phase ib study of immune biomarker modulation with ... ·...

Cancer Therapy: Clinical

Phase Ib Study of Immune Biomarker Modulationwith Neoadjuvant Cetuximab and TLR8Stimulation inHeadandNeckCancer toOvercomeSuppressive Myeloid SignalsGulidanna Shayan1, Benjamin A. Kansy2, Sandra P. Gibson3, Raghvendra M. Srivastava4,James Kyle Bryan5, Julie E. Bauman3, James Ohr3, Seungwon Kim3,4,Umamaheswar Duvvuri3,4, David A. Clump3, Dwight E. Heron3, Jonas T. Johnson3,4,Robert M. Hershberg5, and Robert L. Ferris3,4

Abstract

Purpose: The response rate of patients with head and necksquamous cell carcinoma (HNSCC) to cetuximab therapy is only15% to 20%, despite frequent EGFR overexpression. Becauseimmunosuppression is common in HNSCC, we hypothesizedthat adding a proinflammatory TLR8 agonist to cetuximab ther-apymight result in enhanced T-lymphocyte stimulation and anti–EGFR-specific priming.

Experimental Design: Fourteen patients with previouslyuntreated HNSCC were enrolled in this neoadjuvant trial andtreated preoperatively with 3 to 4 weekly doses of motolimod(2.5 mg/m2) and cetuximab. Correlative tumor and peripheralblood specimens were obtained at baseline and at the time ofsurgical resection and analyzed for immune biomarker changes.Preclinical in vitro studies were also performed to assess the effectof cetuximab plus motolimod on myeloid cells.

Results: TLR8 stimulation skewed monocytes toward an M1phenotype and reversed myeloid-derived suppressor cell

(MDSC) suppression of T-cell proliferation in vitro. These datawere validated in a prospective phase Ib neoadjuvant trial, inwhich fewer MDSC and increased M1 monocyte infiltrationwere found in tumor-infiltrating lymphocytes. Motolimodplus cetuximab also decreased induction of Treg and reducedmarkers of suppression, including CTLA-4, CD73, and mem-brane-bound TGFb. Significantly increased circulating EGFR-specific T cells were observed, concomitant with enhancedCD8þ T-cell infiltration into tumors. These T cells manifestedincreased T-cell receptor (TCR) clonality, upregulation of thecostimulatory receptor CD27, and downregulation of inhibi-tory receptor TIGIT.

Conclusions: Enhanced inflammatory stimulation in thetumor microenvironment using a TLR agonist overcomes sup-pressive myeloid and regulatory cells, enhancing the cellularantitumor immune response by therapeutic mAb in HNSCC.Clin Cancer Res; 24(1); 62–72. �2017 AACR.

IntroductionHead and neck squamous cell carcinoma (HNSCC) is the sixth

leading cancer by incidence worldwide, with a 5-year survival rateof around 40% to 50% despite recent advances in surgery,chemotherapy, and radiotherapy (1–3). As HNSCC has a highprevalence of EGFR overexpression (80%–90%), anti-EGFRmAbs remain an important targeted therapy for treatment of thedisease (3, 4). In addition to blocking EGFR-mediated growthsignals in tumor cells, cetuximab can also mediate antibody-

dependent cell-mediated cytotoxicity (ADCC; ref. 5) and facilitatecross-talk between natural killer (NK) cells and dendritic cells(DCs), which in turn may induce expansion of EGFR-specificcytotoxic T lymphocytes (CTL; refs. 6, 7). However, the responserate to cetuximab therapy in patients with HNSCC is only about15% to 20% (8). Our recent findings suggest that suppressiveimmune cells contribute to this poor prognosis (9) and demon-strate the potential to improve clinical outcome by reversing thesuppressive tumor microenvironment (TME).

Given the impact of innate immune cells on clinical responseto immunotherapy (10), the antitumor properties of moleculestargeting the innate immune system are an emerging areaof interest (11). For instance, myeloid-derived antigen-presentingcells (APCs) have been shown to play an essential role in shapingthe TME, engaging in antitumor functions as well as the enhance-ment of tumor progression (12, 13). Their activity can be mod-ulated by Toll-like receptors (TLRs), proinflammatory innatepattern recognition receptors that induce NF-kB–dependent sig-naling and release of cytokines, which promote antitumor immu-nity (14, 15). Motolimod (formerly VTX-2337) is a novel, selec-tive, small-molecule TLR8 agonist thatmimics viral single-strand-ed ribonucleic acid (ssRNA), the natural ligand of TLR8 (16).TLR8 in humans is expressed in the endosomal compartment ofmonocytes and myeloid DCs (mDC; ref. 17). Recent findings

1School of Medicine, Tsinghua University, Beijing, China. 2Department of Oto-laryngology, Essen University Hospital, Essen, Germany. 3UPMC Hillman CancerCenter, Pittsburgh, Pennsylvania. 4Department of Otolaryngology, University ofPittsburgh, Pittsburgh, Pennsylvania. 5VentiRx Pharmaceuticals Inc., Seattle,Washington.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

CorrespondingAuthor:Robert L. Ferris, UPMCHillman Cancer Center, ResearchPavilion, 5117 Centre Avenue, Room 2.26c, Pittsburgh, PA 15213. Phone: 412-623-0327; Fax: 412-623-4840; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-17-0357

�2017 American Association for Cancer Research.

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demonstrate that TLR8 activation stimulates the release of distinctinflammatory mediators, including Th1-polarizing cytokines andchemokines (18). The combination of cetuximab plus motoli-modwas safe in a recently completed phase I trial of patients withrecurrent/metastatic HNSCC (19). We hypothesized that moto-limod-stimulated APC that express TLR8, such as monocytes andmyeloid-derived suppressor cells (MDSC), could result in a moreinflamed tumor site, leading to a stronger cellular immuneresponse to cetuximab by enhancingNK:DC cross-talk and induc-tion of adaptive immunity. To validate these findings, we per-formed a prospective neoadjuvant clinical trial combining moto-limod with cetuximab, with the primary endpoint to analyzemodulation of immune biomarkers in myeloid and regulatorysuppressor T cells (Treg) and the impact on adaptive cellularimmunity.

Materials and MethodsStudy design

This was a single-center, open-label, phase Ib "windowof opportunity" trial (UPCI #14-002, NCT02124850) approvedby the University of Pittsburgh Institutional Review Board (IRB).The study was performed in accordance with Good ClinicalPractice guidelines and the ethical principles outlined in theDeclaration of Helsinki. All subjects provided written, informedconsent before enrollment. The trial recruited patients with stageII–IVA, previously untreated HNSCC who were candidates forsurgical resection of their disease and had no prior history of headand neck cancer. The primary endpoint of this study was tomeasure quantitative changes in activation and numbers ofimmune effector cell biomarkers (including NK cells, tumorantigen–specific CTL, reduction in Treg, and T-cell receptor [TCR]repertoire diversity) after the combination of motolimod andcetuximab in the 3 to 4weeks prior to scheduled surgery for HNC.Secondary endpoints included measuring modulation of induc-tion of inflammatory markers.

Study treatments and assessmentsTreatment consisted of neoadjuvant doses of subcutaneous

motolimod injection (2.5 mg/m2) and intravenous cetuximab(initial dose of 400 mg/m2 followed by weekly infusions at250 mg/m2) administered once weekly starting on day 1 for aminimumof3weeks andamaximumof4weeks (i.e., days1, 8, 15,and 22) prior to scheduled surgery, which was to be performedwithin 2 days of the last dose of motolimod received. Safetymonitoring and assessment of adverse events (AEs) were per-formed at each study visit. AEswere summarizedusing theMedicalDictionary for Regulatory Activities and graded for severity usingNCI CTCAE Version 4.03. Physical exams were conducted prior tothe first treatment and on the final treatment day (day 15 or 22,depending on surgical scheduling). Hematology and chemistrylaboratory tests were performed on all patients at screening andprior to the first treatment day; in addition, patients were assessedon day 15 for hypomagnesemia, hypocalcemia, and hypokalemia.Tumor imaging assessmentswere carriedout at screening andpriorto surgery within 36 hours of the last dose of neoadjuvant therapy.Patients were evaluated for response and progression per theRECIST Version 1.1. End of study and follow-up assessmentsincluded hematologic and chemistry laboratory evaluations, inaddition to physical examinations and AE monitoring.

Collection of peripheral blood lymphocytes and tumor-infiltrating lymphocytes

Tumor and peripheral blood specimens were collected atscreening and again at the time of surgery under an IRB-approvedtissue banking protocol (UPCI 99-069). Briefly, venous bloodfrompatients withHNSCCwas drawn into heparinized tubes andcentrifuged on Ficoll–Hypaque gradients (GE Healthcare LifeSciences). Peripheral blood lymphocytes (PBL) were recovered,washed in RPMI-1640 medium (Sigma), and either used imme-diately for experiments or resuspended in freezingmedia contain-ing 10% DMSO, transferred to Mr. Frosty containers (ThermoScientific), and stored at -80�C until flow cytometry analysis. Fortumor-infiltrating lymphocyte (TIL) isolation, fresh tumors frompatients with HNSCC were minced into small pieces manually orusing a gentleMACS Dissociator (Miltenyi Biotec), then trans-ferred to 70-mm cell strainers, and mechanically separated usingthe plunger of a 5-mL syringe. The cells passing through the cellstrainer were collected and subjected to Ficoll–Hypaque gradientcentrifugation. After centrifugation, TILs were recovered andstored at –80�C until flow cytometry analysis.

Cell lines and cultureHPV�HNC cell line JHU029 andHPVþ cell line UPCI:SCC090

were grown in RPMI-1640 (Sigma) supplemented with 10% FBS(Corning Mediatech, Inc.) and 1% penicillin/streptomycin (Invi-trogen). JHU029 was a gift from Dr. James Rocco in January of2006 (Ohio StateUniversity). UPCI:SCC090was developed at theUniversity of Pittsburgh through the explant/culture method,authenticated, and validated using short tandem repeat (STR)profiling and HLA genotyping (20, 21). HNC lines were testedevery 6 months and were free of Mycoplasma infection.

Antibodies and flow cytometryThe following anti-human mAbs were used for staining: HLA-

DR-APC, CD11b-PerCP-Cy5.5, CD163-BV421, CD33-BV421,CD8-APC, PD-1-PE-Cy7, TIM-3-BV421, TIGIT-APC, CD27-AlexaFluor 700, LAP-BV421, FOXP3-PerCP-Cy5.5, and PD-L1-PE

Translational Relevance

Patients with head and neck squamous cell carcinoma(HNSCC) only show a response rate of 15% to 20% toanti-EGFR treatment despite the nearly universal overexpres-sion of EGFR, and the suppressive tumor microenvironment(TME) may contribute to this low response rate. Harnessingthe immune-stimulating effect of the selective TLR8 agonistmotolimod, we performed a neoadjuvant trial of motolimodplus cetuximab in patients with resectable HNSCC, includingextensive immune profiling of tumor-infiltrating lymphocytes(TILs). Indeed, motolimod skewed monocytes toward a moreM1 phenotype. Furthermore, by analyzing immune biomark-er changes in patient TIL and peripheral blood lymphocytefrom a phase Ib clinical trial, cetuximab plus motolimodtreatment resulted in a more immunogenic TME and reversedsuppressive signals mediated by myeloid cells and immunecheckpoint receptors. More antigen-specific T cells weredetected using T-cell receptor sequencing and flow cytometrictetramer staining, indicative of enhanced T-cell priming. Ourwork provides insight into potential immunomodulation incombination with therapeutic mAb treatment.

Neoadjuvant Cetuximab and TLR8 Stimulation in HNC

www.aacrjournals.org Clin Cancer Res; 24(1) January 1, 2018 63




purchased from Biolegend; CD11b-PE, CD1c-APC-Cy7, CD141-BV711, CD4-PerCP-Cy5.5, CD56-Alexa Fluor 700, CD3-AlexaFluor 700, CD19-Alexa Fluor 700, CD16-PE-Cy7, HLA-A2-APC-H7, CD73-PE, purchased from BD Biosciences; CD14-PE-TR pur-chased from Life Technologies; CD86-FITC purchased from R&DSystems; CTLA-4–FITC purchased from Ancell; and the PE-labeledHLA-A�0201-EGFR853-861 tetramer obtained from the TetramerFacility of the National Institute of Allergy and Infectious Disease.

Intracellular staining of FOXP3 was performed as follows:PBL or TILs were stained with surface marker antibodies, fixedwith fixation/permeabilization buffer (eBioscience), washed, andstained for intracellular antigens in 1X permeabilization buffer.Cells were analyzed on an LSR Fortessa (BD) flow cytometer, anddata were analyzed using Flow Jo (Treestar). Dead cells wereexcluded based on viability dye staining (Zombie Aqua FixableViability Dye; Biolegend).

Luminex assaysPeripheral blood of study patients was collected using standard

venipuncture techniques prior to neoadjuvant therapy and on theday of surgery. Blood was drawn into heparinized tubes andcentrifuged on Ficoll-Paque PLUS gradients. Peripheral bloodplasmawas recovered fromabove thePBL layer, aliquoted, frozen,and stored in a dedicated -80�C freezer. Luminex assays of plasmaspecimens were performed in duplicate using a MILLIPLEX MAP29-plex human cytokine/chemokine magnetic bead panel for thefollowing analytes: EGF, VEGF, TNFb, TNFa, MIP-1b, MIP-1a,MCP-1, IP-10, IL17, IL15, IL13, IL12 (p70), IL12 (p40), IL10, IL8,IL7, IL6, IL5, IL4, IL3, IL2, IL1ra, IL1b, IL1a, IFNg , IFNa2,GM-CSF, G-CSF, and Eotaxin. Specimens were analyzed by theUPCI IMCPL using the Bio-Plex Multiplex system. Results foreach specimen were averaged together and plotted for eachanalyte. Values that were below the sensitivity of the assay werechanged to the lowest detection limit (e.g., values <18 pg/mLchanged to 18 pg/mL for G-CSF).

TCR sequencingDNA was extracted from matched PBL, at both pretreatment

and posttreatment timepoints, using the DNeasy Blood andTissue kit (Qiagen), and submitted for immunoSEQ profiling(Adaptive Biotechnologies) to characterize TCRB gene rearrange-ments. Acquired data were analyzed using immunoSEQ Analyzersoftware.

Statistical analysisAverages were calculated as means. Two-group comparison

was performed using paired Wilcoxon signed-rank text inGraphPad Prism (GraphPad). Experimental data with twocomparison groups were analyzed using the paired t test.�, P < 0.05; ��, P < 0.01.

ResultsPatients and treatment

A total of 14 patients with HNSCC were enrolled and treated.Eleven patients received a total of four weekly administrations ofneoadjuvant motolimod and cetuximab; two received a total ofthree weekly administrations of motolimod and cetuximab. Onepatient received two weekly administrations of motolimod andcetuximab before withdrawing from the study due to cetuximabtoxicity. Baseline patient characteristics are described in Table 1.

Safety and efficacyOverall, most treatment-related adverse events (TEAEs) were

grade 1 or 2. No patients experienced grade 4 or 5, nor were anynew TEAEs observed based on a prior phase Ib trial using thiscombination (19). The most frequently reported TEAEs weredermatitis acneiform (79%), injection site reaction (79%), andflu-like symptoms (36%; Table 2). All subjects received theplanned resection without delays in performance or extent ofsurgery.

In vitro and correlative studiesTLR8 stimulation skews monocytes toward anM1 phenotype.Moto-limod is a small-molecule TLR8 agonist that can activate mono-cytes, DC, and NK cells (22) through NF-kB–mediated signaling.In order to investigate how TLR8 stimulation affects the activationstatus ofmyeloid lineage cells, we culturedHNSCCpatient PBL orpurifiedCD14þmonocyteswithmotolimod(250nmol/L; ref. 22)for 3 days, and then analyzed the myeloid cells for phenotypicactivationmarkers. CD14þ cells manifested greatest upregulationof HLA-DR and CD86, indicating a more robust antigen presen-tation capacity (Fig. 1A). Interestingly, these cells also showed areduction of CD163, which is a scavenger receptor expressed onM2-suppressive macrophages. Moreover, strong upregulation of

Table 2. TEAEs occurring in at least 20% of patients

(n ¼ 14)

Event Number of patients (%)Injection site reaction 11 (79)Rash acneiform 11 (79)Flu-like symptoms 5 (36)Fatigue 3 (21)Diarrhea 3 (21)Malaise 2 (14)Fever 2 (14)Infusion reaction 2 (14)Headache 2 (14)

Table 1. Characteristics at baseline

(n ¼ 14)

Age, years; median (range) 61 (43–75)Sex, n (%)Female 5 (36)Male 9 (64)

Clinical stage, n (%)Stage III 3 (21)Stage IVA 11 (79)

T stage, n (%)T1 1 (7)T2 0 (0)T3 4 (29)T4 9 (64)

N stage, n (%)N0 5 (36)N1 1 (7)N2A 1 (7)N2B 4 (29)N2C 3 (21)

Tumor site, n (%)Oral cavity 10 (71)Oropharynx 1 (7)Hypopharynx 1 (7)Larynx 2 (14)

Shayan et al.

Clin Cancer Res; 24(1) January 1, 2018 Clinical Cancer Research64




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Figure 1.

A, HNSCC patient PBL and monocytes alone were treated with 250 nmol/L motolimod for 18 hours. Summary data show induction of M1-type macrophage-associated surface molecules HLA-DR and CD86, M2-type–associated markers CD163, and immune-suppressive marker PD-L1; mean fluorescence intensity(MFI) values are normalized to the isotype control of eachmarker (n¼ 5). B, Purified CD14þmonocytes from patients with HNSCCwere isolated and cocultured withUPCI:SCC090 cells or (C) JHU029 cells at a 10:1 ratio. Cetuximab (10 mg/mL) and motolimod (250 nmol/L) were then added, with hIgG1 used as a control forcetuximab. Summary data show expression changes of CD86, CD16, CD163, and PD-L1, where fold change indicates MFI normalized to IgG1 isotype control treatmentgroup (n ¼ 5). Significance was calculated with repeated measures one-way ANOVA. � , P < 0.05; ��, P < 0.01.






PD-L1 was also seen, possibly due to activation of monocytes.Thus, we conclude that TLR8 activation can skewmyeloid cells toan M1-like phenotype, implying potential value in augmentingantitumor immunity.

Motolimod attenuates suppressive effects of MDSC.Next, we inves-tigated whether the combination of cetuximab and motolimodcould modulate M1/M2 macrophage phenotypes. PurifiedCD14þ monocytes from HNSCC patient PBL were coculturedwith the EGFRþ HNSCC cell lines UPCI:SCC090 and JHU029,incubated in the presence of either IgG1 or cetuximab aloneor with motolimod (250 nmol/L) for 3 days. Using flowcytometry, we found that addition of motolimod to cetuximabresulted in higher expression of CD86 (an M1 marker) andreduced expression of CD163 (M2 marker). Downregulationof CD16 (evidence of cross-linking by cetuximab-boundHNSCC cells) was further reduced in the presence of motoli-mod (Fig. 1B–C), indicating stronger ADCC mediated bycetuximab when TLR8 is activated. Notably, expression of

macrophage PD-L1 was also increased when incubated withcetuximab plus motolimod, likely due to stronger activation.Based on our finding that motolimod can skew monocytes to aM1 phenotype, we explored whether activation of TLR8 onMDSC could reverse their suppressive activity. Indeed, sup-pression of T-cell proliferation was reduced after coculturewith MDSC under these conditions (Fig. 2A and B). Thisfinding, together with our recent discovery that signalingthrough CD16 can reverse MDSC suppression of T-cell prolif-eration (23), suggests that the combination of cetuximab andmotolimod can contribute to reversing myeloid cell suppres-sion in the TME.

Cetuximab plus motolimod results in increased mDC infiltrationinto the tumor site. DCs play an important role in processingexogenous antigen and inducing adaptive immune responses.mDCs are categorized into two subsets based on surface CDmarkers: CD1cþ mDCs mediate chemokine production uponactivation, whereas CD141þ mDCs mainly mediate antigen

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HNSCC patient monocytes were cultured in T-75 flasks at 5� 105 cells/mL in complete mediumwith or without 10 ng/mL GM-CSF and IL6 for 7 days in the presenceof motolimod or poly I:C. Medium and cytokines were refreshed every 2 to 3 days. Cells were then harvested and CD33þ cells purified by FACS sorting. AutologousT cells were isolated, labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE), and seeded in 96-well plates at 105/well. CD33þ cells from theabove cultures were added to these cells at a 1:2 ratio. T-cell stimulation was provided by anti-CD3/CD28 stimulation beads (bead:T cell¼ 1:1). Proliferation of T cellswas analyzed by flow cytometry after 3 days. Representative flow plot (A) and (B) summary data are shown. Significance was calculated using one-wayANOVA followed by Tukey test. � , P < 0.05; �� , P < 0.01.

Shayan et al.





cross-presentation (24). Based on our in vitro findings, wehypothesized that the combination of cetuximab plus moto-limod would enhance activation status of innate immunecells. In clinical trial tumor specimens, infiltration by bothCD141þ (P ¼ 0.036) and CD1cþ mDC (P ¼ 0.022) increasedafter combination treatment (Fig. 3A). The activation markerCD80 was upregulated in both subsets (P ¼ 0.024 and P ¼0.033, respectively; Fig. 3B), as was surface expression ofCD16 (P ¼ 0.027 and P ¼ 0.017, respectively; Fig. 3C),indicating a more inflammatory TME with augmented innateimmunity-promoting mature myeloid APC.

Cetuximab plus motolimod reduces suppressive markers of Tregin the TME and increases inflammatory cytokines in patient plasma.We previously demonstrated that cetuximab monotherapyincreases the frequency of intratumoral Treg preferentiallyin non-responders (25). These Treg cells may suppress NK-cell–mediated ADCC and CTL, and are associated with poorclinical outcome. In contrast, patients treated with cetuximabin combination with motolimod had reduced phenotypicmarkers of suppression in Treg (Fig. 4A), including lowerlevels of CTLA-4 (P ¼ 0.002), CD73 (P ¼ 0.005), and mem-brane-bound TGFb (P ¼ 0.012), resulting in a less suppressive

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Clinical trial patient TILs were collectedpre- and posttreatment regimen andmonocytic populations analyzed usingflow cytometry. A, Summary data ofpercentage of CD14þHLA-DRhiCD68þ

and CD14þCD33þHLA-DRlow monocytesbefore and after treatment areshown (n ¼ 9). Summary data of CD80and CD16 expression before and aftertreatment on (B) CD141þ DC and (C)CD1cþ DC are shown (n¼ 9). Significancewas calculated with the paired t test.� , P < 0.05.






TME. By detecting plasma cytokines using multiplex ELISA(Fig. 4B), we found an increase of type 1 inflammatory cyto-kines such as TNFa and IP10, which are known to promoteantitumor immune responses and prevent angiogenesis.

Cetuximab plus motolimod results in reduced ICR expressionon CD8þ TIL. Adaptive antitumor immunity is of significanttherapeutic value, and treatments such as anti–PD-1 haveyielded promising results in multiple cancer types includingHNSCC (26). In order to better assess the antitumor immuneresponse, we compared T-cell phenotypic changes in TIL beforeand after treatment with cetuximab plus motolimod. No sig-nificant changes were observed in Treg (P ¼ 0.309) or CD4

effector populations (P ¼ 0.766; Fig. 5A). In addition toincreased CD8 T-cell infiltration (P ¼ 0.049), CD8 T cellsshowed significant upregulation of stimulatory immune recep-tor CD27 (P ¼ 0.025) together with a downregulationof multiple inhibitory immune checkpoint receptors (ICR)such as TIGIT (P ¼ 0.047), PD-1 (P ¼ 0.041), and CTLA-4(P ¼ 0.053; Fig. 5A–C; 95% confidence intervals for thesemarkers are summarized in Supplementary Table S1). Becausean important feature of T-cell exhaustion is expression ofmultiple inhibitory receptors, our results suggest an enhancedCD8þ T-cell activation, indicated by reduced expression of ICRand upregulated stimulatory receptors following therapy withcetuximab plus motolimod.

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A, Clinical trial patient TILs werecollected pre- and posttreatmentregimen and analyzed for suppressivemarkers using flow cytometry.Representative flow plots and summarydata of percentage of CTLA-4, CD73,and LAP on CD4þCD25hiFOXP3þ Tregare shown (n ¼ 9). B, Luminex analysisof clinical trial patient plasma specimenswas performed in duplicate using aMILLIPLEX MAP 29-plex humancytokine/chemokine bead panel. Datashow plasma concentrations ofTNFa and IP-10 (n ¼ 14). Significancewas calculated with the paired t test.� , P < 0.05; �� , P < 0.01.

Shayan et al.





Cetuximab plus motolimod increases circulating tumor antigen-specific T cells and phenotypic activation. In the setting of strongerfunctionality of tumor-infiltrating CD8 T cells, we investigatedwhether the enhanced immune response was tumor antigen-specific. TCR sequencing in peripheral blood showed that T-cellproductive clonality was significantly increased after treatment(P ¼ 0.0012; Fig. 6B). These data indicate amplified T-cellclones are induced in patients receiving cetuximab plus moto-limod, potentially mediating an antigen-specific antitumorimmune response. Corroborating this finding, we used fluo-rescent multimers to observe an expansion of EGFR-specificCD8þ T cells in peripheral blood in HLA-A2þ patients (n ¼ 5)after treatment with cetuximab plus motolimod (P¼ 0.048; Fig.

6A). These results suggest expansion of circulating antigen-specific CD8 T cells after treatment with cetuximab plusmotolimod.

DiscussionBuilding on a cetuximab monotherapy trial in which poor

clinical response was associated with induction of CTLA-4þ

Treg (9), we added a proinflammatory TLR8 agonist to skewsuppressive myeloid cells and effector T lymphocytes toward amore activated and functionally antitumor phenotype. In thepresent study, we hypothesized that generation of inflamma-tion using motolimod might enhance type 1 CTL responses and

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Figure 5.

Clinical trial patient TILs were collectedboth pre- and posttreatment regimenand lymphocyte populations analyzedusing flow cytometry. A, Summary datashowing percentage of CD8, CD4effector, and Treg population in TIL (n¼8 or 9). B, Representative flow plot andsummary data showing MFI changes ofCD27 before and after treatment inCD8þTIL (n¼9).C,Representative flowplot showing expression of TIGIT andsummary data showing TIGIT, PD-1, andCTLA-4 MFI changes before and aftertreatment in CD8þ TIL (n ¼ 9).Significance was calculated by thepaired t test. � , P < 0.05; ��, P < 0.01.






NK:DC priming. Selective TLR8 expression on suppressivemyeloid cells and MDSC provided the basis for targeting theseAPC to promote the immune-stimulating effects mediated bycetuximab (5, 7, 27).

In this phase Ib clinical trial, active modulation in the TMEwasobserved using TLR8 stimulation to enhance cetuximab-mediatedcellular immunemodulation over 4 weeks. A longer windowmaybe needed for tumor efficacy analysis to correlate tumor responseand survival with these biomarker changes.However, ourfindingsto date provide evidence that cetuximab plus motolimodenhances modulation of host immunity, which was previouslyobserved with cetuximab alone (25). Beneficial effects were seen

in both innate and adaptive immunity. Larger studies need to beperformed by assessing clinical efficacy of adding additional ICRmodulation.

The anti-EGFR antibody cetuximabbinds to EGFR expressed ontumor cells and blocks downstream signaling to interrupt tumorcell growth (28). However, despite relatively universal EGFRexpression on HNSCC cells, single-agent cetuximab results inonly a 15% to 20% response rate. In this context, we consideredthat cetuximab can also mediate ADCC for a more specific tumorcell killing, triggering a cascade of adaptive immune cell stimu-lation through NK:DC priming. Our previous studies using speci-mens from a cetuximab monotherapy neoadjuvant clinical trial

Figure 6.

A, Representative flow plot and summary data showing percentage of EGFR tetramerþ CD8 T cells in PBL of HLA-A2þ patients before and after treatment (n¼ 5).B, DNA from clinical trial patient PBL was analyzed using TCR sequencing (Adaptive). Data show productive clonality before and after treatment (P ¼ 0.0012,Wilcoxon signed-rank test; n ¼ 14). C, Summary data showing percentage of PD-1hi and PD-1int populations within EGFR tetramerþ cells (n ¼ 5). Significancewas calculated using the paired t test. � , P < 0.05; �� , P < 0.01.

Shayan et al.





by Jie and colleagues (25) and Li and colleagues (23) suggest thatcetuximab skews monocytes toward a beneficial macrophagephenotype, but also resulted in more CTLA-4þ Treg infiltrationin the TME, which was correlated with a poor prognosis ofpatients. Moreover, Li and colleagues also showed that duringcetuximab monotherapy treatment, increased circulating MDSCswere observed in clinical nonresponders. Thus, we aimed toevaluate combinatorial therapy adding a proinflammatory TLR8stimulus, due to its expression on monocytes and MDC. Wehypothesized that this combination may generate beneficialinflammatory signals in themicroenvironment, skewing suppres-sive populations such as macrophages and MDSC toward anantitumor phenotype. Functionally, this might result in lowerTreg, which has been correlated with poor clinical outcome incetuximab monotherapy–treated patients with HNSCC.

MDSCs are known to be induced by multiple tumor-derivedsoluble factors. Upon activation, these cells can mediate suppres-sion of both innate and adaptive immune responses in the TME(29–31). Secretion of IL10 and TGFß by MDSC can skew tumor-associated macrophages toward an M2-like phenotype, and alsosuppress NK-cell functions (32, 33). Furthermore, MDSC can alsosuppress T-cell proliferation and activation, as well as inducingTreg, promoting tumor cell evasion from immune surveillance(34–36). In order to induce a robust antitumor immune response,reversing the suppressive microenvironment is essential. Modu-lation of immune cells using mAbs is becoming an importantregimen of cancer therapy, and late-stage cancer patients remainhighly sensitive to TLR8 agonist motolimod as reported byGregory and colleagues (37). By conducting in vitro incubationof chemokine-induced MDSC with motolimod, we found thataddition of motolimod reversed suppression on T-cell prolifera-tion by MDSC. Our previous findings by Stephenson and collea-gues revealed that TLR8 stimulation enhances cetuximab-medi-atedNK-cell lysis of HNSCC cells (22); we thenwished to find outif cetuximab plus motolimod could result in better antitumorimmunity.

Using a novel neoadjuvant trial, our findings suggest thatcetuximab plus TLR8 stimulation using motolimod inducesinflammatory changes in the TME and peripheral blood. GreaterAPC frequency and activation status were seen, corresponding tohigher frequencyofCD8þT cells and reducedMDSCinfiltration inTIL. Treg-suppressivemediators were reduced after the addition ofmotolimod, overcoming a negative prognostic biomarker (Treginduction) observed previously with cetuximab monotherapy(25). Furthermore, enrichment of EGFR antigen–specific CD8 Tcells in peripheral circulation and increased clonality of T cells inperipheral blood suggest that priming of a stronger cellular adap-tive immune response is a consequence of the inflammatorymodulation. Importantly, Srivastava and colleagues previouslyreported that cetuximab monotherapy resulted in increased cir-culating EGFR-specific CD8þ T cells (5). Further study is needed toconfirm whether motolimod has an additive effect in inducingtumor antigen-specific CD8þ T cells. Upregulated costimulatoryCD27 and downregulated expression of inhibitory TIGIT receptor

suggests that CD8þ T cells in the microenvironment might bemore functional and less exhausted. Interestingly, increases ofIL10 and IL1RA were also detected in peripheral circulation(Supplementary Fig. S1A) despite the increase of IP-10 and TNFa,indicative that although a skew toward suppressive TME isobserved, it is important to further overcome the anti-inflamma-tory signals for better antitumor immune response. We alsoobserved a decrease of eotaxin in the peripheral circulation (Sup-plementary Fig. S1B), which is an eosinophil chemotactic protein,the roleofwhich shouldbe further established in future study.Ourfindings also suggest that the addition of a PD-1 inhibitor to thecombination of cetuximab and motolimod would further aug-ment the antitumor response. As the PD-1:PD-L1 pathway isenriched in HNSCC, and anti–PD-1 treatment is shown to beeffective in 15% to 20% of patients, it is likely that addition ofPD-1 blockade to the combination of cetuximab and motolimodwill further augment the antitumor immune response.

Disclosure of Potential Conflicts of InterestR.M. Hershberg is an employee of and has ownership interests (including

patents) at Celgene, and reports receiving commercial research grants fromVentiRx. R.L. Ferris is a consultant/advisory board member for AstraZenecaMedImmune, Bristol-Myers Squibb, Lilly, Merck, and Pfizer. No potentialconflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: G. Shayan, B.A. Kansy, J.E. Bauman, J.T. Johnson,R.M. Hershberg, R.L. FerrisDevelopment of methodology: G. Shayan, B.A. Kansy, R.M. Srivastava,R.L. FerrisAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): G. Shayan, B.A. Kansy, J.E. Bauman, J. Ohr, S. Kim,U. Duvvuri, J.T. Johnson, R.L. FerrisAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): G. Shayan, B.A. Kansy, S.P. Gibson, J.K. Bryan,J.E. Bauman, D.E. Heron, J.T. Johnson, R.M. Hershberg, R.L. FerrisWriting, review, and/or revision of the manuscript: G. Shayan, B.A. Kansy,S.P. Gibson, J.K. Bryan, J.E. Bauman, S. Kim, U. Duvvuri, D.A. Clump,D.E. Heron, J.T. Johnson, R.M. Hershberg, R.L. FerrisAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): B.A. Kansy, S.P. Gibson, R.M. Srivastava,J.T. Johnson, R.L. FerrisStudy supervision: B.A. Kansy, S.P. Gibson, R.M. Srivastava, J.T. Johnson,R.L. Ferris

AcknowledgmentsThis study was supported by NIH grants R01 CA206517, DE019727, P50

CA097190, and T32 CA060397, and the University of Pittsburgh CancerInstitute award P30 CA047904. This project used the UPCI Flow CytometryFacility that is supported in part by award P30 CA047904. G. Shayan wassupported by the China Scholarship Council.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received February 10, 2017; revised May 18, 2017; accepted October 17,2017; published OnlineFirst October 23, 2017.

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Shayan et al.




2018;24:62-72. Published OnlineFirst October 23, 2017.Clin Cancer Res Gulidanna Shayan, Benjamin A. Kansy, Sandra P. Gibson, et al. Overcome Suppressive Myeloid SignalsCetuximab and TLR8 Stimulation in Head and Neck Cancer to Phase Ib Study of Immune Biomarker Modulation with Neoadjuvant

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