{Cell Adhesion &' iViigrationZ2:3, 208213; lnlylAugust/Septernber 2008];
`
`@2008 Landes Bioscience
`
`Speciol Focus: Gliomo Therapy
`
`”hissed” ssrslytir herpes simpler viruses let hrsis raiser therapy
`
`Translational Research Advonceirtem Center and Department of Neurosurgery, The University oi Tokyo; Tokyo lopon
`
`Abbreviations: HSV-l, herpes simplex virus typ ‘ l; FKR, double~stranded RNA~dependent protein ltinase; lCPé, infectedw‘ll protein 6;
`phi, plaque—forming units; EL, interleukin; Chi-(38E grantilocyte macrophage colony'stirnulating factor; GAlNltiis. gibbon ape leukemia
`virus en
`velope iiisogenie nietnhrrane glycoprotein; yCD, yeast cytosine deaniinase; TNFtX, tumor necrosis factor alpha
`
`Key words: oncolytic virus therapy, gene therapy, herpes simplex virus, viral vectors, GINA, G207, antiturnor immunity
`
`Genetically engineered, conditionally replicating; herpes simplex
`viruses type I (HSV~ l) are promising therapeutic agents for brain
`tumors and other solid cancers. They can replicate in situ, spread
`and exhibit oncolytic activity via a direct cytocidal eliect. One of
`the advantages of HSVLI is the capacity to incorporate large and/
`or multiple transgeries within the viral genome. Oncolytic HSVvl
`can therefore be “armed” to add certain functions. Recently, the
`field of armed oncolytic HSV~1 has drastically advanced, due to
`development of recombinant l'l SVui generation systems that utilize
`hacterial artificial chromosome and multiple DNA reconihinases.
`Because antitumor immunity is induced in the course of oneolytic
`activities of HSVrl,
`transgenes flflCOdlilg immunornodnlatory
`molecules have been most frequently used, for arming. Other
`arrned oncolytic HSVvl include those that express antiangiogenic
`factors. fusogenic memhrane glyeoproteins. suicide gene products,
`and proapoptotic proteins. Provided that the transgene product
`does not interfere with viral replication, such arming of oncolytic
`HSV~l results in augmentation of antitunior efficacy. lnimediate~
`early viral promoters are often used to control
`the arming
`transgenes, hut strict-late viral promoters have been shown useful
`to restrict the expression in the late stage of viral replication when
`desirable. Seine artned oncolytic HSV~l have been created for the
`purpose of noninvasive in vivo imaging of viral infection and repli-
`cation. Development of a wide variety of armed oncolytic HSV~1
`will lead to an establishment of a new genre of therapy for brain
`tumors as well as other cancers.
`
`introduction
`
`Oi colytic virus therapy is an attractive and rapidly developing
`means for treating cancer.1 Genetically engineered viruses, such as
`herpes simplex virus typel (HS\’- 1)- and adenovirtas are designed
`so that virus replication is restricted to turnor cells and tlieretore
`iniection causes no harm to normal tissues . in principle, infected
`
`
`
`tumor CCellsa €Cl€5l
`IOVéCl l')V a (lll‘CCt O‘HCO'Vth activity Ol lllé Vll’lJ.S€S.
`ltnportantly, oncolytic viruses can als0 act as vectors that provide
`amplified transgene deliveiy
`HSV-l, especially in comparison with 2ulenovirus, has suitable
`features for cancer therapy: (1) HSVrlin lects most tumor cell types.
`‘2)
`i
`A relatively low multiplicity old infection (is needed for total
`cell filling. b) Antti—viral drugs are avaiiable
`)A large genome
`[.413.. kh) allows the insertion of large and/or multiple trans»
`13
`
`(5) The host immune reactions enhance antituinor eiiects.
`genes,
`(6) Circnl
`aing anti—H5‘v l antibodies do not ai’Tect
`cell-—to2—cell
`sensitive mouse and
`There are l‘ls‘vrl
`spread of the virus.
`7
`nonhtirnan primate rnodels Toor preclinical evaluation. (8‘) Viral DNA
`is not integrated.into the host genome. l'l SV—lis neut’otropic and the
`genes necessary for neuropathogenicity have been identified and can
`he mutated. Therefore, the use of HSV—~l
`is especially advantageous
`
`for brain tumor therapy.
`in order to target l-lSV—l
`replication to turnor cells, viral genes
`th t are essential t”or viral replication in normal cells iout dispensable
`in :i-tnior cells are inactivated or deleted 2 lhis principle usesteatures
`cotnrnort fora ll tytpS (if cancer, therefore the application oioncolytic
`HS"~l
`is not restricted to brain tumors, but also rtcludes a Wide
`
`variety old cancer. The key for successful and practical development
`of oncolytic l-iSV—l
`is to achieve a wide therapeutic window by the
`use of genetic engineering technology.
`
`§etonduGeneretion Gnrolpit H§V~l
`
`£32.07 was the first “oncolytic l-iSV—l used in a clinical trial in the
`United S1ates. 3 This second-generation oi colyttic HSV- i has double
`mutations createdin the l-lS"—l
`genome.4 G207 has deletionsin
`the tnaior determinant of HSV—-l
`hoth copies oi teh “#45 ene,
`u
`vectors are considerably
`llSV—‘i
`neurovirulence.S “[34.5—deticient
`attenuated
`in normal cells but retain their ability to replicate within
`neoplastic cells. in norrnal cells, l-lSV—l intection induces activation
`oi double— stranded RNA—dependent protein kittase (PKR), which
`in turn leads to phosphorylation of the tX—subunit of eul<aryoic
`initiation actor 2 and a subsequent shutdown of host and viral
`protein synthesis "’ lhe product of the 754.5 gene antagonizes this
`PKR activity. However 1nmor cellsnav3 low PKR aCtivities, thereby
`—deiicient ilSVl veto replicate/7’8 (3207 also
`allowing V345
`has an1insertion of the E mlz' ZarZ gene in the intectei—c
`ell protein 6
`
`208
`
`Cell Adhesion 81 lx/ligration
`
`20C8; Voi.2 issue 3
`
`

`

`Armed oncolytic l-lS\7»-‘i for brain tumor therapy
`
`)\
`(7C:.776) coding region (Ulj‘),1, inactivating ribonucleotide reductase,
`a ltey enzyme For viral DNA
`synthesis in non—dividing cells but not
`9
`in dividing cell.’
`1preclinical
`the
`studies using imrnunocompetent animals,
`with G207 1a.
`most remarkable hncling
`that11 induced systemic
`antitutnor immunity in the crouse of <1nto1v11L activ1'ty‘O 11 For
`example, in All mice bearing bilateral subcutaneous N18 (syngeneic
`neuroblastoma) tumors, intraneoplaStic C1207 inoculation into the
`le1tttun11or alone caused growth reduction not only oltthe inoculated
`tumors but also of the non—inoculated contralateral
`The
`tumors.
`
`antitumor immunity was assoc-1ated wi h an
`elevated. cytotoxic T
`lymphocyte activity specilic to N1- 8 tumor cells that persisted for at
`least 15 months,
`
`After an extensive in vivo safety evaluation using HSV— l -
`susceptible mice and non—human primates, the G207 phase l clinical
`trial was performed between 1998 and 2.900 at two i111stitutior11s.5
`Twenty-one patients with recurrent malignant gliorna were treattel,
`and G207 was administered directly into the tumor‘ via stereotactic
`inoculat'tion l7'his dose escalation study started From 106 pla ue—
`forming units {phi} and increased to 3’1 X l09 pin, with three patients
`at each dose, As a result, no acute, moderate to severe adver‘se events
`
`attributable to (3207 were observed. Eight oF 20 patients that had
`serial lVlRl evaluations had a decrease in tumor volume between four"
`
`days and one month post<-inoculation. and two patients survived. for
`ears.
`tnore than live
`
`Third-:Gensrallen Gutaly‘llt HSVJ
`
`The nh:2se l clinical trial proved the salety o1{1207 and hi-tn ed
`its elficacy for human brain tumors. However, in order to iur‘ther
`
`improve the ellicacy without compromising its safety, :1 third— ener»
`ation oncolytic l-lS‘vCl termed (347/3 was newly created From G207
`g
`g
`by introdut:in another
`L e 1c alteration, letthe deletion of the
`1
`0117 gene and the overlapping USE I pr‘omoter region in the ($207
`genome.” BCCBU‘Sfii {1‘36 03/; £16116 pI‘EXlUCE inlli )llfi transporte ElSSOCl“
`ated with antigen presentation, which translocates peptides across
`the endoplasmic reticulum, the lownregulation oF MHC cl ss 1 that
`Ftera infection with l-lSV—l does not
`normally occurs in human cells
`occur when the 01/7 gene is deleted 3 {1477‘1-111LL1 human cells
`in tact presented higher levels of l‘x/ll-lC class 1 expreession than cells
`intec :ed with oth3r HSV-—llvectors 12 Further,hurnan melanotna cells
`infected with G47A were
`better at stimulatiing their matched tumor—
`t 1an those inFected with G207. The
`infiltrating lymphocytes111 vitro
`deletion also places the late USU gene under control of the imme—
`
`ion of the reduced
`diate-e rly (X47 promoter, which results in suppres
`growth phenotype of ’y345—deficient
`l-lSV—l mutants including
`
`'2ted better
`l'
`1
`1207 1‘ ln the ma ority of cell lines tested,
`(3471/51 reulic
`than G207, resulting in the generation or higher virus titers, and
`exhibiting greater cytopathic “llect. 12 ln athyrnic n11ixf3l)earing subcu—
`taneous‘71V(1human glioina and A/l mice bearing subcutaneous
`Neurolla neuroblastoma, lyl'/7A was signihcantly more LlFcac1otts
`than G207 at inhibiting the tumor growth when inoculated intraneo—
`plas11c2tl1y,12 (1117/51 was also more ellicta:ious than G267 in athymic
`mice bearing intracerebral 1118711413 tumors (lno Y et al., manuscript
`in preparation). Nevertheless, the salety oFGtiZ/fi rem:2in-ed unchanged
`A/l
`from G207 following iniection into the brain of l-lSV—l—sensitive
`
`trial 0F (rd/17.33111 recurrent glioblastom:2
`13.12 In japan, a clinical
`patients is underway (34713 has been shown efficacious in animal
`
`21'ariety of cancers inclu12
`tumor models ol':2
`cancer‘, breast cancer and neurofihromaa.
`
`ding brain tumors, prostate
`17.417
`
`Construction al”1lrmsrl” flatalytle l‘ESVMl
`
`Gr1e ol
`
`the advantages ol HSV l is the capacity to incorporate
`l rge and/or multiple transgenes within the viral genome. Lertain
`antitutnor functions may be added to oncolytic activities oll7 l—lSV—l
`The use oF replica ion-competent llSVl for transgene expression
`has multiple attractive advantages over replicationrinconlpetent or
`deFective llS‘7-: 1 vectors: ll) A continuous generation ofa birth—titer,
`loomogenous veLtor stocl<'is possible which allows manufacturing oF
`a la1ge amount with a better quality control. (2)- An amplinel gene
`delivery can he obtained111 v1-v0 And, (5) transgene expression may
`lower administering doses required, therefore decrease toxicity. On
`the other hand, potential demerits of using replicationrconipetent
`viruses For expressing Foreign proteins are that the transgene expresfl
`sion may increase the toxicity of the vector, and may also interfere
`with viral
`replication. 171ansgene expression by armed oncolytic
`H3V~l could be shorterrn durration than replication«incornpetent
`’ectors due to destruction of the host cell by viral replication
`However, we have observed that, because continuous viral spread
`and. infection occur within the tumor, a larger number of tumor
`cells consequently express the transgene,
`therefore a, much higher
`total amount of transgene product is achieved compared with non—
`replica ting vectors
`,arecombinant l'lSV-i t’a
`s constructed by conven-
`ln the past,a
`tional homologous recombination techniques that required selection
`of a correctly structured clone From millions oF candidates. lt often
`toolta:‘ few years until the intended l—lSV-l was obtained. ln order
`{0 Cll‘C‘llBV6llt Ellrl6 {11136'CO‘H. ‘U‘Glflg [3101368863,
`'WC l131V6
`developed
`1..
`an innovative a'tned"
`oncolytic l—lfiV—l constr uetion syst-ern using
`(7717/52 as the bacltbone ’8 liesi'desits Favorable Features for human
`
`LanLer thera-py, ii1’ludmc1 the salety, ligh yields of virus, improved
`oncolytic activity and potent stimulation of antitumor immune
`cells,
`(7147131 is especially suited as a replicrtriron—competent baeh~
`bone for expressing any Foreign protein molecules, because of the
`wide therapeutic window and preclusion of the sh utofl ol protein
`The system,
`termed lTLBAC
`synthesis in the infected host ceesll
`chromosome and two DNA
`svstem, utilizes bacterial
`ratii1
`recombinase sytents (Crlxello17’ and lllj’er’77'l {Fig.1 .lt allows
`(‘1) a construction of armed oncolytic HSV-l
`in a short period
`(usuallydull months), (2/) a simultaneous construciton of multiple
`he
`vectors,
`('5) an accurate ins
`retion ola desired transgene into
`le transgenes using the
`deleted ZCPGlocuss, (4) an insertion ormultip
`same elllort as inserting a single transgene, and (5) a direct conipar«
`l'lSVLl with hte same haclrb
`ison ofmultiple “armed” oncolytic
`done
`A similar system, termed l-lSv’Quil< system, has been also developec
`19,20
`using a G207~lil<e backbone,
`
`Gumlyfie H§Vul Armed with lmmurtasfimalareny Genes
`1‘
`Aside rron11 the extent of replication capability within the tumor‘,
`the Ltlitacy oF :2n oncolyti-c H517 1 depends on ltte extent o1 anti--
`wll‘1,‘1.rlll'l’lel‘lbfi
`tumor itinmunity inductior1e,while any transgene that
`loe not interFere with HSV—l replication rnay le used, the genes
`encoding immunomodulatory molecules would be reasonable candi—
`lm rnunostimulatory lunct ions
`dates for arming oncolytic HSV-l.
`that adds to
`should augment the antitumor immunity induction
`
`
`
` wwwlandesb extencmn
`
`Cell Adhesion 1’31 lx/ligrarion
`
`1.09
`
`

`

`Armed oncoiytic HSV»! for brain tumor therapy
`
`c\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\“\“\“\“\“\“\“\“\
`
`c\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\
`
`i\
`
`Recombination by Cre
`
`\\\\\\“““““““““““\\
`Eiectroporated into E. coli
`Selection with
`Cm and Ken
`
`a,,,,,,,,,,,,,,,,,””,””””””””/
`
`oxP
`
`FRT
`FP?
`
`2
`
`Co—transtection with
`FLP plasmid
`Excision of BAC
`
`
`
`to c1;2th ‘3207—iihe sccon-dgenera—
`The HSVQuih was usei
`
`ytic i‘iSV—i armed with murine iL—4
`tion oncoi
`CD40 ligand or
`
`6CK (Fig
`[10 in BALE/c tnice >earing 4T1 breast cancer in the
`brain, all of these armed HSV—i showed hette1 antitumor et11cacy
`
`than the controi virus. Using the HSVQuik system, we aiso created
`oncoiytic HSV-‘i armed with iL-iZ EL48 or soi-uhie B7- 1.19 Ni
`of these atmed HSV ] de monstr.. .d repiicative capa'oi'1iiities stmiilat
`to the parental Virus in vi1ro The in ViVO efficacy was tested in
`r‘o’j mice harboring subcutaneous tumors oi s1,nrreneic and pooriy
`imtnunogenic Neurolla neurohiastoma. iL-iZ was the most etiica
`cious among the immunostimuiatory moieeuies investigated when
`expressed by the L3207-iikei‘iHSV—i. The tripie eoirthinatiort of the
`three '2rn1ed Viruses exhibited the highest etiicacy amongsraali singie
`Viruses or combinations of two Vi ruses Com'ooining i 1; 105111711 each
`oi the three armed Viruses showed stronger antitumor activities than
`any single armed virus at .3 x 105 pin in inoculated tumors as wcii as
`non-inocuiated remote tumors.
`
`Using the NeuroZa subcutaneous tumor model, another research
`group demonstrated that the antitumor efficacy of MOOZ a first—
`ge11eratiot“
`{34. 5—deiicient
`iiSV—i
`that exptesses iL— 12, couid he
`augmentedw1hen usedin combination with lViOiO, the same backbone
`HSV—l
`that expresses chemokine CC.1 2“ The group I2iso demon-—
`strated that the Virus selected afterin Vivo serial
`:1sage of R4002in
`tumors of a i:)5/i—i\/iG human ntaiignant ghoma celi line improved
`survi'vai in two independent murine brain tumor models compared to
`the parent MOQZ.27 Ti is enhanced antitumor eiip cacy was not due to
`
`resroration of protein synthesis or e:2riy (.751 1' expression.
`Recently. using the T—BAC syst .m, we generated a G47’A2-hack»
`honeo ncolytic i'iSVi-i armed with. mouse iiision-type H.112, termed
`i7—mt7iL7iZ (Fig. 3}.
`in Al} mice hearing hiiaterai subcutaneous
`NeuroZa tumors,
`intrancopiastic inoeuiation with 7111111711312 into
`
`the icft tumor aiorte ied to a significantly better a
`unior activity
`than the unarmed :ontrol Virus, T—Ol, not only in the inocuiate i. ieit
`tumors hut aisoin the non~inoculated remote tumors (Miyamoto
`S, et ai.,manusc1ipt
`in prepa1atio11). We aiso c1eateda G47A—
`hachhone HSV— i a1111ed with both iL—i'b8a11d soiuhie B731.)8 iiiis
`
`douhic-arrned oneoiytic HSV—i showed a significant enhancement
`ot antitumor efficacy via T-ceii mediated immune responses in A/I
`mice with subcutaneous NeuroZa tumors avs11I ii as in C5781.[(1 mic
`
`hearingsubcutaneous [RAMP C2 prostate cancer
`A11 armed oncolyt1c HSV-l has not been tested in patients with
`'rain tumors, hIowever a phase i ciinicai trial with a second-genera:
`tion oncoiytic HS117-1 expressing GM(:81: was conducted in patients
`with cutaneous or subcutaneous deposits of breast, head and neck
`and gastrointestinai cancers and recurrent malignant irtcianoma.28
`OncoV/EXUV‘ ”'51“ has a deletion in the 014/
`"7 gene and the “(34.5
`promoter
`gene rcpiaced with the G.I—W (35F gene driven hy'221LJMV7
`(iig. 31A singie dose (13 patients) 01 muitipie doses (i7 patients),
`rmging trom it)6 to 108 pfu/irti/dose. were in; 1;ted intratumoraiiy.
`{ocai inflammation, erythema and febrile responses were the main
`side eifects. and the ioc22i reaction to injection was dose1imiti11g in
`HSV’?i.»seronegative patients at 107 pfu/mi. Some of biopsy speci»
`mens after treatment showed areas of necrosis that strongly stained
`for HSV—i Thr epatients had stahie disease, six patients shIowed
`flattened iniectcd and/or uninjeetcd tumors. and tour patients
`showed inflammation of uniniected tumors.
`
`Virus formation ‘
`
`rigure i. A scheme describing the T—BAC system tor constructing "ortned"
`oncolytic HSV—i with the (54718 hock'oone. The desired tronsgene ror ”orm-
`rig" is inserted11110 the multiplec! on-ing site oi the shuttle vector (SV—Ol}. The
`tirst step is to insert the entire sequence ott.he shuttie vector into the ioxP site
`of T—BAC by (1 1.remediated recombination toiioved by on eiectropoiotion
`
`into E. coli "the se-onci stuep is to cotronsect the co-integrote with ootiosnid
`efpressing. onto Vere ceiis to excise the SAC sequencertionked by the
`IR: 5112511121: objective armed ortcoiytliC HSV—'1 oppeor (is C. negative orid
`iochosi'ive virus pioques Non recombined viruses do not appear. due to
`the presence oi the iomhdo SYUYfer sequence iimd) causing on overs:ze oi
`the genome.18
`
`direct oncoiytic activityofthe Virus. resuiting in enhanced antitumot
`activities (Fig. 2) 2 3’3411.5 dciicie1t HSV—
`1 containing the murine
`i11terieui<i11 4 (FL—4) gene displayed 2.
`significantiy higher a11titu111or
`activity and proionged survival oi’ mice wi h intracranial
`tumors
`compared with its parentai Virus or the one expressing iL—iO.” First—
`generation oncolytic HSV7—i expressing 11-12 (MOO): and NViO/il)
`showed improvedin vivo cf11cacy against 4C8 giioma in syngeneic
`861.72.}. mice22 and''11air1 tumors otNeuroZa neuro'o'1lastoma'm syrige—
`11cic Ali]- 1n1ce,‘3‘22ndaiso against rnurine squamous ccii carcinoma“:
`and. mutine coiorectai
`tumor.2
`immunoh1sochemicai auaiyses of
`tumors treated with these iL— 2wexprcssing HEW-11 l reveaied a signiii~
`cant influx oi‘CDtii (3138* itesii
`and macrophages 1he oncolytic
`)42} we.5 more eriicaciot-1s than the
`HSV-J expressing iLt—lZ (NV/7H
`"oiony—stimuiating iactor
`one expressing granui:cyte rnacronhage
`in thesz
`(GM-CSF)
`>:la<l<)one (NV7015 4) in mice with subcuta—
`neous squamous ceiincarcinon11a.2Z“The rnice cured by NV'1042 had
`a higher r:2te of rejecting rechailenged tumor cells than those cured
`i1v7\ViiV’1/ 2‘
`
`2 l0
`
`Cell Adhesion (7'1 ivligrarion
`
`2068 V70.1.215911%
`
`

`

`Armed oncolytic l-lSVd for brain tumor therapy
`
`Armed Gnmly’tlr HSVJE with Other Autitumar Functions
`i-nw
`Various types o ' transgenes other than imrnunornodulatory genes
`rave been use 1. to arm oncolytic HSV-~] .Theoretically antiangiogenic
`fac ors can augment the antitumor activities of oncolytic HSV—l
`without compromising the viral replication and antitumor immunity
`induction. Early generation oncolytic HS‘ 11, such as (3207, was
`shown to retain the ability of wild type HS‘ ’«l
`to increase infeCted
`.
`.
`~
`t
`.
`w l-
`a
`
`tissue vascularity, whereas third—generation G47A showed suppressed
`vascularity in infected tiltfl<§f$.2) By using the (ta/A-BAL system,
`2
`preliminary version of the 'llBiL‘iC system, G47A—baclibone oncolytic
`H3V—~l armed with platelet Factor 4 or dominant: negative fibroblast
`«p.
`111, fr...’
`a
`f
`1:
`has
`. .
`rad 310731 B~.‘
`F i
`,
`a ‘
`growt i actor receptor rave
`een create .
`oth or [1656 armed
`oncolytic HSV—l we e more etiicacious in inhibiting the tumor
`
`growth and angiogenesis than the control virus in both human
`
`US7MG glioma and mouse
`3-l8-4 malignant peripheral nerve
`,
`sheath tumor models. By using the l-lSVQuil: system, an oncolytic
`Ht V—l armed with tissue inhibitor of rrtetalloproteinases 3, termed
`rQ143, has been created.32 in athymic mice bearing human neuro—
`blastoma or malignant peripheral nerve sheath tumor, treatment with
`rQTB) caused delayed tumor growth, increased peak levels of infec—
`tious virus, and immature collagen extracellular matrix. Remarkably
`rQ13 treatment caused reduced tumor vascular density, which was
`associated with reducec circulating enclothelial progenitors,
`Another approach for arming oncolytic l-lSV—l is the use of fuso—
`gemc membrane glycoproteins. Expression of fusogenic proteins by
`infected tumor cells could cause involvement of surrounding unin—
`fected cells to form syncytium and facilitate tumor cell ltilling, but
`might also increase toxicity in the normal tissue. Eu et al. constructed
`an oncolytic HSV—i armed with a truncated form of the gihbon ape
`leukemia virus envelope fusogenic membrane glycoprotein {Ci/iLl/f
`fm3.33 ln athymic mice bearing human Hep 3B hepatocellular carci--
`noma xenografts, the expression of Gflllffiu significantly enhanced
`the antitumor effect of the virus. Furthermore, by using a strict: late
`viral promoter instead of a Cit/{V promoter,
`(314129:fits glycoprotein
`could be expressel. only in tumor cells and not
`in normal non--
`dividing cells.
`So-called suicide genes have beet used from early stages of armed
`oncolytic l-iSV—l development. Expression of a suicide gene by an
`
`in ’ected tumor crll should el
`.it byStantler killing of surrounding
`tumor cells via extracellular diffusion of activated
`uninfecte
`
`prodrug, but premature killing of the host cell could also suppress
`viral replication.
`l-‘iSV—i naturally expresses thymidine kinase that
`activates the prodrug ganciclovir. However:
`a combination with
`systemic ganciclovir administration did not significantly enhance
`the efi’icacy of (3207 in Al] tnice with intracerebral 1 Ti 8 neuroblas-—
`toma.33 er450 was engineered by replacing the 1/452 gene within
`the [(1736 locus of the first—generation oncolytic HSV—l, hrRS,
`with the gene encoding rat cytochrome P450 2131 (C‘r’l’ZBl), a
`member of the cytochrome P450 iamily responsible for activating
`the prodrug cyclophosphamide.33 in rat 9L and human U87AEG1‘ R
`glioma models, systemic administration of both cyclophosph»
`amide and ganciclovir in combination with er430 showed the
`most efficacy compared with any other combinations.36 By using
`the l-iSVQuih system, an oncolytic l-lSV—l,
`termed MGHZ, was
`created that expressed both CYPZB'! and secreted human intestinal
`carborevlesterase.3‘7 The latter enzyme converts irinotecan into an
`
`Oncolytic HSV~1 armed
`with tL—‘t 2
`
`\&
`
`lot 2 secretion §
`
`Immune cells ®
`
`
`
`\‘\
`“\
`1 g 3
`\1\\\\\
`
`Viral replication + Antitumor immunity
`
`Figure 2. Concept oi ontiturnor etticocy augmentation using oncolytic t'lSV—l
`orrned with on immunostimulotor’y gene. When oncolytic HSV—l armed with
`the ii» .72 gene intects tumor cells,
`lL»l 2 is secreted in the course oi virol repli-
`cation and stimulates the immune cells. in addition to direct tumor cell killing
`vio viral replicotion and spread,
`tumor cells ore destroyed by augmented
`ontiturnor immune responses, resulting in enhanced dntitumor activities.
`
`in athymic mice bearing GliSGAEFGR glioma
`active metabolite.
`in the brain, l‘lel‘lZ displayed increased antitumor efficacy when
`combined with cyclophosphamide and irinotecan. lhe researchers
`found that, unlilte ganciclovir, cyclophosphamide, irinotecan or the
`combination of both did not significantly affect virus replication.
`Ht VlyCD was created bf replacing t e [C336 gene of HSV—l with
`the gene encoding yeast cytosine deaminase (yCID).38 yCD converts
`the prodrug 5<-fluorocytosine {5— PC) to a cytotoxic agent, S—fluorou»
`racil. This research group also observed that the approach enhanced
`
`cytotoxicity without significantly reducing viral
`replication and
`oncolysis. in BALE/c mice bearing subcutaneous tumors or diffuse
`liver metastases of MCZQ colon cancer, anti—neoplastic activity of
`HSVlyCD combined with systemic Si—FC administration was
`“we
`greater than HSVvaD alone. By utilizing the same backbone
`{1 xCM-Lor
`as OncoV
`(lig 33, an oncolytic HSV—l
`termed
`OncoVEXGALV/CD double—armed with yCD/uracil phospho—
`rihosyltransferase fusion and GALVlus has been created.39 in
`-
`v
`1
`~
`,
`.
`‘r
`r V
`“’
`Fischer i544 rats bearing subcutaneous 91, glioma, Onco‘t/EXCALV’
`CD proved most efficacious compares with the control viruses
`avatar
`"1.11
`t/
`,
`,
`,, p}
`.
`.
`( Onco‘J 12X, OncoV 1sz1333 or anthX‘rL) when combined wrth
`systemic S-FC administration.
`
`
`
` wwwlandesb encocum
`
`Cell Adhesion 81 li/ligrarion
`
`1‘) -.t
`
`

`

`Armed oncolyric l-lSVd for brain tumor therapy
`
`T-BAC-derived
`
`
`
`Figure'b Slruclutires or reprLseritolive ctrde oricolylic HSV—l The LiSV—l
`genome consists oflong oncl shorII uniq-re regions lUI oI'Ioi Usl each bounded
`by terminal (T) and Inernol (I) ereot regions -’RLand R5,. Armed oncolyric
`HSV—l created by using the T-BAC (or G475.BACl sysIem has the backbone
`structure or’347A, o third—generation oncolytic HSV—l.
`it has triple deletions
`n.he 734. 5, r‘CPéand d47genes. The Ironsgene is inserted into the deleIed
`lens locu3. As (1 marl:er, it ol3o expres3es Ihe LocZ gene driven by the iCPé
`promoter. Armed oncolylIc HSV—l created by using the HSVQuik systert
`hos the backbone structure similar to G207 or MGHl _, second-generation
`oncolyric HSV—l.
`it has double deletions in the 733.5 and 2076- gene5.
`The Ironsgene is inserted irIIo Ihe deleted I‘C‘Pé locus. As o. marker,I. also
`expre5ses the GF”Gene driven by the 10% promoter. The Onco‘v’EX series
`has the bockborre structure or o second-generol‘lort oncolyric HSV-l will
`double deletions in Ihe 734.5 and Ir47 genes. The Ironsgehe .3 in3erlecl into
`the deleted «1311.5 loci.
`
`recerttly created air oricolytic raw 1 with double
`Han et al
`deletionsin the {34. 51nd (X47 genes, armed with tumor necrosis
`Ar) 5
`.
`“ lNFtXIs a cytokinc with at)otcnt Irrititttmor
`factoralp‘na(”lNFcr)
`activity
`toxicity
`buta locla delivery of TNFOI is ltnovvn to cause
`and its ability to induce tLtmor c1llarpoptosis could iirtcrtere with
`viral replication. To avoid these problems, they used the USZ 1 true
`latc HSVLl promoter to drive the YEN/RX geric.
`\X'rlicrcas the virus
`armed with USZ Z-Idriven TNFOI expressed lower amounts ol‘rlTNFQ’
`it, carirbrtcd higher antrtumor effects and less toxicity-trigIn the virus
`that used t Ie immediate—early CMV promoter.
`
`Armed Gnmlyfit HSVa-Il lot in viva imaging
`
`u
`With the advancement ofoncolytic virus therapy developtnent,
`there has been an increasing need tor tron—invasive methods of
`imaging or monitoring of viral infection and replication. Oncolytic
`HiV—l can be armed not only for the purpose of augmenting the
`therapeutic eTi'icacy but also for realizing such non—invasive in vivo
`
`imaginglrt precil nical settings one*Lpproathis to utili7e a luciI-e
`based hioluminescent system. Two l-lSVQuilt—based oncolytic HSV—l
`
`[I
`,..
`were generated that expres: liretly luc'l3rase under tie control
`of the imimdiare—mrli (If)
`/5 promoter or gC promoter.h ihe
`[E .. ,1/) promoter act5 inttn-edi:tely alter viral intection, whereas t re
`
`strict late gC promotcta.acts in the late stage or the replication cycle.
`\‘Vhen athymic mice bearing subcutaneous tumors of GliSGAEGFR
`glioma were observed under a sLIpctserisitive charged coupled device
`.
`.
`.
`.
`r
`.
`.
`.
`:amera. the expression oilucilhrasc controlled. bythe [E4/5 promoter
`correlated wtttt Viral intectton and that by the go promoter With vtral
`replication.
`
`$ystamlt: Delivery til Armed Gntnlyfir lIESle
`
`\Whereas the most common route oi delivery olioncolytic HSV—l
`has been a direct intratumoIal inoculation, an intravenous delivery
`would lurthcr broaden the clinical application of oncolytic l-lSV—l
`if proven effective. The tnain hurdle for intravenous delivery is that
`only a small percentage of the administered virus reaches tlrto tumor".
`By arming of oncolytic HSV—l, a large antitumor effect can be
`induced from a small number of virus that initiates replication at the
`tumor.
`'We observed that intravenous delivery of ll_.—12.Lexpressing
`T—mflLlZ caused a significant inhibition of-Ltmot growth compared
`with mock and the unarmed C(Hiti’Olvirusl(I—O l) treatrrients in [V]
` {l1
`
`e bearing subcutaneous Neurola tumors (Guan et al.., tnanus ript
`in preparation;I When A.'l mice bearing int racerehral tumors were
`treated by repeated intravenous injections, .[Lmtl E514,out not T-Ol,
`signilicantlv prolonged the survival compared with moclt. Also, in a
`renal cancer lung metastases model usirigb‘bAlll/c mice and syrig'en ‘ic
`ReriCa cells3, intravenous administrations oli’lmlll.l2 sigtiiiicarItly
`”TI
`inhibite l the nIIitnber of metastases compared with nt-:otl<: and T-(ll
`treatments tlsurumaxi fit ill. manuscript in preparation).
`
`Summary
`
`in summary, “arming” ol’ottcolytic HSV—l wi h transgenes leads
`to development ola *ariety or“ oncolytic l'lSVLl with certain litnc»
`tions resultingtn enhancement of antitumor efficacy and/or in vivo
`irnaging caprhility. in the futurca cries or armed oircolvtic HSV l
`suited tor ceitain tumor types oi certain administration routes may
`be used di'Tlitretttially or in combination according to conditions of
`patients. Armed oncolytic l-lSV—l has high potential as a new genre
`of therapy for brain tumors as well as other cancers
`
`Acknowledgements
`
`g);
`
`thank the current members of» my laboratory especially Drs.
`l
`
`Yasushi lrto and Hiroshi Fukuhara. Our research '
`been supported
`in part by grants to TJT. From the Ministry of Education, Culture,
`Sports. Science and Technolo y oflapan.
`References
`Aghi lvi. Nlartuza KL. Oncol ’tic viral therapies-write clinical experience Oncogene 2005;
`2427802413.
`2. Torin ’T.lOncolyric virus therapy using genetically engineered herpes simplex viruses. Front
`Bosci 230‘ 8" l3:2.06‘3-~4.
`
`iV1arl<ertjiVL Merll oclc iVlD Rabkir‘ ‘3‘)), Gillespie CI, Torin T, Hunter W'D, Palmer CA,
`lax
`.
`'
`:nbaum F, Turnaroreti, libero lI, Martuza Rn. Conditionally replicating herpes sin
`
`
`
`
` :an a phase .I trial l...
`
`.. mutant, G’
`ror the
`earment of... gnanr gliorna: resun.
`roentsl. Gene. Ther 2-300
`967-74
`
`
`Attenuated naulri-murated
`4. hiincra 'l,
`Rahnin SiJ, \azatti T Hunter W'3, Marruza RL.
`
`herpes Simplex virus-l. rot the treatment of malignant gliomas. Nat Med 19
`l :93 43.
`
`S. Chou j, Kern ER, \Whitlcy R], Roizman B. ivlapping of harp
`simplex virus—l neurovtru-
`urc. Science 1990; 2'30 262-6.
`
`
`lt‘l’lCC CO gamma _
`. 3 gene nonessential for 8,117th in Cl
`
`
`6. He B, Chou ], drandtrfiarrr
`Mohr l. Gliizman Y.
`.. B. Suppression of~ the
`'
`phenotype of gammalil34.5herpes siIItfleV virus l‘ failure. of activared RNA-dependent
`
`
`s assocrated with a deletion in the domain of
`protein kiiiase to shut oi ‘
`otcirr synrhe
`/ 2CD?
` Elf.
`
`the alp
`jV.. .i ”9/". /l 2604,.
`.
`
`2 l2
`
`Cell Adhesion 81 lI/ligrarion
`
`2008; Voi.2 issue 3
`
`

`

`Armed oncolvtic HSV»! for brain tumor therapy
`
`host—cell per-
`
`be\J
`
` \. prerger MC, Baird‘IX
`
`.tor of metalloproteinase—3 ‘. a oncolytic herpes 'irus inhibits tumor
`
`
`'s. Can ‘ r Res ZOUS; 1'82l170»9.
`growth and vascular PngCiil
`
`F11 X, Tao L, Jin A, Vile R. Brenner M
`
`glycoprotein by arr oncolytic herpes simplex virus poten'etiats the vi1
`Ther 2003; 7‘//48»‘,I:.
`.odo T Rabkin SD, Martuza RT. EV luarion of ganciclovirmeedJated.enha..cement orreh
`anritumoraI erfect in o..colytic, multi...utatedherpes simplex ij‘llS tVIrIe 3 {11207; Iherapy
`or brain tuIIIo sCaJJcer Gene TheI5000 /2939-41).
`
`Chase M, Chung, R, {hIocca EA,
`.r‘. nncolytic Viral mutant that delivers the CY?"
`
`
`tra Jgene a
`.Li augments prlQPhOSP<9n11uL chern.1‘herapy. Natore
`
`
`ll). 44-8.
`
`modal cancer treatment
`K, Rrealrefield X0, Chiocca in . 1S4ul
`‘
`Aghi lv’l. Chou TC, Suling
` . 111$
`
`
`i
`ted by a replicating o11c-3lytic virus that d i
`the oxazanliosplrorirre/rat cytoch
`
`
`e kinas
`
`.5) 281 and gan 1c:lIIvir.’hetpes si...plex "irus thymILii
`gene rherapies. Cancer Res
`W99; 5‘J'Q86l-5,
`PM, SaeJ'iI,
`i E, leroy S, Terada K Finkelstein DJVT, Hyatt JiJ, Da..ks MK, TorreI~
`
`1s
`
`
`
`
`implex
`TAJ Brain tumor oncolysis w h riepl
`.n—conclitiL'ial herp J
`typel
`expressinI> the proditt'—ac.1va.1ng genes, CYYl’ZBl and 'ecreted human intestinal
`Larboxyleste1ase,II11. combination With cyclophospl‘Iamid) arrd1rir1-3t carr. Cancer Res 209 J,
`135168-54} /.
`
`
`Nakamura‘lJ ,l.v4‘..lle..JT, Chan.ira3ekhaIS
`
`lawlik1‘.v4, ‘monSS, Tanal)el\K. MultimIIdaI .)
`.K])T{"‘3€S yeast
`therapy WIIh a replication-cor‘rrditionall‘r‘.e,IIrIes smplet Virus l
`rI‘Iutar‘t that
`
`to S-tluo ourat‘JJl.
`CerSlR'f deaminase TOP inrratumoral COanil'.iOn OT J-UHOTOCYT'
`
`Cancer Res 200i; 6l254
`
`I'npson GR, Han Z, Lin B,Wnag, 1', Cam
`I'npbell G. Collin