the invention will now be described with reference to the following figures . fig1 shows an example of a serum sample from an hcc ( hepatocellular carcinoma ) patient reacting with different hcc - derived cdna clones . clones 45 and 56 represent clones carrying different portions of bmi - 1 . fig2 shows elispot results examining whether there are t cell responses to predicted bmi - 1 epitopes in pbmc preparations from hepatocellular carcinoma patients . the peptides are : a predicted a0201 peptide —‘ tlq ’ ( tlqdivykl ; seq id no : 1 ); the predicted b2702 / 05 peptides —‘ vry ’ ( vryletsky ; seq id no : 2 ) and ‘ kry ’ ( krylrcpaa ; seq id no : 3 ); an hla - a02 - restricted epitope from alpha - fetoprotein —‘ gva ’ ( gvalqtmkq ; seq id no : 4 ); and a b7 - restricted peptide from ebna3a ( negative control )—‘ rppi ’ ( rppifirrl ; seq id no : 5 ); ‘ t only ’ is t cells only and ‘ pbl ’ is peripheral blood lymphocytes alone . fig3 shows elispot results examining whether there are t cell responses to predicted bmi - 1 epitopes in pbmc preparations from a normal volunteer and an hepatocellular carcinoma patient . pbmc were untreated or infected with the adenoviruses indicated ( ade3c , ebna3c expressing adenovirus ; adbgal , β - galactosidase expressing adenovirus , adbmi , bmi - 1 expressing adenovirus ). fig4 shows elispot results examining whether there are t cell responses to predicted ezh2 epitopes in pbmc preparations from normal volunteers . the peptides are the predicted a0201 peptides , ymc — ymccsflfnl ; ( seq id no : 6 ); sqa — sqadalkyv ; ( seq id no : 7 ) or frk — frkaqiqgl ( seq id no : 8 ) an hla - b27 restricted peptide from ebna3c ( negative control ). fig5 shows immunohistochemical staining of tumour sections for bmi - 1 . the sections shown are a ) hepatocellular carcinoma ( arrow n indicates positively staining nuclei of hepatocellular carcinoma cells ), b ) gastric cancer ( arrow s indicates stromal cells showing significantly less staining , arrow c indicates glandular epithelial gastric cancer cells staining positive ), c ) breast cancer ( arrow b indicates breast cancer cells staining highly positive for bmi - 1 ) and d ) prostate cancer ( arrow p indicates prostate cancer cells staining strongly for bmi - 1 compared to surrounding poorly / negatively staining fibroblasts ). fig6 shows ctl responses to ezh2 polycomb protein epitopes in liver cancer patients ( a ) and normal controls ( b ) as measured by elispot assays . fig7 shows the results of chromium release assays demonstrating ezh2 peptide - specific ctl lysis of target cells . the serological detection of antigens using a recombinant cdna expression library derived from a hepatocellular carcinoma ( hcc , purchased from novagen and confirmed with our own ‘ in house ’ library ) was performed using serum from hcc patients and normal healthy controls . the sera were preabsorbed against both e . coli lysate and a phage λ lysate and then used to probe filters carrying the cdna library . briefly , a culture of bl21 cells are grown overnight and resuspended in 10 mm mgso 4 . 600 ul of these cells are incubated with 3 μl of the cdna hcc library for 15 minutes and then mixed with nzy agarose and plated out onto nzy agar plates . these are incubated for 6 - 8 hours until plaques are visible and then overlaid with iptg soaked hybond - c filters . the plates are stored overnight at 4 ° c . and incubated at 37 ° c . for 4 hours the next day . the filters are removed from the plates , washed 3 times in tbs - t and then blocked for an hour in tbs - t 5 % milk . they are then washed again before incubating with the absorbed serum ( 1 in 1000 dilution in tbs - t 5 % milk ) for 2 . 5 hours room temperature , followed by washing and then incubating with the second antibody 1 in 1000 ( goat anti - human igg alkaline phosphatase conjugate , sigma ) for 2 hours at room temperature . filters are washed again and stained using the biorad alkaline phosphatase kit for no more than 10 minutes . once the filters have dried positive plaques are identified and picked from the plates , eluted , rescreened and cloned to monoclonality . the insert is screened to check it is not patient specific by screening with a number of different patient sera and with normal sera . screening controls include second step antibody alone to exclude anti - immunoglobulin responses and screening alongside a negative clone . positive clones are then excised and sequenced . the sequences are compared to known sequences using the blast nucleotide search on the ncbi database . three sequences were identified as different cdna clones of bmi - 1 using this approach . all 9 hcc patients screened had strong antibody responses to at least one of the clones . normal donors were also screened and 5 out of 6 had responses to bmi - 1 but these were weaker than those observed in the hcc patients . epitopes were predicted from the bmi - 1 protein sequence using the bimas ( bioinformatics and molecular analysis section ) web site ( parker , kc et al 1994 scheme for ranking potential hla - a2 binding peptides based on independent binding of individual peptide side - chains . j immunol 152 : 163 ). the peptides were selected according to scores compared to known epitopes from epstein - barr virus . a0201 peptides tlqdivykl ( seq id no : 1 ) and clpspstpv ; ( seq id no : 9 ) b2702 / 05 peptides vryletsky ( seq id no : 2 ) and krylrcpaa ; ( seq id no : 3 ) b4402 / 03 peptides yeeeplkdy ( seq id no : 10 ) and keevndkry . ( seq id no : 11 ) elispots were performed using these peptides on peripheral blood mononuclear cells ( pbmcs ) from hcc patients and normal donors . frozen pbmc were recovered overnight before use and the assays were performed in duplicate where possible using 2 - 4 × 10 5 cells per well . peptides were used at concentration of 100 μg / ml adding 10 μl per well . the final well volume was 100 μl . the plates were incubated overnight and then washed , antibodies added and stained according to usual protocols . the plates were counted using an automated system ( aid , strassburg , germany ). specific t cell responses ( ifnγ secretion ) can be seen to a single hla - ao2 restricted peptide in an hla - ao2 patient ( fig2 a ), and in response to an hla - b27 restricted peptide in a hla - b27 patient ( fig2 b ). elispots were performed using peripheral blood mononuclear cells ( pbmcs ) from an hcc patient and a normal donor . these assays were performed in duplicate where possible using 2 - 4 × 10 5 cells per well . a replication - defective adenovirus containing the bmi - 1 gene was produced in 293 cells and titred . pbmc were left to recover overnight if frozen samples were used , and then used in the elispot . adenovirus was added at a moi of 100 . the plates were incubated overnight and then washed , antibodies added and stained according to usual protocols . the plates were counted using an automated system ( aid , strassburg , germany ). see fig3 . experiments are underway to generate cd8 + bmi - 1 specific t cells by reactivation in vitro . a variety of restimulation protocols may be being employed , including the use of autologous dendritic cells or langerhans cells , either pulsed with peptide epitopes or infected with the recombinant adenovirus expressing the whole protein . a novel method of stimulation labels autologous b cells with a streptavidin - conjugated anti - cd20 antibody followed by a biotin - labelled monomeric . hla - a2 / peptide complex . once successfully isolated , bmi - 1 specific cytotoxic t lymphocytes are used to confirm that target cells endogenously processing the antigen are able to be efficiently recognised . epitopes were predicted for ezh2 using the same protocol as example 2 . a0201 peptides ymcsflfnl ( seq id no : 12 ) and sqadalkyv . ( seq id no : 7 ) b2702 / 05 peptides krfrradev ( seq id no : 13 ) and yrysqadal . ( seq id no : 14 ) b4402 / 03 peptides eelfvdyry ( seq id no : 15 ) and kesrpprkf . ( seq id no : 16 ) mice are vaccinated against bmi - 1 . methodologies for vaccination against an antigen are well known in the art and include , but are not limited to , the use of protein vaccines , peptide vaccines and genetic vaccines . methodologies for genetic vaccination are well known to individuals skilled in the field and can include , but are not limited to , i ) vaccination using bmi - 1 encoding plasmid dna ( by needle injection using intradermal , subcutaneous or intramuscular routes ), ii ) gene gun delivery of bmi - 1 encoding plasmid dna ( by the subcutaneous route ), iii ) by recombinant , bmi - 1 encoding , viral vectors , an example of such a vector would be a first generation adenovirus encoding bmi - 1 , or iv ) by a combination of the above approaches , for example the commonly used prime boost approach where the first administration , priming administration , is with naked dna , and the subsequent boost is with a recombinant viral vector . common immunisation protocols involve at least two administrations at least 6 days apart . naked dna administrations by a needle normally involve injection of 1 - 100 μg dna by the intramuscular route . gene gun administrations use 01 - 10 μg of dna delivered by the intradermal / subcutaneous route . when using a replication defective ( e1 / e3 deleted ) adenovirus such as that described in xiang et al . virology ( 1996 ) 219 : 220 - 227 , one can administer 1 × 10 7 to 1 × 10 10 pfu by the intramuscular , oral , intranasal or subcutaneous route . following the last administration mice are usually left at least 5 days prior to harvesting serum to measure humoral responses or the spleen or lymph nodes to measure cellular responses . humoral responses can be detected using a number of standard procedures well known to those skilled in the art . these methods include immunoblotting , western blotting , a bmi - 1 specific elisa and radioimmunoprecipatation . these procedures are well - known to those skilled in the art . the following western blotting protocol can be used to detect anti - bmi - 1 antibodies in murine sera . mice immunised with bmi - 1 were bled at various time points following the vaccination . for each sample , protein extracts from a cell line expressing high amounts of bmi - 1 protein ( mcf - 7 ) are run on nupage bis - tris pre - cast gel ( invitrogen ), and transferred to a pvdf membrane using the xcell ii blot module ( invitrogen ) according to manufacturer instructions ( 30v constant for 1 h in nupage transfer buffer + 10 % methanol ). after 1 h blocking with casein at rt , membranes are blocked to avoid non - specific binding of biotin / avidin . this blocking step is performed using the vector blocking kit by 10 min incubation at rt in avidin d solution ( 2 drops of avidin d from the kit in 10 ml tbs ) followed by a brief wash and 10 minutes incubation at rt in biotin solution ( 2 drops of biotin from the kit in 10 ml tbs ). after blocking , membranes are stained using vectastain abc - amp kit ( vector ) according to the manufacturer &# 39 ; s instructions . briefly , membranes are incubated for 30 min with various dilutions of mouse serum in pbs and washed . for all washings , 3 incubations of 4 min in casein solution ( vector ) are performed , and all incubations are done at rt . membranes are then incubated for 30 min with 10 ml of biotinylated anti - mouse igg ( vector ) diluted at 1 . 5 μg / ml in casein solution . after washing , membranes are incubated 10 min with vectastain abc - amp reagent diluted 1 : 500 in casein solution , washed , equilibrated by 5 min incubation in 0 . 1m tris buffer ph 9 . 5 , and stained with the chromogenic substrate development kit ( vector , cat no . ak - 6401 ). for this staining , membranes are incubated in the staining solution ( 4 drops of each reagent from the kit in 10 ml of 0 . 1m tris buffer ph 9 . 5 ) for 5 to 30 min and washed with water . to detect the anti - bmi - 1 antibodies by radio - immunoprecipitation following can be used . labeled recombinant bmi - 1 can be produced by in vitro transcription and translation ( promega , uk ) with 35 s - methionine ( amersham , uk ) from a plasmid encoding bmi - 1 under the sp6 or t7 promoter . in vitro translated 35 s - bmi - 1 ( 20 , 000 cpm ) is incubated overnight at 4 ° c . with 2 μl of diluted hyperimmune murine serum ( 1 : 25 to 1 : 500 ). autoantibody - bound antigen is precipitated with 25 μl of 25 % protein a - sepharose with 25 % protein g - sepharose ( amersham - pharmacia , uk ) in multiscreen - dp opaque 96 - well filtration plates ( millipore , uk ) and is washed 8 times with washing buffer ( 20 mm tris - hcl , ph 7 . 4 , containing 150 mm nacl , 0 . 1 % bsa , and 0 . 15 % tween - 20 ) using a millipore vacuum - operated 96 - well plate washer ( millipore , uk ). after washing , scintillation fluid is added directly to the 96 - well plate and radioactivity counted on a topcount 96 - well plate beta counter ( packard ). cellular immune responses can be demonstrated by demonstrating t cell - derived antigen specific cytolytic activity or ifnγ secretion in response to bmi - 1 presenting target cells . spleens are removed from mice 10 days after the last bmi - 1 immunisation . the spleen cells are immediately cocultured with syngenic bone marrow - derived dendritic cells ( bmdc ) transfected with a bmi - 1 encoding construct using a non - viral transfection system , or electroporation . bmdc transfected with an irrelevant cdna are used as negative controls , as well as spleen cells from mice immunised with irrelevant cdna and adenovirus . coculture is performed in elispot plates ( millipore ) coated with ifn - γ mab with 5 × 10 5 splenocytes / well and up to 1 × 10 5 bmdc / well . after 24 h co - culture in 200 μl of rpmi 10 % fcs , elispot plates are washed and stained according to manufacturer instructions and spots are counted using digital image analyser software . bmdc are generated by culturing mouse bm cells ( flushed from femurs and tibia ), depleted of t cells and erythrocytes ( cocktail of anti - cd4 , - cd8 , - cd45r followed by lysis with guinea pig complement and red cell lysis buffer . ), in 500 u / ml murine gmcsf and 1000 u / ml murine il - 4 . mice are vaccinated against bmi - 1 using protocols and procedures as described in example 6 . the vaccinated mice are then challenged with a bmi - 1 expressing syngeneic tumour cell line . the anti - tumour activity of the anti - bmi - 1 immunisation is demonstrated by observed protection from challenge with the bmi - 1 expressing tumour . an appropriate tumour model for this study is the murine mammary cell line 4t1 in balb / c mice . embedded tissue sections were stained as follows : the sections were soaked in xylene for 5 mins , alcohol for 5 mins , returned to water , and then treated with 0 . 3 % h 2 o 2 in h 2 o for 15 mins followed by a wash in water . the sections were placed in edta buffer ph8 plus tween20 @ 65 ° c . on a hotplate stirrer ( 500 rpm ) overnight ( alter technique by g m reynolds : reynolds , g . m ., billingham , l . j ., gray , l . j ., flavell , j . r ., cocker , j ., scott , k ., young , l . s . and murray , p . g . ( 2002 ) il - 6 expression in hodgkin / reed - sternberg cells is associated with the presence of ‘ b ’ symptoms and failure to achieve complete remission in patients with advanced hodgkin &# 39 ; s disease . br . j . haematol . 118 ; 195 - 201 ). slides were rinsed in water and were mounted onto a sequenza ™ ( thermoshandon , runcorn , uk ) and washed in tbs ( tris - buffered saline ) ph 7 . 6 . they were then incubated in primary anti - bmi - 1 ab ( either a 1 : 10 dilution in tbs for 4 hrs for mc69 ab ( gift from arie otte , university of amsterdam ), or a 1 : 100 dilution in tbs for 1 hr for clone 229f6 mab ( upstate cell signalling solutions , milton keynes , uk ). slides were then washed in tbs plus tween20 and incubated in dako envision ™ secondary ab ( dako uk ltd , ely , uk ) for 30 mins . slides were washed in tbs plus tween20 and visualised by incubating in vector novared ™ ( vector laboratories , peterborough , uk ) for 10 mins . the slides were then counterstained in mayers haematoxylin for 10 secs , dehydrated , cleared and mounted . fig5 shows the over - expression of bmi - 1 in a number of tumour specimens . in all sections the nuclei of tumour cells can be seen to clearly stain positive for the presence of bmi - 1 . positive staining tumour cells are indicated by an arrow ( s ) on each of the slides .