Patent Application: US-53100203-A

Abstract:
the invention provides an improved method of inducing an immune response against targeted cells . using gene therapy to express both a toxin or prodrug converting enzyme as a means of killing a targeted cell type and also stress response protein enhances the subsequent immune response directed against such cells . the method is particularly applicable to inducing an immune response against cancer cells . also provided are polynucleotides , products and vectors for use in such a method .

Description:
the invention is described in detail by the use of the following examples . these are by way of illustration only and are not to be taken as limiting . 4t1 , a mouse breast cancer cells were obtained from atcc ( crl - 2539 ). ej - 6 - 2 - bam - 6a was obtained from atcc ( crl - 1888 ) and was generated by transfecting nih / 3t3 with dna from the human ej bladder carcinoma . per . c6 cells ( lit ) were obtained from introgene ( leiden , the netherlands ). 911 cells were kindly provided by prof . l . young ( crc institute for cancer studies , university of birmingham , uk ) and were maintained in dmem containing 10 % fcs , 10 mm mgcl 2 and antibiotics . 4t1 and ej - 6 - 2 - bam - 6a were cultured as recommended by the supplier . ptx0374 was constructed by cloning a 1 . 6 kb bglii - bamhi fragment containing the human cmv promoter fused to the e . coli ntr gene ( ntr : e . coli b / r nitroreductase gene amplified from genomic dna ) into psw107 . praj 43 bp4 is a puc19 plasmid containing the mouse gm - csf cdna and was kindly provided by prof . l . young ( crc institute for cancer studies , university of birmingham , uk . cet902 is a puc19 plasmid containing the mouse cmv ie enhancer / promoter and the sv40 late poly ( a ) signal . cet902 / mcmv - mhsp70 contains an expression cassette for stress inducible mouse hsp70 protein and was constructed as described . first , the mouse full length ( 1 . 4 kb ) cmv promoter was cloned into a puc19 - based vector . second , the sv40 late poly ( a ) signal was cloned downstream from the promoter . finally , the cdna coding for mouse hsp70 was cloned between the promoter and the poly ( a ) signal using a smal site . a plasmid containing the cdna for mouse hsp70 was kindly provided from dr . r . vile ( molecular medicine program , mayo clinic , 200 first street sw , rochester , minn ., usa ) and was cut out as nhel / bamhi fragment and blunted using t4 dna polymerase . cet902 / mcmv - mgm - csf was prepared by digesting praj 43 bp4 with ecori and bamhi releasing a 465 bp fragment . the fragment was blunted and ligated into smai prepared cet902 . pps1128 was kindly provided by dr . p . searle , crc institute of cancer studies , university of birmingham . pps1128 contains adenoviral sequences from the left hand itr to nt 359 and from nt 3525 to 10 , 589 and is therefore an e1 - deleted vector . pps1128 was constructed by cloning a 917 bp fragment of the human beta - globin gene ( bamhi site in exon2 to the ecori site in exon3 ) coupled to a 240 bp hincii - bamhi fragment containing the poly ( a ) addition and transcriptional termination signals of the human complement c2 gene into pbluescript ( stratagene ). this plasmid was constructed in two stages . in the first , the left hand ecori site of pps971 ( weedon et al , int . j . cancer , in press ) was converted to a swal site to create pps115 . in the second , the 350 bp spe1 - amf / ii fragment of pps115 was replaced with a linker prepared by annealing the two oligonucleotides : pps1022 was constructed from pps972 by conversion of the right hand ecori site to a swai site . ptx0375 , the transfer vector used to generate ctl102 , was constructed by cloning a spei fragment spanning the whole expression cassette ( hcmv - ntr - ivsii - p ( a )) from ptx0374 into spei digested pps1128 and identification of a clone containing the cassette in the left to right orientation . pps1128 / mcmv - mhsp70 and ptxo375 / mcmv - mhsp70 was constructed by transferring the mcmv - mhsp70 - sv40 p ( a ) expression cassette from cet902 / mhsp70 into blunted pps1128 or into the blunted paci site of ptx0375 , respectively . the mcmv - mhsp70 - sv40 p ( a ) expression cassette was prepared by digestion of cet902 / mhsp70 plasmid with xmni , asci and bstz171 and blunting with t4 dna polymerase . therefore the final cassette only contains about 500 bp of the mouse cmv promoter , this piece provides most of its activity . pps118 / mcmv - mgm - csf was constructed by cloning the complete expression cassette ( 1 . 3 kb ) prepared from cet902 / mcmv - mgm - csf ( bstz171 and asci digested and blunted ) into pps1128 . the adenoviral “ backbone ” vector pps160 was constructed by paci linearisation of pps128 , ligation with a paci - compatible adaptor ( oligo1 : 5 ′- tacatctagatmt - 3 ′, oligo2 : 5 ′- ttatctagatgta - 3 ′) containing an xbai site followed by xbai digestion to release a ca . 7 kb xbai fragment containing ad5 sequences 3524 - 10589 . this was then cloned into xbai linearised pps1022 ( dr . peter searle ) a puc18 - based plasmid containing ad5 sequences from nt 10 , 589 to the right hand itr but lacking nt 28 , 592 to 30 , 470 ( e3 region ). the recombinant viruses ctl102 ( ad . hcmv - ntr ), ctl102 / mcmv - mhsp70 , ad . mcmv - mhsp70 and ad . mcmv - mgm - csf were constructed by homologous recombination in per . c6 cells . these cells were cotransfected with an equimolar mixture of ptx0375 , ptxo3751mcmv - mhsp70 , pps1128 / mcmv - hsp70 or pps1128 / mcmv - mgm - csf , respectively , and pps1160 into 90 % confluent per . c6 cells . the recombinant viruses were harvested about 7 days later by 3 freeze - thaw cycles in infection medium ( dmem , 1 % fcs , 2 mm mgcl 2 ). by repeated infection / harvesting cycles the viruses were grown to large scale and then purified by standard cscl density centrifugation , dialysed against excess of storage buffer ( 10 mm tris ph 7 . 4 , 140 mm nacl , 5 mm kcl , 0 . 6 mm na 2 hpo 4 , 0 . 9 mm cacl 2 , 0 . 5 mm mgcl 2 and 5 % sucrose ) and finally snap - frozen in liquid nitrogen and stored at − 80 ° c . particle concentrations were determined using the bca protein assay reagent ( perbio science uk , ltd , tattenhall , cheshire , uk ). plaque forming units ( p . f . u .) titres were determined by plaque assays on 911 cells . 4t1 cells were infected at a cell density of 1 × 10 7 / ml for about 2 . 5 hours in a humidified co 2 incubator with the indicated p . f . u . per cell ( moi ) in infection medium ( normal medium but containing only 1 % fcs ). during infections cells were gently mixed every 30 minutes . then , cells were pelleted ( 300 × g , 5 minutes ) and washed before being resuspended in pbs . in vaccinations experiments the moi used was 200 to 300 and where combination viruses were used , the final dose of vector was kept constant to avoid a cytopathic dose - dependent effect . 3 × 10 5 hela cells were infected with the indicated mois in infection medium by incubation for 2 hours at 37 ° c . in 5 % co 2 . cells were then fed with complete medium ( 10 % fcs ) and cultured for 24 hours . cells were then washed in pbs and then a whole cell lysate was prepared by adding 200 μl ripa buffer ( 10 mm tris ph 8 . 0 , 150 mm nacl , 1 . 0 % np40 , 0 . 1 % sds , 0 . 5 % na - desoxycholat plus complete protease inhibitor cocktail , roche ) per 6 - well . cells were incubated for 10 min at rt and then centrifuged at 13 , 000 rpm at 4 c . the cleared lysate was transferred to a new tube and protein concentration determined using the biorad dc protein assay kit ( hercules , calif ., usa ). 30 μg of each lysate sample was resolved by 11 % sds - page using high rainbow protein size marker ( amersham pharmacia , piscataway , n . j ., usa ). proteins were then transferred to a nitrocellulose membrane ( gelman sciences , ann arbor , mich ., usa ). the membrane was blocked in tbs ( 10 mm tris ph 7 . 5 , 150 mm nacl )/ 0 . 1 % tween20 / 5 % milk powder for 1 hour at rt . primary antibodies were diluted in blocking buffer as follows : sheep anti - ntr ( polyclonal antibodies , dyfed , uk ): 1 : 2000 , mouse anti - hsp70 ( spa - 810 , stressgen biotechnologies , victoria bc , canada ) 1 : 1000 and goat anti - gm - csf ( santacruz , calif ., usa ; sc - 1322 ): 1 : 1000 . membranes were incubated with primary antibody for 1 hour , rt and then extensively washed and then incubated for 30 min at rt with secondary antibodies in tbs / 0 . 1 % tween20 / 0 . 5 % milk powder as following : donkey anti - sheep - hrp ( 1 : 7 , 500 ; sigma ), anti - mouse - hrp ( 1 : 10 , 000 ; sigma a - 9917 ) and anti - goat - hrp ( sigma , a - 5420 ). after extensive washings , enhanced chemiluminescence was carried out using pierce “ supersignal west pico chemiluminescence substrate ” ( perbio science uk ltd ., tattenhall , uk ) and analysed with alpha innotech imager model # 2 . 3 . 1 . female balb / c mice ( h - 2 d , 6 - 8 weeks old ) were obtained from harlan ( oxion , uk ) and were maintained in a temperature - controlled , light - cycled room with food and water ad libitum . mice were allowed to rest for one week before any treatment . care as well as all experimental procedures were conducted in full compliance with the uk home office regulations . 4t1 is a highly aggressive and metastatic cell line established from a mammary adenocarcinoma , which arose spontaneously in a balb / cfc 3 h mouse ( dexter et al , 1978 ). 4t1 cell line which expresses h - 2 d class i but not class ii molecules , is a nonimmunogenic tumour model , which does not stimulate a syngeneic antitumour response in vitro or in vivo . the minimal 100 %- tumour - inducing dose in balb / c mice is 5 × 10 2 cells . 4t1 cells produce aggressive solid tumours when injected subcutaneously into balb / c mice that can spontaneously metastasise primarily to the lung while the primary tumour is growing in situ . the ej - 6 - 2 - bam - 6a ( shih et al ., 1981 ), a fibroblast cell line was used as a control tumour . an indicated number of adenovirus - transduced 4t1 cells were injected s . c . into the right flank of each naïve mouse in a volume of 100 μl total ( day 0 ). different experimental groups were injected with cells transduced with single or different combinations of adenovirus containing the nitroreducatse and or co - stimulatory genes . after allowing for transgene expression to proceed in vivo till day 2 , a time at which a maximal transgene expression was demonstrated to occur , a solution of 400 μm cb1954 was injected peritumourally in a total volume of 500 μl . unless otherwise stated , on day 21 all mice were challenged with a second contralateral injection of 5 . 0 × 10 3 parental 4t1 cells . this dose is 10 - fold higher than the minimal lethal dose in naïve mice . a naïve group of mice was also injected with these cells at the same time . the induction of a long - term antitumoural immune response was demonstrated by challenging mice 80 days following vaccination . all groups of mice in any one experiment were challenged on the same day using the same preparation of cells . animals were examined regularly for the appearance of tumours by palpation ; whereafter tumours were measured in two perpendicular dimensions using a vernier calliper 2 - 3 times / week . tumour size was expressed as the product of the two diameters of individual tumours . mice were culled for humane reasons when they exhibited signs of distress , the tumours becoming too necrotic or when tumours exceeded a size of 160 mm 2 . ctl102 / cb1954 killing of 4t1 can induce significant and prolonged protection against 4t1 cell challenge to test the possibility that ntr / cb1954 tumour cell killing in situ may contribute to the generation of specific antitumour immune , vaccination experiments were conducted . in optimisation experiments to establish the optimum killing conditions where dose ranging of cell inoculation ( 5 . 0 × 10 2 to 5 . 0 × 10 6 ntr - expressing 4t1 cells ) and subsequently in vivo treatment with escalating cb1954 doses ranging from 100 to 400 μm were tried ( data not shown ), tumour - free mice were vaccinated . mice that rejected the initial inoculation of tumour cells from two groups vaccinated with either 5 . 0 × 10 3 or 5 . 0 × 10 4 ntr - expressing 4t1 cells respectively were challenged with 5 . 0 × 10 3 unmodified 4t1 cells on the opposite flank at 21 days following cell inoculation . challenging mice 80 days following vaccination was also conducted to assess the induction of a long - term antitumoural immune response . all naïve mice developed progressively growing tumours by day 10 and reached compulsory sacrifice by day 25 post - challenge . a vaccine dose - dependent response of rejecting the challenge was observed in mice vaccinated with ntr / cb1954 killed cells irrespective of the time of challenge ( fig4 a ). vaccination with a low dose of 5 . 0 × 10 3 cells conferred a low level protection compared to naïve mice , while a higher vaccination dose ( 5 . 0 × 10 4 ) enhanced considerably the immunogenecity of 4t1 cells and protected the majority of mice from challenge . to assess the specificity of the protection seen with the ntr / cb1954 killing vaccine , mice vaccinated with either 5 . 0 × 10 3 or 5 . 0 × 10 4 ntr - expressing 4t1 cells were challenged with another syngeneic tumour , ej6 , a mouse fibrosarcoma . immunisation with 4t1 cells failed to protect all mice against challenge with ej6 cells ( fig4 b ). enhancement of antitumour immunity of ntr / cb1954 killing by expression of murine hsp70 costimulatory molecules to determine whether the effects of ntr / cb1954 killing in the vaccination model could be improved by expression of the costimulatory molecule hsp70 , mice received a single immunisation consisting of 4t1 cells transduced with either the single recombinant virus carrying the ntr gene or the double recombinant virus carrying both ntr and hsp70 genes . animals were injected peritumourally with cb1954 on day 2 and animals were then challenged with 5 . 0 × 10 3 unmodified 4t1 cells on the opposite flank ( see immunisation protocol ). two different vaccination doses were used , a low dose of 5 . 0 × 10 4 and a high dose of 5 . 0 × 10 5 . all animals were challenged with 5 . 0 × 10 3 unmodified 4t1 cells at 21 days post - cell inoculation and followed for tumour appearance , tumour growth and survival . the results suggest that ntr / cb1954 killing of cells expressing hsp70 is more potent in priming immune response than mice vaccinated with ntr / cb1954 killed cells alone ( fig5 a ). a higher number of vaccinating cells seem to be better in protecting mice against a subsequent challenge with 4t1 cells . consequently , animals vaccinated with ntr - hsp70 - expressing cells showed significant survival rate ( fig5 c ). by contrast animals receiving ntr - expressing cells show only a modest survival advantage with one mouse tumour - free surviving more than 3 months . immunisation with cells expressing hsp70 alone and treated with cb1954 did not protect any mice against the emergence of tumours at the inoculation site where a progressively growing tumours developed that took approximately 30 days to reach the size of compulsory sacrifice and mice had to be sacrificed not long after the immunisation time of 21 days post - 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