SITC 2016

116 Pages·2016·8.44 MB·English

by  Casey Ager

31st Annual Meeting and Associated Programs of
the Society for Immunotherapy of Cancer (SITC
2016): part two: National Harbor, MD, USA. 9-13
November 2016
Citation
Ager, C., M. Reilley, C. Nicholas, T. Bartkowiak, A. Jaiswal, M. Curran, T. C. Albershardt, et
al. 2016. “31st Annual Meeting and Associated Programs of the Society for Immunotherapy
of Cancer (SITC 2016): part two: National Harbor, MD, USA. 9-13 November 2016.” Journal
for Immunotherapy of Cancer 4 (Suppl 1): 107-221. doi:10.1186/s40425-016-0173-6. http://
dx.doi.org/10.1186/s40425-016-0173-6.
Published Version
doi:10.1186/s40425-016-0173-6
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http://nrs.harvard.edu/urn-3:HUL.InstRepos:29739091
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JournalforImmunoTherapyofCancer2016,4(Suppl1):73
DOI10.1186/s40425-016-0173-6
MEETING ABSTRACTS Open Access
31st Annual Meeting and Associated
Programs of the Society for
Immunotherapy of Cancer (SITC 2016):
part two
NationalHarbor,MD,USA.9-13November2016
Published:16November2016
Aboutthissupplement
TheseabstractshavebeenpublishedaspartofJournalforImmunoTherapyofCancerVolume4Suppl1,2016.Thefullcontentsofthesupplement
areavailableonlineathttp://jitc.biomedcentral.com/articles/supplements/volume-4-supplement-1.Pleasenotethatthisispart2of2.
Combinations: Immunotherapy/ intratumoral Flt3L, although administration at both flanks is re-
Immunotherapy quired for full effect. Intratumoral low-dose αCTLA-4, αPD-1, and
α4-1BB at a single flank induces abscopal effects in 20 % of mice,
and concurrent administration of CDG enhances systemic immun-
P189
ity to cure up to 50 % of mice. We observe that the level of
RationalcombinationsofintratumoralTcellandmyeloidagonists
STING activation required to mediate rejection without inducing
mobilizeabscopalresponsesinprostatecancer
ulcerative toxicity is proportional to initial tumor size. Function-
CaseyAger1,MatthewReilley2,CourtneyNicholas1,ToddBartkowiak1,
ally, local STING activation complements intratumoral checkpoint
AshvinJaiswal1,MichaelCurran1
modulation to reduce local MDSC infiltration, enhance CD8:Treg
1DepartmentofImmunology,UniversityofTexasMDAndersonCancer
ratios, and downregulate the M2 macrophage marker CD206. In
Center,Houston,TX,USA;2DepartmentofCancerMedicine,Universityof
contrast, local Flt3L robustly enhances immune infiltration of
TexasMDAndersonCancerCenter,Houston,TX,USA
injected and distal tumors, but therapeutic benefit is attenuated
Correspondence:CaseyAger([email protected])
due to concomitant induction of FoxP3+ Treg.
JournalforImmunoTherapyofCancer2016,4(Suppl1):P189
Conclusions
Intratumoral STING activation via CDG or DC expansion with Flt3L
Background potentiatesthetherapeuticeffectsofsystemically-deliveredαCTLA-4,
Despite the success of checkpoint blockade immunotherapy in αPD-1, and α4-1BB against multi-focal TRAMP-C2 prostate cancer.
characteristically immunogenic cancers such as melanoma, these
TheabscopalpotentialofCDGaloneisweak,incontrasttopriorob-
antibodies remain ineffective against poorly T cell-infiltrated malig-
servations, but combining CDG with low-dose checkpoint blockade
nanciesincludingprostatecancer.Sensitizingthese“cold”tumorsto
intratumorallycaninducesystemicimmunity,suggestinganalterna-
immunotherapywillrequireinterventionswhichenhancetumoranti-
tive approach for clinical implementation of combination immuno-
genpresentationandTcellpriming,whilesuppressingmicroenviron-
therapiesatreduceddoses.
mentalsignalswhichconstrainTcellexpansion,survival,andeffector
function independent of coinhibitory signaling. We investigated
whetherintratumoraladministrationofeithertheSTINGagonistc-di-
GMP (CDG) or dendritic cell (DC) growth factor Flt3-ligand can po- P190
tentiatethetherapeuticeffectsofTcellcheckpointmodulationwith Multi-genomereassortantdendriticcell-tropicvectorplatform
αCTLA-4, αPD-1, and α4-1BB in a bilateral subcutaneous model of (ZVex®-Multi)allowsflexibleco-expressionofmultipleantigens
prostate adenocarcinoma. Additionally, we tested whether intratu- andimmunemodulatorsforoptimalinductionofanti-tumorCD8+
moraldeliveryoflow-dosecheckpointmodulatorswithCDGatasin- Tcellresponses
glelesioncanachieveabscopalcontrolofdistallesions. TinaCAlbershardt,AnshikaBajaj,JacobFArcher,RebeccaSReeves,Lisa
Methods YNgo,PeterBerglund,JanterMeulen
Male C57BL/6 mice were challenged subcutaneously on both flanks ImmuneDesign,Seattle,WA,USA
withTRAMP-C2prostateadenocarcinoma,andtreatmentwasadmin- Correspondence:TinaCAlbershardt
istered intraperitoneally and/or intratumorally for 3 doses every ([email protected])
4 days, beginning on day 14 post-implantation for survival experi- JournalforImmunoTherapyofCancer2016,4(Suppl1):P190
mentsorday31forflowanalysisexperiments.
Results Background
Intratumoral delivery of STING agonist CDG alone potently rejects Inductionofimmuneresponsesagainstmultipleantigensexpressed
all injected TRAMP-C2 tumors, but fails to generate systemic con- from conventional vector platforms is often ineffective for reasons
trol of uninjected lesions. Systemic administration of αCTLA-4, notwellunderstood.Commonmethodsofexpressingmultipleanti-
αPD-1, and α4-1BB cures 40 % of mice with bilateral TRAMP-C2, gens within a single vector construct include the use of fusion pro-
and concurrent administration of CDG at one or both flanks en- teins, endoprotease cleavage sites, or internal ribosome entry sites.
hances survival to 75 %. Similar effects are observed with These methods often lead to decreased expression of antigens-of-
©TheAuthor(s).2016OpenAccessThisarticleisdistributedunderthetermsoftheCreativeCommonsAttribution4.0
InternationalLicense(http://creativecommons.org/licenses/by/4.0/),whichpermitsunrestricteduse,distribution,and
reproductioninanymedium,providedyougiveappropriatecredittotheoriginalauthor(s)andthesource,providealinkto
theCreativeCommonslicense,andindicateifchangesweremade.TheCreativeCommonsPublicDomainDedicationwaiver
(http://creativecommons.org/publicdomain/zero/1.0/)appliestothedatamadeavailableinthisarticle,unlessotherwisestated.
JournalforImmunoTherapyofCancer2016,4(Suppl1):73 Page108of221
interest and/or reduced induction of T cell responses against the Methods
encoded antigens. Circumventing these limitations, we have devel- To assess the effect of combined blockade of NKG2A/HLA-E and
oped a novel production process for our integration-deficient, den- PD-1/PD-L1 in vivo, anti-mouse NKG2A and PD-1 antibodies were
dritic cell-targeting lentiviral vector platform, ZVex, enabling highly used in mice engrafted with A20 mouse B lymphoma cell line.
flexible and effective multigene delivery in vivo, making it possibly For in vitro assays, anti-PD-L1 antibody durvalumab, and monali-
themostversatilevectorplatformintheindustry. zumab were tested in human PBMC staphylococcal enterotoxin b
Methods assays.
Up to five vector genome plasmids, each encoding one full-length Results
antigen or immuno-modulator, were mixed with four constant plas- Whenculturedinvitro,theA20cellsexpressligandsforPD-1butnot
mids,eachencodingvectorparticleproteins,priortotransfectionof forNKG2A.ExposuretoIFN-γinvitro,orsubcutaneousinjectioninto
production cells. Due to the propensity of lentiviruses forming gen- mice, induced expression of Qa-1b, resulting in a tumor model co-
omic reassortants, the resulting vector preparations hypothetically expressing PD-L1 and Qa-1b. Monotherapy with PD-1 or NKG2A
contain a mix of homozygous and heterozygous vector particles. blockers resulted in moderate anti-tumor efficacy while treatment
qRT-PCR was used to determine total and antigen-specific titers of with combination of NKG2A and PD-1 blockers resulted in a signifi-
ZVex-Multivectors,definedasvectorgenomecounts.Micewereim- cantly higher anti-tumor immunity, and an increased rate of
munizedwithZVex-Multi vectorsor monozygousvectorsexpressing complete tumor regression. Depletion of either NK, or CD8+ T cells,
multipleantigensfromthesamebackbonetocompareimmunogen- or IFN-γ was enough to abrogate the efficacy of PD-1 and NKG2A
icityviaintracellularcytokinestaining.Twotumormodelswereused blockade, indicatingthatbothoftheseeffectorpopulationscontrib-
to evaluate therapeutic efficacy: 1) a B16 melanoma model, where uteto theefficacyofthecombinationtreatment.Tofurtherexplore
tumorcellswereinoculatedintheflankandmeasured2–3timesper this possibility and to assess the potential therapeutic relevance in
week;and2)ametastaticCT26coloncarcinomamodel,wheretumor humans,well-validatedPBMC-basedassayswereusedwhichshowed
cellswereinoculatedintravenously,andlungnoduleswereenumer- thatblockingbothaxeswithacombinationofdurvalumabandmon-
ated17–19dayspost-tumorinoculation. alizumabledtoincreasedproductionofcytokinesbybothTandNK
Results cells. Furthermore, the magnitude of the increase in cytokine secre-
Titrations by qRT-PCR of multiple ZVex-Multi vector lots demon- tionwasdependentontheproductionofhighlevelsofIFN-γ.Since
stratedthatproductionyieldsofZVex-Multiexpressinguptofourdif- IFN-γ is known to induce HLA-E this suggests that blockade of
ferenttumor-associatedantigens(e.g.,NY-ESO-1,MAGE-A3)andtwo NKG2A could have a beneficial role in activation of immune cells
immuno-modulators (e.g., IL-12, anti-CTLA-4 or anti-PD-L1) were throughthecombinedblockadeofPD-1/PD-L1.
highly reproducible. Compared to mice immunized with vectors ex- Conclusions
pressing multiple antigens from the same backbone, mice immu- Together, these data indicate that blocking NKG2A in conjunction
nizedwithZVex-Multi vectorsconsistentlydevelopedT cells against withPD-1/PD-L1checkpointinhibitorsprovidesincreasedanti-tumor
alltargetedTAAsandexhibitedimprovedtumorgrowthcontroland efficacy mediated by IFN-γ and support the rationale for assessing
survival. thiscombinationinclinicaltrials.
Conclusions
ZVex-Multi is a next generation DC-tropic vector platform designed
to overcome limitations of single-genome vector platforms with re-
P192
specttoefficientco-expressionofanycombinationofdesiredgenes.
Pharmacokineticsandimmunogenicityofpembrolizumabwhen
Unlikeothervectorplatforms,ZVex-Multieliminatesmultiplecloning
givenincombinationwithipilimumab:datafromKEYNOTE-029
steps modifying the vector backbone, which can often result in un-
MichaelBAtkins1,MatteoSCarlino2,AntoniRibas3,JohnAThompson4,
predictableexpressionpatternsofcodedgeneproducts.Itsversatility
ToniKChoueiri5,FStephenHodi5,Wen-JenHwu6,DavidFMcDermott7,
andagilitymakesZVex-Multipotentiallythebest-in-classvectorplat-
VictoriaAtkinson8,JonathanSCebon9,BernieFitzharris10,MichaelB
form for co-expression of multiple tumor antigens and immuno-
Jameson11,CatrionaMcNeil12,AndrewGHill13,EricMangin14,Malidi
modulators for enhanced cancer immunotherapy against a broad
Ahamadi14,MariannevanVugt15,MariëllevanZutphen15,Nageatte
rangeoftumors.
Ibrahim14,GeorginaVLong16
1Georgetown-LombardiComprehensiveCancerCenter,Washington,DC,
USA;2WestmeadandBlacktownHospitals,MelanomaInstituteAustralia,
P191 andtheUniversityofSydney,Westmead,NewSouthWales,Australia;
NK,TcellsandIFN-gammaarerequiredfortheanti-tumorefficacy 3UniversityofCalifornia,LosAngeles,CA,USA;4Universityof
ofcombination-treatmentwithNKG2AandPD-1/PD-L1checkpoint Washington,Seattle,WA,USA;5Dana-FarberCancerInstitute/Brigham
inhibitorsinpreclinicalmodels andWomen’sHospital,HarvardUniversity,Boston,MA,USA;6University
CarolineDenis1,HormasGhadially2,ThomasArnoux1,FabienChanuc1, ofTexasMDAndersonCancerCenter,Houston,TX,USA;7BethIsrael
NicolasFuseri1,RobertWWilkinson2,NicolaiWagtmann1,YannisMorel1, DeaconessMedicalCenter,Boston,MA,USA;8GallipoliMedicalResearch
PascaleAndre1 Foundation,GreenslopesPrivateHospital,andtheUniversityof
1InnatePharma,Marseille,Provence-Alpes-Coted'Azur,France; Queensland,Greenslopes,Queensland,Australia;9OliviaNewton-John
2MedImmune,Cambridge,England,UK CancerResearchInstitute,Heidelberg,Victoria,Australia;10Canterbury
Correspondence:PascaleAndre([email protected]) DistrictHealthBoard,ChristchurchHospital,Christchurch,NewZealand;
JournalforImmunoTherapyofCancer2016,4(Suppl1):P191 11WaikatoHospitalRegionalCancerCentre,Hamilton,NewZealand;
12RoyalPrinceAlfredHospital,MelanomaInstituteAustralia,the
Background UniversityofSydney,andChrisO’BrienLifehouse,Camperdown,New
Monalizumab (IPH2201) is a first-in-class humanized IgG target- SouthWales,Australia;13TasmanOncologyResearch,SouthportGold
4
ing NKG2A, which is expressed as heterodimer with CD94 on the Coast,Queensland,Australia;14Merck&Co.,Inc.,Kenilworth,NJ,USA;
surface of NK, γδT and tumor infiltrating CD8+ T cells. This inhibi- 15QuantitativeSolutions,aCertaracompany,Oss,Netherlands;
tory receptor binds to HLA-E in humans and to Qa-1b in mice. 16MelanomaInstituteAustralia,theUniversityofSydney,MaterHospital,
HLA-E is frequently up-regulated on cancer cells, protecting from andRoyalNorthShoreHospital,Wollstonecraft,NewSouthWales,
killing by NKG2A+ cells. Monalizumab blocks binding of CD94- Australia
NKG2A to HLA-E, reducing inhibitory signaling thereby enhancing Correspondence:MichaelBAtkins([email protected])
NK and T cell responses. PD-1/PD-L1 inhibitors are successfully JournalforImmunoTherapyofCancer2016,4(Suppl1):P192
being used to treat patients with a wide variety of cancers. Com-
bined blockade of NKG2A/HLA-E and PD-1/PD-L1 may be a prom- Background
ising strategy to better fight cancer by activating both the The pharmacokinetics of pembrolizumab given as monotherapy
adaptive and innate immune systems. are well characterized. Consistent with other monoclonal
JournalforImmunoTherapyofCancer2016,4(Suppl1):73 Page109of221
antibodies, pembrolizumab has low clearance (0.202 L/day), lim-
ited central (3.53 L) and peripheral (3.85 L) volume of distribu-
tion, and low variability in the central volume of distribution
(19 % coefficient of variation). Pembrolizumab monotherapy has
low immunogenicity potential, with an observed incidence of
treatment-emergent anti-drug antibodies (ADA) of<1 %. We
present data on the pharmacokinetics and immunogenicity of
pembrolizumab when given in combination with ipilimumab in
the phase I KEYNOTE-029 study.
Methods
KEYNOTE-029 included 2 cohorts that assessed the safety and
antitumor activity of pembrolizumab plus ipilimumab: a safety
run-in that included patients with advanced melanoma or renal
cell carcinoma (RCC) (N=22) and a melanoma expansion cohort
(N=153). In both cohorts, patients received 4 doses of pembroli-
zumab 2 mg/kg plus ipilimumab 1 mg/kg Q3W followed by
pembrolizumab2mg/kgQ3Wforupto2years.Pembrolizumabserum
concentrationwasquantifiedwithanelectrochemiluminescence-based
immunoassay(lowerlimitofquantitation,10ng/mL).Avalidatedbridg-
ing electrochemiluminescence immunoassay using a standard 3-
tiered approach (drug tolerance level, 124 μg/mL) was used to
detect ADA in serum.
Results
Across cohorts, 175 patients received pembrolizumab plus ipili-
mumab: 165 with melanoma and 10 with RCC. At least 1 evalu-
Fig.2(abstractP192).Observedpembrolizumabserum
able sample for pharmacokinetic assessment was available for all
concentrationsfrompatientswithmelanomatreatedwith
10 patients with RCC and 162 patients with melanoma. The pre-
pembrolizumabplusipilimumabinrelationtothepredicted
dose serum concentration versus time profiles for pembrolizumab
concentrationinterval(gray)forpembrolizumab2mg/kgQ3W
were similar in patients with RCC and melanoma (Fig. 1). Ob-
monotherapy(logscale)
served serum concentrations were within the range predicted for
pembrolizumab 2 mg/kg Q3W given as monotherapy (Fig. 2). Of
the 160 patients with melanoma who provided postdose ADA
samples, 156 (97.5 %) were negative, 2 (1.3 %) were inconclusive,
and 2 (1.3 %) were positive for treatment-emergent ADA. Best
P193
overall response in the ADA-positive patients was stable disease
EstablishingamodelforsuccessfulimmunotherapywithT-Vec
in one and progressive disease in the other. No patient with RCC
combinedwithBRAFinhibitionandanti-PD-1ingenetically
had treatment-emergent ADA.
engineeredmurinemelanoma
Conclusions
RobynGartrell1,ZoeBlake1,InesSimoes2,YichunFu1,TakuroSaito3,
The addition of ipilimumab does not appear to impact pembrolizu-
YingzhiQian1,YanLu1,YvonneMSaenger4
mabserumconcentration or increasethe riskof developingADAin
1ColumbiaUniversityMedicalCenter,NewYork,NY,USA;2Institut
patientswithadvancedmelanomaorRCC.
d'InvestigacionsBiomediquesAugustPiiSunyer,Barcelona,Catalonia,
TrialRegistration
Spain;3IcahnSchoolofMedicineatMountSinai,NewYork,NY,USA;
ClinicalTrials.govidentifierNCT02089685.
4NewYorkPresbyterian/ColumbiaUniversityMedicalCenter,NewYork,
NY,USA
Correspondence:ZoeBlake([email protected])
JournalforImmunoTherapyofCancer2016,4(Suppl1):P193
Background
Talimogene laherparepvec (T-Vec) is the first oncolytic virus to be
U.S. Food and Drug Administration (FDA) approved for the treat-
ment of cancer. T-Vec, a modified herpes simplex type I (HSV I)
virus, has two proposed mechanisms of action: direct cell lysis
and immune activation. Combination immunotherapy using T-Vec
and checkpoint blockade has shown promise in clinical trials. In
preliminary work, our laboratory has shown that T-Vec causes up-
regulation of programmed cell death protein 1 (PD-1) on infiltrat-
ing T cells in mice, suggesting potential synergy of T-Vec and
anti-PD-1 (αPD-1).
Methods
In a temporally and spatially regulated murine model of BRAFCA
PTEN−/− spontaneous melanoma [1], tumors are induced on right
flank. When tumors reach >5 mm in diameter, mice are random-
ized into 6 treatment groups comparing combinations of BRAF
inhibition (BRAFi), αPD-1, and T–Vec (Table 1). Tumor growth is
Fig.1(abstractP192).Arithmeticmean(SE)predoseserum
measured twice a week until end of study. Flow cytometry is per-
concentration-timeprofileofpembrolizumabfollowingmultiple
formed on tumor, lymph node, and spleen to assess immune
dosesofpembrolizumabplusipilimumab(linear-linearscale)
microenvironment.
JournalforImmunoTherapyofCancer2016,4(Suppl1):73 Page110of221
Results
Mean tumor volume and survival was plotted to compare
groups (Figs. 3 and 4). Mice treated with triple combination
have decreased tumor growth. Mice treated with combination
T-Vec+BRAFi with or without αPD-1 have longer survival com-
pared to mice treated with control or single drug arms. Flow
cytometry shows increase in percent CD3+/CD45+ cells in tu-
mors of mice treated with combination αPD-1+T-Vec compared
to the control and single drug arms. Percent CD8+/CD3+ cells
in tumors treated with immunotherapy appears to be increased
compared to the control and BRAFi only group (Fig. 5). Add-
itionally, percent of FOXP3+/CD4+ cells in tumors appears to be
decreased in groups receiving T-Vec (Fig. 6) while no change in
FOXP3+/CD4+ populations was observed in tumors from groups
receiving αPD-1 without T-Vec or in draining lymph node or
spleen.
Conclusions Fig.4(abstractP193).Survivalcomparisonoftreatmentgroups
Initial findings show that combination therapy of BRAFi+αPD-1
+T-Vec is more effective than any single treatment.
Combination immunotherapy increases infiltration of T cells into
tumor. Furthermore, oncolytic virus appears to decrease
regulatory T cells infiltrating tumor. This study is ongoing and
further analysis will continue as we further evaluate the
immune microenvironment using flow cytometry and
immunohistochemistry.
Acknowledgements
ThestudywasfundedbytheMelanomaResearchAllianceandAmgen
(Amgen-CUMC-MRAEstablishedInvestigatorAcademic-IndustryPartnership
Award).
Reference
1.Dankort, Curley, Cartlidge, et al.: Braf(V600E) cooperates with
Pten loss to induce metastatic melanoma. Nature Genetics 2009,
41:544–552.
Table1(abstractP193).Treatmentgroups
Group Treatment
Group1(Red) ControlChow+IP2A3+ITPBS
Group2(Orange) BRAFiChow+IP2A3+ITPBS
Group3(Yellow) BRAFiChow+Ipα-PD1+ITPBS Fig.5(abstractP193).FlowcytometrydataofCD8+cellsperCD3
+cellpopulations
Group4(Green) BRAFiChow+IP2A3+ITT-Vec
Group5(Blue) BRAFiChow+IPα-PD1+ITT-Vec
Group6(Purple) ControlChow+IPα=PD1+ITT-Vec
IPintraperitoneal,ITintratumoral,BRAFibriefinhibiotor,α-PD1anti
programmedcelldeath1,T-VectalimogeneLeherparepvec
Fig.6(abstractP193).FlowcytometrydataofCD4+/FOXP3+cells
Fig.3(abstractP193).Tumorvolumecomparisonofallmice perCD4+cellpopulations
JournalforImmunoTherapyofCancer2016,4(Suppl1):73 Page111of221
P194 Background
Phosphatidylserinetargetingantibodyincombinationwith In the tumor microenvironment, T cell inhibitory checkpoint recep-
checkpointblockadeandtumorradiationtherapypromotesanti- torstriggersignalsthatsuppressTcelleffectorfunction,resultingin
canceractivityinmousemelanoma tumorimmuneevasion.Clinicalantibodiesblockingoneofthesere-
SadnaBudhu1,OlivierDeHenau1,RobertaZappasodi1,Kyle ceptors,PD-1, yield positiveresponses inmultiplecancers;however,
Schlunegger2,BruceFreimark2,JeffHutchins2,ChristopherABarker1, their efficacy is limited. Simultaneously targeting more than one in-
JeddDWolchok1,TahaMerghoub1 hibitorycheckpointreceptorhasemergedasapromisingtherapeutic
1MemorialSloanKetteringCancerCenter,NewYork,NY,USA;2Peregrine strategy.Insupportofthisconcept,micedeficientinPD-1andLAG-
Pharmaceuticals,Inc.,Tustin,CA,USA 3,aninhibitorycheckpointreceptoroftenco-expressedwithPD-1in
Correspondence:SadnaBudhu([email protected]) the tumor microenvironment, exhibit enhanced anti-tumor activity.
JournalforImmunoTherapyofCancer2016,4(Suppl1):P194 Here, we demonstrate increased anti-tumor efficacy of a combined
anti–human PD-1 (hPD-1) and anti–human LAG-3 (hLAG-3) therapy
Background usingfullyhumanmonoclonalantibodiesindualhumanizedPD-1x
Phosphatidylserine(PS)isaphospholipidthatisexposedonthesur- LAG-3mice.Thepharmacokineticsandtoxicologyof thenovelanti-
faceofapoptoticcells,sometumorcellsandtumorendothelium.PS hLAG-3 antibody were assessed in non-human primates to support
hasbeenshowntopromoteanti-inflammatoryandimmunosuppres- clinicaldevelopment.
sive signals in the tumor microenvironment. Antibodies that target Methods
PShavebeenshowntoreactivateanti-tumorimmunitybyrepolariz- REGN2810, a high affinity anti-hPD-1 monoclonal antibody that
ingtumorassociatedmacrophagestoaM1-likephenotype,reducing blocks PD-1 interaction with PD-L1 and PD-L2, and a novel high
thenumberofMDSCsintumorsandpromotethematurationofden- affinity monoclonal anti–hLAG-3 antibody, which blocks the LAG-
driticcellsintofunctionalAPCs.InaB16melanomamodel,targeting 3/MHC II interaction were generated. Dual humanized PD-1 x
PS in combination with immune checkpoint blockade has been LAG-3 mice were engineered by replacing the extracellular do-
shown to have a significantly greater anti-cancer effect than either mains of mouse Pdcd1 and Lag3 with the corresponding regions
agent alone. This combination was shown to enhance CD4+ and of hPD-1 and hLAG-3 and were used for testing antibody efficacy
CD8+ T cell infiltration and activation in the tumors of treated ani- in a MC38.ova syngeneic tumor model. Expression of humanized
mals. Radiation therapy is an effective focal treatment of primary PD-1 and LAG-3 were analyzed by flow cytometry. Binding of
solidtumors, butislesseffectiveintreatingmetastatic solidtumors hLAG-3 to mouse MHC II was confirmed with a cell adhesion
asamonotherapy.Thereisevidencethatradiationinducesimmuno- assay, and binding of hPD-1 to mouse PD-L1 was confirmed
genictumorcelldeathandenhancestumor-specificTcellinfiltration using surface plasmon resonance. The pharmacokinetics of anti-
inirradiatedtumors.Inaddition,theabscopaleffect,aphenomenon hLAG-3 antibody following a single i.v. dose, and the safety pro-
in which tumor regression occurs outside the site of radiation ther- file in a 4-week weekly i.v. dose regimen of up to 50 mg/kg/
apy,hasbeenobservedinbothpreclinicalandclinicaltrialswiththe dose, were determined in cynomolgus monkeys.
combinationofradiationtherapyandimmunotherapy. Results
Methods TreatmentofMC38.ovatumor-bearinghumanizedmicewithacom-
Weexaminedtheeffectsofcombiningtumorradiationtherapywith bination of anti-hPD-1 and anti-hLAG-3 antibodies triggered activa-
an antibody that targets PS (1 N11) and an immune checkpoint tion of intratumoral and peripheral T cells. Importantly, the
blockade (anti-PD-1) usingthe mouseB16 melanoma model. Tumor combination treatment exhibited an additive, dose dependent anti-
surfaceareaandoverallsurvivalofmicewereusedtodetermineeffi- tumoreffectcomparedtotherespectivemonotherapies.Anti-hLAG-
cacyofthecombinations. 3 antibody pharmacokinetics in cynomolgus monkeys followed a
Results standardmean concentration-time profile characterized by an initial
We examined the expression of PS on immune cells infiltrating B16 briefdistributionphaseandalinearbetaeliminationphase.Exposure
melanomas.CD11b+myeloidcellsexpressedthehighestlevelsofPS toanti-hLAG-3increasedinadose-proportionalmanner,withelimin-
ontheirsurfacewhereasTcellsandB16tumorcellsexpresslittleto ationhalf-livesrangingfrom10.8to11.5days.Anti-hLAG-3antibody
noPS.ThesedatasuggestthattargetingPSinB16melanomawould was well tolerated, and no-observed-adverse-effect level (NOAEL)
induce a pro-inflammatory myeloid tumor microenvironment. We couldbeestablishedupto50mg/kg.
hypothesizethattherapiesthatinduceapoptoticcelldeathontumor Conclusions
cells would enhance the activity of PS-targeting antibodies. We Preclinical anti-tumor efficacy of combined REGN2810 and anti-
thereforeexaminedtheeffectsofcombiningaPS-targetingantibody hLAG-3antibodytreatment,togetherwithfavorablepharmacokinetic
withlocaltumorradiation.WefoundthatthePS-targetingantibody and safety data for anti-hLAG-3 antibody in cynomolgus monkeys,
synergizes with both anti-PD-1 and radiation therapy to improve support clinical development of this cancer combination
anti-cancer activity and overall survival. In addition, the triple com- immunotherapy.
binationofthePS-targetingantibody,tumorradiationandanti-PD-1
treatmentdisplayedevengreateranti-cancerandsurvivalbenefit.
Conclusions
P196
Thisfindinghighlightsthepotentialofcombiningthesethreeagents
CombinationofPD-L1blockadewithoncolyticvaccinesre-shapes
toimproveoutcomeinpatientswithadvanced-stagemelanomaand
thefunctionalstateoftumorinfiltratinglymphocytes
may inform the design of future clinical trials with PS targeting in
CristianCapasso1,FedericaFrascaro2,SaraCarpi3,SiriTähtinen1,Sara
melanomaandothercancers.
Feola4,ManlioFusciello1,KaritaPeltonen1,BeatrizMartins1,Madeleine
Sjöberg1,SariPesonen5,TuuliRanki5,LukaszKyruk1,ErkkoYlösmäki1,
VincenzoCerullo1
P195 1UniversityofHelsinki,Helsinki,Uusimaa,Finland;2UniversityofSiena,
Anovelanti-humanLAG-3antibodyincombinationwithanti- Supersano(LE),Puglia,Italy;3UniversityofPisa,Pisa,Toscana,Italy;
humanPD-1(REGN2810)showsenhancedanti-tumoractivityin 4UniversityofNapoliFedericoII,Helsinki,Uusimaa,Finland;5PeptiCRAd
PD-1xLAG-3dual-humanizedmiceandfavorablepharmacokinetic Oy,Helsinki,Uusimaa,Finland
andsafetyprofilesincynomolgusmonkeys Correspondence:CristianCapasso([email protected])
ElenaBurova,OmairaAllbritton,PeterHong,JieDai,JerryPei,MattLiu, JournalforImmunoTherapyofCancer2016,4(Suppl1):P196
JoelKantrowitz,VenusLai,WilliamPoueymirou,DouglasMacDonald,Ella
Ioffe,MarkusMohrs,WilliamOlson,GavinThurston Background
Regeneron,Tarrytown,NY,USA Theimmunologicalescapeoftumorsrepresentsoneofthemainob-
Correspondence:ElenaBurova([email protected]) stacles to the treatment of malignancies. The blockade of PD-1 or
JournalforImmunoTherapyofCancer2016,4(Suppl1):P195 CTLA-4 receptors represented a milestone in the history of
JournalforImmunoTherapyofCancer2016,4(Suppl1):73 Page112of221
immunotherapy.However,immunecheckpointinhibitorsseemtobe P197
effective in specific cohorts of patients. It has been proposed that Invitroevaluationofimmunotherapyprotocolsthroughalabel-
their efficacy relies on the presence of an immunological response. freeimpedance-basedtechnologyallowsdynamicmonitoringof
Thus,wehypothesizedthatdisruptionofthePD-L1/PD-1axiswould immuneresponseandreagentefficacy
synergizewithouroncolyticvaccineplatformPeptiCRAd. FabioCerignoli,BiaoXi,GarretGuenther,NaichenYu,LincolnMuir,
Methods LeynaZhao,YamaAbassi
We used murine B16OVA in vivo tumor models and flow cytometry ACEABiosciencesInc.,SanDiego,CA,USA
analysistoinvestigatetheimmunologicalbackground. Correspondence:FabioCerignoli([email protected])
Results JournalforImmunoTherapyofCancer2016,4(Suppl1):P197
First, we found that high-burdenB16OVA tumors were refractory to
combination immunotherapy. However, with a more aggressive Background
schedule,tumorswithalowerburdenweremoresusceptibletothe Invitrocharacterization of reagent efficacy in the context of can-
combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cer immunotherapy is a necessary step before moving to more
cantly increased the median survival of mice (Fig. 7). Interestingly, expensive animal models and clinical studies. However, current
the reduced growth of contralaterally injected B16F10 cells sug- in vitro assays like Chromium-51, ATP-based luminescence or flow
gested the presence of a long lasting immunological memory also cytometry are either difficult to implement in high throughput
against non-targeted antigens. Concerning the functional state of environments or are mainly based on endpoint methodologies
tumor infiltrating lymphocytes (TILs), we found that all the immune that are unable to capture the full dynamic of the immune re-
therapies would enhance the percentage of activated (PD-1pos TIM- sponse. Here, we present the adaptation of an impedance-based
3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos platform to monitor cytotoxic activity of immune cells activated
TIM-3pos) cells compared to placebo. As expected, we found that trough different means.
PeptiCRAdmonotherapycould increase the numberofantigenspe- Methods
cific CD8+ T cells comparedto other treatments. However, only the Impedance technology detects cell death and proliferation of ad-
combinationwithPD-L1blockadecouldsignificantlyincreasethera- herent cells by measuring changes in conductance of microelec-
tio between activated and exhausted pentamer positive cells (p= trodes embedded in 96 and 384-wells cell culture plates. We
0.0058),suggestingthatbydisruptingthePD-1/PD-L1axiswecould utilized adherent and B cell leukemia/lymphoma cell lines as well
decreasetheamountofdysfunctionalantigenspecificTcells.Weob- as primary tumor cells as in vitro models for immunotherapy re-
served that the anatomical location deeply influenced the state of agent evaluation. We seeded the cells on electrodes coated 96-
CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- well plates and monitored cell adhesion and proliferation for
creasedby2foldonTILscomparedtosplenicandlymphoidTcells. 24 hours. The following day effector cells were added at multiple
In the CD8+ compartment, the expression of PD-1 on the surface effector:target ratios in presence of BiTEs antibodies and/or anti
seemedtoberestrictedtothetumormicro-environment,whileCD4 PD-1/PD-L1 antibodies. Impedance signal was monitored for up
+ T cells had a high expression of PD-1 also in lymphoid organs. to seven days. Control wells were set up with effector cells only
Interestingly, we found that the levels of PD-1 were significantly or with target plus effector cells but without antibodies. We
higher on CD8+ T cells than on CD4+ T cells into the tumor micro- adapted such adhesion-based technology to monitor non-
environment(p<0.0001). adherent B-leukemia/lymphoma cells, by developing a strategy
Conclusions where the wells are coated with an anti-CD40 antibody. The coat-
In conclusion, we demonstrated that the efficacy of immune check- ing allows specific adhesion and retention of B cells and meas-
point inhibitors might be strongly enhanced by their combination urement of changes in impedance that are proportional to cell
withcancervaccines.PeptiCRAdwasabletoincreasethenumberof number.
antigen-specific T cells and PD-L1 blockade prevented their exhaus- Results
tion, resulting in long-lasting immunological memory and increased Using increasing concentrations of EpCAM/CD3 BiTE, we demon-
mediansurvival. strated the suitability of an impedance-based approach to quantita-
tively monitor the efficacy of immune cells-mediated cancer cell
killing both under different effector:target ratios and antibody con-
centrations.Combinationtreatmentswithcheckpointreducedtiming
andincreasedamountofkilledcancercells.Similarresultswerealso
obtained with engineered CAR-T cells against CD19 or NK cell lines,
demonstratingspecifickillingoftumorBcellsatveryloweffector:tar-
getratios.Theresultswerealsoconfirmedbyflowcytometry.
Conclusions
Overall, our results demonstrate the value of an impedance-based
approach in measuring the cytotoxic response across the temporal
scale, an aspect that is otherwise very difficult to assess with more
canonical end point assays. Furthermore, the availability of 384-well
format and minimal sample handling place the technology in an
ideal spot for applications in large reagent validation screening or
personalized medicine, like therapeutic protocol validation directly
onpatientsamples.
P198
Tumornecrosisfactoralphaandinterleukin-2expressing
adenovirusplusPD-1blockadeasaboostforTcelltherapyinthe
contextofsolidtumortherapies
VíctorCervera-Carrascón1,MikkoSiurala1,JoãoSantos1,RiikkaHavunen2,
SuviParviainen1,AkseliHemminki1
Fig.7(abstractP196).SurvivalofC57micebearingB16OVA
1TILTBiotherapeutics,Helsinki,Uusimaa,Finland;2UniversityofHelsinki,
tumorsandtreatedonday6post-implantationwitheitherPBS,
Helsinki,Uusimaa,Finland
PDL1blockade,OVA-targetingPeptiCRAdorthecombinationof
Correspondence:VíctorCervera-Carrascón([email protected])
PDL1-blockadeandOVA-PeptiCRAd.
JournalforImmunoTherapyofCancer2016,4(Suppl1):P198
JournalforImmunoTherapyofCancer2016,4(Suppl1):73 Page113of221
Background IMM-101, no response (initial SD). 2011 given Ipilimumab. Patient
Becauseoftheimmunosuppressivetumormicroenvironment,theim- 2: 2011 50 F axillary lump removed, melanoma (no primary).
munesystemisunabletodevelopeffectiveresponsesagainsttumor Concomitant mediastinal, lung, gastric and peritoneal deposits.
cells. This phenomenon also acts against the effectiveness of adop- Gastric surgery, decarbazine. Commenced IMM-101 with cyber-
tiveTcelltherapy.Inordertoovercomethissituationinthetumor, knife to lung lesion. 2013 Small bowel obstruction from new dis-
anattractivetherapeuticcombinationisthecombinationofoncolytic ease. Started ipilimumab. Patient 3: 2014 79 M melanoma, left
viruses and immune checkpoint inhibitors. In this case, besides the cheek, BT 2.4 mm. Regional lymph node recurrence, treated with
last two therapies mentioned above, combinations with T cell ther- a left neck dissection in April 2014. Developed paracardiac
apy were also included. The virus used was engineered to express nodes, adrenal, lung and multiple large subcutaneous deposits.
tumor necrosis factor α (TNFα) and interleukin (IL)-2, two cytokines Commenced IMM-101 with initial shrinkage. However, new large
that will boost the immunogenicity of the virus and thus its antitu- subcutaneous lesions. Commenced pembrolizumab.
morproperties.Ontheotherhand,theuseofanti-PD-1willavoidex- Results
haustionontumorinfiltratingTcellsandhenceremovethebarriers Patient 1 - CR on Pet CT, maintained through 2016. Patient 2 - CR
thatcoulddampenthedesiredimmuneresponseagainstthetumor. maintainedfor2years.Patient3-CRofsubcutaneousdepositsfour
Methods daysafterfirstinjection.
In the study of the antitumor effect of this three armed treat- Conclusions
ment we used an in vivo model of subcutaneous B16-OVA TheCRratetoCPI’sisdisappointing,<1%forIpilimumab.PDL-1ex-
melanoma-bearing mice. Two experiments were carried out; the pressionispredictiveforPD-1responsesandalthoughCPIcombina-
first one (n=47) to establish the differences between the triple, tions are clearly needed, most are very toxic. IMM-101 is relatively
double, and single armed combination therapies and the second free of toxicity, enhances PD-1expressionin pre-clinical models but
experiment (n=84) was focused on study the differences be- mayalsoprimetumourresponsetocheckpointinhibitorsbyitsac-
tween the groups that showed the best outcomes in the first tion on macrophage function. Based on these observations, we
one and also optimize viral and anti-PD-1 administration regimes. speculatethatIMM-101primesfor CPI’s and propose a trialpriming
Results withIMM-101,followedbyanti-PD-1antibodies.
Preliminaryresultsshowastatisticallysignificantpositiveeffectcom-
ing out from the combination of virus therapy and immune check- References
point blockade with regard to both tumor progression and overall 1. FowlerD,etal.:MycobacteriaactivateγδT-cellanti-tumourresponses
survival, with up to 43 % complete tumor regression achieved in viacytokinesfromtype1myeloiddendriticcells:amechanismof
someofthegroupsafter96daysposttreatment.Ontheotherhand, actionforcancerimmunotherapy.CeII2012,61(4):535–547.
the effect of adoptive cell therapy in this combination is not com- 2. Stebbing J, et al.: An intra-patient placebo-controlled phase I trial to
pletelyclear.Moreresultswillbepresentedafteranalyzingbiological evaluatethesafetyandtolerabilityofintradermalIMM-101inmelan-
samplescollectedduringbothexperiments. oma.AnnOncol2012,23(5):1314–1319.
Conclusions 3. Dalgleish,etal.:Randomisedopen-label,phaseIIstudyofGemcitabine
Preclinical studies are a key step to detect which combinations are withandwithoutIMM-101foradvancedpancreaticcancer(IMAGE-1
moresuitableforsuccessinhumantrials.Inthisstudywedeveloped Trial).BJC2016,inpress.
arationaleforthecombinationrelyingontwoconcepts:tomakesi-
lent tumors more visible to the immune system and to counter im-
munosuppressivemechanismstounleashthefullpotentialofTcells
P200
against the tumor, rendering in a modification of the tumor micro-
Immunologicalimpactofcheckpointblockadeondendriticcell
environmentthatmakesitmoresusceptibleforTcellmediatedkill-
drivenTcellresponses:acautionarytale
ing. According to the results displayed from these experiments, the
MarkDeBenedette,AnaPlachco,AliciaGamble,ElizabethWGrogan,
combinationofthisgeneticallymodifiedadenovirusandPD-1block-
JohnKrisko,IrinaTcherepanova,CharlesNicolette
ade is an efficient combination to be considered for future applica-
ArgosTherapeuticsInc.,Durham,NC,USA
tioninhumans.
Correspondence:MarkDeBenedette
([email protected])
JournalforImmunoTherapyofCancer2016,4(Suppl1):P200
P199
IMM-101primesforincreasedcompleteresponsesfollowing Background
checkpointinhibitorsinmetastaticmelanoma;3casereports AGS-003 is an individualized, autologous, tumor antigen-loaded,
AngusDalgleish1,SatvinderMudan2 dendritic cell (DC) immunotherapy currently in phase III develop-
1StGeorge'sUniversityofLondon,London,UK;2TheRoyalMarsden ment for the treatment of metastatic renal cell carcinoma (mRCC)
HospitalandImperialCollegeLondon,London,UK in combination with standard-of-care. Antibodies to PD-1 on acti-
Correspondence:AngusDalgleish([email protected]) vated T cells or PD-L1 expressed on APCs have now been ap-
JournalforImmunoTherapyofCancer2016,4(Suppl1):P199 proved for treatment of several cancer indications including RCC.
While there is a strong mechanistic rationale for the potential
Background synergy of these agents in combination, data supporting the im-
IMM-101, a heat-killed borate-buffered whole cell product of Myco- portance of sequencing the administration of these agents are
bacteriumobuensehasbeenshowntoenhancecellmediatedcyto- limited. Since the DC-based immunotherapy, AGS-003, expresses
kine responses and innate immune responses involving NK and high levels of PD-L1, combinations with checkpoint blockade may
gammadeltacells[1].Completeresponses(CR)inpatientswithmel- remove a critical signal protecting DCs during the early CTL acti-
anomalungmetastasesdemonstrated.Followupoforiginalpublica- vation phase in vivo. Concurrent administration of checkpoint in-
tion [2] has shown a 30 % 5-year survival. Combined with hibitors with AGS-003 may, therefore, impede the proposed
gemcitabine in metastatic pancreatic cancer a significant survival mechanism of action of AGS-003, which is the induction of
advantageovergemcitabinemonotherapyisseen[3]. tumor-specific CTL responses. Results derived from in vitro model-
Methods ing of DCs inducing T cell responses can demonstrate how to
We present 3 patients with metastatic melanoma, progressed better mobilize the immune system to overcome the immunosup-
after initial stabilisation with IMM-101, who showed CR after pressive environment of cancer. Therefore, it was of interest to
check point inhibitors (CPI) ipilimumab (n=2), pembrolizumab test anti-PD-1/anti-PD-L1 antibody therapy in vitro in combination
(n=1). Patient 1: 2006 46 M melanoma left forearm, BT 3.7 mm, with DCs representative of AGS-003, to observe the effects com-
1 positive lymph node. Recurrent disease treated with surgery, bination therapy would have on antigen-specific CTL proliferation
Aldara and low dose IL-2. 2010 pulmonary mets, commenced and functional responses.
JournalforImmunoTherapyofCancer2016,4(Suppl1):73 Page114of221
Methods anti-PD-1 treated group as compared to the untreated. Anti-PD-1
DCs derived from monocytes were co-electroporated with MART-1 treatment led to a significant increase in the number of NK cells
RNAandCD40ligandRNAtorepresentAGS-003DCproducts.Invitro and macrophages in the OS lung tumors suggesting these cells
co-cultures were set up with autologous CTLs and MART-1/CD40L to have a potential therapeutic benefit against OS lung metasta-
DCs in the presence of anti-PD-1 or anti-PD-L1 antibodies. In some ses. In addition, anti-PD-1 therapy caused a decrease in PD-L1 ex-
instances,PD-1expression washyperexpressedonCTLsbyelectro- pression in the lung tumors, possibly due to a decrease in p-
poratingMART-1-specficCTLswithPD-1RNA.Subsequentexpansion ERK1/2 and p-Stat3 expression.
of MART-1-specific CTLs and multi-functional responses in the pres- Conclusions
ence of checkpoint blockade were mapped using multi-color flow We conclude that targeting the PD-1/PD-L1 axis could be used to
cytometry. treat OS lung metastasis. Therapeutic efficacy of anti-PD-1 may be
Results due to an increased activity of NK cells and/or macrophages in the
Combination with anti-PD-1 antibody did not did not negatively lung tumors and that inhibition of the p-Stat3/PD-L1 pathway may
affect the expansion of MART-1-specific CTL responses; however, if be the mechanismimplicatedin OS lung metastases afteranti-PD-1
PD-1 was hyper-expressed on previously stimulated MART-1-specific treatment.
CTLs responses were diminished. Anti-PD-1 antibody blocking re-
storedCTLfunctioninthepresenceofhighlevelsofPD-1expression.
Interestingly,anti-PD-L1antibodyblockingresultedinsuppressionof
P202
earlyMART-1-specificCTLexpansionandsubsequentdownstreamef-
EffectoftheclassI-HDACinhibitorentinostatandthepan-HDAC
fectorfunction.
inhibitorvorinostatonperipheralimmunecellsubsets
Conclusions
ItaliaGrenga,LaurenLepone,SofiaGameiro,KarinMKnudson,Massimo
Our results suggest that the sequencing of AGS-003 therapy and
Fantini,KwongTsang,JamesHodge,ReneeDonahue,JeffreySchlom
checkpointblockadeisimportantto allowfullCTLactivationbythe
LaboratoryofTumorImmunologyandBiology,NationalCancerInstitute,
DCsprior to anti-PD-1/PD-L1therapy.Moreoverthe high expression
ofPD-L1onDCsmayserveasa“don’tkillthemessenger”signal,crit- NationalInstitutesofHealth,Bethesda,MD,USA
Correspondence:ReneeDonahue([email protected])
icaltopreventdeletionoftheDCpriortofullsignaldeliveryduring
JournalforImmunoTherapyofCancer2016,4(Suppl1):P202
earlyphasesofCTLactivation.
Background
Cancer immunotherapy requires effective recognition and elimin-
P201 ation of tumor cells identified as non-self; however, tumors can
TargetingthePD-1/PD-L1signalingpathwayforthetreatmentof evade host immune surveillance through multiple mechanisms,
OSlungmetastasis including epigenetic silencing of genes involved in antigen pro-
PoojaDhupkar,LingYu,EugenieSKleinerman,NancyGordon cessing and immune recognition. Epigenetic therapy with histone
UniversityofTexasMDAndersonCancerCenter,Houston,TX,USA deacetylase (HDAC) inhibitors has shown limited benefit as a
Correspondence:PoojaDhupkar([email protected]) monotherapy in patients with solid tumors; however, recent re-
JournalforImmunoTherapyofCancer2016,4(Suppl1):P201 ports suggest the potential for synergy when combined with im-
munotherapy. Entinostat is a class I-HDAC inhibitor undergoing
Background trials for the treatment of various cancers, while vorinostat is a
Osteosarcoma (OS) is a primary bone malignancy, commonly cul- pan-HDAC inhibitor approved in the United States for the treat-
minating into aggressive pulmonary metastasis. Despite chemo- ment of cutaneous T cell lymphoma. The aim of this study was
therapy advances, the 5-year survival of pulmonary metastatic OS to extensively evaluate the effects of entinostat and vorinostat on
remains 25-30 %. Immunotherapy is one of the promising novel human peripheral immune cell subsets in order to examine the
approaches to target minimal residual and relapsed disease. The potential for combination of HDAC inhibitors with cancer
objective of this study is to determine if blocking the PD-1/PD-L1 immunotherapy.
immunosuppressive signaling pathway using a PD-1 checkpoint Methods
inhibitor will have an effect in OS lung metastasis. Anti-PD-1 and Peripheral blood mononuclear cells (PBMCs) from metastatic
anti-PD-L1 antibodies have exhibited therapeutic benefit in mel- breast cancer patients (n=7) were exposed in vitro for 48 hours
anoma, and non-small cell lung carcinoma. We hypothesize that to clinically relevant exposures of entinostat, vorinostat, or ve-
disruption of the PD-1/PD-L1 signaling pathway using anti-PD-1 hicle control. PBMCs were then analyzed by multicolor flow cy-
antibody has an effect against OS lung metastasis and improves tometry using 27 unique markers to identify 123 immune cell
overall survival. subsets, which included 9 classic cell types [CD4+ and CD8+ T
Methods cells, regulatory T cells (Treg), B cells, conventional dendritic
Flow cytometry and western blotting were used to analyze PD-L1 cells (cDC), plasmacytoid dendritic cells (pDC), natural killer cells
expression in 7 different OS cell lines. Immunohistochemistry (NK), natural killer T cells (NKT), and myeloid derived suppressor
(IHC) analysis was used to determine PD-L1 expression in OS lung cells (MDSC)], and 114 refined subsets relating to their matur-
metastases from patients and mice. LM7 human OS mouse model ation and function.
was used to test the effect of blocking murine PD-1 in OS lung Results
metastases. Therapeutic effect of anti-PD-1 treatment was mea- Treatmentwithentinostatandvorinostatinducedseveralnotableal-
sured by the number of macro and micro-metastases. IHC was terationsinperipheralimmunecells,suggestingmainlyimmuneacti-
used to measure cell proliferation (Ki-67), apoptosis (TUNEL) and vatingproperties.Exposureto entinostatincreasedthefrequencyof
cleaved-caspase 3 expression in addition to NK cells and macro- activatedCD4+Tcells,activatedmatureNKcells,antigenpresenting
phages infiltration. Western blotting was used to address the cells(cDCs),andhighlyimmatureMDSCs,aswellasdecreasedtotal
downstream components of the signaling pathway such as p- Tregs and those with a suppressive phenotype. Exposure to vorino-
Stat3 and p-Erk1/2. The Simple PCI software was used to quantify statinducedfewerchangesthanentinostat,includingincreasingthe
the IHC data. frequency of activated CD4+ T cells, highly immature MDSCs, and
Results NKTcells.
Our studies revealed surface and total PD-L1 expression in five Conclusions
out of seven human OS cell lines. Primary and metastatic OS Thesefindingsshowthatwhileentinostatandvorinostathaveoverall
lung tumor samples from patients demonstrated membranous immune activating properties, entinostat induced a greater number
and cytoplasmic PD-L1 expression. Using a human OS mouse changes than vorinostat. This study supports the combination of
model we demonstrated therapeutic effect of anti-PD-1 therapy HDAC inhibitors with immunotherapy, including therapeutic cancer
as the number of macro and micro-metastases decreased in the vaccinesand/orcheckpointinhibitors.
JournalforImmunoTherapyofCancer2016,4(Suppl1):73 Page115of221
P203 andinhibittumorprogression.PhaseIb/IIatrialsofcombinationther-
Shifting the balance of tumor-mediated immune suppression apywithimmunecheckpointinhibitionareplanned.
and augmenting immunotherapy with antibody blockade
of semaphorin 4D to facilitate immune-mediated tumor
rejection P204
ElizabethEvans1,HolmBussler1,CrystalMallow1,ChristineReilly1, CombinationofaglycomimeticantagonisttoE-selectinand
SeboldTorno1,MariaScrivens1,CathieFoster1,AlanHowell1,Leslie CXCR4,GMI-1359,withananti-PD-L1antibodyattenuates
Balch1,AlyssaKnapp1,JohnELeonard1,MarkParis1,TerryFisher1, regulatoryTcellinfiltrationandacceleratestimetocomplete
SiwenHu-Lieskovan2,AntoniRibas2,ErnestSmith1,MauriceZauderer1 responseinthemurineCT26tumormodel
1Vaccinex,Rochester,NY,USA;2UniversityofCalifornia,LosAngeles, WilliamFogler1,MarilynFranklin2,MattThayer2,DanSaims2,JohnL.
LosAngeles,CA,USA Magnani1
Correspondence:ElizabethEvans([email protected]) 1GlycoMimetics,Inc.,Rockville,MD,USA;2MIBioresearch,AnnArbor,MI,
JournalforImmunoTherapyofCancer2016,4(Suppl1):P203 USA
Correspondence:WilliamFogler([email protected])
Background JournalforImmunoTherapyofCancer2016,4(Suppl1):P204
We report a novel role for semaphorin 4D (SEMA4D, CD100) in
modulatingthetumormicroenvironment(TME)toexcludeactivated Background
antigen presenting cells and cytotoxic T lymphocytes so as to pro- RegulatoryT cells(T ) modulate anti-tumorimmunitybysuppress-
reg
motetumorgrowth.AntibodyblockadereducesexpansionofMDSC, ingTcellactivation.T areinducedandmaintainedbyimmunoreg-
reg
shiftsthebalanceofM1/M2,Teffector/Tregulatorycellsandassoci- ulatory receptors, such as PD-L1, and respond to homing signals
atedcytokinesandchemokines,andaugmentstumorrejectionwith withintheinflamedtumormicroenvironmentthatincludetheendo-
immunecheckpointinhibition. thelialcellprotein,E-selectin,andtheCXCR4ligand,SDF-1.GMI-1359
Methods is a small molecule glycomimetic beginning clinical evaluation with
Anti-SEMA4D antibodies were evaluated, alone and in combination dual inhibitory activity against E-selectin and SDF-1. The aim of the
with immune checkpoint antibodies. Immune response was charac- currentstudywastodetermineifGMI-1359aloneorincombination
terized by immunohistochemistry, flow cytometry, functional assays, withanti-mPD-L1antibodyaffectedtheinvivogrowthofCT26colon
and cytokine, chemokine and gene expression analysis. Anti-tumor carcinomaandtoassesspercentagesofinfiltrativeintratumoralcells
activitywasevaluatedinvariouspreclinicalmodels.AphaseItrialfor expressingimmunemarkers.
singleagentVX15/2503wascompleted. Methods
Results Female Balb/c mice were implanted subcutaneously with 5x105
SEMA4D restricts migration of macrophages and promotes expan- CT26.WT tumor cells. Three days post tumor injection, mice (n=
sion of suppressive myeloid cells in vitro. Strong expression of 15/group) were treated with saline, GMI-1359 (40 mg/kg for 12
SEMA4D at the invasive margins of actively growing tumors consecutive days), isotype control antibody (anti-KLH) or anti-
in vivo modulates the infiltration and polarization of leukocytes in mPD-L1 antibody (10 F.9G2, 10 mg/kg on days 3, 6, 10, 13, and
the TME. Antibody neutralization facilitated recruitment of acti- 17), or the combination of GMI-1359 and anti-mPD-L1 or anti-
vated APCs and T lymphocytes into the TME in preclinical KLH. On day 15, tumors and spleens (n=5/group) were excised
models. M-MDSCs were significantly reduced in both tumor and and T cells (total CD4+ and CD8+, and CCR7+/CD62L+ subsets of
blood following treatment. This was accompanied by a significant each), regulatory T cells (T ; CD4/CD25/FoxP3), and myeloid de-
reg
shift towards increased Th1 cytokines and CTL-recruiting chemo- rived suppressor cells (MDSC; CD11b+/Gr1+) were determined by
kines, with concurrent reduction in Treg-, MDSC-, and M2- flow cytometry. The remaining mice were followed for tumor
macrophage promoting chemokines (CCL2, CXCL1, CXCL5). response.
Accordingly, an increase in Teff:Treg ratio (3x, p<0.005) and CTL Results
activity (4x, p<0.0001) was observed. NanoString gene expression Treatments were well tolerated. Mice in control groups and single
analysis of on-treatment tumors confirms an increase in the agent GMI-1359 were all identified with progressive disease. In
gamma-inflammatory gene signature (Ribas, ASCO 2015), includ- contrast, treatment with anti-mPD-L1 alone or in combination with
ing significant increases in CXCL9, Gzmb, CCR5, Stat1, Lag3, Ptprc, GMI-1359produceda40%completeresponse(CR)rate.Themedian
Ciita, Pdcd1 (PD-1), and Itga1. These coordinated changes in the time to CR was shorter when anti-mPD-L1 was combined with GMI-
tumoral immune context are associated with durable tumor rejec- 1359comparedtoanti-mPD-L1alone(14vs.23days,respectively,p<
tion and immunologic memory in preclinical colon, breast, and 0.0471).Evaluationoftumorinfiltratingcellsshowedthatcombination
melanoma models. Importantly, anti-SEMA4D antibody can further therapy with GMI-1359 and anti-mPD-L1 reduced the percentage of
enhance activity of immune checkpoint inhibitors and chemother- T compared to treatment with saline, GMI-1359 or anti-mPD-L1 as
reg
apy. Strikingly, the combination of anti-SEMA4D with anti-CTLA-4 singletreatments(0.9%vs.3.3%,2.9%and1.9%,respectively).No
acts synergistically, with maximal increase in survival (p<0.01) otherTcellsubsetswereaffected.Inspleens,themedianpercentage
and complete tumor regression in 100 % of mice, as compared ofT wereunaffectedbyanyofthetreatmentsandsuggestthatthe
reg
to 22 % with monotherapy (p<0.01). SEMA4D antibody treat- reductioninintratumoralT bycombinedtreatmentwithanti-PD-L1
reg
ment was well tolerated in nonclinical and clinical studies; includ- andGMI-1359wasanattenuatedresponsetomaintenanceandhom-
ing a phase I multiple ascending dose trial in patients with ingsignalsinthetumormicroenvironment.
advanced refractory solid tumors. Patients with the longest dur- Conclusions
ation of treatment, 48–55 weeks, included colorectal, breast, and In conclusion, these studies demonstrate that the dual E-selectin/
a papillary thyroid patient, who had a partial response by RECIST. CXCR4 antagonist, GMI-1359, in combination with anti-mPD-L1 anti-
Conclusions body attenuates the induction and distribution of intratumoral T
reg
InhibitionofSEMA4Drepresentsanovelmechanismandtherapeutic and this reduction in T is associated with a more rapid immuno-
reg
strategy to promote functional immune infiltration into the tumor therapeuticanti-tumorresponse.