPMI at a cell density of 5x106 cells / ml prior to cryopreservation at -1800C
- a K1735 tumour grown in a C3H mouse was excised and chopped into small pieces before being forced through a 70um gauze, irradiated at 100 Gy and formulated in 10%DMSO in RPMI at a cell density of 5x106 cells / ml, then cryopreserved at -1800C
- B16 F10 mouse melanoma clone G10 was grown in RPMI 1640 medium supplemented with 10% FCS and 2 mM L-glutamine, trypsmised and used to moculate groups of 5 C57BL6 mice at a viable dose of 1 x104 cells in 200ul of medium Three days following viable tumour challenge the mice were dosed with 1 x106 of either in vitro grown K1735 or in vivo grown tumour or PBS injected mice and subsequently on three other occasions on days 6 10 and 13 post tumour challenge Tumour volume and survival were measured (sacrifice when tumour attained 15x15mm) at three day intervals starting at day 1 1 post tumour challenge Results
- Cell line DU145 was seeded at 1 x 10 6 cells onto confluent monolayers of normal human prostatic stromal fibroblasts (NHPSF) in T175 culture flasks Co-cultures were maintained for 2 days using in MEM medium supplemented with 10% FCS and 2 mM L-glutamine Cells were harvested by scraping from the surface of the plastic and washed in Hanks balanced salt solution before RNA extraction
- Normal human prostatic stromal fibroblasts were grown to confluency in a T175 flask in MEM medium supplemented with 10% FCS and 2 mM L-glutamme Cells were harvested by scraping from the surface of the plastic and washed in Hanks balanced salt solution before RNA extraction
- Cell line DU145 was seeded at 1 x 10 6 cells into T175 flasks and grown in MEM medium supplemented with 10% FCS and 2 mM L-glutamine and grown to confluency Cells were harvested by scraping from the surface of the plastic and washed in Hanks balanced salt solution before RNA extraction
- RNA precipitated with isopropanol RNA pellets were washed with 75% ethanol, dried and re-suspended in TE buffer
- RNA sample was treated with Deoxynbonuclease I (Life Technologies #18068-015) to ensure there was no contamination with genomic DNA Reactions were incubated for 15 minutes at room temperature then the DNase inactivated by the addition of 25mM EDTA and heating to 65°C for 10 minutes
- tumour cell lines which are up or down regulated by growth of tumour cell lines on stromal cells from various organ compartments
- tumour cell lines with varying degrees of mvasivity or metastatic capability in combination with stromal cells such as prostatic fibroblasts, bone stromal fibroblasts, or endothelial cells it is possible to identify significant genes which are temporally and spatially expressed as a result of the intimate contact between tumour and stromal cell and thus enable the identification of antigens which are thus meaningful and viable potential targets for immunotherapy or other therapeutic strategies
- Cell line DU145 was seeded at 1 x 10 6 cells onto confluent monolayers of normal human bone stromal fibroblasts (NHBSF) in T175 culture flasks Co-cultures were maintained for 2 days using in MEM medium supplemented with 10% FCS and 2 mM L-glutamine Cells were harvested by scraping from the surface of the plastic and washed in Hanks balanced salt solution before RNA extraction .
- NBSF normal human bone stromal fibroblasts
- Normal human bone stromal fibroblasts (NHBSF) were grown to confluency in a T175 flask in MEM medium supplemented with 10% FCS and 2 mM L-glutamine. Cells were harvested by scraping from the surface of the plastic and washed in Hanks balanced salt solution before RNA extraction
- Cell line DU145 was seeded at 1 x 10 6 cells into T175 flasks and grown in MEM medium supplemented with 10% FCS and 2 mM L-glutamine and grown to confluency. Cells were harvested by scraping from the surface of the plastic and washed in Hanks balanced salt solution before RNA extraction
- RNA precipitated with isopropanol RNA pellets were washed with 75% ethanol, dried and re-suspended in TE buffer
- RNA sample was treated with Deoxynbonuclease I (Life Technologies #18068-015) to ensure there was no contamination with genomic DNA Reactions were incubated for 15 minutes at room temperature then the DNase inactivated by the addition of 25mM EDTA and heating to 65°C for 10 minutes.
- Reverse transcription was performed using the 1 st strand cDNA synthesis kit for RT-PCR (AMV) from Boehnnger Mannheim (#1 483 188) Reactions were incubated at 25°C for 10 minutes then at 42°C for 1 hour The AMV enzyme was denatured by heating to 99°C for 5 minutes then the reaction was cooled to 4°C 32P or 33P deoxynucleotides were used to label the cDNA for subsequent probing of the arrays RNA from DU145 and NHBSF cultures were mixed in equal proportions and the same quantity of RNA used in the reverse transcription as was used for the co-culture RNA
- Labelled cDNA was used to probe a Clontech AtlasTM array (human array II) following the manufacturers protocol for hybridisation and washing Images were recorded utilising a phosphor imager and recorded files analysed with the Clontech Atlas ImageTM software
- tumour cell lines which are up or down regulated by growth of tumour cell lines on stromal cells from various organ compartments
- tumour cell lines with varying degrees of mvasivity or metastatic capability in combination with stromal cells such as prostatic fibroblasts, bone stromal fibroblasts, or endothelial cells it is possible to identify significant genes which are temporally and spatially expressed as a result of the intimate contact between tumour and stromal cell and thus enable the identification of antigens which are thus meaningful and viable potential targets for immunotherapy or other therapeutic strategies
- Results are shown for patient numbers 1 , 2, 3, and 4 who had received a series of vaccinations with human prostate cell lines, PNT-2, NIH1542, DU145 and LnCap in a clinical trial to evaluate the safety and immunogenicity of these cell lines
- Results are given for cell lysates namely, NIH1542, LnCap, DU-145, PNT-2 and also a co- culture of DU145 and NHBSF cells in such a way that the same cell number equivalent is used in each proliferation assay
- the co- culture lysate has led to a significant improvement in CD4 and CD8 prohferative response in 3 out of 4 patients This is indicative of a more representative spectrum of antigens being produced in co-culture than in the mono-culture of the DU-145 alone
- the rat PA3 prostate tumour line is a relatively weak allogeneic immunogen in the Copenhagen rat challenged with autologous MatLyLu prostate cells
- PA3 Clone 1 cells (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 trypsm/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, centnfuged at 18,000 rpm for 3 minutes The resultant pellet was re-suspended into 10ml PBS and stored on ice, prior to irradiation
- PA3 Clone 1 cells (1 x 10 5 ) and YPEN cells (1 x 10 5 ) were seed simultaneously into a T75 tissue culture flask containing 15ml RPMI 1640 culture medium supplemented with 10% FCS/2mM Glutamine Cells were allowed to grow until confluent and harvested by cell scraping, pelleted by centrifugation at 18,000 rpm for 3 mm, and re-suspended in 10ml PBS Cells were stored on ice prior to irradiation
- PA3 Clone 1 cells (1 x 10 5 ) , YPEN cells 1 x 10 5 and RBMS (1 x 10 5 ) were seeded simultaneously into a T75 tissue culture flask containing 15ml RPMI 1640 culture medium supplemented with 10% FCS/2mM Glutamine Cells were allowed to grow until confluent and harvested by cell scraping, pelleted by centrifugation at 18,000 rpm for 3 mm, and re-suspended in 10ml PBS Cells were stored on ice prior to irradiation PA3 / RBMS 2D Co-Culture
- PA3 Clone 1 cells (1 x 105) and (1 x 105) RBMS were seeded simultaneouslymto a T75 tissue culture flask containing 15ml RPMI 1640 culture medium supplemented with 10% FCS/2mM Glutamine Cells were allowed to growuntil confluent and harvested by cell scraping, pelleted by centrifugation at18,000 rpm for 3 mm, and re-suspended in 10ml PBS Cells were stored once prior to irradiation
- 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 10 ⁇ l of each cell suspension in mixed with 90 ⁇ l of tryphan blue and approx 10 ⁇ l was loaded under the coverslip of the haemocytometer A cell count was performed using a x20 objective lens Each cell suspension was then centnfuged 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 ahquoted into cryovials at 1 2ml per vial, vials were then placed into a -80°C freezer overnight prior to storage in liquid nitrogen
- vaccine freezing medium RPMI 1640/8%FCS/8%DMSO
- 1 vial is sufficient to inject 5 animals (1 experimental group) See below for injection regime for protection and therapy
- Mat-Ly-Lu cells (1 x 10 6 ) 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 trypsm/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, centnfuged 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 10 5 per ml and ahquoted into cryovials at 1 2 ml per vial Vials were then held on ice
- Tumor 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 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
- the rat PA3 prostate tumour line is a relatively weak allogeneic immunogen in the Copenhagen rat challenged with autologous MatLyLu prostate cells
- PA3 rat prostate tumour cells grown in 3D microgravity culture and PA3 cells co- cultured with bone stromal fibroblasts and YPEN endothelial cells in 3D microgravity culture as a vaccine in rat therapy and protection models
- the roller cassette were filled with 35 ml culture medium (RPMI 1640 10% FCS/2mM Glutamine) through the screw cap, to this 6 x 10 5 cells suspended 6 ml of culture medium was added The roller was then topped up to full (approx 50ml) with -10 ml culture medium and the screw cap replaced A 20ml syringe fitted with a 20 ⁇ m filter attached was screwed onto the outlet valve of the roller cassette A second 20ml syringe fitted with a 20 ⁇ m was filled with culture medium and screwed onto the inlet valve of the roller 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 roller cassettes were attached to the roller 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
- 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 10ul of each cell suspension is mixed with 90 ⁇ l of tryphan blue and approx 10 ⁇ l was loaded under the coverslip of the haemocytometer A cell count was performed using a x20 objective lens Each cell suspension was then centnfuged 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 ahquoted into cryovials at 1 2ml per vial, vials were then placed into a -80°C freezer overnight prior to storage in liquid nitrogen
- vaccine freezing medium RPMI 1640/8%FCS/8%DMSO
- 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) Mat-Ly-Lu cells(1 x 10 6 ) 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 trypsm/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, centnfuged 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 10 5 per ml and
- Tumor 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 C02 and cervical dislocation Kill curves were then generated.
- LRP low-density hpoprotein receptor-related protein 1 precursor
- CD91 antigen 9 15 1 glutathione synthetase (GSH synthetase, GSH-S), glutathione synthase 19 32 1 paxiihn
- MIF macrophage migration inhibitory factor
- GEF glycosylation- hibiting factor
- basigm precursor BSG
- leukocyte activation antigen M6, 8 143 metalloproteinase inhibitor 1 precursor TGFP1
- TGF8 TGF8
- AIGF antigen-induced growth factor precursor
- CDKN1A cyclin-dependent kinase inh 1
- IRF3 interferon regulatory factor 3
- poly(ADP-ribose) polymerase PARP poly(ADP-ribose) polymerase PARP
- lymphocyte function-associated antigen 3 precursor (LFA3), CD58 antigen
- HMG-CoA reductase HMGCR
- apo-D apolipoprotein D precursor
- DHFR 15652 10956 0.699974 homolog of yeast D2-isopentenylpyrophosphate isomerase (IPP isomerase) 10036 1888 0.188123 LOX (Protein-lysine 6-oxidase, Lysyl-Oxidase)
- nucleolysin TIAR 15136 10044 0.663584 TIA-1 related protein; nucleolysin TIAR
- NPY5-R Y5 RECEPTOR
- NPYY5 16000 8672 0.542 neuron-derived orphan receptor 1 (NOR1 );
- PP1 -gamma 1 catalytic subunit 26140 16248 0.621576 ras-related protein RAP-1 B; GTP-binding protein
- EHOC-1 transmembrane protein 1 13184 6080 0 461 165 DNA polymerase gamma (POLG), mitochondnal DNA polymerase
- MVP major vault protein
- LRP lung resistance-related protein
- CHAIN 13484 21 100 1.564818 phosphatidylethanolamine-binding protein; neuropolypeptide 556 13308 23.93525 cystathionine beta-synthase
- CYP3 cyclophilin 3 protein
- CTSB cathepsin B precursor
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Priority Applications (7)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AT00931400T ATE259655T1 (en) | 1999-05-21 | 2000-05-19 | WHOLE CELL CANCER VACCINE CONTAINING CO-CULTURED MALIGINAL AND NON-MALIGINAL CELLS |
KR1020017014806A KR20020027318A (en) | 1999-05-21 | 2000-05-19 | New vaccine formulations-2 |
EP00931400A EP1178822B1 (en) | 1999-05-21 | 2000-05-19 | Whole cell cancer vaccine comprising co-cultured malignant and non-malignant cells |
AU49356/00A AU4935600A (en) | 1999-05-21 | 2000-05-19 | New vaccine formulations - 2 |
DE60008368T DE60008368T2 (en) | 1999-05-21 | 2000-05-19 | WHOLE CELLS OF CANCER VACCINE, CONTAINING CO-CULTIVATED MALIGNE AND NON-MALIGNE CELLS |
JP2000619457A JP2003500366A (en) | 1999-05-21 | 2000-05-19 | New vaccine preparation 2 |
CA002374294A CA2374294A1 (en) | 1999-05-21 | 2000-05-19 | New vaccine formulations - 2 |
Applications Claiming Priority (8)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GBGB9911823.4A GB9911823D0 (en) | 1999-05-21 | 1999-05-21 | New vaccine formulations |
GBGB9911824.2A GB9911824D0 (en) | 1999-05-21 | 1999-05-21 | New vaccine formulations |
GB9911824.2 | 1999-05-21 | ||
GB9911823.1 | 1999-05-21 | ||
GB0008032A GB0008032D0 (en) | 2000-04-01 | 2000-04-01 | New prostate cell lines |
GB0008029.1 | 2000-04-01 | ||
GB0008029A GB0008029D0 (en) | 2000-04-01 | 2000-04-01 | New vaccine formulations |
GB0008032.5 | 2000-04-01 |
Publications (2)
Publication Number | Publication Date |
---|---|
WO2000071155A2 true WO2000071155A2 (en) | 2000-11-30 |
WO2000071155A3 WO2000071155A3 (en) | 2001-02-22 |
Family
ID=27447826
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/GB2000/001918 WO2000071155A2 (en) | 1999-05-21 | 2000-05-19 | New vaccine formulations - 2 |
Country Status (9)
Country | Link |
---|---|
EP (1) | EP1178822B1 (en) |
JP (1) | JP2003500366A (en) |
KR (1) | KR20020027318A (en) |
AT (1) | ATE259655T1 (en) |
AU (1) | AU4935600A (en) |
CA (1) | CA2374294A1 (en) |
DE (1) | DE60008368T2 (en) |
ES (1) | ES2215662T3 (en) |
WO (1) | WO2000071155A2 (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2001075073A2 (en) * | 2000-04-01 | 2001-10-11 | Onyvax Limited | Prostate cell lines and their use |
WO2005011729A1 (en) * | 2003-07-23 | 2005-02-10 | Onyvax Limited | Human prostate cell lines in cancer treatment |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR20130055591A (en) * | 2010-04-13 | 2013-05-28 | 가부시키가이샤 르네상스 에너지 인베스트먼트 | Composition for treatment of cancer which is produced from cancer-tissue-derived cell mass or cancer cell aggregate, and process for production of immunotherapeutic agent and method for evaluation of efficacy of immunotherapy both using the composition |
Citations (4)
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WO1997024132A1 (en) * | 1995-12-28 | 1997-07-10 | The Johns Hopkins University School Of Medicine | Allogeneic paracrine cytokine tumor vaccines |
WO1997028255A1 (en) * | 1996-02-02 | 1997-08-07 | The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services | Immortal human prostate epithelial cell lines and clones and their applications in the research and therapy of prostate cancer |
US5804443A (en) * | 1997-02-28 | 1998-09-08 | Bernofsky; Carl | Human monocytic leukemia cell line |
WO1999019462A1 (en) * | 1997-10-10 | 1999-04-22 | The Regents Of The University Of California | Enhanced immunogenic cell populations prepared using h2 receptor antagonists |
-
2000
- 2000-05-19 ES ES00931400T patent/ES2215662T3/en not_active Expired - Lifetime
- 2000-05-19 EP EP00931400A patent/EP1178822B1/en not_active Expired - Lifetime
- 2000-05-19 WO PCT/GB2000/001918 patent/WO2000071155A2/en not_active Application Discontinuation
- 2000-05-19 JP JP2000619457A patent/JP2003500366A/en active Pending
- 2000-05-19 CA CA002374294A patent/CA2374294A1/en not_active Abandoned
- 2000-05-19 DE DE60008368T patent/DE60008368T2/en not_active Expired - Fee Related
- 2000-05-19 AT AT00931400T patent/ATE259655T1/en not_active IP Right Cessation
- 2000-05-19 KR KR1020017014806A patent/KR20020027318A/en not_active Application Discontinuation
- 2000-05-19 AU AU49356/00A patent/AU4935600A/en not_active Abandoned
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1997024132A1 (en) * | 1995-12-28 | 1997-07-10 | The Johns Hopkins University School Of Medicine | Allogeneic paracrine cytokine tumor vaccines |
WO1997028255A1 (en) * | 1996-02-02 | 1997-08-07 | The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services | Immortal human prostate epithelial cell lines and clones and their applications in the research and therapy of prostate cancer |
US5804443A (en) * | 1997-02-28 | 1998-09-08 | Bernofsky; Carl | Human monocytic leukemia cell line |
WO1999019462A1 (en) * | 1997-10-10 | 1999-04-22 | The Regents Of The University Of California | Enhanced immunogenic cell populations prepared using h2 receptor antagonists |
Non-Patent Citations (2)
Title |
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FRESHNEY R. I.: "Culture of animal cells" 1987 , ALAN R. LISS , US, NEW YORK XP002152704 154920 page 295, left-hand column, line 26 -right-hand column, line 2 page 314, left-hand column, line 3 - line 17 * |
OLUMI ARIA F ET AL: "A novel coculture technique demonstrates that normal human prostatic fibroblasts contribute to tumor formation of LNCaP cells by retarding cell death." CANCER RESEARCH, vol. 58, no. 20, 15 October 1998 (1998-10-15), pages 4525-4530, XP002152739 ISSN: 0008-5472 * |
Cited By (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2001075073A2 (en) * | 2000-04-01 | 2001-10-11 | Onyvax Limited | Prostate cell lines and their use |
WO2001075073A3 (en) * | 2000-04-01 | 2002-02-14 | Onyvax Ltd | Prostate cell lines and their use |
US7303911B2 (en) | 2000-04-01 | 2007-12-04 | Onyvax Limited | Prostate cell lines |
WO2005011729A1 (en) * | 2003-07-23 | 2005-02-10 | Onyvax Limited | Human prostate cell lines in cancer treatment |
GB2419530A (en) * | 2003-07-23 | 2006-05-03 | Onyvax Ltd | Human prostate cell lines in cancer treatment |
GB2419530B (en) * | 2003-07-23 | 2008-01-02 | Onyvax Ltd | Human prostate cell lines in cancer treatment |
US7438922B2 (en) | 2003-07-23 | 2008-10-21 | Onyvax Limited | Human prostate cell lines in cancer treatment |
US8097244B2 (en) | 2003-07-23 | 2012-01-17 | Onyvax Limited | Human prostate cell lines in cancer treatment |
US8545835B2 (en) | 2003-07-23 | 2013-10-01 | Onyvax Limited | Human prostate cell lines in cancer treatment |
Also Published As
Publication number | Publication date |
---|---|
ES2215662T3 (en) | 2004-10-16 |
WO2000071155A3 (en) | 2001-02-22 |
EP1178822B1 (en) | 2004-02-18 |
JP2003500366A (en) | 2003-01-07 |
KR20020027318A (en) | 2002-04-13 |
DE60008368T2 (en) | 2004-12-09 |
DE60008368D1 (en) | 2004-03-25 |
EP1178822A2 (en) | 2002-02-13 |
AU4935600A (en) | 2000-12-12 |
ATE259655T1 (en) | 2004-03-15 |
CA2374294A1 (en) | 2000-11-30 |
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