adjuvant是什么意思uvant在线翻译读音例

DesignandSynthesisofPotentQuillajaSaponinVaccine

Adjuvants

,PayalDamani,,AnnieWon,FengHong,

ston,*GovindRagupathi,**

MolecularPharmacologyandChemistryProgram,andtheMelanomaandSarcomaSerVice,

DepartmentofMedicine,MemorialSloan-KetteringCancerCenter,1275YorkAVenue,

NewYork,NewYork10065

ReceivedSeptember29,2009;E-mail:gind@;ragupatg@;livingsp@

Abstract:Thesuccessofantitumorandantiviralvaccinesoftenrequirestheuseofanadjuvant,asubstance

thatsignifiandfulofadjuvants

havebothsuffimisingadjuvantis

QS-21,asaponinnaturalproductthatistheimmunopotentiatorofchoiceinmanycancerandinfectious

r,thetherapeuticpromiseofQS-21adjuvantiscurtailedbyseveral

factors,includingitsscarcity,difficultyinpurificationtohomogeneity,dose-limitingtoxicity,andchemical

,wereportthedesign,synthesis,andevaluationofchemicallystablesyntheticsaponins.

Thesenovel,amide-modified,non-naturalsubstancesexhibitimmunopotentiatingeffectsinvivothatrival

orexceehly

convergentsyntheticpreparationofthesenovelsaponinsestablishesnewavenuesfordiscoveringimproved

molecularadjuvantsforspecificallytailoredvaccinetherapies.

Introduction

Thedevelopmentofvaccinestocombatcancerandvaccine-

resistantinfectiousdiseaseshasreliedsignificantlyonsubunit

efinedmolecularantigensoffer

advantagesintermsofsafetyandprecisioninimmuneresponse

targeting,,these

vaccineformulationsrequireanadjuvant,asubstancethat

potentiatesimmuneresponse.1,2Thecriticalrolesofvaccine

adjuvantslieintheirabilityto:(1)enabletheuseofotherwise

impotentantigens;(2)extendthebenefitsofvaccinationtopoor

,olderorimmune-compromisedpatients);and

(3)effectdose-sparingofrareandexpensiveantigensinshort

,duringanepidemic).Giventhatthemajorityof

FDA-approvedsubunitvaccinesrelyonanadjuvantcomponent,

thechallengetodiscovernovelimmunopotentiatorsremainsat

-21(Chart1),apurifiedsaponinfractionfromthe

barkextractsofQuillajasaponaria(QS),3,4isapromising

adjuvantinnumerousprophylacticandtherapeuticvaccines.5

Thissaponinfractioncomprisestwoprincipalisomersthatshare

atriterpene,abranchedtrisaccharide,andaglycosylated

pseudodimericacylchain.6Thetwoisomericformsdifferin

theconstitutionoftheterminalsugarwithinthelineartetrasac-

charidesegment,whereinthemajorisomer,QS-21-Api(1,

∼65%abundance),incorporatesa-D-apioseresidue,andthe

minorisomer,QS-21-Xyl(2,∼35%abundance),terminatesin

a-D-xylosesubstituent.7

NumerousclinicaltrialshavebeenconductedwithQS-21

adjuvantinvaccinesagainstinfectiousdiseases(malaria,8,9

HIV,10,11hepatitis,12tuberculosis13)andcancer(melanoma,14,15

(1)Jiang,Z.H.;Koganty,.2003,10,1423–1439.

(2)Kwissa,M.;Kasturi,S.P.;Pulendran,es2007,

6,673–684.

(3)Dalsgaard,.1970,44,327–331.

(4)Kensil,C.R.;Patel,U.;Lennick,M.;Marciani,l.1991,

146,431–437.

(5)Kensil,rrierSyst.1996,13,1–55.

(6)Jacobsen,N.E.;Fairbrother,W.J.;Kensil,C.R.;Lim,A.;Wheeler,

D.A.;Powell,.1996,280,1–14.

(7)Cleland,J.L.;Kensil,C.R.;Lim,A.;Jacobsen,N.E.;Basa,L.;

Spellman,M.;Wheeler,D.A.;Wu,J.Y.;Powell,.

Sci.1996,85,22–28.

(8)Kester,K.E.;McKinney,D.A.;Tornieporth,N.;Ockenhouse,C.F.;

Heppner,D.G.;Hall,T.;Wellde,B.T.;White,K.;Sun,P.;Schwenk,

R.;Krzych,U.;Delchambre,M.;Voss,G.;Dubois,M.C.;Gasser,

R.A.;Dowler,M.G.;O’Brien,M.;Wittes,J.;Wirtz,R.;Cohen,J.;

Ballou,W.R.;Rts,e2007,25,5359–5366.

(9)Abdulla,S.;Oberholzer,R.;Juma,O.;Kubhoja,S.;Machera,F.;

Membi,C.;Omari,S.;Urassa,A.;Mshinda,H.;Jumanne,A.;Salim,

N.;Shomari,M.;Aebi,T.;Schellenberg,D.M.;Carter,T.;Villafana,

T.;Demoitie,M.A.;Dubois,M.C.;Leach,A.;Lievens,M.;

Vekemans,J.;Cohen,J.;Ballou,W.R.;Tanner,.

2008,359,2533–2544.

(10)Evans,T.G.;McElrath,M.J.;Matthews,T.;Montefiori,D.;Weinhold,

K.;Wolff,M.;Keefer,M.C.;Kallas,E.G.;Corey,L.;Gorse,G.J.;

Belshe,R.;Graham,B.S.;Spearman,P.W.;Schwartz,D.;Mulligan,

M.J.;Goepfert,P.;Fast,P.;Berman,P.;Powell,M.;Francis,D.;

Grp,e2001,19,2080–2091.

(11)Kennedy,J.S.;Co,M.;Green,S.;Longtine,K.;Longtine,J.;O’Neill,

M.A.;Adams,J.P.;Rothman,A.L.;Yu,Q.;Johnson-Leva,R.;Pal,

R.;Wang,S.X.;Lu,S.;Markham,e2008,26,4420–4424.

(12)Vandepapeliere,P.;Horsmans,Y.;Moris,P.;VanMechelen,M.;

Janssens,M.;Koutsoukos,M.;VanBelle,P.;Clement,F.;Hanon,

E.;Wettendorff,M.;Garcona,N.;Leroux-Roels,e2008,

26,1375–1386.

(13)Garcon,N.;Chomez,P.;VanMechelen,es2007,

6,723–739.

(14)Ragupathi,G.;Meyers,M.;Adluri,S.;Howard,L.;Musselli,C.;

Livingston,2000,85,659–666.

(15)Ragupathi,G.;Livingston,P.O.;Hood,C.;Gathuru,J.;Krown,S.E.;

Chapman,P.B.;Wolchok,J.D.;Williams,L.J.;Oldrield,R.C.;

Hwu,Res.2003,9,5214–5220.

PublishedonWeb01/20/2010

10.1021/ja9082842.2010,132,1939–194591939

breast,16,17smallcelllung,18prostate19).Despitethepromise

ofQS-21,however,thereareseveralliabilitiesassociatedwith

,highvariabilityinmolecular

compositionofsaponinsisseenevena三生石下原唱 mongQStreesofsimilar

ageandlocalenvironment.20Purificationofthemulticomponent

QS-21fractionentailselaboratelow-yieldingextractionand

HPLCprotocols.21Notsurprisingly,thissaponinadjuvantis

,potencyofQS-

21isproportionaltodose,butthetolerateddoseofQS-21in

cancerpatientsdoesnotexceed150g,abovewhichsignificant

localerythemaandsystemicfly,

QS-21wasreportedtodegradeinamatterofdaysonstorage

insolutionsofphysiologicalpHatambienttemperature,7

whereinthelabileestergroupwithintheacylchain(Chart1)

ofthemoleculeundergoesspontaneoushydrol出塞古诗全诗 ysistoproduce

byproductswithseverelyattenuatedadjuvantactivity.22,23

Importantly,thelatterissueofchemicalinstabilityisasignificant

factorinprecludingtheadvancementofQS-21aloneasan

adjuvantforvaccinesindevelopingcountries,theepidemic

strongholdsofmalaria,HIV,stoaddress

theseimpedimentshavelargelyfocusedontheincorporation

ofadditives(copolymers,lipids,etc.)13,24toimpartvarying

r,suchcomplexformula-

tionsconferadditionaldimensionsofheterogeneitytothe

vaccineandintroducechallengesassociatedwithmaintaining

formulationconsistencyforadvancementtoclinicalevaluation.

Analternateapproachtoovercomingalloftheproblems

associatedwithQS-21istoimprovethisadjuvantthrough

controlledstructuralmodifi

extentoftheseeffortshasbeenquitelimited,however,asthe

chemicalsensitivityofthenaturalproductpresentsanexceed-

inglynarrowwindowforitsfunctionalgroupderivatization.

Thisbarriercanbeovercomethroughchemicalsynthesisof

cessfulvalidationofthisapproachis

exemplifiedhereinbythedesign,synthesis,andevaluationof

novelsyntheticsaponinsbasedontheQS-21parentarchitecture.

Thesenewhydrolyticallystablemolecularadjuvantsexhibitin

vivoimmunostimulatingpotenciesrivalingorexceedingthat

er,whencomparedtothe

naturalproduct,thesemolecularentitiescanbeobtainedin

homogeneousformwithhigherefficiency,andsignalthe

potentialformodulationoftoxicitybyfurtherstructural

modification.

Results

servationfromearlystability

studiesonQS-21revealedthattheremovaloftheacylchainof

thenaturalproductfurnishedadeacylatedsaponinbyproduct

thatwasacutelycompromisedinitsabilitytostimulateantibody

andCTLresponsesagainstOVAantigeninmice.22The

establishmentoftheunstableacylchainasacriticalcomponent

forthebioactivityofQS-21promptstheinvestigationsherein,

whichfocusonthestructuralmodificationofthisquadrantto

context,simple

replacementoftheunstableesterlinkageswithintheacylchain

withmorerobustamidelinkageswouldenhancethestability

oftheadjuvantwiththeintentionofincreasingpotencyand/or

durationoftherapeuticactivityperdose.

Accesstotheseamide-stabilizedstructuralvariantswould

requireanovelmonosaccharidemoietytoserveasastructural

mimictotheacylatedfucosesugar(Chart1,Fuc-residuewithin

1and2).Accordingly,theprincipaldesigncriterioninthis

carbohydratecomponentisthereplacementoftheC4-oxygen

functionalitywithanappropriatenitrogengroup,asexemplified

byC4-deoxy-C4-azidogalactopyranoside6(Scheme1).The

syntheticsequencetopreparethefucosesurrogate6(Scheme

1)wasinitiatedwith3,6-di-O-benzoyl-4-O-methanesulfonyl-

D-glucal(3),derivedinonestepfromD-glucalaccordingto

theprocedureofPiancatelli.25ThisC4-mesylate3wasa

competentelectrophileforS

N

2displacementwithsodiumazide

(16)Gilewski,T.;Ragupathi,G.;Bhuta,S.;Williams,L.J.;Musselli,C.;

Zhang,X.F.;Bencsath,K.P.;Panageas,K.S.;Chin,J.;Hudis,C.A.;

Norton,L.;Houghton,A.N.;Livingston,P.O.;Danishefsky,S.J.

.U.S.A.2001,98,3270–3275.

(17)Musselli,C.;Ragupathi,G.;Gilewski,T.;Panageas,K.S.;Spinat,

Y.;Livingston,2002,97,660–667.

(18)Krug,L.M.;Ragupathi,G.;Ng,K.K.;Hood,C.;Jennings,H.J.;

Guo,Z.W.;Kris,M.G.;Miller,V.;Pizzo,B.;Tyson,L.;Baez,V.;

Livingston,Res.2004,10,916–923.

(19)Ragupathi,G.;Slovin,S.F.;Adluri,S.;Sames,D.;Kim,I.J.;Kim,

H.M.;Spassova,M.;Bornmann,W.G.;Lloyd,K.O.;Scher,H.I.;

Livingston,P.O.;Danishefsky,.,.1999,

38,563–566.

(20)Kamstrup,S.;SanMartin,R.;Doberti,A.;Grande,H.;Dalsgaard,K.

Vaccine2000,18,2244–2249.

(21)Kensil,.6,231,859,

2001.

(22)Kensil,C.R.;Soltysik,S.;Wheeler,D.A.;Wu,ure/function

studiesonQS-21,auniqueimmunologicaladjuvantfromQuillaja

saponariaInSaponinsUsedinTraditionalandModernMedicine;

Waller,G.R.,Yamasaki,K.,Eds.;PlenumPress:NewYork,1996;

pp165-172.

(23)Liu,G.;Anderson,C.;Scaltreto,H.;Barbon,J.;Kensil,e

2002,20,2808–2815.

(24)Drane,D.;Gittleson,C.;Boyle,J.;Moraskovsky,es

2007,6,761–772.

(25)Squarcia,A.;Vivolo,F.;Weinig,H.G.;Passacantilli,P.;Piancatelli,

edronLett.2002,43,4653–4655.

Chart1

.9VOL.132,NO.6,2010

ARTICLESAdamsetal.

toprovidethecorresponding关于中秋节的优美诗词 azide,whoseestergroupswere

subsequentlyexchangedforbenzylethersbyZemplensaponi-

fication(NaOMe)andalkylationwithbenzylbromidetofurnish

azido-galactal4(51%from3).Stereoselective1,2-bis(acyloxy-

lation)26ofglycal4(PhI(OAc)

2

,BF

3

OEt

2

)wasaccomplished

in85%yield,allowingforsubsequentacetatemethanolysis(5,

84%).SelectiveprotectionofthehemiacetalwithTIPSCl

providedtheC4-azidogalactoside6(59%),whichservesasthe

isworthnotingthatthefucosemimic6bearsan“extra”C6-

benzyloxygroupascomparedtothenaturalfucosemoiety,

tifactofthe

synthesisnotonlyallowsforarelativelyshortsynthetic

sequenceto6,butalsoimpartsanadditionalfunctionalhandle

withwhichtoexplorefuturenovelcompositionsofmatter.

Glycosylationoftheazido-sugar6withthetrisaccharide

hemiacetal8,derivedfromanomericdesilyationoftheprevi-

ouslypreparedtrisaccharide7,27proceededunderthedehydra-

tivecouplingprotocol28(Ph

2

SO,Tf

2

O)toaffordtheselectively

protectedtetrasaccharide9(70%).Subsequentremovalofthe

tri-iso-propylsilylacetalwithin9wasaccomplishedwithTBAF

toprovidethecorrespondinghemiacetal(93%),whichwasthen

convertedtotheanomericR-trichloroacetimidate(10,95%).

Thisconstitutesasuccessfulroutetoafullyelaboratedtet-

rasaccharidefragmentthatincorporatestherequiredgalacto-

C4-azidogrouponwhichtoanchoravarietyofamideacyl

chains.

Initialeffortstowardthepreparationofacylchainvariants

ofQS-21focusedon19(Scheme2),22,and23(Scheme3)as

promisingacylationagentstoaccessnovelhydrolyticallystable

tcomplexamideacylchainofthese

(26)Shi,L.;Kim,Y.J.;Gin,.2001,123,6939–

6940.

(27)Deng,K.;Adams,M.M.;Gin,.2008,130,

5860–5861.

(28)Garcia,B.A.;Poole,J.L.;Gin,.1997,119,

7597–7598.

Scheme1a

aReagentsandconditions:(a)NaN

3

,Bu

4

NCl,PhMe,110C,66%;(b)

NaOH,MeOH,23C;NaH,BnBr,DMF,23C,78%;(c)PhI(OAc)

2

,

BF

3

OEt

2

,CH

2

Cl

2

,-50f-25C,85%;(d)K

2

CO

3

,MeOH,H

2

O,23C,

84%;(e)TIPSCl,imidazole,DMAP,DMF,23C,59%;(f)TBAF,THF,

23C,98%;(g)8,Tf

2

O,Ph

2

SO,TBP,CH

2

Cl

2

,-55C;add6,-78f23

C,67%;(h)TBAF,THF,0C,93%;(i)CCl3

CN,DBU,CH

2

Cl

2

,0f23

C,95%.

Scheme2a

aReagentsandconditions:(a)NaOH,BnOCOCl,H

2

O,0f23C,>99%;

(b)EtOCOCl,Et

3

N,THF;CH

2

N

2

,0f23C,78%;(c)CF

3

CO

2

Ag,Et

3

N,

THF,-50f23C,79%;(d)CDI,THF,23C;13,THF,-78C,60%;

(e)H

2

,RuCl

2

(S)-BINAP,MeOH,23C,89%;(f)TBSOTf,2,6-lutidine,

CH

2

Cl

2

,-78C,94生字组词 %;(g)Pd/C,H

2

,MeOH,23C,97%;(h)17,EtOCOCl,

Et

3

N,THF,0C;add16,23C,80%;(i)TMSOTf,2,6-lutidine,CH

2

Cl

2

,

0f23C,76%.

Scheme3a

aReagentsandconditions:(a)21,Tf

2

O,Ph

2

SO,TBP,CH

2

Cl

2

,-45C;

add20,-78f0C,91%(1.1:1,R:,SiO

2

separation);(b)BaOH8H

2

O,

MeOH,23C,81%.

.9VOL.132,NO.6,20101941

PotentQuillajaSaponinVaccineAdjuvantsARTICLES

istheglycosylatedpseudodimericamide19(Scheme2),

incorporatingthemostconservativestructuralvariationsofthe

bstrateservesasanisostericmimicofthe

naturalacylsubstituent,differingonlyinthecentralester-to-

amidereplacement,whichshouldservetofurtherenhance

addition,twosimplifiedlipophilicamideacylchains(Scheme

3),,22)ora

,23)linearaliphaticmoiety,werealso

ticQSsaponinsbearingthelattertwohydro-

phobicchainswouldnotonlybepreparedbyconsiderably

shortersyntheticsequencesascomparedtothatof19,butalso

provideaclearindicationofwhethertheelaboratestereochem-

icalarraywithinthenativeacylchainisrequiredforadjuvant

activity.

Thesynthesisoftheisostericamideacylchain19(Scheme

2)beganwithD-alloisoleucine(11),obtainedfromL-isoleucine

bythethree-stepepimerization-resolutionprotocolofSakai.29

Followingaminoprotectionof11asitsbenzylcarbamate

(BnOCOCl,>99%),thecarboxylicacidwassubjectedto

Arndt-Eisterthomologationviaitsderivatizationtothecor-

respondingR-diazoketoneandWolffrearrangementtoprovide

the-aminoacid12(62%).Subsequentactivationofthe

carboxylicacidin12withcarbonyldiimidazoleallowedfor

ClaisencondensationwiththeLi-enolateoft-butylacetate(13)

toprovide-ketoester14(60%),asuitablesubstrateforNoyori

catalyticasymmetrichydrogenation.30Thisproceededwith

RuCl

2

(S)-BINAPandH

2

,providingthe-hydroxyester15

(89%)withcompletecatalyst-controlleddiastereoselectivity.

Hydroxylgroupsilylation(TBSOTf,94%)wasfollowedby

hydrogenolyticunmaskingofcarbamate15toaffordamine16

in97%antly,thedeliberateselectionofthet-butyl

esterprotectivegroupinthissequenceprecludedunproductive

lactamizationofamine16,allowingforitsacylation(80%)with

theglycosylatedacylchainfragment17,previouslyprepared

inthesynthesisofQS-21.31-33Thistransformationprovided,

afteracid-catalyzedt-butylesterremoval(76%),theselectively

protectedfullyintactacylchain19,thedirectOfNamide

mimicoftheQS-21acylchain.

Fortunately,syntheticaccesstothetworemainingacylchain

fragments22and23(Scheme3)wassignificantlylesseffort-

intensivegiventheabsenceofstereochemicalcomplexitywithin

mple,theglycosylatedacylchain

22couldbereadilyobtainedinathree-stepsequence34involving

dehydrativeglycosylationofallyl12-hydroxydodecanoate(20)

with2,3,5-tri-O-TBS-arabinofuranose(21)31,32toprovidethe

intermediateglycoside(91%)asaseparablemixtureofanomers

(1.1:1,R:).TheR-anomerwasthenadvancedviaBa(OH)

2

-

mediateds围魏救赵的主人公是谁 aponificationoftheallylestertoprovidetheacyl

chain22(81%).Finally,thenonglycosylatedaliphaticacylchain

variantwastheeasiesttoprocuregiventhatlaurylchloride(23)

iscommerciallyavailable.

Thelate-stageconvergentassemblyoftheamideacylchain

variantsofQS-21(Scheme4)capitalizedontherecent

disclosureofoursemisyntheticapproachtoaccesshomogeneous

samplesofQS-saponinadjuvants.27Althoughtheacylchain

andlineartetrasaccharidequadrantsoftheQSsaponinsare

synthesizeddenovo,theremaininghalfofthenaturalproduct,

comprisingthetrisaccharide-triterpeneconjugate,couldbe

isolatedbycontrolledchemicaldegradationofQS-extractsand

selectiveprotectiontoyieldtheprotectedprosapogenin24

(Scheme4)inonlythreesteps.27Thus,Schmidtglycosylation35

oftheC28carboxylicacidin24withthetetrasaccharide

glycosyltrichloroacetimidatedonor10providedtheglycosyl

ester25(82%)e

azidegroupin25wasresponsivetoreductionwithbenzene-

selenoltorevealthecorrespondingamine26(91%),ontowhich

asesof

N-acylationwiththeglycosylatedacylchaincarboxylicacid

derivatives19and22,couplingswereaccomplishedbyinitial

roduction

ofthealiphaticlaurylchain,directacylationwithlaurylchloride

(23)uentsequentialhydro-

genolysisandacidhydrolysiseffectedglobaldeprotectionof

theresultingadvancedintermediatestoprovidetheamideacyl

chainSQS-saponinvariants27(SQS-0101),28(SQS-0102),

and29(SQS-0103)in79%,69%,and68%yields,respectively,

fromtheamineprecursor26.

tly,

thereexistsnorapidinvitrobiologicalscreenforassessingthe

potentialefficacyofsaponinvaccineadjuvants,giventhatthe

mechanismbywhichsaponinsaugmentimmuneresponseis

ult,evaluationofthesenovelsaponinsas

immunostimulantsproceededdirectlytopreclinicalstudies

involvingmousevaccinationwiththemelanomaantigenGD3

gangliosideconjugatedtotheKLHcarrierprotein(GD3-KLH,

Chart2).Thisisaclinicallyrelevantvaccinemodelthathas

provenusefulforcomparingtheimmunopotentiatingabilityof

variousadjuvants.36,37Monitoringantibodyresponsestoboth

thecarbohydrateantigenandtheproteincarrierprovidesauseful

assessmentofadjuvantperformancetoantigensofdifferent

immunogenicity,rangingfromapoorlyimmunogenicglycolipid

(GD3)toahighlyimmunogenicprotein(KLH).Moreover,

previousexperiencewiththisvaccinationprotocolhasconsis-

tentlycorrelatedantibodytitersagainstKLHtothatofT-cell

responsetothisantigen.37

Groupsoffivemice(C57BL/6J,female,6-8weeksofage)

werevaccinatedwithGD3-KLHata10gdose(Figure1).

Theantigenwascoadministeredwiththeadjuvantofinterest

inavaccinationprotocolinvolvingthreesubcutaneousinjections

at1-weekintervals(days0,7,and14)plusaboosteratday65.

Asthenegativecontrol,micewerevaccinatedwiththeGD3-

itivecontrol,vaccinationswere

performedwithnaturallyderivedQS-21(NQS-21),obtained

byfractionatingamixtureofsaponinsfromQuillaja

saponaria.38Theadjuvantdoseemployedinthesecomparative

studieswas10g,aquantityofQS-21knowntoinduce

measurableantibodyresponseswithacceptabletoxicityeffects

(29)Noda,H.;Sakai,K.;Murakami,edron:Asymmetry2002,

13,2649–2652.

(30)Noyori,R.;Ohkuma,T.;Kitamura,M.;Takaya,H.;Sayo,N.;

Kumobayashi,H.;Akutagawa,.1987,109,5856–

5858.

(31)Wang,P.;Kim,Y.J.;Navarro-Villalobos,M.;Rohde,B.D.;Gin,

.2005,127,3256–3257.

(32)Kim,Y.J.;Wang,P.;Navarro-Villalobos,M.;Rohde,B.D.;

Derryberry,J.;Gin,.2006,128,11906–11915.

(33)Deng,K.;Adams,M.M.;Damani,P.;Livingston,P.O.;Ragupathi,

G.;Gin,.,.2008,47,6395–6398.

(34)Lee,R.E.;Mikusova,K.;Brennan,P.J.;Besra,.

Soc.1995,117,11829–11832.

(35)Schmidt,R.R.;Kinzy,m.1994,

50,21–123.

(36)Kim,S.K.;Ragupathi,G.;Musselli,C.;Choi,S.J.;Park,Y.S.;

Livingston,e2000,18,597–603.

(37)Kim,S.K.;Ragupathi,G.;Cappello,S.;Kagan,E.;Livingston,P.O.

Vaccine2001,19,530–537.

(38)Kensil,.2000,42,259–271(Vaccine

Adjuvants).

.9VOL.132,NO.6,2010

ARTICLESAdamsetal.

dytitersagainstGD3(IgMandIgG)andKLH

(IgG)ably,allofthe

syntheticSQS-adjuvants(27-29)areatleastasactiveasNQS-

21inimmunopotentiatingability,asillustratedbytheIgMand

IgGresponsetoGD3(Figure1AandB)measured1week

followingthebooster(day72).Ineachcase,antibodytiters

weresignificantlyhigherthanthegroupwithGD3-KLHalone.

Inaddition,theIgGantibodyresponseagainstKLHwasalso

strikinglyelevated(Figure1C)withallofthenovelSQS-saponin

dysubtypingoftheanti-GD3IgGisotype

(Figure1D)revealedasignificantbiastowardthemouseIgG2b

subtypewithallofoursyntheticsaponinadjuvants(SQS-0101,

-0102,and-0103),aresultsimilartothatofnaturallyderived

tionofothermouseanti-GD3IgGsubtypes,

includingIgG1,IgG2a,andIgG3,waslowornegligibleas

indicatedbyclass-specificELISAemployingthestandard0.1

absorbancelevelthresholdforpositivereactivity.

Theactivityoftheseanti-GD3antibodieswasfurther

investigatedfortheirabilitytoeffecttumorcellbindingand

ropertieswerefirstassessed有关母亲的诗 byflowcytometry,

monitoringthecellsurfacereactivityofatumorcell-line

expressingGD3antigen(Figure1E).Seradrawn7daysafter

theboosterwereevaluatedbyFACSusingtheSK-Mel-28(GD3

positive)ividualFACSresultsarepresented

at10ci-

nationserafrommiceshowedlessthan10%positivecells,while

seraobtainedaftervaccinationwithallthreesyntheticadjuvants

showedsignifi

medianpercentpositivecellreactivitieswere15%fortheno-

adjuvantcontrol,87%forNQS-21(1and2),78%forSQS-

0101(27),83%forSQS-0102(28),and64%forSQS-0103

(29).Thesecell-surfacereactivityresultsfurtherreinforcethe

comparableadjuvantactivityofsyntheticSQS-0101,-0102,and

-tion,cellsurface

reactivityagainstSK-Mel-28wasfurthercharacterizedby

complement-dependentcytotoxicity(CDC)assays(Figure1F)

usingrabbitcomplementandserafromthemicevaccinatedwith

GD3-KLH(10g)plusSQS-101(27),SQS-102(28),orSQS-

103(29),eachat20an6%medianreactivity

wasdetectedinprevaccinationseraandinmicevaccinatedwith

GD3-KLHalone,r,

seraobtainedfrommiceimmunizedwithGD3-KLHplusSQS-

101,SQS-102,andSQS-103allshowedsignificantlyenhanced

cytotoxicityagainstSK-Mel-28overthenegativecontrols.

Asastandardinitialoverallassessmentoftoxicity,theweight

lossofthemicewasmonitoredat0,1,2,3,and7daysafter

thefirstvaccination(Figure2).Forthenegativecontrol

involvingvaccinationswithGD3-KLHonly(noadjuvant),no

positivecontrol,

thepresenceofNQS-21(10g)elicitednotableandexpected

medianweightlossof9%up

Scheme4a

aReagentsandconditions:(a)BF

3

OEt

2

,4ms,CH

2

Cl

2

,-78f23C,82%;(b)PhSeH,Et

3

N,30C,91%;(c)19,EtOCOCl,Et

3

N,THF,0C;add26,

10C,93%;(c′)22,EtOCOCl,Et

3

N,THF,0C;add26,12C,89%;(c′′)23,TBP,CH

2

Cl

2

,23C,98%;(d)Pd/C,H

2

,THF,EtOH,23C;TFA,H

2

O,

23C,RP-HPLC,85%(for27),78%(for28),69%(for29).

Chart2

.9VOL.132,NO.6,20101943

PotentQuillajaSaponinVaccineAdjuvantsARTICLES

vaccinatedwithSQS-0102hadan11%medianweightloss,

whilethemaximumweightlossoftheremaininggroups

incorporatingSQS-0101andSQS-0103waslessthan5%.

Overall,weightlosswith10gSQS-0102wasslightly,but

notsignificantly,morethanwithNQS-21(p>0.15),while

weightlosswithSQS-0101(p)0.05)andSQS-0103(p<

0.025)wassignificantlylessthanthatwithNQS-21.

Discussion

QS-21remainstheimmunopotentiatorofchoiceinmany

cancerandinfectiousdiseasevaccinetrialsdespiteitsliabilities,

whichincludescarcity,difficultyinpurificationtohomogeneity,

dose-limitingtoxicity,llenges

associatedwithQS-21havepromptedthegenerationofalternate

structuresinspiredbyQSsaponinsforthediscoveryofadjuvants

withgreaterstabilityandmorefavorabletherapeuticprofiles.

WiththecurrentsemisyntheticapproachtocomplexQS

saponins,27thepreparationofnoveladjuvantswithunpre-

thisstrategy,thefirstgenerationofSQS-amideanalogues

(27-29)thesestructures

exhibitsatleastcomparableadjuvantactivitytothatofnatural

NQS-21,asevidencedbyinitialELISA,FACS,andCDCdata

(Figure1).ForthestronglyimmunogenicKLHproteincarrier,

atofNQS-

21,theanti-KLHtitersforalloftheSQS-analogueswereat

least20-foldhigherthanthenegativecontrolgroupcomprising

theweaklyimmunogenicGD3antigen,initialIgMproduction

followedbyclassswitchingtoIgGwasobservedwithallthree

subtypingtheseantibodies,theIgG2bsubclasswasshownto

seIgG2bandIgG2asubclassesareknown

toinducepotentimmunotherapeuticeffectorfunctions,39includ-

ingcomplement-dependentcytotoxicity(CDC)andantibody-

(39)Nimmerjahn,F.;Ravetch,e2005,310,1510–1512.

nicalimjuvant,plusano-adjuvantnegativecontrol,

wasevaluatedbyvaccinationofagroupoffivemice(C57BL/6J,female).Vaccinationstooktheformofweeklysubcutaneousinjectionofantigen(10g)

andvarioussaponinadjuvants(10gforELISAandFACSresults;20gforCDCresults)for3weeks(days0,7,and14),followedbyaboosteratday

ostserologicaldataatday72arepresented.(A)Anti-GD3titers(IgM)aftervaccination;medianvaluesincurlybrackets.(B)Anti-GD3titers

(IgG)aftervaccination;medianvaluesincurlybrackets.(C)Anti-KLHtiters(IgG)aftervaccination;medianvaluesincurlybrackets.(D)Anti-GD3IgG

subtypingasassessedbyclass-specificindirectELISA;medianabsorbancelevels>0.1areconsideredpositive.(E)Cellsurfacereactivityofanti-GD3

againstSK-Mel-28tumorcelllineexpressingGD3antigenfollowingvaccination;medianvaluesincurlybrackets.(F)Complement-dependentcytotoxicity

(CDC)activityofanti-GD3againstSK-Mel-28tumorcelllineexpressingGD3antigen.

ltoxicity

assessmentwasperformedbytrackingmedianweightlossofeachgroup

ofmicefollowingthefirstvaccinationwithGD3-KLH(10g)antigenwith

varioussyntheticsaponinadjuvants(10g).

.9VOL.132,NO.6,2010

ARTICLESAdamsetal.

dependentcellularcytotoxicity(ADCC).Importantly,the

cytotoxicactivityoftheseanti-GD3antibodieswasunambigu-

ouslyverifiedtoeffectCDContheSK-Mel-28(GD3positive)

tumorcellline.

Onthebasisoftheseinitialimmunologicaldata,ourfirst

fewhydrolyticallystablestructuralvariants(27-29)clearly

showthattheelaboratestereochemicalarraywithinthenative

QS-21acylchainisnotessentialtomaintainadjuvantactivity

finding,incombination

withtheintegrationofsemisyntheticprosapogenin2427intothe

synthesis,shortensthesyntheticroutetopotentsaponin

adjuvantslike28and29bymorethan30stepswhencompared

totheoriginalchemicalsynthesisofQS-21.31–33Finally,

differencesintoxicityprofilesbetweentheamidevariantsand

NQS-21,basedontrackingtheweightlossinmiceduring

vaccination,stoxicityofSQS-0102

(28)isatleastaspronouncedasthatofNQS-21(1and2),

SQS-0101(27)andSQS-0103(29)exhibitloweroveralltoxicity

,thespecificreasonsbehindthisstructure-

dependenttoxicityremaintobedefiinvestigations

onthisfrontwouldentailadjuvantdose-escalationstudies,

followedbydetailedbiochemicaltoxicityevaluationsofthose

miceexhibiting>10%he-

less,theweightlossdifferentialobservedevenatthe10g

adjuvantdose(Figure2)isastrongindicationthattoxicityof

thesaponincouldbeindependentlymodulatedthroughspecific

structuralperturbationswhilemaintainingpotentadjuvant

nitialresultsspeaktothelikelihoodofthefuture

discoveryofevensimplermolecularvariantsthatpossesspotent

immunostimulatoryactivitiesandattenuatedtoxicity.

ItisworthnotingthatthemechanismbywhichQS-21(or

ourSQS-analogues)potentia远上寒山石径斜寒山什么意思 tesimmuneresponseisunknown.

Hypotheseshavebeenputforththatthesaponin,throughlectin-

mediatedcellmembraneinteractions,mayfacilitateuptakeof

theantigenintoantigen-presentingcells(APCs),leadingto

specificcytokineprofilesthatenhanceT-and/orB-cell

responses.5,40ThelonealdehydegroupwithintheQStriterpene

hasalsobeensuggestedtoreactwithputativeT-cellsurface

receptorsviaSchiffbaseformation,providingT-cellswitha

costimulatorysignalforT-cellactivation.40Withinthelimited

availabledataconcerningthemechanismofactionofQS-21,

thereisstrongevidencetosuggestthatadepoteffectisnot

operative5andthatthesaponinisnotaligandforToll-like

receptors2and4(TLR-2,TLR-4),41proteinsthatactivateinnate

immuneresponsesbyrecognizingpathogen-associatedmolec-

hesefindingsarehighlysignificant,there

remainaninfinitenumberofmechanisticpossibilitiesto

ethestrikingefficacyofQS-21andrelated

saponinsasimmunologicaladjuvants,thereislittleguidance

(beyondbroadimmunologicalintuition)withwhichtomarshal

ucity

ofdatahasplaguedthesaponinadjuvantfield,wherebyseminal

mechanisticinformationinthisarenaisessentiallynonexistent,

despitethedecades-longuseofsaponinadjuvantsinclinical

,thisdemonstrationofhighadjuvantpotencywith

simpler,hydrolyticallystableSQS-adjuvants(27-29)notonly

signalsthepotentialfordiscoveryofnovelimmunotherapeutics,

butalsoshouldenablethecontrolledinstallationofchemical,

optical,andradio-isotopicreportersasvaluablemolecularprobes

ontheadjuvantmoleculetoaddresslong-standingmechanistic

questionsconcerningsaponinimmunopotentiation.

rkwassupportedbygrantsfrom

theNIH(R01GM058833,PO1CA052477,P50AT002779),the

DODProstateCancerResearchProgram#W81XWH-05-1-0085,

oodwinandtheCom-

monwealthFoundationforCancerResearchandTheExperimental

TherapeuticsCenterofMemorialSloan-KetteringCancerCenter.

SupportingInformationAvailable:Experimentaldetailsfor

syntheticproceduresandanalyticaldataforisolablesynthetic

terialisavailablefreeofchargeviathe

Internetat.

JA9082842

(40)Marciani,scoVeryToday2003,8,934–943.(41)Pink,J.R.;Kieny,M.描写黄河的诗歌 -e2004,22,2097–102.

.9VOL.132,NO.6,20101945

PotentQuillajaSaponinVaccineAdjuvantsARTICLES