IL-21 Enhances Natural Killer Cell Response to Cetuximab-Coated … · Cancer Therapy: Preclinical IL-21 Enhances Natural Killer Cell Response to Cetuximab-Coated Pancreatic Tumor

Cancer Therapy: Preclinical

IL-21 Enhances Natural Killer Cell Response toCetuximab-Coated Pancreatic Tumor CellsElizabeth L. McMichael1, Alena Cristina Jaime-Ramirez2, Kristan D. Guenterberg3,Eric Luedke3, Lakhvir S. Atwal4, Amanda R. Campbell1, Zhiwei Hu4, Armika S. Tatum4,Sri Vidya Kondadasula4, Xiaokui Mo5, Susheela Tridandapani6, Mark Bloomston3,E. Christopher Ellison3, Terence M.Williams7, Tanios Bekaii-Saab8, andWilliam E. Carson III3,9

Abstract

Purpose: Alternative strategies to EGFR blockage by mAbs isnecessary to improve the efficacy of therapy in patients withlocally advanced or metastatic pancreatic cancer. One such strat-egy includes the use of NK cells to clear cetuximab-coated tumorcells, as need for novel therapeutic approaches to enhance theefficacy of cetuximab is evident. We show that IL-21 enhances NKcell-mediated effector functions against cetuximab-coated pan-creatic tumor cells irrespective of KRAS mutation status.

Experimental Design: NK cells from normal donors or donorswithpancreatic cancerwere used to assessADCC, IFN-g release, andT-cell chemotaxis toward human pancreatic cancer cell lines. The invivo efficacy of IL-21 in combinationwith cetuximabwas evaluatedin a subcutaneous and intraperitoneal model of pancreatic cancer.

Results: NK cell lysis of cetuximab-coated wild-type andmutant kras pancreatic cancer cell lines were significantly higher

following NK cell IL-21 treatment. In response to cetuximab-coated pancreatic tumor cells, IL-21–treated NK cells secretedsignificantly higher levels of IFN-g and chemokines, increasedchemotaxis of T cells, and enhanced NK cell signal transductionvia activation of ERK and STAT1. Treatment of mice bearingsubcutaneous or intraperitoneal EGFR-positive pancreatic tumorxenografts with mIL-21 and cetuximab led to significant inhibi-tion of tumor growth, a result further enhanced by the additionof gemcitabine.

Conclusions: These results suggest that cetuximab treat-ment in combination with IL-21 adjuvant therapy in patientswith EGFR-positive pancreatic cancer results in significantNK cell activation, irrespective of KRAS mutation status, andmay be a potential therapeutic strategy. Clin Cancer Res; 23(2);489–502. �2016 AACR.

IntroductionOverexpression of the EGFR is observed in greater than 70% of

pancreatic adenocarcinomas and is associated with disease pro-gression and poor prognosis (1–4). Cetuximab (Erbitux) is amonoclonal antibody that binds to the extracellular domain ofthe EGFR molecule and prevents ligand binding. Cetuximab-mediated inhibition of EGFR-mediated signal transduction intumor cells leads to G1 arrest and the induction of apoptosis

in vitro (5, 6) and in murine xenograft models (7, 8). Cetuximabhas been approved by the FDA alone or in combination with thetopoisomerase inhibitor irinotecan for the treatment of patientswith irinotecan-refractory colorectal carcinoma. This regimen ledto a significant increase in progression-free survival in colorectalcancer patients and led to complete or partial tumor shrinkagein over 20% of patients (9, 10). However, cetuximab, likeother EGFR-directed therapies, has produced objective clinicalresponses in only a minority of pancreatic cancer patients withEGFR-positive tumors (11). One explanation for this could be thepresence of mutations in the KRAS oncogene, which results inconstitutive activation of the MAPK pathway. This activatingmutation stimulates the MAPK pathway downstream of EGFR,resulting in reduced cetuximab effectiveness.

NK cells are bone marrow–derived, large granular lympho-cytes that contain abundant cytolytic granules and expressnumerous cellular adhesion molecules (12, 13). NK cells areunique in their constitutive expression of receptors for numer-ous cytokines (i.e. IL-12, -15, -18 and -21) and an activatingreceptor for the Fc region of IgG (FcgRIIIa; ref. 14–16). Inaddition to their ability to mediate antibody-dependent cellu-lar cytotoxicity(ADCC), FcR-activated NK cells also secretefactors such as IFN-g , TNF-a and chemokines that inhibit tumorcell proliferation, enhance antigen presentation and stimulatethe chemotaxis of T cells (17, 18). NK cells constitutivelyexpress receptors for a number of cytokines, includingthe IL-21 receptor. IL-21 promotes the maturation of murineNK cells and increases their expression of activating receptors

1Biomedical Sciences Graduate Program, College of Medicine, The Ohio StateUniversity, Columbus, Ohio. 2Department of Neurosurgery, The Ohio StateUniversity, Columbus, Ohio. 3Division of Surgical Oncology, Department ofSurgery, The Ohio State University, Columbus, Ohio. 4Comprehensive CancerCenter, The Ohio State University, Columbus, Ohio. 5Center for Biostatistics, TheOhio StateUniversity, Columbus, Ohio. 6Division of PulmonaryMedicine, Depart-ment of Medicine, The Ohio State University, Columbus, Ohio. 7Department ofRadiation Oncology, The Ohio State University, Columbus, Ohio. 8Division ofMedical Oncology, Department of Internal Medicine, The Ohio State University,Columbus, Ohio. 9Department of Molecular Virology, Immunology, and MedicalGenetics, The Ohio State University, Columbus, Ohio.

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

Corresponding Author: William E. Carson III, The Ohio State University, N924Doan Hall, 410 West 10th Avenue, Columbus, OH 43210. Phone: 614-293-6306;Fax: 614- 293-3465; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-16-0004

�2016 American Association for Cancer Research.

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(19, 20). It was hypothesized that IL-21–mediated enhance-ment of NK cell FcR effector function would be a potentialmethod of enhancing the effectiveness of cetuximab irrespec-tive of the KRAS mutational status of the tumor cells.

In the present study, it was shown that NK cell ADCC andcytokine release in response to cetuximab-coated pancreatic can-cer cells was significantly increased following IL-21 treatment.This effect was present for both wild-type and mutant KRASpancreatic cancer cells, and the combination of IL-21 and cetux-imab had robust in vivo antitumor efficacy. Notably, treatment oftumor bearing mice with gemcitabine and cetuximab in combi-nation led to only a modest reduction in tumor burden, but thiseffect was markedly enhanced by the addition of IL-21. Further-more, pancreatic patient–derived NK cells exhibited significantlyhigher ADCC against cetuximab-coated pancreatic tumor cellsfollowing IL-21 stimulation. These findings support a role forcytokine adjuvant therapy and cetuximab treatment in the settingof EGFR-positive pancreatic cancer patients.

Materials and MethodsCell lines, NK cells, and reagents

The human pancreatic adenocarcinoma cell lines AsPc1,BxPc3, MiaPaCa2 and Panc-1 were a gift from Dr. Mark Bloom-ston (The Ohio State University). MDA-MB-453 (human breastadenocarcinoma, negative control) was obtained from theATCC. The murine pancreatic cancer cell line Panc02 was a giftfrom Michael Hollingsworth (University of Nebraska MedicalCenter). Colorectal cancer cell lines HCT-116MUT andHCT-116WT were a gift from Dr. Terrence Williams (The Ohio StateUniversity). Cell lines were grown as previously described(21). Human natural killer (NK) cells were isolated from freshperipheral blood leukopacks (American Red Cross, Columbus,OH) or pancreatic cancer patients (OSU IRB Protocol2006C0046) by 30minutes. incubationwith RosetteSep cocktail(StemCell Technologies) before Ficoll Hypaque (Sigma) densitygradient centrifugation and cultured as previously described(22). Recombinant human interleukin-21 (rhu-IL-21) was sup-plied by ZymoGenetics, Inc.

Immunoblot analysisThe expression of EGFR was verified via immunoblot analysis.

Lysates were prepared from human pancreatic cancer cell lines aspreviously described (23, 24) and assayed for the expression ofEGFR (Santa Cruz Biotechnology) or b-actin, as a loading control(Sigma-Aldrich).

Flow cytometry of tumor cell linesThe expression of EGFR was evaluated by extracellular flow

cytometry (21). Tumor cells were harvested by trypsanization andincubated on ice for 30 minutes in flow buffer (5% FBS in PBS)with EGFR-PE or isotype control antibodies (Santa Cruz Biotech-nology). Cells were then washed and fixed in 1% formalin. Non-specific staining by an isotype control Ab was used to determinethe percentage of positive population.

Antibody-dependent cellular cytotoxicity assaysNK cells from normal donors or donors with pancreatic cancer

were treated with IL-21 (10 ng/mL) overnight in RPMI-1640media supplemented with 10% human AB serummedia at 37�C.Eighteen hours later, cetuximab- or IgG-coated 51Cr-labeledtumor cells were incubated with NK cells at various effector:target(E:T) ratios. Following a 4-hours incubation, supernatants wereharvested and the percentage of lysis was calculated as previouslydescribed (25).

NK cell cytokine secretionTumor cells were treated with 100 mg/mL of cetuximab or IgG

for 1 hours at 37�C. Purified NK cells were then added to wellsat 2 � 105 cells per well in RPMI supplemented with 10%human AB serum media with or without IL-21 for varyinglengths of time. Cell-free supernatants were harvested at theindicated time points and the cytokine and chemokine levelswere measured using commercially available ELISA kits (R&DSystems; ref. 26).

T-cell chemotaxisNormal T cells were activated for 48 hours with 1 mg/mL

phytohemagglutinin and for 72 hours with 500 pmol/L huIL-2.NK cell co-culture supernatants were placed in the lower chambersofa24-wellflatbottomplate.Mediumsupplementedwith1mg/mLmonokine induced by gamma interferon served as a positivecontrol. Migration experiments were conducted by placing 2 �105 purified activated T cells in 100 mL of 10%HABmedium in theupper chambers of 5-mmpore size Transwell inserts (Corning Inc.).The plates were incubated for 4 hours at 37�C followed by a 10-minutes incubation at 4�C. The number of migrated T cells wasdetermined by trypan blue exclusion.

Intracellular flow cytometryIntracellular levels of phospho-ERK and phospho-STAT1 with-

in NK cells were detected using a pERK-FITC mAb or a pSTAT1-FITC mAb in combination with the NK cell marker CD56 (BDBiosciences; ref. 27). The percentage of positively staining cellswas calculated for the specified cell population.

In vitro generation of human myeloid-derived suppressorcells

Peripheral blood mononuclear cells (PBMC) were isolateddirectly from fresh peripheral blood leukopacks (American

Translational Relevance

Although pancreatic cancers overexpress the EGFR, 95% ofpatients have mutations in the KRAS oncogene, rendering theuse ofmonoclonal antibodies (mAb) against EGFR ineffective.Given the interactions between the innate immune systemandantibody therapy, our group has suggested here that theefficacy of mAb therapy can be enhanced via the administra-tion of immune stimulatory cytokines, mainly interleukin-21(IL-21), with the capacity to activate NK cells. The presentstudy shows that the activation of natural killer (NK) cells byIL-21 enhances NK cell mediated effector functions againstcetuximab-coated pancreatic tumor cells irrespective of KRASmutation status. Notably, the addition of IL-21 to a regimen ofcetuximab and gemcitabine further reduced tumor burden invivo, suggesting that the addition of cytokines to standardchemotherapy-antibody regimens could have beneficialeffects for patients with pancreatic cancer that have limitedtherapeutic options.

McMichael et al.

Clin Cancer Res; 23(2) January 15, 2017 Clinical Cancer Research490




Red Cross) as previously described (22). Monocytes wereisolated from PBMC by positive selection using CD14MicroBeads (Miltenyi Biotec) and cultured in RPMI-1640media supplemented with 10% human AB serum and 10ng/mL IL-6 and GM-CSF (Peprotech) to generate myeloid-derived suppressor cells (MDSC). Cytokines were replenishedevery 2 to 3 days.

Transfection of Panc02 cells with human EGFR, luciferase, andGFP

Human EGFR vectors were a gift from Dr. Anil Rustgi (Uni-versity of Pennsylvania). The empty pFb-neo vector was obtainedfrom Stratagene. After plasmids were expanded in bacterial cells,they were transiently transfected into the Phoenix-Ampho viralpackaging cell line using the calcium phosphate chloroquinemethod (22). Luciferase/GFP plasmid DNA vectors (ACT PBaseand PB Transposon) were obtained from Dr. Tian Xu (YaleUniversity, New Haven, CT). The presence of the EGFR proteinwas confirmed by immunoblot analysis and extracellular flowcytometry. The presence of the luciferaseþGFP vector was con-firmed by fluorescent microscopy.

Murine tumor modelThe ability or cetuximab to bind to tumor-expressed EGFR

in murine models in vivo has been previously demonstrated(28–31). Age-matched female 01B74 nude athymic nude mice(NCI-Frederick, MD) were injected subcutaneously in the rightflank with 1 � 106 Panc02EGFR, Panc02neo, or Panc02 cells in100 mL PBS or were injected intraperitoneally with 5 � 105

Panc02EGFR/lucþGFP cells in 100 mL PBS. Tumors became pal-pable approximately 2 to 3 days later. All treatments wereadministered intraperitoneally thrice weekly in 200 mL of PBS.Subcutaneous tumor volumes were calculated 3x/week asfollows: tumor volume ¼ 0.5 � [(large diameter) x (smalldiameter)2]. Quantification of intraperitoneal tumor burdenwas performed using Xenogen IVIS software to quantify non-saturated bioluminescence in regions of interest. Treatmentscontinued until tumors were greater than 2 cm in maximumdimension (subcutaneous model) or until average radianceexceeded 1.5 � 109 p/s/cm2/sr via IVIS Bioluminescence imag-ing (intraperitoneal model), at which time mice were sacrificed.Mice received an intraperitoneal injection of an anti-asialoGM1 Ab (for depletion of NK cells) or normal rabbit serum(control) on days -3, -1, þ1, and þ3 with respect to tumorinoculation and every 4 days thereafter or an intraperitonealinjection of a clodronate-containing liposome at 1.0 mg/kg (fordepletion of monocytes) or PBS-containing liposomes (con-trol) on day 0 with respect to tumor inoculation, and given a0.5 mg/kg dose every 4 days thereafter. The efficiency ofdepletion was >98% as confirmed by flow cytometric analysisof tumor cells and splenocytes. All animal protocols wereapproved by the Ohio State University Animal Care and UseCommittee and mice were treated in accordance with institu-tional guidelines for animal care.

Statistical analysisTumor cell lysis as measured by ADCC, IFN-g , and chemokine

release as measured by ELISA, and T-cell chemotaxis as measuredby a Transwell assay system was analyzed by ANOVA. Tumorgrowth data was analyzed by mixed effect models, incorporatingrepeated measures for each tumor.

ResultsHuman pancreatic cell lines express EGFR

The expression of EGFR protein was validated in four humanpancreatic cancer cell lines via immunoblot analysis and flowcytometry. There was expression of the EGFR protein in all celllines tested irrespective of KRAS mutational status (Fig. 1A). Ingeneral, the expression of EGFR as determined by immunoblotanalysis was consistent with surface expression of EGFR as mea-sured by flow cytometric analysis (Fig. 1B).

IL-21 enhances NK cell lytic activity against cetuximab-coatedEGFR-positive pancreatic cancer cells

It was hypothesized that the lytic activity of normal human NKcells against cetuximab-coated pancreatic cancer cell lines wouldbe enhanced in vitro following the pre-treatment of NK cells withIL-21, irrespective of KRAS mutation status. ADCC assays wereperformed using normal donor NK cells and various EGFR-positive pancreatic cancer tumor targets. There was a statisticallysignificant enhancement of NK cell-mediated ADCC againstcetuximab-coated targets following IL-21 activation as comparedwith control conditions (P < 0.001). The lysis of wild-type KRAS(wtKRAS) BxPc3 tumor cells (Fig. 2A) was on the same order asthat of mutant KRAS (mutKRAS) AsPc1 (Fig. 2B), MiaPaCa2 (Fig.2C), and Panc1 (Fig. 2D) pancreatic tumor cells (P < 0.001). Ineach instance, lysis of the tumor cells by untreated NK cells wasloweven at the 50:1 E:T ratio. StimulationofNK cellswith IL-21ortreatment of tumor cells with cetuximab led to modest gains intumor cell killing. However, the combination of IL-21 and cetux-imab treatment led to marked levels of ADCC as compared tocontrols (P < 0.001). A graphical representation of this cytotox-icity data at the 50:1 E:T ratio is presented in Fig. 2E.

IL-21 enhances the NK cell response to KRASmutant and KRASwild-type colorectal cancer cell lines

To further determine the efficacy of cetuximab and IL-21 in thetreatment of KRAS mutant cancers, NK cells were obtained fromhealthy donors and used in an ADCC assay as described above.EGFR expressionwas validated in colorectal cancer cell linesHCT-116 MUT (mutKRAS) and HCT-116 WT (wtKRAS) via flowcytometry (Supplementary Fig. S1A). In experiments similar tothose in Fig. 2, it was determined that there was amarked increasein ADCCwhen NK cells were treated with IL-21 and cetuximab incombination, as compared to either treatment alone, irrespectiveof KRAS mutation status (Supplementary Fig. S1B-C, P < 0.001).Notably, the overall level of cytotoxicity in the combination groupwas similar for both cell lines. This result suggests that ADCC byIL-21–activated NK cells is largely unaffected by the KRAS muta-tional status of these cancer cells.

NK cells from pancreatic cancer patients exhibit enhancedADCC in the presence of IL-21

To further determine the efficacy of cetuximab and IL-21 in thesetting of pancreatic cancer, NK cells were obtained from pan-creatic cancer patients before surgical resection and used in ADCCassays as described above. Of the 17 patients included in thisanalysis, 9 had received neoadjuvant chemotherapy and 8 had nochemotherapy before surgical resection (Fig. 3A and B). In allcases, treatment of patient NK cells with IL-21 resulted in greatermean percent lysis of cetuximab-coated AsPc1 tumor (mutKRAS)compared with controls (P < 0.001). Patients that underwent

IL-21 Enhances NK Cell Killing of Ab-Coated Panc Tumor Cells

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neuroendocrine tumor resection exhibited significant ADCCactivity and served as a control (Fig. 3C).

IL-21 enhances production of IFN-g and T-cell attractingchemokines by NK cells co-cultured with cetuximab-coatedEGFR-positive pancreatic cancer cells

Although the cytolytic potential of NK cells is widely appre-ciated, the capability of NK cells to initiate and shape the adaptiveimmune response by providing an early source of IFN-g hasbecome evident (32). It was hypothesized that there would beelevated production of IFN-g by NK cells in response to co-stimulation of the NK cell IL-21R and FcR. As predicted, exposureof IL-21-activated NK cells to cetuximab-coated wtKRAS BxPc3(Fig. 4A) and mutKRAS AsPc1 and MiaPaca2 cells (Fig. 4B)resulted in increased production of IFN-g (P<0.001) as comparedwith control conditions. Dual stimulation of NK cells resulted inrobust NK cell IFN-g production for up to 72 hours (Supplemen-tary Fig. S2AandB,P<0.001).Cetuximab-coatedmutKRASAsPc1tumor cells were also a potent co-stimulus for NK cell production

of the chemokines IL-8, MIP-1a, MIP-1b, and RANTES (Supple-mentary Fig. S3A and D, P < 0.001).

NK cell supernatants from co-stimulated NK cells inducechemotaxis of activated T cells

To confirm the functional activity of NK cell-derived chemo-kines, supernatants from co-cultures of tumor cells with NK cellswere tested for their ability to stimulate T-cell migration. A greaterpercentage of T cells were recruited to supernatant factors releasedfrom the culture of IL-21–activated NK cells with cetuximab-coated tumor cells as compared to control conditions (Fig. 4C,P < 0.001). Importantly, chemotaxis was not greatly affected bythe mutational status of KRAS.

Myeloid-derived suppressor cells inhibit NK cell ADCC andproduction of IFN-g

Recently, our group has shown that MDSCs induced by tumor-derived factors are elevated in patients with gastrointestinalmalignancies, inhibit T-cell proliferation and effector functions,

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Expression of EGFR in human pancreaticcancer cell lines. A, Lysates wereprepared from human pancreatic cancercell lines and subjected to immunoblotanalysis with antibodies directed againstEGFR. Nitrocellulose membranes wereprobed for b-actin to control for loading.HeLa andMDA-MB-453 servedas positiveand negative controls, respectively. B,The percentage of cells expressing EGFRwas assessed by flow cytometry usingan anti–EGFR-PE Ab. Experiments arerepresentative of two separatedeterminations.

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IL-21 enhances antibody-dependent cellular cytotoxicity of NK cells against pancreatic cancer cells. Purified human NK cells were incubated overnight inmedium alone or in medium supplemented with 10 ng/ml IL-21. The lytic activity of IL-21–activated NK cells was then assessed in a standard 4 hourschromium release assay using cetuximab-coated KRAS wild-type (A) or KRAS mutant (B–D) pancreatic cancer cells as targets. E, Graphical summary ofcytotoxicity data of four pancreatic cancer cell lines at the 50:1 E:T ratio. zDenotes presence of a KRAS mutation. The percentage of lysis wascalculated as previously described. Each graph depicts the results from one representative donor � SD. Three normal donors were tested per cell line.The asterisk (�) denotes P < 0.001 versus all conditions shown.






and correlatewith progressive disease (33). Therefore, autologousPBMC-derived MDSCs, or control PBMCs, and healthy donor NKcells were cultured at a 0.5:1 ratio and added to co-culture withAsPc1 (mutKRAS) tumor cells. Co-culture conditions included IL-21–activated NK cells, cetuximab-coated tumor cells or the com-bination. When MDSCs were added to the co-culture, IFN-gproduction by NK cells cultured with mutKRAS AsPc1 tumorcellswasmarkedly inhibited as comparedwith control-treatedNKcells (Fig. 5A). Furthermore,MDSCs impaired IL-21–activatedNKcell ADCC activity against cetuximab-coated mutKRAS AsPc1tumor cells (Fig. 5B).

Co-stimulation of NK cells with Ab-coated pancreatic cancercells and IL-21 results in enhanced activation of ERK and STAT1

It was hypothesized that the interaction of the human NK cellFcR with cetuximab-coated pancreatic tumor cells and the IL-21receptor in the presence of IL-21 would activate the MAPK andJAK/STAT signaling pathways and result in enhanced phosphor-ylation of ERK and STAT1, respectively, within NK cells. HumanNK cells were co-cultured with cetuximab-coated pancreatic can-cer cells in the presence or absence of IL-21. After a 30-minutesincubation period, culture supernatants were collected and cellswere dual stained for CD56 and activated (phosphorylated) ERK

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IL-21 enhances antibody-dependent cellular cytotoxicity of pancreatic patient NK cells against KRAS mutant pancreatic cancer cells. Purified NK cells frompatients with pancreatic adenocarcinoma were incubated overnight with medium alone or in medium supplemented with 10 ng/mL IL-21. The lytic activityof IL-21–activated NK cells was then assessed in a standard 4 hours chromium release assay using cetuximab-coated KRAS mutant AsPc1 tumor cells. The meanpercent lysis at 25:1 E:T ratio for 9 patients who received neoadjuvant chemotherapy (A) or the 8 patients who had not received chemotherapy beforesurgical resection (B) is shown in box plots. The box plots represent themedian and interquartile rangewith I bars showing the range for each group.C,NKcell lysis ofa patient who underwent neuroendocrine tumor resection � SD. Experiments are representative of two separate determinations. The asterisk (�) denotesP < 0.001 versus all conditions shown.

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(pERK) or activated (phosphorylated) STAT1 (pSTAT1). As pre-dicted, NK cells stimulatedwith antibody-coatedmutKRAS AsPc1tumor cells and IL-21 demonstrated the highest level of ERKactivation with 53.5% of cells staining positively for pERK (Fig.6A; Supplementary Fig. S4A). NK cells co-cultured with cetux-

imab-coatedmutKRASAsPc1pancreatic cancer cells or stimulatedwith IL-21 alone exhibited 42.8% and 36.3% staining, respec-tively, whereas only 17.9% of resting NK cells demonstratedactivation of ERK (Fig. 6A; Supplementary Fig. S4A). Thus, cetux-imab-coated tumor cells are a strong stimulus that results in

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Human NK cells secrete high levels ofIFN-g and NK culture supernatantsstimulate the migration of T cells. TheKRAS wild-type BxPc3 (A) and KRASmutant AsPc1 and MiaPaCa2 (B) cell lineswere culturedwith humanNK cells in an invitro tumor co-culture assay. Controlconditions consisted of tumor cells andNK cells cultured with medium alone, IgGalone, cetuximab alone, IL-21 alone or thecombination of IgG and IL-21. Culturesupernatants were harvested at 48 hoursand analyzed for IFN-g by ELISA. C,Chemotaxis of activated T cells inresponse to culture supernatants derivedfrom NK cells costimulated withcetuximab-coated KRAS wild-type BxPc3or KRAS mutant AsPc1 tumor cells and IL-21. Culture medium supplemented withmonokine induced by gamma-interferon(MIG) was included as a positive control.Each graph depicts the results from onerepresentative donor � SD. Three normaldonors were tested per cell line. Theasterisk (�) denotes P < 0.001 versus allconditions shown.






significant induction of the MAPK signaling pathway. Similarly,NK cells stimulated with antibody-coated tumor cells and IL-21demonstrated the highest level of STAT1 activation with 44.1% ofcells staining positively for pSTAT1 (Fig. 6B; Supplementary Fig.S4C). NK cells co-cultured with cetuximab-coated mutKRASAsPc1 pancreatic cancer cells or stimulated with IL-21 aloneexhibited 9.98% and 21.5% staining, respectively, whereas only9.26% of resting NK cells demonstrated activation of STAT1 (Fig.6B; Supplementary Fig. S4C). Thus, IL-21 is a strong stimulus thatresults in significant induction of the JAK/STAT signaling path-way. The percentage of increase in NK cells for both pERK andpSTAT1was greater with the combination of IL-21 and cetuximabversus either condition alone (Fig. 6; Supplementary Fig. S4).Similar results were obtained for the wtKRAS cell line BxPc3 (Fig.4; Supplementary Fig. S4B, Supplementary Fig. S4D). Thus, thecombination treatment led to concurrent activation ofNK cells via

the MAPK and JAK/STAT pathways that was not present in thecontrol conditions.

IL-21 enhances the therapeutic efficacy of cetuximab in amurine model of pancreatic cancer

A murine subcutaneous tumor model of EGFR-positive pan-creatic cancer was used to determine the therapeutic efficacy of IL-21 and cetuximab in vivo. The murine pancreatic tumor cell linePanc02 was engineered to express the human EGFR protein(PANC02EGFR). Tumors were generated in athymic nu/nu miceby subcutaneous injection of 1 � 106 PANC02EGFR tumor cells.There was no significant difference in tumor growth or tumorhistology at the microscopic level as compared with non-trans-formed tumors or those expressing the empty vector (Supple-mentary Fig. S5). Mice bearing PANC02EGFR tumors were thentreated with PBS, IL-21 (5 mg), cetuximab (0.5 mg/kg) or IL-21

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MDSCs inhibit NK cell IFN-g productionand ADCC. Autologous PBMC-derivedMDSCs or control PBMCswere co-culturedwith healthy donor NK cells at a 0.5:1 ratio.In vitro generatedMDSCswere found to besuppressive to T-cell proliferation by CFSEassay (data not shown). A, Purified IL-21–activated NK cells were co-cultured withcetuximab-coated KRAS mutant AsPc1cancer cells alone or in the presence ofautologous PBMCs or MDSCs. Controlconditions consisted of medium alone,IL-21 alone, or cetuximab alone. Culturesupernatants were harvested at 48 hoursand analyzed for IFN-g by ELISA. B,Purified human NK cells were incubatedovernight in medium alone in mediumcontaining 10 ng/mL of IL-21. The lyticactivity was tested against cetuximab-coated KRAS mutant AsPc1 cancer cells inthe presence of autologous PBMCs orMDSCs in a standard 4 hours chromiumrelease assay. Each graph depicts theresults from one representative donor �SD. Three normal donors were tested percell line. The asterisk (�) denotes P < 0.001versus all conditions shown.

McMichael et al.





plus cetuximab intraperitoneally three times per week. There wasno significant difference in tumor volumes at baseline. However,by day 24 post tumor inoculation, there was a significant reduc-tion in tumor volume inmice receiving the combination regimenas compared with the other treatment groups (Fig. 7A, P < 0.01).To further evaluate the therapeutic efficacy of IL-21 and cetuximabin vivo, a murine intraperitoneal tumor model of EGFR-positivepancreatic cancer was used. Here, plasmid vectors encodingluciferase and GFP were transfected into the murine pancreatictumor cell line that was modified to express human EGFR(Panc02EGFR/lucþGFP). Tumor burden was quantified by com-paring average radiance in mice treated with PBS, IL-21 orcetuximab alone, to the combination of IL-21 and cetuximab.By day 11, there was a significant reduction in tumor burden ofmice treated with IL-21 and cetuximab versus mice treated withPBS or either agent alone (Fig. 7B and C, P < 0.001). Expressionof EGFR was necessary for NK cell-mediated ADCC (Supple-mentary Fig. S6A) and the efficacy of the combination of IL-21and cetuximab was dependent on the expression of humanEGFR in the Panc02 cell line (Supplementary Fig. S6B).

IL-21 further enhances the therapeutic efficacy of a gemcitabineand cetuximab regimen in amurinemodel of pancreatic cancer

Given that gemcitabine is widely used in the treatment ofpancreatic cancer, it was determined whether the addition ofIL-21 could enhance the anti-tumor effects of cetuximab andgemcitabine combination therapy against EGFR-positive pancre-atic tumor cells. Therapy with gemcitabine plus cetuximabresulted in modest tumor reduction by day 26; however, theaddition of IL-21 to this regimen led to a further and significantreduction in tumor volume (Fig. 7D, P < 0.007).

The antitumor effects of IL-21 and cetuximab are dependent onNK cells

To determine whether the therapeutic efficacy of IL-21 andcetuximab was dependent on the presence of NK cells or mono-cytes,mice bearing Panc02EGFR tumorswere depleted of eitherNKcells (via administration of anti-asialo GM1) or monocytes (viaadministration of clodronate-containing liposomes) and thentreated with PBS or with IL-21 and cetuximab (Fig. 7E, Supple-mentary Fig. S7). The effectiveness of combination therapy was

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NK cell signal transduction is enhanced byIL-21 receptor and Fc receptor engagementby cetuximab-coated mutant KRAS AsPc1tumor cells. Purified human NK cells werestimulated with 10 ng/ml of IL-21 andco-cultured with cetuximab-coated KRASmutant AsPc1 pancreatic cancer cells for30 minutes. Control conditions consisted ofmedia alone, IL-21–stimulated NK cells alone,or cetuximab-coated tumor cells alone.Following incubation, cells were collectedand underwent dual flow cytometry stainingusing anti-CD56 and (A) anti–phospho-ERK1/2 or (B) anti–phospho-STAT1antibodies. Percentages reported are of dualpositive populations (Q2). Each plot depictsthe results from one representative donor.Three normal donors were tested per cellline.






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McMichael et al.





noticeably reduced following NK cell depletion as comparedwith mock-depleted mice (Fig. 7E, P < 0.002). In contrast, theefficacy of combination therapy did not appear to be greatlydependent on the presence of the monocyte compartment(Supplementary Fig. S7)

DiscussionIn this study, it was demonstrated that IL-21 can enhance NK

cell–mediated effector functions against cetuximab-coated pan-creatic tumor cells. Stimulation of NK cells with IL-21 elicitedgreater NK cell-mediated ADCC against Ab-coated pancreaticcancer cells compared with control conditions in both healthydonors and pancreatic cancer patients. In addition, costimulationof NK cells in this fashion induced the secretion of IFN-g andchemokineswith the ability to direct the chemotaxis of activated Tcells. An increase in levels of phosphorylated STAT1were found inNK cells following IL-21:IL-21R interactions, and heightenedlevels of phosphorylated ERK were identified following stimula-tion of NK cell FcR with cetuximab-coated tumor cells. Thus, themechanism for the observed synergistic actions of the combina-tion therapy likely lies in the induction of non-overlapping path-ways of cellular activation within NK cells. Co-administration ofIL-21 and cetuximab to tumor-bearing mice resulted in reducedtumor burden in both a subcutaneous and intraperitoneal modelof pancreatic adenocarcinoma. Notably, the addition of IL-21 to aregimen of cetuximab and gemcitabine further reduced tumorburden in vivo, suggesting that the addition of cytokines tostandard chemotherapy–antibody regimens couldhave beneficialeffects. The effectiveness of combination therapy was dependenton NK cells, and minimally affected by the depletion of mono-cytes. The enhancement of the NK cell response to EGFR-expres-sing pancreatic tumor targets by IL-21 occurred irrespective ofKRAS mutational status. Indeed, IL-21 enhanced NK cell lyticactivity against a cetuximab-coated colorectal cancer cell lineexpressing wtKRAS, as well as the identical cell line transfectedwith mutKRAS. These data demonstrate the ability of IL-21 toenhance NK cell activation in response to cetuximab-coatedtargets, independently of the intracellular signaling pathways thatare operational within the cancer cell.

Alternative strategies are necessary to improve the efficacy ofmAb therapy in patients with locally advanced or metastaticEGFR-positive pancreatic cancer. One such strategy would be tomake use of the immune system to clear cetuximab-coated tumorcells. Given the ability of the innate immune system to recognize

targets coated in IgG, our group has suggested that the efficacy ofmAb therapy could be enhanced via the administration ofimmune stimulatory cytokines with the capacity to activateFcR-bearing NK cells. IL-21 is a pleiotropic cytokine producedby activated CD4þ T cells that affects the differentiation, matu-ration, and function of a number of lymphoid and myeloid cells.It has also been shown to enhance the proliferation and cytotox-icity of NK cells and CD8þ T cells and increase their secretion ofIFN-g (34). Systemic expression of IL-21 in vivo by plasmid-mediated delivery revealed that IL-21 could inhibit the growthof melanoma and fibrosarcoma tumors, and this inhibition wasfound to be mediated through enhanced cytolytic activity of NKcells (35). A study comparing the activity of intraperitoneallydelivered IL-2, IL-15, and IL-21 against syngeneic tumors in miceshowed that IL-21 had the most potent antitumor activity andresulted in substantially enhanced long-term survival (36). IL-21was found to enhance rituximab activity in a cynomolgusmonkeymodel of B cell depletion and inmouse B-cell lymphomamodelswhere IL-21–activated innate immune effectors, increased theADCC of rituximab-coated targets, mobilized B cells into periph-eral blood and worked in vivo in synergy with rituximab to yieldsignificant survival benefits over either agent alone (37). Theseprior results support the use of IL-21 to enhance cetuximabtherapy of pancreatic cancers.

IL-21 has now entered phase I and II clinical testing andpromising efficacy has been reported. In a phase I trial of subcu-taneous recombinant human IL-21 in patients with metastaticmelanoma or renal cell carcinoma, 69% of patients achieved anoverall response of stable disease or better, which correlated withsignificant enhancement of granzyme B expression in NK cellsfollowing administration of IL-21, with similar trends beingobserved for IFN-g and perforin mRNA (38). An examination ofpatient blood samples from two phase I trials of intravenous rhIL-21 in metastatic melanoma and renal cell carcinoma confirmedincreases in perforin and granzyme B mRNA in CD8þ T cells andCD56þNK cells, suggesting enhanced cytotoxic potential by thesecell types (39). An expanded phase IIA trial of IL-21 in 10additional patients with stage IV malignant melanoma resultedin one complete response and one partial response, with correl-ative studies showing increases in serum soluble CD25,mRNA forIFN-g , perforin, and granzyme B in CD8þ and NK cells, andincreased frequencies of CD25þ NK and CD8þ T cells (40). Amultianalyte profiling of serum proteins in patients withadvanced metastatic melanoma or renal cell carcinoma showedincreased levels of biomarkers indicative of lymphocyte

Figure 7.The combination of IL-21 and cetuximab enhances tumor regression in a mouse xenograft model. A, Nude mice were inoculated subcutaneously with1 � 106 Panc02EGFR tumor cells. Once tumors reached 50 to 100 mm3 in size, treatment began thrice weekly with intraperitoneal injections of PBS, 5 mg IL-21,0.5 mg/kg cetuximab, or the combination. Tumor growth was measured by calipers thrice weekly and tumor volumes were calculated as described in Materials andMethods. Results shown are average tumor volumes of n¼ 7 mice per group� SD. The asterisk (�) denotes P < 0.01 versus all conditions shown. B, Nude mice wereinoculated intraperitoneally with 5 � 105 Panc02EGFR/LucþGFP tumor cells. Treatment began on day 3 and was administered as described above. Mice wereanesthetized on days 3, 11, and 17 using isoflurane. Approximately 10 minutes after luciferin injection, mice were imaged at an exposure time of 1 second.C, Xenogen IVIS software was used to quantify nonsaturated bioluminescence in regions of interest. Radiance greater than 5.5� 106 p/s/cm2/sr was assumed to beindicative of viable luciferase-labeled tumor cells whereas emissions below this range were considered as background. Results shown are average radianceof n¼ 5mice per group� SD. The asterisk (�) denotes P <0.001 versus all conditions shown.D,Nudemice bearing Panc02EGFR tumorswere treated intraperitoneallywith low-dose gemcitabine (15 mg/kg) and cetuximab (0.5 mg/kg) with or without mIL-21 (5 mg) thrice weekly. Tumor volume was calculated as describedabove. Results shown are average tumor volumes of n ¼ 7 mice per group � SD. The asterisk (�) denotes P < 0.007 versus control condition shown. E, Nude micebearing Panc02EGFR tumors were depleted of NK cells by an intraperitoneal injection of anti-asialo GM1 or mock depleted by an intraperitoneal injection ofnormal rabbit serum and treated with PBS or the combination of IL-21 and cetuximab. Results shown are average tumor volumes of n ¼ 5 mice per group � SD.The asterisk (�) denotes P < 0.002 versus control condition shown.






activation, acute phase response, myeloid activation and leuko-cyte chemotaxis/trafficking following treatment with IL-21 (41).Thus, IL-21 appears to have profound effects on both innate andspecific immune effector cells that translates into clinical efficacy.

The use of interleukins in combination with monoclonalantibody therapy to boost immune function against cancer cellshas been the topic of a few clinical studies. An analysis of NK cellfunctionality inpatientswithCLL in response to IL-21 stimulationshowed an increase in CD25 expression, IFN-g production, nat-ural cytotoxicity, and ADCC against rituximab-coated targets(42). The ability of IL-21 to enhance the activity of gemcita-bine/cetuximab therapy in vivo is notable and suggests that ther-apeutic combinations or mAbs with chemotherapy and immuneactivating agents canmediate both direct cytotoxicity and indirectimmunologic tumor cell destruction. In support of this concept,our group conducted a phase I trial of paclitaxel and trastuzumabin combination with IL-12 in patients with HER2/neu-expressingmalignancies and showed that there was increased activation ofextracellular signal-regulated kinases in peripheral blood mono-nuclear cells and increased levels of IFN-g and several chemokinesin patientswith clinical benefit (43). Correlative studies in a phaseI trial of rhIL-21 in combination with cetuximab in patients withmetastatic colorectal cancer, where 9 of 15 patients achievedstable disease, revealed that patient NK cells exhibited increasedcytotoxicity against K562 target cells at the 1-week time point aswell as a significant increase in soluble CD25 (44). Overall, theseclinical trials of cytokines in combination with a mAb show thatIL-21 has potential as an immune-stimulating therapy andhas theability to enhance cytolytic activity of NK cells in combinationwith mAb therapy. The ineffectiveness of standard chemotherapyand even newly developed checkpoint inhibitors provides signif-icant impetus to develop strategies for optimizing antibody-basedtherapies in the setting of pancreatic cancer.

Despite promising preclinical work, phase II and phase III trialshave consistently failed to showefficacy of cetuximab treatment inadvanced pancreatic cancer either alone or in combination withcytotoxic agents (45). One of the defining features of pancreaticadenocarcinomas is a high rate of activating mutations in theKRAS oncogene (>90%).When constitutively activated, KRAS canactivate MAPK signal transduction independent of EGFR, thusrendering EGFR inhibitors (e.g., erlotinib or gefitinib) ineffective.Genetic expression of mutant KRASG12D or KRASG12V specificallyin the murine pancreas proved to be sufficient to initiate acinar-ductal metaplastic lesions and pancreatic intraepithelial neoplas-tic lesions, which progressed with long latency to invasive met-astatic pancreatic adenocarcinoma (46). With the ability of onco-genic KRAS to drive/promote pancreatic cancer cell survival, therehave been considerable efforts to develop direct inhibitors of thispathway. However, clinical attempts to directly interrupt KRASactivity have failed, andKRAS is considered by some researchers tobe undruggable (47). Although strides have been made to targetmolecules downstream of RAS through RAF inhibitors or MEKinhibitors, it has become clear that activation of RAS results in therecruitment ofmultiple branching signaling pathways that will bedifficult to abrogate. Despite failure in targeting molecules down-streamofRAS, a recent report suggested that primingof activated Tcells with a bispecific anti-EGFR and anti-CD3 antibody couldsuppress MDSC differentiation and attenuation of their suppres-sive activity in the tumor microenvironment, even when thetumors express amutant form of KRAS (48). This finding suggeststhat immunotherapy could be beneficial in pancreatic cancer

treatments and that immune-based therapies targeting the EGFRmay be a way to overcome KRAS mutations.

Although mutations in the KRAS oncogene can bypass thedirect downstream effects of anti-EGFR mAbs like cetuximab, thepresent study shows that the immune response to cetuximabtherapy can be significantly enhanced by the addition of IL-21in the settingof oncogenicKRAS. IL-21markedly enhancesNKcelllysis of cetuximab-coated targets in vitro and improves the elim-ination of cetuximab-coated mutKRAS cells. IL-21 is also able toaugment antigen-specific T cell responses and, at the same time,promote NK cell survival, functioning as a link between innateimmune responses and adaptive immune responses (49). Theability of IL-21 activated NK cells to lyse cetuximab-coated pan-creatic and colorectal tumor cells irrespective of KRAS mutationstatus suggests that the oncogenic pathways driving pancreaticcancer growth do not greatly affect, in a positive or negativefashion, the ability of NK cells to lyse those cells. In addition,the secretion of IFN-g and T cell chemotactic factors such as IL-8,MIP-1a, MIP-1b and RANTES by NK cells that come into contactwith cetuximab-coated mutKRAS pancreatic cell lines wasmarkedly enhanced in the presence of IL-21. This would indicatethat the combination therapy has the ability to attract T cells to themutKRAS tumor microenvironment. Considering that IL-21 acti-vates NK cells against mAb-coated tumor cells, an examination ofnegative regulators of NK cell function is also instructive. Tumorcells induce immune suppressor cells, like MDSCs, that canproduce reactive oxygen species to influence the tumor microen-vironment. We show that MDSCs significantly inhibit NK cellADCC and IFN-g production in the current model. Therefore,inhibition or depletion of MDSC function could enhance anti-body therapy. These in vitro results of IL-21 and cetuximab againstmutKRAS cancer cells were confirmed in twomodels of pancreaticcancer and results indicate that IL-21 can activate NK cells to lysecetuximab-coated EGFR-positive tumor cells. Yang and colleagues(50) created a novel EGFR-positive murine tumor cell line anddemonstrated that cetuximab-induced tumor regression dependson both innate and adaptive immunity components, includingCD8þ T cells, MyD88, and FcgR. Figure 7E shows that the efficacyof the combination therapy was almost fully dependent on NKcells, as a depletion of NK cells via the administration of anti-asialo GM1 Ab abrogated the effects of IL-21 and cetuximab. Inaddition, Supplementary Fig. S6 shows that monocytes had littleimpact on tumor growth. Because of the limitations of the presentmodel, we did not evaluate the role played by T cells. However,given the ability of IL-21R/FcR dual-activated NK cells to producechemokines with the ability to stimulate T-cell chemotaxis, webelieve that T cells would likely contribute to the antitumor effectsof IL-21 and cetuximab in an immunocompetent host. Continueddevelopment ofmethods to enhanceNK cell activity in the settingof cetuximab therapy of pancreatic cancer are warranted.

In the present article, it has been demonstrated that the admin-istration of IL-21 is able to activate FcR-bearing NK cells, andenhance their ability to recognize and eliminate cetuximab-coat-ed tumor cells via the induction of ADCC and the release ofcytokines with antitumor activity. In addition, there is evidencethat NK cell elimination of Ab-coated targets proceeds largelyindependent of target cell KRAS mutational status. It was effec-tively demonstrated in vitro, with healthy donor and pancreaticcancer patient NK cells, and in vivo that this interplay betweenantibody therapy and the innate immune system can lead toimproved antitumor effects.

McMichael et al.





Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: E.L. McMichael, A.C. Jaime-Ramirez, K.D. Guenter-berg, S.V. Kondadasula, W.E. Carson IIIDevelopment of methodology: E.L. McMichael, A.C. Jaime-Ramirez,K.D. Guenterberg, Z. Hu, S.V. Kondadasula, T.M. WilliamsAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): E.L. McMichael, A.C. Jaime-Ramirez, K.D. Guenter-berg, E. Luedke, A.R. Campbell, A.S. Tatum, S.V. Kondadasula, S. Tridandapani,M. Bloomston, E.C. Ellison, T. Bekaii-SaabAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): E.L. McMichael, A.C. Jaime-Ramirez, K.D. Guenter-berg, L.S. Atwal, A.R. Campbell, Z. Hu, A.S. Tatum, S.V. Kondadasula, X. Mo,S. Tridandapani, T. Bekaii-Saab, W.E. Carson III

Writing, review, and/or revision of the manuscript: E.L. McMichael, A.C.Jaime-Ramirez, A.R. Campbell, Z. Hu, X. Mo, T. Bekaii-Saab, W.E. Carson IIIAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): A.C. Jaime-Ramirez, A.S. Tatum, T.M. WilliamsStudy supervision: A.C. Jaime-Ramirez, W.E. Carson III

Grant SupportThis work was supported by NIH Grants P01 CA095426 and P30 CA016058

(toM. Caligiuri), CA84402, K24CA93670 (toW.E. Carson III), T32GM068412(to A.C. Jaime-Ramirez) and T32 CA009338.

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 January 7, 2016; revised June 3, 2016; accepted June 23, 2016;published OnlineFirst July 19, 2016.

References1. Ang KK, Berkey BA, Tu X, ZhangHZ, Katz R, Hammond EH, et al. Impact of

epidermal growth factor receptor expression on survival and pattern ofrelapse in patients with advanced head and neck carcinoma. Cancer Res2002;62:7350–6.

2. Bloomston M, Bhardwaj A, Ellison EC, Frankel WL. Epidermal growthfactor receptor expression in pancreatic carcinoma using tissue microarraytechnique. Dig Surg 2006;23:74–9.

3. Hemming AW,Davis NL, Kluftinger A, Robinson B, Quenville NF, LisemanB, et al. Prognostic markers of colorectal cancer: an evaluation of DNAcontent, epidermal growth factor receptor, and Ki-67. J Surg Oncol 1992;51:147–52.

4. Mayer A, Takimoto M, Fritz E, Schellander G, Kofler K, Ludwig H. Theprognostic significance of proliferating cell nuclear antigen, epidermalgrowth factor receptor, and mdr gene expression in colorectal cancer.Cancer 1993;71:2454–60.

5. Fan Z, Shang BY, Lu Y, Chou JL, Mendelsohn J. Reciprocal changes in p27(Kip1) and p21(Cip1) in growth inhibition mediated by blockade oroverstimulation of epidermal growth factor receptors. Clin Cancer Res1997;3:1943–8.

6. Wu X, Fan Z, Masui H, Rosen N, Mendelsohn J. Apoptosis induced by ananti-epidermal growth factor receptor monoclonal antibody in a humancolorectal carcinoma cell line and its delay by insulin. J Clin Invest1995;95:1897–905.

7. Bruns CJ, Harbison MT, Davis DW, Portera CA, Tsan R, McConkey DJ, et al.Epidermal growth factor receptorblockadewithC225plusgemcitabineresultsin regression of human pancreatic carcinoma growing orthotopically in nudemice by antiangiogenic mechanisms. Clin Cancer Res 2000;6:1936–48.

8. Huang SM, Harari PM. Modulation of radiation response after epidermalgrowth factor receptor blockade in squamous cell carcinomas: inhibition ofdamage repair, cell cycle kinetics, and tumor angiogenesis. Clin Cancer Res2000;6:2166–74.

9. Saltz LB, Meropol NJ, Loehrer PJ, Needle MN, Kopit J, Mayer RJ. Phase IItrial of cetuximab in patientswith refractory colorectal cancer that expressesthe epidermal growth factor receptor. J Clin Oncol 2004;22:1201–8.

10. Bonner JA, Harari PM, Giralt J, Azarnia N, Shin DM, Cohen RB, et al.Radiotherapyplus cetuximab for squamous-cell carcinomaof the head andneck. N Engl J Med 2006;354:567–78.

11. Faloppi L, Andrikou K, Cascinu S. Cetuximab: still an option in thetreatment of pancreatic cancer? Expert Opin Biol Ther 2013;13:791–801.

12. RobertsonMJ, Ritz J. Biology and clinical relevance of human natural killercells. Blood 1990;76:2421–38.

13. Vilches C, Parham P. KIR: diverse, rapidly evolving receptors of innate andadaptive immunity. Annu Rev Immunol 2002;20:217–51.

14. Carson WE, Giri JG, Lindemann MJ, Linett ML, Ahdieh M, Paxton R, et al.Interleukin (IL) 15 is a novel cytokine that activates human natural killercells via components of the IL-2 receptor. J Exp Med 1994;180:1395–403.

15. Carson WE, Lindemann MJ, Baiocchi R, Linett M, Tan JC, Chou CC, et al.The functional characterization of interleukin-10 receptor expression onhuman natural killer cells. Blood 1995;85:3577–85.

16. Fehniger TA, Shah MH, Turner MJ, VanDeusen JB, Whitman SP, CooperMA, et al. Differential cytokine and chemokine gene expression by humanNK cells following activation with IL-18 or IL-15 in combination with IL-12: implications for the innate immune response. J Immunol 1999;162:4511–20.

17. Bluman EM, Bartynski KJ, Avalos BR, Caligiuri MA. Human natural killercells produce abundant macrophage inflammatory protein-1 alpha inresponse to monocyte-derived cytokines. J Clin Invest 1996;97:2722–7.

18. Somersalo K, Carpen O, Saksela E. Stimulated natural killer cells secretefactors with chemotactic activity, including NAP-1/IL-8, which supportsVLA-4- and VLA-5-mediated migration of T lymphocytes. Eur J Immunol1994;24:2957–65.

19. Brady J, Hayakawa Y, Smyth MJ, Nutt SL. IL-21 induces the functionalmaturation of murine NK cells. J Immunol 2004;172:2048–58.

20. Burgess SJ, Marusina AI, Pathmanathan I, Borrego F, Coligan JE. IL-21down-regulates NKG2D/DAP10 expression on human NK and CD8þ Tcells. J Immunol 2006;176:1490–7.

21. Roda JM, Joshi T, Butchar JP, McAlees JW, Lehman A, Tridandapani S, et al.The activation of natural killer cell effector functions by cetuximab-coated,epidermal growth factor receptor positive tumor cells is enhanced bycytokines. Clin Cancer Res 2007;13:6419–28.

22. Kondadasula SV, Roda JM, Parihar R, Yu J, Lehman A, Caligiuri MA, et al.Colocalization of the IL-12 receptor and FcgammaRIIIa to natural killer celllipid rafts leads to activation of ERK and enhanced production of inter-feron-gamma. Blood 2008;111:4173–83.

23. Lesinski GB, Badgwell B, Zimmerer J, Crespin T, Hu Y, AboodG, et al. IL-12pretreatments enhance IFN-alpha-induced Janus kinase-STAT signalingand potentiate the antitumor effects of IFN-alpha in a murine model ofmalignant melanoma. J Immunol 2004;172:7368–76.

24. Lesinski GB, Kondadasula SV, Crespin T, Shen L, Kendra K, Walker M, et al.Multiparametric flow cytometric analysis of inter-patient variation inSTAT1 phosphorylation following interferon Alfa immunotherapy. J NatlCancer Inst 2004;96:1331–42.

25. CarsonWE, Parihar R, LindemannMJ, PersoneniN,Dierksheide J,MeropolNJ, et al. Interleukin-2 enhances the natural killer cell response to Her-ceptin-coated Her2/neu-positive breast cancer cells. Eur J Immunol2001;31:3016–25.

26. Roda JM, Parihar R, Lehman A, Mani A, Tridandapani S, Carson WE 3rd.Interleukin-21 enhances NK cell activation in response to antibody-coatedtargets. J Immunol 2006;177:120–9.

27. Parihar R,Dierksheide J,HuY,CarsonWE. IL-12 enhances the natural killercell cytokine response to Ab-coated tumor cells. J Clin Invest 2002;110:983–92.

28. Prewett M, Rockwell P, Rockwell RF, Giorgio NA, Mendelsohn J, Scher HI,et al. The biologic effects of C225, a chimeric monoclonal antibody to theEGFR, on human prostate carcinoma. J Immunother Emphasis TumorImmunol 1996;19:419–27.

29. Prewett M, Rothman M, Waksal H, Feldman M, Bander NH, Hicklin DJ.Mouse-human chimeric anti-epidermal growth factor receptor antibody






C225 inhibits the growth of human renal cell carcinomaxenografts in nudemice. Clin Cancer Res 1998;4:2957–66.

30. Ciardiello F, BiancoR,DamianoV,DeLorenzo S, Pepe S,DePlacido S, et al.Antitumor activity of sequential treatment with topotecan and anti-epi-dermal growth factor receptormonoclonal antibodyC225.ClinCancer Res1999;5:909–16.

31. Overholser JP, PrewettMC,Hooper AT,WaksalHW,HicklinDJ. Epidermalgrowth factor receptor blockade by antibody IMC-C225 inhibits growthof a human pancreatic carcinoma xenograft in nude mice. Cancer2000;89:74–82.

32. Raulet DH.Interplay of natural killer cells and their receptors with theadaptive immune response. Nat Immunol 2004;5:996–1002.

33. Markowitz J, Brooks TR, Duggan MC, Paul BK, Pan X, Wei L, et al. Patientswithpancreatic adenocarcinoma exhibit elevated levels ofmyeloid-derivedsuppressor cells upon progression of disease. Cancer Immunol Immun-other 2015;64:149–59.

34. Parrish-Novak J, Dillon SR, Nelson A, Hammond A, Sprecher C, Gross JA,et al. Interleukin 21 and its receptor are involved in NK cell expansion andregulation of lymphocyte function. Nature 2000;408:57–63.

35. WangG, TschoiM, Spolski R, Lou Y,Ozaki K, FengC, et al. In vivo antitumoractivity of interleukin 21 mediated by natural killer cells. Cancer Res2003;63:9016–22.

36. Moroz A, Eppolito C, Li Q, Tao J, Clegg CH, Shrikant PA. IL-21 enhancesand sustains CD8þ T cell responses to achieve durable tumor immunity:comparative evaluation of IL-2, IL-15, and IL-21. J Immunol 2004;173:900–9.

37. Krejsa CM, Holly RD, Heipel M, Bannink KM, Johnson R, Roque R, et al.Interleukin-21 enhances rituximab activity in a cynomolgus monkeymodel of B cell depletion and in mouse B-cell lymphoma models. PLoSONE 2013;8:e67256.

38. Schmidt H, Brown J, Mouritzen U, Selby P, Fode K, Svane IM, et al. Safetyand clinical effect of subcutaneous human interleukin-21 in patients withmetastatic melanoma or renal cell carcinoma: a phase I trial. Clin CancerRes 2010;16:5312–9.

39. Davis ID, Skrumsager BK, Cebon J, Nicholaou T, Barlow JW, Moller NP,et al. An open-label, two-arm, phase I trial of recombinant human inter-leukin-21 in patients with metastatic melanoma. Clin Cancer Res 2007;13:3630–6.

40. Davis ID, Brady B, Kefford RF, Millward M, Cebon J, Skrumsager BK, et al.Clinical and biological efficacy of recombinant human interleukin-21 inpatients with stage IV malignant melanoma without prior treatment: aphase IIa trial. Clin Cancer Res 2009;15:2123–9.

41. Dodds MG, Frederiksen KS, Skak K, Hansen LT, Lundsgaard D, ThompsonJA, et al. Immune activation in advanced cancer patients treated withrecombinant IL-21: multianalyte profiling of serum proteins. CancerImmunol Immunother 2009;58:843–54.

42. Eskelund CW, Nederby L, Thysen AH, Skovbo A, Roug AS, Hokland ME.Interleukin-21 and rituximab enhance NK cell functionality in patientswith B-cell chronic lymphocytic leukaemia. Leuk Res 2011;35:914–20.

43. Bekaii-Saab TS, Roda JM, Guenterberg KD, Ramaswamy B, Young DC,Ferketich AK, et al. A phase I trial of paclitaxel and trastuzumab incombination with interleukin-12 in patients with HER2/neu-expressingmalignancies. Mol Cancer Ther 2009;8:2983–91.

44. Steele N, Anthony A, Saunders M, Esmarck B, Ehrnrooth E, Kristjansen PE,et al. A phase 1 trial of recombinant human IL-21 in combination withcetuximab in patients with metastatic colorectal cancer. Br J Cancer2012;106:793–8.

45. Luedke E, Jaime-Ramirez AC, Bhave N, Carson WE. Monoclonal antibodytherapy of pancreatic cancer with cetuximab: potential for immune mod-ulation. J Immunother 2012;35:367–73.

46. Hingorani SR, Petricoin EF, Maitra A, Rajapakse V, King C, Jacobetz MA,et al. Preinvasive and invasive ductal pancreatic cancer and its earlydetection in the mouse. Cancer Cell 2003;4:437–50.

47. Berndt N, Hamilton AD, Sebti SM. Targeting protein prenylation for cancertherapy. Nat Rev Cancer 2011;11:775–91.

48. Thakur A, Schalk D, Tomaszewski E, Kondadasula SV, Yano H, Sarkar FH,et al. Microenvironment generated during EGFR targeted killing of pan-creatic tumor cells by ATC inhibits myeloid-derived suppressor cellsthrough COX2 and PGE2 dependent pathway. J Transl Med 2013;11:35.

49. KasaianMT,Whitters MJ, Carter LL, Lowe LD, Jussif JM, Deng B, et al. IL-21limits NK cell responses and promotes antigen-specific T-cell activation: amediator of the transition from innate to adaptive immunity. Immunity2002;16:559–69.

50. Yang X, Zhang X, Mortenson ED, Radkevich-Brown O, Wang Y, Fu YX.Cetuximab-mediated tumor regression depends on innate and adaptiveimmune responses. Mol Ther 2013;21:91–100.


McMichael et al.




2017;23:489-502. Published OnlineFirst July 19, 2016.Clin Cancer Res Elizabeth L. McMichael, Alena Cristina Jaime-Ramirez, Kristan D. Guenterberg, et al. Pancreatic Tumor CellsIL-21 Enhances Natural Killer Cell Response to Cetuximab-Coated

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