Prostate Oncolytic Virus Rodriguez

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1997;57:2559-2563. Published online July 1, 1997.Cancer ResRon Rodriguez, Eric R. Schuur, Ho Yeong Lim, et al.Antigen-positive Prostate Cancer CellsCN706: A Selective Cytotoxic for Prostate-specificProstate Attenuated Replication Competent Adenovirus (ARCA)

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[CANCERRESEARCH 57, 25592563,u ly 1 , 1 99 71Advances in Brief

Prostate A ttenuated Replication Com petent A denovirus (A RCA ) C N706:A Sele ctive Cyto toxic for Prosta te -spe cific Antigen -pos itiveP ro sta te Cancer Cells'Ron Rod rig uez,2 E ric R . S ch uu r,2 Ho Yeong L im , Gail A . Hen der so n, J on ath an W . S imon s, a ndDaniel R . Hender son3Brad y U rol og ic al I ns ti tu te , J oh ns Hopk in s Un iv er si ty On co lo gy C en te r, B a lt imo re , Ma ry la nd 21287 ( R. R ., H . 1 '.L , J . W . S . ); a nd Ca ly don , I nc ., Me nl o Pa rk , C a li fo rn ia 9 4025[E. R . S ., G . A . H ., D . R . H .]

AbstractProstate-specific antigen (PSA) is a widely used marker for the dingnosis and m anagem ent of prostate cancer. M inim al enhancer/prom oterconstructs derived from the 5' flank of the hum an P SA gene (prostatespecific enhancer) were inserted into adenovirus type 5 DN A so as to drivet he EJA gene , the reby c reat ing a pro st at e- spec if ic enhance r- con ta in ingv irus , CN706. EJA was expressed a t high leve ls in CN706- infec ted humanP SA -producing L NC aP cells but not in C N706-infected D U145 cells,which are human prostate cells that do not express PSA. The titer ofCN706 was significantly higher in LNCaP cells compared to severalhuman cell lines that do not produce PSA (HBL100, PANC-1, M CF-7,DU145 , and OVCAR3) . Furthe rmore , i n LNCaP cel ls , t he y ie ld o f CN706wa s dependent on exogenous androg en (R1881 ). CN706 de st ro yed lar ge

LNCaP tumors (1 x iO@ c ells) and abolished PSA production in nu/num ouse xenograft m odels w ith a sin gle intra tum oral injection.IntroductionP SA 4 is th e m ost w id ely u sed serum m ark er for th e d iagn osis an dm an agem en t of an y form of can cer. It is p rod uced in P CA cells an dp ro st at e du ct al e pi th eli a (wh ic h r ep re se nt s l es s t ha n 5% o f t he c ell s o fthe prostate); it is also produced in m uch sm aller am ounts in theperiurethral glands and, very rarely, in tum ors of the skin, salivary,and breas t (13) .W ith a dv an ces in P SA s cre en in g follow ed b y tissu eb iopsy, new ly diagnosed P CA represents 43% of all diagn oses ofn on sk in c an ce r in m en . D esp ite th ese a dv an ce s in PCA d ia gn osis a ndmanag ement , e ff ec ti ve s ys tem ic t re atment r ema ins e lu si ve ; me ta st at icP CA is the second leading cause of cancer death of m en in the U nitedS ta te s ( 2) . Th er e is n o c ur at iv e t re atment f or PCA onc e t he d is ea se hasprogressed beyond the organ (4, 5). Human gene therapy usingtissu e-sp ecific ex pressio n o f c yto tox ic g ene s m ay b e a ration al tre atmen t stra teg y. T he re gu la to ry re gio ns o f th e P SA g en e a re a r ea so nab le ch oice for su ch an ap proach . R ecen fly, w e d escrib ed an an drog en -r es po ns iv e a nd t is su e- sp ec if ic PSE lo ca te d ups tr eam o f t he PSAp romote r, w hic h, w hen fu se d to th e p romo ter in a m in im al e nh an cer /p romo te r uni t, c au se s PSA t o b e e xp re ss ed a t a lmos t w il d- ty pe le ve ls(6).Becauseheprosta tesanaccessoryrgan,emovalrablatio nft he e nt ir e g la nd has no s er io us h ea lt h r ep er cu ss io ns (7 1 0) .Mo st c ur re nt m e thod s o f g en e th er apy u se v ir al v ec to rs b ec au se t heReceived 4/7/97; accepted 5/22/97.The costs of publ icat ionof th is ar tic lewere defrayed in partby the paymentof pagecharges.This articlem ustthereforebe herebymarkedadvertisementin accordancewith1 8 U .S .C . S ec ti on 1 73 4 s ol ely t o i nd ic at e t his f ac t.I R . R., H. Y . L., and J. w . S. have no financial inte res t in Calydon, Inc., and w eresupport ed by NIH Special ized Programs of Research Excel lence in Prost at e Cancer GrantCA-58230 andthe CapCureFoundat ion .2 T hese authors made equal contributions to this work.3 To whom requests for reprints should be addressed, at Calydon, Inc., 1014 HamiltonC ou rt, M en lo P ark , C A 9 40 25 .4 The abbreviations used are: PSA, prostate-specific antigen; PCA, prostate cancer;PSE, prostate-specificenhancer;ARC A, attenuatedreplication-competentadenovirus;AdS, human adenov irus 5 ; p fu, p l aque- forming un it (s ) ; i t. , i n tra tumora l.

e ff ic ie nc y o f g en e t ra ns fe r w it h v ir us es is s up er io r t o t ha t a c hi ev ed bynonv ir al s ys tems (1 113 ).W e chose an adenoviral vector. W e reasoned that placing its EJA gene under the control of the PSE wouldc re at e a v ir us , t he r ep li ca ti on o f wh ic h wou ld b e r es tr ic te d p rima ri lyto P SA -p ro du cing cells w ith in the p ros ta te an d P SA -ex pressin g P CAcells. Here, we describe the construction of such an ARCA andd em on str ate its se le ctive cy to tox icity to wa rd P SA -e xp re ssin g PCAcells in vitro and in vivo.M aterials and M ethods

C ells an d C ell C ultu re. T he fo llo wing ce ll lin es, all ob tain ed fro m theAmericanTypeCultureCollectionwere used:LNCaP,a humanPCA cell l ined erive d fro m a ce rv ica l lym ph n od e m etasta sis th at p ro duc es P SA a nd amuta ted bu t func tiona land rogen receptor (14, 15 );HBLIOO,a human lung ce llli ne ; MCF -7 , a h um an b re as t c an ce r c ell li ne ; PANC -l , a h um an p an cr ea ticcancer cell line; D U145, a hum an P CA cell line that lacks the androgenr ec ep to r a nd doe s n ot p ro du ce PSA , a s d et erm in ed by r ev er se t ra ns cr ip ta sePCR; a nd OVCAR3 , a h uman ova ri an c an ce r c el l l in e. Th e human embryoni ckidney cell line, 293, which expresses the adenovirus E1A and E1B geneproducts, was obtained from Microbix Biosystems, Inc . (Toronto , Canada; Ref.1 6) . C el ls w er e m ai nta in ed in DMEM, w it h th e e xc ep tio n o f LNCaP c ell s(ma intainedin RPM! 1640) and suspensioncul ture293 ce ll s (ma intainedinJ ok li k's MEM) . Cu lt ur es we re s uppl ement ed w it h 1 0% FCS , w it h t he e xc eption of suspension culture 293 cells, which were supplem ented w ith 10% horses er um . A ll m ed ia w er e s up pl em en te d w it h 1 00 u nit s/mI p en ic il lin a nd 1 [email protected]/mlt re pt omycin a nd ma in ta in ed a t 3 7Cn 5% CO2 .Cons tr uc ti on a nd Pu ri fi ca ti on o fCN7O6 and CN702. pXC . 1 and BHG1O(17, 18) were purchased from M icrobix Biosystem s. pXC. 1 contains hum anadenovirus 5 (A dS ) sequences from base pairs 22 to 5790 (17). B HG IOcontains AdS sequences with two deletions: an El deletion of base pairs1 88 -1 33 9 a nd a n E 3 d eletio n o f b ase p airs 2 8,1 33 3 0,8 18 . B HGIO DNA isnon in fectiou s, w hereas cotran sfection of p XC . 1 a nd B HG 1O creates in fectio usv iru s by homologous recombina tion (18) . A un iqueAgeI res tr ic tion s ite in thepromoter of adenovirus E1A at AdS nucleotide 522 was created in pXC. I . T hefirst se t o f P CR p rim ers, 1 5.l3 la (5 '-T CG TC TT CA AG AA TT CT CA ) an d15.133d (5'-lTFCAGTCACCGGTGTCGGA), produced a 927-bp PCR fragm en t fro m th e u niq ue E co RI site in th e p BR 32 2 b ac kbo ne o f p XC .l to th eu niq ue A ge ! s ite a t A d S n uc le ot id e 5 52 . A s ec on d s et o f PCR p rime rs , l5 .1 33 c(5'-T CC GA CA CC GG TG AC TG AA A) and l5.133b (5'-G CA TfC TC TA GACACAGGTG),produceda 787-bpfragmentfromtheAge! site to theXba! sitea t AdS nucleo tide 1339 .Combin ing equa l amoun tso f the 927-bp p roduct wi ththe 787-bp product, a second PCR w as perform ed w ith the tw o outsidep rimer s, t o y ie ld a p ro du ct o f 1 714bp tha t c ou ld b e cut w it hAge ! i nt o t he twosmal le r f ra gment s. Th e l 7l 4- bp f ra gment wa s c le av ed w it h EcoPJ a nd XbaIa nd clo ned in to sim ila rly clea ved p XC .l to y ield C N9 5.CN65 contains the enhancer and prom oter of the human PSA gene, consisting of the enhancer at base pairs 5322 to 3738 fused to the PSApromotera tbasepa ir s541o + 12 , separa tedby 76 bp of a multip lec loningsite from BSKSII+ (Stratagene; Ref. 6). The 22l3-bp PSE with Age! ends wasp re pa re d b y P CR o fC N6 5 (6 ) w ith p rim ers 1 5.1 76 a ( 5'-C AT FA AC CG GT ACCTCTAGAAAATCTAGC),which introducesanAge! s ite a t the 5 '-end of the

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Tab le 1T i t er of CN 7O 2 an d C N70 6in h um an cellinesCell

lineVirusCN702

I @ -@uffer 0-@N7060 7 14 21 28 35Da@e- .N702

A17ENUATED ADENOVIRUS FOR PROSTATECANCERtected in either cell line prior to infection or in cells infected w ithA d5 Lac Z. CN 70 2 ex pres sed E1A at sig nific ant lev els in bo th LNCaPand DU-145 cells, as did CN 706 in LNCaP cells (Fig. 2). In CN 706-infec te d DU1 5 ce lls, ho wev er, E1 A ex pres sio n w as reduc ed by 99%.Northe rn bl ot anal ys is o f CN7 06 -i nf ec te d LNCaP c el ls di d no t de te ctE 1A m R NA fr om t he en dogen ou s vir al en ha ncer /p rom oter , w hichw as d isp la ced 1681 b p u pst rea m (d at a n ot sh ow n). T hu s, t he P SE inCN70 6 pro vide d E1A e xpre ssio n se le ctiv ely in PSA-pro ducing ce lls(LNCaP) but not in non-PSA-producing cell s (DU145) .Ability o f CN702 and CN76 to Multiply in S ev eral Ce ll Type s.Dif fe renti al ti te rs hav e been used to c ompare g rowth o f mutan t v iruse s

in d iffer en t cell lin es (24 30).T h e d iffer en tia l t it er of C N 702 a ndCN706 in various human cel l l ines i s shown in Table 1 .The abso lutenumberof plaqueswas normalizedto 5.0 X l0@,and relativetiterswere calculated. The results show that CN702 and CN706 greweq ua lly w ell in L N Ca P cells. H ow ever , in a ll ot her cell lin es, C N 702g av e a higher titer than CN 70 6; the titer of CN 70 6 w as reduc ed3000:1 in HBL100 (human lung) cel ls , 20 :1 in MCF-7 (human breas tcarcinoma) cells, 20: 1 in PANC-l (human pancreatic carcinoma)cells, 2000: 1 in D U145 (h um an n on -P SA p r od ucin g p r ost at e ca rcin om a ) cells, a nd 60: 1 in OVCAR 3 (h um a n ova r ia n ca r cin om a ) cells.These differential titer data may reflect a potential therapeutic indexf or ARCA in dif fe rent tis sue type s i n v ivo. Thus , depending on cellty pe , the the rape utic ratio o f CN706 v arie s from a low o f 2 0:1 to ahigh of 3000: 1. Therapeutic ratios of many conventional cytotoxicd ru gs r an ge fr om 1.5:1 to a h igh of 6:1 (31).

T h e a ct ivit y of t he P SE h as b een sh ow n t o b e in du cib le b y a nd r ogen( 6) . An and rogen -inducednc r e a sein CN706 t i t er a s compa r edtoC N702 t it er w as sh ow n in L NC aP cells. L NC aP cells w er e in fect edw ith a con stan t am oun t of C N702 or C N706, a nd cu ltu r es wer eo ve r la ye d w it h a ga r c on t ain in g in cr e asin g c on ce nt r a tion s o f t h e n onmetabolizable synthetic androgen R1881. CN 702 titer w as not effect ed b y a nd rogen , w her ea s 1 n t@ t nd 10 n r s@R l881 in du ced a dd itional5- and 7-foldincreases,respectively,in CN706 titercomparedto n o t rea tm en t w it h R 1881 (d ata n ot sh ow n).Treatment o f Pro state Tumo rs w ith CN 70 6. LNCaP tumors

w er e in ject ed i.t . w it h 5 X 108 p fi@CN 706 on d ay 0 (F ig. 3A ). T u m or sw er e m ea su red a t t he in dica ted t im es. T her e w as a sligh t in cr ea se intumor v olume duri ng the fi rs t 2 w ee ks af te r i .t. inje cti on, fo llowe d bya r ap id d ecr ease. After 6 week s, 5 of 10 m ice wer e visu ally fr ee oft umo r .These experiments show tumor cell selectivity and tumor cell

ki lli ng , but, whe re as w il d- ty pe AdS c an i nfe ct and o cc as io nal ly transfo rm mous e c ell s, AdS c anno t pro pag ate s ig ni fic antly i n mous e c ell s.T h us, t issu e select ivit y of C N 706 m u st b e sh ow n in d ir ect ly in m ou sexenografts. Indeed, as expected, CN 702 can also eliminate humanLN CaP xenografts in nude mice (data not show n). D U145 is a PCAcell lin e t ha t d oes n ot p r od uce P SA or a nd r ogen r ecep tor . I n con tr a st ,all, o r ne arly all, human PCAs produc e PSA in situ (1 , 7 , 3 2 3 4).H ow ever , P SA exp r ession is lost w it hin h ou r s in p r im a r y cu lt ur es of

A

I Iuffer 0-CN706

I

B

F i g. 3 . T r e a tm en t of t umo r x e nogr a f tsw i th r e comb in a n tv ir u s es . T umo r x enogr a f tswe r e g r own s . c. i n BALB /c nu / n uma l em i c e t o a p p r o x im a t e lyI cm i n d i am e t er .T umo r swe r e t r e a t e dwith r e combinan tv ir u se sby i t . i n j ec t ionon day 0 , and mea su r emen t swe r et a k enwe e k ly .A , LNCaP t umo r swer e t r e a te dwi th PBS- lO% g ly ce r ol ( b u ff er , n = 5 ) o rCN706in buffer(n 10).Averagetumorvolumeswerenormalizedo 100%on day0.B , D U 1 45 tu m o rs w e re tre ate d w ith b u ffe r (n 5 ), C N 7 02 (n 5 ), o r C N 7 06 (n 5 ),and w ee kl y tumor meas ureme nts w ere take n as in A .

PCA cells.5 Tumors of D U145 cells w ere induced in nude mice andch allen ged with b uffer , C N702, an d C N706 (F ig. 3B; n = 5 for eachgroup o f m ic e). The re sults s how CN702 inhibits g row th o f DU145tu mor s, wh er eas C N706 h as n o effect on tu mor gr owth . T hu s, th eprostate-specif ic CN706 virus shows selectivity for cell s producingP SA in vivo.B lood samples w ere harvested from the mice show n in Fig. 3A at

t he sa m e t im e a s t um or volu m es a nd ser u m P SA levels w er e m ea su r ed(Fig. 4) . PSA levels also increased slighf ly , as did tumor volume, afterin fect ion b ut t hen fell r ap id ly. T he fa ll in P SA levels p reced ed th e

CN706293 5.0 X lO@ 5.0 X 10'LNCaP 2.0 x l0@ 1.5 X iO@HBL100 5.0 X 10' 5.0 X l0@M CF -7 2.0 X l0@ 1.0 X l0@PANC-1 2.0 X l0@ 1.0 X l0@DU145 2.0 X l0@ ' 1.0 x l0@OVCAR3 6.0 X l0@ 1.0 X l0@

Cellswereplat edan dinfectedas descr ibedn M ater ialsndMethods.nfectedellswere overlayed with nutrient agarose and plaques counted at 5 days. Titers were normalized to 5 x 1 0' pfu/m l in 2 93 cells. 5 D. P ech l, p er sona l com mu nica tion .2561



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--Bu ffer eCN706

A T ItN U A T ED AD ENO V IR U S F OR PRO ST A T E C A NC E Rcells lack ing or containing m utated p53. S uch a v irus could conceiva b ly b e a P CA a n tin eo pla st ic. H owe ver , on ly 8 20%o f h um a n P CA scarry m utations in p53 (41, 42), w hereas m ore than 95% of PCA s arePSA- po sit iv e (3 2, 4 3) .V e ct ors l ik e CN7 06 h av e s ev e ral ad van tag es f or c li nic al e valu at io n:

t hey ca n b e en gin eer ed for sp ecific cell t yp e t ar get in g; t hey a m plifyd ose a nd P CA cell k illin g b y r ep lica tin g; t hey exp r ess vir a l a nt igen st hat may e li ci t immune ce ll k i ll ing o f de si red t arge t cel ls ; and on ly oned ose m a y b e r eq uir ed t o elim in at e t um or s. H u m an sa fet y h as a lr ea dyb een est ab lish ed for r ep lica tion -com p et en t a den ovir u ses; in 1956,Sm ith et a!. d em on strated re sp onses in tu m ors o f 2 6 o f 4 0 p atien tsw it h ce r vica l ca n cer in j ec ted w it h w ild -t yp e a d en ov ir u s (44 ). S tu d ie sof optim um dose and route of adm inistration of C N 706 are ongoing.H um an clinical testing of C N 706 and v ectors lik e it w ill pennite val uat ion o f t he sel ec ti v e t ox i ci ty and the rapeut ic po tent ial o f at tenu at ed a den ovir u s in vivo cyt or ed uct ive t her a py for P CA.Acknowledgments

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B io ph y s. R e s. C omm u n. , 1 73 : 5 34 54 0, 19 90 .16. G r ah am , F . L ., S miley, J ., R u ssell, W . C ., a nd N air n , R . C h ar a ct er ist ics of a h um ancell lin e t r an sfor m ed b y D NA fr om h um an a den ovir us typ e 5. J . G en . V ir ol., 36:5972,1977.17. M cK innon, R . D ., B acchetti, S ., and Graham , F. L . T n 5 m utagenesis of the

t r an sfo r min g g en es o f h u m an a de no vir u s t yp e 5 . G e ne, 1 9: 3 3 42 ,1 98 2.1 8. B eU , A . J . , H a d d a r a,W . , P r ev ec , L ., a n d G r a h am ,F . L . A n e ff ic ie nt a n d fle xib les ys te m fo r c on st r uc tio n o f a de no vir u s v ect or s w it h in ser t io ns o r d elet io ns in ea r lyreg io ns I and 3 . P ro c. Na il . A cad. S c i. USA , 9 1: 8 8028806,9 94.19 . McGro ry ,W. J . ,Bau t i s t a ,D . S ., a nd Graham ,F . L . A s imp le t e chn ique fo r the r e scueo f e ar ly r eg io n I m u ta tio ns in to in fe ct io us h u m an a d en ov ir u s t yp e 5 . V ir o lo gy , 1 63 :614617,1988.2 0. G rah am , F. L . , a nd V a nD e r E b , A . J. A n ew te ch ni qu e f or t h e as say o f i nf ec tiv ity o fh u m an a de no vir u s 5 DNA. V ir o lo gy , 5 2: 4 56 4 67 . 1 97 3.

21. L im , D. J., L iu, X -l., S utk ow sk i, D. M ., B raun, E . J., L ee, C ., and K oz low sk i, J. M .Growthof an androgen-sensitiveumanprostatecancercellline,L NC aP,in nudemice . Prosta te ,22: 10918, 1993.2 2. L ow e, S . W . , B o dis , S . , M cC latc he y , A . , R em i ng to n, L . , R u le y ,H . E ., Fis he r. D . E .,2 5 6 2

35 0

30 0

250

0IH

20 0

15 0

1 0 0

50

0 8 14 21 28 35Days

F ig. 4. S er u m P SA L evels of m ou se L NC aP xen ogr aft s t rea ted w it h C N 706. W eek lyblood sam ples w ere tak en f rom the m ice in the ex perim ent sho wn in Fig. 3A at the tim eo f t h e t umo rme a s u r em e n t s.S er um f r om t h e s amp le swa s u s ed t o me a su r e PSA l ev el su si ng a PSA EL!SA as d es cri bed i n Ma te ri al snd Methods.

re du ctio n in tum or v o lum e b y 1 we ek . A l th ou gh th es e s im u ltan eo ussm all in cre as es in tum or v olum e an d PSA le ve ls w e re w ith in e xp erim en tal e rro r, th e d elay in th e d ec re as e in tumo r s iz e c ompare d to th ed ecr ea se in P SA levels m a y r eflect t he t im e r eq uir ed for r ea dsor p tiona nd clea r an ce of d ea d cells. L at e r ecu r ren ce of t um or s h as n ot b eend etected th ou gh 70 d ays. All C N706-t r ea ted a nim als wer e fr ee ofserum PS A by 6 w eek s af ter inf ection. C ontrol tum ors injected w ithbuf f er, Ad5LacZ , o r UV- irrad iat ed CN706 con ti nu ed to g row .Discussion

In vivo th era py w ith r ep lica tio n-d efic ie nt ad en oviru se s in vo lv es ab ala nce b et ween a ch ievin g a u sefu l t her a peu tic en d p oin t b efor e t hep at ien t clea r s t he vir u s (35) a nd t he im m u ne syst em p r oh ib it s fu r th erv irus use (36). Even changing adenov irus serotypes can only beex pe ct ed t o a ch ie ve 25 30%o f t h e o r ig in a l t r ea t m en t e ffic ac y (3 7).T hus, an ef fectiv e adenov iral therapeutic w ill need as high a therap eu tic p oten tia l a s p ossib le, fr om a s low a n im m un ogen ic d ose aspossible. R epeat doses are encum bered as the host def ense im m unes ys tem i s ac ti vat ed ( 38 4 0) .

An AR CA d esign ed to a tta ck a sp ecific th er ap eu tic ta rget m aya d dr e ss som e of t h es e co nc er n s. B y r e plic at in g p r efe r en t ia lly in t a r ge tc ell s, t he th erap eu tic w i ll d eli ve r a th erap eu tic d os e at t he i nte nd ed s it eof a ct ion , w it h a sm a ll in pu t im m un ogen ic d ose, a nd r eq uir e a m in im u m n um b er of t her a peu tic d oses. B y p r od ucin g vir a l a nt igen s sp ecif ically at the site of the desired therapeutic action, the patient'simmune s y stem may be e lic ite d to e nhan ce th e t arg et c ell k illi ng o f th ec yt oly ti c r e p lic a tin g a d en o vi r u s ( 36 ).R ecen tly , B isch of f et a!. (1 2) co nstru cted an ad en ov iru s w ith amu tate d EJB gen e th at re pl ic ate s in and s ele ctiv e ly d es tro y s c an ce r



6/6

ATFENUATED ADENOVIRUSFOR PROSTATE CANCERHousman , D . E ., a nd J acks , T . p 53 s ta tu s and t he e ff ic acy o f c an cer t he rapy i n v iv o.Sc ience (Wash ing ton DC) , 266: 807810,994 .23. G leav e, M . E ., H sieh , J -T ., W u , H . C ., v on E sh en bach , A . C ., an d C hu ng, L . W . K .Serum prostate-specific ant igen levels in mice bearing human prostate LNCaP tumorsar e d ete rm in ed b y tu mo r v olu me a nd en do crin e a nd g ro wth f ac to rs . C an ce r R es ., 5 2:15981605,992.2 4. G r o dz ic k er ,T . , L e w is , J . B ., a n d A n d e rs on , C . W . C o nd it io n al le th a l m u t a n ts o fadenovi ru s t yp e 2 -s im i an v ir us 40 hyb ri ds . U . Ad2+ND! hos t- rang e mu tant s t ha tsynthes ize f ragmen ts o f the theAd2+ND1 30K protein .J . V i ro l ., 19 : 55957!,1976.2 5. B re id in g. D . E ., E db au er , C . A ., T on g, J . Y ., B yr d, P ., G ra nd , R . J . A ., G af fimo re ,P . H . , a n d W il li am s , J . !s ola ti on a n d c ha ra ct er iz a tio no f a d en o vir u s t yp e 1 2 E lhost -range mutan ts defec tive fo r g rowth in nont ransfo rmed human cell s. V i ro logy ,164: 390402,1988 .2 6. H i ts , M . M . , a n dG r a h am ,F . L A d e n ov ir u sE 1 A u n d er t h e c on t ro l of h et er o lo go u sp ro m ote rs : w id e v ar ia ti on i n E 1A e xp re ss io n l ev el s h as l it tl e e ff ec t o n v ir us r ep li cat ion . V irology , 179: 667678,990.2 7. T el li ng , G . C ., P er er a, S ., S za tk ow sk i-Oz er s, M ., a nd W ill iam s, J . A bs en ce o f a ne ss en tial re gu la to ry in flu en ce o f th e ad en ov in is E 1B l9 -k ilo dalto n p ro tein o n v ir algrow th and early gene exp ression in hum an diploid W !38. H eL a, and A 549 cells.J . V irol ., 68: 541547,994.2 8. H u i, M . B . V . , L ie n, E . J ., a nd T r ou sd ale , M . D . I nh ib it io n o fh u m an a den ov ir u se s b yl-(2'-hydroxy-5'-methoxybenzylidene)amino-3-hydroxyguanidine tosylate. AntiviralRca. ,24: 261273,994.29. J on e s, N . , and Sh ank, T . An adenovi ru s t yp e 5 ea rl y g en e fun ct io n r egul at es exp re ss io n o f o th er e ar ly v ir al g en es . P ro c. N at l. A c ed . S ci. U SA , 7 6: 3 66 5 3 66 9, 19 79 .30. Mineta,1., Rabkin,S. D.,Tazaki,1., Hunter,W. D.,and Martuza,R. L Attenuatedmu lt i-mut at ed h erpe s s imp lex v ir us -! f or t he t re atmen t o f ma li gn ant g li omas. Na t.M ed.,1: 938943,995.3 1. E ll io tt@W . L , R o b er t s, B . I ., H ow ar d ,C . T . , a n d L e op o ld , W . R . 1 . C h em o t h er a pywith [SP-43-(R)J-[l,l-cyclobutanedicarboxylato(2)](2-methyl-1,4-butanediamineN,N ')platin umC!-973,NK 121)ncom binationithstandardgentsagainstmu r in et umors in v ivo .Cancer Res ., 54 : 44124418 ,1994.32 . Ghaz i zadeh ,M . ,K agawa ,S ., M aebayash i,K . , I z um i ,K . , and Ku rokawa ,K . Prosta t icoriginof metastases:immunoperoxidaseocalizationof prostate-specificntigen.Uro l. h i t ., 39 : 912 , 984 .33. A n dr iole, G . L ., a nd C at alo na, W . J ., M . D . T h e d iagn osis an d t reat m en t of p rost at ecancer.Annu.Rev. Me d.,42: 915,991.3 4. P a rt in ,A . W . , Y o o, I ., C a r t er ,H . B . , P ea r so n ,J .D . , C h a n ,D. W . , E p s t ei n ,J . L . , a n d

Walsh , P . C. The use o f p ros ta te -speci fi c an tigen . c l in i ca l s tage and Gleason score topredic tpathologicals tage in men with localizedprosta tecancer.J . Urol ., 150:110114,993.35. W o rgall, S ., W o lf f , G ., Falck -P en der sen , E ., an d C ry st al, R . G . I nn at e im m u nem ec ha ni sm s d om in at e e li mi na tio n o f a de no vi ra l v ec to rs f ol lo wi ng i n v iv o a dm in istration.Hum.GeneTher.,8: 3744,997.3 6. Y a n g, Y . , L i , Q . , E r t l ,J ., a n dW i ls on , 3 .M .C e l lu l ar imm un i ty t o v i r al a n t ig en s lim i tsE l-de le ted ad enov ir us es f or g en e t he rapy. P ro c. Na il . Aced . Sc i. USA , 91: 4 407 4411,1994.37. M a ck , C . A ., S on g, W - R ., C ar pen ter , H . , W i ck h am , T . J ., K ov esd i, I ., H ar vey , B -G .,M agovern, C . J., bo rn, 0 . W ., R osengart, T ., F aick-P ed erso n, E ., H ack ett, N . R .,Crys ta l, R . G . , an d Mast rang e i, A . C ir cumvent io n o f ant i- ad enovi ru s n eu tr al iz in gi mm un it y b y a dm in is tr ati on o f a n a de no vi ra l v ec to r o f o n a lt er na te s er ot yp e. H um anGene The r . , 8 : 99109, 997.38 . V i l qu in ,J - T . ,Gure t t e ,. ,K ino sh i t a ,I . , Roy , B . , Gou l e t,M . ,G rav e l,C ., Roy , R . , andT rem b lay ,J .P . FK506 !m mu nosuppre ss ionocon t ro lt h e im mu ne reac t ion s t r iggere dby f i r st - ge ne rat i onadenov iru s -m ed ia t e dgene t ran s f er .Hum . Gene The r . , 6 : 13911401,1995.39. E n gelh ar dt ,J . F ., Y e , X ., D or an z, B ., an d W i lson , J . M . A b lat ion of E 2A inrecombinant adenov iruses improves t ransgene pers is tence and decreases in fl ammatory respon se m ouse liver. Proc. N atI. A ced. S ci. U SA , 91: 6196-6200, 1994.40 . Y ang , Y ., X iang, Z ., E rtl, H . C. J., an d W ilson, J. M . U pregulatio n of class I m ajorhis tocompat ib i i tycomp lex ant igensby inter fe ronys ne c es sa ry fo r T c e ll -med ia t edel imination of recombinantadenovirus-infectedhepatocytes in vivo.Proc. Nail .A ced.Sc i. USA, 92 : 72577261 ,995 .4 1. D ah iy a, R . , D e n g, G . , C h e n , K . M . , C h u i , R . M . , H a u gh n ey ,P . C . , a n d N a r ay an ,P .p53 tumor -suppressor gene muta ti ons a re mainl y l oca li sed on exon 7 in humanp r im a ry a nd m e ta st at ic c an cer . B r . J . C an cer , 7 4: 2 64 -2 68 , 1 99 6.42 . Brook s , J .D ., Bova ,G . S . ,E w ing ,C .M . ,P ian tados i,S . ,Car t e r ,B . S . ,R ob in son ,J .C . ,E p s t ei n ,J . I . , a n d I s aa cs ,W . B . A n u n c er t ai nr o le f o r p 5 3 g en e a lt e ra t io n si n h umanprosta tecancers .CancerR es ., 56: 38143822,996.4 3. D e gu c h i, T . , D o i, T . , E h a r a,H ., I t o, S - i. , T a k a h a sh i ,Y . , N i sh i n o, Y . , F u j ih i r o, S .,Kawamur a, T ., K omed a, H ., H on e, M ., K aj i, H ., S himo kawa , K ., T an ak a, T ., a ndKawada, Y . De te ct io n o f m i cr ome ta st at ic p ro st at e c ancer c el ls i n l ymph node s byreverse transcriptase-polymerase chain reaction. Cancer Res., 53: 53505354,1993.4 4. S mith , R . R ., H ue bn er, R . 3 ., R o we, W . P ., S ch atte n, W . E ., an d T ho ma s, L B . S tu dieson theuseof virusesn thetreatm entf carcinomaf thecervix .CancerPhila.),9:12111218 ,956 .

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