[CANCER RESEARCH59,4200–4203,September1,1999]
Advances in Brief
The Addition of Adenovirus Type5Region E3Enables Calydon Virus787to Eliminate Distant Prostate Tumor Xenografts
De-Chao Yu,Yu Chen,Monica Seng,Jeanette Dilley,and Daniel R.Henderson1
烤包子
Calydon,Inc.,Sunnyvale,California94089
Abstract
CV787,a novel highly prostate-specific replication-competent adenovi-rus with improved efficacy,was constructed.CV787contains the prostate-specific rat probasin promoter,driving the adenovirus type5(Ad5)E1A gene,and the human prostate-specific enhancer/promoter,driving the E1B gene.To improve efficacy,we constructed CV787such that it also contains the entire Ad5E3region.CV787replicates in prostate-specific antigen(PSA)؉cells as well as wild-type adenovirus,but in PSA؊cells, CV787replicates104–105times less efficiently.CV787destroys PSA؉prostate cancer cells10,000times more efficiently than PSA؊cells.Incor-poration of the Ad5E3regi
on significantly improves the target cell killing ability or efficacy of CV787.In nu/nu mice LNCaP xe-nografts,a single i.v.tail vein injection of CV787eliminates300-mm3 tumors within4weeks.CV787could be a powerful therapeutic for human metastatic prostate cancer.
Introduction
Prostatic cancer is the cond leading cau of cancer-related deaths in men in the United States(39,000deaths in1998;Ref.1).Current treatment for metastatic prostate cancer is androgen ablation therapy, which,in70%of men,provides relief from otherwi uncontrollable bone pain and increas life expectancy by6–18months.Clearly,a new approach to metastatic prostate cancer treatment is needed(2–4). We previously constructed a prostate-specific ARCA,2CN706,in which the Ad5E1A gene is driven by PSE(5,6)706destroys human PSAϩcells400times more efficiently than PSAϪcells and eliminates LNCaP xenografts in nu/nu mice with a injection. To improve the specificity of CN706,we placed both the Ad5E1A and E1B genes under the control of prostate-specific transcriptional regulatory elements.CV764contains the PSE,driving the Ad5E1A gene,and the promoter/enhancer of a cond human prostate-specific gene,the hKLK2gene,driving the Ad5E1B gene.CV764destroys PSAϩcells10,000times more efficiently than PSAϪcells and cannot productively infect PSAϪcells(7).However,CN706and CV764 could not eliminate distant preexistent L
NCaP xenograft tumors in nu/nu mice by i.v.tail vein administration(data not shown).
To improve efficacy by systemic i.v.administration,we,led by preliminary evidence(data not shown),restored the Ad5E3region (nucleotides28133–30818).The E3region had been deleted in both CN706and CV764(6–8).The E3region has long been considered unnecessary for replication of adenovirus in vitro and has been uni-versally deleted from Ad5gene therapy constructs until recent efforts to reduce the immune respon to the vector(9–14).The Ad5E3region encodes proteins that play a role in assisting virus relea and evading or slowing host immune respons to the virus(15–25). CV764did not have the genome space required to include the entire E3region,but we wished to retain the specificity of using two independent prostate-specific transcriptional regulatory elements driv-ing the Ad5E1A and E1B genes.Thus,we constructed CV787using the rat probasin prostate-specific promoter(26–28),driving the E1A gene,and the PSE,driving the E1B gene,and retained the entire Ad5 E3region.The CV787genome length is105%the length of wt Ad5, yet the virus replicates well and is completely stable.CV787is an ARCA that replicates like wt Ad5in cells that express PSA but is attenuated10,000–100,000times,with respect to replication in PSAϪcells.CV787is highly cytopathogenic in PSAϩcells and is capable of eliminating distantly located preexisting prostate tumors following i.v. injection.
Materials and Methods
Virus Constructions.CV787was constructed by inrtion of the rat pro-basin promoter and the human PSE,driving the Ad5E1A and E1B genes, respectively.pXC1,pBHG10,and pBHGE3were purchad from Microbix Biosystems(Ontario,Canada;Refs.8and29).pXC1contains the Ad5bp 22–5790,including the inverted terminal repeat,the packaging quence,and the E1A and E1B genes inrted into pBR322(29).pBHGE3contains the entire Ad5genome,except for a deletion between Ad5bp188and1339, inrted into pBR322(8).pBHG10is similar to pBHGE3,except that it is E3 deleted(⌬Ad5,nucleotides28133–30818).pXC1was modified to contain an Age I site at bp547between the E1A mRNA cap site and the E1A translation initiation site by inrting a T between Ad5bp551and552,yielding CP95(6). CP95was modified to create an Eag I site at Ad5bp1681between the E1B promoter and the E1B mRNA cap site.The Eag I site was created by inrting a G between Ad5bp1681and1682into CP95using overlap PCR with the following two ts of primers.The first t[primer i,5Ј-TCGTCTTCAA-GAATTCTC-3Ј;and primer ii,5Ј-GCCCA CGGCCG CATTATATAC-3Ј; Eag I site is italicized),amplifies a2090-bp fragment in CP95,and the cond t(primer iii,5Ј-GTATATAATG CGGCCG TGGGC-3Ј;and primer iv,5Ј-CCAGAAAATCCAGCAGGTACC-3Ј),amplifies a399-bp fragment from the same plasmid.The two P
CR products were annealed in equal molar ratios and ud as template for PCR with primers i and iv.The2468-bp overlap product was digested with Eco RI and Kpn I and ligated to similarly cut CP95,creating CP124.CP125was constructed by cloning the PSA5Ј-flanking quence, containing the enhancer domain fromϪ5322toϪ3875from the transcription start site and the promoter fromϪ230toϩ7,into the Eag I site of CP124(6). CP257was constructed by cloning the rat probasin promoter(positionsϪ426 toϩ28;Ref.26)into the Age I site of CP125.The rat probasin promoter was amplified by PCR from rat genomic DNA(Clontech,Palo Alto,CA)with primers(5Ј-GATC ACCGGT AAGCTTCCACAAGTGCATTTAGCC-3Јand 5Ј-GATC ACCGGT CTGTAGGTATCTGGACCTCACTG-3Ј)containing Age I sites(italicized).The PCR product was digested with Pin AI(an isoschizomer of Age I)and cloned into a similarly cut CP125,creating CP257.CV739, CV787,and CV802were generated by homologous recombinations of CP257 and pBHG10,CP257and pBHGE3,and pXC1and pBHGE3,respectively(8, 10,30).Fig.1shows the structures of CN702,CN706,CV739,CV787,and CV802.Virus were grown and purified as described previously(6,7).The particle:pfu ratio of all virus was20:1.
Received4/21/99;accepted7/20/99.
The costs of publication of this article were defrayed in part by the payment of page charges.This arti
cle must therefore be hereby marked advertiment in accordance with 18U.S.C.Section1734solely to indicate this fact.
1To whom requests for reprints should be addresd,at Calydon Inc.,1324Chesa-peake Terrace,Sunnyvale,CA94089.Phone:(408)734-0733;Fax:(408)734-2808; E-mail:
2The abbreviations ud are:ARCA,attenuated replication-competent adenovirus; Ad5,adenovirus type5;P,virus particle;PSA,prostate-specific antigen;PSE,PSA promoter and ,intratumoral(ly);wt,wild-type;pfu,plaque-forming unit(s); MTT,3-[4,5-dimethylthiazole-2–4]-2,5-diphenyl-2H-tetrazolium bromide.
Cell Culture.Cells were obtained and maintained as described previously (6,7).Plaque assays were determined as described previously (6,7).Burst sizes of CN706,CV739,CV787,and CV802(wt Ad5)in PSA ϩand PSA Ϫcells were determined as follows.Cell monolayers (2ϫ105cells/well)in six-well plates were inoculated with 4ϫ105pfu of CN706,CV739,CV787,or CV802(wt Ad5;Refs.7and 8).Virus yields were titrated at 48h after infection.Cells were infected in duplicate;assays were carried out in triplicate.One-step growth curves of CV787and CV802(wt Ad5)were performed in
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human microvascular endothelial cell (hMVEC)monolayers.Monolayers of hMVEC cells were infected at a multiplicity of 2pfu/cell with either CV787or CV802(wt Ad5).At the indicated times thereafter,duplicate cell samples were harvested and lyd by three cycles of freeze-thawing,and the virus in the supernatants was assayed in triplicate in 293cell monolayers.One-step growth curves of CV787in LNCaP cells using medium containing charcoal-stripped rum with or without R1881(methyltrienolone)were performed similarly.Cytopathogenity of CV787and CV802(wt Ad5)was also determined in hMVEC monolayers,as described previously (7)钻木取火
Cell survival of PSA ϩand PSA Ϫcells infected with either CV787or CV802(wt Ad5)was determined.Monolayers of LNCaP,HBL-100,and OVCAR-3cells were infected at a multiplicity of 1pfu/cell with either CV787or CV802(Ad5).Cell survival (viability)comparing the effect of the E3region with CV739and CV787in LNCaP cells was assd by measuring mito-chondrial activity using MTT (17).
In Vivo Animal Experiments.LNCaP xenograft tumors in nu /nu were induced in 6–7-week-old BALB/c nu /nu mice and allowed to grow to an average volume of 300mm 3(6).Fifty l of CV787in PBS-10%glycerol or PBS-10%glycerol alone were injected into the tail vein.Tumor size was measured weekly,as described previously (6).Serum samples were collected by tail vein incision.PS
A levels were measured using an immunoassay kit (Genzyme Diagnostics,San Carlos,CA).
Results and Discussion
Specificity of CV787.The target cell specificity of CV787,rela-tive to that of wt Ad5,was tested in Ad5E1A ϩand E1B ϩ293cells,PSA ϩhuman LNCaP prostate cells,and a panel of PSA Ϫcells,including human breast epithelial HBL-100cells,ovarian cancer OVCAR-3cells,and PA-1cells.Fig.2shows the 48-h burst sizes of CN706,CV739,CV787,and CV802(wt Ad5).The burst size of CV802(wt Ad5)in the cells ranged from 3ϫ103to 2ϫ104,
and
Fig.1.Structure of virus706contains the PSE,driving the Ad5E1A gene,but is Ad5E3region deleted;CV739contains the rat probasin promoter,driving the Ad5E1A gene,and the PSE,driving the Ad5E1B gene,but is Ad5E3region deleted;CV787is identical to CV739but contains the Ad5E3region;and CV802is a constructed wt Ad5containing a normal Ad5E1region and a normal Ad5E3
region.
Fig.2.Burst sizes of CN706,CV739,CV787,and CV802(wt Ad5)in PSA ϩand PSA Ϫcells.Cells were infected at a multiplicity of infection of 2,virus was harvested 48h after infection,and virus yield was determined by plaque assay on 293
cells.
Fig.3.Growth curves of CV787and CV802(wt Ad5)in hMVEC cells.Cells were infected at a multiplicity of infection of 2,virus was harvested at indicated times after infection,and virus yield was determined by plaque assay on 293
cells.
Fig.4.Plaque morphology of CV739and CV787.293cells were infected with CV739and CV787.After a 4-h adsorption period,plates were overlayed with agar and incubated for 10days.After 10days,the agar overlay was removed,and the cells were stained with crystal
violet.
Fig.5.Comparison of CV739and CV787on mitochondrial activity of infected LNCaP
cells.Cells were infected with CV739and CV787at a multiplicity of infection of 1,and mitochondrial activity was measured by the MTT assay at the indicated time intervals.
the burst size of CN706ranged from 3ϫ103to 1ϫ104in 293cells and the PSA ϩcell line LNCaP;however,in all PSA Ϫcell lines,the burst size ranged from 1ϫ101to 8ϫ101.The burst sizes of CV739and CV787were ϳ104in 293cells and the PSA ϩcell line LNCaP and were less than 1to 1ϫ10Ϫ2in all PSA Ϫcell lines.Failure of CV787to reach a burst size of 1by 48h following infection with a multi-plicity of 2indicates an esntially complete inability to replicate in cells lacking transcription factors that are capable of interacting with PSE.CV787is capable of replicating but to an extremely limited extent in PSA Ϫcells,as shown by its growth curve in hMVEC cells (Fig.3).In the cells,CV787replicated 10–20-fold during 4days following infection with a multiplicity of 2pfu/cell,but in which the burst size,in agreement with the results prented above,failed to reach 1.In contrast,CV802(wt Ad5)replicated 106-fold and reached a burst size of 104.
Specificity of CV787was also shown by cell survival of PSA ϪHBL-100and OVCAR-3cells,as measur
ed by the MTT assay,compared with the destruction of PSA ϩLNCaP cells.Whereas CV802destroyed all three cell types,CV787only destroyed PSA ϩLNCaP cells (data not shown).Specific cell destruction was also measured in hMVEC monolayers infected for 10days with multiplic-ities ranging from 0.01to 10.Data similar to tho previously pub-lished for CV764(7)show that,even at an infecting multiplicity of 0.01,CV802(wt Ad5)caud very substantial cytopathic effects;in contrast,CV787caud minimal cytopathic effects in 10days,even at an infecting multiplicity of 10(data not shown).Together,the
results confirm the conclusion that CV787is almost totally attenuated for PSA Ϫcells but replicates normally in PSA ϩcells.
Incread Efficacy of CV787Due to the Ad5E3region.The incread efficacy resulting from the incorporation of the Ad5E3region was first shown by in vitro tests.A reprentative plaque assay of CV739(E3Ϫ)and CV787(E3ϩ)on 293cells is shown in Fig.4.Plaques of E3-deleted Ad5are substantially smaller and less distinct than tho of E3-containing adenovirus 10days after infection.Plaques of CV787appear more than 3-fold larger than plaques of CV739.E3ϪCN702and E3ϪCN706produced plaques similar to tho shown for E3ϪCV739,whereas E3ϩCV802produced plaques similar to tho shown for E3ϩCV787(data not shown).A similar picture of efficacy emerged
when LNCaP cell survival was assd by measuring mitochondrial activity (17).CV787completely eliminated mitochondrial activity 7days after infection,whereas CV739cells still had 67%the mitochondrial activity of uninfected cells 7days after infection (Fig.5).
The incread efficacy of incorporation of the Ad5E3region was also shown in vivo ,as measured in the LNCaP nu/nu mou xenograft model.BALB/c nu /nu mice 300-mm 3LNCaP xe-nografts located on the back were injected i.v.into their tail veins with either vehicle (50l of PBS-10%glycerol)or the same volume of vehicle containing CN706,CV739,or CV787.Fig.6shows the result of a atment of LNCaP tumors with 1ϫ108particles/mm 3tumor CN706or 1ϫ106particles/mm 3tumor CV787.Both virus yielded the same degree of tumor reduction,implying a 100-fold increa in efficacy of CV787,compared with CN706.In addition,1ϫ108particles/mm 3tumor CV787completely eliminated LNCaP tumors by a injection (data not shown).
Differential
Fig. 6.Comparison of injection activity toward LNCaP xenografts.nu /nu mice LNCaP tumors (average size,300mm 3)were injected once into the tumor with 1ϫ108particles/mm 3tumor of CN706or 1ϫ106particles/mm 3tumor of
CV787.
Fig.7.Comparison of CV739and CV787i.v.injection activity toward LNCaP xenografts.nu /nu mice LNCaP tumor xenografts (average size,300mm 3)were injected once into the tail vein with 5ϫ1010particles of CV739or
CV787.
真好的拼音Fig.8.LNCaP xenograft tumors in nu /nu mice following one i.v.tail vein injection of CV787.A ,tumor si
ze,measured weekly.B ,rum PSA,measured weekly by immuno-assay.C ,replication of adenovirus in LNCaP tumors determined by immunohistochem-istry with rabbit polycolonal antibody to
Ad5.
Fig.9.Growth of CV787in LNCaP cells with or without R1881.LNCaP cells were infected with CV787at a multiplicity of infection of 2.Following adsorption,the cells were washed twice with PBS,and medium with or without R1881was added.Duplicate cultures were harvested at the indicated time points and subjected to three cycles of freeze-thaw,and virus titers were determined in triplicate on 293cells.
efficacy toward LNCaP tumors between CV739and CV787was also shown following a single i.v.administration of5ϫ1010particles.Fig. 7shows that CV739could stop tumor growth at this do level but that CV787caud a4-fold reduction in tumor volume.
玫瑰籽油i.v.Administration of CV787.Perhaps most importantly,CV787 could eliminate preexistent distantly located LNCaP tumors.Six weeks following a single i.v.injection of1ϫ1011CV787particles, the sizes of the tumors were reduced to less than5%of their original size(Fig.8A),and8of14mice were visually free of tumors.Tho tumors that were still prent were immunohistologically devoid of PSA(data not shown).The rum PSA levels in mice injected with buffer incread(Fig.8B),whereas the levels in mice injected with CV787decread toϳ5%of their starting values within3weeks. Virus replication w
ithin LNCaP xenografts could be shown at both7 and28days after injection,as evidenced by Ad5immunostaining (Fig.8C).As controls,similar experiments were carried out with nu/nu mice carrying LoVo(colon cancer)or Hep3B(hepatoma) xenografts.The xenografts continued to grow normally following tail vein injection of CV787(data not shown).
Finally,hormone-refractory patients continue hormone therapy, even if such hormone ablation therapy is no longer effective(2,3).If CV787is to be considered to treat end-stage metastatic hormone-refractory prostate cancer,it is important to know whether CV787can grow in the abnce of testosterone.Fig.9shows the one-step growth curve of CV787in LNCaP cells in the prence of the stable but biologically active testosterone analogue R1881at1n M and in the abnce of R1881.CV787grows normally in the abnce of R1881 and achieved a burst size in excess of104pfu/cell.
In summary,CV787is an ARCA that replicates like wt Ad5in cells that express PSA but that is attenuated10,000–100,000-fold with respect to replication in PSAϪcells.CV787is highly cytopathogenic in PSAϩcells and is capable of eliminating distantly located preex-isting prostate tumors following i.v.injection.The addition of the Ad5 E3region increas efficacy10–100-fold both in vitro and in vivo. Furthermore,CV787with the complete E3region can be expected to resist many of t
he host antiviral MHC-1defen mechanisms of wt Ad5(15–17,25).Finally,CV787can replicate normally in the ab-nce of testosterone and could,thereby,help patients continuing hormonal ablation therapy.CV787is a potentially powerful therapeu-tic for human metastatic cancer.
Acknowledgments
We thank Young Kim,Heather Conners,and Andy Little for technical assistance.We also thank Dr.W.K.Joklik for suggestions in preparing and editing the manuscript.
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