characterization of mk-4166, a clinical agonistic antibody that … · 2017. 8. 2. ·...

Therapeutics, Targets, and Chemical Biology

Characterization of MK-4166, a Clinical AgonisticAntibody That Targets Human GITR and Inhibitsthe Generation and Suppressive Effects of TRegulatory CellsSelvakumar Sukumar1, Douglas C.Wilson1, Ying Yu1, Jerelyn Wong1,Saraswathi Naravula1, Grigori Ermakov1, Romina Riener1, Bhagyashree Bhagwat1,Antoaneta S. Necheva2, Jeff Grein1, Tatyana Churakova1, Ruban Mangadu1,Peter Georgiev2, Denise Manfra2, Elaine M. Pinheiro2, Venkataraman Sriram1,Wendy J. Bailey3, Danuta Herzyk3, Terrill K. McClanahan1, Aarron Willingham1,Amy M. Beebe1, and Svetlana Sadekova1

Abstract

GITR is a T-cell costimulatory receptor that enhances cellularand humoral immunity. The agonist anti-mouse GITR anti-body DTA-1 has demonstrated efficacy in murine models ofcancer primarily by attenuation of Treg-mediated immunesuppression, but the translatability to human GITR biologyhas not been fully explored. Here, we report the potentialutility of MK-4166, a humanized GITR mAb selected to bind toan epitope analogous to the DTA-1 epitope, which enhancesthe proliferation of both na€�ve and tumor-infiltrating T lym-phocytes (TIL). We also investigated the role of GITR agonismin human antitumor immune responses and report here thepreclinical characterization and toxicity assessment of MK-4166, which is currently being evaluated in a phase I clinical

study. Expression of human GITR was comparable with that ofmouse GITR in tumor-infiltrating Tregs despite being drasticallylower in other human TILs and in many human peripheralblood populations. MK-4166 decreased induction and sup-pressive effects of Tregs in vitro. In human TIL cultures, MK-4166induced phosphorylation of NFkB and increased expressionof dual specificity phosphatase 6 (DUSP6), indicating thatMK-4166 activated downstream NFkB and Erk signaling path-ways. Furthermore, MK-4166 downregulated FOXP3 mRNA inhuman tumor infiltrating Tregs, suggesting that, in addition toenhancing the activation of TILs, MK-4166 may attenuate theTreg-mediated suppressive tumor microenvironment. CancerRes; 77(16); 1–11. �2017 AACR.

IntroductionThe significant clinical benefit obtained from the use of

immune checkpoint inhibitors in the treatment of cancer andthe durability of such responses has generated tremendous inter-est in immunotherapy of cancers (1–4). Therapeutic antibodiesdesigned to block the inhibitory feedbackmechanisms of CTLA-4or PD-1 have been approved for the treatment of cancer. Inaddition, several agonist antibodies that target costimulatory

molecules on T cells like GITR, CD27, 4-1BB, CD40, and OX40are in various stages of pre-clinical or clinical development (5, 6).

Glucocorticoid-induced tumor necrosis factor receptor-relatedprotein (GITR), also referred to as TNFRSF18, is a type I trans-membrane protein of the tumor necrosis factor receptor super-family that is expressed on human T lymphocytes (preferentiallyon Tregs; refs. 7–10) and natural killer (NK) cells (11–13). Expres-sion of GITR is significantly increased upon T-cell activation, andligation of GITR provides a costimulatory signal that positivelymodulates antigen-specific T-cell responses, leading to enhancedcellular and humoral immunity (11, 12, 14). The counter struc-ture, GITR-Ligand (GITR-L), is expressed on antigen-presentingcells (dendritic cells, B cells, and macrophages; refs. 15–18).

Engagement of murine GITR by either an agonistic anti-mouseGITR antibody (clone DTA-1) or GITR-L, in the presence of aprimary TCR signal, results in enhanced T-cell proliferation andcytokine production (11, 12). In mice, DTA-1 abrogates Treg-mediated suppression either by eliminating GITR-expressingtumor-infiltrating Tregs (19, 20) or by causing them to becomeunstable thereby attenuating their suppressive activity (21, 22).Agonist anti-GITR antibodies can prevent tumor growth or cureestablished tumors in murine tumormodels (21, 23–28), but thequestion of whether or not human GITR biology parallels that ofthemouse remains largely unanswered (29). Several humanGITR

1Merck Research Laboratories, Palo Alto, California. 2Merck Research Labora-tories, Boston, Massachusetts. 3Merck Research Laboratories, West Point,Pennsylvania.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

S. Sukumar and D.C. Wilson contributed equally this article.

Current address for S. Sukumar: CSL Behring, King of Prussia, PA; and currentaddress for V. Sriram, Pionyr Immunotherapeutics, San Francisco, CA.

Corresponding Author: Selvakumar Sukumar, Merck Research Laboratories,901 South California Avenue, Palo Alto, CA 94304. Phone: 610-878-4271; Fax:650-496-1200; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-16-1439

�2017 American Association for Cancer Research.

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agonists are currently in early clinical development for thetreatment of solid tumors (NCT01239134, NCT01216436,NCT02583165, NCT02628574, NCT02697591, NCT02132754,NCT02437916).

On the basis of the potent antitumor efficacy ofDTA-1 inmurinemodels (21, 23–26), we developed the humanized IgG1 agonistanti-GITR mAb MK-4166, focusing our selection on binding to anepitopeonhumanGITRanalogous to the epitope thatDTA-1bindsonmouseGITR.We show that engagement of GITR byMK-4166 isa high-affinity interaction that enhances TCR-driven in vitro prolif-eration of human and cynomolgus monkey na€�ve CD4þ T cellssimilar to the effect of DTA-1 on mouse T cells. In addition, MK-4166 enhanced the proliferation of human tumor-infiltrating Tcells. Toaddress the translatabilityofmurine efficacydata tohumancancers, we evaluated GITR expression in mouse and human. Theexpression of GITR on tumor-infiltrating Tregs was comparablebetweenmouse and human despite differences in GITR expressionon many peripheral blood populations and tumor-infiltratingCD4þ and CD8þ T cells. Because Tregs have been shown to playa critical role in antitumor efficacy of DTA-1, we focused oncharacterization of MK-4166 activity on human Tregs. MK-4166significantly inhibited the generation of Tregs in mixed lymphocytereactions (MLR) and decreased the suppressive effects of Tregs.Moreover, MK-4166 induced phosphorylation of NFkB, upregu-lated ERK pathway genes, and decreased expression of FOXP3 intumor infiltrating T cells in ex vivo human tumor cultures.MK-4166did not induce any adverse events when administered intravenous-ly to cynomolgus macaques at a broad range of doses. The safetyand tolerability ofMK-4166 inpatientswith advanced solid tumorsis currently being evaluated in a phase I study (NCT02132754).

Materials and MethodsAntibodies

Humanized IgG1, anti-human GITR (MK-4166) and human-ized IgG4, anti-human GITR (MK-1248, which has the same CDRregions but significantly reduced Fc effector function) were devel-oped at Merck Research Laboratories, and for certain uses labeledwith DyLight650 (Thermo Fisher). Generation of MK-4166 isdescribed in the Supplementary Material. A murinized IgG2aversion of rat anti-mouse GITR antibody DTA-1 was generatedon the basis of published sequences (30). Antibodies used forflow cytometry are listed in the Supplementary Material.

Mice and cell linesC57BL/6J, BALB/cAnN, DBA/2J, or C3H/HeN mice (4 weeks

old) were purchased from The Jackson Laboratory or Taconic andmaintained in specific pathogen-free facilities according toapproved IACUC protocols. The cell lines MC38 (NCI, 2015),MB49 (University of Iowa, 2015), B16F10 (ATCC, 2015), LL/2(ATCC, 2015), TC-1 (Johns Hopkins University, 2015), 4T1(ATCC, 2015), CT26 (ATCC, 2015), RENCA (ATCC, 2011), EMT6(ATCC, 2015), CM3 (ATCC, 2016) were all authenticated byIDEXX CellCheck authentication service. The source of each cellline and year of authentication, includingMycoplasma testing, areindicated in parentheses. Tumor cell lines were implanted atpassages 4–6 after thawing.

Murine and human tissuesApproximately 200 mL of blood from healthy female C57BL/6J

mice or C57BL/6J mice bearing MC38 syngeneic tumors of

approximately 100mm3was collected by cardiac puncture. Bloodfrom cynomolgus monkeys (Bioreclamation IVT) and patientswith cancer (MT Group) was collected into K2-EDTA tubes. Bloodfrom healthy human subjects was collected as part of the volun-tary blood donor program at Merck Research Laboratories.Human buffy coats from healthy volunteers were obtained fromthe Stanford Blood Center. All blood samples were collected fromvoluntary donors in accordance with Institutional Review Board(IRB) protocols after obtaining an informed consent. The IRBs forMT Group, Merck volunteer blood donor program, and StanfordBlood Center are Sterling IRB, Merck Research Laboratories PaloAlto IRB, and Panel on Medical Human Subjects, respectively. Allhuman blood and tumor samples were collected in accordancewith the Declaration of Helsinki ethical guidelines.

Mouse tumor cell lines were injected subcutaneously into thehind flanks of syngeneic female mice and tumors were harvestedwhen they reached a volume of 100mm3. Single-cell suspensionswere obtained by fine mincing with a scalpel, followed by a 60-minute incubation at 37�C in 2.5 mL of digestion media contain-ing DMEM, 0.208 WU/mL liberase TM (Roche), and 400 U/mLDNase I (Worthington). Human tumor specimens were obtainedfrom commercial sources (MT Group, Cureline) or University ofRochester in accordance with IRB protocols after obtaininginformed consent. The IRBs for MT Group, Cureline, and Uni-versity of Rochester are Sterling IRB, Western IRB, and ResearchSubjects Review Board, respectively. Single-cell suspensions fromtumors were obtained by finemincing with a scalpel, followed bya 30-minute incubation at 37�C in digestion medium containing8 mL of RPMI1640 medium, 40 mL of 100 mg/mL Collagenase I(Thermo Fisher Scientific), and 320 mL of 10,000 U/mL DNase I.

ELISAGITR variants with E-tag were serially diluted and added to

ELISA plates coated with the parental mouse clone of MK-4166.HRP-conjugated anti-E-tag mAb was used as the detectionreagent. The reaction was developed with 2, 2'-Azino-di(3-ethyl-benzthiazoline-6-sulfonate; ABTS) and read at 405 nm.

T-cell proliferation assaysNa€�ve CD4þ T cells were isolated from blood by negative

selection using EasySep Human Na€�ve CD4þ T-cell Enrichmentkits (Stemcell Technologies) per kit instructions. L cells expressingCD32, CD58, and CD80 on their surface were irradiated with7000 rads, plated at 2.5 � 104 cells per well, and then coculturedwith 2 � 104 na€�ve CD4þ T cells in Yssel's medium with 1%human AB serum. The T cells were stimulated with 0.3 ng/mL ofanti-human CD3mAb (clone UCHT1) in the presence or absenceof MK-4166. T-cell proliferation was assessed after 4 or 5 days byincorporation of tritiated thymidine (3H-thymidine) added 18 to24 hours before harvesting. T-cell proliferation in cynomolgusmonkeys was assayed similarly, except that a NHPNa€�ve CD4þ T-cell kit (Miltenyi Biotec)was used to enrichna€�veCD4þT cells andclone FN-18 at 0.2 ng/mLwas used to stimulate T cells. The degreeof response to MK-4166 costimulation was variable; 6 of 12human donors and 5 of 10 cynomolgus monkeys had dose–response curves sufficient to calculate an EC50 value.

Mouse na€�veCD4þT cellswere isolated fromspleenbynegativeselection using an EasySep mouse na€�ve CD4þ T-cell kit and 2 �104 cells were stimulated with 100 ng/mL of anti-mouse CD3mAb (clone 145-2C11) and cultured with or without DTA-1 incomplete RPMI-1640. Irradiated mouse splenocytes (2,000 rads,

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1� 105), depleted of T cells using CD90.2microbeads, were usedto provide costimulation and T-cell proliferation was quantifiedon day 3 using 3H-thymidine incorporation.

Single-cell suspensions of human tumors were layered on 25mL of Histopaque and centrifuged at 2,000 RPM to remove deadcells and to enrich for TILs. A total of 0.5� 105 enriched TILs werestimulated with 5 ng/mL of soluble anti-CD3mAb (clone OKT3)for 7 days in the presence of 10 mg/mL of MK-4166, MK-1248 orcorresponding isotype matched control antibodies in completeDMEM. Samples were stimulated in triplicates and proliferationwas quantified on day 7 using 3H-thymidine incorporation.

Generation of iTregs in MLR culturesMonocytes enriched from human PBMCs (RosetteSep human

monocyte enrichment kit) were cultured in complete DMEM with10% FBS (SAFC Biosciences), 1,000 U/mL GM-CSF (PeproTech)and 400U/mL IL4 (R&DSystems) for 7 days to generatemonocytederived DCs (mo-DCs). LPS (0.5 mg/mL) was added to the cultureduring the last 2 days to mature the mo-DCs. MLR was set up byculturing PBMCs (2 � 106 cells/mL) with g-irradiated (30 Gy)allogeneicmo-DCs (0.2� 106 cells/mL) in the presence of IL2 (100U/mL) and IL15 (5ng/mL).MK-4166,MK-1248, or isotype controlmAb was added to the cultures and the relative abundance ofCD4þFoxP3high Tregs was evaluated at day 7 using flow cytometry.

nTreg suppression assayTotal CD3þ T cells, Tregs and HLA-DRþ cells were isolated from

PBMCs of the same donor with EasySep Human T-cell isolationkit (Stemcell Technologies), CD4þCD25þCD127dim/� Regula-tory T-Cell Isolation Kit II, human (Miltenyi Biotec) and humananti–HLA-DR MicroBeads (Miltenyi Biotec), respectively. Teffs(Total CD3þ) were labeled with 1.5 mmol/L CFSE (CellTrace CellProliferation Kits, CFSE, Invitrogen) and Tregs were labeled with1.5 mmol/L CellTrace Violet (CellTrace Cell Proliferation Kits,Violet, Invitrogen). After labeling, cells were counted and culturedat indicated Teff:nTreg ratios without the addition of exogenousIL2. Teffs were stimulated with autologous HLA-DRþ cells (HLA-DRþ:Teff ratio of 2:1) and 2 mg/mL of anti-CD3 (clone OKT3) inthe presence of indicated amounts ofMK-4166 or control Ab for 4days. Cells were labeled with anti-CD3, anti-CD4, and anti-CD8,and proliferation of specific T-cell subsets was measured as adecrease in CFSE-labeling intensity by flow cytometry.

Analysis of early signaling events triggered by MK-4166Nine-day-old MLR cultures were frozen for future analysis.

Upon thawing cells were rested for 24 hours to allow for stabi-lization of basal phosphoprotein levels. Tregs were then stimulatedwith either DyLight650-labeled MK-4166, DyLight650-labeledMK-1248 or DyLight650-labeled isotype matched control mAbat a concentration of 5 mg/mL for 5, 10, 15, or 30 minutes. Afterstimulation, cells were immediately chilled on ice and centri-fuged. Supernatant media were aspirated from each well and cellswere incubatedwith a viability dye andphenotypic antibodies. Allsamples were fixed in 100 mL of 1.5% formaldehyde solution for15minutes followed by incubation with 200 mL of ice-cold 100%methanol for 30 minutes. Cells were then washed twice withDPBS, blocked 10 minutes with 2% normal mouse serum, andsubsequently incubated for 30 minutes on ice with mAbs specificfor FoxP3 (clone PCH101), phosphorylated NFkB p65 (clone93H1), and phosphorylated Erk1/2 (clone D13.14.4E).

Gene expression in CD4þ TILs after stimulation of tumorcultures with MK-4166

Dissociated tumor cells were cultured in complete RPMI medi-um supplemented with 62.5 ng/mL of IL2 and 10 mg/mL of eitherDyLight650-labeled MK-4166 or DyLight650-labeled isotypecontrol antibody at 37�C,with5%CO2 in ahumidified incubator.After 1 or 7 days in culture, CD4þ T cells were sorted usingFACSAriaII (BD Biosciences) into individual wells of a 96-wellplate containing 5mLof preamplification bufferwith gene-specificprimers and probes for RT-PCR as detailed in the SupplementaryMaterial.

ResultsSelection of MK-4166 and epitope matching with DTA-1

On the basis of several reports that indicate that GITR signalingenhances antitumor immunity in mouse models, a humanizedIgG1 agonistmAb against humanGITR,MK-4166, was developedto treat patients with advanced malignancies. Because the anti-mouseGITRmAbDTA-1has shown impressive antitumor efficacyin rodent models, the drug candidate was selected to bind to anepitope that is analogous to that of DTA-1. To this end, the aminoacid residues important for DTA-1 and MK-4166 binding tomurine GITR and human GITR, respectively, were determinedby domain swapping and site-directed mutagenesis experimentsas detailed in the Supplementary Material. An alignment of theregion containing the amino acids identified as important forantibody binding is shown in Fig. 1A. Six out of the 7 key residueswere different in mice compared with humans, and when theseresidues on mouse GITR were replaced with corresponding resi-dues from human GITR, MK-4166 was able to bind the modifiedmouse GITR, albeit with lower affinity (Fig. 1B). The averageaffinities of MK-4166 for human and cynomolgus monkey GITRat 25�C as determined by cell-based KinExA were comparable at5.5 and 7.6 pmol/L, respectively (Supplementary Table S1). MK-4166 did not bind to mouse GITR (Fig. 1B) as determined byELISA. The affinity ofDTA-1 formouseGITRwas determined to be26 pmol/L (Supplementary Table S1) by cell-based KinExA, andwas comparable with that of MK-4166 for human and cynomol-gus monkey GITR.

MK-4166 and DTA-1 costimulate na€�ve T cells, resulting inenhanced proliferation

A dose-dependent augmentation of T-cell proliferation wasobserved when human or cynomolgus monkey na€�ve CD4þ Tcells were costimulated with anti-CD3 and L-cells in the presenceof MK-4166 (Fig. 2A and B). A dose-dependent enhancement ofmurine T-cell proliferation was observed in an analogous in vitroproliferation assay using splenic na€�ve CD4þ T cells costimulatedwith T-cell–depleted, irradiated splenocytes and anti-CD3 in thepresence of DTA-1 (Fig. 2C). The bioactivity of MK-4166 wascomparable in human and cynomolgus monkey T cells, withmedian EC50 values of 7.7 and 7.9 pmol/L, respectively (Supple-mentary Table S2). Themedian EC50 value for DTA-1 inmouse Tcells was 99 pmol/L (Supplementary Table S2). Considering therelative high potency (picomolar range) of both MK-4166 andDTA-1, and the variability attributable to the differences betweenrodent and primate assay formats, the bioactivities of both mAbswere deemed fairly comparable.

The ability ofMK-4166 orMK-1248 (which has the same CDRsas MK-4166, but is an IgG4 with a different FcgR binding profile

Characterization of MK-4166, an Agonistic Anti-GITR mAb

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andminimal Fc effector function) to costimulate human TILs wasdetermined in single-cell suspensions obtained from NSCLCtumor tissues. In the presence of either MK-4166 or MK-1248,increasedproliferation of anti–CD3-stimulated TILswas observed(Fig. 2D).

Expression of GITR in peripheral blood cells is significantlydifferent between mice and humans or non-human primates

Translational approaches in pursuing GITR as a target forimmunotherapy require knowledge ofGITR expression in healthyand tumor-bearing mice, non-human primates (NHP), healthyhuman donors, and cancer patients. GITR expression wasobserved on blood CD4þ T cells, NK cells, and NKT cells inhealthy human donors and cancer patients, but was very low toundetectable on CD8þ T cells, B cells, monocytes, and granulo-cytes (Fig. 3A and B). Among the CD4þ T-cell subsets evaluated,Tregs, TH17 and effector memory T cells (TEM) had the highestfrequencies of GITRþ cells (Fig. 3C; gating scheme depicted inSupplementary Fig. S1). In addition, we observed that the upre-gulation of GITR upon activation of sorted CD4þ T-cell subsetswas highest on TEM, followed by central memory (TCM) and thenna€�ve T cells (Fig. 3C and D). In cynomolgus monkeys, GITRexpression was similar with the exception of NK cells, which didnot express GITR (Fig. 3A). The discrepancy in expression of GITRon human and cynomolgus monkey NK cells was further con-firmed by gene expression analysis of sorted NK cells (Supple-mentary Fig. S2A and S2B). In marked contrast, the vast majorityof murine CD4þ T cells, CD8þ T cells, NK cells, and NKT cellsexpressed GITR (Fig. 3A). Moreover, a large proportion of B cellsand a subset of monocytes and granulocytes also expressed GITRin mice (Fig. 3B).

Expression of GITR on tumor-infiltrating lymphocytes issignificantly different between mice and humans

Expression of GITR on CD4þ and CD8þ TILs obtained fromnon–small cell lung carcinoma (NSCLC), melanoma, and renalcell carcinoma (RCC) tumor tissues was evaluated by flow cyto-metry and compared with that on mouse TILs from 10 syngeneicmouse tumormodels (MC38,MB49, LL/2, B16F10, TC1, RENCA,4T1, CT26, EMT6, and CM3). Similar to the profile in peripheralblood, a significant difference in the frequency of GITRþ TILs wasobserved between human and mouse tumors (Fig. 4A and B). Inthe 3 human tumor types analyzed, GITR was expressed on 22%to 42% of CD4þ TILs, whereas it was typically expressed on lessthan 11% of the CD8þ TILs (Fig. 4A). In contrast, GITR wasexpressed on nearly 100%of CD4þ andCD8þ TILs in all analyzedsyngeneic mouse tumor models (Fig. 4B). Both, frequency (Fig.4C; Supplementary Fig. S3A and S3B) and intensity (Fig. 4D) ofGITR expressionwere considerably higher on Tregs compared withnon-Treg CD4þ TILs from NSCLCs and were similar to GITRexpression on Tregs infiltrating mouse tumors (Fig. 4E and F).Furthermore, almost all of the tumor-infiltrating CD4þCD25þ

cells expressed high levels of FoxP3 protein (Supplementary Fig.S4A), high mRNA levels of Treg markers such as Helios and Eos(Supplementary Fig. S4B) and were able to suppress proliferationof CD8þ TILs isolated from NSCLC tissues by cell sorting (FACS;Supplementary Fig. S4C), thus confirming their identity as Tregs.

MK-4166 decreases both the induction and suppressive effectsof Tregs

Since Tregs have been shown to play a central role in antitumorefficacy of DTA-1 and the expression of GITR was the highest onhuman intratumoral Tregs as compared with the other TIL

Figure 1.

Epitopes of DTA-1 and MK-4166. A, Partial sequence alignment of mouse and human GITR. Residues critical for DTA-1 binding to mouse GITR and MK-4166binding to human GITR are boxed. The connecting lines represent disulphide connectivity predicted using homology modeling. Residue numbering isaccording to Nocentini and colleagues (42). B, Binding of the parental mouse clone of MK-4166 to GTR80 (modified mouse GITR with six residues mutated tomatch human GITR - K59R, Y61H, D74K, I75F, V76S, R80Q), human GITR-His, and mouse GITR-His as determined by ELISA. The mean and SD of threeindependent experiments are shown.

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populations, we focused on characterization of the ability of MK-4166 to affect the induction of human Tregs and their suppressiveeffects in vitro.

The effect of MK-4166 on the induction of Tregs (iTregs) in MLRcultures was assessed. The iTregs were identified as CD4þ CD25þ

FoxP3high and the expression of GITR in this population wasconfirmed by flow cytometry (Supplementary Fig. S5A and S5B).Because activated T cells can express low levels of FoxP3, weconfirmed that the iTregs were functionally suppressive (Supple-mentary Fig. S5C and S5D). The addition of MK-4166 to MLRcultures on day 0 resulted in a dose-dependent decrease inabundance of iTregs after 7 days, measured either as relativeabundance (Fig. 5A) or as absolute numbers (Supplementary Fig.S6A). This decrease in iTregswasobservedonlywhenMK-4166waspresent from the start of culture. When it was added on day 7 afteriTregs were already established, the effect was not observed, indi-cating that the decrease in the number of iTregs is due to lack ofinduction as opposed to loss of established iTregs (SupplementaryFig. S6B and S6C). A similar dose-dependent decrease in theinduction of iTregs was observed with MK-1248 (Fig. 5B), indi-cating that Fc effector functions do not likely play a role in thisassay, even though MK-4166 does demonstrate the potential toinduce ADCC in humanMLR-derived iTregs using a Jurkat reportercell line (Supplementary Fig. S7A and S7B).

To determine whether MK-4166 could reduce the suppressiveeffects of human natural Tregs (nTregs), a nTreg suppression assaywas used inwhich donor-matchedCD4þCD25þCD127low nTregsand CD4þ and CD8þ Teffs isolated from blood were stimulatedwith anti-CD3 and autologous HLA-DRþ cells and proliferationmeasured as dilution of CFSE (Fig. 5C and D; Supplementary Fig.S8A and S8B). This was further confirmed in independent experi-ments using a MLR suppression assay where T cells were stimu-lated with allogeneic-DCs and T-cell proliferation was tracked

using 3H-thymidine incorporation (Supplementary Fig. S8C). Thepurity of nTregs (CD4þFoxP3þCD127low) used in the nTreg sup-pression assay was >85% (Supplementary Fig. S9A) and in theMLR suppression assay was 40 to 70% (Supplementary Fig. S9B).Addition of MK-4166 increased T-cell proliferation comparedwith isotype-matched controls at several Teff:nTreg ratios tested(Fig. 5C and D), indicating that MK-4166 partially attenuates thesuppressive effects of Tregs. MK-4166 did not enhance prolifera-tion of Teffs alone (Teff:nTreg ratio1:0; Fig. 5C and D; Supplemen-tary Fig. S8A–S8C), indicating that there was no costimulatoryeffect ofGITR agonism in this assay. In addition,MK-4166did notenhance the proliferation of nTregs in an independent experimentwhere nTregs were stimulated by anti-CD3/anti-CD28–coatedbeads (Supplementary Fig. S10A and S10B).

MK-4166 engagement triggers NFkB phosphorylation in Tregsand Teff

Published reports indicate that GITR signaling is mediated byNFkB and MAP kinases p38, JNK, and ERK in mice (31–35). Todetermine whether binding of the agonist mAb, MK-4166, toGITR elicits similar early signaling events, phosphorylation ofMAPK/Erk and NFkB was evaluated by flow cytometry. MK-4166andMK-1248 bound at similar levels to Tregs (CD4þFoxP3þ) or toTeffs (CD4þFoxP3�) as determined by flow cytometry (Fig. 6A).Following stimulation with MK-4166, a rapid induction of phos-pho-NFkB p65 was observed inMLR-derived GITRþCD4þ T cellsbut not in GITR� CD4þ T cells (Fig. 6B). Maximal phosphoryla-tion of NFkB p65 was observed at 5 minutes following engage-ment of GITR by MK-4166, and this time point was chosen forfurther analysis. Increases in phosphorylation of NFkB p65 wereobserved in both MLR-derived and intratumoral Tregs and Teffs(Fig. 6C) following stimulation with MK-4166 but not withisotype control mAb. DyLight650-labeled MK-4166 was used in

Figure 2.

Costimulation of na€�ve peripheral T cellsfrom human, cynomolgus monkey ormouse blood, and from human TILs byagonist GITR antibodies. Na€�ve CD4þ Tcells from peripheral blood of humanhealthy donors (A) or cynomolgusmonkeys (B) were incubated with L cellsexpressing CD32a, CD58, and CD80. MK-4166 or an isotype-matched controlantibody was added in a dose-titrationmanner in the presence of anti-CD3 (0.2to 0.3 ng/mL of anti-CD3mAb) and T-cellproliferation was measured after 4 or 5days. C, Na€�ve CD4þ T cells from mousespleens were incubated with irradiatedsplenocytes depleted of T cells in thepresence of 100 ng/mL of anti-CD3 mAb.DTA-1 or an isotype-matched controlantibody was added in a dose-titrationmanner and T-cell proliferation wasmeasured. D, Human TILs from fourindependent donorswere stimulatedwithanti-CD3 antibody (10 ng/mL) in thepresence 10 mg/mL of MK-1248 orMK-4166 or an isotype-matched controlantibody for 7 days and proliferation wasmeasured.






these experiments to identify T cells that bound to MK-4166(GITR expressing) and T cells that did not (GITR non-expressing).Within each sample stimulated with DyLight650-labeled MK-4166, T cells that expressed GITR had significantly higher phos-pho-NFkB levels compared with T cells that did not (Fig. 6D).Furthermore, all of these results were reproduced with MK-1248,indicating that GITR signaling is independent of isotype in thisassay (Fig. 6C and D). Phosphorylation of Erk1/2 in CD4þ T cellswas not detected in this assay upon addition of MK-4166 (datanot shown).

Gene-expression changes in human TILs upon engagement ofGITR by MK-4166

To determine the downstream effects of GITR signaling, humantumor single-cell suspensions were cultured either withDyLight650-labeled MK-4166 or DyLight650-labeled isotypematched control mAb. CD4þ T cells were sorted after 24 hoursor 7 days, and gene expression was analyzed by RTqPCR. Eventhough phospho-Erk1/2 protein was not detected in TILs fromNSCLCs, an increase in the expression of DUSP6, a gene inducedby theErk signaling pathway (36),was observed inCD4þT cells inRCC and colorectal carcinoma after 24 hours of incubation withDyLight650-labeled MK-4166 (Fig. 7A). Cells from NSCLC werenot available for this experiment. Some reports indicate that GITR

signaling among murine intratumoral Tregs decreases expressionof FoxP3 and causes instability of Treg lineage commitment(21, 22). We also observed a decrease in FOXP3 mRNA inCD4þ T cells sorted from human RCC andNSCLC tumor culturestreated with MK-4166 for 7 days (Fig. 7B).

Safety and toxicity assessments in cynomolgus monkeysPotential toxicity of MK-4166 was characterized in a 1-month

study in cynomolgus monkeys with a 2-month post-dosingmonitoring period, where a broad range of doses (0.03, 1, 30,or 200 mg/kg/dose) was administered once weekly by intrave-nous infusion. MK-4166 was well tolerated at all doses and notreatment-related toxicity was detected. Details can be found inthe Supplementary Materials.

DiscussionIncreased understanding of the immunosuppressive mechan-

isms in cancers has identified several molecular pathways, includ-ingmembers of the TNF/TNFR family, as potential targets for anti-cancer therapies (6). In particular, the agonist anti-GITR antibodyDTA-1 has impressive antitumor efficacy in murine syngeneictumormodels (21, 23–26).On the basis of these observations, wedeveloped MK-4166, a humanized agonist mAb against human

Figure 3.

Expression of GITR in blood of humans, cynomolgus monkeys, and mice. A and B, The frequency of GITRþ cells on the indicated cell types from healthyhuman donors (n¼ 3–15), cancer patients (n¼ 7–22), cynomolgus monkey (n¼ 15–30), healthy mice (n¼ 5–10), and tumor-bearing mice (n¼ 5) was evaluated byflow cytometry. C, The frequency of GITRþ cells on the indicated T-cell subtypes from healthy human donors (n ¼ 8). The gating scheme used to identifythese cells is shown in Supplementary Fig. S1. D, The frequency of GITRþ cells on na€�ve CD4þ T cells (CD45RAþCCR7þ), CD4þ TCM (CD45RA�CCR7þ), andCD4þ TEM (CD45RA�CCR7�). The dark histograms show labeling with control Ab, whereas light gray histograms show labeling with MK-4166. Na€�ve, TCM and TEMwere FACS-sorted on the basis of the gating scheme shown, stimulated with anti-CD3 and anti-CD28 microbeads (at a bead-to-cell ratio of 1:2) in completeRPMI-1640, and expression of GITR on their surface was determined on day 2 post-activation by flow cytometry. One of two representative donors is shown.

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Figure 4.

Expression of GITR on human and mouse tumor-infiltrating lymphocytes.A,The frequencyofGITRþhumanCD4þ and CD8þ TILs in NSCLC (n¼ 21), melanoma (n¼ 7),and renal cell carcinoma (n ¼ 9) was determined by flowcytometry. B, The frequency of GITRþ CD4þ and CD8þ

TILs from 10 different mouse syngeneic tumors(6 mice/model) was determined by flow cytometry. Thefrequency (C) and the geometric mean fluorescenceintensity (D) of GITR expression in Teffs (CD4

þCD25�) andTregs (CD4

þCD25þ) in tumor tissues obtained frompatients with NSCLC. �� , P < 0.001 calculated by pairedStudent t test. The trend lines in D show donor-matched populations. Representative histograms ofexpression of GITR on human (E) or mouse (F) CD4þ,CD4þCD25þ, and CD8þ TILs are also shown. Error bars,where shown, indicate SD.






GITR for the treatment of solid tumors. To take advantage of therobust efficacy seen with DTA-1, we ensured that MK-4166 andDTA-1 bound to highly analogous epitopes in the respectivespecies. This innovative approach of ensuring that the clinicalcandidate binds to an analogous epitope as that of its mousesurrogate enabled us to make translational comparisons betweenthe observations in the two species.

Although the role of GITR is studied extensively in murinemodels, there is a dearth of information on the role GITR plays inhumans (29), and the experiments reported here not only provideinsights on the translatability of observations made in murinemodels, but extend our understanding of GITR biology inhumans. DTA-1 has been shown to deplete intratumoral Tregs invivo (20) and to decrease the induction of Tregs in murine sple-nocytes treated in vitrowith TGFb (21). In contrast, GITR agonism(with DTA-1 or mouse GITRL) has been reported to enhanceproliferation of Tregin vitro and in vivo (37, 38). This suggests thatthe effects of GITR agonism can be context dependent (12). It is

unclear whether our observation that MK-4166 did not enhancenTreg proliferation in vitro is due to conditions of testing or tospecies-specific differences. We found that the expression ofhuman GITR is comparable to that of mouse GITR in tumorinfiltrating Tregs, despite being drastically lower in other TILpopulations and in peripheral blood. MK-4166 decreased thenumber of Tregs induced in aMLR culture. This is likely not due toADCC because the decrease in the number of Tregs was also seenwith MK-1248 (an IgG4, which has the same CDRs but possessminimal Fc effector functions) and only when MK-4166 wasadded at the beginning of the coculture. Furthermore, the NKcell-to-target cell ratio inMLR cultures is insufficient for significantADCC. However, in an assay optimized to detect ADCC potential(Supplementary Fig. S7A and B), we show that Treg-bound MK-4166 has the potential to induce ADCC of human Tregs. Takentogether these data suggest that MK-4166 may deplete intratu-moral Tregs as well as potentially inhibiting their de novo gener-ation in the tumor.

Figure 5.

Effect of MK-4166 on iTreg generation and nTreg-mediated suppression of Teff. A and B, Human PBMCs were stimulated with irradiated allogeneic DCs in thepresence of MK-4166, MK-1248, or isotype-matched control mAb for 7 days, and the relative abundance of iTregs (CD4þCD25þFoxP3High) generated in theseallo-MLR cultures was measured by flow cytometry and is plotted as a fraction of the total CD4þ population. Aggregate data from 7 individual donors areshown and one-way ANOVA/Kruskal–Wallis test was used to determine statistical significance. Experiments with MK-4166 and MK-1248 were conductedusing blood from the same 7 donors. C andD,CFSE-labeled Teffs were stimulatedwith anti-CD3 in cultures containing lethally irradiated autologous HLA-DRþ feedercells. The ability of responder matched nTregs to inhibit proliferation of CD4þ (C) or CD8þ (D) Teffs at indicated Teff:nTreg ratios was measured as dilutionof CFSE-labeled intensity in the presence of MK-4166 or isotype-matched control mAb. Data shown are representative of three donors. � , P < 0.05 calculated bypaired Student t test. � , P < 0.05; �� , P < 0.01; and �� , P < 0.0001, respectively.

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Furthermore, using an in vitro nTreg suppression assay, we showfor the first time that an agonist anti-human GITR antibody (MK-4166) attenuates the suppressive effects of human nTregs on Teffproliferation. Unlike in assays where TCR signaling is suboptimal(Fig. 2A–D), a direct proliferative effect of MK-4166 on Teffs wasnot observed in these assays where a very strong primary TCRsignal is present (Teff:nTreg ratio 1:0; Fig. 5C andD; SupplementaryFig. S8A-C). However, we cannot rule out the possibility that MK-4166 might act directly on Teffs in these cultures to increase their

refractoriness to Treg suppression as has been suggested formouseGITR agonism (21, 23, 39, 40). In either case, MK-4166 decreasesthe suppressive effects of nTregs. Analysis of proximal signalingevents showed that NFkB p65 is phosphorylated immediatelyupon engagement of GITRon intratumoral Tregs and Teffs (Fig. 6B–D) by MK-4166, suggesting that direct GITR signaling may con-tribute to the effects of MK-4166 on both populations.

Some studies indicate that DTA-1 causes lineage instability anddedifferentiation of Tregs, and this instability is mechanistically

Figure 6.

Effects of MK-4166 engagement of GITR on CD4þ T cells derived from human MLR cultures and tumors. A, The fraction of Tregs or Teffs expressing GITR andability to bind DyLight650-labeled MK-4166 or MK-1248 were evaluated using flow cytometry. B, DyLight650-labeled MK-4166 or DyLight650-labeled isotype-matched control antibodywas added toMLR cultures and the level of phosphorylated NFkBp65 in CD3þCD4þ T cellswasmeasured using flow cytometry after 5, 10,15, or 30 minutes. The level of NFkB phosphorylation in GITRþ CD4þ T cells was compared against GITR� CD4þ T cells. C, Induction of phosphorylatedNFkB, 5 minutes after treatment with MK-4166, MK-1248 or isotype-matched control antibody is shown as fold change over media control. D, In the samplestreated with MK-4166 or MK-1248, the geometric mean fluorescence intensity of phosphorylated NFkB labeling at 5 minutes was compared between T cellsexpressing GITR (DyLight650-positive) and T cells not expressing GITR (DyLight650-negative). Aggregate data from 4 (MLR) or 5 (NSCLC) donors is shownin A, C, and D. � , P < 0.05 and �� , P < 0.01, using the paired, two-tailed t test.






tied to its anti-tumor and pro-inflammatory activity (21, 22),though other studies do not (19, 20, 41). We observed a MK-4166-mediated decrease in the expression of FOXP3, a transcrip-tion factor linked to Tregs function, suggesting that MK-4166 mayfunction by attenuating the suppressive activity of Tregs.

MK-4166 has been shown to inhibit growth of established SK-MEL-5 tumors in humanized mice (19). In this model, MK-4166reduced the number of Tregs in the spleen and to a lesser extent intumor. In both tissues, a decrease in the activation marker ICOSwas observed on Tregs, indicating that MK-4166 can attenuatehuman Treg function in vivo.

The data presented here elucidate the effects of GITR agonismon human T cells and provide a strong scientific and translationalrationale for the clinical development of MK-4166 to treatadvanced malignancies. These are very early insights into theeffects of agonist mAbs on human GITR using ex vivo modelsand suggest that MK-4166 offers an alternative immune-modu-latory mechanism to the reversal of checkpoint inhibition in thetreatment of cancer. TNFR-agonist antibodies elicit a higher

concern for safety, however MK-4166 was well tolerated innon-human primates at all doses administered and no treat-ment-related toxicity was detected. The safety and tolerability ofMK-4166 antibody either as a monotherapy or in combinationwithpembrolizumab (anti–PD-1mAb) inpatientswith advancedsolid tumors is currently being evaluated in a phase I study(NCT02132754).

Disclosure of Potential Conflicts of InterestS. Sukumar has ownership interest (including patents) in Merck & Co.

G. Ermakov has ownership interest (including patents) in WO 2011/028683A1and14/261,152.Nopotential conflicts of interest were disclosedby the otherauthors.

Authors' ContributionsConception and design: S. Sukumar, D.C. Wilson, Y. Yu, B. Bhagwat,P. Georgiev, V. Sriram, A. Willingham, A.M. Beebe, S. SadekovaDevelopment of methodology: S. Sukumar, D.C. Wilson, Y. Yu, S. Naravula,G. Ermakov, R. Riener, B. Bhagwat, A.S. Necheva, T. Churakova, R. Mangadu,P. Georgiev, D. Manfra, E.M. Pinheiro, S. SadekovaAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): S. Sukumar, D.C. Wilson, Y. Yu, J. Wong, S. Naravula,G. Ermakov, R. Riener, B. Bhagwat, A.S. Necheva, J. Grein, T. Churakova,R. Mangadu, P. Georgiev, V. SriramAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): S. Sukumar, D.C. Wilson, Y. Yu, S. Naravula,G. Ermakov, R. Riener, B. Bhagwat, A.S. Necheva, J. Grein, T. Churakova,R. Mangadu, P. Georgiev, W.J. Bailey, A. Willingham, S. SadekovaWriting, review, and/or revision of the manuscript: S. Sukumar, D.C. Wilson,Y. Yu, S. Naravula, G. Ermakov, R. Riener, A.S. Necheva, P. Georgiev,W.J. Bailey,D. Herzyk, A. Willingham, A.M. Beebe, S. SadekovaAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): W.J. Bailey, T.K. McClanahan, S. SadekovaStudy supervision: D. Manfra, E.M. Pinheiro, W.J. Bailey, T.K. McClanahan,S. Sadekova

AcknowledgmentsThe cell lines MB49 and TC-1 were a generous gift from Dr. Michael

O'Donnell (University of Iowa) and Dr. Tzyy-Choou Wu (Johns HopkinsUniversity). We thank Gary Starling for a critical review of the article.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received June 4, 2016; revised November 21, 2016; accepted June 6, 2017;published OnlineFirst June 13, 2017.

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MK-4166 affects gene expression in ex vivo–treated human TILs. Single-cellsuspensions from human RCC (n ¼ 6), NSCLC (n ¼ 3), or colorectal carcinoma(CRC; n ¼ 1) specimens were treated either with DyLight650-labeled MK-4166(10 mg/mL) or DyLight650-labeled isotype control for 24 hours or 7 days andCD4þ T cells were sorted by FACS. Changes in mRNA expression of DUSP6on day 1 (A) and FOXP3 on day 7 (B) in CD4þ T-cell populations was determinedby qPCR and graphed as fold change over isotype-matched control mAb.

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Published OnlineFirst June 13, 2017.Cancer Res Selvakumar Sukumar, Douglas C. Wilson, Ying Yu, et al. Suppressive Effects of T Regulatory CellsThat Targets Human GITR and Inhibits the Generation and Characterization of MK-4166, a Clinical Agonistic Antibody

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