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Immunotherapy of L1210 Leukemia using neuraminidase-modified plasma membranes combined with chemotherapy

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Immunotherapy of L1210 Leukemia using neuraminidase-modified plasma membranes combined with chemotherapy
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  1981;41:3082-3086. Published online August 1, 1981. Cancer Res  Jack H. Pincus, Alice K. Jameson and Alan E. Brandt ChemotherapyNeuraminidase-modified Plasma Membranes Combined withImmunotherapy of L1210 Leukemia Using  Updated Version  http://cancerres.aacrjournals.org/content/41/8/3082Access the most recent version of this article at: E-mail alerts  related to this article or journal.Sign up to receive free email-alerts SubscriptionsReprints and .pubs@aacr.orgDepartment atTo order reprints of this articleor to subscribe to the journal, contact the AACR Publications Permissions .permissions@aacr.orgDepartment atTo request permission to re-use all orpart of this article, contact the AACR Publications American Association for Cancer ResearchCopyright © 1981on August 9, 2011cancerres.aacrjournals.orgDownloaded from   [CANCERRESEARCH41,3082-3086.August1981]0008-5472/81/0041-OOOOS02.00 ImmunotherapyofL1210LeukemiaUsingNeuraminidase-modifiedPlasmaMembranesCombinedwithChemotherapy1 JackH.Pincus,AliceK.Jameson,andAlanE.Brandt LifeSciencesDivision,SRIInternational,MentoPark,California94025 ABSTRACT PurifiedL1210plasmamembranestreatedwithVibriochol- eraeneuraminidase(VCN)wereusedforactiveimmunotherapy ofL1210tumorsinDBA/2Jmice.ImmunotherapywithVCN-treatedmembraneswaseffectiveonlywhencombinedwith1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea(Me-CCNU).SuccessfultherapywasafunctionofthedoseofMeCCNU,thedoseofVCN-treatedmembranes,andthetimeafterMeCCNUtreatmentwhenVCN-treatedmembraneswereadministered.Optimumconditionsfortreatinganimalswithtumorsinitiatedwith10"cellswereMeCCNU(20mg/kg)given3daysaftertumorinoculationand0.25mgVCN-treatedmembranesgiven1dayafterchemotherapy.Controlmembranes,nottreatedwithVCN,thatwereadministered1dayafterMeCCNUwereineffective;whengiven4daysafterchemotherapy,theycausedacceleratedmortality,suggestingimmu-nologicalenhancementoftumorgrowth.OurresultsindicatethatVCN-treatedplasmamembranescanbeusedforactiveimmunotherapyofestablishedtumorsandunderscoretheimportanceofcarefullydesigningimmunotherapyprotocolstoachieveoptimumdesirableeffects. INTRODUCTION VCN2-treated,nondividing,viabletumorcellshavebeenusedforactiveimmunotherapyofestablishedtumorsinanimalsandhumans.Administrationofnondividing,VCN-treated,methyl-cnolanthrene-inducedfibrosarcomacellstotumor-bearinganimalsslowsthegrowthofthetumor(28,29)andinsomeanimalscausesittocompletelyregress(27).VCN-treatedtumorcellsalsoinduceregressionofspontaneouscaninemammarytumors(25),B16melanoma(16),L1210leukemia(2,3),andmurinepulmonary(1)andsquamouscell(16)carcinomas.VCN-treatedtumorcellshavebeenusedtotreatresidualtumorsinpatientswithmammarycarcinomas,gastrointestinaltumors,skincancers(20),andbronchogeniccarcinomas(30)afterthemaintumormasseshavebeensurgicallyremoved.Theyhavealsobeencombinedwithchemotherapytotreatpatientswithacutemyelogenousleukemia(2,3).Thedisease-freeintervalintreatedpatientswaslongerthanthatincontrolpatientsinalloftheabovecases.ImmunotherapywithVCN-treatedtumorcellsiseffectiveonlywhentumorsaresmall(17)orwhenthetumorburdenhasbeenreducedbysurgery(17)orchemotherapy.ViableVCN-treatedcellsarenotessentialfortherapy;enzyme-treated, 1ThisworkwassupportedbyContractN01-CB-64053fromtheNationalCancerInstitute.2Theabbreviationsusedare:VCN,Vibriocho/eraeneuraminidase;NANA.N-acetylneuramicacid;MeCCNU.1-(2-chloroethyl)-3-<4-methylcyclohexyl)-1-nitrosourea;HBSS.calcium-andmagnesium-freeHanks'balancedsaltsolution.ReceivedDecember3.1980;acceptedMay7.1981. frozen-and-thawedcellsareequallyeffective(13,18).TheseobservationssuggestthatVCN-treated,purifiedtumorcellmembranescouldbeeffectivevaccinesforactiveimmunotherapyofsmalltumorsorresidualtumorsaftersurgeryorchemotherapy.Inthepreviouspaper(4),wedemonstratedthattreatmentofDBA/2Jmicewithpurified,VCN-treatedL1210plasmamembranesprotectedthemagainstasupralethalchallengeofL1210cells.WedescribedthepreparationofmodifiedplasmamembranesanddemonstratedthattheonlymodificationwastheremovalofNANA.Inthisreport,wedescribethetreatmentofestablishedL1210tumorswithVCN-treatedL1210plasmamembranescombinedwithMeCCNUchemotherapy. MATERIALSANDMETHODSAnimals.FemaleDBA/2Jmiceweighingapproximately20 gwerepurchasedfromTheJacksonLaboratory(BarHarbor,Maine). L1210Tumor.TheL1210tumorlineused(LE50B01)was obtainedfromtheNationalCancerInstitutetumorbankthroughArthurD.LittleCo.,Inc.(Cambridge,Mass.).Thetumorwasmaintainedbyweeklyi.p.passageof105cellsinDBA/2Jmice. PlasmaMembranePreparation.Plasmamembraneswere preparedfromL1210ascitestumorsasdescribedinthepreviouspaper(4). VCNTreatmentofPlasmaMembranes.Theconditionsfor treatingL1210plasmamembraneswithVCNtoremovesialicacidfromglycoproteinandglycolipidoligosaccharidesidechainsaredescribedinthepreviouspaper(4).Control(untreated)membraneswereincubatedunderthesameconditionswithoutVCN. Chemotherapy.MeCCNUwasobtainedfromtheDrugSyn thesisandChemistryBranch,DivisionofCancerTreatment,NationalCancerInstitute.Itwasstoredat—85°.Suspensionsofthedrugwerepreparedfreshbeforeeachusebywarmingittoroomtemperatureinadesiccator,rapidlyweighingtheamountrequired,andsonicatingitin0.8%NaCIsolution-0.05%Tween80for30to60sec.Micereceivingchemotherapyweregiveni.p.injectionsof0.5-mlsuspensionsofthedrug. Immunotherapy.WeusedaprotocolsimilartothatofSan- singandKollmorgen(23)forimmunotherapywithoutchemotherapy.SixtyfemaleDBA/2Jmicewereeachinoculatedwith100L1210cellsandrandomlydividedinto3groupsof20.Fivedaysaftertumorinoculation,onegroupreceivedi.p.injectionsof0.2mgofVCN-treatedmembranessuspendedin0.1mlofHBSS,asecondgroupreceiveduntreatedmembranessuspendedin0.1mlofHBSS,andthethirdgroupreceived0.1mlofHBSSonlyandservedastheno-treatmentcontrolgroup. 3082CANCERRESEARCHVOL.41  American Association for Cancer ResearchCopyright © 1981on August 9, 2011cancerres.aacrjournals.orgDownloaded from   ImmunotherapyofL1210Leukemia Forchemoimmunotherapyexperiments,80micewereeachgiveni.p.injectionsof104L1210cells.SixtyoftheseweretreatedwithMeCCNUasdescribedabove;theremaining20receivedi.p.injectionsof0.8%NaCIsolution-0.05%Tween80andservedastheno-treatmentcontrolgroup.Thosereceivingchemotherapywererandomlydividedinto3groupsof20.ThemiceineachgroupreceivedeitherVCN-treatedmembranessuspendedin0.1mlofHBSS,untreatedmembranessuspendedin0.1mlofHBSS,or0.1mlofHBSSonly(chemotherapycontrol).ThedosesofMeCCNUormembranesandthetimeaftertumorinoculationwhenthedrugormembraneswereadministeredwerevaried.Inboththeimmunotherapyandthechemoimmunotherapyexperiments,theanimalsintheexperimentalandcontrolgroupswereobserveddaily,andmortalitywasrecordedasafunctionoftimeaftertumorinoculation. RESULTSImmunotherapywithoutChemotherapy.Weusedcondi tionssimilartothoseofSansingandKollmorgen(23)todeterminewhetheri.p.injectionsofVCN-treatedoruntreatedL1210plasmamembranesinducedaremissionofestablishedL1210tumors.Theamountofuntreatedmembraneproteinadministered(0.2mg)waschosensothattheamountofNANAthatitcontainedwouldbethesameasthatin1.5x107L1210cells(23);0.2mgofVCN-treatedmembraneshadthatamountofNANAremovedfromthem.Themediansurvivaltimeofmicereceivingnotreatmentwas16days;thisvalueagreeswiththatreportedbySansingandKollmorgen(23).AdministrationofeitherVCN-treatedoruntreatedmembranesdidnotprolongsurvivalorinduceregressionoftumors.Therefore,usingtheconditionsreportedbySansingandKollmorgen(23),wefoundthatVCN-treatedoruntreatedL1210membranepreparationsdidnotinduceregressionofestablishedtumorsinDBA/2Jmice. ChemotherapyofL1210Tumor-bearingMicewithMeCCNU.Bekesiefal.(2)andLeFeveretal.(13)foundthat immunotherapywithVCN-treated,nondividing(2),orfrozen-and-thawedcells(13)waseffectiveonlyifmicewerefirstgivenchemotherapy.Intheirexperiments,chemotherapyincreasedthemediansurvivaltimeoftumor-bearingmice,whichwasfurtherincreasedbytheuseofVCN-treatedcells.Wewereunabletoreproducetheresultsobtainedbyeitherofthesegroupswithchemotherapyonly.Usingtheirconditionsofinitialdoseoftumor,doseofdrug,andtheintervalbetweentumorinoculationanddrugadministration,wefoundthatthemediansurvivaltimeoftreatedmicewasthesameasthatofcontrolmice.Thissuggestedthat,inourhands,thechemotherapyprotocolsofBekesiefal.(2)andLeFeverefal.(13)didnotreducethetumorburden.Therefore,weperformedchemotherapytrialswithMeCCNUtofindconditionsthatwouldhavethedesiredeffectwhenMeCCNUwascombinedwithtreatmentwithVCN-treatedanduntreatedmembranes.Thedesiredeffectwasdefinedasprolongationofthemediansurvivaltimeoftreatedmicewithoutcuringahighpercentage.Undertheseconditions,apositiveeffectofadditionaltherapy(VCN-treatedoruntreatedtumorcellmembranes)wouldmanifestitselfasfurtherprolongationofsurvival.WeperformedchemotherapytrialsinDBA/2Jmicebearingascitestumorsinitiatedwith104L1210cells.MeCCNUwasadministeredatdifferenttimesaftertumorinoculation,micewereobserveddaily,anddeathswererecordedastheyoccurred.AsillustratedinChart1,60%ofmicereceivingMeCCNU(20mg/kg)1dayaftertumorinoculationwerecured.Becauseofthishighpercentageofcures,theseconditionsofchemotherapyareunsuitableforourexperiments.NoadditionalimprovementwasnotedintheseanimalswhenchemotherapywascombinedwithimmunotherapyusingVCN-treatedL1210plasmamembranes(datanotshown).Incontrast,whenMeCCNUwasadministeredatadoseof20mg/kg3daysaftertumorinoculationoratadoseof10mg/kg3,4,or6.5daysaftertumorinoculation,themediansurvivaltimeoftreatedmicewas8,6,and4dayslongerthanthatofuntreatedmice.Inaddition,alargepercentageofmicewerenotcured.WeusedalloftheseprotocolsforcombinationtherapywithMeCCNUandVCN-treatedanduntreatedL1210plasmamembranes. CombinationTherapyofL1210Tumor-bearingMicewithMeCCNUandVCN-treatedorUntreatedL1210PlasmaMembranes.Chart2illustratestheresultsofanexperimentinwhich L1210ascitestumor-bearingmiceweregivenMeCCNU(10mg/kg)onDay4afterinoculationof104L1210cellsandVCN-treatedoruntreatedL1210plasmamembranes(0.2mg/mouse)onDay6.Chemotherapyimprovedthemediansurvivaltime.However,combiningMeCCNUwithVCN-treatedoruntreatedmembranesdidnotresultinadditionalprolongationofsurvival.Similarresults(datanotshown)wereobtainedwhenMeCCNUwasadministeredonDay3andthemembranesweregivenonDay6orwhenthedrugwasadministeredonDay6.5andthemembranesweregivenonDay8.TheseresultscouldbeduetoaninsufficientdoseofMeCCNU,orplasmamembranescouldbeineffective.Todistinguishbetweenthesepossibilities,weperformedcombinationtherapyusingahigherdoseofMeCCNU.Chart3illustratestheresultsofacombinationtherapyexperimentinwhichthedoseofMeCCNUwasincreasedto20mg/kg.Inthisexperiment,thedrugwasadministeredonDay3,andVCN-treatedoruntreatedplasmamembranes(0.2mg)wereadministeredonDay6.ThemediansurvivaltimeofmicereceivingtheMeCCNUandVCN-treatedmembraneswas5dayslongerthanthatofmicereceivingchemotherapyonly.ThisdemonstratesthatVCN-modifiedL1210plasmamembranesareeffectivewhenusedincombinedtherapy.ThefailureofexperimentsdescribedaboveandillustratedinChart2wasapparentlyduetoaninsufficientdoseofMeCCNU. 3024283236DAYSAFTERTUMORINOCULATION Chart1.ChemotherapyofDBA/2Jmicebearingtumorsinitiatedwith104Ll210cells.Sixgroupsof10miceeachwereinoculatedwithtumorcellsonDay0.Thecontrolgroup(A)receivednofurthertreatment.IndividualgroupsoftheremainingmicereceivedMeCCNU,10mg/kgonDay3(A),4(D),or6.5(O)aftertumorinoculationor20mg/kgonDay1(•)r3(•)ftertumorinoculation.AUGUST1981 3083  American Association for Cancer ResearchCopyright © 1981on August 9, 2011cancerres.aacrjournals.orgDownloaded from   J.H.Pincusetal. Chart3alsoillustratestheresultsofcombinationtherapyusingMeCCNUanduntreatedL1210plasmamembranes.MicereceivingMeCCNUanduntreatedmembranesdiedsoonerthandidthosereceivingMeCCNUonly.Thus,undertheconditionsofthisexperiment,untreatedmembranesenhancedtumorgrowth.Wealsovariedthedoseofplasmamembranesusedincombinedtherapy.Intheseexperiments,MeCCNU(20mg/kg)wasadministeredonDay3,andmembraneswereadministeredonDay4.TheresultsoftheseexperimentsareillustratedinChart4.UnliketheexperimentillustratedinChart3,untreatedmembranesatdosesof0.25mg(Chart4X\),0.125mg(Chart 4B),or0.025mg(Chart4C),whengiven1dayafterchemo therapy,didnotenhancetumorgrowth.Themediansurvivaltimeofthesemicewasthesameasthatofmicereceivingchemotherapyonly.However,thepercentageofmicereceivingchemotherapyand0.25or0.125mgofuntreatedL1210plasmamembranesthatsurvivedlongerthan60dayswasgreaterthanthatofmicetreatedwithchemotherapyonly.VCN-treatedmembranesweremoreeffectivethanuntreatedmembranes.ThemosteffectivedoseofVCN-treatedmembranesadministered1dayafterchemotherapywas0.25mg(Chart4A)',themediansurvivaltimeofthisgroupwas17dayslongerthanthatofthegroupreceivingchemotherapyonly.BecausethisprotocolwasmoreeffectivethanthatusedfortheexperimentillustratedinChart3,itsuggeststhatthetimingoftherapyisimportant.Moreover,becauselowerdosesofVCN-treatedmembranes(Chart4,BandC)wereonlymargin-iiiiii 202428323640«48DAYSAFTERTUMORINOCULATION Chart2.ChemoimmunotherapyofL1210tumor-bearingmicewithMeCCNU(10mg/kg)andVCN-treatedoruntreatedL1210plasmamembranes.Fourgroupsof20miceeachwereinoculatedwith10'L1210cellsonDay0.ThreeofthegroupsreceivedMeCCNUonDay4.Thefourthgroupwasdesignatedastheno-treatmentgroup(A)andreceived0.8%NaCIsolution-0.05%Tween80.Miceinthe3groupsreceivingMeCCNUwererandomizedandonDay6received0.125mgofVCN-treatedmembranes(*),oruntreatedmembranessuspendedinHBSSOorHBSSonly(O). 81216202428323640DAYSAFTERTUMORINOCULATION812162024283236404448DAYSAFTERTUMORINOCULATION04812162024283236DAYSAFTERTUMORINOCULATION Chart4.ChemoimmunotherapyofL1210tumor-bearingmicewithMeCCNU(20mg/kg)anddifferentamountsofVCN-treatedoruntreatedL1210plasmamembranes.Eightgroupsof20miceeachwereinoculatedwith10*L1210cellsonDay0.SevenofthegroupsreceivedMeCCNUonDay3.Theeighthgroupwasdesignatedastheno-treatmentgroup(A)andreceived0.8%NaCIsolution-0.05%Tween80.Miceinthe7groupsreceivingMeCCNUwererandomizedandonDay4receivedVCN-treatedmembranes(*),oruntreatedmembranessuspendedinHBSS(D),orHBSSonly(O).Thedosesofmembranesadministeredwere:A,0.25mg;B,0.125mg;C.0.025mg. allyeffective,theremaybeathresholddoseforeffectivetherapy. DISCUSSION Intheprecedingpaper(4),wedemonstratedthatVCNtreatmentofpurifiedL1210plasmamembranesenhancedtheir Charts.ChemoimmunotherapyofL1210tumor-bearingmicewithMeCCNU(20mg/kg)andVCN-treatedoruntreatedplasmamembranes.Fourgroupsof20miceeachwereinoculatedwith104L1210cellsonDay0.ThreeofthegroupsreceivedMeCCNUonDay3.Thefourthgroupwasdesignatedastheno-treatmentgroup(A)andreceived0.8%NaCIsolution-0.05%Tween80.Miceinthe3groupsreceivingMeCCNUwererandomizedandonDay6received0.2mgofVCN-treatedmembranes(O),untreatedmembranessuspendedinHBSS(•),orHBSSonly(•). 681012141618202224262830323436DAYSAFTERTUMORINOCULATION3340424446485060 3084CANCERRESEARCHVOL.41  American Association for Cancer ResearchCopyright © 1981on August 9, 2011cancerres.aacrjournals.orgDownloaded from   ImmunotherapyofL7270Leukemia therapeuticpotential.Weshowedthatthemostprobablereasonforthisisexposureofgalactoseand/V-acetylgalactosa-mineterminionplasmamembraneglycoproteinoligosaccha-rides.Moreover,themodifiedmembranesprotectedDBA/2JmiceagainstchallengebyviableL1210cells.Thepresentstudydemonstratesthatthesepreparationscanalsobeusedtotreatactivelygrowingtumors.SansingandKollmorgen(23)reportedcompleteremissionoftumorsin80%ofmicegiveninjectionsofVCN-treated,nonviableL1210cells5daysafteri.p.inoculationofviableL1210cells.WeusedVCN-treatedL1210cellplasmamembranesundersimilarconditionsandfoundVCN-treatedL1210plasmamembranesinhibitedthegrowthofestablishedL1210tumorsinDBA/2JmiceonlywhenusedinconjunctionwithMeCCNUchemotherapy.ThisisconsistentwiththefindingsofothersthatVCN-treatedcellsareeffectiveonlyagainstsmalltumorsorafterthetumorburdenhasbeenreduced(17).However,becausetheVCN-treatedmembraneswereineffectivewithoutchemotherapywhenthetumorwasinitiatedwithasfewas100L1210cells,chemotherapymaybeneededforreasonsotherthanreductionoftumorburden.Forexample,inthereportsofKillionandKollmorgen(11)andKillion(10),itwasshownthatsubpopulationsofL1210cellsdifferintheirimmunogenicity,andMihichandKitano(15)demonstratedthatL1210tumorcellsthatsurvivechemotherapyaremoreimmu-nogenicthanistheuntreatedtumor.Therefore,chemotherapymayeliminatelessimmunogeniccells;hence,thosethatsurvivethistreatmentaremoresusceptibletotherapywithmodifiedplasmamembranes.ConditionsforsuccessfulcombinationtherapyofgrowingL1210tumorswithMeCCNUandVCN-treatedmembraneswerearrivedatempirically.Thedosesofdrugandmembranesandthetimeaftertumorinoculationwhenthedrugandmembranesareadministeredareimportantvariables.UntreatedmembraneseitherenhancedtumorgrowthorwerelesseffectivethanVCN-treatedmembranes,demonstratingtheimportanceofVCNmodificationinmaximizingthetherapeuticpotentialofL1210plasmamembranes.Factorssuchastumorburden,changesinthecellpopulationofagrowingtumor,andtheimmunestatusofatumor-bearinghostmayalsoinfluencetheoutcomeofthistypeoftherapy.AdditionalworkisneededtodefineallparametersthataffectsuccessfultherapywithVCN-treatedplasmamembranes;thiswouldbeofaidindevelopingprotocolsthataremaximallybeneficialintreatinggrowingtumors.OurconditionsforsuccessfulchemoimmunotherapyofL1210leukemiawithVCN-treatedplasmamembranesdifferfromthoseofBekesiefal.(2,3),whousedVCN-treated,nondividingL1210cellscombinedwithMeCCNUchemotherapy,andfromthoseofLeFeverefal.(13),whousedfrozen-and-thawed,VCN-treatedL1210cellscombinedwith1,3-bis(2-chloroethyl)-1-nitrosoureachemotherapy.Oneexplanationforthisisthattheseinvestigatorsuseddifferentinbredstrainsofmiceintheirexperimentsthanweusedinours.Bekesietal.(2,3)usedDBA/2Hamice,asublineofDBA/2micedifferentthantheoneinwhichtheL1210tumororiginated(12).Moreover,DBA/2HamiceimmunetoL1210cellsshowacceleratedrejectionofDBA/2Jskin,suggestingthatthereisanantigenicincompatibilitybetweentheL1210tumorandtheDBA/2Haline(14).Similarly,LeFeverefal.(13)usedC57BL/6xDBA/2F,(hereaftercalledBD2F,)hybridsintheirexperiments.L1210tumorcellsgrowninthesemicehavealonger.doublingtimethandoL1210cellsgrowninDBA/2Jmice(8).ThisphenomenonhasbeenascribedtoallogeneicinhibitionoftumorgrowthinBD2Fimice(6)andsuggeststhatthereisalsoanantigenicincompatibilitybetweentheL1210tumorandtheBD2F,host.Thetumor-hostincompatibilityintheexperimentalsystemsofBekesiefal.(2,3)andLeFeverefal.(13)couldinfluencetheoutcomeoftheirexperimentsaswellastheoptimalprotocolforsuccessfultherapyandmayaccountforourinabilitytoreproducetheirresults.Incontrasttotheirexperimentalsystems,ourexperimentswereperformedusingDBA/2Jmice,thesamesublineinwhichthetumororiginated(12);therefore,theoutcomeofourexperimentsisnotinfluencedbyatumor-hostincompatibility.VCNtreatmentmayenhancethetherapeuticpotentialofplasmamembranesbyoneofseveralmechanisms.Theenzymemayincreasethecontentofcarbohydrate-associatedantigens(5,7,9,22)bymodifyingoligosaccharidesidechainssothatimmunizationwithmodifiedcellsormembranescouldenhancetheimmuneresponsetoantigensonthecellsurfacethatarenotnormallypresentinlargeenoughamountstoelicitastrongimmuneresponse.Alternatively,cytotoxinstoVCN-treatedcellsthatarefoundinmanynormalsera(8,19,21,26)mayenhancethebindingofenzymaticallytreatedcellsinperitonealmacrophages(14,24),resultinginmacrophagesthatarecytotoxic(26).Additionalexperimentsareneededtodistinguishwhichofthesemechanismsismoreimportantintumorimmunity.Vaccinespreparedfrompurifiedplasmamembraneshaveanadvantageoverthosepreparedfromwholecellsinthatfewerextraneouscontaminantsareintroducedintothehost.Inaddition,becausetheyaremorestablethanarewholecells,theyshouldbemoreapplicableforclinicalmedicine.Moreover,becausetheyareasubcellularcomponentthecompositionofwhichcanbedefined,theyshouldprovideanimprovedsystemoverwholecellsforanalyzingthemodificationsresultingfromVCNtreatmentandrelatingthesetobiologicalactivity. REFERENCES 1.Alley,C.D..andSnodgrass.M.J.Effectivenessofneuraminidaseinexperimentalimmunotherapyoftwomurinepulmonarycarcinomas.CancerRes.,37.95-101,1977.2.Bekesi,J.G.,Roboz,J.P..andHolland,J.F.Therapeuticeffectivenessofneuraminidase-treatedtumorcellsasanimmunogeninmanandexperimentalanimalswithleukemia.Ann.N.Y.Acad.Sci.,277.311-331,1976.3.Bekesi,J.G.,Roboz,J.P.,Walter,L.,andHolland,J.F.Stimulationofspecificimmunityagainstcancerbyneuraminidase-treatedtumorcells.BehringInst.Mitt.,55.309-321,1974.4.Brandt,A.E..Jameson,A.K..andPincus,J.H.Characterizationanduseofneuraminidase-modifiedLi210plasmamembranesforprotectionagainsttumorgrowth.CancerRes.,41:3077-3081,1981.5.Burnet,F.M..andAnderson,S.G.The"T"antigenofguineapigandhumanredcells.Ausi.J.Exp.Biol.Med.Sci.,25.213-217,1947.6.Choquet,C..Chavaudre,N.,andMalarie,E.P.TheinfluenceofallogeneicinhibitionandtumorageonthekineticsofL1210leukemiainvivo.Eur.J.Cancer,6:373-378,1970.7.Drzeniek.R.,Saber,M.S..andRoh,R.VeränderungderErythrozytenob-erflächedurcheNewcastleDiseaseVirus.II.Mitteilung.AuftreteneinesForsmann-undeinesmononucleose-antigensanNewcastleDiseaseVirusbehandeltenErythrozyten.Z.Naturforsch.,21:254-260,1966.8.Hughes,R.C.,Sanford,B.,andJeanloz,R.W.Regenerationofthesurfaceglycoproteinsofatransplantablemousetumorcellaftertreatmentwithneuraminidase.Proc.Nati.Acad.Sei.U.S.A.,69.942-945,1972.9.Kassulke,J.T.,Stutman,O.,andTunis,E.J.Blood-groupisoantigensinleukemiccells;reversibilityofisoantigenicchangesbyneuraminidase.J.Nati.CancerInst.,46:1201-1208,1971.10.Killion,J.J.Immunotherapywithtumorcellsubpopulations.CancerImmunol.AUGUST19813085  American Association for Cancer ResearchCopyright © 1981on August 9, 2011cancerres.aacrjournals.orgDownloaded from 

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