Anti-HIV-1 Antibodies and CD4-IgG Breadth and Potency of Neutralization of Antibody Conjugation ...

44 Pages·2013·1.38 MB·English

Antibody Conjugation Approach Enhances
Breadth and Potency of Neutralization of
Anti-HIV-1 Antibodies and CD4-IgG

Julia Gavrilyuk, Hitoshi Ban, Hisatoshi Uehara, Shannon J.
D
Sirk, Karen Saye-Francisco, Angelica Cuevas, Elise o
w
Zablowsky, Avinash Oza, Michael S. Seaman, Dennis R.
n
Burton and Carlos F. Barbas III lo
a
J. Virol. 2013, 87(9):4985. DOI: 10.1128/JVI.03146-12. d
Published Ahead of Print 20 February 2013. ed

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Antibody Conjugation Approach Enhances Breadth and Potency of
Neutralization of Anti-HIV-1 Antibodies and CD4-IgG
JuliaGavrilyuk,aHitoshiBan,a,bHisatoshiUehara,a,cShannonJ.Sirk,aKarenSaye-Francisco,dAngelicaCuevas,dEliseZablowsky,e
AvinashOza,eMichaelS.Seaman,eDennisR.Burton,dCarlosF.BarbasIIIa D
o
DepartmentsofChemistryandMolecularBiologyandtheSkaggsInstituteforChemicalBiology,TheScrippsResearchInstitute,LaJolla,California,USAa;Dainippon w
n
SumitomoPharma,Osaka,Japanb;MitsubishiChemicalGroupScienceandTechnologyResearchCenter,Yokohama,Japanc;DepartmentofImmunologyandMicrobial lo
ScienceandIAVINeutralizingAntibodyCenter,TheScrippsResearchInstitute,LaJolla,California,USAd;BethIsraelDeaconessMedicalCenter,HarvardMedicalSchool, a
d
Boston,Massachusetts,USAe e
d

f
BroadlyneutralizingantibodiesPG9andPG16effectivelyneutralize70to80%ofcirculatingHIV-1isolates.Inthisstudy,the r
o
neutralizationabilitiesofPG9andPG16werefurtherenhancedbybioconjugationwithaplaviroc,asmall-moleculeinhibitorof m

virusentryintohostcells.Anovelair-stablediazoniumhexafluorophosphatereagentthatallowsforrapid,tyrosine-selective h
t
t
functionalizationofproteinsandantibodiesundermildconditionswasusedtoprepareaseriesofaplaviroc-conjugatedanti- p
:
bodies,includingb12,2G12,PG9,PG16,andCD4-IgG.TheconjugatedantibodiesblockedHIV-1entrythroughtwomecha- //
jv
nisms:bybindingtothevirusitselfandbyblockingtheCCR5receptoronhostcells.Chemicalmodificationdidnotsignificantly i.
a
alterthepotencyoftheparentantibodiesagainstnonresistantHIV-1strains.Conjugationdidnotalterthepharmacokineticsof s
m
amodelIgGinblood.ThePG9-aplavirocconjugatewastestedagainstapanelof117HIV-1strainsandwasfoundtoneutralize
.
o
100%oftheviruses.PG9-aplavirocconjugateIC swerelowerthanthoseofPG9inneutralizationstudiesof36ofthe117HIV-1 r
50 g
strains.TheseresultssupportthisnewapproachtobispecificantibodiesandofferapotentialnewstrategyforcombiningHIV-1 /
o
therapies. n

A
p
r
Innovative new approaches to HIV-1 prophylaxis and therapy demonstrating that catalytic monoclonal antibodies covalently il 9
,

are desperately needed. Despite the successes of highly active linkedtodesignedligandspossesspotentbiologicalactivitiesina 2
0
antiretroviraltherapy(HAART),morethan2millionpeopledie varietyofanimalmodelsofdisease(15–19).Severaloftheseare 1
3
each year and more than 33 million individuals are infected nowinclinicaldevelopment(20).Thesestudiesrevealedthemany
b
worldwide (http://aids.gov/hiv-aids-basics/hiv-aids-101/global advantagesofcouplingactivesmallmoleculesandpeptideswith y

-statistics/).AlthoughHAARTistypicallyeffective,itisnotwith- antibodies. In contrast to bispecific-antibody approaches based K
r
outproblems,includingcomplicateddrug-druginteractions,ad- on protein engineering, such as the dual-variable-domain e
s
herenceissues,andamyriadofsideeffects.Thedevelopmentof (DVD)-Ig (21) or single-chain variable fragment (scFv)-Ig (22) g
e
potentandbroadlyactingbiologicdrugsmightofferasolutionto fusionapproaches,amongothers,laboriousproteinengineering L
someoftheseproblemsandcomplementtraditionalHAART. is not required to endow a second specificity when the desired ib
r
Broadly neutralizing monoclonal antibodies (BNmAbs) that ligand is chemically coupled to the antibody. Furthermore, ex- a
r
y
recognizefeaturesconservedacrosscladesofHIVarepromising pressionissuesarebypassed,sincedevelopmentofanewcellline ,

startingpointsforthedevelopmentofimmunotherapeuticagents isnotrequired. T
h
againstHIV-1(1–8).Severalstudieshaveshownthatthetransfer A promising additional blockade to HIV-1 infection that e

S
of sufficient quantities of broadly neutralizing antibodies can shouldcomplementthetargetingofviralproteinsisthetargeting
c
achievesterilizingimmunityagainstintravenous,vaginal,orrec- ofhostproteinsrequiredforviralentryandreplication.Unlike rip
talchallengeinmacaquemodels(9,10).Thedeliveryofbroadly viral proteins, host proteins are not under selective pressure to p
s
neutralizing antibodies using gene-based approaches has also evolvetoevadethetherapeuticagent.Anumberofsmall-mole- R
beenshowntobeeffectiveinanimalmodels(11,12).Indeed,soon culeinhibitorsoftheHIV-1coreceptorsCCR5andCXCR4have e
s
after our discovery of BNmAb b12, we developed protein engi- beendeveloped(23,24),andoneCCR5-targetingdrughasbeen e
a
neeringmethodstoincreasethepotencyandbreadthofneutral- approved (25–28). Here we covalently linked a CCR5-targeting r
c
ization by b12 with the original aim of developing evolved b12 small molecule, aplaviroc, to BNmAbs and CD4-IgG. This ap- h

variants for HIV-1 therapy (13, 14). Collectively, these studies proach provided rapid access to bispecific proteins with excep- In
s
suggestthatBNmAbscouldbeeffectiveHIV-1prophylacticand t
it
therapeuticagents.Unfortunately,eventhemostbroadlyneutral- u
t
izingantibodyisvulnerabletoviralescape,becauseasingleamino Received9November2012 Accepted2February2013 e
acidchangeonthetargetproteincanalterthebindingepitope.If Publishedaheadofprint20February2013
aBNmAbcouldbemodifiedtoinhibitHIVinmultipleways,the AddresscorrespondencetoCarlosF.BarbasIII,[email protected].
evolutionary hurdle for escape would be significantly elevated. Supplementalmaterialforthisarticlemaybefoundathttp://dx.doi.org/10.1128
Furthermore, by combining multiple inhibitory functions in a /JVI.03146-12.
singlemolecule,theregulatoryandcostissuesforabiologiccom- Copyright©2013,AmericanSocietyforMicrobiology.AllRightsReserved.
plementtocombinatorialdrugtherapymightbeminimized. doi:10.1128/JVI.03146-12
Recently,wedevelopedanewclassoftherapeuticmoleculesby
May2013 Volume87 Number9 JournalofVirology p.4985–4993 jvi.asm.org 4985
Gavrilyuketal.
tional breadth in their abilities to neutralize diverse isolates of finalconcentrationof20(cid:1)g/ml;eachsamplewastestedintriplicate.The
HIV-1. cellswereincubatedonicefor1h,washedtwicewithstainbuffer,and
resuspendedin100(cid:1)lofstainbuffer.Afluorescence-labeledsecondary
antibodywasaddedataconcentrationrecommendedbymanufacturer.
MATERIALSANDMETHODS
Theplatewasincubatedonice,protectedfromlight,for1h.Thecellswere
Antibodies.Antibodiesb12,2G12,andDEN3wereprovidedbyDennis washedtwicewiththestainbuffer,resuspendedin200(cid:1)lofstainbuffer,
R.Burton(ScrippsResearchInstitute);antibodiesPG9andPG16were
and transferred to filter-top fluorescence-activated cell sorter (FACS)
providedbytheIAVI;theCD4-IgG2immunoadhesionproteinwasob- tubes(BDBiosciences)containing300(cid:1)lofbuffer(2%fetalbovinese- D
tainedfromProgenics(PRO542).Antibodieswerestoredat4°C.Thera-
rum[FBS]inphosphate-bufferedsaline[PBS]).Cellcountingwasper- o
peutic-gradetrastuzumab(Genentech)wasusedwithoutadditionalpu- w
formedusingadigitalLSRIIcytometer.DatawereanalyzedwithFlowJo
rification. n
software,version8.7.1. lo
Synthesisoflabelingreagents.Thesynthesisandcharacterizationof
Pharmacokineticstudy.Thepharmacokineticexperimentwascar- a
aplavirocwithalinkerhavebeendescribedpreviously(29). d
riedoutasdescribedpreviously(19).Inbrief,allanimalexperimentswere e
Antibodylabelingprocedure.Ina1.5-mltube,anantibodysolution d
(99(cid:1)l;1.5mg/mlin0.1MNa HPO [pH8.0])and10equivalentsof performedaccordingtoDivisionofAnimalResources(TSRI)guidelines f
4-formylbenzenediazoniumhex2afluor4ophosphate(FBDP;1(cid:1)l;10mM followingapprovedprotocols.Femaleathymicnudemice(8weeksofage) ro
wereinjectedsubcutaneouslywith100(cid:1)gtrastuzumabortrastuzumab- m
solutioninCH3CN)werecombined(30).Thesolutionwasmixedgently aplavirocin100(cid:1)lsterilePBS(4animalspergroup).Bloodwascollected h
andwasallowedtoreactfor30minatroomtemperaturewithintermit- t
tentmixing.Thesolutionturnedyellowuponcompletionofthereaction. fromtailveinsafter5min,2h,4h,8h,24h,48h,72h,96h,168h,and tp
After 30 min, excess FBDP was removed using a Zeba Spin desalting 336h.Inordertominimizebloodloss,only10(cid:1)lofbloodwaswithdrawn ://
column(molecularweightcutoff[MWCO],7,000[7K];Pierce),andbuf- per bleed; blood was diluted 1/5 in PBS containing 1% bovine serum jvi.
ferwasexchangedwith0.1MNa2HPO4(pH6.0).Aplaviroc-oxyamine albumin(BSA).Sampleswereallowedtochillonicefor20min.Insoluble as
(20equivalents;2(cid:1)l;10mMsolutioninCH CN)wasadded,andthe componentswereremovedbycentrifugation,andsampleswerestoredat m
solutionwasincubatedovernightat4°C.Exces3saplaviroc-oxyaminewas (cid:3)20°Cuntilanalysis. .o
removed using a Zeba Spin desalting column (MWCO, 7K), and free Thebindingandwashbufferforenzyme-linkedimmunosorbentas- rg
aplavirocwasremovedbyusingaproteinAspincolumn(GEHealthcare) says(ELISA)wasPBScontaining1%BSA.Half-areaELISAplates(Corn- o/
accordingtothemanufacturer’sinstructions. ing)werecoatedwithanti-humanIgGFc(Pierce)at750ng/wellover- n

Trypticdigestionandquadrupoletimeofflighttandemmassspec- night at 4°C. After wells were blocked with 3% BSA, serum samples A
p
trometry (Q-TOF MS-MS) characterization of antibody conjugates. dilutedinbindingbufferwereadded,andsampleswereincubatedfor1h r
Purifiedantibodysamples(100(cid:1)l;1mg/ml)wereexchangedinto6M at37°C.Trastuzumabandtrastuzumab-aplavirocweredetectedbyincu- il 9
guanidine,0.1MTris(pH8.0)byusing0.5-mlZebaSpindesaltingcol- bationwithdonkeyanti-humanIgGconjugatedwithhorseradishperox- ,
2
umns (MWCO, 7K) according to the manufacturer’s instructions. To idase(HRPO)(JacksonImmunoResearch).Todeterminetheserumdi- 0
eachsample,1Mdithiothreitol(DTT)(Fisher)wasaddedtoafinalcon- lution at which 80% saturation was reached, the 5-min samples were 13
centrationof20mM.Thesampleswereincubatedfor1hat37°Cwith seriallydilutedandanalyzed;80%saturationoftheELISAsignalattime b
gentleshaking,anda1Msolutionofiodoacetamide(Fisher)wasaddedto zerowasreachedatadilutionof1/500fortheanti-humanIgG.Inall y
K
eachsampletoafinalconcentrationof40mM.Sampleswereincubatedat subsequentexperiments,theopticaldensity(OD)attimezerowassetas r
roomtemperatureinthedarkfor40min.Thealkylationreactionswere 100%,andtheabsorptionatalllatertimepointswasdisplayedasaper- es
quenchedbyadding1MDTTtoa40mMfinalconcentration.Sample centageofthatattimezero.TheresultingcurveswerefittedusingGraph- g
e
bufferwasthenexchangedfortrypsindigestionbuffer(50mMTris[pH PadPrismone-phaseexponentialdecay. L
7.5],5mMCaCl2)using0.5-mlZebaSpindesaltingcolumns(MWCO, Virusneutralizationassay.Neutralizationassayswithsingle-round ib
7K).TrypsinGold(0.5mg/ml;Pierce)wasdissolvedin50mMaceticacid. r
infectious pseudovirus were performed as described elsewhere (10) by a
Thetrypsinsolutionwasaddedtoeachsampleatanenzyme-to-protein usingU87.CD4.CCR5targetcellsobtainedfromtheNIHAIDSResearch ry
ratioof1:20(wt/wt),andsampleswereincubatedat37°Cwithshakingat andReferenceReagentProgram(contributedbyHongKuiDengandDan , T
600rpmforapproximately18h.Digestionwasstoppedbytheadditionof Littman).Briefly,1(cid:2)104targetcellsinavolumeof100(cid:1)lwereseeded h
trifluoroaceticacid(TFA)toapproximately0.1%. e
Samples(with20(cid:1)gproteininjected)wereanalyzedusinganAgilent intowellsof96-wellplates(Corning)andwereincubatedovernightat S
37°Cunder5%CO .Afterovernightincubation,themediumwasre- c
6510Q-TOFmassspectrometerequippedwithaZorbaxSBC18column moved,and50(cid:1)loff2reshmediumwasaddedtoeachwell.Seriallydiluted rip
((cid:1)nmar)ro(Awgbiloernet;)i.nHneigrhd-ipaemrfeotremr,a2n.1cemlmiqu;liedncghthro,1m5a0tomgmra;pphayrt(iHclPeLsiCze),p3a.5- samples(50(cid:1)l)weretransferredtoplatedtargetcellsandwereincubated ps
for1hat37°C.Anequalvolumeofviruspreviouslydeterminedtoyield R
rameterswereasfollows:flowrate,0.2ml/min;agradientfrom0to40%
2(cid:2)105relativelightunits(RLU)wasadded(100(cid:1)l),andplateswere e
mobilephaseBover80min,followedbyagradientto0%Bfrom80to90 s
incubatedforanadditional72h.Tomeasureluciferaseactivity,theme- e
a9mc0ie%nto.naMciteortbiolienlei(tpvriohlela/sv(eovAol)l,/wvaoansld)0.m.M05oS%bdilTaetFapAhwa(esvreoelB/cvoowllla)escitn0e.dH04fPo%LrC2T-0Fg0rAatdo(ev2oH,l5/2v0Oo0,l)m2%i/nz dfrieuemphwoassprheamteo-bveudff,etrheedwsaellilnsew,aenredw5a0s(cid:1)heldofoannceapwpirthopCriaa2t(cid:4)el-yadnidluMtedg2l(cid:4)u-- arch

and 100 to 2,000 m/z, positive polarity, a gas temperature of 325°C, a ciferase cell culture lysis reagent (Promega) was added and mixed by In
nebulizerpressureof30lb/in2,andacapillaryvoltageof3,500V.Data pipettingvigorouslyupanddown.Aliquots(20(cid:1)l)weretransferredto st
wereanalyzedusingMassHuntersoftware(Agilent)andGPMAWsoft- opaque96-wellassayplates(Corning),andluciferaseactivitywasmea- itu
ware(version8.20;ChemSW). suredonaluminometer(EG&GBertholdLB96V;Perkin-Elmer)usinga te
Flowcytometry.Flowcytometryexperimentswereperformedasde- luciferaseassaysubstrate(Promega).Thepercentageofvirusneutraliza-
scribedpreviously(29).ThecelllinesusedwereA431cells,whichdonot tionatagivenantibodyconcentrationwasdeterminedbycalculatingthe
express CCR5, and TZM-bl cells, which do express CCR5. In brief, a reductioninluciferaseactivityinthepresenceofanantibody(relativeto
single-cellsuspensionwasprepared,andcellswerewashedtwicewithcold thatinvirus-onlywells).
stainbuffer(BDPharmingen)andwerepelletedbycentrifugation(300(cid:2) High-throughput virus neutralization assay. High-throughput
g).Thecellpelletwasresuspendedincoldstainbuffertoafinalconcen- screeningofthePG9-aplavirocconjugatewasperformedinthelaboratory
trationof2(cid:2)107cells/ml.Aliquotsof50(cid:1)lweredistributedtoV-bottom ofMichaelSeaman,HarvardMedicalSchool.Thehighestconcentration
wellsofmicrowellplates(Corning).Primaryantibodieswereaddedtoa ofPG9-aplaviroctestedwas50(cid:1)g/ml;seven5-folddilutionswerealso
4986 jvi.asm.org JournalofVirology
Potent,BroadlyActiveBispecificAnti-HIVAntibodies
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FIG1 Schematicrepresentationofthechemicalmodificationofanantibodywithadiazoniumhexafluorophosphate(FBDP)reagent.Theantibodyisfirst h
t
modifiedwiththeFBDPreagentatasurface-exposedtyrosineresidue(s),introducinganaldehydetagontotheantibody.Inthesecondstep,chemoselective tp
conjugationofthealdehydewithoxyamineisperformed,resultingintheintroductionoftheaplavirocmoietyontothesurfaceoftheantibody. :/
/
jv
i.
a
s
testedinduplicatewells.Thehighestconcentrationofaplaviroctestedwas ofHer2;however,meanfluorescenceintensitywasconsiderably m
.
100nM. higherfortrastuzumab-aplavirocboundtoTZM-blcellsthanfor o
r
g
theparentantibody,indicatingthattheCCR5bindingactivityof /
RESULTS aplavirocwasmaintainedfollowingconjugation. o
n
Model trastuzumab-aplaviroc conjugate. In order to indepen- Theanti-HIV-1activityoftrastuzumab-aplavirocwasassessed A
dentlyassesstheactivityofaplavirocconjugatedtoacarrierIgG byneutralizationassayswithasingleroundofinfectiouspseudo- p
r
without anti-HIV-1 activity, trastuzumab, an anti-Her2 mono- virus. Trastuzumab-aplaviroc neutralized HIV-1 strains JR-FL il
9
clonalantibody(MAb),wasusedasamodelhumanIgGforchem- andYU2with50%inhibitoryconcentrations(IC50s)of2.3nM , 2
icalmodificationandtesting.Bioconjugationconditionswerese- and 4.9 nM, respectively; unmodified trastuzumab showed no 0
lected on the basis of our previous study (30) and were further 1
neutralizingactivity(Fig.2B).ThepotentCCR5bindingantibody 3
optimizedhere(forfulloptimizationdetails,seethetablesinsec- 2D7neutralizedHIV-1strainsJR-FLandYU2withIC sof0.2 b
tionS3inthesupplementalmaterial).Thetwo-stepconjugation nMand0.1nM,respectively.ToensuretheCCR5-based50mecha- y K
methodology involved modification of the IgG with FBDP (4- nismoftrastuzumab-aplaviroc,itwasalsotestedforneutraliza- re
formylbenzene diazonium hexafluorophosphate) followed by s
tionofCXCR4-tropicHIVHXB2andwasfoundtobeinactive g
ligationoftheantibodytoaplaviroc-oxyamine(Fig.1).Thetras- e
(seethesupplementalmaterial).

tuzumab-aplavirocconjugatewassubjectedtoextensivepurifica- L
tion,includinggelfiltrationandproteinApurification,toensure ModificationoftheantibodyoccurredprimarilyatTyr319in ib
completeremovalofunreactedaplaviroc. theCH2regionoftheheavychain.Becausetheinvivohalf-lives rar
(t )ofantibodiesaremediatedprimarilybycontactsintheC 2 y
Characterizationoftrastuzumab-aplavirocbymatrix-assisted 1/2 H ,
regionoftheIgGwiththeneonatalFcreceptorFcRn,weevaluated T
laser desorption ionization–time of flight (MALDI-TOF) mass h
spectrometryrevealedtheincorporationofanaverageof1apla- thehalf-livesoftheparentandtheconjugateinmice.Thetrastu- e
zumab-aplavirocconjugatehadpharmacokineticpropertiessim- S
virocmoietyperIgGmolecule(seethesupplementalmaterial). c
Tryptic digestion and MS-MS characterization of the trastu- ilar to those of the unmodified parent antibody (Fig. 2C). The rip
zumab-aplavirocindicatedthatthediazoniummodificationoc- half-lifeoftrastuzumabinathymicnudemicewas115h,whereas p
the half-life of the trastuzumab-aplaviroc conjugate was 168 h. s
curredpreferentiallyattheconservedsurface-exposedtyrosinein R
theFcdomainatposition319byKabatnumbering.Someminor Thehalf-lifeofaplavirocinmiceis30min(33).Therefore,con- e
jugationofaplavirocwithtrastuzumabdramaticallyextendedthe s
modificationwasalsoobservedintheheavychainofFab(seethe e
half-life of aplaviroc in vivo and did not negatively impact the a
supplemental material). This observation is in agreement with r
c
previous studies that demonstrated preferential diazene forma- half-lifeofthescaffoldantibody. h

tionwiththemostaccessibletyrosines(22,31,32).Whentrastu- Conjugationofaplaviroctobroadlyneutralizingmonoclo- In
zumabthatwasnotmodifiedwithFBDPwasincubatedwithapla- nalantibodiesandCD4-IgG.BNmAbsb12,2G12,PG9,andPG16 st
viroc-oxyamine,noconjugationofaplaviroc-oxyamineoccurred, and the immunoadhesin protein CD4-IgG were conjugated with itu
t
asshownbyMALDI-TOFmassspectrometry.Thissamplehadno aplaviroc, and the conjugates were characterized by MALDI-TOF e
activityintheHIVneutralizationassays,indicatingthatthepuri- massspectrometry.Weobservedtheincorporationof0.5to2apla-
fication protocol used removed unconjugated aplaviroc-oxy- virocmoietiesperproteinmolecule(Table1).Trypticdigestionand
amine. MS-MSanalysisofconjugatesindicatedthattheheavy-chaincon-
Flow cytometry demonstrated that trastuzumab-aplaviroc stantregionTyr319wastheprimarysiteofchemicalmodificationin
bound to Her2-positive A431 cells and, to a lesser extent, to eachBNmAb,althoughminormodificationsiteswerealsoidentified
CCR5-positiveTZM-blcells(Fig.2A).Theparentantibody,tras- (seethesupplementalmaterial).Nomodificationswereobservedin
tuzumab,alsoboundtoTZM-blcells,duetolow-levelexpression theFvregionsoftheantibodies.
May2013 Volume87 Number9 jvi.asm.org 4987
Gavrilyuketal.
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A
FIG 2Evaluation of the model antibody conjugate trastuzumab-aplaviroc. (A) Flow cytometry analysis of binding to CCR5-positive TZM-bl cells and
p
CCR5-negativeA431cells.ThepotentCCR5bindingantibody(Ab)2D7servedasapositivecontrolinTZM-blcellassays.MFI,meanfluorescenceintensity.(B) r
NeutralizationofHIV-1JR-FLandYU2bytrastuzumabandthetrastuzumab-aplavirocconjugate.(C)Pharmacokineticprofilesoftrastuzumabandthe il
9
trastuzumab-aplavirocconjugateinathymicmice(4miceperexperimentalgroup).Allexperimentaldatarepresenttwoormoreindependentexperiments ,

performedintriplicate.Errorbarsrepresentstandarddeviations. 2
0
1
3

b
HIV-1 neutralization studies showed that the potencies of isolate, PG9 and PG16 were inactive except as aplaviroc conju- y

K
BNmAbconjugatesagainstHIV-1isolatesJR-FLandYU2were gates.CD4-IgGpotentlyneutralizesbothJR-FLandYU2.Conju-
r
e
significantlyhigherthanthoseoftheunconjugatedparentalanti- gationofaplaviroctoCD4-IgGdidnothaveanotableeffectonthe s
bodiesortheCD4fusionprotein(Table1;Fig.3).Improvements neutralizationoftheseviruses,sincetheimmunoadhesinprotein g
e
in potency over the parent antibody ranged from (cid:5)3-fold for itselfcanneutralizeJR-FLandYU2morepotentlythanaplaviroc L
2G12-aplaviroc against the JR-FL isolate to (cid:6)400-fold for b12- alone. To determine whether aplaviroc conjugation can have a ib
r
aplavirocagainsttheYU2isolate.Differencesbetweenconjugates positiveeffectontheactivityofthisprotein,weadditionallyas- a
r
y
and parent antibodies were smallest for the most potent of the sayedthecladeAvariant92RW020.5,astrainrelativelyresistant ,

parental antibodies. In the neutralization assay with the JR-FL toneutralizationbyCD4-IgG.Againstthisstrain,theconjugation T
h
resultedina20-foldimprovementintheIC .CD4-IgGneutral- e
50 S
izedthe92RWpseudoviruswithanIC of10nM,whereasCD4-
50 c
TABLE1NeutralizationofHIV-1JR-FLandYU2byaplaviroc- IgG–aplavirocneutralizedthisisolatewithanIC50of0.5nM.Sig- rip
conjugatedBNmAbsandCD4-Ig nificantly,noevidenceofenhancedinfectionwasnotedwithany p
s
Avgno.of conjugatedproteinstudied. R
IC (nM)for:
aplavirocmoieties 50 We also studied the neutralization activity of a 1:1 molar e
s
Antibodya perIgGmolecule JR-FL YU2 mixtureofaplavirocandPG9againstHIV-1strainsJR-FLand e
a
b12-apl 0.5 0.1 0.1 YU2,andweobservedapotencyequaltothatofaplaviroc.The r
c
b12 0 0.3 46.7 invitroassay,however,doesnotallowustoassessthepotential h

2G12-apl 2 0.3 25.5 clinicalbenefitoftheextendedhalf-lifeoftheantibody-apla- In
2G12 0 0.8 (cid:6)100 virocconjugaterelativetothe1:1mixtureoftwoagents(vide st
PG9-apl 1 11.6 1.2 infra). itu
PG9 0 (cid:6)100 22.6 NeutralizationofHIV-1strainsbyPG9-aplaviroc.ThePG9 te
PG16-apl 2 5.9 0.4
conjugatewaschosenformorein-depthstudybecausetheparent
PG16 0 (cid:6)100 1.5
antibody,PG9,isrepresentativeofarecentlydescribedgroupof
CD4-IgG–apl 1 0.6 0.6
BNmAbsthatdemonstratetremendousbreadthofneutralization
CD4-IgG 0 0.6 0.7
Aplaviroc 0.95 0.86 activity.ELISAstudiesofPG9andPG9-aplavirocperformedus-
2D7(positivecontrol) 0 0.2 0.1 inggp120fromHIVstrain16055,acladeCvirus,indicatednoloss
DEN3(negativecontrol) 0 (cid:6)1,000 (cid:6)1,000 ofbindingactivityforPG9followingconjugation.Theneutraliz-
aapl,aplaviroc. ing ability of PG9-aplaviroc was assessed against a panel of 117
4988 jvi.asm.org JournalofVirology
Potent,BroadlyActiveBispecificAnti-HIVAntibodies
D
o
w
n
lo
a
d
e
d

f
r
o
m

h
t
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:
/
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FIG3 EvaluationoftheneutralizationabilitiesofantibodyconjugatesagainstHIV-1strainsJR-FLandYU2andoftheCD4-IgG2–aplavirocconjugateagainst il
9
HIV-1strainsJR-FL,YU2,and92RW.HIV-1JR-FLisresistanttoneutralizationbyparentantibodiesPG9andPG16.Thebottomrightgraphshowsresultsfor ,

controlsonly:2D7,positive-controlMAb;DEN3,negative-controlMAb;aplaviroc,small-moleculepositivecontrol. 2
0
1
3

b
pseudoviruses representing major circulating HIV-1 subtypes neutralizedbyPG9-aplavirocwithameanIC of6(cid:1)g/ml(Table y
50
(Table2;seealsosectionS5inthesupplementalmaterial).PG9 3).PG9-aplavirocneutralizedall117ofthesubtypeswithIC s K
50 r
neutralized101ofthe117virusestestedwithIC sbelow50(cid:1)g/ below17(cid:1)g/mlandwithameanIC 28(cid:1)g/ml(seethesupple- e
50 50 s
ml. The 16 viruses that were not well neutralized by PG9 were mentalmaterial).PG9-aplavirocneutralized94.9%ofthe117vi- g
e

L
ib
r
TABLE2Summaryofneutralizationdataforapanelof117HIV-1strains a
r
y
,

T
h
e

S
c
r
ip
p
s

R
e
s
e
a
r
c
h

I
n
s
t
it
u
t
e
aAplavirocneutralizationwithIC sof(cid:7)100nMandIC sof(cid:7)100nM.
50 80
May2013 Volume87 Number9 jvi.asm.org 4989
Gavrilyuketal.
TABLE3SummaryofthepseudovirusesneutralizedbyPG9-aplaviroc TABLE4SummaryofthepseudovirusesneutralizedbyPG9-aplaviroc
withIC sof(cid:7)50 (cid:1)g/mlthatarenotneutralizedbyPG9(IC s,(cid:6)50 withIC sof(cid:7)50 (cid:1)g/mlthatarenotneutralizedbyPG9(IC s,(cid:6)50
50 50 80 80
(cid:1)g/ml) (cid:1)g/ml)
IC titerinTZM-blcells IC titerinTZM-blcells
50 80
((cid:1)g/ml)c ((cid:1)g/ml)c
VirusIDa Cladeb PG9 PG9-aplaviroc VirusIDa Cladeb PG9 PG9-aplaviroc
QH0692.42 B (cid:6)50 10.817 WEAU_d15_410_5017 B(T/F) (cid:6)50 19.772
D
1054_07_TC4_1499 B(T/F) (cid:6)50 6.916 HIV-16845-2.22 C (cid:6)50 18.415 o
6244_13_B5_4576 B(T/F) (cid:6)50 7.674 620345.c01 CRF01_AE (cid:6)50 14.409 w
62357_14_D3_4589 B(T/F) (cid:6)50 4.305 0815.v3.c3 ACD (cid:6)50 7.406 nlo
ZM214M.PL15 C (cid:6)50 2.136 6480.v4.c25 CD (cid:6)50 8.097 a
Ce1086_B2 C(T/F) (cid:6)50 1.183 6952.v1.c20 CD (cid:6)50 11.544 de
Ce2010_F5 C(T/F) (cid:6)50 11.145 6811.v7.c18 CD (cid:6)50 24.897 d

246FC1G C(T/F) (cid:6)50 1.262 X2088_c9 G (cid:6)50 30.361 fr
o
7030102001E5(Rev-) C(T/F) (cid:6)50 6.763 3016.v5.c45 D (cid:6)50 1.571 m
CNE30 BC (cid:6)50 9.645 191821_E6_1 D(T/F) (cid:6)50 0.976 h
T251-18 CRF02_AG (cid:6)50 16.982 246FC1G C(T/F) (cid:6)50 3.500 tt
X2088_c9 G (cid:6)50 10.453 7030102001E5(Rev-) C(T/F) (cid:6)50 29.579 p:
/
6480.v4.c25 CD (cid:6)50 1.476 CNE30 BC (cid:6)50 41.902 /jv
6952.v1.c20 CD (cid:6)50 1.666 Ce1086_B2 C(T/F) (cid:6)50 4.069 i.
a
6811.v7.c18 CD (cid:6)50 5.080 ZM214M.PL15 C (cid:6)50 18.933 s
0815.v3.c3 ACD (cid:6)50 1.399 PVO.4 B (cid:6)50 8.239 m
.
aID,identification. TRO.11 B (cid:6)50 35.584 or
bT/F,transmitted/foundervirus. RHPA4259.7 B (cid:6)50 7.092 g/
cThePG9dataarehistoricaldataforthis117-viruspanelbridgedforthecurrent THRO4156.18 B (cid:6)50 20.071 o
etixteprerinimTeZnMtw-bitlhcaelslsetiso5f.r9e2p1re(cid:1)sge/nmtalt,iavnedvitrhuesems.eFaonr(cid:8)PGst9a-nadpalardvirdoecv,iathtieonmiesd6ia.1n8I1C(cid:8)50 16025444__0173__TB5C_44_5174699 BB((TT//FF)) (cid:6)(cid:6)5500 3268..069440 n Ap
4.703(cid:1)g/ml. 62357_14_D3_4589 B(T/F) (cid:6)50 17.254 ril
aID,identification. 9
,
ruseswithIC80sof(cid:7)50 (cid:1)g/ml;for22ofthepseudovirusstrains bcTTh/Fe,PtGra9ndsmataittaerde/hfoisutonrdicearlvdiartuasf.orthis117-viruspanelbridgedforthecurrent 20
1
naneutitbroadlizye,dPGby9,thweaPsGab9o-avpela5v0ir(cid:1)ocg/cmonlj(uTgaabtele,t4h)e.IUCn8l0ikoeftthheeppaarreenntt eTxZpMer-imblecnetllwsiisth17a.8se3t4o(cid:1)fgre/mprle,saenndtatthiveemvieraunse(cid:8)s.FstoarnPdaGr9d-dapevlaivatirioonc,itsh1e7m.6e5d5i(cid:8)an1IC2.8106t0iterin 3 b
antibody,theconjugateneutralizedallcladeBandCviruses,includ- (cid:1)g/ml. y
K
ingtransmitted/founderviruses.CladeCispredominantinSouthern r
bolictoxicity.Bothoftheselimitationsmightbeaddressedbythe e
andEastAfrica,India,andNepalandisresponsibleforabouthalfof s
developmentofaHAARTequivalentbasedonproteindrugs.For g
worldwideinfections.CladeBisthemajorcauseofinfectioninthe e
Americas,Europe,Japan,andAustralia.Thus,conjugationtoaplavi- example, antibodies have long in vivo half-lives. Native human L
IgG1hasat of23daysinhumans,andengineeredvariantsof ib
rocsignificantlyimprovedthebreadthofneutralizationability. 1/2 r
a
The PG9-aplaviroc conjugate demonstrated a dramatic im- r
y
provementovertheparentantibodyinthemaximumpercentage ,
TABLE5SummaryofMPIsaforPG9andPG9-aplaviroc T
ofinhibition(MPI)acrossthe117-viruspanel(Table5;seealso h
e
thesupplementalmaterial).PG9issensitivetotheglycosylation No.ofviruses
S
profile of a virus and cannot completely neutralize certain viral No.of neutralizedwithan c
strainsevenathighantibodyconcentrations(34).ThePG9-apla- viruses MPIof100% AvgMPI rip
viroc conjugate did not display this glycan sensitivity and was Cladeb tested PG9 PG9-aplaviroc PG9 PG9-aplaviroc ps
foundtobemorepotentthannativePG9whenMPIswerecom- A 8 3 6 97 99 R
pared.PG9andPG9-aplavirocneutralized40and84ofthe117 e
A(T/F) 3 3 3 100 100 s
viralisolatestestedatMPIsof(cid:6)99.5%,respectively. B 12 1 4 77 95 ea
B(T/F) 9 0 2 63 94 rc
DISCUSSION C 16 7 12 88 99 h

The success of HAART is predicated on the fact that although C(T/F) 15 10 13 76 97 In
s
HIV-1isahypermutatingvirus,atherapyconsistingofacombi- DandD(T/F) 5 1 3 83 99 t
nationofinhibitorstargetingthreeviralenzymes—reversetrans- G 7 1 4 85 98 itu
t
AC 4 1 3 98 100 e
criptase,protease,andintegrase—presentsastringentbarrierto
AG 9 3 7 92 97
viralreplication.HAARToftenreducesviralreplicationtounde-
AE 10 5 7 96 99
tectablelevelsinpatients.HAARTdoesnot,however,presentan
AE(T/F) 4 0 2 93 100
insurmountable barrier to viral replication, and drug resistance
ACD 2 0 0 29 84
canevolverapidlywhenpatientcomplianceispoor.Twokeyfac- BC 8 5 6 89 98
torsthatcontributetodrugcompliancefailurearefrequencyof CD 5 0 2 47 97
administration, which has been reduced to once daily for some aMPIsof(cid:6)99.5%wereroundedupto100%bytheanalysissoftware.
HAARTregimens,andadversesideeffects,oftendrivenbymeta- bT/F,transmitted/foundervirus.
4990 jvi.asm.org JournalofVirology
Potent,BroadlyActiveBispecificAnti-HIVAntibodies
theFcregionsofantibodiespromiset sof(cid:6)6months(35, 36). rocderivativesthatareamenabletolinkagetoproteinsorlong-
1/2
ThemetabolicliabilityofHIV-1small-moleculedrugsisapartic- livedcarriershavenotyetbeendescribed.
ular challenge, because the drugs are dosed chronically at very Wepreparedaplavirocconjugateswiththebroadlyneutral-
highcompoundloadinglevelsduetotheirshortpharmacokinetic izing antibodies b12, 2G12, PG9, PG16, and CD4-IgG. The
profiles.Themetabolismofantibodiesdoesnotcreatenewactive incorporationofaplavirocsignificantlyimprovedtheneutral-
metabolites. ization abilities of BNmAbs for strains resistant to or weakly
Both the high genetic variability and the diversity of HIV-1 neutralized by the parent antibodies. The antiviral activity of
pose challenges to the creation of a HAART therapy based on PG9-aplaviroc was analyzed against a panel of 117 different D
o
protein therapeutic agents such as antibodies. The extracellular viralstrains.Theconjugateneutralizedtheactivitiesof100%of w
targets on which this approach focuses are the HIV-1 envelope viralstrainswithIC50slowerthan50(cid:1)g/ml.Tothebestofour nlo
andthehostcellreceptorsCD4,CCR5,andCXCR4.Despitethe knowledge, none of the currently known BNmAbs neutralize a
highgeneticvariabilityandpreexistingdiversityoftheHIVenve- allofthese117cross-cladepseudoviruseswithIC50slowerthan de
lopeprotein,anumberofBNmAbsthatpossessexceptionalanti- 50 (cid:1)g/ml. Notably, compared to parent PG9-aplaviroc had d
f
HIV-1activityandbreadthhavebeencharacterized(1–8).Target- lower IC50s against 13 out of 32 tested transmitted/founder ro
ing the host cell receptors CD4, CCR5, and CXCR4 has the virusesofdifferentcladesandwasmorepotentthananyofthe m

advantageofgeneticstabilitybutcouldhavedeleteriouseffectsif parentmoleculesaloneagainst10oftheseviruses(seethesup- h
t
the therapeutic agent negatively impacts the host cells. Certain plementalmaterial).PG9-aplavirocalsodisplayedMPIssignif- tp
:
tshtuedraiepseuantidcaapnptiebaordtioespotasrsgesestipnrgomCDis4ingansdafeCtyCpRr5ofiarlees;inhocwlinevicearl, icanTthlyeimmepdrioanveIdCo50veorftthhoesPeGo9f-tahpelapvairreonctcaonntjiubgoadtey.against the //jvi.
panelwas2.76nM,whereasthatoftheparentantibody,PG9,was a
noneareapproveddrugs(37–39). s
0.77 nM. On a panel of 76 of 117 viruses, PG9-aplaviroc had m
The road to an approved drug cocktail is long and requires .
higherIC sthanthenakedPG9antibody(seesectionS5inthe o
independenttestingandapprovalofeachspecies.Oneapproach 50 r
supplementalmaterial),withmedianIC sof0.674nMforPG9 g
tostreamliningthisprocessistoconstructsingleentitiesthatpos- 50 /
and1.737nMforPG9-aplaviroc.Notably,aplavirocitselfwasnot o
sessmultipleactivities.Thefirststeptowardthisgoalisthecre- n
verypotentonthissetoftheviruses,withamedianIC of4.6nM.
ation of bispecific-antibody therapeutic agents. To address this 50 A
Wehypothesizethatduringtheneutralizationassaythereiscom- p
challenge,modifiedantibodyformats(e.g.,knobsintoholes[40], r
IgG-scFv[41],andDVD-Ig[21])havebeencreatedusingprotein petitionbetweenbindingtoCCR5andbindingtotheHIV-1en- il 9
velope, and thus, the conjugate is not always used in the most ,
engineeringmethods.Oftentheseapproachessufferfromprotein 2
instability and low expression yields, although the recently de- efficientneutralizationpathway.Sincethesetwomechanismsof 0
neutralizationareincompetitionwhentheconjugateisassayed 1
scribedpeptidefusionapproachovercomesmanyoftheseprob- 3
againstvirusesthatareverysensitivetoPG9neutralization,some b
lems(42).Theapproachusedhereinvolveschemicalconjugation reductioninpotencyagainsttheseisolatesisexpected. y
of a peptide or small molecule to a monoclonal antibody that K
CombiningpotentmoleculesthatpreventHIVentrythrough r
possessesadesiredspecificity.Theconjugationisperformedusing e
differentmechanismsintoasinglemultifunctionalmoleculemay s
amethodologythatallowsboththeMAbandtheprogramming g
createaninsurmountableevolutionarychallengelimitingthede- e
agenttobindtheirrespectivetargets.
velopmentofresistance.Thishypothesisissupportedbyarecent L
HereweconjugatedtheCCR5-specificdrugaplaviroctosev- reportbyZhouetal.showingthatHIVescapemutantsselectedby ib
r
eralbroadlyneutralizingantibodiesandCD4-IgG.Aplavirocwas a
exposure to entry inhibitors were much more sensitive to BN- r
withdrawnfromclinicalstudyfollowingtheobservationofidio- y
mAbsthantheoriginalvirus(44).Thechemicalapproachtothe ,

syncratic hepatotoxicity in phase IIb clinical trials (43). In hu- T
synthesisofmultispecificBNmAbsdescribedherewillallowready h
mans,aplavirochasahalf-lifeofapproximately3h,makinghigh access to various combinations of neutralizing antibodies and e

dosesanunavoidablerequirement.Wehypothesizedthatinthe S
host-protectingsmall-moleculedrugsthatbindtothereceptors c
contextofanantibodyconjugate,thehalf-lifeofaplavirocwould crucialforHIVentry(CCR5,CXCR4,CD4).Intelligentdesignof rip
be considerably extended. To test this, we prepared a trastu- thelinkerarchitecturewillallowthepreparationoftri-andtetras- p
s
zumab-aplavirocconjugatethroughatyrosine-selectivereaction. pecificantibodyconjugates. R
The plasma half-life of the trastuzumab-aplaviroc conjugate in e
s
micewassignificantlylonger(168h)thanthatofaplavirocalone ACKNOWLEDGMENTS e
a
(30min).Ithasbeenshownpreviouslythatchemicalprogram-
r
This work was supported by NIH grants AI095038 (C.F.B.), AI33292 c
ming of an antibody resulted in a 1,000-fold reduction of the h
(D.R.B.),AI100663(D.R.B.),andIAVI(D.R.B.).
amountofasmallmoleculerequiredfortherapeuticeffect(17). WethankB.Hahn,F.McCutchan,G.Shaw,D.Montefiori,M.Thom- In
Reductionoftheeffectivedosewilllikelymitigatethemetabolic son,J.Overbaugh,R.Swanstrom,L.Morris,J.Kim,L.Zhang,D.Ellens- stit
toxicityobservedwithanti-HIVdrugssuchasaplaviroc,although berger,andC.WilliamsonforcontributingtheHIV-1envelopeplasmids u
t
thisremainstobedemonstrated.Thetrastuzumab-aplaviroccon- usedintheextendedneutralizationpanel. e
jugatewasabletorecognizetheCCR5receptoronthesurfacesof
CCR5-expressing cells and efficiently blocked HIV entry at low REFERENCES
nanomolar concentrations. Thus, attributes of both the parent 1. BinleyJM,WrinT,KorberB,ZwickMB,WangM,ChappeyC,Stiegler
antibodyandthesmallmoleculeweremaintainedinthecontext G,KunertR,Zolla-PaznerS,KatingerH,PetropoulosCJ,BurtonDR.
2004. Comprehensive cross-clade neutralization analysis of a panel of
of the conjugate. Success with aplaviroc suggests that clinically
anti-humanimmunodeficiencyvirustype1monoclonalantibodies.J.Vi-
approved CCR5 inhibitors, such as maraviroc, can potentially
rol.78:13232–13252.
benefitfromevaluationasBNmAbconjugates.However,maravi- 2. DiskinR,ScheidJF,MarcovecchioPM,WestAP,Jr,KleinF,GaoH,
May2013 Volume87 Number9 jvi.asm.org 4991
Gavrilyuketal.
GnanapragasamPN,AbadirA,SeamanMS,NussenzweigMC,Bjork- 17. PopkovM,RaderC,GonzalezB,SinhaSC,BarbasCF,III.2006.Small
manPJ.2011.IncreasingthepotencyandbreadthofanHIVantibodyby molecule drug activity in melanoma models may be dramatically en-
usingstructure-basedrationaldesign.Science334:1289–1293. hancedwithanantibodyeffector.Int.J.Cancer119:1194–1207.
3. FalkowskaE,RamosA,FengY,ZhouT,MoquinS,WalkerLM,WuX, 18. Rader C, Sinha SC, Popkov M, Lerner RA, Barbas CF, III. 2003.
SeamanMS,WrinT,KwongPD,WyattRT,MascolaJR,PoignardP, Chemically programmed monoclonal antibodies for cancer therapy:
BurtonDR.2012.PGV04,anHIV-1gp120CD4bindingsiteantibody,is adaptorimmunotherapybasedonacovalentantibodycatalyst.Proc.Natl.
broadandpotentinneutralizationbutdoesnotinduceconformational Acad.Sci.U.S.A.100:5396–5400.
changescharacteristicofCD4.J.Virol.86:4394–4403. 19. WuellnerU,GavrilyukJI,BarbasCF,III.2010.Expandingtheconcept
4. ScheidJF,MouquetH,UeberheideB,DiskinR,KleinF,OliveiraTY, of chemically programmable antibodies to RNA aptamers: chemically D
PietzschJ,FenyoD,AbadirA,VelinzonK,HurleyA,MyungS,Boulad programmed biotherapeutics. Angew. Chem. Int. Ed. Engl. 49:5934– o
F,PoignardP,BurtonDR,PereyraF,HoDD,WalkerBD,SeamanMS, 5937. w
BjorkmanPJ,ChaitBT,NussenzweigMC.2011.Sequenceandstruc- 20. DoppalapudiVR,TryderN,LiLN,AjaT,GriffithD,LiaoFF,RoxasG, n
turalconvergenceofbroadandpotentHIVantibodiesthatmimicCD4 Ramprasad MP, Bradshaw C, Barbas CF, III. 2007. Chemically pro- lo
a
binding.Science333:1633–1637. grammedantibodies:endothelinreceptortargetingCovX-Bodies.Bioorg. d
5. WalkerLM,HuberM,DooresKJ,FalkowskaE,PejchalR,JulienJP, Med.Chem.Lett.17:501–506. e
d
WangSK,RamosA,Chan-HuiPY,MoyleM,MitchamJL,Hammond 21. Wu C, Ying H, Grinnell C, Bryant S, Miller R, Clabbers A, Bose S,
f
PW,OlsenOA,PhungP,FlingS,WongCH,PhogatS,WrinT,Simek McCarthy D, Zhu RR, Santora L, Davis-Taber R, Kunes Y, Fung E, ro
MD,KoffWC,WilsonIA,BurtonDR,PoignardP.2011.Broadneu- SchwartzA,SakorafasP,GuJ,TarcsaE,MurtazaA,GhayurT.2007. m
tralizationcoverageofHIVbymultiplehighlypotentantibodies.Nature Simultaneoustargetingofmultiplediseasemediatorsbyadual-variable- h
477:466–470. domainimmunoglobulin.Nat.Biotechnol.25:1290–1297. tt
p
6. WalkerLM,PhogatSK,Chan-HuiPY,WagnerD,PhungP,GossJL, 22. KovacsEW,HookerJM,RomaniniDW,HolderPG,BerryKE,Francis :
/
Wrin T, Simek MD, Fling S, Mitcham JL, Lehrman JK, Priddy FH, MB.2007.Dual-surface-modifiedbacteriophageMS2asanidealscaffold /jv
OlsenOA,FreySM,HammondPW,KaminskyS,ZambT,MoyleM, foraviralcapsid-baseddrugdeliverysystem.Bioconjug.Chem.18:1140– i.
KoffWC,PoignardP,BurtonDR.2009.Broadandpotentneutralizing 1147. a
s
antibodiesfromanAfricandonorrevealanewHIV-1vaccinetarget.Sci- 23. ChenW,ZhanP,DeClercqE,LiuX.2012.Recentprogressinsmall m
ence326:285–289. molecule CCR5 antagonists as potential HIV-1 entry inhibitors. Curr. .
o
7. WuX,YangZY,LiY,HogerkorpCM,SchiefWR,SeamanMS,Zhou Pharm.Des.18:100–112. r
g
T,SchmidtSD,WuL,XuL,LongoNS,McKeeK,O’DellS,LouderMK, 24. LiuT,WengZ,DongX,HuY.2010.Recentadvancesinthedevelop- /

WycuffDL,FengY,NasonM,Doria-RoseN,ConnorsM,KwongPD, mentofsmall-moleculeCCR5inhibitorsforHIV.MiniRev.Med.Chem. o
n
RoedererM,WyattRT,NabelGJ,MascolaJR.2010.Rationaldesignof 10:1277–1292.
A
envelopeidentifiesbroadlyneutralizinghumanmonoclonalantibodiesto 25. DorrP,WestbyM,DobbsS,GriffinP,IrvineB,MacartneyM,MoriJ,
p
HIV-1.Science329:856–861. RickettG,Smith-BurchnellC,NapierC,WebsterR,ArmourD,Price r
8. WuX,ZhouT,ZhuJ,ZhangB,GeorgievI,WangC,ChenX,Longo D,StammenB,WoodA,PerrosM.2005.Maraviroc(UK-427,857),a il 9
NS,LouderM,McKeeK,O’DellS,PerfettoS,SchmidtSD,ShiW,Wu potent, orally bioavailable, and selective small-molecule inhibitor of ,
2
L, Yang Y, Yang ZY, Yang Z, Zhang Z, Bonsignori M, Crump JA, chemokinereceptorCCR5withbroad-spectrumanti-humanimmunode-
0
Kapiga SH, Sam NE, Haynes BF, Simek M, Burton DR, Koff WC, ficiencyvirustype1activity.Antimicrob.AgentsChemother.49:4721– 1
Doria-Rose NA, Connors M, Mullikin JC, Nabel GJ, Roederer M, 4732. 3

ShapiroL,KwongPD,MascolaJR.2011.FocusedevolutionofHIV-1 26. HuntJS,RomanelliF.2009.Maraviroc,aCCR5coreceptorantagonist b
y
neutralizingantibodiesrevealedbystructuresanddeepsequencing.Sci- thatblocksentryofhumanimmunodeficiencyvirustype1.Pharmaco-
K
ence333:1593–1602. therapy29:295–304. r
e
9. Hessell AJ, Hangartner L, Hunter M, Havenith CE, Beurskens FJ, 27. KromdijkW,HuitemaAD,MulderJW.2010.TreatmentofHIVinfec- s
BakkerJM,LaniganCM,LanducciG,ForthalDN,ParrenPW,Marx tionwiththeCCR5antagonistmaraviroc.ExpertOpin.Pharmacother. g
e
PA,BurtonDR.2007.Fcreceptorbutnotcomplementbindingisimpor- 11:1215–1223.
L
tantinantibodyprotectionagainstHIV.Nature449:101–104. 28. LorenzenT.2010.Profileofmaraviroc:aCCR5antagonistintheman- ib
10. ParrenPW,MarxPA,HessellAJ,LuckayA,HarouseJ,Cheng-Mayer agement of treatment-experienced HIV patients. HIV AIDS (Auckl.) r
a
C, Moore JP, Burton DR. 2001. Antibody protects macaques against 2:151–156. r
y
vaginalchallengewithapathogenicR5simian/humanimmunodeficiency 29. GavrilyukJ,UeharaH,OtsuboN,HessellA,BurtonDR,BarbasCF, ,

virusatserumlevelsgivingcompleteneutralizationinvitro.J.Virol.75: III.2010.PotentinhibitionofHIV-1entrywithachemicallyprogrammed T
8340–8347. antibodyaidedbyanefficientorganocatalyticsynthesis.Chembiochem h
e
11. Balazs AB, Chen J, Hong CM, Rao DS, Yang L, Baltimore D. 2012. 11:2113–2118.
S
Antibody-based protection against HIV infection by vectored immu- 30. Gavrilyuk J, Ban H, Nagano M, Hakamata W, Barbas CF, III. 2012. c
noprophylaxis.Nature481:81–84. Formylbenzene diazonium hexafluorophosphate reagent for tyrosine- rip
12. JohnsonPR,SchneppBC,ZhangJ,ConnellMJ,GreeneSM,YusteE, selectivemodificationofproteinsandtheintroductionofabioorthogonal p
DesrosiersRC,ClarkKR.2009.Vector-mediatedgenetransferengenders aldehyde.Bioconjug.Chem.23:2321–2328. s

long-livedneutralizingactivityandprotectionagainstSIVinfectionin 31. HookerJM,KovacsEW,FrancisMB.2004.Interiorsurfacemodification R
e
monkeys.Nat.Med.15:901–906. ofbacteriophageMS2.J.Am.Chem.Soc.126:3718–3719.
s
13. Saphire EO, Parren PW, Barbas CF, III, Burton DR, Wilson IA. 32. SchlickTL,DingZ,KovacsEW,FrancisMB.2005.Dual-surfacemod- e
a
2001. Crystallization and preliminary structure determination of an ificationofthetobaccomosaicvirus.J.Am.Chem.Soc.127:3718–3723. r
intacthumanimmunoglobulin,b12:anantibodythatbroadlyneutral- 33. NakataH,MaedaK,MiyakawaT,ShibayamaS,MatsuoM,TakaokaY, ch
izesprimaryisolatesofHIV-1.ActaCrystallogr.DBiol.Crystallogr. ItoM,KoyanagiY,MitsuyaH.2005.Potentanti-R5humanimmuno-
I
57:168–171. deficiencyvirustype1effectsofaCCR5antagonist,AK602/ONO4128/ n
s
14. ZwickMB,BonnycastleLL,MenendezA,IrvingMB,BarbasCF, III, GW873140,inanovelhumanperipheralbloodmononuclearcellnon- tit
ParrenPW,BurtonDR,ScottJK.2001.Identificationandcharacteriza- obesediabetic-SCID,interleukin-2receptorgamma-chain-knocked-out u
t
tionofapeptidethatspecificallybindsthehuman,broadlyneutralizing AIDSmousemodel.J.Virol.79:2087–2096. e
anti-humanimmunodeficiencyvirustype1antibodyb12.J.Virol.75: 34. DooresKJ,BurtonDR.2010.Variableloopglycandependencyofthe
6692–6699. broadandpotentHIV-1-neutralizingantibodiesPG9andPG16.J.Virol.
15. Gavrilyuk JI, Wuellner U, Barbas CF, III. 2009. Beta-lactam-based 84:10510–10521.
approach for the chemical programming of aldolase antibody 38C2. 35. HintonPR,XiongJM,JohlfsMG,TangMT,KellerS,TsurushitaN.
Bioorg.Med.Chem.Lett.19:1421–1424. 2006.AnengineeredhumanIgG1antibodywithlongerserumhalf-life.J.
16. Gavrilyuk JI, Wuellner U, Salahuddin S, Goswami RK, Sinha SC, Immunol.176:346–356.
BarbasCF,III.2009.Anefficientchemicalapproachtobispecificanti- 36. IgawaT,TsunodaH,KuramochiT,SampeiZ,IshiiS,HattoriK.2011.
bodiesandantibodiesofhighvalency.Bioorg.Med.Chem.Lett.19:3716– Engineering the variable region of therapeutic IgG antibodies. MAbs
3720. 3:243–252.
4992 jvi.asm.org JournalofVirology
Potent,BroadlyActiveBispecificAnti-HIVAntibodies
37. Bruno CJ, Jacobson JM. 2010. Ibalizumab: an anti-CD4 monoclonal 42. LaFleurDW,AbramyanD,KanakarajP,SmithRG,ShahRR,WangG,
antibodyforthetreatmentofHIV-1infection.J.Antimicrob.Chemother. YaoX-T,KankanalaS,BoydE,ZaritskayaL,NamV,PufferBA,Buasen
65:1839–1841. P,KaithamanaS,BurnetteAF,KrishnamurthyR,PatelD,RoschkeVV,
38. Song R, Franco D, Kao CY, Yu F, Huang Y, Ho DD. 2010. Epitope Kiener P, Hilbert DM, Barbas CF, III. 2013. Monoclonal antibody
mapping of ibalizumab, a humanized anti-CD4 monoclonal antibody therapeuticswithuptofivespecificities:functionalenhancementthrough
withanti-HIV-1activityininfectedpatients.J.Virol.84:6935–6942. fusionoftarget-specificpeptides.mAbs5:208–218.
39. TenorioAR.2011.ThemonoclonalCCR5antibodyPRO-140:theprom- 43. Nichols WG, Steel HM, Bonny T, Adkison K, Curtis L, Millard J,
iseofonce-weeklyHIVtherapy.Curr.HIV/AIDSRep.8:1–3. KabeyaK,ClumeckN.2008.Hepatotoxicityobservedinclinicaltrials
40. RidgwayJB,PrestaLG,CarterP.1996.‘Knobs-into-holes’engineering of aplaviroc (GW873140). Antimicrob. Agents Chemother. 52:858–
D
ofantibodyCH3domainsforheavychainheterodimerization.Protein 865. o
Eng.9:617–621. 44. Zhou N, Fan L, Ho HT, Nowicka-Sans B, Sun Y, Zhu Y, Hu Y, w
41. Bostrom J, Yu SF, Kan D, Appleton BA, Lee CV, Billeci K, Man W, McAuliffeB,RoseB,FangH,WangT,KadowJ,KrystalM,Alexander n
PealeF,RossS,WiesmannC,FuhG. 2009.Variantsoftheantibody L,ColonnoR,LinPF.2010.IncreasedsensitivityofHIVvariantsselected lo
a
herceptinthatinteractwithHER2andVEGFattheantigenbindingsite. byattachmentinhibitorstobroadlyneutralizingantibodies.Virology402: d
Science323:1610–1614. 256–261. e
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May2013 Volume87 Number9 jvi.asm.org 4993