Novel anti-Sialyl-Tn monoclonal antibodies and antibody-drug conjugates demonstrate tumor

14 Pages·2017·1.53 MB·English

by  Jillian M. Prendergast

Novel anti-Sialyl-Tn monoclonal antibodies and
antibody-drug conjugates demonstrate tumor
specificity and anti-tumor activity
Citation
Prendergast, Jillian M., Ana Paula Galvao da Silva, David A. Eavarone, Darius Ghaderi, Mai
Zhang, Dane Brady, Joan Wicks, Julie DeSander, Jeff Behrens, and Bo R. Rueda. 2017. “Novel
anti-Sialyl-Tn monoclonal antibodies and antibody-drug conjugates demonstrate tumor
specificity and anti-tumor activity.” mAbs 9 (4): 615-627. doi:10.1080/19420862.2017.1290752.
http://dx.doi.org/10.1080/19420862.2017.1290752.
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doi:10.1080/19420862.2017.1290752
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MABS
2017,VOL.9,NO.4,615–627
http://dx.doi.org/10.1080/19420862.2017.1290752
REPORT
Novel anti-Sialyl-Tn monoclonal antibodies and antibody-drug conjugates
demonstrate tumor specificity and anti-tumor activity
JillianM.Prendergasta,AnaPaulaGalvaodaSilvaa,*,DavidA.Eavaronea,*,DariusGhaderi a,MaiZhanga,DaneBradyb,
JoanWicksb,JulieDeSandera,JeffBehrensa,andBoR.Ruedac,d
aSiamabTherapeutics,Inc.,Newton,MA,USA;bAliz(cid:1)eePathology,LLC,Thurmont,MD,USA;cVincentCenterforReproductiveBiology,Departmentof
ObstetricsandGynecology,MassachusettsGeneralHospital,Boston,MA,USA;dHarvardMedicalSchool,Boston,MA,USA
ABSTRACT ARTICLEHISTORY
Targetedtherapeuticsthatcandifferentiatebetweennormalandmalignanttumorcellsrepresenttheideal Received16December2016
standard for the development of a successful anti-cancer strategy. The Sialyl-Thomsen-nouveau antigen Revised25January2017
(STn or Sialyl-Tn, also known as CD175s) is rarely seen in normal adult tissues, but it is abundantly Accepted30January2017
expressedinmanytypesofhumanepithelialcancers.Wehaveidentifiednovelantibodiesthatspecifically KEYWORDS
target with high affinity the STn glycan independent of its carrier protein, affording the potential to AntibodyDrugConjugate
recognize a wider array of cancer-specific sialylated proteins. A panel of murine monoclonal anti-STn (ADC);breastcancer;CD175s;
therapeutic antibodies were generated and their binding specificity and efficacy were characterized in coloncancer;ovariancancer;
vitroandininvivomurinecancermodels.Asubsetoftheseantibodieswereconjugatedtomonomethyl sialyl-Tn;STn;Tumor-
auristatin E (MMAE) to generate antibody-drug conjugates (ADCs). These ADCs demonstrated in vitro AssociatedCarbohydrate
efficacy in STn-expressing cell lines and significant tumor growth inhibition in STn-expressing tumor Antigen(TACA)
xenograftcancermodelswithnoevidenceofoverttoxicity.
Introduction
leading to more aggressive cancer cell behaviors.6-9 Truncated
Cancer is the second leading cause of death in the US, with O-glycansareoneclassoftumor-associatedcarbohydrateanti-
595,690deathsand1.69millionnewcasesexpectedin2016.1A gens(TACAs)10-12thatcanbetargetedbycancertherapy,par-
targeted therapy that reliably differentiates between normal ticularlywhenpresentedoncellsurfaceglycoproteins.
andmalignanttumorcellsrepresentsthegoldstandardforcan- STn is expressed in numerous human adenocarcinomas,
cer treatment. Glycosylation of proteins is one of the most includingbreast,ovarian,bladder,cervical,colon,pancreaticand
abundant and diverse post-translational modifications, with lungcancers.3,5,13-15Thepresenceofcellsurface/membraneSTn
more than half of all human proteins estimated to be glycosy- intumorsisassociatedwithtumorigenesis,metastaticpotential,
lated.2 Aberrant glycosylation has been implicated in an array immunesuppression,chemoresistanceandpoorprognosis;3,14,16
of human diseases and is a common feature of cancer cells.3,4 therefore, STn is an attractive therapeutic target. Therapeutic
One altered glycosylation pathway associated with malignancy approaches targeting STn have consisted primarily of STn vac-
isO-glycanbiosynthesis.IncompleteO-glycosylationresultsin cines. The most advanced clinical candidate was Theratope, a
truncated glycans, such as Thomsen-nouveau (Tn; GalNAc- therapeuticvaccineconsistingofSTncoupledtokeyholelimpet
a1-O-Ser/Thr) antigen and its sialylated version sialyl-Tn hemocyanin (KLH). In murine mammary carcinoma models,
(STn; Siaa2–6GalNAc-a1-O-Ser/Thr, also known as CD175s). Theratope immunization induced a potent antibody response
AberrantSTnexpressionisassociatedwithdysregulationofthe that delayed tumor growth.17 However, Theratope failed to
O-glycosylation machinery, including imbalanced expression achieve its primary end point ina Phase 3 clinicaltrial not due
of molecular chaperone Cosmc/T-synthase and STn synthase totoxicitybuttolackofefficacyinpartpossiblyduetothebroad
(ST6GalNAc-I).Cosmc is a chaperone of T-synthase and both variabilityofSTnexpressioninbreastcancertissues.3,18
are necessary for the addition of glycan structures to Tn in TACAsarepoorlyimmunogenic,andthusmakingeffective
mucin-type core-1 O-glycans.5 STn synthase is an a2,6-sialyl- vaccines or antibodies against these targets has proven diffi-
transferasethatcatalyzesthetransferofaterminalsialicacidto cult.14Previousantibodydevelopmenteffortsusedpurifiedgly-
Tn, effectively capping O-glycans and preventing any further coproteins from cancer samples and Freund’s adjuvant, or
glycan additions. De novo STn expression via STn synthase mucin-coated heat-inactivated bacteria, for mouse immuniza-
transfection can change a tumor’s malignant phenotype, tion. These approaches have resulted in the development of
CONTACT JillianM.Prendergast [email protected] 90BridgeSt.Suite100,Newton,MA02458,USA.
*Theseauthorscontributedequallytothiswork.
Supplementaldataforthisarticlecanbeaccessedonthepublisher’swebsite.
PublishedwithlicensebyTaylor&FrancisGroup,LLC.©SiamabTherapeutics,Inc.
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttribution-NonCommercial-NoDerivativesLicense(http://creativecommons.org/licenses/by-nc-nd/
4.0/),whichpermitsnon-commercialre-use,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited,andisnotaltered,transformed,orbuiltupon
inanyway.
616 J.M.PRENDERGASTETAL.
several murine anti-STn monoclonal antibodies (mAbs), ADCs consisting of anti-STn mAbs, conjugated to MMAE,
including B72.319 (and its successor antibody CC4920), which demonstrate high affinity, specificity and anti-tumor
TKH2,21andHB-STn1(clone3F121,22),andothers.14Thetarget activityinvitroandinvivo.
specificity of these mAbs have come into question as these
mAbs bind additional glycan targets and may have glycopro-
tein preferences for antigen recognition.23 Advances in adju- Results
vant technology and immunization strategies have enabled
Antibodyspecificityandepitopemapping
high titer and desirable antibody maturation responses to his-
torically difficult immunization targets.24 We used immune Generatinghighspecificityandaffinityanti-STnmAbsdepends
modulatoryandenhanceddeliveryofaTLR9agonist(CpGoli- ontheabilitytopreciselyprofiletheglycanbindingspecificities
godeoxynucleotides) and AbISCO, an adjuvant composed of ofcandidateantibodies,independentoftheproteincarrier.We
saponin, phospholipid and cholesterol that acts both as an developedaglycanarrayincollaborationwithDr.AjitVarki31
immunostimulant and delivery agent. These immunization thatcontains 71chemically synthesizedandwell-characterized
optimizationstrategiesandsynergisticadjuvants(AbISCO-100 glycans (Table S1). The glycan array includes several STn-
and ODN 2395) enabled the generation of high affinity, STn- related structures, allowing determination of the exact binding
specificmAbs. epitope of each antibody (Fig. 1). The information generated
Antibody-drugconjugates(ADCs)utilizeamAbasatarget- from the glycan array, and other binding assays, enables us to
ingtoolfordeliveringapotentcytotoxicpayloadspecificallyto determinetheprecisespecificityoftheanti-STnantibodiesgen-
cancer cells. An STn-specific ADC may overcome shortcom- erated and identify antibodies that do not cross-react with
ings of previous attempts to target STn with therapeutic vac- relatedglycanspresentintheglycanarray.
cines. ADCs enable dosing at therapeutic concentrations, do The glycan array technology utility was demonstrated by
notrelyonvariableimmunesystemresponses,andadditionally characterizationof commerciallyavailableanti-STn mAbs.The
offer the promise of companion diagnostic development to binding epitope for these mAbs is purported to be ‘Neu5Ac-
identifypatientsmostlikelytobenefitfromtherapy.Thespeci- a-2,6-galNAc’ (as per manufacturer’s description). However,
ficity and targeting capabilities of ADCs have resulted in commercialmAbsrecognizemultipleSTn-relatedoligosacchar-
numerousdrugswithclinicalefficacyandfavorablesafetypro- idesincludingtheTnantigen(GalNAc)(Fig.2).Anti-STnanti-
files.25-27 We used the microtubule disrupting agent mono- body 3H1951 did not recognize STn or Tn structures, but
methylauristatinE(MMAE)withaMC-vc-PABlinkersystem, instead bound Neu5,9Ac2-ST/Neu5Gc-ST (Neu5Gca3Gal
whichhasbeendemonstratedeffectiveinkillingtumorantigen b3GalNAc/Neu5,9Ac2a3Galb3GalNAc).In contrast, ourinter-
expressingcellsinvitroalongwithneighboringnegativetumor nally generated anti-STn mAb 8C2–2D6 selectively binds to
cells through bystander killing,28 and successful in vivo and STn, but no related structures (Fig. 2). Additionally, we used
humanclinicalstudies,leadingtotheFoodandDrugAdminis- flow cytometry and enzyme-linked immunosorbent assays
tration (FDA)’s approval of the product brentuximab vedotin (ELISA) to map the antibodies’ binding epitope and determine
(Adcetris(cid:1)).29,30 Here, we report the development of novel relative affinities. We tested for sensitivity to mild periodate
Figure1. Bindinggroupsdeterminedforanti-STnantibodies.mAbsweregroupedbasedontheirbindingspecificitiestowardO-glycans.Group1:mAbsbindSTnonly
(Neu5Aca2,6GalNAcaO).Group2:mAbsbindtoNeu5Aca2,6Gal(NAc)aO.Group3:mAbsbindtoNeu5Aca2,6Gal(NAc)aOandNeu5Aca2,6Gal(NAc)b1,4Glc(NAc)bO.Group
4:mAbsbindtoSTnandTn(GalNAcaO).AllmAbsalsobindthecorresponding9-O-acetylatedandNeu5GcO-glycans.Theredshadedcirclesinthefigureandnon-greyed
outglycansinthetablerepresentthedetectedepitopeforeachgroup.
MABS 617
Figure2.Representativebindingprofilesofanti-STnantibodiesontheglycanarray.Anti-STncommercialantibodieswerepurchasedfromvendors(B72.3ThermoScien-
tific;CC49and3H1951,SantaCruzBiotechnology;STn219Abcam).Allantibodiesweretestedusing1mg/mLconcentration.Commercialantibodiesrecognizemultiple
STn-relatedoligosaccharidesincludingtheTnantigen(GalNAc).
treatment to determine whether the antibodies bound to the immunization,includingthefinalboost.Serawasscreenedwith
side chain of terminal sialic acids. This periodate oxidation of ELISAs and glycan arrays to identify mice with desirable anti-
terminalsialicacidsremovesthesidechain,givingrisetotheC- STnbindingprofiles,andtoidentifyoptimaltimingforsplenic
7 analogs of these sialic acids.32 The commercially available collection. Splenocytes were harvested and fused to establish
mAbsB72.3andCC49boundequallywelltomucinsafterperi- hybridomas and an anti-STn mouse antibody panel. Hybrido-
odatetreatment,suggestingthatthebindingofthesemAbsdoes mas were generated and screened for specificity and affinity to
notrelyonthesidechainnoracetylationofsialicacid. STnantigenbyFACS,ELISAandglycanarray.Selectedhybrid-
Together,ELISA,flowcytometryandglycanarrayswereused omas were expanded for antibody production and cell pelleted
tocharacterizethebindingofthepanelof8anti-STnmAbs.The for protein sequencing. Antibodies were purified and in some
resultsfromELISA and flowcytometrydemonstratednanomo- cases made recombinantly to enhance production, purification
larEC50s,suggestingrobustbindingaffinity(Table1).Thegly- andanalysis.Anexampleoftheevolutionoftheanti-STnanti-
can binding profile determined from the glycan array further bodyresponseleadingtocandidate2G12–2B2isrepresentedin
demonstratedSTnspecificbindingformostmAbstested. Fig. 3. Additionally, serum ELISA immune responses are
detailedinTableS2.ArobustpanelofmAbswasnarrowedto8
forfullbindingcharacterization(Table1).
Generationandcharacterizationofanti-STncandidate
mAbs
Immunohistochemistrytoconfirmcancerspecificity
Six to eight-week-old female mice (BALB/c) were immunized
subcutaneously(50mLpersite,4sites,200mL/mouse)ondays Tissue microarrays (TMAs) consisting of a panel of formalin-
0, 14, 28 and 42 with immunogen and an adjuvant cocktail fixedneoplasticandnormaltissueswerestainedwithanti-STn
(AbISCO-100andODN2395).Bloodwascollectedbeforeeach mAbstodeterminereactivitywithneoplasticandnormalcells.
Table1.Anti-STnmAbbindingresultssummary.
ELISAEC50(nM) GlycanArray Internalization
FACSEC50(nM)
mAb Isotype BSM1 OSM2 PSM3 (%)4 BindingGroup5 MDA-MB-231STnC MeanFI p-value
8C2–2D6 IgG2a,k 0.4 0.5 0.3 99 1 2.8 7.6 0.03
2G12–2B2 IgG2a,k 0.1 0.1 0.1 99 1 0.3 6.7 0.01
4G8–1E3 IgG2a,k 1.2 0.6 0.1 99 1 0.7 7.4 0.01
S3F(cid:1) IgG2a,k 0.3 0.1 0.2 98 1 3.1 8.1 0.001
5G2–1B3 IgG1,k 0.4 0.2 10.2 92 4 0.2 ND ND
2C2–2C5 IgG3,k 1 0.2 0.2 100 1 0.8 6.2 0.03
5E6–2E7 IgG3,k 5.8 0.8 0.4 100 1 1.8 7.5 0.04
9F11–1F7 IgG3,k 4.6 0.9 0.9 99 1 0.8 5.5 0.05
SummaryTableofbindingresults.
1BSMisbovinesubmaxillarymucin.2PSMisPorcineSubmaxillaryMucin.3OSMisovinesubmaxillarymucin.4%signalontheglycanarray;thehigherthepercentagethe
moreSTnspecifictheantibody.5ThebindinggroupseeFig.1.NDisnotdetermined.ELISAEC50sweredeterminedaftersubtractingsignalfromperiodatetreatedwells
fromnon-treatedwells.(cid:1)S3Fwasnotgeneratedduringourimmunizations–thisisderivedfromHB-STnclone3F1.WeclassswitchedfrommouseIgG1andrecombi-
nantlyexpressedasamouseIgG2a,werefertothisengineeredantibodyas“S3F”inthetext.
618 J.M.PRENDERGASTETAL.
abroadrangeofhumanepithelialcancers.Examplesof2G12–
2B2 staining of normal tissue and neoplastic lung, pancreas
and colon tissue are detailed in Fig. 4. Urinary bladder and
ovariancanceralsoyieldedastrongmembranestainingpattern
with high frequency. A summary of positive IHC-scored neo-
plastic TMA cores with the other 7 mAbs tested is shown in
Table2.
Invitrointernalizationassays
To determine whether anti-STn mAbs were internalized upon
binding to the cell surface, and therefore candidates for cyto-
Figure 3.Immunization resulted in STn specific antibodies as seen in sera and toxicpayloadconjugation,allmAbsweretestedforinternaliza-
purifiedhybridomasupernatants.Immuneresponseandantibodyevolutionfrom
asingleanimaldemonstratedsignificantandspecificSTnbinding.Theantibody tion in STn-expressing human breast cancer cells. Eight anti-
bindstoall4variantsofSTnincludedonthearray(Neu5Ac/Gcand9-Oacetylated STn mAbs and an isotype control were conjugated to a pH
forms).Antibodygenerationwasmonitoredinserumafterimmunization(Day35, reactive dye per manufacturer’s recommendations (pHAb
49and63)usingtheglycanarray.STn-specificantibodieswereobservedstarting
onDay63,nosignificantbindingwasobservedtonon-STnglycans.Thismouse Reactive Dye, Promega catalog number G9845). This dye
wasusedtogeneratehybridomasandclone2G12–2B2wasselectedforSTnspeci- becomes fluorescent only upon internalization into lower pH
ficity.Purifiedantibodiesfromthishybridomasupernatantdemonstratedsignifi- organelles such as lysosomes. Six of eight mAbs (S3F, 4G8–
cantandspecificSTnbindingontheglycanarray.
1E3, 2G12–2B2 P<0.01; 8C2–2D6, 2C2–2C5, and 5E6–2E7
P<0.05)showedsignificantinternalizationintoSTn-expressing
Optimal immunohistochemistry (IHC) staining conditions MDA-MB-231 cells as compared with non-expressing cells
were determined and validated on appropriate STn-positive (Fig. 5A, Table 1). Poor 5G2–1B3 recovery after conjugation
and STn-negative control cells and tissues before testing on didnotallowforcomparisontoothertestedmAbs.Theisotype
TMAs (data not shown). The IHC-stained TMAs were evalu- control MOPC173 mAb did not internalize (p>0.05) into
atedandscoredforcelltype,subcellularlocalization,andstain- eitherSTnCorSTn-cells.
ing intensity and frequency by a board-certified pathologist Internalization also was examined using an Alexa 488-
(JW)withextensiveexperienceinimmunohistochemicalanaly- labeled S3F antibody in STn-expressing and non-expressing
sis. The specific reactivity of candidate anti-STn mAbs was MDA-MB-231 cells. The Alexa 488-labeled S3F antibody only
evaluatedacross13differenttumortypesand30normaltissues stained STnC cells and optimal staining was achieved with
(Table 2). For a given tissue, the staining patterns for all anti- 5 mg/mL (Fig. 5B). Internalization appeared as early as
STnmAbstestedweresimilarintermsofcelltypeandsubcel- 15minutesandwasstronglyevidentat60minutes.
lular location (e.g., cytoplasm versus membrane), and differed
by intensity and frequency of staining. There was infrequent
ADCconjugationandinvitroviabilityassays
membrane staining of the normal, non-neoplastic tissue. Any
staining observed in normal tissues was generally cytoplasmic, Effects of unconjugated and MMAE-conjugated mAbs on cell
or involved staining of apical membranes. Neither of these viability were compared in both transfected STn-expressing
locations are expected to be readily accessible to anti-STn (STnC)andparental(STn-)MDA-MB-231humanbreastcan-
ADCs in the circulation. In comparison, there was positive cer cell lines. While the MMAE-conjugated isotype control
membrane reactivity (not limited to apical membranes) across mAb had no effect on cell viability, MMAE-conjugated
Table2.BindingspecificityofantibodypanelonhumanTMA.
S3F 4G8–1E3 8C2–2D6 5G2–1E3
Ovarian: 3/9 Ovarian: 5/9 Ovarian: 4/9 Ovarian: 2/9
Urinarybladder: 5/9 Urinarybladder: 3/9 Urinarybladder: 3/9 Urinarybladder: 2/9
Colorectal: 7/9 Colorectal: 7/9 Colorectal: 6/9 Colorectal: 7/9
Pancreatic: 9/10 Pancreatic: 8/10 Pancreatic: 8/10 Pancreatic: 7/10
Lung: 2/2 Lung: 2/2 Lung: 2/2 Lung: 1/2
Stomach: 9/10 Stomach: 9/10 Stomach: 9/10 Stomach: 2/10
5E6–2E7 2G12–2B2 9F11–1F7 2C2–2C5
Ovarian: 1/9 Ovarian: 5/9 Ovarian: 1/9 Ovarian: 2/9
Urinarybladder: 2/9 Urinarybladder: 3/9 Urinarybladder: 1/9 Urinarybladder: 1/9
Colorectal: 5/9 Colorectal: 7/9 Colorectal: 4/9 Colorectal: 5/9
Pancreatic: 6/10 Pancreatic: 6/10 Pancreatic: 3/10 Pancreatic: 7/10
Lung: 1/2 Lung: 2/2 Lung: 0/2 Lung: 0/2
Stomach: 6/10 Stomach: 8/10 Stomach: 2/10 Stomach: 5/10
Antibodycandidates’stainingoftumorcellsispresentedasafractionofstainedTMAfromtotalTMAstested.Thirteencommontumortypesweretested,oftheseovarian,
urinarybladder,colorectal,pancreatic,lungandstomacharedescribedinTable2.Neoplasticsubtypesasfollows:ovarianwasacombinationofserous(8)andmucinous
(1)adenocarcinoma;urinarybladdertransitionalcellcarcinoma;colorectaladenocarcinomawell(2)andmoderately(7)differentiated;pancreasductaladenocarcinoma
moderatelydifferentiated;lungadenocarcinomawelldifferentiated;stomachwasacombinationofadenocarcinomapoorly(5),moderatelydifferentiated(2),signetring
cellcarcinoma(1),andmucinousadenocarcinoma(1).
MABS 619
Figure4.Bindingspecificityof2G12–2B2onhumanTMAs.(A)Normal(non-neoplastic)lung:Therewaslumenmembranestainingofrarealveolarliningcells(arrow-
heads).Imageisfromaregionwithmorefrequentstaining.BarD40mm.(B)Lungsquamouscellcarcinoma:Membranestainingofneoplasticepithelialcells(arrow-
heads).Asterisksindicatesmononuclearinflammatorycellinfiltratearoundneoplasticcellinfiltrate.BarD100mm.(C)Normal(non-neoplastic)pancreas:Therewasno
stainingofanytissueelements.ArrowsDisletofLangerhans.ArrowheadDPancreaticductule.BarD100mm.(D)Pancreaticductaladenocarcinoma:Moderateto
intensestainingofneoplasticcellsinfiltratingintoadjacentstroma.BarD50mm.(E)Normal(non-neoplastic)colon:Cytoplasmicstainingofgobletcells(arrowheads).
CytoplasmicC/¡membranestainingofscatteredendothelialcells.BarD100mm.(F)Ascendingcolonadenocarcinoma,moderatelydifferentiated:Frequentmembrane
andstainingofneoplasticepithelialcells(arrowheads).Cytoplasmicstainingalsopresent.BarD40mm.
anti-STnmAbsinducedcelldeathofSTnCMDA-MB-231cells as percent mean treated tumor volume/mean vehicle control
(Fig.6).Fourof5MMAE-conjugatedanti-STn mAbshadsin- tumorvolume(%T/C)(errorbarsDSEM).
gle digit nanomolar IC50 values. No cell death was observed OnDay22,thevehiclecontrolgroupwaskilledafteritmet
when parental STn- MDA-MB-231 cells were treated with the group mean tumor volume end point of 1000 mm3, at
MMAE-conjugated, nor when STnC MDA-MB-231 cells were which time the unconjugated mAb treatment produced a
treatedwithunconjugatedanti-STnmAbs(Fig.S1). 60.0% T/C (p D 0.1). At the same time point, isotype control
ADC MOPC173-CL–MMAE showed 16.0% T/C (p D 0.001),
indicatingthatthepayloaditselfhadsomeanti-tumor activity.
Thismaybeduetobindingoftheisotypecontroltounknown
ADCefficacyandsafetyinaninvivohumanbreastcancer
target(s) in vivo, or could indicate an effect of some toxin is
model
being shed in the system. The anti-STn ADCs 4G8–1E3- and
Three anti-STn ADCs (S3F-, 2G12–2B2- and 4G8–1E3- 2G12–2B2-CL–MMAE were the most robust treatments, with
CL–MMAE)wereevaluatedassingleagentsinICRSCIDmice 3.6% and 3.0% T/C, respectively (both p < 0.001). Significant
implanted subcutaneously with STnC MDA-MB-231. differences between isotype control MOPC173-CL–MMAE
MOPC173-CL–MMAE was used as an ADC isotype control. and 2G12–2B2- (p D 0.003) and 4G8–1E3-CL–MMAE (p D
Unconjugated antibody was an equal mixture of 2G12–2B2 0.004) were alsoobserved on Day 22. Nosignificant difference
and4G8–1B3.Allantibodiesweretestedat2.5mg/kgfinalcon- was observed between S3F-CL–MMAE and MOPC173-CL–
centration.InadditiontoevaluatingtheefficacyoftheseADCs, MMAE isotype control on Day 22. S3F-CL–MMAE anti-STn
body weight was monitored as an indicator of toxicity, with a ADC demonstrated modest inhibition (11.0% T/C (p <
decrease of > 5% considered significant. Tumor volumes are 0.001)).Onday43,4weeksafterthelastADCdosewasadmin-
plottedinFig.7,andinhibitionoftumorgrowthwasexpressed istered, mean tumor volumes in the MOPC173- and S3F-CL–
620 J.M.PRENDERGASTETAL.
Figure5. InternalizationofSTnantibodiesinSTn§MDA-MB-231cells.(A)Internalizationoflabeledanti-STnmAbsweretestedusingSTn-expressing(filledbars)and
non-expressing(emptybars)humanbreastcancercells.Eightanti-STnmAbsandanisotypecontrolwereconjugatedtoapHreactivedyethatbecomesfluorescentupon
internalizationintolowerpHorganelleslikelysosomes.SixofeightmAbsshowedsignificantinternalizationintoSTn-expressingcellsascomparedwithnon-expressing
cells((cid:1)P<0.05,(cid:1)(cid:1)P<0.01,ND6).5G2–1B3wasalsoconjugated,butpoorrecoveryafterconjugationdidnotallowforcomparisontoothertestedantibodies.Theiso-
typecontrolMOPC173antibodydidnotsignificantlyinternalizeintoeitherexpressingornon-expressingcells.Errorbarsdenotestandarderror.P-valuesweredetermined
byone-sidedstudent-ttestwithunequalvariance.(B)RepresentativeimagesforantibodyAlexa488-labeledS3Finternalization.Cellswereincubatedwith5mg/mLS3F-
Alexa488for1hourat4(cid:3)C,thenincubatedatvarioustimesat37(cid:3)C(0,15,30,60minutes).Cellsweretreatedwith5-minuteacidwash(150mMNaCl/HClpH2.5)to
removesurfaceboundantibodies.NosurfacestainingnorinternalizationwereseeninMDA-MB-231STn-cells.InternalizationofS3FwasseeninSTnCcellsasearlyas
15minutesandwasstronglyevidentat60minutes.
MMAE-treated groups increased to »850 mm3, while the expression levels of these cell lines compared with transfected
groups treated with the 4G8–1E3- and 2G12–2B2-CL–MMAE lines is detailed in Table 3. Approximately 60% of cultured
exhibitedsuppressedmeantumorvolumesof97and146mm3, COLO205 cells express STn on the cell surface in in vitro
respectively. Effect of treatment on average body weight for monolayer culture (EC50 D 10 nM). A preliminary xenograft
eachofthetreatmentgroups(Fig.S2)showednoovertsignsof study using the COLO205 cell line and the anti-STn ADC is
toxicityinanyofthecontrolortreatedgroups.Whilethemean shown in Fig. 8. The anti-STn ADC S3F-CL–MMAE demon-
bodyweightlossobservedtemporarilyspikedtoamaximumof strated significant tumor growth inhibition (%T/C 44.5%, p D
¡1.8to ¡2.5%for theADCandunconjugatedmAbs after the 0.008)invivocomparedwiththeother3treatmentgroups.
firstdose,thesemicerecoveredbytheseconddose.
Discussion
Anti-STnADCefficacyinanon-transfectedhuman
STnisknowntobeexpressedbymorethan80%ofhumancar-
colorectalcancermodel cinomas,includingpancreatic,ovarian,andcolorectalcancers.3
Wehaveidentifiedseveralcelllinesbyflowcytometrythatnat-
urally express STn without requiring transfection. STn
Figure7.Effectofanti-STnADCsontumorgrowthinsubcutaneoushumanbreast
cancerxenograftmodel.AnICRSCIDsubcutaneousxenograftmousemodelwas
usedwithMDA-MB-231STnCtransfectedhumanbreastcancercells.Thedosing
schedulewasQ7Dx3(one2.5mg/kgdoseaweekfor3weeks,bluearrows).When
Figure6.Cellviabilityassaywithmouseanti-STnADCs.Anti-STnADCantibodies tumorsreachedendpointvolume((cid:2)1000mm3)thegroupwasterminated.ND
killSTnCMDA-MB-231cells(singledigitnanomolarIC50s),whilenakedanti-STn 10miceforallgroups.Unconjugatedantibodycontrolwasanequalmixtureof
antibodiesdonot(Fig.S1). 2.5mg/kgunconjugated2G12–2B2and4G8–1E3.
MABS 621
Table3.STnexpressioninvariouscancercelllines. immuneresponseandgeneratedglycantarget-specificantibod-
ies. The combination of these immunogens and adjuvants
NaturallySTn-linesstablytransfectedwithST6GalNAc-IcDNAtoexpressSTn
resulted in a more robust immune response and the discovery
%STnExpression CellLine TumorType ofglycantarget-specificantibodies.
Attempts to target the STn antigen have included both vac-
98.7 MDA-MB-231 HumanBreast
STnC Adenocarcinoma cine-andantibody-basedapproaches.Themostadvancedagent
90.0 SKOV3-H HumanOvarianCarcinoma targetingSTnwasavaccine(Theratope),whichwasevaluatedin
HighExpressingLine
breast cancer patients in a Phase 3 clinical trial that concluded
50.0 SKOV3-M HumanOvarianCarcinoma
MediumExpressingLine in2003.34Theanti-STnmAbsB72.3andCC49alsoweretested
20.0 SKOV3-L HumanOvarianCarcinoma in the clinic; while these mAbs are described as specific to the
LowExpressingLine
STn-glycoprotein TAG-72, they also cross react with the non-
NaturallySTn+lines:ExpressionNaturallyVariesfromHightoLow sialylatedglycan,Tn.3,35Oncoscint(cid:1) (B72.3antibodyconjugated
to indium-111)wasapprovedbyFDAasadiagnosticin199236
%STnExpression CellLine TumorType
forwholebodyimagingforrecurrencesofovarianandcolorectal
(cid:1)
99.5 OV-90 HumanOvarianCarcinoma cancers.WhileOncoscint wasfoundtobesafeandeffectivein
79.4 SNU-16 HumanGastricCarcinoma detecting tumors, this product was eventually discontinued due
75.0 OVCAR8 HumanOvarianCarcinoma
to PET (positron emission tomography) technology advance-
69.9 LS174T HumanColonCarcinoma
67.7 COLO-205 HumanColonCarcinoma mentsindetectingmetastaticlesions.37Theregulatoryapproval
35.0 OVCAR3 HumanOvarianCarcinoma andstrongsafetyprofileofOncoscint(cid:1)38ledtothedevelopment
28.9 Jurkat HumanTCellLeukemia
ofthesecondgenerationTAG-72murineantibodyCC49,which
26.4 OVCAR4 HumanOvarianCarcinoma
20.0 OVCAR5 HumanOvarianCarcinoma wasevaluatedforefficacyinPhase2clinicaltrials.39Thesetrials
evaluated an interferon adjuvant to enhance tumor antigen
%STnexpressionwasdeterminedusingS3Fantibodyandflowcytometryofcul-
turedcells,STnCcellswereindicatedasa%oftotalcellsinthetableabove. expression and 131I-labeled CC49 to target both soft tissue and
bonemetastasesinhormone-resistantmetastaticprostatecancer
DenovoexpressionofSTnviaSTnsynthase(a2,6-sialyl-trans- patients. The addition of the adjuvant therapy enhanced tumor
ferase) transfection has been reported to lead to more malig- uptake and anti-tumor effects of 131I-labeled CC49 alone based
nant phenotypes.6-9 Normal adult tissue expression of STn is onapreviousPhase2clinicaltrialof131I-labeledCC49inmeta-
rare and largely restricted to cell types specialized in secretion, static prostate cancer patients where pain relief was reported,
generally on the apical surface, which would likely restrict its but no objective antitumor response was observed.40 While the
accessibilitytoanti-STnADCsadministeredintheblood.3 experimental therapy was generally well tolerated, the results
Historically, glycans have been difficult targets to raise a weremodest,anddiseaseprogressionwasnotdelayedformore
robustimmuneresponseagainst.14,33Wehavecompletedmul- than 6 months.Moreover,human anti-mouse antibodies devel-
tiple rounds of mouse STn immunization to optimize the opedinallpatients,whichprecludedrepeattherapyby8weeks.
immunogen and adjuvant selected. In previous immunization ItispossiblethatahumanizedversionofthemurineCC49may
attempts, we used various mouse strains, synthetic glycan and have improved efficacy. Regardless, therapeutic modalities have
mucin-based immunogens, and traditional adjuvants (CFA/ since shifted away from antibody-radionuclide conjugates
IFA),whichresultedinfewglycan-specificantibodycandidates. toward ADCs.41 Advances in ADC formats are hypothesized to
In our most successful immunizations, we immunized using allowformorerobustanddurableresultsthanpreviousmurine
various intact and digested mucins with newer adjuvants radiotherapies because they utilize potent cytotoxic payloads
(ODN and AbISCO). Intact mucins produced a more robust insteadofradionuclides.42
WithrecentadvancesinADClinkertechnologypairedwith
various options in cytotoxic payloads, many new potential
methods to target glycans are available. Variable immune
responsesexhibitedinvaccinetherapycanbecircumventedby
usingADCswithproperlinkerandpayloadstoachieverobust
bystander killing in heterogeneous tumors. The payload used
inouranti-STnADCisMMAE,whichisalsothesamepayload
(cid:1)
incorporated in Adcetris , an FDA-approved drug for the
treatmentofHodgkinlymphomaandsystemicanaplasticlarge
cell lymphoma.29,30 ADCs using the same payload format are
currently in ongoing Phase 1/2 trials targeting solid tumors
such as prostate, breast, lung, pancreatic, ovarian, and blad-
der.42,43 This toxin is shed at very low levels and is acceptable
in safety assessments, with multiple MMAE ADC molecules
Figure 8. Proof-of-concept human colorectal cancer naturally expressing STn
havingbeenevaluatedforsafetyandtoxicityinvariousclinical
modelusinganti-STnS3Fand2differentADCformats.Anathymicnudemouse
subcutaneousxenograftmousemodelwasusedwithCOLO205humancolorectal trials.41-45 The ADC-linker technology used was the MC-vc-
cancercells.AnimalsweredosedQ7Dx3at5mg/kg(one5mg/kgdoseaweekfor PAB-MMAE, a cleavable linker (CL) that allows for release of
3weeks,bluearrows).Antibodyalonewithnotoxin(red),S3F-CL–MMAE(MC-vc-
MMAEuponinternalizationintocells.MMAEisamembrane-
PAB-MMAE, green) and S3F-NC-MMAF (NC-MMAF, purple), all were compared
withvehiclealone(blue). permeabletoxinthatcanbereleasedfromdyingcells,resulting
622 J.M.PRENDERGASTETAL.
in a bystander killing effect that produces greater efficacy in observedSTn-specificreactivityacrossawidearrayofsolidepi-
tumorswithheterogeneoustargetexpression.28STnexpression thelialtumortypes.Ourpreliminarydatashowsstrikingreduc-
is reported to be heterogeneous in stomach, colon, ovary, cer- tion in tumor volumes in multiple in vivo models. Further
vix,breast,andothercarcinomas,withSTnexpressionranging development and humanization of our anti-STn ADC offers
from 5% to 100%.3 Another ADC-linker technology used was promise of effectively targeting this O-glycan modification in
MMAF with a maleimidocaproyl (MC) linker. MMAF is a multiplecancers.
non-cell membrane permeable toxin and must be internalized
tokilleachtumorcell,thusitisineffectiveinbystanderkilling.
Materialsandmethods
BasedontheresultsoftheCOLO205xenograftstudies(Fig.8),
whereinS3FwascomparedwithvariationsinADC-linkertech- GenerationofSTnspecificantibodies
nologies, MMAF resulted in no effects on tumor growth com-
Mouseimmunizationswereusedtogenerateapanelofanti-STn
pared with controls. This may suggest bystander killing or the
antibodies.Fourmousegroups(ND 10per group)wereestab-
toxin/linkercombinationitselfcouldbeimportantforeffective
lished using 6- to 8-week-old female BALB/c mice immunized
STn tumor targeting. Previously published studies with other
withdifferentantigens,but sharingthesameadjuvant/immuni-
targets using the MC-vc-PAB-MMAE system also demon-
strated that bystander killing results in additional efficacy in zationscheduletocompareeffectivenessofSTnpresentinganti-
heterogeneous tumors.28 Understanding bystander killing and gens.All groups usedAbISCOincombinationwith ODN-2395
effectsofthelinker/toxinofourantibodieswouldbebeneficial as adjuvants to promote humoral as well as cellular immune
responses. The first dose on Day 0 for all groups used 12 mg
totheirfurthercharacterization.
AbISCO and 50 mg ODN-2395. Unacceptable toxicity was evi-
ADC binding to shed antigens can be a concern because
dentwiththisstrategyandtheadjuvantdosesforallsubsequent
theseantigensactasadrugsink,effectivelydepletingtherapeu-
boost regimes were reduced by half. Group 1 was immunized
tic away from the intended tumor target. While shed STn has
with 10 mg porcine submaxillary mucin (PSM) on Days 0, 14,
been reported to be present in the circulation, it has not been
28, and 42. Group 2 was immunized with alternating mucins:
viewed as a limiting drug sink in related ADC clinical studies.
STn in the serum and its effect as a possible drug sink have 10mgPSMonDay0andDay28and10mgovinesubmaxillary
been described in the clinical study of an anti-MUC16 ADC mucin (OSM) on Days 14 and 42. Group 3 was immunized
(Genentech; Clinical Trials ID: NCT01335958).46 MUC16 with 10 mg digested PSM on Days 0, 14, 28, and 42. Group 4
(CA125) is a known carrier of STn on cancer cells.47 In this was immunized with OSM on Days 0, 14, 28, and 42. Each
mouse was immunized with mucin/adjuvant subcutaneously
study,thecirculatinglevelsofCA125hadnoeffectonthephar-
(SC)aroundthearmpitsandinguinalregions(50mLpersite,4
macokineticsoftheanti-MUC16ADC,eveninpatientswitha
sites, 200 mL/mouse) on Days 0, 14, 28, and 42 as described
high level of circulating CA125. This suggests that the soluble
formofthedrugtargetinbloodmaynotresultinasignificant above.Approximately0.2mLofwholebloodwascollectedfrom
drug-sinkeffect. mice on Days 0, 14, 28, 42, and 51 and processed for serum.
A number of related TACAs have been preclinically vali- Serum was screened for STn binding specificity using ELISAs
dated. For example, an anti-Tn ADC48 and MUC1-Tn CAR- and glycan arrays. Mice displaying desirable binding properties
T49haverecentlybeenevaluated. Tn,alsoknown asCD175,is were immunized again with 10 mg mucin (without adjuvants),
3 d later their immune response was probed by screening their
thenon-sialylatedformofSTn.Tnisnormallyabuildingblock
sera. If the serum showed the desired robust and specific
forothercomplexsugars,anditsexpressionisgenerallyabsent
immuneresponse,thecorrespondingmicewerekilledandtheir
innormaladulttissues.Tnisexpressedoncancercell surfaces
spleenswereharvestedandusedforfusiontoestablishhybrido-
in a similar heterogeneous manner to STn when glycosylation
is altered, particularly in metastatic lesions.48 The anti-Tn- masandtheanti-STnmouseantibodypanel.
ADC,calledChi-Tn,isachimericantibodygeneratedfroman We used the HB-STn clone 3F121,51 as a proof-of-concept
original murine IgM (83D450) that uses the MMAF payload, for some studies. We class switched from a mouse IgG1 and
relyingoncancercellinternalizationforefficacy.WhileChi-Tn recombinantly expressed this antibody as a mouse IgG2a; this
demonstrated efficacy in vitro cytotoxic and in vivo xenograft engineered antibody is referred to as “S3F” in the text. B72.3
models, its efficacy was only observed in high Tn-expressing (ThermoFisher Scientific,catalognumberMS138P),CC49and
models.48Chi-Tndidnotdemonstrateefficacyinthesemodels 3H1951 (Santa Cruz Biotechnology, catalog numbers sc-20043
and sc-70558, respectively), STn219 (Abcam, catalog number
when using cells with lower, more heterogeneous Tn expres-
sion, perhaps due to the absence of bystander killing with ab115957) and MOPC173 (BioLegend, catalog number
MMAF. In addition to ADC therapies, genetically modified T 400264)wereusedasassaycontrols.
cells expressing chimeric antigen receptors (CARs) targeting
Tnalsohavebeendescribed.ACAR-Ttargetingtheglycopro-
QuantificationofSTnbindingspecificityELISA
tein MUC1 with the Tn modification was based upon the 5E5
MUC-Tn specific antibody. This therapy was described as The antigens bovine (BSM), porcine (PSM) or ovine (OSM)
effective in targeting T-cell lymphomas and pancreatic cancer submaxillarymucinwerecoatedonCorningCostarhigh-bind-
inpreclinicalinvitroandinvivomodels.49 ing plates (Corning, catalog number 9018) by overnight incu-
Our anti-STn antibodies target the glycan itself, not a spe- bation of antigen solution in coating buffer (50 mM sodium
cific glycopeptide, offering broad potential to bind multiple carbonate/bicarbonate pH 9.5) at 48C. When the sialic acid
STn-glycosylated proteins on the surface of cancer cells. We composition of these mucins is examined by HPLC, BSM
MABS 623
contains a mixture of Neu5Ac and Neu5Gc, while PSM con- acetylated STn (Neu5,9Ac2a6GalNAca and Neu5-
tainsmostlyNeu5Gc,andOSMcontainsmainlyNeu5Acsialic Gc9Aca6GalNAca) and Tn (GalNAca) were compared along
acids (data not shown).52 After washing with phosphate-buff- with theremaining 66glycans onthearray.Bindingwas com-
ered saline (PBS), a subset of wells had their sialic acids oxi- pared with mouse monoclonal anti-STn antibody 3F1, B72.3
dizedbytreatment with2mMperiodate,wellswereincubated andCC49.
for 20 minutes at 48C. Plates were washed with 1x PBS and
additional binding sites were blocked with 1% ovalbumin
QuantificationofSTnatthecellsurfacebyflowcytometry
(OVA)inPBSfor1hour.AfteraPBSwash,theanti-STnanti-
bodies were added to wells in a serial dilution (0–100 nM), Thebindingaffinitiesofanti-STnantibodiestoSTnonthesur-
using1%OVAasthediluentandwereincubatedfor2hoursat faceofcellswasdeterminedbyflowcytometry.Anti-STnanti-
room temperature. Plates were washed 3 times with 0.05% bodies were screened for binding to MDA-MB-231 cells stably
Tween-20 –PBS followed by incubation with 0.08 mg/mL per- transfected to express STn9 and absence of binding on non-
oxidase-conjugated goat anti-mouse antibody (Jackson Immu- STnexpressingparentalcells.CellswereharvestedusingStem-
noResearch, catalog number 115–035–071) for 1 hour. Next, ProAccutase buffer, cells were resuspended to a concentration
wells were washed 3 times with 0.05% Tween-20 –PBS and of 5£106 cells/mL in staining buffer (5% heat-inactivated fetal
then wells were incubated with 100 mL of enzyme substrate bovine serum in PBS). 50 mL of cells and 50 mL of staining
(0.5 mg/mL o-phenylenediamine; 0.03% H O in citric/phos- buffer with anti-STn antibodies were combined. Antibodies
2 2
phate buffer pH 5.5). The enzyme reaction was terminated by werescreenedusingaserialdilutionoveraconcentrationrange
addition of an equal volume of 1.6 M sulfuric acid. Optical of0–300nM.Antibodiesandcellswereincubatedfor1hourat
Density (OD) readings of periodate and non-periodate treated 48C.Cellswerewashed3timeswithstainingbuffer.Bindingof
wells were determined at 490nm, log transformed and then fit antibodieswasdeterminedusingananti-mouseIgGallophyco-
to a nonlinear regression model (4-parameter logistic regres- cyanin(APC)-conjugatedsecondaryantibodyat1:1500(South-
sion) to obtain a dose response curve. Data from periodate ern Biotech 1031–11L) diluted in staining buffer. A total of
treated wells were subtracted from non-periodate treated wells 5,000 events were acquired per sample using BD FACSverse
toobtaintheperiodate-sensitive,STnbindingcurveandcorre- and data was analyzed using FlowJo software. Mean of APC
sponding EC50 values. Binding affinities of anti-STn mouse fluorescence and % APC positive cells were calculated. These
mAbs were compared with the well-characterized mouse data were log transformed then fit to a nonlinear regression
monoclonalanti-STnantibodies3F1,B72.3andCC49. modeltoobtainadoseresponsecurveandEC50bindingcalcu-
lations using Prism software. Mouse isotype MOPC173 anti-
body was used as an isotype control. The anti-epidermal
QualificationofSTnspecificityusingglycanarray
growthfactorreceptor(EGFR)antibodyLA22(EMDMillipore
The glycan array was used to determine the glycan binding catalognumber05–104)wasusedasapositivecontrol.
specificity of anti-STn antibodies. This array was developed in
collaboration with Dr. Ajit Varki.31 STn and related glycans
Tumormicroarray(TMA)–Immunohistochemistryanalysis
were synthesized and printed as described in the literature.31
ofSTnexpression
Briefly, 71 glycans were synthesized using one-pot 3-enzyme
chemoenzymatic approach;53 structures were confirmed by Commercially available FFPE TMAs containing a broad range
DMB-HPLC,NMRormassspectrometry.Glycansincluded25 of common human cancer specimens were scored microscopi-
matched Neu5Ac/Gc pairs (including 9-O acetylated versions) cally by a qualified pathologist for antibody staining intensity,
along with several un-sialylated versions (Table S1). Glycans frequency and localization. Normal TMAs containing 60 sam-
weredilutedtoafinalconcentrationof100mM(300mMphos- ples (2 human donors each for 30 organs or subregions of
phatebuffer,pH8.4)andprintedin4replicates.Mousewhole organs,SuperBioChips,Seoul,Korea,ProductAC1),aswellas
IgGwasprintedat0.05mg/mLfinalconcentrationandprinted commonhumancancerTMAscontainingatotalof118donor
in 4 replicates across the array matrix as a control. STn-speci- tumorsamples(SuperBioChips,Seoul,Korea,Products:MA2
ficitywasdeterminedcomparingSTnvs.non-STnglycanbind- and MA4), were immunohistochemically stained using 10 mg/
ing. Epoxy slides were blocked (0.1 M Tris, 0.05 M ethanol mLofeachcandidatemAbusingastandardABCstainingpro-
amine,pH9.0)for1hourat508C.Slideswerewashedwithdis- cedure following antigen retrieval. Cells and tissues known to
tilled water, and blocked (PBS with 1% OVA) for 1 hour. either express, or not express STn were used as positive and
Blockingbufferwasaspiratedandanti-STnantibodywasadded negative control tissues, respectively. A total of 13 different
at1mg/mLconcentrationdilutedinblockingbufferfor1hour. common tumor types were tested overall, with numerous sub-
SlideswerewashedtwicewithPBSwith0.1%Tween,thenonce classification annotations captured for each tumor type. All
with PBS alone. Next, secondary antibody (Cy3 goat anti- commercially available human normal and cancer TMAs used
mouse, Jackson ImmunoResearch catalog number 115–165– in this study had been declassified by the manufacturer, and
071) was added at a final concentration 1.5 mg/mL in PBS for patientdonorswerenotidentifiable.
1 hour. Slides were washed with PBS then distilled water and
air-dried before reading on the GenePix 4000B (Molecular
Invitrointernalizationassay
Devices). Fluorescence intensities were measured and back-
ground was subtracted using GenePix Pro software. Intensity The pHAb amine reactive dye (Promega catalog number
of STn (Neu5Aca6GalNAca and Neu5Gca6GalNAca), 9-O G9845) was conjugated to a subset of antibodies to determine