Immune-modulating Effects of the Newest Cetuximab-based Chemoimmunotherapy Regimen in Advanced

Colorectal Cancer Patients

Cirino Botta,* Elena Bestoso,* Serena Apollinari,* Maria Grazia Cusi,w Pierpaolo Pastina,*Alberto Abbruzzese,z Pasquale Sperlongano,y Gabriella Misso,z Michele Caraglia,z

Pierfrancesco Tassone,8 Pierosandro Tagliaferri,8 and Pierpaolo Correale*

Summary: Cetuximab is a human–murine chimeric monoclonalantibody to the epidermal growth factor receptor, active for ad-vanced colorectal cancer treatment in combination with chemo-therapy. Cetuximab mainly acts by inhibiting epidermal growthfactor receptor-mediated pathways in cancer cells; however, in thehuman host, its IgG1 backbone may o!er additional antitumoractivity that includes FcgRs-mediated antibody-dependent cell cy-totoxicity, phagocytosis, cross priming, and tumor-specific T-cell–mediated immune response. These mechanisms are still under ac-tive investigation. At this purpose, we have performed an im-munologic investigation in advanced colon cancer patients enrolledin an ongoing phase II trial aimed to test the toxicity and thebiological and antitumor activity of a novel biochemotherapyregimen combining polychemotherapy with gemcitabine, irinote-can, levofolinic acid, and fluorouracil with cetuximab and withsubcutaneous low-dose metronomic aldesleukin (GILFICet regi-men). The peripheral blood mononuclear cells of the first 20 pa-tients enrolled in the GILFICet trial were collected at baseline andafter 6 treatment cycles and examined for immune-phenotypechange by flow cytometry. Colon cancer-specific T-cell lines werealso generated ex vivo from these samples and subsequently char-acterized for immune phenotype, functional activity, and antigenspecificity. We found a treatment-related increase of circulatingdendritic cells, natural killer cells, central memory T cells, andactivated T cells with a T-helper 1 (Th1)-cytotoxic phenotype. Inaddition, the ex-vivo characterization of antigen-specific T cellsderived from the treated patients revealed a significant increase inproliferating cytotoxic T-lymphocyte precursors specific for carci-noembryonic antigen and thymidylate synthase derivative epitopepeptides. On these basis, we concluded that the GILFICet regimenexerts substantial immune-modulating activity that significantlya!ects tumor antigen-specific T-cell compartment with potentialantitumor activity.

Key Words: cetuximab, epidermal growth factor receptor, color-ectal cancer, polychemotherapy

(J Immunother 2012;00:000–000)

Epidermal growth factor receptor (EGFR) is a tyrosinekinase receptor belonging to the human epidermal

growth factor receptor (HER) family. The binding of thisreceptor to its ligands (epidermal growth factor, trans-forming growth factor-a, etc.) leads to receptor homo-dimerization and heterodimerization.1 These events, inturn, promote EGFR autophosphorylation and subsequentactivation of biochemical intracellular pathways able tomaintain malignant phenotype. In fact, EGFR cascadecontrols tumor cell proliferation and survival, resistance toproapoptotic stimuli, as well as angiogenesis and metasta-tization.2,3

Cetuximab is a chimeric IgG1 monoclonal antibody(mAb) capable of preventing the ligand-mediated activationof EGFR pathway by binding the extracellular domain ofthis receptor. Preclinical and clinical results demonstratedadditive antitumor activity when cetuximab is used in com-bination with cytotoxic drugs such as gemcitabine, irino-tecan, 5-fluorouracil (5-FU), and taxanes.4,5 These resultsled to approval of cetuximab+chemotherapy combinationfor the treatment of advanced colorectal cancer not bearingactivating K-ras mutations.6

However, the lack of clinical activity of intracellularEGFR kinase inhibitors in colon cancer patients has fo-cused attention on the immunologic properties of the ce-tuximab human IgG1 backbone. Preclinical studies andFcgIIIR polymorphism analysis have already shown thecritical role played by human IgG1 backbone in the ultimateantitumor activity of cetuximab in colon cancer patients.7

In fact, it was shown that mAbs containing this kind ofbackbone may trigger both complement-dependent cyto-toxicity and natural killer (NK)/granulocyte/macrophage-mediated antibody-dependent cell cytotoxicity (ADCC)against cancer cells expressing su"cient amounts of theantigen targeted by the mAb. We have more recently shownthe capability of a novel polychemotherapy regimen with5-FU, irinotecan, and levofolinic acid (ILF) or gemcita-bine+ ILF (GILF) to upregulate EGFR expression oncolon cancer cells in vitro, thus leading to an enhancedcetuximab-dependent NK-cell–mediated ADCC.8 This ef-fect seemed to be independent from k-ras mutational statusof tumor target cells, and it was not correlated to a directantitumor e!ect of this mAb.8 Additional studies have alsoshown that the immunologic e!ects mediated by cetux-imab-IgG1 backbone are not limited to ADCC. In fact, ithas been shown that it also promotes the trogocytosisand phagocytosis of cetuximab-coated tumor cells by pro-fessional antigen-presenting cells (APC) such as dendriticcells (DCs) and macrophages.9,10 This event, in turn, may

Received for publication February 12, 2011; accepted March 3, 2012.From the *Medical Oncology Unit, Oncology Department; wMicro-

biology Section, Department of Molecular Biology, Siena Uni-versity Hospital, Istituto Toscano Tumori, Siena; zDepartment ofBiochemistry and Biophysics; yDepartment of Anesthesiology andspecial surgery, Second University of Naples, Naples; and 8MedicalOncology Unit, Campus “Salvatore Venuta,” Magna Græcia Uni-versity and Tommaso Campanella Cancer Center, Catanzaro, Italy.

Reprints: Michele Caraglia, Department of Biochemistry and Bio-physics, Second University of Naples, Via Costantinopoli, 16 80138Naples, Italy (e-mail: michele.caraglia@unina2.it).

Copyright r 2012 by Lippincott Williams & Wilkins

CLINICAL STUDY

J Immunother ! Volume 00, Number 00, ’’ 2012 www.immunotherapy-journal.com | 1

lead to an e"cient cross priming with consequent triggeringof an e"cient tumor-specific cytotoxic T-lymphocyte (CTL)response.11 In this view, we have recently shown that chemo-therapy pretreatment of colon cancer cells with 5-FU-basedchemotherapy may greatly enhance this e!ect by: (i) upre-gulating EGFR expression7; (ii) inducing antigen remod-eling, with increase of carcinoembryonic antigen (CEA)and12 thymidylate synthase (TS)13; and (iii) enhancing thesynthesis of heat shock protein 90 and calreticulin thatrepresent powerful danger signals able to alert both DCsand CTL precursors.

On these bases, we have designed anticancer regimensaimed to increase the possible immunologic antitumor ac-tivity of cetuximab and other IgG1/mAbs through thecombination with di!erent inflammatory cytokines as hu-man recombinant interleukin (IL)-2 (aldesleukin).14 Thelatter is a cytokine, produced by activated T cells, knownfor its ability to enhance NK-cell activity and to induceantigen-specific CTL amplification both in vitro andin vivo.15 In this context a number of clinical trials arecurrently ongoing in order to evaluate this hypothesis.16,17

The above-mentioned data represented the rationaleto design a phase II trial in metastatic colorectal cancerpatients that aimed to investigate the toxicity and the bio-logical and antitumor activity of the newest biochemo-therapy regimen called GILFICet that combines 5-FU-basedpolychemotherapy (with gemcitabine, irinotecan, levofo-linic acid, and 5-FU designated as GILF) with cetuximab,followed by subcutaneous metronomic low-dose aldesleu-kin. On these bases, we carried out a planned ex-vivo studyaimed to investigate possible immune–biological changesoccurring in the first 20 patients enrolled in the study.

MATERIALS AND METHODS

Ethical Considerations and Study DesignThe study was authorized by the University Commit-

tee (equivalent to Human Subject Committee of Inves-tigational Review Board) and registered as GILFICet trial.This phase II study was planned on the basis of the Simon’s2-stage optimal design. The study was planned to evaluatethe antitumor activity and toxicity of the GILFICet regi-men in advanced colon cancer patients who had received atleast 2 chemotherapy lines. We selected a target activity of40%–60% response rate with a 0.05 a-error and a 0.20 b-error. The primary endpoint of the study was the responserate and the secondary endpoints were occurrence of ad-verse events and biological activity.

Treatment ScheduleAll patients signed an informed consent and received

biweekly chemotherapy with gemcitabine (1000mg/m2 onday 1), irinotecan (75mg/m2 on day 2), levofolinic acid(100mg/m2 on days 1 and 2), 5-FU (400mg/m2 as a bolusand 800mg/m2 as a 24-h infusion on days 1 and 2), andcetuximab (450mg/m2 on day 3), followed by subcutaneousvery low-dose IL-2 (1.5"106 IU twice a day on days 3–14).

Peripheral Blood Mononuclear Cell (PBMC)Sampling and Storage

PBMCs were isolated from heparinized blood samplesobtained from HLA-A(*)02.01-typed healthy human do-nors and colorectal cancer patients undergone GILFICet or

FOLFOX polychemotherapy (at baseline and after thesixth cycle of therapy), using the Ficoll-Hypaque (CelbioS.P.A., Italy) gradient separation and then cryopreserved at#801C in 10% dimethyl sulfoxide.

Generation of DCsThe DCs used for CTL stimulation in vitro were

generated from autologous PBMCs (107 cell/mL) seeded incomplete RPMI-1640 medium with the addition of 10%heat-inactivated human AB serum, 2mM of L-glutamine,and 100U/mL of penicillin/streptomycin (Invitrogen,Carlsbad, CA) at 371C in 5% CO2 humidified atmospherefor 4 hours. Nonadherent cells were removed, whereasadherent cells were cultured for 7 days in medium con-taining 50 ng/mL of granulocyte–macrophage colony-stimulation factor and 0.5 ng/mL of IL-4 (IL-4) (R&DSystem, Minneapolis, MN).

Generation of T-cell LinesT-cell lines were generated from human PBMCs ac-

cording to a modified protocol of in-vitro stimulation (IVS)described in previous studies.7 PBMCs were maintained incomplete AIM-V medium containing 5% human AB se-rum, 2mM of L-glutamine, and 100U/mL of penicillin/streptomycin. PBMCs were cocultured with autologous ir-radiated DCs loaded with irradiated WiDr colon carcinomacells at a PBMC/DC/tumor cell ratio of 5/1/1. The co-cultures of PBMCs and DCs loaded with tumor cells wereincubated for 5 days at 371C in 5% CO2 humidified at-mosphere in the presence of granulocyte–macrophage col-ony-stimulation factor and IL-4. Then, the cultures werefed with low concentration (25U/mL) of IL-2 (aldesleukin;Novartis Co.) replenished every 2 days for 10 days. Finally,the T-cell lines were restimulated as previously described.One IVS was represented by an incubation of PBMC withDCs loaded with tumor cells for 5 days followed by 10 daysof IL-2 treatment. Established T-cell lines were evaluatedfor immune phenotype and cytotoxic activity. Prior beingloaded on DCs as antigen source, colon cancer cells wereeither irradiated (300Gy) or irradiated and then exposed tocetuximab (10mg/mL). WiDr human colon carcinoma celllines were purchased from American Type Culture Collection(Manassas, VA) and maintained in complete RPMI-1640(Hyclone Europe, Cramlington, UK) with the addition of10% fetal bovine serum, 1% glutamine, 1% penicillin–streptomycin, 2% HEPES, and 1% ciprofloxacin. Adherentcells were removed using trypsin–EDTA solution [0.05%trypsin and 0.02% EDTA in phosphate-bu!ered saline(PBS) without calcium and magnesium].

Flow CytometryThe procedure for flow-cytometric analysis has been

previously described.7 Cancer cells were harvested withtrypsin–EDTA solution, washed twice with PBS containing0.02% sodium azide (Sigma) and distributed into 3-mLtubes (106 cell/tube). The cocultures of PBMCs and DCswere labeled with anti-CD3, CD4, CD8, CD62L, CD11c,CCR7, CD45RA, CD40, CD80, Ki67 (all purchased fromeBioscence, San Diego, CA), 6B11, and FoxP3 (purchasedfrom BioLegend, San Diego, CA) antibodies, accordingto producer’s guidelines. Cells were then fixed with 100mLof 2% paraformaldehyde in PBS and analyzed by flow

Botta et al J Immunother ! Volume 00, Number 00, ’’ 2012

2 | www.immunotherapy-journal.com r 2012 Lippincott Williams & Wilkins

cytometry (FACScan; Becton Dickinson) at 488 nm. Foreach sample, 1"104 events were acquired.

Interferon-c (IFN-c) and IL-10 Enzyme-linkedImmunosorbent Assay (ELISA)

IFN-g and IL-10 cytokine ELISA were performedaccording to the procedures provided by the manufacturer(Bender Systems, Vienna, Austria). Briefly, PBMCs wereplaced in 24-well plates at a final concentration of 106 cells/mL and subsequently stimulated with immune-recon-stituted influenza virosome at a concentration of 1 mg/100 mL or irradiated (300Gy) WiDr tumor cells (1"106 forwell). Forty-eight hours later, 100 mL of the culture super-natant was evaluated in triplicate to detect IFN-g and IL-10concentration.

Enzyme-linked Immuno Spot (ELISPOT) AnalysisThe cytokine ELISPOT assay is a technique designed

to evaluate in T-cell cultures the frequency of activated/memory T cells able to secrete IFN-g in response to specificantigen peptides.18 According to the procedure proposed bythe kit provider (U-CyTech Bioscience, Utrecht, theNetherlands; #CT230-PB2, Human IFN-g ELISPOT Kit),5"104 lymphocytes were seeded into ELISPOT multiwellplates precoated with a high-a"nity mAb to IFN-g andstimulated with antigen peptide-loaded autologous DCs.Forty-eight hours later, cells were washed away. The areasin which the cytokines had been bound were detected by acombination of biotinylated anticytokine detection mAband f-labeled goat anti-biotin mAbs. A final reagent wasadded to the assay in order to promote precipitation ofsilver on f, revealing the site of cytokine secretion. In ourexperiments, T-cell lines in the wells were stimulated withautologous DCs (1:5, DC/lymphocyte ratio) loaded with25 mg/mL of the following antigen peptides: CAP-1, derivedfrom CEA, and TS/1, TS/3, and TS/PP, derived from TS,as described in previous studies.12,13,19,20 DCs loaded withmumps 1 peptide (10mg/mL) or fresh medium containingPhytohemagglutinin (10mg/mL) were used as positive con-trols, whereas unloaded DCs in fresh medium was used as anegative control. The spots were finally evaluated by usingan ELISPOT reader (A.EL.VIS Gmbh, Hannover, Ger-many) whose software (ELISPOT analysis software V 5.1,Roxio creator 2009 License #13413) also performed counts.Results were expressed as number of spots/field.

Statistical AnalysisThe between-mean di!erences were statistically analyzed

by using a GraphPad Instat 3.2 statistical software. Theresults were expressed as the mean+/#SD of 4 determi-nations made in 3 di!erent experiments, and the di!erencesdetermined using the 2-tailed Student t test. A P-value of 0.05or less was considered statistically significant.

RESULTS

Patient Demography and TreatmentBetween January 2009 and August 2010, 20 patients,

15 males and 5 females, with a mean age of 59 years wereenrolled in the study. All of them had a histologic diagnosisof colorectal carcinoma, presented an ECOG performancestatus r1, and had received at least 2 previous treatmentlines for advanced disease. Moreover, they had received nochemotherapy or biochemotherapy in the 30 days beforethe first treatment course. Treatment was administered on

biweekly bases according to the GILFICet regimen asdescribed in the “Patients and Methods” section.

Effect of GILFICet Regimen on Patients’ PBMCs:An Immunocytofluorimetric Analysis

As a side study of the GILFICet trial, we carried outan immunologic investigation aimed to evaluate the abilityof this regimen to a!ect the expression of di!erent immune-competent cell lineages like NK/NKT cells, monocytes/macrophages, DCs, and T cells, which may be influenced bycetuximab human IgG1 backbone as described by severalauthors.21,22 We performed an immune-cytofluorimetricassay on the PBMCs derived from these patients before andafter 6 treatment courses, as previously described. PBMCsfrom healthy donors and metastatic colon cancer patientswho had received standard chemotherapy with 5-FU andoxaliplatin (FOLFOX-4) were used as control groups.

Our analysis did not reveal any significant treatment-related change in the number and percentage of CD3+

CD8+ and CD3+CD4+ T-lymphocyte subsets (baselinevs. sixth cycle: 20.85% vs. 18.83% and 30.15% vs. 30.83%,respectively), a slight increase in peripheral CD4+CD25hi+

FoxP3+ immune-regulatory T (Treg) cells that did not ach-ieve statistical significance, and a trend in reduction of NKTcells (CD3+anti-6B11+ cells) counterbalanced by a signif-icant increase in CD3+CD56+ lymphocytes. Moreover, wefound a significant treatment-related increase in: (i) lympho-cyte subsets expressing NK-cell phenotype and (ii) CD14+

CD11c+ APCs (Table 1). We also found in the GILFICetregimen a significant increase in peripheral DCs expressinghigher levels of HLA-DR, CD80 (baseline vs. sixth cycle:2.3% vs. 4.7%, respectively; P=0.04). All together, theseresults are in line with our previous preclinical findings thathighlighted the ability of ILF/GILF+cetuximab-treatedcancer cells to induce DCs maturation and activation.23

An additional immune-cytofluorimetric analysis alsoshowed a significant treatment-related increase in lympho-cyte subsets carrying a migrant lymphocyte subpopulationphenotype expressing CD62L (Table 1), a homing moleculethat binds to L-selectine24 and mediates cell migrationthrough high endothelial venules. In parallel, our analysisdetected a progressive increase in T lymphocytes expressingCD8+CD45RA+CCR7+ (naıve T lymphocytes) and CD8+

CD45RA#CCR7+ (central memory T lymphocytes) immunephenotype (Fig. 1). CCR7, in particular, is a chemokine re-ceptor involved in chemotaxis, driven by the chemokinesCCL-19 and 21, which are produced by immune-competentcells in inflammatory sites and tumor-infiltrated tissues.25

These T cells represent a fresh source of activated e!ectorlymphocytes ready to counteract either cancer cell progress-ion or viral infections. In fact, these lymphocytes, underspecific microenvironmental conditions occurring in the im-mune-attack site, maturate as highly cytotoxic e!ectors orlong-lasting memory T cells (central memory T cells).26

These findings suggest that GILFICet regimen pro-duces a significant increase in the number of lymphocyteprecursors with potential antitumor activity, capable ofmigrating from primary lymphoid organs (like nodes andbone marrow) to the tissues where the malignant develop-ment occurs.

All of the above-mentioned immune–biological e!ectswere not observed in the PBMCs derived from a controlgroup of patients who received standard chemotherapyaccording to the FOLFOX-4 regimen.

J Immunother ! Volume 00, Number 00, ’’ 2012 Newest Cetuximab-based Chemoimmunotherapy

r 2012 Lippincott Williams & Wilkins www.immunotherapy-journal.com | 3

Effect of GILFICet Regimen on Patients’ T Cells: AFunctional Analysis of the T-helper (Th) Subset

To investigate possible treatment-related modulationsin the Th1/Th2 functional phenotype of the PBMCs ofthese patients, we carried out a cytokine ELISA studyaimed to measure possible changes in the production ofeither IFN-g or IL-10 in response to known antigenicsubstances at baseline and after 6 treatment cycles. IFN-g isa cytokine primarily produced by cells belonging to theinnate immune system (such as NK lymphocytes andmacrophages) and by CD4+ and CD8+ cells in responseto specific antigens. Its production is commonly associatedwith the occurrence of a Th1 polarization.27 IL-10, on thecontrary, is a pleiotropic cytokine, responsible for the in-hibition of Th1-associated cytokine production and for thereduction of NK and DCs proliferation and maturation. Itis commonly produced under a Th2 response.27 The bal-ance between these 2 cytokines may be used as an index ofthe polarization of the Th-response. In details,ex-vivo cul-tures of patients’ PBMCs were exposed to influenza viro-somes (Vir) used as a positive control and irradiated coloncarcinoma (WiDr) cells. Our analysis revealed no changesin the levels of IL-10, which was, as expected, higher than inthe control group, and a significant increase in the antigen-specific production of IFN-g in the supernatants of thePBMCs derived from patients who had received the GIL-FICet treatment (Fig. 2A). These results suggest that theGILFICet regimen may promote a Th2-to-Th1 polar-ization switch with potential antitumor activity.

All of the above-mentioned immune–biological e!ectswere not observed in the PBMCs derived from a controlgroup of patients who received standard chemotherapyaccording to the FOLFOX-4 regimen (data not shown).

Effect of GILFICet Regimen on Patients’ T Cells: AFunctional Analysis of Antigen-specific CTL

To evaluate the influence of GILFICet regimen on thegeneration of an antitumor antigen-specific CTL response,we performed an ex-vivo study where T cells, were sensi-tized in vitro with autologous DCs loaded with coloncarcinoma (WiDr and HT29) cells as described in theMaterials and methods section. T-cell lines were establishedby performing multiple IVS of PBMCs of the above-men-tioned patients who presented an HLA-A(*)02.01+ pheno-type, collected at baseline and after 6 treatment courses.

In our series, T-cell lines derived from PBMCs ofpatients who had undergone standard FOLFOX chemo-therapy presented a significant decrease in immune-sup-pressive Tregs as described in previous studies28 associatedwith a significant decrease in proliferating antigen-specificCTL e!ectors (Fig. 3A). In contrast, T-cell lines derivedfrom patients undergone 6 courses of GILFICet regimenstill presented a decrease in Treg lymphocytes and associatedto a substantial increase in proliferating CTLs (CD8+

Ki67+) (Fig. 3B). To evaluate the specificity of these T-celllines, we also performed an IFN-g ELISPOT assay. Thisassay was specifically designed to evaluate the capability ofthe above-mentioned T-cell lines to recognize epitope pep-tides derived from antigenic structures such as CEA(CAP1) and TS (TS1, TS3, and TSPP), which are com-monly upregulated in colon cancer cells exposed to 5-FU-based chemotherapy.12,13,19 This assay detected a muchgreater number of IFN-g-secreting lymphocyte precursors’colonies, specific for TS1, TS3, TSPP, and CAP1 in theT-cell lines derived from patients who had received theGILFICet regimen compared with those generated fromthe same patients at the baseline (Figs. 4A, B). This increasewas similarly much greater than that detected in T-cell linesderived from HLA-A(*)02.01+ normal donors generatedin vitro according to the same modality previously de-scribed. The specificity of these T cells was confirmed by thefact that no significant intergroup di!erence could bedemonstrated when the ELISPOT assay was designed toevaluate the e!ector response to Phytohemagglutinin orfresh medium, which were used as controls (Fig. 4).

The results of these experiments suggest that T-celllines generated from PBMCs of patients who had receivedthe GILFICet regimen presented a more e"cient responseto antigen peptides commonly over-expressed in coloncancer cells.

DISCUSSIONThe results of the present study suggest that the

sequential administration of gemcitabine+FOLFIRI(GILF) polychemotherapy, followed by cetuximab andlow-dose metronomic aldesleukin is associated with aprominent immune-modulating activity in colon cancerpatients that improves a tumor antigen-specific antitumorresponse. These e!ects were similar to those observed in our

TABLE 1. Shows the Immunocytofluorimetric Analysis Performed on the Peripheral Blood Mononuclear Cell (PBMCs) Isolated FromNormal Healthy Donors and From PBMCs of Colon Cancer Patients Enrolled in the GILFICet Trial or Receiving Standard FOLFOXTreatment, Isolated at Baseline and After 6 Treatment Courses

GILFICet Group FOLFOX Group

Normal Donors Pretreatment Posttreatment P Pretreatment Posttreatment P

CD3+CD8+ 15.22±2.20 20.85±3.26 18.83±2.81 0.66 20.23±3.11 16.52±3.71 0.08CD3+CD4+ 33.11±3.94 30.15±4.36 30.83±5.38 0.99 29.95±5.30 29.02±4.03 0.86CD3+CD62L+ 10.55±0.73 11.8±2.61 17.12±2.59 0.048 4.27±2.65 8.49±4.32 0.13CD8+CD62L+ 3.55±0.73 2.89±0.67 3.83±0.39 0.076 2.65±0.57 4.03±0.67 0.09CD4+CD25hi+ FoxP3+ 0.31±0.07 3.76±1.40 5.73±1.38 0.081 2.01±0.35 1.85±0.92 0.75CD3#CD56+ 11.21±2.08 13.17±2.26 16.36±3.73 0.042 12.37±2.72 13.46±5.22 0.73CD3+CD56+ 3.69±0.61 6.84±1.23 11.19±2.12 0.038 6.07±1.13 5.02±1.37 0.52CD11c+CD14+ 4.5±5.33 6.11±2.73 9.20±4.34 0.044 5.45±1.85 6.82±0.82 0.44

No statistical di!erences were seen between patients’ baseline characteristics. It was detected a statistically significant treatment-related increase, only in theGILFiCet group, in: (i) activated T lymphocytes with migrating phenotype (CD3+/CD62L+); (ii) natural killer cells (CD 56+); (iii) CD3+CD56+ cells; and (iv)activated monocyte (CD11c+CD14+).

Botta et al J Immunother ! Volume 00, Number 00, ’’ 2012

4 | www.immunotherapy-journal.com r 2012 Lippincott Williams & Wilkins

in-vitro models that represented the translational rationaleof the GILFICet trial.

Our studies take advantage by a large series of pre-clinical and clinical results that describe the FcR-mediatedimmunologic antitumor properties of IgG1 mAbs like ce-tuximab, rituximab, and trastuzumab. These mAbs arecapable to promote additional tumor cell killing by acti-vating an e"cient myeloid cell/NK-cell–mediated ADCC29

and by igniting a powerful antitumor immune responsewith generation of long-lasting memory,8,10,30,31 triggeredby an enhanced tumor antigen cross priming by DCs toCTL precursors.7,32 In fact, DCs and other myeloid APCsnormally express several subtypes of FCgR, whose en-gagement may trigger phagocytic/trogocytic signal trans-duction pathways.23,33 In addition, the FcgIIIaR-mediatedactivation of NK cells, which follows its interaction with

mAb-covered tumor cells, stimulates both the productionand release of cytokines such as IFN-g and IL-12, which,in turn, cause the occurrence of a tumor-specific immuneresponse by facilitating NK cell–DCs cross talking andinducing a cytotoxic Th1 response polarization.34,35

In this context, we have recently shown that coloncancer cells exposed to GILF polychemotherapy and thento cetuximab become highly susceptible to NK-cell–medi-ated ADCC.8 We also observed that colon cancer cellstreated with GILF chemotherapy and cetuximab are avidlyphagocyted and degraded by human DCs which, in turn,are able to cross present the derivative antigen to CTLprecursors, thus giving rise to a tumor antigen-specific im-mune response.8 In our in-vitro model, we found that hu-man PBMCs stimulated in vitro with autologous DCsloaded with colon cancer cells previously exposed to GILF

FIGURE 1. Representative flow-cytometric analysis carried out on the peripheral blood mononuclear cells (PBMCs) of 1 single patientcollected at baseline (A) and after 6 treatment courses (B). The test was designed to identify possible changes in the expression ofCD45RA and/or CCR7 on CD8+T cells. Number in the quadrants represent the average results of 20 examined patients as reported (C).C, The average of results obtained in the PBMCs of the whole patient population, collected at baseline [’] and after 6 treatment courses[&]. The histograms show a significant increase in T lymphocytes expressing a CCR7+/CD45RA+ naive (column II and upper right in thedot plot) and a CCR7+/CD45RA# central memory (column III and lower right in the dot plot) T-cell phenotype; a significant decrease inCCR7#/CD45RA+ T cells (column I and upper left in the dot plot) and minimal reduction in CCR7#/CD45RA# T-cell subset (column IVand lower left in the dot plots). *Statistically significantly different compared with control.

J Immunother ! Volume 00, Number 00, ’’ 2012 Newest Cetuximab-based Chemoimmunotherapy

r 2012 Lippincott Williams & Wilkins www.immunotherapy-journal.com | 5

chemotherapy and then to cetuximab gave rise to a robustcolon cancer-specific CTL response with multiepitope-spe-cific antitumor activity. This e!ect was much greater thanthat observed in T-cell lines sensitized against tumor cellsexposed to chemotherapy alone (GILF or ILF), cetuximabalone, or simply irradiation.8 This finding was explained bythe hypothesis that cetuximab improves DC-mediatedphagocytosis and processing of EGFR-expressing tumorcells. Moreover, GILF polychemotherapy is necessary, be-cause it induces: (i) apoptosis; (ii) upregulation of tumorantigens like CEA and TS; (iii) powerful immunologicdanger signals mediated by heat shock protein 90 and cal-reticulin; and ( iv) EGFR upregulation that makes tumorcells still more susceptible to cetuximab binding and opso-nization.23 In addition, we found that the interaction ofGILF+cetuximab-exposed colon cancer cells with humanDCs promotes their maturation and antigen-presentingability represented by an increased expression of HLA-DRand costimulatory molecules such as CD80, CD83, andCD86.8 Our findings were additionally supported by theresults of Lopez-Albaitero and colleagues who clearly

demonstrated that the events occurring after tumor cellphagocytosis/trogocytosis mediated by human DCs areessential processes to obtain an e"cient cross priming andCTL response.29,36,37

The immune–biological results of our clinical trial,which was designed to investigate in advanced colon cancercells the e!ects of the newest regimen based on the com-bination of GILF chemotherapy with cetuximab and low-dose metronomic IL-2, presented strong analogies withthese preclinical findings. In fact, we found that the treat-ment was followed by a significant increase in NK cells,myeloid APCs, and peripheral mature DCs. It is interestingto note that we found a trend to decrease in NKT cellscounterbalanced by a statistically significant increase inCD3+CD56+ cells; this result may be correlated to atreatment-mediated increase in gd-T lymphocytes; however,further investigations are needed to clarify this point. Inaddition, we found that the treatment produced a profoundinfluence on the T-cell compartment, and even if we didnot observe a reduction in the production of IL-10, wefound a significant increase in IFN-g secretion in response

NDPre

Post NDPre

Post0

30

60

90

120A B

Virosome Tumor cells

[IL-1

0], p

g/m

l # *# *

FIGURE 2. The histograms represent the results of an enzyme-linked immunosorbent assay aimed to detect the concentration ofinterleukin (IL)-10 (A) and interferon (IFN)-g (B) in the supernatant of patients’ peripheral blood mononuclear cells (PBMCs) in vitrostimulated with influentia virosomes or irradiated colon cancer cells. The test was performed on the PBMCs of normal healthy donor(ND) and those of colon cancer patients enrolled in the GILFICet trial isolated at baseline (Pre) and after 6 treatment courses (Post). Ouranalysis revealed that patient treatment did not influence the production of IL-10 by PBMCs upon antigen stimulation. However, itsproduction in cancer patients was much greater than that observed in the PBMCs of normal healthy donors. In contrast, the PBMCs ofpatients who had received 6 GILFICet courses showed a significant increase in the production of IFN-g, which was much greater thanthat detected in the supernatant of PBMCs derived from normal healthy donors and the same colon cancer patients at baseline(*P < 0.05).

#

# *

*

A B

*

# *

I II III IV I II III IV

FIGURE 3. This figure shows the results of an immune-cytofluorimetric analysis aimed to evaluate the percentage of differentT-lymphocyte subsets: CD3+CD4+ (column I); CD4+FoxP3+ (Treg) (column II); CD3+CD8+ (column III); and CD8+Ki67+ [proliferatingcytotoxic T lymphocytes (CTLs)] (column IV) in tumor-specific T-cell lines derived from HLA-A(*)02.01 + colon cancer patients whoreceived standard (FOLFOX) chemotherapy (A) and patients enrolled in the GILFICet trial (B). T-cell lines were in vitro generated, asdescribed in the Materials and methods section, from peripheral blood mononuclear cells taken from colon cancer patients at baseline[’] and after 6 treatment courses (either FOLFOX or GILFICet treatment) [&]. A, Significant reduction in Treg and proliferating CTLs inthose patients who had received standard (FOLFOX) chemotherapy. B, Significant decrease in Treg and a substantial increase inproliferating CTLs in those patients who had received the GILFICet treatment (*P < 0.05).

Botta et al J Immunother ! Volume 00, Number 00, ’’ 2012

6 | www.immunotherapy-journal.com r 2012 Lippincott Williams & Wilkins

to di!erent tumor-associated antigens after 6 courses oftreatment, thus supporting an e!ective PBMCs’ switchtoward an antitumor cytotoxic functional Th1 phenotype.The last event was also supported by a significant increasein the percentage of T cells with migrant (CD62L+) andcentral memory T-cell phenotype and cytotoxic naıveT cells. These subpopulations of T lymphocytes represent afresh source of e!ectors capable of di!erentiating intohighly specific antitumor CTLs or T cells with long-lastingmemory and to produce Th1-associated cytokines.23,24 Ourstudy also revealed the ability of this regimen to empowerthe antitumor antigen-specific CTL compartment. We ob-served that tumor-specific T-cell lines derived from thePBMCs of patients exposed to the GILFICet regimen,compared with those derived from the PBMCs of the samepatients at the baseline and from normal healthy donors orpatients undergone to standard chemotherapy with 5-FU,levofolinic acid, and oxaliplatin (FOLFOX-4), presenteda significant reduction in immune-suppressive Treg, a muchgreater amount of proliferating CTLs (CD8+Ki67+),and a significant increase in the number of TS and CEAepitope-specific T-cell precursors.

Altogether these findings suggest that our bio-chemotherapy regimen exerted immune-modulating e!ectsin advanced colon cancer patients, resulting in: (i) an im-proved NK-cell expression; (ii) increased DC-mediatedantigen presentation; and (iii) empowered antitumor-spe-cific CTL response with long-lasting memory.

These results provide strong support to the emerginghypothesis of a possible involvement of CTL-dependentimmunity in cetuximab mediated anticancer e!ects, whosee!ective therapeutic role and potential applications needfurther studies in cancer patients.

CONFLICTS OF INTEREST/FINANCIAL DISCLOSURES

All authors have declared there are no financial conflictsof interest in regard to this work.

REFERENCES

1. Herbst RS. Review of epidermal growth factor receptorbiology. Int J Radiat Oncol Biol Phys. 2004;59:21–26.

2. Yarden Y. The EGFR family and its ligands in human cancer:signalling mechanisms and therapeutic opportunities. Eur JCancer. 2001;37:S3–S8.

3. Mass RD. The HER receptor family: a rich target fortherapeutic development. Int J Radiat Oncol Biol Phys.2004;58:932–940.

4. Mendelsohn J, Fan Z. Epidermal growth factor receptor familyand chemosensitization. J Natl Cancer. 1997;89:341–343.

5. Prewett MC, Hooper AT, Bassi R, et al. Enhanced antitumoractivity of anti-epidermal growth factor receptor monoclonalantibody IMC-C225 in combination with irinotecan (CPT-11)against human colorectal tumour xenografts. Clin Cancer Res.2002;8:994–1003.

6. Karapetis CS, Khambata-Ford S, Jonker DJ, et al. K-rasmutations and benefit from cetuximab in advanced colorectalcancer. N Engl J Med. 2008;359:1757–1765.

7. Correale P, Cusi MG, Del Vecchio MT, et al. Dendritic cell-mediated cross-presentation of antigens derived from coloncarcinoma cells exposed to a highly cytotoxic multidrugregimen with gemcitabine, oxaliplatin, 5-fluorouracil, andleucovorin, elicits a powerful human antigen-specific CTLresponse with antitumor activity in vitro. J Immunol.2005;175:820–828.

8. Correale P, Marra M, Remondo C, et al. Cytotoxic drugs up-regulate epidermal growth factor receptor (EGFR) expressionin colon cancer cells and enhance their susceptibility to EGFR-targeted antibody-dependent cell-mediated-cytotoxicity (ADCC).Eur J Cancer. 2010;46:1703–1711.

9. Clynes R. Antitumor antibodies in the treatment of cancer:Fc receptors link opsonic antibody with cellular immunity.Hematol Oncol Clin North Am. 2006;20:585–612.

10. Kalergis AM, Ravetch JV. Inducing tumor immunity throughthe selective engagement of activating Fcg receptors ondendritic cells. J Exp Med. 2002;195:1653–1659.

11. Liu YJ, Kanzler H, Soumelis V, et al. Dendritic cell line-age, plasticity and cross-regulation. Nat Immunol. 2001;2:585–589.

12. Tsang KY, Zaremba S, Nieroda CA, et al. Generation ofhuman cytotoxic T cells specific for human carcinoembryonicantigen epitopes from patients immunized with recombinantvaccinia-CEA vaccine. J Natl Cancer Inst. 1995;87:982–990.

*

*

*

*

Pre-treatment Post-treatment Healty donor

Pep

tide

Ant

igen

PHA

A B

TS1

TS3

TSPP

CAP1

Pre-treatment Post-treatment Healty donor

FIGURE 4. The figure shows the results of an interferon (IFN)-g Enzyme-linked Immuno Spot (ELISPOT) assay aimed to evaluate thefrequency of antigen peptide-specific T-cell precursors in tumor-specific T-cell lines derived from HLA-A(*)02.01 + patients enrolled inthe GILFICet trial and healthy donors. T-cell lines were in vitro generated as described in the Materials and methods section fromperipheral blood mononuclear cells taken from patients at baseline and after 6 treatment courses and normal healthy donors. A,Representative of 1 experiment and shows differences in the number of IFN-g peptide antigen-specific spots among T cells derived fromnormal healthy donors and cancer patients before and after treatment. B, Histogram of the average results performed on multiplepatients and normal donors. T cells derived from patients undergone 6 treatment courses, compared with those derived from patients atbaseline or normal donors had a much greater number of lymphocyte precursors, specific for TS1 [’], TS3 [’], TSPP[’], andCAP1[&]peptides (*P < 0.05). No statistical differences among the different groups were conversely observed when a T-cell responsewas measured against PHA [’] used as an internal control. PHA indicates Phytohemagglutinin.

J Immunother ! Volume 00, Number 00, ’’ 2012 Newest Cetuximab-based Chemoimmunotherapy

r 2012 Lippincott Williams & Wilkins www.immunotherapy-journal.com | 7

13. Correale P, Del Vecchio MT, Di Genova G, et al. 5-fluorouracil-based chemotherapy enhances the antitumoractivity of a thymidylate synthase-directed polyepitopic peptidevaccine. J Natl Cancer Inst. 2005;97:1437–1445.

14. Hara M, Nakanishi H, Tsujimura K, et al. Interleukin-2potentiation of cetuximab antitumor activity for epidermalgrowth factor receptor-overexpressing gastric cancer xeno-grafts through antibody-dependent cellular cytotoxicity. Can-cer Sci. 2008;99:1471–1478.

15. Correale P, Campoccia G, Tsang KY, et al. Recruitment ofdendritic cells and enhanced antigen-specific immune reactivityin cancer patients treated with hr-GM-CSF (Molgramostim)and hr-IL-2. results from a phase Ib clinical trial. Eur J Cancer.2001;37:892–902.

16. Roda JM, Joshi T, Butchar JP, et al. The activation of naturalkiller cell effector functions by cetuximab-coated, epidermalgrowth factor receptor positive tumor cells is enhanced bycytokines. Clin Cancer Res. 2007;13:6419–6428.

17. Milani V, Stangl S, Issels R, et al. Anti-tumor activity ofpatient-derived NK cells after cell-based immunotherapy—acase report. J Transl Med. 2009;7:50–68.

18. Carter LL, Swain SL. Single cell analyses of cytokineproduction. Curr Opin Immunol. 1997;9:177–182.

19. Correale P, Aquino A, Giuliani A, et al. Treatment of colonand breast carcinoma cells with 5-fluoruracil enhancesexpression of carcinoembryonic antigen and susceptibilityto HLA-A(*)02.01 restricted, CEA-peptide-specific cytotoxicT cells in vitro. Int J Cancer. 2003;104:437–445.

20. Correale P, Sabatino M, Cusi MG, et al. In vitro generation ofcytotoxic T lymphocytes against HLA-A2.1-restricted peptidesderived from human thymidylate synthase. J Chemother.2001;13:519–526.

21. Ferrone C, Dranoff G. Dual roles for immunity in gastro-intestinal cancers. J Clin Oncol. 2010;28:4045–4051.

22. Correale P, Cusi MG, Tagliaferri P. Immunomodula-tory properties of anticancer monoclonal antibodies: is the‘magic bullet’ still a reliable paradigm? Immunotherapy. 2011;3:1–4.

23. Correale P, Botta C, Cusi MG, et al. Cetuximab+/#chemotherapy enhances dendritic cell-mediated phagocytosisof colon cancer cells and ignites a highly efficient colon cancerantigen-specific cytotoxic T-cell response in vitro. Int J Cancer.2011;130:1577–1589.

24. Klinger A, Gebert A, Bieber K, et al. Cyclical expression ofL-selectin (CD62L) by recirculating T cells. Int Immunol.2009;21:443–455.

25. Beauvillain C, Cunin P, Doni A, et al. CCR7 is involved in themigration of neutrophils to lymph nodes. Blood. 2011;117:1196–1204.

26. Janssen EM, Lemmens EE, Wolfe T, et al. CD4+ T cells arerequired for secondary expansion and memory in CD8+

T lymphocytes. Nature. 2003;421:852–856.27. Romagnani S. Type 1 T helper and type 2 T helper cells:

functions, regulation and role in protection and disease. Int JClin Lab Res. 1991;21:152–158.

28. Correale P, Rotundo MS, Del Vecchio MT, et al. Regulatory(FoxP3+) T-cell tumor infiltration is a favorable prognosticfactor in advanced colon cancer patients undergoing chemo orchemoimmunotherapy. J Immunother. 2010;33:435–441.

29. Lopez-Albaitero A, Lee SC, Morgan S, et al. Role ofpolymorphic Fc gamma receptor IIIa and EGFR expressionlevel in cetuximab mediated, NK cell dependent in vitrocytotoxicity of head and neck squamous cell carcinoma cells.Cancer Immunol Immunother. 2009;58:1853–1864.

30. Kang X, Patel D, Ng S, et al. High affinity Fc receptor bindingand potent induction of antibody-dependent cellular cytotox-icity (ADCC) in vitro by anti-epidermal growth factor receptorantibody cetuximab. J Clin Oncol. 2007;25:3041. Abstract.

31. Mimura K, Kono K, Hanawa M, et al. Trastuzumab-mediatedantibody-dependent cellular cytotoxicity against esophagealsquamous cell carcinoma. Clin Cancer Res. 2005;11:4898–4904.

32. McDonnell AM, Robinson BW, Currie AJ. Tumor antigencross-presentation and the dendritic cell: where it all begins?Clin Dev Immunol. 2010;2010:539519. Abstract.

33. Beum PV, Mack DA, Pawluczkowycz AW, et al. Binding ofrituximab, trastuzumab, cetuximab, or mAb T101 to cancercells promotes trogocytosis mediated by THP-1 cells andmonocytes. J Immunol. 2008;181:8120–8132.

34. Roda JM, Parihar R, Magro C, et al. Natural killer cellsproduce T cell-recruiting chemokines in response to antibody-coated tumor cells. Cancer Res. 2006;66:517–526.

35. Kalinski P, Mailliard RB, Giermasz A, et al. Natural killer-dendritic cell cross-talk in cancer immunotherapy. Expert OpinBiol Ther. 2005;5:1303–1315.

36. Shaw DR, Griffin FM. Phagocytosis requires repeatedtriggering of macrophage phagocytic receptors during particleingestion. Nature. 1981;289:409–411.

37. Bibeau F, Lopez-Crapez E, Di Fiore F, et al. Impact ofFcgRIIa-FcgRIIIa polymorphisms and KRAS mutations onthe clinical outcome of patients with metastatic colorectalcancer treated with cetuximab plus irinotecan. J Clin Oncol.2009;27:1122–1129.

Botta et al J Immunother ! Volume 00, Number 00, ’’ 2012

8 | www.immunotherapy-journal.com r 2012 Lippincott Williams & Wilkins